Hydrogen-producing Acetogens Research Articles

Enhanced syntrophic propionate degradation and methane production by granular activated carbon in AnSBR

Syntrophic degradation of propionate has been regarded as a limiting factor for methane formation in anaerobic digestion (AD) processes, due to its easier production but harder degradation than other volatile fatty acids. In the present study, 20 g/L granular activated carbon (GAC) was introduced into an anaerobic sequence batch reactor (AnSBR) and the enhancement of propionate degradation and methane production was evaluated with another AnSBR without GAC as a control. Inoculated with the same excess activated sludge, both AnSBRs were synchronously started up at 35 °C with the fed propionate increased gradually from the initial 340 mg/L to the final 1700 mg/L. The results showed that the introduction of GAC made the AnSBR get steady state 6 days earlier. With the fed propionate of about 1700 mg/L in the steady state, the average specific methane production and biomass was enhanced from 0.20 to 0.25 L/L·d, and 7.72 to7.96 g/L, respectively, by the introduced GAC. The results suggested that the GAC had functioned in stimulating microbial growth and enhancing direct interspecies electron transfer between hydrogen-producing acetogens and methanogens, which had resulted in the enhanced propionate degradation and methane production.

Variable sediment methane production in response to different source-associated sewer sediment types and hydrological patterns: Role of the sediment microbiome

Production of methane (CH4), an essential anthropogenic greenhouse gas, from municipal sewer sediment is a problem deserving intensive attention. Based on long-term laboratory batch tests in conjunction with 16 s rRNA gene sequencing and metagenomics, this study provides the first detailed assessment of the variable sediment CH4 production in response to different pollution source-associated sewer sediment types and hydrological patterns, while addressing the role of the sediment microbiome. The high CH4-production capability of sanitary sewer sediment is shaped by enriched biologically active substrate and dominated by acetoclastic methanogenesis (genus Methanosaeta). Moreover, it involves syntrophic interactions among fermentation bacteria, hydrogen-producing acetogens and methanogens. Distinct source-associated microbial species, denitrifying bacteria and sulfate-reducing bacteria occur in storm sewer and illicit discharge-associated (IDA) storm sewer sediments. This reveals their insufficient microbial function capabilities to support efficient methanogenesis. Hydrogenotrophic methanogenesis (genus Methanobacterium) prevails in both these sediments. In this context, storm sewer sediment has an extremely low CH4-production capability, while IDA storm sewer sediment still shows significant carbon emission through a possibly unique mechanism. Hydrological connections promote the sewer sediment biodegradability and CH4-production capability. In contrast, hydrological disconnection facilitates the prevalence of acetoclastic methanogenesis, sulfate-reducing enzymes, denitrification enzymes and the sulfur-utilizing chemolithoautotrophic denitrifier, which drastically decreases CH4 production. Turbulent suspension of sediments results in relative stagnation of methanogenesis. This work bridges the knowledge gap and will help to stimulate and guide the resolution of ‘bottom-up’ system-scale carbon budgets and GHG sources, as well as the target CH4 abatement interventions.

Effect of Hydraulic Retention Time on Anaerobic Baffled Reactor Operation: Enhanced Biohydrogen Production and Enrichment of Hydrogen-producing Acetogens

Biohydrogen production by fermentation has become a promising technology developed in the world. In this study, diluted normal molasses wastewater is used as the raw material, and biohydrogen production efficiency and operation characteristics of an anaerobic baffled reactor (ABR) are studied. The effect of hydraulic retention time (HRT) on biohydrogen production efficiency and operating characteristics of ABR is extensively discussed. Experimental results showed that methanogen residuals were still observed in the last three compartments under HRT of 24 h and COD(Chemical oxygen demand) concentration of 8000 mg/L. Meanwhile, the first three compartments presented an ethanol fermentation type. The characteristics of butyric acid fermentation in Compartment IV were also enhanced. The average removal efficiency of COD was reduced to 15.4%. The average rates of biohydrogen production and specific biohydrogen production were 12.85 and 360.22 L/kg COD, respectively. The extension of HRT was beneficial to enrich hydrogen-producing acetogens and could increase the production rate of biohydrogen by a factor of 1.65. However, with the decrease in the bioactivity of acidogenic fermentation bacteria, the biohydrogen production efficiency of ABR was significantly reduced when HRT was longer than 30 h. The specific biogas production rate decreased from 191 to 92 L/(kg MLVSS·d). The specific biohydrogen production rate also decreased from 24.34 to 2.7 L/(kg MLVSS·d).

Bioaugmentation with Mixed Hydrogen-Producing Acetogen Cultures Enhances Methane Production in Molasses Wastewater Treatment.

Hydrogen-producing acetogens (HPA) have a transitional role in anaerobic wastewater treatment. Thus, bioaugmentation with HPA cultures can enhance the chemical oxygen demand (COD) removal efficiency and CH4 yield of anaerobic wastewater treatment. Cultures with high degradation capacities for propionic acid and butyric acid were obtained through continuous subculture in enrichment medium and were designated as Z08 and Z12. Bioaugmentation with Z08 and Z12 increased CH4 production by glucose removal to 1.58. Bioaugmentation with Z08 and Z12 increased the COD removal rate in molasses wastewater from 71.60% to 85.84%. The specific H2 and CH4 yields from COD removal increased by factors of 1.54 and 1.63, respectively. Results show that bioaugmentation with HPA-dominated cultures can improve CH4 production from COD removal. Furthermore, hydrogen-producing acetogenesis was identified as the rate-limiting step in anaerobic wastewater treatment.

Microbial community dynamics at high organic loading rates revealed by pyrosequencing during sugar refinery wastewater treatment in a UASB reactor

The performance and microbial community structure in an upflow anaerobic sludge blanket reactor (UASB) treating sugar refinery wastewater were investigated. The chemical oxygen demand (COD) removal reached above 92.0% at organic loading rates (OLRs) of 12.0–54.0 kgCOD/(m3∙d). The volatile fatty acids (VFAs) in effluent were increased to 451.1 mg/L from 147.9 mg/L and the specific methane production rate improved by 1.2–2.2-fold as the OLR increased. The evolution of microbial communities in anaerobic sludge at three different OLRs was investigated using pyrosequencing. Operational taxonomic units (OTUs) at a 3% distance were 353, 337 and 233 for OLR12, OLR36 and OLR54, respectively. When the OLR was increased to 54.0 kgCOD/(m3∙d) from 12.0 kgCOD/(m3∙d) by stepwise, the microbial community structure were changed significantly. Five genera (Bacteroides, Trichococcus, Chryseobacterium, Longilinea and Aerococcus) were the dominant fermentative bacteria at the OLR 12.0 kgCOD/(m3∙d). However, the sample of OLR36 was dominated by Lactococcus, Trichococcus, Anaeroarcus and Veillonella. At the last stage (OLR = 54.0 kgCOD/(m3∙d)), the diversity and percentage of fermentative bacteria were markedly increased. Apart from fermentative bacteria, an obvious shift was observed in hydrogen-producing acetogens and nonacetotrophic methanogens as OLR increased. Syntrophobacter, Geobacter and Methanomethylovorans were the dominant hydrogen-producing acetogens and methylotrophic methanogens in the samples of OLR12 and OLR36. When the OLR was increased to 54.0 kgCOD/(m3∙d), the main hydrogen-producing acetogens and hydrogenotrophic methanogens were substituted with Desulfovibrio and Methanospirillum. However, the composition of acetotrophic methanogens (Methanosaeta) was relatively stable during the whole operation period of the UASB reactor.

The effect of mixing on fermentation of primary solids, glycerol, and biodiesel waste.

In this study, the effect of mixing on volatile fatty acid (VFA) production and composition was investigated through running five identical bench-scale reactors that were filled with primary solid and dosed with either pure glycerol or biodiesel waste. Experimental results revealed that there was an inverse correlation between the mixing intensity and the VFA production. The total VFA production in the un-mixed reactor was 9,787 ± 3,601 mg COD/L, whereas in the reactor mixed at 100 rpm this dropped to 3,927 ± 1,175 mg COD/L, while both types of reactor were dosed with pure glycerol at the beginning of each cycle to reach the initial concentration of 1,000 mg/L (1,217 mg COD/L). Propionic acid was the dominant VFA in all the reactors except the reactor mixed at 30 rpm. It is hypothesized that low mixing facilitated hydrogen transfer between obligate hydrogen producing acetogens (OHPA) and hydrogen consuming acidogens in these non-methanogenic reactors. Also, in a narrower range of mixing (0 or 7 rpm), the total VFA production in biodiesel waste-fed reactors was considerably higher than that of pure glycerol-fed reactors.

Enhanced methane production through bioaugmentation of butyrate‐oxidizing hydrogen‐producing acetogens in anaerobic wastewater treatment

A feasible and readily available source of hydrogen‐producing acetogens (HPAs) was developed by obtaining microflora D83 dominated by butyrate‐oxidizing HPAs through enrichment and subculture of anaerobic sludge with the use of butyric acid. The effect of bioaugmentation of D83 both on methane production through glucose fermentation and molasses wastewater treatment was evaluated. At a bacterial number‐to‐activated sludge ratio of 1:9, inoculation of microflora D83 could enhance methane yield and production rate from glucose by a factor of 2.1 and 2.0, respectively. In treatment of normal molasses wastewater, chemical oxygen demand (COD) removal efficiency improved from 75.6% to 88.2%, and the accumulated methane yield and methane yield obtained through COD removal increased by a factor of 2.3 and 2.0, respectively. Hydrogen‐producing acetogenesis is apparently a rate‐limiting step in methanogenesis because bioaugmentation of HPAs during methane production improved not only hydrogen‐producing acetogenesis but also hydrolysis/acidogenesis and methanogenesis. © 2017 American Institute of Chemical Engineers Environ Prog, 37: 367–374, 2018

A syntrophic propionate-oxidizing microflora and its bioaugmentation on anaerobic wastewater treatment for enhancing methane production and COD removal

Methane fermentation process can be restricted and even destroyed by the accumulation of propionate because it is the most difficult to be anaerobically oxidized among the volatile fatty acids produced by acetogenesis. To enhance anaerobic wastewater treatment process for methane production and COD removal, a syntrophic propionate-oxidizing microflora B83 was obtained from an anaerobic activated sludge by enrichment with propionate. The inoculation of microflora B83, with a 1:9 ratio of bacteria number to that of the activated sludge, could enhance the methane production from glucose by 2.5 times. With the same inoculation dosage of the microflora B83, COD removal in organic wastewater treatment process was improved from 75.6% to 86.6%, while the specific methane production by COD removal was increased by 2.7 times. Hydrogen-producing acetogenesis appeared to be a rate-limiting step in methane fermentation, and the enhancement of hydrogen-producing acetogens in the anaerobic wastewater treatment process had improved not only the hydrogen-producing acetogenesis but also the acidogenesis and methanogenesis. Open image in new window

Limitation of syntrophic coculture growth by the acetogen.

The syntrophic cooperation between hydrogen-producing acetogens and hydrogenotrophic methanogens relies on a critical balance between both partners. A recent study, provided several indications for the dependence of the biomass-specific growth rate of a methanogenic coculture on the acetogen. Nevertheless, final experimental proof was lacking since biomass-specific rates were obtained from a descriptive model, and not from direct measurement of individual biomass concentrations. In this study, a recently developed quantitative PCR approach was used to measure the individual biomass concentrations in the coculture of Desulfovibrio sp. G11 and Methanospirillum hungatei JF1 on lactate, formate or both. The model-derived growth yields and biomass-specific rates were successfully validated. Experimental findings identified the acetogen as the growth-limiting partner in the coculture on lactate. While the acetogen was operating at its maximum biomass-specific lactate consumption rate, the hydrogenotrophic methanogen showed a significant overcapacity. Furthermore, this study provides experimental evidence for different growth strategies followed by the syntrophic partners in order to maintain a common biomass-specific growth rate. During syntrophic lactate conversion, the biomass-specific electron transfer rate of Methanospirillum hungatei JF1 was three-fold higher compared to Desulfovibrio sp. G11. This is to compensate for the lower methanogenic biomass yield per electron-mole of substrate, which is dictated by the thermodynamics of the underlying reaction.

Physioecologic and Metabolic Properties of a Ayntrophic Acetogenesis Coculture

In anaerobic digestion system, the functional niche of hydrogen-producing acetogens (HPAs) is between acidogenic fermentation bacteria and methanogens. HPAs are quite difficult to be isolated by pure and ampliative cultures due to the obligate anaerobic metabolism property in syntrophic association with methanogenic. In order to enhance the HPAs activities in anaerobic disgestion, the physioecologic and metabolic properties of HPAs under different medium conditions, such as carbon and nitrogen type, influent temperature and pH was investigated in batch cultivation experiments. The result illustrated that under the cultivation temperature of 45°C and pH of 8.0, the butyrate, propionate and benzoate could be utilized by the syntrophic acetogenesis coculture, of which the uptake rate of butyrate was highest. The glucose and sucrose were repulsed however. The metabolic activity of HPAs could be enormously simulated as the mixture of tryptone and yeast was taken as the nitrogen source.

Survey of Combined Hydrogen-Methane Production in Anaerobic Baffled Reactor (ABR)

Anaerobic fermentative technology is an important route to solving environmental pollution and resources problems. Combined hydrogen and methane production in a two-stage process is a concept which has been developed in recent years Anaerobic biological treatment organic wastewater can produce large amounts of hydrogen and methane，which can be used as energy carrier. At present，the research focusing on the adjustment of fermentation hydrogen-methane production has been conducted. Furthermore, the simultaneous hydrogen-methane production was tested and optimized. However, it lacked combined hydrogen-methane production in an anaerobic reactor in literature so far. Based on preview experiment, the paper studied the simultaneous hydrogen-methane fermentation in an anaerobic baffled reactor (ABR) system. ABR has the advantage of biomass phase separation and integration of acidogenic and methanogenic processes to simultaneously conduct hydrogen-methane production during wastewater treatment. Through deep biohydrogen production, it can enhance the activity of hydrogen-producing acetogens (HPA) and the efficiencies of the combined hydrogen-methane fermentation system. It showed to enhance the activity of HPA was the key to the combined hydrogen-methane production.

Metabolic Pathways of Hydrogen Production in Fermentative Acidogenic Microflora

Biohydrogen production from organic wastewater by anaerobically activated sludge fermentation has already been extensively investigated, and it is known that hydrogen can be produced by glucose fermentation through three metabolic pathways, including the oxidative decarboxylation of pyruvic acid to acetyl-CoA, oxidation of NADH to NAD+, and acetogenesis by hydrogen-producing acetogens. However, the exact or dominant pathways of hydrogen production in the anaerobically activated sludge fermentation process have not yet been identified. Thus, a continuous stirred-tank reactor (CSTR) was introduced and a specifically acclimated acidogenic fermentative microflora obtained under certain operation conditions. The hydrogen production activity and potential hydrogen-producing pathways in the acidogenic fermentative microflora were then investigated using batch cultures in Erlenmeyer flasks with a working volume of 500 ml. Based on an initial glucose concentration of 10 g/l, pH 6.0, and a biomass of 1.01 g/l of a mixed liquid volatile suspended solid (MLVSS), 247.7 ml of hydrogen was obtained after a 68 h cultivation period at 35 +/- 1 degrees C. Further tests indicated that 69% of the hydrogen was produced from the oxidative decarboxylation of pyruvic acid, whereas the remaining 31% was from the oxidation of NADH to NAD+. There were no hydrogen-producing acetogens or they were unable to work effectively in the anaerobically activated sludge with a hydraulic retention time (HRT) of less than 8 h.

Effect of illumination on the hydrogen-production capability of anaerobic activated sludge

To investigate the influence of illumination on the fermentative hydrogen production system, the hydrogen production efficiencies of two kinds of anaerobic activated sludge (floc and granule) from an anaerobic baffled reactor were detected under visible light, dark and light-dark, respectively. The 10 mL floc sludge or granular sludge was respectively inoculated to 100 mL diluted molasses (chemical oxygen demand of 8000 mg·L−1) in a 250 mL serum bottle, and cultured for 24 h at 37°C under different illumination conditions. The results showed that the floc was more sensitive to illumination than the granule. A hydrogen yield of 19.8 mL was obtained in the dark with a specific hydrogen production rate of 3.52 mol·kg−1MLVSS·d−1 (floc), which was the highest among the three illumination conditions. Under dark condition, the hydrogen yield of floc sludge reached the highest with the specific hydrogen production rate of 3.52 mol·kg−1MLVSS·d−1, and under light-dark, light, the specific hydrogen production rate was 3.11 and 2.21 mol·kg−1MLVSS·d−1, respectively. The results demonstrated that the illumination may affect the dehydrogenase activity of sludge as well as the activity of hydrogen-producing acetogens and then impact hydrogen production capacity.

Identification of putative benzene-degrading bacteria in methanogenic enrichment cultures

Anaerobic benzene-degrading enrichment cultures performing methanogenesis were obtained from non-contaminated lotus field soil. Stable isotope probing with 13C benzene was used to detect the bacteria that were involved in benzene degradation. Denaturing gradient gel electrophoresis (DGGE) of fractionated samples exhibited an obvious shift of some DGGE bands to a heavier DNA fraction. An almost full-length 16S rRNA gene sequence corresponding to the DGGE band, namely Hasda-A, was obtained by constructing a clone library of the heavier fraction. The Hasda-A sequence showed only 85.1% identity with the closest identified bacterium, Syntrophus gentianae. Hasda-A may be an important bacterium involved in the initial steps of benzene degradation under methanogenic conditions, as it was the most prominent bacterium that assimilated labeled benzene early in the process of benzene degradation. A primer set was designed to quantify the gene copies of Hasda-A by quantitative PCR. Hasda-A was present at a concentration of (3.5+/-0.8) x 10(6) copies/mL and represented 8.4% of gene copies among bacteria in the enrichment culture. The enrichment culture consisted of three dominant bacterial groups: Hasda-A and both aceticlastic and hydrogenotrophic methanogens. Methane is believed to be produced from benzene by the sequential degradation of benzene by fermenting bacteria, hydrogen-producing acetogens, and methanogens.

Enhanced sulfate reduction with acidogenic sulfate-reducing bacteria

Sulfate reduction in a continuous flow, acidogenic reactor using molasses wastewater as the carbon source was studied at varying chemical oxygen demand/sulfate (COD/SO 4 2−) ratios. At a critical COD/SO 4 2− ratio of 2.7, neither COD nor sulfate were in excess for extra production of ethanol or acetate in the reactor. An acetic-type microbial metabolism was established with sulfate-reducing bacteria (SRB) significantly consuming hydrogen and volatile fatty acids produced by acidogenic bacteria and hydrogen producing acetogens in degrading COD, thereby yielding sulfate removal rate >94.6%. A low critical COD/SO 4 2− ratio of 1.6 was also observed with the enriched ASRB population in reactor which overcomes the barrier to the treatment capability of sulfate-laden wastewater treatment with limited COD supply.

Inhibitory effect of ammonia nitrogen on specific methanogenic activity of anaerobic granular sludge

A series of batch experiments were conducted in 125 mL serum bottles to assess the toxicity of different concentrations of ammonia nitrogen to the specific methanogenic activity of anaerobic granular sludge from upflow anaerobic sludge bed(UASB) and expanded granular sludge bed(EGSB) reactors. The effects of pH value and temperature on toxicity of ammonia nitrogen to anaerobes were investigated. The results show that the specific methanogenic activity of anaerobic granular sludge suffers inhibition from ammonia nitrogen, the concentrations of ammonia nitrogen that produce 50% inhibition of specific methanogenic activity for sludge from UASB and EGSB reactor are 2.35 and 2.75 g/L, respectively. Hydrogen utilizing methanogens suffers less inhibition from ammonia nitrogen than that of acetate utilizing methanogens. Hydrogen-producing acetogens that utilize propionate and butyrate as substrates suffer serious inhibition from ammonia nitrogen. The toxicity of ammonia nitrogen to anaerobic granular sludge enhances when pH value and temperature increase. Anaerobic granular sludge can bear higher concentrations of ammonia nitrogen after being acclimated by ammonia nitrogen for 7 d.

Effect of iron on the sensitivity of hydrogen, acetate, and butyrate metabolism to inhibition by long-chain fatty acids in vegetable-oil-enriched freshwater sediments

Freshwater sediment microbial communities enriched by growth on vegetable oil in the presence of a substoichiometric amount of ferric hydroxide (sufficient to accept about 12% of the vegetable-oil-derived electrons) degrade vegetable oil to methane faster than similar microbial communities that develop when sediments are enriched by growth on vegetable oil in the absence of ferric hydroxide. This study examined the effects of enrichment in the presence of Fe(III) on the fatty-acid sensitivity of several important members of anaerobic triglyceride-degrading microbial communities in freshwater sediments. The fatty-acid sensitivity of three groups of microorganisms—hydrogenotrophic methanogens, acetate consumers, and hydrogen-producing acetogens—were investigated by comparing the rates of hydrogen, acetate, or butyrate consumption in the presence and absence of oleic acid. Methanogenesis from hydrogen was not affected by sediment enrichment conditions or by the presence of oleic acid, suggesting that hydrogenotrophic methanogens were insensitive to fatty acid inhibition in these sediments. Oleic acid inhibited the anaerobic degradation rates of acetate and butyrate by 38% and 63%, respectively, but enrichment in the presence of Fe(III) eliminated the fatty-acid sensitivity of acetate degradation and reduced the sensitivity of butyrate degradation by about half. These results suggest that iron-reducing bacteria may provide an alternative pathway through which vegetable oil can be converted to methane in anaerobic freshwater sediments.

In situ substrates for sulfidogens and methanogens in municipal anaerobic sewage digesters with different levels of sulfate

To clarify the interaction between sulfidogens and methanogens in municipal anaerobic digesters, in situ substrate utilization by the two microbial groups was characterized by the accumulation rates of substrates after the addition of specific inhibitors; 20 mM of molybdate for sulfidogens and 0.01% (v/v) of chloroform for methanogens in three anaerobic sludge digesters with different concentrations of sulfate (A, 1.2; B, 1.8; C, 2.7 mM). The rates of sulfate reduction and methane production in sludge C were the highest and the lowest, respectively among the three sludges. The major substrates for methanogens were acetate (35–58%), and propionate (17–22%) in all the sludges. Propionate should be utilized by methanogens after conversion to acetate and H 2 by syntrophic hydrogen-producing acetogens, as no methanogens are known to directly utilize propionate. Detectable accumulated substrates for sulfidogens (<20%) were propionate in sludges A and B and acetate in C. Still approximately 80% of substrates for sulfidogens were unknown. These results show that acetate or propionate is a common substrate for sulfidogens and methanogens but sulfidogens can mainly grow on the other substrates in the municipal anaerobic sludges, implying nutritional segregation between sulfidogens and methanogens.

Interactions between methanogenic, sulfate-reducing and syntrophic acetogenic bacteria in the anaerobic degradation of benzoate

Effect of sulfate on the anaerobic degradation of benzoate was investigated by using the chemostat-type reactors at 35°C. The benzoate concentrations were equivalent to 1250–10000 mg.l −1 in COD (chemical oxygen demand) and the sulfate concentrations were equivalent to 167–1670 mg.l −1 in sulfur (S). Interactions between the methane-producing bacteria (MPB) and sulfate-reducing bacteria (SRB) were dependent strongly on the ratio of COD/S in wastewater. The MPB consumed 99% of the available electron donors at COD/S ratio of 60, but consumed only 69% at ratio of 1.5, and 13% at 0.75. The biochemical reactions and the bacterial composition in the biomass were also governed by the COD/S ratio. At high COD/S ratios (3.0 or higher), benzoate was degraded mainly to methane via acetate and hydrogen/formate. The degradation of benzoate required the syntrophic association between the hydrogen-producing acetogens such as Syntrophus buswellii and hydrogen-consuming MPB, plus Methanothrix-like MPB. On the other hand, at low COD/S ratio (1.5 or lower), benzoate was consumed mainly by SRB, converting sulfate into sulfide and suppressing the methane production. The anaerobic degradation of benzoate was partially inhibited when sulfide concentration was high.

Microstructural analysis of UASB granules treating brewery wastewater

The microstructure of granules from a full-scale UASB reactor treating brewery wastewater was examined using light microscopy as well as the scanning and transmission electron microscopies. The granules typically have a complex, layered structure. The outer layer of the granule was densely packed with various types of bacteria, including cocci, bacilli, Methanosarcina, and Methanothrix. The second layer was composed of a matrix of Methanothrix with two types of microcolony showing evidence of syntrophic associations between hydrogen-producing acetogens and hydrogen-consuming methanogens. The interior of the granule, on the other hand, was predominantly Methanothrix with scattered microcolonies of syntrophic associations, which became sparse and disappeared toward the centre core of the granule.