Hydraulic fracturing is a method of oil and gas extraction from shale in which substantial volumes of water return to the surface containing chemicals and microorganisms. This paper begins to address the microbial composition and aqueous chemistry and the potential for intrinsic and enhanced bioremediation of these waters. The waters from a gas and oil shale in the Marcellus and Bakken regions, respectively, were analyzed for inorganic elements, organic chemicals, microbial taxonomic composition, and biodegradative capabilities. The waters were highly saline, reaching NaCl concentrations up to 3.5 N, but no significant levels of radioactive elements were detected. More than 1,000 organic compounds were separated and identified by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry. The major classes of organic compounds, in order of decreasing abundance, were aliphatics, cycloaliphatics, single-ring aromatics, and polycyclic aromatic compounds. The bacterial genera found natively in the waters were identified by sequencing the 16S rRNA genes within the extracted DNA. The major genera identified included strains known to thrive under saline conditions (Halanaerobium, Marinobacter, Oceanimonas, Streptohalobacillus) and degrade petroleum hydrocarbons (Thauera, Pseudomonas, Marinobacterium, Williamsia, Colwellia). Microbial populations were extracted from the Bakken shale waters, encapsulated within silica gels, and then reintroduced into their waters of origin. Both intrinsic biodegradation from the free native microorganisms and enhanced biodegradation with the addition of encapsulated bacteria were observed. In total, this paper begins to better define the properties of waters derived from hydraulic fracturing and suggests a potential for the application of bioremediation to remove organic contaminants.

Encapsulation of recombinant Escherichia coli cells expressing a biocatalyst has the potential to produce stable, long-lasting enzyme activity that can be used for numerous applications. The current study describes the use of this technology with recombinant E. coli cells expressing the atrazine-dechlorinating enzyme AtzA in a silica/polymer porous gel. This novel recombinant enzyme-based method utilizes both adsorption and degradation to remove atrazine from water. A combination of silica nanoparticles (Ludox TM40), alkoxides, and an organic polymer was used to synthesize a porous gel. Gel curing temperatures of 23 or 45 °C were used either to maintain cell viability or to render the cells non-viable, respectively. The enzymatic activity of the encapsulated viable and non-viable cells was high and extremely stable over the time period analyzed. At room temperature, the encapsulated non-viable cells maintained a specific activity between (0.44 ± 0.06) μmol/g/min and (0.66 ± 0.12) μmol/g/min for up to 4 months, comparing well with free, viable cell-specific activities (0.61 ± 0.04 μmol/g/min). Gels cured at 45 °C had excellent structural rigidity and contained few viable cells, making these gels potentially compatible with water treatment facility applications. When encapsulated, non-viable cells were assayed at 4 °C, the activity increased threefold over free cells, potentially due to differences in lipid membranes as shown by FTIR spectroscopy and electron microscopy.

The most problematic hydrocarbons in hydraulic fracturing (fracking) wastewaters consist of fused, isolated, bridged, and spiro ring systems, and ring systems have been poorly studied with respect to biodegradation, prompting the testing here of six major ring structural subclasses using a well-characterized bacterium and a silica encapsulation system previously shown to enhance biodegradation. The direct biological oxygenation of spiro ring compounds was demonstrated here. These and other hydrocarbon ring compounds have previously been shown to be present in flow-back waters and waters produced from hydraulic fracturing operations. Pseudomonas sp. strain NCIB 9816-4, containing naphthalene dioxygenase, was selected for its broad substrate specificity, and it was demonstrated here to oxidize fundamental ring structures that are common in shale-derived waters but not previously investigated with this or related enzymes. Pseudomonas sp. NCIB 9816-4 was tested here in the presence of a silica encasement, a protocol that has previously been shown to protect bacteria against the extremes of salinity present in fracking wastewaters. These studies demonstrate the degradation of highly hydrophobic compounds by a silica-encapsulated model bacterium, demonstrate what it may not degrade, and contribute to knowledge of the full range of hydrocarbon ring compounds that can be oxidized using Pseudomonas sp. NCIB 9816-4.