Cellulase production was examined in two strains of Neocallimastixfrontalis, namely, PN-1 isolated from the ovine rumen, and PN-2 from the bovine rumen. For both strains, carboxymethylcellulase (CMCase) had a pH optimum of 6.0 and a temperature optimum of 50°C. CMCase resided mainly in the culture fluid, and activities up to 170 U ml-' (1 U represents 1 ,Ig of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of cellulose ml-'. For resting cultures of strain PN-1, the yield of CMCase increased from 9.9 x 103 to 10.4 x 104 U per g of cellulose degraded, as the initial cellulose concentration decreased from 10 to 0.58 mg ml-'. The range for PN-2 was 8.1 x 103 to 11 x 104 U g-1. Shaking cultures improved yields for strain PN-1 but not for PN-2. Decreased CMCase production at high initial cellulose concentrations concurred with accumulation of glucose, and addition of glucose (4 mg ml-') to cultures grown on low cellulose in which none of the sugar accumulated repressed CMCase. Adsorption of CMCase was excluded as a likely explanation for decreased yields at high initial cellulose as only a low proportion (<20%) of the enzyme was adsorbed onto the growth substrate. Exoglucanase, measured with alkali-treated Sigmacell or Avicel, gave low levels of activity in the culture fluid (<2 U ml-') and did not appear to be associated with the fungal rhizoid, as treatment with various solubilizing agents failed to give increased activity. Activity of the culture fluid towards long-chained cello-oligosaccharide, prepared by partial acid hydrolysis of cellulose, was almost sixfold higher than that for Avicel or Sigmacell, suggesting that the oligosaccharide was more suitable for the measurement of exoglucanase. Both CMCase and exoglucanase production increased when fungi were cocultured with Methanospirillum hungatei, and soluble sugars were found to be less effective than cellulose as inducers of cellulase. Yields of CMCase were higher than previously reported for some other anaerobes.

An investigation of carbon and electron flow in mud and sandflat intertidal sediments showed that the terminal electron acceptor was principally sulfate and that the carbon flow was mainly to CO 2 . Studies with thin layers of sediment exposed to H 2 showed that methane production accounted for virtually none of the H 2 utilized, whereas sulfate reduction accounted for a major proportion of the gas uptake. At all sampling sites except one (site B7), rates of methanogenesis were low but sulfate concentrations in the interstitial water were high (>18 mM). At site B7, the sulfate concentrations declined with depth from 32 mM at 2 cm to <1 mM at 10 cm or below, and active methanogenesis occurred in the low-sulfate zone. Sulfate-reducing activity at this site initially decreased and then increased with depth so that elevated rates occurred in both the active and nonactive methanogenic zones. The respiratory index (RI) [RI = 14 CO 2 /( 14 CO 2 + 14 CH 4 )] for [2- 14 C]acetate catabolism at site B7 ranged from 0.98 to 0.2 in the depth range of 2 to 14 cm. Addition of sulfate to sediment from the low-sulfate zone resulted in an increase in RI and a decrease in methanogenesis. At all other sites examined, RI ranged from 0.97 to 0.99 and was constant with depth. The results suggested that although methanogenesis was inhibited by sulfate (presumably through the activity of sulfate-reducing bacteria), it was not always limited by sulfate reduction.

The fermentation of cellulose by a rumen anaerobic fungus in the presence of Methanobrevibacter sp. strain RA1 and Methanosarcina barkeri strain 227 resulted in the formation of 2 mol each of methane and carbon dioxide per mol of hexose fermented. Coculture of the fungus with either Methanobrevibacter sp. or M. barkeri produced 0.6 and 1.3 mol of methane per mol of hexose, respectively. Acetate, formate, ethanol, hydrogen, and lactate, which are major end products of cellulose fermentation by the fungus alone, were either absent or present in very low quantities at the end of the triculture fermentation (≤0.08 mol per mol of hexose fermented). During the time course of cellulose fermentation by the triculture, hydrogen was not detected (<1 × 10 −5 atm; <0.001 kPa) and only acetate exhibited transitory accumulation; the maximum was equivalent to 1.4 mol per mol of hexose at 6 days which was higher than the total acetate yield of 0.73 in the fungus monoculture. The effect of methanogens is interpreted as a shift in the flow of electrons away from the formation of electron sink products lactate and ethanol to methane via hydrogen, favoring an increase in acetate, which is in turn converted to methane and carbon dioxide by M. barkeri . The maximum rate of cellulose degradation in the triculture (3 mg/ml per day) was faster than previously reported for bacterial cocultures and within 16 days degradation was complete. The triculture was used successfully also in the production of methane from cellulose in the plant fibrous materials, sisal (fiber from leaves of Agave sisalona L.) and barley straw leaf.

Minimal growth of Methanosarcina barkeri strain DM occurred when sulfide was omitted from the growth medium, and addition of either sodium sulfate or coenzyme M to sulfide-depleted media failed to restore growth. Optimal growth occurred in the presence of 1.25 mM added sulfide, giving a molar growth yield (YCH4) of 4.4 mg (dry weight) of cells per mmol of CH4 produced. Increasing sulfide to 12.5 mM led to a decrease in YCH4 (1.9 mg [dry weight]/mmol of CH4), in the specific growth rate and in the intracellular levels of adenosine triphosphate. However, the specific rate of methane production increased. The data suggested that at elevated sulfide levels (12.5 mM) the decrease in YCH4 might be a result of an increase in the relative energy needed for maintenance and of uncoupling of growth from energy production.

In an anaerobic digestor which was fed daily with bovine waste, during the early stages after feeding (4 to 7 h) acetate (via the methyl group) accounted for almost 90% of the methane produced. As time after feeding increased, acetate declined as a precursor so that in the 12- to 14-h and 21- to 23-h periods, after feeding the methyl group accounted for 80 and 73% of the methane produced, respectively. Measurements of methane production from CO2 reduction showed that in the 2- to 12-h period after feeding, CO2 accounted for 14% of the methane produced, whereas in the 12- to 24-h period it accounted for 27-5%. These results show that the percentages of methane accounted for by acetate and CO2 vary with time after feeding the digestor.