Acidovorax benzenivorans sp. nov., a novel aromatic hydrocarbon-degrading bacterium isolated from a xylene-degrading enrichment culture.

A Gram-stain-negative strain, designated as D2M1T was isolated from xylene-degrading enrichment culture and characterized using a polyphasic approach to determine its taxonomic position. The 16S rRNA gene sequence analysis revealed that strain D2M1T belongs to the genus Acidovorax, with the highest 16S rRNA gene similarity to Acidovorax delafieldii DSM 64T (99.93 %), followed by Acidovorax radicis DSM 23535T (98.77 %) and Acidovorax kalamii MTCC 12652T (98.76 %). The draft genome sequence of strain D2M1T is 5.49 Mb long, and the G+C content of the genome is 64.2 mol%. Orthologous average nucleotide identity and digital DNA-DNA hybridization relatedness values between strain D2M1T and its closest relatives were below the threshold values for species demarcation confirming that strain D2M1T is distinctly separated from its closest relatives. The whole genome analysis of the strain revealed a phenol degradation gene cluster, encoding a multicomponent phenol hydroxylase (mPH) together with a complete meta-cleavage pathway including an I.2.C-type catechol 2,3-dioxygenase (C23O) gene. The strain was able to degrade benzene and ethylbenzene as sole sources of carbon and energy under aerobic and microaerobic conditions. Cells were facultatively aerobic rods and motile with a single polar flagellum. The predominant fatty acids (>10 % of the total) of strain D2M1T were summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c), C16 : 0 and summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c). The major ubiquinone of strain D2M1T was Q8, while the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Based on polyphasic data, it is concluded that strain D2M1T represents a novel species of the genus Acidovorax, for which the name of Acidovorax benzenivorans sp. nov. is proposed. The type strain of the species is strain D2M1T (=DSM 115238T=NCAIM B.02679T).

Combined Omics Approach Reveals Key Differences between Aerobic and Microaerobic Xylene-Degrading Enrichment Bacterial Communities: Rhodoferax─A Hitherto Unknown Player Emerges from the Microbial Dark Matter.

Among monoaromatic hydrocarbons, xylenes, especially the ortho and para isomers, are the least biodegradable compounds in oxygen-limited subsurface environments. Although much knowledge has been gained regarding the anaerobic degradation of xylene isomers in the past 2 decades, the diversity of those bacteria which are able to degrade them under microaerobic conditions is still unknown. To overcome this limitation, aerobic and microaerobic xylene-degrading enrichment cultures were established using groundwater taken from a xylene-contaminated site, and the associated bacterial communities were investigated using a polyphasic approach. Our results show that the xylene-degrading bacterial communities were distinctly different between aerobic and microaerobic enrichment conditions. Although members of the genus Pseudomonas were the most dominant in both types of enrichments, the Rhodoferax and Azovibrio lineages were only abundant under microaerobic conditions, while Sphingobium entirely replaced them under aerobic conditions. Analysis of a metagenome-assembled genome of a Rhodoferax-related bacterium revealed aromatic hydrocarbon-degrading ability by identifying two catechol 2,3-dioxygenases in the genome. Moreover, phylogenetic analysis indicated that both enzymes belonged to a newly defined subfamily of type I.2 extradiol dioxygenases (EDOs). Aerobic and microaerobic xylene-degradation experiments were conducted on strains Sphingobium sp. AS12 and Pseudomonas sp. MAP12, isolated from the aerobic and microaerobic enrichments, respectively. The obtained results, together with the whole-genome sequence data of the strains, confirmed the observation that members of the genus Sphingobium are excellent aromatic hydrocarbon degraders but effective only under clear aerobic conditions. Overall, it was concluded that the observed differences between the bacterial communities of aerobic and microaerobic xylene-degrading enrichments were driven primarily by (i) the method of aromatic ring activation (monooxygenation vs dioxygenation), (ii) the type of EDO enzymes, and (iii) the ability of degraders to respire utilizing nitrate.

Isolation of Pseudomonas aromaticivorans sp. nov from a hydrocarbon-contaminated groundwater capable of degrading benzene-, toluene-, m- and p-xylene under microaerobic conditions.

Members of the genus Pseudomonas are known to be widespread in hydrocarbon contaminated environments because of their remarkable ability to degrade a variety of petroleum hydrocarbons, including BTEX (benzene, toluene, ethylbenzene and xylene) compounds. During an enrichment investigation which aimed to study microaerobic xylene degradation in a legacy petroleum hydrocarbon-contaminated groundwater, a novel Gram-stain-negative, aerobic, motile and rod-shaped bacterial strain, designated as MAP12T was isolated. It was capable of degrading benzene, toluene, meta- and para- xylene effectively under both aerobic and microaerobic conditions. The 16S rRNA gene sequence analysis revealed that strain MAP12T belongs to the genus Pseudomonas, with the highest 16S rRNA gene similarity to Pseudomonas linyingensis LYBRD3-7 T (98.42%), followed by Pseudomonas sagittaria JCM 18195 T (98.29%) and Pseudomonas alcaliphila JCM 10630 T (98.08%). Phylogenomic tree constructed using a concatenated alignment of 92 core genes indicated that strain MAP12T is distinct from any known Pseudomonas species. The draft genome sequence of strain MAP12T is 4.36 Mb long, and the G+C content of MAP12T genome is 65.8%. Orthologous average nucleotide identity (OrthoANI) and digital DNA-DNA hybridization (dDDH) analyses confirmed that strain MAP12T is distinctly separated from its closest neighbors (OrthoANI < 89 %; dDDH < 36%). Though several members of the genus Pseudomonas are well known for their aerobic BTEX degradation capability, this is the first report of a novel Pseudomonas species capable of degrading xylene under microaerobic conditions. By applying genome-resolved metagenomics, we were able to partially reconstruct the genome of strain MAP12 T from metagenomics sequence data and showed that strain MAP12 T was an abundant member of the xylene-degrading bacterial community under microaerobic conditions. Strain MAP12T contains ubiquinone 9 (Q9) as the major respiratory quinone and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine as major polar lipids. The major cellular fatty acids of strain MAP12T are summed feature 3 (C16:1ω6c and/or C16:1ω7c), C16:0 and summed feature 8 (C18:1ω6c and/or C18:1ω7c). The results of this polyphasic study support that strain MAP12T represents a novel species of the genus Pseudomonas, hence the name of Pseudomonas aromaticivorans sp. nov. is proposed for this strain considering its aromatic hydrocarbon degradation capability. The type strain is MAP12T (=LMG 32466, =NCAIM B.02668).

Microaerobic enrichment of benzene-degrading bacteria and description of Ideonella benzenivorans sp. nov., capable of degrading benzene, toluene and ethylbenzene under microaerobic conditions.

In the present study, the bacterial community structure of enrichment cultures degrading benzene under microaerobic conditions was investigated through culturing and 16S rRNA gene Illumina amplicon sequencing. Enrichments were dominated by members of the genus Rhodoferax followed by Pseudomonas and Acidovorax. Additionally, a pale amber-coloured, motile, Gram-stain-negative bacterium, designated B7T was isolated from the microaerobic benzene-degrading enrichment cultures and characterized using a polyphasic approach to determine its taxonomic position. The 16S rRNA gene and whole genome-based phylogenetic analyses revealed that strain B7T formed a lineage within the family Comamonadaceae, clustered as a member of the genus Ideonella and most closely related to Ideonella dechloratans CCUG 30977T. The sole respiratory quinone is ubiquinone-8. The major fatty acids are C16:0 and summed feature 3 (C16:1 ω7c/iso-C15:0 2-OH). The DNA G + C content of the type strain is 68.8 mol%. The orthologous average nucleotide identity (OrthoANI) and in silico DNA-DNA hybridization (dDDH) relatedness values between strain B7T and closest relatives were below the threshold values for species demarcation. The genome of strain B7T, which is approximately 4.5 Mb, contains a phenol degradation gene cluster, encoding a multicomponent phenol hydroxylase (mPH) together with a complete meta-cleavage pathway including a I.2.C-type catechol 2,3-dioxygenase (C23O) gene. As predicted by the genome, the type strain is involved in aromatic hydrocarbon-degradation: benzene, toluene and ethylbenzene are degraded aerobically and also microaerobically as sole source of carbon and energy. Based on phenotypic characteristics and phylogenetic analysis, strain B7T is a member of the genus Ideonella and represents a novel species for which the name Ideonella benzenivorans sp. nov. is proposed. The type strain of the species is strain B7T (= LMG 32,345T = NCAIM B.02664T).

Pinisolibacter aquiterrae sp. nov., a novel aromatic hydrocarbon-degrading bacterium isolated from benzene-, and xylene-degrading enrichment cultures, and emended description of the genus Pinisolibacter.

Two Gram-reaction-negative strains, designated as B13T and MA2-2, were isolated from two different aromatic hydrocarbon-degrading enrichment cultures and characterized using a polyphasic approach to determine their taxonomic position. The two strains had identical 16S rRNA gene sequences and were most closely related to Pinisolibacter ravus E9T (97.36 %) and Siculibacillus lacustris SA-279T (96.33 %). Cells were facultatively aerobic rods and motile with a single polar flagellum. The strains were able to degrade ethylbenzene as sole source of carbon and energy. The assembled genome of strain B13T had a total length of 4.91 Mb and the DNA G+C content was 68.8 mol%. The predominant fatty acids (>5 % of the total) of strains B13T and MA2-2 were C18 : 1 ω7c/C18 : 1 ω6c, C16 : 1 ω7c/C16 : 1 ω6c and C16 : 0. The major ubiquinone of strain B13T was Q10, while the major polar lipids were phosphatidyl-N-methylethanolamine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and a phospholipid. Based on phenotypic characteristics and phylogenetic data, it is concluded that strains B13T and MA2-2 are members of the genus Pinisolibacter and represent a novel species for which the name Pinisolibacter aquiterrae sp. nov. is proposed. The type strain of the species is strain B13T (=LMG 32346T=NCAIM B.02665T).

Evaluating the aerobic xylene-degrading potential of the intrinsic microbial community of a legacy BTEX-contaminated aquifer by enrichment culturing coupled with multi-omics analysis: uncovering the role of Hydrogenophaga strains in xylene degradation.

To develop effective bioremediation strategies, it is always important to explore autochthonous microbial community diversity using substrate-specific enrichment. The primary objective of this present study was to reveal the diversity of aerobic xylene-degrading bacteria at a legacy BTEX-contaminated site where xylene is the predominant contaminant, as well as to identify potential indigenous strains that could effectively degrade xylenes, in order to better understand the underlying facts about xylene degradation using a multi-omics approach. Henceforward, parallel aerobic microcosms were set up using different xylene isomers as the sole carbon source to investigate evolved bacterial communities using both culture-dependent and independent methods. Research outcome showed that the autochthonous community of this legacy BTEX-contaminated site has the capability to remove all of the xylene isomers from the environment aerobically employing different bacterial groups for different xylene isomers. Interestingly, polyphasic analysis of the enrichments disclose that the community composition of the o-xylene-degrading enrichment community was utterly distinct from that of the m- and p-xylene-degrading enrichments. Although in each of the enrichments Pseudomonas and Acidovorax were the dominant genera, in the case of o-xylene-degrading enrichment Rhodococcus was the main player. Among the isolates, two Hydogenophaga strains, belonging to the same genomic species, were obtained from p-xylene-degrading enrichment, substantially able to degrade aromatic hydrocarbons including xylene isomers aerobically. Comparative whole-genome analysis of the strains revealed different genomic adaptations to aromatic hydrocarbon degradation, providing an explanation on their different xylene isomer-degrading abilities.

Microaerobic conditions caused the overwhelming dominance of Acinetobacter spp. and the marginalization of Rhodococcus spp. in diesel fuel/crude oil mixture-amended enrichment cultures.

The aim of the present study was to reveal how different microbial communities evolve in diesel fuel/crude oil-contaminated environments under aerobic and microaerobic conditions. To investigate this question, aerobic and microaerobic bacterial enrichments amended with a diesel fuel/crude oil mixture were established and analysed. The representative aerobic enrichment community was dominated by Gammaproteobacteria (64.5%) with high an abundance of Betaproteobacteriales (36.5%), followed by Alphaproteobacteria (8.7%), Actinobacteria (5.6%), and Candidatus Saccharibacteria (4.5%). The most abundant alkane monooxygenase (alkB) genotypes in this enrichment could be linked to members of the genus Rhodococcus and to a novel Gammaproteobacterium, for which we generated a high-quality draft genome using genome-resolved metagenomics of the enrichment culture. Contrarily, in the microaerobic enrichment, Gammaproteobacteria (99%) overwhelmingly dominated the microbial community with a high abundance of the genera Acinetobacter (66.3%), Pseudomonas (11%) and Acidovorax (11%). Under microaerobic conditions, the vast majority of alkB gene sequences could be linked to Pseudomonas veronii. Consequently, results shed light on the fact that the excellent aliphatic hydrocarbon degrading Rhodococcus species favour clear aerobic conditions, while oxygen-limited conditions can facilitate the high abundance of Acinetobacter species in aliphatic hydrocarbon-contaminated subsurface environments.

Hydrogenophaga aromaticivorans sp. nov., isolated from a para-xylene-degrading enrichment culture, capable of degrading benzene, meta- and para-xylene.

A benzene, para- and meta-xylene-degrading Gram-stain-negative, aerobic, yellow-pigmented bacterium, designated as D2P1T, was isolated from a para-xylene-degrading enrichment culture. Phylogenetic analyses based on 16S rRNA genes showed that D2P1T shares a distinct phyletic lineage within the genus Hydrogenophaga and shows highest 16S rRNA gene sequence similarity to Hydrogenophaga taeniospiralis NBRC 102512T (99.2 %) and Hydrogenophaga palleronii NBRC 102513T (98.3 %). The draft genome sequence of D2P1T is 5.63 Mb long and the genomic DNA G+C content is 65.5 %. Orthologous average nucleotide identity (OrthoANI) and digital DNA-DNA hybridization (dDDH) analyses confirmed low genomic relatedness to its closest relatives (OrthoANI <86 %; dDDH <30 %). D2P1T contains ubiquinone 8 (Q-8) as the only respiratory quinone and phospholipid, phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol as major polar lipids. The main whole-cell fatty acids of D2P1T are summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c), C16 : 0 and summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c). The polyphasic taxonomic results indicated that strain D2P1T represents a novel species of the genus Hydrogenophaga, for which the name Hydrogenophaga aromaticivorans sp. nov. is proposed. The type strain is D2P1T (=LMG 31780T=NCAIM B 02655T).