Microbiology of Hydraulic Fracturing Wastewater

The extraction of natural gas and oil from shale formations using hydraulic fracturing generates large volumes of wastewater, often termed produced water. One of the biggest challenges associated with produced water management is microbial activity. Microorganisms growing in produced water may have the ability to form biofilms and produce acids and sulfides, which can contribute to biocorrosion and gas souring. This dissertation investigates the microbial ecology of microorganisms living in produced water by studying their community structure and metabolic potential as well as the active, genetic response of Pseudomonas biofilms to the biocide sodium hypochlorite to inform microbial control. First, storage guidelines for hydraulic fracturing produced waters intended for microbiological analysis were developed. Results suggested microbial communities in produced water samples to remain stable when stored at 4oC for three days or less. Next, the microbial ecology of 42 Marcellus Shale produced water samples was analyzed. Samples were dominated by the taxa Halanaerobiales, specifically the genus Halanaerobium. Subsequently, metagenomic sequencing and binning allowed the recovery and annotation of a Halanaerobium draft genome. Annotation results suggested Halanaerobium to have the metabolic potential for acid production and sulfide production through thiosulfate reduction. Microbiological assessment of produced waters from 18 Bakken Shale wells, sampled across a six-month time frame, confirmed the presence of Halanaerobium in produced water. However, the microbial community structure was found to change temporally, and the majority of the samples were dominated by the order Bacillales. Finally, the active, genetic response to the broad-spectrum biocide sodium hypochlorite, which is also used for microbial control in hydraulic fracturing operations, was assessed. Pseudomonas fluorescens biofilms were exposed to sublethal concentrations of sodium hypochlorite and differential genes expression was analyzed. Results suggested genes involved in oxidative stress response pathways and multidrug efflux mechanisms to be upregulated, demonstrating genetic components to be involved in sodium hypochlorite resistance. Ultimately, findings from this dissertation enhance the current understanding of microbial community dynamics in produced water and may help to limit corrosion, control fouling and souring issues, protect well infrastructure, and minimize unnecessary biocide application

The Effects of Sample Storage Conditions on the Microbial Community Composition in Hydraulic Fracturing Produced Water

The petroleum industry has an increasing interest in understanding the microbial communities driving biofouling and biocorrosion in reservoirs, wells, and infrastructure. However, sampling of the relevant produced fluids from subsurface environments for microbiological analyses is often challenged by high liquid pressures, workplace regulations, operator liability concerns, and remote sampling locations. These challenges result in infrequent sampling opportunities and the need to store and preserve the collected samples for several days or weeks. Maintaining a representative microbial community structure from produced fluid samples throughout storage and handling is essential for accurate results of downstream microbial analyses. Currently, no sample handling or storage recommendations exist for microbiological analyses of produced fluid samples. We used 16S rRNA gene sequencing to monitor the changes in microbial communities in hypersaline produced water stored at room temperature or at 4 oC for up to 7 days. We also analyzed storage at -80 oC across a 3-week period. The results suggest ideal handling methods would include placing the collected sample on ice as soon as possible, but at least within 24 h, followed by shipping the samples on ice over 2–3 days, and finally, long-term storage in the -20 oC or -80 oC freezer

Microbial Diversity and Biogeochemical Interactions in the Seismically Active and CO2-Rich Eger Rift Ecosystem

The Eger Rift subsurface is characterized by frequent seismic activity and consistently high CO2 concentrations, making it a unique deep biosphere ecosystem and a suitable site to study the interactions between volcanism, tectonics, and microbiological activity. Pulses of geogenic H2 during earthquakes may provide substrates for methanogenic and chemolithoautotrophic processes, but very little is currently known about the role of subsurface microorganisms and their cellular processes in this type of environment. To assess the impact of geologic activity on microbial life, we analyzed the geological, geochemical, and microbiological composition of rock and sediment samples from a 238 m deep drill core, running across six lithostratigraphic zones. We evaluated the diversity and distribution of bacterial and archaeal communities. Our investigation revealed a distinct low-biomass community, with a surprisingly diverse archaeal population, providing strong support that methanogenic archaea reside in the Eger subsurface. Geochemical analysis demonstrated that ion concentrations (mostly sodium and sulfate) were highest in sediments from 50 to 100 m depth and in weathered rock below 200 m, indicating an elevated potential for ion solution in these areas. Microbial communities were dominated by common soil and water bacteria. Together with the occurrence of freshwater cyanobacteria at specific depths, these observations emphasize the heterogenous character of the sediments and are indicators for vertical groundwater movement across the Eger Rift subsurface. Our investigations also found evidence for anaerobic, autotrophic, and acidophilic communities in Eger Rift sediments, as sulfur-cycling taxa like Thiohalophilus and Desulfosporosinus were specifically enriched at depths below 100 m. The detection of methanogenic, halophilic, and ammonia-oxidizing archaeal populations demonstrate that the unique features of the Eger Rift subsurface environment provide the foundation for diverse types of microbial life, including the microbial utilization of geologically derived CO2 and, when available, H2, as a primary energy source

The Role of Anthropogenic Influences on a Tropical Lake Ecosystem and Its Surrounding Catchment: A Case Study of Lake Sentani

Lake Sentani is a tropical lake in Indonesia, consisting of four interconnected sub-basins of different water depths. While previous work has highlighted the impact of catchment composition on biogeochemical processes in Lake Sentani, little is currently known about the microbiological characteristics across this unique ecosystem. With recent population growth in this historically rural area, the anthropogenic impact on Lake Sentani and hence its microbial life is also increasing. Therefore, we aimed to explore the influence of environmental and anthropogenic factors on the microbial diversity of Lake Sentani. Here, we present a detailed microbiological evaluation of Lake Sentani, analyzing 49 different sites across the lake, its tributary rivers and their river mouths to assess diversity and community structure using 16S rRNA gene sequencing. Our results reveal distinct communities in lake and river sediments, supporting the observed geochemical differences. Taxonomic assessment showed the potential impact of anthropogenic pressure along the northern, urbanized shore, as river and river mouth samples revealed high abundances of Bacteroidota, Firmicutes, and Cyanobacteria, which could be attributed to pollution and eutrophication. In contrast, lake sediment communities were dominated by Thermodesulfovibrionia, Methanomethylicia, Bathyarchaeia, and Thermoplasmata, suggesting sulfate reducing, thermophilic, acidophilic bacteria and methanogenic archaea to play an important role in tropical lake systems. This study provides novel insights into ecological functions of tropical lakes and contributes to the optimization of management strategies of Lake Sentani, ensuring its holistic preservation in the future

Microbial Hotspots in Lithic Macrohabitats Inferred from DNA Fractionation and Metagenomics in the Atacama Desert

The existence of microbial activity hotspots in temperate regions of Earth is driven by soil heterogeneities, especially the temporal and spatial availability of nutrients. Here we investigate whether microbial activity hotspots also exist in lithic microhabitats in one of the most arid regions of the world, the Atacama Desert in Chile. While previous studies evaluated the total DNA fraction to elucidate the microbial communities, we here for the first time use a DNA separation approach on lithic microhabitats, together with metagenomics and other analysis methods (i.e., ATP, PLFA, and metabolite analysis) to specifically gain insights on the living and potentially active microbial community. Our results show that hypolith colonized rocks are microbial hotspots in the desert environment. In contrast, our data do not support such a conclusion for gypsum crust and salt rockenvironments, because only limited microbial activity could be observed. The hypolith community is dominated by phototrophs, mostly Cyanobacteria and Chloroflexi, at both study sites. The gypsum crusts are dominated by methylotrophs and heterotrophic phototrophs, mostly Chloroflexi, and the salt rocks (halite nodules) by phototrophic and halotolerant endoliths, mostly Cyanobacteria and Archaea. The major environmental constraints in the organic-poor arid and hyperarid Atacama Desert are water availability and UV irradiation, allowing phototrophs and other extremophiles to play a key role in desert ecology

Draft Genome Sequence of <i>Pseudomonas</i> sp. BDAL1 Reconstructed from a Bakken Shale Hydraulic Fracturing-Produced Water Storage Tank Metagenome

ABSTRACT We report the 5,425,832 bp draft genome of Pseudomonas sp. strain BDAL1, recovered from a Bakken shale hydraulic fracturing-produced water tank metagenome. Genome annotation revealed several key biofilm formation genes and osmotic stress response mechanisms necessary for survival in hydraulic fracturing-produced water. </jats:p

Comparative metagenomics of coalbed methane microbial communities reveals biogenic methane potential in the Appalachian Basin

Natural gas is a major source of global energy, and a large fraction is generated in subsurface coalbed deposits. Microbial communities within coalbed deposits impact methane production, and as a result contribute to global carbon cycling. The process of biogenic coal-to-methane conversion is not well understood. Here we demonstrate the first read- and assembly-based metagenome profiling of coal-associated formation waters, resulting in the recovery of over 40 metagenome-assembled genomes (MAGs) from eight individual coalbed methane wells in the Appalachian Basin. The majority of samples contained hydrogenotrophic methanogens, which were present in higher relative abundances than was previously reported for other coalbed basins. The abundance of Archaea and salinity were positively correlated, suggesting that salinity may be a controlling factor for biogenic coalbed methane. Low-abundance coalbed microbial populations were functionally diverse, while the most dominant organisms exhibit a high degree of genomic and functional similarities. Basin-specific pan-metagenome clustering suggests lower abundant and diverse bacterial communities are shaped by local basin parameters. Our analyses show Appalachian Basin coalbed microbial communities encode for the potential to convert coal into methane, which may be used as an indicator of potential biogenic methane production for future well performance and increased well longevity.</jats:p