Fungal Biodegradation of Melanoidin in Distillery Spentwash

8/10/2019 Fungal Biodegradation of Melanoidin in Distillery Spentwash

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FUNGAL DECOLOURIZATION OF MELANOIDIN IN

MOLASSES SPENT WASH

SEMINAR REPORT

Submitted by

SITARA D.

in partial ful fi lment for the award of the degree of

MASTER OF ENGINEERING IN

ENVIRONMENTAL ENGINEERING

CENTER FOR ENVIRONMENTAL STUDIES

DEPARTMENT OF CIVIL ENGINEERING

ANNA UNIVERSITY CHENNAI: CHENNAI 600 025

AUGUST 2012


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ABSTRACT

Decolourization and degradation of recalcitrants using fungi is a widely

pursued study area. This report presents information regarding the nature of molasses

spent wash and its major recalcitrantmelanoidin, followed by a description of fungal

decolourization of melanoidin, where some basic information regarding the

mechanism of fungal degradation, progress in work on potential decolourizing fungi,

factors affecting fungal degradation and scope for future research are discussed.


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LIST OF TABLES

TABLE TITLE PAGE

NO.

1 Characteristics of raw spent wash and post biomethanated

spent wash

2


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1. A BRIEF DESCRIPTION OF MOLASSES SPENT WASH

In 1963, Hubert Olbrich published detailed information on molasses. The Latin

adjective mellacea refers to honey like. Molasses denotes the final effluent

obtained in the preparation of sugar by repeated crystallization of sugarcane or sugar -

beet juice. The theoretically final molasses is a mixture of sugar (not more than 49%),

some non-sugars and water, from which no saccharose crystallizes under any

conceivable physical and technically optimum conditions, with no regard to time. It is

a commonly used raw material in most distilleries for the commercial production of

ethanol in India due to its easy availability and low cost. Ethanol production involves

the yeast fermentation of diluted molasses (wash) to yield ethanol and distillation offermented wash containing ethanol to separate ethanol as a final product. The

fermented wash separated from ethanol during distillation is the spent wash.

The spent wash is dark brown in colour, with temperature around 90oC110

oC

and contains about 10-11% w/w of solids. It is high in Bio-chemical Oxygen Demand

(BOD) / Chemical Oxygen Demand (COD) content and inorganics, as shown in table

1. Its main recalcitrant is the brown polymer, melanoidinand the others are caramel

and variety of sugar decomposition products, anthocyanins, tannins and different

xenobiotic compounds. The unpleasant odour of the effluent is due to the presence of

skatole, indole and other sulphur compounds present in the raw material - final

molasses, which are not effectively decomposed by yeast during the fermentation

process (Sarayu Mohana et al, 2009). The production and characteristics of spent wash

depend on nature of feedstock, process water and the process used.

CPCB reports that, in India, by 2011, there were about 350 molasses based

distillery units that could generate 60 billion litres of spent annually. Presently around

56% of Indian distilleries, as reported by J. Singh and S. Gu in 2010, opt for

biomethanation of spent wash to recover biogas energy and the treated effluent is still

dark brown in colour and has a considerable organic load and so it is subjected to

aerobic processes and/or facultative lagoons and tertiary membrane processes. The

Pollution Control Board mandates a BOD of 30 mg/l in the effluent for disposal insurface water and 100 mg/L for disposal on land.


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Table 1: Characteristics of raw spent wash and post biomethanated spent wash

(Sarayu Mohana et al, 2009)

Parameters

( in mg/L, except for pH)

Values of distillery

effluent

Values of anaerobically

treated effluent

pH 3.04.5 7.58

BOD5 50,00060,000 800010,000

COD 110,000190,000 45,00052,000

Total solid (TS) 110,000190,000 70,00075,000

Total volatile solid (TVS) 80,000120,000 68,00070,000

Total suspended solid (TSS) 13,00015,000 38,00042,000

Total dissolved solids (TDS) 90,000150,000 30,00032,000

Chlorides 80008500 70009000

Phenols 800010,000 70008000

Sulphate 75009000 30005000

Phosphate 25002700 15001700

Total nitrogen 50007000 40004200

Untreated or partially treated effluent very often finds access to inland waters

and poses a serious threat to the water quality. High COD and nutrient content of

even the secondary treated effluent may result in eutrophication of natural water

bodies, while the highly coloured components of the spent wash reduce sunlight

penetration and decrease both photosynthetic activity and dissolved oxygen

concentration affecting aquatic life. Disposal of distillery spent wash on land is

reported to reduce soil alkalinity and manganese availability, inhibiting seed

germination (Sarayu Mohana et al, 2009). Use of biomethanated spent wash for

irrigation without proper monitoring can affect groundwater quality by altering its

physiochemical properties such as colour, pH, electrical conductivity etc. due to

leaching down of organic and inorganic ions (CPCB, 2010-2011).


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2. MELANOIDINS IN SPENT WASH

The main colourants in the molasses spent wash are the brown polymers,

melanoidins, which are formed by Maillard reaction i.e. interaction between reducing

sugars and free amino acids or amino groups of peptides in the fermented wash, at

high temperatures during distillation (Sarayu Mohana et al, 2009). Maillard reactions

are natural condensation, non-enzymatic browning reactions.

A study found the quantities of melanoidin and caramel in a typical South

Indian molasses spent wash to be 82.48 g/mL and 17.98 g/mL at 30% dilution by

observed changes in peaks in IR spectra analysis following UV- VIS spectral analysis

(Naik et al, 2010) i.e. around 274.76 g/mL melanoidin and 59.93 g/mL caramel in

raw spent wash.

Apart from being discharged in large volumes by various agro-based industries

especially from cane molasses based distilleries and fermentation industries as

environmental pollutants, melanoidins are also found naturally in food, drinks and

impart commercial (colour, flavour), nutritional (antioxidant, antiallergenic) and

toxicological (antimicrobial, cytotoxic) significance to those products including bread

and beer varieties.

2.1 Chemistry of melanoidins

Ram Chandra et al, have made a review of melanoidin chemistry and

degradation in 2008. Melanoidins are assumed not to have a definite elemental

composition as it depends on the nature and molar concentration of parent reacting

compounds and reaction conditions as pH, temperature, heating time and solvent

system used. . They are high molecular weight aminocarbonyl compounds. The

nitrogen contents, acidities and electrophoretic behavior of the polymers all reflect

functional group distributions inherited from the amino acids. The melanoidins

chromophore has not been yet identified.

Melanoidins are water soluble, recalcitrant and antimicrobial, thus qualifying it

as a potent contaminant of the soil and water bodies they are disposed into.


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Melanoidins degradation and decolourization of wastewater by physico-chemical

treatments such as flocculation, ozonation and activated carbon adsorption are

efficient, but not economic on large scale. Also these methods require high reagent

dosages and generate large amount of sludge whereas biological decolourisation by

using certain microbes have been successfully achieved and thus can be applied as

bioremediation.

2.2 Environmental significance of melanoidins

Melanoidins can inhibit microbial growth by cross-linking polypeptide chains

and sequestering essential multivalent metal cations. Melanoidins are found in natural

fresh and coastal waters as a key link in the transformation of organic matter

(polysaccharides, amino acids) that are relatively easily degradable into more

recalcitrant humic material in nature/environment. They are potential buffer

compounds for metallic ions. When discharged into water bodies, they reduce sunlight

penetration and decrease both photosynthetic activity and dissolved oxygen

concentration affecting aquatic life. Similiarly, it is also reported to adversely affect

soil microflora.

2.3 Degradation of melanoidins

Both chemical and biological methods of melanoidin have been attempted.

Research work includes those carried out using synthetic melanoidins.

2.3.1 Chemical methods

Products of hydrolysis reactions of melanoidins depend on the startingcompounds used in melanoidin synthesis. Using di- and oligosaccharides as carbonyl

components in Maillard reaction, a significantly higher amount of sugars than in

monosaccharide model melanoidins was released by acid hydrolysis. Pyrolytic

degradation resulted in various furans and methyl pyrazines. Synthetic melanoidins

prepared from glucose-glycine system had been decolourized using hydrogen

peroxide and ozone treatments (Ram Chandra et al, 2008).


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2.3.2 Biodegradation of melanoidins

Biological methods for the treatment of industrial wastes involves the

exploitation of the natural or induced unique metabolism capabilities of certain

microorganisms to degrade substances that are recalcitrant and toxic to many other

micro and macro organisms. This has a long history of use due to its eco-friendly and

cost effectiveness when compared to chemical methods (Sarayu mohana et al, 2009).

This is largely dependent on the enzymatic setup, nutrient requirement of microbes as

well as nature and chemical structure of recalcitrant compounds and environmental

conditions.

The molasses spent wash is highly acidic in nature and has a variety of

recalcitrant colouring compounds as melanoidins, phenolics and metal sulphides

which are mainly responsible for the dark colour of distillery effluent and give a high

inhibitory and antimicrobial activity to this wastewater, thus slowing down the

anaerobic digestion process of distillery spent wash. Ram Chandra et al, 2008,

compiled information on decolourization mechanisms of melanoidin. Microbial

decolourisation of melanoidins is due to two decomposition mechanisms; in the first

the smaller molecular weight melanoidins are attacked and in the second the larger

molecular weight melanoidins are attacked. Sarayu mohana et al, in 2009, have

reviewed aerobic treatment of post methanated spent wash using potential

decolourizing bacteria (pure and mixed culture), cyanobacteria, yeast, fungi have been

widely studied.


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3. MYCOREMEDIATION OF MELANOIDINS

3.1 Mechanism of fungal degradation of recalcitrants

Generally, fungi are found to be more adaptable in the face of environmentalconstraints. Fungi are major decomposers in most ecosystems as they secrete

extracellular enzymes such as laccase, Mn peroxidase and lignin peroxidase (LiP), and

acids that break down lignin and cellulose, the two main building blocks of plant

fibres. These are organic compounds composed of long chains of carbon and

hydrogen, structurally similar to many organic pollutants. The key to

mycoremediation is determining the right fungal species to target a specific pollutant.

The ability of the white rot fungi to degrade dye can be directly correlated with

its ability to degrade lignin; the dye molecules are degraded along with lignin

(Tripathi et al, 2007).

It is suggested that decolourisation by fungi takes place due to the destruction

of coloured molecules and partially because of sorption phenomena. A mechanism

proposed for decolourization of melanoidins by Rhizoctonia sps. D-90 suggests that

the pigments were accumulated in the cytoplasm and around the cell membrane as

melanoidin complex, which was then gradually decolourized by intracellular enzymes.

3.2 Significance as a pre-treatment for anaerobic digestion

Fungi have been used effectively as a pre-treatment for anaerobic digestion of

materials with high phenolic content, such as molasses and olive mill wastewater. The

phenolic compounds in such materials exert antimicrobial activity inside biologicalwastewater treatment systems, inhibiting the effectiveness of the treatment. In such

cases, fungal pre-treatment under aerobic conditions makes it possible to obtain 51 -

100% phenol removal; good decolourisation (31 - 100%);


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3.3 Use of fungal systems for melanoidin degradation

3.3.1 Aspergillus species

One of the most studied fungi having the ability to degrade and decolorizedistillery effluent is Aspergillus sps. Aspergillus fumigatus G-2-6, Aspergillus niger,

A. niveus, A. fumigates UB260 brought about an average of 6975% decolorization

along with 7090% COD reduction (Sarayu mohana et al, 2009). Aspergillus niger is

reported to have biologically eliminated 83% of the total colour removed and the

remaining 17% by adsorption on the mycelium under optimal nutrient concentration

(Ram Chandra et al, 2008).

3.3.2 Penicillium species

Penicillium spp. such asPenicillium decumbens,Penicillium lignorumresulted

in about 50% reduction in color and COD, and 70% phenol removal. Penicillium

pinophilumTERI DB1,Alternaria gaisenTERI DB6 andPleurotus floridaEM 1303

produce ligninolytic enzymes and decolourization efficiency is reported to be up to

50%, 47% and 86%, respectively (Ram Chandra et al, 2008).

3.3.3 Marine fungi

Marine fungi have been least exploited for melanoidin degradation compared to

their terrestrial counterparts. A marine white rot fungus, Flavodon flavus has been

reported by Raghukumar C and Rivonkar G., 2001 to be effective in decolourizing

raw molasses spent wash. The colour removal was 80% after 8 days of incubation,

when used at concentrations of 10% and 50%. The decolorization was not effectivewhen F. flavus was immobilized in calcium alginate beads. Decolorization was

achieved best in oxygenated cultures. Besides colour, total phenolics and COD were

reduced by 50%, suggesting its potential in the bioremediation of effluents.

Previously, Raghukumar et al, 1999, had also found that it produces all

important lignocellulosic enzymes that could decolorize several individual synthetic

dyes. Mtui and Nakamura, 2008 showed that the enzymes from F. flavus under

seawater conditions could also decolorize 94% of raw textile wastewater and almost


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completely decolorized RBB-R dye, Congo red, Brilliant green, Reactive black and

Reactive yellow at low carbon culture medium. This confirmed extracellular enzymes

from F. flavus to be potential degraders of organic pollutants and showed that

facultative marine fungi that live under harsh seawater conditions are suitable for

bioremediation of recalcitrant environmental pollutants.

3.3.4 White rot fungi

White rot fungi are widely preferred candidates for bioremediation as they

produce various extracellular oxidizing enzymes directly involved in the degradation

of various xenobiotic compounds and dyes. Coriolus versicolor, Trametes versicolor,

Phanerochaete chrysosporium were studied in various works, under laboratory

conditions involving addition of nutrients and dilution of effluent, along with some

other members of this species.

Fungal pre-treatment of wine distillery wastewater with Trametes pubescens

led to a significant reduction in CODs and polyphenols in the studied WDW. The

CODs removal efficiency after fungal pre-treatment reached 53.3 %. The pH of the

fungally pre-treated wastewater reached 6.7, reducing the pH buffering requirements

for anaerobic digestion. The latter was conducted under pH buffering using a mixture

of CaCO3 and K2HPO4, which provided stable environment inside the bioreactor

system for efficient CODs. The total CODs removal efficiency reached 99.5%, and

the system proved able to eliminate shock loads of high input CODs concentrations

(Melamane et al, 2007).

3.3.5 Cell extracts

The possibility of using dead yeast cells for decolourization of distillery

effluents has also been reported where dry yeast powder decolourized the effluents of

biomethanation plant by more than 70%.

3.3.6 Yeasts

Sirianuntapiboon and Tondee isolatedIssatchenkia orientalisyeast from a from

fruit sample in 2008. It showed 60% melanoidin decolorization at 30 C in 7 days


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under aerobic condition. In 2011, they found that the melanoidin degradation ability

was induced by melanoidin, while it did not influence the adsorption ability. Then, the

cultivated living cell showed the highest MP-adsorption yield of 27.81.3% - about 15

to 20% higher than the dead (autoclaved) cell. The deteriorated cell (MP-adsorbed

cell) could be reused after washing with diluted-H2SO4 solution, where the capacity

increased by 100 to 150% due to elution adsorbed matter.. The harvested-biomass

from this treatment system could be used as the protein source for animal feed due to

the high protein content of 36.381.12%.

Candida tropicalis, isolated from soil near a distillery showed maximum

decolorization of diluted effluent (75%) at 45C using a little amount of carbon and

nutrient supplements at pH-5.5 within 24 h of incubation under static condition

(Tiwari et al, 2012).

Neethu S Kumar et al, 2012 isolated a Cunninghamella blakesleeana from

distillery site which showed a decolourization zone of 61mm diameter in 1% synthetic

melanoidin and 69mm diameter in 10% distillery effluent after 48hours of incubation,

and further research is being continued.

3.3.7 Mixed consortiums

Various microbial consortiums are reported, including that of Sumit Pal and

Vimala in 2012, who used a consortium of Phanerochaete chrysosporium MTCC787

along with Psedomonas aeruginosa and Aspergillus niger to decolourize distillery

effluent. P. chrysosporium showed 78.30%; A. niger 52.5%; Pseudomonas aeruginosa

70.8%, and the consortium showed 87.80% decolourization efficiency.


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4. FACTORS AFFECTING FUNGAL DEGRADATION

Nagaraj M. Naik, 2007 in his thesis, mentioned important findings reported to

factors influencing fungal degradation.

4.1 Carbon source

In Coriolus versicolour Ps-4, melanoidin decolourizing ability was due to sugar

dependent enzyme which was induced by melanoidin. For another white rot fungus,

glucose, starch, maltose and cellobiose were found to be good carbon sources or co

substrates, while sucrose, lactose, xylon, xylose, methanol and glyoxol were poor

carbon sources. For Aspergillus niger VM2, addition of carbon source to culture

media was necessary for the decolorization of primary effluent and addition of 1 per

cent glucose was essential for co-metabolism of melanoidin complex.

4.2 Nitrogen

For Aspergillus sojae B-10, Sodium nitrate was the optimal nitrogen source,

and the highest colour removal occurred with NH4NO3at 1.8 g per litre. Nitrogen had

no effect on decolorization of dyes by the fungus Cyathus bulleri. As decolorization of

dyes by P. chrysosporium occurs in secondary metabolic conditions, the important

enzyme LiP was released by the fungal cells under either carbon or nitrogen limitation

it was also suggested that the organic nitrogen sources were considered essential

medium supplements for the regeneration of NADH that acted as electron donor for

the reduction of azodyes by microorganisms.

4.3 pH

Raghukumar et al. 1999 worked on the effect of pH on colour removal by three

marine fungi and found pH 4.5 to be effective. Also, other workers reported the

optimum pH for decolourizing fungi to be in the range of 4 to 5, which concurs with

the pH of raw spent wash.


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4.4 Oxygen

No adverse effect was observed due to shaking during the bio-decolorization

process by fungi. During intermittent aeration three times a day, DO (dissolved

oxygen) ranged between 0.5 and 1.0 mg per L; prior to aeration DO was zero and

lignin breakdown was found to be enhanced for Schizophyllum communewith colour

removal between 72% on the first day and 80% on the third day. Agitation was

reported essential for keeping a high rate of decolorization by Trametes villosa.

Raghukumar et al. observed that Flavodon flavus, required higher dissolved

oxygen (DO) levels for decolorization. When spentwash was flushed with oxygen gas,

the fungus decolourized spentwash (50% diluted) upto 79 per cent in five days.


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5. CONCLUSION: FUTURE RESEARCH FOCUS

Under nutrient limiting conditions, fungal cells generally cannot remain active

during a long-term cultivation. Therefore, the continuous-culture method is not

practical and the semi-batch or repeated-batch method can be an alternative for long-

term cultivation. Nevertheless, the feasibility of application of the process to full-scale

would need further research in this continuous culture set-up, in order to minimize the

added nutrients and extend the biomass activity for a longer period.

The application of fungi for decolourization as well as COD removal in

industrial wastewaters including distillery spent wash on a large scale has been

impeded owing to lack of an appropriate reactor system capable of coping with

relatively slow fungal degradation, loss of extracellular enzymes and mediator with

discharged water (Jiranuntipon et al, 2008).

An understanding of complete profile of the effluent and the structures of

recalcitrant colouring compounds would also be helpful in achieving the appropriate

treatment solutions.


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6. REFERENCES

Central Pollution Control Board - Central Zonal Office - Bhopal, 2010 2011,

A Report on Assessment of grain based fermentation technology, waste treatment

options, disposal of treated effluents.

J. Singh and S. Gu, 2010, Biomass conversion to energy in IndiaA critique,

Renewable and Sustainable Energy Reviews, Vol 14, pg 13671378

Jiranuntipon et al, 2008, Decolorization of synthetic melanoidins-containing

wastewater by a bacterial consortium, Journal of Industrial Microbiology and

Biotechnology, vol. 3, No 11, pg. 1313-1321

Molasses from Wikipedia,http://en.wikipedia.org/wiki/MolassesMelamane et al, 2007, Anaerobic digestion of fungally pre-treated wine

distillery wastewater, African Journal of Biotechnology Vol. 6 (17), pp. 1990-1993

Mtui and Nakamura, 2008, Lignocellulosic enzymes from Flavodon flavus,

afungus isolated from Western Indian Ocean off thecoast of Dar es Salaam, Tanzania,

African Journal of Biotechnology Vol. 7, No 17, pg. 3066-3072

Nagaraj M. Naik, 2007, Decolorization of biomethanated spentwash by native

microorganisms, Ph. D thesis, University of agricultural sciences, Dharwad

Naik et al, 2010, Enhanced Degradation of Melanoidin and Caramel in

Biomethanated Distillery Spentwash by Microorganisms Isolated from Mangroves,

Iranica Journal of Energy & Environment, Vol 1, No 4, pg 347-351

Neethu S Kumar et al, 2012, Isolation of a novel soil fungus VT-NSK capable

of utilizing the distillery spentwash and synthetic melanoidin a preliminary report,

Research in Biotechnology, Vol 3, No 1, pg 01-08

Olbrich H., 1963, The molasses,http://kempetrade.de/Molasses_OLBRICH.pdf

Raghukumar et al, 1999, Lignin-modifying enzymes of Flavodon flavus, a

basidiomycete isolated from a coastal marine environment, Applied and

Environmental Microbiology, Vol.65, pg 2103-2111

Raghukumar C and Rivonkar G., 2001, Decolorization of molasses spent wash

by the white-rot fungus Flavodon flavus, isolated from a marine habitat, Applied

Microbiology and Biotechnology, Vol 55, No 4, pg 510-4
http://en.wikipedia.org/wiki/Molasseshttp://en.wikipedia.org/wiki/Molasseshttp://en.wikipedia.org/wiki/Molasseshttp://kempetrade.de/Molasses_OLBRICH.pdfhttp://kempetrade.de/Molasses_OLBRICH.pdfhttp://kempetrade.de/Molasses_OLBRICH.pdfhttp://kempetrade.de/Molasses_OLBRICH.pdfhttp://en.wikipedia.org/wiki/Molasses


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Ram Chandra et al, 2008, Melanoidins as major colourant in sugarcane

molasses based distillery effluent and its degradation, Bioresource Technology, Vol

99, Pg 46484660

Sarayu Mohana et al, 2009, Distillery spent wash: Treatment technologies and

potential applications, Journal of Hazardous Materials, Vol. 163, pg. 1225

Sirianuntapiboon and Tondee, 2011, Melanoidin decolorization mechanism and

some properties of Issatchenkia orientalis No.SF9-246, African Journal of

Biotechnology Vol. 10, No 47, pg. 9683-9693

Sumit Pal and Vimala Y, 2012, Bioremediation and decolourization of

Distillery effluent by novel Microbial Consortium, European Journal of Experimental

Biology, Vol 2, No 3, pg 496-504

Tiwari et al, 2012, Decolorization of a recalcitrant organic compound

(Melanoidin) by a novel thermotolerant yeast, Candida tropicalis RG-9, BMC

Biotechnology, Vol 12, No 30,

Tripathi et al, 2007, Fungal treatment of industrial effluents: a mini-review,

Life Science Journal, Vol 4, No 2, pg 7881

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Fungal Biodegradation of Melanoidin in Distillery Spentwash