Robert D. Fallon

Characterizing microbial diversity in production water from an Alaskan mesothermic petroleum reservoir with two independent molecular methods

The phylogenetic diversity of Bacteria and Archaea within a biodegraded, mesothermic petroleum reservoir in the Schrader Bluff Formation of Alaska was examined by two culture-independent methods based on fosmid and small-subunit rRNA gene PCR clone libraries. Despite the exclusion of certain groups by each method, there was overall no significant qualitative difference in the diversity of phylotypes recovered by the two methods. The resident Bacteria belonged to at least 14 phylum-level lineages, including the polyphyletic Firmicutes, which accounted for 36.2% of all small-subunit rRNA gene-containing (SSU(+)) fosmid clones identified. Members of uncultured divisions were also numerous and made up 35.2% of the SSU(+) fosmid clones. Clones from domain Archaea accounted for about half of all SSU(+) fosmids, suggesting that their cell numbers were comparable to those of the Bacteria in this microbial community. In contrast to the Bacteria, however, nearly all archaeal clones recovered by both methods were related to methanogens, especially acetoclastic methanogens, while the plurality of bacterial fosmid clones was affiliated with Synergistes-like acetogenic Firmicutes that possibly degrade longer-chain carboxylic acid components in the crude oil to acetate. These data suggest that acetate may be a key intermediary metabolite in this subsurface anaerobic food chain, which leads to methane production as the primary terminal electron sink.

Purification, cloning, sequencing and over-expression in Escherichia coli of a regioselective aliphatic nitrilase from Acidovorax facilis 72W

A regioselective aliphatic nitrilase from Acidovorax facilis 72W was purified and characterized, and the corresponding gene was cloned and sequenced. This nitrilase gene was over-expressed in Escherichia coli, generating a microorganism that efficiently and regioselectively catalyzes the conversion of aliphatic dinitriles to cyanocarboxylic acids. The high yields obtained, mild reaction conditions used, and robustness observed make this biocatalyst suitable for industrial applications.

A Pseudomonas putida capable of stereoselective hydrolysis of nitriles

Pseudomonas putida NRRL-18668 contains a nitrile hydratase capable of stereoselective hydrolysis of 2-(4-chlorophenyl)-3-methylbutyronitrile at more than 90 % enantiomeric excess (ee) to the (S)-amide. This soil isolate was recovered from enrichments using (R,S)-2-methylglutaronitrile as the sole nitrogen source. Enzyme expression is constitutive and does not show a high level of catabolite repression. The organism is capable of growth on a wide variety of aliphatic mono- and dinitrile compounds. The hydrolysis activity on propionitrile is approximately 10.3 μmole h−1 mg wet cells−1. The enzyme in cell-free preparations is inhibited by a number of heavy metals, phenylhydrazine, and cyanide. Substrate specificity is broad with highest rates shown on C4 and C5 aliphatic mononitriles. The strain appears somewhat unusual in its dependence on cobalt supplementation for maximum enzyme activity and the ability to hydrolyze some aromatic nitriles. This strain is also capable of a two-step hydrolysis of 2-(4-isobutylphenyl)-propionitrile and 2-(6-methoxy-2-napthyl)-propionitrile to the (S)-acids (ibuprofen and naproxen respectively) with stereoselectivity residing primarily in the aliphatic amidase. This appears to be the first description of a steroselective nitrile hydratase from a gram-negative organism.

A Gram-negative bacterium producing a heat-stable nitrilase highly active on aliphatic dinitriles

Abstract A Gram-negative bacterial strain, identified as Acidovorax facilis strain 72W, has been isolated from soil by enrichment using 2-ethylsuccinonitrile as the sole nitrogen source. This strain grows on a variety of aliphatic mono- and dinitriles. Experiments using various heating regimes indicate that nitrile hydratase, amidase and nitrilase activities are present. The nitrilase is efficient at hydrolyzing aliphatic dinitriles to cyanoacid intermediates. It has a strong bias for C3–C6 dinitriles over mononitriles of the same chain length. Whole, resting cell hydrolysis of 2-methylglutaronitrile results in 4-cyanopentanoic acid and 2-methylglutaric acid as the major products. Heating, at least 20 min at 50 °C, eliminates nitrile hydratase and amidase activities, resulting in greater than 97% selectivity to 4-cyanopentanoic acid. The nitrilase activity has good heat stability, showing a half-life of 22.7 h at 50 °C and a temperature optimum of at least 65 °C for activity. The strain has been deposited as ATCC 55746.

Chemoenzymatic production of 1,5-dimethyl-2-piperidone

Abstract A chemoenzymatic process for the preparation of 1,5-dimethyl-2-piperidone (1,5-DMPD) from 2-methylglutaronitrile (MGN) has been demonstrated. MGN was first hydrolyzed to 4-cyanopentanoic acid (4-CPA) ammonium salt using the nitrilase activity of immobilized Acidovorax facilis 72W cells. The hydrolysis reaction produced 4-CPA ammonium salt with greater than 98% regioselectivity at 100% conversion, and at concentrations of 170–210 g 4-CPA/l. Catalyst productivities of at least 1000 g 4-CPA/g dry cell weight (dcw) of immobilized cells were achieved by recycling the immobilized-cell catalyst in consecutive stirred-batch reactions. After recovery of the immobilized cell catalyst for reuse, the 4-CPA ammonium salt in the aqueous product mixture was directly converted to 1,5-DMPD by low-pressure catalytic hydrogenation in the presence of added methylamine.

Bacterial production in freshwater sediments: Cell specific versus system measures

Estimates of bacterial production based on total trichloroacetic acid (TCA)-precipitable [methyl-3H]thymidine incorporation and frequency of dividing cell (FDC) techniques were compared to sediment respiration rates in Lake George, New York. Bacterial growth rates based on thymidine incorporation ranged from 0.024 to 0.41 day−1, while rates based on FDC ranged from 1.78 to 2.48 day−1. Respiration rates ranged from 0.11 to 1.8μmol O2·hour−1·g dry weight sediment−1. Thymidine incorporation yielded production estimates which were in reasonable agreement with respiration rates. Production estimates based on FDC were 4- to 190-fold higher than those predicted from respiration rates.

Molecular tritium uptake in Southeastern U.S. soils

Molecular tritium deposition occurred in all soils tested. Deposition velocities for tritium ranged from 0.0025 to 0.11cm s−1 with a geometric mean of 0.028 cm s−1. Coring surveys from six different sites showed that deposition rates were highest in the upper 20 cm of soil, with little activity evident below this depth. Plant cover did not appear to have a strong direct influence on the profile of tritium deposition activity.

Microbial EOR -- Critical Aspects Learned From The Lab

DuPont and BP have been working together to develop Microbial EOR targeted at viscous oil in the Schrader Bluff formation on the North Slope of Alaska. The goal of this program was a 5% increase in the recovery factor. Mechanisms to be assessed in the original agreement included 1. Viscosity reduction of the oil by transformation or degradation of heavy components in the oil – thus improving the oil water mobility ratio. 2. Drastic reduction (to ~<0.01 dynes/cm) in the interfacial tension between water and the oil After extensive fundamental research we have learned many critical aspects of microbial EOR that made the application of these two mechanisms to the Schrader Bluff formation impractical. Instead, we have demonstrated two site appropriate mechanisms that achieved, in the lab, the targeted increase in the recovery factor. 1. Improved flow conformance and increased sweep efficiency by preferential plugging of high permeable zones thereby forcing water to produce oil from previously unswept parts of the reservoir. 2. Reduced oil / rock surface tension and a subsequent reduction in the oil “wetting” the rock. This results in changes in the relative permeability of the oil and the water and ultimately lower residual oil saturation. This paper describes the key laboratory tests used to evaluate these four mechanisms. The cornerstones of our work have been the detailed characterization of the waters, the oil, the formation matrix and the microbial community. In addition we describe our search for useful microbes isolated from a variety of environmental samples collected from the Milne Point Unit (MPU) of the Alaskan North Slope. These samples were taken over several years and included injection, production and power fluid waters. These samples were used to understand the temporal changes in the microbial populations and to provide inoculum for our enrichment cultures. Our ongoing research has provided many insights into the appropriate application of microbial EOR. The unique aspects of each production area, the nature of the oil, the water, the formation matrix, and the background microbial population and their complex interactions must all be assessed when considering the potential application of microbial EOR. The amount of work discribed below for assessing potential MEOR mechanisms is extensive. However, this process has been streamlined and we have been able to assess new target reservoirs for potential MEOR treatments in about 6 months.

Sedimentary sulfides in the Nearshore Georgia Bight

Abstract Sedimentary sulfide, iron, and organic matter were measured in neritic sediments from the Georgia Bight. The two measured depth integrated sulfur pools, FeS + HS − and FeS 2 , tended to decrease with increasing distance from shore out to 33 km. Total iron and organic matter were strongly correlated and both tended to decrease with increasing distance from shore. Sediment depth profiles of organic matter/reduced sulfur suggest relatively constant rates of sulfate reduction over the top 40 cm of sediment. Differences in within-station variance indicated regions of lower and higher spatial/temporal heterogeneity, that may be related to tidally driven circulation patterns. No seasonal cycles were evident in sedimentary sulfides.

Considerations for Field Implementation of Microbial Enhanced Oil Recovery

For the last 6 years DuPont with different partners has done extensive fundamental research into the application of Microbial Enhanced Oil Recovery technology (MEOR). We have demonstrated two mechanisms that have shown in the lab, more than a 10% increase in the recovery factor. 1. Increased sweep efficiency by plugging of high permeable zones thereby forcing water to produce oil from previously unswept parts of the reservoir. 2. Reduced oil / rock surface tension resulting in lower residual oil saturation. This paper describes the key laboratory tests and preliminary field data used to evaluate these two mechanisms. Our approach has been to inoculate the reservoir with a microbe that under the optimal nutrient conditions will expressed the needed function -- bioplugging or reduced oil saturation. The microbe and the nutrients are tailored to the conditions of each reservoir thus giving MEOR the greatest chance for success. This paper presents challenges that were raised as a result of extensive lab work that are relevant to the implementation of MEOR on a field level. Our ongoing research has provided many insights into the appropriate application of microbial EOR. The unique