FUNGAL DECOLOURIZATION OF MELANOIDIN IN

MOLASSES SPENT WASH

SEMINAR REPORT

Submitted by

SITARA D.
in partial fulfilment 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 recalcitrant melanoidin, 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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TABLE OF CONTENTS
CHAPTER TITLE

PAGE NO.

A BRIEF DESCRIPTION OF MOLASSES SPENT WASH 1

MELANOIDINS IN SPENT WASH

2.1

Chemistry of melanoidins

2.2

Environmental significance of melanoidins

2.3

Degradation of melanoidins

2.3.1

Chemical methods

2.3.2

Biodegradation of melanoidins

MYCOREMEDIATION OF MELANOIDINS

3.1

Mechanism of fungal degradation of recalcitrants

3.2

Significance as a pre-treatment for anaerobic digestion

3.3

Use of fungal systems for melanoidin degradation

3.3.1

Aspergillus species

3.3.2

Penicillium species

3.3.3

Marine fungi

3.3.4

White rot fungi

3.3.5

Cell extracts

3.3.6

Yeasts

3.3.7

Mixed consortiums

FACTORS AFFECTING FUNGAL DEGRADATION

10

4.1

Carbon source

10

4.2

Nitrogen

10

4.3

Ph

10

4.4

Oxygen

11

CONCLUSION: FUTURE RESEARCH FOCUS

12

REFERENCES

13

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LIST OF TABLES
TABLE

TITLE

Characteristics of raw spent wash and post biomethanated

spent wash

PAGE
NO.
2

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 of
fermented 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 90oC 110oC
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, melanoidin and 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 in
surface water and 100 mg/L for disposal on land.

Table 1: Characteristics of raw spent wash and post biomethanated spent wash
(Sarayu Mohana et al, 2009)
Parameters

Values of distillery Values of anaerobically

( in mg/L, except for pH)

effluent

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).

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.

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 starting
compounds 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).

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.

3. MYCOREMEDIATION OF MELANOIDINS
3.1 Mechanism of fungal degradation of recalcitrants
Generally, fungi are found to be more adaptable in the face of environmental
constraints. 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 biological
wastewater 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%); <85.4% BOD reductions,
and production of enzymes capable of degrading xenobiotics e.g., laccase (Melamane
et al, 2007).

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 decolorize
distillery 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 as Penicillium decumbens, Penicillium lignorum resulted
in about 50% reduction in color and COD, and 70% phenol removal. Penicillium
pinophilum TERI DB1, Alternaria gaisen TERI DB6 and Pleurotus florida EM 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 effective
when 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

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 isolated Issatchenkia orientalis yeast from a from
fruit sample in 2008. It showed 60% melanoidin decolorization at 30 C in 7 days

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 NH4NO3 at 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 commune with 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 longterm 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/Molasses
Melamane 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

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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 78 81