Anaerobic fungi in the digestive tract of herbivores are one of the critical types of fiber-degrading microorganisms present in the rumen. They degrade lignocellulosic materials using unique rhizoid structures and a diverse range of fiber-degrading enzymes, producing metabolic products such as H2/CO2, formate, lactate, acetate, and ethanol. Methanogens in the rumen utilize some of these products (e.g., H2 and formate) to produce methane. An investigation of the interactions between anaerobic fungi and methanogens is helpful as it provides valuable insight into the microbial interactions within the rumen. During the last few decades, research has demonstrated that anaerobic fungi stimulate the growth of methanogens and maintain methanogenic diversity. Meanwhile, methanogens increase the fiber-degrading capability of anaerobic fungi and stimulate metabolic pathways in the fungal hydrogenosome. The ability of co-cultures of anaerobic fungi and methanogens to degrade fiber and produce methane could potentially be a valuable method for the degradation of lignocellulosic materials and methane production.
Rumen in vitro fermentation was used to evaluate the capacity of nitrooxy compounds to mitigate rumen methane production. The following three nitrooxy compounds, each with different molecular structures, were evaluated: 2,2-dimethyl-3-(nitrooxy) propanoic (DNP), N-[2-(Nitrooxy)ethyl]-3-pyridinecarboxamide (NPD), and nitroglycerin (NG). All three compounds substantially decreased the total gas production, methane production, and the acetate:propionate ratio, while increasing hydrogen production. The growth of methanogens was specifically inhibited by all three compounds, without affecting the abundance of bacteria, anaerobic fungi, or protozoa. However, inhibition of methanogenesis required a much higher dose of DNP when compared to NPD or NG. Further investigations were conducted on NG to determine its effects on the methanogenic community. NG reduced the relative abundance of Methanomassiliicoccales, while increasing the relative abundance of Methanobrevibacter and Methanosphaera. Overall, the results suggested that all three of these nitrooxy compounds could specifically inhibit rumen methanogenesis, but NPD and NG were much more efficient than DNP at rumen methane mitigation.
Background Lignocellulose biomass is the most abundant and renewable material in nature. The objectives of this study were to characterize two endoglucanases TrepCel3 and TrepCel4, and determine the effect of the combination of them (1.2 mg TrepCel3, 0.8 mg TrepCel4) on in vitro rumen fermentation characteristics. In this study, three nature lignocellulosic substrates (rice straw, RS; wheat straw, WS; leymus chinensis, LC) were evaluated for their in vitro digestibility, gas, NH3-N and volatile fatty acid (VFA) production, and microbial protein (MCP) synthesis by adding enzymatic combination. Methods Two endoglucanases’ genes were successfully expressed in Escherichia coli (E. coli) BL21 (DE3), and enzymatic characteristics were further characterized. The combination of TrepCel3 and TrepCel4 was incubated with lignocellulosic substrates to evaluate its hydrolysis ability. Results The maximum enzymatic activity of TrepCel3 was determined at pH 5.0 and 40 °C, while TrepCel4 was at pH 6.0 and 50 °C. They were stable over the temperature range of 30 to 60 °C, and active within the pH range of 4.0 to 9.0. The TrepCel3 and TrepCel4 had the highest activity in lichenan 436.9 ± 8.30 and 377.6 ± 6.80 U/mg, respectively. The combination of TrepCel3 and TrepCel4 exhibited the highest efficiency at the ratio of 60:40. Compared to maximum hydrolysis of TrepCel3 or TrepCel4 separately, this combination was shown to have a superior ability to maximize the saccharification yield from lignocellulosic substrates up to 188.4% for RS, 236.7% for wheat straw WS, 222.4% for LC and 131.1% for sugar beet pulp (SBP). Supplemental this combination enhanced the dry matter digestion (DMD), gas, NH3-N and VFA production, and MCP synthesis during in vitro rumen fermentation. Conclusions The TrepCel3 and TrepCel4 exhibited the synergistic relationship (60:40) and significantly increased the saccharification yield of lignocellulosic substrates. The combination of them stimulated in vitro rumen fermentation of lignocellulosic substrates. This combination has the potential to be a feed additive to improve agricultural residues utilization in ruminants. If possible, in the future, experiments in vivo should be carried out to fully evaluate its effect.
This study aimed to investigate the effects of fumarate and nitroglycerin on rumen fermentation, methane and hydrogen production, and microbiota. In vitro rumen fermentation was used in this study with four treatment groups: control (CON), fumarate (FA), nitroglycerin (NG) and fumarate plus nitroglycerin (FN). Real-time PCR and 16S rRNA gene sequencing were used to analyze microbiota. The results showed that nitroglycerin completely inhibited methane production and that this resulted in hydrogen accumulation. Fumarate decreased the hydrogen accumulation and improved the rumen fermentation parameters. Fumarate increased the concentration of propionate and microbial crude protein, and decreased the ratio of acetate to propionate in FN. Fumarate, nitroglycerin and their combination did not affect the abundance of bacteria, protozoa and anaerobic fungi, but altered archaea. The PCoA showed that the bacterial (Anosim, R = 0.747, p = 0.001) and archaeal communities (Anosim, R = 0.410, p = 0.005) were different among the four treatments. Compared with CON, fumarate restored Bacteroidetes, Firmicutes, Spirochaetae, Actinobacteria, Unclassified Ruminococcaceae, Streptococcus, Treponema and Bifidobacterium in relative abundance in FN, but did not affect Succinivibrio, Ruminobacter and archaeal taxa. The results indicated that fumarate alleviated the depressed rumen fermentation caused by the inhibition of methanogenesis by nitroglycerin. This may potentially provide an alternative way to use these chemicals to mitigate methane emission in ruminants.
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