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Two protocols involving batch cultures were used to investigate the bioaugmentation of methane production by Pecoramyces ruminantium, and Methanobrevibacter thaueri. Protocol I examined the effect of altering the proportion of the microbial constituents in inoculum on alfalfa stalk fermentations and showed a 25 % improvement in dry matter loss in cultures where the inoculum contained just 30 % of co-culture and 70 % of fungal monoculture. Protocol II involved consecutive cultures and alternating inoculations. This protocol resulted in 17-22 mL/g DM methane production with co-cultures a 30 % increase in methane relative to the fungal monoculture. Both protocols indicate that the co-culture rapidly dominated and was more resilient than the monoculture. Synergistic interaction between fungus and methanogen, promoted more efficient lignocellulose degradation and higher methane yield. This study highlighted the potential of microbial co-cultures for enhancing methane production from lignocellulosic biomass, offering a promising bioaugmentation strategy for improving biogas yields and waste valorization., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In this study, rumen content was used to obtain three enrichments of anaerobic fungi and methanogens (F + M enrichment), bacteria and methanogens (B + M enrichment), and whole rumen content (WRC enrichment), to evaluate their respective ability to degrade lignocellulose and produce methane. Among the treatments, F + M enrichment elicited the strongest lignocellulose degradation and methane production ability with both rice straw and wheat straw as substrates. Quantitative real-time PCR analysis and diversity analyses of methanogens in the three enrichment treatments demonstrated that F + M had larger number of 16S rRNA gene copies of methanogens and higher relative abundance of Methanobrevibacter, the predominant methanogen found in all enrichments. Caecomyces was the main anaerobic fungal genus for co-culturing to provide substrates for methanogens in this enrichment. Importantly, the F + M enrichment was stable and could be maintained with transfers supplied every 3 days, confirming its potential utility in anaerobic digestion for lignocellulose degradation and methane production.

The effects of nitroglycerine (NG) on the rumen methane emission, fermentation, and microbial community of Hu sheep were investigated. Eight sheep were fed NG (100 mg/head/day); another eight sheep served as controls. NG decreased methane emission of Hu sheep by ~ 19.3% (P < 0.05) without adversely affecting the production performance or rumen fermentation (P > 0.05). The alpha and beta diversity indexes of the bacterial and archaeal community showed no significant differences (P > 0.05). The dominant methanogenic species was the Methanobrevibacter gottschalkii clade, accounting for ~ 60%, followed by the Methanobrevibacter boviskoreani and Methanobrevibacter ruminantium clades. Prevotella 1 was the most dominant bacterial genus, accounting for ~ 42%, followed by the Rikenellaceae RC9 and Bacteroidales BS11 gut groups. In addition, pearson correlation analysis showed a few Methanomassiliicoccales species significantly correlated with several bacterial genera (P < 0.05).

Anaerobic fungi (phylum Neocallimastigomycota), an early branching family of fungi, are commonly encountered in the digestive tract of mammalian herbivores. To date, isolates from ten described genera have been reported, and several novel taxonomic groupings are detected using culture-independent molecular methods. Anaerobic fungi are recognized as playing key roles in the decomposition of lignocellulose (up to 50% of the ingested and untreated lignocellulose), with their physical penetration and extracellular enzymatical secretion of an unbiased diverse repertoire of cell-wall-degrading enzymes. The secreted cell-wall-degrading enzymes of anaerobic fungi include both free enzymes and extracellular multi-enzyme complexes called cellulosomes, both of which have potential as fiber degraders in industries. In addition, anaerobic fungi can provide large amounts of substrates such as hydrogen, formate, and acetate for their co-cultured methanogens. Consequently, large amounts of methane can be produced. And thus, it is promising to use the co-culture of anaerobic fungi and methanogens in the biogas process to intensify the biogas yield owing to the efficient and robust degradation of recalcitrant biomass by anaerobic fungi and improved methane production from co-cultures of anaerobic fungi and methanogens.

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.

Although the scheme of metabolic pathways involved in the production of the major end products has been described, the dynamic profile of metabolites of anaerobic fungi co-cultured with methanogens is limited, especially for the intermediate metabolites. In the present study, the fermentation of the co-culture of Piromyces sp. F1 and Methanobrevibacter thaueri on glucose was investigated. The presence of methanogens shortened the growth lag time of anaerobic fungi and enhanced the total gas production. The occurrence of the maximum cell dry weight and the disappearance of most of the substrate were observed at 24 h for the co-culture and 48 h for the fungal mono-culture. In the co-culture, hydrogen was detected at a very low level during fermentation, and formate transitorily accumulated at 24 h and disappeared at 48 h, resulting in an increase of pH. Acetate was higher during the fermentation in the co-culture (P < 0.05), while lactate and ethanol were higher only in the initial stage of fermentation (P < 0.05). After 48 h, lactate in the mono-culture became much higher than that in the co-culture (P < 0.05), and ethanol tended to remain the same in both cultures. Moreover, malate tended to be exhausted in the co-culture, while it accumulated in the mono-culture. Citrate was also detected in both co-culture and mono-culture. Collectively, these results suggest that methanogen enhanced the malate pathway and weakened the lactate pathway of anaerobic fungus.

Objective: We studied the microbial interaction between anaerobic fungi and methanogens in the rumen of Holstein Cow., Methods: Co-cultures of anaerobic fungi with indigenously associated methanogen were isolated by Hungate roll-tube technique. The anaerobic fungi were identified by morphology and 4', 6 diamidino-2-phylindole nucleus staining and the methanogens were identified by 16S rRNA gene sequencing., Results: A total of 28 co-cultures of anaerobic fungus with indigenously associated methanogen were obtained. The anaerobic fungi in the co-cultures were identified as monocentric genera Piromyces, Neocallimastix and Caeomyces. The indigenously associated methanogens were Methanobrevibacter olleyae like and Methanobrevibacter thaueri like strains. Four different phylotypes of fungus-methanogen co-cultures were obtained, which were Piromyces/Methanobrevibacter olleyae like strains, Neocallimastix/ Methanobrevibacter olleyae like strains, Neocallimastix/Methanobrevibacter thaueri like strains and Caecomyces/ Methanobrevibacter olleyae like strains., Conclusion: Our study isolated and identified 28 co-cultures of anaerobic fungus and associated methanogens, which provided new materials for further study the mechanism of methane emission in the rumen.

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. [ABSTRACT FROM AUTHOR]

• Steam explosion mainly decomposed the hemicellulose of corn stover. • Steam explosion did not increase the methane production. • The co-culture could be used to degrade un-pretreated lignocellulose. The aim of this study was to investigate the effects of steam explosion on lignocellulose digestibility of, and methane production from corn stover by a co-culture of anaerobic fungus and methanogen. The cumulative methane production at 72 h of incubation from the steam-exploded corn stover was 32.2 ± 1.74 mL, which not significantly different (P > 0.05) from that of the untreated corn stover (37.1 ± 1.09 mL). However, steam explosion decreased the hemicellulose contents of corn stover by 28.0 ± 0.39% and increased the neutral detergent solute by 23.5 ± 0.25%. While this treatment did not affect the dry matter digestibility (64.1 ± 0.26%, and 64.1 ± 0.28%, respectively). In conclusion, the co-culture of anaerobic fungus and methanogen can degrade the crude fibrous portion of corn stover without any pretreatments. It possesses promising biotechnological prospects for conversion of crop residue based straw resources to obtain biofuel in the form of methane. [ABSTRACT FROM AUTHOR]