Understanding rice growth-promoting potential of Enterobacter spp. isolated from long-term organic farming soil in India through a supervised learning approach.

This study addresses the plant beneficial enterobacteria present in rice rhizosphere and their efficiency for enhancing nitrogen uptake in rice plant. Using culturable approaches, the population of total diazotrophs present in rhizosphere samples collected from different organic rice fields of Sikkim were studied and recorded in the range between 4.62 to 4.97 log 10 CFU/g soil. All the isolated commonly occurred diazotrophic bacterial isolates were screened based on their ability to fix nitrogen in milligram per gram of sugar consumed under in-vitro condition with the reference check. In addition to nitrogen fixation, plant growth promoting traits such as production of indole-3-acetic acid and gibberellic acid were estimated using spectrophotometric approaches and compared against Bacillus subtilis as reference multi-potent plant growth promoting strain. In-vivo evaluation of these diazotrophic species in rice found improvement in both above and below ground responses in rice plant evaluated by estimating changes in chlorophyll concentration, plant biomass, root architecture, nitrogen uptake, microbial biomass and associated biochemical activity of soil. Further, the selected isolates were identified through DNA targeted analysis of 16S rRNA gene present in diazotrophs and which identified that the isolates belonged to the Enterobacter genus. Statistical models were prepared for deciphering the dynamics of plant growth improvement due to selective enrichment of rhizosphere bacteria and found significant (p<0.05) correlation between soil and plant parameters. This study concludes that Enterobacter spp. present in organic paddy soils of Sikkim having good nitrogen fixing abilities and whose selective enrichment in rhizosphere improved nitrogen uptake and plant growth promotion in rice plant.

Isolation and characterization of plant growth promoting rhizobacteria isolated from organically grown high yielding pole type native pea (Pisum sativum L.) variety Dentami of Sikkim, India.

Organic farming is an eco-friendly and sustainable farming practice that enhances soil fertility and helps in improving soil quality. But with the commencement of more sophisticated advances in agricultural techniques, organic farming has gradually become limited in the world. Culture-dependent plant growth-promoting bacterial isolates were isolated from the bulk and rhizospheric soil, of the native high yielding pole type organic pea (Pisum sativum L.) cultivar Dentami of Dentam, West Sikkim, India. Based on the 16S rRNA gene sequencing identification of these isolates, it was found that from the bulk soil, Actinobacteria (58%) was the dominant phyla followed by Firmicutes (28%), and Proteobacteria (14%). In the rhizospheric soil it was dominated by Proteobacteria (56%), followed by Firmicutes (33%), and Bacteriodetes (11%). A total of 40 bacterial isolates were initially screened for the plant growth-promoting (PGP) activity and out of them only four bacterial isolates i.e., Bacillus cereus P8, Arthrobacter woluwensis DP2, Paenarthrobacter nitroguajacolicus PP3, and Bacillus mycoides PP10 with accession numbers MN589697, MN559516, MN519462 and MN589696 respectively were found to possess higher PGP activity (i.e. phosphorous, potassium solubilization and nitrogen-fixing activity) as compared to the other bacteria present in the soil. Based on the indole-3-acetic acid (IAA) quantitative assay and siderophore production assay, it was found that Bacillus cereus (MN589697) produced the highest IAA (65.5 µg mL-1) and siderophore (71%) when compared with the other isolates. The statistical correlation suggests that pH and available phosphorus were the strongest influencing factors for the distribution of Proteobacteria in the rhizospheric soil. The results indicate that these isolates can be potential plant growth promoter under the agro-climatic conditions of Sikkim, India. To the best of our knowledge the present study is the first report of its kind and showcases significant findings pertaining to the assessment of diversity, isolation and identification of plant growth-promoting rhizobacteria of organic pea grown in Sikkim.

Isolation, Characterization, and Evaluation of Native Rhizobacterial Consortia Developed From the Rhizosphere of Rice Grown in Organic State Sikkim, India, and Their Effect on Plant Growth.

Eight rhizospheric bacteria were isolated from the organic paddy fields of Sikkim, India, and identified as Pseudomonas kribbensis KSB, Burkholderia cenocepacia SRD, Kosakonia oryzendophytica YMA7, Pseudomonas rhodesiae SRB, Bacillus sp. ARA, Paenibacillus polymyxa COW3, Bacillus aryabhattai PSB2, and Bacillus megaterium PSB1. They showed plant growth-promoting attributes in rice and have bio-control potential against phytopathogen Colletotrichum gloeosporioides of large cardamom (Amomum subulatum). Burkholderia cenocepacia SRD showed production of indole acetic acid and ammonia and solubilization of phosphate and potassium and also possessed nitrogen fixation potential. It showed antagonistic activity against two other plant pathogens of large cardamom, viz., Curvularia eragrostidis and Pestalotiopsis sp., under in vitro conditions. The liquid bacterial consortium was prepared using the bacterial strains SRB, PSB1, and COW3 (Consortia-1); PSB2, SRD, and COW3 (Consortia-2); and COW3, KSB, and YMA7 (Consortia-3) to increase the growth and yield of rice plants under organic farming conditions. Greenhouse and field studies showed that the Consortia-3 had the highest plant growth-promoting activity. Consortia-3 demonstrated better agronomic performance in terms of root length (9.5 cm),number of leaflets per plant (5.3), grains per panicle (110.6), test grain weight (27.4 g), dry root weight per plant (0.73 g), and total dry biomass per plant (8.26 g).

Antagonistic and plant-growth promoting novel Bacillus species from long-term organic farming soils from Sikkim, India.

Three bacteria namely Bacillus luciferensis K2, Bacillus amyloliquefaciens K12 and Bacillus subtilis BioCWB possessing plant growth promotion and biocontrol potential against phytopathogens and rice leaf folder were identified from organic soils of Sikkim, India. The results revealed significant higher production of phytohormones IAA (97.1 µg mL-1) and GA3 (10.6 µg mL-1) was found in K2, whereas BioCWB had higher phosphate solubilization (570.0 µg mL-1) efficacy and also possessed nitrogen fixation ability (5.34 log copy number mL-1 culture). All these bacteria had higher antagonistic activities against phytopathogens viz. Rhizoctonia solani, Fusarium proliferatum, Athelia rolfsii and Colletotrichum gloeosporioides and also had higher larvicidal activity against rice leaf folder Cnaphalocrocis medinalis (Guenne) under in vitro conditions. Molecular insights into the antagonistic mechanisms of Bacillus strains deciphered the presence of several antimicrobial peptides (ericin, subtilin, surfactin, iturin, bacilysin, subtilosin, fengycin and bacillomycin), volatiles (dimethyl disulphide, methyl-Furan, acetic acid, Z-1,3-pentadiene and 3-hexyn-2-ol) and soluble metabolites (9-octadecenamide, E-15-heptadecenal, E-3-eicosene and 5-octadecene). Furthermore, liquid microbial inoculum prepared using the bacterial strains (K2, K12 and BioCWB) were evaluated under glass house (rice) and field condition (capsicum), which significantly enhanced plant growth in rice and yield in capsicum compared to control. The present study revealed the combination of Bacillus spp. (K2, K12 and BioCWB) can be used as bio-inoculants for improving agricultural production in Sikkim. Moreover, for the first time, we demonstrated plant growth promoting (PGP) traits, antifungal and insecticidal properties of B. luciferensis.

Biomedical applications of microbially engineered polyhydroxyalkanoates: an insight into recent advances, bottlenecks, and solutions.

Biopolymeric polyhydroxyalkanoates (PHAs) are fabricated and accumulated by microbes under unbalanced growth conditions, primarily by diverse genera of bacteria. Over the last two decades, microbially engineered PHAs gained substantial interest worldwide owing to their promising wide-range uses in biomedical field as biopolymeric biomaterials. Because of non-hazardous disintegration products, preferred surface alterations, inherent biocompatibility, modifiable mechanical properties, cultivation support for cells, adhesion devoid of carcinogenic impacts, and controllable biodegradability, the PHAs like poly-3-hydroxybutyrate, 3-hydroxybutyrate and 3-hydroxyvalerate co-polymers, 3-hydroxybutyrate and 4-hydroxybutyrate co-polymers, etc., are available for various medical applications. These PHAs have been exploited to design in vivo implants like sutures as well as valves for direct tissue repairing as well as in regeneration devices like bone graft substitutes, nerve guides as well as cardiovascular patches, etc. Furthermore, they are also emerged as attractive candidates for developing effective/novel drug delivery systems because of their biocompatibility and biodegradability with the ability to deliver and release the drugs at a specific site in a controllable manner and, therefore widen the therapeutic window with reduced side effects. However, there still remain some bottlenecks related to PHA purity, mechanical properties, biodegradability, etc., that are need to be addressed so as to make PHAs a realistic biomaterial. In addition, innovative approaches like PHAs co-production with other value-added products, etc., must be developed currently for economical PHA production. This review provides an insight toward the recent advances, bottlenecks, and potential solutions for prospective biomedical applications of PHAs with conclusion that relatively little research/study has been performed presently toward the viability of PHAs as realistic biopolymeric biomaterials.

Poly-ß-hydroxybutyrate accumulation in cyanobacteria under photoautotrophy.

Poly-ß-hydroxybutyrate (PHB) is a biodegradable and biocompatible polymer that has immense potential in the field of environmental, agricultural and biomedical sciences. An alternative host system has been explored in this study for low-cost production. Examination of 25 cyanobacterial species from 19 different genera for photoautrophic production of polyhydroxyalkanoates (PHAs) under batch culture demonstrated that 20 species were poly-ß-hydroxybutyrate (PHB) accumulators, while others were found to be negative. Presence of PHB was confirmed by UV-spectroscopy, (1)H-NMR spectroscopy and GC-MS analysis. Accumulation of PHB in cyanobacteria was found to be species specific. The PHB extracted from Nostoc muscorum exhibited comparable material properties with the commercial PHB, thus advocating its potential applications in various fields.

Poly-beta-hydroxybutyrate accumulation in Nostoc muscorum: effects of metabolic inhibitors.

Poly-beta-hydroxybutyrate (PHB) accumulation in Nostoc muscorum was studied in presence of various metabolic inhibitors. Supplementation of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was found to suppress PHB accumulation in phosphate-limited N. muscorum under photoautotrophic growth condition. PHB accumulation increased up to 21% and 17% from an initial PHB content of 8.5% of dry weight, respectively, under carbonylcyanide m-chlorophenylhydrazone (CCCP) and dicyclohexylcarbodiimide (DCCD) treatment, whereas 2,4 -dinitrophenol (DNP) supplementation depicted insignificant effect on PHB pool of the test cyanobacterium. Supplementation of l-methionine-dl-sulfoximine (MSX) and azaserine was also found to increase PHB accumulation in N(2) -fixing and NH(4)(+) -grown N. muscorum, but not in NO(3)(-) -grown cells. The stimulatory action of monofluoroacetate on PHB accumulation was suppressed in presence of alpha-ketoglutarate and DCMU. Interestingly, 2,3 -butanedione supplementation was not only found inhibitory for accumulation of PHB in P-deficient, N-deficient and chemoheterotrophically grown N. muscorum but suppression of PHB synthesis was also evident in control cultures in presence of 2,3 -butanedione. The possible mechanisms are discussed.

Process optimization for poly-beta-hydroxybutyrate production in a nitrogen fixing cyanobacterium, Nostoc muscorum using response surface methodology.

A five-level-four-factor central composite rotary design was employed to find out the interactive effects of four variables, viz. concentrations of acetate, glucose and K2HPO4, and dark incubation period on poly-beta-hydroxybutyrate (PHB) production in a N2-fixing cyanobacterium, Nostoc muscorum. Acetate, glucose and dark incubation period exhibited positive impacts on PHB yield. Using response surface methodology (RSM), a second order polynomial equation was obtained by multiple regression analysis. A yield of 45.6% of dry cell weight (dcw) was achieved at reduced level of nutrients, i.e. 0.17% acetate, 0.16% glucose and 5 mg l(-1) K2HPO4 at a dark incubation period of 95 h as compared to 41.6% PHB yield in 0.4% acetate, 0.4% glucose and 40 mg l(-1) K2HPO4 at a dark incubation period of 168 h under single factor optimization strategy.

Studies on poly-beta-hydroxybutyrate synthase activity of Nostoc muscorum.

This study compares the PHB synthase activity of Nostoc muscorum, a N(2)-fixing cyanobacterium under control (grown in usual BG-11 medium), nitrogen (N) and phosphorus (P) deprivation and chemoheterotrophic conditions. Specific activity of PHB synthase did not depict significant variations in the latter three types of cultures, except for the control one, where a significantly lower activity was recorded. PHB synthase activity was detected only in the soluble fractions of both the control as well as cells incubated under chemoheterotrophic conditions. A K(m) of 80.2 microM DL-beta-hydroxybutyryl-CoA and V(max) of 197.5 nmol thiobenzoate (TNB) mg protein(-1)min(-1) were observed for the enzyme. PHB synthase remained insensitive to acetyl-CoA, ATP, NADP, NADPH supplementation under in vitro condition. Addition of acetyl phosphate was found to activate the enzyme and the level of activation was dependent on the concentration of acetyl phosphate supplementation. Inhibition of PHB synthase in 2,3-butanedione supplemented cultures and reactivation following acetyl phosphate addition proved the post-translational control of acetyl phosphate over PHB synthase.

Optimization of cultural and nutritional conditions for accumulation of poly-beta-hydroxybutyrate in Synechocystis sp. PCC 6803.

Poly-beta-hydroxybutyrate (PHB) accumulation in the unicellular cyanobacterium, Synechocystis sp. PCC 6803, was studied under various cultural and nutritional conditions. Under controlled condition, cells harvested at the stationary phase of growth depicted maximum accumulation of PHB, i.e., 4.5% (w/w of dry cells) as compared to lag (1.8%) or logarithmic (2.9%) phases of cultures. A temperature range of 28-32 degrees C and pH between 7.5 and 8.5 were preferred for PHB accumulation. Cells cultivated under regular light-dark cycles accumulated more PHB (4.5%) than those grown under continuous illumination (2.4%). Nitrogen and phosphorus starvation stimulated PHB accumulation up to the tune of 9.5 and 11% (w/w of dry cells), respectively. Synechocystis cells pre-grown in glucose (0.1%)-supplemented BG-11 medium when subjected to P-deficiency in presence of acetate (0.4%), PHB accumulation was boosted up to 29% (w/w of dry cells), the value almost 6-fold higher with respect to photoautotrophic condition. Fishpond discharges were found as suitable media for PHB accumulation in the test cyanobacterium.

Accumulation of poly-beta-hydroxybutyrate in Nostoc muscorum: regulation by pH, light-dark cycles, N and P status and carbon sources.

Accumulation of poly-beta-hydroxybutyrate (PHB) in Nostoc muscorum was studied. Cells harvested at stationary phase of growth depicted maximum accumulation i.e. 8.6% (w/w) of dry cells as compared to lag (4.1%) or logarithmic (6.1%) phases of cultures. In contrast to alkaline pH, acidic pH, continuous illumination and cells grown in presence of combined nitrogen sources, such as NH(4)Cl and KNO(3), were found to affect PHB accumulation negatively. However, P-deficiency and addition of exogenous carbon sources (acetate, glucose, maltose, fructose and ethanol) were found stimulatory for PHB accumulation. In this report PHB accumulation in N. muscorum was boosted up to 35% (w/w) of dry cells when cells supplemented with 0.2% acetate were subjected to dark incubation for 7 days. Further studies are needed at metabolic engineering level or to apply genetic engineering techniques to improve the expression level of PHB photoproduction in cyanobacteria.

Enhancement of poly-beta-hydroxybutyrate accumulation in Nostoc muscorum under mixotrophy, chemoheterotrophy and limitations of gas-exchange.

Nostoc muscorum, a heterocystous cyanobacterium, produced poly-beta-hydroxybutyrate ( PHB) up to 8% (w/w) dry cells when grown photoautotrophically but 35% when grown mixotrophically with 0.4% (w/v) glucose and acetate after 21 d. Gas-exchange limitations under mixotrophy and chemoheterotrophy with 0.4% (w/v) acetate enhanced the accumulation up to 40-43% (w/w) dry cells, the value almost 5-fold higher with respect to photoautotrophic condition.

Poly-beta-hydroxybutyrate accumulation in Nostoc muscorum and Spirulina platensis under phosphate limitation.

Nostoc muscorum and Spirulina platensis were grown under phosphate deficiency in order to investigate the role of internal phosphate pool and activity of alkaline phosphatase on poly-beta-hydroxybutyrate (PHB) accumulation. PHB accumulation in N. muscorum increased to 22.7% of dry weight (dw) after 4 day of phosphate deficiency, while the internal phosphate pool reduced to the level of 0.02 microM mg dw(-1) at a maximum APase activity of 2.57nM PNP mg dw(-1) h(-1). In contrary, S. platensis depicted maxima of 1.39nM PNP mg dw(-1) h(-1) on day 30 of incubation, which was about 2 fold lower than the observed value of N. muscorum. PHB content in S. platensis remained low even after prolonged phosphate starvation, and a rise only up to 3.5% of dw was recorded on day 60 of phosphate deficiency. Supplementation of NADPH exogenously to S. platensis cultures grown under phosphate deficiency favoured PHB accumulation in 10, 20 and 30 days old cultures, but not in the cultures grown under phosphate deficiency for 60 days. The possible role of phosphate limitation on PHB accumulation is discussed.

Antioxidative role of nitric oxide on copper toxicity to a chlorophycean alga, Chlorella.

The response of Chlorella vulgaris to copper exposure was investigated under laboratory batch culture conditions. Increased toxicity of Cu with respect to photosynthetic carbon fixation, O(2) evolution, chlorophyll fluorescence, and oxidative burst was observed for N-NH(4)(+)-grown cultures. The addition of sodium nitroprusside, a nitric oxide (NO) donor, in combination with Cu to N-NH(4)(+)-grown Chlorella not only lowered the inhibition levels of carbon fixation, O(2) evolution, and maximum quantum yield of PS II, but also significantly reduced the oxidative burst. The protective action of sodium nitroprusside was, however, arrested in cultures in which sodium nitroprusside was supplemented in combination with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a specific scavenger of NO in the experimental system. The N-NO(3)(-)-grown Chlorella depicted less sensitivity to Cu compared to its N-NH(4)(+)-grown counterpart. The N-NO(3)(-)-, N-NH(4)(+)-, and N-NH(4)(+)+sodium nitroprusside-grown Chlorella did not show any significant differences with respect to their Cu uptake potential. The role of NO as an antioxidant is discussed.