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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 Hg(2+) 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.

The fermentative production of biosurfactants by five Bacillus strains in a bench-scale bioreactor and evaluation of biosurfactant-based enhanced oil recovery using sand pack columns were investigated. Adjusting the initial dissolved oxygen to 100% saturation, without any further control and with collection of foam and recycling of biomass, gave higher biosurfactant production. The microorganisms were able to produce biosurfactants, thus reducing the surface tension and interfacial tension to 28 mN/m and 5.8–0.5 mN/m, respectively, in less than 10 hours. The crude surfactant concentration of 0.08–1.1 g/L, and critical micelle concentration (CMC) values of 19.4–39 mg/L, corresponding to the biosurfactants produced by the different Bacillus strains, were observed. The efficiency of crude biosurfactant preparation obtained from Bacillus strains for enhanced oil recovery, by sand pack column studies, revealed it to vary from 30.22–34.19% of the water flood residual oil saturation. The results are indicative o...

Nitrosomonas genus belongs to beta-subclass of Proteobacteria and encompasses closely related species. Sequence independent techniques like single strand confirmation polymorphism (SSCP) was attempted in the present study to resolve AOB using ammonia monooxygenase (amoA) and hydroxylamine oxidoreductase (hao) gene fragments, unique to AOB. Variation in hydroxylamine oxidoreductase (HAO) enzyme zymogram of isolates observed in the study was also explored as an additional sequence independent method to substantiate the observations. Nitrosomonas europaea (standard strain) and 12 isolates, obtained by enriching environmental samples, were differentiated into six and four groups by SSCP analyses of amoA and hao gene fragments, respectively, whereas they could be resolved into six distinct groups through activity staining of HAO enzyme. amoA gene fragment was therefore found to be better than hao gene fragment in resolving the studied AOB based on richness and evenness with Simpson's index of diversity - 0.85. However, the ensembled use of these molecular methods (SSCP of amoA and hao gene fragments) and HAO enzyme zymogram in fingerprinting AOB provide better resolution and evenness, contributing significantly in AOB diversity studies. Grouping of AOB isolates by hao gene SSCP analysis followed almost the same pattern as that by 16S rRNA gene based sequence analysis, hence it is suitable as a phylogenetic marker.

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 46.6 mg/L COD at 2.31 days 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.

Cloning and expression of two siderophore receptor genes, pfeA , and fpvA of Pseudomonas aeruginosa origin, encoding outer membrane receptor for enterobactin, a catechol type siderophore and for pyoverdin, a mixed ligand type siderophore, respectively, were studied in two Cajanus cajan rhizobial isolates, IC3123 and ST1. Expression of an approximately 80/89 kDa protein band was seen in all the transformants while the corresponding parental strains failed to express the same. Functionality of the construct, pAKEnt (plasmid containing pfeA ) and pAKFpvA (plasmid harbouring fpvA ), was checked by transforming it in P. aeruginosa K407Δ pfeA and P. aeruginosa K437Δ fpvA , respectively and monitoring phenotypic complementation. Positive cross utilization of cognate siderophores by respective transformant and failure to utilize the same by corresponding parent strains, confirmed the expression of the respective genes. The constructs, pAKEnt and pAKFpvA, were mobilized in to Rhizobium sp. strains IC3123 and ST1 and expression of the corresponding gene in transformants led to utilization of the respective siderophores and stimulation in the growth of the transformants in the presence of respective siderophore producing strains in a co-inoculation study. Peanut and pigeon pea plants inoculated with transformants harbouring pAKEnt and pAKFpvA constructs, not only showed better rhizospheric colonization but also showed better plant growth with respect to increase in root weight, shoot weight, nodule number and chlorophyll content as compared to the parental strains. Amongst the two constructs, pAKEnt showed relatively higher increase in growth response as compared to pAKFpvA, which could be attributed to higher affinity of enterobactin for Fe 3+ ( K = 10 52 M −1 ) as compared to pyoverdine ( K = 10 32 M −1 ).

Osmolytes KCl, glycerol, mannitol, trehalose, sucrose, betaine, proline and Na-glutamate at different concentrations (5-30%) were investigated as effective solutes for retaining the activity of Halobacterium sp. SP1(1) protease in the absence of NaCl. Maximum activity was observed in the presence of 30% Na-glutamate. Kinetic and thermodynamic parameters for casein hydrolysis revealed that the protease was equally efficient in the presence of Na-glutamate as in NaCl. The enzyme was active over a broader range of temperature (20-80 degrees C) and was highly stable even at 80 degrees C with Na-glutamate. Thermodynamic parameters (DeltaH*, DeltaS*, G*) for irreversible inactivation of protease at different temperatures (20-80 degrees C) were determined in the presence of Na-glutamate and NaCl. The efficiency of these osmolytes for thermal stability of protease was 30% (1.6 M) Na-glutamate4 M ( approximately 25%) NaCl2 M (approximately 10%), suggesting that the effect exerted by the osmolyte depends not only on its chemical nature but also on its concentration. Na-glutamate was thus found to play an important role in thermal stabilization of enzyme substituting for NaCl. Moreover, substitution of NaCl by Na-glutamate may increase the applicability of halophilic enzymes in biotechnology and industry, which is otherwise limited to high NaCl concentrations.

Superoxide dismutase (SOD) and catalase expression is associated with oxidative stress. Existing techniques for the individual staining of SOD and catalase have been described in the past. The objective of this study was to achieve a simple and rapid technique for the double staining of bacterial SOD and catalase on the same polyacrylamide gel. SOD detection was carried out using nitro-blue tetrazolium (NBT) dye reduction followed by ferricyanide precipitation for negative staining of the catalase enzyme on the same gel. The staining procedure resulted in pale blue SOD bands while catalase appeared as yellow bands against a greenish blue background on the same gel. This technique was used to detect changes in the polymorphic forms of these enzymes in Deinococcus radiodurans R1 and Kocuria sp. C2 subjected to stresses like UV and gamma radiation and desiccation. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

The inability to utilize a fungal siderophore as source of iron nutrition by most of the rhizobial cultures isolated from pigeon pea, could be considered a negative fitness factor since hydroxamate siderophores are found in significant amounts in natural soils. Thus these cultures were engineered to use ferrichrome a prototype of hydroxamate type siderophore. Pigeon pea Rhizobium spp. IC3123 and ST1 harboring Escherichia coli fhuA gene, responsible for uptake of Fe3+-ferrichrome, were obtained by transformation with pGR1, a broad host range plasmid carrying the fhuA gene under the control of the lac promoter of E. coli. Expression of fhuA in transformed rhizobial strains IC3123::pGR1 and ST1::pGR1 was confirmed by the ability of the plasmid-bearing strains to utilize iron bound to ferrichrome. Inoculation of pigeon pea plants with fhuA expressing rhizobial strains in pot experiments showed a significant increase in plant growth as well as nodule density as compared to those inoculated with the parent as well as the empty vector-bearing strain. Inoculation of pigeon pea seedlings with IC3123::pGR1 and ST1::pGR1 led to marked increase in shoot fresh weight, nodule number per plant, chlorophyll content of leaves and effective nodule symbiosis when compared with plants inoculated with the parent strains IC3123 and ST1. The positive effect of IC3123::pGR1 and ST1::pGR1 treatment on plant growth was more significantly observed when ferrichrome producing Ustilago maydis, known to secrete ferrichrome, was co-inoculated along with the transformed rhizobia. The presence of fhuA gene in rhizobial strains also led to an increased survival and root colonization.

Problem statement: Rhizobial isolates belonging to genera (Rhizobium sp. and Mesorhizobium sp.) in our laboratory produced only catecholate type of siderophores. Although FhuA and FegA (ferrichrome receptors) homologs were found to be present in the sequenced genomes of few rhizobia (e.g., 1 in R. etli and 2 in Mesorhizobium sp. BNC1), laboratory isolates of the corresponding genera failed to utilize ferrichrome, a siderophore which is present in nanomolar concentrations in the soil. This inability was considered as a negative fitness factor with respect to rhizospheric colonization by these rhizobia. Approach: The 2.4 kb fegA gene (encoding ferrichrome receptor) was amplified along with its native promoter from Bradyrhizobium japonicum 61A152 and cloned in a broad host range plasmid vector pUCPM18. The plasmid construct pFJ was transferred by conjugation into Rhizobium sp. ST1 to give transconjugant ST1pFJ12. The consequence of FegA expression on the transconjugant was tested under lab and soil conditions, using physiological experiments. Results: Ability of the transconjugant ST1pFJ12 to utilize ferrichrome and expression of a 79 kD protein band on the outer membrane of the transconjugant confirmed FegA expression. Transconjugant ST1pFJ12 exhibited increased growth rate as compared to the parent strain ST1, in minimal media containing ferrichrome as the sole iron source, confirming the positive effect of FegA expression. Inoculation of pigeon pea seedlings with transconjugant ST1pFJ12 led to a marked increase in plant growth parameters as compared to plants inoculated with the parent strain ST1, the effect being more pronounced when Ustilago maydis, a ferrichrome producer was co-inoculated in the systems. Nodule occupancy on pigeon pea plant when inoculated with the transconjugant ST1pFJ12 alone was 57% which increased to 66% when co-inoculated with U. maydis as compared with 37 and 30% respectively, seen with parental strain ST1 inoculation. Conclusion: The clear increase in nodule occupancy and higher rhizospheric colonization by the fegA transconjugants, presented in this study together with the previous research reported from our laboratory, led us to conclude that ferrichrome utilization ability played an important role in the rhizospheric colonization of the bioinoculant strains. Testing the ability to utilize hydroxamate siderophores therefore, holds prime importance in selecting an efficient biofertilizer strain.

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.

Mesorhizobium sp. GN25 failed to utilize ferri-ferrichrome as iron source. The fegA gene (encoding ferrichrome receptor in Bradyrhizobium japonicum 61A152) was amplified and cloned in a broad host range expression vector. The plasmid construct (pFJ) was transformed into Escherichia coli MB97ΔfhuA and transferred by conjugation into Mesorhizobium sp. GN25 and the resulting strains were named MB97pFJ and GN25pFJ4, respectively. Ability to cross-utilize ferrichrome and presence of a 79-kDa protein band in the outer membranes of GN25pFJ4 and MB97pFJ confirmed the expression of FegA on the outer membrane. Rhizobial transconjugant GN25pFJ4 showed growth stimulation when grown in the presence of pure ferrichrome as well as upon co-inoculation with ferrichrome producing Ustilago maydis, as compared to the parent strain Mesorhizobium sp. GN25, confirming positive effect of fegA expression. Peanut plant inoculation with GN25pFJ4 led to marked increase in plant growth parameters. Nodule occupancy on peanut plant when inoculated with GN25pFJ4 alone was 61% which increased to 66% when co-inoculated with U. maydis as compared with 50%, shown by the parent strain GN25 alone or in co-inoculation with U. maydis. A clear increase in nodule occupancy upon peanut plant inoculation of GN25pFJ4, alone and in combination with U. maydis indicated that ferrichrome utilization ability may play a role in increasing the rhizospheric colonization of the bioinoculants. Our results provide evidence that engineering rhizobial strains to utilize heterologous siderophores could provide them with better iron acquisition ability and consequently, rhizospheric stability.

Methylobacterium sp. ZP24 produced polyhydroxybutyrate (PHB) from disaccharides like lactose and sucrose. As Methylobacterium sp. ZP24 showed growth associated PHB production, an intermittent feeding strategy having lactose and ammonium sulfate at varying concentration was used towards reaching higher yield of the polymer. About 1.5-fold increase in PHB production was obtained by this intermittent feeding strategy. Further increase in PHB production by 0.8-fold could be achieved by limiting the dissolved oxygen (DO) levels in the fermenter. The decreased DO is thought to increase flux of acetyl CO-A towards PHB accumulation over TCA cycle. Cheese whey, a dairy waste product and being a rich source of utilizable sugar and other nutrients, when used in the bioreactor as a main substrate replacing the lactose, led to further increase in the PHB production by 2.5-fold. A total of 4.58-fold increase in the PHB production was obtained using limiting DO conditions with processed cheese whey supplemented with ammonium sulfate in fed batch culture of Methylobacterium sp. ZP24. The present investigation therefore reflects on the possibility of developing a cheap biological route for production of green thermoplastics.

Nodule isolates from the cowpea miscellany group of legumes produced varying concentrations of catecholate and hydroxamate types of siderophores under iron-limiting conditions. The nodule isolates differed with respect to siderophore cross-utilizing abilities; some were proficient at using siderophores of other nodule isolates (homologous siderophores) while others could utilize siderophores produced by other rhizospheric bacteria (heterologous siderophores). Utilization of siderophore of rhizospheric bacterium PsB, a plant pathogen, benefited the nodule isolate G11 in terms of growth under iron-limiting laboratory conditions, while PsB was clearly inhibited in the presence of G11. Plate assays showed that siderophore of G11 could withhold iron from PsB and hence PsB was inhibited in the presence of G11. Isolates G11 and PsB when applied simultaneously to peanut seedlings under sterile soil conditions, provided a clear advantage to the plant in terms of reduction in the inhibitory effect of PsB. The count of the nodule isolate G11 increased in the soil when co-inoculated with PsB, as compared to when inoculated alone. Thus, the increased growth of the plant can be attributed to the iron sequestration and plant growth promoting properties of G11. The isolate G11 could utilize the siderophores produced by many other rhizospheric isolates while the siderophore of G11 was not being utilized by these rhizospheric isolates.

Biosurfactants have gained attention because they exhibit some advantages such as biodegradability, low toxicity, ecological acceptability and ability to be produced from renewable and cheaper substrates. They are widely used for environmental applications for bioremediation and also in biomedical field. However, the high cost of production is the limiting factor for widespread industrial applications. Thus, optimization of the growth medium for biosurfactant-lichenysin production by Bacillus licheniformis R2 was carried out using response-surface methodology. A preliminary screening phase based on a two-level fractional factorial design led to the identification of NH4NO3, glucose, Na2HPO4 and MnSO4·4H2O concentrations as the most significant variables affecting the fermentation process. The 24 full-factorial central composite design was then applied to further optimize the biosurfactant production. The optimal levels of the aforementioned variables were (g/l): NH4NO3, 1.0; glucose, 34.0; KH2PO4, 6.0; Na2HPO4, 2.7; MgSO4·7H2O, 0.1; CaCl2, 1.2 × 10−3; FeSO4·7H2O, 1.65 × 10−3; MnSO4·4H2O, 1.5 × 10−3 and Na–EDTA, 2.2 × 10−3. With the optimization procedure, the relative lichenysin yield expressed as the critical micelle dilution (CMD) was fourfold higher than that obtained in the non-optimized reference medium.

The biosurfactant produced by Bacillus licheniformis R2 was characterized and studied for enhancing the heavy crude oil recovery at 80 °C in coreflood experiments. The strain was found to be nonpathogenic and produced biosurfactant, reducing the surface tension of medium from 70 to 28 mN/m with 1.1 g/l yield. The biosurfactant was quite stable during exposure to elevated temperatures (85 °C for 90 days), high salinity (10 % NaCl), and a wide range of pH (5–12) for 10 days. It was characterized as lipopeptide similar to lichenysin-A, with a critical micelle concentration of about 19.4 mg/l. The efficiency of crude biosurfactant for enhanced oil recovery by core flood studies revealed it to recovering additional 37.1 % oil from Berea sandstone cores at 80 °C. The results are indicative of the potential for the development of lipopeptide biosurfactant-based ex situ microbial enhanced heavy oil recovery from depleting oil fields with extreme temperatures.

Cajanus cajan rhizobial isolates were found to be unable to utilize iron bound to ferrichrome, desferrioxamine B or rhodotorulic acid, all being hydroxamate type siderophores. A broad host range expression vector containing the Escherichia coli fhuA gene, encoding the outer membrane receptor for Fe-ferrichrome, was constructed. The plasmid construct (pGR1), designed to express fhuA under the lac promoter of E. coli, complemented E. coli MB97 DeltafhuA mutant for ferri-ferrichrome utilization and also allowed Rhizobium spp. ST1 and Rhizobium spp. IC3123 to grow using iron bound to ferrichrome. Sensitivity to the antibiotic albomycin, transported via the FhuA receptor, was found in case of MB97 as well as rhizobial transformants harboring pGR1. The rhizobial transformants expressing fhuA showed growth stimulation when co-inoculated with Ustilago maydis, a fungal species known to produce ferrichrome under iron starved conditions. Growth stimulation was also observed in the presence of externally supplied ferrichrome. The significance of these findings in terms of the potential for improving the survivability of rhizobial bioinoculant strains in natural soils is discussed.

Siderophores are ferric specific ligands that are involved in receptor specific iron transport into bacteria. Partially purified siderophores from 37 different rhizospheric bacterial isolates, and 17 Cajanus cajan root nodule isolates were tested for their utilization by the 17 nodule isolates as test organisms. Significant variation in the siderophore cross-utilization pattern was observed. Catecholate siderophores were produced predominantly by nodule isolates whereas rhizospheric isolates produced catecholates, as well as hydroxamates. The quantity of hydroxamate produced was higher than that of catecholates. Considerable variation was observed with respect to siderophore cross-utilization amongst nodule isolates obtained from a particular location. Isolates collected from different nodules of same plant also showed considerable variation with respect to siderophore cross-utilization. Isolates, which showed, higher siderophores cross-utilizing ability, when tested for homologous (from other nodule bacteria) siderophores also gave good cross-utilization of heterologous (from non-nodule rhizospheric bacteria) siderophores. Isolate GHU(iii), the highest cross-utilizer of homologous and heterologous siderophores showed a significant increase in growth in the presence of exogenously added siderophores. Similar results were observed during the co-inoculation studies with rhizospheric isolate under iron-limited condition. Both isolates GAU(1)1 and AP(6)4, low cross-utilizers of siderophores, failed to show growth increase in response to co-inoculation and addition of siderophores. The iron regulated outer membrane protein (IROMP) profiles of strains showing maximum or least cross-utilization did not differ. The significance of these findings in context to survivability under natural conditions and the possible reasons accounting for high siderophore cross-utilizing status of isolate GHU(iii) is discussed.

A bioreactor for the removal of nitrate nitrogen (NO3-N) from industrial effluent is described which is comprised of a glass column (60 cm × 6 cm) packed with alginate beads containing denitrifying organisms Pseudomonas stutzeri and Comamonas testosteroni. The effluent containing high concentrations of nitrate (600–950 mg l−1) from the fertilizer industry and fusel oil (methanol as a major component) as organic carbon were used in the process. The reactor is operated in the continuous mode by injecting the pretreated nitrate-containing effluent at the top of the column. The Hydraulic retention time (HRT) was adjusted by changing the flow rates. When nitrate-containing wastewater was treated with immobilized cells, the nitrate removal rate reached a maximum 1.66 ± 0.07 Kg NO3-N m−3d−1 at an influent NO3-N concentration of 850 mg NO3M-N l−1within 12 h. The denitrification activity of the immobilized cells was compared with that of the free cells.

The extracellular poly(β-hydroxybutyrate) (PHB) depolymerase of Aspergillus fumigatus Pdf1 was purified by a new, simple, one-step affinity chromatography method using the substrate PHB. The purified enzyme was glycosylated, with the molecular mass of ≃40 KD, and exhibited a novel self-aggregation behavior by means of hydrophobic interaction that was resolved by Triton X-100 (TX-100) pretreatment of enzyme and also TX-100 incorporation in the native gel. The apparent Km value of purified enzyme for PHB was 119 μg/mL and 3-hydroxybutyrate was detected as the main endproduct of PHB hydrolysis. The depolymerase was insensitive to phenylmethyl sulfonyl fluoride (PMSF), sodium azide, ethylenediaminetetraacetic acid (EDTA), and para-chloromercuric benzoic acid (PCMB), but was inactivated by dithioerythritol (DTT) and showed specificity for short chain-length poly(β-hydroxyalkanoates) (PHAs) such as PHB, poly(hydroxyvalerate) (PHV), and copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). Medium-chain-length PHA failed to get hydrolyzed. The enzyme, however, exhibited strong cross reactivity with the Comamonas sp. PHB depolymerase antibodies, but not with PHV depolymerase antibodies of Pseudomonas lemoignei. Southern hybridization and dot blot analysis of A. fumigatus Pdf1 genomic DNA with alkaline phosphatase labeled probes of P. lemoignei PHB and PHV depolymerase genes revealed no homology, although the enzyme hydrolyzed both PHB and PHV.

Production of the polymer, poly 3-hydroxybutyrate (PHB), by Methylobacterium sp. ZP24 increased when sucrose-containing medium was partially substituted with low concentrations of several organic acids. Low concentrations of formate could enhance the PHB yield 2·4-fold and bring down the production time by 12 h. The use of formate as a growth enhancer could be successfully scaled up to the 18 litre level. The possible mechanism of enhancement is discussed.

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.

Cowpea miscellany group of rhizobia are generally broad host range. Transconjugants of these cowpea rhizobial isolates having nodABC-lacZ fusion were monitored for flavonoid/root exudate-induced activation of the nod genes in terms of beta-galactosidase activity, thus determining the potential host range of the rhizobial isolates.

Slow-growing cowpea Rhizobium sp. S2, a pigeon pea isolate, showed excessive synthesis of exopolysaccharide (EPS) and nod factor(s) only when grown in the presence of corresponding host root exudate (RE) or naringenin (a flavonoid present in RE of pigeon pea). muc- strain, a plasmid-cured derivative of the parent strain, showed negligible EPS production and failed to synthesize the nod factor(s) under similar growth conditions. The nod factor(s) was extracted and partially purified from the total EPS of the culture supernatant of S2 grown in the presence of either RE or naringenin by the butanol-soluble, ethyl acetate-insoluble method. The parent strain also showed ex-planta nitrogenase activity, whereas in muc- it was not detectable. In contrast to observations of the location of nod, nif- and EPS-synthesizing genes on the chromosome in commonly studied cowpea Rhizobia and Bradyrhizobium sp. colony blot and dot blot hybridization studies revealed the presence of nod (common nod genes), nif (nif KDH); and EPS (pss) determinants to be on the large plasmid in the parent strain and therefore absent in muc-, which is a plasmid-cured derivative.

A petrochemical wastewater isolate, capable of utilizing high concentrations of acetonitrile and acetamide as the sole source of carbon and nitrogen was identified as Rhodococcus erythropolis A10. Cell-free extracts of acetonitrile-grown cells exhibited activities corresponding to nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4), which mediate the two-step breakdown of acetonitrile into acetic acid and ammonia. Studies indicated that both these enzymes in R. erythropolis A10 are intracellular, inducible and capable of hydrolysing a wide range of nitriles, including simple (acetonitrile, propionitrile), branched-chain (isobutyronitrile) and dinitrile (succinonitrile). The specific activity of the amidase was found to be several-fold higher than nitrile hydratase.

Polyethylene glycol 6000 mediated protoplast fusion between an alkane degrader Acinetobacter sp. A3, and a naphthalene degrader, Pseudomonas putida DP99 , resulted in fusants capable of degrading both hydrocarbons and were morphologically similar to Acinetobacter sp. A3. While fusant F4/13 and Pseudomonas putida DP99 degraded over 98% of naphthalene provided by the end of five days, tetradecane degradation by fusant F4/13 was 82% compared to 77% by Acinetobacter sp. A3 in the same time period. Also, while from naphthalene +tetradecane mixture, fusant F4/13 could degrade 99% and 53% of naphthalene and tetradecane respectively, both the parent strains together could degrade over 99% naphthalene but only about 16% tetradecane.

The utilization of Bombay High crude oil by Acinetobacter sp. A3 was not accompanied by the production of any surface active agents. Uptake of crude oil was therefore thought to be mediated by direct cell-oil interaction as the cell surface was hydrophobic in nature. SDS-PAGE of the outer membrane proteins of Acinetobacter sp. A3 grown on crude oil as sole source of carbon showed induction of two proteins/polypeptides of approximate molecular mass of 26.5 kDa and 56 kDa.

Siderophore produced by cowpea Rhizobium GN1 (Peanut isolate) was shown to be involved in iron uptake by this organism. Siderophore enhanced iron uptake in iron-starved cells. SDS-PAGE analysis of the outer membrane proteins showed two iron repressible outer membrane proteins with approximate molecular mass of 80 kDa and 76 kDa. A siderophore non-producing mutant, which was unable to grow on a medium containing synthetic iron chelators unless and until iron was added exogenously in the medium, could use siderophore of the wild-type for iron uptake indicating that the receptor for Fe-siderophore complex was intact in the mutant.

Conventional primary and secondary oil recovery processes recover only one third of the oil present in the oil reservoir. EOR technologies produce an additional oil recovery after these processes cease to produce oil. Microbial enhanced oil recovery (MEOR) includes the tertiary oil recovery process where microorganisms or their metabolites are used to retrieve the unrecoverable oil from oil reservoirs. Oil recovery mechanisms facilitated by microorganisms are attributed to the metabolites produced by them. Biosurfactants, biomass, biopolymers, solvents, acids, gases, etc. are notably the products of microbial growth in the reservoir. MEOR approaches are either in situ or ex situ. In in situ approach growth of the indigenous microbes is stimulated or isolated microbes having specific traits are injected into oil reservoir along with the nutrients where production of the metabolites causes oil release. The approach where microbes are cultivated outside in a fermentor on inexpensive raw materials and they and/or their crude metabolites, viz. biosurfactant, biopolymer, etc. are injected into oil reservoir in order to retrieve the unrecoverable oil, is known as ex situ oil recovery. The technical feasibility of oil recovery was assessed in porous model sand packed columns with biosystems developed separately comprising of the specially isolated B. licheniformis TT42, K125 and TT33. The crude biosurfactant of B. licheniformis K125 gave 43 ± 3.3% additional oil recovery upon application to sand column which is 13.7% and 8.5% higher in comparison to the standard strains, Bacillus mojavensis JF-2 and Bacillus licheniformis TT42 respectively. B. licheniformis when tested in sand packed column by injecting the biomass and the nutrients gave 27.7 ± 3.5% oil recovery. Lack of holistic approach allowing for a critical evaluation of economics, applicability and performance of MEOR is missing. For a thorough assessment of this process a multidisciplinary approach is warranted.

Iron is critical for life and poses the living system with a number of challenges. Its low solubility in the environment is of major concern which tends to create problems during its acquisition and transport. Its rich redox chemistry that underlies many of iron’s essential biological functions has resulted in highly evolved mechanisms of iron transport in living system. Iron, although available in plenty in rhizospheric soil, is seldom in its bioavailable form at neutral and alkaline pH, hence there always prevails an “iron stressed” condition. Siderophore is the Greek phrase for “iron bearer” and is applied to molecules that can bind metal at very high affinity. One of the major mechanisms evolved for iron acquisition to combat the iron-stressed condition is synthesis of high-affinity siderophore-mediated iron transport systems. Two main components – siderophore production, and synthesis of membrane receptor molecules which mediate internalization of iron bound to siderophores – form part of this high-affinity transport system. Better exploration and exploitation of soil resources require increasing the efficiency of nutrient uptake by plants/microorganisms and decreasing nutrient augmentation in the soil from outside. Achieving this condition requires understanding of intimate processes and factors that govern nutrient availability to plants and microorganisms. The present chapter addresses this issue with special emphasis on siderophore-mediated iron acquisition system, the significance of iron–siderophore affinity in niche colonization by specific group of microorganisms, significance of possessing diverse ferri-siderophore uptake system and its impact in competitive survival of microorganisms in the rhizosphere, and how all these factors contribute to the plant growth.

A rapid and simple technique has been developed for screening potential crude oil degrading bacteria using the redox indicator 2,6-dichlorophenol indophenol in Bushnell and Haas medium with crude oil and a microtitre plate. Bacteria possessing high crude oil degrading potential turn the medium colourless after 24 h incubation but, depending upon the time taken for the change in colour, relative abilities of different cultures can be ascertained.

Surfactants and biosurfactants are amphipathic molecules with both hydrophilic and hydrophobic moieties that partition preferentially at the interface between fluid phases that have different degrees of polarity and hydrogen bonding which confers excellent detergency, emulsifying, foaming and dispersing traits, making them most versatile process chemicals. One of the major applications of (bio)surfactants is in environmental bioremediation field. Most synthetic organic compounds present in contaminated soils are only weakly soluble or completely insoluble in water, so they exist in the subsurface as separate liquid phase, often referred as a non-aqueous phase liquids (NAPL), which poses as threat to environment. Several studies have revealed the use of surfactants for remediation; however, several factors limit the use of surfactants in environmental remediation, mainly persistence of surfactants or their metabolites and thus potentially pose an environmental concern. Biosurfactants may provide a more cost-effective approach for subsurface remediation when used alone or in combination with synthetic surfactants. There are several advantages of biosurfactants when compared to chemical surfactants, mainly biodegradability, low toxicity, biocompatibility and ability to be synthesized from renewable feedstock. Despite having many commercially attractive properties and clear advantages compared with their synthetic counterparts, biosurfactants have not yet been employed extensively in industry because of their low yields and relatively high production and recovery costs. However, the use of mutants and recombinant hyperproducing microorganisms along with the use of cheaper raw materials and optimal growth and production conditions and more efficient recovery processes, the production of biosurfactant can be made economically feasible. Therefore, future research aiming for high-level production of biosurfactants must be focused towards the development of appropriate combinations of hyperproducing microbial strains, optimized cheaper production media and optimized process conditions, which will lead to economical commercial level biosurfactant production.

Surfactants and biosurfactants are amphipathic molecules with both hydrophilic and hydrophobic moieties that partition preferentially at the interface between fluid phases that have different degrees of polarity and hydrogen bonding which confers excellent detergency, emulsifying, foaming and dispersing traits, making them most versatile process chemicals. One of the major applications of (bio)surfactants is in environmental bioremediation field. Most synthetic organic compounds present in contaminated soils are only weakly soluble or completely insoluble in water, so they exist in the subsurface as separate liquid phase, often referred as a non-aqueous phase liquids (NAPL), which poses as threat to environment. Several studies have revealed the use of surfactants for remediation; however, several factors limit the use of surfactants in environmental remediation, mainly persistence of surfactants or their metabolites and thus potentially pose an environmental concern. Biosurfactants may provide a more cost-effective approach for subsurface remediation when used alone or in combination with synthetic surfactants. There are several advantages of biosurfactants when compared to chemical surfactants, mainly biodegradability, low toxicity, biocompatibility and ability to be synthesized from renewable feedstock. Despite having many commercially attractive properties and clear advantages compared with their synthetic counterparts, biosurfactants have not yet been employed extensively in industry because of their low yields and relatively high production and recovery costs. However, the use of mutants and recombinant hyperproducing microorganisms along with the use of cheaper raw materials and optimal growth and production conditions and more efficient recovery processes, the production of biosurfactant can be made economically feasible. Therefore, future research aiming for high-level production of biosurfactants must be focused towards the development of appropriate combinations of hyperproducing microbial strains, optimized cheaper production media and optimized process conditions, which will lead to economical commercial level biosurfactant production.

Azospirillum lipoferum M was found to produce catechol-type of siderophores under iron-starved conditions. Chemical characterization of siderophores revealed the presence of salicylic acid, 2,3-dihydroxybenzoic acid (DHBA), and 3,5-DHBA conjugated with threonine and lysine. Siderophore production was found to be maximum after 28 h of growth. In addition to their established role in iron transport, the siderophores exhibited antimicrobial activity against various bacterial and fungal isolates.

In an iron-depleted broth culture of cowpeaRhizobium (a peanut isolate), phenolate type of compounds were detected. Chemical characterization showed the presence of 2,3-dihydroxy benzoic acid (DHBA) and 3,4-DHBA in the siderophore extract. Lysine and alanine were identified as conjugated amino acids of the siderophore. Maximum concentration of the siderophore in the culture supernatant was found after 24 h of growth. The compounds in the extracted siderophore induced growth ofRhizobium in a medium containing EDTA. Addition of lysine and alanine in the growth medium (20 mM each) led to a fourfold increase in siderophore production.

Aims: Application of Halobacterium sp. SP1(1) for the acceleration of fish sauce fermentation. Methods and Results: Traditional fish sauce fermentation was mimicked using Halobacterium sp. SP1(1) as starter culture. Protease activity, peptide release and α-amino content (parameters used to monitor the progress of the fermentation) were high at day 10 in tests and day 20 in un-inoculated controls. The total protein and nitrogen contents were also high in tests compared with controls. The amino acid profile observed at the end of fermentation in experimental samples, when compared with the commercial sauce preparation, was found to be better with respect to flavour and aroma contributing amino acids as well as essential amino acid lysine. Microflora analysis of the final fish sauce revealed the absence of any nonhalophilic or halotolerant micro-organisms. The protease-producing halophilic isolates obtained from the fish sauce of eviscerated and uneviscerated controls were identified as Halobacterium sp. F1 and F2, respectively, by 16S rDNA sequence analysis. Conclusions: Exogenous augmentation of Halobacterium sp. SP1(1) accelerated the fish sauce fermentation process with an additive effect on the existing natural microflora present in the fish during fermentation. Halobacterium sp SP1(1), therefore, can be used as an important starter culture for accelerating the fish fermentation process, which is attributed to its extracellular protease. Significance and Impact of the Study: The present study is the first report on use of Halobacterium species as a starter culture for accelerating fish sauce fermentation. Use of halobacterial starter cultures may revolutionize the process in fish sauce industries by reducing the fermentation time and making the process more economical with improved nutritive value of product.

Rhodococcus erythropolis A10 metabolizes acetonitrile by a two step process involving nitrile hydratase (NHase) and amidase. Both the enzymes were inducible and low basal levels of activities were observed in the cells grown in the absence of acetonitrile (AN). Cobalt and iron enhanced NHase, while amidase showed iron dependence. Presence of glucose or ammonium sulphate (AS) failed to affect acetonitrile utilization.

Aims: Optimization of medium components for extracellular protease production by Halobacterium sp. SP1(1) using statistical approach. Methods and Results: The significant factors influencing the protease production as screened by Plackett–Burman method were identified as soybean flour and FeCl3. Response surface methodology such as central composite design was applied for further optimization studies. The concentrations of medium components for higher protease production as optimized using this approach were (g l−1): NaCl, 250; KCl, 2; MgSO4, 10; tri-Na-citrate, 1·5; soybean flour, 10 and FeCl3, 0·16. This statistical optimization approach led to production of 69·44 ± 0·811 U ml−1 of protease. Conclusions: Soybean flour and FeCl3 were identified as important factors controlling the production of extracellular protease by Halobacterium sp. SP1(1). The statistical approach was found to be very effective in optimizing the medium components in manageable number of experimental runs with overall 3·9-fold increase in extracellular protease production. Significance and Impact of the Study: The present study is the first report on statistical optimization of medium components for production of haloarchaeal protease. The study also explored the possibility of using extracellular protease produced by Halobacterium sp. SP1(1) for various applications like antifouling coatings and fish sauce preparation using cheaper raw material.

The nutritional medium requirement for biosurfactant production by Bacillus licheniformis K51 was optimized. The important medium components, identified by the initial screening method of Plackett-Burman, were H3PO4, CaCl2, H3BO3, and Na-EDTA. Box-Behnken response surface methodology was applied to further optimize biosurfactant production. The optimal concentrations for higher production of biosurfactants were (g/l): glucose, 1.1; NaNO3, 4.4; MgSO4 x 7H2O, 0.8; KCl, 0.4; CaCl2, 0.27; H3PO4, 1.0 ml/l; and trace elements (mg/l): H3BO3, 0.25; CuSO4, 0.6; MnSO4, 2.2; Na2MoO4, 0.5; ZnSO4, 6.0; FeSO4, 8.0; CoCl2, 1.0; and Na-EDTA, 30.0. Using this statistical optimization method, the relative biosurfactant yield as critical micelle dilution (CMD) was increased from 10x to 105x, which is ten times higher than the non-optimized rich medium.

A biosurfactant producing strain, Bacillus subtilis 20B, was isolated from fermented food in India. The strain also showed inhibition of various fungi in in-vitro experiments on Potato Dextrose Agar medium. It was capable of growth at temperature 55 degrees C and salts up to 7%. It utilized different sugars, alcohols, hydrocarbons and oil as a carbon source, with preference for sugars. In glucose based minimal medium it produced biosurfactant which reduced surface tension to 29.5 mN/m, interfacial tension to 4.5 mN/m and gave stable emulsion with crude oil and n-hexadecane. The biosurfactant activity was stable at high temperature, a wide range of pH and salt concentrations for five days. Oil displacement experiments using biosurfactant containing broth in sand pack columns with crude oil showed 30.22% recovery. The possible application of organism as biocontrol agent and use of biosurfactant in microbial enhanced oil recovery (MEOR) is discussed.

Endophytic bacteria which are known to reside in plant tissues have often been shown to promote plant growth. Present study deals with the isolation of putative endophytes from the surface sterilized root nodules of pigeon pea (Cajanus cajan) designated as non-rhizobial (NR) isolates. Three of these non-rhizobial isolates called NR2, NR4 and NR6 showed plant growth promotion with respect to increase in plant fresh weight, chlorophyll content, nodule number and nodule fresh weight when co-inoculated with the rhizobial bioinoculant strain IC3123. The three isolates were neither able to nodulate C. cajan nor did they show significant plant growth promotion when inoculated alone without Rhizobium spp. IC3123. All the three isolates were gram positive rods with NR2 and NR4 showing endospore formation and formed one single cluster in Amplified Ribosomal DNA Restriction Analysis (ARDRA). Partial sequences of 16S rRNA genes of NR4 and NR6 showed 97% similarity to Bacillus megaterium. The Bacillus strains NR4 and NR6 were able to produce siderophores which the rhizobial bioinoculant IC3123 was able to cross-utilize. Under iron starved conditions IC3123 showed enhanced growth in the presence of the Bacillus isolates indicating that siderophore mediated interactions may be underlying mechanism of beneficial effect of the NR isolates on nodulation by IC3123.

Acinetobacter sp. A3 is able to extensively degrade Bombay High Crude Oil (BHCO) and utilize it as the sole source of carbon. A total degradation of 70% BHCO was noted by the end of 120 h of growth of Acinetobacter sp. A3 under shake flask condition, 60% of which was due to biodegradation. In crude oil-contaminated soil (5%) amended with Acinetobacter sp. A3, there was both an increase in colony-forming units (CFU) and crude oil degradation. This is in contrast to a decrease in CFU of the indigenous microorganisms and lower degradation in unamended soil within the same 30-day period. Also, Acinetobacter sp. A3-treated soil permitted better germination of Mung beans (Phaseolus aureus) and growth as evidenced by better length and weight of the plants and chlorophyll content of its leaves, which was attributed to the reduction in phytotoxicity of the crude oil owing to its degradation. This crude oil degradative capability of Acinetobacter sp. A3 could be exploited for bioremediation purposes.

A modified and rapid technique has been developed for activity staining of a fungal poly(3-hydroxybutyrate) depoly-merase by incorporating poly(3-hydroxybutyrate) (2.5% w/v) in the polyacrylamide gel under polymerisation and incubating in 100 mM Tris/HCl (pH 8.0) containing 1 mM CaCl at 30°C. This decreased the time required for detection to 4 h from the conventional 12–18 h using solid-plate method. The enzyme preparation failed to penetrate the gel on conventional electrophoresis but treatment with 0.5% (v/v) Triton X-100 facilitated enzyme migration into the gel without its activity being affected.

The majority of bacteria isolated from rhizospheres of Arachis hypogea (Groundnut) and Vigna radiata (Mung bean) predominantly produced catechol-type siderophores except for a few fluorescent pseudomonads that produced hydroxamates in addition to catecholates. The rhizospheric isolates differed in their ability to cross-utilize siderophores produced by other rhizospheric isolates (heterologous); some were highly proficient at utilizing heterologous siderophores, while others were poor cross-utilizers. Isolate G9, which utilized hydroxamate as well as catecholate siderophores, was found to be an efficient siderophore cross-utilizer, while isolates G2 and G6 were poor-utilizers of catecholate and non-utilizers of hydroxamate siderophores. Growth stimulation of two isolates G9 and G6 was seen when grown in the presence of externally supplied heterologous siderophores, which they cross-utilized. The iron-regulated outer membrane protein (IROMP) profiles differed for the most cross-utilizer and the least cross-utilizer strains, but in both the cases no new outer membrane proteins (OMP) were induced in response to the exogenous siderophores supplied. The growth of the organisms in the presence of heterologous siderophores that they failed to cross-utilize led to growth inhibition in the case of isolate G9. This appears to be due to a lower affinity of the siderophore of G9 as compared to the exogenously supplied G6 siderophore. A simple method was devised to measure relative affinities of respective siderophores for iron based on CAS solution decolorization by the siderophore preparations. The effect on the growth of the differential affinities of the siderophores for iron and the interactions of the organisms through cross-utilization is also discussed.

Biosurfactant production was studied by Bacillus licheniformis K51, B. subtilis 20B, B. subtilis R1 and Bacillus strain HS3 using molasses or cheese whey as a sole source of nutrition at 45 °C. The isolates were able to grow and produce biosurfactant under shaking as well as static conditions. Maximum biosurfactant production was achieved with molasses at 5.0–7.0% (w/v). The biosurfactant retained its surface-active properties after incubation at 80 °C at a wide range of pH values and salt concentrations for nine days. Oil displacement experiments in sand pack columns with crude oil showed 25–33% recovery of residual oil.

The chemotactic response of Rhizobium sp. S2, a slow-growing Cajanus cajan isolate, towards its host root exudate was examined. Two classes of mutants, one nonchemotactic towards nutrients (amino acids and sugars) and signal compounds like flavonoids and the other, nonchemotactic towards amino acids and sugars but positive towards naringenin, the flavonoid present in Cajanus cajan root exudate, were obtained. The plasmid-cured derivative of the parent showed positive response towards amino acids and sugars but was nonchemotactic towards naringenin. A possible presence of dual chemotaxis pathways, one towards nutrients and the other for sensing signal compounds, was thus demonstrated. The possible involvement of naringenin as a chemoattractant in the preliminary stages of this Rhizobium-legume interaction was also established.

Pseudomonas aeruginosa GS3 produced rhamnolipid biosurfactants during growth on carbohydrates, higher chain length n-alkanes and l-alkenes, petroleum crude oil and vegetable oils. With glucose as the substrate, maximum surfactant production (0.44 g/l) was observed during the stationary phase of growth. Partially purified rhamnolipids showed excellent surface-active properties in terms of reduction in the interfacial tension between them and a variety of hydrocarbons, hydrocarbon mixtures and vegetable oils and formation of stable emulsion.

Cell-free extracts of cowpea Rhizobium GN1 (peanut isolate) possessed reductase activity towards various ferric siderophores including its own. This activity was heat- and O2-sensitive and required NADH and flavins. Whereas NADPH could replace NADH with half-efficiency, succinate could not serve as reductant. Activity was insensitive to antimycin A and rotenone but was completely inhibited by HgCl2 (1 mM). Mg2+ (1 mM) enhanced reductase activity but Zn2+ and Cu2+ at the same concentration were inhibitory. The enzyme was located in the periplasmic fraction.