In situ biosurfactant production by Bacillus strains injected into a limestone petroleum reservoir.

Biosurfactant-mediated oil recovery may be an economic approach for recovery of significant amounts of oil entrapped in reservoirs, but evidence that biosurfactants can be produced in situ at concentrations needed to mobilize oil is lacking. We tested whether two Bacillus strains that produce lipopeptide biosurfactants can metabolize and produce their biosurfactants in an oil reservoir. Five wells that produce from the same Viola limestone formation were used. Two wells received an inoculum (a mixture of Bacillus strain RS-1 and Bacillus subtilis subsp. spizizenii NRRL B-23049) and nutrients (glucose, sodium nitrate, and trace metals), two wells received just nutrients, and one well received only formation water. Results showed in situ metabolism and biosurfactant production. The average concentration of lipopeptide biosurfactant in the produced fluids of the inoculated wells was about 90 mg/liter. This concentration is approximately nine times the minimum concentration required to mobilize entrapped oil from sandstone cores. Carbon dioxide, acetate, lactate, ethanol, and 2,3-butanediol were detected in the produced fluids of the inoculated wells. Only CO(2) and ethanol were detected in the produced fluids of the nutrient-only-treated wells. Microbiological and molecular data showed that the microorganisms injected into the formation were retrieved in the produced fluids of the inoculated wells. We provide essential data for modeling microbial oil recovery processes in situ, including growth rates (0.06 +/- 0.01 h(-1)), carbon balances (107% +/- 34%), biosurfactant production rates (0.02 +/- 0.001 h(-1)), and biosurfactant yields (0.015 +/- 0.001 mol biosurfactant/mol glucose). The data demonstrate the technical feasibility of microbial processes for oil recovery.

In situ growth and activity and modes of penetration of Escherichia coli in unconsolidated porous materials.

Statistically reliable data on the in situ rates of growth, substrate consumption, and product formation are required to test the validity of the mathematical models developed for microbially enhanced oil recovery and in situ bioremediation processes. A simple, replicable porous-core system that could be aseptically divided into sections at various times was developed to follow the kinetics of microbial growth and metabolism in situ. This core system was used to study the kinetics of growth and the mode of penetration of strains of Escherichia coli through anaerobic, nutrient-saturated, fine Ottawa sand (permeability of 7.0 microns2 and porosity of 37%) under static conditions. The in situ rate of growth of a wild-type, motile, chemotactic strain, RW262, was two times slower inside cores than it was in liquid cultures. The mode of metabolism of galactose by strain RW262 was not altered inside cores, as acetate was the only product detected either inside the cores or in liquid cultures. Without applied advective force, strain RW262 grew exponentially and moved through cores at a rate of about 0.1 m/day. The cell population moved through cores in a band-like fashion, as the front of the moving cells consisted of high cell concentrations (greater than 10(5) cells per ml). Until the breakthrough of the cells occurred, galactose consumption and acetate production were observed only in the proximal sections of the core, showing that the cell propagation preceded the complete depletion of the substrate or the accumulation of large amounts of products.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of nitrate on biogenic sulfide production.

The addition of 59 mM nitrate inhibited biogenic sulfide production in dilute sewage sludge (10% [vol/vol]) amended with 20 mM sulfate and either acetate, glucose, or hydrogen as electron donors. Similar results were found when pond sediment or oil field brines served as the inoculum. Sulfide production was inhibited for periods of at least 6 months and was accompanied by the oxidation of resazurin from its colorless reduced state to its pink oxidized state. Lower amounts of nitrate (6 or 20 mM) and increased amounts of sewage sludge resulted in only transient inhibition of sulfide production. The addition of 156 mM sulfate to bottles with 59 mM nitrate and 10% (vol/vol) sewage sludge or pond sediment resulted in sulfide production. Nitrate, nitrite, and nitrous oxide were detected during periods where sulfide production was inhibited, whereas nitrate, nitrite, and nitrous oxide were below detectable levels at the time sulfide production began. The oxidation of resazurin was attributed to an increase in nitrous oxide which persisted in concentration of about 1.0 mM for up to 5 months. The numbers of sulfate-reducing organisms decreased from 10 CFU ml sludge to less than detectable levels after prolonged incubation of oxidized bottles. The addition of 10 mM glucose to oxidized bottles after 14.5 weeks of incubation resulted in rereduction of the resazurin and subsequent sulfide production. The prolonged inhibition of sulfide production was attributed to an increase in oxidation-reduction potential due to biogenic production of nitrous oxide, which appeared to have a cytotoxic effect on sulfate-reducing populations.

Isolation of halotolerant, thermotolerant, facultative polymer-producing bacteria and characterization of the exopolymer.

Over 200 bacterial strains were selected for anaerobic growth at 50 degrees C and extracellular polysaccharide production in a sucrose-mineral salts medium with NaNO(3) and up to 10% NaCl. The predominant cell type was an encapsulated gram-positive, motile, facultative sporeforming rod similar to Bacillus species. Strain SP018 grew and produced the polysaccharide on a variety of substrates at salinities up to 12% NaCl. Good polymer production only occurred anaerobically and was optimal between 4 and 10% NaCl. The ethanol-precipitated SP018 polymer was a charged heteropolysaccharide that contained glucose, mannose, arabinose, ribose, and low levels of allose and glucosamine. The SP018 polymer showed pseudoplastic behavior, was resistant to shearing, and had a higher viscosity at dilute concentrations and at elevated temperatures than xanthan gum. High-ionic-strength solutions reversibly decreased the viscosity of SP018 polymer solutions. The bacterium and the associated polymer have many properties that make them potentially useful for in situ microbially enhanced oil recovery processes.

Effect of Sterilization by Dry Heat or Autoclaving on Bacterial Penetration through Berea Sandstone.

A study was undertaken to determine why bacteria could penetrate lengths of consolidated sandstone (Berea) faster when the sandstone was sterilized by autoclaving than when dry heat (150 degrees C, 3 h) was used. Changes in permeability, porosity, and pore entrance size of the rock as a result of autoclaving were not sufficient to explain the differences in penetration times observed, but electron dispersion spectroscopy and electron microscopy of the rock revealed changes in mineral composition and clay morphology. Autoclaved cores contained more chloride than dry-heated cores, and the clays of autoclaved cores were aggregated and irregularly shaped. Therefore, the decreases in bacterial penetration rates caused by autoclave sterilization were probably the result of a change in surface charge of the pores of the rock and of a reduction in surface area of clays available for adhesion. The results implied that dry-heat sterilization was preferable to autoclaving when examining biotic and abiotic interactions in a native-state rock model.

Graduate medical education: financing at the crossroads.

Teaching hospitals are concerned about the new competitive environment because their costs are generally higher than those of nonteaching hospitals. Many of the higher costs of teaching hospitals derive from their educational programs, the nature of the patient diagnostic case mix; losses on charity care; and their role in the introduction of new and more effective methods for prevention, diagnosis, and treatment of illness. All of these functions are important to the missions of teaching hospitals, and all make teaching hospitals more expensive to operate than nonteaching hospitals. While a solution to the problem of financing graduate medical education will not ensure teaching hospital financial health, a solution to the financing of graduate medical education will provide a more equitable environment in which teaching hospitals can compete. The basic question to be answered in the price competitive environment is, "can the teaching hospital continue to attract patients at a competitive price and maintain financial support for its educational programs at current levels?" Teaching hospitals are a diverse group of highly complex institutions performing medical education and research services for the nation and providing both basic and tertiary patient care. The current emphasis on reexamining national policies in light of more limited public resources places teaching hospitals and their vital activities at significant risk if their special nature and role are not appreciated.

Anaerobic Production of a Biosurfactant by Bacillus licheniformis JF-2.

Bacillus licheniformis JF-2 anaerobically produced a biosurfactant when grown in a glucose-mineral salts medium containing yeast extract and NaNO(3). Surface tension of the medium was reduced from 70 to 74 mN/m to as low as 28 mN/m due to the production of an anionic biosurfactant.

Microbial Penetration through Nutrient-Saturated Berea Sandstone.

Penetration times and penetration rates for a motile Bacillus strain growing in nutrient-saturated Berea sandstone cores were determined. The rate of penetration was essentially independent of permeabilities above 100 mdarcys and rapidly declined for permeabilities below 100 mdarcys. It was found that these penetration rates could be grouped into two statistically distinct classes consisting of rates for permeabilities above 100 mdarcys and rates for those below 100 mdarcys. Instantaneous penetration rates were found to be zero order with respect to core length for cores with permeabilities above 100 mdarcys and first order with respect to core length for cores with permeabilities below 100 mdarcys. The maximum observed penetration rate was 0.47 cm . h, and the slowest was 0.06 cm . h; however, these rates may be underestimates of the true penetration rate, since the observed rates included the time required for growth in the flask as well as the core. The relationship of penetration time to the square of the length of the core suggested that cells penetrated high-permeability cores as a band and low-permeability cores in a diffuse fashion. The motile Enterobacter aerogenes strain penetrated Berea sandstone cores three to eight times faster than did the nonmotile Klebsiella pneumoniae strain when cores of comparable length and permeability were used. A penetration mechanism based entirely on motility predicted penetration times that were in agreement with the observed penetration times for motile strains. The fact that nonmotile strains penetrated the cores suggested that filamentous or unrestricted growth, or both, may also be important.