Due to their high resistance against environmental challenges, bacterial biofilms are ubiquitous and are frequently associated with undesired phenomena in environmental industry (e. g. biofouling). However, because of the high phylogenetic and functional diversity, bacterial biofilms are important sources of biotechnologically relevant microorganisms, e.g. those showing bioremediation potential. In our previous work, the high phylogenetic and metabolic diversity of a clogging biofilm, developed in a simple aromatic hydrocarbon (BTEX)-contaminated groundwater well was uncovered. The determination of relationships between different groups of biofilm bacteria and certain metabolic traits has been omitted so far. Therefore, by setting up new biofilm-based enrichment microcosms, the research goal of the present study was to identify the aerobic/hypoxic BTEX-degrading and/or prolific biofilm-forming bacteria. The initial bacterial community composition as well as temporal dynamics due to the selective enrichment has been determined. The obtained results indicated that the concentration of dissolved oxygen may be a strong selective force on the evolution and final structure of microbial communities, developed in hydrocarbon-contaminated environments. Accordingly, members of the genus Malikia proved to be the most dominant community members of the aerobic BTEX-degrading enrichments. Acidovorax spp. dominated the oxygen-limited/hypoxic setup. During the study, a strain collection of 23 different bacterial species was obtained. Non-pathogenic members of this strain collection, with outstanding biodegradation (e.g. Pseudomonas, Variovorax isolates) and biofilm-forming potential (e.g. Rhizobium), may potentially be applied in the development of biofilm-based semipermeable reactive biobarriers.

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.

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.

Dissimilatory iron-reducing bacteria are commonly found in microbial communities of aromatic hydrocarbon-contaminated subsurface environments where they often play key role in the degradation of the contaminants. The Siklós benzene, toluene, ethylbenzene, and xylene (BTEX)-contaminated area is one of the best characterized petroleum hydrocarbon-contaminated sites of Hungary. Continuous monitoring of the microbial community in the center of the contaminant plume indicated the presence of an emerging Geobacter population and a Rhodoferax phylotype highly associated with aromatic hydrocarbon-contaminated subsurface environments. The aim of the present study was to make an initial effort to enrich Rhodoferax-related and other dissimilatory iron-reducing bacteria from this environment. Accordingly, four slightly different freshwater media were used to enrich Fe(III) reducers, differing only in the form of nitrogen source (organic, inorganic nitrogen or gaseous headspace nitrogen). Although enrichment of the desired Rhodoferax phylotype was not succeeded, Geobacter-related bacteria were readily enriched. Moreover, the different nitrogen sources caused the enrichment of different Geobacter species. Investigation of the diversity of benzylsuccinate synthase gene both in the enrichments and in the initial groundwater sample indicated that the Geobacter population in the center of the contaminant plume may not play a significant role in the anaerobic degradation of toluene.

Organisms of the candidate phylum Saccharibacteria have frequently been detected as active members of hydrocarbon degrading communities, yet their actual role in hydrocarbon degradation remained unclear. Here, we analyzed three enrichment cultures of hydrocarbon-amended groundwater samples using genome-resolved metagenomics to unravel the metabolic potential of indigenous Saccharibacteria. Community profiling based on ribosomal proteins revealed high variation in the enrichment cultures suggesting little reproducibility although identical cultivation conditions were applied. Only 17.5 and 12.5% of the community members were shared between the three enrichment cultures based on ribosomal protein clustering and read mapping of reconstructed genomes, respectively. In one enrichment, two Saccharibacteria strains dominated the community with 16.6% in relative abundance and we were able to recover near-complete genomes for each of them. A detailed analysis of their limited metabolism revealed the capacity for peptide degradation, lactate fermentation from various hexoses, and suggests a scavenging lifestyle with external retrieval of molecular building blocks. In contrast to previous studies suggesting that Saccharibacteria are directly involved in hydrocarbon degradation, our analyses provide evidence that these organisms can be highly abundant scavengers acting rather as organic carbon sinks than hydrocarbon degraders in these communities.