Effect of oxygen limitation on the enrichment of bacteria degrading either benzene or toluene and the identification of Malikia spinosa (Comamonadaceae) as prominent aerobic benzene-, toluene-, and ethylbenzene-degrading bacterium: enrichment, isolation and whole-genome analysis.

The primary aims of this present study were to evaluate the effect of oxygen limitation on the bacterial community structure of enrichment cultures degrading either benzene or toluene and to clarify the role of Malikia-related bacteria in the aerobic degradation of BTEX compounds. Accordingly, parallel aerobic and microaerobic enrichment cultures were set up and the bacterial communities were investigated through cultivation and 16S rDNA Illumina amplicon sequencing. In the aerobic benzene-degrading enrichment cultures, the overwhelming dominance of Malikia spinosa was observed and it was abundant in the aerobic toluene-degrading enrichment cultures as well. Successful isolation of a Malikia spinosa strain shed light on the fact that this bacterium harbours a catechol 2,3-dioxygenase (C23O) gene encoding a subfamily I.2.C-type extradiol dioxygenase and it is able to degrade benzene, toluene and ethylbenzene under clear aerobic conditions. While quick degradation of the aromatic substrates was observable in the case of the aerobic enrichments, no significant benzene degradation, and the slow degradation of toluene was observed in the microaerobic enrichments. Despite harbouring a subfamily I.2.C-type C23O gene, Malikia spinosa was not found in the microaerobic enrichments; instead, members of the Pseudomonas veronii/extremaustralis lineage dominated these communities. Whole-genome analysis of M. spinosa strain AB6 revealed that the C23O gene was part of a phenol-degrading gene cluster, which was acquired by the strain through a horizontal gene transfer event. Results of the present study revealed that bacteria, which encode subfamily I.2.C-type extradiol dioxygenase enzyme, will not be automatically able to degrade monoaromatic hydrocarbons under microaerobic conditions.

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).

Sphingobium aquiterrae sp. nov., a toluene, meta- and para-xylene-degrading bacterium isolated from petroleum hydrocarbon-contaminated groundwater.

A Gram-negative, aerobic, slightly yellow-pigmented bacterium, designated as SKLS-A10T, was isolated from groundwater sample of the 'Siklós' petroleum hydrocarbon contaminated site (Hungary). Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain SKLS-A10T formed a distinct phyletic lineage within the genus Sphingobium. It shared the highest 16S rRNA gene homology with Sphingobium abikonense DSM 23268T (97.29 %), followed by Sphingobium lactosutens DSM 23389T (97.23 %), Sphingobium phenoxybenzoativorans KCTC 42448T (97.16 %) and Sphingobium subterraneum NBRC 109814T (96.74 %). The predominant fatty acids (>5 % of the total) are C18 : 1ω7c, C14 : 0 2-OH, C16 : 1ω7c/iso C15 : 0 2-OH, C17 : 1ω6c and C16 : 0. The major ubiquinone is Q-10. The predominant polyamine is spermidine. The major polar lipids are sphingoglycolipid and diphosphatidylglycerol. The DNA G+C content of strain SKLS-A10T is 65.9 mol%. On the basis of evidence from this taxonomic study using a polyphasic approach, strain SKLS-A10T represents a novel species of the genus Sphingobium for which the name Sphingobiumaquiterrae sp. nov. is proposed. The type strain is SKLS-A10T (=DSM 106441T=NCAIM B. 02634T).