Rajeev Kaushik

Diversity and phylogeny of plant growth-promoting bacilli from moderately acidic soil

The molecular diversity of aerobic endospore‐forming bacteria, typically Bacillus and its derived genera, has been investigated in various environments. However, there have been few investigations concerning Bacillus in acidic soils. In this study, the genotypic diversity and phylogenetic relationships among plant growth‐promoting (PGP) bacilli isolated from the rice rhizosphere growing in acidic soils of Kerala (pH varying from 6.3 to 6.8) were investigated. For assessing their biocontrol potential and PGP attributes, 115 isolates were randomly selected and 49 isolates that were positive for multiple traits were selected. Metabolic characterization of representative strains, using the Biolog GP2 (Gram Positive) MicroPlateTM, revealed a large versatility with respect to carbohydrate utilization. Amplified ribosomal DNA restriction analysis revealed 13 clusters at 65% similarity level, which consisted of 1–21 strains. 16S rDNA partial sequencing assigned all the isolates, except for one, to the Bacillus genus, with close relatedness to Bacillus humi, B. megaterium, B. drentensis, B. pocheonensis, B. aestuarii, B. arbutinivorans, B. niacini, and Brevibacterium casei. The Bacillus species with different metabolic capabilities, PGP abilities, and genetic diversity found in this study are likely to have ecological relevance. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Biological delignification of paddy straw and Parthenium sp. using a novel micromycete Myrothecium roridum LG7 for enhanced saccharification.

A new lignolytic micromycete fungus Myrothecium roridum LG7 was isolated and selected for biological delignification of agro residue-paddy straw and herbaceous weed Parthenium sp. Physical and chemical modifications in the biomass following pretreatment with M. roridum LG7 for 7 days in term of structural modification and lignin removal, changes in lignin skeleton, and alteration of cellulose crystallinity was observed through SEM-EDXA, FTIR and XRD analysis, respectively. Colonization of the fungus led to high amount of lignin removal (5.8-6.98mg/gds) from pretreated biomass which could be recovered as a value added product. Enzymatic hydrolysis of M. roridum LG7 pretreated biomass released significantly higher amount of reducing sugars (455.81-509.65 mg/gds) as compared to respective raw biomass within 24h. This study illustrates the promise of M. roridum LG7 for biological pretreatment through structural and chemical alteration of biomass beside creation of alkaline environment which prevent the growth of other contaminants.

Cold active hydrolytic enzymes production by psychrotrophic Bacilli isolated from three sub-glacial lakes of NW Indian Himalayas.

The diversity of culturable, cold‐active enzymes producing Bacilli was investigated from three sub‐glacial lakes of north western Indian Himalayas. Amplified ribosomal DNA restriction analysis (ARDRA) using three restriction enzymes Alu I, Msp I, and Hae III led to the clustering of 136 Bacilli into 26, 23, and 22 clusters at 75% similarity index from Chandratal Lake, Dashair Lake, and Pangong Lake, respectively. Phylogenetic analysis based on 16S rRNA gene sequencing led to the identification of 35 Bacilli that could be grouped in seven families viz.: Bacillaceae (48%), Staphylococcaceae (14%), Bacillales incertae sedis (13%), Planococcaceae (12%), Paenibacillaceae (9%), Sporolactobacillaceae (3%), and Carnobacteriaceae (1%), which included twelve different genera Bacillus, Desemzia, Exiguobacterium, Jeotgalicoccus, Lysinibacillus, Paenibacillus, Planococcus, Pontibacillus, Sinobaca, Sporosarcina, Staphylococcus, and Virgibacillus. Based on their optimal temperature for growth, 35 Bacilli were grouped as psychrophilic (11 strains), psychrotrophic (17 strains), or psychrotolerant (7 strains), respectively. The representative isolates from each cluster were screened for cold‐active enzyme activities. Amylase, β‐glucosidase, pectinase, and protease activities at 4 °C were detected in more than 80% of the strains while approximately 40, 31, 23, 14, 11, and 9% of strains possessed cellulase, xylanase, β‐galactosidase, laccase, chitinase, and lipase activity, respectively. Among 35 Bacilli, Bacillus amyloliquefaciens, Bacillus marisflavi, Exiguobacterium indicum, Paenibacillus terrae, Pontibacillus sp., Sporosarcina globispora, and Sporosarcina psychrophila were efficient producers of different cold‐active enzymes. These cold‐adapted Bacilli could play an important role in industrial and agricultural processes.

Cold‐active hydrolases producing bacteria from two different sub‐glacial Himalayan lakes

Microorganisms, native to the cold environments have successfully acclimatized their physiological, metabolic, and biological features, exhibiting uniqueness in their enzymes, proteins, and membrane structures. These cold‐active enzymes have immense biotechnological potential. The diversity of culturable bacteria in two different water lakes (the sub‐glacial freshwater and the brackish) of Himalayas was analyzed using SYBR green staining and cultural methods. A total of 140 bacteria were isolated and were grouped as psychrophiles, psychrotrophs, and psychrotolerant organisms, based on their optimal temperature for growth. The amplified ribosomal DNA restriction analysis using three restriction enzymes facilitated the grouping of these isolates into 96 genotypes at ≥85% polymorphism. Phylogenetic analysis using 16S rRNA gene sequences revealed that the bacterial strains from both lakes belonged to Firmicutes, Proteobacteria (α, β, and γ) or Actinobacteria. Screening of the germplasm for the activity of different cold‐active hydrolases such as protease, amylase, xylanase, and cellulase, revealed that about 16 isolates were positive, and exhibiting a wide range of stability at various temperature and pH. Our results suggest that the distinctly different ecosystems of sub‐glacial freshwater and brackish water lakes have diverse groups of bacteria, which can be an excellent source of extracellular hydrolases with a wide range of thermal stability.

Selection of Tn5::lacZ mutants isogenic to wild type Azospirillum brasilense strains capable of growing at sub-optimal temperature.

Azospirillum brasilense strains, CDJA and A40, capable of growing at sub-optimal temperature were tagged with stable chromogenic marker Tn5-lacZ. Mutants were screened for plant growth promoting activities at 20, 22, 25, 30 and 37 °C. Mutants MC48 and MA3 were found to fix nitrogen upto 85% and produced indole acetic acid (IAA) and siderophore in isogenic manner to their respective wild type strains, CDJA and A40, at sub-optimal temperatures. Co-inoculation of mutants with their respective parent (1:1 ratio) to the wheat revealed that colonization potential of the mutants was affected greatly. Tn5-lacZ tagged mutants MC48 and MA3 were found isogenic to their respective wild type Azospirillum strain, with regards to plant growth promoting activities and root colonization ability.

Haloarchaea Endowed with Phosphorus Solubilization Attribute Implicated in Phosphorus Cycle.

Archaea are unique microorganisms that are present in ecological niches of high temperature, pH and salinity. A total of 157 archaea were obtained from thirteen sediment, water and rhizospheric soil samples collected from Rann of Kutch, Gujarat, India. With an aim to screen phosphate solubilizing archaea, a new medium was designed as Haloarchaea P Solubilization (HPS) medium. The medium supported the growth and P solubilization activity of archaea. Employing the HPS medium, twenty isolates showed the P-solubilization. Phosphate solubilizing archaea were identified as seventeen distinct species of eleven genera namely Haloarcula, Halobacterium, Halococcus, Haloferax, Halolamina, Halosarcina, Halostagnicola, Haloterrigena, Natrialba, Natrinema and Natronoarchaeum. Natrinema sp. strain IARI-WRAB2 was identified as the most efficient P-solubilizer (134.61 mg/L) followed by Halococcus hamelinensis strain IARI-SNS2 (112.56 mg/L). HPLC analysis detected seven different kinds of organic acids, namely: gluconic acid, citric acid, formic acid, fumaric acid succinic acid, propionic acid and tartaric acid from the cultures of these isolates. These phosphate solubilizing halophilic archaea may play a role in P nutrition to vegetation growing in these hypersaline soils. This is the first report for these haloarchaea to solubilize considerable amount of P by production of organic acids and lowering of pH.

Deciphering Diversity of Salt-Tolerant Bacilli from Saline Soils of Eastern Indo-gangetic Plains of India

The intensive use of chemical fertilizers, monoculture and irrigation with surface saline water has resulted in the deterioration of soil health by enhancing the level of salinity in the Eastern Indo-Gangetic Plains of India. Therefore, diversity of halotolerant bacteria adapted to that environment and possessed the ability to produce plant growth hormones was explored, that could be used for salt stress amelioration. The 16S rRNA gene sequencing and fatty acid methyl ester (FAME) were used for diversity analysis of salt-tolerant bacilli. Among the 95 isolates, 55 strains showed plant growth promotion traits, production of industrially important enzymes (amylase, protease and cellulase) and tolerance to more than 4% NaCl. Using partial 16S rRNA sequences and FAME comparisons, 21 different species of Bacillus and Bacillus-derived genera were identified, viz. Bacillus megaterium, B. subtilis, B. licheniformis, B. firmus, B. horikoshii, B. pumilus, Bacillus sp., B. safensis, B. thuringiensis, B. simplex, B. agri, B. flexus, B. oceanisediminis, B. cereus, B. arsenicus, Paenibacillus dendritiformis, Lysinibacillus sp., L. sphaericus, B. marisflavi, Terribacillus sp., and B. mycoides. These isolates possess the ability to tolerate high salt, form endospores, withstand harsh environments, and also have the potential for plant growth promotion, which could be useful in formulation of new inoculants to enhance the availability of nutrients for crop growth under saline conditions.

Genetic and functional diversity of fluorescent Pseudomonas from rhizospheric soils of wheat crop

Wheat rhizospheric soils were collected from different part of northern and eastern Indo‐Gangetic plains, which is being irrigated from water of Ganga River. Isolation of fluorescent Pseudomonas species was carried out from the soil samples collected. The percentage of isolates positive for indolic compound, P‐solubilisation, siderophore production and ACC deaminase activity were 64.0, 38.6, 63.5, and 19.7, respectively. A total of 543 isolates were randomly selected for studies based on the genus specific confirmation by the Pseudomonas specific primer. Among the 543 isolates, 26 different clusters were formed from 16S rDNA‐RFLP whereas 27 clusters were generated by the rpoB‐RFLP with similarity percent ranging from 3 to 100%. 16S rDNA sequencing showed 9 different species of Pseudomonas whereas, rpoB sequencing showed 13 different species of Pseudomonas. Phylogenetic analysis based on 16S rDNA gene sequences generated 15 branches showing the more than 70% of boot strap value, whereas 18 branches in the rpoB based phylogenetic tree were supported by bootstrap values above 70%. Diversity indices based on rpoB were higher than the ribosomal RNA gene.

Characterization of halophilic bacteria from environmental samples from the brackish water of Pulicat Lake, India

Culture dependent phenotypic characterization and 16S rDNA based phylogenetic analyses were applied to study the aerobic halophilic bacterial population present in the Pulicat brackish-water Lake of India. Five different media were employed for isolation of bacteria. A total of 198 morphotypes were recovered, purified and screened for salt tolerance in nutrient agar medium amended with 5–25% NaCl. Based on 16S rDNA restriction fragment length polymorphism analysis with three restriction endonucleases, 51 isolates tolerant to 5% or more NaCl were grouped into 29 clusters. Phylogenetic analysis using 16S rRNA gene sequences revealed that 29 strains could further be allocated into two clades: 19 to Firmicutes and 10 to γ-Proteobacteria. Firmicutes included low G+C Gram-positive bacteria related to family Bacillaceae, which included five genera Bacillus, Virgibacillus, Rummelibacillus, Alkalibacillus and Halobacillus. Another genera included in Firmicutes was Salimicrobium halophilum. In the γ-Proteobacteria group, all the isolates belonged to one genus Halomonas, represented by six different species Halomonas salina, H. shengliensis, H. salifodinae, H. pacifica, H. aquamarina and H. halophila. Most of the isolates exhibited cellulase, xylanase, amylase and protease activities.

Biochemical and molecular characterization of thermo-alkali tolerant xylanase producing bacteria from thermal springs of Manikaran

One hundred ten alkalo-tolerant thermophilic bacteria were isolated from 17 samples (water and sediment) collected from Manikaran. Of 110 isolates, 70 showed the production of xylanases and were further screened for growth and production of xylanases at different temperature ranging from 40 to 75°C. Eleven isolates that showed growth and xylanase production at temperatures ≥50°C were selected for quantitative estimation in modified Reese mineral liquid medium containing wheat bran. Maximum xylanase activity was produced by isolate H-7 followed by H-9 and R-9 and was statistically superior to other isolates. The microscopic observation showed that the isolates possessed the typical rod with endospore, characteristic of genus Bacillus. The isolates were found to be oxidase and catalase positive. Using BIOLOG Microlog 3 software, the isolates H7, H9 and R9 were identified as Paenibacillus ehemensis, Bacillus cereus/B. thuringiensis and B. subtilis respectively, based on utilization of 95 carbon sources. PCR-RFLP analysis of 16S rDNA indicated that the isolates were genetically different from each other. DNA sequencing of the three isolates and phylogenetic analysis revealed that all the isolates obtained from Manikaran thermal springs showed 97 to 100% similarity with the sequences within the GenBank. The closest phylogenetic neighbours according to the 16S rRNA gene sequence data for the three isolates H-7, H-9, and R-9 were Paenibacillus ehemensis, Bacillus cereus and Bacillus subtilis, respectively.