The genome sequence of Desulfatibacillum alkenivorans AK-01: a blueprint for anaerobic alkane oxidation.

Desulfatibacillum alkenivorans AK-01 serves as a model organism for anaerobic alkane biodegradation because of its distinctive biochemistry and metabolic versatility. The D. alkenivorans genome provides a blueprint for understanding the genetic systems involved in alkane metabolism including substrate activation, CoA ligation, carbon-skeleton rearrangement and decarboxylation. Genomic analysis suggested a route to regenerate the fumarate needed for alkane activation via methylmalonyl-CoA and predicted the capability for syntrophic alkane metabolism, which was experimentally verified. Pathways involved in the oxidation of alkanes, alcohols, organic acids and n-saturated fatty acids coupled to sulfate reduction and the ability to grow chemolithoautotrophically were predicted. A complement of genes for motility and oxygen detoxification suggests that D. alkenivorans may be physiologically adapted to a wide range of environmental conditions. The D. alkenivorans genome serves as a platform for further study of anaerobic, hydrocarbon-oxidizing microorganisms and their roles in bioremediation, energy recovery and global carbon cycling.

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.

Benzoate fermentation by the anaerobic bacterium Syntrophus aciditrophicus in the absence of hydrogen-using microorganisms.

The anaerobic bacterium Syntrophus aciditrophicus metabolized benzoate in pure culture in the absence of hydrogen-utilizing partners or terminal electron acceptors. The pure culture of S. aciditrophicus produced approximately 0.5 mol of cyclohexane carboxylate and 1.5 mol of acetate per mol of benzoate, while a coculture of S. aciditrophicus with the hydrogen-using methanogen Methanospirillum hungatei produced 3 mol of acetate and 0.75 mol of methane per mol of benzoate. The growth yield of the S. aciditrophicus pure culture was 6.9 g (dry weight) per mol of benzoate metabolized, whereas the growth yield of the S. aciditrophicus-M. hungatei coculture was 11.8 g (dry weight) per mol of benzoate. Cyclohexane carboxylate was metabolized by S. aciditrophicus only in a coculture with a hydrogen user and was not metabolized by S. aciditrophicus pure cultures. Cyclohex-1-ene carboxylate was incompletely degraded by S. aciditrophicus pure cultures until a free energy change (DeltaG') of -9.2 kJ/mol was reached (-4.7 kJ/mol for the hydrogen-producing reaction). Cyclohex-1-ene carboxylate, pimelate, and glutarate transiently accumulated at micromolar levels during growth of an S. aciditrophicus pure culture with benzoate. High hydrogen (10.1 kPa) and acetate (60 mM) levels inhibited benzoate metabolism by S. aciditrophicus pure cultures. These results suggest that benzoate fermentation by S. aciditrophicus in the absence of hydrogen users proceeds via a dismutation reaction in which the reducing equivalents produced during oxidation of one benzoate molecule to acetate and carbon dioxide are used to reduce another benzoate molecule to cyclohexane carboxylate, which is not metabolized further. Benzoate fermentation to acetate, CO(2), and cyclohexane carboxylate is thermodynamically favorable and can proceed at free energy values more positive than -20 kJ/mol, the postulated minimum free energy value for substrate metabolism.

Foot care practices, services and perceptions of risk among medicare beneficiaries with diabetes at high and low risk for future foot complications.

A cohort of Medicare beneficiaries with diabetes was identified from inpatient and outpatient claims data and their risk for foot complications was estimated based on claims reflecting services for recent foot problems. A telephone survey of a random sample from this cohort was conducted to assess their foot care practices, barriers, and perceptions of risk. Eight percent of respondents reported a history of foot ulcers and 7% a history of lower extremity amputation. Based on claims data, 30% of respondents were at high risk for future foot complications. Compared to those at low risk, those at high risk were more likely to report having an annual foot exam, using protective footwear, and perceiving themselves to be high risk for future foot complications. However, 50% of those with claims indicating a high risk perceived themselves to be at low risk for future foot complications. Overall, 20% of respondents seldom checked their feet daily for sores or irritations. Among this group, 60% felt that it was unimportant and 9% reported they were limited by poor vision or physical problems. Our findings suggest that strategies are needed to improve the delivery of preventive foot care services to older persons with diabetes. Additionally, emphasis is needed to help individuals understand their risk and seek and perform appropriate preventive foot care.

Signature metabolites attesting to the in situ attenuation of alkylbenzenes in anaerobic environments.

Accurate assessment of the fate of hydrocarbons spilt in aquifers is essential for gauging associated health and ecological risks. Regulatory pressure to actively remediate such contaminated ecosystems can be substantially diminished if solid evidence for in situ microbial destruction of pollutants is obtained. In laboratory incubations, sediment-associated microorganisms from a gas condensate-contaminated aquifer anaerobically biodegraded toluene, ethylbenzene, xylene, and toluic acid isomers with stoichiometric amounts of sulfate consumed or methane produced. The activation of the alkylated aromatic contaminants involved conversion to their corresponding benzylsuccinic acid derivatives, a reaction known to occur for toluene and m-xylene decay, but one previously unrecognized for ethylbenzene, o- and p-xylene, and m-toluate metabolism. Benzylsuccinates were further biodegraded to toluates, phthalates, and benzoate. In laboratory incubations, these metabolites were transiently produced. Several of the metabolites were also detected in groundwater samples from an aquifer where alkylbenzene concentrations decreased over time, suggesting that anaerobic microbial metabolism of these contaminants also occurs in situ. Our studies confirm the utility of the aforementioned compounds as signature metabolites attesting to the natural attenuation of aromatic hydrocarbons in anaerobic environments.

Relationship of hydrogen bioavailability to chromate reduction in aquifer sediments.

Biological Cr(VI) reduction was studied in anaerobic sediments from an aquifer in Norman, Okla. Microcosms containing sediment and mineral medium were amended with various electron donors to determine those most important for biological Cr(VI) reduction. Cr(VI) (about 340 microM) was reduced with endogenous substrates (no donor), or acetate was added. The addition of formate, hydrogen, and glucose stimulated Cr(VI) reduction compared with reduction in unamended controls. From these sediments, an anaerobic Cr(VI)-utilizing enrichment was obtained that was dependent upon hydrogen for both growth and Cr(VI) reduction. No methane was produced by the enrichment, which reduced about 750 microM Cr(VI) in less than six days. The dissolved hydrogen concentration was used as an indicator of the terminal electron accepting process occurring in the sediments. Microcosms with sediments, groundwater, and chromate metabolized hydrogen to a concentration below the detection limits of the mercury vapor gas chromatograph. In microcosms without chromate, the hydrogen concentration was about 8 nM, a concentration comparable to that under methanogenic conditions. When these microcosms were amended with 500 microM Cr(VI), the dissolved hydrogen concentration quickly fell below the detection limits. These results showed that the hydrogen concentration under chromate-reducing conditions became very low, as low as that reported under nitrate- and manganese-reducing conditions, a result consistent with the free energy changes for these reactions. The utilization of formate, lactate, hydrogen, and glucose as electron donors for Cr(VI) reduction indicates that increasing the availability of hydrogen results in a greater capacity for Cr(VI) reduction. This conclusion is supported by the existence of an enrichment dependent upon hydrogen for growth and Cr(VI) reduction.

Metabolism of benzoate, cyclohex-1-ene carboxylate, and cyclohexane carboxylate by "Syntrophus aciditrophicus" strain SB in syntrophic association with H(2)-using microorganisms.

The metabolism of benzoate, cyclohex-1-ene carboxylate, and cyclohexane carboxylate by "Syntrophus aciditrophicus" in cocultures with hydrogen-using microorganisms was studied. Cyclohexane carboxylate, cyclohex-1-ene carboxylate, pimelate, and glutarate (or their coenzyme A [CoA] derivatives) transiently accumulated during growth with benzoate. Identification was based on comparison of retention times and mass spectra of trimethylsilyl derivatives to the retention times and mass spectra of authentic chemical standards. (13)C nuclear magnetic resonance spectroscopy confirmed that cyclohexane carboxylate and cyclohex-1-ene carboxylate were produced from [ring-(13)C(6)]benzoate. None of the metabolites mentioned above was detected in non-substrate-amended or heat-killed controls. Cyclohexane carboxylic acid accumulated to a concentration of 260 microM, accounting for about 18% of the initial benzoate added. This compound was not detected in culture extracts of Rhodopseudomonas palustris grown phototrophically or Thauera aromatica grown under nitrate-reducing conditions. Cocultures of "S. aciditrophicus" and Methanospirillum hungatei readily metabolized cyclohexane carboxylate and cyclohex-1-ene carboxylate at a rate slightly faster than the rate of benzoate metabolism. In addition to cyclohexane carboxylate, pimelate, and glutarate, 2-hydroxycyclohexane carboxylate was detected in trace amounts in cocultures grown with cyclohex-1-ene carboxylate. Cyclohex-1-ene carboxylate, pimelate, and glutarate were detected in cocultures grown with cyclohexane carboxylate at levels similar to those found in benzoate-grown cocultures. Cell extracts of "S. aciditrophicus" grown in a coculture with Desulfovibrio sp. strain G11 with benzoate or in a pure culture with crotonate contained the following enzyme activities: an ATP-dependent benzoyl-CoA ligase, cyclohex-1-ene carboxyl-CoA hydratase, and 2-hydroxycyclohexane carboxyl-CoA dehydrogenase, as well as pimelyl-CoA dehydrogenase, glutaryl-CoA dehydrogenase, and the enzymes required for conversion of crotonyl-CoA to acetate. 2-Ketocyclohexane carboxyl-CoA hydrolase activity was detected in cell extracts of "S. aciditrophicus"-Desulfovibrio sp. strain G11 benzoate-grown cocultures but not in crotonate-grown pure cultures of "S. aciditrophicus". These results are consistent with the hypothesis that ring reduction during syntrophic benzoate metabolism involves a four- or six-electron reduction step and that once cyclohex-1-ene carboxyl-CoA is made, it is metabolized in a manner similar to that in R. palustris.

Is interspecies hydrogen transfer needed for toluene degradation under sulfate-reducing conditions?

Sediments from a hydrocarbon-contaminated aquifer, where periodic shifts between sulfate reduction and methanogenesis occurred, were examined to determine whether the degradation of toluene under sulfate-reducing conditions depended on interspecies hydrogen transfer. Toluene degradation under sulfate-reducing conditions was inhibited by the addition of 5 mM sodium molybdate, but the activity was not restored upon the addition of an actively growing, hydrogen-using methanogen. Toluene degradation was not inhibited in microcosms where hydrogen levels were maintained at a level theoretically sufficient to inhibit toluene degradation if the process proceeded via interspecies hydrogen transfer. Finally, the addition of carbon monoxide, a potent inhibitor of hydrogenase activity, inhibited hydrogen but not toluene consumption in sulfate-reducing microcosms. These results suggest that toluene is degraded directly by sulfate-reducing bacteria without the involvement of interspecies hydrogen transfer. The sequence of experiments used to reach this conclusion could be applied to determine the role of interspecies hydrogen transfer in the degradation of a variety of compounds in different environments or under different terminal electron-accepting conditions.

Direct mail intervention to increase retinal examination rates in Medicare beneficiaries with diabetes.

The objective of this study was to identify the baseline frequency of eye examinations for Medicare beneficiaries with diabetes in Montana and to determine whether a direct mail reminder increased eye examinations. Using Medicare Part A and Medicare Part B claims data, a cohort of Medicare beneficiaries with diabetes was defined. Eye examination claims were identified using billing codes specific for retinal examinations, as well as visits to ophthalmologists and optometrists during which retinal exams were likely to have been performed. A random sample of the identified beneficiaries with diabetes received a letter encouraging regular annual retinal examinations. In the first 3-month period after the mailing, the billed eye examination rate for those to whom letters were sent was 2.2 percentage points greater than the rate for those to whom letters were not sent (19.4% vs 17.2%; relative risk, 1.13; 95% confidence interval, 1.01-1.26). However, 6 months after the letters were sent, there was no longer a significant difference in the rates for these 2 groups (32.9% vs 32.4%; relative risk, 1.02; 95% confidence interval, 0.94-1.10). In this study, direct mail outreach initially influenced the proportion of Medicare beneficiaries receiving an eye examination, but this pattern was not sustained over the 6-month follow-up period.

Improving the pneumococcal immunization rate for patients with diabetes in a managed care population: a simple intervention with a rapid effect.

BACKGROUND: Pneumococcal disease kills more people in the United States than any other vaccine-preventable bacterial disease, and a national health objective for the year 2000 is that at least 60% of eligible persons be immunized with pneumococcal vaccine. METHODS: An electronic care monitoring system was used to track immunization of patients with diabetes in a managed care plan who were receiving their care through a staff-model primary care clinic in Guam. In November 1998 a letter was sent to all patients not known to be immunized. The letter invited these patients to attend immunization clinics and waived usual copayment. Standing orders were also created for the clinic nurses to administer pneumococcal vaccines. In addition, a diabetes care status report was placed on each patient's medical record. RESULTS: The immunization rate for the 1,278 actively enrolled patients with diagnosed diabetes increased from 42% in October 1998 to 62% in January 1999. Compared to November 1995, 1996, and 1997, the number of pneumococcal immunizations increased more than 15-fold in November 1998. DISCUSSION: The combined use of patient outreach letters, special immunization clinics, standing orders, and practitioner reminders on medical records resulted in a rapid, marked increase in the pneumococcal immunization rate for patients with diabetes. The electronic care monitoring system is being used to target get interventions for improvement opportunities for an array of diabetes care measures, including regular foot care and eye exams.

Thiosulfate disproportionation by Desulfotomaculum thermobenzoicum.

Desulfotomaculum thermobenzoicum, but not Desulfotomaculum nigrificans, Desulfotomaculum ruminis, or Desulfosporosinus orientis, grew by disproportionation of thiosulfate, forming stoichiometric amounts of sulfate and sulfide; sulfite was not disproportionated. The addition of acetate enhanced growth and thiosulfate disproportionation by D. thermobenzoicum compared to those observed with thiosulfate alone.

Haloanaerobium kushneri sp. nov., an obligately halophilic, anaerobic bacterium from an oil brine.

Three strains, designated VS-751T, VS-511 and VS-732, of a strictly anaerobic, moderately halophilic, Gram-negative, rod-shaped bacterium were isolated from a highly saline (15-20%) brine from an oil reservoir in central Oklahoma, USA. The optimal concentration of NaCl for growth of these three strains was 2 M (12%), and the strains also grew in the presence of an additional 1 M MgCl2. The strains were mesophilic and grew at a pH range of 6-8. Carbohydrates used by all three strains included glucose, fructose, arabinose, galactose, maltose, mannose, cellobiose, sucrose and inulin. Glucose fermentation products included ethanol, acetate, H2 and CO2, with formate produced by two of the three strains. Differences were noted among strains in the optimal temperature and pH for growth, the maximum and minimum NaCl concentration that supported growth, substrate utilization and cellular fatty acid composition. Despite the phenotypic differences among the three strains, analysis of the 16S rRNA gene sequences and DNA-DNA hybridizations showed that these three strains were members of the same genospecies which belonged to the genus Haloanaerobium. The phenotypic and genotypic characteristics of strains VS-751T, VS-511 and VS-732 are different from those of previously described species of Haloanaerobium. It is proposed that strain VS-751T (ATCC 700103T) be established as the type strain of a new species, Haloanaerobium kushneri.

Syntrophus aciditrophicus sp. nov., a new anaerobic bacterium that degrades fatty acids and benzoate in syntrophic association with hydrogen-using microorganisms.

Strain SBT is a new, strictly anaerobic, gram-negative, nonmotile, non-sporeforming, rod-shaped bacterium that degrades benzoate and certain fatty acids in syntrophic association with hydrogen/formate-using microorganisms. Strain SBT produced approximately 3 mol of acetate and 0.6 mol of methane per mol of benzoate in coculture with Methanospirillum hungatei strain JF1. Saturated fatty acids, some unsaturated fatty acids, and methyl esters of butyrate and hexanoate also supported growth of strain SBT in coculture with Desulfovibrio strain G11. Strain SBT grew in pure culture with crotonate, producing acetate, butyrate, caproate, and hydrogen. The molar growth yield was 17 +/- 1 g cell dry mass per mol of crotonate. Strain SBT did not grow with fumarate, iron(III), polysulfide, or oxyanions of sulfur or nitrogen as electron acceptors with benzoate as the electron donor. The DNA base composition of strain SBT was 43.1 mol% G+C. Analysis of the 16 S rRNA gene sequence placed strain SBT in the delta-subdivision of the Proteobacteria, with sulfate-reducing bacteria. Strain SBT was most closely related to members of the genus Syntrophus. The clear phenotypic and genotypic differences between strain SBT and the two described species in the genus Syntrophus justify the formation of a new species, Syntrophus aciditrophicus.

Geovibrio ferrireducens, a phylogenetically distinct dissimilatory Fe(III)-reducing bacterium.

A new, phylogenetically distinct, dissimilatory, Fe(III)-reducing bacterium was isolated from surface sediment of a hydrocarbon-contaminated ditch. The isolate, designated strain PAL-1, was an obligately anaerobic, non-fermentative, motile, gram-negative vibrio. PAL-1 grew in a defined medium with acetate as electron donor and ferric pyrophosphate, ferric oxyhydroxide, ferric citrate, Co(III)-EDTA, or elemental sulfur as sole electron acceptor. PAL-1 also used proline, hydrogen, lactate, propionate, succinate, fumarate, pyruvate, or yeast extract as electron donors for Fe(III) reduction. It is the first bacterium known to couple the oxidation of an amino acid to Fe(III) reduction. PAl-1 did not reduce oxygen, Mn(IV), U(VI), Cr(VI), nitrate, sulfate, sulfite, or thiosulfate with acetate as the electron donor. Cell suspensions of PAL-1 exhibited dithionite-reduced minus air-oxidized difference spectra that were characteristic of c-type cytochromes. Analysis of the 16S rRNA gene sequence of PAL-1 showed that the strain is not related to any of the described metal-reducing bacteria in the Proteobacteria and, together with Flexistipes sinusarabici, forms a separate line of descent within the Bacteria. Phenotypically and phylogenetically, strain PAl-1 differs from all other described bacteria, and represents the type strain of a new genus and species, Geovibrio ferrireducens.

The importance of hydrogen in landfill fermentations.

Forty-two samples taken from two landfills were monitored for CH(inf4) production and apparent steady-state H(inf2) concentration. The rates of methanogenesis in these samples ranged from below the detection limit to 1,900 (mu)mol kg (dry weight)(sup-1) day(sup-1), and the median steady-state hydrogen concentration was 1.4 (mu)M in one landfill and 5.2 (mu)M in the other. To further investigate the relationship between hydrogen concentration and methanogenesis, a subset of seven landfill samples was selected on basis of their rates of CH(inf4) production, H(inf2) concentrations, sample pHs, and moisture contents. Samples with H(inf2) concentrations of <20 nM had relatively small amounts of volatile fatty acids (VFAs) (undetectable to 18.6 mmol of VFA kg [dry weight](sup-1)), while samples with H(inf2) concentrations of >100 nM had relatively high VFA levels (133 to 389 mmol of VFA kg [dry weight](sup-1)). Samples with high H(inf2) and VFA contents had relatively low pH values (<=6.3). However, methanogenic and syntrophic bacteria were present in all samples, so the lack of methanogenesis in some samples was not due to a lack of suitable inocula. The low rates of methanogenesis in these samples were probably due to inhibitory effects of low pH and VFA accumulation, resulting from a thermodynamic uncoupling of fatty acid oxidation. As in other anaerobic ecosystems, H(inf2) is a critical intermediate that may be used to monitor the status of landfill fermentations.

Interspecies acetate transfer influences the extent of anaerobic benzoate degradation by syntrophic consortia.

Benzoate degradation by an anaerobic, syntrophic bacterium, strain SB, in coculture with Desulfovibrio sp. strain G-11 reached a threshold value which depended on the amount of acetate added and ranged from about 2.5 to 29.9 (mu)M. Increasing acetate concentrations also uncompetitively inhibited benzoate degradation. The apparent V(infmax) and apparent K(infm) for benzoate degradation decreased with increasing acetate concentration, but the benzoate degradation capacities (V(infmax)/K(infm)) of cell suspensions remained comparable. The addition of an acetate-using bacterium to cocultures after the threshold was reached resulted in the degradation of benzoate to below the detection limit. Mathematical simulations showed that the benzoate threshold was not predicted by the inhibitory effect of acetate on benzoate degradation kinetics. With nitrate instead of sulfate as the terminal electron acceptor, no benzoate threshold was observed in the presence of 20 mM acetate even though the kinetics of benzoate degradation were slower with nitrate rather than sulfate as the electron acceptor. When strain SB was grown with Desulfovibrio sp. strain DG2 that had a fourfold-lower V(infmax) for hydrogen use than strain G-11, the V(infmax) for benzoate degradation was 37-fold lower than that of strain SB-G-11 cocultures. The Gibb's free energy for benzoate degradation was less negative in cell suspensions with a threshold than in suspensions without a threshold. These studies showed that the threshold was not a function of the inhibition of benzoate degradation by acetate or the toxicity of the undissociated form of acetate. Rather, a critical or minimal Gibb's free energy may exist where thermodynamic constraints preclude further benzoate degradation.

Evidence for anaerobic syntrophic benzoate degradation threshold and isolation of the syntrophic benzoate degrader.

An anaerobic, motile, gram-negative, rod-shaped, syntrophic, benzoate-degrading bacterium, strain SB, was isolated in pure culture with crotonate as the energy source. Benzoate was degraded only in association with an H2-using bacterium. The kinetics of benzoate degradation by cell suspensions of strain SB in coculture with Desulfovibrio strain G-11 was studied by using progress curve analysis. The coculture degraded benzoate to a threshold concentration of 214 nM to 6.5 microM, with no further benzoate degradation observed even after extended incubation times. The value of the threshold depended on the amount of benzoate added and, consequently, the amount of acetate produced. The addition of sodium acetate, but not that of sodium chloride, affected the threshold value; higher acetate concentrations resulted in higher threshold values for benzoate. When a cell suspension that had reached a threshold benzoate concentration was reamended with benzoate, benzoate was used without a lag. The hydrogen partial pressure was very low and formate was not detected in cell suspensions that had degraded benzoate to a threshold value. The Gibbs free energy change calculations showed that the degradation of benzoate was favorable when the threshold was reached. These studies showed that the threshold for benzoate degradation was not caused by nutritional limitations, the loss of metabolic activity, or inhibition by hydrogen or formate. The data are consistent with a thermodynamic explanation for the existence of a threshold, but a kinetic explanation based on acetate inhibition may also account for the existence of a threshold.

Geobacter sulfurreducens sp. nov., a hydrogen- and acetate-oxidizing dissimilatory metal-reducing microorganism.

A dissimilatory metal- and sulfur-reducing microorganism was isolated from surface sediments of a hydrocarbon-contaminated ditch in Norman, Okla. The isolate, which was designated strain PCA, was an obligately anaerobic, nonfermentative nonmotile, gram-negative rod. PCA grew in a defined medium with acetate as an electron donor and ferric PPi, ferric oxyhydroxide, ferric citrate, elemental sulfur, Co(III)-EDTA, fumarate, or malate as the sole electron acceptor. PCA also coupled the oxidation of hydrogen to the reduction of Fe(III) but did not reduce Fe(III) with sulfur, glucose, lactate, fumarate, propionate, butyrate, isobutyrate, isovalerate, succinate, yeast extract, phenol, benzoate, ethanol, propanol, or butanol as an electron donor. PCA did not reduce oxygen, Mn(IV), U(VI), nitrate, sulfate, sulfite, or thiosulfate with acetate as the electron donor. Cell suspensions of PCA exhibited dithionite-reduced minus air-oxidized difference spectra which were characteristic of c-type cytochromes. Phylogenetic analysis of the 16S rRNA sequence placed PCA in the delta subgroup of the proteobacteria. Its closest known relative is Geobacter metallireducens. The ability to utilize either hydrogen or acetate as the sole electron donor for Fe(III) reduction makes strain PCA a unique addition to the relatively small group of respiratory metal-reducing microorganisms available in pure culture. A new species name, Geobacter sulfurreducens, is proposed.

Haloanaerobium salsugo sp. nov., a moderately halophilic, anaerobic bacterium from a subterranean brine.

A strictly anaerobic, moderately halophilic, gram-negative bacterium was isolated from a highly saline oil field brine. The bacterium was a non-spore-forming, nonmotile rod, appearing singly, in pairs, or occasionally as long chains, and measured 0.3 to 0.4 by 2.6 to 4 microns. The bacterium had a specific requirement for NaCl and grew at NaCl concentrations of between 6 and 24%, with optimal growth at 9% NaCl. The isolate grew at temperatures of between 22 and 51 degrees C and pH values of between 5.6 and 8.0. The doubling time in a complex medium containing 10% NaCl was 9 h. Growth was inhibited by chloramphenicol, tetracycline, and penicillin but not by cycloheximide or azide. Fermentable substrates were predominantly carbohydrates. The end products of glucose fermentation were acetate, ethanol, CO2, and H2. The major components of the cellular fatty acids were C14:0, C16:0, C16:1, and C17:0 cyc acids. The DNA base composition of the isolate was 34 mol% G+C. Oligonucleotide catalog and sequence analyses of the 16S rRNA showed that strain VS-752T was most closely related to Haloanaerobium praevalens GSLT (ATCC 33744), the sole member of the genus Haloanaerobium. We propose that strain VS-752 (ATCC 51327) be established as the type strain of a new species, Haloanaerobium salsugo, in the genus Haloanaerobium.

Effect of grain size on bacterial penetration, reproduction, and metabolic activity in porous glass bead chambers.

We determined the effects of grain size and nutritional conditions on the penetration rate and metabolic activity of Escherichia coli strains in anaerobic, nutrient-saturated chambers packed with different sizes of glass beads (diameters, 116 to 767 mum) under static conditions. The chambers had nearly equal porosities (38%) but different calculated pore sizes (range, 10 to 65 mum). Motile strains always penetrated faster than nonmotile strains, and nutrient conditions that resulted in faster growth rates (fermentative conditions versus nitrate-respiring conditions) resulted in faster penetration rates for both motile and nonmotile strains for all of the bead sizes tested. The penetration rate of nonmotile strains increased linearly when bead size was increased, while the penetration rate of motile strains became independent of the bead size when beads having diameters of 398 mum or greater were used. The rate of H(2) production and the final amount of H(2) produced decreased when bead size was decreased. However, the final protein concentrations were similar in chambers packed with 116-, 192-, and 281-mum beads and were only slightly higher in chambers packed with 398- and 767-mum beads. Our data indicated that conditions that favored faster growth rates also resulted in faster penetration times and that the lower penetration rates observed in chambers packed with small beads were due to restriction of bacterial activity in the small pores. The large increases in the final amount of hydrogen produced without corresponding increases in the final amount of protein made indicated that metabolism became uncoupled from cell mass biosynthesis as bead size increased, suggesting that pore size influenced the efficiency of substrate utilization.