Predictions based on a general model of surface components involved in gliding motility of Cytophaga johnsonae along with characteristics of previously isolated nonspreading mutants of C. johnsonae led us to suspect that isolation of such mutants could be accomplished by selecting for nonadherent or nonhydrophobic mutants. Accordingly, conditions were devised to select for mutants that failed to attach to cheesecloth suspended in growth media and for mutants that failed to adhere to n-hexadecane droplets. Populations of cells obtained from both selection procedures were screened for mutants producing nonspreading colonies. Both techniques resulted in enrichment for nonspreading mutants that were classified by previously established criteria as MNS (motile nonspreading), CNS (conditional nonspreading) or TNM (truly nonmotile). When assayed for adherence and hydrophobicity, all TNM mutants were nonadherent and nonhydrophobic, compared with wild-type cells. Most mutants of the other two classes were unchanged with respect to these properties. Results from subjecting cells of four other strains of gliding bacteria to selection by the same procedures indicate that the methods will be broadly applicable for isolating nonspreading mutants of gliding bacteria. Abbreviations: EC, enriched cytophaga medium; PB, phosphate buffer (5 mM, pH 7-1); AI, adhesion index; HI, hydrophobicity index ; CNS, conditional nonspreading mutants; MNS, motile nonspreading mutants; TNM, truly nonmotile mutants. 0001-2132 0 1985 SGM

Methanosarcina barkeri was adapted to grow on carbon monoxide by sequential transfer of the culture in medium that contained CO (100% of culture headspace). These experiments document the ability of the organism to grow slowly (65-h doubling time) and to produce methane and CO2 either on CO as the sole carbon and energy source or by the simultaneous consumption of methanol and CO. During growth on CO as carbon and energy source, net hydrogen formation occurred when the CO partial pressure in the culture headspace was greater than 20% CO, but hydrogen was consumed when the CO concentration was below this value.Carbon monoxide is an abundant atmospheric pollutant generated by incomplete conbustion of fossil fuels. Carbon monoxide is also formed as a metabolite of microbial metabolism, especially heme degradation by aerobic microorganisms (5,19). The physiological features of anaerobic bacteria that are able to grow on various one-carbon substrates as the sole carbon and electron source (i.e., unicarbonotrophic growth) and to grow mixotrophically on mixtures of onecarbon substrates, including CO, have been reviewed (20). Several genera of anaerobic bacteria have been shown to grow on CO as the energy source, including: Rhodopseudomonas (18), Methanobacterium (2), Butyribacterium (4), Eubacterium (13), Clostridium (9), and Acetobacterium (8).Kluyver and Schnellen (10) demonstrated that Methanosarcina barkeri produced 1 mol of methane and 3 mol of CO2 from 4 mol of CO when cell suspensions were incubated under 1 atm (ca. 101.29 kPa) (100%) of this gas. Later, Daniels et al.(2) showed that M. barkeri oxidized small amounts of added CO during growth on H2-CO2 and that cell extracts contained a methyl viologen-linked CO dehydrogenase activity. The significance of CO metabolism by some anaerobes like Clostridium pasteurianum is enigmatic because this anaerobe consumes CO via CO dehydrogenase activity, but it is not capable of growth on this substrate (3). However, it was established that acetogenic species, like Clostridium thermoaceticum, made acetate during growth with CO as the carbon and energy source (9). Carbon monoxide dehydrogenase has been proposed to function in carbonylation of a methyl group during acetyl-coenzyme Aacetate synthesis in both acetogenic (6) and methanogenic (7, 17) anaerobes. Krzycki and Zeikus (11) reported that the level of this enzyme increased fivefold when M. barkeri was grown on acetate versus on H2-CO2 or methanol as the sole carbon and energy source. This result suggested that CO dehydrogenase of M. barkeri may also function in acetate dissimilation by a biochemical mechanism that involves the production and consumption of a carbonyl group as an intermediary step in methane and CO2 formation. In the present note, we show that M. barkeri is able to grow either * Corresponding author. unicarbonotrophically with CO as the sole carbon and energy source or mixotrophically with CO and methanol.M. barkeri neotype strain MS was routinely cultivated under strictly anoxic conditions in a phosphat...

We examined the unitrophic metabolism of acetate and methanol individually and the mixotrophic utilization of these compounds by using detailed 14C-labeled tracer studies in a strain of Methanosarcina barkeri adapted to grow on acetate as the sole carbon and energy source. The substrate consumption rate and methane production rate were significantly lower on acetate alone than during the unitrophic or mixotrophic metabolism of methanol. Cell yields (in grams per mole of substrate) were identical during exponential growth on acetate and exponential growth on methanol. During unitrophic metabolism of acetate, the methyl moiety accounted for the majority of the CH4 produced, but 14% of the CO2 generated originated from the methyl moiety. This correlated with the concurrent reduction of equivalent amounts of the C-1 of acetate to CH4. 14CH4 was also produced from added 14CO2, although to a lesser extent than from reduction of the C-1 of acetate.

Clostridium thermocellum was compared in batch and continuous cultures. Maximum specific growth rates per hour obtained on cellulosic substrates were 0.1 in batch culture and >0.13 in continuous culture. Cell yields (grams of cells per gram of substrate) in batch culture were 0.17 for pretreated wood and 0.15 for Avicel. Ethanol and acetate were the main products observed under all conditions. Ethanol:acetate ratios (in grams) were approximately 1.8:1 in batch culture and generally slightly less than 1:1 in continuous culture. Utilization of cellulosic substrates was essentially complete in batch culture. A prolonged lag phase was initially observed in batch culture on pretreated wood; the length of the lag phase could be shortened by addition of cell-free spent medium. In continuous culture with-5 g of glucose equivalent per liter in the feed, substrate conversion relative to theoretical ranged from 0.86 at a dilution rate (D) of 0.05/h to 0.48 at a D of 0.167/h for Avicel and from 0.75 at a D of 0.05/h to 0.43 at a D of 0.11/h for pretreated wood. At feed concentrations of <4.5 g of glucose equivalent per liter, conversion of pretreated wood was 80 to 90% at D = 0.083/h. Lower conversion was obtained at higher feed substrate concentrations, consistent with a limiting factor other than cellulose. Free Avicelase activities of 12 to 84 mU/ml were observed, with activity increasing in this order: batch celiobiose, batch pretreated wood < batch Avicel, continuous pretreated wood < continuous Avicel. Free cellulase activity was higher at increasing extents of substrate utilization for both pretreated wood and Avicel under all conditions tested. The results indicate that fermentation parameters, with the exception of free cellulase activity, are essentially the same for pretreated mixed hardwood and Avicel under a variety of conditions. Hydrolysis yields obtained with C. thermocellum cellulase acting either in vitro or in vivo were comparable to those previously reported for Trichoderma reesei on the same substrates.

Selective enrichment culture techniques were employed to obtain mixed cultures of methanogenic rods and sarcina from surface flooding waters and deep subsurface (-1650 m) oil-bearing sedimentary rocks and formation waters sampled from an old oil field in the U.S.S.R. previously reported to display active biological methanogenesis. The methanogens were selectively isolated as colonies on agar petri dishes that were incubated in a novel container. The general cellular and growth features of three Methanobacterium isolates were determined. These strains grew optimally at 37 to 45°C in anaerobic pressure tube cultures with a doubling time of 16 to 18 h on H2-CO2 and proliferated as autotrophs. Acetate addition significantly enhanced the final cell yield. Growth of these strains was completely inhibited by either 0.6 g of sodium sulfide per liter or 31.0 of sodium chloride per liter, but growth was not inhibited by either 0.3 g of sodium sulfide per liter or 1.0 g of sodium sulfate per liter. One novel isolate, Methanobacterium sp. strain ivanov, was grown on H2-CO2, and the stable-carbon isotopic fractionations that occurred during synthesis of methane, cell carbon, and lipids were determined. The results of this study were used to examine the anomalous relationship between the isotopic and chemical compositions of natural gas occurring in the deep subsurface environment of the oil field.