Biogenic methane formation in marine and freshwater environments: CO2 reduction vs. acetate fermentation—Isotope evidence

Soil microorganisms as controllers of atmospheric trace gases (H2, CO, CH4, OCS, N2O, and NO).

Denitrification occurs in essentially all river, lake, and coastal marine ecosystems that have been studied. In general, the range of denitrification rates measured in coastal marine sediments is greater than that measured in lake or river sediments. In various estuarine and coastal marine sediments, rates commonly range between 50 and 250 µmol N m−2 h−1, with extremes from 0 to 1,067. Rates of denitrification in lake sediments measured at near-ambient conditions range from 2 to 171 µmol N m−2 h−1. Denitrification rates in river and stream sediments range from 0 to 345 µmol N m−2 h−1. The higher rates are from systems that receive substantial amounts of anthropogenic nutrient input. In lakes, denitrification also occurs in low oxygen hypolimnetic waters, where rates generally range from 0.2 to 1.9 µmol N liter−1 d−1. In lakes where denitrification rates in both the water and sediments have been measured, denitrification is greater in the sediments.

The major source of nitrate for denitrification in most river, lake, and coastal marine sediments underlying an aerobic water column is nitrate produced in the sediments, not nitrate diffusing into the sediments from the overlying water. During the mineralization of organic matter in sediments, a major portion of the mineralized nitrogen is lost from the ecosystem via denitrification. In freshwater sediments, denitrification appears to remove a larger percentage of the mineralized nitrogen. N2 fluxes accounted for 76–100% of the sediment-water nitrogen flux in rivers and lakes, but only 15–70% in estuarine and coastal marine sediments. Benthic N2O fluxes were always small compared to N, fluxes.

The loss of nitrogen via denitrification exceeds the input of nitrogen via N2 fixation in almost all river, lake, and coastal marine ecosystems in which both processes have been measured.

Denitrification is also important relative to other inputs of fixed N in both freshwater and coastal marine ecosystems. In the two rivers where both denitrification measurements and N input data were available, denitrification removed an amount of nitrogen equivalent to 7 and 35% of the external nitrogen loading. In six lakes and six estuaries where data are available, denitrification is estimated to remove an amount of nitrogen equivalent to between 1 and 36% of the input to the lakes and between 20 and 50% of the input to the estuaries.

https://aslopubs.onlinelibrary.wiley.com/doi/pdf/10.4319/lo.1988.33.4part2.0702

Denitrification in freshwater and coastal marine ecosystems: Ecological and geochemical significance

Gliding motility, ultrastructure and nutrition of two newly isolated filamentous sulfate-reducing bacteria, strains 5ac10 and 4be13, were investigated. The filaments were always attached to surfaces. Growth was supported by addition of insoluble aluminium phosphate or agar as substrata for gliding movement. Electron microscopy of ultrathin sections revealed cell walls characteristic of Gramnegative bacteria; the undulated structure of the outer membrane may pertain to the translocation mechanism. Intracytoplasmic membranes were present. Acetate, higher fatty acids, succinate or fumarate served as electron donors and carbon sources. Strain 5ac10 grew also with lactate, but not with benzoate that was used only by strain 4be13. Strain 5ac10 was able to grow slowly on H2 plus CO2 or formate in the presence of sulfate without additional organic carbon source. The capacity of complete oxidation was shown by stoichiometric measurements with acetate plus sulfate. Both strains contained b- and c-type cytochromes. Desulfoviridin was detected only in strain 5ac10. The two filamentous gliding sulfate reducers are described as new species of a new genus, Desulfonema limicola and Desulfonema magnum.

Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids

Biological methanogenesis plays a major role in the carbon cycle on Earth. Methanogenesis is the terminal step in carbon flow in many anaerobic habitats, including marine and freshwater sediments, marshes and swamps, flooded soils, bogs, geothermal habitats, and animal gastrointestinal tracts. CH4 escaping from anaerobic habitats can serve as a carbon and energy source for aerobic methanotrophic bacteria, and can escape to the atmosphere, where it is a major participant in atmospheric chemical reactions and is an important greenhouse gas.

Physiological Ecology of Methanogens

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 = CO(2)/(CO(2) + CH(4))] for [2-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.

Carbon and Electron Flow in Mud and Sandflat Intertidal Sediments at Delaware Inlet, Nelson, New Zealand

Cellulase production was examined in two strains of Neocallimastix frontalis, 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 degrees C. CMCase resided mainly in the culture fluid, and activities up to 170 U ml-1 (1 U represents 1 microgram of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of cellulose ml-1. For resting cultures of strain PN-1, the yield of CMCase increased from 9.9 X 10(3) to 10.4 X 10(4) U per g of cellulose degraded, as the initial cellulose concentration decreased from 10 to 0.58 mg ml-1. The range for PN-2 was 8.1 X 10(3) to 11 X 10(4) 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-1) 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 (less than 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 (less than 2 U ml-1) and did not appear to be associated with the fungal rhizoid, as treatment with various solubilizing agents failed to give increased activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Production and regulation of cellulase by two strains of the rumen anaerobic fungus Neocallimastix frontalis.

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 x 10 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.

https://aem.asm.org/content/aem/44/1/128.full.pdf

Fermentation of Cellulose to Methane and Carbon Dioxide by a Rumen Anaerobic Fungus in a Triculture with Methanobrevibacter sp. Strain RA1 and Methanosarcina barkeri.

Minimal growth of Methanosarcina barkeri strain DM occurred when sulfide was omitted fromthe 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 decrease in YCH4 (1.9 mg [dry weight]/mmol of CH4), in the specific growth rate and in be 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 maintnenace and of uncoupling of growth from energy production.

Effect of inorganic sulfide on the growth and metabolism of Methanosarcina barkeri strain DM.

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.

Rodney A. Asher

Papers

Carbon and Electron Flow in Mud and Sandflat Intertidal Sediments at Delaware Inlet, Nelson, New Zealand

Production and regulation of cellulase by two strains of the rumen anaerobic fungus Neocallimastix frontalis.

Fermentation of Cellulose to Methane and Carbon Dioxide by a Rumen Anaerobic Fungus in a Triculture with Methanobrevibacter sp. Strain RA1 and Methanosarcina barkeri.

Effect of inorganic sulfide on the growth and metabolism of Methanosarcina barkeri strain DM.

Changes in proportions of acetate and carbon dioxide used as methane precursors during the anaerobic digestion of bovine waste