Isolation and characterization of distillery spent wash color reducing bacteria and process optimization by Taguchi approach

The ability of five different types of bacterial strains isolated from a distillery mill site was analyzed for decolorization of distillery spent wash. 16S rDNA based denaturing gradient gel electrophoresis (DGGE) and amplified random DNA restriction analysis (ARDRA) were used to characterize the bacterial strains. One of the isolates had higher capability to reduce color (21%) and chemical oxygen demand (COD) (30%) was finally identified by 16S rDNA sequence analysis as Bacillus sp. Different parameters such as pH, temperature, aeration,% carbon,% nitrogen, inoculum size and incubation time were optimized by the Taguchi approach to achieve maximum decolorization of distillery spent wash by the Bacillus sp. Reduction in color (85%) and COD (90%) was observed within 12 h after optimization by the Taguchi method. The significant factor in the optimization process was duration followed by inoculum size to attain maximum color reduction. The Taguchi approach proved to be a reliable tool in optimizing culture conditions and analyzing interaction effects of process parameters in achieving the best possible combination for maximum decolorization of the distillery spent wash.     

Isolation and characterization of alkalotolerant bacteria and optimization of process parameters for decolorization and detoxification of pulp and paper mill effluent by Taguchi approach

Four different bacterial strains were isolated from pulp and paper mill sludge in which one alkalotolerant isolate (LP1) having higher capability to remove color and lignin, was identified as Bacillus sp. by 16S RNA sequencing. Optimization of process parameters for decolorization was initially performed to select growth factors which were further substantiated by Taguchi approach in which seven factors, % carbon, % black liquor, duration, pH, temperature, stirring and inoculum size, at two levels, applying L-8 orthogonal array were taken. Maximum color was removed at pH 8, temperature 35°C, stirring 200 rpm, sucrose (2.5%), 48 h, 5% (w/v) inoculum size and 10% black liquor. After optimization 2-fold increase in color and lignin removal from 25–69% and 28–53%, respectively, indicated significance of Taguchi approach in decolorization and delignification of lignin in pulp and paper mill effluent. Enzymes involved in the process of decolorization of effluent were found to be xylanase (54 U/ml) and manganese peroxidase (28 U/ml). Treated effluent was also evaluated for toxicity by Comet assay using Saccharomyces cerevisiae MTCC 36 as model organism, which indicated 58% reduction after treatment by bacterium.     

Decolorization of phenolic effluents by soluble and immobilized phenol oxidases

Summary Colour removal from phenplic industrial effluents by phenol oxidase enzymes and white-rot fungi was compared. Soluble laccase and horseradish peroxidase (HRP) removed colour from pulp mill (E), cotton mill hydroxide (OH) and cotton mill sulphide (S) effluents, but rapid and irreversible enzyme inactivation took place. Entrapment of laccase in alginate beads improved decolorization by factors of 3.5 (OH) and 2 (E); entrapment of HRP improved decolorization by 36 (OH), 20 (E) and 9 (S). Beads were unsuitable for continuous use because the enzymes were rapidly released into solution. Co-polymerization of laccase or HRP with L-tyrosine gave insoluble polymers with enzyme activity. Entrapment of the co-polymers in gel beads further increased the efficiency of decolorization of E by 28 (laccase) and by 132 (HRP) compared with soluble enzymes. Maximum decolorization of all three effluents by batch cultures of Coriolus versicolor (70%–80% in 8 days) was greater than the maximum enzymic decolorization (48% of OH in 3 days by entrapped laccase). Soluble laccase (222 units ml^–1) precipitated 1.2 g l^–1 phenol from artificial coal conversion effluent at pH 6.0 and the rate of precipitation and enzyme inactivation was faster at pH 6.0 than at pH 8.5.Offprint requests to: R. G. Burns     

Biodegradation of Cyanide by a White Rot Fungus, Trametes versicolor

The cyanide degradation abilities of three white rot fungi, Trametes versicolor ATCC 200801, Phanerochaete chrysosporium ME 496 and Pleurotus sajor-caju, were examined. T. versicolor was the most effective with 0.35 g dry cell/100 ml degrading 2 mm KCN (130 mg/l) over 42 h, at 30°C, pH 10.5 with stirring at 150 rpm.     

Biodegradation of disperse dye brown 3REL by microbial consortium of Galactomyces geotrichum MTCC 1360 and Bacillus sp. VUS

The consortium-GB (Galactomyces geotrichum MTCC 1360 and Bacillus sp. VUS) exhibited 100% decolorization ability with the dye Brown 3REL within 2 h at shaking condition with optima of pH 7 and at 50°C. However, G. geotrichum MTCC 1360 showed 39% decolorization within 24 h and Bacillus sp. VUS took 5 h for 100% decolorization, when incubated individually. Additional carbon and nitrogen sources like, starch, peptone, and urea were found to enhance decolorization. Induction in lignin peroxidase, tyrosinase, and riboflavin reductase was observed in consortium as that of individual organisms. GCMS identification showed different metabolites formed using consortium (2-(6,8-dichloro-quinazolin-4yloxy)-acetyl-urea and 2-(6,8-dichloro-quinazolin-4yloxy)-acetyl-formamide) and Bacillus sp. VUS (6,8-dichloro-4 methoxy-quinazoline) after 2 h of incubation with Brown 3REL. G. geotrichum MTCC 1360 showed minor modifications in structure of Brown 3REL. Phytotoxicity revealed non toxic nature of metabolites. This consortium-GB was also able to decolorize various industrial dyes.     

Production of Laccase and Optimization of Its Production by Bacillus sp. Using Distillery Spent Wash as Inducer

A bacillus sp. isolated from the sediments of a distillery mill was used for laccase production under optimized culture conditions. The distillery effluent was used as an inducer for overproduction of laccase by employing the Taguchi approach. Screening of different medium components and their effect on laccase production was studied using an M-16 orthogonal array. The formation of laccase was considerably increased by addition of 1 mM copper sulfate (51.95 U/ml), which was further enhanced by the use of different inducers. The usefulness of the Taguchi method for optimization of culture conditions was investigated with five selected factors at four levels, and it was observed that the optimized medium resulted in a 9-fold increase in extracellular laccase production compared with the control. The optimized medium composition for laccase production was dextrose (1%), tryptone (0.1%), CuSO₄ (1 mM), and an inducer (distillery effluent 10% [v/v]) at pH 7, which altogether resulted in 107.32 U/ml extracellular laccase activity. Hence, the Taguchi approach proved to be a reliable tool in optimizing culture conditions and achieving the best possible combination for enhanced laccase production.     

Isolation and characterization of aerobic bacteria capable of the degradation of synthetic and natural melanoidins from distillery effluent

Melanoidins, complex biopolymer of amino-carbonyl compounds are the major coloring and polluting constituents of distillery wastewaters. In this study, three aerobic melanoidin-degrading bacteria (RNBS1, RNBS3 and RNBS4) were isolated from soil contaminated with distillery effluent and characterized as Bacillus licheniformis (RNBS1), Bacillus sp. (RNBS3) and Alcaligenes sp. (RNBS4) by biochemical tests and 16S rRNA gene sequence analysis. The degradation of synthetic and natural melanoidins was studied by using the axenic and mixed bacterial consortium. Results have revealed that the mixed consortium was more effective compared to axenic culture decolorizing 73.79 and 69.83% synthetic and natural melanoidins whereas axenic cultures RNBS1, RNBS3 and RNBS4 decolorized 65.88, 62.56 and 66.10% synthetic and 52.69, 48.92 and 59.64% natural melanoidins, respectively. The HPLC analysis of degraded samples has shown reduction in peak areas compared to controls, suggesting that decrease in color intensity might be largely attributed to the degradation of melanoidins by isolated bacteria.     

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.     

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     

Isolation of a new flavonol glycoside and its effects on the blue color of seed coats ofOphiopogon jaburan

From the blue seed coats ofOphiopogon jaburan, a new flavonol glycoside was isolated as needles and determined to be kaempferol 3-O-β-d-galactoside-4′-O-β-d-glucoside (OK-2) by UV and NMR spectral analyses. OK-2 and kaempfrol 3, 4′-di-O-β-d-glucoside (OK-1), which was detected previously, in the blue seed coat were present in a molar ratio of about 13:7. OK-2 was newly found as a factor causing the blueing effects on ophionin which is a main anthocyanin in the blue seed coats. The mixture of 4.8×10⁻³ M OK-2 and 2.5×10⁻³ M ophionin in Mcllvaine's buffer solution (pH 5.6) showed stable blue color, and the absorption spectrum of the mixture showed two absorption peaks and a shoulder in visible reasion, coinciding with that of the fresh blue seed coat. The effect of ophionin and OK-2 co-pigmentation on the blue color of seed coat ofO. jaburan was discussed.     

Purification and characterization of laccase from <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> and decolorization of an anthraquinone dye by the enzyme

The white rot fungus Pycnoporus sanguineus produced high amount of laccase in the basal liquid medium without induction. Laccase was purified using ultrafiltration, anion-exchange chromatography, and gel filtration. The molecular weight of the purified laccase was estimated as 61.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme oxidized typical substrates of laccases including 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate), 2,6-dimethoxyphenol, and syringaldazine. The optimum pH and temperature for the purified laccase were 3.0 and 65°C, respectively. The enzyme was stable up to 40°C, and high laccase activity was maintained at pH 2.0–5.0. Sodium azide, l-cysteine, and dithiothreitol strongly inhibited the laccase activity. The purified enzyme efficiently decolorized Remazol Brilliant Blue R in the absence of added redox mediators. The high production of P. sanguineus laccase as well as its decolorization ability demonstrated its potential applications in dye decolorization.     

Enhanced ligninolytic enzyme production and degrading capability of Phanerochaete chrysosporium and Trametes versicolor

Ligninolytic enzyme production by the white-rot fungi Phanerochaete chrysosporium and Trametes versicolor precultivated with different insoluble lignocellulosic materials (grape seeds, barley bran and wood shavings) was investigated. Cultures of Phanerochaete chrysosporium precultivated with grape seeds and barley bran showed maximum lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP) activities (1000 and 1232 U/l, respectively). Trametes versicolor precultivated with the same lignocellulosic residues showed the maximum laccase activity (around 250 U/l). For both fungi, the ligninolytic activities were about two-fold higher than those attained in the control cultures. In vitro decolorization of the polymeric dye Poly R-478 by the extracellular liquid obtained in the above-mentioned cultures was monitored in order to determine the respective capabilities of laccase, LiP and MnP. It is noteworthy that the degrading capability of LiP when P. chrysosporium was precultivated with barley bran gave a percentage of Poly R-478 decolorization of about 80% in 100 s, whereas control cultures showed a lower percentage, around 20%, after 2 min of the decolorization reaction.     

Selection of salt-tolerant Rhizobium isolates of Acacia nilotica

Among 35 Rhizobium isolates of Acacia nilotica, from different agro-climatic zones, two, ANG4 and ANG5, tolerated up to 850 mm NaCl and one, ANG3, was sensitive to NaCl above 250 mm. Nodulation and nitrogenase activity of the three isolates decreased with increasing concentration of salt up to 150 mm. Nodulation by ANG3 was 15% at 75 mm NaCl and nil at 100 mm. With ANG4 and ANG5, nodulation was only slightly decreased at 150 mm NaCl. Nitrogenase activity associated with plants inoculated with ANG3 was halved at 25 mm NaCl compared with salt-free controls, whereas isolates ANG4 and ANG5 retained 25% and 15% activity, respectively, even at 100 mm NaCl. Salt-tolerant Rhizobium isolates can therefore nodulate and fix N₂ in saline soils.     

Decolorization of Fast red by metabolizing cells of <Emphasis Type="Italic">Oenococcus oeni</Emphasis> ML34

Three different azo dyes such as Fast red, metanil yellow and Fast orange were examined for their decolorization by O. oeni ML34. Fast red (FR) was decolorized by 68%, whereas the other dyes were removed by only about 30%. The effects of glucose addition, substrate (dye) concentration and environmental factors (temperature, pH) on decolorization were investigated by two-level factorial design. The statistical analyses revealed that glucose specifically increases the extent of FR decolorization. A glucose level of 5 g/l was the optimum concentration for removal of, FR reaching a decolorization percentage of up to 93%.     

Isolation and Characterization of a Serine Protease from the Nematophagous Fungus, <Emphasis Type="Italic">Lecanicillium psalliotae</Emphasis>, Displaying Nematicidal Activity

Lecanicillium psalliotae produced an extracellular protease (Ver112) which was purified to apparent homogeneity giving a single band on SDS-PAGE with a molecular mass of 32 kDa. The optimum activity of Ver112 was at pH 10 and 70 °C (over 5 min). The purified protease degraded a broad range of substrates including casein, gelatin, and nematode cuticle with 81% of a nematode (Panagrellus redivivus) being degraded after treating with Ver112 for 12 h. The protease was highly sensitive to PMSF (1 mM) indicating it to be a serine protease. The N-terminal amino acid residues of Ver112 shared a high degree of similarity with other cuticle-degrading proteases from nematophagous fungi which suggests a role in nematode infection.     

Effect of n-dodecane on Crypthecodinium cohnii fermentations and DHA production

The potential use of n-dodecane as an oxygen vector for enhancement of Crypthecodinium cohnii growth and docosahexaenoic acid (DHA) production was studied. The volumetric fraction of oxygen vector influenced the gas–liquid volumetric mass transfer coefficient k _L a positively. The k _L a increased almost linearly with the increase of volumetric fraction of n-dodecane up to 1%. The stirring rate showed a higher influence on the k _L a than the aeration rate. The effects of this hydrocarbon on C. cohnii growth and DHA production were then investigated. A control batch fermentation without n-dodecane addition (CF) and a batch fermentation where n-dodecane 1% (v/v) was added (DF) were carried out simultaneously under the same experimental conditions. It was found that, before 86.7 h of fermentation, the biomass concentration, the specific growth rate, the DHA, and total fatty acids (TFA) production were higher in the CF. After this fermentation time, the biomass concentration, the DHA and TFA production were higher in the DF. The highest DHA content of biomass (6.14%), DHA percentage of TFA (51%), and DHA production volumetric rate r _DHA (9.75 mg l⁻¹ h⁻¹) were obtained at the end of the fermentation with n-dodecane (135.2 h). The dissolved oxygen tension (DOT) was always higher in the DF, indicating a better oxygen transfer due to the oxygen vector presence. However, since the other C. cohnii unsaturated fatty acids percentages did not increase with the oxygen availability increase due to the n-dodecane presence, a desaturase oxygen-dependent mechanism involved in the C. cohnii DHA biosynthesis was not considered to explain the DHA production increase. A selective extraction through the n-dodecane was suggested.     

Isolation and characterization of membrane-associated proteoglycans from normal and malignant human mammary epithelial cells

The plasma membrane-associated proteoglycans of a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal cell line (HBL-100). The labeled proteoglycans were isolated from the plasma membranes of cells grown in the presence of [³H]glucosamine and [³⁵S]Na₂SO₄ by extraction with guanidine hydrochloride and subsequently purified by DEAE-ion exchange chromatography. Their structural properties were established by treatment with nitrous acid, heparitinase and chondroitinase ABC, and by gel filtration before and after alkaline -elimination. About 18% of the proteoglycans synthesized by these cell lines were associated with the plasma membranes. The HBL plasma membranes contained 80% heparan sulfate and 20% chondroitin sulfate proteoglycans whereas MDA plasma membranes had 50% heparan sulfate and 50% chondroitin sulfate proteoglycans. The MDA plasma membrane contained two heparan sulfate proteoglycans, both having nearly the same molecular size as the two species secreted into the medium by these cells. The HBL plasma membrane also contained two hydrodynamic size heparan sulfate proteoglycans. The larger hydrodynamic size species has a slightly lower molecular size than that secreted into the medium, and the smaller hydrodynamic size species was not detectable in the medium. Even though the major chondroitin sulfate proteoglycans from MDA plasma membranes were smaller in size than those from HBL plasma membrane, a larger proportion of the glycosaminoglycan chains of the former were bigger than those from the latter.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate - Di-OS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-d-galactose - Di-4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-4-O-sulfo-d-galactose - Di-6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-6-O-sulfo-d-galactose - Gdn-HCl guanidine hydrochloride - WGA wheat germ agglutinin     

Ethylenediamine uptake and metabolism in the cyanobacterium Anabaena variabilis

The cyanobacterium Anabaena variabilis showed a pH dependent uptake of ethylenediamine. No uptake of ethylenediamine was detected at pH 7.0. At higher pH values (e.g. pH 8.0 and pH 9.0) accumulation did occur and was attributed to diffusion of uncharged ethylenediamine in response to a pH gradient. A biphasic pattern of uptake was observed at these higher pH values. Treatment with l-methionine-d,l-sulphoximine (MSX) to inactivate glutamine synthetase (GS) inhibited the second slower phase of uptake without any significant alteration of the initial uptake. Therefore for sustained uptake, metabolism of ethylenediamine via GS was required. NH ₄ ⁺ did not alter the uptake of ethylenediamine. Ethylenediamine was converted in the second phase of uptake to an analogue of glutamine which could not be detected in uptake experiments at pH 7.0 or in uptake experiments at pH 9.0 following pretreatment of cells with MSX. Ethylenediamine treatment inhibited nitrogenase activity and this inhibition was greatest at high pH values.Abbreviations EDA 1,2-diaminoethane (ethylenediamine) - GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1 piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine - membrane potential - Tricine N-tris(hydroxymethyl) methylglycine