Functional Genetic Diversity and Plant Growth Promoting Potential of Polyphosphate Accumulating Bacteria in Soil.

Polyphosphate (polyP) accumulation is an important trait of microorganisms. Implication of polyP accumulating bacteria (PAB) in enhanced biological phosphate removal, heavy metal sequestration, and dissolution of dental enamel is well studied. Phosphorous (P) accumulated within microbial biomass also regulates labile P in soil; however, abundance and diversity of the PAB in soil is still unexplored. Present study investigated the genetic and functional diversity of PAB in rhizosphere soil. Here, we report the abundance of Pseudomonas spp. as high PAB in soil, suggesting their contribution to global P cycling. Additional subset analysis of functional genes i.e., polyphosphate kinase (ppk) and exopolyphosphatase (ppx) in all PAB, indicates their significance in bacterial growth and metabolism. Distribution of functional genes in phylogenetic tree represent a more biologically realistic discrimination for the two genes. Distribution of ppx gene disclosed its phylogenetic conservation at species level, however, clustering of ppk gene of similar species in different clades illustrated its environmental condition mediated modifications. Selected PAB showed tolerance to abiotic stress and strong correlation with plant growth promotary (PGP) traits viz. phosphate solubilization, auxin and siderophore production. Interaction of PAB with A. thaliana enhanced the growth and phosphate status of the plant under salinity stress, suggestive of their importance in P cycling and stress alleviation. IMPORTANCE Study discovered the abundance of Pseudomonas genera as a high phosphate accumulator in soil. The presence of functional genes (polyphosphate kinase [ppk] and exopolyphosphatase [ppx]) in all PAB depicts their importance in polyphosphate metabolism in bacteria. Genetic and functional diversity reveals conservation of the ppx gene at species level. Furthermore, we found a positive correlation between PAB and plant growth promotary traits, stress tolerance, and salinity stress alleviation in A. thaliana.

Application of Cell-Free Protein Synthesis System for the Biosynthesis of l-Theanine.

l-Theanine, as an active component of the leaves of the tea plant, possesses many health benefits and broad applications. Chemical synthesis of l-theanine is possible; however, this method generates chiral compounds and needs further isolation of the pure l-isoform. Heterologous biosynthesis is an alternative strategy, but one main limitation is the toxicity of the substrate ethylamine on microbial host cells. In this study, we introduced a cell-free protein synthesis (CFPS) system for l-theanine production. The CFPS expressed l-theanine synthetase 2 from Camellia sinensis (CsTS2) could produce l-theanine at a concentration of 11.31 µM after 32 h of the synthesis reaction. In addition, three isozymes from microorganisms were expressed in CFPS for l-theanine biosynthesis. The γ-glutamylcysteine synthetase from Escherichia coli could produce l-theanine at the highest concentration of 302.96 µM after 24 h of reaction. Furthermore, CFPS was used to validate a hypothetical two-step l-theanine biosynthetic pathway consisting of the l-alanine decarboxylase from C. sinensis (CsAD) and multiple l-theanine synthases. Among them, the combination of CsAD and the l-glutamine synthetase from Pseudomonas taetrolens (PtGS) could synthesize l-theanine at the highest concentration of 13.42 µM. Then, we constructed an engineered E. coli strain overexpressed CsAD and PtGS to further confirm the l-theanine biosynthesis ability in living cells. This engineered E. coli strain could convert l-alanine and l-glutamate in the medium to l-theanine at a concentration of 3.82 mM after 72 h of fermentation. Taken together, these results demonstrated that the CFPS system can be used to produce the l-theanine through the two-step l-theanine biosynthesis pathway, indicating the potential application of CFPS for the biosynthesis of other active compounds.

Production of optically pure (-)-borneol by Pseudomonas monteilii TCU-CK1 and characterization of borneol dehydrogenase involved.

(-)-Borneol is a bicyclic plant secondary metabolite. Optically pure (-)-borneol can only be obtained from plants, and demand exceeds supply in China. In contrast, chemically synthesized borneol contains four different stereoisomers. A strain of Pseudomonas monteilii TCU-CK1, isolated in Hualien, Taiwan, can accumulate (-)-borneol in growth culture and selectively degrades the other three isomers when chemically synthesized borneol is used as sole carbon source. This (-)-borneol production method can be scaled-up for production of large quantities in the future. More importantly, laborious plant cultivation and harvest is no longer required. The main enzyme that appears in this degradation pathway, borneol dehydrogenase (BDH), and the genome sequence of TCU-CK1 are reported. The kcat/Km values of TCU-CK1 BDH on (+)- and (-)-borneol are 538.4 ± 38.4 and 17.7 ± 1.1 (s-1 mM-1), respectively. About ∼30 fold difference in the kcat/Km value between (+)-borneol and (-)-borneol was observed, in good agreement with the fact that TCU-CK1 prefers to degrade (+)-borneol, rather than (-)-borneol. A BDH isozyme was identified in a strain in which the primary BDH gene had been knocked out. (-)-Camphor can work as an inhibitor of BDH with a Ki of 1.03 ± 0.11 mM at pH 7.0, leading to the accumulation of (-)-borneol in culture. (Patent pending).

Control of pyrimidine nucleotide formation in Pseudomonas aurantiaca.

The control of pyrimidine nucleotide formation in the bacterium Pseudomonas aurantiaca ATCC 33663 by pyrimidines was studied. The activities of the pyrimidine biosynthetic pathway enzymes were investigated in P. aurantiaca ATCC 33663 cells and from cells of an auxotroph lacking orotate phosphoribosyltransferase activity under selected culture conditions. All activities of the pyrimidine biosynthetic pathway enzymes in ATCC 33663 cells were depressed by uracil addition to the minimal medium when succinate served as the carbon source. In contrast, all pyrimidine biosynthetic pathway enzyme activities in ATCC 33663 cells were depressed by orotic acid supplementation to the minimal medium when glucose served as the carbon source. The orotidine 5'-monophosphate decarboxylase activity in the phosphoribosyltransferase mutant strain increased by more than sixfold in succinate-grown cells and by more than 16-fold in glucose-grown cells after pyrimidine limitation showing possible repression of the decarboxylase by a pyrimidine-related compound. Inhibition by ATP, GTP, UTP and pyrophosphate of the in vitro activity of aspartate transcarbamoylase in ATCC 33663 was observed. The findings demonstrated control at the level of pyrimidine biosynthetic enzyme synthesis and activity for the P. aurantiaca transcarbamoylase. The control of pyrimidine synthesis in P. aurantiaca seemed to differ from what has been observed previously for the regulation of pyrimidine biosynthesis in related Pseudomonas species. This investigation could prove helpful to future work studying pseudomonad taxonomic analysis as well as to those exploring antifungal and antimicrobial agents produced by P. aurantiaca.

Lipase of Pseudomonas guariconesis as an additive in laundry detergents and transesterification biocatalysts.

A newly isolated culture, Pseudomonas guariconesis, is reported for the first time for lipase production. Various process parameters affecting enzyme production were optimized through statistical design experiments. The Plackett-Burman experimental design was used for screening 10 parameters for lipase production, which was further optimized using the central composite design of response surface methodology. Maximum lipase activity of 220 U/ml was obtained after 24 h of incubation in shake-flask cultures with an inoculum concentration of 0.6% v/v, incubation temperature of 30°C, and medium pH 9.0. Castor oil (0.5% v/v) was used as the inducer for lipase production. The enzyme was found to be compatible with five different commercial detergents, indicating its potential to be used in detergent formulations. It also acted as a biocatalyst in a transesterification process. The alkaline enzyme was found to be stable in the presence of bleaching agents, metal ions, and organic solvents as well.

Phenol hydroxylase from Pseudomonas sp. KZNSA: Purification, characterization and prediction of three-dimensional structure.

A 61.3 kDa Phenol hydroxylase (PheA) was purified and characterized from Pseudomonas sp. KZNSA (PKZNSA). Cell free extract of the isolate grown in mineral salt medium supplemented with 600 ppm phenol showed 21.58 U/mL of PheA activity with a specific activity of 7.67 U/mg of protein. The enzyme was purified to 1.6-fold with a total yield of 33.6%. The purified PheA was optimally active at pH 8 and temperature 30 °C, with ≈95% stability at pH 7.5 and temperature 30 °C after 2 h. The Lineweaver-Burk plot showed the vmax and Km values of 4.04 µM/min and 4.03 µM, respectively, for the substrate phenol. The ES-MS data generated from the tryptic digested fragments of pure protein and PCR amplification of a ≈600 bp gene from genomic DNA of PKZNSA lead to the determination of complete amino acid and nucleotide sequence of PheA. Bioinformatics tools and homology modelling studies indicated that PheA from PKZNSA is likely a probable protein kinase UbiB (2-octaprenylphenol hydroxylase) involving Lys and Asp at positions 153 and 288 for binding and active site, respectively. Characterization and optimization of PheA activity may be useful for a better understanding of 2,4-dichlorophenol degradation by this organism and for potential industrial application of the enzyme.

Isolation, purification and characterization of a novel solvent stable lipase from Pseudomonas reinekei.

The Pseudomonas sp. have been long recognized for their exogenous lipolytic activities yet the genus still contains a lot of unexplored strains. Due to the versatile metabolic machinery and their potential for adaptation to fluctuating environmental conditions Pseudomonas sp. are of great interest for biotechnological applications. In this study, a new extracellularly produced lipolytic enzyme from Pseudomonas sp. (P. reinekei) was purified and characterized. The production of lipase from P. reinekei (H1) was enhanced 10-fold by optimizing the nitrogen source. The 50 kDa H1 lipase was purified using negative and positive mode anion exchange chromatography. The purified lipase was active over a broad pH range (5.0-9.0) and was stable for 24 h at 40 °C. The lipase showed significant stability, and indeed activation, in the presence of organic solvents with log P ≥ 2.0. These features render this lipase of interest as a biocatalyst for applications such as biodiesel production, detergent formulations and biodegradation of oil in the environment.

Promoting the treatment of crude oil alkane pollution through the study of enzyme activity.

Microbes appear to play a key role in bioremediation of petroleum hydrocarbons pollution and little attention has been paid to the enzyme activity in the process of alkane bioremediation. Oil field bacterium identified as Pseudomonas synxantha LSH-7' was chosen as the tested strain. Periodically collected samples were analyzed by GC-FID (Gas Chromatography- Flame Ionization Detector) and RT-qPCR (Quantitative-Real-Time-PCR). GC-FID results showed this bacterial strain has great degradation ability on crude oil n-alkanes and RT-qPCR data indicated the differences between the three genes expression including AlkB-, Cytochromes P450-, and almA- related when grown on different-chain alkanes. Meanwhile, enzyme activity like alkane hydroxylase, alcohol dehydrogenase, dehydrogenase, protease, phosphatase, catalase and lipase were measured. Extracellular alkane hydroxylase was induced in a higher degree than intracellular in the early incubation time, alcohol dehydrogenase increased/decreased along with alkane hydroxylase, and the pH of the medium obviously decreased. Other enzymes were also described including dehydrogenase activity that reached a highest point that was slower than alcohol dehydrogenase, protease activity started multiplying after a period of culture while biomass was immediately increased, catalase activity dramatically enhanced in the presence of alkanes, phosphatase activity was closely linked to pH approximately but lipase activity was found to be moderate.

Alternative Oils Tested as Feedstocks for Enzymatic FAMEs Synthesis: Toward a More Sustainable Process.

Previously isolated and characterized Pseudomonas lipases were immobilized in a low-cost MP-1000 support by a re-loading procedure that allowed a high activity per weight of support. Immobilized LipA, LipC, and LipCmut lipases, and commercial Novozym® 435 were tested for fatty acid methyl ester (FAMEs) synthesis using conventional and alternative feedstocks. Triolein and degummed soybean oils were used as model substrates, whereas waste cooking oil and M. circinelloides oil were assayed as alternative, low cost feedstocks, whose free fatty acid (FFA), and acylglyceride profile was characterized. The reaction conditions for FAMEs synthesis were initially established using degummed soybean oil, setting up the best water and methanol concentrations for optimum conversion. These conditions were further applied to the alternative feedstocks and the four lipases. The results revealed that Pseudomonas lipases were unable to use the FFAs, displaying a moderate FAMEs synthesis, whereas a 44% FAMEs production was obtained when M. circinelloides oil was used as a substrate in the reaction catalysed by Novozym® 435, used under the conditions established for degummed soybean oil. However, when Novozym® 435 was tested under previously described optimal conditions for this lipase, promising values of 85 and 76% FAMEs synthesis were obtained for waste cooking oil and M. circinelloides oil, respectively, which might result in promising, nonfood, alternative feedstocks for enzymatic biodiesel production. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1209-1217, 2017.

Borneol Dehydrogenase from Pseudomonas sp. Strain TCU-HL1 Catalyzes the Oxidation of (+)-Borneol and Its Isomers to Camphor.

Most plant-produced monoterpenes can be degraded by soil microorganisms. Borneol is a plant terpene that is widely used in traditional Chinese medicine. Neither microbial borneol dehydrogenase (BDH) nor a microbial borneol degradation pathway has been reported previously. One borneol-degrading strain, Pseudomonas sp. strain TCU-HL1, was isolated by our group. Its genome was sequenced and annotated. The genome of TCU-HL1 consists of a 6.2-Mbp circular chromosome and one circular plasmid, pTHL1 (12.6 kbp). Our results suggest that borneol is first converted into camphor by BDH in TCU-HL1 and is further decomposed through a camphor degradation pathway. The recombinant BDH was produced in the form of inclusion bodies. The apparent Km values of refolded recombinant BDH for (+)-borneol and (-)-borneol were 0.20 ± 0.01 and 0.16 ± 0.01 mM, respectively, and the kcat values for (+)-borneol and (-)-borneol were 0.75 ± 0.01 and 0.53 ± 0.01 s-1, respectively. Two plant BDH genes have been reported previously. The kcat and kcat/Km values of lavender BDH are about 1,800-fold and 500-fold lower, respectively, than those of TCU-HL1 BDH. IMPORTANCE: The degradation of borneol in a soil microorganism through a camphor degradation pathway is reported in this study. We also report a microbial borneol dehydrogenase. The kcat and kcat/Km values of lavender BDH are about 1,800-fold and 500-fold lower, respectively, than those of TCU-HL1 BDH. The indigenous borneol- and camphor-degrading strain isolated, Pseudomonas sp. strain TCU-HL1, reminds us of the time 100 years ago when Taiwan was the major producer of natural camphor in the world.

Mechanism of Antiactivation at the Pseudomonas sp. Strain ADP σN-Dependent PatzT Promoter.

UNLABELLED: PatzT is an internal promoter of the atzRSTUVW operon that directs the synthesis of AtzT, AtzU, AtzV, and AtzW, components of an ABC-type cyanuric acid transport system. PatzT is σ(N) dependent, activated by the general nitrogen control regulator NtrC with the assistance of protein integration host factor (IHF), and repressed by the LysR-type transcriptional regulator (LTTR) AtzR. We have used a variety of in vivo and in vitro gene expression and protein-DNA interaction assays to assess the mechanisms underlying AtzR-dependent repression of PatzT Here, we show that repression only occurs when AtzR and NtrC interact simultaneously with the PatzT promoter region, indicating that AtzR acts as an antiactivator to antagonize activation by NtrC. Furthermore, repression requires precise rotational orientation of the AtzR and NtrC binding sites, strongly suggesting protein-protein interaction between the two proteins on the promoter region. Further exploration of the antiactivation mechanism showed that although AtzR-dependent repression occurs prior to open complex formation, AtzR does not alter the oligomerization state of NtrC or inhibit NtrC ATPase activity when bound to the PatzT promoter region. Taken together, these results strongly suggest that PatzT-bound AtzR interacts with NtrC to prevent the coupling of NtrC-mediated ATP hydrolysis with the remodeling of the interactions between E-σ(N) and PatzT that lead to open complex formation. IMPORTANCE: Here, we describe a unique mechanism by which the regulatory protein AtzR prevents the activation of the σ(N)-dependent promoter PatzT Promoters of this family are always positively regulated, but there are a few examples of overlapping negative regulation. The mechanism described here is highly unconventional and involves an interaction between the repressor and activator proteins to prevent the action of the repressor protein on the RNA polymerase-promoter complex.

The diversity and abundance of phytase genes (ß-propeller phytases) in bacterial communities of the maize rhizosphere.

UNLABELLED: The ecology of microbial communities associated with organic phosphorus (P) mineralization in soils is still understudied. Here, we assessed the abundance and diversity of bacteria harbouring genes encoding ß-propeller phytases (BPP) in the rhizosphere of traditional and transgenic maize cultivated in two Brazilian soils. We found a soil-dependent effect towards a higher abundance of phytase genes in the rhizosphere, and an absence of any impact of plant genotype. Phylogenetic analyses indicated members of the genera Pseudomonas, Caulobacter, Idiomarina and Maricaulis, close to 'uncultured bacteria', to constitute the dominant bacteria hosting this gene. The results obtained validate a methodology to target bacteria that are involved in the organic P cycle, and depict the responsiveness of such bacteria to the rhizosphere, albeit in dependency of the soil in which maize is cultivated. The data also identified the major bacterial groups that are associated with the organic P mineralization function. SIGNIFICANCE AND IMPACT OF THE STUDY: Micro-organisms play a key role in nutrient balance in soil ecosystems that are essential to life on the planet. However, some processes such as organic phosphorus mineralization, an important source of phosphorus supply in soil, is poorly studied mainly due the absence of an efficient methodology to assess the phytase-producing micro-organisms. In this study, a method to assess beta-propeller phytase (BPP)-carrying bacteria in soil was validated. This method may contribute to the knowledge of how these micro-organisms behave in the environment and contribute for plant growth promotion.

Investigation, expression, and molecular modeling of ORF2, a metagenomic lipolytic enzyme.

One clone exhibiting lipolytic activity was selected among 30 positives from a metagenomic library of a microbe consortium specialized in petroleum hydrocarbon degradation. From this clone, a sublibrary was constructed and a metagenome contig was assembled and analyzed using the ORF Finder; thus, it was possible to identify a potential ORF that encodes a lipolytic enzyme, denoted ORF2. This ORF is composed of 1035-bp 345 amino acids and displayed 98 % identity with an alpha/beta hydrolase from Pseudomonas nitroreducens (accession number WP024765380.1). When analyzed against a metagenome database, ORF2 also showed 76 % of sequence identity with a hypothetical protein from a marine metagenome (accession number ECT55726.1). The ProtParam analyses indicated that the recombinant protein ORF2 has a molecular mass approximately 39 kDa, as expected from its amino acid sequence, and based on phylogenetic analysis and molecular modeling, it was possible to suggest that ORF2 is a new member from family V. This enzyme exhibits the catalytic triad and conserved motifs typical from this family, wherein the serine residue is located in the central position of the conserved motif GASMGG. The orf2 gene was cloned in the expression vector pET28a, and the recombinant protein was superexpressed in Escherichia coli BL21(DE3) cells. The lipolytic activity of protein bands presented in a SDS-PAGE gel was confirmed by zymogram analyses, indicating ORF2 activity. These discoveries raise the possibility of employing this protein in biotechnological applications, such as bioremediation.

Directed arginine deiminase evolution for efficient inhibition of arginine-auxotrophic melanomas.

Arginine deiminase (ADI) is a therapeutic protein for cancer therapy of arginine-auxotrophic tumors. However, ADI's application as anticancer drug is hampered by its low activity for arginine under physiological conditions mainly due to its high "K M" (S0.5) values which are often 1 magnitude higher than the arginine concentration in blood (0.10-0.12 mM arginine in human plasma). Previous evolution campaigns were directed by us with the aim of boosting activity of PpADI (ADI from Pseudomonas plecoglossicida, k cat = 0.18 s(-1); S0.5 = 1.30 mM), and yielded variant M6 with slightly reduced S0.5 values and enhanced k cat (S0.5 = 0.81 mM; k cat = 11.64 s(-1)). In order to further reduce the S0.5 value and to increase the activity of PpADI at physiological arginine concentration, a more sensitive screening system based on ammonia detection in 96-well microtiter plate to reliably detect ≥0.005 mM ammonia was developed. After screening ~5,500 clones with the ammonia detection system (ADS) in two rounds of random mutagenesis and site-directed mutagenesis, variant M19 with increased k cat value (21.1 s(-1); 105.5-fold higher compared to WT) and reduced S0.5 value (0.35 mM compared to 0.81 mM (M6) and 1.30 mM (WT)) was identified. Improved performance of M19 was validated by determining IC50 values for two melanoma cell lines. The IC50 value for SK-MEL-28 dropped from 8.67 (WT) to 0.10 (M6) to 0.04 µg/mL (M19); the IC50 values for G361 dropped from 4.85 (WT) to 0.12 (M6) to 0.05 µg/mL (M19).

PsasM2I, a type II restriction-modification system in Pseudomonas savastanoi pv. savastanoi: differential distribution of carrier strains in the environment and the evolutionary history of homologous RM systems in the Pseudomonas syringae complex.

A type II restriction-modification system was found in a native plasmid of Pseudomonas savastanoi pv. savastanoi MLLI2. Functional analysis of the methyltransferase showed that the enzyme acts by protecting the DNA sequence CTGCAG from cleavage. Restriction endonuclease expression in recombinant Escherichia coli cells resulted in mutations in the REase sequence or transposition of insertion sequence 1A in the coding sequence, preventing lethal gene expression. Population screening detected homologous RM systems in other P. savastanoi strains and in the Pseudomonas syringae complex. An epidemiological survey carried out by sampling olive and oleander knots in two Italian regions showed an uneven diffusion of carrier strains, whose presence could be related to a selective advantage in maintaining the RM system in particular environments or subpopulations. Moreover, carrier strains can coexist in the same orchards, plants, and knot tissues with non-carriers, revealing unexpected genetic variability on a very small spatial scale. Phylogenetic analysis of the RM system and housekeeping gene sequences in the P. syringae complex demonstrated the ancient acquisition of the RM systems. However, the evolutionary history of the gene complex also showed the involvement of horizontal gene transfer between related strains and recombination events.

Production and characterization of medium-chain-length polyhydroxyalkanoates by Pseudomonas mosselii TO7.

The polyhydroxyalkanoate (PHA) production and growth of Pseudomonas mosselii TO7, a newly isolated Pseudomonas species from the wastewater of a vegetable oil manufacturing facility, was analyzed. Phenotypic analysis and phylogenetic analysis of the 16S rRNA gene revealed that it is closely related to Pseudomonas mosselii. In the presence of palm kernel and soybean oils, P. mosselii TO7 produced up to 50% cell dry weight (CDW) medium-chain-length (MCL) PHAs comprising high poly(3-hydroxyoctanoate) (P(3HO)) content; P(3HO) content increased to 45% CDW when grown in octanoate using a single-step culture process. The PHA monomer was identified by (13)C nuclear magnetic resonance spectroscopy. The average molecular weight and polydispersity index of PHA were 218.30 ± 31.73 and 2.21 ± 0.18, respectively. The PHA produced by P. mosselii TO7 in the presence of palm kernel oil had two melting temperature (Tm) values of 37.2°C and 55.7°C with melting enthalpy (ΔHm) values of 51.09 J g(-1) and 26.57 J g(-1), respectively. Inhibition analyses using acrylic and 2-bromooctanoic acids revealed ß-oxidation as the primary pathway for MCL-PHA biosynthesis using octanoic acid. Moreover, Pseudomonas putida GPp104 PHA(-), harboring the PHA synthase genes of P. mosselii (phaC1pm and phaC2pm) was used for heterologous expression, which demonstrated that phaC1pm is the main PHA synthesis enzyme, and 3-hydroxyoctanoyl-CoA is its major substrate. This was the first report of a P. mosselii TO7 isolate producing high-yield P(3HO) through utilization of plant oils.

Quantitative approach to track lipase producing Pseudomonas sp. S1 in nonsterilized solid state fermentation.

UNLABELLED: Proliferation of the inoculated Pseudomonas sp. S1 is quantitatively evaluated using ERIC-PCR during the production of lipase in nonsterile solid state fermentation an approach to reduce the cost of enzyme production. Under nonsterile solid state fermentation with olive oil cake, Pseudomonas sp. S1 produced 57·9 IU g(-1) of lipase. DNA fingerprints of unknown bacterial isolates obtained on Bushnell Haas agar (BHA) + tributyrin exactly matched with that of Pseudomonas sp. S1. Using PCR-based enumeration, population of Pseudomonas sp. S1 was proliferated from 7·6 × 10(4) CFU g(-1) after 24 h to 4·6 × 10(8) CFU g(-1) after 96 h, which tallied with the maximum lipase activity as compared to control. Under submerged fermentation (SmF), Pseudomonas sp. S1 produced maximum lipase (49 IU ml(-1) ) using olive oil as substrate, while lipase production was 9·754 IU ml(-1) when Pseudomonas sp. S1 was grown on tributyrin. Optimum pH and temperature of the crude lipase was 7·0 and 50°C. Crude enzyme activity was 71·2% stable at 50°C for 360 min. Pseudomonas sp. S1 lipase was also stable in methanol showing 91·6% activity in the presence of 15% methanol, whereas 75·5 and 51·1% of activity were retained in the presence of 20 and 30% methanol, respectively. Thus, lipase produced by Pseudomonas sp. S1 is suitable for the production of biodiesel as well as treatment of oily waste water. SIGNIFICANCE AND IMPACT OF STUDY: This study presents the first report on the production of thermophilic organic solvent tolerant lipase using agro-industry waste in nonsterile solid state fermentation. Positive correlation between survival of Pseudomonas sp. S1 and lipase production under nonsterile solid state fermentation was established, which may emphasize the need to combine molecular tools and solid state fermentation in future studies. Our study brings new insights into the lipase production in cost-effective manner, which is an industrially relevant approach.

Enriched microbial community in bioaugmentation of petroleum-contaminated soil in the presence of wheat straw.

The bioaugmentation of petroleum-contaminated soil using Enterobacter cloacae was profiled from the evolution of microbial community, soil dehydrogenase activity, to the degradation of petroleum contaminants. The seeding and proliferation of inoculant and the consequential microbial community were monitored by denaturing gradient gel electrophoresis analysis of the amplification of V3 zone of 16S rDNA. Degradation process kinetics was characterized by the degradation ratio of nC17 to nC18. The dehydrogenase activity was also determined during the degradation process. An abrupt change in the microbial community after inoculation was illustrated as well as successive changes in response to degradation of the petroleum contaminants. Seeding with E. cloacae stimulated the growth of other degrading stains such as Pseudomonas sp. and Rhodothermus sp. The application of wheat straw as a representative lignin waste, at 5% (w/w), induced an increase in the total dehydrogenase activity from 0.50 to 0.79, an increase in the microbial content of 130% for bacteria and 84% for fungi, and an increase of the overall degradation ratio from 44% to 56% after 56 days of treatment. The above mentioned results have provided a microbial ecological insight being essential for the design and implementation of bioaugmentation processes.

Use of immobilized Pseudomonas sp. as whole cell catalyst for the transesterification of used cotton seed oil.

Lipase enzyme producing bacteria, Pseudomonas sp., have been grown at varying oil concentrations to make it adaptive for high oil concentrations and it was found to show the maximum growth and maximum lipase activity when 40- and 30-vol% of oil respectively was used as a source of carbon in growth medium. Bacteria was immobilized with sodium alginate and used as whole cell catalyst for the transesterification of used cotton seed oil. Preliminary experiments resulted about 70% transesterification of used cotton seed oil with methanol as calculated by proton NMR technique.

Pseudomonas diversity in crude-oil-contaminated intertidal sand samples obtained after the Prestige oil spill.

The Galicia seashore, in northwestern Spain, was one of the shorelines affected by the Prestige oil spill in November 2002. The diversity of autochthonous Pseudomonas populations present at two beaches (Carnota municipality) was analyzed using culture-independent and culture-dependent methods. The first analysis involved the screening of an rpoD gene library. The second involved the isolation of 94 Pseudomonas strains that were able to grow on selective media by direct plating or after serial enrichments on several carbon sources: biphenyl, gentisate, hexadecane, methylnaphthalene, naphthalene, phenanthrene, salicylate, xylene, and succinate. Eight denitrifying Pseudomonas strains were also isolated by their ability to grow anaerobically with nitrate. The calculated coverage index for Pseudomonas species was 89% when clones and isolates were considered together, and there were 29 phylospecies detected. The most abundant were members of the species P. stutzeri, P. putida, P. anguilliseptica, and P. oleovorans. Thirty-one isolates could not be identified at the species level and were considered representatives of 16 putative novel Pseudomonas species. One isolate was considered representative of a novel P. stutzeri genomovar. Concordant results were obtained when the diversities of the cloned DNA library and the cultured strains were compared. The clone library obtained by the rpoD PCR method was a useful tool for evaluating Pseudomonas communities and also for microdiversity studies of Pseudomonas populations.