Genetic and Comparative Genome Analysis of Exiguobacterium aurantiacum SW-20, a Petroleum-Degrading Bacteria with Salt Tolerance and Heavy Metal-Tolerance Isolated from Produced Water of Changqing Oilfield, China

The genome of Exiguobacterium aurantiacum SW-20 (E. aurantiacum SW-20), a salt-tolerant microorganism with petroleum hydrocarbon-degrading ability isolated from the Changqing Oilfield, was sequenced and analyzed. Genomic data mining even comparative transcriptomics revealed that some genes existed in SW-20 might be related to the salt tolerance. Besides, genes related to petroleum hydrocarbon degradation discovered in genomic clusters were also found in the genome, indicating that these genes have a certain potential in the bioremediation of petroleum pollutants. Multiple natural product biosynthesis gene clusters were detected, which was critical for survival in the extreme conditions. Transcriptomic studies revealed that some genes were significantly up-regulated as salinity increased, implying that these genes might be related to the salt tolerance of SW-20 when living in a high salt environment. In our study, gene clusters including salt tolerance, heavy metal tolerance and alkane degradation were all compared. When the same functional gene clusters from different strains, it was discovered that the gene composition differed. Comparative genomics and in-depth analysis provided insights into the physiological features and adaptation strategies of E. aurantiacum SW-20 in the oilfield environment. Our research increased the understanding of niches adaption of SW-20 at genomic level.

Microbial consortiums of putative degraders of low-density polyethylene-associated compounds in the ocean

Polyethylene (PE) is one of the most abundant plastics in the ocean. The development of a biofilm on PE in the ocean has been reported, yet whether some of the biofilm-forming organisms can biodegrade this plastic in the environment remains unknown. Via metagenomics analysis, we taxonomically and functionally analysed three biofilm communities using low-density polyethylene (LDPE) as their sole carbon source for two years. Several of the taxa that increased in relative abundance over time were closely related to known degraders of alkane and other hydrocarbons. Alkane degradation has been proposed to be involved in PE degradation, and most of the organisms increasing in relative abundance over time harboured genes encoding proteins essential in alkane degradation, such as the genes alkB and CYP153, encoding an alkane monooxygenase and a cytochrome P450 alkane hydroxylase. Weight loss of PE sheets when incubated with these communities and chemical and electron microscopic analyses provided evidence for alteration of the PE surface over time. Taken together, these results provide evidence for the utilization of LDPE-associated compounds by the prokaryotic communities. This study identifies a group of genes potentially involved in the degradation of the LDPE polymeric structure and/or associated plastic additives in the ocean and describes a phylogenetically diverse community of plastic biofilm-dwelling microbes with the potential of utilizing LDPE-associated compounds as carbon and energy source.

Moderate thermophilic chemoorganoheterotrophic bacterium in surface layer of anthropogenic grounds of industrial estate area of Al-Mafraq, Jordan

Surface of oil-contaminated soil from Industrial Estate of Al-Mafraq city, Jordan, was investigated for the presence of aerobic oil-degrading moderately thermophilic bacteria. A pure culture of spore forming aerobic chemoorganogeterotrophic rod shaped bacterial isolate, designated as strain j3n, was obtained. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain j3n is closely related to gram-positive bacteria of kaustophilus thermoleovorans cluster of Geobacillus genus. Strain j3n grew aerobically with oil, hexadecane, benzoate and acetate. Growth data indicated that utilization of hexadecane but not of oil and benzoate might be under catabolite repression control. Possibility of a regulation of alkane degradation by acetate in aerobic thermophilic gram-positive bacteria of Geobacillus spp. was shown for the first time.

Corksorb Enhances Alkane Degradation by Hydrocarbonoclastic Bacteria

Biosorbent materials are effective in the removal of spilled oil from water, but their effect on hydrocarbonoclastic bacteria is not known. Here, we show that corksorb, a cork-based biosorbent, enhances growth and alkane degradation by Rhodococcus opacus B4 (Ro) and Alcanivorax borkumensis SK2 (Ab). Ro and Ab degraded 96 ± 1% and 72 ± 2%, respectively, of a mixture of n-alkanes (2 g L–1) in the presence of corksorb. These values represent an increase of 6 and 24%, respectively, relative to the assays without corksorb. The biosorbent also increased the growth of Ab by 51%. However, no significant changes were detected in the expression of genes involved in alkane uptake and degradation in the presence of corksorb relative to the control without the biosorbent. Nevertheless, transcriptomics analysis revealed an increased expression of rRNA and tRNA coding genes, which confirms the higher metabolic activity of Ab in the presence of corksorb. The effect of corksorb is not related to the release of soluble stimulating compounds, but rather to the presence of the biosorbent, which was shown to be essential. Indeed, scanning electron microscopy images and downregulation of pili formation coding genes, which are involved in cell mobility, suggest that cell attachment on corksorb is a determinant for the improved activity. Furthermore, the existence of native alkane-degrading bacteria in corksorb was revealed, which may assist in situ bioremediation. Hence, the use of corksorb in marine oil spills may induce a combined effect of sorption and stimulated biodegradation, with high potential for enhancing in situ bioremediation processes.

Ecological Response in the Integrated Process of Biostimulation and Bioaugmentation of Diesel-Contaminated Soil

The study applied microbial molecular biological techniques to show that 2.5% to 3.0% (w/w) of diesel in the soil reduced the types and number of bacteria in the soil and destroyed the microbial communities responsible for the nitrogen cycle. In the meantime, the alkane degradation gene alkB and polycyclic aromatic hydrocarbons (PAHs) degradation gene nah evolved in the contaminated soil. We evaluated four different remediation procedures, in which the biostimulation-bioaugmentation joint process reached the highest degradation rate of diesel, 59.6 ± 0.25% in 27 days. Miseq sequencing and quantitative polymerase chain reaction (qPCR) showed that compared with uncontaminated soil, repaired soil provides abundant functional genes related to soil nitrogen cycle, and the most significant lifting effect on diesel degrading bacteria γ-proteobacteria. Quantitative analysis of degrading functional genes shows that degrading bacteria can be colonized in the soil. Gas chromatography-mass spectrometry (GC-MS) results show that the components remaining in the soil after diesel degradation are alcohol, lipids and a small amount of fatty amine compounds, which have very low toxicity to plants. In an on-site remediation experiment, the diesel content decreased from 2.7% ± 0.3 to 1.12% ± 0.1 after one month of treatment. The soil physical and chemical properties returned to normal levels, confirming the practicability of the biosimulation-bioaugmentation jointed remediation process.

Horizontal gene transfer of copper-containing membrane-bound monooxygenase (CuMMO) and di-iron soluble monooxygenase (SDIMO) in ethane- and propane-oxidizing Rhodococcus

The families of copper-containing membrane-bound monooxygenases (CuMMOs) and soluble di-iron monooxygenases (SDIMOs) are not only involved in methane oxidation but also in short-chain alkane oxidation. Herein, we describe Rhodococcus sp. ZPP, a bacterium able to grow with ethane or propane as the sole carbon and energy source and report on horizontal gene transfer (HGT) of actinobacterial hydrocarbon monooxygenases (HMO) of the CuMMO family and sMMO (soluble methane monooxygenase)-like SDIMO in the genus Rhodococcus. The key function of HMO in strain ZPP for propane oxidation was verified by allylthiourea inhibition. The HMO genes (designated hmoCAB) and those encoding sMMO-like SDIMO (designated smoXYB1C1Z) are located on a linear mega-plasmid (pRZP1) of strain ZPP. Comparative genomic analysis of similar plasmids indicated mobility of these plasmids within the genus Rhodococcus. The plasmid pRZP1 in strain ZPP could be conjugatively transferred to a recipient R. erythropolis in a mating experiment and showed similar ethane and propane consuming activities. Finally, our findings demonstrate that horizontal transfer of plasmid-based CuMMO and SDIMO genes confers the ability to use ethane and propane on the recipient. Importance CuMMOs and SDIMOs initiate the aerobic oxidation of alkanes in bacteria. Here, the supposition that horizontally transferred plasmid-based CuMMO and SDIMO genes confer on the recipient the similar ability to use ethane and propane was proposed and confirmed in Rhodococcus. This study is a living example of HGT of CuMMOs and SDIMOs and outlines the plasmid-borne properties responsible for gaseous alkane-degradation. Our results indicate that plasmids can support rapid evolution of enzyme-mediated biogeochemical processes.

New evidence for a hydroxylation pathway for anaerobic alkane degradation supported by analyses of functional genes and signature metabolites in oil reservoirs

Microbial degradation of recalcitrant alkanes under anaerobic conditions results in the accumulation of heavy oil fraction in oil reservoirs. Hydroxylation of alkanes is an important activation mechanism under anaerobic conditions, but the diversity and distribution of the responsible microorganisms in the subsurface environment are still unclear. The lack of functional gene polymerase chain reaction (PCR) primers and commercially available intermediate degradation chemical compounds are the major obstacles for this research. In this investigation, PCR primers for the ahyA gene (encoding alkane hydroxylase) were designed, evaluated, and improved based on the nucleotide sequences available. Using microbial genomic DNA extracted from oil-contaminated soil and production water samples of oil reservoirs, ahyA gene nucleotide sequences were amplified and retrieved successfully from production water sample Z3-25 of Shengli oilfield. Additionally, the signature biomarker of 2-acetylalkanoic acid was detected in both Shengli and Jiangsu oilfields. These results demonstrate that anaerobic hydroxylation is an active mechanism used by microorganisms to degrade alkanes in oxygen-depleted oil reservoirs. This finding expands the current knowledge of biochemical reactions about alkane degradation in subsurface ecosystems. In addition, the PCR primers designed and tested in this study serve as an effective molecular tool for detecting the microorganisms responsible for anaerobic hydroxylation of alkanes in this and other ecosystems.

Phylogeny and diversity of alkane-degrading enzyme gene variants in the laurentian great lakes and western atlantic

ABSTRACT Many aquatic environments are at risk for oil contamination and alkanes are one of the primary constituents of oil. The alkane hydroxylase (AlkB) is a common enzyme used by microorganisms to initiate the process of alkane-degradation. While many aspects of alkane bioremediation have been studied, the diversity and evolution of genes involved in hydrocarbon degradation from environmental settings is relatively understudied. The majority of work done to-date has focused on the marine environment. Here we sought to better understand the phylogenetic diversity of alkB genes across marine and freshwater settings using culture-independent methods. We hypothesized that there would be distinct phylogenetic diversity of alkB genes in freshwater relative to the marine environment. Our results confirm that alkB has distinct variants based on environment while our diversity analyses demonstrate that freshwater and marine alkB communities have unique responses to oil amendments. Our results also demonstrate that in the marine environment, depth is a key factor impacting diversity of alkB genes.

Chemical Profiling Provides Insights into the Metabolic Machinery of Hydrocarbon-Degrading Deep-Sea Microbes

ABSTRACT Marine microbes are known to degrade hydrocarbons; however, microbes inhabiting deep-sea sediments remain largely unexplored. Previous studies into the classical pathways of marine microbial metabolism reveal diverse chemistries; however, metabolic profiling of marine microbes cultured with hydrocarbons is limited. In this study, taxonomic (amplicon sequencing) profiles of two environmental deep-sea sediments (>1,200 m deep) were obtained, along with taxonomic and metabolomic (mass spectrometry-based metabolomics) profiles of microbes harbored in deep-sea sediments cultured with hydrocarbons as the sole energy source. Samples were collected from the Gulf of México (GM) and cultured for 28 days using simple (toluene, benzene, hexadecane, and naphthalene) and complex (petroleum API 40) hydrocarbon mixtures as the sole energy sources. The sediment samples harbored diverse microbial communities predominantly classified into Woeseiaceae and Kiloniellaceae families, whereas Pseudomonadaceae and Enterobacteriaceae families prevailed after sediments were cultured with hydrocarbons. Chemical profiling of microbial metabolomes revealed diverse chemical groups belonging primarily to the lipids and lipid-like molecules superclass, as well as the organoheterocyclic compound superclass (ClassyFire annotation). Metabolomic data and prediction of functional profiles indicated an increase in aromatic and alkane degradation in samples cultured with hydrocarbons. Previously unreported metabolites, identified as intermediates in the degradation of hydrocarbons, were annotated as hydroxylated polyunsaturated fatty acids and carboxylated benzene derivatives. In summary, this study used mass spectrometry-based metabolomics coupled to chemoinformatics to demonstrate how microbes from deep-sea sediments could be cultured in the presence of hydrocarbons. This study also highlights how this experimental approach can be used to increase the understanding of hydrocarbon degradation by deep-sea sediment microbes. IMPORTANCE High-throughput technologies and emerging informatics tools have significantly advanced knowledge of hydrocarbon metabolism by marine microbes. However, research into microbes inhabiting deep-sea sediments (>1,000 m) is limited compared to those found in shallow waters. In this study, a nontargeted and nonclassical approach was used to examine the diversity of bacterial taxa and the metabolic profiles of hydrocarbon-degrading deep-sea microbes. In conclusion, this study used metabolomics and chemoinformatics to demonstrate that microbes from deep-sea sediment origin thrive in the presence of toxic and difficult-to-metabolize hydrocarbons. Notably, this study provides evidence of previously unreported metabolites and the global chemical repertoire associated with the metabolism of hydrocarbons by deep-sea microbes.