Embed Size (px)
Citation preview
Pretreatment of cellulosic waste and high-rate
biogas production
Solmaz Aslanzadeh
ii
Copyright © Solmaz Aslanzadeh School of Engineering University of Borås SE-501 90 Borås (Sweden) Handle-ID http://hdl.handle.net/2320/12853 ISBN 978-91-87525-10-0 (Printed) ISBN 978-91-87525-11-7 (pdf) ISSN 0280-381X, Skrifter från Högskolan i Borås, nr. 47 Printed in Sweden by Ineko AB Borås 2014
iii
Abstract
The application of anaerobic digestion technology is growing worldwide, mainly because of its
environmental benefits. Nevertheless, anaerobic degradation is a rather slow and sensitive process.
One of the reasons is the recalcitrance nature of certain fractions of the substrate (e.g.,
lignocelluloses) used for microbial degradation; thus, the hydrolysis becomes the rate-limiting step.
The other reason is that the degradation of organic matter is based on a highly dynamic, multi-step
process of physicochemical and biochemical reactions. The reactions take place in a sequential and
parallel way under symbiotic interrelation of a variety of anaerobic microorganisms, which all
together make the process sensitive. The first stage of the decomposition of the organic matter is
performed by fast growing (hydrolytic and acid forming) microorganisms, while in the second stage
the organic acids produced are metabolized by the slow growing methanogens, which are more
sensitive than the acidogens; thus, methanogenesis becomes the rate-limiting step.
The first part of this work evaluates the effects of a pretreatment using an organic solvent, N-
methylmorpholine-N-oxide (NMMO), on cellulose-based materials in order to overcome the
challenge of biomass recalcitrance and to increase the rate of the hydrolysis. NMMO-pretreatment
of straw separated from the cattle and horse manure resulted in increased methane yields, by 53%
and 51%, respectively, in batch digestion tests. The same kind of pretreatment of the forest residues
led to an increase by 141% in the methane production during the following batch digestion assays.
The second part of this work evaluates the efficacy of a two-stage process to overcome the second
challenge with methanogenesis as the rate-limiting step, by using CSTR (continuous stirred tank
reactors) and UASB (up flow anaerobic sludge blanket) on a wide variety of different waste
fractions in order to decrease the time needed for the digestion process. In the two-stage semi-
continuous process, the NMMO-pretreatment of jeans increased the biogas yield due to a more
efficient hydrolysis compared to that of the untreated jeans. The results indicated that a higher
organic loading rate (OLR) and a lower retention time could be achieved if the material was easily
degradable. Comparing the two-stage and the single-stage process, treating the municipal solid
waste (MSW) and waste from several food processing industries (FPW), showed that the OLR
could be increased from 2 gVS/l/d to 10 gVS/l /d, and at the same time the HRT could be decreased
from 10 to 3 days, which is a significant improvement that could be beneficial from an industrial
point of view. The conventional single stage, on the other hand, could only handle an OLR of 3
gVS/l/d and HRT of 7 days.
Keywords: Biogas, Two-stage anaerobic digestion, N-methylmorpholine-N-oxide (NMMO)
pretreatment, Lignocelluloses, Textile waste
iv
v
List of Publications
This thesis is mainly based on the results presented in the following articles:
I. Aslanzadeh S, Taherzadeh MJ and Sárvári Horváth I. (2011): Pretreatment of straw fraction
of manure for improved biogas production. Bioresources 6: 5193-5205.
II. Aslanzadeh S, Berg A, Taherzadeh MJ and Sárvári Horváth I. (2014): Biogas production
from N-Methylmorpholine-N-oxide (NMMO) pretreated forest residues. Applied
Biochemistry and Biotechnology, in press.
III. Jeihanipour A, Aslanzadeh S, Rajendran K, Balasubramanian G and Taherzadeh MJ.
(2013): High-rate biogas production from waste textiles using a two-stage process.
Renewable Energy 52: 128-135.
IV. Aslanzadeh S, Rajendran K, Jeihanipour A and Taherzadeh MJ. (2013): The Effect of
Effluent Recirculation in a Semi-Continuous Two-Stage Anaerobic Digestion System.
Energies 6: 2966-2981
V. Aslanzadeh S, Rajendran K and Taherzadeh MJ. A comparative study between
conventional and two-stage anaerobic processes: Effect of organic loading rate and
hydraulic retention time (Submitted).
Statement of Contribution
Paper I: Performed the experimental work of the pretreatments and anaerobic digestion assays
and responsible for the data analyses and manuscript writing.
Paper II: Responsible for parts of the experimental work and data analyses. Active participant
in the preparation and organization of the manuscript.
Paper III: Responsible for parts of the experimental work and involved in the manuscript
preparation and its revision.
Paper IV: Responsible for parts of the experimental work and for the manuscript preparation.
Paper V: Responsible for major part of the experimental work and data analyses as well as the
manuscript preparation.
vi
List of Publications not included in this thesis
Articles:
I. Rajendran K., Aslanzadeh S., Taherzadeh M.J. (2012): Household Biogas Digesters—A
Review. Energies 5, 2911-2942.
II. Rajendran K., Aslanzadeh S., Johansson F., Taherzadeh M.J. (2013): Experimental and
Economical Evaluation of a Novel Biogas Digester. Energy Conversion & Management 74:
183-191.
Book chapters:
I. Aslanzadeh S, Ishola MM, Richards T, Taherzadeh,MJ, (2014): An Overview of Existing
Individual Unit Operations in Biological and Thermal platforms of Biorefineries, In: N.
Qureshi, D. Hodge & A.V. Vertes (Eds): Biorefineries: Integrated Biochemical Processes
for Liquid Biofuels (Ethanol and Butanol), Elsevier, Chapter 1, in press
II. Aslanzadeh S, Rajendran K, Taherzadeh MJ. (2013): Pretreatment of Lignocelluloses for
Biogas and Ethanol Processes, In: Ram Sarup Singh, Ashok Pandey and Christian Larroche
(Eds): Advances in Industrial Biotechnology, Asiatech Publishers Inc, New Delhi, India,
Chapter 8, Pages 125-150.
vii
Table of content
Abstract ............................................................................................................................... iii
List of Publications .............................................................................................................. v
List of Publications not included in this thesis ................................................................ vi
Chapter 1. Introduction ..................................................................................................... 1
Chapter 2. Anaerobic Digestion ....................................................................................... 5
2.1. Biogas industry: current status and challenges ....................................................................... 5
2.2. The AD process and its complexities ...................................................................................... 7
2.2.1. Factors influencing the AD process ............................................................................................. 10
2.3. Bottlenecks of anaerobic digestion ........................................................................................ 12
2.3.1. Organic loading rate ..................................................................................................................... 12
2.3.2. Retention time .............................................................................................................................. 13
2.4. Phase separation ................................................................................................................... 14
Chapter 3. Substrates for biogas production ................................................................ 17
3.1. Substrate composition and its effect on AD ........................................................................... 17
3.1.1. Lignocellulosics-structural carbohydrates .................................................................................... 18
3.1.2. Textile waste-cellulose and synthetic fibers ................................................................................. 20
3.1.3. Starch-non structural carbohydrates ............................................................................................ 21
3.1.4. Organic fraction of municipal solid waste ..................................................................................... 22
3.2. Remarks on theoretical and experimental methods for determination of biogas potential ... 23
3.2.1. Theoretical methods .................................................................................................................... 23
3.2.2. Experimental methods ................................................................................................................. 25
Chapter 4. Approaching the challenge of biomass recalcitrance ................................ 27
4.1. Definition of substrate biodegradability .................................................................................. 27
4.2. Challenges with lignocellulosic recalcitrance ......................................................................... 27
4.3. Microbial strategy for lignocellulose recalcitrance: Cellulosome ........................................... 29
4.4. Goal of pretreatment .............................................................................................................. 30
4.5. Effect of pretreatment on biogas production .......................................................................... 30
4.6. Pretreatment technologies ..................................................................................................... 31
4.6.1. Physical pretreatment .................................................................................................................. 31
4.6.2. Physiochemical pretreatments ..................................................................................................... 31
4.6.3. Biological pretreatment ................................................................................................................ 32
4.6.4. Chemical pretreatments ............................................................................................................... 33
Chapter 5. High-rate anaerobic treatment systems ...................................................... 39
5.1. Background and Status ......................................................................................................... 39
5.2. Upflow anaerobic sludge blanket reactor .............................................................................. 40
5.2.1. Biogranulation of microorganisms ................................................................................................ 42
5.2.2. Factors influencing anaerobic granulation.................................................................................... 43
viii
5.2.3. Characteristics of anaerobic granules .......................................................................................... 46
5.1. Two-stage process for high-rate methane production ........................................................... 47
5.1.1. Batch process- single vs. two-stage ............................................................................................. 48
5.1.2. Two-stage semi-continuous process ............................................................................................ 51
5.1.3. Two-stage- open system vs. closed system ................................................................................. 54
5.1.4. Semi-continuous process- Single vs. two stage ........................................................................... 56
Concluding Remarks ......................................................................................................... 59
Future work ........................................................................................................................ 61
Nomenclature ..................................................................................................................... 64
Acknowledgments ............................................................................................................. 65
References ......................................................................................................................... 68
1
Chapter 1. Introduction
The ultimate aspiration of energy conversion systems is to achieve steady energy output at the
maximum possible conversion rate. Actually, in reality this is easier said than done because of the
recalcitrance nature of the substrate, in addition to the complexity of the anaerobic digestion (AD)
process. The rate of the biogas production is a function of the biochemical processes [1]. The
presence of difficult to degrade material fractions slows down the hydrolysis rate, which in turn
limits the rate of the overall anaerobic digestion process. However, for the easily degradable
materials the methanogenesis is considered as being the rate-limiting step due to the slow growth
rate of methanogens [2, 3].
Attaining the maximum biogas yield, by complete degradation of the substrate, would require a
long retention time of the substrate inside the digester and an equally large digester size. Putting this
into practice, the choice of a system design or of an applicable retention time is often based on a
compromise between receiving the highest achievable biogas yield and having a reasonable plant
economy [4]. In this regard, the organic load is a significant operational parameter, which indicates
how much organic dry matter can be fed into the digester, per volume and time unit. Today, the
total degradation time of the solid organic waste is normally about 30 days for the biogas process.
Nevertheless, it can be even longer depending on the specific substrate and the operational
temperature [4-6]. At lower HRTs (hydraulic retention times), the risk for a washout of certain
microorganisms is high. This makes it difficult to preserve the effective number of useful
microorganisms in the system. To maintain the population of anaerobes, large reactor volumes or
higher retention times is essential [7]. Today, this problem has been solved in the wastewater
treatment systems due to the introduction of the modern ―high-rate‖ reactors, in which the HRT is
decreased dramatically, usually to less than 1 day [8, 9]. Biomass immobilization is the key factor
for the successful applications of the high-rate anaerobic systems in wastewater treatment processes
[8]. However, the drawback of this technique is that it cannot handle a higher total solid content;
2
hence, only dissolved or soluble materials can be used as feed, which is the reason why this
technology has been successful in the wastewater treatment processes [10, 11]. On the other hand,
for the utilization of substrates with a high solid content, it is mandatory to divide the process into
two stages in order to take advantage of the high-rate reactors. Two-stage processes are divided into
two steps in order to optimize the conditions for different groups of microorganisms that are active
in the digestion process. The first step is a hydrolysis reactor, and the conditions are optimized there
to get the solid matter to be solubilized, while in the second step a high-rate reactor is used to
convert the solubilized material into biogas [11]. However, it should be mentioned that regardless of
the process configuration used, the rate of the biogas production will largely depend on the
composition of the substrate, and particularly, on its biodegradability. The hydrolysis of difficult to
degrade substrate fractions is one of the challenges the biogas industry is facing today. Although
materials, such as lignocelluloses, are available in large amounts and receive special attention for
utilization in the biogas production, they are prone to slow degradation; hence, they require some
kind of pretreatment to increase their degradation rate.
In this thesis, the potential of using an organic solvent N-methylmorpholine-N-oxide (NMMO) for
the pretreatment is studied in order to deal with the reluctant nature of lignocellulose- and cellulose-
based substrates and to increase the rate of hydrolysis during the following anaerobic digestion
process. The second part of this thesis focuses on a two-stage process and investigates the
performance at various organic loading rates and hydraulic retention times. For this propose, a wide
variety of substrates with a high total solid content and different degradability was used. The
following studies were performed:
o The effects of an organic solvent called N-methylmorpholine-N-oxide (NMMO)
used for the pretreatment of the straw fraction from the manure and forest residues
were evaluated by measuring the biogas potential during the following anaerobic
digestion process (paper I and II).
o The long-term effects of the best pretreatment conditions used for the forest residues
determined by batch digestion assays were also examined in a semi-continuous
anaerobic digestion system (paper II).
o Application of the NMMO–pretreatment on cellulose-based textile waste and their
subsequent digestion in a high-rate two-stage anaerobic digestion process was
examined at various organic loading rates and hydraulic retention times (paper III).
o The effect of effluent recirculation in a two-stage anaerobic process using
carbohydrate-based starch and cotton as the substrate at various organic loading rates
and hydraulic retention times was evaluated (paper IV).
3
o The effect of an organic loading rate and hydraulic retention time comparing single
stage and two stage processes using municipal solid waste and food processing waste
was evaluated (paper V).
4
5
Chapter 2. Anaerobic Digestion
2.1. Biogas industry: current status and challenges
There is a variety of waste produced by human activities, and the amount of waste generated is on
the rise [12]. Anaerobic digestion of organic waste is of increasing interest as it offers an
opportunity to deal with some of the problems regarding the reduction of the amount of organic
waste, while diminishing the environmental impact and facilitating a sustainable development of the
energy supply [3, 13]. Long-term successful practice and understanding have made anaerobic
digestion to be one of the favorite treatment technologies for the organic fraction of MSW, applying
a range of technological approaches and systems [14].
Anaerobic digestion technology has been developed in the last 20 years. With a total of 244 plants
and a capacity of nearly 8 million tons of organic treatment capacity, anaerobic digestion is already
taking care of about 25% of the biological treatment in Europe [14]. By the year 2015, the
Netherlands and Belgium are expected to convert 80% of the composting plants into anaerobic
digestion as the primary treatment technology [14].
In comparison to other biofuels, in biogas production a wide range of substrates can be utilized as
long as they are biodegradable, which is one of the great advantages [13]. AD systems are
employed in a wide variety of wastewater treatment plants for sludge degradation and stabilization,
and are used in highly engineered anaerobic digesters to treat high-strength industrial and food
processing wastewaters before discharge. In addition, there are many cases of AD systems applied
in the agricultural sector at animal feeding operations and dairies to alleviate some of the impacts of
manure and for energy production [15]. The majority of these AD systems in operation are single
stage. The European market has shown a large inclination toward single-stage over two-stage
digesters [15]. The number of plants treating MSW using two-phase digestion has continued to
6
decline since the beginning of the 90s. It is predicted that no change is expected in this trend,
mainly due to the higher investment and operating costs of running two-stage processes [14]. There
are studies arguing that two-stage anaerobic digestion could provide great advantages over the
single-stage digestion due to a more rapid and more stable treatment achieved [16]. In practice,
however, it is argued that the two-stage digestion has not been able to validate its claimed
advantages in the market, and the added benefits in increasing the rate of hydrolysis and
methanization have not been confirmed [17]. Industrial applications, therefore, have displayed little
acceptance for the two-stage systems so far [18].
Anaerobic digestion systems are often appropriate for all wastewater treatment systems, given that
the solids can be introduced to the system at an acceptable concentration, which includes new
installations as well as retrofits. In fact, a great deal of the existing research on anaerobic digestion
is aimed at retrofitting multi-stage systems into facilities where single-stage processes are already
present. The most important factor in determining whether a multi-stage anaerobic digestion
process is achievable for a system is the concentration of the feed solids. Given that a multi-stage
process could be sensitive to variation in the feed solids, it might not be practicable if the
characteristics of the feed solids concentrations fluctuate extensively [19].
Figure 1. Outline of the development and ratio of 1-phase and 2-phase digestion capacity in Europe. Adapted from [20,
21]
Figure 1 illustrates an overview of the development and the ratio of the one-phase and two-phase
digestion capacity in Europe, respectively. As noticeable, the vast majority is one-phase system [14,
22].
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0
1000
2000
3000
4000
5000
6000
19
90
19
91
19
92
19
93
19
94
19
95
19
96
19
97
19
98
19
99
20
00
20
01
20
02
20
03
20
04
20
05
20
06
20
07
20
08
20
09
20
10
Cu
mu
lati
ve (
Kto
n/y
ear
)
One cumulative Two cumulative Two stage % Single stage %
7
In order to get an overview of the status of anaerobic digestion of the organic fraction of MSW in
Europe, taking into account a wide variety of criteria, a quantitative analysis was performed on the
installed annual capacity up to the year 2014 [14]. It was estimated that the cumulative percentage
of the one-phase processes would add up to 93%, with only 7% remaining for the two-phase
capacity installed in 2014 [14].
Nonetheless, the future role of biogas in Europe is based on the availability of the substrates. The
technology development concerning biofuel production has opened up a larger substrate supply
base. On the other hand, for the same substrate, it generates more rivalry with the other related
technologies [23]. There is an abundant availability of cellulose-based waste, which could be
appropriate for biogas production e.g., lignocelluloses and waste textiles. These materials are
carbohydrate-rich and could be used as a substrate for biogas production. However, the reluctant
nature of these substrates makes them very difficult to digest, as their structure opposes microbial
hydrolysis in biogas production [12, 24]. Today, the application of lignocellulosic materials in
biogas production is limited and for waste textiles, it is nonexistent [12, 24].
The main goal of this thesis is to increase the rate of the biogas production as well as to investigate
the possibilities of difficult-to-degrade cellulose-based materials, utilized as a substrate for the
biogas production. In order to achieve this goal, one must first overcome the difficulties of the
degradation by using a pretreatment to make the material available for the following microbial
degradation, which was the focus in the first part of this thesis. Furthermore, the extent of the
increase in the organic loading and the decrease in the retention time while developing a two-stage
process, utilizing different waste fractions including cellulose-based materials, was evaluated in the
second part of this work.
2.2. The AD process and its complexities
Anaerobic digestion is often considered to be a complex process. The digestion itself is based on a
reduction process consisting of a number of biochemical reactions taking place under anoxic
conditions. By the actions of a variety of anaerobic and facultative anaerobic microorganisms, multi
molecular organic substances are degraded into simpler, chemically stabilized compounds, and the
final products are primarily methane and carbon dioxide and some smaller amounts of other gases,
such as hydrogen sulfide, hydrogen, carbon monoxide, nitrogen, ammonia NH3, and water [25, 26].
These reactions can be divided into four phases of degradation: hydrolysis, acidogenesis,
8
acetogenesis, and methanogenesis [25]. The AD process involves four fundamental steps, as
outlined in Figure 2. The individual phases are carried out in parallel; however, in each phase
different groups of microorganisms are involved, which partially stand in a syntrophic relation to
each other, with dissimilar requirements on the environment. Normally, the first and the second
phase are closely linked to each other while the third phase is closely connected to the fourth phase
[27].
Due to the small amount of energy available in methanogenic conversion, the microorganisms are
compelled to be part of a very complex, well-organized and efficient cooperation, which could be
the primary reason that this step is the final step to occur in the anaerobic digestion process [28].
The mutual reliance of the partner bacteria regarding energy limitations can go so far that neither
group of microorganisms can function without the other and that together they show a metabolic
activity that neither group could carry out on its own. This type of cooperation is called syntrophic
relationship [28]. Syntrophism is a special case of symbiotic collaboration between two
metabolically different types of microorganisms relying on each other, often for energetic reasons in
order to degrade a certain substrate. The term was created to express the close interrelation of fatty
acid oxidizing, fermenting bacteria with hydrogen oxidizing methanogens [28].
Hydrolysis / acidogenesis process
Undissolved compounds like carbohydrates, proteins, and fats are degraded into monomers, which
usually are water-soluble fragments, by exoenzymes. The microorganisms involved are facultative
and obligatorily anaerobic bacteria. In this phase, the covalent bonds are broken down with water
in a chemical reaction. The monomers produced in the hydrolytic phase are taken up by different
facultative and obligatorily anaerobic bacteria and are degraded further into short-chain organic
acids, such as butyric acid, propionic acid, acetic acid, alcohols, hydrogen, and carbon dioxide. The
concentration of the hydrogen formed as an intermediate product in this stage influences the type of
final products produced during the fermentation process. For example, if the partial pressure of the
hydrogen were too high, it would decrease the amount of reduced compounds (e.g., acetate). In
general, during this phase, simple sugars, fatty acids, and amino acids are converted into organic
acids and alcohols [29].
Acetogenesis /Methanogenesis
The products produced in the acidogenic phase are consumed as substrates for the other
microorganisms, active in the third phase. In the third phase, also called acetogenic phase, anaerobic
9
oxidations are performed. It is important that the organisms, which carry out the anaerobic
oxidation reactions, collaborate with the next group, the methane forming microorganisms. This
collaboration depends on the partial pressure of the hydrogen present in the system. Under
anaerobic oxidation, protons are used as the final electron acceptors, which lead to the production of
H2. However, these oxidation reactions can only occur if the partial pressure of H2 is low, which
explains why the collaboration with the methanogens is very important, since they will
continuously consume the H2 to produce methane. Hence, during this symbiotic relationship ―inter-
species hydrogen transfer‖ occurs [5, 27, 28, 30].
In the fourth phase, or the methanogenic phase, the methane is formed under strict anaerobic
conditions. These reactions are exergonic. The most important substrates for these microorganisms
are H2, CO2, and acetic acid. The methanogenic microorganisms can be divided into three main
groups:
(1) Acetoclastic methanogenesis Acetate → CH4 + CO2
(2) Hydogenotrophic methanogenesis H2 + CO2 → CH4
(3) Methylotrophic methanogenesis Methanol→ CH4 + H2O
Acetogenesis
Complex organic matter
Biodegradable organic matter
CarbohydratesProteins Lipids
Amino acids,
Sugars Fatty Acids
5% 21%21% 40%
100%
0%20%
Intermediate products
Propionate, Butyrate, etc.
Acetate H2, CO2
46% 34%
35% 11%
Methane, CO2
30%70%
20%8%12%
11% 23%
Hydrolysis
Acidogenesis
Methanogenesis Rate-limiting step
Figure 2. Schematic diagram of the anaerobic digestion process. Adapted from [31]
10
2.2.1. Factors influencing the AD process
As is the case for all biological processes, the steadiness of the living conditions is of great
importance. Factors that affect the anaerobic digestion could be physical, chemical, or biological.
An alteration in the temperature, the composition, and/or amounts of substrates can have fatal
consequences for the gas production. The microbial metabolism processes are reliant on many
parameters. In order to achieve optimal conditions for the degradation process, apart from the
organic loading rate and the hydraulic retention time, various other parameters ought to be
considered and controlled. Given that the environmental requirements of the fermentative bacteria
vary from those of the methane forming microorganisms, the only way that the optimum
environmental conditions for all microorganisms involved can be achieved is in a two-stage system,
i.e., one stage for hydrolysis/acidification and one stage for acetogenesis/methanogenesis [27].
However, if the complete degradation process has to happen in the same reaction system (one-
phase), the requirements for methanogenesis must be prioritized, if not, it would be tough for the
methanogens to continue to be active within the mixed culture, due to their lower growth rate and
higher sensitivity to environmental factors.
Temperature
The time-span of the fermentation period is dependent on the temperature. The temperature of the
digester, even a few degrees, has an effect on nearly all the biological activities, especially on the
methane-forming archaea. The majority of the methane formers are active at two temperature
ranges: mesophilic range (30–35 °C) and the thermophilic range (50–60 °C) [27].
The methanogens are very responsive to thermal fluctuations. Thus, any rapid alterations in the
operating temperature should be avoided. In comparison to the psychrophilic and mesophilic
ranges, the thermophilic operation offers a shorter degradation time, better pathogens reduction,
higher gas production, and enhanced sludge separation. The drawback is that it is more difficult to
control the process [29]. The experiments in papers I and II were performed both at thermophilic
and mesophilic conditions, respectively. The operational temperature in the two-stage continuous
process, investigated in papers III, IV, and V, was in the thermophilic range in the first stage while
the second phase was under mesophilic conditions.
pH
pH is an important parameter in the AD process. It has an extensive influence on the performance
and growth of the various microorganisms involved in the different stages of the process [32, 33].
The pH of the digester can be maintained at a desired range (7.0–8.5) by feeding the system at an
11
optimal organic loading rate (OLR). A pH outside this range could cause disturbances to the system
by affecting most of the microorganisms including the methanogens. The pH of the system relies on
the rate of the intermediates formed (e.g., volatile fatty acids) during fermentation. Upon starting up
a biogas process, the pH in the digester can drop below 6.0 due to the production of volatile acids
during the first degradation steps. However, as methane-forming microorganisms consume the
volatile acids, the pH of the digester increases and then stabilizes [5, 34].
Volatile fatty acid
Volatile fatty acids (VFAs) are important intermediates of the anaerobic digestion process. They
exist in two forms: undissociated and dissociated. The dissociated form takes over at a high pH
level, whereas the undissociated fraction dominates at a lower pH [27]. An increase in the VFAs
leads to a drop in the pH; hence, the undissociated form of VFAs (free fatty acids) will dominate,
which in turn will inhibit the methanogenesis [27, 35]. Apart from the pH-value, the amount of
VFAs therefore is commonly used as an indicator of the performance of anaerobic digesters. It
should be noted that the level of inhibition of total VFA and individual VFAs differ from each other
[36, 37]. In order to monitor the stability of the process in papers II, III, IV, and V, the total volatile
fatty acid concentration was monitored. In paper V, the effect of the individual acid was analyzed as
well.
C/N ratio and ammonia
A C/N ratio in the range of 20 to 30 is considered to be an optimum level for anaerobic digestion
[32]. If the C/N ratio is too high, microorganisms will quickly consume the nitrogen in order to
meet their protein requirements and will no longer take care of the available carbon content of the
material, which would accordingly decrease the gas production. Conversely, if the C/N ratio is too
low, due to the degradation of the proteins and other nitrogenous materials, nitrogen will be
released and build up in the form of ammonium ion (NH4+) or ammonia (NH3) in the system [30,
38]. The chemical equilibrium between the ammonium and the ammonia is controlled by the
temperature and the pH. An increase in the temperature or the pH would shift this equilibrium more
toward NH3. The free ammonia could be a source of inhibition as it is capable of diffusing into the
cell, causing proton imbalance or leading to a potassium loss [39]. Moreover, it should be noted that
microorganisms are capable of adapting to higher levels [27]. The C/N ratio can be adjusted by
feeding the digester with a proper substrate mixture [30, 38]. In papers III and IV, NH4Cl was
added as an ammonium supplement to keep the C/N ratio at 25. In papers III, IV, and V the
concentration of ammonium was monitored.
12
Substrate
The biogas yield and composition are directly affected by the composition of the feed materials
with respect to carbohydrate, fat, and protein contents [5]. Moreover, physical and chemical
characteristics of the substrate used such as pH, moisture content, total and volatile solids (VS),
particle size, and biodegradability play a considerable role in the anaerobic digestion process.
2.3. Bottlenecks of anaerobic digestion
Controlling the anaerobic digestion process is a complicated task. Because of the complex mixed
microbial and substrate spectrum, advanced studies and development are necessary to eliminate
various bottlenecks in the degradation chain. However, practical experience shows that there are
several factors that can be attributed to the process failures in anaerobic digestion. These factors
include: microbiological limitations, affecting automatically the microbial community (e.g.,
ammonia inhibition, trace element insufficiency, etc.) or technical weaknesses of the equipment,
such as insufficient mixing caused by the inappropriate particle size or rheological limitations [40].
For a balanced and stable process, the reaction rate in both stages must be equivalent. If the rate of
the degradation in the first stage is too fast, the concentration of the acids increases, causing an
inhibition of methanogenic microorganisms in the second phase. On the other hand, if the second
phase runs too fast, the production rate of methane becomes limited by the hydrolytic stage [41].
Other bottlenecks related to the process performance include extended reactor start-up times and
process instability, as a result of the slow growth rates and sensitivity to changes in the
environmental conditions of the microorganisms involved in the process. Hence, monitoring the
process by measurements aiming to attain and maintain effective and robust microbial communities
are considered necessary to guarantee stable performance with high efficiencies [42]
2.3.1. Organic loading rate
Organic loading rate (OLR) is defined as the amount of substrate expressed as e.g., total solids (TS),
volatile solids (VS) or chemical oxygen demand (COD) fed to the system per unit volume per unit
time. It is a helpful criterion used for measuring the biological performance of the AD system [18],
since it is very sensitive to the organic loading rate (OLR) and the waste composition [43, 44].
13
It is well-known that easily degradable substrates can be quickly converted into volatile fatty acids
(VFA), which can cause the inhibition of methanogenesis as a consequence of the rapid hydrolysis
rate and accumulation of VFAs. At high OLRs, there is a risk for overloading the system/reactor,
particularly during the period of reactor start-up. In such cases, the feeding rate to the system should
be reduced [45, 46]. Higher OLRs can permit smaller reactor volumes, thus, reducing the capital
cost.
2.3.2. Retention time
Another parameter that basically controls the rate of the substrate conversion into biogas is the
retention time [18]. It is an important parameter in terms of evaluating the conversion efficiency in
the process. Normally, shorter retention times are desired in order to reduce the system costs [30].
The retention time is usually expressed as: the hydraulic retention time (HRT), which states the
approximate time that the liquid sludge remains in the digester, and the solid retention time (SRT),
which is the time that the microorganisms /solids spend in the digester [47]. In general, HRT is
more important if the substrate is complex and slowly degradable, whereas SRT is significant for
easily degradable biomass [13]. In addition, at high OLRs, the retention times should be long
enough for the microorganisms to be able to utilize the substrate. Thus, there is a balance between
the OLR and HRT that must be determined in order to optimize the digestion efficiency and reactor
volume [48].
The different steps in the digestion process are directly connected to the SRT. Reducing the SRT
would decrease the extent of the reactions and vice versa. Whenever the sludge (mixture of biomass
solids and water) is removed from the digester, a portion of the bacterial population is also removed
[49]. Since methanogenic microorganisms have a significantly longer generation time compared to
hydrolytic and acid forming microorganisms, shorter HRTs would cause a washout of slow growing
biomass from the system, which would ultimately jeopardize the process stability and decrease the
conversion efficiency of the process [30]. Therefore, in order to avoid process failure and keep a
steady state condition, the rate of the cell growth must at least compensate the rate of the cell
removal [47, 49]. In general, the hydraulic retention times must be at least 10 – 15 days to avoid
washout from the system [27]. However, the risk for a washout of the microorganisms from the
system can be prevented with phase separation. In order to reach high cell densities of the slow
growing methanogenic microorganisms, the hydraulic and solid retention time should be uncoupled
in the second stage, and it is necessary to raise the solid content in the methanogenic reactor [18]. In
this way, the digestion rate can be increased for a given substrate and reactor volume, and the
14
conversion to methane can be achieved at shorter HRTs. Consequently, a greater amount of
substrate can be converted into methane in a given period of time, thus, increasing the productivity
[30].
2.4. Phase separation
Generally, in an anaerobic digestion process, the rate-limiting step can be defined as the step that
causes process failure under imposed kinetic stress. In other words, in a context of a continuous
culture, kinetic stress is defined as the imposition of a constantly reducing value of the SRT until it
is lower than its limiting value; hence, it will result in a washout of the microorganism [50].
The AD process can be divided into two phases as illustrated in Figure 3. The microorganisms
carrying out the degradation reactions in each of these phases differ widely regarding physiology,
nutritional needs, growth kinetics, and sensitivity to environment. Very often, it is difficult to keep a
delicate balance between these two groups: the acid forming and the methane forming
microorganisms, which lead to reactor instability and consequently low methane yield [51]. Poland
and Gosh [17] were the first to propose that two main groups of microorganisms could physically
be separated with the intention of making use of the difference in their growth kinetics. In order to
accomplish phase separation, several techniques have been employed such as membrane separation,
kinetic control, and pH control [52-56].
Liquification Acidification Methane Formation
Suspended Solids
Dissolved Solids
Organic Acids Acetate Methane
Gas (Methane) PhaseAcid phase
Acetification
Figure 3. Phase separation of the anaerobic digestion system. Adapted from [19]
A two-phase process allows for the selection and enrichment of the microorganisms corresponding
to each of the phases independently from each other. Thus, the first phase can operate at optimal
conditions for the growth of hydrolytic and acidogenic microorganisms, while the second phase can
be optimized for the acetate and methane formation [57].
15
The two-phase process has numerous potential advantages. First of all, it allows for a decrease in
the total reactor volume. Another advantage is the appropriate control of the acidification, which
improves the stability owing to the more heterogeneous bacterial population. The process would
tolerate organic and hydraulic overloading and fluctuations, as the first-phase will function as a
metabolic buffer. Toxic materials and substances that can affect the more sensitive methanogenic
microorganisms will possibly also be eliminated in the first phase [58]. Moreover, fast growing
acidogenic microorganisms may be disposed of, thus, avoiding the loss /washout of the
methanogens.
16
17
Chapter 3. Substrates for biogas production
3.1. Substrate composition and its effect on AD
The substrate composition is extremely important for the microorganisms in the AD process, as it
affects the process stability, gas production, and composition. The substrate should meet the
nutritional requirements of the microorganisms, regarding the energy sources and various
components, vital for building new cells. The substrate should also include a wide variety of
components necessary for the activity of microbial enzyme systems, such as trace elements and
vitamins. When it comes to the decomposition of organic material in the AD process, the ratio of
carbon to nitrogen (C/N ratio) is also regarded to be of great importance [59]. Therefore, the
performance of the AD process is shown to be enhanced by using substrates from different sources
and with the right proportions. Investigations show that co-digestion of substrates from different
sources produce more gas than predicted compared to gas production from the individual substrates
[33, 60, 61]. Substrates that are complex and not too homogeneous encourage the growth of the
numerous types of microorganisms in the digester. A continuous process that is fed with a uniform
composition of substrate, for instance, carbohydrates, for a longer period will lead to a buildup of a
consortium of microorganisms, which will find it difficult to digest the proteins and fat, since most
of the organisms that had the ability to break down the fat and proteins have been washed out from
the process. Therefore, feeding the reactor with a diverse substrate is advantageous, as it amplifies
the build-up of diverse microbiological composition, hence, resulting in the possibility of a stable
and robust process [5].
18
3.1.1. Lignocellulosics-structural carbohydrates
Lignocellulose is the most abundant renewable biomass worldwide [62] with an estimated annual
production between 10–50 billion tons [63]. Since both the cellulose and hemicelluloses are
polymers of sugars, they are potential sources of fermentable sugars. While the hemicelluloses can
be readily hydrolyzed, the cellulose fraction is more unwilling toward the hydrolysis due to the
presence of a lignin shield as well as its crystallinity. A more rigorous pretreatment, therefore, is
required to access the sugars [62]. Consequently, various pretreatment methods have been used to
improve the rate of the hydrolysis of lignocelluloses toward biogas production [24].
Lignocellulosic waste is produced by several sectors including industries, forestry, agriculture, and
municipalities [64]. A large fraction of animal manures consist of straw, which is used as bedding
material in animal cultivation. Straw is a lignocellulosic material; therefore, it makes these kinds of
manure fractions difficult to degrade. A pretreatment is needed to improve the rate as well as the
degree of enzymatic hydrolysis during the degradation process.
Forest residues, another example of lignocellulosic waste, have a potential for energy production.
Forest residues are the biomass material remaining in the forests that have been harvested for
timber, and are more or less identical in composition to forest thinning [65]. Forest residuals consist
of tops and branches, needles, bark, roots, logging residues, etc. It is estimated that in Sweden forest
residues have the energy potential of between 49–59 TWh/year [24]. Today, a major part of these
residues are not used for biogas production due to a high lignin content, which makes it hard to
digest.
In this thesis the effect of the NMMO-pretreatment on the straw fraction of manure (paper I) and
forest residues (paper II) and its subsequent effect on the hydrolysis and biogas production were
investigated.
Cellulose
Cellulose is the main structural component of plant cell walls. Typically, a plant cell wall consists
of up to 35 to 50% cellulose [66], which is a linear polysaccharide polymer of glucose molecule
linked together through β-1,4 glucosidic bonds. The character of the β-1, 4 glucosidic bonds allows
the polymer to build long and straight chains. The level of polymerization of the cellulose, which
refers to the number of glucose units making up one polymer molecule, can range from 800–10,000
units [67]. Cellulose occur in two forms: an unorganized amorphous form and an organized
crystalline form. Within the cell wall, however, the crystalline form of cellulose dominates, which
are less vulnerable to enzymatic degradation than the amorphous cellulose [30]. In crystalline
21
that are removed in order to achieve a high quality fiber for textile manufacturing, which means that
the cotton fibers in the waste textile can be considered to be more or less pure cellulose [12]. Still,
after extensive research, the use of cellulose as a platform for industrial production for different by
products has failed. The challenge of using cellulose is that it is highly crystalline, which opposes
microbial degradation, and a cost effective pretreatment method to overcome the crystallinity has up
to now been elusive. Jeihanipour et al. [81] showed the possibility for hydrolyzing the cotton using
enzymes or acids to achieve glucose and subsequently utilize it as a carbon source in the ethanol
fermentation.
Cotton based blue jeans is a textile waste, basically made of pure cellulose. In this thesis, the
possibility of using pure cotton and blue jeans with and without pretreatment as a substrate in two-
stage semi-continuous high-rate biogas production was investigated in papers III and IV.
3.1.3. Starch-non structural carbohydrates
Apart from sugars, starch is one of the most commonly found non-structural carbohydrates in
anaerobic digesters. Starch is present in food, coming mainly from grains, such as corn and wheat,
and tubers, such as yam and cassava. Starch comprises of two primary biopolymers: amylose,
which is a linear chain of α- 1,4-linked D-glucose units, and amylopectin, which is a chain of α-1,4-
linked D-glucose with branches of α-1,6-linked D-glucose [82]. Starch, which is partially water
soluble, is the primary polysaccharide for storing energy in higher plants. Some forms of starches
are insoluble and resistant to degradation (e.g., wheat breads), whereas others are partially
bioavailable [83]. In this thesis, pure starch was used as an easily degradable substrate to compare
with cotton in order to evaluate the semi-continuous two-stage process (paper IV).
Given that carbohydrates vary in their nature, they are degraded at different rates in the AD process.
Simple sugars and disaccharides are broken down easily and rapidly; this might appear
advantageous, but it can cause instability problems as a result of the accumulation of fatty acids as
intermediary degradation products [84-86]. In addition, carbohydrate-rich materials used to have
poor buffering capacity, and there is a risk of process instability due to a decrease in the alkalinity
of the system [51].
22
3.1.4. Organic fraction of municipal solid waste
Municipal solid waste (MSW) is the waste generated from residential sources, for instance,
households and from institutional and commercial sources such as offices, schools, hotels, and other
sources. The main components of MSW are food, garden waste, paper, board, plastic, textile, metal,
and glass waste [87]. The global production of municipal solid waste (MSW) reached
1.3 billion tons /year in 2010, and it is predicted to increase to more than 2 billion tons/ year by
2025 [88, 89]. The disposal of this increasing volume of waste in a sustainable manner is a major
challenge. It is estimated that the major fraction of the global municipal solid waste consists of food
waste [88]. The application of the anaerobic digestion for the treatment of the organic fraction of
municipal solid waste (OFMSW) has been of interest because of its high content of fats /lipids and
proteins. The main obstacle in the treatment of this type of organic waste is its conversion, due to
the complexity of the organic material [90, 91].
Fats are a major part of the OFMSW and food processing waste (FPW). There are numerous
different lipids (fats, oils, greases), with a varying composition depending on their origin. Lipids are
distinguished by the length of their fatty acid chain, extent of chemical saturation, which refers to
the number of double bonds, and also their physical state, i.e., liquid or solid. Fats are classified as
saturated (found in meat and dairy products), monounsaturated (in vegetable oils and nuts), or
polyunsaturated fats (in fish and corn oil). Saturated fats are less biodegradable than unsaturated
fats. Triglycerides, the most common type of fat, are primarily hydrolyzed into glycerol and long
chain fatty acids (LCFAs) in the AD process[5, 92]. The degradation of fats is generally both easy
and fast [93]. However, while glycerol is rapidly converted into acetate by acidogenesis, the
degradation of LCFA is more complicated. The inhibitory effect of fats is usually connected to the
LCFAs [93, 94]. Fats are a very promising substrate for anaerobic digestion, since high methane
yields can be achieved.
Proteins are present in many organic materials such as OFMSW and FPW, which are rich in energy
and produce a relatively high amount of methane in the AD process. Proteins are linear polymers,
consisting of a string of subunits called amino acids. Proteins are primarily hydrolyzed into
individual amino acids or peptides by the action of an extracellular enzyme called protease [50].
Amino acids are then further broken down to amine groups while releasing ammonia (NH3) or
ammonium (NH4+) in the process. Ammonia and ammonium are in balance with each other, and the
form that would be present in the AD process is dependent on the pH and the temperature. At high
concentrations, ammonia (NH3) could cause inhibition in the AD process, as it can be lethal to
many microorganisms. Methane-producing archaea is the first to become inhibited, as the
23
concentration of ammonia begins to increase [86, 95, 96]. How this inhibition happens is not
completely understood yet. There are hypotheses that ammonia, as an uncharged compound, is
capable of entering the cell and changing the pH inside the cell leading to cell disruption [95]. The
rate and extent of protein degradation is dependent on many factors such as solubility, the category
of end group, pH, and tertiary structure. In general, the rate of protein hydrolysis under an anaerobic
environment is slower than the hydrolysis rate of carbohydrates [50, 97]. In this thesis, OFMSW
and waste from the FPW have been used as a substrate in a high-rate two-stage biogas production
system. Rapid processing was achieved by increasing the loading rate and decreasing the digestion
time (paper V).
3.2. Remarks on theoretical and experimental methods for
determination of biogas potential
3.2.1. Theoretical methods
Biogas production from the organic substrates engages internal redox reactions that convert organic
molecules into CH4 and CO2. The fraction of these two gases are defined by the composition as well
as the biodegradability of the substrates [98].
During the conversion of the carbohydrates, such as sugars, starch, or cellulose, an equal amount of
CH4 and CO2 is produced [27]:
C6H12O6 3 CH4+ 3 CO2 (1)
For proteins, the process can be described as follows:
C13H25O7N3S + 6 H2O 6.5 CH4 + 6.5 CO2 + 3 NH3+ H2S (2)
The degradation of fats and vegetable oils (triglycerides) can be summarized by the following
equation:
C12H24O6 + 3 H2O 7.5 CH4 + 4.5 CO2 (3)
The ideal stoichiometry, for a two-phase digestion, considering the simple case of carbohydrate
degradation, can theoretically be defined as follows:
First stage: C6H12O6 + 2 H2O 4 H2 + 2 C2H4O2 (acetic acid) + 2 CO2 (4)
24
Second stage: 2 C2H4O2 2 CH4 + 2 CO2 (5)
4 H2+CO2 CH4 + 2 H2O (6)
With the remaining sugars present in the substrate being converted into a more reduced form of
products such as propionic acid, butyric acid, ethanol, etc.:
First stage: C6H12O6 C4H8O2 (butyric acid) + 2 CO2 + 2 H2 (7)
Second stage: C4H8O2 + H2O 2.5 CH4 + 1.5 CO2 (8)
These simplified examples can vary according to the effects of numerous factors [27, 98]. For
instance, the reactions are often not complete e.g., up to half of the cellulose is refractory to
microbial degradation, and lignin is entirely inert. Part of the substrates is utilized by the
microorganisms for growth; consequently, there is also some biomass produced.
The theoretical methane potential in practice can be determined, for instance, by using the elemental
composition (C,H,O,S,N) and the Buswell formula (papers III and IV):
CcHhOoNnSs+ yH2O xCH4 + nNH3 + sH2S+ (c-x) CO2 (9)
Where: x= 1/8 (4c+h-2o-3n-2s)
The component composition e.g., carbohydrate, fat, and protein content of the substrate (papers I
and V) can also be used for the calculations according to the data presented in Table 1 below:
Table .1 Buswell’s formula for theoretical methane potential
Component Chemical formula Theoretical methane yield
(m3CH4 /kg VS)
Carbohydrates C6H10O5 0.42
Lipids/fats C57H104O6 0.50
Proteins C5H7O2N 1.01
For liquid substrates, such as wastewater, with low particulate organic content, the chemical oxygen
demand (COD) is followed by:
CH4 + 2 O2 CO2 + H2O (10)
25
One mole of CH4 needs two moles of O2 to oxidize carbon to carbon dioxide and water. Thus, one
kilogram COD is equal to 0.35 m3
CH4.
All these methods presented above are based on the assumption that the substrate is completely
degraded, and the utilization of the substrate for microbial growth is negligible [13, 27, 99].
3.2.2. Experimental methods
Theoretical methods assume complete degradation of the organic material. However, in practice the
actual digestibility is lower, which means that the calculated methane potential is usually higher
than the measured methane potential. The reason for that is that several factors, such as the presence
of inhibitors, the lack of a growth factor, nutrients, and suboptimal conditions during the actual
digestion process will limit the degradation and the biogas production will not reach its theoretically
calculated potential. Therefore, a digestion test of a substrate should be performed as a tool for the
actual biogas potential. Digestion tests can be performed in different scales and modes. In this
thesis, all the experiments were done in a lab scale. In batch single-stage and batch two-stage mode,
the biogas potential of the untreated and the pretreated materials were tested; in semi-continuous
single-stage and two-stage modes, the long-term effects of the digestion process were evaluated.
Batch digestion
Batch digestion assay is a method that is normally used for determining the methane potential and
for kinetic evaluation of the substrate. The substrate and the inoculums (different ratios) are placed
in the reactor, which is sealed thereafter. To provide an anaerobic environment, the head space is
flushed by a mixture of carbon dioxide and nitrogen and the reactor is then placed in an incubator at
a temperature depending on the inoculums optimum. Normally, these kinds of assays take 50 days
or more, to make sure there is a complete degradation of the substrate, as the anaerobic digestion is
a slow process [13]. Usually, many tests are performed in parallel with different substrates or to
compare different pretreatment methods and conditions.
Semi-continuous digestion
Semi-continuous systems are used to study the performance and stability of the anaerobic digesters,
where microbial populations are adapted for specific substrates; hence, product inhibition can be
accurately assessed over a long-term period with daily supervision. The testing period of the semi-
continuous process is usually several months. CSTR (Continuously stirred tank reactor) is the
extensively used technology in the lab as well as large-scale processes. In a single-stage semi-
26
continuous process, only a CSTR with a retention time between 10 – 50 days is used in order to
avoid washout of the slow growing microbial population[13]. In two-stage, the process is divided
into two parts: the first step digestion tank (CSTR) where the process is focused on hydrolysis and
fermentation. However, biogas is normally also produced, since a complete separation is not always
accomplished. In the second step, the fermentation and hydrolysis products are transferred to
another digestion tank that is adapted for methanogenesis. The high-rate reactors can be utilized in
the second step.
In this thesis, the CSTR reactors were used in the single-stage experiments (paper II); however, for
the two-stage experiments, the CSTR reactors were used in the first stage where fermentation and
hydrolysis occur while a high-rate upflow anaerobic sludge blanket (UASB) reactor using
granulated sludge was used in the second stage for methanogenesis. The two configurations, one
with effluent recirculation from the UASB to the CSTR (closed system) (papers III and V) and the
other without recirculation (open system), (paper IV) are illustrated in Figure 5.
Figure 5. The setup of the semi-continuous two-stage system. (Left) with recirculation (Closed system), and (Right) without recirculation (Open system) (paper IV)
27
Chapter 4. Approaching the challenge of biomass recalcitrance
4.1. Definition of substrate biodegradability
Substrate biodegradability is typically described in terms of rate and degree of degradation. Rate is
the speed of substrate utilization (degradation), which under ideal and steady-state conditions in
absence of inhibition is directly related to the rate of intermediate or product(s) formation. The total
biodegradability stands for the maximum biological degradation, accomplished at solid retention
time equal to infinity. In batch conditions, the ultimate biodegradability is assumed to be attained
when the degradation rate moves towards zero, that is, the stabilization is considered to be
completed. Ultimate biodegradability of organic substrates is decided by physicochemical and
biochemical factors. The characteristics of the bio-molecules, of the influent material, and the
interaction between them, define the degree of complexity of the substrate and its surface area
accessible for enzymatic hydrolysis; therefore, constitute a physicochemical limitation for
biodegradability. Additionally, biochemical inhibition forms a significant factor that determines the
substrate biodegradability, by influencing the rate and finally the extent of any biologically-
mediated reaction taking place in the anaerobic digestion process [83].
4.2. Challenges with lignocellulosic recalcitrance
The plant cell wall offers mechanical strength, upholds the cell shape, controls cell expansion,
regulates transport, gives protection, and accumulates food reserves. Plant biomass has different
layers shielding its cellulose. Cell walls contain the cellulose microfibrils arranged together with
28
polymers, as hemicelluloses, lignin, and pectin. Cells are linked by lamellae, a lignin-rich layer.
Primary cell walls contain cellulosic microfibrils, which are randomly arranged. The secondary cell
wall is composed of three layers, including S1 (outer), S2 (middle), and S3 (inner) layers. S2 is the
thickest layer, comprising the major part of the cell wall. Additionally, the arrangements of these
layers are alternates of horizontal and vertical. S1 is arranged horizontally, while S2 is vertical
followed by S3, which is again horizontal. This arrangement is the reason for the mechanical
strength and the complexity of the plant cell walls [64, 100, 101].
Lignin is an extremely branched, hydrophobic polyphenolic aromatic compound, mainly placed in
the cell walls of vascular plants [102, 103]. Lignin provides rigidity to the plant cell walls and
resistance to biodegradation, and makes up the most significant factor restraining biodegradability
of lignocelluloses in anaerobic digestion systems [104]. Lignin is closely linked to hemicellulose,
which covers cellulose and constructs a physical barrier for hydrolytic enzymes [105]. In fact, the
biodegradability of hemicellulose is directly connected with that of cellulose and inversely related
to lignification [104, 106]. Lignin in itself is considered to be recalcitrant in anaerobic environments
[107]. However, earlier studies have revealed that the degradation of lignin is achievable under
anaerobic conditions, predominantly by the rumen microorganisms [102, 108].
Even after breaking down the lignin shield and exposing the cellulose to the microbial enzymes, a
second challenge appears, which is called cellulose crystallinity [64]. The rate-limiting step in the
hydrolysis of cellulose is not the cleavage of β-1,4 glucosidic bonds between the monomers, but
rather the interruption of a single chain of the substrate from its native crystalline matrix to facilitate
the contact with the active site of enzymes [109]. The cellulose microfibrils are twisted in their
native state. Cellulose is also able to take on a variety of crystalline forms of which two allomorphs
are most important: cellulose I and cellulose II. Cellulose II can be obtained from the cellulose I
after pretreatment, for example [110]. Cellulase enzymes easily hydrolyze the cellulose II portion
because of its lower crystallinity, making it more accessible, whereas the enzyme is not so efficient
when it comes to the crystalline part. It is, therefore, predictable that high-crystallinity cellulose will
be more opposing to enzymatic hydrolysis, and it is commonly acknowledged that decreasing the
crystallinity would enhance the digestibility of the lignocelluloses [64].
In order to improve the enzymatic hydrolysis, it is necessary to eliminate the lignin and
hemicelluloses to increase the accessible surface area of the cellulose. The first requirement for
degradation of the cellulose into simple sugars is the physical attachment of the cellulase enzymes
onto the surface of the cellulose. Thus, physical contact between the cellulytic enzyme and cellulose
is vital for enzymatic hydrolysis [64, 111].
29
Investigations show [111] that the rate of hydrolysis is generally very high at first, but decreases
later. The slowdown of the hydrolysis in the later stages is not because of the lack of available
surface area, but because of the difficulty in the hydrolysis of the crystalline part of the cellulose.
Consequently, a lower rate of hydrolysis might be expected after the hydrolysis of the amorphous
cellulose is completed [64, 111]. Accumulation of only small amounts of hydrolyzed products in the
reactor shows that the conversion of the cellulosic material into soluble products was the rate-
limiting step in the overall AD process [90].
4.3. Microbial strategy for lignocellulose recalcitrance:
Cellulosome
Degradation of cellulosic substrate in nature is accomplished by a variety of microorganisms. In
some cases, microorganisms aid higher animals e.g., ruminants, in transforming the polysaccharides
to more digestible components. Microbial degradation of cellulosic material is one of the most
significant processes in nature. Different microorganism e.g., bacteria and fungi approach the task
in their own specific ways. While aerobes normally produce copious amounts of relevant enzymes
e.g., cellulases and hemicellulases, the anaerobic microorganisms, on the other hand, are much
more frugal in their output of such enzymes. The energy yield per unit sugar hydrolyzed in the
aerobes is much higher than for anaerobes. As a result, the anaerobes tend to adopt other strategies
for degradation of the recalcitrant plant material. Among these strategies, the organization of
enzymes into cellulosome in anaerobic microorganisms is shown to be the most outstanding [112].
The cellulosome consists of an essential set of structural and some enzymatic multi-modular
components. A key non-catalytic subunit called scaffoldin locks different enzymatic subunits into a
complex, using the interaction between cohesin-dockerin. Therefore, the main scaffoldin needs a
series of functional modules, cohesins that are involved in the enzyme attachment. The scaffoldin
consists of the cellulose specific carbohydrate binding module (CBM) used for substrate targeting.
The different enzyme subunits, cellulases and hemicellulases, in particular, contain a specialized
doctrine module, which is complementary to the scaffoldin-based cohesins. The specificity of the
binding between the scaffoldin-based cohesin modules and the enzyme–borne dockerin domains
gives an idea about the supra-molecular construction of the cellulosome. These multi-enzyme
complexes anchor to the cell envelope and to the substrate, and mediate the proximity between the
cells and the cellulose. Binding to the scaffoldin stimulates the activity of each individual
30
component toward the crystalline substrate [113]. The range in cellulosome architecture among the
known cellulosome-producing microorganisms is dependent on the arrangement of their genes
[113].
4.4. Goal of pretreatment
The advantages of the pretreatment of lignocellulosic materials are widely recognized. The aim of
the pretreatment process is to get rid of the lignin and degrade the hemicellulose, decrease the level
of crystallinity in the cellulose, and enhance the porosity of the lignocellulosic materials.
Pretreatment should meet some important requirements [69]:
(1) Enhance the formation of sugars or facilitate the hydrolysis process after the pretreatment
(2) Avoid the degradation or loss of carbohydrates
(3) Not cause the formation of by products that could possibly give rise to inhibition in the
subsequent hydrolysis and fermentation processes
(4) Be economical
4.5. Effect of pretreatment on biogas production
It has already been mentioned that the degradation of complex materials is slow, and the AD
process is therefore usually limited by the long retention times [47]. The anaerobic digestion is rate-
limited by the hydrolysis step; pretreatment methods are therefore often used to support the
solubilization of organic matter. However, it was discovered that sometimes regardless of the high
solubilization after the pretreatment, the anaerobic conversion into methane did not improve. The
poor anaerobic biodegradability performances were accredited to the soluble molecules produced
after the pretreatment had been inhibitory to the anaerobic microorganisms [45]. There are authors
who argue that the rate of the hydrolysis of particulate organic matter is decided by the adsorption
of hydrolytic enzymes to the biodegradable surface sites [114]. Some substrates are either very
resistant against anaerobic digestion due to their compact, complex structure, or they contain
inhibitors [13, 64]. In some cases, the main idea of the pretreatment is to improve the degradation
31
rate and efficiency, as well as improve the bioavailability of the feedstock [64]. In other cases, the
goal is to eliminate the undesirable compounds such as inhibitors. Therefore, the selection of a
suitable pretreatment method should always go hand in hand with the properties of the substrate.
4.6. Pretreatment technologies
In general, pretreatment methods are divided into three distinct categories, namely, physical,
chemical, and biological pretreatments. Combination pretreatment, such as physicochemical
pretreatment by including two or more pretreatment techniques from the same or different
categories is quite common as well. However, combination pretreatment is not considered as an
individual pretreatment category [64, 115-117].
4.6.1. Physical pretreatment
The goal of physical pretreatments is to physically /mechanically decrease the particle size and
reduce the crystallinity. As a result, the accessible surface area and the pore sizes of the biomass
should be increased by these methods. Enhanced hydrolysis is achieved as crystallinity is reduced,
and mass transfer characteristics are improved due to the reduction of the particle size. Milling,
grinding, irradiation, ultrasound and hydrothermal pretreatments are some of the methods that
belong in this category [64, 118]. Forest residues (paper II), blue jeans (paper III), and cotton (paper
IV) were milled to 2 mm particle size prior to the pretreatment and digestion. The OFMSW was
crushed prior to the pretreatment (paper V).
4.6.2. Physiochemical pretreatments
In order to prevail over the recalcitrance of the lignocellulosic biomass, physiochemical
pretreatments, which combine chemical and physical processes, have emerged. The processes that
belong to this group are: ammonia fiber explosion (AFEX), CO2 explosion, SO2 explosion, steam
pretreatment/autohydrolysis, hydrothermolysis, and wet oxidation [13]. These pretreatment methods
have been successfully introduced prior to the biogas production.
32
4.6.3. Biological pretreatment
Biological pretreatments make use of microorganisms’ (e.g., fungi) natural ability to degrade the
lignin and hemicellulose, leaving the cellulose intact [119-121]. The most studied microorganism is
white-rot fungi, which is considered to be promising due to the substrate specificity of its
ligninolytic enzymes [120]. Lignin is then degraded through the action of lignin degrading enzymes
secreted by the fungi. Although biological pretreatments involve mild conditions and are
economically beneficial, the drawbacks connected to these pretreatment methods, such as low rates
of hydrolysis and long pretreatment times, weigh against their advantages compared to other
technologies [122, 123]. However, there are studies combining the biological pretreatment with
other pretreatment technologies, as well as developing novel microorganisms for more rapid
hydrolysis [62, 118-120]. Oat straw treated with white-rot fungi during 28 days of incubation
increased the initial rate of hydrolysis during 10 days of digestion with and without a nutrient-rich
medium, while the total methane yield was slightly higher for the nutrient-rich medium after 28
days with white-rot fungi compared with those of the untreated straw (Figure 6). The total lignin
content decreased from almost 22% for the untreated oat straw to 17% for the treated straw (data
not published). The food processing waste (FPW) used in paper V was partially pretreated and pre-
hydrolyzed as well as acidified to some extent by the microorganisms present in the storage tank for
3–4 days.
Figure 6. Rm0=Rich medium (nutrient added) untreated, RM7=Rich medium 7 days pretreatment, RM14=Rich
medium 14 days pretreatment, RM28=Rich medium 28 days pretreatment, PM 28=Poor medium (without nutrient) 28
days pretreatment
0
0,05
0,1
0,15
0,2
0,25
0,3
0,35
0,4
0,45
0 10 20 30 40 50 60
Vo
lum
e m
3C
H4/
kg V
S
Days
RM0 RM7 RM14 RM28 PM28
33
4.6.4. Chemical pretreatments
Chemical pretreatments involve chemical reactions for the disruption of the biomass structure [67].
Chemical pretreatment is comprised of alkali, dilute acid, alkaline peroxide, oxidizing agents, and
organic solvents [69]. Acid and alkaline pretreatments are the most commonly used chemical
pretreatments today. For the pretreatment of lignocellulosic material, strong acids e.g., sulfuric or
nitric acids are usually used for the removal of lignin and hemicelluloses. For the alkaline
pretreatment, sodium, potassium, calcium, and ammonium hydroxide are reused as a base causing
the degradation of the ester and glycosidic side chains, and thereby modifying the structure of the
lignin, as well as resulting in the cellulose swelling and its partial decrystallization [124]. In this
thesis, chemical pretreatment using N-methylmorpholine-N-oxide (NMMO), which is a cellulose
solvent, has been used for pretreatment prior to the biogas production (Papers I, II, and III).
NMMO- pretreatment
NMMO is a cyclic organic amine oxide, known as a non-derivatizing solvent for cellulose. It has
the ability to dissolve cellulose, while generating a solution with great rheological properties for
fiber spinning. This solvent is already commercially used in the textile industry for the production
of regenerated cellulosic fibers under different trade names, such as Tencel, Lyocell, and Newcell.
In addition to being almost recoverable as well as recyclable, NMMO is considered to be
environmentally friendly due to its non-toxicity and biodegradability potential [12, 125-127]. The
solubilization extent of cellulose in the NMMO-water mixture relies on the concentration of the
NMMO [128]. Optical microscopy investigations of the free floating fibers in the NMMO-water
mixtures using different concentrations of the NMMO showed four modes for the dissolution of
cotton fibers, depending on the concentration of the NMMO [129]:
(1) Fast dissolution by fragmentation with no major swelling (> 83% NMMO)
(2) Swelling by ballooning and dissolution (76–82% NMMO)
(3) Swelling by ballooning and partial dissolution (70–75% NMMO)
(4) Low homogenous swelling and no dissolution (below 65% NMMO)
The dissolution of cellulose entails breaking the hydrogen bonds. The crystallinity of cellulose is
also significant regarding the solubilization. The lower energy level of the crystalline form is more
difficult to dissolve than the amorphous form with a higher energy level. The reason is that the level
34
of solubility corresponds to the difference in the energy level involving the solid and the solution
state [130].
The effect of the NMMO-pretreatment on the rate of the pure cellulose solubilization in batch
anaerobic digestion has been performed earlier [131]. The swelling and ballooning mode showed a
complete degradation during 15 days of digestion. NMMO-pretreatment has been successfully
applied to lignocelluloses, aiming for improvements during the following biogas and bioethanol
production [24, 132-134]. NMMO degrades the intra-molecular hydrogen as well as van der Waals
interactions, and opens up the lignocellulosic structure and reduces its crystallinity [129];
consequently, it improves the methane and ethanol yield from different types of lignocellulosic
materials [24, 132]. Textile wastes, unlike waste from lignocellulosic materials, do not contain any
lignin or hemicelluloses but have a higher crystallinity; thus, the main goal of treating cotton-based
waste textile is simply to reduce its crystallinity [81]. Studies show that regenerated cellulose from
the NMMO-water-cellulose solution is three times more reactive in the hydrolysis reactions than the
untreated cellulose, as the result of the conversion of crystalline cellulose to amorphous cellulose
[135]. In this work, the use of the NMMO as a pretreatment method prior to the biogas production
has been applied on the straw fraction of manure (paper I), forest residues with high lignin content
(paper II) and textile wastes obtained from blue jeans (paper III).
Effect of NNMO-pretreatment on biogas production
The effect of the NMMO-pretreatment on the straw fraction of horse and cattle manure was
investigated (Paper I). The pretreatment was carried out for 5 and 15 h at 120 °C, with 85%
NMMO, and the effects were evaluated by batch digestion assays. The kinetic of the degradation
process was evaluated using the first-order kinetic model, according to Jiménez et al. [136]. The
results showed that the NMMO-pretreatment of the straw fraction of manure could improve the
degradation rate of the manure, and the specific rate constant, k0, was increased from 0.041 to 0.072
(d-1
) for the cattle and from 0.071 to 0.086 (d-1
) for the horse manure (Figure 7).
35
Figure 7. Variation of ln[Gm/ (Gm-G)] values for different retreatment conditions. A-cattle manure, b-horse manure. G =
ln[Gm/ (Gm-G)] =k0t. Where G (ml) is the volume of methane accumulated after a period of time t (days), Gm (ml) is the
maximum accumulated gas volume at an infinite digestion time, k0 (day-1
) is the specific rate constant, and t (days) is
the digestion time (paper I)
Analysis of the pretreated straw showed that the structural lignin content decreased by
approximately 10% for both the samples. Furthermore, the structural changes caused by the
NMMO-pretreatment were confirmed by Fourier transform infrared spectroscopy (FTIR) (Figure
8). The weaker intra- and intermolecular hydrogen bonding as well as van der Waals interactions
could be the reason for improved gas production as increasing the accessible surface area for the
enzyme attachment. This is further confirmed by the increase in the carbohydrate levels after the
pretreatment, which was around 13% for the straw separated from cattle and 9% for the straw
separated from the horse manure. The crystallinity index, or the lateral order index (LOI), was
calculated as the absorbance ratio of the bands around 1,420 and 898 cm-1
. The results showed that
the crystallinity of the cellulose was affected by the pretreatment, as it decreased with increasing
pretreatment time. Consequently, the NMMO-pretreatment for 15 h resulted in an increase of
methane yield by 53 and 51% for the cattle and horse manure, respectively (Paper I).
Time(days)
0 2 4 6 8 10 12 14
ln [
Gm
/(G
m-G
)]
-0,2
0,0
0,2
0,4
0,6
0,8
1,0
Untreated5h treatment15h treatment
0 2 4 6 8 10 12 14
ln[G
m/(
Gm
-G)]
-0,2
0,0
0,2
0,4
0,6
0,8
1,0
1,2
untreated5h treatment15h treatment
(b)
(a)
Time(days)
36
Figure 8. FTIR spectrum of treated and untreated straw in (a) cattle manure and (b) horse manure. Inside the spectrum
a-untreated, b-5 h treatment, c-15 h treatment
Previous studies showed that the water content in the NMMO affect the behaviour of wood and
cotton cellulose fibers [129]. The effect of NMMO concentration (75 and 85%), temperature (120
and 90 °C) and times (3h and 15 h) on the methane yield using forest residues as substrate with high
lignin content was investigated (Paper II). The forest residues were first milled to 0.5 – 2 mm in size
prior to the pretreatments. The following batch anaerobic digestion assays showed that all three
pretreatment conditions had positive effects on the initial reaction rates as well as the total methane
yields comparing to those of the untreated forest residues (Figure 9).
Anaerobic digestion of untreated forest residues resulted in 42 NmL CH4/gVSadded, and the initial
reaction rate of untreated forest residues obtained within the first 10 days of digestion was 0.83
Nml/gVS/d. The pretreatment with the highest NMMO concentration of 85%, temperature of 120°C
and duration time of 15h resulted in higher methane yield of 109 NmL CH4/gVSadded comparing to
that after the treatment with the same NMMO concentration but lower temperature of 90°C and
duration time of 3h (87 NmL CH4/gVSadded). Furthermore, the results indicate that regarding the
methane production the concentration and the pretreatment time are inversely proportional to each
37
other. For instance, the higher the concentration of NMMO 85%, the shorter the pretreatment time
3h and vice versa. This observation seem to be in accordance to previous study of NMMO-
pretreatment of lignocelluloses [132, 137].
Figure 9. Accumulated methane production of NMMO-pretreated and untreated forest residues at mesophilic batch
conditions. The pretreatment conditions are described in the figure
Based on the results from the batch experiment, the best pretreatment (75% NMMO at 120 °C for
15 h) regarding the methane production rate (4.27 NmL CH4/gVS/day) was further studied during
the semi-continuous digestion experiment (Paper II).
The results obtained in our study demonstrated that the NMMO-pretreated forest residues could be
used as a potential substrate in a continuous biogas process. However, forest residues are carbon-
rich substrates; hence, for a nutritional balance they can be utilized in the co-digestion with other
materials [138].
In textile wastes, the lignin or hemicelluloses are negligible; thus, the utilization of these materials
as a substrate for biogas production should be less complex compared to the lignocellulosic
materials. However, there are a wide variety of fibers and colors used in the textile waste that can
cause problems in the anaerobic digestion [12]. Therefore, the goal of a pretreatment is to reduce
the crystallinity, and to enhance the accessible surface area as well as get rid of colors during
washing after pretreatment. Previous studies have already confirmed the effects of the NMMO-
pretreatment on pure cellulose.
0
20
40
60
80
100
120
0 10 20 30 40 50 60
Vo
lum
e N
ml/
gVS
Time (day)
untreated
90, 3h , 85 %
120, 3h, 85%
120, 15h, 75%
38
39
Chapter 5. High-rate anaerobic treatment systems
5.1. Background and Status
Development of high-rate reactor systems demands a separation of the solid retention time from the
hydraulic retention time. This separation can be accomplished by different methods of sludge
retention, for instance, sedimentation, immobilization on a fixed matrix or moving carrier material,
and recycling of the biomass and granulation. Hence, high-rate systems can be divided into
suspended growth and attached-growth processes with expanded/fluidized bed reactors and fixed-
film processes. In an expanded/fluidized bed reactor, sand or porous inorganic particles are used to
build up an attached film. Fixed film processes count on the bacteria to attach to a fixed media, like
rocks, plastic rings, modular cross-flow media, etc. Some systems, such as the anaerobic hybrid
process, unite suspended- and attached-growth processes in a single reactor in order to make use of
the advantages of both types of biomass [139-141]. Current high-rate processes are anchored in the
concept of retaining high viable biomass. A variety of reactor designs has been developed in order
to achieve this goal [142]:
i) Development of biomass aggregates with high settling capabilities, e.g., UASB reactor and
anaerobic baffled reactor.
ii) Attachment of high density viable biomass to certain types of carrier materials e.g., fluidized bed
reactors and anaerobic expanded bed reactors.
iii) Entrapment of biomass aggregates between the packing materials provided for the reactor, e.g.,
down flow anaerobic filter and upflow anaerobic filter. Recent investigations on the development of
new techniques for cell immobilization by using specific capsules made of a membrane, permeable
to nutrients and metabolites with no leakage of the biomass, revealed a promising result towards
applications in the biogas production [143, 144].
40
The application of these high-rate anaerobic treatment systems has been successful in the treatment
of industrial wastewater. The development of this technology was crucial, since large volumes of
wastewater effluent needs to be treated in optimally designed bioreactors aiming to reduce the
treatment time and to increase the treatment efficiency [145-147]. High-rate reactors meet the
conditions for attaining the high retention of viable biomass under high organic loading rates, and
achieving high contact between the biomass and the incoming effluent, resulting in a reduced
reactor size and low process energy requirements [148, 149].
The most recognized sludge bed bioreactors are: Upflow Anaerobic Sludge Blanket (UASB)
Reactor, Expanded Granular Sludge - Bed (EGSB) Reactor, and Internal Circuit (IC) Reactor. The
IC reactor and the EGSB reactor are basically modified forms of the UASB [27]. The worldwide
number of plants in operation using these kinds of techniques between 1980–2007 was estimated to
be 2,266; however, this number declined to 610 between 2002–2007 (Figure 10) [150]. It should be
pointed out that the granular sludge based technologies (UASB, IC, and EGSB) were leading
technologies in the market during the past few decades.
Figure 10. Anaerobic digestion technologies for industrial wastewater for different periods. UASB: upflow anaerobic sludge blanket; EGSB: expanded granular sludge bed; Hybrid: combined system with sludge bed at the bottom part and a filter in the top; IC: internal circulation reactor; AF: anaerobic filter; FB: fluidized bed reactor; CSTR: continuous stirred tank reactor
5.2. Upflow anaerobic sludge blanket reactor
Regardless of its introduction early on, the awareness of anaerobic systems as the main biological
step in wastewater treatment was quite limited until the UASB reactor was developed by Dr. Gatze
Lettinga during the early 70s in the Netherlands, although a rather similar system, called the
UASB 50%
IC 15%
EGSB 12%
CSTR 7%Af 6%
LAGOON 5%
HYBRID 3% FB 2%
1981-2007
UASB 34%
IC 33%
EGSB 22%
CSTR 4%
HYBRID 2%
FB 2% Af 1% LAGOON 1%
2002-2007
41
―biolytic tank,‖ was already studied earlier in 1910 [151]. Today, the UASB reactor is widely used
for the treatment of several types of wastewater from different sources, such as distilleries, food
processing units, tanneries, and municipal wastewater [142, 148, 152, 153].
The reason that the UASB concept became successful is attributable to the establishment of a dense
sludge bed in the bottom of the reactor, where all biological processes occur. The bed is principally
created by the accumulation of incoming suspended solids and bacterial growth. In upflow
anaerobic systems, the microorganisms can aggregate naturally in the flocs and build granules under
specific conditions. These aggregates are quite dense and this characteristic gives the granules good
settling properties, which are not vulnerable to the wash-out from the system under practical reactor
conditions.
Retention of the active biomass, either granular or flocculent, inside the UASB reactor allows for a
good treatment performance at high organic loading rates. The flow of the influent at the bottom of
the UASB system and the biogas produced causes a natural turbulence providing a good contact
between the wastewater and the biomass [154].
One of the main advantages of the UASB technologies is that it has relatively less investment
requirements in comparison to the anaerobic filter or fluidized bed systems. It is worth mentioning
that a long start-up period or a necessity for an adequate amount of granular seed sludge for more
rapid start-up is considered to be the drawbacks of this system [153].
The UASB reactor is typically separated into four compartments: (i) the granular sludge bed, (ii) the
fluidized zone, (iii) the gas-solids separator, and (iv) the settling section (Figure 11). The granular
sludge bed is located in the bottom of the reactor. The wastewater is pumped in at the bottom of the
reactor and moved upward through the granular sludge bed. At this point the organic materials are
biologically degraded and biogas is produced. Just above the granular sludge bed, a fluidized zone
will develop, owing to the production of the biogas. Further biological degradation can occur at this
zone as well. The gas-liquid separator divides the biogas from the liquid. Strong granules with high
settling abilities will settle back to the granular sludge bed, whereas flocculated and dispersed
microorganisms are washed out of the reactor together with the effluent [155].
42
Biogas outlet
Effluent
Tri-phase separator
Gas deflector
Sludge bed
Sludge blanket
Influent
Figure 11. Illustration of an Upflow Anaerobic Sludge Blanket reactor (UASB). Adapted from [156]
5.2.1. Biogranulation of microorganisms
There are numerous theories trying to shed light on the mechanisms of anaerobic sludge
granulation. These theories could be divided into three groups, that is, the physical, microbial, and
thermodynamical approaches, which are believed to be the main factors responsible for the granule
formation [157]. To put it in a simple expression, it is a conglomeration of biomass as a result of the
self-immobilization of the anaerobic microorganisms occurring under hydrodynamic conditions
[158]. However, these theories are not entirely firm as some theories have features that could fit in
to other classifications [157]. One of these theories for the initiation of the granulation process is
illustrated in Figure 12.
In general, it is considered that the extracellular polymeric substances (ECP) play an essential role
in the process of anaerobic biogranulation [159, 160]. Microbial adhesion, i.e., when a cell attaches
to a surface or another is conceded to be the reason for the cell granulation (Figure 22). Under a
proper physiological environment, ECP is excreted by the microbial cells and exposed on their
surfaces [155]. The ECP consists of a complex combination of polymeric substances excreted by
the microorganisms, lysis, and hydrolysis products, and adsorbed organic matter from the
surrounding environment. Proteins, polysaccharides, humic acids, uronic acids along with small
amount of lipids and nucleic acids are found to be the main components of ECP. So far, the exact
role of the components in the formation and the functions of ECP are not understood. Earlier
investigations pointed out that proteins, humic substances, and carbohydrates alter the cell surface
charge, hydrophobicity, and viscosity, which in turn will have an effect on the surface properties of
the bacterial flocs, which aid the adhesion of the flocculated sludge particles together [160].
45
Addition of polymers
One of the key factors for the granule development from non-granular sludge is the existence of a
nuclei or bio carriers for the microbial attachment. Synthetic and natural polymers have been
commonly used during the coagulation/flocculation processes. The addition of polymers, such as
water absorbing polymers (WAP), hybrid polymers, and cationic polymers, encourages particle
agglomeration and enhances the formation of the anaerobic granules considerably [161]. These
polymers behave as ECP substances in the aggregating process of anaerobic sludge. Polymeric
chains form a link between the cells and this encourages the formation of the initial microbial
nuclei, which in turn serves as the first step toward microbial granulation [160].
Addition of cations
There is strong proof that divalent and trivalent cations, such as Ca2+
, Mg2+
, Fe2+
, and Fe3+
could
bind to negatively charged cells and aid the formation of a microbial nuclei [161]; hence, the
presence of cations can be a key factor in the granulation processes. Calcium-enhanced granulation
can be caused by physicochemical and biological effects. The Ca2+
binds with the ECP produced by
microorganisms, thus, promoting anaerobic granulation [161].
Reactor temperature
The performance of an anaerobic system is strongly associated with variations in the temperature.
Methanogenic archaea are the core microbial components of the UASB granules, hence, grow
slowly. There are studies that show that their generation time could range from 3 days at 35 °C to
50 days at 10 °C [162]. This suggests that temperatures below 30 °C would seriously cause an
inhibition in growth of the methanogens. This is the reason that the mesophilic UASB reactors
ought to be operated at a temperature range of 30–35 °C. Even though a relatively high temperature
encourages the growth of the microorganisms, extremely high temperatures would cause a loss of
metabolic activity [161, 163, 164].
Reactor pH
Investigations on the effects of the pH on anaerobic granulation explained that the strength of the
anaerobic granules decreased when the pH increased to a range of pH 8.5–11.0, indicating that high
pH conditions would weaken the granular structure. From pH 5.5 to 8.0, the strength of the granules
was unaffected and relatively stable. However, in the range of pH 5.0 to 3.0, a sharp decline in the
strength of the granule was observed [165]. These results suggest that a somewhat acidic condition
46
would assist the maintenance of the granular structure. As a result, the pH of the reactor needs to be
regularly monitored and kept stable at a very narrow pH range of 6.7–7.4 [161, 162].
5.2.3. Characteristics of anaerobic granules
The microstructure of the anaerobic granules are proposed to be a multi-layered structural model
with acidogenic bacteria dominating the outer layer, methanogenic archaea at the center, and H2-
producing and H2-utilizing microorganisms in the middle layer [166, 167]. Filamentous
microorganisms were also found to be dominant not only on the surface of the granules but also in
the center.
Anaerobic granules in general have a black or dark brown color on their surface. When the OLR
and liquid upflow velocity are low, the granules are found to become lighter (gray or white) with a
hollow core, which makes them extremely soft and very weak under mechanical stress. On the other
hand, at a high OLR and liquid upflow velocity, granules were found to be dark black and had a
dense structure [168]. It has been suggested [168] that the color change and the hollowing of the
granules depends on different mechanisms. The "hollowing" of the granules is most likely
connected to the size of the granules. The feed may penetrate the granules simply by diffusion so
when the size of the granule goes beyond a certain limit, the concentration of the feed becomes too
small in the center of the granules, leading to starvation of the microbial population and
consequently autolysis to occur. Since the autolysis products are not as densely packed compared to
the viable cells, the gas produced will be captured inside the granules, reducing the density, which
would lead to the floatation of the granules. The change in color, on the other hand, is suggested to
be dependent both on the composition of the feed around the granules as well as on the hydro-
dynamic conditions in the reactor. When granules are packed densely at the bottom of the reactor,
nutrient deficiency may arise around the granules for a long period of time. This may cause
irreversible alteration in the granules’ composition and structure. This phenomenon occurs more
likely at low specific loading rates and at low upflow velocities. Higher loading rates and higher
upflow velocities increase the access of the granules to the nutrients and possibly slows down the
color change of the granules [168].
The density of the anaerobic granules stands for the compactness of the microbial community. A
higher density is linked to a faster settling velocity of sludge. The geometric dimension of the
granules has duel effects on the performance of the UASB system. A too small sized granule would
increase the possibility of a washout from the system and thus cause operational instability.
Conversely, for those large-size granules, the efficiency of mass transfer inside the granule would
47
be reduced. Furthermore, the size and density of the anaerobic granules are dependent on many
factors e.g., hydrodynamic conditions, OLR, and microbial species [169, 170]. The mechanical
strength of the granules influences the stability, and it reveals a more compact and stable structure
of the anaerobic granules. The higher the strength of the anaerobic granules, the more attractive
they become for large-scale industrial applications [161].
Figure.13 Size distribution of the granules from the UASB reactor digesting starch
5.1. Two-stage process for high-rate methane production
Besides introducing a pretreatment step for increasing the rate of the degradation, the rate of the
methane production can be accelerated by increasing the rate of conversion of the VFAs into
methane as well; this can be achieved by increasing the concentration of the methanogens in the
reactor. In this work, a two-stage system was developed, where a continuous stirred tank reactor
(CSTR) was used for the first stage and an UASB reactor filled with the granules was used for the
second stage (Papers III, IV, and V). The performance of this two-stage system was evaluated in
different configurations (Figure 5). The CSTR was operated at thermophilic conditions, while the
UASB at mesophilic conditions. The substrates used in the different two-stage processes were
blended fibers, viscose/ polyester, (60/40) and cotton/polyester (50/50) (Paper III); untreated and
48
NMMO-pretreated jeans (Paper III); cotton and starch (Paper IV); and organic fraction of municipal
solid waste (OFMSW), and food processing waste (FPW) (Paper V).
5.1.1. Batch process- single vs. two-stage
The differences between the digestion performances of two untreated cellulosic textile wastes, i.e.,
viscose and cotton blended with polyester, using a single stage CSTR or a two-stage (CSTR and
UASB) system, both in batch operation mode were investigated. In all of these experiments the
same initial cellulose concentration was applied. In the single stage process, cotton/polyester
showed a much longer lag phase compared to the viscose/polyester (Figure 14); furthermore, during
the first 10 days of digestion, 80% of the theoretical yield of methane could be achieved in the case
of viscose/polyester. An earlier batch study [171] showed that anaerobic batch digestion of
regenerated cellulose after the NMMO-pretreatment of viscose/polyester reached 53% of the
theoretical methane within 6 days of digestion; thus, viscose/polyester might not need any
pretreatment at all, as the rate of gas production was not different from the untreated material. In
contrast, for cotton/polyester, after a long lag phase period, only 17% of the theoretical yield of
methane was achieved. In an earlier study on the same material, only 5% of the theoretical yield
was observed after 6 days of digestion [171].
Figure 14. Cumulative methane production in a single stage batch process - Viscose/polyester and Cotton/polyester
(paper III)
0
10
20
30
40
50
60
0 5 10 15 20 25 30
Me
than
e (
ml/
gVS/
day
)
Days
Viscose/polyester
Cotton/polyester
53
Figure 18. Methane volume in semi-continuous two-stage process at different OLR. ― methane volume, --- % share in
CSTR, and - - -% share in the UASB (paper III)
Figure 19. Total VFA concentration in (♦) the UASB and (▲) CSTR for the untreated jeans and the pretreated jeans
(paper III)
0
10
20
30
40
50
60
70
80
90
100
0
100
200
300
400
500
Shar
e o
f re
acto
r in
me
than
e p
rod
uct
ion
(%
)
Me
than
e (
mL/
gVS/
day
)
Untreated Jeans
0
10
20
30
40
50
60
70
80
90
100
0
100
200
300
400
500
0 10 20 30 40 50 60 70 80
Shar
e o
f re
acto
r in
me
than
e p
rod
uct
ion
(%
)
Me
than
e (
mL/
gVS/
day
)
Days
Pretreated Jeans
0
0,4
0,8
1,2
1,6
0
1
2
3
4
5
6
7
Tota
l VFA
in U
ASB
(g/L
)
Tota
l VFA
in C
STR
(g/L
)
Untreated Jeans
0
0,4
0,8
1,2
1,6
0
1
2
3
4
5
6
7
0 10 20 30 40 50 60 70 80
Tota
l VFA
in U
ASB
(g/L
)
Tota
l VFA
in C
STR
(g
/L)
Days
Pretreated Jeans
54
The digestion of starch, on the other hand, showed a much more stable methane production during
an OLR of 2 and 2.7 gVS/l/d and an HRT of 10 and 7 days, respectively (paper IV). However, a
sharp decrease in the methane yield was observed when the OLR was increased to 4gVS/l/d and the
HRT was decreased to 5 days. This is an indication that the hydrolysis process was not inhibited
like methanogenesis in the CSTR, and the volatile fatty acids could still be produced (Figure 22A),
but it was converted into methane in the UASB (Figure 22C) and therefore, the major share of the
methane production was shifted to the UASB without accumulating in the process and causing
failure. This shows that the microbial degradability and the structure of the substrate play a rather
significant role in handling a higher OLR and shorter retention times HRT.
Figure 20. Total methane production in the closed system for the cotton and starch with - - -% share in the CSTR and ---
% share in the UASB (paper IV)
5.1.3. Two-stage- open system vs. closed system
The degradation of starch and cotton was also investigated in the two-stage system in two different
configurations to study the effect of the process on accelerating the digestion. Two-stage processes,
one with recirculation (closed system) and the other without recirculation (open system), were run
in parallel (Figure 5). Starch and cotton were chosen as a substrate since both contain glucose as
monomers, but at opposite ends of the degradability scale. The goal was also to evaluate how these
two configurations would respond to the degradability of these substrates at different OLRs and
0
10
20
30
40
50
60
70
80
90
100
0
50
100
150
200
250
300
350
400
450
Shar
e o
f m
eth
ane
pro
du
ctio
n (
%)
CH
4vo
lum
e (m
l/gV
S/d
ay)
Cotton
0
10
20
30
40
50
60
70
80
90
100
0
50
100
150
200
250
300
350
400
450
0 10 20 30 40 50 60 70 80 90 100
Shar
e o
f m
eth
ane
pro
du
ctio
n (
%)
CH
4vo
lum
e (m
l/gV
S/d
ay)
Days
Starch
55
HRTs. The results of this study suggest that the recirculation has a positive effect regarding the
stability of the two-stage system, especially when higher OLRs and lower HRTs were applied. A
transition pattern observed in both the open and the closed systems showed that the major share of
the methane production shifted from the CSTR to the UASB (Figures 20 and 21). This shift,
however, emerges at earlier stages in the open system compared to the closed system. During this
transition stage, a decrease in the methane yield is observed, which is due to the increase in the
accumulation of the VFAs in the CSTR (Figure 22). The increase in the acids in the CSTR
decreases the pH; thereby, an inhibition of the methanogenic archaea occurs in the CSTR. All the
VFAs produced in the first stage are then converted into methane in the second stage or the
methanogenic phase. This shows that the recirculation could support the hydrolysis step as well as
avoid nutrient loss at a higher OLR, thus, improving the performance and the stability of the process
significantly [172].
Figure 21. Total methane production in the open system for the cotton and starch with --- % share in the CSTR and - - -
% share in the UASB (paper IV)
The capacity of the CSTR to hydrolyze cotton and starch is limited. In the open system, the cotton
did not handle more than 4 gVS/l/d while starch handled an OLR of 10 gVS/l/d, even though the
gas production decreased, but the process could continue until the methane production stopped.
0
10
20
30
40
50
60
70
80
90
100
0
50
100
150
200
250
300
350
400
450
0 10 20 30 40 50 60 70 80 90 100
Shar
e o
f m
eth
ane
pro
du
ctio
n (
%)
CH
4vo
lum
e (m
l/gV
S/d
ay)
Days
Starch0
10
20
30
40
50
60
70
80
90
100
0
50
100
150
200
250
300
350
400
450
Shar
e o
f m
eth
ane
pro
du
ctio
n (
%)
CH
4vo
lum
e (m
l/gV
S/d
ay)
Cotton
56
This also further confirms that the more degradable the substrate, the higher OLR and a lower HRT
can be applied.
Figure 22. Total VFA concentration.●- Starch ♦- Cotton in closed and open system
5.1.4. Semi-continuous process- Single vs. two stage
Anaerobic digestion (AD) under controlled conditions is one suitable technique for the treatment of
OFMSW. This technique is at present in use in large scale applications mostly in Europe [173].
Many attempts have been made to introduce anaerobic digestion processes for treating the organic
fraction of the industrial solid waste [174]. However, the main barrier in spreading this technology
is the lower biodegradation rate of the solid wastes, owing to the complexity of the organic material,
in comparison to the liquid ones [90]. Anaerobic digestion of the OFMSW usually requires a long
retention time of more than 20 days in conventional single stage digesters with a connected large
reactor volume requirements [175]. Pretreatment of the OFMSW to improve the hydrolysis can be
used to solubilize the organic matter prior to the digestion process in order to improve the
performance of the overall AD process, in terms of faster rates and degree of degradation, hence,
decreasing the HRT and increasing the methane production [176]. The application of the OFMSW
0 10 20 30 40 50 60 70 80 90 100
0
0,2
0,4
0,6
0,8
1
1,2
Days
Tota
l VFA
(g/
L)
C-UASB Closed system
0 10 20 30 40 50 60 70 80 90 100
0
0,2
0,4
0,6
0,8
1
Days
Tota
l V
FA (g
/L)
D-UASB -Open system0
2
4
6
8
10
Tota
l V
FA (g
/L)
A-CSTR-Closed system
0
2
4
6
8
10
Tota
l VFA
(g/
L)
B-CSTR-Open system
57
and FPW in a two-stage anaerobic digestion process was investigated (paper V). The objective was
to investigate the optimum OLR and HRTs that could be achieved using the OFMSW and FPW as
substrates in a single stage and two-stage process. The OLR was increased slowly from 2 gVS/l/d to
6 gVS/l/d and the HRT was decreased from 10 days to 3 days and then kept stable at 3 days to
provide a sufficient time for the breakdown of the organic matter in the CSTR, while the OLR was
further increased to 14 gVS/l/d.
The results of this study (Figure 24) show that the two-stage process could enable the possibility of
operating the anaerobic digestion at higher OLRs and lower HRTs. On the other hand, the single
stage reactors could handle an OLR of 3 gVS/l/d as the maximum; moreover, the HRT could not be
decreased to lower than 7 days. In contrast, the two-stage process was stable up to an OLR of 10
gVS/l/d and an HRT of 3 days for both substrates. The increase in the OLR to 12 and 14 gVS/l/d at
an HRT of 3 days showed a decrease in the gas production, but the process did not completely fail
as it did in the case of the single stage system.
Figure 24. Total methane production in the single stage and two-stage process at different organic loading rate and
hydraulic retention times for the organic fraction of municipal solid waste (OFMSW) and food processing waste (FPW)
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
2gVS/l/d 3gVS/l/d 4g VS/l/d 5gVS/l/d 6gvs/l/d 8gVS/l/d 10gvs/l/d 12gvs/l/d 14gvs/l/d
10 d 7 d 5 d 3 d 3 d 3 d 3 d 3 d 3 d
Vo
lum
e C
H4
m3 /
Kg
VS
OLR and HRT
one-stage MSW one-stage FPW two-stage MSW two-stage FPW
58
59
Concluding Remarks
In spite of its advantages, the potential of biogas technology is not fully harnessed, as certain
limitations are associated with it. Most common among these are the long degradation time of the
anaerobic digestion. One of the reasons is attributed to substrates with a recalcitrance structure that
oppose biological degradation and consequently increase the hydrolysis time, and thereby affect the
overall process; thus, hydrolysis becomes the limiting step. Lignin shield together with the compact
cellulose structure of the lignocellulosic material and high crystalline structure of the cellulose in
textile waste is a challenge in the AD process. These problems could be solved by the pretreatment
prior to the biogas production. In this thesis, the use of NMMO as a pretreatment improved the
hydrolysis of the straw fraction of manure and increased the methane yield by 53% for the cattle
and 51% for the horse manure. It also increased the methane yield of the forest residues by 141%
compared to the untreated material. The crystallinity of the cellulose was also affected by the
pretreatment, as it decreased with increasing pretreatment time. However, easily degradable
materials face another challenge in the anaerobic process. If the material is easily degradable, it
causes a problem by hydrolyzing too fast, however, slow growing methanogens are not able to
ferment the hydrolyzed products at the same rate as they are produced, which ultimately leads to the
accumulation of intermediate products, which results in a failed process; thus, the methanogenesis
becomes the limiting step. This means that a decrease in the retention time would lead to a biomass
washout. This challenge is solved by using high-rate systems by separating the process into two
phases. Two-stage processes make it possible to disconnect the dependence of the organic loading
and the retention time. Pretreatment of blue jeans showed that the hydrolysis rate was increased, but
the two-stage process could handle the intermediate products produced without the process failing.
The organic loading rate of the highly crystalline cotton and easily degradable starch could
successfully be increased to 4 gVS/l/d and to 10 gVS/l/d and the retention time could be decreased
to 5 and 2 days, respectively. Application of the two-stage process using the organic fraction of the
municipal solid waste (OFMSW) and the food processing waste (FPW) as inhomogeneous material
showed that the two-stage process could handle an organic loading rate between 8–10g VS /l/d and
60
a retention time of 3 days while the single stage could not handle more than 3g VS/l/d and a
retention time of 7 days.
61
Future work
Research has demonstrated that a two-stage system has several advantages over a conventional
single stage system. However, most of these investigations have been carried out on a lab scale
process. The majority of the two-stage, full-scale, processes have been applied to wastewater
treatment systems with a low solid content. After decades of research, the advantages of the two-
phase anaerobic digestion are yet to be demonstrated.
More pretreatment methods should be investigated for biogas production in order to
decide which pretreatment method is most suitable for the biogas production.
There is still work to be done on the lab scale to investigate the textile waste with
blended fibers. The preliminary work in this thesis shows a potential for digesting
the types of material without pretreatment. However, further work is needed.
Furthermore, the feasibility of using textile waste in the anaerobic digestion should
also be evaluated further.
There is still little known about the application of high solid content materials to
two-stage process on both lab scale and pilot scale. Furthermore, to use the results of
the laboratory scale experiments, it is essential to know whether the results are
transferable to a pilot scale and whether the experiments are reproducible or not.
In reality, the most important benefit claimed for the two-phase digestion, that is, the
reduction in the overall tank sizes, have still not been confirmed. To confirm this
doubt, further investigation is needed in this area especially from an industrial point
of view.
62
The economics of the two-stage process are also one of the obstacles that decrease
the interest in the commercialization of the two-stage process. Economic process
evaluation based on pilot scale data is necessary.
Biogas could be included within a broader category of biomass-related technologies,
and its possibilities will mainly depend on the availability of the biomass
(feedstock). The application of new feedstock (e.g., waste textile and the
lignocelluloses based material in this thesis) to this technology could result in it
having a better standing in the market.
More investigations and attention are needed regarding the by-products of the
anaerobic digestion process such as polyester in blended fibers and lignin left after
the digestion of forest residues in the process. This would be beneficial for the
economics of the process.
63
64
Nomenclature
AD Anaerobic digestion
CSTR Continuous stirred tank reactor
UASB Upflow anaerobic sludge blanket
OLR Organic loading rate
HRT Hydraulic retention time
LCFAs Long-chain fatty acids
SRT Solid retention time
TS Total solids
VS Volatile solids
VFA Volatile fatty acids
COD Chemical oxygen demand
OFMSW Organic fraction of municipal solid waste
FPW Food processing waste
NMMO N-methylmorpholine-N-oxide
ECP Extracellular polymeric substances
65
Acknowledgments
Pursuing my Ph.D. has been a long journey full of emotional ups and downs. Much has happened
and changed during these past four years that could have blocked my path of reaching the end of
this incredible journey. The support and encouragement of many people gave me the strength and
motivation to stay strong through the most difficult periods.
I would like to take this opportunity to acknowledge everyone whose contribution, help and
guidance made this thesis possible and a memorable experience for me in many ways.
I would like to express my deepest gratitude to my supervisor Professor Mohammad Taherzadeh
who gave me this opportunity, guided, and supported me throughout my studies. Thank you for
always being available at any time of the day, at any place in the world, which is one of many
positive qualities I realized about you as a supervisor. It has been a great privilege to have had your
guidance and support throughout this journey.
I wish to express my heartfelt appreciation and gratitude to my co-supervisor Dr. Ilona Sárvári
Horváth for being so kind and helpful. Thank you for your constant motivation, support, and advice,
both professionally and emotionally, especially through those difficult periods. You are an
invaluable friend.
I extend my acknowledgment to my examiner Professor Michael Skrivars, for supporting me in this
thesis.
To Karthik Rajedran, Massoud Salehi, and Håkan Romeborn: thank you for all your help and
support with this thesis. Karthik, your professional support, calming nature, and technical
knowledge as well as your kindhearted words “It will be OK!” during the most stressful times have
all been invaluable to me.
I would also like to declare my greatest appreciation to Dr. Gergely Forgács and Dr. Patrik
Lennartsson for their valuable scientific support during these four years. Thank you for giving me
your precious time and sharing your knowledge.
66
To my fellow lab-mates, and colleagues who supported me in one way or another, some whom I
have spent most of my time with during the past four years, Maryam, Jhosane, Päivi, Johan,
Supansa, Anna, Behnaz, Abbas, Farzad, Wikan, Jorge, Martin, Azam, Akram, Pour, Kamran,
Haike, Adib, Dan, Tomas, Tatiana, Isroi, Khamdan, Mofoluwake, and Julius. Thank you for
creating an environment that made me enjoy the long hours of working.
I also extend my gratitude to Jonas Hanson and Kristina Laurila, the past and the present lab
supervisor, for their technical and practical support in the lab.
I would like to express my profound gratitude to so many people at the Department of Engineering
at the University of Borås for their support and concern, specially the present and former Head of
the Department, Dr. Peter Axelberg and Dr. Hans Björk, Dr. Peter Therning, and Dr. Thomas
Wahnström. I am also very grateful to my teachers, Dr.Magnus Lundin and Dr.Elizabeth Feuk
Lagerstedt, for all the support and concern.
I also want to give great thanks to all the administrative staff, especially Susanne Borg, Sari
Sarhamo, Solveigh Klug, Louise Holmgren, and Thomas Södergren for their practical support.
I would like to extend a special thanks and gratitude to Borås Energy and Miljö AB for financing
this thesis, including Per Karlsson and Anna-Karin Schön. I am also very grateful to Rakel
Martinsson and Camilla Ölander for providing me with information, material, and resources
necessary for my studies.
Above and beyond all, I would like to thank the people that mean the world to me: my parents, my
brother, and my sister. I consider myself fortunate to have such an understanding and loving family.
I cannot imagine a life without your love and support.
67
68
References
[1] Korres NE. Storage and distribution of biomethane. In: N. E. Korres, P. O'Kiely, J. Benzie, J. Wes editors. Bioenergy production by anaerobic digestion: Using agricultural biomass and organic wastes Canada: Taylor & Francis 2013. p. 183-192.
[2] Noike T, Endo G, Chang J-E, Yaguchi J-I, Matsumoto J-I. Characteristics of carbohydrate degradation and the rate-limiting step in anaerobic digestion. Biotechnology and Bioengineering 1985;27:1482-1489.
[3] Jeihanipour A, Niklasson C, Taherzadeh MJ. Enhancement of solubilization rate of cellulose in anaerobic digestion and its drawbacks. Process Biochemistry 2011;46:1509-1514.
[4] Nayono SE. Anaerobic digestion of organic solid waste for energy production vol. 46: KIT Scientific Publishing; 2009. p. 11-23.
[5] Schnürer A, Jarvis A. Microbiological handbook for biogas plants. Swedish waste management , Swedish gas center; 2009. p. 1-74.
[6] Rajendran K, Aslanzadeh S, Taherzadeh MJ. Household Biogas Digesters—A Review. Energies 2012;5:2911-2942.
[7] Bal AS, Dhagat NN. Upflow Anaerobic Sludge Blanket Reactor- A Review. Indian Journal of Environmental Health 2001;43:1-82.
[8] Skiadas IV, Gavala HN, Schmidt JE, Ahring BK. Anaerobic Granular Sludge and Biofilm Reactors. Biomethanation II vol. 82: Springer Berlin Heidelberg; 2003. p. 35-67.
[9] Van Lier JB, Tilche A, Ahring BK, Macarie H, Moletta R, Dohanyos M, Hulshoff Pol LW, Lens P, Verstraete W. New perspectives in anaerobic digestion. Water Science and Technology 2001;43:1–18.
[10] Abbasi T, Abbasi SA. Formation and impact of granules in fostering clean energy production and wastewater treatment in upflow anaerobic sludge blanket (UASB) reactors. Renewable and Sustainable Energy Reviews 2012;16:1696-1708.
[11] Lettinga G, van Velsen AFM, Hobma SW, de Zeeuw W, Klapwijk A. Use of the upflow sludge blanket (USB) reactor concept for biological wastewater treatment, especially for anaerobic treatment. Biotechnology and Bioengineering 1980;22:699-734.
[12] Jeihanipour A. Waste textiles bioprocessing to ethanol and biogas. Sweden: Chalmers University of Technology; Doctoral thesis; 2011.
[13] Forgács G. Biogas production from citrus wastes and chicken feather: pretreatment and co-digestion. Sweden: Chalmers University of Technology; Doctoral thesis; 2012.
[14] De Baere L, Mattheeuws B. Anaerobic Digestion of the Organic Fraction of Municipal Solid Waste in Europe ; Status, Experience and Prospects. Waste Management 2012; 3:517-526.
[15] Rapport J, Zhang R, Jenkins BM, Williams RB. Current Anaerobic Digestion Technologies Used for Treatment of Municipal Organic Solid Waste. California Environmental Protection Agency; 2008.
[16] De Bere L. Anaerobic digestion of solid waste: state-of-the-art. Water Science and Technology 2000;41:283-290.
[17] Pohland FG, Ghosh S. Developments in Anaerobic Stabilization of Organic Wastes - The Two-Phase Concept. Environmental Letters 1971;1:255-266.
[18] Vandevivere P, De Baere L, Verstraete W. Types of anaerobic digester for solid wastes. In: J. Mata-Alvarez editor. Biomethanization of the organic fraction of municipal solid wastes; 2003. p. 111-137.
69
[19] US EPA. Biosolids Technology Fact Sheet- Multi-Stage Anaerobic Digestion. US Environmental Protection Agency 2006;Washington DC: EPA 832-F-806-031.
[20] De Baere L, Mattheeuws B. State-of-the-art 2008-Anaerobic digestion of solid waste Belgium: 2008 http://www.waste-management-world.com.
[21] European Bioplastics. anaerobic digestion. Germany: 2010 http://en.european-bioplastics.org/ [22] De Baere L, Mattheeuws B. Anaerobic digestion of MSW in Europe; 2010 Updates and trends. 25th
Annual Biocycle West Coast Conference. 2010. p. 29. [23] van Foreest F. Perspectives for Biogas in Europe. Oxford Institute for Energy Studies 2011. [24] Teghammar A. Biogas Production from Lignocelluloses: Pretreatment, Substrate Characterization, Co-
digestion and Economic Evaluation. Sweden: Chalmers University of Technology; Doctoral thesis; 2013.
[25] Ziemioski K, Frąc M. Methane fermentation process as anaerobic digestion of biomass: Transformations, stages and microorganisms. African Journal of Biotechnology 2012;11:4127-4139.
[26] Naik SN, Goud VV, Rout PK, Dalai AK. Production of first and second generation biofuels: a comprehensive review. Renewable and Sustainable Energy Reviews 2010;14:578-597.
[27] Deublein D, Steinhauser A. Biogas from waste and renewable resources: An introduction. Weinheim: Wiley-VCH; 2008. p. 89-290.
[28] Schink B. Energetics of syntrophic cooperation in methanogenic degradation. Microbiology and Molecular Biology Reviews 1997;61:262-280.
[29] Gerardi MH. The microbiology of anaerobic digesters. USA: Wiley 2003. p. 89-92. [30] Chandra R, Takeuchi H, Hasegawa T. Methane production from lignocellulosic agricultural crop wastes:
A review in context to second generation of biofuel production. Renewable and Sustainable Energy Reviews 2012;16:1462-1476.
[31] van Haandel AC, van der Lubbe J. Handbook of Biological Wastewater Treatment: Design and Optimisation of Activated Sludge Systems. Netherlands: IWA Publishing; 2007. p. 379-469.
[32] Mittal KM. Biogas Systems: Principles and Applications. New Age International Limited Publishers; 1996. p. 412.
[33] Yadvika, Santosh, Sreekrishnan TR, Kohli S, Rana V. Enhancement of biogas production from solid substrates using different techniques––a review. Bioresource Technology 2004;95:1-10.
[34] Dague RR. Application of digestion theory to digester control. Journal of Water Pollution Control Federation 1968:2021-2032.
[35] Siegert I, Banks C. The effect of volatile fatty acid additions on the anaerobic digestion of cellulose and glucose in batch reactors. Process Biochemistry 2005;40:3412-3418.
[36] Stafford DA. The effects of mixing and volatile fatty acid concentrations on anaerobic digester performance. Biomass 1982;2:43-55.
[37] Wang Q, Kuninobu M, Ogawa HI, Kato Y. Degradation of volatile fatty acids in highly efficient anaerobic digestion. Biomass and Bioenergy 1999;16:407-416.
[38] Hobson PN, Bousfield S, Summers R. Methane production from agricultural and domestic wastes. Springer; 1981. p. 91-163.
[39] Chen Y, Cheng JJ, Creamer KS. Inhibition of anaerobic digestion process: A review. Bioresource Technology 2008;99:4044-4064.
[40] Braun R, Drosg B, Bochmann G, Weiß S, Kirchmayr R. Recent developments in bio-energy recovery through fermentation. In: B. D. R. Braun, G. Bochmann, S. Weiß, R. Kirchmayr editor. Microbes at work: From wastes to resources: Springer; 2010. p. 35-58.
[41] Lee S, Shah YT. Biofuels and Bioenergy: Processes and Technologies. CRC Press; 2012. [42] Martin-Ryals AD. Evaluating the potential for improving anaerobic digestion of cellulosic waste via
routine bioaugmentation and alkaline pretreatmentUniversity of Illinois; 2012. [43] Zuo Z, Wu S, Zhang W, Dong R. Effects of organic loading rate and effluent recirculation on the
performance of two-stage anaerobic digestion of vegetable waste. Bioresource Technology 2013;146:556-561.
[44] Sharma A, Unni BG, Singh HD. A novel fed-batch digestion system for biomethanation of plant biomasses. Journal of Bioscience and Bioengineering 1999;87:678-682.
70
[45] Mata-Alvarez J, Macé S, Llabrés P. Anaerobic digestion of organic solid wastes. An overview of research achievements and perspectives. Bioresource Technology 2000;74:3-16.
[46] Bouallagui H, Touhami Y, Ben Cheikh R, Hamdi M. Bioreactor performance in anaerobic digestion of fruit and vegetable wastes. Process Biochemistry 2005;40:989-995.
[47] Appels L, Baeyens J, Degrève J, Dewil R. Principles and potential of the anaerobic digestion of waste-activated sludge. Progress in Energy and Combustion Science 2008;34:755-781.
[48] Demirer GN, Chen S. Two-phase anaerobic digestion of unscreened dairy manure. Process Biochemistry 2005;40:3542-3549.
[49] Turovskiy IS, Mathai PK. Wastewater sludge processing. USA: John Wiley & Sons, Inc.; 2006. p. 30-60. [50] Pavlostathis SG, Giraldo‐Gomez E. Kinetics of anaerobic treatment: A critical review. Critical Reviews in
Environmental Control 1991;21:411-490. [51] Demirel B, Yenigün O. Two-phase anaerobic digestion processes: a review. Journal of Chemical
Technology and Biotechnology 2002;77:743-755. [52] Ghosh S, Pohland FG. Kinetics of substrate assimilation and product formation in anaerobic digestion.
Journal of Water Pollution Control Federation 1974:748-759. [53] Massey ML, Pohland FG. Phase separation of anaerobic stabilization by kinetic controls. Journal of
Water Pollution Control Federation 1978:2204-2222. [54] Cohen A, Zoetemeyer RJ, Van Deursen A, Van Andel JG. Anaerobic digestion of glucose with separated
acid production and methane formation. Water Research 1979;13:571-580. [55] Pohland FG, Mancy KH. Use of pH and pE measurements during methane biosynthesis. Biotechnology
and Bioengineering 1969;11:683-699. [56] Fernandes IAP. Application of Porous Membranes for Biomass Retention in a Two-phase Anaerobic
Process. University of Newcastle upon Tyne; 1986. [57] Ince O. Performance of a two-phase anaerobic digestion system when treating dairy wastewater.
Water Research 1998;32:2707-2713. [58] Zoetemeyer RJ. Acidogenesis of Soluble Carbohydrate-Containing Wastewaters. Universiteit van
Amsterdam; 1982. p. 132. [59] Yen H-W, Brune DE. Anaerobic co-digestion of algal sludge and waste paper to produce methane.
Bioresource Technology 2007;98:130-134. [60] Pagés Díaz J, Pereda Reyes I, Lundin M, Sárvári Horváth I. Co-digestion of different waste mixtures from
agro-industrial activities: Kinetic evaluation and synergetic effects. Bioresource Technology 2011;102:10834-10840.
[61] Ahring B, Angelidaki I, Macario EC, Gavala HN, Hofman-Bang J, Macario AJL, Elferink SJWHO, Raskin L, Stams AJM, Westermann P, Zheng D. Perspectives for Anaerobic Digestion. Biomethanation I vol. 81: Springer Berlin Heidelberg; 2003. p. 1-30.
[62] Sanchez OJ, Cardona CA. Trends in biotechnological production of fuel ethanol from different feedstocks. Bioresource Technology 2008;99:5270-5295.
[63] Claassen PAM, van Lier JB, Lopez Contreras AM, van Niel EWJ, Sijtsma L, Stams AJM, de Vries SS, Weusthuis RA. Utilisation of biomass for the supply of energy carriers. Applied Microbiology and Biotechnology 1999;52:741-755.
[64] Taherzadeh MJ, Karimi K. Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review. International Journal of Molecular Sciences 2008;9:1621-1651.
[65] U. S. Environmental Protection Agency and Combined Heat and Power Partnership. Biomass Combined Heat and Power, Catalog of Technologies. 2007.
[66] Gopalakrishnan K, van Leeuwen J, Brown RC, Takara D, Khanal S. Biomass Pretreatment for Biofuel Production. Sustainable Bioenergy and Bioproducts: Springer London; 2012. p. 59-70.
[67] Harmsen P, Huijgen W, Bermudez L, Bakker R. Literature review of physical and chemical pretreatment processes for lignocellulosic biomass. Wageningen UR, Food & Biobased Research; 2010.
[68] Eriksson KEL, Babel W, Blanch HW, Cooney CL, Enfors SO, Fiechter A, Klibanov AM, Mattiasson B, Primrose SB, Rehm HJ, Rogers PL, Sahm H, Schügerl K, Tsao GT, Venkat K, Villadsen J, Stockar U, Wandrey C, Kuhad R, Singh A, Eriksson K-E. Microorganisms and enzymes involved in the degradation of plant fiber cell walls. Biotechnology in the Pulp and Paper Industry vol. 57: Springer Berlin Heidelberg; 1997. p. 45-125.
71
[69] Kumar P, Barrett DM, Delwiche MJ, Stroeve P. Methods for Pretreatment of Lignocellulosic Biomass for Efficient Hydrolysis and Biofuel Production. Industrial & Engineering Chemistry Research 2009;48:3713-3729.
[70] Mohnen D, Bar-Peled M, Somerville C. Cell Wall Polysaccharide Synthesis. In: M. E. Himmel editor. Biomass recalcitrance :Deconstructing the plant cell wall for bioenergy. USA: Blackwell Publishing Ltd.; 2009. p. 94-187.
[71] Van Dyk JS, Pletschke BI. A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes—Factors affecting enzymes, conversion and synergy. Biotechnology Advances 2012;30:1458-1480.
[72] Pérez J, Munoz-Dorado J, de la Rubia T, Martinez J. Biodegradation and biological treatments of cellulose, hemicellulose and lignin: an overview. International Microbiology 2002;5:53-63.
[73] Peng P, Bian J, Sun R-C. Extractives. Cereal Straw as a Resource for Sustainable Biomaterials and Biofuels. Amsterdam: Elsevier; 2010. p. 49-72.
[74] Kostamo A, Holmbom B, Kukkonen JVK. Fate of wood extractives in wastewater treatment plants at kraft pulp mills and mechanical pulp mills. Water Research 2004;38:972-982.
[75] Leiviskä T, Rämö J, Nurmesniemi H, Pöykiö R, Kuokkanen T. Size fractionation of wood extractives, lignin and trace elements in pulp and paper mill wastewater before and after biological treatment. Water Research 2009;43:3199-3206.
[76] Rubin EM. Genomics of cellulosic biofuels. Nature 2008;454:841-845. [77] Rosa Estela Q-C, Jorge Luis F-M. Hydrolysis of Biomass Mediated by Cellulases for the Production of
Sugars. 2013. p. 121-141. [78] Quiroz-Castañeda RE, Folch-Mallol JL. Hydrolysis of biomass mediated by cellulases for the production
of sugars. In: A. K. Chandel,S. Silvério da Silva editors. Sustainable degradation of lignocellulosic biomass - Techniques, applications and commercialization InTech; 2013. p. 120-141.
[79] Food and Agriculture Organization Of The United Nations and International Cotton Advisory Committee. World apparel fibre consumption survey July 2013.
[80] Palm. David. Improved waste management of textiles IVL Swedish Environmental Research Institute Ltd, vol. IVL Report B1976 Stockholm Sweden 2011.
[81] Jeihanipour A, Taherzadeh MJ. Ethanol production from cotton-based waste textiles. Bioresource Technology 2009;100:1007-1010.
[82] Kaplan DL. Biopolymers from renewable resources. Springer; 1998. [83] Labatut RA. Anaerobic biodegradability of complex substrates: performance and stability at mesophilic
and thermophilic conditions.USA: Cornell University; Doctoral thesis; 2012. [84] Nallathambi Gunaseelan V. Anaerobic digestion of biomass for methane production: A review. Biomass
and Bioenergy 1997;13:83-114. [85] Bouallagui H, Torrijos M, Godon JJ, Moletta R, Ben Cheikh R, Touhami Y, Delgenes JP, Hamdi M. Two-
phases anaerobic digestion of fruit and vegetable wastes: bioreactors performance. Biochemical Engineering Journal 2004;21:193-197.
[86] Schnürer A, Nordberg Å. Ammonia, a selective agent for methane production by syntrophic acetate oxidation at mesophilic temperature. Water Science and Technology 2008;57:735-740.
[87] Malik A, Grohmann E, Alves M, Albanna M. Anaerobic Digestion of the Organic Fraction of Municipal Solid Waste. Management of Microbial Resources in the Environment: Springer Netherlands; 2013. p. 313-340.
[88] Hoornweg D, Bhada-Tata P. What a Waste: A Global Review of Solid Waste Management. Urban development series Washington, DC: World Bank; 2012.
[89] Rajendran K, Björk H, Taherzadeh MJ. Borås, a zero waste city in Sweden. Journal of Development and Management 2013;1:3-8.
[90] Fdez.-Güelfo LA, Álvarez-Gallego C, Sales Márquez D, Romero García LI. Biological pretreatment applied to industrial organic fraction of municipal solid wastes (OFMSW): Effect on anaerobic digestion. Chemical Engineering Journal 2011;172:321-325.
[91] Cirne DG, Paloumet X, Björnsson L, Alves MM, Mattiasson B. Anaerobic digestion of lipid-rich waste—Effects of lipid concentration. Renewable Energy 2007;32:965-975.
72
[92] Sousa DZ, Pereira MA, Stams AJM, Alves MM, Smidt H. Microbial communities involved in anaerobic degradation of unsaturated or saturated long-chain fatty acids. Applied and Environmental Microbiology 2007;73:1054-1064.
[93] Angelidaki I, Ahring BK. Effects of free long-chain fatty acids on thermophilic anaerobic digestion. Applied Microbiology and Biotechnology 1992;37:808-812.
[94] Woolley P, Petersen SB. Lipases: their structure, biochemistry and application. Cambridge University Press Cambridge; 1994.
[95] Sprott GD, Patel GB. Ammonia toxicity in pure cultures of methanogenic bacteria. Systematic and Applied Microbiology 1986;7:358-363.
[96] Hashimoto AG. Ammonia inhibition of methanogenesis from cattle wastes. Agricultural Wastes 1986;17:241-261.
[97] Breure AM, Mooijman KA, Andel JG. Protein degradation in anaerobic digestion: influence of volatile fatty acids and carbohydrates on hydrolysis and acidogenic fermentation of gelatin. Applied Microbiology and Biotechnology 1986;24:426-431.
[98] Krich K, Augenstein D, Batmale J, Benemann J, Rutledge B, Salour D. Biomethane from Dairy Waste: A Sourcebook for the Production and Use of Renewable Natural Gas in California; 2005. p. 179.
[99] Møller HB, Sommer SG, Ahring BK. Methane productivity of manure, straw and solid fractions of manure. Biomass and Bioenergy 2004;26:485-495.
[100] Bidlack J, Malone M, Benson R. Molecular structure and component integration of secondary cell walls in plants. Proceedings of the Oklahoma Academy of Science 1992;72:51-56.
[101] Himmel ME, Picataggio SK. Our Challenge is to Acquire Deeper Understanding of Biomass Recalcitrance and Conversion. In: M. E. Himmel editor. Biomass recalcitrance :Deconstructing the plant cell wall for bioenergy. USA: Blackwell Publishing Ltd.; 2009. p. 1-6.
[102] Colberg PJ. Anaerobic microbial degradation of cellulose, lignin, oligolignols, and monoaromatic lignin derivatives. In: A. J. B. Zehnder editor. Biology of Anaerobic Microorganisms. USA: Wiley; 1988. p. 333-372.
[103] Hendriks ATWM, Zeeman G. Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresource Technology 2009;100:10-18.
[104] Van Soest PJ. Nutritional Ecology of the Ruminant ; Ruminant Metabolism, Nutritional Strategies, the Cellulolytic Fermentation and the Chemistry of Forages and Plant Fibers. O & B Books; 1982. p. 374.
[105] Angelidaki I, Ahring BK. Methods for increasing the biogas potential from the recalcitrant organic matter contained in manure. Water Science and Technology 2000;41:189-194.
[106] Keys JE, Van Soest PJ, Young EP. Comparative Study of the Digestibility of Forage Cellulose and Hemicellulose in Ruminants and Nonruminants. Journal of Animal Science 1969;29:11-15.
[107] Zeikus JG. Chemical and fuel production by anaerobic bacteria. Annual Reviews in Microbiology 1980;34:423-464.
[108] Hu Z-H, Liu S-Y, Yue Z-B, Yan L-F, Yang M-T, Yu H-Q. Microscale analysis of in vitro anaerobic degradation of lignocellulosic wastes by rumen microorganisms. Environmental Science & Technology 2007;42:276-281.
[109] Bayer EA, Chanzy H, Lamed R, Shoham Y. Cellulose, cellulases and cellulosomes. Current Opinion in Structural Biology 1998;8:548-557.
[110] Taherzadeh MJ, Jeihanipour A. Recalcitrance of Lignocellulosic Biomass to Anaerobic Digestion. In: A. Mudhoo editor. Biogas Production:Pretreatment methods in anaerobic digestion. USA: Wiley 2012. p. 27-54.
[111] Fan LT, Lee Y-H, Beardmore DH. Mechanism of the enzymatic hydrolysis of cellulose: Effects of major structural features of cellulose on enzymatic hydrolysis. Biotechnology and Bioengineering 1980;22:177-199.
[112] Bayer EA, Henrissat B, Lamed R. The cellulosome: A natural bacterial strategy to combat biomass recalcitrance. In: M. E. Himmel editor. Biomass Recalcitrance: Deconstructing the Plant Cell Wall for Bioenergy. USA: Blackwell Publishing Ltd.; 2008. p. 407-435.
[113] Bayer EA, Belaich J-P, Shoham Y, Lamed R. The cellulosomes: multienzyme machines for degradation of plant cell wall polysaccharides. In: M. E. Himmel editor. Biomass recalcitrance :Deconstructing the plant cell wall for bioenergy. USA: Blackwell Publishing Ltd.; 2004. p. 521-554.
73
[114] Veeken A, Hamelers B. Effect of temperature on hydrolysis rates of selected biowaste components. Bioresource Technology 1999;69:249-254.
[115] McMillan James D. Pretreatment of Lignocellulosic Biomass. Enzymatic Conversion of Biomass for Fuels Production vol. 566: American Chemical Society; 1994. p. 292-324.
[116] Wyman C. Handbook on Bioethanol: Production and Utilization. Taylor & Francis; 1996. [117] Zheng Y, Pan Z, Zhang R. Overview of biomass pretreatment for cellulosic ethanol production.
International Journal of Agricultural and Biological Engineering 2009;2:51-68. [118] Brodeur G, Yau E, Badal K, Collier J, Ramachandran KB, Ramakrishnan S. Chemical and
Physicochemical Pretreatment of Lignocellulosic Biomass: A Review. Enzyme Research 2011 2011:1-17
[119] Kumar R, Wyman CE. Effects of cellulase and xylanase enzymes on the deconstruction of solids from pretreatment of poplar by leading technologies. Biotechnology Progress 2009;25:302-314.
[120] Sánchez C. Lignocellulosic residues: biodegradation and bioconversion by fungi. Biotechnology Advances 2009;27:185-194.
[121] Shi J, Chinn MS, Sharma-Shivappa RR. Microbial pretreatment of cotton stalks by solid state cultivation of Phanerochaete chrysosporium. Bioresource Technology 2008;99:6556-6564.
[122] Sun Y, Cheng J. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource Technology 2002;83:1-11.
[123] Johnson DK, Elander RT. Pretreatments for Enhanced Digestibility of Feedstocks. In: M. E. Himmel editor. Biomass recalcitrance :Deconstructing the plant cell wall for bioenergy. USA: Blackwell Publishing Ltd.; 2009. p. 436-453.
[124] Cheng Y-S, Zheng Y, Yu C, Dooley T, Jenkins B, VanderGheynst J. Evaluation of High Solids Alkaline Pretreatment of Rice Straw. Applied Biochemistry and Biotechnology 2010;162:1768-1784.
[125] Tormo J, Lamed R, Chirino AJ, Morag E, Bayer EA, Shoham Y, Steitz TA. Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose. The EMBO Journal 1996;15:5739.
[126] Liu R-G, Shen Y-Y, Shao H-L, Wu C-X, Hu X-C. An Analysis of Lyocell Fiber Formation as a Melt–spinning Process. Cellulose 2001;8:13-21.
[127] Woodings CR. The development of advanced cellulosic fibres. International Journal of Biological Macromolecules 1995;17:305-309.
[128] Eckelt J, Eich T, Röder T, Rüf H, Sixta H, Wolf BA. Phase diagram of the ternary system NMMO/water/cellulose. Cellulose 2009;16:373-379.
[129] Cuissinat C, Navard P. Swelling and Dissolution of Cellulose Part 1: Free Floating Cotton and Wood Fibres in N‐Methylmorpholine‐N‐oxide–Water Mixtures. Macromolecular Symposia vol. 244: Wiley Online Library; 2006. p. 1-18.
[130] Lindman B, Karlström G, Stigsson L. On the mechanism of dissolution of cellulose. Journal of Molecular Liquids 2010;156:76-81.
[131] Jeihanipour A, Karimi K, Taherzadeh MJ. Enhancement of ethanol and biogas production from high‐crystalline cellulose by different modes of NMO pretreatment. Biotechnology and Bioengineering 2010;105:469-476.
[132] Kabir MM, del Pilar Castillo M, Taherzadeh MJ, Horváth IS. Effect of the N-Methylmorpholine-N-Oxide (NMMO) Pretreatment on Anaerobic Digestion of Forest Residues. Bioresources 2013;8:5409-5423.
[133] Shafiei M, Karimi K, Taherzadeh MJ. Pretreatment of spruce and oak by N-methylmorpholine-N-oxide (NMMO) for efficient conversion of their cellulose to ethanol. Bioresource Technology 2010;101:4914-4918.
[134] Lennartsson P. Zygomycetes and cellulose residuals: hydrolysis, cultivation and applications.Sweden: Chalmers University of Technology; Doctoral thesis; 2012.
[135] Zhao H, Kwak JH, Wang Y, Franz JA, White JM, Holladay JE. Interactions between cellulose and N-methylmorpholine-N-oxide. Carbohydrate Polymers 2007;67:97-103.
. A comparative kinetic evaluation of the anaerobic digestion of untreated molasses and molasses previously fermented with Penicillium decumbens in batch reactors. Biochemical Engineering Journal 2004;18:121-132.
74
[137] Shafiei M, Karimi K, Taherzadeh MJ. Techno-economical study of ethanol and biogas from spruce wood by NMMO-pretreatment and rapid fermentation and digestion. Bioresource Technology 2011;102:7879-7886.
[138] Teghammar A, Forgács G, Sárvári Horváth I, Taherzadeh MJ. Techno-economic study of NMMO pretreatment and biogas production from forest residues. Applied Energy 2014;116:125-133.
[139] Lier JBv. Anaerobic industrial wastewater treatment; perspectives for closing water and resource cycles. Proceedings of ACHEMA 2006, 28th International Exhibition Conference on Chemical Technology, Environmental Protection and Biotechnology. Frankfurt, Germany; 2006.
[140] Lens P, Verstraete W. New perspectives in anaerobic digestion. Biological Treatment Processes 2001;43:1.
[141] Nordberg Å, Jarvis Å, Stenberg B, Mathisen B, Svensson BH. Anaerobic digestion of alfalfa silage with recirculation of process liquid. Bioresource Technology 2007;98:104-111.
[142] Pol LH, Lettinga G. New technologies for anaerobic wastewater treatment. Water Science and Technology 1986;18:41-53.
[143] Youngsukkasem S, Rakshit SK, Taherzadeh MJ. Biogas production by encapsulated methane-producing bacteria. Bioresources 2011;7:56-65.
[144] Talebnia F. Ethanol Production from Cellulosic Biomass by Encapsulated Saccharomyces cerevisiae.Sweden: Chalmers University of Technology; Doctoral thesis; 2008.
[145] Barber WP, Stuckey DC. The use of the anaerobic baffled reactor (ABR) for wastewater treatment: a review. Water Research 1999;33:1559-1578.
[146] Chynoweth DP, Owens JM, Legrand R. Renewable methane from anaerobic digestion of biomass. Renewable Energy 2001;22:1-8.
[147] Van Lier J, Van der Zee F, Tan N, Rebac S, Kleerebezem R. Advances in high rate anaerobic treatment: staging of reactor systems. Water Science and Technology 2001;44:15-25.
[148] Lettinga G. Anaerobic digestion and wastewater treatment systems. Antonie van Leeuwenhoek 1995;67:3-28.
[149] Hulshoff Pol L. The phenomenon of granulation of anaerobic sludgeLandbouwuniversiteit te Wageningen; 1989.
[150] Henze M. Biological wastewater treatment: principles, modelling and design. IWA publishing; 2008. [151] Winslow C-EA, Phelps EB. Investigations on the Purification of Boston Sewage. The Journal of
Infectious Diseases 1911;8:259-288. [152] Lettinga G. Sustainable integrated biological wastewater treatment. Water Science and Technology
1996;33:85-98. [153] Rajeshwari KV, Balakrishnan M, Kansal A, Lata K, Kishore VVN. State-of-the-art of anaerobic digestion
technology for industrial wastewater treatment. Renewable and Sustainable Energy Reviews 2000;4:135-156.
[154] Seghezzo L, Zeeman G, van Lier JB, Hamelers HVM, Lettinga G. A review: The anaerobic treatment of sewage in UASB and EGSB reactors. Bioresource Technology 1998;65:175-190.
[155] Schmidt JE, Ahring BK. Granular sludge formation in upflow anaerobic sludge blanket (UASB) reactors. Biotechnology and Bioengineering 1996;49:229-246.
[156] Chan YJ, Chong MF, Law CL, Hassell DG. A review on anaerobic–aerobic treatment of industrial and municipal wastewater. Chemical Engineering Journal 2009;155:1-18.
[157] Hulshoff Pol LW, de Castro Lopes SI, Lettinga G, Lens PNL. Anaerobic sludge granulation. Water Research 2004;38:1376-1389.
[158] Tay J, Xu H, Teo K. Molecular Mechanism of Granulation. I: H+ Translocation-Dehydration Theory. Journal of Environmental Engineering 2000;126:403-410.
[159] Schmidt JEE, Ahring BK. Extracellular polymers in granular sludge from different upflow anaerobic sludge blanket (UASB) reactors. Applied Microbiology and Biotechnology 1994;42:457-462.
[160] Zhou W, Imai T, Ukita M, Li F, Yuasa A. Effect of loading rate on the granulation process and granular activity in a bench scale UASB reactor. Bioresource Technology 2007;98:1386-1392.
[161] Liu Y, Xu H-L, Show K-Y, Tay J-H. Anaerobic granulation technology for wastewater treatment. World Journal of Microbiology and Biotechnology 2002;18:99-113.
[162] Gabriel B. Wastewater Microbiology. Wiley; 1999.
75
[163] Lepistö R, Rintala J. Extreme thermophilic (70°C), VFA-fed UASB reactor: performance, temperature response, load potential and comparison with 35 and 55°C UASB reactors. Water Research 1999;33:3162-3170.
[164] Fang HHP, Lau IWC. Startup of thermophilic (55°C) UASB reactors using different mesophilic seed sludges. Water Science and Technology 1996;34:445-452.
[165] Teo K-C, Xu H-L, Tay J-H. Molecular mechanism of granulation. II: Proton translocating activity. Journal of Environmental Engineering 2000;126:411-418.
[166] Guiot SR, Pauss A, Costerton JW. A structured model of the anaerobic granule consortium. Water Science and Technology 1992;25:1-10.
[167] MacLeod FA, Guiot SR, Costerton JW. Layered structure of bacterial aggregates produced in an upflow anaerobic sludge bed and filter reactor. Applied and Environmental Microbiology 1990;56:1598-1607.
[168] Kosaric N, Blaszczyk R, Orphan L, Valladarfs J. The characteristics of granules from upflow anaerobic sludge blanket reactors. Water Research 1990;24:1473-1477.
[169] Jian C, Shi-yi L. Study on mechanism of anaerobic sludge granulation in UASB reactors. Water Science and Technology 1993;28:171-178.
[170] Pereboom J, Vereijken T. Methanogenic granule development in full scale internal circulation reactors. Water Science and Technology 1994;30:9-21.
[171] Jeihanipour A, Karimi K, Niklasson C, Taherzadeh MJ. A novel process for ethanol or biogas production from cellulose in blended-fibers waste textiles. Waste Management 2010;30:2504-2509.
[172] Alimahmoodi M, Mulligan CN. Anaerobic bioconversion of carbon dioxide to biogas in an upflow anaerobic sludge blanket reactor. Journal of the Air & Waste Management Association 2008;58:95-103.
[173] Eastman JA, Ferguson JF. Solubilization of particulate organic carbon during the acid phase of anaerobic digestion. Journal of Water Pollution Control Federation 1981:352-366.
[174] Wu M-c, Sun K-W, Zhang Y. Influence of temperature fluctuation on thermophilic anaerobic digestion of municipal organic solid waste. Journal of Zhejiang University SCIENCE B 2006;7:180-185.
[175] Climent M, Ferrer I, Baeza MdM, Artola A, Vázquez F, Font X. Effects of thermal and mechanical pretreatments of secondary sludge on biogas production under thermophilic conditions. Chemical Engineering Journal 2007;133:335-342.
[176] Mata-Alvarez J. Biomethanization of the organic fraction of municipal solid wastes. UK: IWA publishing; 2003. p. 201-261.
I
PEER-REVIEWED ARTICLE bioresources.com
Aslanzadeh et al. (2011). “Pretreatment for better biogas” BioResources 6(4), 5193-5205. 5193
PRETREATMENT OF STRAW FRACTION OF MANURE FOR IMPROVED BIOGAS PRODUCTION
Solmaz Aslanzadeh,* Mohammad J. Taherzadeh, and Ilona Sárvári Horváth
Pretreatment of straw separated from cattle and horse manure using N-methylmorpholine oxide (NMMO) was investigated. The pretreatment conditions were for 5 h and 15 h at 120 °C, and the effects were evaluated by batch digestion assays. Untreated cattle and horse manure, both mixed with straw, resulted in 0.250 and 0.279 Nm3 CH4/kgVS (volatile solids), respectively. Pretreatment with NMMO improved both the methane yield and the degradation rate of these substrates, and the effects were further amplified with more pretreatment time. Pretreatment for 15 h resulted in an increase of methane yield by 53% and 51% for cattle and horse manure, respectively. The specific rate constant, k0, was increased from 0.041 to 0.072 (d-1) for the cattle and from 0.071 to 0.086 (d-1) for the horse manure. Analysis of the pretreated straw shows that the structural lignin content decreased by approximately 10% for both samples and the carbohydrate content increased by 13% for the straw separated from the cattle and by 9% for that separated from the horse manure. The crystallinity of straw samples analyzed by FTIR show a decrease with increased time of NMMO pretreatment.
Keywords: Anaerobic digestion; Manure; Straw; Pretreatment; N-Methylmorpholine Oxide Contact information: School of Engineering, University of Borås, 501 90, Borås, Sweden *Corresponding author: Tel: +46 33 435 46 20, Fax: +46 33 435 40 08, E-mail: [email protected]
INTRODUCTION A reliance on fossil fuels as the main energy source has caused several environmental and economical challenges (Budiyono et al. 2010). Thus, there is a steadily rising worldwide interest in investigating renewable sources for energy production (Amon et al. 2007). Anaerobic digestion (AD) is a technology generally used for management of organic waste for biogas production, since it offers a renewable source of energy and at the same time solves ecological and agrochemical problems (Budiyono et al. 2010). A variety of raw materials, among others energy crops and animal manure, can be utilized as organic matter for biogas production (Neves et al. 2009).
Methane is produced during the anaerobic degradation of the organic components such as carbohydrates, proteins, and lipids present in the manure. The ultimate methane yield is affected by several factors, such as the feed, species, breed, and growth stage of the animals as well as the amount and type of the bedding material, together with the pre-storage conditions prior to biogas production (Møller et al. 2004). The composition, i.e., the protein, fat, fiber, cellulose, hemicellulose, starch, and sugar content, are also important factors that influence the methane yield (Comino et al. 2009).
PEER-REVIEWED ARTICLE bioresources.com
Aslanzadeh et al. (2011). “Pretreatment for better biogas” BioResources 6(4), 5193-5205. 5194
Straw, when used as bedding material in proper ratios or after appropriate pre-treatment, can beneficially affect the methane yield by enabling a more advantageous carbon to nitrogen (C/N) ratio for the substrate (Hashimoto 1983). Since straw belongs to the class of difficult-to-degrade lignocellulosic materials, a pretreatment step is needed to improve the rate and degree of enzymatic hydrolysis during the degradation process. Lignocelluloses are composed of cellulose, hemicellulose, lignin, extractives, and several inorganic materials. The cellulose and hemicellulose are sheltered by lignin, which provides integrity and structural rigidity. The content and distribution of lignin is responsible for the restricted enzymatic degradation of lignocelluloses, by limiting the accessibility of enzymes (Taherzadeh and Karimi 2008). Therefore, to improve biogas formation, often an effective and economically feasible pretreatment step is necessary. However, most of the reported methods such as dilute acid, hot water, AFEX, ammonia recycle percolation, and lime treatments are costly and have strong negative environmental effects, while others such as biological pretreatments are time consuming (Taherzadeh and Karimi 2008).
A study on the efficiency of biogas production of plant residues in co-digestion with cattle manure (Hassan Dar and Tandon 1987) showed that pretreatment of plant residues resulted in increased biogas yield by 31 to 42%. On the other hand, the rate of the bioconversion was very slow. They have also reported that caustic soda pretreatment has a promising effect on the delignification process compared to other pretreatments using sulphuric acid, phosphoric acid, ammonia, sodium hypochlorate, and acetic acid. However chemical pretreatments also can have strong negative environmental effects.
N-Methylmorpholine-N-oxide (NMMO) is one of the non-derivatizing solvents that can break the intermolecular interactions in cellulose, and it is mainly used in the textile industry for spinning of cellulose fibers (Lyocell process). It is considered to be environmentally friendly, since it does not generate toxic pollutants and it is recyclable with more than 98% recovery. Furthermore, NMMO is known to modify the highly crystalline structure of cellulose, while leaving the composition of wood intact and causing no hydrolysis of the hemicellulose (Lennartsson et al. 2011). Another study (Shafiei et al. 2009) showed that NMMO pretreatment of oak and spruce resulted in an increase of the digestibility during a following enzymatic hydrolysis.
The objective of this study was to investigate the effects of NMMO pretreatment on biogas production from horse and cattle manures, both with a high content of straw. The manure samples were first separated to obtain the straw fraction for the NMMO pretreatment. The pretreated fractions were then mixed back with the rest of the samples, and the biogas production was determined and compared with that of the untreated samples using anaerobic batch digestion tests. MATERIAL AND METHODS
Materials Two different deep litter manures obtained from a horse farm and a cattle farm outside Borås (Sweden) were investigated. The characterization of the substrates was
PEER-REVIEWED ARTICLE bioresources.com
Aslanzadeh et al. (2011). “Pretreatment for better biogas” BioResources 6(4), 5193-5205. 5195
carried out by Analys- & Konsulatlaboratoreiet Borås, Sweden, according to standard methods and the data are summarized in Table 1.
Table 1. Characterization of the Horse and Cattle Manures Mixed with Straw
Analyses Horse manure Cattle manure
Total Solids(wt%) 81.5 23.0
Volatile Solids(wt%) 61.8 18.1
Protein(wt%) 11.0 4.80
Kjeldahl Nitrogen(wt%) 1.70 0.76
Ammonium Nitrogen(wt%) 0.047 0.017
Fat Content(wt%) 1.60 0.30
Carbohydrates(wt%) 49.2 13.0 Pretreatment Procedure
The straw fraction of the manure was separated for each pretreatment condition according to a procedure shown in Fig. 1, where 7.5 g of manure was washed with 150 mL hot tap water and then filtered using a coarse vacuum filter with 1 mm pore size. The filtrate was collected and stored at -20ºC until further utilization.
The pretreatment of each straw fraction was carried out using a commercial grade NMMO (50% w/w in aqueous solution) solution (BASF, Ludwig-Shafen, Germany). In order to achieve a concentration of 85%, the NMMO solution was evaporated in a vacuum evaporator. Then, propylgallate was added to a concentration of 0.6 g/L. It is an antioxidant and prohibits oxidation and deterioration of the solvent during the following pretreatment procedure (Shafiei et al. 2009). Each straw fraction was then pretreated with 92.5 g of 85% NMMO solvent in an oil bath at 120ºC for 5 h or 15 h. Under the 5 h pretreatment, the suspension was mixed every 15 min, while during the 15 h pretreatment, the suspension was left overnight without mixing. After the pretreatments, the NMMO was separated by adding boiling tap water and then filtered through a coarse vacuum filter. This washing and filtering step was repeated a few times, until the filtrate was clear, indicating that the NMMO solvent was completely washed out. The filtrate was then centrifuged (5 min, 5000 rpm) in order to obtain fine particles, which had passed through the filter, and the supernatant was discarded. The pellet was also repeatedly washed with hot boiling water and centrifuged until the supernatant was clear and the NMMO solvent was completely washed out. The pretreated straw together with the fine particles were dried in a freeze dryer and kept at 4ºC until use.
Anaerobic Batch Digestions
The anaerobic batch digestion experiments were carried out according to a previously published method (Hansen et al. 2004). The digesters used were 118 mL glass bottles closed with a rubber septum and aluminum caps. The inoculum was obtained from a 3000-m3 municipal solid waste digester operating under thermophilic conditions (Borås Energi och Miljö AB, Sweden), and was incubated and stabilized at 55ºC for three days before use.
PEER-REVIEWED ARTICLE bioresources.com
Aslanzadeh et al. (2011). “Pretreatment for better biogas” BioResources 6(4), 5193-5205. 5196
Fig. 1. Schematic presentation of the pretreatment process
The filtrate collected after the separation of straw was centrifuged at 5000 rpm for
5 min. The supernatant was discarded, and the sediment was mixed with the NMMO-pretreated straw fraction and used as substrate for the biogas production. Each reactor contained 40 mL inoculum, 0.3 g volatile solid of pretreated or untreated manure straws, and tap water to bring the total volume to 45 mL. In order to determine the methane production from the inoculum itself, blanks containing only inoculum and tap water were also examined in order to determine the biogas production from the substrate. In order to facilitate anaerobic conditions and to prevent pH-change, the head space of each reactor was finally flushed with a gas mixture of 80% N2 and 20% CO2. All experimental set-ups were performed in triplicates, and the reactors were then incubated at 55ºC for 52 days. During this experimental period, the reactors were shaken once per day.
The methane produced was measured by taking gas samples regularly from the headspace, using a pressure-tight gas syringe. During the first two weeks, samplings and measurements were carried out every third day, followed by weekly sampling for the rest of the experimental period. The pH in the reactors was measured at the end of the experiment.
PEER-REVIEWED ARTICLE bioresources.com
Aslanzadeh et al. (2011). “Pretreatment for better biogas” BioResources 6(4), 5193-5205. 5197
Kinetic Modeling The kinetics of the degradation process were evaluated using the following first-
order kinetic model (Jiménez et al. 2004):
G = Gm [1 exp( k0t)] (1) or ln[Gm/ (Gm-G)] =k0t (2) where G (mL) is the volume of methane accumulated after a period of time t (days), Gm (mL) is the maximum accumulated gas volume at an infinite digestion time, k0 (day-1) is the specific rate constant, and t (days) is the digestion time. Plotting the calculated data of ln[Gm/ (Gm G)] vs. time, t, gives a straight line with a slope equal to k0 with intercept of zero. The value of Gm was considered equivalent to the volume of accumulated methane at the end of the experiments. Analytical Methods
The total solids (TS) and volatile solids (VS) were determined according to Sluiter at al. (2005). Kjeldahl nitrogen and protein content were determined according to Swedish standard method ISO 25663 (Swedish Standard Institute, 1984), in which the materials are treated with a strong acid in order to release nitrogen, which can be then determined by titration. Since the Kjeldahl method does not measure the protein content, an average conversion factor of 6.4 is used to convert the measured nitrogen concentration to a protein concentration. For determination of ammonium nitrogen, the SIS 028134-1 method (Swedish Standard Institute 1976) was used. It is based on sparging the samples with deionized water and mixed it with ammonium citrate and reagents containing sodium nitroprusside, phenol, and sodium hypochlorite before analysis. Fat content was determined according to Method no. 131 (Nordic Committee on Food Analysis 1989). The method is based on treatment with hot concentrated hydro-chloric acid to release fat bound to protein, prior to extraction of the fat with diethylether. The structural carbohydrates and lignin content of the pretreated and untreated straw fractions were determined using a two-step hydrolysis method that has been used for lignocelluloses (Sluiter et al. 2008). The acid-soluble lignin was measured using a UV spectrophotometer, while acid-insoluble lignin was determined after ignition of the samples at 575ºC. The quantification of the sugars formed was performed by HPLC (Waters 2695, Millipore and Milford, USA) equipped with a refractive index (RI) detector (Waters 2414, Millipore and Milford, USA), using a Pb-based ion exchange column (Aminex HPX-87P, Bio-Rad, USA) with 0.6 mL/min pure water at 85°C, or a H-based ion exchange column (Aminex HPX-87H, Bio-Rad) at 60°C with 0.6 mL/min 5 mM H2SO4 as eluents.
The NMMO-pretreated straws were analyzed using a Fourier transform infrared (FTIR) spectrometer (Impact, 410, Nicolet Instrument Corp., Madison, WI). The spectra were achieved with an average of 32 scans and a resolution of 4 cm-1 in the range from 600 to 4000 cm-1 and controlled by Nicolet OMNIC 4.1 analyzing software (Jeihanipour,
PEER-REVIEWED ARTICLE bioresources.com
Aslanzadeh et al. (2011). “Pretreatment for better biogas” BioResources 6(4), 5193-5205. 5198
et al., 2009). The methane and carbon dioxide analyses were carried out using a gas chromatograph (Auto System, Perkin Elmer, USA) equipped with a packed column (Perkin Elmer, 6’ x 1.8” OD, 80/100, Mesh, USA) and a thermal conductivity detector (Perkin Elmer) with inject temperature of 150°C. Nitrogen was used as carrier gas at 75ºC with a flow rate of 20 mL/min. For gas sampling, a 250μL pressure-tight syringe (VICI, Precision Sampling Inc., USA) was used. The results are presented as gas volume per kilogram volatile solids at standard conditions (0°C, atmospheric pressure). RESULTS AND DISCUSSION
The straw fraction of horse and cattle manure was separated and pretreated with 85% NMMO for 5 and 15h at 120ºC in order to open up the lignin shield and make the cellulose accessible for enzymatic degradation prior to biogas production. After the pretreatment, the NMMO was washed out, and the pretreated straw samples were dried in a freeze dryer and mixed with the rest of the manure samples and used for biogas production. The effect of the pretreatment was evaluated using anaerobic batch digestion assays. Moreover, the changes in the structure of the separated straw fraction due to the pretreatment were investigated by FTIR analysis. Biogas Production
The biogas potential of horse and cattle manures mixed with the fraction of straw, before and after NMMO-pretreatment, was investigated in batch digestion experiments. Figure 2 shows the average values of accumulated methane production of triplicate samples measured during 52 days of incubation. The pretreatment improved the methane potential of every pretreated material. The methane yield increased by 22% and 53%, after the pretreatment of the straw fraction for 5 h and 15 h, respectively. The specific methane production for untreated cattle manure was 0.250 Nm3 CH4/kgVS, which increased to 0.305 Nm3 CH4/kgVS after 5 h pretreatment and further to 0.382 Nm3
CH4/kgVS after the 15 h pretreatment (Table 2). The same pattern was observed for the horse manure. The specific methane production increased to 0.350 and 0.422 Nm3
CH4/kgVS after 5 h, respective, 15 h pretreatments, while the methane yield of the untreated horse manure was 0.279 Nm3 CH4/kgVS. This means an increase in the methane yields by 25% and 51% for 5 h and 15 h pretreatments, respectively (Table 2).
The theoretical methane yield for manure samples was calculated using the general formula presented previously based on the fat, protein, and carbohydrate contents of the substrate (Davidsson 2007). According to the data presented at Table 1, the theoretical yield for cattle and horse manure was calculated to be 0.447 m3 CH4/kgVS and 0.445 m3 CH4/kgVS, respectively. These results are in accordance with the theoretical methane yield for dairy cattle manure of 0.469 m3 CH4/kgVS reported previously (Møller et al. 2004). These authors also calculated the theoretical yield of methane of manure mixed with straw, based on the composition of this mixture regarding to carbohydrates, lipids, and proteins and concluded that in comparison with manure without straw, 1 kg straw mixed with 100 kg manure would increase the yield of methane by approximately 10% considering the compositional variation in such biomass.
PEER-REVIEWED ARTICLE bioresources.com
Aslanzadeh et al. (2011). “Pretreatment for better biogas” BioResources 6(4), 5193-5205. 5199
Table 2. Lignin and Carbohydrate Content, and Lateral Order Index (LOI) in IR Spectra of Straw Separated from Horse and Cattle Manure before and after Pretreatments with NMMO at 120ºC for 5 h and 15 h, Respectively. Specific methane yields and specific rate constants (k0) were obtained during batch digestion of manure mixed with untreated vs. pretreated straw.
Sample
Total Lignin (% of TS)
Total Carbohydrates (%) LOIa
Specific Methane Yield(Nm3/kgVS)b
Specific rate constant k0(day-1)
Horse manure untreated 32.92 44.68 2.68 0.279±0.002 0.071 5h-treatment 25.90 50.92 0.88 0.350±0.01 0.064 15h-treatment 22.72 53.84 0.66 0.422±0.05 0.086
Cattle manure untreated 39.53 25.44 5.36 0.250±0.09 0.041 5h-treatment 31.67 36.68 1.47 0.341±0.01 0.063 15h-treatment 29.75 38.68 1.26 0.382±0.08 0.072
a Lateral order index A1420cm-1/A898cm-1
b Accumulated methane per gram volatile solids produced after 52 days of incubation together with two standard deviations on accumulated methane production d Specific rate constant during the first 12 days of incubation
Fig. 2. Methane yield obtained after 52 days of anaerobic batch digestion from untreated and pretreated horse manure and cattle manure
A batch assay provides information about the methane yield from certain
substrates as well as the kinetics of the degradation process. The results showed that not only the accumulated methane production, but also the degradation rate was improved as a result of the treatments. Figures 3a and 3b illustrate the variation of specific rate constant (k0) for treated vs. untreated horse and cattle manures, respectively.
PEER-REVIEWED ARTICLE bioresources.com
Aslanzadeh et al. (2011). “Pretreatment for better biogas” BioResources 6(4), 5193-5205. 5200
As shown in Fig. 3, k0 increased with the pretreatment time for both the cattle and the horse manure samples (Table 2). After 15 h pretreatment, k0 increased from 0.071 (untreated) to 0.086 d-1 in the case of horse manure, while similar treatment conditions resulted in an increase from 0.041 (untreated) to 0.072 d-1 for cattle manure. The pH was around 7 at the end of each digestion setup.
Time(days)
0 2 4 6 8 10 12 14
ln [G
m/(G
m-G
)]
-0,2
0,0
0,2
0,4
0,6
0,8
1,0
Untreated5h treatment15h treatment
0 2 4 6 8 10 12 14
ln[G
m/(G
m-G
)]
-0,2
0,0
0,2
0,4
0,6
0,8
1,0
1,2
untreated5h treatment15h treatment
(b)
(a)
Time(days)
Fig. 3. Values of ln [Gm/ (Gm G)] in function of time for (a) Untreated and pretreated cattle manure. (b) Untreated and pretreated horse manure
The Effects of Pretreatment on the Composition and Structure of Straw Separated from Manure
Lignin and carbohydrate contents of the untreated vs. pretreated straw fractions are shown in Table 2. The total lignin content (acid-soluble and insoluble) for untreated straw separated from cattle manure was 39.53%(w/w), which decreased to 31.67% and 29.75% following 5 h and 15 h pretreatment with NMMO, respectively. Consequently, the total carbohydrates of the straw from untreated cattle manure increased from 25.44% to 36.68% and 38.68% after the 5 and 15 h pretreatments, respectively. Similarly, with investigations of the straw separated from horse manure, a decrease in the lignin content was observed from 32.92 (wt %) to 25.90 (wt %) and to 22.72 (wt %) after 5 and 15 h
PEER-REVIEWED ARTICLE bioresources.com
Aslanzadeh et al. (2011). “Pretreatment for better biogas” BioResources 6(4), 5193-5205. 5201
pretreatments, respectively (Table 2). The total carbohydrate for untreated straw from horse manure was 44.68%, which increased to 50.92% and 53.84% after the pretreatments. This shows that the pretreatment reduced the structural lignin content by approximately 10% for both the separated straw samples and increased the carbohydrate content by 13% for straw separated from cattle manure and by 9% for that from horse manure. Additionally, an increase in the pretreatment time made the delignification more effective and further improved the following digestion process. This is because the pretreatment opened up the lignin that shields the cellulose and hemicelluloses, which in turn limits the accessibility of enzymes involved in further degradation during the following digestion process.
Table 3. Assignments of FT-IR Absorption Bands (cm-1) with Related ReferencesBands (lit.) cm-1 Assignment Reference 3500-3100 OH –stretching vibrations (Denise S. Ruzene 2007)
2919-2925 Methyl, methylene, and methine group vibrations
(Lawther et al. 1996)
2850,2920 CH2-streching bands (Kristensen et al. 2008)
1727 Aliphatic carboxyl groups (Buta, Zadrazil et al 1989)
1665-1680 Carbonylgroup (C=O) conjugated to aromatic ring
(MacKay, O'Malley et al. 1997)
1595-1605 Aromatic skeletal vibrations (Buta, Zadrazil et al. 1989)
1595 Aromatic ring with C=O stretching
(MacKay, et al. 1997)
1505-1515 Aromatic skeletal vibrations (Buta, Zadrazil et al. 1989)
1510 Aromatic ring with C—O stretching
(MacKay et al. 1997)
1420 Aromatic skeletal vibrations (Buta, Zadrazil et al. 1989)
1245-1519 Guaiacyl and syringyl (Niu, Chen et al. 2009)
1040 Dialkylether linkages linking cinnamyl alcohol subunits
(MacKay, et al. 1997)
1035 polysaccharide vibrations (Lawther, Sun et al. 1996)
The change in the structure of the straws, caused by the pretreatment, was investigated by FTIR analysis. The interesting bands studied are summarized in Table 3, and the absorbance spectra are shown in Fig. 4. The comparison of these FTIR spectra shows that NMMO pretreatment resulted in reducing the absorption band around 1420 cm-1 and in increasing the absorption band at 898 cm-1. These two bands are characteristic for the crystalline cellulose I and amorphous cellulose II, respectively (Nelson and O'Connor 1964). The crystallinity index, which is also called the lateral order index (LOI), was calculated as the absorbance ratio of the bands around 1420 and 898 cm-1 (He et al. 2008; Zhao et al. 2009). The results in Table 2 show that the
PEER-REVIEWED ARTICLE bioresources.com
Aslanzadeh et al. (2011). “Pretreatment for better biogas” BioResources 6(4), 5193-5205. 5202
crystallinity index decreases as the time of the pretreatment increases This implies that there is a breakdown in the structure of straw, and as a result more sugars are hydrolyzed after the pretreatment, which improved the biogas production.
Fig. 4. FTIR spectrum of treated and untreated straw in (a) cattle manure and (b) horse manure (a) untreated, (b) 5 h treatment, (c) 15 h treatment
The lignin IR spectra have a strong broad band between 3500 and 3100 cm-1, which is related to OH stretching vibrations caused by the presence of alcoholic and phenolic hydroxyl groups involved in hydrogen bonds (Adney et al. 2008). The OH stretching band of the hydroxyl groups around 3300 cm-1 was changed to a higher wavenumber and somewhat broadened as a result of the pretreatment, which is an indication of weaker intra- and intermolecular hydrogen bonding and thereby a lower crystallinity (Jeihanipour et al. 2009). This result confirms the analysis data showing that the pretreatment reduced the structural lignin content in the straw (Table 2).
PEER-REVIEWED ARTICLE bioresources.com
Aslanzadeh et al. (2011). “Pretreatment for better biogas” BioResources 6(4), 5193-5205. 5203
An additional effect of the pretreatment was the elimination of waxes, which can be observed from the reduced CH2- stretching bands at about 2850 and 2920 cm-1 for the pretreated straw, suggesting a decrease in the amount of the aliphatic fractions of waxes (Kristensen et al. 2008). Several other changes were also observed in the structure, as is shown by changes in many other regions given in Table 3. These changes are more obvious as the pretreatment time increases (Fig. 4). CONCLUSIONS
1. The aim of this study was the pretreatment of straw fraction separated from cow and horse manure, since the accumulation of this low digested lignocellulosic material can cause problems when manure is utilized for biogas production, and resulting in low methane yields.
2. NMMO pretreatment of straw separated from cattle and horse manure improved the methane yield during the following digestion of both manure substrates and these improvements were increased by increased the pretreatment times. Treating the straw fraction for 15 h increased the methane yield by 53% and 51% for cattle and horse manure, respectively, compared to that of when untreated straw was present in the manure samples.
3. The kinetics of the degradation process were evaluated using a specific rate constant, k0, which was also improved when the straw fractions separated from both manure samples were pretreated for 15 h.
4. The effects of the pretreatment were evaluated by chemical and structural characterizations of the separated straw fractions. The total lignin content decreased by about 10% and the carbohydrate content increased by about 9% for straw separated from horse manure and by 13% for straw separated from cattle manure.
5. A reduction of crystallinity, obtained by FTIR, in the structure of the treated straw fractions, indicates an increase of the accessible surface area on the lignocellulosic material for further microbial degradation, improving the methane yield.
ACKNOWLEDGMENTS
This work was financially supported by the Swedish Rural Economy and Agricultural Societies in Sjuhärad and Borås Energi & Miljö AB (Sweden). REFERENCES CITED Adney, W. S., McMillan, J. D., Mielenz, J., Klasson, K. T., Ruzene, D. S., Silva, D. P.,
Vicente, A. A., Gonçalves, A. R., and Teixeira, J. A. (2008). "An alternative application to the Portuguese agro-industrial residue: Wheat straw," Biotechnologyfor Fuels and Chemicals, Humana Press, 453-464.
PEER-REVIEWED ARTICLE bioresources.com
Aslanzadeh et al. (2011). “Pretreatment for better biogas” BioResources 6(4), 5193-5205. 5204
Amon, T., Amon, B., Kryvoruchko, V., Zollitsch, W., Mayer, K., and Gruber, L. (2007). "Biogas production from maize and dairy cattle manure. Influence of biomass composition on the methane yield," Agriculture, Ecosystems & Environment 118(1-4), 173-182.
Budiyono, I., Widiasa, N., Johari, S., and Sunarso (2010). "The kinetic of biogas production rate from cattle manure in batch mode," Internationa Journal of Chemical and Biomolecular Engineering 3(1), 39-44.
Comino, E., Rosso, M., and Riggio, V. (2009). "Development of a pilot scale anaerobic digester for biogas production from cow manure and whey mix," BioresourceTechnology 100(21), 5072-5078.
Davidsson, Å. (2007). "Increase of biogas production at wastewater treatment plants, Addition of urban organic waste and pre-treatment of sludge," Ph.D. Thesis, Department of Chemical Engineering, Lunds University, Sweden
Nordic Committee on Food Analysis (1989). "Fat, determination according to SBR (Schmid-Bondzynski- Ratslaff) in meat and meat products," NMKL method no. 131, NMKL, Oslo.
Hansen, T. L., Schmidt, J. E., Angelidaki, I., Marca, E., Jansen, J. l. C., Mosbæk, H., and Christensen, T. H. (2004). "Method for determination of methane potentials of solid organic waste," Waste Management 24(4), 393-400.
Hashimoto, A. G. (1983). "Conversion of straw-manure mixtures to methane at mesophilic and thermophilic temperatures," Biotechnology and Bioengineering 25(1), 185-200.
Hassan Dar, G., and Tandon, S. M. (1987). "Biogas production from pretreated wheat straw, lantana residue, apple and peach leaf litter with cattle dung," Biological Wastes 21(2), 75-83.
He, J., Cui, S., and Wang, S.-y. (2008). "Preparation and crystalline analysis of high-grade bamboo dissolving pulp for cellulose acetate," Journal of Applied Polymer Science 107(2), 1029-1038.
Jeihanipour, A., Karimi, K., and Taherzadeh, M. J. (2009). "Enhancement of ethanol and biogas production from high-crystalline cellulose by different modes of NMO pretreatment," Biotechnology and Bioengineering 105(3), 469-476.
Jiménez, A. M., Borja, R., and Martín, A. (2004). "A comparative kinetic evaluation of the anaerobic digestion of untreated molasses and molasses previously fermented with Penicillium decumbens in batch reactors," Biochemical Engineering Journal, 18(2), 121-132.
Kristensen, J., Thygesen, L., Felby, C., Jorgensen, H., and Elder, T. (2008). "Cell-wall structural changes in wheat straw pretreated for bioethanol production," Biotechnology for Biofuels 1(1), 5.
Lawther, J. M., Sun, R., and Banks, W. B. (1996). "Fractional characterization of wheat straw lignin components by alkaline nitrobenzene oxidation and FT-IR spectroscopy," Journal of Agricultural and Food Chemistry 44(5), 1241-1247.
Lennartsson, P. R., Niklasson, C., and Taherzadeh, M. J. (2011). "A pilot study on lignocelluloses to ethanol and fish feed using NMMO pretreatment and cultivation with zygomycetes in an air-lift reactor," Bioresource Technology 102(6), 4425-4432.
PEER-REVIEWED ARTICLE bioresources.com
Aslanzadeh et al. (2011). “Pretreatment for better biogas” BioResources 6(4), 5193-5205. 5205
MacKay, J. J., O'Malley, D. M., Presnell, T., Booker, F. L., Campbell, M. M., Whetten, R. W., and Sederoff, R. R. (1997). "Inheritance, gene expression, and lignin characterization in a mutant pine deficient in cinnamyl alcohol dehydrogenase," Proceedings of the National Academy of Sciences of the United States of America 94(15), 8255-8260.
Møller, H. B., Sommer, S. G., and Ahring, B. K. (2004). "Methane productivity of manure, straw and solid fractions of manure," Biomass and Bioenergy 26(5), 485-495.
Nelson, M. L., and O'Connor, R. T. (1964). "Relation of certain infrared bands to cellulose crystallinity and crystal latticed type. Part I. Spectra of lattice types I, II, III and of amorphous cellulose," Journal of Applied Polymer Science 8(3), 1311-1324.
Neves, L. C. M. d., Converti, A., and Penna, T. C. V. (2009). "Biogas production: New Trends for alternative energy sources in rural and urban zones," ChemicalEngineering & Technology 32(8), 1147-1153.
Shafiei, M., Karimi, K., and Taherzadeh, M. J. (2009). "Pretreatment of spruce and oak by N-methylmorpholine-N-oxide (NMMO) for efficient conversion of their cellulose to ethanol," Bioresource Technology 101(13), 4914-4918.
Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., and Templeton, D. (2005). "Determination of ash in biomass," National Renewable Energy Laboratory.
Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., and Crocker, D. (2008). "Determination of structural carbohydrate and lignin in biomass." National Renewable Energy Laboratory.
Taherzadeh, M., and Karimi, K. (2008). "Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: A review," International Journal of Molecular Sciences 9(9), 1621-1651.
Swedish Standard Institute (1976). "Water quality- Determination of ammonia nitrogen content of water," SIS 28134, Article No STD-883, Stockholm, Sweden.
Swedish Standard Institute (1984). "Water quality - Determination of Kjeldahl nitrogen - Method after mineralization with selenium," ISO 5663,Stockholm, Sweden.
Zhao, H., Baker, G. A., and Cowins, J. V. (2009). "Fast enzymatic saccharification of switchgrass after pretreatment with ionic liquids," Biotechnology Progress 26(1), 127-133.
Article submitted: July 15, 2011; Peer review completed: September 24, 2011; Revised version received and accepted: October 27, 2011; Published: November 1, 2011.
II
1
Biogas production from N-Methylmorpholine-N-
oxide (NMMO) pretreated forest residues
Solmaz Aslanzadeha*, Andreas Berg
b, Mohammad J. Taherzadeh
a, Ilona Sárvári Horváth
a
a School of Engineering, University of Borås, Borås, Sweden
b Scandinavian Biogas Fuels AB, Linköping, Sweden
Corresponding author: *E-mail: [email protected]
Keywords; Biogas, Anaerobic digestion, Forest residues, Batch experiment, Continuous
experiment
2
ABSTRACT
Lignocellulosic biomass represents a great potential for biogas production. However, a suitable
pretreatment is needed to improve their digestibility. This study investigates the effects of an
organic solvent, NMMO at temperatures of 120 °C and 90 °C, NMMO concentrations of 75%
and 85% and treatment times of 3h and 15 h on the methane yield. The long-term effects of the
treatment were determined by a semi-continuous experiment. The best results were obtained
using 75% NMMO at 120 °C for 15 h, resulting in 141% increase in the methane production.
These conditions led to a decrease by 9% and an increase by 8% in the lignin and in the
carbohydrate content, respectively. During the continuous digestion experiments a specific
biogas production rate of 92 NmL/gVS/day was achieved while the corresponding rate from the
untreated sample was 53 NmL/gVS/day. The operation conditions were set at 4.4 gVS/L/day
organic loading rate (OLR) and hydraulic retention time (HRT) of 20 days in both cases.
NMMO-pretreatment has substantially improved the digestibility of forest residues. The present
study shows the possibilities of this pre-treatment method, however an economic and technical
assessment of its industrial use needs to be performed in the future.
3
INTRODUCTION
As a consequence of the enormous wood exploitation, a large amount of forest residues, mainly
consisting of leaves, small branches, and bark, is generated. Up to date, the majority of these
forest residues are abandoned in situ, thus originating important environmental problems.
Among these problems, soil acidification (due to the accumulation of organic matter) and
increased risk of forest fires, especially during dry periods with high temperatures can be
mentioned (1).
Forest residues are one of the most abundant lignocellulosic waste streams in Sweden with 1.6
million tons total solids per year reported for 2008. This amount is predicted to more than double
by the year 2018 (2). This makes forest residues a potential biomass for biogas production and
an energy production on a scale of 59 TWh/year in Sweden (3). There is a large demand for
alternative fuels produced from renewable resources worldwide especially for the transport
sector, and biogas is one of the alternatives which can be used. However, in order to meet these
increasing requirements, new sources of substrates are needed to be utilized for biogas
production (4). Lignocellulosic biomass, such as forest residues, is primarily composed of
cellulose, hemicelluloses, and lignin. These kinds of materials can serve as an inexpensive
substrate for biofuel production, avoiding the moral dilemma connected with the utilization of
potential food resources (5). However, the enzymatic conversion of the cellulose and
hemicelluloses in lignocelluloses is slow if the biomass is not exposed to some kind of
pretreatment.
4
Several pretreatment methods have been investigated, including ammonia fiber explosion
(AFEX), wet oxidation, and liquid hot water (LHW), among others, which are shown to be more
successful for agricultural and forestry residues. However, all these pretreatments performed on
forest residues were carried out prior to ethanol production. Furthermore, none of these
pretreatment methods are considered to be enough efficient today (6).
Hendriks and Zeeman (7) reviewed the effect(s) of several pretreatment methods on the three
main parts of the lignocellulosic biomass to improve its digestibility. Steam pretreatment, lime
pretreatment, liquid hot water pretreatments, and ammonia-based pretreatments are concluded to
be pretreatments with a high potential. Their main effects are dissolving hemicellulose and
alteration in the lignin structure, providing an improved accessibility to the cellulose for
hydrolytic enzymes. However, it was also concluded that many of these methods give rise to
different inhibitory products, which especially in high concentrations can possibly be very
harmful to microorganisms in anaerobic digestion. On the other hand, it was also concluded that
during anaerobic digestion the microorganisms have a potential to adapt to these inhibitory
products, when presented at very low concentrations.
Recent studies (8-10) used an organic solvent N-methylmorpholine-N-oxide (NMMO) for
regeneration of cellulose in the industrial Lyocell process, and show that this solvent has a great
potential for the pretreatment of lignocelluloses. The melting point of this industrial solvent is
about 70 °C, while it decomposes at temperatures higher than 130 °C. Consequently, most of the
pretreatment studies with NMMO are performed between these temperatures (11).
5
Earlier studies focusing on NMMO-treatment of lignocellulosic materials aimed either to
improve the ethanol production rate (7-9) or to determine the effects of the treatment on biogas
production through anaerobic batch digestion assays (12, 10).
Previous studies showed that the behavior of wood and cotton cellulose fibers pretreated with
NMMO is highly affected by the water content in the solvent (13). In a recent study, it was
shown that pretreatment of cotton with NMMO corresponding to NMMO concentrations of 85%,
79%, and 73% resulted in dissolution, ballooning, and swelling of the cellulose fibers (14). All of
the experiments were carried out at both 90 °C and 120 °C during treatment times of 0.5 – 15 h.
The study showed that the dissolution (85%) mode had the best effect on the following
enzymatic hydrolysis of cellulose. However, the swelling (73%) and ballooning (79%) mode
resulted in the highest yields of methane production during the following anaerobic digestion.
Another study on NMMO-pretreatment of softwood spruce and hardwood oak with 85% at 90,
110, and 130 °C for 1–3 h showed that the temperatures as well as the treatment times had
significant effects on the performance of the following enzymatic hydrolysis (9). Furthermore,
pretreatment with 85% NMMO at 130 °C for 1–15 h on lignocellulosic materials, such as spruce
chips from the Swedish forests, triticale straw from the Swedish farmland, and rice straw from
the Indonesian fields indicated that increasing the pretreatment time can improve the methane
yield during the following anaerobic digestion (10).
All previous studies used batch digestion assays to determine the effects of different treatment
conditions on methane yield and methane production rate. This study was performed to
investigate the long-term effects of the NMMO-treatment using continuous digestion
experiments. The substrate utilized was forest residues in Sweden, which is a heterogeneous
6
material with high lignin content. Biogas production from the treated vs. untreated materials
were compared after different treatment conditions as well as at different operational conditions.
EXPERIMENTAL SECTION
Raw materials
The forest residues were delivered by Norrskog (Östersund, Sweden). It was an inhomogeneous
material consisting of a mixture of both spruce and pine with a high amount of bark. The
material was first milled to 0.5 – 2 mm in size using a laboratory mill (Retsch SM100, Retch,
Germany) prior to characterization, treatment, and digestion. The characterization of the
untreated material showed that it consisted of 45.75% lignin and 41.05% carbohydrates.
NMMO-pretreatment
NMMO-pretreatments at different conditions were carried out using an industrial grade, 50%
w/w, NMMO solution obtained from BASF (Ludwigshafen, Germany). In order to concentrate
the solution up to 75% and 85%, a rotary evaporator (Laborata 20 eco, Heidolph, Germany),
operating at a pressure of 0.10 bar and a maximum temperature of 130 °C was used. The
pretreatments were performed in 5 L beakers containing 6% forest residues in either 75% or 85%
NMMO solution. During the pretreatments, the reaction mixtures were heated in an oil bath at
120 or 90 °C for 3 and 15 h at atmospheric pressure, while mixing constantly with a mixer. After
the pretreatments, the reaction was stopped by adding 1 L of boiling water to the beakers. The
pretreated materials were then filtered and washed with hot tap water, which made it easier to
7
dissolve and wash away the NMMO until no traces of NMMO was observed in the filtrate. The
pretreated materials were stored at 6 °C until further use.
Biogas production
Batch digestion experiments
The anaerobic batch digestion experiments were carried out at mesophilic (37 °C) conditions
according to a method that was published earlier by Hansen et al (15). The inoculum used in
mesophilic experiments was obtained from a large scale digester treating municipal wastewater
sludge (Tekniska Verken, Linköping, Sweden). The batch assays were performed using sealed
serum glass bottles with a volume of 118 ml, and all experimental setups were prepared in
triplicates. All assay bottles contained 40 mL inoculum, pretreated or untreated forest residues as
substrate in amounts to achieve a VS ratio inoculum to substrate of 2:1 and tap water to give a
final volume of 45 mL. To determine the methane production from the inoculum itself, blanks
containing only inoculum and tap water without any substrate addition were also examined. In
order to create anaerobic conditions and to avoid pH-change, the headspace of each reactor was
finally flushed with a gas mixture containing 80% N2 and 20% CO2. During the experimental
period of 52 days, the reactors were shaken and moved around in the incubator once a day.
The production of methane was measured by taking gas samples regularly from the headspace,
using a pressure-tight gas syringe. During the first two weeks, samplings and measurements were
carried out in every third day, followed by once a week for the rest of the experimental period.
The pH in the reactors was measured at the end of the experiment.
8
Semi-continuous anaerobic digestion experiments
The semi-continuous experiments were carried out at mesophilic conditions (37 °C) in 2 L glass
digesters, equipped with plastic tubes protruding the top of the reactor and ending in the liquid
phase; one for addition and withdrawal and one for the impeller. A rubber stopper with an outlet
for gas covered the top of the reactor. Two experiments were performed in parallel; one with
milled forest residues and one with NMMO-treated milled forest residues. Both digesters were
fed with reject water and digested sludge obtained from a municipal wastewater treatment plant
(WWTP) (Linköping, Sweden). These experiments continued for 118 days and were performed
in two ways: fed batch during start up, and thereafter as semi-continuously fed digesters.
Initially, 540 mL of digester sludge from a municipal WWTP together with 260 mL of reject
water was added to a 2 L glass reactor. Forest residues (pretreated or untreated) together with
reject water and digester sludge were then added daily to the digesters until they reached a
working volume of 1500 mL on day 26. Thereafter, the digesters were kept at a constant
hydraulic retention time of 20 days by daily withdrawal of digester fluid and addition of
substrate, reject water, and digester sludge (90% of OLR from forest residues) as explained
above. The digesters were both initially loaded with 2.0 g VS/L/day. Every fifth day, the OLR
was increased with 0.5 g VS/L/d until reaching 4.2 g VS/L/day. Due to process disturbances, the
OLR was decreased to between 2.4 and 3.7 g VS/L during days 48–55. However, from day 56 to
118, the OLR was kept at 4.4 g VS/ L. Because of the process disturbance, 350 mL digester fluid
was removed from the digester and replaced with 100 mL digested sludge and 100 mL of reject
water from the same source as described above, on day 49. In addition, from day 49 onward, the
daily amount of reject water was decreased by 10 mL (to 21 mL) and the amount of digested
9
sludge was increased by 10 mL (to 26mL). Stirring was initially (up to day 12) performed with a
magnet and thereafter with a metal impeller at regular intervals, five times a day. Volatile fatty
acids (VFA’s), pH, Total solids (TS), and Volatile solids (VS) in the digestate residue were
measured weekly. The gas production was measured continuously using gas meters (own
design), working according to the gas displacement method. All gas volumes are given at
standard conditions.
Analytical methods
Total carbohydrate and lignin contents of the pretreated and untreated forest residues were
determined according to the NREL procedures (16). In these methods, a two-step acid hydrolysis
with concentrated and diluted sulfuric acid was performed to release the sugars from the
hemicellulose and cellulose fractions. The amount of different liberated sugars was measured
afterward by HPLC (Waters 2695, Millipore, Milford, U.S.A.) equipped with a refractive index
(RI) detector (Waters 2414, Millipore, Milford, U.S.A.) and an ion-exchange column (Aminex
HPX-87P, Bio-Rad, U.S.A.) at 85 °C, using ultra-pure water as eluent with a flow rate of 0.6
ml/min. The acid-soluble and acid-insoluble lignin contents were analyzed using UV
spectroscopy at 205 nm and after drying the material at 575 °C, respectively. All lignin and
carbohydrate analyses were carried out in triplicates.
The methane and carbon dioxide in the anaerobic batch digestion series were analyzed as
described by Teghammar et al (17). using a gas chromatograph (Auto System, Perkin-Elmer,
USA) equipped with a packed column (Perkin-Elmer, 6’x1.8’’ OD, 80/100 Mesh, USA) and a
thermal conductivity detector (Perkin-Elmer, U.S.A.) with the inject temperature of 150 °C. The
10
carrier gas was nitrogen, operated with a flow rate of 20 ml/min at 60 °C. A 250-µl pressure-tight
gas syringe (VICI, Precision Sampling, Inc., USA) was used for gas sampling. The overpressure
in the bottles caused by the excess gas was released through a needle following the gas analyses
in order to avoid overpressure higher than 2 bar in the head space of the flasks. The methane
content in the gas produced during the CSTR experiments was not measured due to the low
amount of gas being produced, which caused too large errors in the measurements. All the results
are presented as gas volume at normal conditions (0 °C and atmospheric pressure) per kilogram
volatile solids.
Total solids and volatile solids were determined by drying the samples to a constant weight at
105 °C and then igniting the dried material at 575°C (18).
The VFA’s obtained during the CSTR experiments was measured using GC-FID as described by
Jonsson and Borén (19). The pH was measured with a pH electrode (WTW Inolab, Germany).
RESULTS
Effects of NMMO-pretreatment on the composition of forest residues
The composition of forest residues before and after NMMO-pretreatments at different conditions
is presented in Table 1. The pretreatment with 75% NMMO at 120 °C for 15 h increased the total
carbohydrate content by 8% (from 41% to 49%) in comparison to the untreated forest residues.
On the other hand, the pretreatment with 85% NMMO for 3 h and at 120 °C increased the
carbohydrate content from 41 wt% for untreated material to approximately 45 wt% for the
treated materials. The decrease in the pretreatment temperature from 120 to 90 °C did not have a
11
considerable effect, so the carbohydrate content remained at the same level as it was obtained for
the untreated material. However, when the temperature was decreased from 120 to 90°C, while
all the other parameters for the pretreatment remained the same, i.e., 85% NMMO and 3 h, a
decrease by 3.5% in the carbohydrate content was observed.
The NMMO-pretreatment resulted in a decrease in total lignin content. The highest decrease in
lignin was achieved when the forest residues were pretreated with 75% NMMO at 120 °C for 15
h, reducing the lignin content by over 9% (from 45.75% to 36.48%). The pretreatment with 85%
NMMO for 3 h decreased the lignin content by 7% and by 5%, when the forest residue was
treated at 120 °C and at 90 °C, respectively.
Batch digestion of NMMO-pretreated vs. untreated forest residues
Anaerobic digestion of untreated forest residues resulted in 42 NmL CH4/gVSadded (Figure 1).
Furthermore, the initial reaction rate of untreated forest residues obtained within the first 10 days
of digestion was 0.83 Nml/gVS/d (Figure 1 and Table 1). The pretreatment at 120 °C with 85%
for 3 h and with 75% NMMO for 15 h had a positive effect on the methane production. The
methane yield increased more than twofold, achieving up to 109 NmL CH4/gVSadded, and 100
NmL CH4/gVSadded, respectively. The initial reaction rate was also improved, achieving 4.27
Nml/g VS/day and 3.65 Nml/g VS/day after 15h and 3h pretreatment, respectively. The material
pretreated with 85% NMMO for 3h and at 90 °C showed a slightly lower methane yield of 87
NmLCH4/g VSadded, and methane production rate of 2.75 Nml/g VS/day. The highest methane
production rate, i.e., 4.27 NmL CH4/gVS/day, was observed after pretreatment with 75%
12
NMMO at 120 °C for 15 h. Therefore the forest residues pretreated at these conditions were
further investigated during the CSTR experiments.
Anaerobic digestion forest residues in fed-batch and semi-continuous mode
The specific biogas production was 53 NmL /gVS /day day (n = 63; SD ± 7) for digester 1
(Figure 2), where untreated forest residues were included in the feed. In digester 2, where the
treated forest residues were digested, specific biogas production of 92 NmL /gVS /day (n = 51;
SD ± 24) was obtained (Figure 3). Both digesters were operating at maximum OLR of 4.4
gVS/L/day with HRT of 20 days. Under these conditions, the mean VS-reduction was 8% (SD ±
4) for digester 1 and 20% (SD ± 6) for digester 2. The pH was measured at between 7.4 – 7.8 in
reactor 1 and between 7.1 – 7.6 in reactor 2 during the experiments (Figure 3).
Acetate and propionate were the main VFAs detected, and the values varied both between the
processes and over time. Starting on day 41, the amount of acetate and propionate increased in
reactor 2, reaching at most 7.1 mM acetate and 1.3 mM propionate on day 49. On day 51, both
acetate and propionate started to decrease to concentrations lower than 0.6 mM. In reactor 1,
both acetate and propionate was below 0.6 mM during the experimental period.
DISCUSSION
In accordance with previous studies on NMMO-pretreated lignocellulosic materials, it was found
that the pretreatment had positive effects, resulting in increased methane yields during the
subsequent anaerobic batch digestion assays. A previous study on NMMO-pretreatment of
13
lignocellulosic materials such as spruce, rice straw, and triticale straw showed an increase
between 400 and 1200% in the methane production after the treatment (10). However, the
substrates that were used there were homogenous with lower lignin content (between 19 and 29
wt%) compared to the heterogeneous forest residues with much higher lignin content of 46 wt%.
The pretreatment (75% NMMO, 120 °C for 15h) decreased the total lignin content by more than
9% and consequently, increased the total carbohydrates by 8% resulting in an increase in
methane production by 141%.
The long-term effects of the most effective pretreatment conditions were further investigated in
semi-continuous anaerobic digestion using pretreated vs. untreated forest residues as substrates.
The results obtained in our study clearly demonstrate that a continuous biogas process can also
be based on NMMO-treated forest residues with a low addition of supplemental material to keep
the nutritional balance in the system. To our knowledge, this is the first stable continuous
digestion of NMMO-treated forest residues to be shown.
The biogas yield was also improved from 53 to 92 NmL/gVS/day, during continuous digestion
of untreated and treated forest residues, respectively (Figure 2). The rapid increase in VFA’s
obtained in digester 2 is probably due to the higher level of organic material available for
digestion in the NMMO-treated forest residues, which in turn inhibits hydrogenotrophic
methanogens due to a drop in pH, demanding a higher amount of active microorganisms and/or
enzyme activity (20, 21).
During continuous operation, an OLR of at most 4.4 g VS/ L/ day could be achieved. Around
90% of the OLR came from forest residues where 45% of VS is lignin. Since lignin is not
digested in the biogas process, the OLR calculated from the remaining 55% of the VS is 2.2 g
14
VS/ L/ day. This is a more moderate OLR and given the stable process shown in the present
study, it points toward the possibility of using a higher OLR, while still maintaining stable
conditions. A higher OLR would mean a better utilization of any given biogas plant.
Furthermore, with an OLR of 2.2 g VS/ L /day, the VS-reduction will increase from 8% and 20%
to 15% (SD ± 6) and 33% (SD ± 10) for digesters 1 and 2, respectively. Hence, NMMO-
treatment enables a doubling of the digestibility of forest residues. Nonetheless, there is still two-
thirds of the total digestible VS left in the digestate residue; thus, further work needs to be done
to increase the VS-reduction during the anaerobic digestion of forest residues. The lignin-rich
digestate residue contains a high heating value, and can be used as fuel for combustion in
combined heat and power (CHP) plants (22). The water content of the digetate should be
decreased to 45% TS prior to combusion (23).
In a previous study performed by Shafiei et al. (24), a techno-economic analysis for ethanol
production from wood based on NMMO pretreatment was developed and the process was
designed to utilize 200,000 tons of spruce wood per year. The wastewater from this process with
a large amount of unutilized pentoses was directed to an anaerobic digestion process for
production of biogas. According to this study the bioethanol production in combination with
biogas production using NMMO pretreated spruce as feedstock would be a feasible process. The
total energy output in form of ethanol, lignin, and methane were calculated to be 134 MW/year
and the share of the heat value generated from lignin residues is around 66 MW/year (24). The
total energy output based on the results in this present study can be calculated to about 85
MW/year, when 200,000 tons (dry weight) forest residues are utilized for biogas production in a
continuous process. The energy output from lignin is about 80 MW/year which is 21% higher
than for spruce 66 MW/year considering that spruce has higher carbohydrate content and lower
15
lignin content than the forest residues used in this study. The production of biogas from
lignocelluloses however would have a higher overall energy efficiency comparing to that for
ethanol production, hence pentoses can also be utilized in biogas production (25).
The methane yield obtained during the continuous process is approximately 60% lower than the
methane potential measured in the batch assay for both pretreated and untreated forest residues
(Figure 2 vs Figure 1). The lower yield could be due to the lower retention time of 20 days used
for the digestion of the substrate in the continuous process compared with 52 days digestion
period in the batch process. The accumulated methane yields observed after 20 days of digestion
time in the batch assay were about 25 Nml/g VS added for untreated and 64 Nml/g VS added for the
pretreated material (Figure 1). Comparing these data with the yield of biogas production of 53
and 92 NmL/gVS/day, during continuous digestion of untreated and treated forest residues,
respectively (Figure 2), and assuming 50% methane in the produced biogas from carbohydrates
(26), it can be concluded that the results from batch and continuous digestions are in accordance
with each other. This also explains the relatively low VS reduction obtained.
Moreover, addition of carbon-rich materials, such as lignocelluloses, to digesters treating waste
mixtures with low C/N ratios has previously shown to enhance the nutritional balance and
stabilize sensitive processes (27). It was also shown (28) that the material and the ratio, by which
the forest residues are co-digested with, would have a significant effect for the economy of the
process. Using OFMSW for co-digestion instead of sludge and decreasing the ratio of forest
residues in the mixture would increase the methane yield considerably, since OFMSW has higher
methane potential than sludge.
16
CONCLUSION
Today, forest residues are available for energy production in Sweden on a scale of 59 TWh/year
(3). However, the use of this feedstock for biogas production is limited due to the lack of an
efficient pre-treatment enabling digestion of the cellulose and hemi-cellulose in the forest
residue. The present study shows the possibility of one pre-treatment method; however, an
economic and technical assessment of its industrial use needs to be performed in the future. One
aspect not evaluated in this study is the quality of the digestate. Since 45% of the substrate is
lignin that is not degradable, hence remains in the digestate residue, which would after de-
watering have a potential value as a fuel for combustion.
17
NOMENCLATURE
NMMO = N-Methylmorpholine-N-oxide (NMMO)
OLR= Organic loading rate
HRT= Hydraulic retention time
VFA= volatile fatty acids
VS= volatile solids
TS= total solids
SD= Standard deviation
HPLC= High performance liquid chromatography
GC= Gas chromatography
CSTR= Continuous stirred tank reactor
WWTP= Wastewater treatment plant
18
REFERENCES
1. Núñez-Regueira, L., Rodrıíguez-Añón, J., Proupín, J. and Romero-García, A. (2003)
Bioresour. Technol. 88, 121-130.
2. Kabir, M. M., del Pilar Castillo, M., Taherzadeh, M. J. and Sárvári Horváth, I. (2013)
BioResources. 8, 5409-5423.
3. Swedish Waste Management. (2008:2) Den svenska biogaspotentialen från inhemska
råvaror Swedish Waste Management, Sweden.
4. Skinner, I., Essen, H., Smokers, R. and Hill, N. (2010) Final report produced under the
contract ENV.C.3/SER/2008/0053 between European Commission Directorate-General
Environment and AEA Technology,.
5. Ferreira, S., Gil, N., Queiroz, J. A., Duarte, A. P. and Domingues, F. C. (2010) Bioresour.
Technol. 101, 7797-7803.
6. Hahn-Hägerdal, B., Galbe, M., Gorwa-Grauslund, M. F., Lidén, G. and Zacchi, G. (2006)
Trends Biotechnol. 24, 549-556.
7. Hendriks, A. T. W. M. and Zeeman, G. (2009) Bioresour. Technol. 100, 10-18.
8. Lennartsson, P. R., Niklasson, C. and Taherzadeh, M. J. (2011) Bioresour. Technol. 102,
4425-4432.
9. Shafiei, M., Karimi, K. and Taherzadeh, M. J. (2010) Bioresour. Technol. 101, 4914-
4918.
10. Teghammar, A., Karimi, K., Sárvári Horváth, I. and Taherzadeh, M. J. (2012) Biomass
Bioenergy. 36, 116-120.
11. Rosenau, T., Potthast, A., Sixta, H. and Kosma, P. (2001) Prog. Polym. Sci. 26, 1763-
1837.
12. Aslanzadeh, S., Taherzadeh, M. J. and Sárvári Horváth, I. (2011) Bioresources. 6, 5193-
5205.
13. Cuissinat, C. and Navard, P. (2006) Macromolecular Symposia. 244, 1-18.
14. Jeihanipour, A., Karimi, K. and Taherzadeh, M. J. (2010) Biotechnol. Bioeng. 105, 469-
476.
15. Hansen, T. L., Schmidt, J. E., Angelidaki, I., Marca, E., Jansen, J. l. C., Mosbæk, H. and
Christensen, T. H. (2004) Waste Manage. (Oxford). 24, 393-400.
16. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D. and Crocker, D.
(2008) Determination of Structural Carbohydrates and Lignin in Biomass. Standard
Biomass Analytical Procedures. National Renewable Energy Laboratory.
17. Teghammar, A., Yngvesson, J., Lundin, M., Taherzadeh, M. J. and Sárvári Horváth, I.
(2010) Bioresource Technology. 101, 1206-1212.
18. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J. and Templeton, D. (2005)
Determination of Ash in Biomass. Standard Biomass Analytical Procedures. National
Renewable Energy Laboratory.
19. Jonsson, S. and Borén, H. (2002) J. Chromatogr. A. 963, 393-400.
20. Jeihanipour, A., Aslanzadeh, S., Rajendran, K., Balasubramanian, G. and Taherzadeh, M.
J. (2013) Renewable Energy. 52, 128-135.
21. Schink, B. (1997) Microbiology and Molecular Biology Reviwes. 61, 262-280.
19
22. Larsen, J., Østergaard Petersen, M., Thirup, L., Wen Li, H. and Krogh Iversen, F. (2008)
Chemical Engineering & Technology. 31, 765-772.
23. Henriksson, G., del Pilar Castillo, M., Jakubowicz, I., Enocksson, H., Contreras, J. A. and
Lundgren, P. (2010) Miljöeffekter av polymerer inom biogasbranschen-Förstudie.
Projektnummer WR-33.
24. Shafiei, M., Karimi, K. and Taherzadeh, M. J. (2011) Bioresour. Technol. 102, 7879-
7886.
25. Murphy, J. D. and Power, N. (2009) Applied Energy. 86, 25-36.
26. Davidsson, Å. (2007) Ph.D. , Lunds University, Lund.
27. Teghammar, A., Castillo, M. D. P., Ascue, J., Niklasson, C. and Sárvári Horváth, I.
(2013) Energy Fuels. 27, 277-284.
28. Teghammar, A., Forgács, G., Sárvári Horváth, I. and Taherzadeh, M. J. (2014) Applied
Energy. 116, 125-133.
20
FIGURES
Figure 1. Accumulated methane production on NMMO-pretreated and untreated forest residue at
mesophilic batch condition. The pretreatment conditions are described in the figure.
0
20
40
60
80
100
120
0 10 20 30 40 50 60
Vo
lum
e N
ml/
gVS
Time (day)
untreated
90, 3h , 85 %
120, 3h, 85%
120, 15h, 75%
21
Figure 2. Specific gas production for untreated forest residues (●...) and NMMO-treated forest
residue (o…)
0
20
40
60
80
100
120
140
0 20 40 60 80 100 120 140
Vo
lum
e (N
ml/
gVS)
Time (Day)
22
Figure 3. pH (♦) and organic loading rate (▬) for the reactors fed with (A) untreated forest
residue and (B) NMMO-treated forest residues
0
1
2
3
4
5
7,17,27,37,47,57,67,77,87,9
0 10 20 30 40 50 60 70 80 90 100 110
Org
aniic
load
ing
rate
[g
VS/
L &
day
]
pH
A
0
1
2
3
4
5
7
7,2
7,4
7,6
7,8
8
0 10 20 30 40 50 60 70 80 90 100 110 120 130 Org
aniic
load
ing
rate
[g V
S/L
& d
ay]
pH
Time [Days]
B
23
TABLES.
Table 1. Pretreatment condition, Methane production, Carbohydrates, lignin of untreated and
NMMO- Pretreated forest residue
a Reaction rate during the first 10 days with two standard deviations.
b Accumulated methane gas produced per gram volatile solids after 52 days of incubation with
two standard deviations
Pretreatment conditions
Methane production
Temperature °C
NMMO (%)
Treatment time (h)
Total solids
(%)
Volatile Solids (%)
Total lignin (wt%)
Total carbohydrates
(%)
Initial reaction rate
(Nml/g VS/day)a Yield
(Nml/gVS added)b
120 °C 75 15 30.5 29.69 36.48 49.05 4.27 ± 1.5 100.5±16 120 °C 85 3 26.77 26.04 38.67 45.3 3.65 ± 1.0 109,5±20 90 °C 85 3 27.33 26.48 40.55 41.8 2.75 ± 0.6 87,68±17
Untreated - - 50.7 45.64 45.75 41.05 0.83 ± 0.4 41,53±3,0
III
High-rate biogas production from waste textiles using a two-stage process
Azam Jeihanipour a, Solmaz Aslanzadeh b, Karthik Rajendran b,*, Gopinath Balasubramanian b,Mohammad J. Taherzadeh b
aDepartment of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan 81746-73441, Iranb School of Engineering, University of Borås, 50190 Borås, Sweden
a r t i c l e i n f o
Article history:Received 3 July 2012Accepted 30 October 2012Available online 22 November 2012
Keywords:Rapid digestionBiogasNMMO pretreatmentUASBNMMOWaste textiles
a b s t r a c t
The efficacy of a two-stage Continuously Stirred Tank Reactor (CSTR), modified as Stirred Batch Reactor(SBR), and Upflow Anaerobic Sludge Blanket Bed (UASB) process in producing biogas from waste textileswas investigated under batch and semi-continuous conditions. Single-stage and two-stage digestionswere compared in batch reactors, where 20 g/L cellulose loading, as either viscose/polyester or cotton/polyester textiles, was used. The results disclosed that the total gas production from viscose/polyester ina two-stage process was comparable to the production in a single-stage SBR, and in less than two weeks,more than 80% of the theoretical yield of methane was acquired. However, for cotton/polyester, the two-stage batch process was significantly superior to the single-stage; the maximum rate of methaneproduction was increased to 80%, and the lag phase decreased from 15 days to 4 days. In the two-stagesemi-continuous process, where the substrate consisted of jeans textiles, the effect of N-methyl-morpholine-N-oxide (NMMO) pretreatment was studied. In this experiment, digestion of untreated andNMMO-treated jeans textiles resulted in 200 and 400 ml (respectively) methane/g volatile solids/day(ml/g VS/day), with an organic loading rate (OLR) of 2 g VS/L reactor volume/day (g VS/L/day); underthese conditions, the NMMO pretreatment doubled the biogas yield, a significant improvement. The OLRcould successfully be increased to 2.7 g VS/L/day, but at a loading rate of 4 g VS/L/day, the rate of methaneproduction declined. By arranging a serial interconnection of the two reactors and their liquids in thetwo-stage process, a closed system was obtained that converted waste textiles into biogas.
� 2012 Elsevier Ltd. All rights reserved.
1. Introduction
The annual global production of end-of-life waste textiles issteadily increasing, causing an increasing concern regarding theimpact of the disposal of this enormous amount of waste on theenvironment. In spite of textile waste in fact being a potentially richsource of energy and materials, the current normal routine todispose of this waste is by incineration or as landfilling. Interest-ingly, of the world’s total textile production, around 40% of the fiberconsumption comprises cellulose [1], the same percentage as theaverage content of cellulose in lignocellulosic materials.
Waste textiles are mainly composed of cotton and viscose fibers,and holds, thanks to their cellulose content, a significant potential for
production of different biofuels, such as biogas [2]. For instance, in2008, the influx of clothing and textiles to Swedenwas 131,800 tons[3].Assuming that the total amountofwaste textiles inSwedennearlyequals the amount of imported clothing and textiles, and that 40% ofthe textile fibers consists of cellulose, approximately 53,000 tons ofcellulose iswasted every year. Ayield of415mlmethane (at STP)pergcellulose implies that the amount of waste textiles produced inSweden would suffice as substrate for producing more than 20million Nm3 of methane, equaling in the region of 4 TWh power peryear; to be compared with 11 TWh estimated to be the biogaspotential of leycrops, straw,potato, andsugarbeet tops inSweden[4].
Fossil fuels are currently dominating the global energy market.However, the growing world population along with diminishingfossil fuel reserves have resulted in a global interest in graduallyshifting the energy source from fossil to alternative fuels [5,6]. Inaddition, environmental pollution caused by e.g. the dumping ofwaste materials in the environment, is one of the most importantissues the world is facing today. Biogas, produced by means ofanaerobic digestion of biological waste, is a renewable bioenergyand a potential alternative to petroleum-based fuels [7]. In addition
Abbreviations: CSTR, continuously stirred tank reactor; SBR, stirred batchreactor; UASB, upflow anaerobic sludge blanket bed; GC, gas chromatography; IC,ion-exchange chromatography; HPLC, high performance liquid chromatography;VFA, volatile fatty acid; AMPTS, automatic methane potential testing system.* Corresponding author. Tel.: þ46 33 435 4855; fax: þ46 33 435 4008.
E-mail address: [email protected] (K. Rajendran).
Contents lists available at SciVerse ScienceDirect
Renewable Energy
journal homepage: www.elsevier .com/locate/renene
0960-1481/$ e see front matter � 2012 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.renene.2012.10.042
Renewable Energy 52 (2013) 128e135
to the methane itself, biogas production holds the potential tominimize the waste pollution, thus protecting the environment [8].From the perspective of resource efficiency, biogas production hasa higher outputeinput energy ratio compared to, for example,current ethanol production systems [9]. Furthermore, in terms ofemissions, biogas production might be better for the environmentthan incineration of waste [10,11].
Methane-rich biogas has different applications. It may serve asa source for heat, steam, and electricity, and can be further upgradedto vehicle fuel, or for production of chemicals. It may also be used asa household fuel for cooking and lighting, or in fuel cells. Taking allthese aspects into account, being a well-established technology forgenerating bioenergy, biogas production is one of the most envi-ronmentally beneficial processes for replacing fossil fuels [8,12].Furthermore, development of new technologies has facilitatedbiogas production for combined heat and power (CHP) systems insmall scale (�100 KWe) [13]. Thanks to biogas production being anuncomplicated process, which is a significant advantage, it can belocated near the place where waste is produced, and the wasteproducers can be the end-users of the biogas, hence evading prob-lems related to transport of both wastes and biogas. Such systems(so-called on-farm biogas plants), have in Germany been commer-cially installed in thousands [14], mainly using biomass from agri-culture. Apart for the conventional waste streams such as municipalsolid wastes (MSW) and manure, the recent trend includes thepretreated lignocellulosic biomass, wooden fractions and agricul-tural residues are used for biogas production [15].
However, the potential of other available biological wastes, suchas cellulosic waste textiles, as substrate in small-scale biogas plants,has not been adequately investigated. From the literature, therewas very little work that has been focused on the textile waste asa substrate for anaerobic digestion. Previous work includes pre-treating waste textile containing cellulosic blend fibers and highcrystalline cellulose fibers in a batch assay [16,17] in order toincrease the biogas production. Additionally, textile wastes werenever tested in a two-stage process.
The present study is focused on investigating the feasibility ofusing waste cellulosic textile fibers for production of biogas,employing different processes and comparing their efficacy.
2. Materials and methods
The first step was to examine a one-stage batch process (i.e. in anSBR) and a two-stage (i.e. in an SBR and a UASB) anaerobic digestion,using two different substrates (viscose and cotton fibers, bothblended with polyester fibers), without separating the cellulosicfibers. In the two-stage process, textile was converted into biogas ina closed system, which was obtained by arranging a serial inter-connection of the liquids of both reactors. However, previousattempts have been made to separate cellulose from mixed fibers[18] by e.g. dissolution of cellulose [19] and subsequently regener-ating it. Jeihanipour et al. [16] recently developed a process forseparating the cellulosic part from waste textiles, using an environ-mental friendly cellulose solvent, i.e. N-methylmorpholine-N-oxide(NMMO), in order to facilitate the production of biogas or bioethanolfromwaste textiles [17]. Hence, the second step of the present studycomprised a semi-continuous two-stage anaerobic digestionprocess, comparing biogas yield from NMMO-treated and untreatedcotton-based waste textiles, at different organic loading rates.
2.1. Materials and inoculums
The three waste textiles used in the present study were woventextiles: orange (50% polyester, 50% cotton), blue (40% polyester, 60%viscose), both provided by local shops in Borås (Sweden), and also
used blue jeans textiles (100% cotton). Prior to the experiments, thefirst two textiles were cut into small pieces (approximately 2.5 � 2.5cm2), while the jeans textiles were ground into finematerials. NMMOwas provided by BASF (Ludwigshafen, Germany) as a 50% watersolution.
The inoculum used in the CSTRs and SBRs was obtained froma 3000-m3 municipal solid waste digester, operating under ther-mophilic (55 �C) conditions (Borås Energy & Environment AB,Sweden). The granulated anaerobic sludge used as seed in the UASBreactors was provided from a UASB reactor treating municipalwastewater in Hammarby Sjöstad (Stockholm, Sweden).
2.2. Pretreatment procedure
For pretreatment of the ground jeans textiles, the NMMOsolution was concentrated to 85% in a rotary vacuum evaporator(Laborota 20, Heidolph, Schwabach, Germany), equipped witha vacuum pump (PC 3004 VARIO, Vacuubrand, Wertheim,Germany). The concentrate was mixed with ground jeans textiles(6% w/w dry matter) in an oil bath at 120 �C for 3 h under atmo-spheric conditions, using a mixer for continuous blending in orderto dissolve the cellulose [17]. The resulting celluloseeNMMOsolution was then added to boiling water while mixing continu-ously, thereby regenerating the dissolved cellulose. Using a vacuumfilter, the regenerated cellulose was separated from the NMMOewater solution, and washed with hot water. The washed cellulosewas stored wet at 4 �C until used for anaerobic digestion.
2.3. Experimental setup
2.3.1. ReactorsTwo types of reactors, a continuous flow stirred tank reactor
(CSTR), modified for batch process as stirred batch reactor (SBR)and an upflow anaerobic sludge blanket bed (UASB) reactor, bothmade of polymethylmethacrylate (PMMA), were used in differentconfigurations. The CSTR had a working volume of 3 L (an internaldiameter of 18.5 cm and a height of 18.5 cm), while the workingvolume of the UASB was 2.25 L (an internal diameter of 6.4 cm anda height of 70 cm). Temperature was set at 55 �C for the CSTR andSBR, and at 34 �C for the UASB, using a temperature-controlledwater-bath with water recirculation through the reactor’s doublejacket. Both types of reactors were equipped with a feed inlet,a liquid sampling point, an outlet, and a gas line to the gasmeasuring system, which had a gas sampling port. The CSTR andSBR were equipped with an impeller for continuous mixing of thecontents. The inlet of the UASB reactor had a mesh to avoid largeparticles entering the system (Fig. 1B).
2.3.2. Reactor seeding and start upThe UASB reactors were seeded with 1.28 L of granular anaer-
obic sludge. The remaining volume of the reactor was filled withwater. Upon receipt, the inoculum for the CSTR was stored in anincubator at 55 �C for three days, to degrade easily degradableorganic matter still present in the inoculum, and to remove dis-solved methane. The CSTR and SBR were filled with 2.5 L of inoc-ulum and 0.5 L of nutrient solution to set the C:N:P:S ratio to500:20:5:3, in accordance with the cellulose concentration in thebeginning of the experiment. The final nutrient concentrations forthe basic medium (1 g cellulose/L, containing inorganic macronu-trients) were (in mg/L): NH4Cl (76.4), KH2PO4 (5.18), MgSO4$7H2O(0.27), CaCl2$2H2O, (10.00), and trace nutrients, 1 ml/L [20].
2.3.3. Reactor configurationsIn the present study, the efficacy of single-stage and two-stage
batch processes as well as a two-stage continuous process for
A. Jeihanipour et al. / Renewable Energy 52 (2013) 128e135 129
anaerobic digestion of waste textiles was examined. The arrange-ments of the reactor facilities are schematically illustrated in Fig.1. Inthe one-stage batch process, an SBR was used as a digester. In thetwo-stage batch process, the SBRwas serially connectedwith a UASBreactor. Liquid effluent from the SBR was continuously pumped tothe UASB reactor at a rate of 3 L/day. At this flow rate, the hydraulicretention times (HRT) in the SBR and the UASB reactors were ca. 24and 18 h, respectively. A peristaltic pump with a tube diameter of1.02 mm was used. The effluent of the UASB reactor was continu-ously fed back to the SBR. The SBR of the two-stage batch processwas equipped with a cylindrical filter around the impeller, and thetextile wastes were placed inside the filter. The liquid outlet of theSBR passed through the filter, while the textileswere retainedwithinthe SBR. With this filter, the polyester part of the textile could berecovered after the process was completed.
The configuration of the reactors in the two-stage continuousprocess was quite similar to that in the two-stage batch process.The difference was the removal of the internal filter in the SBR,placing a sedimentation tank (with a volume of 100 ml) in liquidline to the UASB, before the pump, to settle the large particles(Fig. 1).
2.4. Experimental operations
The batch processes were conducted by feeding the SBRs withcotton/polyester (50/50) and viscose/polyester (60/40) textiles, to
establish a cellulose concentration of 20 g/L. After 25 days, theprocess was interrupted, and the remaining textiles were sepa-rated, washed, and studied in a stereomicroscope. In the semi-continuous processes, 2 two-stage systems were used to digestground jeans textiles and NMMO-treated jeans textiles. The OLR ofthe process was increased stepwise from 2 up to 4 g VS/L/day. Oncea day, a certain amount of substrate was fed into the CSTR, inaccordance with the desired OLR. The HRT of UASB was controlledby changing the speed of the pump in the beginning of each step.Each OLR was continued for more than three HRTs in the CSTR,when a steady state condition was attained. Table 1 describes theconditions of the different steps during the process, including theOLRs and their respective HRTs, flow rates, and durations.
No solids were withdrawn from the reactors during the exper-imental period in neither the batch nor the continuous processes,except when sampling for the analyses. Liquid and gas weresampled twice a week throughout the running process, and the
Fig. 1. Schematic diagram of the CSTReUASB combined system with internal recirculation. (A) Batch process equipped with internal filter in the SBR and (B) semi-continuousprocess equipped with sedimentation tank.
Table 1Organic loading rates (OLR), hydraulic retention times (HRT) in the CSTR and theUASB reactor, and phase durations, determined at different stages.
Stage OLR (g VS/L/day) HRT in CSTR (day) HRT in UASB (day) Duration (day)
1 2.0 10.0 7.50 30.02 2.7 7.5 5.62 30.03 4.0 5.0 3.75 15.0
A. Jeihanipour et al. / Renewable Energy 52 (2013) 128e135130
volumes of produced gas were recorded. The gas samples wereanalyzed directly by gas chromatography (GC), while the liquidsamples were stored in the freezer at �20 �C for later analyses.
2.5. Analytical methods
Thecellulose contentof the substrateswasdeterminedaccordingto the method provided by the National Renewable Energy Labora-tory in the USA [21]. The gas production was recorded by using theAutomatic Methane Potential Testing System (AMPTS, Bioprocesscontrol AB, Lund, Sweden), whose function is based on waterdisplacement and buoyancy, with a measuring resolution of 13 ml.The instrument was equipped with a laptop computer and thevolumesof producedgasvs. timewere recorded foreach reactor. Thecomposition of the biogas produced during anaerobic digestionwasmeasured using a gas chromatograph (Auto System Perkin Elmer,Waltham, MA) equipped with a packed column (Perkin Elmer,60 � 1.800OD, 80/100, mesh) and a thermal conductivity detector(Perkin Elmer) set to 200 �C. The inject temperature was set to
0
10
20
30
40
50
60
0 5 10 15 20 25 30
Meth
an
e (m
l/g
VS
/d
ay)
Days
Viscose/polyester
Cotton/polyester
Fig. 2. Rate of methane production from (A) viscose/polyester and (C) cotton/poly-ester in the single-stage batch process.
Fig. 3. Stereomicroscopic pictures of viscose/polyester and cotton/polyester before and after single-stage and two-stage digestions.
A. Jeihanipour et al. / Renewable Energy 52 (2013) 128e135 131
150 �C, and the oven temperature to 75 �C. The carrier gas used wasnitrogen, kept at amaintained pressure of 0.70 bar and a flow rate of40 ml/min at 60 �C. A 250-ml pressure-tight gas syringe (VICI,Precision Sampling Inc., LA) was used for the gas sampling.
Liquid samples were centrifuged at 10,000 rpm for 10 min, andsolidparticleswere removedbyfiltration througha0.2-mmfilterpriorto analyses for pH, soluble chemical oxygen demand (COD), andvolatile fatty acid (VFA) concentrations. The CODwasmeasuredusingan HACH apparatus equipped with a UVevis Spectrophotometer(HACH, Germany), using Digestion Solution COD vials (operatingrange 0e15,000 mg COD/L). The VFA concentrations, comprisingacetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid,and isovaleric acid, were analyzed by HPLC (Waters 2695, WatersCorporation, Milford, MA, USA) with a UV detector (Waters 2414),utilizing an ion-exchange column (Aminex HPX-87H Bio-Rad,Hercules, CA)workingat60 �C, andusing5mMsulfuricacidaseluent,with a flow of 0.6 ml/min. The macronutrients, ammonium andpotassium, were analyzed with an Ion Chromatograph (Metrohm,Herisau, Switzerland), using a cation column holding an eluent flowrate of 1 ml/min; the pressure was set at 7e9 MPa, and the temper-aturewas 35e40 �C. The eluent solution consisted of 4 mmol tartaricacid and0.75mmoldipicolinic acidper Lwater. Sampleswere dilutedwith the eluent, and pH was adjusted to 2e3. They were thencentrifuged at 10,000 rpm for 4 min, and filtered through a 0.45 mmfilter prior to injection. The texture of the textiles before and afterbatch digestion was studied, using a NIKON stereomicroscope(SMZ800, Tokyo, Japan) fitted with a C-DSD230 camera.
3. Results and discussion
3.1. Batch digestion
3.1.1. Single-stage anaerobic digestion in the SBRThe cumulative methane produced during 25 days of single-
stage anaerobic digestion in the SBR is presented in Fig. 2. The
0
10
20
30
40
50
60
70
80
90
100
0
25
50
75
100
125
Sh
are
o
f re
ac
to
r in
m
eth
an
e p
ro
du
ctio
n
(%
)
Me
th
an
e (m
l /g
VS
/d
ay
)
Viscose/polyester
0
10
20
30
40
50
60
70
80
90
100
0
25
50
75
100
125
0 5 10 15 20 25 30
Sh
are
o
f re
ac
to
r in
m
eth
an
e p
ro
du
ctio
n
(%
)
Me
th
an
e (m
l/g
VS
/d
ay
)
Days
Cotton/polyester
Fig. 4. Rate of methane production from (A) viscose/polyester and (C) cotton/poly-ester in the two-stage batch process. The amount of methane, produced in eachreactor, presented as (---) % share of the CSTR and (- - -) % share of the UASB.
0102030405060708090100
0
100
200
300
400
500
Sh
are
of re
acto
r in
m
eth
an
e p
ro
du
ctio
n
(%
)
Meth
an
e (m
L/g
VS
/d
ay)
Untreated Jeans
0102030405060708090100
0
100
200
300
400
500
0 10 20 30 40 50 60 70 80
Sh
are
of re
acto
r in
m
eth
an
e p
ro
du
ctio
n
(%
)
Meth
an
e (m
L/g
VS
/d
ay)
Days
Pretreated Jeans
Fig. 5. Rate of methane production from untreated and pretreated jeans textiles in thesemi-continuous process. Thedifferent line styles represent: (�)methanevolume, (---) %share of methane produced in the CSTR and (- - -) % share of methane produced in theUASB.
0
0,4
0,8
1,2
1,6
0
1
2
3
4
5
6
7
Co
ncen
tratio
n in
U
AS
B (g
/L
)
Co
ncen
tratio
n in
C
ST
R (g
/L
)
Untreated Jeans
0
0,4
0,8
1,2
1,6
0
1
2
3
4
5
6
7
0 10 20 30 40 50 60 70 80
Co
ncen
tratio
n in
U
AS
B (g
/L
)
Co
ncen
tratio
n in
C
ST
R (g
/L
)
Days
Pretreated Jeans
Fig. 6. Variation of VFA concentrations in (:) CSTR and (A) UASB during digestion ofuntreated and pretreated jeans in the semi-continuous process.
A. Jeihanipour et al. / Renewable Energy 52 (2013) 128e135132
reactor fed with viscose/polyester textiles had a 2-day-long lagphase, whereas in the reactor fed with cotton/polyester, the cor-responding lag phase before gas production was triggered wasabout 15 days long. The longer lag phasemay be due to the differenttexture of cotton/polyester, providing a smaller contact surface areafor cellulolytic microorganisms to work on. Once the biogasproduction started, the production rate from cotton/polyester wasslower than from viscose/polyester. The theoretical methane yieldwas calculated according to Buswell formula [22], which is 415ml/gVS for cellulose. Within 12 days of gas production from viscose/polyester, more than 80% of the theoretical yield of methane wasacquired, to be compared with the 17% yield from cotton/polyester,gainedduring the10days following the lagperiod (Fig. 2). Differencesin contact surface area, molecular structure of cellulose, and chem-istry of the dyes and reagents covering the cotton and viscose fibers,are possible reasons for this huge difference in digestion outcomebetween the viscose/polyester and cotton/polyester waste textilesutilized in these experiments. The maximum rate of biogas produc-tion from viscose/polyester reached 55 ml/g VS/day after 8 days.
The appearances of the textiles used in the batch process, asshown in stereomicroscopy before and after digestion, are pre-sented in Fig. 3. Themicroscopy revealed that viscose/polyester haddisintegrated fibers compared to cotton/polyester, consequentlyfacilitating the process of degradation of viscose/polyester by
microorganisms. Single-stage and two-stage digestion of viscose/polyester (Fig. 3C and E) did not differ much, while degradation ofcotton/polyester wasmore successful in the two-stage process thanin the single-stage digestion (Fig. 3D and F).
3.1.2. Two-stage anaerobic digestionThe cumulative methane production acquired over 25 days, and
its share (in percentage) of the SBR and the UASB reactor, is pre-sented in Fig. 4. Though the gas production from both textiles(cotton/polyester and viscose/polyester) started after three days,the initial rate of biogas production from viscose/polyester wassuperior compared to the initial production rate of cotton/poly-ester, where it was low in the single-stage digestion as well. Thetotal gas production from viscose/polyester did not differ betweenthe single-stage process and the two-stage process.
Jeihanipour et al. [16] reported that under batch conditions,methane yield from untreated cotton/polyester was lower thanfrom viscose/polyester; after six days of digestion, only about 4.95%of the theoretical yield was acquired from cotton/polyester, while36.28% was produced from viscose/polyester. In the present study,however, biogas production from cotton/polyester reached 40% ofthe theoretical yield after 10 days of digestion, while 80% of thetheoretical yield was attained from viscose/polyester after 12 daysof digestion. The maximum rate of methane production from
Table 2The COD, the ratio of methane to carbon dioxide in the CSTR and the UASB reactor, and the COD removal efficiency of the UASB reactor, during digestion of untreated andpretreated jeans at different stages. The efficacy of the UASB-digesters (expressed as COD removal efficiency in percent) was calculated by dividing the difference between CODinlet and outlet with the COD inlet.
Substrate OLR (g VS/L/day) COD (mg/L) COD removal efficiency (%) Ratio of methane to carbondioxide
CSTR UASB CSTR UASB
Untreated jeans 2.0 6169 � 1348 2027 � 626 66.8 � 9.7 1.46 � 0.09 5.33 � 0.552.7 4395 � 1234 1198 � 235 72.0 � 3.3 1.97 � 0.26 5.40 � 0.464.0 2873 � 562 1276 � 185 56.6 � 10.5 2.17 � 0.04 5.03 � 0.20
Pretreated jeans 2.0 4377 � 652 2019 � 638 53.4 � 14.4 1.92 � 0.29 4.72 � 0.532.7 3212 � 416 1822 � 239 42.2 � 11.5 2.14 � 0.13 4.18 � 0.154.0 2833 � 267 2220 � 305 22.9 � 14.5 2.18 � 0.06 3.63 � 0.31
0
400
800
1200
1600
2000
2400
2800
Am
mo
niu
m (m
g/L
)
A
0
400
800
1200
1600
2000
2400
2800
0 10 20 30 40 50 60 70 80
Am
mo
niu
m (m
g/L
)
Days
B
0 10 20 30 40 50 60 70 800
100
200
300
400
500
600
700
Days
Po
tassiu
m (m
g/L
)
D
0
100
200
300
400
500
600
700
Po
tassiu
m (m
g/L
)
C
Fig. 7. Variation of ammonium and potassium concentrations in the CSTR (A and C) and the UASB (B and D) during digestion of (A) untreated and (-) pretreated jeans in the semi-continuous process.
A. Jeihanipour et al. / Renewable Energy 52 (2013) 128e135 133
cotton/polyester in the two-stage process reached 30.6 ml/g VS/dayon day 8, and in the single-stage process, this textile produceda maximum methane volume of merely 17 ml/g VS/day, which wasachieved only on day 17. This implies an 80% yield increase whenusing the two-stage process rather than the single-stage SBR(during this time period). This efficacy increase may be due toa more efficient conversion of VFA into methane in the UASBprocess than in a single-stage process. The SBR produced the majorshare of gas from both textiles, as compared to the UASB reactor.Since the textiles were neither milled nor pretreated, the contactsurface area available for the microorganisms’ degradation of thetextiles was probably low.
3.2. Semi-continuous two-stage anaerobic digestion
3.2.1. Gas productionThe accumulated volume of methane produced per gram VS per
day fromuntreated jeans and NMMO-pretreated jeans are presentedin Fig. 5,which also illustrates the share ofmethane production in theCSTR and the UASB reactor, expressed as percentage. The volume ofmethane produced per gram VS per day increased with an increasedOLR. Comparing biogas production from untreated and treated jeansrevealed that anOLRof 2 g cellulose/L/day (stage 1) produced 200ml/g VS/day from untreated jeans, but more than 400 ml/g VS/day fromtreated jeans, i.e. pretreatment increased methane production with100%. Furthermore, an accumulation of VFA in the CSTR withuntreated jeans evidently resulted in a lower methane productionduring the experimental period (Fig. 6). When increasing the OLRfrom 2.0 to 2.7 g VS/L/day, the microorganisms adapted to theconditions, resulting inacquiring91%of the theoreticalmethaneyieldfromuntreated jeansand96% fromtreated jeans.However, increasingthe OLR to 4.0 g VS/L/day did not improve the methane productionany further. The CSTRwas responsible for the largest share of the totalmethane production (w90%) from treated jeans, most likely asa result of the enzymatic degradation of the cellulosic part of thetextiles being facilitated by the pretreatment.
The only comparable information found was the application ofrumen microorganisms in combination with a high-rate UASB usingfilter paper cellulose as substrate produced 438 ml/g VS/day, whichis equivalent to 98% of the theoretical yield. This slightly higher yieldcompared to present study could be explained by presence ofruminant bacteria which have high efficiency to hydrolyze even thecellulose based material with high-crystallinity [23].
3.2.2. COD and COD removal efficiencyThe chemical oxygen demand (COD) during the operation,
measured from the influent and effluent of the UASB reactor, isillustrated in Table 2. The efficacy of the UASB digestion ofuntreated jeans textiles increased from 66.8% to 72.3% whenincreasing the OLR from 2.0 to 2.7 g VS/L/day and decreasing theHRT from 10 to 7.5 days. A further increase in the OLR to 4.0 g VS/L/day decreased, however, the COD removal efficiency to 56.6% whenprocessing untreated jeans in the UASB reactor. When treated jeanstextiles were used, a decrease trend (from 53.4% to 22.9%) in theCOD removal efficiency was observed when the OLR was increasedfrom 2.0 to 4.0 g VS/L/day. The COD in the CSTR decreased withincreasing OLR and decreasing HRT, when processing untreated aswell as pretreated jeans textiles. Furthermore, during the entireprocess, the COD in the UASB reactor processing untreated jeansdecreased from 2027 mg/L to 1276 mg/L while a more stable CODaround 2000 mg/L was established when digesting pretreatedjeans. An increase in the OLR decreased the COD in the CSTR,regardless of textiles having undergone pretreatment or not.However, an increase in OLR resulted in a decreasing efficiency ofthe COD removal in the UASB reactor.
Mahmoud et al. [24] studied the COD removal efficiency ofa single-stage UASB reactor and a combined UASB-digester system,and found that the COD removal efficiency was higher in thecombined system (30%) compared to the single-stage system(about 5%). In the present study, for the complete process, theaverage COD removal efficiency was 65.1% for untreated jeans and39.5% for treated jeans.
3.2.3. Effect of nutrientsThe concentrations of the macronutrients (ammonium and
potassium) during the process, are illustrated in Fig. 7. The nutrientconcentration decreased with time in both digesters for bothtextiles which was due to activity of the cells to remove COD,produce biogas and of course some biomass. The final ammoniumconcentrations (Fig. 7A and B) in the CSTR and the UASBwere in therange of 600e800mg/L, while the potassium concentration (Fig. 7Cand D) decreased to around 150 and 200 mg/L in the CSTR and theUASB, respectively. After decreasing the nutrients to a minimumlevel, in spite of no nutrients or water being added to or removedfrom the system, the two-stage process was still able to producebiogas with a good yield. This observation may be because ofendogenous metabolism which causes autohydrolysis of some ofthe biomass present in the reactor.
3.2.4. Ratio of methane to carbon dioxideThe ratio of methane to carbon dioxide in each stage is illus-
trated in Table 2. By increasing the OLR from 2.0 to 4.0 g VS/L/day,the ratio in the CSTR increased from 1.46 to 2.17 and from 1.92 to2.18 for treated jeans and untreated jeans, respectively. However,the ratio for untreated jeans in the UASB reactor was stable ataround 5 throughout the experimental period, while the ratio fortreated jeans during the same period, decreased from 4.72 until3.63. Accumulation of VFA in the CSTR with untreated jeans,increased the ratio of methane to carbon dioxide in the UASBreactor. Pretreatment with NMMO decreased the crystallinestructure, and increased the digestibility of the material. Conse-quently, during digestion of treated jeans, the ratio of methane tocarbon dioxide increased in the CSTR but decreased in the UASBreactor. The treated jeans textiles were easily degraded to methanein the CSTR with no accumulation of VFA.
4. Conclusions
The comparison of single-stage and two-stage batch digestionprocesses for producing biogas from cotton/polyester and viscose/polyester with no pretreatment or milling revealed that gasproduction efficacy is highly affected by the molecular structure ofthe textile. In the semi-continuous process, pretreatment of textileshad a significant effect on the biogas production, due to a moreaccessible surface area for the degradation of cellulose fibers.Despite the complex structure of cotton/polyester, the initial rate ofbiogas production was higher and the lag phase shorter in the two-stage batch process, in comparison with the single-stage CSTR. Itwas furthermore concluded that when digesting treated oruntreated jeans textiles, the semi-continuous two-stage processwas able to handle a high OLR with a shorter HRT, in the CSTR aswell as in the UASB reactor.
Acknowledgement
This work was financed by Borås Energy & Environment AB andthe Sparbank foundation in Sjuhärad (Sweden). The authors wouldlike to acknowledge Adib Kalantar Mehrjerdi for the design andconstruction of the reactors and Michael Lacintra for technicalsupport in the laboratory.
A. Jeihanipour et al. / Renewable Energy 52 (2013) 128e135134
References
[1] Engelhardt A. The fiber year 2009/10: a world survey on textile and nonwovenindustry. Oerlikon 2010.
[2] Rajendran K, Balasubramanian G. High rate biogas production from wastetextiles. Borås: School of Engineering, University of Borås; 2011.
[3] Carlsson A, Hemström Kristian, Edborg Per, Stenmarck Åsa, Sörme Louise.Kartläggning av mängder och flöden av textilav fall. Norrköping: SvenskaMiljö Emissions Data (SMED); 2011.
[4] Svensson LM, Christensson K, Björnsson L. Biogas production from crop resi-dues on a farm-scale level: is it economically feasible under conditions inSweden? Bioprocess and Biosystems Engineering 2005;28:139e48.
[5] Banerjee S, Mudliar S, Sen R, Giri B, Satpute D, Chakrabarti T, et al.Commercializing lignocellulosic bioethanol: technology bottlenecks andpossible remedies. Biofuels, Bioproducts and Biorefining 2010;4:77e93.
[6] Sivakumar G, Vail DR, Xu J, Burner DM, Lay JO, Ge X, et al. Bioethanol andbiodiesel: alternative liquid fuels for future generations. Engineering in LifeSciences 2010;10:8e18.
[7] Soetaert W, Vandamme EJ. Biofuels. John Wiley & Sons Inc.; 2009.[8] Weiland P. Biogas production: current state and perspectives. Applied
Microbiology and Biotechnology 2010;85:849e60.[9] Börjesson P, Mattiasson B. Biogas as a resource-efficient vehicle fuel. Trends in
Biotechnology 2008;26:7e13.[10] Hill J, Nelson E, Tilman D, Polasky S, Tiffany D. Environmental, economic, and
energetic costs and benefits of biodiesel and ethanol biofuels. Proceedings ofthe National Academy of Sciences 2006;103:11206e10.
[11] Valerio F. Environmental impacts of post-consumer material managements:recycling, biological treatments, incineration. Waste Management 2010;30:2354e61.
[12] Holm-Nielsen JB, Al Seadi T, Oleskowicz-Popiel P. The future of anaer-obic digestion and biogas utilization. Bioresource Technology 2009;100:5478e84.
[13] Ciotola RJ, Lansing S, Martin JF. Emergy analysis of biogas production andelectricity generation from small-scale agricultural digesters. EcologicalEngineering 2011;37:1681e91.
[14] Wilkinson KG. A comparison of the drivers influencing adoption of on-farmanaerobic digestion in Germany and Australia. Biomass and Bioenergy2011;35:1613e22.
[15] Appels L, Lauwers J, Degrève J, Helsen L, Lievens B, Willems K, et al. Anaerobicdigestion in global bio-energy production: potential and research challenges.Renewable and Sustainable Energy Reviews 2011;15:4295e301.
[16] Jeihanipour A, Karimi K, Niklasson C, Taherzadeh MJ. A novel process forethanol or biogas production from cellulose in blended-fibers waste textiles.Waste Management 2010;30:2504e9.
[17] Jeihanipour A, Karimi K, Taherzadeh MJ. Enhancement of ethanol and biogasproduction from high-crystalline cellulose by different modes of NMOpretreatment. Biotechnology and Bioengineering 2010;105:469e76.
[18] Langley KD, Kim YK, Lewis AF, Recycling and reuse of mixed-fiber fabricremnants. Technical report, 2000.
[19] Negulescu II, Kwon H, Collier BJ, Collier JR, Pendse A. Recycling cotton fromcotton/polyester fabrics. Textile Chemist and Colorist 1998;30:31e5.
[20] Osuna MB, Zandvoort MH, Iza JM, Lettinga G, Lens PNL. Effects of traceelement addition on volatile fatty acid conversions in anaerobic granularsludge reactors. Environmental Technology 2003;24:573e87.
[21] Ruiz R, Ehrman T. Determination of carbohydrates in biomass by highperformance liquid chromatography. Laboratory Analytical Procedure 1996;2.
[22] Deublein D, Steinhauser A. Biogas from waste and renewable resources: anintroduction. Wiley-Vch; 2008.
[23] Gijzen HJ, Zwart KB, Verhagen FJM, Vogels GP. High-rate two-phase process forthe anaerobic degradation of cellulose, employing rumen microorganisms foran efficient acidogenesis. Biotechnology and Bioengineering 1988;31:418e25.
[24] Mahmoud N, Zeeman G, Gijzen H, Lettinga G. Anaerobic sewage treatment ina one-stage UASB reactor and a combined UASB-digester system. WaterResearch 2004;38:2348e58.
A. Jeihanipour et al. / Renewable Energy 52 (2013) 128e135 135
IV
Energies 2013, 6, 2966-2981; doi:10.3390/en6062966
energies ISSN 1996-1073
www.mdpi.com/journal/energies
Article
The Effect of Effluent Recirculation in a Semi-Continuous Two-Stage Anaerobic Digestion System
Solmaz Aslanzadeh 1,*, Karthik Rajendran 1, Azam Jeihanipour 2
and Mohammad J. Taherzadeh 1
1 School of Engineering, University of Borås, Borås 501 90, Sweden;
E-Mails: [email protected] (K.R.); [email protected] (M.J.T.) 2 Department of Biotechnology, Faculty of Advanced Sciences and Technologies,
University of Isfahan, Isfahan 81746-73441, Iran; E-Mail: [email protected]
* Author to whom correspondence should be addressed; E-Mail: [email protected];
Tel.: +46-33-435-4620; Fax: +46-33-435-4008.
Received: 5 May 2013; in revised form: 5 June 2013 / Accepted: 9 June 2013 /
Published: 17 June 2013
Abstract: The effect of recirculation in increasing organic loading rate (OLR) and
decreasing hydraulic retention time (HRT) in a semi-continuous two-stage anaerobic
digestion system using stirred tank reactor (CSTR) and an upflow anaerobic sludge bed
(UASB) was evaluated. Two-parallel processes were in operation for 100 days, one with
recirculation (closed system) and the other without recirculation (open system). For this
purpose, two structurally different carbohydrate-based substrates were used; starch and
cotton. The digestion of starch and cotton in the closed system resulted in production of
91% and 80% of the theoretical methane yield during the first 60 days. In contrast, in the
open system the methane yield was decreased to 82% and 56% of the theoretical value, for
starch and cotton, respectively. The OLR could successfully be increased to 4 gVS/L/day
for cotton and 10 gVS/L/day for starch. It is concluded that the recirculation supports the
microorganisms for effective hydrolysis of polyhydrocarbons in CSTR and to preserve the
nutrients in the system at higher OLRs, thereby improving the overall performance and
stability of the process.
Keywords: two-stage anaerobic digestion; recirculation effect; UASB; CSTR;
cotton; starch
OPEN ACCESS
Energies 2013, 6 2967
1. Introduction
Anaerobic digestion is gaining more attention nowadays, both as a solution to environmental
concerns, and also as an energy resource for today’s energy-demanding life style [1]. Biogas is a
product of anaerobic digestion processes, which is produced by a consortium of microorganisms. The
anaerobic digestion process is highly dependent on a variety of different factors such as pH,
temperature, HRT, carbon to nitrogen ratio, etc., [2–4]. However, the anaerobic degradation process is
a quite slow and sensitive process, which is highly affected by environmental stress and alterations in
operating conditions [5], that would lead to a disturbance of the balance in the microbial community.
The consequence of this imbalance is usually process failure. Stability of an anaerobic process,
especially in an industrial scale, is thus a vital factor for evaluation [6].
The microbial community in an anaerobic digestion comprise of fermentative, acetogenic and
methanogenic microorganisms. In general, methanogens have slower growth rates compared to
hydrolytic and acetogenic organisms, and are more sensitive to environmental stress. Efficient
anaerobic digestion requires the development and maintenance of a large, stable and viable population
of methane-forming microorganisms [5]. The most common reactor configuration used for anaerobic
digestion is the continuously stirred tank reactor (CSTR), in which the active biomass is constantly
removed from the system. These conventional systems usually have long retention times. This
drawback has been overcome using a high rate system, which is basically based on immobilization of
the active biomass which enables short retention times. It is because the sludge retention time is more
or less independent of the hydraulic retention time [7–9]. The microorganisms in the upflow anaerobic
sludge blanket (UASB) reactors are kept in the reactor by their ability to flocculate and produce
granules and thereby give the sludge good settling properties [9–11].
Two-stage anaerobic digestion process is considered to be effective when the rate limiting step in
the process is hydrolysis and liquefaction [12]. It consists of two separate reactors; one for
hydrolysis/acidogenesis and one for acetogenesis/methanogenesis. This physical separation makes it
possible to overcome the problem of the differences in the optimum conditions of the microorganisms’
activity and their growth kinetics [12] by optimizing conditions that are favorable to the growth of
each group of microorganisms in each reactor, such as short HRT and low pH for acid formers, which
is inhibitory for methanogens [13]. This type of phase separation would increase the stability of the
process, which is not possible in a conventional anaerobic process, where these two groups of
microorganisms are kept together in a single phase in a delicate balance [14]. Ever since the phase
separation was introduced into anaerobic digestion technology in 1970s, a significant number of papers
and reports have been published on the benefits of treating a variety of wastes at mesophilic as well as
thermophilic conditions such as treating fruits and vegetables [15,16], urban wastewaters [17],
industrial wastes [18], grass [19], coffee pulp juice [20], food wastes [21], cane-molasses alcohol
stillage [22], spent tea leaves [23], dairy wastewater [24–27], olive mill oil [28], and abattoir
wastes [29]. However, two-stage digestion processes have been used for treatment of wastes with very
low solid content [30–33]. The drawback of UASB is that this technology is not able to handle high
solid content [34]. Apart from this, data concerning the optimization of operating conditions, operation
and performance of two-phase configuration are inadequate as well. There is a lack of investigations
Energies 2013, 6 2968
on how effluent recycling affects the two stage process with high solid content and high organic
loading rate [35].
This paper investigates the effect of the effluent recirculation in a high rate semi-continuous two
stage anaerobic process using carbohydrate-based starch and cotton as substrate with high solid content
at various organic loading rates and hydraulic retention times.
2. Materials and Methods
2.1. Materials and Inoculums
The substrates used in this study were pure cotton and starch provided from local shops in Borås
(Sweden). The cotton was ground into fine materials before using them. The volatile solid of the cotton
and starch was 96% and 75%, respectively. The COD of the both materials were 1.19 kgCOD/kg of
the materials [36]. The inoculum used in the CSTR bioreactors was obtained from a 3000-m3 digester
treating municipal solid waste and working under thermophilic (55 °C) condition (Borås Energy &
Environment AB, Borås, Sweden). The UASB reactors were seeded using granulated anaerobic
sludge, which was provided from a pilot scale UASB reactor treating municipal wastewater at
Hammarby Sjöstad (Stockholm, Sweden) operating at 37 °C.
2.2. Experimental Set-Up
2.2.1. Reactors
The CSTR and UASB reactors were made of polymethylmethacrylate (PMMA), and used in
different configurations. The CSTR had a working volume of 3 L with an inner diameter of 18.5 cm
and a height of 18.5 cm, while the working volume of the UASB was 2.25 L with an internal diameter
of 6.4 cm and a height of 70 cm. Temperature of the reactors was sustained at 55 °C for CSTR and
34 °C for UASB by a thermal water-bath with water recirculation through the reactor’s water jacket
during the whole digestion process. Both reactors were equipped with a feed inlet, a liquid sampling
point, an effluent outlet, and a gas line to the gas measuring system which contained a gas sampling
port. The CSTR had an impeller for continuous mixing. The inlet from the bottom of the UASB reactor
was equipped with a net trap to prevent the large particles away from entering the reactor (Figure 1).
2.2.2. Reactors Seeding and Start Up
The UASB reactors were seeded with 1.3 L of granular anaerobic sludge and the remaining volume
of the reactors were filled with water. The inoculum for the CSTR was incubated at 55 °C for three
days in order to get stabilized before use, and remove the dissolved methane. The CSTR’s were filled
with 2.5 L of inoculum and 0.5 L of nutrient solution in which the C:N:P:S ratio was adjusted at
500:20:5:3 at the beginning of the experiment. The nutrient concentration for 1 g cellulose/L contained
basal medium with inorganic macro nutrient (in mg/L): NH4Cl (76.4), KH2PO4 (5.18), MgSO4·7H2O
(0.27), CaCl2·2H2O (10), and 1 mL/L of trace nutrients according to [37].
Energies 2013, 6 2969
Figure 1. Schematic figure of the semi-continuous two-stage system. (A) with
recirculation (Closed system), and (B) without recirculation (Open system).
2.2.3. Reactors Configuration
The arrangement of the two stage closed system and the two stage open system is presented
schematically in Figure 1. The configuration of the closed system and open system continuous process
was quite similar. The difference was that in the closed system the effluent of the UASB reactor was
continuously recirculated back to the CSTR, while the open system did not have any effluent
recirculation from UASB [38]. The recirculation rate of the liquid in the closed system was 91% ± 3%.
The recirculation rate of the liquid is based on the HRT in each OLR, which is controlled by the flow
rate in the pump. In order to separate particulate matter from the CSTR effluent, the outlet of the
Energies 2013, 6 2970
CSTR was equipped with a sedimentation tank consisting of a 100 mL glass bottle, to separate and
settle the large particles before pumping the liquid to the UASB. The feeding to both systems was once
and twice a day depending on the OLR.
2.2.4. Experimental Procedure
The semi-continuous digestions (open and closed) were carried out by feeding the bioreactors with
OLRs increasing from 2 up to 20 gVS/L/day in several steps. Once a day, depending on the OLR, the
substrate was fed into the CSTR. The HRT of UASB was controlled by adjusting the speed of the
pump prior to each step. Each OLR was maintained for more than three HRTs in the CSTR in order to
achieve a steady state condition. The steady state condition in each OLR refers to the constant loading
rate and gas production, which was achieved during three HRT periods. The process conditions;
including the OLR and their respective HRT, flow rate and duration are summarized in Table 1.
During the experiments, no solids/biomass was withdrawn from the reactors, except for the sample
analyses. The volume of biogas produced was recorded continuously by Automatic Methane Potential
Testing System (AMPTS, Bioprocess Control AB, Lund, Sweden) and gas chromatography. The liquid
and gas sampling were performed twice a week during the initial state of the process and increased to
every day from stage 4–6 due to short retention times. The liquid samples were kept at −20 °C until the
analyses were performed.
Table 1. The process conditions including the OLR and their respective HRT, flow rate
and duration in each stage of the experimental period.
Stage OLR (gVS/L/day) HRT in CSTR (day) HRT in UASB (day) Duration (day)
1 2.0 10.0 7.50 30.0 2 2.7 7.5 5.62 30.0 3 4.0 5.0 3.75 15.0 4 8.0 2.5 1.88 8.0 5 10 2.0 1.50 6.0 6 20 1.0 0.75 6.0
2.2.5. Analytical Methods
The production of biogas was recorded using AMPTS, operating based on water displacement. It
was equipped with a computer to record the biogas volume from each reactor. The composition of the
biogas produced during anaerobic digestion was measured using a gas chromatograph (Auto System
Perkin Elmer, Waltham, MA, USA), equipped with a packed column (Perkin Elmer, 6’ × 1.8’’OD,
80/100 Mesh) and a thermal conductivity detector (Perkin Elmer) with an inject temperature of 150 °C,
detection temperature of 200 °C, and oven temperature of 75 °C. The carrier gas used was
nitrogen-operated at a maintained pressure of 0.70 bar and a flow rate of 40 mL/min at 60 °C. A
250 µL pressure-tight gas syringe (VICI, Precision Sampling Inc., Baton Rouge, LA, USA) was used
for the gas sampling.
Liquid samples were analyzed for pH, soluble chemical oxygen demand (COD), and VFA
concentrations after centrifugation at 17,000 g for 10 min and subsequent filtration through a 0.2-µm
Energies 2013, 6 2971
filter to remove solid particles. The COD was measured using a HACH apparatus equipped with a
UV–Vis Spectrophotometer (HACH, Düsseldorf, Germany), with Digestion Solution COD vials
(operating range 0–15,000 mg COD/L). The VFA concentrations, including acetic acid, propionic acid,
butyric acid, isobutyric acid, valeric acid and isovaleric acid, were analyzed by HPLC (Waters 2695,
Waters Corporation, Milford, MA, USA), equipped with an ion-exchange column (Aminex HPX-87H
Bio-Rad, Hercules, CA, USA), working at 60 °C using 5 mM sulfuric acid as eluent with a flow of
0.6 mL/min, and a UV detector (Waters 2414, Milford, MA, USA). The macronutrients, including
ammonium and potassium were analyzed using an Ion Chromatography (Metrohm, Herisau,
Switzerland) working with a cation column at an eluent flow rate of 1 mL/min, pressure of 7–9 MPa,
and temperature of 35–40 °C. The eluent solution was composed of 4 mM/L tartaric acid and
0.75 mM/L dipicolinic acid in water. Before injection, the samples were diluted with eluent, the pH
was adjusted to 2–3, were then centrifuged at 17,000 g for 4 min and filtered through a 0.45 µm filter.
3. Results
Cellulose and starch were used as a substrate in a semi-continuous two-stage anaerobic digestion
process for biogas production. The substrates were digested separately, in two CSTRs with an OLR of
2 gVS/L/day, which was then increased stepwise up to 20 gVS/L/day for starch and 4 gVS/L/day for
cotton. The HRT was decreased in each step, and the reactors were continuously operated for three
consecutive HRTs to obtain steady state condition. The total methane with its percentage share of
methane production in CSTR and UASB produced per gram VS per day for the operational period of
90 days of digestion is presented in Figure 2.
Figure 2. Total methane production in open system for cotton and starch with --- % share
in CSTR and - - - % share in UASB.
0
10
20
30
40
50
60
70
80
90
100
0
50
100
150
200
250
300
350
400
450
0 10 20 30 40 50 60 70 80 90 100
Shar
e of
met
hane
pro
duct
ion
(%)
CH4
volu
me
(ml/
gVS/
day)
Days
Starch0
10
20
30
40
50
60
70
80
90
100
0
50
100
150
200
250
300
350
400
450
Shar
e of
met
hane
pro
duct
ion
(%)
CH4
volu
me
(ml/
gVS/
day)
Cotton
Energies 2013, 6 2972
3.1. Gas Production
3.1.1. Biogas Production in Open System (without Effluent Recirculation)
During the startup for cotton, the maximum methane production reached 363 gVS/L/day at day 12,
and then kept stable for 8 days before it started to decrease to 150 mL/gVS/d. Even after the increase
in OLR up to 2.7 gVS/L/day, the methane production remained stable at 150 mL/gVS/d for 10 days.
However, after day 40, the gas production decreased to less than 100 mL/gVS/d. An additional
increase in OLR to 4 gVS/L/day produced only 84 mL/gVS/d until day 75. Further increase in OLR to
8 gVS/L/day resulted in reactor failure, and the experiment was stopped.
On the other hand for starch, the process could be continued up until OLR 20 gVS/L/day for
95 days, while adding 2 gVS/L/day OLR, 340 mL/gVS/d of methane was produced. However, further
increase in OLR to 2.7 gVS/L/day resulted in decreased biogas production and just 45% of the
theoretical methane yield was achieved. A significant shift in the share of methane production from
CSTR to UASB could be observed at OLR 2.7 gVS/L/day. During the OLRs 4–10 gVS/L/day, the
theoretical methane yield was constant around 55%–60%. Furthermore, the accumulation of VFA in
CSTR was increased to more than 8 g/L during the same period (Figure 3C). In addition, the methane
production decreased from 230 mL/gVS/d to 128 mL/gVS/d when OLR was increased from
10 to 20 gVS/L/day. Starch had a more stable process during the first stage compared to cotton, which
could not reach steady state conditions even at low OLRs.
Figure 3. Volatile fatty acid concentration during the experimental period. ●- Starch;
♦- Cotton. (A) CSTR closed system; (B) UASB closed system; (C) CSTR open system;
(D) UASB open system.
0
0,2
0,4
0,6
0,8
1
0 10 20 30 40 50 60 70 80 90 100
Tota
l VF
A (g
/L)
Days
C
0 10 20 30 40 50 60 70 80 90 100
0
0,2
0,4
0,6
0,8
1
Days
Tota
l VF
A(g
/L)
D0
2
4
6
8
10
Tota
l VF
A (g
/L)
A
0
2
4
6
8
10
Tota
l VF
A(g
/L)
B
Energies 2013, 6 2973
3.1.2. Biogas Production in Closed System (with Effluent Recirculation)
The two-stage process in closed system was more stable compared to the open system. The
accumulated methane volume produced per gram VS per day for cotton and starch in the closed system
are presented in Figure 4. The percentage share of methane production in CSTR and UASB are also
marked in the same figure.
For cotton, the OLR could be increased from 2 to 2.7 gVS/L/day, and it resulted in a theoretical
methane yield of 85% and 76%, respectively. Further increase of OLR to 4 g VS/L/d caused a rapid
decline in the total gas production and the process was stopped.
In the case of starch, the theoretical methane yield was higher than 90% in the OLRs of 2 and
2.7 gVS/L/day. Additional decrease in HRT and increase in OLR up to 10 gVS/L/day stepwise
resulted in a theoretical methane yield of 50%–60%. The transition of the major share of methane
production from CSTR to UASB was observed at OLR 8 gVS/L/day. Though, the methane yield
between the OLR 4 to 10 gVS/L/day, the closed system possessed an overall stability. However, when
the OLR increased to 20 gVS/L/day, the gas production declined rapidly and the process was stopped.
Figure 4. Total methane production in closed system for cotton and starch with --- % share
in CSTR and - - - % share in UASB.
0
10
20
30
40
50
60
70
80
90
100
0
50
100
150
200
250
300
350
400
450
Shar
e of
met
hane
pro
duct
ion
(%)
CH4
volu
me
(ml/
gVS/
day)
Cotton
0
10
20
30
40
50
60
70
80
90
100
0
50
100
150
200
250
300
350
400
450
0 10 20 30 40 50 60 70 80 90 100
Shar
e of
met
hane
pro
duct
ion
(%)
CH4
volu
me
(ml/
gVS/
day)
Days
Starch
Energies 2013, 6 2974
3.2. COD and Its Removal
The COD was analyzed from the influent and effluent of the UASB during the operation. The
UASB digesters performance was examined using COD removal efficiency, calculated by dividing the
differences between COD inlet and outlet of UASB by the COD inlet to UASB. The results are
presented in Table 2. Equation (1) shows the calculation of the COD removal efficiency:
100in out
in
COD CODCOD removal efficiency
COD
−= × (1)
3.2.1. Open System
The COD removal efficiency was greater than 95% throughout the process for starch. The COD
was increased from 4300 to 27,700 mg/L in the CSTR fed with starch by increasing the OLR from
2 to 10 gVS/L/day. Furthermore, when the OLR was increased further to 20 g VS/L/d for starch, the
COD was decreased to approximately 24,000 mg/L. The COD in the UASB on the other hand, kept
stable throughout the entire process between 3000 and 4000 mg/L.
Table 2. The ratio of methane to carbon dioxide, concentration of COD and the COD
removal efficiency for cotton and starch in UASB and CSTR during different organic
loading rates.
Subs
tate
OLR
(gVS/L/day)
COD (mg/L) COD removal
efficiency (%)
COD (mg/L) COD removal
efficiency (%) Open system Closed system
CSTR UASB CSTR UASB
Cot
ton 2 3,259 ± 638 1,194 ± 95 61.9 ± 16.3 4,204 ± 742 1,958 ± 791 49.2 ± 21.6
2.7 3,699 ± 844 229 ± 82 93.3 ± 3.5 2,651 ± 572 1,525 ± 172 39.4 ± 16.5
4 3,241 ± 545 196 ± 33 93.9 ± 1.8 2,571 ± 204 1,476 ± 357 45.6 ± 15.9
Star
ch
2 4,324 ± 1,345 1,308 ± 335 69.7 ± 14.2 5,041 ± 430 3,273 ± 674 35.0 ± 12.8
2.7 9,034 ± 1,127 317 ± 143 96.49 ± 1.2 4,463 ± 626 3,353 ± 374 24.8 ± 16.2
4 19,500 ± 2,493 350 ± 165 98.2 ± 2.1 4,396 ± 565 3,051 ± 494 30.5 ± 7.8
8 21,450 ± 2,185 708 ± 186 96.6 ± 2.7 17,865 ± 2,767 3,091 ± 423 82.69 ± 3.8
10 27,716 ± 1,606 876 ± 270 96.8 ± 1.3 25,833 ± 3,333 3,995 ± 873 84.5 ± 4.2
20 23,800 ± 2,347 898 ± 98 96.2 ± 2.5 12,516 ± 2,171 3,256 ± 658 73.9 ± 9.5
In contrast, for cotton in open system, the COD removal efficiency was as high as 93%.
Interestingly, in the open system the increase in OLR from 2 to 4 gVS/L/day, did not significantly
change the COD of the CSTR fed with cotton which was stable around 3500 mg/L. The COD
concentration in the UASB on the other hand, decreased from 1194 mg/L to 196 mg/L during the
same period.
Energies 2013, 6 2975
3.2.2. Closed System
In the closed system digesting starch, the COD removal efficiency of UASB reactor performance of
starch increased from 35% to 84.5% with increasing in OLR from 2 to 10 g VS/L/d and decreasing in
HRT from 7.5 to 1.5 days. A further increase in OLR up to 20 gVS/L/day decreased the COD removal
efficiency of starch in UASB with more than 10%. The effluent COD out of UASB of starch during
the entire process in closed system was stable, even though the OLR increased and the HRT decreased
compared to the open system, which were more stable between 3000 and 4000 mg/L. A decreasing
trend was observed for the COD removal efficiency in cotton in closed system, in which a reduction
from 49.2% to 45.6% was occurred by increasing the OLR from 2 to 4 gVS/L/day.
3.3. Effect of Nutrients
The effects of macronutrients, including ammonium and potassium were studied and the results of
ammonium and potassium concentration during the entire experimental period are illustrated in
Figures 5 and 6.
A decreasing trend of nutrient concentration was observed for cotton and starch in both the open
and closed systems. In the closed system, the final ammonium concentration in CSTR and UASB was
four times higher than in the open system at OLR of 8 to 10 gVS/L/day for both the substrates. An
interesting observation was obtained in OLR between 10 and 20 gVS/L/day. The concentration of
ammonium show a sudden increase in the CSTR fed with starch in the closed system from 300 mg/L
to more than 1300 mg/L (Figure 5A).
Figure 5. The ammonium concentration during the experimental period. ●- Starch;
♦- Cotton. (A) CSTR closed system; (B) UASB closed system; (C) CSTR open system;
(D) UASB open system.
0
500
1000
1500
2000
2500
3000
0 10 20 30 40 50 60 70 80 90 100
Amm
oniu
m (m
g/L)
Days
B-UASB0
500
1000
1500
2000
2500
3000
Amm
oniu
m (m
g/L)
A-CSTR
0
500
1000
1500
2000
2500
3000
Amm
oniu
m (m
g/L)
C-CSTR
0 10 20 30 40 50 60 70 80 90 100
0
500
1000
1500
2000
2500
3000
Days
Amm
oniu
m (m
g/L)
D-UASB
Energies 2013, 6 2976
The same trend could also be observed in the potassium concentration for both starch and cotton as
the potassium concentration declined in CSTR in both systems. However, in the closed system, the
concentration was maintained between 100 and 500 mg/L (Figures 6A,C). On the other hand, in open
system the concentration of potassium decreased to less 100 mg/L in both CSTR and UASB
(Figure 6B,D).
Figure 6. The potassium concentration during the experimental period.●- Starch;
♦- Cotton. (A) CSTR closed system; (B) UASB closed system; (C) CSTR open system;
(D) UASB open system.
0
100
200
300
400
500
600
0 10 20 30 40 50 60 70 80 90 100
Pota
ssiu
m (m
g/L)
Days
B-UASB0
100
200
300
400
500
600
700
800
Pota
ssiu
m (m
g/L)
A-CSTR
0
100
200
300
400
500
600
700
800
Pota
ssiu
m (m
g/L)
C-CSTR
0 10 20 30 40 50 60 70 80 90 100
0
100
200
300
400
500
600
Days
Pota
ssiu
m (m
g/L)
D-UASB
3.4. Ratio of Methane to Carbon Dioxide
Anaerobic digestion of the carbohydrate in starch and cotton results in 50% methane
(CH4/CO2 = 1 mol/mol) in the biogas formed. However, partial dissolution of carbon dioxide in water
can lead to higher content of methane in the formed biogas. On the other hand, if the earlier steps in
the digestion process (e.g., hydrolysis and acidogenesis) occur and methanogenic bacteria fail to
produce methane, this CH4/CO2 ratio approaches to zero, since CO2 is still produced.
The ratio of methane to carbon dioxide in each stage of the experiments in this work is illustrated in
Table 3. The increase in OLR had a significant effect on the methane to carbon dioxide ratio for both
open and closed systems in CSTR.
In the CSTR open system for starch, the ratio CH4/CO2 ratio decreased from 2 to almost 0.1 at OLR
10 gVS/L/day. During the same period, the closed system could maintain a high ratio around 0.8. In
UASB, in contrast the ratio was stable throughout the process for both systems. However, while
adding 20 gVS/L/day OLR, the ratio was decreased for starch in the open system.
Energies 2013, 6 2977
The CH4/CO2 ratio for cotton in CSTR was very stable in both open and closed system and did not
show any significant change as the OLR increased from 2 to 4 gVS/L/day, being stable around 4.9.
The CH4/CO2 ratio for cotton in UASB was somewhat lower than in CSTR for both systems, being
around 2, and remained stable and during the entire process.
Table 3. The Ratio of methane to carbon dioxide, in open and closed system, for cotton
and starch in UASB and CSTR.
Substrate OLR (gVS/L/day)
Ratio of methane to carbon dioxide
Open system Closed system
CSTR UASB CSTR UASB
Cotton
2 1.8 ± 0.2 4.4 ± 0.8 1.9 ± 0.2 4.4 ± 0.4
2.7 1.7 ± 0.1 4.9 ± 0.6 2.0 ± 0.1 4.2 ± 0.2
4 2.0 ± 0.0 4.9 ± 0.2 2.1 ± 0.0 4.1 ± 0.1
Starch
2 2.0 ± 1.0 4.0 ± 0.5 2.1 ± 0.2 4.1 ± 0.6
2.7 1.3 ± 0.3 4.5 ± 0.6 2.2 ± 0.0 3.4 ± 0.2
4 1.0 ± 0.1 4.9 ± 0.2 1.8 ± 0.5 3.4 ± 0.1
8 0.3 ± 0.2 4.4 ± 0.1 0.9 ± 0.1 4.2 ± 0.3
10 0.1 ± 0.0 4.2 ± 0.2 0.8 ± 0.1 4.7 ± 0.4
20 0.07 ± 0.04 3.5 ± 0.8 0.7 ± 0.1 4.3 ± 0.8
4. Discussion
The results of this comparative study suggest that the recirculation in the closed system increases
the stability and the performance of a two stage system, using substrates at high OLRs. A higher
methane production was also achieved in the closed system comparing to open system for both
substrates. The major share of methane production seems also to be higher in CSTR at lower OLRs
rather than UASB for both processes and substrates. However, an interesting transition pattern is
observed in both systems as the major share of methane production is shifted from CSTR to UASB
Figures 2 and 3. This shift, however appear to occur at earlier stages in the open system comparing to
the closed system.
During OLR 2–2.7 gVS/L/day in the closed system the major share of the methane, produced in the
UASB was around 90% in CSTR for both cotton and starch. However, the increase in OLR to
4 gVS/L/day decreased the total methane yield and the transition of the major share of methane
production from CSTR to UASB begins. Additional increase of OLR to 8 gVS/L/day in closed system
digesting starch shifted the major share of methane produced shifted from CSTR to UASB and it
continued to increase with increasing OLR.
In the open system this transition was also observed, but at lower OLR (2.7 gVS/L/day) for both
substrates. The decrease in methane yield, which is the starting point of the transition, could be
explained by the accumulation of VFA in CSTR from less than 1 g/L to more than 8 g/L. The pH was
more stable in the closed system, which could be due to the effect of effluent recirculation from UASB
with pH around 8 to the CSTR and thereby stabilizing and keeping a stable pH over 6 in CSTR (data
not shown). Furthermore, the VFA produced in the CSTR was converted to biogas in the
UASB without accumulating in the first phase and reaching inhibitory levels for the acidogenesis
Energies 2013, 6 2978
process, which consequently contributes to the stability of the closed system in comparison to the open
system. This transition on the other hand, was never reached in the closed system digesting cotton. A
combination of the composition and efficient hydrolysis and the conversion of the intermediates to
methane in the CSTR due to the effect of recirculation causing higher and stable pH can be the
possible explanation [39].
The COD removal efficiency and the COD concentration in the CSTR were also affected by
recirculation as it started to increase during the same time as the transition occurs. The COD
concentration in the CSTR is highly dependent on the hydrolysis of the organic material to VFA and
follows more or less the same trend. The COD removal efficiency was higher, around 95%, in the open
system comparing to closed system which started for starch at the OLR 8 gVS/L/day to almost 85%.
However the COD efficiency in closed system could also be increased at higher OLR comparing to
open system. This also shows that UASB is more efficient at higher COD concentrations and could
handle high OLRs.
In contrast, the concentration of COD in the UASB decreased as the OLR increased in the open
system compared to closed system. The COD concentration in the closed system stayed stable between
1500 and 2000 mg/L for cotton and around 3000 mg/L for starch. It could be because of some
solubilized material kept recirculating in the system, and thereby keeping the COD both higher and
stable in closed system for both substrates, without having any considerable effects on the process.
Furthermore, when the OLR was increased further to 20 g VS/L/d for starch in open system, the COD
concentration in CSTR was decreased to approximately 1000 mg/L. This observation indicates that the
capacity of the CSTR to hydrolyze cotton and starch is limited. This capacity was obtained as less than
4 gVS/L/day for cotton, and 10–20 gVS/L/day for starch.
The ratio of methane to carbon dioxide was increased in UASB and decreased in CSTR in the
closed system. The increased CH4/CO2 ratio in the UASB could be due to dissolution of some part of
the produced carbon dioxide in the UASB and the capacity of the media in the UASB to capture and
further convert the carbon dioxide to methane by methanogens [39]. In the CSTR open system for
starch, the CH4/CO2 ratio decreased from 2 to almost 0.1 at OLR 10 gVS/L/day. During the same
period, the closed system could maintain a high ratio around 0.8. As the pH falls in the CSTR, the
more CO2 is dissolved to compensate as buffering system. A too strong acidification, consumes the
entire CO2 produced to keep the pH stable, which consequently inhibits the methanogens [40], and
hence, lower CH4/CO2 ratio in the CSTR open system comparing to the CSTR closed system. This is
an indication that recirculation could be able to support the microorganisms for effective hydrolysis in
CSTR. In UASB, the ratio was stable throughout the process for both systems.
In the closed system, the final ammonium concentration in CSTR and UASB was four times higher
than in the open system for both starch and cotton. The closed system supported the maintenance of
the nutrients in the system, compared to the open system, where fresh nutrients were added every day.
Since no liquid was removed or added to the system, the nutrients kept recycling in a closed cycle in
the process, leading to negligible loss of nutrients compared to the open system. An interesting
observation was obtained in OLR between 10 and 20 gVS/L/day in CSTR closed system. The
concentration of ammonium show a sudden increase at OLR 10–20 gVS/L/day in CSTR closed system
fed with starch (Figure 5A). This could be explained by the fact that shorter HRT, which is
accomplished by the increase in flow rate, causes high upflow velocities and thereby turbulence in the
Energies 2013, 6 2979
UASB. The consequence of this high flow rate is granule disintegration as the effect of shearing. The
resulting fragments are then washed out of the reactor [41] and are migrated to the CSTR by
recirculation. The subsequent degradation of the biomass and the release of the proteins into the
medium in CSTR cause an increase in the ammonium concentration [42].
5. Conclusions
The effect of recirculation in a semi-continuous two-stage anaerobic digestion combining CSTR
and UASB was studied using starch and cotton as substrate. The comparison of the closed system with
open system revealed that higher theoretical yield of methane could be achieved in the closed system
compared to the open system. Furthermore, it can be concluded that the recirculation could support the
hydrolysis step as well as avoiding nutrient loss at higher OLR and thus improving the performance
and the stability of the process a great deal.
Acknowledgements
This work was financially supported by Sparbank foundation in Sjuhärad (Sweden) and Borås
Energy and Environment AB (Sweden). The authors acknowledge Gopinath Balasubramanian for
experimental, technical and analytical support.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Asam, Z.-U.-Z.; Poulsen, T.G.; Nizami, A.-S.; Rafique, R.; Kiely, G.; Murphy, J.D. How can we
improve biomethane production per unit of feedstock in biogas plants? Appl. Energy 2011, 88,
2013–2018.
2. Chynoweth, D.P.; Owens, J.M.; Legrand, R. Renewable methane from anaerobic digestion of
biomass. Renew. Energy 2001, 22, 1–8.
3. Yadvika.; Santosh.; Sreekrishnan, T.R.; Kohli, S.; Rana, V. Enhancement of biogas production
from solid substrates using different techniques––A review. Bioresour. Technol. 2004, 95, 1–10.
4. Mahmoud, N.; Zeeman, G.; Gijzen, H.; Lettinga, G. Solids removal in upflow anaerobic reactors,
a review. Bioresour. Technol. 2003, 90, 1–9.
5. Vartak, D.R.; Engler, C.R.; McFarland, M.J.; Ricke, S.C. Attached-film media performance in
psychrophilic anaerobic treatment of dairy cattle wastewater. Bioresour. Technol. 1997, 62,
79–84.
6. Tay, J.-H.; Zhang, X. Stability of high-rate anaerobic systems. I: Performance under shocks.
J. Environ. Eng. 2000, 126, 713–725.
7. Lettinga, G. Anaerobic digestion and wastewater treatment systems. Antonie Van Leeuwenhoek
1995, 67, 3–28.
8. Schmidt, J.E.; Ahring, B.K. Granular sludge formation in upflow anaerobic sludge blanket
(UASB) reactors. Biotechnol. Bioeng. 1996, 49, 229–246.
Energies 2013, 6 2980
9. Lyberatos, G.; Skiadas, I.V. Modelling of anaerobic digestion—A review. Glob. Nest. Int. J.
1999, 1, 63–76.
10. Lettinga, G.; van Velsen, A.F.M.; Hobma, S.W.; de Zeeuw, W.; Klapwijk, A. Use of the upflow
sludge blanket (USB) reactor concept for biological wastewater treatment, especially for
anaerobic treatment. Biotechnol. Bioeng. 1980, 22, 699–734.
11. Bal, A.S.; Dhagat, N.N. Upflow anaerobic sludge blanket reactor—A review. Indian J. Environ.
Health 2001, 43, 1–83.
12. Shin, H.S.; Han, S.K.; Song, Y.C.; Lee, C.Y. Performance of UASB reactor treating leachate from
acidogenic fermenter in the two-phase anaerobic digestion of food waste. Water Res. 2001, 35,
3441–3447.
13. Ince, O. Performance of a two-phase anaerobic digestion system when treating dairy wastewater.
Water Res. 1998, 32, 2707–2713.
14. Demirel, B.; Yenigün, O. Two-phase anaerobic digestion processes: A review. J. Chem. Technol.
Biotechnol. 2002, 77, 743–755.
15. Verrier, D.; Roy, F.; Albagnac, G. Two-phase methanization of solid vegetable wastes.
Biol. Wastes 1987, 22, 163–177.
16. Bouallagui, H.; Torrijos, M.; Godon, J.J.; Moletta, R.; Ben Cheikh, R.; Touhami, Y.;
Delgenes, J.P.; Hamdi, M. Two-phases anaerobic digestion of fruit and vegetable wastes:
Bioreactors performance. Biochem. Eng. J. 2004, 21, 193–197.
17. Chanakya, H.N.; Borgaonkar, S.; Rajan, M.G.C.; Wahi, M. Two-phase anaerobic digestion of
water hyacinth or urban garbage. Bioresour. Technol. 1992, 42, 123–131.
18. Ghosh, S.; Ombregt, J.P.; Pipyn, P. Methane production from industrial wastes by two-phase
anaerobic digestion. Water Res. 1985, 19, 1083–1088.
19. Yu, H.W.; Samani, Z.; Hanson, A.; Smith, G. Energy recovery from grass using two-phase
anaerobic digestion. Waste Manag. 2002, 22, 1–5.
20. Calzada, J.F.; de Porres, E.; Yurrita, A.; de Arriola, M.C.; de Micheo, F.; Rolz, C.; Menchú, J.F.;
Cabello, A. Biogas production from coffee pulp juice: One- and two-phase systems. Agric. Wastes
1984, 9, 217–230.
21. Koster, I.W. Liquefaction and acidogenesis of tomatoes in an anaerobic two-phase solid-waste
treatment system. Agric. Wastes 1984, 11, 241–252.
22. Yeoh, B.G. Two-phase anaerobic treatment of cane-molasses alcohol stillage. Water Sci. Technol.
1997, 36, 441–448.
23. Goel, B.; Pant, D.C.; Kishore, V.V.N. Two-phase anaerobic digestion of spent tea leaves for
biogas and manure generation. Bioresour. Technol. 2001, 80, 153–156.
24. Lo, K.V.; Liao, P.H. Two-phase anaerobic digestion of screened dairy manure. Biomass 1985, 8,
81–90.
25. Liao, P.H.; Lo, K.V. Two-phase thermophilic anaerobic digestion of screened dairy manure.
Biomass 1985, 8, 185–194.
26. Lo, K.V.; Chen, W.Y.; Liao, P.H. Mesophilic digestion of screened dairy manure using anaerobic
rotating biological contact reactor. Biomass 1986, 9, 81–92.
27. Demirer, G.N.; Chen, S. Two-phase anaerobic digestion of unscreened dairy manure.
Process Biochem. 2005, 40, 3542–3549.
Energies 2013, 6 2981
28. Fezzani, B.; Ben Cheikh, R. Two-phase anaerobic co-digestion of olive mill wastes in
semi-continuous digesters at mesophilic temperature. Bioresour. Technol. 2010, 101, 1628–1634.
29. Wang, Z.; Banks, C.J. Evaluation of a two stage anaerobic digester for the treatment of mixed
abattoir wastes. Process Biochem. 2003, 38, 1267–1273.
30. Bhattacharya, S.K.; Madura, R.L.; Walling, D.A.; Farrell, J.B. Volatile solids reduction in
two-phase and conventional anaerobic sludge digestion. Water Res. 1996, 30, 1041–1048.
31. Diamantis, V.; Aivasidis, A. Two-stage uasb design enables activated-sludge free treatment of
easily biodegradable wastewater. Bioproc. Biosyst. Eng. 2010, 33, 287–292.
32. Diamantis, V.I.; Aivasidis, A. Comparison of single- and two-stage UASB reactors used for
anaerobic treatment of synthetic fruit wastewater. Enzym. Microb. Technol. 2007, 42, 6–10.
33. Feng, C.; Shimada, S.; Zhang, Z.; Maekawa, T. A pilot plant two-phase anaerobic digestion system
for bioenergy recovery from swine wastes and garbage. Waste Manag. 2008, 28, 1827–1834.
34. Sabry, T. Application of the UASB inoculated with flocculent and granular sludge in treating
sewage at different hydraulic shock loads. Bioresour. Technol. 2008, 99, 4073–4077.
35. Joubert, W.A.; Britz, T.J.; Lategan, P.M. The effect of effluent recirculation on the performance
of a two stage anaerobic process. Biotechnol. Lett. 1985, 7, 853–858.
36. Suss, H.U.; Kronis, J.D. The Correlation of Cod and Yield in Chemical Pulp Bleaching. Presented
at Tappi Breaking the Pulp Yield Barrier Symposium, Atlanta, GA, USA, 8–12 March 1998.
37. Osuna, M.B.; Zandvoort, M.H.; Iza, J.M.; Lettinga, G.; Lens, P.N.L. Effects of trace element
addition on volatile fatty acid conversions in anaerobic granular sludge reactors. Environ.
Technol. 2003, 24, 573–587.
38. Jeihanipour, A.; Aslanzadeh, S.; Rajendran, K.; Balasubramanian, G.; Taherzadeh, M.
High-rate biogas production from waste textiles using a two-stage process. Renew. Energy 2013,
52, 128–135.
39. Alimahmoodi, M.; Mulligan, C.N. Anaerobic bioconversion of carbon dioxide to biogas in an
upflow anaerobic sludge blanket reactor. J. Air Waste Manag. Assoc. 2008, 58, 95–103.
40. Deublein, D.; Steinhauser, A. Biology. In Biogas from Waste and Renewable Resources;
Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2008; pp. 93–128.
41. Kosaric, N.; Blaszczyk, R.; Orphan, L. Factors influencing formation and maintenance of granules
in anaerobic sludge blanket reactors (UASBR). Water Sci. Technol. 1990, 22, 275–282.
42. Blaszczyk, R.; Gardner, D.; Kosaric, N. Response and recovery of anaerobic granules from shock
loading. Water Res. 1994, 28, 675–680.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).
V
1
1
A comparative study between single and two-stage anaerobic digestion 2
processes: Effect of organic loading rate and hydraulic retention time 3
4
Solmaz Aslanzadeh*, Karthik Rajendran, Mohammad J. Taherzadeh 5
6
School of Engineering, University of Borås, Borås, Sweden 7
8
9
10
* Corresponding author: 11
Phone: +46-33 435 4620 12
Fax: +46-33 435 4008 13
E-mail: [email protected] 14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
2
Abstract 29
30
The effect of organic loading rate (OLR) and hydraulic retention time (HRT) was evaluated 31
by comparing single-stage and two-stage anaerobic digestion processes. Waste from food 32
processing industry (FPW) and organic fraction of municipal solid waste (OFMSW) were 33
used as substrates. The OLR was increased at each step from 2 gVS/l/d to 14 gVS/l/d and the 34
HRT was decreased from 10 days to 3 days. The highest theoretical methane yield achieved in 35
the single-stage process was about 84% for FPW during OLR 3 gVS/l/d at HRT of 7 days and 36
67 % for OFMSW at OLR of 2 gVS/l/d and HRT of 10 days. The single-stage process could 37
not handle further increase in OLR and decrease in HRT and the process was stopped. A more 38
stable operation was observed at higher OLRs and lower HRTs in the two-stage system. The 39
OLR could be increased to 8 gVS/l/d for FPW and to 12 gVS/l/d for OFMSW, operating at a 40
HRT of 3 days. The results show a conclusion of 26 % and 65 % less reactor volume for two-41
stage processes compared to single-stage processes for FPW and OFMSW, respectively. 42
Key words: Anaerobic digestion, Single stage process, Two-stage process, Hydraulic 43
retention time (HRT), Organic loading rate (OLR), Food waste 44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
3
1 Introduction 63
64
The global energy demand is currently being met by coal and oil, which are depleting sources, 65
not to mention the rise in environmental problems that the use of the fossil fuel is causing. It 66
is estimated that the energy demand will increase by a factor between two and three during 67
this century (Weiland, 2010). Biogas from wastes could be a part of the solution in the 68
security of energy supply. According to Forgács (2012), about 10,000 biogas plants are 69
currently operating in Europe and the number of the plants is expected to increase by a factor 70
of five within 10 years. 71
The methane production process of organic wastes such as organic fraction of municipal solid 72
waste (OFMSW), and industrial waste is today carried out by a sequence of biochemical 73
transformations. The process can be generally separated into a first step where hydrolysis, 74
acidification and liquefaction occur and a second step where acetate, hydrogen and carbon 75
dioxide are converted into methane. All these reactions occur simultaneously in a single 76
reactor (Forster-Carneiro et al., 2008). A balanced anaerobic digestion process demands that 77
in both phases, the rates of degradation must be equivalent in size (Angelidaki et al., 1999). 78
Numerous studies have shown to improve the efficiency of single-stage reactors (Cecchi et 79
al., 1991; Climenhaga and Banks, 2008; Forster-Carneiro et al., 2008; Heo et al., 2004). High 80
methane yields have already been achieved during digestion at total solids content less than 81
5% total solid content (Cho et al., 1995; Heo et al., 2004; Verrier et al., 1987; Zhang et al., 82
2007). However, in the single-stage processes, the organic loading rate (OLR) still remains 83
unsatisfactory, at 1–4 kgVS/m3/day (Cho et al., 1995; Heo et al., 2004; Verrier et al., 1987; 84
Zhang et al., 2007). The most important reason for this limitation is that higher OLRs cause 85
inhibition because of accumulated volatile fatty acids (VFAs) (Ahring et al., 1995). In 86
addition, at high OLRs, retention times (HRTs) should be sufficient for the microorganisms to 87
have enough time to degrade the substrate. Thus, there is a balance between OLR and HRT 88
that must be determined in order to optimize digestion efficiency and reactor volume 89
(Demirer and Chen, 2005). 90
91
The development of high rate reactors was based on immobilization of biomass in wastewater 92
treatment systems, which improved the degradation rate of anaerobic treatment systems by 93
decreasing the retention time. However, a drawback of these systems is that they are usually 94
suitable for dilute waste water streams, which contain around 3% total suspended solids with 95
a particle size less than 0.75 mm (Revitt et al., 2010). This means that substrate with high 96
solid content should be solublized before it can be entered in these high rate systems. 97
Therefore, a two-phase system is required in order to achieve a rapid digestion and more 98
stable operation and a higher organic loading capacity. However, there are very little 99
investigations on the application of substrate with high total solid content in two-stage 100
processes. 101
102
Our earlier studies on evaluating the two-stage process, based on pretreated and untreated 103
waste textile (Jeihanipour et al., 2013) and cotton and starch (Aslanzadeh et al., 2013) showed 104
that the two-stage process can be beneficial using substrate that are rather unconventional 105
substrate. These studies indicate that the structure and the degradability of the material is one 106
of the factors deciding the OLR and HRT. 107
108
4
In this work, the effects of OLR and HRT in conventional one stage and two-stage systems 109
using organic fraction of municipal waste (OFMSW) and food processing waste (FPW) with 110
high total solid content were compared. These two substrates are among the most common 111
waste streams that are currently used for biogas production, which principally relies on single-112
stage systems. 113
114
2 Materials and Methods 115
116
2.1 Substrates and inoculums 117
The substrates used in this study includes organic fraction of municipal solid waste 118
(OFMSW), waste from food processing industry (FPW). The inoculum used in the continuous 119
stirred tank reactor (CSTR) were obtained from a 3000-m3 biogas plant
(Borås Energy & 120
Miljö AB, Borås, Sweden), treating municipal solid waste at thermophilic (55 °C) conditions. 121
The UASB reactors were seeded using granulated anaerobic sludge, which was provided from 122
a pilot plant using upflow anaerobic sludge blanket (UASB) reactor treating municipal 123
wastewater at Hammarby Sjöstad (Stockholm, Sweden). The FPW was obtained from storage 124
tank with a retention time of 3-4 days, before it was fed to the digester. 125
126
2.2 Experimental set up 127
2.2.1 Reactors 128 The reactors, both CSTR and UASB were built in house from thermoplastic material 129
polymethylmethacrylate (PMMA). The CSTR had a working volume of 3 l with an inner 130
diameter of 18.5 cm and a height of 18.5 cm, whereas the working volume of the UASB was 131
2.25 l with an internal diameter of 6.4 cm and a height of 70 cm. Temperature of the reactors 132
were kept constant at 55 °C for CSTR and 34 °C for UASB by a thermal water-bath with 133
water recirculation through the reactor’s water jacket during the entire digestion process. The 134
reactors were equipped with a feed inlet, a liquid sampling point, an effluent outlet, and a gas 135
line connected to the gas measuring system containing a gas sampling port. The CSTR was 136
equipped with an impeller for continuous mixing. The inlet from the bottom of the UASB 137
reactor was provided with a mesh to stop the large particles from entering the reactor. 138
2.2.2 Reactor seeding and start up 139 The CSTR inoculums were incubated at 55 °C for three days in order to stabilize it before use. 140
The CSTR’s were filled with 3 l of inoculums at the beginning of the experiment. The UASB 141
reactors were inoculated with 1.3 l of granular anaerobic sludge and the remaining volume of 142
the reactors were filled with water. 143
144
2.2.3 Reactors configuration 145 In the single-phase, digestion a CSTR was employed. The two-stage continuous process, on 146
the other hand consisted of a CSTR connected to a UASB reactor. The liquid of the CSTR 147
was pumped continuously to the bottom of the UASB and the effluent of the UASB reactor 148
was continuously recirculated back to the CSTR. In order to separate particulate matter from 149
the CSTR effluent, the outlet of the CSTR was connected to a sedimentation tank consisting 150
of a 100 ml glass bottle, to separate and settle the large particles before pumping the liquid to 151
the UASB. The reactor configurations are illustrated in Figure 1. Both systems were fed once 152
a day. 153
5
154
2.3 Experimental procedure 155
The semi-continuous digestions were carried out by feeding the bioreactors during the start up 156
period by increasing the OLR by 0.5gVS/l/d until reaching the desired OLR (2gVS/l/d). The 157
HRT of UASB in the two-phase process was controlled by adjusting the speed of the pump 158
prior to each step. To achieve a steady state condition, each OLR was sustained for more than 159
three HRTs in the CSTR. There were no solids withdrawn from the reactors except for the 160
sample analyses during the entire experimental setup. The volume of biogas produced was 161
recorded continuously by Automatic Methane Potential Testing System (AMPTS, Bioprocess 162
Control AB, Sweden) and the composition of the gas was measured by gas chromatography. 163
The liquid and gas sampling were performed 3 times a week during the start of the process. At 164
higher OLRs and lower HRTs, however, the sampling was performed every day. The liquid 165
samples were kept at -20 °C until the analyses were performed. 166
167
2.4 Analytical method 168
The total solid (TS) and the volatile solid (VS) content of the substrates were determined 169
based on drying the samples to constant weight at 105 ºC and 575 ºC respectively (Sluiter et 170
al., 2005). The characterization of the substrate (Table 1) was carried out by Analys-& 171
Konsulat labouratoreiet (AK labbet, Borås, Sweden). The Kjeldahl nitrogen and protein 172
content of the substrates were determined according to Swedish standard method ss-en 173
25663/NMKL 6-4 (Swedish Standard Institute, 1984). Ammonium concentration was 174
measured according to method SIS 028134-1 (Swedish Standard Institute, 1976) and fat 175
content was determined by NMKL method 131 (Nordic Commity on Food Analysis, 1989). 176
177
The biogas production was measured using AMPTS, working based on water displacement. It 178
was equipped with a computer to record the volume of the biogas produced from each reactor. 179
The composition of the biogas produced was determined by a gas chromatograph (Auto 180
System Perkin Elmer, Waltham, MA), equipped with a packed column (Perkin Elmer, 181
6’x1,8’’OD, 80/100 Mesh) was used. The gas chromatograph was set at a thermal 182
conductivity detector (Perkin Elmer) with an inject temperature of 150 °C, detection 183
temperature of 200 °C, and oven temperature of 75 °C. The carrier gas used was nitrogen-184
operated at a pressure of 0.70 bar and a flow rate of 40 ml/min at 60°C. A pressure-tight gas 185
syringe with a volume of 250 μl (VICI, Precision Sampling Inc., LA) was used for the gas 186
sampling. 187
Liquid samples were analyzed for pH, alkalinity, TS, VS, COD (soluble chemical oxygen 188
demand) and VFA. Prior to analysis of alkalinity, COD, VFA, the samples were centrifuged 189
at 17,000 g for 10 min and filtered through a 0.2-µm filter to remove solid particles. The COD 190
and ammonium concentration was measured using a HACH apparatus equipped with a UV–191
Vis Spectrophotometer (HACH, Germany), with digestion solution COD and ammonium 192
vials operating range 0–15,000 mg COD/l and 0-80 mg/l ammonium. The total VFA 193
concentrations, based on acetic acid, propionic acid, butyric acid and valeric acid were 194
analyzed by HPLC (Waters 2695, Waters Corporation, Milford, MA, USA), operating with an 195
ion-exchange column (Aminex HPX-87H Bio-Rad, Hercules, CA), working at 60 °C using 5 196
mM sulfuric acid as eluent with a flow of 0.6 ml/min, and a UV detector (Waters 2414). The 197
total alkalinity was measured according to Forgács et al. (2012) based on an end point 198
potentiometric titration with 0.05 mol/l HCl to pH 4.0. 199
200
6
3 Results and Discussions 201
The effects of increasing OLR and decreasing HRT on the performance of a single-stage and 202
a two-stage process digesting FPW and OFMSW were evaluated. The OLR was increased 203
from 2 gVS/l/d and the HRT was decreased gradually from 10 days in 9 steps to OLR 14 204
gVS/l/d and HRT of 3 days. 205
206
3.1 Methane production 207
The average total methane produced at various OLRs and HRTs in the single-stage is 208
presented in Figure 2A. The average total methane production for FPW increased from 0.42 209
m3/kgVS to 0.44 m
3/kgVS corresponding to 81 % and 84 % of the theoretical methane yield 210
at step 1 (OLR 2 gVS/l/d, HRT 10 days) and step 2 (OLR 3 gVS/l/d, HRT 7 days) 211
respectively. During the same period, the methane production for OFMSW decreased from 212
0.33 m3/kgVS to 0.24 m
3/kgVS, which is 67 % and 48 % of theoretical methane yield, 213
respectively. Further increase to step 3 (OLR 3 gVS/l/d, HRT 5 days) failed the process for 214
both substrates. 215
The two-stage process, on the other hand showed higher overall stability compared to the 216
single-stage process at higher OLRs and lower HRTs (Figure 2B & C). The overall total 217
methane production for the CSTR digesting FPW showed a relatively stable performance up 218
to step 6 (OLR 8 gVS/l/d, HRT 3 days). During this period the theoretical methane yield 219
fluctuated between 82 % and 93 %. Additional increase in OLR decreased the methane yield 220
successively at each step. The digestion of OFMSW between on the other hand showed a 221
relative stable methane production until step 8 (OLR 12 gVS/l/d, HRT 3 days) with a 222
theoretical yield fluctuating between 60 % and 78 %. The process could not handle a further 223
increase in OLR to step 9 (OLR 14 gVS/l/d, HRT 3 days) and the methane yield was 224
decreased. The major share of methane was produced in CSTR in the two-stage process for 225
both substrates. However, this major share of methane production was shifted to UASB at the 226
last step for both FPW and OFMSW. 227
Compared to single-stage process, in two-stage process the OLR was increased by 167 % i.e., 228
(from 3 gVS/l/d to 8 gVS/l/d) for FPW. Similarly, the HRT was decreased by 57 % (i.e. from 229
7 days to 3 days). The overall increase in OLR and the decrease in HRT have result in the 230
need of total reactor volume by 26% less than a single-stage process for FPW. Likewise for 231
OFMSW, the two-stage process could handle higher OLR by 333 % compared to single-stage 232
process (from 2 gVS/l/d to 12 gVS/l/d). The HRT and the overall reactor volume were 233
decreased by 70 % and 65 %, respectively. 234
The process fed with FPW showed less fluctuation and was easier to handle the changes in the 235
operational condition than OFMSW as in both single and two-stage processes. This could be 236
due to the combination of two factors; difference between the substrate and the solubilization 237
efficiency in the single-stage and two-stage processes. A previous study (Jash and Ghosh, 238
1996) shows that the maximum amount of methane produced in anaerobic digestion of solid 239
organic residue depend on the extent of solubilization of the organic material. The FPW was 240
prehydrolyzed and contained readily solubilized material in the feed thus much more stable 241
methane production was observed compared to OFMSW. 242
243
7
3.2 Volatile fatty acid and Alkalinity 244
The concentration of total VFA and individual fatty acids is presented in Figures 3 and 4 for 245
single-stage and two-stage process, respectively. The concentration of total VFA in single-246
stage process at initial step (OLR 2gVS/l/d, HRT 10 days) was high around 4.4 g/l and 3.9 g/l 247
for FPW and OFMSW respectively. However, after the adaptation period the VFA 248
concentration decreased and continued to be stable until the last step (OLR 4 gVS/l/d, HRT 5 249
days) which it started to increase in the CSTR digesting OFMSW started to increase while the 250
CSTR digesting FPW kept stable under 1 g/l. The individual organic acid dominating in the 251
total VFA, with the highest concentration first was; valeric acid, for both substrates and 252
increased at the initial step to 2.2 g/l and 2.3 g/l in CSTR digesting OFMSW and FPW 253
respectively. 254
In the two-stage process on the other hand, the VFA concentration was stable between 0.3 g/l 255
to 0.5 g/l in the CSTR for both substrates until step 4 (OLR 5 gVS/l/d, HRT 3 days). 256
However, the concentration started to increase in the CSTR for both substrates at the end of 257
step 4 (OLR 5 gVS/l/d, HRT 4 days) and continued to increase with each step until the end of 258
the experiment and reached above 14 g/l and 16 g/l in CSTR digesting FPW and OFMSW 259
respectively. The concentration of total VFA in the UASB followed the same trend as CSTR 260
and was stable in the initial steps (OLR 2-5 gVS/l/d, HRT 10-3 days) under 0.2 g/l for both 261
substrates. Even though the concentration was raised in parallel at each step it kept under 1.6 262
g/l for both substrates until the end of the experimental period. 263
The valeric acid was also the dominating acid in the two-stage process as well and shows a 264
sudden increase to 1.3 g/l for in CSTR digesting FPW and to more than 3 g/l for CSTR 265
digesting OFMSW at step 5 (OLR 6 gVs/l/d, HRT 3) for both substrates. The other acids start 266
to rise during the last 3 steps (OLR 10-14 gVS/l/d, HRT 3 days) of the process for both 267
substrates. 268
The high fluctuation of total VFA concentration at the initial step in the single-stage process 269
indicates that the acidification is reversible at lower OLR and higher HRT. The increase in 270
VFA concentration reflects a kinetic disconnection between acid producers and consumers 271
and is characteristic for stress situation (Ahring et al., 1995). In the two-stage process, the 272
total VFA started to increase almost to the same level as single-stage but at much higher OLR 273
and lower HRT for both substrates. Even at higher VFA level than observed in single-stage 274
the methanogenesis activity was still high in the CSTR and the increase in VFA could be 275
handled. In a two-stage process, short HRT could be beneficial, as the optimum conditions of 276
the first stage should aim for the maximum rate of acid production. For a given substrate 277
concentration, the acid production is maximized for short HRTs (Dinopoulou et al., 1988). 278
The total alkalinity is an indication of buffering capacity of the system and was monitored 279
during the entire experimental period in both single and two stage processes and the results 280
are illustrated in Table 2 and Table 3. In the single-stage process, the total alkalinity shows a 281
decreasing trend during entire experimental period for both substrates from 3,188 mg/l to 282
1,695 mg/l in the CSTR digesting FPW and from 3,130 mg/l to 2,354 mg/l in the CSTR 283
digesting OFMSW (Table 1). 284
In the two-stage process the same trend was observed as single-stage for both substrates. In 285
the CSTR the alkalinity was fluctuated between 9,000 and 12,000 mg/l with the highest in the 286
earlier steps of the process, while in UASB it was stable between 11,000 and 12,000 mg/l 287
thorough all steps during the entire experimental period. In the CSTR digesting OFMSW was 288
slightly lower than FPW and fluctuated between 4,000 mg/l and 10,000 mg/l with the highest 289
alkalinity at the initial steps of the process which suggests that enhancement of the OLR 290
8
further would reduce the methanogenesis activity in the CSTR due to accumulation of VFA in 291
the CSTR for both single-stage and two-stage processes. Furthermore, in the UASB the 292
alkalinity decreased from 11,000 mg/l in step 1 to 6,248 mg/l at last step (OLR 14 gVS/l/d, 293
HRT 3 days), which indicates that the methanogenesis were inhibited. The reason for this 294
inhibition could be due to the high concentration of valeric acid. In the two-stage process, the 295
valeric acid concentration in the CSTR, digesting OFMSW, was higher than the concentration 296
in the CSTR for FPW. Previously, it has been shown that at higher concentration of butyrate 297
or valerate can inhibit the methanogenic activity (Ahring et al., 1995). This might be the 298
reason that the alkalinity was reduced at higher OLR and shorter HRTs in the UASB for 299
OFMSW. 300
301
3.3 Volatile solid reduction, Chemical oxygen demand (COD) and COD removal efficiency 302
and Ammonium 303
The VS-reduction in The CSTR was measured at each stage of the process for both substrates 304
In the single-stage process, the VS reduction of 95 % and 93 % was achieved at step 1 (OLR 305
2 gVS/l/d, HRT 10 days) for the CSTR digesting FPW and OFMSW, respectively (Table 2). 306
Further increase in OLR and decrease in HRT at step 2 did not affect the VS reduction 307
considerably, and it was stable about 94 % for both substrates, and increased to 97 % at the 308
last step in the CSTR digesting FPW. 309
The VS reduction was higher at earlier steps of the process in the two-stage process, when the 310
organic loading rate was lower and the HRT was higher (Table 3). The VS reduction for FPW 311
fluctuated between 86.2 % and 95.7 % between steps 1 and 5 (OLR 2-6 gVS/l/d, HRT 10-3 312
days). However, it starts to decrease from step 6 (OLR 8 gVS/l/d, HRT 3 days) with 86.2 % to 313
step 9 (OLR 14 gVS/l/d, HRT 3 days) with 57.7 %. The CSTR digesting OFMSW between 314
step 1 and step 7 (2-10 gVS/l/d, HRT 10-3 days) of the process the VS reduction fluctuated 315
between 95.1 % and 85.42 % with the highest VS reduction at the earlier steps of the process. 316
The VS reduction which is an indication of the treatment efficiency obtained in this study is in 317
accordance to previous study on two-stage process using fruit and vegetable waste which was 318
about 96% (Verrier et al., 1987). The VS reductions were high and stable the single-stage 319
process. In the two-stage process it observed to be higher at earlier steps with lower OLR and 320
higher HRT. The low HRT of 3 days did not affect the VS reduction, however the OLR 321
between 8 and 10 gVS/l/d seem to be the limitation for the system in the two-stage process. 322
The high VS reduction shows that the process is able to dissolve the particulate organic 323
material, which is confirmed by the increase in COD content in the CSTR and the COD 324
removal efficiency of the process. 325
In the single-stage process between step 1 (OLR 2 gVS/l/d, HRT 10 days) and step 3 (OLR 4 326
gVS/l/d, HRT 5 days) the COD concentration showed a slight decrease, but stable between 327
3,000 and 5,000 mg/l FPW. The COD concentration for the CSTR digesting OFMSW 328
decreased from 3,700 mg/l to 3,433 mg/l at step 1 and 2 respectively. In two-stage process, 329
the COD concentration of both CSTR and the UASB was monitored and the COD removal 330
efficiency was calculated based on the difference between the COD content of the influent 331
and the effluent of the UASB (Table 3). 332
The COD removal efficiency rose in the two-stage process with each step both in CSTR and 333
UASB. The COD efficiency fluctuated but observed to be increasing during the entire 334
experimental period for both substrate from approximately 42.3 % and 74 % for FPW and 335
from 21.3 % to 83 % for OFMSW. The COD content in the CSTR during the last 3 steps of 336
the experiment display a significant increase from approximately 6,800 mg/l to 15,000 mg/l 337
while it was stable around 3,000 mg/l in the UASB for both substrates. Earlier studies on the 338
9
hydrolytic and acidogenic process confirm that shorter HRTs slightly improves total 339
solubilization yield (De La Rubia et al., 2009). Increase in COD removal efficiency shows 340
that the methanogenesis in the CSTR is inhibited while the intermediate products were 341
digested in the UASB without accumulating in the system, increasing efficiency of the UASB 342
(Aslanzadeh et al., 2013). 343
The ammonium concentration showed a decreasing trend with increasing OLR and decreasing 344
HRT in both single as well as two-stage process in the single-stage process (Figure 5). In the 345
single-stage process, the concentration of ammonium decreased from 885 mg/l in the initial 346
step to 200 mg/l at the last step for the CSTR digesting OFMSW and from 990 mg/l to 350 347
mg/l for FPW during the same period (Figure 5A). In the two-stage process, the same trend 348
was observed as single-stage process (Figure 5B and 5C). The ammonium concentration 349
decreased slightly in CSTR from 1,860 mg/l to 1,500 mg/l for FPW while for OFMSW 1,830 350
mg/l to 800 mg/l. The ammonium concentration in the UASB was stable between 500 mg/l 351
and 700 mg/l throughout the entire process for both substrates. The decrease in ammonium 352
concentration in both single-stage and two-stage processes indicates that the capacity of the 353
CSTRs to fully degrade the particulate material at higher OLR and lower HRTs is reduced. 354
Ammonium is released during the degradation of organic matter and stand for the extent of 355
the hydrolysis process, primarily protein compounds (Forgács et al., 2013; Rincón et al., 356
2009). However, this capacity limit is reached much sooner in single-stage process than in 357
two stage process. 358
359
4 Conclusion 360
361
The effect of OLR and HRT in single-stage and two-stage process using OFMSW and FPW 362
was evaluated. The single-stage process could at best handle an OLR of 3 gVS/l/d and a HRT 363
of 7 days. A more stable operation could be achieved at higher OLR and lower HRT in the 364
two-stage process. The two-stage process could handle an OLR of 8 gVS/l/d for FPW and up 365
to 12 gVS/l/d for OFMSW, while the HRT could also be decreased to 3 days. Using the two-366
stage process, a higher theoretical yield and a lower reactor volume could be achieved 367
compared to the single-stage process. 368
369
Nomenclature 370
371
FPW-Food processing waste 372
OFMSW- organic fraction of municipal solid waste 373
OLR-Organic loading rate 374
HRT-Hydraulic retention time 375
VFA-volatile fatty acids 376
VS reduction-volatile solids reduction 377
TS-total solids 378
10
VS-Volatile solids 379
HPLC-High performance liquid chromatography 380
GC- Gas chromatography 381
CSTR- Continuous stirred tank reactor 382
UASB Upflow anaerobic sludge blanket 383
384
385
References 386
Ahring, B.K., Sandberg, M., Angelidaki, I., 1995. Volatile fatty acids as indicators of process 387
imbalance in anaerobic digestors. Applied Microbiology and Biotechnology 43, 559-565. 388
Angelidaki, I., Ellegaard, L., Ahring, B.K., 1999. A comprehensive model of anaerobic 389
bioconversion of complex substrates to biogas. Biotechnology and Bioengineering 63, 363-390
372. 391
Aslanzadeh, S., Rajendran, K., Jeihanipour, A., Taherzadeh, M.J., 2013. The Effect of 392
Effluent Recirculation in a Semi-Continuous Two-Stage Anaerobic Digestion System. 393
Energies 6, 2966-2981. 394
Cecchi, F., Pavan, P., Mata Alvarez, J., Bassetti, A., Cozzolino, C., 1991. Anaerobic digestion 395
of municipal solid waste: thermophilic vs. mesophilic performance at high solids. Waste 396
management & research 9, 305-315. 397
Cho, J.K., Park, S.C., Chang, H.N., 1995. Biochemical methane potential and solid state 398
anaerobic digestion of Korean food wastes. Bioresource Technology 52, 245-253. 399
Climenhaga, M., Banks, C., 2008. Uncoupling of liquid and solid retention times in anaerobic 400
digestion of catering wastes. 401
De La Rubia, M.A., Raposo, F., Rincón, B., Borja, R., 2009. Evaluation of the hydrolytic–402
acidogenic step of a two-stage mesophilic anaerobic digestion process of sunflower oil cake. 403
Bioresource Technology 100, 4133-4138. 404
Demirer, G.N., Chen, S., 2005. Two-phase anaerobic digestion of unscreened dairy manure. 405
Process Biochemistry 40, 3542-3549. 406
Dinopoulou, G., Rudd, T., Lester, J.N., 1988. Anaerobic acidogenesis of a complex 407
wastewater: I. The influence of operational parameters on reactor performance. 408
Biotechnology and Bioengineering 31, 958-968. 409
Forgács, G., 2012. Biogas production from citrus wastes and chicken feather: pretreatment 410
and co-digestion, Chalmers University of Technology, Sweden. 411
Forgács, G., Lundin, M., Taherzadeh, M., Sárvári Horváth, I., 2013. Pretreatment of Chicken 412
Feather Waste for Improved Biogas Production. Applied Biochemistry and Biotechnology 413
169, 2016-2028. 414
Forgács, G., Pourbafrani, M., Niklasson, C., Taherzadeh, M.J., Hováth, I.S., 2012. Methane 415
production from citrus wastes: process development and cost estimation. Journal of Chemical 416
Technology & Biotechnology 87, 250-255. 417
Forster-Carneiro, T., Pérez, M., Romero, L.I., 2008. Anaerobic digestion of municipal solid 418
wastes: Dry thermophilic performance. Bioresource Technology 99, 8180-8184. 419
Heo, N.H., Park, S.C., Kang, H., 2004. Effects of mixture ratio and hydraulic retention time 420
on single-stage anaerobic co-digestion of food waste and waste activated sludge. Journal of 421
Environmental Science and Health, Part A 39, 1739-1756. 422
11
Jash, T., Ghosh, D.N., 1996. Studies on the solubilization kinetics of solid organic residues 423
during anaerobic biomethanation. Energy 21, 725-730. 424
Jeihanipour, A., Aslanzadeh, S., Rajendran, K., Balasubramanian, G., Taherzadeh, M.J., 425
2013. High-rate biogas production from waste textiles using a two-stage process. Renewable 426
Energy 52, 128-135. 427
Nordic Commity on Food Analysis, 1989. Fat. Determination according to SBR (Schmid-428
Bondzynski-Ratslaff) in meat and meat products, NMKL method No 131, OSLO. 429
Revitt, M., Scholes, L., Donner, E., 2010. Priority pollutant behaviour in on-site treatment 430
systems for industrial wastewater. Urban Pollution Research Centre, Middlesex University, 431
UK. 432
Rincón, B., Borja, R., Martín, M.A., Martín, A., 2009. Evaluation of the methanogenic step of 433
a two-stage anaerobic digestion process of acidified olive mill solid residue from a previous 434
hydrolytic–acidogenic step. Waste Management 29, 2566-2573. 435
Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., 2005. Determination 436
of Ash in Biomass A national laboratory of the U.S. Department of Energy,Office of Energy 437
Efficiency & Renewable Energy, U.S. 438
Swedish Standard Institute, 1976. "Water quality-Determination of of ammonia nitrogen 439
content" SIS 28134, Artikel No STD-883,, Stochholm, sweden. 440
Swedish Standard Institute, 1984. Warer quality-Determination of kejldahl nitrogen-Method 441
after mineralization with selinium, ISO 5663,, Stockholm, Sweden. 442
Weiland, P., 2010. Biogas production: current state and perspectives. Applied Microbiology 443
and Biotechnology 85, 849-860. 444
Verrier, D., Roy, F., Albagnac, G., 1987. Two-phase methanization of solid vegetable wastes. 445
Biological wastes 22, 163-177. 446
Zhang, R., El-Mashad, H.M., Hartman, K., Wang, F., Liu, G., Choate, C., Gamble, P., 2007. 447
Characterization of food waste as feedstock for anaerobic digestion. Bioresource Technology 448
98, 929-935. 449
450
451
452
453
454
455
456
457
458
459
460
461
462
12
Table 1. Characterization of substrates OFMSW and FWP 463
464
Analyses FPW OFMSW
Total solid (TS) Wt% 7.95 13.1
Volatile solid (VS) Wt % 7.8 12.9
Carbohydrate Wt % 2.6 7.1
Fat Wt % 1.8 2.1
Protein Wt % 2.4 2.6
Kjeldahl nitrogen Wt % 0.38 0.41
Ammonium nitrogen Wt % 0.00056 0.00045
Volatile fatty acid (mg/l) 2133 -
465
466
467
468
469
470
471
472
473
474
475
476
477
478
13
479
Table 2. Operation condition in each step with connected organic loading rate, (OLR) and 480
hydraulic retention time (HRT) and the average chemical oxygen demand (COD), volatile 481
solid (VS) reduction and theoretical yield in the single-stage process. 482
Substrate
Steps
OLR (gVS/l/d
)
HRT (days)
Operation days
COD (mg/l)
Alkalinity (mg/l)
VS reduction
(%)
Theorethical methane
yields1 (%)
FPW
1 2.0 10.0
30 4000±1946 3188±136 95±2.1 81
2 3.0 7.0
21
3067±850 2866±369 94±2.5 84
3 4.0 5.0
15
5000±707 1695±350 97±1.4 2
OFMSW 1 2.0 10.0
30
3700±1212 3130±343 93±1.5 67
2 3.0 7.0
21
3433±321 2354±205 94±2.7 48
1 The theoretical methane yield is calculated based on fat, protein and carbohydrate content of the substrates. The 483
VFA concentration in FPW is not taken into account. 484
485
486
14
Tab
le 3
. O
per
atio
n c
ond
itio
n i
n e
ach
ste
p w
ith c
onnec
ted
org
anic
load
ing r
ate,
(O
LR
) an
d h
yd
rauli
c re
tenti
on t
ime
(HR
T)
and t
he
aver
age
chem
ical
ox
ygen
dem
and
(C
OD
), v
ola
tile
soli
d (
VS
) re
duct
ion a
nd t
heo
reti
cal
yie
ld i
n t
he
single
-sta
ge
pro
cess
1 T
he
theo
reti
cal
met
han
e yie
ld i
s ca
lcula
ted
bas
ed o
n f
at,
pro
tein
and
car
bo
hyd
rate
co
nte
nt
of
the
sub
stra
tes.
The
VF
A c
once
ntr
atio
n i
n F
PW
is
no
t ta
ken
into
acc
ou
nt.
Sub
stra
te
Step
O
LR
HR
T in
C
STR
HR
T in
U
ASB
Op
erat
ion
d
ays
C
OD
(m
g/l)
C
OD
Ef
fici
ency
(%
) A
lkal
init
y (m
g/l)
V
S re
du
ctio
n (
%)
Theo
reth
ical
m
eth
ane
yiel
d(%
)1
(gV
S/l/
d)
(day
) (d
ay)
CST
R
UA
SB
C
STR
U
ASB
C
STR
FPW
1 2
.0
10
.0
7.5
0
30
5
667
±11
50
236
7±2
31
5
6.9
±10
.8
104
68
±24
5
11
71
4±6
14
9
5.2
±1.2
8
2
2 3
.0
7.0
5
.25
2
1
416
7±1
154
2
400
±26
4
42
.3±7
.48
1
190
6±4
04
1
25
06
±57
9
86
.2±8
.2
74
3 4
.0
5.0
3
.75
1
5
300
0±4
00
1
833
±15
2
38
.6±3
.50
1
251
6±2
92
1
17
79
±29
3
92
.5±5
.0
92
4 5
.0
4.0
3
.00
1
2
305
0±2
12
1
700
±56
5
44
.8±1
4.7
1
213
1±5
84
1
21
60
±15
3
89
.4±2
.3
87
5 6
.0
3.0
2
.25
9
3
700
±28
3
170
0±2
52
5
3.6
±11
.2
104
65
±11
6
11
59
8±3
92
9
5.7
±1,9
9
3
6 8
.0
3.0
2
.25
9
3
100
±14
1
135
0±2
12
5
6.5
±4.8
6
104
42
±31
1
11
24
3±1
69
8
6.2
±0.7
8
6
7 1
0.0
3
.0
2.2
5
9
680
0±1
052
3
200
±13
5
53
.7±1
.13
1
048
7±2
65
1
11
30
±10
9
79
.1±2
.1
72
8 1
2.0
3
.0
2.2
5
9
112
50±1
768
3
133
±11
5
72
.0±5
.66
9
50
6±8
1
11
21
0±1
35
6
1.3
±3.8
6
2
9 1
4.0
3
.0
2.2
5
9
150
00±2
854
3
900
±65
7
74
.0±8
.00
9
06
8±5
7
12
11
6±1
62
5
4.7
±5.4
5
7
OFM
SW
1 2
.0
10
.0
7.5
0
30
4
700
±22
53
290
0±2
65
3
1.4
±21
.4
962
5±1
48
8
11
65
2±3
15
9
4.2
±1.2
7
2
2 3
.0
7.0
5
.25
2
1
350
0±9
64
2
733
±70
2
21
.3±6
.1
97
16
±37
7
10
94
2±3
64
9
5.1
±0.5
7
7
3 4
.0
5.0
3
.75
1
5
243
3±3
05
1
733
±30
6
27
.4±2
0.2
1
029
3±1
41
0
10
07
3±6
96
8
6.5
±3.6
6
6
4 5
.0
4.0
3
.00
1
2
195
0±9
19
1
800
±43
5
26
.3±6
.4
904
21
±20
1
86
49
±80
8
85
.4±4
.7
70
5 6
.0
3.0
2
.25
9
2
900
±14
1
170
0±2
15
4
1.3
±2.8
7
42
9±5
38
8
17
5±4
42
8
7.8
±2.1
6
0
6 8
.0
3.0
2
.25
9
3
550
±13
43
150
0±1
38
5
7.7
±8.0
7
709
±10
46
8
55
8±2
90
8
9.5
±0.7
6
7
7 1
0.0
3
.0
2.2
5
9
700
0±1
587
3
300
±64
7
47
.6±7
.4
742
9±2
34
2
72
58
±13
4
87
.8±1
.8
78
8 1
2.0
3
.0
2.2
5
9
870
0±1
997
3
267
±45
1
64
.6±1
3.7
6
858
±12
49
7
39
8±5
43
8
2.2
±0.2
7
1
9 1
4.0
3
.0
2.2
5
9
150
00±3
051
2
500
±58
3
83
.0±9
.5
44
45
±12
3
62
48
±35
4
78
.1±7
.3
40
15
Feed
Bio
gas
Ou
tlet
CST
R (
55
°C
)U
ASB
(3
4°C
)
Pu
mp
Feed
Hyd
roly
sis
Met
han
oge
nes
is
Bio
gas
mea
sure
men
t
Rec
ircu
lati
on
Sed
imen
tati
on
tan
k
CST
R (
55
°C)
Sin
gle-
stag
e p
roce
ss
Two
-sta
ge p
roce
ss
Fig
ure
1.S
chem
atic
sket
ch o
f th
e si
ngle
-sta
ge
and t
wo
-sta
ge
pro
cess
16
Figure 2. Methane production in A- single-stage process B- two-stage process FPW, C-two stage process (OFMSW)
17
Figure 3. Total and individual VFA concentration in single-stage process. A- CSTR –FPW,B-CSTR-OFMSW
18
Fig
ure
4.
Tota
l an
d i
ndiv
idual
vola
tile
fat
ty a
cid c
once
ntr
atio
n i
n t
wo
-sta
ge
pro
cess
fo
r F
PW
and O
FM
SW
A-t
ota
l V
FA
in C
ST
R, B
-tota
l V
FA
in
UA
SB
, C
-indiv
idual
VF
A i
n C
ST
R-F
PW
, D
- in
div
idual
VF
A i
n C
ST
R-O
FM
SW
0
0,3
0,6
0,9
1,2
1,5
1,8
05
01
00
15
02
00
25
0
VFA (g/L)
Day
s
B
0510
15
20
05
01
00
15
02
00
25
0
VFA (g/L)
Day
s
AO
FMSW
tota
l V
FA
FPW
tota
l V
FA
01234567
05
01
00
15
02
00
25
0
VFA (g/L)
Day
s
D
01234567
05
01
00
15
02
00
25
0
VFA (g/L)
Day
s
CA
ceti
c ac
id
Bu
tyri
c ac
id
Pro
pio
nic
aci
d
Val
eri
c ac
id
19
Figure 5. Ammonium concentration in A-single-stage and B- FPW-two-stage, C- OFMSW- two-stage during the entire experimental period at each organic loading rate (OLR) and Hydraulic retention time (HRT).
