Increasing the Production of Volatile Fatty Acids from Corn Stover Using Bioaugmentation of a Mixed Rumen Culture with Homoacetogenic Bacteria

Volatile fatty acids (VFA) are industrially versatile chemicals and have a major market. Although currently produced from petrochemicals, chemical industries are moving towards more bio-based VFA produced from abundant, cheap and renewable sources such as lignocellulosic biomass. In this study, we examined the effect of bioaugmentation with homoacetogenic bacteria for increasing VFA production in lignocellulose fermentation process. The central hypothesis of this study was that inhibition of methanogenesis in an in vitro rumen bioreactor fed with lignocellulosic biomass hydrolysate increases the hydrogen partial pressure, which can be redirected towards increased VFA production, particularly acetic acid, through targeted bioaugmentation with known homoacetogenic bacteria. In this study, methanogenesis during ruminal fermentation of wet exploded corn stover was initially inhibited with 10 mM of 2-bromoethanesulfonate (BES), followed by bioaugmentation with either Acetitomaculum ruminis and Acetobacterium woodii in two separate bioreactors. During the inhibition phase, we found that addition of BES decreased the acetic acid yield by 24%, while increasing headspace hydrogen from 1% to 60%. After bioaugmentation, the headspace hydrogen was consumed in both bioreactors and the concentration of acetic acids increased 45% when A. ruminis was added and 70% with A. woodii added. This paper demonstrates that mixed microbial fermentation can be manipulated to increase VFA production through bioaugmentation.

The role of homoacetogenic bacteria as efficient hydrogen scavengers in microbial electrochemical cells (MXCs)

We evaluated the consumption of hydrogen gas at the anode of a microbial electrolysis cell (MEC) and characterized the significance of new interactions between anode respiring bacteria (ARB) and homo-acetogens. We demonstrated the significance of biofilm limitation for direct consumption of H2 over acetate by ARB, using the deep biofilm model. Selective inhibition of the major competing hydrogen sink at the biofilm anode, methanogenesis, resulted in significant increase in electron recovery as electric current (∼10–12 A/m2). The presence of acetate at high concentration in the anode compartment and detection of formate, a known intermediate of the acetyl-CoA pathway, provide evidence towards the role of homoacetogenic bacteria. We also assessed the activity of homoacetogens with reverse transcription quantitative PCR targeting formyltetrahydrofolate synthetase (FTHFS) transcripts, and observed a comparable decrease in the FTHFS transcript numbers with current density and acetate concentrations as we decreased the HRT below 4.5 h. The biofilm anode community was predominated by Deltaproteobacteria (70% of total readouts) along with a fraction of the homoacetogenic genus, Acetobacterium (4% of total readouts), established by pyrosequencing targeting the V6 region of the 16S rRNA. Homoacetogens seem to play a major role as syntrophic members of the biofilm anode community when electron recovery is high.

Investigation of the diversity of homoacetogenic bacteria in mesophilic and thermophilic anaerobic sludges using the formyltetrahydrofolate synthetase gene

Homoacetogenic bacteria are strict anaerobes capable of autotrophic growth on H2/CO2 or CO, and of heterotrophic growth on a wide range of sugars, alcohols, methoxylated aromatic compounds and one carbon compounds, yielding acetate as their sole metabolic end-product. Batch activity tests on anaerobic granular sludge, using H2/CO2 as a substrate and 2-bromoethanesulfonate (BES) as a specific methanogenic inhibitor revealed that H2/CO2 conversion and concomitant acetate production commenced only after a lag period of 60–100 h. This finding suggests that the homoacetogenic population of digester sludge could be maintained by heterotrophic growth on sugars or other organic compounds, rather than by autotrophic growth on H2/CO2. In the present study, two upflow anaerobic sludge bed (UASB) reactors were operated at 37°C and 55°C for two distinct trial periods, each characterised by the application of influents designed to enrich for homoacetogenic bacteria. Specific primers designed for the amplification of the functional gene encoding formyltetrahydrofolate synthetase (FTHFS), a key enzyme in the acetyl-CoA pathway of acetogenesis, were used as a specific probe for acetogenic bacteria. The diversity of acetogens in the granular sludge cultivated in each reactor was revealed by application of FTHFS targeted PCR. Results show that biomass acetogenic composition was dependent upon the operational temperature of the reactor and the substrate supplied as influent.

Hydrogen-Dependent Oxygen Reduction by Homoacetogenic Bacteria Isolated from Termite Guts

ABSTRACT Although homoacetogenic bacteria are generally considered to be obligate anaerobes, they colonize the intestinal tracts of termites and other environments that are not entirely anoxic in space or time. In this study, we investigated how homoacetogenic bacteria isolated from the hindguts of various termites respond to the presence of molecular oxygen. All strains investigated formed growth bands in oxygen gradient agar tubes under a headspace of H2-CO2. The position of the bands coincided with the oxic-anoxic interface and depended on the O2 partial pressure in the headspace; the position of the bands relative to the meniscus remained stable for more than 1 month. Experiments with dense cell suspensions, performed with Clark-type O2 and H2 electrodes, revealed a large capacity for H2-dependent oxygen reduction in Sporomusa termitida and Sporomusa sp. strain TmAO3 (149 and 826 nmol min−1 mg of protein−1, respectively). Both strains also reduced O2 with endogenous reductants, albeit at lower rates. Only in Acetonema longum did the basal rates exceed the H2-dependent rates considerably (181 versus 28 nmol min−1 mg of protein)−1). Addition of organic substrates did not stimulate O2 consumption in any of the strains. Nevertheless, reductive acetogenesis by cell suspensions of strain TmAO3 was inhibited even at the lowest O2 fluxes, and growth in nonreduced medium occurred only after the bacteria had rendered the medium anoxic. Similar results were obtained with Acetobacterium woodii, suggesting that the results are not unique to the strains isolated from termites. We concluded that because of their tolerance to temporary exposure to O2 at low partial pressures (up to 1.5 kPa in the case of strain TmAO3) and because of their large capacity for O2 reduction, homoacetogens can reestablish conditions favorable for growth by actively removing oxygen from their environment.

Localization and In Situ Activities of Homoacetogenic Bacteria in the Highly Compartmentalized Hindgut of Soil-Feeding Higher Termites (Cubitermes spp.)

ABSTRACT Methanogenesis and homoacetogenesis occur simultaneously in the hindguts of almost all termites, but the reasons for the apparent predominance of methanogenesis over homoacetogenesis in the hindgut of the humivorous species is not known. We found that in gut homogenates of soil-feeding Cubitermes spp., methanogens outcompete homoacetogens for endogenous reductant. The rates of methanogenesis were always significantly higher than those of reductive acetogenesis, whereas the stimulation of acetogenesis by the addition of exogenous H2 or formate was more pronounced than that of methanogenesis. In a companion paper, we reported that the anterior gut regions of Cubitermes spp. accumulated hydrogen to high partial pressures, whereas H2 was always below the detection limit (<100 Pa) in the posterior hindgut, and that all hindgut compartments turned into efficient H2 sinks when external H2 was provided (D. Schmitt-Wagner and A. Brune, Appl. Environ. Microbiol. 65:4490–4496, 1999). Using a microinjection technique, we found that only the posterior gut sections P3/4a and P4b, which harbored methanogenic activities, formed labeled acetate from H14CO3 −. Enumeration of methanogenic and homoacetogenic populations in the different gut sections confirmed the coexistence of both metabolic groups in the same compartments. However, the in situ rates of acetogenesis were strongly hydrogen limited; in the P4b section, no activity was detected unless external H2 was added. Endogenous rates of reductive acetogenesis in isolated guts were about 10-fold lower than the in vivo rates of methanogenesis, but were almost equal when exogenous H2 was supplied. We conclude that the homoacetogenic populations in the posterior hindgut are supported by either substrates other than H2 or by a cross-epithelial H2transfer from the anterior gut regions, which may create microniches favorable for H2-dependent acetogenesis.