Classification of microorganisms on the basis of fi traditional microbiological methods (morphological, physiological and biochemical) creates a blurred image about their taxonomic status and thus needs further clarification. It fi should be based on a more pragmatic approach of deploying a number of methods for the complete characterization of microbes. Hence, the methods now employed for bacterial systematics include, the complete 16S rRNA gene sequencing and its comparative analysis by phylogenetic trees, DNA-DNA hybridization studies with related organisms, analyses of molecular markers and signature pattern(s), biochemical assays, physiological and morphological tests. Collectively these genotypic, chemotaxonomic and phenotypic methods for determining taxonomic position of microbes constitute what is known as the 'polyphasic approach' for bacterial systematics. This approach is currently the most popular choice for classifying bacteria and several microbes, which were previously placed under invalid taxa have now been resolved into new genera and species. This has been possible owing to rapid development in molecular biological techniques, automation of DNA sequencing coupled with advances in bioinformatic tools and access to sequence databases. Several DNA-based typing methods are known; these provide information for delineating bacteria into different genera and species and have the potential to resolve differences among the strains of a species. Therefore, newly isolated strains must be classifi ed fi on the basis of the polyphasic approach. Also previously classifi ed organisms, as and when required, can be reclasfi sified on this ground in order to obtain information about fi their accurate position in the microbial world. Thus, current techniques enable microbiologists to decipher the natural phylogenetic relationships between microbes.

The contamination levels in ground and river water suggest significant run-off from the dumped HCH wastes and contamination of drinking water resources. The extent of dumping urgently needs to be assessed regarding the risks to human and ecosystem health. A plan for securing the waste isomers needs to be developed and implemented together with a plan for their final elimination. As part of the assessment, any polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/PCDF) generated during HCH recycling operations need to be monitored.

The study demonstrates that biostimulation of indigenous HCH-degrading microbial population can be used for decontamination of chronically HCH-contaminated sites.

A Gram-negative, non-spore-forming, cream-yellow-pigmented bacterial strain, IP-10 T , was isolated from soil samples from a waste site highly contaminated with hexachlorocyclohexane in Ummari village, India. The organism showed the highest 16S rRNA gene sequence similarity of 92.7 % with Flavobacterium soli KCTC 12542 T and levels of 87-92 % with the type strains of other recognized species of the genus Flavobacterium. The DNA G+C content of strain IP-10 T was 31 mol%. The predominant fatty acids were iso-C 15 : 0 (22.1 %), iso-C 17 : 0 3-OH (18.5 %) and summed feature 3 (comprising C 16 : 1 v7c and/or iso-C 15 : 0 2-OH; 13.2 %). Strain IP-10 T could be differentiated from recognized species of the genus Flavobacterium based on a number of phenotypic features. Strain IP-10 T is therefore considered to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium lindanitolerans sp. nov. is proposed. The type strain is IP-10 T (5MTCC 8597 T 5CCM 7424 T ).The genus Flavobacterium, the type genus of the family Flavobacteriaceae, was proposed by Bergey et al. (1923) and the description was considerably emended by Bernardet et al. (1996). It belongs to the phylum Bacteroidetes (Bernardet & Bowman, 2006) (formerly the Flavobacterium-Flexibacter-Bacteroides group; Woese et al., 1990). Members of the genus are widely distributed in nature and are routinely isolated from soil (Yoon et al., 2006), sediments, freshwater and marine water ecosystems, microbial mats (Van Trappen et al., 2003, sea ice (Humphry et al., 2001) and infected fish. The genus Flavobacterium comprises Gram-negative, non-sporeforming, strictly aerobic, predominantly gliding, pigmented bacteria with menaquinone-6 as the major respiratory quinone and a DNA G+C content of 30-41 mol% (Bernardet & Bowman. 2006;Park et al., 2006). Flavobacterium aquatile is the type species of the genus.The present study deals with the taxonomic characterization of a novel species of the genus Flavobacterium isolated from soil from a recently created waste site for a lindane manufacturing unit situated in Ummari village, Lucknow city, in the state of Uttar Pradesh, India. Lindane, c-hexachlorocyclohexane (HCH), is the only HCH isomer that has insecticidal properties. Strain IP-10 T was isolated after 7 days incubation at 28 u C by plating serially diluted soil samples on Luria-Bertani agar plates (10 g tryptone, 5 g yeast extract, 5 g NaCl, 1 g D-glucose, 15 g agar per litre) containing nystatin (30 mg ml 21 ) and streptomycin (200 mg ml 21 ), used to isolate HCH-degrading sphingomonads (Vanbroekhoven et al., 2004). 16S rRNA gene sequence analysis, fatty acid profiling and other phenotypic characteristics revealed that the new isolate belonged to the genus Flavobacterium but exhibited sufficient significant differences from other closely related members of the genus to be considered as representing a novel species.16S rRNA gene sequence analysis was carried out as described by Prakash et al. (2007). Briefly, a single colony of strain IP-10 T was...

A yellow-pigmented, hexachlorocyclohexane (HCH)-degrading bacterium, strain IP26 T , was isolated from an HCH dumpsite and subjected to a polyphasic analysis in order to determine its taxonomic position. Strain IP26 T showed maximum 16S rRNA gene sequence similarity with Sphingobium francense Sp+ T (98.5 %), Sphingobium japonicum UT26 T (98.4 %) and Sphingobium indicum B90A T (98.2 %). Phylogenetic analysis based on 16S rRNA gene sequences also showed that strain IP26 T formed a cluster with these three HCH-degrading strains. Chemotaxonomic data (major polyamine, spermidine; major quinone, ubiquinone with ten isoprene units; major polar lipids, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, diphosphatidylglycerol, phosphotidylcholine; and presence of 2-hydroxy fatty acid) supported inclusion of strain IP26 T in the genus Sphingobium. However, the results of DNA-DNA hybridization and morphological and biochemical tests clearly allowed phenotypic and genotypic differentiation of strain IP26 T from recognized species of the genus Sphingobium. Strain IP26 T thus represents a novel species of the genus Sphingobium for which the name Sphingobium chinhatense sp. nov. is proposed. The type strain is IP26 T (5MTCC8598 T 5CCM 7432 T ).Hexachlorocyclohexane (HCH) contamination can occur as a result of the unusual production process of this compound . HCH is prepared by the chlorination of benzene in the presence of UV. This generally leads to the production of four main HCH isomers, a-, b-, c-and d-, in the ratio 60-70, 5-12, 10-12 and 6-10 %, respectively (Willett et al., 1998). Among these, only c-HCH, also called lindane, has insecticidal activity. During the purification process, the remaining isomers (one tonne of lindane produces nine tonnes of HCH waste) are discarded in the open, thus creating an HCH-contaminated dumpsite. We discovered one such dumpsite in the vicinity of an industry that has been producing lindane for the past 10 years (Dadhwal et al., 2009). Around 24 bacterial strains were isolated from these sites and their potential to degrade HCH isomers was evaluated (Dadhwal et al., 2009). Some of these bacterial isolates have been characterized by using a polyphasic approach (Kumar et al., 2008;Singh & Lal, 2009).Strain IP26 T was isolated from highly HCH-contaminated soil by culturing on LB agar using a serial dilution method (Dadhwal et al., 2009). The strain was found to degrade a-, b-, c-and d-HCH isomers faster than Sphingobium indicum B90A T (Dadhwal et al., 2009) (Supplementary Fig. S1, available in IJSEM Online). Strain IP26 T was characterized taxonomically and found to represent a novel species of Sphingobium.Colony size, shape and colour were studied on LB agar plates incubated at 28 uC. Gram-staining and sporestaining were performed using HiMedia kits. Motility of strain IP26 T was assessed by the hanging drop method as well as on motility agar medium. Catalase and oxidase tests were carried out as described by McCarthy & Cross (1984). Hydrolysis of aesculin and Tween 80 and ...

The unusual process of production of hexachlorocyclohexane (HCH) and extensive use of technical HCH and lindane has created a very serious problem of HCH contamination. While the use of technical HCH and lindane has been banned all over the world, India still continues producing lindane. Bacteria, especially Sphingomonads have been isolated that can degrade HCH isomers. Among all the bacterial strains isolated so far, Sphingobium indicum B90A that was isolated form HCH treated rhizosphere soil appears to have a better potential for HCH degradation. This conclusion is based on studies on the organization of lin genes and degradation ability of B90A. This strain perhaps can be used for HCH decontamination through bioaugmentation.

A Gram-negative, non-spore-forming, cream-coloured bacterial strain, UM2 T , was isolated from an open hexachlorocyclohexane (HCH) dump site at Ummari village in Lucknow, India. Data generated from a polyphasic approach including phenotypic, genotypic and chemotaxonomic analyses confirmed that strain UM2 T belonged to the genus Sphingomonas. The highest similarity found to the 16S rRNA gene sequence of strain UM2 T was 99.4 %, with Sphingomonas wittichii DSM 6014 T , whereas the DNA-DNA relatedness value between these strains was 31 %, indicating that they represent separate species. The DNA G+C content of UM2 T was 66.9 mol%. The respiratory pigment ubiquinone Q-10 was present. The predominant fatty acids were summed feature 8 (C 18 : 1 v6c and/or C 18 : 1 v7c; 32.9 %), C 19 : 0 cyclo v8c (15.5 %) and C 16 : 0 (12.1 %). The major polar lipids were phosphatidylcholine, phosphatidylglycerol and phosphatidyldimethylethanolamine. sym-Homospermidine was the major polyamine observed. On the basis of the data reported, it was concluded that UM2 T represents a novel species of the genus Sphingomonas, for which the name Sphingomonas histidinilytica sp. nov. is proposed. The type strain is UM2 T (5MTCC 9473 T 5CCM 7545 T ).The genus Sphingomonas was proposed by Yabuuchi et al. (1990) and its description has subsequently been amended (Takeuchi et al., 2001;Yabuuchi et al., 2002;Busse et al., 2003). At the time of writing, the genus Sphingomonas includes species that produce orange, yellow or off-white or red colonies (Asker et al., 2007) and cells that are nonsporulating, aerobic, Gram-negative, non-motile or motile rods with a single polar flagellum.A polyphasic approach was adopted for the taxonomic classification of a novel bacterium, strain UM2 T , isolated from a hexachlorocyclohexane (HCH) dump site in the village of Ummari, Lucknow, Uttar Pradesh, India. Strain UM2 T was isolated as described by Jit et al. (2008). Biochemical, physiological and morphological characterization was carried out by routine culturing at 28 u C in Luria-Bertani (LB) broth or on LB agar. Morphological features like colony shape, size and colour were observed on LB agar plates after 48-72 h of incubation. To determine the optimal conditions for growth, strain UM2 T was cultured at 0-40 u C, pH 4-10 and with 0-4 % (w/v) NaCl in LB broth (Arden-Jones et al., 1979). Growth of UM2 T was also observed on nutrient, tryptone soya and MacConkey agars. Gram-staining and spore staining were tested using a Gram and spore forming kit (HiMedia). Motility was tested on motility agar. The presence of flagella was determined with a transmission electron microscope (model 269D; Morgagni).16S rRNA gene sequence analysis was carried out using the universal primers 8F (59-AGAGTTTGATCCTGGCTCAG-39) and 1542R (59-AAGGAGGTGATCCAGCCGCA-39) as described by Prakash et al. (2007). After sequencing, a 1394 bp stretch of the 16S rRNA gene was obtained, which was checked manually to ensure sequence quality. Similarity searching was carried out using the Seqmatch tool o...

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 β-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 β- and δ-tetrachlorocyclohexane-1,4-diol, the terminal metabolites of β- 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.