Non-methanogenic Bacteria Research Articles

Fermentation of Cellulose with a Mixed Microbial Rumen Culture with and without Methanogenesis

Ruminal fermentation has been well studied and includes cellulolytic microorganisms to hydrolyze cellulose to monomers, acidogenic microbes including cellulolytic microorganism to convert the monomers to volatile fatty acids (VFA), hydrogen and carbon dioxide and methanogens to convert the acetic acid, hydrogen and carbon dioxide to methane. Notably, methane production in ruminants causes energy loss for the animal and emitted methane contributes significantly to greenhouse gases in the atmosphere. The present study focuses on selectively inhibiting of the methanogens using 2–bromoethanesulfonate (BES) and its effect on ruminal fermentation in an anaerobic rumen bioreactor model system. It was found that BES inhibited methane production (99.7%) and that addition of BES decreased the total VFA productivity from 3 g/L/day to 1.3 g/L/day. Our study also found that addition of BES not only inhibited the methanogens, but also had an impact on the non-methanogenic bacteria as well, resulting in a decrease in the acetic acid productivity from 1.8 g/L/day, in a reactor without BES to 0.8 g/L/day in reactor with BES added. Endoglucanase assay revealed that addition of BES further inhibits cellulolytic microbes, resulting in a decrease in endoglucanase concentration in the reactor supplemented with BES. A notable increase in hydrogen partial pressure was seen in the reactor with BES (from 1.7% to 29.8%).

Mode of action uncovered for the specific reduction of methane emissions from ruminants by the small molecule 3-nitrooxypropanol

Ruminants, such as cows, sheep, and goats, predominantly ferment in their rumen plant material to acetate, propionate, butyrate, CO2, and methane. Whereas the short fatty acids are absorbed and metabolized by the animals, the greenhouse gas methane escapes via eructation and breathing of the animals into the atmosphere. Along with the methane, up to 12% of the gross energy content of the feedstock is lost. Therefore, our recent report has raised interest in 3-nitrooxypropanol (3-NOP), which when added to the feed of ruminants in milligram amounts persistently reduces enteric methane emissions from livestock without apparent negative side effects [Hristov AN, et al. (2015) Proc Natl Acad Sci USA 112(34):10663-10668]. We now show with the aid of in silico, in vitro, and in vivo experiments that 3-NOP specifically targets methyl-coenzyme M reductase (MCR). The nickel enzyme, which is only active when its Ni ion is in the +1 oxidation state, catalyzes the methane-forming step in the rumen fermentation. Molecular docking suggested that 3-NOP preferably binds into the active site of MCR in a pose that places its reducible nitrate group in electron transfer distance to Ni(I). With purified MCR, we found that 3-NOP indeed inactivates MCR at micromolar concentrations by oxidation of its active site Ni(I). Concomitantly, the nitrate ester is reduced to nitrite, which also inactivates MCR at micromolar concentrations by oxidation of Ni(I). Using pure cultures, 3-NOP is demonstrated to inhibit growth of methanogenic archaea at concentrations that do not affect the growth of nonmethanogenic bacteria in the rumen.

The diversity of non-methanogenic bacteria involved in biogas production from tofu-processing wastewater

Sunarto, Tsaaqifah H, Purwoko C, Pangastuti A. 2014. The diversity of non-methanogenic bacteria involved in biogasproduction from tofu-processing wastewater. Biodiversitas 16: 62-68. The energy crisis is an important current issue, so diversificationof energy is needed to solve the problem. Biogas from tofu-processing wastewater is one potential alternative energy to be developedbecause it has high organic matter content. The amount of methane formed by methanogens is also influenced by the presence ofbacteria, so it is necessary to do research on the diversity of non-methanogenic bacterial communities. This study aimed to determine thediversity of non-methanogenic bacteria in anaerobic fermentation using ARDRA (Amplified Ribosomal DNA Restriction Analysis)method. Tofu-processing wastewater was anaerobically fermented, followed by DNA extraction, amplification of 16S rRNA markergene, and cloning. Then, restriction was done using MspI and AluI restriction enzymes, followed by sequencing of different patterns.The data were analyzed to know the diversity of non-methanogenic bacteria and the relative importance of each phylotype in thecommunity using Shannon-Wiener index of diversity (H’) and evenness (E).The results showed that there were 38 positive clones of16S rRNA genes clones. The sequencing using BLASTn analysis showed the presence of 10 species of non-methanogenic bacteria.There were four sequences with 97% similarity, indicating that the bacteria belong to species from GeneBank data. There were 6sequences that had 96% similarity with the closest relative in the database, indicating that these bacteria are new species. The Shanon-Wiener index was 1.291, categorized as low and the evenness was 0.561, categorized as low because there was dominance of a species.

Changes to bacterial community make-up in a two-phase anaerobic digestion system

Changes to microbial populations in a two-phase anaerobic digestion system were studied over 34 weeks. Numbers of autofluorescent methanogenic and non-methanogenic bacteria decreased significantly during start-up, but did not change markedly either in the acid reactor or the upflow anaerobic filter for the remainder of the study. Although the proportion of autofluorescent methanogens increased in the acid reactor, the numbers of viable methanogens decreased 590-fold. The numbers of viable methanogens increased 10-fold in the port, decreased 10-fold in the effluent and there was almost no change in the drain of the upflow anaerobic filter. The data indicated that bacterial attachment in the upflow anaerobic filter gave a 90% COD removal and a methane yield of 0.33 m3 CH4 kg−1 COD removed at an organic loading rate of 7 kg COD m−3day−1. Epifluorescence microscopy of the seed sludge revealed a diverse methanogenic population of equally dominant groups of medium rods and filaments with Methanococcus, short rods, long rods and Methanosarcina also present. The medium rod-shaped species remained the most dominant group in the acid reactor. As the volatile fatty acid concentration increased in the acid reactor the number of Methanosarcina and filament species decreased, becoming the least dominant groups. At the end of the operation, Methanococcus species were the dominant group in the upflow anaerobic filter having been washed from the biofilm. © 2000 Society of Chemical Industry

Purification and characterization of an alcohol:N,N-dimethyl-4-nitrosoaniline oxidoreductase from the methanogen Methanosarcina barkeri DSM 804 strain Fusaro.

Cell-free extracts of Methanosarcina barkeri DSM 804 showed alcohol dehydrogenase activity under aerobic conditions when N,N-dimethyl-4-nitrosoaniline (NDMA) was used as an artificial electron acceptor. The NDMA-dependent alcohol dehydrogenase (NDMA-ADH) was purified to approximate homogeneity by column chromatography. It is most probably a homodimeric enzyme consisting of subunits of 45 kDa, the native molecular mass estimated by gel filtration being about 87 kDa. The purified protein had an isoelectric point of 4.3. It possesses a tightly but noncovalently bound NADP(H) cofactor. Each subunit contains 1 mol NADP(H)/mol, about 2 mol Zn2+/mol and significant amounts of magnesium. The purified enzyme preferably oxidized primary alcohols (including benzyl alcohol). NDMA-ADH from M. barkeri also catalyzed the stoichiometric dismutation of aldehydes, especially higher aliphatic aldehydes, to form equimolar amounts of the corresponding alcohol and acid without addition of an electron carrier. The enzyme did not catalyze the dehydrogenation of methanol or the disproportionation of formaldehyde and therefore is not directly involved in methanogenesis. An alignment of the N-terminal amino acid sequence of the enzyme with the sequences of other alcohol dehydrogenases from methanogenic and nonmethanogenic bacteria indicated no significant identity. Nevertheless there was a quite interesting sequence similarity in the first 30 N-terminal amino acids to plant cinnamyl alcohol dehydrogenase. NDMA-ADH from M. barkeri is a novel type of alcohol dehydrogenase in methanogenic bacteria.

Simulation of constituent processes of anaerobic degradation of organic matter by the "methane" model.

The model of anaerobic digestion described earlier by the authors was used for analysis of the different phases of the process. It was shown that at the glucose conversion a coexistence of hydrogen-producing acidogenic bacteria and hydrogen-utilizing non-methanogenic bacteria causes a hydrogen partial pressure decrease at an increase of solids retention time (i), the intensity of the negative feed-back effect in sulfate-reduction through hydrogen sulfide formation is regulated by the pH level during an oscillation dynamics in acetate/sulfate system (ii), under the toxicity influence the processes of methanogenesis and acetogenesis together with hydrolysis may be rate-limiting steps in the anaerobic system with particulate substrate degradation (iii).

Biomethanation of spent wash: Heavy metal inhibition of methanogenesis in synthetic medium

Five heavy metals detected in distillery waste were lead (1.0–8.8 μg/ml), copper (1.7–15.7 μg/ml), zinc (3.1–11.8 μg/ml), iron (36.0–43.5 μg/ml), and manganese (3.0–5.1 μg/ml). Their toxicity to biomethanogenesis in a synthetic medium containing 1% sodium acetate, propionate, or butyrate was measured by batch fermentation, after cultivating the bacterial biomass semicontinuously. Lead, copper, and zinc in decreasing order were found to be toxic to biomethanogenesis. Lead at the concentration of 10 μg/ml completely stopped methane production. Iron did not produce any notable change in the process while manganese stimulated the rate of methane production. The toxicity of lead, copper, and zinc to methanogenic bacteria and methane production was dose-dependent but the growth of acetogenic bacteria was impaired at higher concentrations (2.5–10.0 μg/ml) of lead, copper, and zinc. Manganese stimulated the growth of only methanogenic bacteria, but not that of non-methanogenic bacteria or acetic acid production. The reduction in the synthesis of acetic acid via butyrate was more in the presence of these three metals than the synthesis of this acid via propionate.

Increase in colonic methanogens and total anaerobes in aging rats.

Methanogens are present in the colons of our local Wistar rat colony. We studied the changes in concentrations of their fecal methanogenic and nonmethanogenic bacteria with age as a model of the development of these communities in humans. We found that the predominant methanogen in the rats is a Methanobrevibacter species. The log of the concentration of total anaerobes increased from 9.8/g (dry weight) at 3.0 weeks of age (shortly after weaning) to 10.7/g (dry weight) at 96 weeks (shortly before the end of the life span). In contrast, the log concentration of methanogens increased from 5.5 to 9/g (dry weight) during the same time period. Therefore, methanogens increased as a percentage of the total anaerobes from 0.005% at 3.0 weeks to 2.0% at 96 weeks. About 12 doublings of the methanogenic population and 3.3 doublings of the nonmethanogenic population took place from weaning until death. The slow increase in the ratio of methanogens to total anaerobes with age followed the same pattern in cecal contents as found in feces. There were no relationships between animal weights or fecal outputs and the increase in total anaerobe and methanogen concentrations in feces. A possible explanation for the slow increase in the Methanobrevibacter species in Wistar rats with age is a gradual shifting of the use of electrons from the reduction of CO2 to acetate by acetogens to the reduction of CO2 to CH4. The results provide the first evidence for an age-related change in the nonmethanogenic bacteria of the colon and supporting microbiological evidence for physiological studies that have shown age-related increases in colonic methane production in humans.

Temporal change of gas metabolism by hydrogen-syntrophic methanogenic bacterial associations in anoxic paddy soil

Interspecies H 2 transfer within methanogenic bacterial associations (MBA) accounted for 95–97% of the conversion of 14CO 2 to 14CH 4 in anoxic paddy soil. Only 3–5% of the 14CH 4 were produced from the turnover of dissolved H 2. The H 2-syntrophic MBA developed within 5 days after the paddy soil had been submerged and placed under anoxic atmosphere. Afterwards, both the contribution of MBA to H 2-dependent methanogenesis and the turnover of dissolved H 2 did not change significantly for up to 7 months of incubation. However, while the rates of H 2-dependent methanogenesis stayed relatively constant, the rates of total methanogenesis decreased. The contribution of MBA to H 2-dependent methanogenesis was further enhanced to 99% when the temperature was shifted from 30°C to 17°C, or when the soil had been planted with rice. This enhancement was partially due to an increased utilization of dissolved H 2 by chloroform-insensitive non-methanogenic bacteria, most probably homoacetogens, so that CH 4 production was almost completely restricted to H 2-syntrophic MBA. The activity of MBA, as measured by the conversion of 14CO 2 to 14CH 4, was stimulated by glucose, lactate, and ethanol to a similar or greater extent than by exogenous H 2. Propionate and acetate had no effect.

Temporal change of gas metabolism by hydrogen-syntrophic methanogenic bacterial associations in anoxic paddy soil

Interspecies H2 transfer within methanogenic bacterial associations (MBA) accounted for 95–97% of the conversion of 14CO2 to 14CH4 in anoxic paddy soil. Only 3–5% of the 14CH4 were produced from the turnover of dissolved H2. The H2-syntrophic MBA developed within 5 days after the paddy soil had been submerged and placed under anoxic atmosphere. Afterwards, both the contribution of MBA to H2-dependent methanogenesis and the turnover of dissolved H2 did not change significantly for up to 7 months of incubation. However, while the rates of H2-dependent methanogenesis stayed relatively constant, the rates of total methanogenesis decreased. The contribution of MBA to H2-dependent methanogenesis was further enhanced to 99% when the temperature was shifted from 30°C to 17°C, or when the soil had been planted with rice. This enhancement was partially due to an increased utilization of dissolved H2 by chloroform-insensitive non-methanogenic bacteria, most probably homoacetogens, so that CH4 production was almost completely restricted to H2-syntrophic MBA. The activity of MBA, as measured by the conversion of 14CO2 to 14CH4, was stimulated by glucose, lactate, and ethanol to a similar or greater extent than by exogenous H2. Propionate and acetate had no effect.

Methanogenesis: surprising molecules, microorganisms and ecosystems.

Methanogenesis involves a novel set of coenzymes as one-carbon and electron carriers. Consequently, metabolic processes of methanogens deviate from those present in non-methanogenic bacteria. Methanogenic bacteria can be classified on the basis of substrate utilization. Group I (24 species) grows at the expense of hydrogen plus CO2 and/or formate and group II (7 species) uses methanol and/or acetate. Hydrogen-consuming methanogens are found as epi- or endosymbionts of anaerobic ciliates.

Detection and Quantitation of Methanogens by Enzyme-Linked Immunosorbent Assay

Antisera to two methanogenic bacteria, Methanosarcina barkeri and a Methanobacterium sp., were raised in rabbits and used to develop an enzyme-linked immunosorbent assay (ELISA) method. ELISA was shown to be a sensitive technique, detecting as little as 4 ng of methanogen protein. The specificities of the antisera toward other methanogens were evaluated, and it was found that the antisera recognized species of the same genus as the immunizing species, but gave very little cross-reaction with methanogens of different genera. ELISA was used to estimate the growth of methanogens in pure culture. In natural environments and in anaerobic digesters methanogens exist as part of a mixed bacterial community, so the possibility of using ELISA to quantitate methanogens in mixed cultures was examined. The two antisera gave very little reaction in ELISA when non-methanogenic bacteria were used as antigens and ELISA was used to quantitate methanogens in an acetate enrichment culture. I conclude that the ELISA is a useful method for quantitating methanogens in defined mixed cultures, but has limited applicability to more complex systems.

Death rates of obligate anaerobes exposed to oxygen and the effect of media prereduction on cell viability

Stationary-phase cells of Methanospirillum hungatei GP1, Methanobacterium thermoautotrophicum ΔH, Methanobacterium bryantii M.o.H., Fusobacterium sp., Bacteroides fragilis, and Eubacterium limosum were aerated in medium devoid of cysteine – sodium sulfide (cysteine–Na2S) reducing agents to determine the death rates caused by exposure to oxygen. D values (time in minutes required to destroy 90% of the initially viable population) for these anaerobes were 208, 293, 543, 239, 410, and 507 min, respectively. Death was exponential throughout the treatment period for all the anaerobes, except B. fragilis and E. limosum which showed no significant death for the first 2 and 9 h of aeration, respectively. A minimum prereduction period of 6 h inside an anaerobic chamber was required for aerobically poured agar medium containing cysteine–Na2S to achieve maximum viable cell recovery of the nonmethanogens not previously exposed to air. The results also showed that agar medium containing reducing agents, poured anaerobically, and previously reduced in an anaerobic chamber for 24 h, could be plated aerobically and reintroduced into the anaerobic chamber within 60 min of inoculation without affecting viable cell recovery of the nonmethanogenic bacteria.

Levels of water-soluble vitamins in methanogenic and non-methanogenic bacteria.

The levels of seven water-soluble vitamins in Methanobacterium thermoautotrophicum, Methanococcus voltae, Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, and Bacteroides thetaiotaomicron were compared by using a vitamin-requiring Leuconostoc strain. Both methanogens contained levels of folic acid and pantothenic acid which were approximately two orders of magnitude lower than levels in the nonmethanogens. Methanobacterium thermoautotrophicum contained levels of thiamine, biotin, nicotinic acid, and pyridoxine which were approximately one order of magnitude lower than levels in the nonmethanogens. The thiamine level in Methanococcus voltae was approximately one order of magnitude lower than levels in the nonmethanogens. Only the levels of riboflavin (and nicotinic acid and pyridoxine in Methanococcus voltae) were approximately equal in the methanogens and nonmethanogens. Folic acid may have been present in extracts of methanogens merely as a precursor, by-product, or hydrolysis product of methanopterin.

Volatile fatty acid cycling in organic-rich marine sediments

Volatile fatty acid (VFA) apparent turnover rates were determined by measuring whole sediment VFA concentrations and the corresponding reaction rate constants. The following ranges of VFA concentrations were measured in Cape Lookout Bight, N.C. sediments (μmole·l s −1): acetate 54–660, propionate 1–24, butyrate <0.5–22, iso-butyrate <0.5–6. Apparent turnover rates measured over a one-year period ranged from 18–600 μmole·l s −1·h −1 for acetate and 0.7–7 μmole·l s −1·h −1 for the carboxyl carbon of propionate. Methane production was observed only with acetate and only in sulfatedepleted sediments; total acetate turnover attained approximately the same maximum value in both sulfate-reducing and sulfate-depleted sediments. Apparent turnover rates for acetate and propionate appeared to be controlled by similar factors: in sulfate-reducing (surface) sediments the turnover rates were stimulated by autumn storm-mediated deposition/resuspension events; in deeper sulfate-depleted sediments the turnover rates followed changes in the ambient temperature. Changes in VFA poolsizes were proportionally much larger than changes in corresponding rate constants. The ratio of CO 2 to CH 4 produced from acetate vs. depth suggested that non-methanogenic bacteria accounted for 60% of the acetate turnover in sulfate-depleted sediments. VFA concentrations were much lower in N.C. continental slope mud than in Cape Lookout sediments; acetate was the only VFA detectable throughout the top 40 cm of the slope sediments. The estimated production rate of CO 2 from acetate decreased rapidly with depth. The surface rate was approximately 20 times less than that measured at similar temperatures in sulfate-reducing Cape Lookout sediments.

Microbiological aspects of anaerobic digestion

The complete degradation of complex organic matter to CO2 and CH4 is the result of the combined and coordinated metabolic activity of the digester population. In this respect, metabolic interactions between methanogenic and non-methanogenic species are of extraordinary importance to the anaerobic digestion process. In spite of several studies on the predominant non-methanogenic bacteria present in anaerobic digesters (Toerien et al., 1967; Mah and Susman, 1968; Kirsch, 1969; Hobson and Shaw, 1974; Ueki et al., 1980), detailed studies on generic and species identification of the hydrolytic and fermentative bacteria have not been reported. Because at this stage it is still not clear whether the predominant organisms present in piggery waste digesters participate actively during anaerobic digestion, microbial studies have been performed with common laboratory fermentors fed with either a sterilized or a conventional input. From the results of our experiments the following conclusions could be drawn: * In balanced mesophilic piggery waste digesters the anaerobic bacterial count is similar to the aerobic and facultative anaerobic count; generally, counts of around 106-10S/ml were obtained. * In digesters fed with a sterilized input the bacterial count is always lower than in a conventional one; counts were of the order of 105-106/ml. * In anaerobic digesters coliforms are only contaminating organisms, whereas some Bacillus species remain active during the digestion process. Indeed, experiments with sterilized input revealed that several Bacillus spp. were still active (104/ml) after more than 20 complete fermentor replacements (retention period = 15 days). The main species were: Bacillus subtilis, B. pumilis, B. cereus, B. licheniformis and B. circulans. * Clostridium spp. are among the most metabolically active bacteria. The asaccbarolytic species C. sporogenes, C. subterminale and C. hastiforme were most frequently isolated. In peptone-containing media these proteolytic species do produce large amounts of iso-fatty acids. Other important species were C. cochlearium, C. malenominatum, C. butyricum and C. tertium. * The dominant anaerobic population of piggery waste digesters (with conventional or sterilized input) consists of species belonging to the genera: Bacteroides, Peptostreptococcus, Peptococcus, Ruminococcus, Coprococcus, Sarcina, Eubacterium, Clostridium, Propionibacterium, Actinomyces and Lactobacillus, the first two genera probably being the most important.

Methanogenesis and methanogenic partnerships

The microbial formation of methane from organic compounds greater than C 2 in chain length demands a mixture of methanogenic and chemoheterotrophic non-methanogenic bacteria. The chemoheterotrophic, non-methanogens initiate the reactions by well established pathways, hydrolysing complex polymers and fermenting the unit constituents to smaller end-product molecules, which are further metabolized by other chemoheterotrophs to acetic acid, H 2 and CO 2 . The methanogens are essential physiological partners in the overall conversion of the initial substrates to this level because they oxidize H 2 and reduce CO 2 to form methane. The ultimate sformation of acetate as the chief intermediate in this fermentation depends on the removal of H 2 by the methanogens. Otherwise, these acetogenic reactions are not thermodynamically feasible. Acetate, the major precursor of CH 4 in fermentation systems, is converted to CH 4 and CO 2 via a unique aceticlastic reaction. Examples of microbial partnerships producing methane from the fermentation of organic com­ pounds are described

Anaerobic digestion: microbial and biochemical aspects of volatile acid production

The production of organic acids has been tested with bacterial flora selected from a municipal sludge digestor. In order to elucidate the basic mechanisms by which glucose is converted to volatile fatty acids, the examination of non-methanogenic bacteria was attempted. Both lactate-producers and lactate-utilizers were found among these bacteria. When mixed isolates were used as the inoculum, the accumulation of lactic acid and its further conversion to propionic and butyric acids was demonstrated at a carbon conversion rate of about 0.75. It is therefore suggested that this metabolic sequence may occur as a normal process in acidogenic fermentation, which is the first step in anaerobic digestion.

Isolation of Methanobacterium bryantii from a Deep Aquifer by Using a Novel Broth-Antibiotic Disk Method

Methanobacterium bryantii was isolated from a mixed-culture enrichment of a water sample from a deep aquifer by using a complex growth medium supplemented with antibiotic susceptibility disks to inhibit the growth of non-methanogenic bacteria.

Methane Production by Microorganisms

Methane fermentation can be utilized as a fuel gas producing process from solar energy because plant-body can become the raw material f or methane fermentation. The bacteria which cause methane fermentation can be grouped into methanogenic bacteria which produce methane and non-methanogenic bacteria which produce substrates f or methanogenic bacteria. The substrates f or methanogenic bacteria are hydrogen and carbon dioxide, acetate, f ormate, and methanol. Acetate is utilized only in the presence of hydrogen by some species of bacteria, and is utilized even in the absence of hydrogen by other species. The existence of bacteria which produce methane from propionate or n-butyrate is doubted. Some examples of symbiotic relation between methanogenic and non-methanogenic bacteria are known. Methane fermentation of algae, water hyacinth, arrowroot, giant kelp etc. has been studied in U.S.A. In Japan methane fermentation of urban wastes has been studied. Now we are needed to carried out not only macroscopic studies on a total system of waste water treatment processes, wastes treatment processes, and solar energy-utilizing processes but also microscopic studies on the phenomenon of methane fermentation itself.