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Stable and efficient hydrocarbon degrading microbial consortia were developed from a refinery sludge through nitrate amendment for their application in enhanced bioremediation of petroleum contaminated waste. Nitrate induced biostimulation of refinery sludge resulted in increased abundance of hydrocarbon degrading Rhodocyclaceae, Xanthomonadaceae, Syntrophaceae and Comamonadaceae members. Repeated subculturing of nitrate stimulated communities in crude oil supplemented basal medium was done under aerobic and anaerobic conditions. Aerobically enriched consortia (composed of Pseudomonadaceae, Pseudoxanthomonadaceae and unclassified Comamonadaceae) showed their ability to utilize alkanes, aromatics and crude oil as growth substrates. Anaerobically enriched consortium was predominated by Bacillaceae, Pseudomonadaceae, Xanthomonadaceae, Porphyromonadaceae and Comamonadaceae members. Anaerobic consortium was found to be relatively less efficient in terms of TPH (total petroleum hydrocarbons) degradation compared to its aerobic counterpart. These enriched microbial consortia were finally tested for their biodegradation performance and stability during bioremediation of highly contaminated refinery sludge using different strategies. A 30 days microcosm based bioremediation trial showed that bioaugmentation of aerobic cultures with refinery sludge was more effective in TPH degradation (~ 65% degradation) compared to the anaerobic consortium (only 36% TPH degradation) and a combination of bioaugmentation and nitrate amendment with sludge resulted in enhanced hydrocarbon attenuation (up to 86% TPH degradation). Subsequent community analysis at the end of bioremediation trial confirmed the stability of the added microbial populations. Thus, the strategy of bioaugmentation of specially enriched native microbial populations in combination with nitrate amendment was successfully used for the enhanced bioremediation of petroleum hydrocarbon contaminated refinery waste.

Background: Sustainable management of voluminous and hazardous oily sludge produced by petroleum refineries remains a challenging problem worldwide. Characterization of microbial communities of petroleum contaminated sites has been considered as the essential prerequisite for implementation of suitable bioremediation strategies. Three petroleum refinery sludge samples from North Eastern India were analyzed using next-generation sequencing technology to explore the diversity and functional potential of inhabitant microorganisms and scope for their on-site bioremediation., Results: All sludge samples were hydrocarbon rich, anaerobic and reduced with sulfate as major anion and several heavy metals. High throughput sequencing of V3-16S rRNA genes from sludge metagenomes revealed dominance of strictly anaerobic, fermentative, thermophilic, sulfate-reducing bacteria affiliated to Coprothermobacter, Fervidobacterium, Treponema, Syntrophus, Thermodesulfovibrio, Anaerolinea, Syntrophobacter, Anaerostipes, Anaerobaculum, etc., which have been well known for hydrocarbon degradation. Relatively higher proportions of archaea were detected by qPCR. Archaeal 16S rRNA gene sequences showed presence of methanogenic Methanobacterium, Methanosaeta, Thermoplasmatales, etc. Detection of known hydrocarbon utilizing aerobic/facultative anaerobic (Mycobacterium, Pseudomonas, Longilinea, Geobacter, etc.), nitrate reducing (Gordonia, Novosphigobium, etc.) and nitrogen fixing (Azovibrio, Rhodobacter, etc.) bacteria suggested niche specific guilds with aerobic, facultative anaerobic and strict anaerobic populations. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) predicted putative genetic repertoire of sludge microbiomes and their potential for hydrocarbon degradation; lipid-, nitrogen-, sulfur- and methane- metabolism. Methyl coenzyme M reductase A (mcrA) and dissimilatory sulfite reductase beta-subunit (dsrB) genes phylogeny confirmed methanogenic and sulfate-reducing activities within sludge environment endowed by hydrogenotrophic methanogens and sulfate-reducing Deltaproteobacteria and Firmicutes members., Conclusion: Refinery sludge microbiomes were comprised of hydrocarbon degrading, fermentative, sulfate-reducing, syntrophic, nitrogen fixing and methanogenic microorganisms, which were in accordance with the prevailing physicochemical nature of the samples. Analysis of functional biomarker genes ascertained the activities of methanogenic and sulfate-reducing organisms within sludge environment. Overall data provided better insights on microbial diversity and activity in oil contaminated environment, which could be exploited suitably for in situ bioremediation of refinery sludge.

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Scope for developing an engineered bioremediation strategy for the treatment of hydrocarbon-rich petroleum refinery waste was investigated through biostimulation and bioaugmentation approaches. Enhanced (46–55%) total petroleum hydrocarbon (TPH) attenuation was achieved through phosphate, nitrate or nitrate+phosphate amendment in the sludge with increased (upto 12%) abundance of fermentative, hydrocarbon degrading, sulfate-reducing, CO 2 -assimilating and methanogenic microorganisms ( Bacillus , Coprothermobacter , Rhodobacter , Pseudomonas, Achromobacter , Desulfitobacter , Desulfosporosinus , T78 , Methanobacterium , Methanosaeta , etc). Together with nutrients, bioaugmentation with biosurfactant producing and hydrocarbon utilizing indigenous Bacillus strains resulted in 57–75% TPH reduction. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis revealed enhanced gene allocation for transporters (0.45–3.07%), ABC transporters (0.38–2.07%), methane (0.16–1.06%), fatty acid (0.018–0.15%), nitrogen (0.07–0.17%), butanoate (0.06–0.35%), propanoate (0.004–0.26%) metabolism and some xenobiotics (0.007–0.13%) degradation. This study indicated that nutrient-induced community dynamics of native microorganisms and their metabolic interplay within oil refinery sludge could be a driving force behind accelerated bioremediation. [ABSTRACT FROM AUTHOR]