Saroj Bhosle

Tolerance of bacteria to organic solvents.

Organic-solvent-tolerant bacteria are a relatively novel group of extremophilic microorganisms. They overcome the toxic and destructive effects of organic solvents due to the presence of various adaptive mechanisms. Extensive studies done on the toluene tolerance of certain Pseudomonas strains have led to an understanding of the mechanisms of organic solvent tolerance involving novel adaptations such as the toluene efflux pumps, cis-trans isomerisation of membrane fatty acids, rapid membrane repair mechanisms, etc. Organic-solvent-tolerant mutants of Escherichia coli have been constructed and genes enhancing such tolerance characterised. However, there is practically no information available on the tolerance mechanisms of the reported Gram-positive organic-solvent-tolerant bacterial strains like Bacillus, Rhodococcus and Arthrobacter. This review discusses the general aspects of organic-solvent-tolerant bacteria, their history, biodiversity, mechanisms of tolerance and proposes certain probable adaptations of Gram-positive bacteria in tolerance to organic solvents.

Industrial potential of organic solvent tolerant bacteria

Most bacteria and their enzymes are destroyed or inactivated in the presence of organic solvents. Organic solvent tolerant bacteria are a relatively novel group of extremophilic microorganisms that combat these destructive effects and thrive in the presence of high concentrations of organic solvents as a result of various adaptations. These bacteria are being explored for their potential in industrial and environmental biotechnology, since their enzymes retain activity in the presence of toxic solvents. This property could be exploited to carry out bioremediation and biocatalysis in the presence of an organic phase. Because a large number of substrates used in industrial chemistry, such as steroids, are water‐insoluble, their bioconversion rates are affected by poor dissolution in water. This problem can be overcome by carrying out the process in a biphasic organic‐aqueous fermentation system, wherein the substrate is dissolved in the organic phase and provided to cells present in the aqueous phase. In bioprocessing of fine chemicals such as cis‐diols and epoxides using such cultures, organic solvents can be used to extract a toxic product from the aqueous phase, thereby improving the efficiency of the process. Bacterial strains reported to grow on and utilize saturated concentrations of organic solvents such as toluene can revolutionize the removal of such pollutants. It is now known that enzymes display striking new properties in the presence of organic solvents. The role of solvent‐stable enzymes in nonaqueous biocatalysis needs to be explored and could result in novel applications.

Isolation and characterization of a lipopeptide bioemulsifier produced by Pseudomonas nitroreducens TSB.MJ10 isolated from a mangrove ecosystem.

Pseudomonas nitroreducens TSB.MJ10 exhibiting growth and bioemulsifier production with 0.5% sodium benzoate as the sole carbon source was isolated from a mangrove ecosystem in the vicinity of a petroleum pump. The bioemulsifier is a lipopeptide that is stable over a pH range of 5-11 and a temperature range of 20-90°C and showed emulsifying activity in the presence of relatively high NaCl concentrations (up to 25%). The bioemulsifier formed stable emulsions with aliphatic (hexadecane, n-heptane, cyclohexane), aromatic (xylene, benzene, toluene) and petroleum (gasoline, diesel, kerosene, crude oil) compounds. It exhibited a maximum emulsification activity with weathered crude oil (97%) and was capable of transforming the rheological behavior of the pseudoplastic to a Newtonian fluid. The results reveal the potential of the bioemulsifier for use in bioremediation of hydrocarbons in marine environments and in enhanced oil recovery.

Effect of metals on a siderophore producing bacterial isolate and its implications on microbial assisted bioremediation of metal contaminated soils

A bacterial isolate producing siderophore under iron limiting conditions, was isolated from mangroves of Goa. Based on morphological, biochemical, chemotaxonomical and 16S rDNA studies, the isolate was identified as Bacillus amyloliquefaciens NAR38.1. Preliminary characterization of the siderophore indicated it to be catecholate type with dihydroxy benzoate as the core component. Optimum siderophore production was observed at pH 7 in mineral salts medium (MSM) without any added iron with glucose as the carbon source. Addition of NaCl in the growth medium showed considerable decrease in siderophore production above 2% NaCl. Fe(+2) and Fe(+3) below 2 μM and 40 μM concentrations respectively, induced siderophore production, above which the production was repressed. Binding studies of the siderophore with Fe(+2) and Fe(+3) indicated its high affinity towards Fe(+3). The siderophore concentration in the extracellular medium was enhanced when MSM was amended with essential metals Zn, Co, Mo and Mn, however, decreased with Cu, while the concentration was reduced with abiotic metals As, Pb, Al and Cd. Significant increase in extracellular siderophore production was observed with Pb and Al at concentrations of 50 μM and above. The effect of metals on siderophore production was completely mitigated in presence of Fe. The results implicate effect of metals on the efficiency of siderophore production by bacteria for potential application in bioremediation of metal contaminated iron deficient soils especially in the microbial assisted phytoremediation processes.

Isolation of an Organic-Solvent-Tolerant Cholesterol-Transforming Bacillus species, BC1 , from Coastal Sediment

Steroid transformation is of great importance in the pharmaceutical industry. The major limiting factor in this process is the extremely poor solubility of steroids in aqueous media, which lowers their transformation rate and increases costs. This problem can be overcome by using organic-solvent-tolerant bacteria (OSTB), which can carry out the desired bioconversions in an organic-solvent-saturated system. OSTB are a relatively novel group of extremophilic microbes that have developed various adaptations to withstand solvent toxicity. The aim of this study was to isolate marine bacteria producing organic-solvent-stable cholesterol-transforming enzymes. A Bacillus species, BC1, isolated from Arabian Sea sediment was found to degrade cholesterol and exhibit excellent solvent tolerance particularly to chloroform. OSTB have tremendous potential in industrial processes involving nonaqueous biocatalysis and transformation in the presence of an organic phase.

Sand aggregation by exopolysaccharide-producing Microbacterium arborescens--AGSB.

In the rhizosphere, exopolymers are also known to be useful to improve the moisture-holding capacity. The ability of the isolates from coastal sand dunes to produce exopolymers was determined. Among which the isolate, showing very high production of exopolysaccharide (EPS), Microbacterium arborescens––AGSB, a facultative alkalophile was further studied for exopolymer production. The isolate a gram-positive non-spore forming, slender rod, catalase positive, oxidase negative, showed growth in 12% sodium chloride. The culture was found to produce exopolymer which showed good aggregation of sand which has an important role in the stabilization of sand dunes. The exopolymer was further analysed. The cold isopropanol precipitation of dialysed supernatants grown in polypeptone yeast extract glucose broth produced partially soluble EPSs with glucose as the sole carbon source. Chemical analysis of the EPS revealed the presence of rhamnose, fucose, arabinose, mannose, galactose and glucose. On optimization of growth parameters (sucrose as carbon source and glycine as nitrogen source), the polymer was found to be a heteropolysaccharide containing mannose as the major component. It was interesting to note that the chemical composition of the exopolymers produced from both unoptimized and optimized culture conditions of Microbacterium arborescens––AGSB is different from those of other species from the same genera. This study shows that marine coastal environments such as coastal sand dunes, are a previously unexplored habitat for EPS-producing bacteria, and that these molecules might be involved in ecological roles protecting the cells against dessication especially in nutrient-limited environments such as the coastal sand dunes more so in the extreme conditions of pH. Such polysaccharides may help the bacteria to adhere to solid substrates and survive during the nutrient limitations.

Siderophore-Producing Bacteria from a Sand Dune Ecosystem and the Effect of Sodium Benzoate on Siderophore Production by a Potential Isolate

Bioremediation in natural ecosystems is dependent upon the availability of micronutrients and cofactors, of which iron is one of the essential elements. Under aerobic and alkaline conditions, iron oxidizes to Fe+3 creating iron deficiency. To acquire this essential growth-limiting nutrient, bacteria produce low-molecular-weight, high-affinity iron chelators termed siderophores. In this study, siderophore-producing bacteria from rhizosphere and nonrhizosphere areas of coastal sand dunes were isolated using a culture-dependent approach and were assigned to 8 different genera with the predominance of Bacillus sp. Studies on the ability of these isolates to grow on sodium benzoate revealed that a pigmented bacterial culture TMR2.13 identified as Pseudomonas aeruginosa showed growth on mineral salts medium (MSM) with 2% of sodium benzoate and produced a yellowish fluorescent siderophore identified as pyoverdine. This was inhibited above 54 μM of added iron in MSM with glucose without affecting growth, while, in presence of sodium benzoate, siderophore was produced even up to the presence of 108 μM of added iron. Increase in the requirement of iron for metabolism of aromatic compounds in ecosystems where the nutrient deficiencies occur naturally would be one of the regulating factors for the bioremediation process.

Rapid Identification of Polyhydroxyalkanoate Accumulating Members of Bacillales Using Internal Primers for phaC Gene of Bacillus megaterium

Bacillus megaterium is gaining recognition as an experimental model and biotechnologically important microorganism. Recently, descriptions of new strains of B. megaterium and closely related species isolated from diverse habitats have increased. Therefore, its identification requires several tests in combination which is usually time consuming and difficult to do. We propose using the uniqueness of the polyhydroxyalkanoate synthase C gene of B. megaterium in designing primers that amplify the 0.9 kb region of the phaC for its identification. The PCR method was optimized to amplify 0.9 kb region of phaC gene. After optimization of the PCR reaction, two methods were investigated in detail. Method I gave an amplification of a single band of 0.9 kb only in B. megaterium and was demonstrated by several strains of B. megaterium isolated from different habitats. The use of Method I did not result in the amplification of the phaC gene with other members of Bacillales. The specificity for identification of B. megaterium was confirmed using sequencing of amplicon and RT-PCR. Method II showed multiple banding patterns of nonspecific amplicons among polyhydroxyalkanoate accumulating members of Bacillales unique to the respective species. These methods are rapid and specific for the identification of polyhydroxyalkanoate accumulating B. megaterium and members of Bacillales.

Microbial Denitrification and Its Ecological Implications in the Marine System

Microbial denitrification is an essential component of the nitrogen cycle and occurs extensively in the estuarine, coastal and marine ecosystems. Denitrifying organisms are unique because they are facultative and can switch between aerobic and anaerobic modes of respiration by utilizing nitrogen oxides as electron acceptors via a series of reductases under conditions of oxygen limitation and nitrate availability. Oxygen plays a regulatory role in aerobic denitrification and controls the electron transport to oxygen or nitrate. Denitrifiers are ubiquitously distributed encompassing a wide array of microorganisms ranging from bacteria and archaebacteria to fungi and foraminifers. Techniques like terminal restriction fragment length polymorphism analysis, functional single-cell isolation method and fluorescent in situ hybridization have provided new insights into the community structure and functioning of these organisms. Pseudomonas, Paracoccus and Alcaligenes are among the most frequently isolated and studied denitrifying bacterial genera. Denitrification acts as an important feedback mechanism and on a global scale has critical impacts on the Earth’s climate. The process operates as a nitrogen sink in estuaries and controls marine biological productivity. The recently discovered anoxic ammonia oxidation process or anammox, which is greatly responsible for the loss of fixed nitrogen in the oxygen minimum zones in the marine system, is also dependent on denitrification for its nitrite. Denitrification also contributes to significant consequences in global warming and hydrocarbon bioremediation.

Presence of a widely disseminated Listeria monocytogenes serotype 4b clone in India.

Details about the members of the Indian Listeria Consortium are provided in the Supplementary Data. Emerging Microbes and Infections (2016) 5, e55; doi:10.1038/emi.2016.55; published online 8 June 2016