Balaram Mohapatra

Biostimulation of Indigenous Microbial Community for Bioremediation of Petroleum Refinery Sludge

Nutrient deficiency severely impairs the catabolic activity of indigenous microorganisms in hydrocarbon rich environments (HREs) and limits the rate of intrinsic bioremediation. The present study aimed to characterize the microbial community in refinery waste and evaluate the scope for biostimulation based in situ bioremediation. Samples recovered from the wastewater lagoon of Guwahati refinery revealed a hydrocarbon enriched [high total petroleum hydrocarbon (TPH)], oxygen-, moisture-limited, reducing environment. Intrinsic biodegradation ability of the indigenous microorganisms was enhanced significantly (>80% reduction in TPH by 90 days) with nitrate amendment. Preferred utilization of both higher- (>C30) and middle- chain (C20-30) length hydrocarbons were evident from GC-MS analysis. Denaturing gradient gel electrophoresis and community level physiological profiling analyses indicated distinct shift in community’s composition and metabolic abilities following nitrogen (N) amendment. High throughput deep sequencing of 16S rRNA gene showed that the native community was mainly composed of hydrocarbon degrading, syntrophic, methanogenic, nitrate/iron/sulfur reducing facultative anaerobic bacteria and archaebacteria, affiliated to γ- and δ-Proteobacteria and Euryarchaeota respectively. Genes for aerobic and anaerobic alkane metabolism (alkB and bssA), methanogenesis (mcrA), denitrification (nirS and narG) and N2 fixation (nifH) were detected. Concomitant to hydrocarbon degradation, lowering of dissolve O2 and increase in oxidation-reduction potential (ORP) marked with an enrichment of N2 fixing, nitrate reducing aerobic/facultative anaerobic members [e.g., Azovibrio, Pseudoxanthomonas and Comamonadaceae members] was evident in N amended microcosm. This study highlighted that indigenous community of refinery sludge was intrinsically diverse, yet appreciable rate of in situ bioremediation could be achieved by supplying adequate N sources.

Enrichment and characterization of hydrocarbon-degrading bacteria from petroleum refinery waste as potent bioaugmentation agent for in situ bioremediation

Intrinsic biodegradation potential of bacteria from petroleum refinery waste was investigated through isolation of cultivable strains and their characterization. Pseudomonas and Bacillus spp. populated the normal cultivable taxa while prolonged enrichment with hydrocarbons and crude oil yielded hydrocarbonoclastic bacteria of genera Burkholderia, Enterobacter, Kocuria, Pandoraea, etc. Strains isolated through enrichment showed assemblages of superior metabolic properties: utilization of aliphatic (C6-C22) and polyaromatic compounds, anaerobic growth with multiple terminal electron acceptors and higher biosurfactant production. Biodegradation of dodecane was studied thoroughly by GC-MS along with detection of gene encoding alkane hydroxylase (alkB). Microcosms bioaugmented with Enterobacter, Pandoraea and Burkholderia strains showed efficient biodegradation (98% TPH removal) well fitted in first order kinetic model with low rate constants and decreased half-life. This study proves that catabolically efficient bacteria resides naturally in complex petroleum refinery wastes and those can be useful for bioaugmentation based bioremediation.

Genome analysis of crude oil degrading Franconibacter pulveris strain DJ34 revealed its genetic basis for hydrocarbon degradation and survival in oil contaminated environment

Franconibacter pulveris strain DJ34, isolated from Duliajan oil fields, Assam, was characterized in terms of its taxonomic, metabolic and genomic properties. The bacterium showed utilization of diverse petroleum hydrocarbons and electron acceptors, metal resistance, and biosurfactant production. The genome (4,856,096bp) of this strain contained different genes related to the degradation of various petroleum hydrocarbons, metal transport and resistance, dissimilatory nitrate, nitrite and sulfite reduction, chemotaxy, biosurfactant synthesis, etc. Genomic comparison with other Franconibacter spp. revealed higher abundance of genes for cell motility, lipid transport and metabolism, transcription and translation in DJ34 genome. Detailed COG analysis provides deeper insights into the genomic potential of this organism for degradation and survival in oil-contaminated complex habitat. This is the first report on ecophysiology and genomic inventory of Franconibacter sp. inhabiting crude oil rich environment, which might be useful for designing the strategy for bioremediation of oil contaminated environment.

Sugarcane as a Potential Biofuel Crop

Sugarcane (Saccharum spp.) belonging to family Poaceae is a tropical perennial grass used widely for sugar production. Research scientists have discovered sugarcane as an alternative biofuel source to conventional petroleum fuels that lead to global warming. The sugars extracted from sugarcane can be easily fermented to produce ethanol. In addition, the bagasse (biomass remaining after the juice is extracted from the stalks) can be further used by sugar mills to generate steam and electricity. The current total global production of renewable fuels is 50 billion liters a year, and sugarcane alone accounts for about 40%, thus becoming a major contributor for biofuel production. The tremendous success of sugarcane industry to produce ethanol as biofuel in Brazil has also enhanced the interest in other parts of the world. With conventional technologies, sugarcane can yield several products from fiber to chemicals. But with the help of genetic recombination, sugarcane would roll to produce the novel biofuels more efficiently. Research scientists have identified the key enzymes that can hasten the process of ethanol production more powerfully. There is tremendous potential of sugarcane as a biofactory which can uplift both socioeconomic status of a country and sustainability of natural resources. Now, it’s time to augment weightage to produce biofuels in developing countries like India which would initiate rural development, create more job opportunities, and also save foreign exchange to great extent.

Taxonomy and physiology of Pseudoxanthomonas arseniciresistens sp. nov., an arsenate and nitrate-reducing novel gammaproteobacterium from arsenic contaminated groundwater, India

Reductive transformation of toxic arsenic (As) species by As reducing bacteria (AsRB) is a key process in As-biogeochemical-cycling within the subsurface aquifer environment. In this study, we have characterized a Gram-stain-negative, non-spore-forming, rod-shaped As reducing bacterium designated KAs 5-3T, isolated from highly As-contaminated groundwater of India. Strain KAs 5-3T displayed high 16S rRNA gene sequence similarity to the members of the genus Pseudoxanthomonas, with P. mexicana AMX 26BT (99.25% similarity), P. japonensis 12-3T (98.9 0%), P. putridarboris WD-12T (98.02%), and P. indica P15T (97.27%) as closest phylogenetic neighbours. DNA-DNA hybridization study unambiguously indicated that strain KAs 5-3T represented a novel species that was separate from reference strains of P. mexicana AMX 26BT (35.7%), P. japonensis 12-3T (35.5%), P. suwonensis 4M1T (35.5%), P. wuyuanensis XC21-2T (35.0%), P. indica P15T (32.5%), P. daejeonensis TR6-08T (32.0%), and P. putridarboris WD12T (22.1%). The DNA G+C content of strain KAs 5-3T was 64.9 mol %. The predominant fatty acids were C15:0 (37.4%), C16:0 iso (12.6%), C17:1 iso ω9c (10.5%), C15:0 anteiso (9.5%), C11:0 iso 3-OH (8.5%), and C16:1 ω7c/ C16:1 ω6c (7.5%). The major polar lipids were diphosphatidylglycerol, phosphatidyldimethylethanolamine, phosphatidylcholine, and two unknown phospholipids (PL1, PL2). Ubiquinone 8 (Q8) was the predominant respiratory quinone and spermidine was the major polyamine of the strain KAs 5-3T. Cells of strain KAs 5-3T showed the ability to use O2, As5+, NO3-, NO2-, and Fe3+ as terminal electron acceptors as well as to reduce As5+ through the cytosolic process under aerobic incubations. Genes encoding arsenate reductase (arsC) for As-detoxification, nitrate- and nitrite reductase (narG and nirS) for denitrification were detected in the strain KAs 5-3T. Based on taxonomic and physiological data, strain KAs 5-3T is described as a new representative member of the genus Pseudoxanthomonas, for which the name Pseudoxanthomonas arseniciresistens sp. nov. is proposed. The type strain is KAs 5-3T (= LMG 29169T = MTCC 12116T = MCC 3121T).

Microbial Soil Enzymes: Implications in the Maintenance of Rhizosphere Ecosystem and Soil Health

Soil enzymes play a crucial role in agriculture as they are important for several vital biochemical reactions necessary for the life processes of soil microbes along with the maintenance of soil structure, decomposition and formation of organic matter and nutrient mineralization. Thus, soil enzymes are instrumental in the maintenance of soil ecosystem. Several factors that affect soil-plant-microorganism and their interaction in turn determine the productivity and activity of soil enzymes. A better understanding of the role of soil enzymes in maintaining soil health, along with the development of rapid and easier protocols for their measurement, will provide us with the opportunity to design novel integrated soil assessment methods leading to more efficient and eco-friendly soil management programmes. Furthermore, harnessing beneficial soil enzymes through currently available technologies will enable in situ applications of desired soil enzymes for restoration of polluted soils. Present article focuses on occurrence of soil enzymes, methods for determining their activity, current developments in mining these enzymes using metagenomic approach and factors affecting the activity of soil enzymes along with the importance of various soil enzymes.

Molecular and eco-physiological characterization of arsenic (As)-transforming Achromobacter sp. KAs 3–5T from As-contaminated groundwater of West Bengal, India

ABSTRACT Molecular and eco-physiological characterization of arsenic (As)-transforming and hydrocarbon-utilizing Achromobacter type strain KAs 3–5T has been investigated in order to gain an insight into As-geomicrobiology in the contaminated groundwater. The bacterium is isolated from As-rich groundwater of West Bengal, India. Comparative 16S rRNA gene sequence phylogenetic analysis confirmed that the strain KAs 3–5T is closely related to Achromobacter mucicolens LMG 26685T (99.17%) and Achromobacter animicus LMG 26690T (99.17%), thus affiliated to the genus Achromobacter. Strain KAs 3–5T is nonflagellated, mesophilic, facultative anaerobe, having a broad metabolic repertoire of using various sugars, sugar-/fatty acids, hydrocarbons as principal carbon substrates, and O2, NO3−, NO2−, and Fe3+ as terminal electron acceptors. Growth with hydrocarbons led to cellular aggregation and adherence of the cells to the hydrocarbon particles confirmed through electron microscopic observations. The strain KAs 3–5T showed high As resistance (MIC of 5 mM for As3+, 25 mM for As5+) and reductive transformation of As5+ under aerobic conditions while utilizing both sugars and hydrocarbons. Molecular taxonomy specified a high genomic GC content (65.5 mol %), ubiquinone 8 (UQ-8) as respiratory quinone, spermidine as predominant polyamine in the bacterium. The differential presence of C12:0, C14:0 2-OH, C18:1 ω7c, and C 14:0 iso 3-OH/ C16:1 iso fatty acids, phosphatidylglycerol (PG), phosphatidylcholine (PC), two unknown phospholipid (PL1, PL2) as polar lipids, low DNA-DNA relatedness (33.0–41.0%) with the Achromobacter members, and unique metabolic capacities clearly indicated the distinct genomic and physiological properties of strain KAs 3–5T among known species of the genus Achromobacter. These findings lead to improve our understanding on metabolic flexibility of bacteria residing in As-contaminated groundwater and As–bacteria interactions within oligotrophic aquifer system.

Genome sequencing reveals the potential of an indigenous arsenotrophic bacterium; Achromobacter sp. KAs 3-5 for sub-surface arsenic mobilization and strategies for bioremediation

Prevalence of toxic arsenic (As) oxyanion species in oligotrophic groundwater of south-east Asiatic regions (India and Bangladesh) has threatened the health of millions of people. As-transforming bacteria alter the mobility, speciation and bioavailability of As in the aquifer ecosystem, hence play important roles in the biogeochemical cycling of As. Till date, only 19 cultivable As-transforming bacterial strains have been reported but with no description on their detail genomic and physiological perspective of As homeostasis. In this study, the draft genome (5.7 Mbp) of an As-transforming, aromatic hydrocarbon utilizing and iron disproportioning indigenous groundwater bacterium KAs 3-5 has been obtained by Ion-Torrent sequencing revealed 65% genomic GC content, 5100 protein coding genes, and taxonomic affiliation to the members of genus Achromobacter, with >85% of genomic completeness. Phylogenomic signatures like MLST of 10 house-keeping genes, cut-off of <95% of average nucleotide/amino acid identity (ANI/OrthoANI/AAI), <0.99 of tetra-nucleotide correlations, and <70% value of DNA-DNA homology with nearest phylogenetic neighbors exhibited its species distinctiveness among all the described Achromobacter sp. members. Pan-genomic analysis confirmed the strain’s potential to adapt wide array of environmental stresses with a higher abundance of unique genes for metabolism of amino acids, polyketide, xenobiotics, nitroso compounds, aromatic hydrocarbons and most necessarily complete operon cluster for As-resistance/transformation/detoxification, as well as genes for transport, and signal transduction mechanisms. The genome analysis also highlighted its genetic determinants for loss of functions for antibiotic resistance, pathogenicity regulations, and gain of new/acquired functions for Fe-transport, fatty acids uptake-metabolism, motility, heavy metal (Cu-Zn-Co) metabolism and several putative/hypothetical proteins owing to its capacity to acquired desired traits through horizontal gene transfer events. The ability of the organism to metabolize mono-poly aromatics like benzene, toluene, naphthalene, anthracene, etc. (by catechol, homogentisate pathways) coupled to As reduction (through arsHBC, arsC, ACR3) found to be well validated by genomic observations. X-ray absorption fine structure (XANES) also enabled us to decipher detailed Fe-based reductive As release process from sediment and its interaction. The obtained genome data provide us with a better understanding of sub-surface mechanisms of hydrocarbon (organic matter) driven As release, which may contribute to the future design of rational strategies for bioremediation of As/other heavy metal contaminated environments. Citation: Mohapatra, B., Dutta, A., Gupta, A., Kazy, S.K. and Sar, P. Genome sequencing reveals the potential of an indigenous arsenotrophic bacterium; Achromobacter sp. KAs 3-5 for sub-surface arsenic mobilization and strategies for bioremediation [Abstract]. In: Abstracts of the NGBT conference; Oct 02-04, 2017; Bhubaneswar, Odisha, India: Can J biotech, Volume 1, Special Issue (Supplement), Page 271. https://doi.org/10.24870/cjb.2017-a255