Decolorization of molasses wastewater by yeast strain, Issatchenkia orientalis No. SF9-246

Among 2402 strains of yeast isolated from various sources in Thailand, a strain No. SF9-246 identified as Issatchenkia orientalis, showed the highest potential for use in decolorization of molasses wastewater. In a malt extract-glucose-peptone broth (MYGP) culture containing melanoidin pigment (MP) at 30 °C a 60.2% decolorization was obtained within 7 days. The strain appeared to enhance both MP-degradation and MP-adsorption. The strain showed MP, chemical oxygen demand (COD) and biological oxygen demand (BOD₅) removal efficiencies of 91.2%, 80.0% and 77.4%, respectively from anaerobic-treated molasses wastewater solution (T-MWW), collected from an anaerobic pond. The wastewater contained 2.5% glucose, 0.1% NH₄Cl, and 0.1% KH₂PO₄. The pH was adjusted to 5.0 at 30 °C for 7 days batch type culture system. The strain showed almost constant decolorization yield of 75–80% over 7 days in a periodical feeding system of 10% fresh T-MWW with the culture system. The strain provided a constant decolorization yield about 70% during 3 replacement cycles. Gel filtration chromatography showed that larger molecular weight fraction of MP solution was rapidly removed, while the smaller molecular weight fraction remained in the effluent.     

Decolorization of a recalcitrant organic compound (Melanoidin) by a novel thermotolerant yeast, Candida tropicalis RG-9

ABSTRACT: BACKGROUND: Sugarcane distilleries use molasses for ethanol production and generate large volume ofeffluent containing high biological oxygen demand (BOD) and chemical oxygen demand(COD) along with melanoidin pigment. Melanoidin is a recalcitrant compound that causesseveral toxic effects on living system, therefore, may be treated before disposal. The aim ofthis study was to isolate a potential thermotolerant melanoidin decolorizing yeast fromnatural resources, and optimized different physico-chemical and nutritional parameters. RESULTS: Total 24 yeasts were isolated from the soil samples of near by distillery site, in which isolateY-9 showed maximum decolorization and identified as Candida tropicalis by Microbial TypeCulture Collection (MTCC) Chandigarh, India. The decolorization yield was expressed as thedecrease in the absorbance at 475 nm against initial absorbance at the same wavelength.Uninoculated medium served as control. Yeast showed maximum decolorization (75%) at 45°C using 0.2%, glucose; 0.2%, peptone; 0.05%, MgSO4; 0.01%, KH2PO4; pH-5.5 within 24 hof incubation under static condition. Decolorizing ability of yeast was also confirmed by highperformance liquid chromatography (HPLC) analysis. CONCLUSION: The yeast strain efficiently decolorized melanoidin pigment of distillery effluent at highertemperature than the other earlier reported strains of yeast, therefore, this strain could also beused at industrial level for melanoidin decolorization as it tolerated a wide range oftemperature and pH with very small amount of carbon and nitrogen sources.     

Enzymatic Decolorization of Melanoidin by Coriolus sp. No. 20

Coriolus sp. No. 20 decolorized a melanoidin solution, a decrease of about 80% in darkness under the optimal conditions. This decolorization occurred with an intracellular enzyme which was prepared from an extract of integrated mycelia, and required aeration and some kinds of sugars, particularly glucose and sorbose. The fraction with melanoidin-decolorizing activity was collected and purified by DEAE-cellulose and Sephadex G-200 column chromatographies. The optimal pH and temperature were pH 4.5 and 35°C, respectively. The molecular weight was found to be about 200,000 by SDS-gel electrophoresis. The purified enzyme was identified as sorbose oxidase; decolorization proceeded in the presence of oxygen and sugars such as maltose, sucrose, lactose, galactose and xylose, besides glucose and sorbose. Glucose in the reaction mixture was converted to gluconic acid. Melanoidin was suggested to be decolorized by the active oxygen formed.     

Decolorization of anaerobically digested molasses spent wash by <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

The distillery wastewater (spent wash) contains dark brown colored recalcitrant organic compounds that are not amenable to conventional biological treatment. The characteristic recalcitrance to decolorization is due to the presence of brown melanoidin polymers. In the present study, feasibility of using Pseudomonas putida putida strain U for decolorization of spent wash was demonstrated. Batch cultures of P. putida decolourized spent wash by 24%, twofold higher decolorization was achieved following immobilization in calcium alginate beads. Glucose concentration was critical for decolourization, and improved color removal efficiency was obtained by periodic replenishment of glucose. Decolourization was also observed with lactose or whey as alternative carbon sources. The results of our study suggest that P.putida could be used for biological decolorization of molasses spent washes and that supplementation with whey (a by-product from cheese industry) can offer economical viability to the process. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2009, Vol. 45, No. 1, pp. 78–83. The text was submitted by the authors in English.     

A new alternative process for Kraft E1 effluent treatment

Lentinus edodes (UEC-2019 strain) was selected after screening 51 ligninolytic strains of fungi for their ability to decolorize phenolic industrial effluent with high content of lignin peroxidases, Mn-peroxidases and beta-glucosidases. This strain removed 73 % of color in theEucalyptus Kraft E1 effluent in 5 days without any additional carbon sources. A 13% mycelial adsorption was found. Correlation between mass loss, COD, TOC and decolorization was observed. When an effluent pre-irradiated (10 min) in the presence of ZnO was treated withL. edodes, a marked enhancement of the decolorization at 48 h was obtained.L. edodes is an active fungus in this pre-treatment and biobleaching process. The combined photo-biological decolorization procedure appears to be an efficient decontamination method with great potential in industrial effluent treatment.Abbreviation COD Chemical oxygen demand - TOC Total organic carbon     

Production of Decolorizing Activity for Molasses Pigment by Coriolus versicolor Ps4a

The distribution of molasses pigment (melanoidin) decolorizing activity (MDA) was investigated in various Basidiomycetes. MDA was only found in some genera of the white-rot-fungi group of which Coriolus versicolor Ps4a showed high activity, a decolorization yield of approximately 80% under the optimal conditions. Production of MDA by C. versicolor was almost completely coincident with the growth of mycelia. The main MDA was due to intracellular enzymes and induced by the molasses pigment. The induced enzyme consisted of two types, namely a sugar dependent enzyme and a sugar independent enzyme. The decolorization by C. versicolor was due to the decomposition of the molasses pigment.     

A Red,Fluorescent Protein from Silkworm

Nondiffusable melanoidin obtained from a glycine-xylose system was heated in aqueous media, and the resulting chemical changes, as affected by the presence of oxygen, pH value, temperature and the addition of transitional metals, were investigated.

Melanoidin, when heated at 90°C in an aqueous solution, caused remarkable discolorization accompanied by the development of fluorescence, oxygen consumption and a noticeable variation of reductone content. Heated melanoidin became polydispersive in molecular weight on gel filtration chromatograms. There appeared reductones, ninhydrin-positive substances, fluorescent substances, aromatic amines, aliphatic carbonyls, and aliphatic primary amines and/or methylene groups in diffusâtes of melanoidin heated in various media.

An increase in pH value favored oxidative discolorization, while an increase in the concentration of transitional metals except Mn²⁺ restrained the discolorization. In the absence of oxygen, heated melanoidin brought about a slight strengthening of color and the accumulation of reductones ca. fifteen times more than the initial level, while in the presence of oxygen the increase of reductone content at the early period was followed by a rapid decrease.

According to the results obtained, the ambivalent reactivity of melanoidin, i.e. polymerization (colorization) and depolymerization (discolorization), was discussed in relation to influencing factors. A mechanism for the production of reductones in heated melanoidin was also proposed.     

Purification and Some Properties of Melanoidin Decolorizing Enzymes,P-III and P-IV,from Mycelia of Coriolus versicolor Ps4a

Melanoidin decolorizing enzymes (MDE) were extracted from mycelia of Coriolus versicolor Ps4a and purified by DEAE-Sephadex, DEAE-Sephacel and Sephadex G-200 column chromatographies. MDE of this strain consisted of a main fraction, P-fraction, and a minor fraction, E-fraction, and the P-fraction was composed of at least five enzymes. P-III and P-IV in the P-fraction were picked as typical enzymes of this strain, and their enzymatic properties were investigated. P-III had a molecular weight of 48,400 ~ 50,000, an optimum pH of 5.5 and an optimum temperature of 30~35°C. P-III required glucose and 02 for the appearance of the activity, and was inhibited by p-CMB, N-BSI, Ag⁺ and o-phenanthroline.

On the other hand, P-IV had a molecular weight of 43,800 ~ 45,000, an optimum pH of 4.0~4.5 and an optimum temperature of 30~35°C. P-IV could decolorize melanoidin in the absence of glucose and O₂, and was inhibited weakly by Ag⁺, p-CMB and N-BSI. P-IV is the enzyme that attacks the melanoidin directly in comparison with P-III which attacks melanoidin indirectly as in the sub-reaction of sugar oxidase.

Incidentally, a multiplicative effect between P-III and P-IV for decolorization was observed.     

Continuous Decolorization of Molasses Waste Water with Mycelia of Coriolus versicolor Ps4a

Continuous decolorization of molasses waste water by mycelia of Coriolus versicolor Ps4a was studied using waste water from a baker’s yeast factory, treated by means of methane fermentation and with activated sludge. Optimum decolorization with bare pellet-type mycelia in shaking flasks needed the addition of glucose (0.5%) and peptone (0.05%) and aerobic conditions (1ppm of dissolved oxygen). Continuous decolorization in a bubbling column reactor showed a decolorization yield of approximately 75% in only 20 hr at a dilution rate (D) of 0.03 hr^?1 under the optimum conditions.

In order to continue the decolorization for a longer time, mycelia immobilized within Caalginate gel were tested in a bubbling column reactor under the optimum conditions. The immobilized mycelia showed an almost constant decolorization yield (65.7%) during continuous decolorization for 16 days at D = 0.22 hr^?1.     

Decolorization and treatment of Kokuto-shochu distillery wastewater by the combination treatment involving biodecolorization and biotreatment by Penicillium oxalicum d, physical decolorization by ozonation and treatment by activated sludge

Kokuto-shochu is a traditional Japanese distilled liquor made from brown sugar. Kokuto-shochu distillery wastewater (KDW) contains high concentrations of organic compounds and brown pigments (called molasses pigments) which are hardly decolorized by general biological wastewater treatment. A fungus, Penicillium oxalicum d, which we isolated in a previous study, decolorizes 47% of the color from KDW without the addition of any nutrients. P. oxalicum d decolorizes KDW by absorbing the pigments into its mycelia. Here we describe a KDW treatment system that combines biodecolorization and biotreatment by P. oxalicum d with treatment by activated sludge and physical decolorization by ozonation. Adding HClO to suppress bacterial growth and replacing fresh seed sludge at regular intervals helped to maintain the dominance and decolorization ability of P. oxalicum d. In a laboratory-scale demonstration, 48 cycles (12 days) achieved a decolorization ratio of 90% and removed more than 97% of dissolved organic carbon (DOC), dissolved total nitrogen (DTN) and dissolved total phosphorus (DTP). A major feature of our system is that it uses only 6% of the water used in an activated sludge-ozonation system.     

Bioremediation Perspective of Navy Blue Rx–Containing Textile Effluent by Bacterial Isolate

The aim of the present study was to investigate the textile effluent degrading potential of an isolated bacterium, Proteus sp. SUK7. The strain had the capacity to decolorize Navy Blue Rx–containing textile effluent up to 83% within 96 h. The maximum decolorization was observed under static conditions at pH 7.0 and 30°C. Reduction in the chemical oxygen demand (COD) and biological oxygen demand (BOD) of textile effluent was observed after treatment with Proteus sp. SUK7. Induction in the activities of laccase and aminopyrine N-demethylase was observed after decolorization, which indicates involvement of these enzymes in the decolorization process. The presence of various inducers was also found to have a modulatory effect on enzyme activities and the decolorization process. Biodegradation was confirmed using various analytical techniques, such as ultraviolet-visible (UV-Vis) spectroscopy, Fourier transform infrared (FTIR), gas chromatography–mass spectrometry (GC-MS), and high-performance liquid chromatography (HPLC). A phytotoxicity study was performed to confirm the nontoxic nature of the degradation metabolites.     

Decolorization of dye and molasses by continuous and semi-continuous jar-fermentor cultures ofGeotrichum candidum Dec 1

Two culture modes, continuous and semi-continuous, of the decolorization fungus,Geotrichum candidum Dec 1, were compared to obtain a high treatment efficiency of molasses decolorization and a large productivity of peroxidase (DyP) to simultaneously decolorize dyes and molasses. The continuous culture ofG. candidum Dec 1 using a 5-l jar-fermentor showed high DyP activity at a low dilution ratio of 0.005h⁻¹, and decolorization ratio of molasses of 80% was obtained concomitantly. Therefore, a semi-continuous culture was performed by repeated refill and draw. In this mode, approximately 1.5 liters of the culture broth was replaced per cycle when the decolorization ratio of molasses was near 80%. The molasses medium (1.0 liter per day) was treated and the peroxidase productivity in the drawn culture broth was 26.6 U/day, whereas the peroxidase productivity was 17.9 U/day in the continuous culture with a dilution rate of 0.005 h⁻¹. The semi-continuous treatment system was an efficient decolorization method for the strain,G. candidum Dec 1.     

Screening of Microorganisms to Decolorize a Model Melanoidin and the Chemical Properties of a Microbially Treated Melanoidin

A microbe of Streptomyces werraensis TT 14, which was newly isolated from soil, decolorized the model melanoidin prepared from glucose and glycine, the decolorization rate being 64% in the optimal medium of pH 5.5 (2.0% starch, 1.0% yeast, 0.3% NaCl, and 0.3% CaC0₃) and 45% in a synthetic medium. There was virtually no difference in the UV-VIS absorption spectra of the microbially treated melanoidin and the control. The peaks of the gel permeation chromatogram for the treated melanoidin and for the control showed the same retention times, but lower molecular weight compounds increased in the decolorized melanoidin. The Cu(II)-chelating activity of the decolorized melanoidin was reduced to about half that of the control. The melanoidin component of pI 2.5 was increased and that of pI 3.5 was reduced by the microbial treatment.     

Fractionation of Nondialyzable Melanoidin into Components by Electrofocusing Electrophoresis

Nondialyzable melanoidin formed in a model system of glucose and glycine was applied on thin layer gel electrofocusing in a polyacrylamide gel. The electrofocusing profile differed according to the reaction time: the pI of the melanoidin formed in the early stage of the reaction was less than 2.9 and, as the reaction progressed, the pI of the melanoidin formed gradually shifted to a less acidic value, from 2.9 to 3.3. Those from the xylose and glycine model system gave a similar profile to that of the glucose system.

Preparative separation of the nondialyzable melanoidin, which was formed by heating the glucose system for 7hr, was performed by flat-bed electrofocusing in Sephadex gel. At least 14 melanoidin bands were clearly electrofocused at a pH range of 2.7-3.3 and about 59% of the melanoidin applied remained unaffected by electrophoresis at the starting position. The major component of electrofocused melanoidin was pI 3.00, this being made up of 12.5% of the total amount of electrofocused melanoidin. The molecular weight of melanoidin affected by electrophoresis was about 25,000, regardless of the pI value of the melanoidin components. The reducing activity, estimated by the potassium ferricyanide method, showed that the lower the pI of melanoidin, the higher was the reducing activity.

The addition of hydrochloric acid to the melanoidin solution caused it to gradually become viscous and the melanoidin was precipitated below pH 3.0-3.5, corresponding approximately to ampholite. This feature can be used as a method to prepare nondialyzable melanoidin in a short time.     

Decolorization of molasses spent wash by the white-rot fungus Flavodon flavus, isolated from a marine habitat

Flavodon flavus (Klotzsch) Ryvarden, a basidiomycete (NIOCC strain 312) isolated from decomposing leaves of a sea grass, decolorized pigments in molasses spent wash (MSW) by 80% after 8 days of incubation, when used at concentrations of 10% and 50%. Decolorizing activity was also present in media prepared with half-strength seawater (equivalent to 15 ppt salinity). Decolorizing activity was seen in low-nitrogen medium, nutrient-rich medium and in sugarcane bagasse medium. The percentage decolorization of MSW was highest when glucose or sucrose was used as the carbon source in the low-nitrogen medium. The production of lignin-modifying enzymes, manganese-dependent peroxidase (MNP) and laccase decreased in a medium containing MSW. MNP production and MSW decolorization were inversely correlated, suggesting no role for MNP in MSW decolorization. The decolorization of MSW was not effective when F. flavus was immobilized in calcium alginate beads. Decolorization was achieved best in oxygenated cultures. Besides color, total phenolics and chemical oxygen demand were reduced by 50% in MSW treated with F. flavus, suggesting its potential in the bioremediation of effluents.     

Degradation of Remazol Red dye by <Emphasis Type="Italic">Galactomyces geotrichum</Emphasis> MTCC 1360 leading to increased iron uptake in <Emphasis Type="Italic">Sorghum vulgare</Emphasis> and <Emphasis Type="Italic">Phaseolus mungo</Emphasis> from soil

Removal of azo dyes from the effluent generated by textile industries is rather difficult. Azo dyes represent a major class of synthetic colorants that are both mutagenic and carcinogenic. Galactomyces geotrichum MTCC 1360, a yeast species, showed more than 96% decolorization of the azo dye Remazol Red (50 mg/L) within 36 h at 30°C and pH 11.0 under static condition with a significant reduction in the chemical oxygen demand (62%) and total organic carbon (41%). Peptone (5.0 g/L), rice husk (10 g/L extract), and ammonium chloride (5.0 g/L) were found to be more significant among the carbon and nitrogen sources used. The presence of tyrosinase, NADH-DCIP reductase, riboflavin reductase and induction in azo reductase and laccase activity during decolorization indicated their role in degradation. High performance thin layer chromatography analysis revealed the degradation of Remazol Red into different metabolites. Fourier transform infrared spectroscopy and high performance liquid chromatography analysis of samples before and after decolorization confirmed the biotransformation of dye. Atomic absorption spectroscopy analysis revealed a less toxic effect of the metabolites on iron uptake by Sorghum vulgare and Phaseolus mungo than Remazol Red dye. Remazol Red showed an inhibitory effect on iron uptake by chelation and an immobilization of iron, whereas its metabolites showed no chelation as well as immobilization of iron. Phytotoxicity study indicated the conversion of complex dye molecules into simpler oxidizable products which had a less toxic nature.     

Ecofriendly degradation of sulfonated diazo dye C.I. Reactive Green 19A using Micrococcus glutamicus NCIM-2168

Micrococcus glutamicus NCIM-2168 exhibited complete decolorization and degradation of C.I. Reactive Green 19A (an initial concentration of 50 mg l⁻¹) within 42 h at temperature 37 °C and pH 8, under static condition. Extent of mineralization was determined with total organic carbon (TOC) and chemical oxygen demand (COD) measurement, showing a satisfactory reduction of TOC (72%) and COD (66%) within 42 h. Enzyme studies shows involvement of oxidoreductive enzymes in decolorization/degradation process. Analytical studies of the extracted metabolites confirmed the significant degradation of Reactive Green 19A into various metabolites. The microbial toxicity and phytotoxicity assay revealed that the degradation of Reactive Green 19A produced nontoxic metabolites. In addition, the M. glutamicus strain was applied to decolorize a mixture of ten reactive dyes showing a 63% decolorization (in terms of decrease in ADMI value) within 72 h, along with 48% and 42% reduction in TOC and COD under static condition.     

A sequential aerobic/microaerophilic decolorization of sulfonated mono azo dye Golden Yellow HER by microbial consortium GG-BL

This study is a part of efforts to develop new batch method with the help of prepared consortium GG-BL using two microbial cultures viz. Galactomyces geotrichum MTCC 1360 and Brevibacillus laterosporus NCIM 2298, varying oxidation conditions for the bio-treatment processes to produce reusable water by decolorization of Golden Yellow HER (GYHER) to less toxic metabolites. Consortium was found to be much faster for decolorization and degradation of GYHER as compared to the individual strains. The intensive metabolic activity of these strains led to 100% decolorization of GYHER (50 mg l⁻¹) within 24 h with significant reduction in chemical oxygen demand (84%) and total organic carbon (63%). The presence of veratryl alcohol oxidase, NADH-DCIP reductase and induction in laccase, tyrosinase, azo reductase and riboflavin reductase during decolorization suggests their role in decolorization process. Substrate staining of nondenaturing polyacrylamide electrophoresis gel (PAGE) also confirms induction of oxidative enzymes during GYHER degradation. The degradation of the GYHER into different metabolites by individual organism and in consortium was confirmed using High Performance Thin Layer Chromatography (HPTLC), High Performance Liquid Chromatography (HPLC), Fourier Transform Infra Red Spectroscopy (FTIR), Gas Chromatography Mass Spectroscopy (GC–MS) analysis. Phytotoxicity studies revealed nontoxic nature of the metabolites of GYHER.     

Decolorization of molasses wastewater by Lactobacillus plantarum No. PV71-1861

Lactobacillus plantarum No. PV71-1861, isolated from pickle samples in Thailand, showed the high potential for use in decolorization of molasses wastewater under both anaerobic and facultative (static) conditions. The strain showed the highest melanoidin pigment (MP) decolorization yield of 68.12% with MP solution (color intensity corresponding to an optical density of 3.5 units at 475 nm) containing 2% glucose, 0.4% yeast extract, 0.1% KH(2)PO(4), 0.05% MgSO(4).7H(2)O and initial pH of 6 under static condition at 30 degrees C within 7 days. But, it showed low growth and MP decolorization yields under aerobic conditions. Gel filtration chromatograms of the MP solutions showed that the small molecular weight fraction of MP solution was decolorized by the strain when the large molecular weight fraction still remained in the effluent. For application, the strain could apply to treat anaerobic treated-molasses wastewater (T-MWW) with high removal efficiency. The highest MP removal efficiencies and growth yield of 76.6% and 2.6 mg/mL, respectively, were observed with the T-MWW within 7 days of culture, and the effluent pH of the system was decreased to lower than 4.0 after 2-3 days operation.     

Degradation and decolorization of monosodium glutamate wastewater with <Emphasis Type="Italic">Coriolus versicolor</Emphasis>

Degradation and decolorization of monosodium glutamate wastewater (MSGW) with Coriolus versicolor were firstly carried out. The effects of various operation parameters namely wastewater concentrations, pH, culture time and incidence of sterilization on maximum percentage of degradation and decolorization of wastewater were investigated. Studies of mycelium and enzyme for C. versicolor degradation and decolorization were estimated in this study. Ten percentage of wastewater concentration and pH = 5.0 were found to be the most suitable ones among the other experiments. The highest degradation and decolorization efficiency of wastewater was obtained at the fifth day of cultivation, which was displayed with more than 70% chemical oxygen demand removal, 83% total sugar removal and 55% color removal, respectively. Sterile operation had no remarkable effect on the degradation and decolorization efficiency for C. versicolor. Mycelium and the extra cellular fungal enzyme were both necessary for the degradation and decolorization of MSGW. C. versicolor possesses great potential and economic advantages in MSGW treatment.