Anuradha S. Nerurkar

Evaluation of bioemulsifier mediated Microbial Enhanced Oil Recovery using sand pack column

Bacillus licheniformis K125, isolated from an oil reservoir, produces an effective bioemulsifier. The crude bioemulsifier showed 66% emulsification activity (E(24)) and reduced the surface tension of water from 72 to 34 mN/m. It contains substantial amount of polysaccharide, protein and lipid. This bioemulsifier is pseudoplastic non-Newtonian in nature. It forms oil in water emulsion which remains stable at wide range of pH, temperature and salinity. It gave 43+/-3.3% additional oil recovery upon application to a sand pack column designed to simulate an oil reservoir. This is 13.7% higher than that obtained from crude lipopeptide biosurfactants produced by the standard strain, Bacillus mojavensis JF2 and 8.5% higher than hot water spring isolate, Bacillus licheniformis TT42. The increased oil recovery obtained by using the crude bioemulsifier can be attributed to its combined surface and emulsification activity. Its mechanism of oil recovery must be similar to the mechanism exhibited by surfactant-polymer flooding process of chemical enhanced oil recovery.

Assessment of denitrifying bacterial composition in activated sludge

The abundance and structure of denitrifying bacterial community in different activated sludge samples were assessed, where the abundance of denitrifying functional genes showed nirS in the range of 10(4)-10(5), nosZ with 10(4)-10(6) and 16S rRNA gene in the range 10(9)-10(10) copy number per ml of sludge. The culturable approach revealed Pseudomonas sp. and Alcaligenes sp. to be numerically high, whereas culture independent method showed betaproteobacteria to dominate the sludge samples. Comamonas sp. and Pseudomonas fluorescens isolates showed efficient denitrification, while Pseudomonas mendocina, Pseudomonas stutzeri and Brevundimonas diminuta accumulated nitrite during denitrification. Numerically dominant RFLP OTUs of the nosZ gene from the fertilizer factory sludge samples clustered with the known isolates of betaproteobacteria. The data also suggests the presence of different truncated denitrifiers with high numbers in sludge habitat.

Development of a simultaneous partial nitrification, anaerobic ammonia oxidation and denitrification (SNAD) bench scale process for removal of ammonia from effluent of a fertilizer industry.

A simultaneous partial nitrification, anammox and denitrification (SNAD) process was developed for the treatment of ammonia laden effluent of a fertilizer industry. Autotrophic aerobic and anaerobic ammonia oxidizing biomass was enriched and their ammonia removal ability was confirmed in synthetic effluent system. Seed consortium developed from these was applied in the treatment of effluent in an oxygen limited bench scale SNAD type (1L) reactor run at ambient temperature (∼30°C). Around 98.9% ammonia removal was achieved with ammonia loading rate 0.35kgNH(4)(+)-N/m(3)day in the presence of 46.6mg/L COD at 2.31days hydraulic retention time. Qualitative and quantitative analysis of the biomass from upper and lower zone of the reactor revealed presence of autotrophic ammonia oxidizing bacteria (AOB), Planctomycetes and denitrifiers as the dominant bacteria carrying out anoxic oxidation of ammonia in the reactor. Physiological and molecular studies strongly indicate presence of anammox bacteria in the anoxic zone of the SNAD reactor.

Carbon sources influence the nitrate removal activity, community structure and biofilm architecture.

Influence of the frequently used carbon sources in nitrate removal processes were evaluated in a lab-scale biofilm reactor. The NO3-N removal efficiency was in the order acetate>glucose>methanol>ethanol. Acetate-fed biofilm reduced nearly 100% NO3-N with negligible amount of NO2-N accumulation. Although 99% NO3-N was reduced in the glucose-fed biofilm, substantial NH3-N and NO2-N accumulated. Methanol-fed biofilm reduced 72% of NO3-N with accumulation of 2.2 mg L(-1) of NO2-N, while biofilm formed in presence of ethanol showed 61% reduction in NO3-N although relatively higher ratio of denitrifiers were observed. Acetate and ethanol-fed biofilm displayed characteristic biofilm architecture with voids, but the former had relatively higher thickness and diffusion distance. In presence of glucose and methanol, a confluent biofilm without characteristic voids was formed. Pseudomonas sp. numerically dominated the acetate and ethanol-fed biofilm, while Enterobacter sp. and Methylobacillus sp., were abundant in glucose and methanol biofilms respectively.

Organic solvent tolerance of Halobacterium sp. SP1(1) and its extracellular protease

Halophilic archaea belonging to three different genera‐ Halobacterium, Haloarcula and Haloferax, were isolated from Kandla salt pans. The isolates had an optimum requirement of 25% NaCl for growth. Increase in organic solvent tolerance of isolates was observed at higher NaCl concentrations. Among the three isolates Halobacterium sp. SP1(1) was found to be more tolerant than Haloarcula sp. SP2(2) and Haloferax sp. SP1(2a). The extracellular protease of Halobacterium sp. SP1(1) showed higher solvent tolerance compared to the organism itself. The enzyme was highly tolerant to toluene, xylene, n‐decane, n‐dodecane and n‐undecane, majority of which are frequently used in paints. These findings may help in understanding the mechanism of organic solvent tolerance in halophilic archaea and their application in antifouling coatings. Also, best to our knowledge the present study is the first report on organic solvent tolerance of haloarchaeal extracellular protease. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Structural and Molecular Characteristics of Lichenysin and Its Relationship with Surface Activity

Lichenysins are most potent anionic cyclic lipoheptapeptide biosurfactants produced by Bacillus licheniformis on hydrocarbonless medium with mainly glucose as carbon source. They have the capacity to lower the surface tension of water from 72 to 27 mN/m. Based on species specific variations they are named lichenysin A, B, C, D, G and surfactant BL86. The lowest ever interfacial tension against decane of 0.006 mN/m is obtained with acid precipitated lichenysin B. Surfactant BL86 and lichenysin B have recorded lowest ever CMC of 10 mg/L by any surfactant under optimal conditions. Surface and interfacial tension lowering ability bears significance in the context of oil recovery from oil reservoir. Similarity exists between structure and biosynthesis of surfactin and lichenysin. Surfactin being the most studied of the two, understanding its structure and biosynthesis gives an insight into the structure and biosynthesis of lichenysin. Lichenysin is synthesized by a multienzyme complex, lichenysin synthetase (LchA/Lic) encoded by 32.4 (26.6 kb) lichenysin operon lchA (lic). The structure of lichenysin and its operon indicate the nonribosomal biosynthesis with the same multifunctional modular arrangement as seen in surfactin synthetase SrfA. The lchA operon consists of lchAA-AC (lic A-C) and lchA TE (licTE) genes encoding the proteins LchAA, LchAB, LchAC and thioesterase LchA-TE. The licA (lchAA) gene is 10,746 bp and codes for a 3,582 amino acids protein, licB (lchAB) gene is 10,764 bp and codes for a similar sized protein, while licC (lchAC) gene is 3,864 bp and codes for protein containing 1,288 amino acid. The biotechnological potential of lichenysin in MEOR has triggered research on structure-activity relationship. Both the nature of peptide and fatty acid dictate the activity of the biosurfactant. Tailormade biosurfactant with desired attributes can be obtained from engineered synthetases. Basic studies are lacking on mechanism of biosynthesis by lichenysin synthetase however, studies on various aspects of lichenysin including regulation are expected to swell in coming years.

Occurrence of Biosurfactant Producing Bacillus spp. in Diverse Habitats

Diversity among biosurfactant producing Bacillus spp. from diverse habitats was studied among 77 isolates. Cluster analysis based on phenotypic characteristics using unweighted pair-group method with arithmetic averages (UPGMAs) method was performed. Bacillus isolates possessing high surface tension activity and five reference strains were subjected to amplified 16S rDNA restriction analysis (ARDRA). A correlation between the phenotypic and genotypic characterization of Bacillus spp. is explored. Most of the oil reservoir isolates showing high surface activity clustered with B. licheniformis and B. subtilis, the hot water spring isolates clustered in two ingroups, while the petroleum contaminated soil isolates were randomly distributed in all the three ingroups. Present work revealed that diversity exists in distribution of Bacillus spp. from thermal and hydrocarbon containing habitats where majority of organisms belonged to B. licheniformis and B. subtilis group. Isolate B. licheniformis TT42 produced biosurfactant which reduced the surface tension of water from 72 mNm−1 to 28 mNm−1, and 0.05 mNm−1 interfacial tension against crude oil at 80°C. This isolate clustered with B. subtilis and B. licheniformis group on the basis of ARDRA. These findings increase the possibility of exploiting the Bacillus spp. from different habitats and their possible use in oil recovery.

Biotreatment of nitrate-rich industrial effluent by suspended bacterial growth

Of the 29 potentially denitrifying organisms isolated from a denitrifying reactor (DNR) of a fertilizer company, two isolates; I-4 and I-5 were recognized as denitrifiers. Under aerobic conditions, with fusel oil as the carbon source, the organisms decreased nitrate from 1200 mg l−1 to 100 mg l−1 in 48 h. Optimal growth conditions for biological removal of nitrate were established in batch culture. The system was scaled up to 4-L and 50-L bioreactors under continuous culture conditions. Up to 95–100% nitrate removal was achieved in the 50-L bioreactor at a COD:NO3–N ratio of 3.45 with a retention time of 48 h. The isolates showed 1.5 fold higher denitrifying activity than reported previously.

Interference of Quorum Sensing by Delftia sp. VM4 Depends on the Activity of a Novel N-Acylhomoserine Lactone-Acylase

Background Turf soil bacterial isolate Delftia sp. VM4 can degrade exogenous N-acyl homoserine lactone (AHL), hence it effectively attenuates the virulence of bacterial soft rot pathogen Pectobacterium carotovorum subsp. carotovorum strain BR1 (Pcc BR1) as a consequence of quorum sensing inhibition. Methodology/Principal Findings Isolated Delftia sp. VM4 can grow in minimal medium supplemented with AHL as a sole source of carbon and energy. It also possesses the ability to degrade various AHL molecules in a short time interval. Delftia sp. VM4 suppresses AHL accumulation and the production of virulence determinant enzymes by Pcc BR1 without interference of the growth during co-culture cultivation. The quorum quenching activity was lost after the treatment with trypsin and proteinase K. The protein with quorum quenching activity was purified by three step process. Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) and Mass spectrometry (MS/MS) analysis revealed that the AHL degrading enzyme (82 kDa) demonstrates homology with the NCBI database hypothetical protein (Daci_4366) of D. acidovorans SPH-1. The purified AHL acylase of Delftia sp. VM4 demonstrated optimum activity at 20–40°C and pH 6.2 as well as AHL acylase type mode of action. It possesses similarity with an α/β-hydrolase fold protein, which makes it unique among the known AHL acylases with domains of the N-terminal nucleophile (Ntn)-hydrolase superfamily. In addition, the kinetic and thermodynamic parameters for hydrolysis of the different AHL substrates by purified AHL-acylase were estimated. Here we present the studies that investigate the mode of action and kinetics of AHL-degradation by purified AHL acylase from Delftia sp. VM4. Significance We characterized an AHL-inactivating enzyme from Delftia sp. VM4, identified as AHL acylase showing distinctive similarity with α/β-hydrolase fold protein, described its biochemical and thermodynamic properties for the first time and revealed its potential application as an anti-virulence agent against bacterial soft rot pathogen Pectobacterium carotovorum subsp. carotovorum based on quorum quenching mechanism.

Mutualism between autotrophic ammonia‑oxidizing bacteria (AOB) and heterotrophs present in an ammonia‑oxidizing colony

Coexistence of an autotrophic ammonia-oxidizing bacterium (Nitrosomonas sp. RA) and heterotrophic bacteria was consistently observed when cultured in an inorganic medium without any external supply of organic carbon. The present study was undertaken to understand the association between autotrophs and the associated heterotrophs for which a system containing active autotrophs and heterotrophs controlled by Hg2+ addition was developed. The study revealed interdependence of heterotrophs and Nitrosomonas sp. RA for growth under iron-limited condition. Growth of Nitrosomonas sp. RA was supported by siderophores produced by the associated heterotroph, Pusillimonas sp., thereby complementing its high iron requirement while the organics (such as pyruvate) excreted by Nitrosomonas sp. RA during its autotrophic growth supported the survival of heterotrophs in the inorganic medium. The study thus sheds light on the nature of the mutual interactions between heterotrophs and autotrophs that play a role in the ammonia-oxidizing system involved in wastewater treatment.