Decolorization and detoxification of Congo red and textile industry effluent by an isolated bacterium <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. SU-EBT

The 16S rRNA sequence and biochemical characteristics revealed the isolated organism as Pseudomonas sp. SU-EBT. This strain showed 97 and 90% decolorization of a recalcitrant dye, Congo red (100 mg l⁻¹) and textile industry effluent with 50% reduction in COD within 12 and 60 h, respectively. The optimum pH and temperature for the decolorization was 8.0 and 40°C, respectively. Pseudomonas sp. SU-EBT was found to tolerate the dye concentration up to 1.0 g l⁻¹. Significant induction in the activity of intracellular laccase suggested its involvement in the decolorization of Congo red. The metabolites formed after decolorization of Congo red, such as p-dihydroxy biphenyl, 8-amino naphthol 3-sulfonic acid and 3-hydroperoxy 8-nitrosonaphthol were characterized using FTIR and GC–MS. Phytotoxicity study revealed nontoxic nature of the degradation metabolites to Sorghum bicolor, Vigna radiata, Lens culinaris and Oryza sativa plants as compared to Congo red and textile industry effluent. Pseudomonas sp. SU-EBT decolorized several individual textile dyes, dye mixtures and textile industry effluent, thus it is a useful strain for the development of effluent treatment methods in textile processing industries.     

Oily sludge degradation by bacteria from Ankleshwar, India

Three bacterial strains, Bacillus sp. SV9, Acinetobacter sp. SV4 and Pseudomonas sp., SV17 from contaminated soil in Ankleshwar, India were tested for their ability to degrade the complex mixture of petroleum hydrocarbons (such as alkanes, aromatics, resins and asphaltenes), sediments, heavy metals and water known as oily sludge. Gravimetric analysis showed that Bacillus sp. SV9 degraded approx. 59% of the oily sludge in 5 days at 30 °C whereas Acinetobacter sp. SV4 and Pseudomonas sp. SV17 degraded 37% and 35%. Capillary gas chromatographic analysis revealed that after 5 days the Bacillus strain was able to degrade oily sludge components of chain length C₁₂–C₃₀ and aromatics more effectively than the other two strains. Maximum drop in surface tension (from 70 to 28.4 mN/m) was accompanied by maximum biosurfactant production (6.7 g l⁻¹) in Bacillus sp. SV9 after 72 h, these results collectively indicating that this bacterial strain has considerable potential for bioremediation of oily sludge.     

Change in colony morphology and kinetics of tylosin production after UV and gamma irradiation mutagenesis of Streptomyces fradiae NRRL-2702

Tylosin is a macrolide antibiotic used as veterinary drug and growth promoter. Attempts were made for hyper production of tylosin by a strain of Streptomyces fradiae NRRL-2702 through irradiation mutagenesis. Ultraviolet (UV) irradiation of wild-type strain caused development of six morphologically altered colony types on agar plates. After screening using Bacillus subtilis bioassay only morphological mutants indicated the production of tylosin. An increase of 2.7±0.22-fold in tylosin production (1500 mg/l) in case of mutant UV-2 in complex medium was achieved as compared to wild-type strain (550 mg/l). Gamma irradiation of mutant UV-2 using ⁶⁰Co gave one morphologically altered colony type γ-1, which gave 2500 mg/l tylosin yield in complex medium. Chemically defined media promoted tylosin production upto 3800 mg/l. Maximum value of q_p (3.34 mg/gh) was observed by mutant γ-1 as compared to wild strain (0.81 mg/gh). Moreover, UV irradiation associated changes were unstable with loss of tylosin activity whereas mutant γ-1 displayed high stability on subsequent culturing.     

Biodegradation of Methyl red by Galactomyces geotrichum MTCC 1360

Galactomyces geotrichum MTCC 1360 can decolorize triphenylmethane, azo and reactive high exhaust textile dyes. At shaking condition this strain showed 100% decolorization of a toxic azo dye Methyl red (100 m gl⁻¹) within 1 h in deionized water at 30 °C. The degradation of Methyl red was possible through a broad pH (3–12) and temperature (5–50 °C) range. Glucose and mycelium concentration had increased the decolorization rate, but the addition of 1 gl⁻¹ molasses in deionized water made decolorization possible in only 10 min. Induction in the NADH–dichloro phenol indophenol (NADH–DCIP) reductase, Malachite green reductase, laccase and lignin peroxidase (Lip) activities were observed in the cells obtained after complete decolorization, showing that there is direct involvement in the degradation of Methyl red. The absence of N-N′-dimethyl-p-phenylenediamine (DMPD) in 5 °C, 2-aminobenzoic acid (ABA) in 50 °C and both the compounds in 30 °C sample have shown the differences in the metabolic fate of Methyl red at different temperatures. The untreated dye at 300 mg l⁻¹ concentration showed 88% germination inhibition in Sorghum bicolor, whereas it was 72% in Triticum aestivum. There was no germination inhibition for both the plants by Methyl red metabolites at 300 mg l⁻¹ concentration.

The scientific relevance of the paper

The azo dye Methyl red (100 mg l⁻¹) was decolorized by G. geotrichum MTCC 1360 within 1 h at shaking condition in deionized water. This organism could decolorize Methyl red at wide pH and temperature ranges. Decolorization time was reduced to 10 min by the addition of molasses to deionized water. There was induction in laccase and Lip, NADH–DCIP reductase and Malachite green reductase activities. The metabolic fate of Methyl red changes with temperature which can be evidenced by the formation of 2-ABA at 5 °C, N-N′-DMPD at 50 °C and both the compounds were absent at 30 °C. Phytotoxicity showed that metabolites of dye had induced shoot and root length of both the tested plants.     

Decolorization of Azo Dye (Orange MR) by an Autochthonous Bacterium, <Emphasis Type="Italic">Micrococcus</Emphasis> sp. DBS 2

Soil and sediment samples obtained from Orange MR dye contaminated habitat were screened for heterotrophic bacterial population. The heterotrophic bacterial density of dye-contaminated soil was 2.14 × 10⁶ CFU/g. The generic composition of heterotrophic bacterial population was primarily composed of 10% of Proteus sp., 15% Aeromonas sp., 20% Bacillus sp., 25% Pseudomonas sp. and 30% Micrococcus sp. The bacterial strain that decolorized the azo dye Orange MR up to 900 ppm was identified as Micrococcus sp. The optimum inoculum load, pH and temperature were found to be 5%, 6 and 35°C, respectively. The rate of decolorization was assessed using spectrophotometer at 530 nm and the percentage of decolorization was ascertained. The autochthonous bacterial isolate was able to utilize the dye as both nitrogen and carbon source.     

Medium optimization for the production of a novel bioflocculant from Halomonas sp. V3a′ using response surface methodology

The novel exopolysaccharide bioflocculant HBF-3 is produced by Halomonas sp. V3a′, which is a mutant strain of the deep-sea bacterium Halomonas sp. V3a. Response surface methodology (RSM) was employed to optimize the production medium for increasing HBF-3 production. Using a Plackett–Burman experimental design to aid in the first step of optimization, edible glucose, MgSO₄·7H₂O, and NH₄Cl were found to be significant factors affecting HBF-3 production. To determine the optimal concentration of each significant variable, a central composite design was employed. Based on response surface and canonical analysis, the optimum concentrations of the critical components were obtained as follows: edible glucose, 16.14 g/l; MgSO₄·7H₂O, 2.73 g/l; and NH₄Cl, 1.97 g/l. HBF-3 production obtained by using the optimized medium was 4.52 g/l, which was in close agreement with the predicted value of 4.55 g/l. By scaling up fermentation from flask to fermenter, HBF-3 production was further increased to 5.58 g/l.     

Pullulanases of alkaline and broad pH range from a newly isolated alkalophilicBacillus sp. S-1 and aMicrococcus sp. Y-1

Summary Two highly alkalophilic bacteria, and potent producers of alkaline pullulanase, were isolated from Korean soils. The two isolates, identified asBacillus sp. S-1 andMicrococcus sp. Y-1, grow on starch under alkaline conditions and effectively secrete extracellular pullulanases. The two isolates were extremely alkalophilic since bacterial growth and enzyme production occurred at pH values ranging from pH 6.0 to 12.0 forMicrococcus sp. Y-1 and pH 6.0 to 10.0 forBacillus sp. S-1. Both strains secrete enzymes that possess amylolytic and pullulanolytic acitivities. Extracellular crude enzymes of both isolates gave maltotriose as the major product formed from soluble starch and pullulan hydrolysis. Compared to other alkalophilic microbes such asMicrococcus sp. (0.57 units ml^–1),Bacillus sp. KSM-1876 (0.56 units ml^–1) andBacillus No. 202-1 (1.89 units ml^–1) these isolates secreted extremely high concentrations (7.0 units ml^–1 forBacillus sp. S-1 and 7.6 units ml^–1 forMicrococcus sp. Y-1) of pullulanases in batch culture. The pullulanase activities from both strains were mostly found in the culture medium (85–90%). The extracellular enzymes of both bacteria were alkalophilic and moderately thermoactive; optimal activity was detected at pH 8.0–10.0 and between 50 and 60°C. Even at pH 12.0, 65% of original Y-1 pullulanase activity and 10% of S-1 pullulanase activity remained. The two newly isolated strains had broad pH ranges and moderate thermostability for their enzyme activities. These result strongly indicate that these new bacterial isolates have potential as producers of pullulanases for use in the starch industry.     

Production of poly-3-hydroxybutyrate by Bacillus sp. JMa5 cultivated in molasses media

A strain of Bacillus sp. coded JMa5 was isolated from molasses contaminated soil. The strain was able to grow at a temperature as high as 45°C and in 250 g/l molasses although the optimal growth temperature was 35–37°C. Cell density reached 30 g/l 8 h after inoculation in a batch culture with an initial concentration of 210 g/l molasses. Under fed-batch conditions, the cells grew to a dry weight of 70 g/l after 30 h of fermentation. The strain accumulated 25–35%, (w/w) polyhydroxybutyrate (PHB) during fermentation. PHB accumulation was a growth-associated process. Factors that normally promote PHB production include high ratios of carbon to nitrogen, and carbon to phosphorus in growth media. Low dissolved oxygen supply resulted in sporulation, which reduced PHB contents and dry weights of the cells. It seems that sporulation induced by reduced supply of nutrients is the reason that PHB content is generally low in the Bacillus strain.     

A novel thermotolerant methylotrophicBacillus sp. and its potential for use in single-cell protein production

A thermotolerant methylotrophicBacillus sp. (KISRI TM1A, NCIMB 40040), isolated from the Kuwaiti environment and belonging to the group II spore-forming, bacilli, could not be correlated with any knownBacillus sp. It may, therefore, be a new species. It grew at temperatures from 37° to 58°C from pH 6.5 to 9.0 and on methanol up to 40 g l^–1. It grew well in a chemostat. Its biomass yield coefficient was improved by about 30% by optimization of medium and growth conditions, reaching a maximum of 0.44g g^–1 at 45°C pH 6.8 to 7.0, dilution rate 0.25 h^–1 with methanol at 10 g l^–1. Average crude protein and amino acid content were 84% and 60%, respectively, and maximum productivity attained under laboratory conditions was 5.06 g l^–1h^–1. It was concluded that this strain has good potential for use in single-cell protein production.     

Genetic and physical mapping of an hydrogenase gene cluster from Rhodobacter capsulatus

Summary An hydrogenase-deficient (Hup^–) mutant of Rhodobacter capsulatus was obtained by adventitious insertion of IS21 DNA into an hydrogenase structural gene (hup) of the wild-type strain 1310. The resulting Hup^– mutant, strain JP91, selected by its inability to grow autotrophically (Aut^– phenotype) together with other Hup^– mutant strains obtained by classical ethyl methane sulphonate mutagenesis were used in R plasmid-mediated conjugation experiments to map the hup/aut loci on the chromosome of R. capsulatus. The hup genes tested in this study were found to cluster on the chromosome in the proximity of the his-1 marker. A cluster of hup genes comprising the structural genes was isolated from a gene bank constructed in the cosmid vector pHC79 with 40 kb insert DNA. The clustered hup genes, characterized by hybridization studies and complementation analyses of the R. capsulatus Hup^– mutants, span 15–20 kb of DNA.     

Correlation between extracellular polysaccharide composition and nodulating ability inRhizobium trifolii

Summary Two auxotrophic mutants ofRhizobium trifolii which are deficient in nodulating ability have been isolated. Both mutants (strain RS 164 His^– and strain RS213 Leu^–) appear to synthesize abnormal extracellular polysaccharides as compared with the wild type strain RS 55. Simultaneous recovery of nodulating ability and wild type polysaccharide composition has been found in a Leu⁺ revertant of strain RS 213.Abbreviation EPS Extracellular Polysaccharide - NIG N-Methyl-N-Nitro-N-Nitrosoguanidine     

Overproduction of Delta-Endotoxins by Sporeless <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">thuringiensis</Emphasis> Mutants Obtained by Nitrous Acid Mutagenesis

Asporogenic and oligosporogenic Bacillus thuringiensis mutants having the ability to overproduce insecticidal crystal protein were generated by using nitrous acid (50 mg/ml), as chemical mutagenic agent. Insecticidal crystal proteins produced by asporogenic mutants remained encapsulated within the cells. Delta-endotoxin production by most of mutants was improved compared to the corresponding wild strains BNS3 and a mutant M26. The overproduction by asporogenic and oligosporogenic mutants was attributed to defect in genes involved in sporulation and to random mutations affecting cell metabolism at different pathways and delta-endotoxin synthesis. Sporeless bioinsecticides could be developed based on stable and environmentally safe Bacillus thuringiensis mutants.     

Changes in photosynthesis and pigmentation in an agp deletion mutant of the cyanobacterium Synechocystis sp

The agp gene encoding ADP-glucose pyrophosphorylase is involved in cyanobacterial glycogen synthesis. By in vitro DNA recombination technology, agp deletion mutant (agp ^–) of cyanobacterium Synechocystis sp. PCC 6803 was constructed. This mutation led to a complete absence of glycogen biosynthesis. As compared with WT (wild type), a 60% decrease in ratio of the c-phycocyanine/chlorophyll a and no significant change in the carotenoid/chlorophyll a were observed in agp ^– cells. The agp ^– mutant had 38% less photosynthetic capacity when grown in light over 600 mol m^–2 s^–1. Under lower light intensity, the final biomass of the mutant strain was only 1.1 times of that of the WT strain under mixotrophic condition after 6 d culture. Under higher light intensity, however, the final biomass of the WT strain under mixotrophic conditions was 3 times that of the mutant strain after 6 d culture and 1.5 times under photoautotrophic conditions. The results indicate that there is a minimum requirement for glycogen synthesis for normal growth and development in cyanobacteria.     

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.     

Strain improvement of <Emphasis Type="Italic">Lactobacillus lactis</Emphasis> for <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid production

Three mutants, isolated by repeated UV mutagenesis of Lactobacillus lactis NCIM 2368, produced increased d-lactic acid concentrations. These mutants were compared with the wild type using 100 g hydrolyzed cane sugar/l in the fermentation medium. One mutant, RM2-24, produced 81 g lactic acid/l which was over three times that of the wild type. The highest d-lactic acid (110 g/l) in batch fermentation was obtained with 150 g cane sugar/l with a 73% lactic acid yield. The mutant utilizes cellobiose efficiently, converting it into d-lactic acid suggesting the presence of cellobiase. Thus, this strain could be used to obtain d-lactic acid from cellulosic materials that are pre-hydrolyzed with cellulase.     

A new subtilisin family: nucleotide and deduced amino acid sequences of new high-molecular-mass alkaline proteases from<Emphasis Type="Italic"> Bacillus</Emphasis> spp.

Six genes encoding high-molecular-mass subtilisins (HMSs) of alkaliphilic Bacillus spp. were cloned and sequenced. Their open reading frames of 2,394–2,424 bp encoded prosubtilisins of 798–808 amino acids (aa) consisting of the prepropeptides of 151–158 aa and the mature enzymes of 640–656 aa. The deduced aa sequences of the mature enzymes exhibited 60–95% identity to those of FT protease of Bacillus sp. strain KSM-KP43, a subtilisin-like serine protease, and a minor serine protease, Vpr, of Bacillus strains. Three of the six recombinant enzymes were susceptible to proteolysis, but the others were autodigestion resistant. All enzymes had optimal pH values of 10.5–11.0, optimal temperatures of 40–45°C for hydrolysis of a synthetic substrate, and were heat labile. These alkaline proteases seem to form a new subtilisin family, as judged by their aa sequences and phylogenetic analysis.Communicated by K. Horikoshi     

H2-Recycling system in mungbean Rhizobium in relation to N2-fixation

Thirty isolates of mungbean Rhizobium were tested for the presence of H₂-recycling system. All the isolates were preliminary screened for detecting H₂-recycling system in free culture using triphenyltetrazolium chloride reduction as screening procedure. The isolates which reduced the dye rapidly at early stages of growth were found to recycle hydrogen both in vivo as well as in vitro. Nitrogen fixing efficiency of hydrogenase positive, hydrogenase negative isolates and Hup^– mutants was compared by green house experiments. There was 13–56% increase in dry matter and 21–46% increase in total nitrogen of the plants inoculated with H₂-recycling isolates over the plants inoculated with non-recycling isolates. There was reduction in dry matter and total nitrogen content of the plants inoculated with Hup^– mutants as compared to plants inoculated with wild type strain. The per cent decrease due to inoculation with Hup^– mutants over wild type strain was 19–22 and 20–26 of dry weight and total nitrogen in plants, respectively.Abbreviations TTC triphenyltetrazolium chloride     

Nitrate and nitrite reductase negative mutants of N2-fixingAzospirillum spp.

Chlorate resistant spontaneous mutants ofAzospirillum spp. (syn.Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants fromA. brasilense and 13 fromA. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr^–). Most of the mutants were also nitrite reductase negative (nir^–), only 3 remaining nir⁺. Two mutants from nr⁺ nir⁺ parent strains lost only nir and became like the nr⁺ nir^– parent strain ofA. brasilense. No parent strain or nr⁺ mutant showed any nitrogenase activity with 10 mM NO ₃ ^– . In all nr^– mutants, nitrogenase was unaffected by 10 mM NO ₃ ^– . Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir^–. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr^– nir^–) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.     

Response surface analysis for enzymatic decolorization of Congo red by manganese peroxidase

The enzymatic decolorization process of manganese peroxidase (MnP) is a complex system, which is greatly affected by the concentrations of H₂O₂, Mn²⁺, dye and enzyme. This work aimed to study these factors and investigate the combined interactions between them by applying response surface methodology (RSM) for decolorization of Congo red with MnP from Schizophyllum sp. F17, meanwhile conventional one-factor-at-a-time analysis was carried out. Through the one-factor-at-a-time analysis the optimized H₂O₂, Mn²⁺, Congo red and MnP extract was 0.2 mM, 0.5 mM, 50 mg/l and 0.8 ml, respectively, and the maximum decolorization attained under such conditions was 24.2%. Response surface analysis was conducted through Box–Behnken design and a second-order polynomial model (R² = 0.8565) was generated to describe the combined effect and the interactions quantificationally. ANOVA analysis indicated that the interactions between H₂O₂ and MnP, between dye and MnP were significant; the optimum condition through RSM was found to be 0.35 mM H₂O₂, 0.5 mM Mn²⁺, 75 mg/l Congo red and 1.4 ml MnP extract, for maximum decolorization of 30.8%.     

Alkaline protease from <Emphasis Type="Italic">Bacillus sp.</Emphasis> isolated from coffee bean grown on cheese whey

Two strains of Bacillus, one from a culture collection (B. subtilis ATCC 6633) and a wild type (Bacillus sp. UFLA 817CF) isolated during coffee fermentation in the south of Minas Gerais, Brazil, were evaluated in relation to secretion of alkaline proteases. The strains were grown on nutrient broth, nutrient broth with sodium caseinate and nutrient broth with three different concentrations of cheese whey powder for 72 h. Samples were collected at 24-h intervals to evaluate the proteolytic activity, protein content and cell population. Maximum protease activity was observed after 24-h growth for both the microorganisms, a period that coincided with the end of the exponential phase. The specific activity values were, respectively, 839.8 U/mg for B. subtilis ATCC 6633 and 975.9 U/mg for Bacillus sp. UFLA 817CF. The 60% saturation presented the best results for specific protease activity in all the growth culture media tested with B. sp. UFLA 817CF. Bacillus sp. UFLA 817CF showed highest enzymatic activity at pH 9.0 and 40°C in the three culture media tested. The protease obtained from culture of the wild Bacillus strain presented stability at pH 7.0 and considerable heat stability at 40°C and 50°C, and could be an alternative for the industry to utilize cheese whey to produce proteolytic enzymes.