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.

Bioaugmentation-Enhanced Remediation of Crude Oil Polluted Water in Pilot-Scale Floating Treatment Wetlands

Floating treatment wetlands (FTWs) are cost-effective systems for the remediation of polluted water. In FTWs, the metabolic activity of microorganisms associated with plants is fundamental to treatment efficiency. Bioaugmentation, the addition of microorganisms with pollutant-degrading capabilities, appears to be a promising means to enhance the treatment efficiency of FTWs. Here, we quantified the effect of bioaugmentation with a four-membered bacterial consortium on the remediation of water contaminated with crude oil in pilot-scale FTWs planted with Phragmites australis or Typha domingensis. The bacteria had been isolated from the endosphere and rhizosphere of various plants and carry the alkane hydroxylase gene, alkB, involved in aerobic hydrocarbon degradation. During a treatment period of 36 days, FTWs planted with P. australis achieved a reduction in hydrocarbon concentration from 300 mg/L to 16 mg/L with and 56 mg/L without bioaugmentation. In the FTWs planted with T. domingensis, respective hydrocarbon concentrations were 46 mg/L and 84 mg/L. The inoculated bacteria proliferated in the rhizoplane and in the plant interior. Copy numbers of the alkB gene and its mRNA increased over time in plant-associated samples, suggesting increased bacterial hydrocarbon degradation. The results show that bioaugmentation improved the treatment of oil-contaminated water in FTWs by at least a factor of two, indicating that the performance of full-scale systems can be improved at only small costs.

Characterization and Transcriptome Analysis of a Long-Chain n-Alkane-Degrading Strain Acinetobacter Pittii SW-1

Strain sw-1, isolated from 7619-m seawater of the Mariana Trench, was identified as Acinetobacter pittii by 16S rRNA gene and whole-genome sequencing. A. pittii sw-1 was able to efficiently utilize long-chain n-alkanes (C18–C36), but not short- and medium-chain n-alkanes (C8–C16). The degradation rate of C20 was 91.25%, followed by C18, C22, C24, C32, and C36 with the degradation rates of 89.30%, 84.03%, 80.29%, 30.29%, and 13.37%, respectively. To investigate the degradation mechanisms of n-alkanes for this strain, the genome and the transcriptome analyses were performed. Four key alkane hydroxylase genes (alkB, almA, ladA1, and ladA2) were identified in the genome. Transcriptomes of strain sw-1 grown in C20 or CH3COONa (NaAc) as the sole carbon source were compared. The transcriptional levels of alkB and almA, respectively, increased 78.28- and 3.51-fold in C20 compared with NaAc, while ladA1 and ladA2 did not show obvious change. The expression levels of other genes involved in the synthesis of unsaturated fatty acids, permeases, membrane proteins, and sulfur metabolism were also upregulated, and they might be involved in n-alkane uptake. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) confirmed that alkB expression was significantly induced by C20, C24, and C32, and almA induction extent by C24 and C32 was higher than that with C20. Furthermore, ladA2 expression was only induced by C32, and ladA1 expression was not induced by any of n-alkanes. In addition, A. pittii sw-1 could grow with 0%–3% NaCl or 8 out of 10 kinds of the tested heavy metals and degrade n-alkanes at 15 °C. Taken together, these results provide comprehensive insights into the degradation of long-chain n-alkanes by Acinetobacter isolated from the deep ocean environment.

Biodegradation of binary mixtures of octane with benzene, toluene, ethylbenzene or xylene (BTEX): insights on the potential of Burkholderia, Ralstonia, Pseudomonas, and Cupriavidus isolates

The contamination of the environment by crude oil and its by-products, which mainly composed of aliphatic and aromatic hydrocarbons, is a widespread problem. Biodegradation by bacteria is one of the processes responsible for the removal of these pollutants. This study was conducted to determine the abilities of Burkholderia sp. B5, Ralstonia sp. B1, Pseudomonas sp. T1, and Cupriavidus sp. X5 to degrade binary mixtures of octane (representing aliphatic hydrocarbons) with benzene, toluene, ethylbenzene, or xylene (BTEX as aromatic hydrocarbons) at a final concentration of 100 ppm under aerobic conditions. These strains were isolated from an enriched bacterial consortium (Yabase or Y consortium) that prefer to degrade aromatic hydrocarbon over aliphatic hydrocarbons. We found that B5 degraded all BTEX compounds more rapidly than octane. In contrast, B1, T1 and X5 utilized more of octane over BTX compounds. B5 also preferred to use benzene over octane with varying concentrations of up to 200 mg/l. B5 possesses alkane hydroxylase (alkB) and catechol 2,3-dioxygenase (C23D) genes, which are responsible for the degradation of alkanes and aromatic hydrocarbons, respectively. This study strongly supports our notion that Burkholderia played a key role in the preferential degradation of aromatic hydrocarbons over aliphatic hydrocarbons in the previously characterized Y consortium. The preferential degradation of more toxic aromatic hydrocarbons over aliphatics is crucial in risk-based bioremediation.

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.

Newly Isolated Alkane Hydroxylase and Lipase Producing Geobacillus and Anoxybacillus Species Involved in Crude Oil Degradation

Isolation and studies of novel, crude oil biodegrading thermophilic strains may provide a wider knowledge in understanding their role in petroleum degradation. In this study, the screening of ten new thermophilic strains revealed that all strains were alkane hydroxylase producers and seven of them produced lipase concurrently. Three best strains were characterized and identified through 16S rRNA sequence analysis as Geobacillus sp. D4, Geobacillus sp. D7, and Anoxybacillus geothermalis D9 with GenBank accession numbers MK615934.1, MK615935.1, and MK615936.1, respectively. Gas chromatography (GC) analysis showed that all three strains were able to breakdown various compounds in crude oil such as alkanes, toxic poly-aromatic hydrocarbons (PAHs), organosulfur, carboxylic acids, alkene, resins, organosilicon, alcohol, organochlorine, and ester. For the first time, alkane hydroxylase and lipase activity as well as crude oil degradation by A. geothermalis species were reported. Geobacillus sp. D7 is the best alkane degrader followed by A. geothermalis D9 and Geobacillus sp. D4 with 17.3%, 13.1%, and 12.1% biodegradation efficiency (BE%), respectively. The potential of thermophiles isolated can be explored further for bioremediation of sites polluted by petroleum and oil spills.

Isolation and characterization of hydrocarbon-degrading bacteria from gas station leaking-contaminated groundwater in the Southern Amazon, Brazil

Twenty-three hydrocarbon-degrading bacteria strains were isolated from gas station leaking-contaminated groundwater located in the Southern Amazon, Brazil. Based on hydrocarbon (diesel, hexadecane, benzene, toluene and xylene) degradation ability, two strains were selected for further study. The amplification and sequencing of the 16S rRNA gene showed that these two strains belonged to the genus Bacillus (Bacillus sp. L26 and Bacillus sp. L30). GC-MS analysis showed that strain L30 was the most effective in degrading n-alkane (C10-C27) from diesel after 7 days of cultivation in mineral medium. Both strains produced biosurfactants and showed emulsification activity, specially the strain L30. Alkane hydroxylase gene (group III), which is important for alkane biodegradation, was present in strains. As a result, this study indicated that these bacteria could have promising applications in hydrocarbon bioremediation.