Bioresource Technology 180 (2015) 177–184

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Bioresource Technology

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Anaerobic co-digestion of sewage sludge and sugar beet pulp lixiviationin batch reactors: Effect of temperature

http://dx.doi.org/10.1016/j.biortech.2014.12.0560960-8524/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +34 660 92 12 71.E-mail address: rocio.montanes@uca.es (R. Montañés).

Rocío Montañés ⇑, Rosario Solera, Montserrat PérezDepartment of Environmental Technologies, University of Cádiz, Spain

h i g h l i g h t s

�Methane productivity is higher under a mesophilic than thermophilic regimen.� Sugar beet pulp lixiviation (SBPL) improves cumulative net methane generation.� Several sludge/SBPL ratios were tested in biochemical methane potential assays.� Initial volatile fatty acid (VFA) content of inocula affects BMP test results.� High VFA content reduces microbial activity.

a r t i c l e i n f o

Article history:Received 31 August 2014Received in revised form 11 December 2014Accepted 15 December 2014Available online 31 December 2014

Keywords:Biochemical methane potential (BMP) testMesophilic rangeThermophilic rangeSewage sludgeSugar beet pulp lixiviation

a b s t r a c t

The feasibility of anaerobic co-digestion of sewage sludge (SS) and sugar beet pulp lixiviation (SBPL) wasassessed. Mesophilic and thermophilic batch assays of five different SS/SBPL ratios were used to investi-gate the effect of temperature, providing basic data on methane yield and reduction in total volatiles.Microbe concentrations (Eubacteria and methanogenic Archaea) were linked to traditional parameters,namely biogas production and removal of total volatile solids (TVS). The relationship between Eubacteriaand Archaea was analysed.

Given equal masses of organic matter, net methane generation was higher in the mesophilic range onthe biochemical methane potential (BMP) test. Methane yield, TVS removal data and high levels ofvolatile fatty acids provided further evidence of the best behaviour of the mesophilic range. At the endof testing the microbial population under of the reactors consisted of Eubacteria and Archaea, withEubacteria predominant in all cases.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

It is essential to develop sustainable energy supply systems tomeet the demand for energy from renewable sources. Reducinggreenhouse gas emissions by increasing production of renewableenergy production is increasingly important. Biogas productiontechnology is critical to the sustainable use of biomass as a renew-able energy source. Biogas can be produced from a wide range ofenergy crops, animal manures and organic wastes and thereforeoffers a flexible source of energy which can be adapted to the spe-cific needs of different locations and farming styles. The residues ofanaerobic digestion are a valuable fertiliser for agricultural crops.

Anaerobic digestion is a biological process in which a group ofmicro-organisms biodegrade organic matter (substrate) in theabsence of free molecular oxygen (O2). This complex biological

process converts organic matter into a mixture composed mainlyof methane (CH4), carbon dioxide (CO2) and new bacterial cells(Romano and Zhang, 2008). Complete bioconversion of organicmatter into stable end products is accomplished by a series ofinterdependent metabolic reactions involving several classes ofmicro-organisms.

The efficiency of anaerobic digestion is highly dependent on thecharacteristics of the waste, the reactor configuration and otheroperational parameters. The temperature, organic strength,buffering capacity and solid and nutrient content of the wasteare important influences on anaerobic biodegradation. Waste canbe treated to improve its digestibility.

Assay of biochemical methane potential (BMP) is a proceduredeveloped to determine how much methane is produced byanaerobic decomposition of a given organic substrate. BMP assayhas proved to be a relatively simple, reliable method for determin-ing the extent and rate at which organic matter is converted tomethane (Chynoweth et al., 1993).

178 R. Montañés et al. / Bioresource Technology 180 (2015) 177–184

Co-digestion can be used to enhance anaerobic degradation ofwastes with certain characteristics. Anaerobic co-digestion is thesynergistic simultaneous biodegradation of different wastes(Mata-Alvarez et al., 2000). The merits of co-digestion include cre-ation of a suitable nutrient ratio, dilution of potentially toxic com-pounds (Sosnowski et al., 2003), provision of buffering capacity(Mshandete et al., 2004), equipment sharing, establishment ofthe required moisture content and easier waste-handling (Mata-Alvarez et al., 2000). Anaerobic co-digestion is also advantageousif the amount of a given waste generated at a particular site isnot sufficient to make anaerobic digestion cost effective(Parawira et al., 2004). There have been numerous studies of anaer-obic co-digestion of various wastes including food industry wastes(Carucci et al., 2005; Murto et al., 2004), animal manure (Gungor-Demirci and Demirer, 2004), municipal solid waste (Zupancic et al.,2008), waste water sludge (Romano and Zhang, 2008), fish wastes(Mshandete et al., 2004) and algal sludge (Yen and Brune, 2007);most showed a remarkable improvement in both treatment effi-ciency and biogas production in comparison with single-wasteanaerobic digestion.

Both thermophilic and mesophilic co-digestion regimes havebeen used successfully, making the technique more flexible thanconventional anaerobic digestion.

Methane is formed over a wide range of temperatures; howeveranaerobic digestion processes are highly temperature dependent.Most BMP assays have been performed at mesophilic tempera-tures. Both pH and temperature have a marked effect on the rateof growth and the composition of the micro-organism populationduring the digestion process (Callaghan et al., 1999).

The majority of methanogens (micro-organisms that producemethane from organic matter) are mesophiles, growing quicklyand converting a higher proportion of organic matter in the meso-philic temperature range. Mesophilic systems are more stable thanthermophilic systems, which has implications for the design of bio-gas plants. The stability and growing conditions in a mesophilicdigester make the process more balanced, more resistant to chem-icals that inhibit digestion (e.g. ammonia) and also more capable oftreating a variety of types of biomass and waste.

Only a minority of methanogens are thermophilic, preferringhigher temperatures. Reaction rates are higher in a thermophilicsystem and the microbial population grows faster. This means thatthermophilic digesters can be smaller (which means lower manu-facturing costs) whilst maintaining very high biogas yields. Ther-mophilic anaerobic digestion also destroys a higher proportion ofthe pathogenic bacteria present in organic wastes.

Despite the advantages of the thermophilic process most biogasplants continue to use mesophilic anaerobic digestion systems.This choice can be justified on the grounds that it is more difficultto control and optimise the thermophilic process. Thermophilicmethanogens are extremely sensitive to changes in the environ-ment for anaerobic digestion; even a small change in operatingparameters can have a negative impact, for example changes ofmore than 1–2 �C in temperature greater significantly reduce bio-gas yield. Anaerobic thermophilic conditions are suitable for asmaller range of waste materials than mesophilic conditions,mainly because of the chemical composition of wastes and thegreater impact of certain digestion inhibitors on the digestionprocess.

Anaerobic co-digestion converts the organic fraction of sewagesludge (SS) and sugar beet pulp lixiviation (SBPL) to methane andcarbon dioxide. It involves coordinated action of several groupsof micro-organisms and is a multi-stage process. The outputs ofthe intermediate stages are volatile fatty acids (VFAs): acetic acid,propionic acid and butyric acid. The conversion of acetate to meth-ane by methanogenic bacteria is the limiting step in the productionof biogas as known methanogens grow slowly meaning that popu-

lations remain relatively small (Zinder, 1993). Methanogens aretypically divided into two main groups based on their substrateconversion capabilities. Acetoclastic methanogens convert acetateinto methane and carbon dioxide; they are the primary methaneproducers: about 70% of the methane produced in digesters comesfrom acetate (Zinder, 1993). Methanogenic bacteria which use H2

also play a critical role in anaerobic digestion since they areresponsible for maintaining the partial pressure of H2 at the verylow level (<10 Pa) required by the intermediate group, which isresponsible for the conversion of organic acids and alcohols tomethane (Pauss et al., 1990).

In this study anaerobic batch reactors were used to determinethe anaerobic biodegradation and biogas generation potential(Owen et al., 1979) of SS and SBPL. Both substrates were subjectedto anaerobic biodegradation in batch reactors. Sugar beet pulp is awaste product of sugar beet processing plants and is known to besuitable for biological degradation so this study investigated thepotential benefits of co-digesting SS and SBPL as well as separatedigestion of these wastes. This study was also the first systematicinvestigation of the effects of variations in temperature on anaero-bic digestion of SS and SBPL.

Abbreviations

BMP

biochemical methane potential OLR organic loading rate COD soluble chemical oxygen demand CODt total chemical oxygen demand H-Ac acetic acid H-Bu butyric acid H-Pr propionic acid TS total solids TVS total volatile solids VFA volatile fatty acid SS sewage sludge SBPL sugar beet pulp lixiviation Series 1 assays under thermophilic conditions Series 2 assays under mesophilic conditions 1-i inoculum series 1 1-1 100% SBPL series 1 1-2 75% SBPL-25% SS series 1 1-3 50% SBPL-50% SS series 1 1-4 25% SBPL-75% SS series 1 1-5 100% SS series 1 2-i Inoculum series 2 2-1 100% SBPL series 2 2-2 75% SBPL-25% SS series 2 2-3 50% SBPL-50% SS series2 2-4 25% SBPL-75% SS series 2 2-5 100% SS series 2

Subscripts

t total s soluble

2. Methods

2.1. Feedstock

The substrates used in the tests were sugar beet pulp, from Azu-carera Ebro company in Jerez de la Frontera (Cádiz), and SS fromthe municipal waste water treatment plant of San Fernando-Cádiz(Spain). Sugar beet pellets were subjected to physical pre-treat-ment before the co-digestion process to promote hydrolysis andsolubilisation of the organic matter and thus improve anaerobicdigestion, biogas yield and possibly also the agronomic value ofthe final residue (Montañés et al., 2013).

2.2. Inoculum

In both series of tests primary sludge from the San Fernando-Cádiz waste water treatment plant was used as the inoculum.

Table 1Inocula characteristics.

Mesophilicinocula

Thermophilicinocula

pH 7.4 7.49CODt (kg/m3) 21.3 45.9COD (kg/m3) 1.2 7.8TS (kg/m3) 14.50 27TVS (kg/m3) 8.58 19.8TS (%) 1.45 2.70TVS (%) 0.86 1.96Alkalinity (kg CaCO3/m3) 2.5 6.6VFA t (mg H-Ac/l) 45.5 7477H-Ac (mg/l) 45.5 649H-Pr (mg/l) 0.0 2909H-Bu (mg/l) 0.0 839Total micro-organism content (cell/

ml)6.5 � 108 1.6 � 108

% Eubacteria 59.4 57.6% Archaea 40.6 42.4

R. Montañés et al. / Bioresource Technology 180 (2015) 177–184 179

Both final methane yield and methane production rate aredependent on the substrates and inoculum. Large inoculation vol-umes ensure high rates of microbial activity and reduce the risks ofoverloading and inhibition (Angelidaki and Sanders, 2004). Theseed culture was the effluent of a completely mixed anaerobicdigester having an hydraulic retention time (HRT) of 20 days andwas capable of operating at mesophilic and thermophilic ranges.

Mesophilic and thermophilic inocula with 1.45% or 2.70% totalsolids (TS) were added to the assays, until the desired conditionswere achieved. Table 1 shows the pH, TS, total volatile solids(TVS), total and soluble carbon oxygen demand (CODt, COD), alka-linity, VFA content and microbial characterisation of inocula used.

Table 2Initial characteristics from substrates in bottle serum.

1-1 1-2 1-3 1-4

pH 8.1 8.1 7.4 7.6CODt (kg/m3) 41.4 47.6 48.1 50.7COD (kg/m3) 11.1 10.8 9.1 6.8TS (kg/m3) 33.1 36.6 36.4 35.7TVS (kg/m3) 24.0 25.0 27.6 27.3TS (%) 3.31 3.66 3.64 3.57TVS (%) 2.40 2.5 2.76 2.73Alkalinity (kg CaCO3/m3) 4.9 6.8 5.4 4.2VFA t (mg H-Ac/l) 4248 5431 3582 2824H-Ac (mg/l) 1536 1518 792 564H-Pr (mg/l) 1180 1353 948 837H-Bu (mg/l) 640 664 439 357

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Fig. 1. Cumulative net methane production (a)

2.3. Experimental set-up and procedures

Separate and co-digestion of SS and SBPL were studied in250 ml serum bottles with an effective volume of 130 ml.

The digesters were loaded with a mixture of inoculum and sub-strate, resulting in a final inoculum concentration of 40% w/w,which is considered optimal for biogas production and substrateacclimatisation, leading to establishment of a TVS concentrationof 8.58 or 19.8 kg/m3 in the mesophilic and thermophilic test sys-tems respectively. Then different amounts of the wastes wereadded to the reactors to give SS/SBPL ratios of approximately0.25, 0.5 or 0.75 (Table 2). Control reactors containing only anaer-obic inocula were also incubated to determine background gas pro-duction. All reactors were run in duplicate and the data presentedhere are average values.

NaOH was added to reactors prior to incubation, to adjust thepH at the beginning of the BMP test and then all 24 reactors werepurged with 100% N2 for 3-4 min to maintain anaerobic conditionsat the appropriate pH. Subsequently the reactors were sealed withnatural rubber stoppers and plastic screw caps. Prepared reactorswere incubated in a temperature-controlled bath at 35 �C or55 �C in mesophilic and thermophilic systems respectively. Reactorcontents were manually mixed three times a day during testing.

Biogas production and biogas composition were determineddaily during the digestion period. At the end of the digestion perioddata on pH, TS, TVS, VFA, alkalinity and CODt and COD were col-lected for all reactors to enable calculation of treatment efficiencyand microbiological analyses.

The parameters of the digestion process were: the degradativecapacity of the system (measured as percentage TVS removal),carbon oxygen demand (COD) and biogas productivity (in termsof cumulative net generation of methane). The mesophilic and

1-5 2-1 2-2 2-3 2-4 2-5

7.3 7.3 7.4 7.3 7.5 7.858.2 29.1 40.3 47.2 63.2 72.13.9 12 10.3 7.8 5.9 1.536.9 17.3 22.6 25.6 31.2 34.529.9 10.7 15.3 18.5 23.4 26.33.69 1.7 2.3 2.6 3.1 3.42.99 1.1 1.5 1.8 2.3 2.64.1 2.2 3.9 3.4 3 2.51705 1503 1271 830 346 152163 371 313.6 209.7 34.5 380498 75.6 62.7 34.5 0 0266 696 585 380 175 91

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Table 3Characteristics of substrates in bottle serum at the end of the biogas methane production test.

1-i 1-1 1-2 1-3 1-4 1-5 2-i 2-1 2-2 2-3 2-4 2-5

pH final 7.6 7.7 6.6 7.7 6.8 6.8 7.5 7.7 7.7 7.6 7.6 7.6COD (kg/m3) 16.8 22.2 13.3 13.4 21.0 21.0 3.8 13.3 12.3 10.7 8.4 6.9% CODt removal 11.7 1.5 34.4 35.3 16.9 21.6 8.3 47.8 49.9 56.1 59 61.5TVS (kg/m3) 7.8 15.3 12.4 10.5 19.4 22.5 6.7 3.9 6.4 9.6 12.3 11.6% TVS removal 60.6 36.4 50.6 61.9 28.9 24.8 22.1 63.5 57.8 48 47.3 56Final VFA (mg Ac/l) 6552 15,091 7840 8120 15,438 11,902 21.6 0 20.2 8.5 0 17Alkalinity(kgCaCO3/m3) 7.1 5.8 7.6 7.8 6.5 6.6 4.7 1.1 2.3 3.0 2.9 2.9ml CH4/g VS added – 52.8 173.1 149 50.7 28.2 – 544.4 520.8 403.4 358.8 255% CH4 59.0 47.6 51.4 59.8 57.4 42.1 62.9 67 63 61.6 63.3 57.5% Of biogas produced in first 10 days 45.4 77.5 44.1 43.0 92.5 95.5 61.2 43.2 47.2 48.4 47 47.2

180 R. Montañés et al. / Bioresource Technology 180 (2015) 177–184

thermophilic inocula were characterised in terms of microbialactivity at the start and end of the BMP tests.

2.4. Analytical methods

Two types of analysis were performed: analysis of the physicaland chemical parameters of the degradation process and quantifi-cation of the microbial population of the reactors.

Total and volatile solids, total and soluble chemical oxygendemand, pH and alkalinity were assessed using the standardmethod (APHA et al., 1995). VFA content and biogas compositionwere determined by gas chromatography. The gases analysed wereH2, CH4, CO2, O2 and N2 (GC-2010 Shimadzu). Those first five com-ponents were analysed using a thermal conductivity detector(TCD) using a Supelco Carboxen 1010 Plot column. Samples weretaken using a 1 ml Dynatech Gastight gas syringe.

The efficiency of organic matter removal was calculated as thepercentage difference between the TVS content of the initial andfinal substrates in the assays. Total acidity was calculated by sum-ming the values for individual fatty acids.

Ideal gas balance was calculated daily for each reactor to deter-mine the amount of methane generated from the stabilisation ofthe waste, net methane generation for experimental reactors wascalculated as the difference between the amount of methane gen-erated in the experimental reactor and the control reactor (Alkayaand Demirer, 2011).

Biogas production was determined indirectly, by measuring thecumulative pressure inside the bottles via pressure transducers.Pressure data were used to infer the volume of biogas at standardtemperature and pressure, according to the ideal law of gases:

P � V ¼ n � R � T

where P is absolute pressure (kPa), V is volume (m3), n is amount ofsubstance (moles), T is temperature (K) and R is the universal gasconstant (8.3145 L kPa/Kmol).

2.5. Microbial analyses

The main steps in fluorescence in situ hybridisation (FISH) ofwhole cells using 16S rRNA-targeted oligonucleotide probes arecell fixation, consequent permeabilisation and hybridisation withthe desired probe(s).

Samples from batch reactors were collected into sterile univer-sal bottles at the end of BMP tests. Absolute ethanol was added tothe bottles in a 1:1 v/v ratio. The samples were stored at �20 �Cuntil they were fixed. Further details of this procedure are givenin Montero et al. (2009).

The technique used for fixing and permeabilising cells wasbased on the one described by Amann et al. (1990a, 1990b). Thefollowing 16S rRNA-targeted oligonucleotide probes were used inthis study: bacteria-universal probe EUB338 (Amann et al.,1990a,b), Archaea-universal probe ARC915 (Speece, 1996),

H2-utilising methanogens probe MB1174 (specific to Methanobac-teriaceae; Stahl and Amann, 1991).

The cellular concentration and percentages of Eubacteria,Archaea and H2-utilising methanogens were obtained using FISH.Total microbial population was estimated as the sum of the popu-lations of Eubacteria and Archaea, because most anaerobic micro-organisms in anaerobic reactors belong to these two groups(Stahl and Amann, 1991). The population of acetate-utilising meth-anogens was calculated as the difference between the populationsof Archaea and a H2-utilising methanogens. Samples were exam-ined visually and the cells were counted using an Axio ImagerUpright epifluorescence microscope (Zeiss) with a 100 W mercurylamp and a 100� oil objective lens. The filter used depended on theidentity of the labelled probe, a B-2A filter (DM 510, Excitation450–490 and Barrer 520) was used for 6-FAM; a G-2A (DM 580,Excitation 510–560 and Barrer 590) filter was used for Cy3.

3. Results and discussion

3.1. Evolution of biogas generation

Reactors were operated until significant biogas production wasno longer taking place. The methane production curves for allassays following removal of the inoculum are presented in Fig. 1.Cumulative methane production followed similar time courses inmesophilic and thermophilic reactors, but the thermophilic curvesare shorter and asymptote was reached before the lag phase.

In anaerobic treatment systems waste stabilisation is achievedby methane production (Speece, 1996) and so the rate of methaneproduction is directly related to the stabilisation rate, which is acrucial factor in the design and operation of anaerobic treatmentsystems.

As is commonly observed in biogas production, cumulative netmethane generation in the BMP test was higher at mesophilic tem-perature (series 2). This explained the relatively poor co-digestionof SS and SBPL at 55 �C. The high VFA levels (Table 3) were consis-tent with this interpretation.

The treatment efficiencies of reactors with similar substratecomposition (SS/SBPL ratio) operating at mesophilic and thermo-philic temperatures can be used to compare the biodegradabilityof various SS/SBPL ratios. The highest methane yield (544.4 ml/g TVSadded) and greatest reduction in TVS (63.5%) were observed inreactor 2-1, which was fed with SBPL only. It is widely acceptedthat SBPL is highly biodegradable because it is made up of solublecarbohydrates, mainly sucrose (Iza et al., 1990). More interestingly,cumulative methane production was greatest in reactor 2-4.

Methane yield, CODt removal and TVS reduction were lower inseries 1. These results reflect the amount of VFAs present at the endof the test. The high values of VFA could be due to the amount ofVFA in the thermophilic inocula (Table 1). This implies a pHreduction in all reactors tested in series 1.

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Fig. 2. Cumulative net methane production at various SS/SBPL ratios in thermophilic conditions (series 1) and mesophilic conditions (series 2).

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Fig. 3. Ratio of total acidity to total alkalinity (VFA/Alk) in (a) thermophilic conditions (series 1); (b) mesophilic conditions (series 2).

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Fig. 1a and b show that methane production was lower in series1 than in series 2 at all SS/SBPL ratios tested. Biodegradation wasinhibited in series 1 reactors; there was some methane production,but it appears that the high VFA content of the thermophilic inocu-lum limits the biodegradability of different mixtures of SS and SBPL.Table 3 gives physical and chemical parameters for the substrate atthe end of the biodegradability test; methane production was lowerin series 1 than series 2 for all reactors and all SS/SBPL ratios inves-

tigated reflecting lower biodegradability caused by the high con-centration of VFAs in the digesters during the degradation testand the resultant decrease in pH. A higher pH is needed for develop-ment of methanogen populations in anaerobic digestion systems.

The following figures compare cumulative net methanegeneration for all substrates tested in both assays and show howtemperature affected methane generation and the productivity ofanaerobic co-digestion of SS and SBPL.

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Table 4Concentrations and percentages of Eubacteria, Archaea, H2-utilising methanogens and acetate-utilising methanogens in series 1 and series 2.

Series 1

1-i 1-1 1-2 1-3 1-4 1-5

Total micro-organism (cell/ml) 1.6 � 108 3.1 � 108 2.7 � 108 2 � 108 2 � 108 1.3 � 108

% Eubacteria 57.7 69.5 74.5 71.5 68.8 77.6% Archaea 42.3 30.5 25.5 28.5 31.2 22.4% H2-utilising methanogens 100 100 100 100 100 100% Acetate-utilising methanogensa 0.0 0.0 0.0 0.0 0.0 0.0

Series 2

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Total micro-organism (cell/ml) 1.2 � 108 7.3 � 107 6.5 � 107 1.3 � 108 1.1 � 108 1.1 � 108

% Eubacteria 52.6 76.1 61.5 64.3 60.3 53% Archaea 47.4 23.9 38.5 35.7 39.7 47% H2-utilising methanogens 100 100 100 100 100 100% Acetate-utilising methanogensa 0.0 0.0 0.0 0.0 0.0 0.0

a Figures for acetate-utilising methanogens have been calculated relative to Archaea.

Table 5Microbial activity.

Series 1

1-i 1-1 1-2 1-3 1-4 1-5

Microbial activity (L CH4/cell) 1.1 � 10�11 1 � 10�11 5.1 � 10�11 4.7 � 10�11 1.5 � 10�11 1.9 � 10�11

Series 2

2-i 2-1 2-2 2-3 2-4 2-5

Microbial Activity (L CH4/cell) 1.1 � 10�11 2.1 � 10�10 2 � 10�10 1 � 10�10 1.2 � 10�10 7.9 � 10�11

182 R. Montañés et al. / Bioresource Technology 180 (2015) 177–184

It is remarkable that although methane production was signifi-cantly greater in series 2, carried out in mesophilic conditions, thepattern of results for different SS/SBPL ratios was the same underthermophilic conditions. In both regimes the adding SBPL to thesystem as a co-substrate increased methane production relativeto separate digestion of the two types of waste, except in reactor1-4.

Fig. 2 shows clearly that the choice of inoculum had a markedeffect on the cumulative production of methane; this effect wasstronger when SS and SBPL were digested separately (reactors 1-1 and 1-5 respectively). These results indicate that even when con-ditions are not optimal for the inoculum co-digestion of SS andSBPL improved biodegradation of the wastes due the synergybetween them except in reactor 1-4.

3.2. Alkalinity and VFAs

VFAs, also known as short chain fatty acids, are widely used asan indicator of stress in anaerobic digestion processes. Accumula-

tion of VFAs results in a drop in pH and may even lead to reactorfailure or severely compromised digestion (Ahring et al., 1995;McCarty, 1964).

The reduction in pH produced by VFAs is normally offset by theactivity of methanogens, which have an alkalising effect as theyproduce carbon dioxide, ammonia and bicarbonate. The pH of adigestion system is determined by CO2 concentration in the gasphase and by HCO3� concentration in the liquid phase. Given aconstant CO2 concentration in the gas phase the addition ofHCO3� will increase the pH in a digester (Turovskiy and Mathai,2006). Fig. 3 shows the relationship between acidifying andalkalising substances in the digesters; it is clear that the digestersin series 2 were operating with good buffering capacity as thequantity of VFAs in the digester remained low or negligible. Thereduction in VFA levels and alkalinity over time in series 2 didnot affect methanogenic activity as the methane concentration inthe digesters did not fall.

The ratio VFA/Alk demonstrated that the acetogens andmethanogens were able to cope with the fluctuations in VFAs

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and alkalinity in the digesters, indicating that conditions were sta-ble and the risk of methanogen inhibition was low. The data on thecomposition and cumulative production of biogas in series 1 reac-tors showed that VFAs nevertheless affected the anaerobic diges-tion process.

In series 1 total acidity values were very high at the beginningof the tests owing to the characteristics of the inoculum used.The concentration of VFAs in the effluent from the anaerobicdigesters was often high, owing to overloading of the digester,entrance of toxic compounds or changes in the temperature orpH. Low pH stimulates acidogenic activity (VFA production) andinhibits methanogenic activity (VFA consumption) which mayexplain the high VFA levels at the end of the tests (Fig. 4).

The accumulation of VFAs has long been associated with distur-bances in the conditions in anaerobic digestion systems (Harperand Pohland, 1986); accumulation of VFAs was reported to bemore pronounced at thermophilic temperatures (Gray et al.,2006), probably because aceticlastic methanogens are tempera-ture-sensitive (Ahring et al., 2001; Leven et al., 2007).

Levels of VFAs were low at the end of testing in series 2 reactorsreflecting the complete biodegradation of wastes that occurred inall series 2 reactors.

The ratios of total acidity/total alkalinity (VFA/Alk) were verylow in series 2 reactors and high in series 1, with the exceptionof reactor 1-i (reactor with inoculum only). Maintenance of anappropriate pH is essential for proper operation of the digesterand optimal VFA degradation; in a strong system the VFA/Alk ratiowill be between 0.0 and 0.1, values between 0.1 and 0.4 indicatefavourable operating conditions and low or no risk of acidification(Sánchez et al., 2005).

3.3. Microbial population dynamics

Data were collected on micro-organism concentrations in thereactors at the end of the BMP tests. Absolute quantities and pro-portions (%) of the main microbial groups are shown in Table 4.

At the end of testing the microbial populations in both reactorseries consisted of Eubacteria and Archaea, with Eubacteria in themajority in all cases. Stable anaerobic reactors have a much largerpopulation of Eubacteria than Archaea (Zahedi et al., 2013). It isnoteworthy that in the reactors there were more H2-utilisingmethanogens than acetate-utilising methanogens.

Biodegradation was limited in all series 1 reactors as pH valueswere not optimum for the methanogenic Archaea as a consequenceof the quantity of VFAs produced. Analysis revealed that althoughthe Archaea populations were higher in series 1 reactors they wereless active than in series 2 reactors.

The pattern of activity and composition of the microbial popu-lation were similar in all series 2 reactors indicating that all sub-strates tested had biodegraded.

The relationship between organic loading rates and microbialactivity which was calculated as the ratio of the volume of CH4 gen-erated to the number of Archaea inside the reactor (assessed usingFISH staining) (Montero et al., 2009). Table 5 shows microbialactivity in both series of reactors; microbial activity was greaterin series 2 reactors, although the pattern of activity for differentSS/SBPL ratios was similar under thermophilic and mesophilicconditions, just as the pattern of change in physicochemical param-eters was similar in both series. The best results were obtainedwhen SBPL was added to SS as a co-substrate.

To evaluate the biochemical activity on initial OLR (in terms ofg TVSadd), it has been considered the parameter to measure meth-anogenic activity. These data are presented in Fig. 5.

Fig. 5a and b show the relationship between Archaea populationdensity and the productivity of the reactor for all SS/SBPL ratiosunder both thermophilic (series 1) and mesophilic (series 2) condi-tions respectively.

Productivity in terms of ml CH4/g TVSadd was lower under ther-mophilic conditions although Archaea population densities were ofthe same order of magnitude.

Fig. 5b shows that the size of the Archaea population isindirectly related to productivity measured in terms of mlCH4/g TVSadd; this relationship is mediated by the proportion oftotal volatile solids in the initial substrate, which is higher. Produc-tivity was not proportional to the size of the Archaea population.

Previous studies have demonstrated links between digesteroperating conditions, physical and chemical performance parame-ters and microbial population dynamics (Montero et al., 2009).These results suggest that the composition and density of micro-bial population may be more closely related to initial organic loadthan to the activity of anaerobic micro-organisms during digestion.

4. Conclusions

Biodegradation was limited under thermophilic conditions,because the VFA concentration increased, but under mesophilicconditions complete biodegradation of the test substratesoccurred.

More methane was produced in reactors containing a substratemade up of a mixture of SS and SPBL (i.e. reactors 1-2, 1-3, 1-4, 2-2,2-3 and 2-4), indicating that combining these wastes had a syner-gistic effect on anaerobic digestion.

Anaerobic co-digestion of SS and SBPL is a promising waste-pro-cessing procedure; addition of SBPL to SS significantly increasesthe rate of biomethanation of SS under suitable pH conditions.

Acknowledgements

The authors wish to express their gratitude to Junta de And-alucía, specifically to Proyecto de Excelencia financed through

184 R. Montañés et al. / Bioresource Technology 180 (2015) 177–184

FEDER funds, with reference P09-TEP-5275, called ‘‘Codigestiónanaerobia de lodos de depuradora y residuos de cultivos vegetalesenergéticos. Estrategias para mejorar la producción de biogás y lavalorización agronómica del residuo final’’.

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