Draft Genome Sequence of Halostagnicola sp. A56, an Extremely Halophilic Archaeon Isolated from the Andaman Islands.

The first draft genome of Halostagnicola sp. A56, isolated from the Andaman Islands is reported here. The A56 genome comprises 3,178,490 bp in 26 contigs with a G+C content of 60.8%. The genome annotation revealed that A56 could have potential applications for the production of polyhydroxyalkanoate or bioplastics.

Exploration of a haloarchaeon, Halostagnicola larsenii, isolated from rock pit sea water, West Coast of Maharashtra, India, for the production of bacteriorhodopsin.

AIMS: The aim of the present investigation was to isolate haloarchaea from rock pit sea water, West Coast of India and to explore their potential in the production of bacteriorhodopsin (BR) which converts light energy into electrical energy. METHODS AND RESULTS: Haloarchaeal strains were isolated from rock pit sea water samples collected from Rock garden, Malvan, West Coast of India. Based on morphological, physiological and biochemical characteristics, and 16S rRNA gene sequencing, all the 11 strains were identified as Halostagnicola larsenii. All the strains require at least 1·5 mol l(-1) NaCl for growth; grow optimally in the range of 3·5-5·2 mol l(-1) NaCl. BR was detected in all the strains ranging from 0·035 to 0·258 g l(-1) . All 11 strains showed conversion of light energy into electrical energy in the range of 0·7-44·2 mV, when exposed to sunlight. CONCLUSIONS: A haloarchaeon, Hst. larsenii is isolated from rock pit sea water and demonstrated to have BR that converted light energy into electrical energy. SIGNIFICANCE AND IMPACT OF THE STUDY: The present investigation is presumably the first report of the isolation of Hst. larsenii from low salinity environment and its potential in production of BR. The haloarchaeon could be explored for the generation of electrical energy.

Production of copolymer, poly (hydroxybutyrate-co-hydroxyvalerate) by Halomonas campisalis MCM B-1027 using agro-wastes.

For cost effective production of PHA, agro-wastes like fruit peels, bagasse and deoiled cakes were screened as a sole source of carbon. Halomonas campisalis MCM B-1027, which was isolated from one of the extreme environment, i.e. Lonar Lake, India, was explored for the production of PHA using fruit peels and bagasse having fermentable sugars. Among the agro-wastes tested, 1% (v/v) aqueous extract of bagasse was found to be the optimum carbon source with 47% PHA production on dry cell weight basis. Significant amount of total sugars are utilized and converted into cell mass and PHA, e.g. 62% sugar utilized from bagasse extract, 84% from orange peel extract and 71% from banana peel extract as compared to 51% in case of maltose. Hence the cost of production would be positively reduced. The detailed characterization of PHA formed by H. campisalis using bagasse extract as sole carbon source revealed that the organism produces a copolymer of PHB-co-PHV (94.4:5.6) having molecular weight M(w) 1.394 × 10(6) and melting temperature 168.9 °C. Production of PHA by H. campisalis using aqueous extract of fruit peels and a copolymer PHB-co-PHV using aqueous extract of bagasse is presumably the first report.

Production and characterization of a biodegradable poly (hydroxybutyrate-co-hydroxyvalerate) (PHB-co-PHV) copolymer by moderately haloalkalitolerant Halomonas campisalis MCM B-1027 isolated from Lonar Lake, India.

Several microorganisms produce polyhydroxyalkanoates (PHA). They are accumulated intracellularly as energy storage compounds. The PHAs are of interest because of their potential in biomedical applications. Halophilic bacteria and archaea are known to produce polyhydroxybutyrate (PHB). This paper describes production of a biodegradable copolymer, PHB-co-PHV by a moderately haloalkalitolerant Halomonas campisalis, isolated from Lonar Lake, India. The production of PHA was in the range of 45-81% on dry cell weight basis when the organism was grown in a production medium containing 1% (w/v) maltose and 0.1% (w/v) yeast extract, at pH ranging from 6 to 9 with an inoculum density of 10(5)-10(7) cells/ml of medium, for incubation period of 15-30 h and at 37 degrees C. The polymer produced by the organism is a hydroxyester with molecular weight of 1.3014 x 10(6). Its melting temperature was 171 degrees C. The (1)H NMR analysis revealed that the polymer was a copolymer of PHB-co-PHV. This could be achieved by providing simple carbon source viz. maltose.

Bioremediation of nitroexplosive wastewater by an yeast isolate Pichia sydowiorum MCM Y-3 in fixed film bioreactor.

Nitroexplosives are essential for security and defense of the nation and hence their production continues. Their residues and transformed products, released in the environment are toxic to both terrestrial and aquatic life. This necessitates remediation of wastewaters containing such hazardous chemicals to reduce threat to human health and environment. Bioremediation technologies using microorganisms become the present day choice. High Melting Explosive (HMX) is one of the nitroexplosives produced by nitration of hexamine using ammonium nitrate and acetic anhydride and hence the wastewater bears high concentration of nitrate and acetate. The present investigation describes potential of a soil isolate of yeast Pichia sydowiorum MCM Y-3, for remediation of HMX wastewater in fixed film bioreactor (FFBR). The flask culture studies showed appreciable growth of the organism in HMX wastewater under shake culture condition within 5-6 days of incubation at ambient temperature (28 +/- 2 degrees C). The FFBR process operated in both batch and continuous mode, with Hydraulic Retention Time (HRT) of 1 week resulted in 50-55% removal in nitrate, 70-88% in acetate, 50-66% in COD, and 28-50% in HMX content. Continuous operation of the reactor showed better removal of nitrate as compared to that in the batch operation, while removal of acetate and COD was comparable in both the modes of operation of the reactor. Insertion of baffles in the reactor increased efficiency of the reactor. Thus, FFBR developed with baffles and operated in continuous mode will be beneficial for bioremediation of high nitrate and acetate containing wastewater using the culture of P. sydowiorum.

Effect of microbial activities on stored raw buffalo hide.

'Keeping qualities' of hides are dependent on the total microbial flora associated with the hides and the biochemical changes brought about by these microorganisms during short-term storage at ambient temperature (28 +/- 2 degrees C). It was evident that within first 24 hr of hide's ambient storage, bacterial load was raised to 8.8 log cfu g(-1) hide from 6.1 log cfu g(-1) hide. Nonlinear parabolic increase in release of hydroxyproline and tyrosine from stored hide was observed starting from 0 hr and confirming proteolytic activities. Continuous release of CO2 from the stored hide suggested its mineralization. Exponential release of free fatty acids during storage indicated simultaneous lipolysis. Thus the process of biodegradation during the course of ambient storage of hide piece was found to progress steadily and seems to be interrelated as well as very complex. During the storage period, the liquefaction of hide piece was also observed visually within 96 hr. Present studies of assessment of bacterial activities on hide with respect to total bacterial load, release of amino acids, free fatty acids and evolved CO2 provide data that can be used to formulate and evaluate hide curing agent(s) other than salt, thus rendering leather industry a platform to design bio-based technologies for efficient and ecofriendly preservation of raw materials.

Moderately halophilic, alkalitolerant Halomonas campisalis MCM B-365 from Lonar Lake, India.

Seven bacterial isolates obtained from sediment and water samples, collected from the alkaline Lonar Lake were identified on the basis of their morphological, physiological and biochemical characteristics and were confirmed by 16S rDNA sequencing to be Halomonas campisalis. They were capable of using a variety of electron donors and were found to grow in the presence of sodium chloride (NaCl) up to 4 M, at pH from 7 to 11, 9 being the optimum. The isolates could grow over a wide range of temperatures (from 4 to 45 degrees C) and showed temperature-dependent salt tolerance. They exhibited requirement of sodium for growth and could grow in any medium where NaCl is replaced by NaNO(3) and Na(2)S(2)O(3) but not in the presence of salts like LiCl, MgCl(2) . 6H(2)O, KCl and NH(4)Cl. One of the seven isolates, ARI 351, was able to produce lipase at pH-9.0, while two isolates, ARI 351 and ARI 360, could accumulate polyhydroxyalkanoic acid (PHA) granules when grown in a medium containing maltose. Thus the H. campisalis isolated from Lonar Lake was different from the previously reported one, with respect to its biotechnological potential for production of Lipase and PHA.

Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326.

Bacillus cereus MCM B-326, isolated from buffalo hide, produced an extracellular protease. Maximum protease production occurred (126.87+/-1.32 U ml(-1)) in starch soybean meal medium of pH 9.0, at 30 degrees C, under shake culture condition, with 2.8 x 10(8) cells ml(-1) as initial inoculum density, at 36 h. Ammonium sulphate precipitate of the enzyme was stable over a temperature range of 25-65 degrees C and pH 6-12, with maximum activity at 55 degrees C and pH 9.0. The enzyme required Ca(2+) ions for its production but not for activity and/or stability. The partially purified enzyme exhibited multiple proteases of molecular weight 45 kDa and 36 kDa. The enzyme could be effectively used to remove hair from buffalo hide indicating its potential in leather processing industry.

Effect of application of different pesticides to soybean on the soil microflora.

Pesticide residues contributing to the contamination of soil may influence microbial population of the soil and in turn fertility of soil. The present paper reports the effect of pesticides applied to soybean i.e. phorate, carbofuran, carbosulfan, thiomethoxam, imidacloprid, chlorpyriphos and monocrotophos on soil microflora. The viable count of rhizobia and phosphate solubilizing bacteria from rhizospheric soil of soybean ranged between 10(7)-10(8) cfu/g soil which was comparable to the count of bacteria from untreated (control) soil. No significant change in the total viable count of any kind of bacteria due to application of pesticides has been found showing their ability to degrade these pesticides.

Optimization of protease activity of alkaliphilic bacteria isolated from an alkaline lake in India.

Alkaliphilic bacteria belonging to the genera Bacillus, Staphylococcus, Micrococcus, Pseudomonas and Arthrobacter isolated from sediment samples of the alkaline Lonar lake situated in the Buldhana District of Maharashtra State, India, were studied for the production of protease activity. Among the 54 isolates obtained, Arthrobacter ramosus and Bacillus alcalophilus exhibited high protease activity using soyacake as a sole source of carbon and nitrogen. Protease activity was optimum at 1% initial substrate concentration, at 30 degrees C and under shake culture condition for both organisms. The enzyme was thermostable (65 degrees C), stable at pH 12 and also active in the presence of commercial detergent. This enzyme removed blood stains from cotton fabric indicating its potential use in detergent formulations.

Biomineralization of an organophosphorus pesticide, Monocrotophos, by soil bacteria.

AIMS: To study biomineralization of Monocrotophos (MCP) and identify the metabolites formed during biodegradation. METHODS AND RESULTS: Two cultures, namely Arthrobacter atrocyaneus MCM B-425 and Bacillus megaterium MCM B-423, were isolated by enrichment and adaptation culture technique from soil exposed to MCP. The isolates were able to degrade MCP to the extent of 93% and 83%, respectively, from synthetic medium containing MCP at the concentration of 1000 mg x l(-1), within 8 d, under shake culture condition at 30 degrees C. The cultures degraded MCP to carbon dioxide, ammonia and phosphates through formation of one unknown compound--Metabolite I, valeric or acetic acid and methylamine, as intermediate metabolites. The enzymes phosphatase and esterase, reported to be involved in biodegradation of organophosphorus compounds, were detected in both the organisms. CONCLUSIONS: Arthrobacter atrocyaneus MCM B-425 and B. megaterium MCM B-423 isolated from soil exposed to MCP were able to mineralize MCP to carbon dioxide, ammonia and phosphates. SIGNIFICANCE AND IMPACT OF THE STUDY: Pathway for biodegradation of MCP in plants and animals has been reported. A microbial metabolic pathway of degradation involving phosphatase and esterase enzymes has been proposed. The microbial cultures could be used for bioremediation of wastewater or soil contaminated with Monocrotophos.

Plasmid-mediated dimethoate degradation in Pseudomonas aeruginosa MCMB-427.

AIMS: To investigate the genetics of dimethoate degradation in Pseudomonas aeruginosa MCMB-427. METHODS AND RESULTS: Pseudomonas aeruginosa MCMB-427 demonstrated the ability to degrade dimethoate, a synthetic organophosphate insecticide. Total DNA preparation of MCMB-427 revealed the presence of a 6.6 kbp plasmid (designated as pDMD427). Escherichia coli NovaBlue transformed with plasmid pDMD427 subsequently acquired the ability to degrade dimethoate. Curing of the plasmid by plumbagin or ethidium bromide resulted in the loss of ability of MCMB-427 to degrade dimethoate. Plasmid pDMD427 was stable in MCMB-427 over 20 passages without selection. Genes encoding resistance to norfloxacin and cobalt were also located on plasmid pDMD427. CONCLUSION: The ability of Ps. aeruginosa MCMB-427 to degrade dimethoate is plasmid-mediated and transferable to other strains. SIGNIFICANCE AND IMPACT OF THE STUDY: As far as is known, this is the first report of plasmid-mediated dimethoate biodegradation. This study contributes significantly towards an understanding of the genetics of bacterial dimethoate degradation.

Biodegradation of methyl violet by Pseudomonas mendocina MCM B-402.

Pseudomonas mendocina MCM B-402 was found to utilize a triphenylmethane dye, methyl violet as the sole source of carbon when incorporated in synthetic medium. Almost complete decolorization of methyl violet by P. mendocina was observed within 48 h of incubation at ambient temperature (28 +/- 2 degrees C) under aerated culture conditions, when the bacteria were inoculated into Davis Mingioli's synthetic medium at a concentration of 100 mg/l medium. Methyl violet was mineralized to CO2 through three unknown intermediate metabolites and phenol. The decolorization of the dye involved demethylation.

Bioremediation of epsilon-caprolactam from nylon-6 waste water by use of Pseudomonas aeruginosa MCM B-407.

Nylon-6, a man-made polymer that finds its application in the manufacture of car tires, ropes, fabrics, automobile parts etc., is manufactured with epsilon-caprolactam. Waste water generated during production of nylon-6 contains the unreacted monomer. Owing to the polluting and toxic nature of epsilon-caprolactam, its removal from waste streams is necessary. Pseudomonas aeruginosa MCM B-407 was isolated from activated sludge used to treat waste from a factory producing nylon-6. This organism was able to remove epsilon-caprolactam with simultaneous reduction in chemical oxygen demand (COD). The degradation of epsilon-caprolactam in waste water was found to be optimal over a wide range of pH from 5.0 to 9.0, temperature of 30 degrees C, and under shake or aerated conditions, with an inoculum density of 10(5) cells/ml and with an incubation period of 24 - 48 h. Thus, Pseudomonas aeruginosa MCM B-407 isolated from the activated sludge exposed to epsilon-caprolactam may play an important role in the bioremediation of epsilon-caprolactam from the waste waters of industries manufacturing nylon-6.

Bioremediation of phenol by alkaliphilic bacteria isolated from alkaline lake of Lonar, India.

Phenol is an industrially important compound which has a wide range of applications. Being highly soluble in water, it appears as the major pollutant in waste waters arising from both phenol manufacturing and from industrial units that utilise phenol. Because of its toxicity, bioremediation of phenol is necessary. Since some of the phenol-bearing industrial waste waters are alkaline in nature, use of alkaliphilic bacteria for bioremediation of phenol was investigated. Alkaliphilic bacteria were isolated from sediments of an alkaline lake in Lonar, Dist. Buldhana, Maharashtra State, India, by phenol enrichment at pH 10.0 and phenol concentration of 500 mg/l. The lake (lat. 19°58'45", long. 76°34') is known to be a unique inland saline lake in Asia. It has a circular periphery and diameter of 2 km around the top of the banks and 1.2 km at the bottom. The lake has a high saline level (∼ 2649 mg/l sodium chloride) and a high level of alkalinity (∼ 2605 mg/l calcium carbonate). Alkaliphilic strains of Arthrobacter spp., Bacillus cereus, Citrobacter freundii, Micrococcus agilis and Pseudomonas putida biovar B were capable of removing phenol from waste waters arising from industries manufacturing methyl violet (using phenol as one of the major raw materials) and cumene-phenol. The waste waters from both these units were alkaline in nature (pH ∼ 9.95-10.1) and had a high phenol content (368-660 mg/l). The alkaliphilic bacteria being studied removed 100% of the phenol from the industrial waste waters within 48 h of incubation under shake culture conditions and at an ambient temperature of 28 ± 2 °C. Bioremediation of phenol by alkaliphilic strains of Arthrobacter spp., B. cereus, C. freundii and M. agilis seems to be the first report.

Evaluation of Acacia nilotica (L.) del. and Casuarina equisetifolia forst. for tolerance and growth on microbially treated dyestuff wastewater.

Two tree species, Acacia nilotica (L.) Del. and Casuarina equisetifolia Forst., were tested for their tolerance and growth on dyestuff wastewater containing phenol, aniline, and methyl violet. The wastewater was treated microbially by using a culture of Pseudomonas alcaligenes in a fixed-film bioreactor. The plants were watered with untreated and treated wastewater and tap water (control) at the rate of 2 litre per plant per week. A. nilotica exhibited 100% survival with both untreated and treated wastewater, and growth (increase in height) was comparable to that of control plants. However, growth of C. equisetifolia was adversely affected. It exhibited 100% survival with treated wastewater but only 87% survival with untreated waste-water and the percentage increase in height was less with both treated and untreated wastewater. There was no effect on soil except for an increase in chloride content.

An activated sludge process to reduce the pollution load of a dye-industry waste.

A laboratory-scale activated sludge process was developed to reduce the pollution load of a dye-industry waste, containing aniline, phenol, methyl violet and rhodamine B as its major components. The waste exerted an organic load of 5576 mg litre(-1) as the chemical oxygen demand (COD), of 896 mg litre(-1) as total organic carbon (TOC), and had a 31.5 mg litre(-1) phenol content. A microbial sludge, capable of growing on the waste, was developed from cattle dung, adapted to the waste and used as a bioinoculum for the process. This resulted in reductions of 60% in COD, 37% in TOC, and 92% in phenol content, and a decrease in optical density of the colour of the waste from an initial 0.915 to 0.360 at 580 nm. Microorganisms isolated from sludge were identified as Pseudomonas alcaligenes and P. mendocina.