Oil type and temperature dependent biodegradation dynamics - Combining chemical and microbial community data through multivariate analysis.

BACKGROUND: This study investigates a comparative multivariate approach for studying the biodegradation of chemically dispersed oil. The rationale for this approach lies in the inherent complexity of the data and challenges associated with comparing multiple experiments with inconsistent sampling points, with respect to inferring correlations and visualizing multiple datasets with numerous variables. We aim to identify novel correlations among microbial community composition, the chemical change of individual petroleum hydrocarbons, oil type and temperature by creating modelled datasets from inconsistent sampling time points. Four different incubation experiments were conducted with freshly collected Norwegian seawater and either Grane and Troll oil dispersed with Corexit 9500. Incubations were conducted at two different temperatures (5 °C and 13 °C) over a period of 64 days. RESULTS: PCA analysis of modelled chemical datasets and calculated half-lives revealed differences in the biodegradation of individual hydrocarbons among temperatures and oil types. At 5 °C, most n-alkanes biodegraded faster in heavy Grane oil compared to light Troll oil. PCA analysis of modelled microbial community datasets reveal differences between temperature and oil type, especially at low temperature. For both oils, Colwelliaceae and Oceanospirillaceae were more prominent in the colder incubation (5 °C) than the warmer (13 °C). Overall, Colwelliaceae, Oceanospirillaceae, Flavobacteriaceae, Rhodobacteraceae, Alteromonadaceae and Piscirickettsiaceae consistently dominated the microbial community at both temperatures and in both oil types. Other families known to include oil-degrading bacteria were also identified, such as Alcanivoracaceae, Methylophilaceae, Sphingomonadaceae and Erythrobacteraceae, but they were all present in dispersed oil incubations at a low abundance (< 1%). CONCLUSIONS: In the current study, our goal was to introduce a comparative multivariate approach for studying the biodegradation of dispersed oil, including curve-fitted models of datasets for a greater data resolution and comparability. By applying these approaches, we have shown how different temperatures and oil types influence the biodegradation of oil in incubations with inconsistent sampling points. Clustering analysis revealed further how temperature and oil type influence single compound depletion and microbial community composition. Finally, correlation analysis of degraders community, with single compound data, revealed complexity beneath usual abundance cut-offs used for microbial community data in biodegradation studies.

Microbial communities related to biodegradation of dispersed Macondo oil at low seawater temperature with Norwegian coastal seawater.

The Deepwater Horizon (DWH) accident in 2010 created a deepwater plume of small oil droplets from a deepwater well in the Mississippi Canyon lease block 252 ('Macondo oil'). A novel laboratory system was used in the current study to investigate biodegradation of Macondo oil dispersions (10 µm or 30 µm median droplet sizes) at low oil concentrations (2 mg l(-1)) in coastal Norwegian seawater at a temperature of 4-5°C. Whole metagenome analyses showed that oil biodegradation was associated with the successive increased abundances of Gammaproteobacteria, while Alphaproteobacteria (Pelagibacter) became dominant at the end of the experiment. Colwellia and Oceanospirillales were related to n-alkane biodegradation, while particularly Cycloclasticus and Marinobacter were associated with degradation of aromatic hydrocarbons (HCs). The larger oil droplet dispersions resulted in delayed sequential changes of Oceanospirillales and Cycloclasticus, related with slower degradation of alkanes and aromatic HCs. The bacterial successions associated with oil biodegradation showed both similarities and differences when compared with the results from DWH field samples and laboratory studies performed with deepwater from the Gulf of Mexico.

Biodegradation of dispersed Macondo oil in seawater at low temperature and different oil droplet sizes.

During the Deepwater Horizon (DWH) accident in 2010 a dispersant (Corexit 9500) was applied at the wellhead to disperse the Macondo oil and reduce the formation of surface slicks. A subsurface plume of small oil droplets was generated near the leaking well at 900-1300 m depth. A novel laboratory system was established to investigate biodegradation of small droplet oil dispersions (10 µm or 30 µm droplet sizes) of the Macondo oil premixed with Corexit 9500, using coastal Norwegian seawater at a temperature similar to the DWH plume (4-5°C). Biotransformation of volatile and semivolatile hydrocarbons and oil compound groups was generally faster in the 10 µm than in the 30 µm dispersions, showing the importance of oil droplet size for biodegradation. These data therefore indicated that dispersant treatment to reduce the oil droplet size may increase the biodegradation rates of oil compounds in the deepwater oil droplets.

Ralstonia sp. U2 naphthalene dioxygenase and Comamonas sp. JS765 nitrobenzene dioxygenase show differences in activity towards methylated naphthalenes.

Methylsubstituted naphthalenes constitute a significant part of light gas oil fractions (LGO). These are toxic compounds with low fuel value, and can potentially be enzymatically modified to increase the fuel value and at the same time reduce toxicity. The first step in the biodegradation of naphthalene involves dioxygenation of the aromatic ring catalysed by naphthalene dioxygenase (NDO). Here we show that recombinantly produced NDO from Ralstonia sp. U2 and the related nitrobenzene dioxygenase (NBDO) from Comamonas sp. JS765 can use several mono-, di-, tri-, and tetramethylated naphthalenes as substrates. For the majority of the substrates both enzymes catalyse the formation of a mixture of mono- and dioxygenated products, and it is only dioxygenated products that are likely to be processed further, leading to ring cleavage. In some cases, like for 1-methylnaphthalene, NDO mainly generates the monooxygenated form, while with NBDO, the dioxygenated form dominates. In other cases, as for 1,4-dimethylnaphthalene, the monooxygenated product dominates with NDO, whereas NBDO generates similar amounts of both forms. Presumably, the best future strategy for bioconversion of methylated naphthalenes in LGO is to develop engineered enzyme that are optimised with respect to the specific composition of naphthalene derivatives found in a given product.

High coverage sequencing of DNA from microorganisms living in an oil reservoir 2.5 kilometres subsurface.

Microorganisms colonize a variety of extreme environments, and based on cultivation studies and analyses of PCR-amplified 16S rDNA sequences, microbial life appears to extend deep into the earth crust. However, none of these studies involved comprehensive characterizations of total DNA. Here we report results of a high-coverage DNA pyrosequencing of an apparently representative and uncontaminated sample from a deep sea oil reservoir located 2.5 km subsurface, attributing a pressure and temperature of 250 bars and 85°C respectively. Bioinformatic analyses of the DNA sequences indicate that the reservoir harbours a rich microbial community dominated by a smaller number of taxa. Comparison of the metagenome with sequences in databases indicated that there may have been contact between the oil reservoir and surface communities late in the sequence of geological events leading to oil reservoir formation. One specific gene, encoding a putative enolase, was synthesized and expressed in Escherichia coli. Enolase activity was confirmed and was found to be much more thermotolerant than for a corresponding E. coli enzyme, consistent with the conditions in the oil reservoir.

Bacterial metabolism of long-chain n-alkanes.

Degradation of alkanes is a widespread phenomenon in nature, and numerous microorganisms, both prokaryotic and eukaryotic, capable of utilizing these substrates as a carbon and energy source have been isolated and characterized. In this review, we summarize recent advances in the understanding of bacterial metabolism of long-chain n-alkanes. Bacterial strategies for accessing these highly hydrophobic substrates are presented, along with systems for their enzymatic degradation and conversion into products of potential industrial value. We further summarize the current knowledge on the regulation of bacterial long-chain n-alkane metabolism and survey progress in understanding bacterial pathways for utilization of n-alkanes under anaerobic conditions.

Identification of novel genes involved in long-chain n-alkane degradation by Acinetobacter sp. strain DSM 17874.

Acinetobacter sp. strain DSM 17874 is capable of utilizing n-alkanes with chain lengths ranging from that of decane (C10H22) to that of tetracontane (C40H82) as a sole carbon source. Two genes encoding AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, have been shown to be involved in the degradation of n-alkanes with chain lengths of from 10 to 20 C atoms in this strain. Here, we describe a novel high-throughput screening method and the screening of a transposon mutant library to identify genes involved in the degradation of n-alkanes with C chain lengths longer than 20, which are solid at 30 degrees C, the optimal growth temperature for Acinetobacter sp. strain DSM 17874. A library consisting of approximately 6,800 Acinetobacter sp. strain DSM 17874 transposon mutants was constructed and screened for mutants unable to grow on dotriacontane (C32H66) while simultaneously showing wild-type growth characteristics on shorter-chain n-alkanes. For 23 such mutants isolated, the genes inactivated by transposon insertion were identified. Targeted inactivation and complementation studies of one of these genes, designated almA and encoding a putative flavin-binding monooxygenase, confirmed its involvement in the strain's metabolism of long-chain n-alkanes. To our knowledge, almA represents the first cloned gene shown to be involved in the bacterial degradation of long-chain n-alkanes of 32 C's and longer. Genes encoding AlmA homologues were also identified in other long-chain n-alkane-degrading Acinetobacter strains.

Utilization of n-alkanes by a newly isolated strain of Acinetobacter venetianus: the role of two AlkB-type alkane hydroxylases.

A bacterial strain capable of utilizing n-alkanes with chain lengths ranging from decane (C10H22) to tetracontane (C40H82) as a sole carbon source was isolated using a system for screening microorganisms able to grow on paraffin (mixed long-chain n-alkanes). The isolate, identified according to its 16S rRNA sequence as Acinetobacter venetianus, was designated A. venetianus 6A2. Two DNA fragments encoding parts of AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, were polymerase chain reaction-amplified from the genome of A. venetianus 6A2. To study the roles of these two alkM paralogues in n-alkane utilization in A. venetianus 6A2, we constructed alkMa, alkMb, and alkMa/alkMb disruption mutants. Studies on the growth patterns of the disruption mutants using n-alkanes with different chain lengths as sole carbon source demonstrated central roles for the alkMa and alkMb genes in utilization of C10 to C18 n-alkanes. Comparative analysis of these patterns also suggested different substrate preferences for AlkMa and AlkMb in n-alkane utilization. Because both single and double mutants were able to grow on n-alkanes with chain lengths of C20 and longer, we concluded that yet another enzyme(s) for the utilization of these n-alkanes must exist in A. venetianus 6A2.

Heme A synthase enzyme functions dissected by mutagenesis of Bacillus subtilis CtaA.

Heme A, as a prosthetic group, is found exclusively in respiratory oxidases of mitochondria and aerobic bacteria. Bacillus subtilis CtaA and other heme A synthases catalyze the conversion of a methyl side group on heme O into a formyl group. The catalytic mechanism of heme A synthase is not understood, and little is known about the composition and structure of the enzyme. In this work, we have: (i) constructed a ctaA deletion mutant and a system for overproduction of mutant variants of the CtaA protein in B. subtilis, (ii) developed anaffinity purification procedure for isolation of preparative amounts of CtaA, and (iii) investigated the functional roles of four invariant histidine residues in heme A synthase by in vivo and in vitro analyses of the properties of mutant variants of CtaA. Our results show an important function of three histidine residues for heme A synthase activity. Several of the purified mutant enzyme proteins contained tightly bound heme O. One variant also contained trapped hydroxylated heme O, which is a postulated enzyme reaction intermediate. The findings indicate functional roles for the invariant histidine residues and provide strong evidence that the heme A synthase enzyme reaction includes two consecutive monooxygenations.