16S rDNA Restriction Analysis Research Articles

Phenotypic and genotypic antimicrobial resistance profiles of fecal lactobacilli from domesticated pigeons in Poland

Lactobacillus species play an important role in the host and although they are non-pathogenic, they could act as reservoirs for antibiotic resistance genes, with the potential risk of transfer to other bacteria inhabiting the gastrointestinal tract. The aim of this study was to identify Lactobacillus species derived from feces of domesticated pigeons and to characterize their phenotypic and genotypic antimicrobial resistance (AMR) profiles. A total of 57 Lactobacillus isolates were classified into six species using the MALDI-TOF technique and 16S rDNA restriction analysis. Strains of L. ingluviei (31%), L. salivarius (28%) and L. agilis (23%) were the dominant species isolated. Determination of antimicrobial susceptibility by the microdilution broth method showed widespread resistance to kanamycin (89%), tetracycline (84%), streptomycin (63%), and enrofloxacin (37%). Less than 30% of the isolates were resistant to erythromycin, lincosamides, gentamycin, chloramphenicol and vancomycin. Over half (51%) of the lactobacilli were classified as multidrug resistant. Tet genes were detected in 79% of isolates; the lnuA, cat, ermB, ermC, ant(6)-Ia, ant(4’)-Ia, and int-Tn genes were found at a lower frequency. Sequence analysis of the quinolone resistance-determining region (QRDR)of the gyrA gene showed that fluoroquinolone resistance in lactobacilli was the result of a mutation that lead to a change in the amino acid sequence (Ser83→Tyr/Leu/Phe). Domesticated pigeons could be a reservoir for AMR Lactobacillus strains and AMR genes.

Susceptibility of chicken Lactobacillus bacteria to coccidiostats.

The aim of this study was to determine the susceptibility of Lactobacillus bacteria to selected coccidiostats. Seventy-five Lactobacillus isolates obtained from chickens were classified by MALDI-TOF mass spectrometry and 16S rDNA restriction analysis into seven species, among which L. salivarius (33%) and L. johnsonii (24%) were dominant. Susceptibility of lactobacilli to coccidiostats was determined by broth microdilution method. The ranges of minimum inhibitory concentrations (MICs) were 0.5–≥128 µg/ml for monensin, 0.125–8 µg/ml for salinomycin, ≤0.03–2 µg/ml for lasalocid A, and 4–16 µg/ml for robenidine. Coccidiostats in low concentrations inhibited in vitro growth of most lactobacilli and therefore there is a high probability that administration of this drugs to chickens would reduce the number of lactobacilli in the gut.

Sistematik Filogenetik Pseudomonas Strain Indigenous Pendegradasi Liniar Alkilbenzen Sulfonat

Linear Alkylbenzene Sulphonate (LAS) was the dominant pollutant in the river ecosystem. Indigenous strains of Pseudomonas in river ecosystem had highly potency to LAS degradation. This research was carried out to study relationship of indigenous strains of LAS degrading to Pseudomonas strains. Indigenous strains of bacteria of LAS degrading were characterized based on ARDRA (Amplified Ribosomal 16S rDNA Restriction Analysis) and 16S rDNA sequence. Result of the research shows that Pseudomonas strain J and R which LAS degrading from detergent polluted river ecosystem based on 16S rDNA sequence, isolate J has 98.37% similarity and it has relationship to P. pseudoalcaligenes LMG 1225T whereas isolate R has 84.86% similarity and related to P. stutzeri phen8.

Isolation and Identification of Pesticides Degrading Bacteria from Farmland Soil

Pesticides are recognized to be the threat to the environment and associated with a wide range of serious diseases including respiratory diseases, cancer and even birth defects. In this study, six-different bacterial strains capable of degrading Carbofuran, Emamectin Benzoate and Thiamethoxam were isolated from eight different soil samples. The isolates were characterized by using different conventional and molecular methods. The strains were identified molecularly into different genotypes using amplified ribosomal DNA restriction analysis (ARDRA) and partial sequencing of 16S rDNA. The ARDRA pattern clustered them into 3 groups. Among the isolates three were identified as Achromobacter spp. and one as Diaphorobacter sp. by biochemical tests. It was further confirmed by the partial 16S rDNA sequencing. The two identified potential bacteria can be used for biodegradation of different pesticides which can have a significant environmental impact in soil farm.
 Bangladesh J Microbiol, Volume 35 Number 2 December 2018, pp 90-94

Identification and antibiotic susceptibility of lactobacilli isolated from turkeys

BackgroundThe aim of this study was to identify Lactobacillus isolates derived from turkeys from six Polish farms and to characterize their phenotypic and genotypic antibiotic resistance profiles.ResultsAmong 62 isolates identified by MALDI-TOF mass spectrometry and restriction analysis of 16S rDNA, the dominant species was L. salivarius (35%), followed by L. crispatus (21%), L. ingluviei (14.5%) and L. johnsonii (10%). A high prevalence of resistance to tetracycline (68% resistant isolates), lincomycin (64.5%) and enrofloxacin (60%) among the lactobacilli tested was observed. Fewer than 50% isolates were resistant to ampicillin (47%), erythromycin (45%), streptomycin (31%), chloramphenicol (29%) and gentamicin (10%). As many as 64,5% of the isolates showed multidrug resistance. High MIC values for ampicillin (≥64 μg/ml) were usually accompanied by elevated MICs for cephalosporins (≥16 μg/ml) and high MICs for tiamulin, i.e. ≥32 μg/ml, were noted in most of the turkey lactobacilli (61%). The occurrence of resistance genes was associated with phenotypic resistance, with the exception of five phenotypically susceptible isolates that contained the tetM, tetL, ermC, ermB or cat genes. The most frequently identified were ermB (45% isolates), tetL (40%), tetW (37%) and tetM (29%), and the occurrence of lnuA (18%), cat (10%), ermC (6%), ant(6)-Ia (5%) and aadE (5%) was less frequent. The mechanism of ampicillin resistance has not been elucidated, but the results of nitrocefin test confirmed that it is not involved in the production of beta-lactamases.ConclusionsThe high rate of antibiotic resistance observed in this study indicates the need to implement the principles of rational use of antibiotics in poultry. The presence of transmissible resistant genes in lactobacilli may contribute to the development of antibiotic resistant pathogenic strains that pose a threat to both poultry and consumers. The results of these studies may be useful for committees providing guidance on antibiotic susceptibility of microorganisms in order to revise and supplement current microbiological cut-offs values within the genus Lactobacillus.

Antibiotic gene specific characterization and ARDRA analysis of native isolates of Pseudomonas spp. from Jammu, India

The genus Pseudomonas is well known biocontrol agent established for its diverse role and antagonistic properties against phytopathogens due its ability of producing antibiotics. The studies were conducted to characterize Pseudomonas spp. for antibiotic production on the basis of gene specific primer. 150 isolates of Pseudomonas spp. were isolated from different agricultural fields of Jammu on King’s B medium using plate dilution method. Out of the total, thirty isolates were selected. The antibiotic i.e. 2,4-Diacetylphloroglucinol (2,4-DAPG) extracted from the various isolates showed spots having Rf values in the range of 0.7–0.89 which is very close to the Rf value of synthetic phloroglucinol (0.89). 2,4-DAPG positive isolates were tested against Fusarium oxysporum using dual culture assay and they were found to inhibit the mycelial growth of F. oxysporum by 52.76–65.45%. PCR analysis with 2,4-DAPG gene (phlD) specific primer indicated that a DNA fragment of approximately 750 bp in size was obtained in all the strains except PS 3 and 5 which is closely corroborated with the already reported size of 2,4-DAPG gene in literature. This confirms that 28 isolates out of 30 isolates have the genetic ability for the production of 2,4-DAPG antibiotic and thus these isolates can be promoted as potential biocontrol agents. The genetic diversity of anti-fungal producing rhizobacteria Pseudomonas spp. was investigated using amplified 16S rDNA restriction analysis. 16S rDNA was amplified using PCR technique and digestion with restriction endonuclease HaeIII and dendrogram analysis was carried out by the SHAN clustering UPGMA method (NTSYS 2.1). ARDRA analysis revealed the variability of the Pseudomonas spp. based on the restriction sites and the amplified 16S rDNA distinguished into 18 clusters/types including twelve minor groups and six major groups.

Isolation, characterization and exploring biotechnological potential of halophilic archaea from salterns of western India.

Thirteen halophilic archaea were isolated from Kandla and Bhayander salt pans. These isolates were grouped into three different genera Halobacterium, Haloferax and Haloarcula based on morphological and biochemical characterization, polar lipid analysis, Amplified 16S rDNA restriction analysis (ARDRA) and 16S rDNA sequence analysis. Biochemical characterization suggested the ability of isolates to produce protease, amylase and poly-hydroxybutyrate (PHB) indicating their biotechnological potential. The isolates were further screened for the amount of extracellular protease produced. Halobacterium sp. SP1(1) showed significant protease production compared to other isolates. Protease producing ability of the isolate was influenced by several factors such as NaCl concentration, type of protein source, metal ions and surfactants, and presence of amino acid supplements in the production medium. Soybean flour, FeCl3 and dicotylsulfosuccinate were found to increase protease production by 2.36, 1.54 and 1.26 folds, respectively compared to production in basal medium. Effect of organic solvents used in paints (n-decane, n-undecane and n-dodecane) was also investigated on protease production by the isolate. Protease production by Halobacterium sp. SP1(1) was enhanced by 1.2 folds in presence of n-decane compared to control. Furthermore, the ability of isolate to hydrolyse fish protein was investigated using three different edible fishes (Pomfret, Flat fish and Seer fish) as sole protein source. Pomfret was found to be a good protein source for protease production by the isolate. These results revealed that Halobacterium sp. SP1(1) may have potential for paint-based antifouling coating preparations and fish sauce preparation by virtue of its extracellular protease.

The ipdC, hisC1 and hisC2 genes involved in indole-3-acetic production used as alternative phylogenetic markers in Azospirillum brasilense.

Plant growth-promoting bacteria of the genus Azospirillum are present in the rhizosphere and as endophytes of many crops. In this research we studied 40 Azospirillum strains isolated from different plants and geographic regions. They were first characterized by 16S rDNA restriction analysis, and their phylogenetic position was established by sequencing the genes 16S rDNA, ipdC, hisC1, and hisC2. The latter three genes are involved in the indole-3-pyruvic acid (IPyA) biosynthesis pathway of indole-3-acetic acid (IAA). Furthermore, the suitability of the 16S-23S rDNA intergenic spacer sequence (IGS) for the differentiation of closely related Azospirillum taxa and development of PCR protocols allows for specific detection of strains. The IGS-RFLP analysis enabled intraspecies differentiation, particularly of Azospirillum brasilense and Azospirillum lipoferum strains. Results demonstrated that the ipdC, hisC1, and hisC2 genes are highly conserved in all the assessed A. brasilense isolates, suggesting that these genes can be used as an alternative phylogenetic marker. In addition, IAA production determined by HPLC ranged from 0.17 to 98.2μgmg(-1) protein. Southern hybridization with the A. brasilense ipdC gene probe did not show, a hybridization signal with A. lipoferum, Azospirillum amazonense, Azospirillum halopreferans and Azospirillum irakense genomic DNA. This suggests that these species produce IAA by other pathways. Because IAA is mainly synthesized via the IPyA pathway in A. brasilense strains, a species that is used worldwide in agriculture, the identification of ipdC, hisC1, and hisC2 genes by PCR may be suitable for selecting exploitable strains.

Comparison of molecular and phenetic typing methods to assess diversity of selected members of the genus Bacillus

A comparison of different molecular typing methods viz. ERIC-PCR, BOX-PCR and ARDRA along with carbohydrate utilization pattern was carried out to analyze their discriminatory power and suitability for assessing diversity of selected Bacillus isolates. ERIC-PCR generated 61 bands ranging from 0.56 to 3.9 kb while BOX-PCR resulted in 127 bands ranging from 0.16 to 3.9 kb. Restriction analysis of 16S rDNA with AluI and HaeIII produced 56 and 67 bands ranging from 0.14 to 0.54 and 0.12 to 0.96, respectively. Clustering of isolates based on the ERIC, BOX and ARDRA pattern clearly showed the superiority of the former two methods to reveal the intrageneric and intraspecific diversity. Carbohydrate utilization pattern showed that most preferred sugar was Fructose while Xylose, Rhamnose and D-Arabinose were least preferred by the isolates used for the study. Clustering based on carbohydrate utilization was also able to differentiate among the isolates which showed 100% similarity based on ARDRA profiles. This study clearly shows that typing methods exploiting the repetitive elements distributed over the genome are more useful for assessing genetic diversity. Moreover, metabolic diversity of the bacterial groups may also be useful instead of using single locus specific marker systems for revealing the diversity.

Plant genotype and nitrogen fertilization effects on abundance and diversity of diazotrophic bacteria associated with maize (Zea mays L.)

This research has investigated the effect of two genotypes of maize (Zea mays L.) on the number and diversity of diazotrophic bacteria under different N-fertilization rates. Cultivars NK940 and PAU871 were grown in soil:sand with 0, 40, and 80 kg N ha−1 added as NH4NO3 under controlled conditions for 60 days. The number of diazotrophic bacteria of rhizosphere (S), disinfected roots (R), and stems (T) was determined by most probable number (MPN) method. Diazotrophic bacteria were isolated by N-free media, confirmed by amplification of nifH gene fragments, characterized by amplified 16S rDNA restriction analysis (ARDRA) and (GTG)5 fingerprinting, and identified by partial 16S ribosomal DNA (rDNA) sequence analysis. The diversity of the diazotrophic community from S and R was determined by Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis considering the nifH gene. Maize genotype had a marked effect on the number and diversity of endophytic communities, the NK940 community being more abundant and diverse than that of PAU871. Additionally, N-fertilization increased the number and diversity of diazotrophs in endophytic communities but not in rhizosphere samples. One hundred and six diazotrophs were isolated from S, R, and T samples. Pseudomonas and Enterobacter were the dominant and ubiquitous genera isolated and detected by culture-independent method. T-RFLP showed that the N-fixing populations of the rhizosphere of both cultivars were more diverse than those of inside roots. Principal component analysis (PCA) separated the samples by cultivar and demonstrated a more marked effect of N-fertilization on the NK940 diazotrophic community than on PAU871. These results support the hypothesis that plant genotype and fertilization conditions should be taken into account when searching for N-fixer inocula.

Prospecting metal-tolerant rhizobia for phytoremediation of mining soils from Morocco using Anthyllis vulneraria L.

The aim of this work was using the legume plant Anthyllis vulneraria L. (ecotype metallicolous) as a trap plant, in order to isolate metal-tolerant rhizobial strains from metal-contaminated soils from Morocco, with pollution indexes spanning three orders of magnitude. As bioindicator, soil bacterial density was inversely correlated to the pollution index. Forty-three bulk soil bacteria and sixty two bacteria from nodules were isolated. The resistance of bacteria from nodules to heavy metals was four to ten times higher than that of bulk soil bacteria, reaching high maximum tolerable concentrations for Cd (2mM), Cu (2mM), Pb (7mM), and Zn (3mM). Besides, some strains show multiple metal-tolerant abilities and great metal biosorption onto the bacterial surface. Amplification and restriction analysis of ribosomal 16S rDNA (ARDRA) and 16S ribosomal DNA (rDNA) sequencing were used to assess biodiversity and phylogenetic position among bacteria present in nodules. Our results suggest that a great diversity of non-rhizobial bacteria (alpha- and gamma-proteobacteria) colonize nodules of Anthyllis plants in contaminated soils. Taking together, our results evidence that, in polluted soils, rhizobia can be displaced by non-rhizobial (and hence, non-fixing) strains from nodules. Thus, the selection of metal-resistant rhizobia is a key step for using A. vulneraria symbioses for in situ phytoremediation.

Deciphering soil diazotrophic diversity in the wheat-maize cropping system of Punjab using morphological, biochemical and molecular techniques

Soil is the critical resource as well as a basic medium for the growth of natural micro flora present in it. The soil fertility depends on its physicochemical properties and microbial population diversity. Diazotrophs are the nitrogen fixing bacteria which possess nif H gene that is responsible for coding the nitrogenase enzyme involved in the reduction of atmospheric nitrogen to ammonia. These nitrogen fixing bacteria play an imperative role, function and significance in the soil. Soil samples were collected from the different wheat-maize cropping system of Punjab and analyzed for physicochemical properties as pH, electrical conductivity, organic carbon, soil texture, ammoniacal as well as nitrate nitrogen. Eighty diazotrophic bacteria were isolated on eight different nitrogen free media and characterized culturally, morphologically, biochemically, functionally and using molecular techniques. The diazotrophic nature of the isolates was confirmed by the amplification of nif H gene using two nifH primers viz. nif H1 and nif H2. The nif H positive isolates were further used for 16S rDNA restriction analysis using different enzymes such as Taq 1, Rsa I and Hae III. Based on UPGMA clustering, the representative strains were sequenced and identified as Bacillus amyloliquifaciens, Pseudomonas aeruginosa, Bacillus subtilis, Paenibacillus sp., Azotobacter vinelendii, Stenotrophomonas maltophilia, Rhizobium larrymorrei, Flavobacterium anhuiense, Sphingomonas paucimobilis, Paenibacillus panacisoli, Azospirillum sp., Pseudomonas putida, Paenibacillus amyloliticus, Bacillus circulans, Paenibacillus polymyxa and Xanthomonas oryzae .

Culturable endophytic bacteria enhance Ni translocation in the hyperaccumulator Noccaea caerulescens

In this work, both culture-dependent and independent approaches were used to identify and isolate endophytic bacteria from roots of the Ni hyperaccumulator Noccaea caerulescens. A total of 17 isolates were cultured from root samples, selected for tolerance to 6mM Ni and grouped by restriction analysis of 16S rDNA. Bacterial species cultivated from roots belonged to seven genera, Microbacterium, Arthrobacter, Agreia, Bacillus, Sthenotrophomonas, Kocuria and Variovorax. The culture-independent approach confirmed the presence of Microbacterium and Arthrobacter while only other five clones corresponding to different amplified ribosomal DNA restriction patterns were detected. Five selected highly Ni-resistant bacteria showing also plant growth promoting activities, were inoculated into seeds of N. caerulescens, and in vivo microscopic analysis showed rapid root colonisation. Inoculated plants showed increased shoot biomass, root length and root-to-shoot Ni translocation. Root colonisation was also evident, but not effective, in the non-hyperaccumulating Thlaspi perfoliatum. Seed inoculation with selected Ni-resistant endophytic bacteria may represent a powerful tool in phytotechnologies, although transferring it to biomass species still requires further studies and screening.

A survey on cultivable heterotrophic bacteria inhabiting a thermally unstratified water column in an Atlantic Rainforest lake.

Due to the importance of heterotrophic bacteria in biogeochemical cycles and their influence on water quality, many studies have assessed the composition of the bacterial community. Most of these were made in temperate freshwaters. Eighteen heterotrophic bacteria communities distributed over time and space in the water column of Carioca Lake, not exposed to anthropogenic activities, were analyzed to characterize their composition. A polyphasic approach was used, including 16S rDNA restriction analysis, 16S rRNA gene sequence analysis, BIOLOG Ecoplates and statistical methods. The physiological profiles among the 18 microbial communities were diverse. Clustering analysis and the metabolic fingerprint of the Biolog EcoplateTM system data separated the communities based on temporal scale. A set of 673 isolates were recovered on high nutrient medium. The 673 isolates obtained yielded 360 Amplified Ribosomal DNA Restriction Analysis (ARDRA) Operational Taxonomic Units (OTUs). Most (313) of the ARDRA patterns, OTUs, were from isolates obtained in a single sampling point, in temporal and spatial scales, indicating changes in the bacterial community. A subset of representative isolates for each ARDRA OTU was identified by 16S rRNA gene fragment sequencing and categorized into five phyla, Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, and Deinococcus-Thermus, represented by 38 genera. The results of this work contribute to a better understanding about the phylogeny of tropical freshwater heterotrophic bacteria.

Diversity and Molecular Characterization of Dominant Bacillus amyloliquefaciens (JNU-001) Endophytic Bacterial Strains Isolated from Native Neem Varieties of Sanganer Region of Rajasthan

Higher plants are ubiquitously colonized with bacterial endophytes that often lack readily detectable structures. In the present study seven novel endophytic bacterial species viz; Bacillus amyloliquefaciens (JNU-001), Burkholderia denitrificans (JNU-002), Pseudomonas aeruginosa (JNU-003), Xanthomonas campestris (JNU-004), Azotobacter tropicalis (JNU-005), Acetobacter xylinum (JNU-006) and Azospirillum lipoferum (JNU-007) has been recovered from native neem varieties at Sanganer areas namely Khonagorion, Muhana, Jagatpura, Goner, Watika, Kalwara and Bagru of Rajasthan state. In which Bacillus amyloliquefaciens was found dominant and associated endophytically in all the plants. Molecular characterization of the isolates was performed using 16S r-DNA restriction analysis. Similarity index in unweighted pair group method with arithmetic mean programme clustered the isolates according to their geographical distribution and confirmed the endophytic association between these novel bacterial strains and neem varieties.

Genotypic diversity in native rhizobial population nodulating Vicia faba in arid and semi-arid regions of Haryana state (India)

A Rhizobium–legume interaction stands out from other plant–microbe interactions as one in which a true developmental mutualism occurs. To study the genotypic diversity in native population of rhizobia-nodulating Vicia faba plants, we retrieved 64 rhizobial isolates from root nodules of faba bean grown in pots holding soils collected from arid and semi-arid regions of the state of Haryana, India. The amplification of nodC in all the isolates authenticated these as rhizobia. The nitrogen-fixing potential of the isolates was tested by the amplification of the nifH gene. Only 50 isolates out of 64 showed nifH gene amplification. The characterization of the isolates by amplified 16S rDNA restriction analysis (ARDRA) categorized these into 36 16S rDNA genotypes using a combination of MspI and HaeIII restriction enzymes. Majority of the isolates resolved into separate genotypes, indicating a wide diversity among them, which seemed to arise from their geographical origin and soil characteristics. These findings may be immensely useful in agriculture towards developing rhizobial inoculants specific for faba beans under arid and semi-arid conditions.

Cellulolytic bacterial biodiversity in long-term manure experimental sites

To study the Influence of long-term application of manure on cellulolytic bacterial diversity, 54 efficient cellulolytic bacterial cultures were isolated from two long-term manure experimental field sites of Berlin-Dahlem (established in 1923) and BadLauchstadt (established in 1902). The sequence divergence of highly conserved region of 16S rDNA was exploited by restriction analysis of PCR-amplified 16S rDNA using restriction enzyme ScrFI to assay the evolutionary relatedness of isolates. Restriction analysis identified 10 genetically diverse pattern groups comprising five groups each of bacterial and actinomycetal domain. Irrespective of manuring, a dominant pattern group (H2) was identified, containing 31.48% of total isolates. On the other hand, two site-specific pattern groups highly specific for the brown soil (H2, 9.25% of total isolates) and for black soil (J2, 11.11% of total isolates), respectively were identified. In general, the composition of cellulolytic isolates in two sites displayed differences with respect manure application and soil properties. Manure strongly influenced the abundance of cellulolytic bacterial diversity in brown soil. The terminal restriction fragment length polymorphism (T-RFLP) data revealed a distinct relationship of total bacterial diversity with long-term manure application. This influence is more prominent in nutrient poor brown soil. Based on 16S rDNA sequence analysis, isolates of the dominant as well as the specific pattern groups could be assigned to the genus Streptomyces comprising species of diverse phylogenetic affiliation. Furthermore, sequencing of 16S rDNA of isolates of five bacterial pattern groups revealed a high phylogenetic diversity among these isolates, including Streptococcus, Paenibacillus, Bacillus, Bacillus megaterium and Bacillus pumilus. Key words: Cellulolytic bacteria, 16SrDNA gene, restriction analysis, restriction fragment length polymorphism (T-RFLP), terminal restriction fragment length polymorphism (T-RFLP), phylogenetic diversity.

Ferric iron amendment increases Fe(III)-reducing microbial diversity and carbon oxidation in on-site wastewater systems

Onsite wastewater systems, or septic tanks, serve approximately 25% of the United States population; they are therefore a critical component of the total carbon balance for natural water bodies. Septic tanks operate under strictly anaerobic conditions, and fermentation is the dominant process driving carbon transformation. Nitrate, Fe(III), and sulfate reduction may be operating to a limited extent in any given septic tank. Electron acceptor amendments will increase carbon oxidation, but nitrate is toxic and sulfate generates corrosive sulfides, which may damage septic system infrastructure. Fe(III) reducing microorganisms transform all major classes of organic carbon that are dominant in septic wastewater: low molecular weight organic acids, carbohydrate monomers and polymers, and lipids. Fe(III) is not toxic, and the reduction product Fe(II) is minimally disruptive if the starting Fe(III) is added at 50–150mgL−1. We used 14C radiolabeled acetate, lactate, propionate, butyrate, glucose, starch, and oleic acid to demonstrate that short and long-term carbon oxidation is increased when different forms of Fe(III) are amended to septic wastewater. The rates of carbon mineralization to 14CO2 increased 2–5times (relative to unamended systems) in the presence of Fe(III). The extent of mineralization reached 90% for some carbon compounds when Fe(III) was present, compared to levels of 50–60% in the absence of Fe(III). 14CH4 was not generated when Fe(III) was added, demonstrating that this strategy can limit methane emissions from septic systems. Amplified 16S rDNA restriction analysis indicated that unique Fe(III)-reducing microbial communities increased significantly in Fe(III)-amended incubations, with Fe(III)-reducers becoming the dominant microbial community in several incubations. The form of Fe(III) added had a significant impact on the rate and extent of mineralization; ferrihydrite and lepidocrocite were favored as solid phase Fe(III) and chelated Fe(III) (with nitrilotriacetic acid or EDTA) as soluble Fe(III) forms.

Biochemical and molecular characterisation of the bacterial endophytes from native sugarcane varieties of Himalayan region.

Seven endophytic bacterial isolates were finally recovered from native sugarcane varieties at hilly areas namely Berinag, Champawat and Didihat of Uttarakhand state in northern Himalayan region. New isolates and two standard cultures—Azospirillum brasilense and Gluconacetobacter diazotrophicus, were evaluated for their morphological, biochemical and molecular characteristics. Morphologically all were rod shaped, Gram-negative bacteria. Their plant growth promotory properties were also assessed which proved isolates RtBn and StBn as IAA producing. Except isolate StBn, all were phosphate solubilising and except RtBn all produced siderophores. Molecular characterisation of the isolates was performed using amplified 16S r-DNA restriction analysis. Similarity index in unweighted pair group method with arithmetic mean programme clustered the isolates according to their geographical distribution. Native isolates showed insignificant similarity with South American strains used as standards. nifH amplification was observed with all the isolates used in the study which again establish them as potential N-fixers.

Occurrence of Biosurfactant Producing Bacillus spp. in Diverse Habitats.

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