Laboratory and field scale bioremediation of hexachlorocyclohexane (HCH) contaminated soils by means of bioaugmentation and biostimulation.

Hexachlorocyclohexane (HCH) contaminated soils were treated for a period of up to 64 days in situ (HCH dumpsite, Lucknow) and ex situ (University of Delhi) in line with three bioremediation approaches. The first approach, biostimulation, involved addition of ammonium phosphate and molasses, while the second approach, bioaugmentation, involved addition of a microbial consortium consisting of a group of HCH-degrading sphingomonads that were isolated from HCH contaminated sites. The third approach involved a combination of biostimulation and bioaugmentation. The efficiency of the consortium was investigated in laboratory scale experiments, in a pot scale study, and in a full-scale field trial. It turned out that the approach of combining biostimulation and bioaugmentation was most effective in achieving reduction in the levels of α- and ß-HCH and that the application of a bacterial consortium as compared to the action of a single HCH-degrading bacterial strain was more successful. Although further degradation of ß- and δ-tetrachlorocyclohexane-1,4-diol, the terminal metabolites of ß- and δ-HCH, respectively, did not occur by the strains comprising the consortium, these metabolites turned out to be less toxic than the parental HCH isomers.

Sphingopyxis flava sp. nov., isolated from a hexachlorocyclohexane (HCH)-contaminated soil.

A Gram-negative-staining, aerobic, non-motile, non-spore-forming, rod-shaped and yellow-pigmented bacterium, designated R11HT, was isolated from a soil sample collected from a hexachlorocyclohexane dumpsite located at Ummari village, Lucknow, Uttar Pradesh, India. The 16S rRNA gene sequence similarity between strain R11HT and the type strains of species of genus Sphingopyxis with validly published names ranged from 93.75 to 97.85 %. Strain R11HT showed the highest 16S rRNA gene sequence similarity to Sphingopyxis indica DS15T (97.85 %), followed by Sphingopyxis soli JCM15910T (97.79 %), Sphingopyxis ginsengisoli KCTC 12582T (97.77 %) and Sphingopyxis panaciterrulae KCTC 22112T (97.34 %). The DNA G+C content of strain R11HT was 63.5 mol%. DNA-DNA relatedness between strain R11HT and its closest phylogenetic neighbours was well below the threshold value of 70 %, which suggested that strain R11HT represents a novel species of the genus Sphingopyxis. The major polar lipids of strain R11HT were sphingoglycolipid and other lipids commonly reported in this genus, phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol and phosphatidylmonomethylethanolamine. Spermidine was detected as the major polyamine. The chemotaxonomic markers in strain R11HT confirmed its classification in the genus Sphingopyxis, i.e. Q-10 as the major ubiquinone and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and C14 : 0 2-OH as the predominant fatty acids. Results obtained from DNA-DNA hybridization and chemotaxonomic and phenotypic analyses clearly distinguished strain R11HT from its closest phylogenetic neighbours. Thus, strain R11HT represents a novel species of the genus Sphingopyxis, for which the name Sphingopyxis flava sp. nov. is proposed. The type strain is R11HT ( = DSM 28472T = MCC 2778T).

Parapedobacter indicus sp. nov., isolated from hexachlorocyclohexane-contaminated soil.

Strain RK1(T), a Gram-stain-negative, non-spore-forming, rod-shaped, non-motile bacterium was isolated from a hexachlorocyclohexane (HCH) dumpsite, Lucknow, India. 16S rRNA gene sequence analysis revealed that strain RK1(T) belongs to the family Sphingobacteriaceae and showed highest sequence similarity to Parapedobacter koreensis Jip14(T) (95.63%). The major cellular fatty acids of strain RK1(T) were iso-C15:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c), iso-C17:0 3-OH, summed feature 9 (10-methyl C16:0 and/or iso-C17:1ω9c), iso-C15:0 3-OH and C16 : 0. The major respiratory pigment and polyamine of RK1(T) were menaquinone (MK-7) and homospermidine, respectively. The main polar lipids were phosphatidylethanolamine and sphingolipid. The G+C content of the DNA was 44.5 mol%. The results of physiological and biochemical tests and 16S rRNA sequence analysis clearly demonstrated that strain RK1(T) represents a novel species of the genus Parapedobacter, for which the name Parapedobacter indicus sp. nov. is proposed. The type strain is RK1(T) ( = DSM 28470(T) =MCC 2546(T)).

Thermus parvatiensis RL(T) sp. nov., Isolated from a Hot Water Spring, Located Atop the Himalayan Ranges at Manikaran, India.

A Gram negative, yellow pigmented, rod shaped bacterium designated as RL(T) was isolated from a hot water spring (90-98 °C) located at Manikaran in Northern India. The isolate grows at 60-80 °C (optimum, 70 °C) and at pH 7.0-9.0 (optimum pH 7.2). Phylogenetic analysis of 16S rRNA gene sequences and levels of DNA-DNA relatedness together indicate that the new isolate represents a novel species of the genus Thermus with closest affinity to Thermus thermophilus HB8(T) (99.5 %) followed by Thermus arciformis (96.4 %). A comparative analysis of partial sequences of housekeeping genes (HKG) further revealed that strain RL(T) is a novel species belonging to the genus Thermus. The melting G+C content of strain RL(T) was calculated as 68.7 mol%. The DNA-DNA relatedness value of strain RL(T) with its nearest neighbours (>97 %) was found to be less than 70 % indicating that strain RL(T) represents a novel species of the genus Thermus. MK-8 was the predominant respiratory quinone. The presence of characteristic phospholipid and glycolipid further confirmed that strain RL(T) belongs to the genus Thermus. The predominant fatty acids of strain RL(T) were iso-C17:0 (23.67 %) and iso-C15:0 (24.50 %). The results obtained after DNA-DNA hybridization, biochemical and physiological tests clearly distinguished strain RL(T) from its closely related species. Thus, strain RL(T) represents a novel species of the genus Thermus for which the name Thermus parvatiensis is proposed (=DSM 21745(T)= MTCC 8932(T)).

Pontibacter lucknowensis sp. nov., isolated from a hexachlorocyclohexane dump site.

A Gram-negative, orange-pigmented, rod-shaped, motile and aerobic bacterial strain designated DM9(T) was isolated from hexachlorocyclohexane (HCH)-contaminated soil (Lucknow, India) and its taxonomic position was determined using a polyphasic approach. 16S rRNA gene sequence analysis showed that the isolate belonged to the phylum Bacteroidetes and confirmed its placement in the genus Pontibacter, with sequence similarity ranging from 93.92 to 96.21 % with other members of the genus Pontibacter. The major cellular fatty acids of the novel strain were iso-C(17 : 0) 3-OH (6.00 %), iso-C(15 : 0) (21.54 %) and summed feature 4 (comprising C(17 : 1) iso I/anteiso B; 32.3 %). The polar lipid profile of strain DM9(T) showed the presence of phosphatidylethanolamine, an unidentified aminophospholipid, two unknown aminolipids and four unknown polar lipids. Strain DM9(T) contained MK-7 as the predominant menaquinone and its DNA G+C content was 49.2 mol%. sym-Homospermidine was the major polyamine observed in the cell. The results obtained on the basis of phenotypic characteristics, phylogenetic analysis, biochemical and physiological tests clearly distinguished DM9(T) from closely related members of the genus Pontibacter. It is proposed that DM9(T) represents a novel species, Pontibacter lucknowensis sp. nov.; the type strain is DM9(T) (= CCM 7955(T) = MTCC 11079(T)).

Draft genome sequence of Thermus sp. strain RL, isolated from a hot water spring located atop the Himalayan ranges at Manikaran, India.

Thermus sp. strain RL was isolated from a hot water spring (90°C to 98°C) at Manikaran, Himachal Pradesh, India. Here we report the draft genome sequence (20,36,600 bp) of this strain. The draft genome sequence consists of 17 contigs and 1,986 protein-coding sequences and has an average G+C content of 68.77%.

Complete Genome Analysis of Thermus parvatiensis and Comparative Genomics of Thermus spp. Provide Insights into Genetic Variability and Evolution of Natural Competence as Strategic Survival Attributes.

Thermophilic environments represent an interesting niche. Among thermophiles, the genus Thermus is among the most studied genera. In this study, we have sequenced the genome of Thermus parvatiensis strain RL, a thermophile isolated from Himalayan hot water springs (temperature >96°C) using PacBio RSII SMRT technique. The small genome (2.01 Mbp) comprises a chromosome (1.87 Mbp) and a plasmid (143 Kbp), designated in this study as pTP143. Annotation revealed a high number of repair genes, a squeezed genome but containing highly plastic plasmid with transposases, integrases, mobile elements and hypothetical proteins (44%). We performed a comparative genomic study of the group Thermus with an aim of analysing the phylogenetic relatedness as well as niche specific attributes prevalent among the group. We compared the reference genome RL with 16 Thermus genomes to assess their phylogenetic relationships based on 16S rRNA gene sequences, average nucleotide identity (ANI), conserved marker genes (31 and 400), pan genome and tetranucleotide frequency. The core genome of the analyzed genomes contained 1,177 core genes and many singleton genes were detected in individual genomes, reflecting a conserved core but adaptive pan repertoire. We demonstrated the presence of metagenomic islands (chromosome:5, plasmid:5) by recruiting raw metagenomic data (from the same niche) against the genomic replicons of T. parvatiensis. We also dissected the CRISPR loci wide all genomes and found widespread presence of this system across Thermus genomes. Additionally, we performed a comparative analysis of competence loci wide Thermus genomes and found evidence for recent horizontal acquisition of the locus and continued dispersal among members reflecting that natural competence is a beneficial survival trait among Thermus members and its acquisition depicts unending evolution in order to accomplish optimal fitness.

Draft Genome Sequence of Sphingobium lactosutens Strain DS20T, Isolated from a Hexachlorocyclohexane Dumpsite.

Sphingobium lactosutens DS20(T) has been isolated from the hexachlorocyclohexane (HCH) dumpsite in Lucknow, India, but does not degrade any of the HCH isomers. Here, we present the ~5.36-Mb draft genome sequence of strain DS20(T), which consists of 110 contigs and 5,288 coding sequences, with a G+C content of 63.1%.

Comparative metagenomic analysis of soil microbial communities across three hexachlorocyclohexane contamination levels.

This paper presents the characterization of the microbial community responsible for the in-situ bioremediation of hexachlorocyclohexane (HCH). Microbial community structure and function was analyzed using 16S rRNA amplicon and shotgun metagenomic sequencing methods for three sets of soil samples. The three samples were collected from a HCH-dumpsite (450 mg HCH/g soil) and comprised of a HCH/soil ratio of 0.45, 0.0007, and 0.00003, respectively. Certain bacterial; (Chromohalobacter, Marinimicrobium, Idiomarina, Salinosphaera, Halomonas, Sphingopyxis, Novosphingobium, Sphingomonas and Pseudomonas), archaeal; (Halobacterium, Haloarcula and Halorhabdus) and fungal (Fusarium) genera were found to be more abundant in the soil sample from the HCH-dumpsite. Consistent with the phylogenetic shift, the dumpsite also exhibited a relatively higher abundance of genes coding for chemotaxis/motility, chloroaromatic and HCH degradation (lin genes). Reassembly of a draft pangenome of Chromohalobacter salaxigenes sp. (∼8X coverage) and 3 plasmids (pISP3, pISP4 and pLB1; 13X coverage) containing lin genes/clusters also provides an evidence for the horizontal transfer of HCH catabolism genes.