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.

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.

Influence of lasalocid and supplement level on productivity of gestating ewes grazing winter range.

Two winter feeding trials (1985-86; 1986-87) were conducted to evaluate the productivity of gestating ewes fed lasalocid (L) and two supplement levels while grazing Montana winter range. Five hundred range ewes were randomized within age and breed each year and allotted to .15 or .23 kg hd-1.d-1 of a 20% CP supplement and either no L or L at 70 mg hd-1.d-1. Feed treatments began on 18 Dec. approximately 100 d before the first expected lambing date and continued for 84 d. Ewes fed .23 kg of supplement per day gained more (P less than .01) total weight (4.9 vs 4.0 kg) during the 84-d experiment and had higher (P less than .05) grease fleece weights (4.2 vs 4.0 kg) than those fed .15 kg of supplement. Lasalocid had no effect (P greater than .05) on ewe weight change or grease fleece weights. Supplement level had no effect (P greater than .05) on reproduction, lamb mortality and lamb performance. Ewes fed L had a greater (P less than .05) percentage of lambs born per ewe than those not fed L (120.7 vs 112.1%); lamb performance was similar (P greater than .05) between treatments. However, because a greater percentage of lambs were born per ewe starting the experiment, ewes fed L produced more (P less than .05) kilograms of weaned lamb than those not fed L (25.9 vs 23.4 kg). In conclusion, ewes fed L and grazing winter range weaned more kg of lamb than did controls because of an improved lambing percentage.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of body condition and lasalocid during late gestation on blood metabolites, lamb birth weight and colostrum composition and production in Finn-cross ewes.

The objective of this experiment was to examine the effects of body condition (BC) and of lasalocid (L) the last 4 wk of gestation on blood metabolite profiles, lamb birth weight and colostrum composition and production. Twenty-eight 3-yr-old Finn-Targhee ewes (73 kg) were assigned randomly within BC grouping (2.5 or 3.5) and individually fed a diet of 90% alfalfa pellets and 10% of a supplement containing either no L (C = control) or L. Gestation and 24-h postlambing weights were higher (P less than .05) for 3.5 BC versus 2.5 BC ewes. Lasalocid had no effect (P greater than .05) on ewe weight. Average number of lambs born within treatment groups were similar (P greater than .05). Total kilograms of lamb born were greater (P less than .10) for 3.5 BC ewes. Body condition 3.5 ewes had greater concentrations of total protein (P less than .10) and albumin (P less than .05) the last 4 wk of gestation than those in the 2.5 BC group. Feeding L decreased (P less than .05) blood urea-N in comparison with C ewes. Colostrum composition and production were not influenced (P greater than .05) by BC, L or number of lambs born. Serum 3-hydroxybutyrate seemed to be a good indicator of energy metabolism; albumin and blood urea-N concentrations reflected dietary protein intake. Lasalocid had a minimal effect on nutrient metabolism and productivity of ewes fed in excess of the NRC (1985) protein and energy requirements.

The effects of ruminal escape protein or fat on nutritional status of pregnant winter-grazing beef cows.

This study was designed to determine the influence of soybean meal supplementation, with or without additional ruminal escape protein or fat, on the nutritional status of pregnant winter-grazing beef cows. During two winters (Trials 1 and 2), approximately 60 prepartum beef cows grazed native foothills range each year. Cows were allotted randomly to five groups and supplemented (g/d) with either none (control); 570 soybean meal (SOY); 450 soybean meal plus 230 blood meal (SOY + BM); 140 soybean meal, 16 urea plus 450 corn gluten meal (SOY + CGM); or 570 soybean meal plus 210 animal fat (SOY + FAT). These supplements were designed to supply similar quantities of ruminal degraded protein while varying in escape protein quantity and source (SOY + BM and SOY + CGM). Condition scores and body weights were determined at trial initiation (mid-December) and conclusion (early March). Eight blood samples obtained over 4 d during three periods (9, 4 and 1 wk prior to parturition) were analyzed for concentrations of glucose, urea nitrogen (N), total bilirubin, creatinine, albumin, total protein and cholesterol. Cows in the control treatment experienced the greatest BW loss in both trials. In Trial 2, escape protein tended to decrease (P less than .06) BW loss compared to SOY, though loss tended to be greater (P less than .08) with SOY + CGM than with SOY + BM. Escape protein can enhance nutritional status when supplemented with .6 kg/d of soybean meal.

Influence of biostimulation by mature bulls on occurrence of puberty in beef heifers.

The objective of this study was to determine if biostimulation of prepuberal beef heifers by mature bulls would alter proportions of heifers exhibiting puberty, or age or weight at puberty. Angus (A), A X Hereford (H) and Tarentaise X HA heifers (n = 103) were stratified by age and weight within breed-type and location of birth and allotted randomly to the following treatments: 1) heifers exposed to mature bulls (T1; n = 52) or 2) heifers isolated from bulls (T2; n = 51). At the start of the experiment, heifers in T1 and T2 were 287 +/- 2 and 286 +/- 2 d of age, respectively. Male-to-female ratio for T1 was 1:26. Heifers in T1 and T2 were maintained in drylots separated by .5 km. Heifers were observed for estrus twice daily for 152 d. Puberty was characterized by the following criteria: 1) behavioral estrus, 2) presence of a palpable corpus luteum (d 9; estrus = d 0) and 3) a rise in serum progesterone above 1 ng/ml (d 9). Proportions of heifers reaching puberty by 11, 12, 13, 14 and 15 mo of age did not differ (P greater than .10) between treatments. Percentages of heifers reaching puberty by the end of the experiment were 84 and 89% for T1 and T2, respectively. Age and weight at puberty did not differ (P greater than .10) between treatments and averaged 370 +/- 7 d and 293 +/- 4 kg, respectively. Results from this experiment indicated that presence of mature bulls did not alter proportions of beef heifers reaching puberty, or age and weight at puberty.

Substitution of DL-methionine for soybean meal as a winter supplement for gestating cows grazing native range.

A winter grazing study was conducted to determine whether DL-methionine could replace soybean meal as a N supplement for gestating beef cows. During two winters (Trial 1, n = 51; Trial 2, n = 60), crossbred beef cows grazed native foothill range. Three treatment groups were supplemented with either none (CON), DL-methionine (7.5 g Trial 1 and 9 g Trial 2) in .5 kg beet pulp carrier (BPM) or .4 kg soybean meal (SBM). Cows were supplemented individually every other day. Small differences were noted in cow BW, condition score and blood metabolites. Unsupplemented cows lost the greatest amount of BW (P less than .01) in both trials and lost more (P less than .05) condition during Trial 1 than cows fed BPM or SBM supplements. Blood samples were obtained on two consecutive days during each trial (45 d and 25 d prepartum) and analyzed for blood urea N, total bilirubin, creatinine, albumin, total protein and cholesterol. A treatment x day preparatum interaction (P less than .05) was noted for blood urea. Blood urea nitrogen declined as gestation length increased for CON and SBM cows, but blood urea of BPM-supplemented cows remained low and unchanged. In situ forage digestion was measured in 12 ruminally cannulated cows (four/treatment). In both trials, in situ rate of NDF disappearance was greater (P less than .05) for SBM than for BPM. In Trial 2, a treatment x sampling hour interaction was detected for purine concentration of whole ruminal contents; SBM maintained greater purine concentrations throughout the 48-h supplementation cycle than BPM did. Principal component analysis suggested that ruminal ammonia limited the microbial growth response to DL-methionine. Therefore, alternate-day supplementation of DL-methionine plus beet pulp did not effectively substitute for soybean meal in these trials.

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.

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.

Nutritional Features of Syntrophomonas wolfei.

Syntrophomonas wolfei subsp. wolfei grew poorly in a defined medium with crotonate as the energy source in the absence of rumen fluid. Thiamine, lipoic acid, biotin, cyanocobalamin, and para-aminobenzoic acid were required for growth comparable to that obtained with the rumen fluid-based medium. Iron and cobalt were also required for the growth of S. wolfei in the chemically defined medium.

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.

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.

Effects of Organic Acid Anions on the Growth and Metabolism of Syntrophomonas wolfei in Pure Culture and in Defined Consortia.

The effects of organic acid anions on the growth of Syntrophomonas wolfei was determined by varying the initial concentration of the acid anion in the medium. The addition of 15 mM acetate decreased the growth rate of a butyrate-catabolizing coculture containing Methanospirillum hungatei from 0.0085 to 0.0029 per hour. Higher initial acetate concentrations decreased the butyrate degradation rate and the yield of cells of S. wolfei per butyrate degraded. Inhibition was not due to the counter ion or the effect of acetate on the methanogen. Initial acetate concentrations above 25 mM inhibited crotonate-using pure cultures and cocultures of S. wolfei. Benzoate and lactate inhibited the growth of S. wolfei on crotonate in pure culture and coculture. Lactate was an effective inhibitor of S. wolfei cultures at concentrations greater than 10 mM. High concentrations of acetate and lactate altered the electron flow in crotonate-catabolizing cocultures, resulting in the formation of less methane and more butyrate and caproate. The inclusion of the acetate-using methanogen, Methanosarcina barkeri, in a methanogenic butyrate-catabolizing coculture increased both the yield of S. wolfei cells per butyrate degraded and the efficacy of butyrate degradation. Butyrate degradation by acetate-inhibited cocultures occurred only after the addition of Methanosarcina barkeri. These results showed that the metabolism of S. wolfei was inhibited by high levels of organic acid anions. The activity of acetate-using methanogens is important for the syntrophic degradation of fatty acids when high levels of acetate are present.

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.

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.

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.

Anaerobic Degradation of Lactate by Syntrophic Associations of Methanosarcina barkeri and Desulfovibrio Species and Effect of H(2) on Acetate Degradation.

When grown in the absence of added sulfate, cocultures of Desulfovibrio desulfuricans or Desulfovibrio vulgaris with Methanobrevibacter smithii (Methanobacterium ruminantium), which uses H(2) and CO(2) for methanogenesis, degraded lactate, with the production of acetate and CH(4). When D. desulfuricans or D. vulgaris was grown in the absence of added sulfate in coculture with Methanosarcina barkeri (type strain), which uses both H(2)-CO(2) and acetate for methanogenesis, lactate was stoichiometrically degraded to CH(4) and presumably to CO(2). During the first 12 days of incubation of the D. desulfuricans-M. barkeri coculture, lactate was completely degraded, with almost stoichiometric production of acetate and CH(4). Later, acetate was degraded to CH(4) and presumably to CO(2). In experiments in which 20 mM acetate and 0 to 20 mM lactate were added to D. desulfuricans-M. barkeri cocultures, no detectable degradation of acetate occurred until the lactate was catabolized. The ultimate rate of acetate utilization for methanogenesis was greater for those cocultures receiving the highest levels of lactate. A small amount of H(2) was detected in cocultures which contained D. desulfuricans and M. barkeri until after all lactate was degraded. The addition of H(2), but not of lactate, to the growth medium inhibited acetate degradation by pure cultures of M. barkeri. Pure cultures of M. barkeri produced CH(4) from acetate at a rate equivalent to that observed for cocultures containing M. barkeri. Inocula of M. barkeri grown with H(2)-CO(2) as the methanogenic substrate produced CH(4) from acetate at a rate equivalent to that observed for acetate-grown inocula when grown in a rumen fluid-vitamin-based medium but not when grown in a yeast extract-based medium. The results suggest that H(2) produced by the Desulfovibrio species during growth with lactate inhibited acetate degradation by M. barkeri.

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.

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)

Formate auxotroph of Methanobacterium thermoautotrophicum Marburg.

A formate-requiring auxotroph of Methanobacterium thermoautotrophicum Marburg was isolated after hydroxylamine mutagenesis and bacitracin selection. The requirement for formate is unique and specific; combined pools of other volatile fatty acids, amino acids, vitamins, and nitrogen bases did not substitute for formate. Compared with those of the wild type, cell extracts of the formate auxotroph were deficient in formate dehydrogenase activity, but cells of all of the strains examined catalyzed a formate-carbon dioxide exchange activity. All of the strains examined took up a small amount (200 to 260 mumol/liter) of formate (3 mM) added to medium. The results of the study of this novel auxotroph indicate a role for formate in biosynthetic reactions in this methanogen. Moreover, because methanogenesis from H2-CO2 is not impaired in the mutant, free formate is not an intermediate in the reduction of CO2 to CH4.