Characterization and complete genome analysis of Klebsiella phage Kp109 with lytic activity against Klebsiella pneumoniae.

Klebsiella pneumonia is a serious pathogen involved in a range of infections. The increasing frequency of infection associated with K. pneumoniae and accelerated development of antimicrobial resistance has limited the available options of antibiotics for the treatment of infection. Bacteriophages are an attractive substitute to alleviate the problem of antibiotic resistance. In this study, isolation, microbiological and genomic characterization of bacteriophage Kp109 having the ability to infect K. pneumoniae has been shown. Phage Kp109 showed good killing efficiency and tolerance to a broad range of temperatures (4-60 °C) and pH (3-9). Transmission electron microscopy and genomic analysis indicated that phage Kp109 belongs to the genus Webervirus and family Drexlerviridae. Genomic analysis showed that the Kp109 has a 51,630 bp long double-stranded DNA genome with a GC content of 51.64%. The absence of known lysogenic, virulence, and antibiotic-resistant genes (ARGs) in its genome makes phage Kp109 safer to be used as a biocontrol agent for different purposes including phage therapy. The computational analysis of the putative endolysin gene revealed a binding energy of - 6.23 kcal/mol between LysKp109 and ligand NAM-NAG showing its potential to be used as an enzybiotic. However, future research is required for experimental validation of the in silico work to further corroborate the results obtained in the present study. Overall, phenotypic, genomic, and computational characterization performed in the present study showed that phages Kp109 and LysKp109 are promising candidates for future in vivo studies and could potentially be used for controlling K. pneumoniae infection.

Comparative genomics of an extensively drug resistant strain Klebsiella pneumoniae IITR008 with international high-risk clonal lineage ST147 isolated from river water.

Carbapenem resistant Klebsiella pneumoniae causing severe infection resulting in morbidity and mortality have become a global health concern. K. pneumoniae with sequence type ST147 is an international high-risk clonal lineage, genomic studies have been done on K. pneumoniae ST147 isolated from clinical origin but genomic data for environmental K. pneumoniae ST147 is very scarce. Herein, K. pneumoniae IITR008, an extensively drug resistant and potentially hypervirulent bacterium, was isolated from Triveni Sangam, the confluence of three rivers where religious congregations are organized. Phenotypic, genomic and comparative genomic analysis of strain IITR008 was performed. Antibiotic susceptibility profiling revealed resistance to 9 different classes of antibiotics including ß-lactams, ß-lactam combination agents, carbapenem, aminoglycoside, macrolide, quinolones, cephams, phenicol, and folate pathway antagonists and was found to be susceptible to only tetracycline. The strain IITR008 possesses hypervirulence genes namely, iutA and iroN in addition to numerous virulence factors coding for adherence, regulation, iron uptake, secretion system and toxin. Both the IITR008 chromosome and plasmid pIITR008_75 possess a plethora of clinically relevant antibiotic-resistant genes (ARGs) including blaCTX-M-15, blaTEM-1, and blaSHV-11, corroborating the phenotypic resistance. Comparative genomic analysis with other ST147 K. pneumoniae provided insights on the phylogenetic clustering of IITR008 with a clinical strain isolated from a patient in Czech with recent travel history in India and other clinical strains isolated from India and Pakistan. According to the 'One Health' perspective, surveillance of antibiotic resistance in the environment is crucial to impede its accelerated development in diverse ecological niches.

Biodegradation of polyethylene terephthalate (PET) by Brucella intermedia IITR130 and its proposed metabolic pathway.

Accumulation of polyethylene terephthalate (PET) polyester in ecosystems across the globe is a major pollution of concern. Microbial degradation recently generated novel insights into the biodegradation of varieties of plastics. In this study, a PET degrading bacterium Brucella intermedia IITR130 was isolated from a contaminated lake ecosystem at Pallikaranai, Chennai, India. Incubation of the bacterium along with the PET sheet (0.1 mm thickness) for 60 days resulted in 26.06% degradation, indicating a half-life of 137.8 days. Considerable changes in the surface morphology of the PET sheet were found as holes, pits, and cracks on incubation with strain IITR130, as revealed by scanning electron microscopy (SEM). After bacterial treatment of PET, the formation of new functional groups, most notably in the area of 3326 cm-1 suggestive of O-H stretch, leading to carboxylic acid and alcohol as products were suggested by fourier transform infrared (FTIR) analysis. Monomethyl terephthalate (MMT) and terephthalic acid (TPA) were identified by gas chromatography-mass spectrometry (GC-MS) analysis as PET degradation metabolites. Tributyrin clearance assay confirmed the presence of a lipase/esterase enzyme in the strain IITR130. In this study, a degradation pathway for PET by an isolated and identified bacterium Brucella intermedia IITR130 was characterized in detail.

Unlocking bioremediation potential for site restoration: A comprehensive approach for crude oil degradation in agricultural soil and phytotoxicity assessment.

Crude oil contamination has inflicted severe damage to soil ecosystems, necessitating effective remediation strategies. This study aimed to compare the efficacy of four different techniques (biostimulation, bioaugmentation, bioaugmentation + biostimulation, and natural attenuation) for remediating agricultural soil contaminated with crude oil using soil microcosms. A consortium of previously characterized bacteria Xanthomonas boreopolis, Microbacterium schleiferi, Pseudomonas aeruginosa, and Bacillus velezensis was constructed for bioaugmentation. The microbial count for the constructed consortium was recorded as 2.04 ± 0.11 × 108 CFU/g on 60 d in augmented and stimulated soil samples revealing their potential to thrive in chemically contaminated-stress conditions. The microbial consortium through bioaugmentation + biostimulation approach resulted in 79 ± 0.92% degradation of the total polyaromatic hydrocarbons (2 and 3 rings âˆ¼ 74%, 4 and 5 rings âˆ¼ 83% loss) whereas, 91 ± 0.56% degradation of total aliphatic hydrocarbons (C8-C16 ∼ 90%, C18-C28 ∼ 92%, C30 to C40 ∼ 88% loss) was observed in 60 d. Further, after 60 d of microcosm treatment, the treated soil samples were used for phytotoxicity assessment using wheat (Triticum aestivum), black chickpea (Cicer arietinum), and mustard (Brassica juncea). The germination rates for wheat (90%), black chickpea (100%), and mustard (100%) were observed in 7 d with improved shoot-root length and biomass in both bioaugmentation and biostimulation approaches. This study projects a comprehensive approach integrating bacterial consortium and nutrient augmentation strategies and underscores the vital role of innovative environmental management practices in fostering sustainable remediation of oil-contaminated soil ecosystems. The formulated bacterial consortium with a nutrient augmentation strategy can be utilized to restore agricultural lands towards reduced phytotoxicity and improved plant growth.

Insights into molecular mechanism of plasticizer biodegradation in Dietzia kunjamensis IITR165 and Brucella intermedia IITR166 isolated from a solid waste dumpsite.

AIMS: Isolation of phthalate esters (PAEs) degrading bacteria from a solid waste dumpsite could degrade many plasticizers efficiently and to investigate their degrading kinetics, pathways, and genes. METHODS AND RESULTS: Based on their 16S rRNA gene sequence the strains were identified as Dietzia kunjamensis IITR165 and Brucella intermedia IITR166, which showed a first-order degradation kinetic model under lab conditions. The quantification of phthalates and their intermediate metabolites identification were done by using ultra-high-performance liquid chromatography (UHPLC) and gas chromatography-tandem mass-spectrometry (GC-MS/MS), respectively. Both the bacteria utilized >99% dibutyl phthalate at a high concentration of 100-400 mg L-1 within 192 h as monitored by UHPLC. GC-MS/MS revealed the presence of metabolites dimethyl phthalate (DMP), phthalic acid (PA), and benzoic acid (BA) during DBP degradation by IITR165 while monobutyl phthalate (MBP) and PA were identified in IITR166. Phthalate esters degrading gene cluster in IITR165 comprised two novel genes coding for carboxylesterase (dkca1) and mono-alkyl phthalate hydrolase (maph), having only 37.47% and 47.74% homology, respectively, with reported phthalate degradation genes, along with the terephthalate dioxygenase system (tphA1, A2, A3, and B). However, IITR166 harbored different gene clusters comprising di-alkyl phthalate hydrolase (dph_bi), and phthalate dioxygenase (ophA, B, and C) genes. CONCLUSIONS: Two novel bacterial strains, Dietzia kunjamensis IITR165 and Brucella intermedia IITR166, were isolated and found to efficiently degrade DBP at high concentrations. The degradation followed first-order kinetics, and both strains exhibited a removal efficiency of over 99%. Metabolite analysis revealed that both bacteria utilized de-methylation, de-esterification, and decarboxylation steps during degradation.

Holistic approach to waste mobil oil bioremediation: Valorizing waste through biosurfactant production for soil restoration.

The combustion of mobil oil leads to the emission of toxic compounds in the environment. In this study, the aromatic and aliphatic hydrocarbon fractions present in a waste mobil oil collected from automobile market were comprehensively identified and their toxicity was evaluated using wheat grain. Lysinibacillus sphaericus strain IITR51 isolated and characterized previously could degrade 30-80% of both aliphatic and aromatic hydrocarbons in liquid culture. Interestingly, the strain IITR51 produced 627 mg/L of rhamnolipid biosurfactant by utilizing 3% (v/v) of waste mobil oil in the presence of 1.5% glycerol as additional carbon source. In a soil microcosm study by employing strain IITR51, 50-86% of 3-6 ring aromatic hydrocarbons and 63-98% of aliphatic hydrocarbons (C8 to C22) were degraded. Addition of 60 µg/mL rhamnolipid biosurfactant enhanced the degradation of both aliphatic and aromatic hydrocarbons from 76.88% to 61.21%-94.11% and 78.27% respectively. The degradation of mobil oil components improved the soil physico-chemical properties and increased soil fertility to 64% as evident by the phytotoxicity assessments. The findings indicate that strain IITR51 with degradation capability coupled with biosurfactant production could be a candidate for restoring hydrocarbon contaminated soils.

Simultaneous Biodegradation of Polyaromatic Hydrocarbons by a Stenotrophomonas sp: Characterization of nid Genes and Effect of Surfactants on Degradation.

A polyaromatic hydrocarbon degrading bacterium was isolated from a petroleum contaminated site and designated as Stenotrophomonas sp. strain IITR87. It was found to utilize pyrene, phenanthrene and benzo(a)pyrene as sole carbon source, but not anthracene, chrysene and fluoranthene. Gas chromatography and mass spectroscopy analysis resulted in identification of pyrene metabolites namely monohydroxypyrene, 4-oxa-pyrene-5-one, dimethoxypyrene and monohydroxyphenanthrene. Southern hybridization using naphthalene dioxygenase gene (nidA) as probe against the DNA of strain IITR87 revealed the presence of nidA gene. PCR analysis suggests dispersed occurrence of nid genes in the genome instead of a cluster as reported in a PAH-degrading Mycobacterium vanbaalenii PYR-1. The nid genes in strain IITR87, dioxygenase large subunit (nidA), naphthalene dioxygenase small subunit (nidB) and aldehyde dehydrogenase gene (nidD) showed more than 97 % identity to the reported nid genes from Mycobacterium vanbaalenii PYR-1. Most significantly, the biodegradation of PAHs was enhanced 25-60 % in the presence of surfactants rhamnolipid and Triton X-100 due to increased solubilization and bioavailability. These results could be useful for the improved biodegradation of high-molecular-weight PAHs in contaminated habitats.

Isolation and characterization of a novel Gram-negative bacterium Chromobacterium alkanivorans sp. nov., strain IITR-71T degrading halogenated alkanes.

The taxonomic position of a Gram-stain negative, non-violaceinpigmented bacterium isolated from an insecticide-contaminated site was characterized by a polyphasic approach. The bacterium was able to grow on three different halogenated compounds namely 1-hlorobutane, 1-hloropropane and 1,2-ichloroethane. As a critical step in the degradation of these haloalkanes, stoichiometric amounts of dechlorination were estimated. Based on selective enrichment method for three months, using a highly contaminated mixed chemical soil, a bacterium was obtained and designated as IITR-71T. Its versatility and novelty led us to further characterize it by polyphasic taxonomy. The 16S rRNA gene sequence (1446 bases) comparison showed highest similarity with those of members of the genus Chromobacterium with the most closely related species to strain IITR-71T being Chromobacterium aquaticum (99.3 %) followed by Chromobacterium haemolyticum (98.6 %) and Chromobacterium piscinae (97.1 %). The major ubiquinone was Q-8. Predominant polar lipids are phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG). The DNA G+C content of IITR-71T was estimated to be 61.2 mol%. The genotypic and phenotypic distinctiveness of IITR-71T and its phylogenetic relationships indicate that IITR-71T represents a novel species, for which the name Chromobacterium alkanivorans sp. nov. is proposed. The type strain is IITR-71T (=MTCC 11059T=JCM 30068T=KCTC 52433T).

Taxonomic description and genome sequence of Rheinheimera mesophila sp. nov., isolated from an industrial waste site.

A novel Gram-staining-negative gammaproteobacterium, designated IITR-13T, was isolated from a pesticide-contaminated soil and characterized using a polyphasic approach. On the basis of 16S rRNA gene sequence analysis, the strain showed the closest similarity (98.7 %) to Rheinheimera tangshanensis JA3-B52T followed by Rheinheimera texasensis A62-14BT (97.7 %) and Rheinheimera soli BD-d46T (97.3 %). The 16S rRNA gene sequence similarity of the novel strain to other members of the genus Rheinheimera was < 97.3 %. However, DNA-DNA hybridization between strain IITR-13T and the type strains of R. tangshanensis, R. texasensis and R. soli was 47.5 ± 0.6, 42.4 ± 0.4 and 39.8 ± 0.3 %, respectively; these values are less than 70 %, a threshold value for delineation of a novel species. The strain had C12 : 0 3-OH, C16 : 0, C17 : 1ω8c, summed feature 3 (C16 : 1ω6c/C16 : 1ω7c) and C18 : 1ω6c as the major fatty acids. The major isoprenoid quinones detected for strain IITR-13T were ubiquinone Q-8 and menaquinone MK-7.The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and seven unknown phospholipids. Based on phenotypic and chemotaxonomic characteristics and analysis of the 16S rRNA gene sequence, the novel strain should be assigned to a novel species, for which the name Rheinheimera mesophila sp. nov. is proposed, with the type strain IITR-13T ( = MTCC 12064T = DSM 29723T). Also, we report the draft genome sequence of Rheinheimera mesophila IITR-13T; the draft genome sequence includes 3 749 903 bases and comprises 3449 predicted coding sequences, with a G+C content of 47.8 %. It consists of 102 contigs (>1000 bp).

Bacillus mesophilum sp. nov., strain IITR-54T, a novel 4-chlorobiphenyl dechlorinating bacterium.

The taxonomic position of a Gram-positive, endospore-forming bacterium isolated from soil sample collected from an industrial site was analyzed by a polyphasic approach. The strain designated as IITR-54T matched most of the phenotypic and chemical characteristics of the genus Bacillus and represents a novel species. It was found to biodegrade 4-chlorobiphenyl through dechlorination and was isolated through enrichment procedure from an aged polychlorinated biphenyl-contaminated soil. Both resting cell assay and growth under aerobic liquid conditions using 4-chlorobiphenyl as sole source of carbon along with 0.01% yeast extract, formation of chloride ions was measured. 16S rRNA (1,489 bases) nucleotide sequence of isolated strain was compared with those of closely related Bacillus type strains and confirmed that the strain belongs to the genus Bacillus. Strain IITR-54T differs from all other species of Bacillus by at least 2.1% at the 16S rRNA level, and the moderately related species are Bacillus oceanisediminis (97.9%) followed by Bacillus infantis (97.7%), Bacillus firmus (97.4%), Bacillus drentensis (97.3%), Bacillus circulans (97.2%), Bacillus soli (97.1%), Bacillus horneckiae (97.1%), Bacillus pocheonensis (97.1%) and Bacillus bataviensis (97.1%), respectively. The cell wall peptidoglycan contained meso-diaminopimelic acid and the major isoprenoid quinone was MK-7. Major fatty acids are iso-C15:0 (32.4%) and anteiso-C15:0 (27.4%). Predominant polar lipids are diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The results of physiological and biochemical tests allowed the genotypic and phenotypic distinctiveness of strain IITR-54T with its phylogenetic relatives and suggest that the strain IITR-54T should be recognized as a novel species, for which the name Bacillus mesophilum sp. nov. is proposed. The type strain is IITR-54T (=MTCC 11060T=JCM 19208T).

Bioremediation of polycyclic aromatic hydrocarbons in crude oil by bacterial consortium in soil amended with Eisenia fetida and rhamnolipid.

The present study investigated the concerted effort of Eisenia fetida and rhamnolipid JBR-425 in combination with a five-member bacterial consortium exhibiting elevated degradation levels of low and high molecular weight polycyclic aromatic hydrocarbons (PAH) from soil contaminated with Digboi crude oil. Application of bacterial consortium (G2) degraded 30-89% of selected PAH from the artificial soil after a 45-day post-exposure, in which chrysene showed the highest level of degradation with 89% and benzo(a)pyrene is the lowest with 30%, respectively. Moreover, an acute exposure study observed that earthworm biomass decreased, and mortality rates increased with increasing crude oil concentrations (0.25 to 2%). Earthworms with a 100% survival rate at 1% crude oil exposure suggest the tolerance potential and its mutual involvement in the bioremediation of crude oil with selected bacterial consortia. Bacterial consortium assisted with E. fetida (G3) showed 98% chrysene degradation with a slight change in benzo(a)pyrene degradation (35%) in crude oil spiked soil. Besides, the most dominant PAH in crude oil found in the current work, fluoranthene, undergoes 93% and 70% degradation in G3 and G5 groups, respectively. However, rhamnolipid JBR-425 coupled with the bacterial consortium (G5) has resulted in 97% degradation of chrysene and 33% for benzo(a)pyrene. Overall, bacterial consortium assisted with earthworm group has shown better degradation of selected PAH than bacterial consortium with biosurfactant. Catalase (CAT), glutathione reductase (GST) activity and MDA content was found to be reduced in earthworms after sub-lethal exposure, suggesting oxidative stress prevalence via reactive oxygen species (ROS). Hence, the findings of the present work suggest that the application of a bacterial consortium, along with earthworm E. fetida, has huge potential for field restoration of contaminated soil with PAH and ecosystem sustainability.

Bacterial remediation of pesticide polluted soils: Exploring the feasibility of site restoration.

For decades, reclamation of pesticide contaminated sites has been a challenging avenue. Due to increasing agricultural demand, the application of synthetic pesticides could not be controlled in its usage, and it has now adversely impacted the soil, water, and associated ecosystems posing adverse effects on human health. Agricultural soil and pesticide manufacturing sites, in particular, are one of the most contaminated due to direct exposure. Among various strategies for soil reclamation, ecofriendly microbial bioremediation suffers inherent challenges for large scale field application as interaction of microbes with the polluted soil varies greatly under climatic conditions. Methodically, starting from functional or genomic screening, enrichment isolation; functional pathway mapping, production of tensioactive metabolites for increasing the bioavailability and bio-accessibility, employing genetic engineering strategies for modifications in existing catabolic genes to enhance the degradation activity; each step-in degradation study has challenges and prospects which can be addressed for successful application. The present review critically examines the methodical challenges addressing the feasibility for restoring and reclaiming pesticide contaminated sites along with the ecotoxicological risk assessments. Overall, it highlights the need to fine-tune the available processes and employ interdisciplinary approaches to make microbe assisted bioremediation as the method of choice for reclamation of pesticide contaminated sites.

Functional genomic analysis of an efficient indole degrading bacteria strain Alcaligenes faecalis IITR89 and its biodegradation characteristics.

Indole is a nitrogenous heterocyclic aromatic pollutant often detected in various environments. An efficient indole degrading bacterium strain IITR89 was isolated from River Cauvery, India, and identified as Alcaligenes faecalis subsp. phenolicus. The bacterium was found to degrade ~ 95% of 2.5 mM (293.75 mg/L) of indole within 18 h utilizing it as a sole carbon and energy source. Based on metabolite identification, the metabolic route of indole degradation is indole → (indoxyl) → isatin → (anthranilate) → salicylic acid → (catechol) → (Acetyl-CoA) → and further entering into TCA cycle. Genome sequencing of IITR89 revealed the presence of gene cluster dmpKLMNOP, encoding multicomponent phenol hydroxylase; andAbcd gene cluster, encoding anthranilate 1,2-dioxygenase ferredoxin subunit (andAb), anthranilate 1,2-dioxygenase large subunit (andAc), and anthranilate 1,2-dioxygenase small subunit (andAd); nahG, salicylate hydroxylase; catA, catechol 1,2-dioxygenase; catB, cis, cis-muconate cycloisomerase; and catC, muconolactone D-isomerase which play an active role in indole degradation. The findings strongly support the degradation potential of strain IITR89 and its possible application for indole biodegradation.

Assessing the half-life and degradation kinetics of aliphatic and aromatic hydrocarbons by bacteria isolated from crude oil contaminated soil.

Pollution from the oil industries and refineries has worsened various environmental compartments. In this study, indigenous oil degrading bacteria were isolated from crude oil obtained from an Oil and Natural Gas Corporation (ONGC) asset in Ankleshwar, Gujarat, India. Based on 16S rRNA phylogeny, they were identified as Pseudomonas boreopolis IITR108, Microbacterium schleiferi IITR109, Pseudomonas aeruginosa IITR110, and Bacillus velezensis IITR111. The strain IITR108, IITR109, IITR110, and IITR111 showed 80-89% and 71-78% degradation of aliphatic (C8-C40) and aromatic (4-5 ring) hydrocarbons respectively in 45 d when supplemented with 3% (v/v) waste crude oil. When compared to individual bacteria, the consortium degrades 93.2% of aliphatic hydrocarbons and 85.5% of polyaromatic hydrocarbons. It was observed that the total aliphatic and aromatic content of crude oil 394,470 µg/mL and 47,050 µg/mL was reduced up to 9617.75 µg/mL and 4586 µg/mL respectively in 45 d when consortium was employed. The rate kinetics analysis revealed that the biodegradation isotherm followed first order kinetics, with a linear correlation between concentration (hydrocarbons) and time intervals. The half-life of aliphatic (C8-C40) and aromatic hydrocarbons ranged from 200 to 453 h and 459-714 h respectively. All the bacteria efficiently produced catabolic enzymes such as alkane monooxygenase, alcohol dehydrogenase, and lipase during the degradation of crude oil. These findings indicated that the bacterial consortium can be a better candidate for bioremediation and reclamation of aliphatic and aromatics hydrocarbon contaminated sites.

Nitratireductor lucknowense sp. nov., a novel bacterium isolated from a pesticide contaminated soil.

A straw-yellow pigmented bacterium, strain IITR-21(T) was isolated from a pesticide contaminated site and characterized by using a polyphasic taxonomic approach. The organism had morphological and chemotaxonomic properties consistent with its classification in the genus Nitratireductor. Phylogenetic analysis of the 16S rRNA gene sequence showed that the strain IITR-21(T) belongs to the genus Nitratireductor and was moderately related to Nitratireductor indicus C115(T) (97.7%) and Nitratireductor pacificus pht-3B(T) (97.4%), whereas sequence similarity value with the other species including the type species of the genus Nitratireductor, Nitratireductor aquibiodomus showed less than 97.0% similarity. However, the DNA-DNA relatedness values between strain IITR-21(T) and the moderately related taxa N. indicus (59.1%) and N. pacificus (40.4%) were well below the 70% threshold value recommended for the delineation of bacterial species. The G+C content of the DNA was 62.4 mol%. Based on physiological, biochemical tests and genotypic differences between the strain IITR-21(T) and the other two validly published species of the genus Nitratireductor, it is proposed that the strain be classified as a new species of Nitratireductor as Nitratireductor lucknowense sp. nov. The type strain is IITR-21(T) (=MTCC 8354(T )= DSM 24322(T)).

Surfactant mediated enhanced biodegradation of hexachlorocyclohexane (HCH) isomers by Sphingomonas sp. NM05.

Environmental biodegradation of several chlorinated pesticides is limited by their low solubility and sorption to soil surfaces. To mitigate this problem we quantified the effect of three biosurfactant viz., rhamnolipid, sophorolipid and trehalose-containing lipid on the dissolution, bioavailability, and biodegradation of HCH-isomers in liquid culture and in contaminated soil. The effect of biosurfactants was evaluated through the critical micelle concentration (CMC) value as determined for each isomer. The surfactant increased the solubilization of HCH isomers by 3-9 folds with rhamnolipid and sophorolipid being more effective and showing maximum solubilization of HCH isomers at 40 µg/mL, compared to trehalose-containing lipid showing peak solubilization at 60 µg/mL. The degradation of HCH isomers by Sphingomonas sp. NM05 in surfactant-amended liquid mineral salts medium showed 30% enhancement in 2 days as compared to degradation in 10 days in the absence of surfactant. HCH-spiked soil slurry incubated with surfactant also showed around 30-50% enhanced degradation of HCH which was comparable to the corresponding batch culture experiments. Among the three surfactants, sophorolipid offered highest solubilization and enhanced degradation of HCH isomers both in liquid medium and soil culture. The results of this study suggest the effectiveness of surfactants in improving HCH degradation by increased bioaccessibility.

Sudden deaths due to accidental leakage of Lindane from a storage tank in a village, Sitapur, Uttar Pradesh, India, 2020: A field epidemiological investigation.

Background: Chemical leakages cause devastating health effects on humans. On 6 February 2020, seven deaths were reported following a hazardous chemical leakage in a village in Uttar Pradesh, India. We investigated the event to identify the cause and propose recommendations. Methods: We defined a case as sudden onset of breathlessness, headache, or death in the village, 6-7 February 2020. We conducted a house-to-house case search and calculated attack rate (AR) and case-fatality rate (CFR) by age and gender. We conducted an environmental investigation at the leakage site and sent the chemicals for forensic analysis. We obtained the cause of death through autopsy reports. Results: Out of 2,942 residents, we identified 23 cases (AR = 8/1,000) and seven deaths (CFR = 30%). The median age of the case was 42 years (range, 2-64 years). The AR was higher among males (14/1,000 [19/1,402]). All the 23 case-patients who were sleeping at the chemical leakage site or visited to witness the event developed symptoms, and all seven cases who were sleeping within 150 meters of the leakage site died. The environmental investigation revealed leakage of hazardous substances from the storage tank. Toxicology analysis confirmed the leaked chemical as Lindane (gamma-hexachlorocyclohexane), and autopsy reports confirmed the cause of death as asphyxia. Conclusions: Asphyxia following the leakage of Lindane from the storage tank possibly led to sudden deaths. We recommend using leak-proof tanks to ensure safe storage and disposal, law enforcement, and regulations to prevent people from staying close to chemical storage sites.

Profiling of biodegradation and bacterial 16S rRNA genes in diverse contaminated ecosystems using 60-mer oligonucleotide microarray.

We have developed an oligonucleotide microarray for the detection of biodegradative genes and bacterial diversity and tested it in five contaminated ecosystems. The array has 60-mer oligonucleotide probes comprising 14,327 unique probes derived from 1,057 biodegradative genes and 880 probes representing 110 phylogenetic genes from diverse bacterial communities, and we named it as BiodegPhyloChip. The biodegradative genes are involved in the transformation of 133 chemical pollutants. Validation of the microarray for its sensitivity specificity and quantitation were performed using DNA isolated from well-characterized mixed bacterial cultures also having non-target strains, pure degrader strains, and environmental DNA. Application of the developed array using DNA extracted from five different contaminated sites led to the detection of 186 genes, including 26 genes unique to the individual sites. Hybridization of 16S rRNA probes revealed the presence of bacteria similar to well-characterized genera involved in biodegradation of various pollutants. Genes involved in complete degradation pathways for hexachlorocyclohexane (lin), 1,2,4-trichlorobenzene (tcb), naphthalene (nah), phenol (mph), biphenyl (bph), benzene (ben), toluene (tbm), xylene (xyl), phthalate (pht), Salicylate (sal), and resistance to mercury (mer) were detected with highest intensity. The most abundant genes belonged to the enzyme hydroxylases, monooxygenases, and dehydrogenases which were present in all the five samples. Thus, the array developed and validated here shall be useful in assessing not only the biodegradative potential but also the composition of environmentally useful bacteria, simultaneously, from hazardous ecosystems.

Plants exert beneficial influence on soil microbiome in a HCH contaminated soil revealing advantage of microbe-assisted plant-based HCH remediation of a dumpsite.

Persistence of hexachlorocyclohexane (HCH) pesticide is a major problem for its disposal. Soil microflora plays an important role in remediating contaminated sites. Keeping concepts of microbial- and phyto-remediation together, the difference between soil microflora with and without association of HCH accumulating plant species was studied. Metagenomic analysis among the non-plant soil (BS) (∑HCH 434.19 mg/g), rhizospheric soil of shrubs (RSS) (∑HCH 157.31 mg/g), and rhizospheric soil of trees (RSD) (∑HCH 105.39 mg/g) revealed significant differences in microbial communities. Shrubs and trees occurred at a long-term dumpsite accumulated α- and ß- HCH residues. Plant rhizospheric soils exhibited high richness and evenness with higher diversity indices compared to the non-plant soil. Order Rhizobiales was most abundant in all soils and Streptomycetales was absent in the BS soil. Proteobacteria and Ascomycota were highest in BS soil, while Actinobacteria was enriched in both the plant rhizospheric soil samples. In BS soil, Pseudomonas, Sordaria, Caulobacter, Magnetospirillum, Rhodospirillum were abundant. While, genera Actinoplanes, Streptomyces, Bradyrhizobium, Rhizobium, Azospirillum, Agrobacterium are abundant in RSD soil. Selected plants have accumulated HCH residues from soil and exerted positive impacts on soil microbial communities in HCH contaminated site. This study advocates microbe-assisted plant-based bioremediation strategy to remediate HCH contamination.

Characterization and properties of the biosurfactant produced by PAH-degrading bacteria isolated from contaminated oily sludge environment.

The aim of the present study was to investigate biosurfactant production ability of five different polyaromatic hydrocarbon (PAH)-metabolizing bacteria, such as Ochrobactrum anthropi IITR07, Pseudomonas mendocina IITR46, Microbacterium esteraromaticum IITR47, Pseudomonas aeruginosa IITR48, and Stenotrophomonas maltophilia IITR87. These bacteria showed biosurfactant production using 2% glucose as rich substrate; strain IITR47 yielded the highest with 906 and 534 mg/L biosurfactant in the presence of naphthalene and crude oil as the unique carbon sources. P. aeruginosa IITR48 showed the least surface tension at 29 N/m and the highest emulsification index at 63%. The biosurfactants produced were identified as glycolipid and rhamnolipid based on Fourier transform infrared spectroscopy analysis. In particular, the biosurfactant produced by bacteria S. maltophilia IITR87 efficiently emulsified mustard oil with an E24 value of 56%. It was observed that, all five biosurfactants from these degrader strains removed 2.4-, 1.7-, 0.9-, 3.8-, and 8.3-fold, respectively, crude oil from contaminated cotton cloth. Rhamnolipid derived from IITR87 was most efficient, exhibiting highest desorption of crude oil. These biosurfactants exhibited good stability without significantly losing its emulsification ability under extreme conditions, thus can be employed for bioremediation of PAHs from diverse contaminated ecosystem. Graphical Abstract.