Microbial response to oil contamination in freshwater ecosystems, especially lacustrine habitats, is poorly studied. Studies on the microbial community composition and metabolic landscape of polluted lacustrine environments contribute to our understanding of the dynamics of microbial response and in turn, facilitate better conservation of water resources. Recently, high-throughput sequencing has facilitated the advancement of microbial ecological studies in polluted habitats with bioinformatic approaches being used to reveal metagenomic characteristics of bacterial response to petroleum hydrocarbon contamination in diverse ecosystems. Using 16S rRNA gene sequences as an input in PICRUSt (Douglas et al. 2019) and other similar bioinformatic tools (Aßhauer et al. 2015), it is possible to predict metagenomics features and functional compositions of microbiomes; subsequently, critically important and enriched taxa and functional pathways can be inferred as biomarkers that can be effectively used to distinguish diverse oil-polluted environments (Mukherjee et al. 2017).
Proteobacteria dominate the oil-polluted lacustrine microbiome
In the present study, Proteobacteria were found to be the dominant phylum in the oil polluted Lake Pertusillo microbiome. The Proteobacterial dominance of an oil-contaminated Lake Pertusillo microbiome is in agreement with previous observations from other ecosystems which suffered oil pollution events (Mukherjee et al. 2017). For example, Yang et al. have previously reported a similar shift in microbial community structure towards phylum Proteobacteria upon oil contamination in permafrost habitats (Yang et al. 2014). Members of this phylum, such as those belonging to the classes Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria, are known to be capable of using various hydrocarbons as their sole carbon source (Kostka et al. 2011). Unsurprisingly, when evaluated at the genus level, contaminated samples from Lake Pertusillo were found to be enriched for alphaproteobacterial and betaproteobacterial genera.
Hydrogenophaga sp., a member of the Betaproteobacteria family Comamonadaceae, was found to be notably enriched in the two contaminated samples 1B and 2B. The genus has previously been isolated from microbial consortia in benzene-contaminated sites (Fahy et al. 2008) along with Acidovorax sp. and Pseudomonas sp., both of which were also detected in our study. The presence of Hydrogenophaga sp. was also described in microcosms set up using groundwater from a BTEX (benzene, toluene, ethylbenzene, and xylenes) contaminated site as inoculum with a mixture of toluene and benzene used as sole carbon sources (Aburto and Peimbert 2011). Hydrogenophaga sp. have additionally been reported to play an important role in PAH-degrading (polycyclic aromatic hydrocarbons) microbial communities (Martin et al. 2012). Furthermore, a role for Hydrogenophaga in polychlorinated biphenyls (PCBs) degradation has been proposed previously (Lambo and Patel 2006). These data are largely in agreement with a previous study, which detected PCBs in ichthyic fauna of Lake Pertusillo (De Pace 2015) and with the ARPAB (ARPAB 2017b) that stresses that PCBs such as PAH are present in the sediment of the entire reservoir with higher concentrations in 2017. Acidovorax, another betaproteobacterial genera, was well represented in the oil-contaminated samples. Acidovorax has previously been found to be the most abundant dominant bacterial species in the sludge of an Alberta oil sand tailing pond (Singleton et al. 2009). Like other members of Comamonadaceae, Acidovorax is frequently encountered in association with PAH degradation (Singleton et al. 2018). Denitrifying Acidovorax sp. has been isolated from terrestrial subsurface sediments exposed to mixed-waste contamination; in particular, this genus has been detected in nitrate-reducing microbial consortia cultivated with alkylated aromatic compounds, revealing their important role in hydrocarbons and nitrate-polluted waters (Sperfeld et al. 2018). The versatile betaproteobacterial genus Variovorax sp. was enriched only in sample 1B; the genus has been previously reported for hydrocarbonoclastic properties such as biosynthesis of biosurfactants (Franzetti et al. 2012) and PAH degradation in the presence of nitrates (Eriksson et al. 2003).
Afipia sp., belonging to class Alphaproteobacteria, was enriched in the two contaminated Lake Pertusillo samples. Similar to Acidovorax sp., Afipia sp. is a well-known degrader of polycyclic aromatic substances (Willumsen et al. 2005) and can degrade PAHs using nitrates as oxidizing agents; such combination of PAH degradation with denitrification by Afipia sp. has been observed in nitrate-contaminated groundwater (Green et al. 2010). Reyranella sp., an alphaproteobacterial genus which was found to be enriched in oil-contaminated samples, has been previously reported in degradation of asphaltenes, a particularly recalcitrant fraction of crude oil (Song et al. 2018). In samples analyzed along the water column, i.e., samples 1B and 2B, the alphaproteobacterial genera Sphingopyxis and Hirschia were detected. Both these genera have been previously identified to play important roles in microbiome successions in relation to oil spills (Rodriguez et al. 2015). In the first phase (during the oil spill), sand microbiomes in beaches affected by the Deep Water Horizon oil spill showed a prevalence of microbes that degrade aliphatic hydrocarbons and were replaced in the second phase (2–3 months after the oil spill) with a PAH-degrading microbial community enriched in Sphingopyxis and Hirschia (Rodriguez et al. 2015).
Dyadobacter sp., a member of Phylum Bacteroidetes, was found to be enriched only in the oil-contaminated sample 1B; members of the genus are particularly specialized in degradation of complex aromatic hydrocarbons such as azaarenes, which are relatively water soluble, nitrogen-containing heterocyclic aromatic hydrocarbons that have been reported to leach from contaminated soils and sediments into aqueous habitats (Pereria et al. 1983). The isolation and study of these enriched species identified herein, among others, could help us better understand lacustrine microbial response to oil pollution and may serve as excellent tools for future bioremediation interventions and as microbial proxies for oil pollution in lakes; taken together, these will contribute to protection of freshwater ecosystems and reduction of public health risks. Indeed, recent further studies concentrated on the hydrocarbonoclastic biofilms formed on the water surface after the Lake Pertusillo oil pollution event have revealed unique electrogenic structural and biochemical properties of the biofilm and sheds further light on possible mechanisms of microbial detoxification of oil-contaminated lacustrine habitats (D’Ugo et al. 2021). Overall, the presence of a hydrocarbonoclastic microbial community characterized by an abundance of proteobacterial genera such as Hydrogenophaga, Acidovorax, Reyranella, and Variovorax among others, highlighted the lake's pollution status, indicating a community shaped by residual recalcitrant hydrocarbons derived from the oil spill 3 months earlier and with significant complex hydrocarbon degradation potential.
Metabolic landscape of polluted lacustrine microbiome reveals microbial hydrocarbonoclastic potential
Detection of hydrocarbonoclastic pathways involved in the degradation of complex aromatic hydrocarbons in metagenomes predicted through PICRUSt is consistent with the hydrocarbon degradation potential of several genera found to be enriched in petroleum hydrocarbon-contaminated Pertusillo samples. Indeed, microbial genera such Hydrogenophaga, Acidovorax, Reyranella, Variovorax, and others, found to be enriched in polluted samples as described above, exhibit multiple metabolic pathways involved in degradation of aromatic hydrocarbons. For example, mineralization pathways for pyrene and benzo-[a]-pyrene along with other high-molecular weight PAHs have been reported previously for Hydrogenophaga sp. (Yan et al. 2017). Additionally, metabolic pathways for the degradation of chloroaromatics have also been identified in Hydrogenophaga sp., including transformation of 2,4′-dichlorobiphenyl (2,4′-DCB) into 2- and 4-chlorobenzoic acid (2- and 4-CBA; Lambo and Patel 2006). Furthermore, degradation of sulfo-nitroaromatic compounds have also been reported for Hydrogenophaga sp.; indeed, Gan et al. reported the mineralization of 4-aminobenzenesulfonate (4-ABS) by Hydrogenophaga sp. strain PBC involving enzymatic conversions catalyzed by 4-sulfocatechol 1,2-dioxygenase, 3-sulfomuconate cycloisomerase, and 3,4-dioxygenase enzymes (Gan et al. 2011). The versatility of Hydrogenophaga sp. as a degrader of PAHs could explain the increased abundance of the genus in oil-contaminated samples, as mentioned above (Supplementary Table S1). PAH degrading capabilities have also been reported for other genera found to be abundant in oil-contaminated samples, i.e., Acidovorax, Reyranella, and Variovorax, with metabolic pathways employing diverse hydrocarbonoclastic enzymes such as ring-hydroxylating dioxygenase (Singleton et al. 2009), catechol 1,2-dioxygenase (El Azhari et al. 2010), and hydrolytic esterases (Wang and Gu 2006), among others, being identified in them.
Although it was not possible to analyze the microbial community structure of Lake Pertusillo during the oil spill, our results indicate that hydrocarbonoclastic capacities of the lacustrine microbiome in the aftermath of the oil spill were highly skewed towards mineralization of complex hydrocarbons including PAHs, choroaromatics, nitroaromatics, and sulfonated aromatic compounds with few metabolic pathways for degradation of aliphatic petroleum hydrocarbons detected. This suggests that there may have been a shift in the microbiome towards a specific hydrocarbonoclastic competence at this stage, i.e., 3 months after the oil spill, where the structure of the Pertusillo microbiome could be shaped by the residual presence of more recalcitrant, complex hydrocarbons such as benzene derivatives, toluene, PAH, PCB, and BTEX, among others, after most aliphatic hydrocarbons had been degraded. Such an observation is substantiated further by detection of several genera of microbes known to specialize in PAH and complex hydrocarbon degradation, as described above, as well as previously reported similar observations from the Deep Water Horizon oil spill where the second stage of microbial successions was dominated by PAH degrading microbes (Rodriguez et al. 2015; Martirani-Von Abercron et al. 2017). Importantly, predictive metabolic reconstruction using PICRUSt demonstrated that advanced bioinformatic pipelines can be successfully employed in dissecting complex freshwater ecosystem metabolomes, where hydrocarbonoclastic contributions of members of microbial communities in polluted habitats can be inferred and in turn, provide important insights.