Giovanni Pilloni

Biodegradation of petroleum hydrocarbons in groundwater occurs naturally and can be enhanced to support contaminated site management and remediation. Molecular biological tools can be used to assess the occurrence of biodegradation and monitor bioremediation efforts; however, the use of specific genes (i.e., biomarkers) to identify the rate constants of contaminant removal has not been explored for monoaromatic hydrocarbons. In this study, an approach was developed to estimate an apparent first‐order rate constant for anaerobic biodegradation of toluene based on toluene concentration and the abundance of the alpha subunit of the benzylsuccinate synthase gene (i.e., bssA ). The utility of the approach was evaluated by comparing the distribution of estimated rate constants to those from a published compilation at benchmark sites. There was good agreement between the distribution of rate constants calculated from the abundance of gene copies from soil cores and the distribution of rate constants at benchmark sites, while rate constants calculated from the abundance of gene copies in groundwater were lower than those from the benchmark sites. Given that groundwater samples are more common and convenient to obtain, it is proposed to use the rate constants estimated from groundwater samples to document whether the microbial community has acclimated for biodegradation of toluene. When practitioners use models to evaluate risk at petroleum release sites, they often assume a “typical” rate constant for biodegradation. If the abundance of the bssA biomarker demonstrates that the microbial community has acclimated, then it is appropriate to select and use a rate constant from benchmark sites to forecast biodegradation and attenuation of toluene at a site being evaluated.

Growth rates and biomass yields are key descriptors used in microbiology studies to understand how microbial species respond to changes in the environment. Of these, biomass yield estimates are typically obtained using cell counts and measurements of the feed substrate. These quantities are perturbed with measurement noise however. Perhaps most crucially, estimating biomass from cell counts, as needed to assess yields, relies on an assumed cell weight. Noise and discrepancies on these assumptions can lead to significant changes in conclusions regarding the microbes response. This article proposes a methodology to address these challenges using probabilistic macrochemical models of microbial growth. It is shown that a model can be developed to fully use the experimental data, relax assumptions and greatly improve robustness to a priori estimates of the cell weight, and provides uncertainty estimates of key parameters. This methodology is demonstrated in the context of a specific case study and the estimation characteristics are validated in several scenarios using synthetically generated microbial growth data.

Endospores of thermophilic bacteria are widespread in cold seabed environments where they remain dormant during initial burial in accumulating sediments. The temperature increase during sedimentation can be simulated in experimental heating of sediments, resulting in the temperature-dependent activation of different endospore populations from the microbial seed bank. Here we investigated the response of endospore populations to heating at extreme high temperature (80– 99°C). Metabolites for germination and organic matter degradation (dipicolinic acid and organic acids) revealed both endospore germination and subsequent metabolism at ≥80°C. Endospore-forming Firmicutes with the genomic potential for organic carbon and nitrogen transformation were recovered by genome-resolved metagenomics. Genomes from Symbiobacteriales, Thermosediminibacteriales, Moorellales and Calditerricolales encode multiple mechanisms for high temperature degradation of sedimentary organic carbon and features of necromass that accumulate during sediment burial including saccharides, amino and nucleic acids. The results provide insight into the metabolism of novel carbon cycling microorganisms activated at high temperature, and suggest that extremely thermophilic Firmicutes dispersed in the ocean are poised to germinate in response to sediment heating during burial and transform a wide range of organic substrates.

The unpredictability of microbial growth and subsequent localized corrosion of steel can cause significant cost for the oil and gas industry, due to production downtime, repair, and replacement. Despite a long tradition of academic research and industrial experience, microbial corrosion is not yet fully understood and thus not effectively controlled. In particular, biomarkers suitable for diagnosing microbial corrosion which abstain from the detection of the classic signatures of sulfate-reducing bacteria are urgently required. In this study, a natural microbial community was enriched anaerobically with carbon steel coupons and in the presence of a variety of physical and chemical conditions. With the characterization of the microbiome and of its functional properties inferred through predictive metagenomics, a series of proteins were identified as biomarkers in the water phase that could be correlated directly to corrosion. This study provides an opportunity for the further development of a protein-based biomarker approach for effective and reliable microbial corrosion detection and monitoring in the field.

Summary: Thermophilic endospores are widespread in cold marine sediments where the temperature is too low to support growth and activity of thermophiles in situ . These endospores are likely expelled from warm subsurface environments and subsequently dispersed by ocean currents. The endospore upper temperature limit for survival is 140°C, which can be tolerated in repeated short exposures, potentially enabling transit through hot crustal fluids. Longer‐term thermal tolerance of endospores, and how long they could persist in an environment hotter than their maximum growth temperature, is less understood. To test whether thermophilic endospores can survive prolonged exposure to high temperatures, sediments were incubated at 80–90°C for 6, 12 or 463 days. Sediments were then cooled by 10–40°C, mimicking the cooling in subsurface oil reservoirs subjected to seawater injection. Cooling the sediments induced sulfate reduction, coinciding with an enrichment of endospore‐forming Clostridia . Different Desulfofundulus , Desulfohalotomaculum , Desulfallas , Desulfotomaculum and Desulfofarcimen demonstrated different thermal tolerances, with some Desulfofundulus strains surviving for >1 year at 80°C. In an oil reservoir context, heat‐resistant endospore‐forming sulfate‐reducing bacteria have a survival advantage if they are introduced to, or are resident in, an oil reservoir normally too hot for germination and growth, explaining observations of reservoir souring following cold seawater injection.

Aquifers are typically perceived as rather stable habitats, characterized by low biogeochemical and microbial community dynamics. Upon contamination, aquifers shift to a perturbed ecological status, in which specialized populations of contaminant degraders establish and mediate aquifer restoration. However, the ecological controls of such degrader populations, and possible feedbacks between hydraulic and microbial habitat components, remain poorly understood. Here, we provide evidence of such couplings, via 4 years of annual sampling of groundwater and sediments across a high-resolution depth-transect of a hydrocarbon plume. Specialized anaerobic degrader populations are known to be established at the reactive fringes of the plume. Here, we show that fluctuations of the groundwater table were paralleled by pronounced dynamics of biogeochemical processes, pollutant degradation, and plume microbiota. Importantly, a switching in maximal relative abundance between dominant degrader populations within the Desulfobulbaceae and Desulfosporosinus spp. was observed after hydraulic dynamics. Thus, functional redundancy amongst anaerobic hydrocarbon degraders could have been relevant in sustaining biodegradation processes after hydraulic fluctuations. These findings contribute to an improved ecological perspective of contaminant plumes as a dynamic microbial habitat, with implications for both monitoring and remediation strategies in situ.

Slow sand filtration (SSF) is an effective low-tech water treatment method for pathogen and particle removal. Yet despite its application for centuries, it has been uncertain to which extent pathogenic microbes are removed by mechanical filtration or due to ecological interactions such as grazing and competition for nutrients. In this study, we quantified the removal of bacterial faecal indicators, Escherichia coli and Enterococcus faecalis, from secondary effluent of a wastewater treatment plant and analysed the microbial community composition in compartments of laboratory model SSF columns. The columns were packed with different sand grain sizes and eliminated 1.6–2.3 log units of faecal indicators, which translated into effluents of bathing water quality according to the EU directive (E. coli per 100 ml) for columns with small grain size. Most of that removal occurred in the upper filter area, the Schmutzdecke. Within that same zone, total bacterial numbers increased however, thus suggesting a specific elimination of the faecal indicators. The analysis of the microbial communities also revealed that some taxa were removed more from the wastewater than others. These results accentuate the contribution of biological mechanisms to water purification in SSF.