Sulfide formation by oil field sulfate-reducing bacteria (SRB) can be diminished by the injection of nitrate, stimulating the growth of nitrate-reducing bacteria (NRB). We monitored the field-wide injection of nitrate into a low temperature (approximately 30 degrees C) oil reservoir in western Canada by determining aqueous concentrations of sulfide, sulfate, nitrate, and nitrite, as well as the activities of NRB in water samples from 3 water plants, 2 injection wells, and 15 production wells over 2 years. The injection water had a low sulfate concentration (approximately 1 mM). Nitrate (2.4 mM, 150 ppm) was added at the water plants. Its subsequent distribution to the injection wells gave losses of 5-15% in the pipeline system, indicating that most was injected. Continuous nitrate injection lowered the total aqueous sulfide output of the production wells by 70% in the first five weeks, followed by recovery. Batchwise treatment of a limited section of the reservoir with high nitrate eliminated sulfide from one production well with nitrate breakthrough. Subsequent, field-wide treatment with week-long pulses of 14 mM nitrate gave breakthrough at an additional production well. However, this trend was reversed when injection with a constant dose of 2.4 mM (150 ppm) was resumed. The results are explained by assuming growth of SRB near the injection wellbore due to sulfate limitation. Injection of a constant nitrate dose inhibits these SRB initially. However, because of the constant, low temperature of the reservoir, SRB eventually grow back in a zone further removed from the injection wellbore. The resulting zonation (NRB closest to and SRB further away from the injection wellbore) can be broken by batch-wise increases in the concentration of injected nitrate, allowing it to re-enter the SRB-dominated zone.

Acetate, propionate, and butyrate, collectively referred to as volatile fatty acids (VFA), are considered among the most important electron donors for sulfate-reducing bacteria (SRB) and heterotrophic nitrate-reducing bacteria (hNRB) in oil fields. Samples obtained from a field in the Neuquén Basin, western Argentina, had significant activity of mesophilic SRB, hNRB, and nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB). In microcosms, containing VFA (3 mM each) and excess sulfate, SRB first used propionate and butyrate for the production of acetate, which reached concentrations of up to 12 mM prior to being used as an electron donor for sulfate reduction. In contrast, hNRB used all three organic acids with similar kinetics, while reducing nitrate to nitrite and nitrogen. Transient inhibition of VFA-utilizing SRB was observed with 0.5 mM nitrite and permanent inhibition with concentrations of 1 mM or more. The addition of nitrate to medium flowing into an upflow, packed-bed bioreactor with an established VFA-oxidizing SRB consortium led to a spike of nitrite up to 3 mM. The nitrite-mediated inhibition of SRB led, in turn, to the transient accumulation of up to 13 mM of acetate. The complete utilization of nitrate and the incomplete utilization of VFA, especially propionate, and sulfate indicated that SRB remained partially inhibited. Hence, in addition to lower sulfide concentrations, an increase in the concentration of acetate in the presence of sulfate in waters produced from an oil field subjected to nitrate injection may indicate whether the treatment is successful. The microbial community composition in the bioreactor, as determined by culturing and culture-independent techniques, indicated shifts with an increasing fraction of nitrate. With VFA and sulfate, the SRB genera Desulfobotulus, Desulfotignum, and Desulfobacter as well as the sulfur-reducing Desulfuromonas and the NR-SOB Arcobacter were detected. With VFA and nitrate, Pseudomonas spp. were present. hNRB/NR-SOB from the genus Sulfurospirillum were found under all conditions.

Bacterial degradation of biphenyl and polychlorinated biphenyls proceeds by a well-studied pathway which produces benzoate and 2-hydroxypent-2,4-dienoate (or, in the case of polychlorinated biphenyls, the chlorinated derivatives of these compounds). Pseudomonas cepacia P166 utilizes 4-chlorobiphenyl for growth and produces 4-chlorobenzoate as a central intermediate. In this study we found that strain P166 further transforms 4-chlorobenzoate to 4-chlorocatechol, which is mineralized by a meta cleavage pathway. Key metabolites which we identified include the meta cleavage product (5-chloro-2-hydroxymuconic semialdehyde), 5-chloro-2hydroxymuconate, 5-chloro-2-oxopent-4-enoate, 5-chloro-4-hydroxy-2-oxopentanoate, and chloroacetate. Chloroacetate accumulated transiently, and slow but stoichiometric dehalogenation was observed.

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There is great interest in engineering human growth factors as potential therapeutic agonists and antagonists. We approached this goal with a synthetic DNA recombination method. We aligned a pool of "top-strand" oligonucleotides incorporating polymorphisms from mammalian genes encoding epidermal growth factor (EGF) using multiple polymorphic "scaffold" oligonucleotides. Top strands were then linked by gap filling and ligation. This approach avoided heteroduplex annealing in the linkage of highly degenerate oligonucleotides and thus achieved completely random recombination. Cloned genes from a human-mouse chimeric library captured every possible permutation of the parental polymorphisms, creating an apparently complete recombined gene-family library, which has not been previously described. This library yielded a chimeric protein whose agonist activity was enhanced 123-fold. A second library from five mammalian EGF homologs possessed the highest reported recombination density (1 crossover per 12.4 bp). The five-homolog library yielded the strongest-binding hEGF variant yet reported. In addition, it contained strongly binding EGF variants with antagonist properties. Our less biased approach to DNA shuffling should be useful for the engineering of a wide variety of proteins.