Bioresource Technology 97 (2006) 2096–2102

Decolourisation and detoxiWcation of synthetic molasses melanoidinsby individual and mixed cultures of Bacillus spp.

Praveen Kumar, Ram Chandra ¤

Environmental Microbiology Section, Industrial Toxicology Research Centre, P.O. Box No. 80, M.G. Marg, Lucknow 226001, (UP), India

Received 12 January 2005; received in revised form 29 September 2005; accepted 1 October 2005Available online 29 November 2005

Abstract

The decolourisation of synthetic melanoidins (i.e., GGA, GAA, SGA, and SAA) by three Bacillus isolates (Bacillus thuringiensis(MTCC 4714), Bacillus brevis (MTCC 4716) and Bacillus sp. (MTCC 6506)) was studied. SigniWcant reduction in the values of physico-chemical parameters was noticed alongwith the decolourisation of all four melanoidins (10% v/v). B. thuringiensis (MTCC 4714) causedmaximum decolourisation followed by B. brevis (MTCC 4716) and Bacillus sp. (MTCC 6506). A mixed culture comprised of these threestrains was capable of decolourising all four melanoidins. The medium that contained glucose as a sole carbon source showed 15% moredecolourisation than that containing both carbon and nitrogen sources. Melanoidin SGA was maximally decolourised (50%) while mela-noidin GAA was decolourised least (»06%) in the presence of glucose as a sole energy source. The addition of 1% glucose as a supplemen-tary carbon source was essential for co-metabolism of melanoidin complex. The decolourisation of synthetic melanoidin by three Bacillusspp. signiWcantly reduced the toxicity to the tubiWcid worm (Tubifex tubifex, Müller).© 2005 Elsevier Ltd. All rights reserved.

Keywords: Melanoidin; Bacillus; Decolourisation; Distillery eZuent; Bio-assay

1. Introduction

Melanoidins are dark brown complex bio-polymersformed by Maillard amino-carbonyl reaction taking placebetween the amino and carbonyl groups in organic sub-stances (Wedzicha and Kaputo, 1992; Reynolds, 1968).They are widely distributed in nature and discharged byvarious agro-based industries, especially sugar cane molas-ses based fermentation industry, as environmental pollu-tants. Melanoidin’s environmental toxicity is due to its highbiological oxygen demand (BOD) (40,000 mg/L), chemicaloxygen demand (COD) (90,000 mg/L) and other toxic com-ponents (FitzGibbon et al., 1995; Chandra and Pandey,2001).

* Corresponding author. Tel.: +91 522 2220107/2220207/2214118/2227332; fax: +91 522 228227/228471.

E-mail addresses: pkp234@yahoo.com (P. Kumar), ramchandra_env@indiatimes.com (R. Chandra).

0960-8524/$ - see front matter © 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2005.10.012

A densely coloured melanoidin may be formed duringthe processing of sugarcane molasses for alcohol produc-tion in distilleries. When discharged to surface water, thismelanoidin may cause reduction of sunlight penetration inrivers, lakes or lagoons which in turn decreases both photo-synthetic activity and dissolved oxygen concentrationthereby severely aVecting the aquatic life (Kumar et al.,1997a). Disposal on land is equally detrimental, causinginhibition of seed germination and depletion of vegetationby reducing the soil alkalinity and manganese availability(Agarwal and Pandey, 1994; Kannabiran and Pragasam,1993).

Recently, decolourisation of molasses melanoidin hasbeen attempted, but with limited success (Sirianuntapiboonet al., 2004; Dahiya et al., 2001). Decolourisation of melanoi-din containing eZuent through chemical methods (Chandraand Singh, 1999), Xocculation treatments (Migo et al., 1993)and physicochemical treatments such as ozonation (Ichikawaet al., 1996; Kim et al., 1985) and activated carbon adsorp-tion (Chandra and Pandey, 2001) have been accomplished

P. Kumar, R. Chandra / Bioresource Technology 97 (2006) 2096–2102 2097

but these methods are not economically feasible on a largescale due to cost limitation. Biological treatments using fungisuch as Coriolus (Aoshima et al., 1985; Ohmomo et al.,1985b), Aspergillus (Ohmomo et al., 1987, 1988a), Phanero-chaete (Fahy et al., 1997) and certain bacteria such as Bacillus(Murata et al., 1992) and Lactobacillus (Ohmomo et al.,1988b) have been successfully achieved and thus can beapplied as a bioremediation technique.

Hence, biological decolourisation of melanoidin con-taining distillery wastewater prior to its disposal into theenvironment and the assessment of its toxicity using anaquatic sediment dweller and a sensitive pollution indicatororganism is essential. Thus, we were inspired to developBacillus cultures to economically and eYciently decolourisesynthetic melanoidins and assess the toxicity of treatedmelanoidin using tubiWcid worm (Tubifex tubifex, Müller),which is a cosmopolitan aquatic oligochaete and a sensi-tive pollution indicator, to allow safer disposal in theenvironment.

2. Methods

2.1. Preparation of synthetic melanoidin

Synthetic melanoidin was prepared following themethod described by Ohmomo et al. (1985a), in which 1.0molar (M) sugar (glucose or sucrose), 1.0 M amino acid(aspartic acid and glutamic acid) and 0.5 M sodium carbon-ate were dissolved in 1.0 L of distilled water and the solu-tion was reXuxed at 100 °C for 6 h.

Four types of melanoidins i.e., glucose-glutamic-acid(GGA), glucose-aspartic-acid (GAA), sucrose-glutamic-acid (SGA), and sucrose-aspartic-acid (SAA) were synthe-sized. GGA was prepared utilizing glucose, glutamicacid and sodium carbonate; GAA from glucose, asparticacid and sodium carbonate; SGA from sucrose, glutamic acidand sodium carbonate and SAA was synthesizedusing sucrose, aspartic acid and sodium carbonate underabovesaid conditions. After adjustment of the reaction mix-ture to pH 7.0 with 1 N NaOH, the solution was Wlteredthrough a 0.22 �m Wlter.

2.2. Physico-chemical analysis of synthetic melanoidin

The analysis for diVerent physico-chemical parameters(i.e., Colour, BOD, COD, total solids (TS), total suspendedsolids (TSS), total dissolved solids (TDS), phenol, sulphate,phosphate, chloride and metals) was accomplished asdescribed in standard methods for examination of waterand wastewater (APHA, 20th ed., 1998).

2.3. Screening for melanoidin tolerant microorganisms

Tolerance studies were carried out in glucose-peptone-yeast extract-melanoidin (GPYM) medium (Ohmomoet al., 1988b) amended with diVerent concentrations (10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, v/v)

of melanoidin. The GPYM medium used in this studyconsisted of (w/v): 1% glucose, 0.3% peptone, 0.2% yeastextract, 0.1% K2HPO4, 0.05% MgSO4 · 7H2O, tap water(0–90%, v/v) and diVerent (10–100%, v/v) concentrationof melanoidin. The pH was adjusted to 7.0§ 0.1 using1 N-NaOH. The isolates were then spread on pre-sterilizedplates of melanoidin amended GPYM medium containing1.5% (w/v) agar. Each plate was incubated at 37 °C for 48 hand observed for growth of tolerant colonies. The highlytolerant isolates were selected, re-streaked and maintainedon GPYM slants at 4 °C.

2.4. Inoculum preparation and decolourisation studies

The inoculum was prepared in two steps. In the Wrststage, a pure culture was transferred to a 50 ml modiWedGPYM broth consisting of (w/v): 1% glucose, 0.05% pep-tone, 0.1% K2HPO4 and 0.05% MgSO4 · 7H2O, incubatedfor 24 h in shaking Xasks (125 rpm, Innova 4230 Refriger-ated Incubator shaker, New Brunswick, Edison, New Jer-sey, USA) under aerobic condition at 37 °C. Subsequently,it was transferred into shaking Xasks containing 10% (v/v)melanoidin and 90% (v/v) medium.

The decolourisation experiments were carried out by theaddition of 1.1e+6 colony forming units (CFU) of bacterialinoculum in 250 ml Erlenmeyer Xasks containing 50 mlmodiWed GPYM broth under shaking Xask conditions(150 rpm) at pH 7.0 and 37 °C. A separate set of uninocu-lated Xasks was maintained in parallel as control. Theexperiments were conducted in two diVerent sets underaforementioned conditions. The Set-I was devoid of anyadditional nitrogen source while Set-II was supplementedwith a nitrogen source. The nitrogen supplemented inthe medium included either peptone (0.3% w/v) as organicor NaNO3 or NH4Cl (0.3% w/v) as inorganic source.Experiments were performed in triplicate and samples werewithdrawn at regular 24 h intervals for decolourisationmeasurements.

2.5. Decolourisation assay

Melanoidin decolourisation was measured as thedecrease in colour density at 475 nm after 10X dilution withsterile 0.1 M acetate buVer (pH 7.0) as adopted by Ohmomoet al. (1988b). Decolourisation yield was expressed as thedegree of decrease in the absorbance at 475 nm against ini-tial absorbance at the same wavelength using a UV–Visiblespectrophotometer (GBC Cintra-40, Australia). The con-trol/reference consisted of uninoculated modiWed GPYMmedium.

2.6. Toxicity assessment (ASTM, 1994)

TubiWcid worms, Tubifex tubifex (Müller, 1774) werecollected from a local reservoir of Gheru campus of Indus-trial Toxicology Research Centre (ITRC), Lucknow, Indiaand cultured in laboratory conditions for 15 days prior to

2098 P. Kumar, R. Chandra / Bioresource Technology 97 (2006) 2096–2102

the experiment. Toxicity tests were conducted in 250 mlbeakers containing 50 ml of melanoidin (10%, v/v). TentubiWcid worms were exposed to untreated (raw) andtreated melanoidins. Test worms were considered nonviableonly when complete loss of mobility and no response topricking/pressing with a blunt glass-rod was observed (Rat-hore and Khangarot, 2003; Popovic and Popovic, 1977).Further, nonviability was conWrmed by transferring tubiW-cid worms back to fresh control tap water. The nonviablespecimens were removed and enumerated at intervals of 24,48, 72, 96 and 120 h. The test-samples (melanoidins) wererenewed every 24 h.

2.7. Statistical analysis

The statistical signiWcance (pD 0.05) of the experimentaldata was analyzed using one-way ANOVA using softwareSPSS 8.0. All experiments were performed in triplicate.

3. Results

The decolourisation of synthetic melanoidin by mixedBacillus culture was evaluated using four types (GGA,GAA, SGA and SAA) of melanoidins diVering in theircomposition (Table 1). Preliminary physicochemical analy-ses revealed that each type of melanoidin has very high col-our, (up to 4.5£ 105 Chloroplatinate [Co–Pt] units), BOD(up to 9.0£ 104 mg/L), COD (up to 1.87£ 105 mg/L) and TS(up to 2.4£103 mg/L) values, as shown in Tables 1 and 2.

The tolerance studies at diVerent concentration of mela-noidins revealed that only three isolates ITRC PIII, PIVand PV, were able to tolerate up to 80% (v/v) of type GGA,SGA and SAA melanoidins. These isolates were subse-quently identiWed as Bacillus thuringiensis (MTCC 4714),Bacillus brevis (MTCC 4716) and Bacillus sp. (MTCC6506), respectively, by Microbial Type Culture Collectionand Gene Bank (MTCC), IMTECH, Chandigarh, India.

Decolourisation results were mixed. The degree ofdecolourisation of all four melanoidins separately by eachisolate varied between 1% and 31%. The isolate B. thuringi-ensis (MTCC 4714) decolourised melanoidin GGA up to14%, GAA 3.5%, SGA 22% and SAA up to 15% in presenceof additional nitrogen (0.3% w/v) along with glucose as car-bon source. It also caused decolourisation of melanoidinGGA up to 21%, type GAA 10.5%, SGA 31% and SAA upto 23.5% in the absence of additional nitrogen along withglucose as carbon source. Isolates B. brevis (MTCC 4716)and Bacillus sp. (MTCC 6506) achieved moderate decolo-urisation (2.1–27.4%) under both nitrogen supplementedand deWcient conditions.

A mixed culture comprised of B. thuringiensis (MTCC4714), B. brevis (MTCC 4716) and Bacillus sp. (MTCC6506), was developed for further investigations. Themixed bacterial culture exhibited increased decolourisation(2–50%) eYciency compared to that shown by any singleisolate (Fig. 1). Among all four types of melanoidins, theSGA, (sucrose + glutamic acid), was maximally decolour-ised (50%), whereas GAA (glucose + aspartic acid) was

Table 1Physico-chemical characteristics of melanoidin GGA and GAA before (untreated) and after (treated) bacterial decolourisation

All values are in mg/L except colour (Co–Pt unit). Mean (n D 3) § SD; ANOVA, p < 0.05 (for each parameter separately). Superscripts “a” indicatessigniWcant (p < 0.05) diVerence between means in a row (for each parameter of respective melanoidins). ND D not detected (minimum detection limit:Cd—2 �g/L, Mn—5 �g/L, Pb—30 �g/L, chloride—0.15 mg/L).¤ Values in parenthesis indicates percent reduction in the values of respective parameters.

Sl. no. Parameters Melanoidin GGA(untreated)

Melanoidin GGA(treated)¤

Melanoidin GAA(untreated)

Melanoidin GAA(treated)¤

1. Composition Glucose + glutamicacid + sodium carbonate

Glucose + glutamicacid + sodium carbonate

Glucose + asparticacid + sodium carbonate

Glucose + asparticacid + sodium carbonate

2. Colour 205,000a § 7500 112,500a § 4907.47 (45.12) 225,000a § 6614.38 160,000a § 7810.25 (28.88)3. BOD 57,500a § 1802.77 31,200a § 482.18 (45.73) 90,000a § 2179.44 64,250a § 2839.45 (28.61)4. COD 117,500a § 3905.12 54,150a § 653.83 (53.91) 187,500a § 7500 119,750a § 2179.44 (36.13)5. TS 2410a § 104.43 995.38 § 16.02 (58.7) 2260a § 87.04 1320a § 52.2 (41.59)6. TDS 2385a § 104.19 985.25a § 15.83 (58.68) 2175a § 87.64 1294a § 52.30 (40.5)7. TSS 25a § 0.88 10.05a § 0.48 (59.8) 45a § 2.23 26.0a § 1.0 (42.22)8. Nitrogen 261.5a § 2.64 151a § 6.24 (42.23) 345.2a § 13.49 194.6a § 6.12 (43.49)9. Sulphate 6263a § 66.3 3595a § 126.16 (42.59) 6025.2a § 63.83 4404a § 45.59 (26.9)

10. Phosphate 2.53a § 0.11 1.171a § 0.02 (53.69) 2.66a § 0.085 1.744a § 0.052 (34.35)11. Phenol 430a § 18.02 236.7a § 6.64 (44.95) 460.8a § 20.32 333b § 14.79 (27.73)12. Chloride ND ND ND ND13. Metals

Cd ND ND ND NDCr 0.37a § 0.01 0.16a § 0.0025 (56.75) 0.65a § 0.013 0.435a § 0.0026 (33.07)Cu 0.05a § 0.002 0.02a § 0.00087 (60) 0.03a § 0.001 0.019a § 0.00062 (36.66)Fe 1.90a § 0.05 0.81a § 0.013 (57.36) 0.75a § 0.034 0.49a § 0.0060 (34.66)Mn ND ND ND NDNi 0.32a § 0.01 0.165a § 0.0065 (48.43) 0.44a § 0.02 0.315a § 0.0043 (28.4)Pb 0.1a § 0.005 0.062a § 0.00095 (38) ND NDZn 0.02a § 0.001 0.0086a § 0.00013 (57) 0.08a § 0.003 0.054a § 0.00086 (32.5)

P. Kumar, R. Chandra / Bioresource Technology 97 (2006) 2096–2102 2099

decolourised least (20%) in absence of an additional nitro-gen but with glucose as carbon source.

Evaluation of the eVect of additional separate organic(peptone) and inorganic (NaNO3 and NH4Cl) nitrogensources on each melanoidin along with glucose followed asimilar pattern. The decolourisation of diVerent melanoi-dins was in the range of 6.0–34.5%, (GGA 22%, GAA 6%,SGA 34.5% and SAA 25.1%) in the presence of the addi-tional organic (peptone) nitrogen source (Fig. 1). Decolo-urisation of 35.5% and 31.7% was attained in type GGAand 47.3% and 41.5% decolourisation was attained in SGAmelanoidin using NaNO3 and NH4Cl as inorganic nitrogensources (Fig. 1).

The physicochemical analyses of treated (decolourised)melanoidins revealed that melanoidin decolourisation wasaccompanied by simultaneous reduction in diVerent physi-cochemical parameters. The mixed culture signiWcantly(p < 0.05) reduced the BOD (45% in GGA, 28% in GAA,56% in SGA and 46% in SAA), COD (54% in GGA, 41% inGAA, 63% in SGA and 54.5% in SAA) and total solids(58.7% in GGA, 41.5% in GAA, 70.5% in SGA and 59.8% inSAA). Similarly, signiWcant (ANOVA p < 0.05) reduction inphenol (44.95% in GGA, 27.7% in GAA, 54.6% in SGA and45.6% in SAA) and metals (32–64%, in diVerent melanoi-dins) was also obtained using mixed culture (Tables 1 and 2).

The assessment of environmental toxicity using tubiWcidworms after melanoidin decolourisation revealed that incontrol sets tubiWcid worms were active (live) throughoutthe test period. Tubifex were clustered at the bottom of the

test container and showed tubiWcid movement. In untreated(raw) melanoidins up to 12 h, the organisms demonstratedtubiWcid movement and remain clustered at the bottom ofthe container. Later, (i.e., after 12 h), worms remain sepa-rated and exhibited rapid twisting movement. While in thetreated melanoidins no such deviation from their naturalbehaviour was seen until the end (120 h) of experiment atall concentrations.

After 48 h, with raw melanoidins, reduced tactile move-ment of the worm was seen. Subsequently, segmentationand degeneration of the body took place and deathappeared without other noticeable signs. In case of treatedmelanoidins 50% mortality at concentration greater than80% (v/v) with above mentioned symptoms was noted onlyin melanoidin GAA in 120 h (Fig. 2).

4. Discussion

Physicochemical characterisation of melanoidinsrevealed them to be a complex polymer of Maillard reac-tion, where melanoidin GAA-polymer of glucose andaspartic acid, was found most recalcitrant to decolourisa-tion than other melanoidins (GGA, SGA and SAA) (Tables1 and 2; Fig. 2). This suggests that chemical complexities ofdiVerent melanoidins depend on the type of amino-acid(s)and sugar moieties reacted at diVerent temperature. Similarobservations were reported by Ohmomo et al. (1985a, 1988)and Hayashi and Namiki (1986) during their studies on syn-thetic melanoidins. The selection of sugars (glucose and

Table 2Physico-chemical characteristics of melanoidin SGA and SAA before (untreated) and after (treated) bacterial decolourisation

All values are in mg/L except colour (Co–Pt unit). Mean (n D 3)§ SD; ANOVA, p < 0.05 (for each parameter separately). Superscript “a” indicatessigniWcant (p < 0.05) diVerence between means in a row (for each parameter of respective melanoidins). ND D not detected (minimum detection limit:Cd—2 �g/L, Mn—5 �g/L, Pb—30 ng/L, chloride—0.15 mg/L).¤ Values in parenthesis indicates percent reduction in the values of respective parameters.

Sl. no. Parameters Melanoidin SGA(untreated)

Melanoidin SGA(treated)¤

Melanoidin SAA(untreated)

Melanoidin SAA(treated)¤

1. Composition Sucrose + glutamicacid + sodium carbonate

Sucrose + glutamicacid + sodium carbonate

Sucrose + asparticacid + sodium carbonate

Sucrose + asparticacid + sodium carbonate

2. Colour 17,800a § 702.38 8800a § 360.55 (50.56) 62,500a § 638.35 33,700a § 519.61 (46.08)3. BOD 52,500a § 2610.07 23,000a § l 145.64 (56.19) 80,000a § 2179.5 43,200a § 1391.94 (46.0)4. COD 112,000a § 5105.14 41,000a § l 767.76 (63.39) 166,000a § 1750 75,500a § 3500 (54.51)5. TS 2055 § 73.46 607a § 7.0 (70.46) 2090a § 80.266 840a § 21.79 (59.8)6. TDS 1985a § 77.20 586a § 7.93 (70.47) 1995a § 73.01 802a § 9.16 (59.79)7. TSS 70a § 3.31 21.0a § 1.0 (70.0) 95a § 3.97 38a § 1.5 (60)8. Nitrogen 182a § 8.15 63a § 3.12 (65.29) 272a § 8.18 160.48a § 3.95 (40.92)9. Sulphate 6255a § 50.74 3033a § 36.98 (51.51) 5765a § 136.15 3275a § 108.97 (43.19)

10. Phosphate 2.36a § 0.087 0.961a § 0.044 (59.29) 2.45a § 0.091 1.1251a § 0.031 (54.06)11. Phenol 583.5a § 16.68 264.9a § 6.64 (54.6) 574.8a § 14.53 312a § 8.18 (45.64)12. Chloride ND ND ND ND13. Metals

Cd ND ND ND NDCr 0.41a § 0.01 0.1648 § 0.0026 (60.0) 0.48a § 0.009 0.24a § 0.01 (50.0)Cu 0.015a § 0.005 0.0053a § 0.00024 (64.2) 0.055a § 0.0015 0.025a § 0.0005 (54.44)Fe 0.5a § 0.013 0.182a § 0.00086 (63.6) 0.85a § 0.029 0.038a § 0.00065 (55.29)Mn ND ND ND NDNi 0.29a § 0.008 0.137a § 0.0005 (52.75) 0.37a § 0.012 0.204a § 0.0025 (44.86)Pb 0.08a § 0.002 0.04634a § 0.00047 (42.12) 0.03a § 0.000% 0.02a § 0.00036 (33.33)Zn 0.041a § 0.001 0.016a § 0.00060 (60.97) 0.053a § 0.0012 0.024a § 0.00049 (54.71)

2100 P. Kumar, R. Chandra / Bioresource Technology 97 (2006) 2096–2102

sucrose) and amino acids (aspartic and glutamic-acid) forsynthetic melanoidin was made on the basis of their highconcentration (glutamic-acid 1.02–1.04 mg/G molasses andaspartic acid 0.9–1.65 mg/G molasses) in comparison ofother amino acids in sugarcane molasses (Paturau, 1982;Binkley and Wolform, 1953).

The mixed culture of Bacillus spp. exhibited a two- tofour-fold increase in melanoidin decolourisation over thatshowed by any individual Bacillus isolate. This may be dueto the enhanced eVect of coordinated metabolic interac-tions on melanoidin decolourisation.

The analyses on the eVects of an additional carbon (glu-cose) source emphasized the necessity of a readily availablecarbon source in the medium. The increased decolour-isation following addition of glucose as external carbonindicated the major role played by sugar-oxidases and

Fig. 1. Decolourisation of synthetic melanoidins using diVerent energysources with Bacillus spp. (A) GGA, (B) GAA, (C) SGA and (D) SAA.

0

10

20

30

40

50

0 24 48 72 96 120 144

Time (hours)

Dec

olou

risa

tion

(%

)

GGA G GGA G+P GGA G+NaNO3 GGA G+NH4Cl

0

10

20

30

40

50

0 24 48 72 96 120 144

Time (hours)

Dec

olou

risa

tion

(%

)

GAA G GAA G+P GAA G+NaNO3 GAA G+NH4Cl

0

10

20

30

40

50

0 24 48 72 96 120 144

Time (hours)

Dec

olou

risa

tion

(%

)

SGA G SGA G+P SGA G+NaNO3 SGA G+NH4Cl

0

10

20

30

40

50

0 24 48 72 96 120 144

Time (hours)

Dec

olou

risa

tion

(%

)

SAA G SAA G+P SAA G+NaNO3 SAA G+NH4Cl

A

B

C

D

peroxidases in melanoidin decolourisation as some studies(Aoshima et al., 1985; Hayase et al., 1984; Watanabe et al.,1982) have linked glucose supplementation to the intracel-lular hydrogen-peroxide (H2O2) producing sugar-oxidaseenzymes. The H2O2 produced, by the action of peroxidases,oxidizes melanoidin (Miyata et al., 1998; Hayase et al.,1984), thus causing its decolourisation.

The studies on the eVect of additional organic (peptone)or inorganic (NaNO3/NH4Cl) nitrogen sources on melanoi-din decolourisation along with glucose suggested that anadditional nitrogen source was unnecessary for melanoidindecolourisation. Nitrogen supplementation, particularlythe organic (peptone) source, resulted in luxuriant growth(A620–2.72) of the bacterial culture but it did not supportthe melanoidin decolourisation. This may be due to imbal-ance of the C/N ratio because the presence of excess nitro-gen, especially organic, becomes inhibitory for induction ofperoxidases which may resulted in decreased melanoidindecolourisation (Miyata et al., 2000; Kumar et al., 1997b;Ohmomo et al., 1988a).

The reduction in physico-chemical parameters of mela-noidin may be related to bacterial degradation of melanoi-dins in presence of supplementary carbon and nitrogensources through co-metabolism. Similar results by other

Fig. 2. Toxicity of synthetic melanoidins (A) before and (B) after bacterialdecolourisation to Tubifex tubifex (Müller) (¤p < 0.05).

0

20

40

60

80

100

24 48 72 96 120

Time (hours)

Mor

talit

y (%

)

GGA Pre-Decolourised GAA Pre-Decolourised

SGA Pre-Decolourised SAA Pre-Decolourised

A

* *

* **

* * *

*

*

*** *

*

0

20

40

60

80

100

24 48 72 96 120Time (hours)

Mor

talit

y (%

)

GGA Post-Decolourised GAA Post-Decolourised

SGA Post-Decolourised SAA Post-Decolourised

B

* *

***

P. Kumar, R. Chandra / Bioresource Technology 97 (2006) 2096–2102 2101

workers on Bacillus spp. have been previously reported(Roane et al., 2001; Nakajima-Kambe et al., 1999).

Mortality of tubifex worms serves as a sensitive endpoint to assess the eco-toxicological risk caused by the envi-ronmental pollutants in an aquatic eco-system (Cairns andNiederlehner, 1994). The results of the toxicity assessmentsuggested that untreated (raw) melanoidins caused nonspe-ciWc, toxic action on tubiWcid worms which could be due tothe high BOD and COD melanoidin values (Tables 1 and 2)and presence of such pollutants as phenol and sulphateformed as byproducts during melanoidin formation (Sibleyet al., 1997). The reduction in toxicity to tubiWcid wormsmay, thus, be due to evident reduction in BOD and CODvalues of decolourised melanoidins.

In conclusion, selected bacterial strains may possesssigniWcant potential to decolourise diVerent type of mela-noidins e.g., B. thuringiensis (MTCC 4714) (ITRC PIII)decolourising all four melanoidins while B. brevis (MTCC4716) (ITRC PIV) and Bacillus sp. (MTCC 6506) (ITRCPV) decolourising type GGA and type SGA more eVec-tively. Also, the developed mixed bacterial culture could beutilized for the decolourisation of natural melanoidins pres-ent in various industrial eZuents for their safe disposal intonearby soil and aquatic ecosystems.

Acknowledgement

The Wnancial assistance by DBT, Govt. of India, ishighly acknowledged.

References

Agarwal, C.S., Pandey, G.S., 1994. Soil pollution by spent wash discharge:depletion of manganese (II) and impairment of its oxidation. J. Envi-ron. Biol. 15, 49–53.

Aoshima, I., Tozawa, Y., Ohmomo, S., Udea, K., 1985. Production of deco-lourising activity for molasses pigment by Coriolus versicolor Ps4a.Agric. Biol. Chem. 49, 2041–2045.

APHA, 1998. In: Standard Methods for the Examination of Water andWaste Water, 20th ed. American Public Health Association, Washing-ton, DC.

ASTM, 1994. Standard guide for conducting sediment toxicity tests withfreshwater invertebrates. E 1383-94a. In: Annual Book of ASTM stan-dards. vol. 11.4. Philadelphia, pp. 1–30.

Binkley, N.W., Wolform, M.L., 1953. Composition of cane juice and caneWnal molasses. Adv. Carbohydrate Chem. 8, 291–314.

Cairns Jr., J., Niederlehner, B.R., 1994. Ecological Toxicity Testing. CRC,Boca Raton, Fla., USA.

Chandra, R., Pandey, P.K., 2001. Decolourisation of anaerobically treateddistillery eZuent by activated charcoal adsorption method. Ind. J.Environ. Prot. 2, 132–134.

Chandra, R., Singh, H., 1999. Chemical decolourisation of anaerobicallytreated distillery eZuent. Ind. J. Environ. Prot. 19 (11), 833–837.

Dahiya, J., Singh, D., Nigam, P., 2001. Decolourisation of synthetic andspentwash melanoidins using the white-rot fungus Phanerochaetechrysosoprium. Bioresour. Technol. 78 (1), 95–98.

Fahy, V., FitzGibbon, F.J., Macmulla, G., Singh, D., Marchant, R., 1997.Decolourisation of molasses spent wash by Phaenerochaete chrysospo-rium. Biotechnol. Lett. 19 (1), 97–99.

FitzGibbon, F.J., Nigam, P., Singh, D., Marchant, R., 1995. Biologicaltreatment of distillery waste for pollution-remediation. J. Basic Micro-biol. 35, 293–301.

Hayase, F., Kim, S.B., Kato, H., 1984. Decolourisation and degradationproducts of the melanoidin by hydrogen peroxide. Agric. Biol. Chem.48, 2711–2717.

Hayashi, T., Namiki, M., 1986. Role of sugar fragmentation in an earlystage browning of amino-carbonyl reaction of sugar with amino acid.Agric. Biol. Chem. 50 (8), 1965–1970.

Ichikawa, H., Taira, N., Wada, S., Tatsumi, K., 1996. Treatment of molas-ses waste water by ozonation and biological treatment. J. Jpn. Soc.Water Environ. (Mizu Kankyo Gakkaishi) 19, 1004–1008 (in Japa-nese).

Kannabiran, B., Pragasam, A., 1993. EVect of distillery eZuent on seedgermination, seedling growth and pigment content of Vigna mungo (L.)Hepper (C.V.T.9). Geobios 20, 108–112.

Kim, S.B., Hayase, F., Kato, H., 1985. Decolourisation and degradationproducts of melanoidins on ozonolysis. Agric. Biol. Chem. 49, 785–792.

Kumar, V., Wati, L., FitzGibbon, F.J., Nigam, P., Banat, I.M., Singh, D.,Marchant, R., 1997a. Bioremediation and decolourisation of anaerobi-cally digested distillery spent wash. Biotechnol. Lett. 19 (4), 311–313.

Kumar, V., Wati, L., Nigam, P., Banat, I.M., MacMullan, G., Singh, D.,Marchant, R., 1997b. Microbial decolourisation and bioremediation ofanaerobically digested molasses spent wash eZuent by aerobic bacte-rial culture. Microbios 89, 81–90.

Migo, V.P., Matsumura, M., Rosario, E.J.D., Kataoka, H., 1993. De-colourisation of molasses waste water using an inorganic Xocculant.J. Ferment. Bioeng. 75, 438–442.

Miyata, N., Iwahori, K., Fujita, M., 1998. Manganese independent anddependent decolourisation of melanoidin by extracellular hydrogenperoxide and peroxidases from Coriolus hirsutus pellets. J. Ferment.Bioeng. 85 (5), 550–555.

Miyata, N., Mori, T., Iwahori, K., Fujita, M., 2000. Microbial decolourisa-tion of melanoidin containing waste waters: combined use of activatedsludge and the fungus Coriolus hirsutus. J. Biosci. Bioeng. 89 (2), 145–150.

Murata, M., Terasawa, N., Homma, S., 1992. Screening of microorganismsto decolourise a model melanoidin and the chemical properties of amicrobially treated melanoidin. Biosci. Biotechnol. Biochem. 56, 1182–1187.

Nakajima-Kambe, T., Shimomura, M., Nomura, N., Chanpornpong, T.,Nakahara, T., 1999. Decolourisation of molasses wastewater by Bacil-lus sp. under thermophilic and anaerobic conditions. J. Biosci. Bioeng.87 (1), 119–121.

Ohmomo, S., Aoshima, I., Tozawa, Y., Sakurada, N., Kiyomoto, U., 1985a.PuriWcation and some properties of melanoidin decolourisationenzymes, PIII and PIV, from mycelia of Coriolus versicolor Ps4a. Agric.Biol. Chem. 49 (7), 2047–2053.

Ohmomo, S., Itoh, N., Wantanabe, Y., Kaneko, Y., Tozawa, Y., Udea, K.,1985b. Continuous decolourisation of molasses waste water with myce-lia of Coriolus versicolor Ps4a. Agric. Biol. Chem. 49 (9), 2551–2555.

Ohmomo, S., Kaneko, Y., Sirianantapiboon, S., Somachi, P., Attha-sampunna, P., Nakamura, I., 1987. Decolourisation of molasses wastewater by a thermophilic strain Aspergillus fumigatus G-2-6. Agric. Biol.Chem. 52 (12), 3339–3346.

Ohmomo, S., Kainuma, M., Kamimura, K., Sirianuntapiboon, S., Aoshima,I., Atthasampunna, P., 1988a. Adsorption of melanoidin to the myceliaof Aspergillus oryzae Y-2-32. Agric. Biol. Chem. 52 (2), 381–386.

Ohmomo, S., Daengasubha, W., Yoshikawa, H., Yui, M., Nozaki, K.,Nakajima, T., Nakamura, I., 1988b. Screening of anaerobic bacteriawith the ability to decolourise molasses melanoidin. Agric. Biol. Chem.52 (10), 2429–2435.

Paturau, J.M., 1982. Sugar Series 3. By Products of the Cane-Sugar Indus-try. Elsevier ScientiWc Publication Company, Amsterdam, The Nether-lands ( Chapter 9).

Popovic, B., Popovic, M., 1977. EVects of heavy metals on survival and res-piration rate of tubiWcid worms: Part I—eVects on survival. Environ.Pollut. 13, 65–72.

Rathore, R., Khangarot, B.S., 2003. EVects of water hardness and metalconcentration on a freshwater Tubifex tubifex Muller. Wat. Air SoilPollut. 142, 341–356.

2102 P. Kumar, R. Chandra / Bioresource Technology 97 (2006) 2096–2102

Reynolds, T.M., 1968. Chemistry of nonenzymatic browning. I. The reac-tion between aldoses and amines. Adv. Food. Res. 12, 1–52.

Roane, T.M., Josephson, K.L., Pepper, I.L., 2001. Dual bioaugmentationstrategy to enhance remediation of contaminated soil. Appl. Environ.Microbiol. 67, 3208–3215.

Sibley, P.K., Leger, J., Dixon, D.G., Barton, D.R., 1997. Environmentalhealth assessment of the benthic habitat adjacent to a pulp milldischarge. I. Acute and chronic toxicity of sediments to benthicmacroinvertibrates. Arch. Environ. Contam. Toxicol. 32 (3), 274–284.

Sirianuntapiboon, S., Phothilangka, P., Ohmomo, S., 2004. Decolourisa-tion of molasses wastewater by a strain no. BP103 of acetogenic bacte-ria. Bioresour. Technol. 92 (1), 31–39.

Watanabe, Y., Sugi, R., Tanaka, Y., Hayashida, S., 1982. Enzymatic deco-lourization of melanoidin by Coriolus sp. no. 20. Agric. Biol. Chem. 46,1623–1630.

Wedzicha, B.L., Kaputo, M.T., 1992. Melanoidins from glucose and gly-cine: composition, characteristics and reactivity towards sulphite-ion.Food chem. 43, 359–367.