Roughage biodegradation by natural co-cultures of rumen fungi and methanogens from Qinghai yaks

Diversity of the fungus–methanogen co-cultures from the rumen of grazing yaks

In this study, 31 natural fungus–methanogen co-cultures were first obtained from the rumen fluid of grazing yaks in spring in Qinghai Province, China, comprising 5 combination types: N. frontalis + M. ruminantium, N. frontalis + M. gottschalkii, O. joyonii + M. ruminantium, C. communis + M. ruminantium, and C. communis + M. millerae. In 2015, we isolated 20 natural fungus–methanogen co-cultures from the rumen fluid of grazing yaks in spring in a Wushaoling pasture of Tianzhu Tibetan Autonomous Prefecture in Gansu Province, China, including 4 combination types: N. frontalis + M. ruminantium, O. joyonii + M. ruminantium, O. joyonii + M. millerae, and Piromyces + M. ruminantium (Wei et al. 2015, 2016). Thus, there were many types of natural fungus–methanogen co-cultures in the rumen of grazing yaks. Furthermore, when compared to the reported natural fungus-methanogen co-cultures isolated from the rumen or faeces of ruminants and non-ruminants by Bauchop et al. (1981), Jin et al. (2011), Leis et al. (2014), Sun et al. (2014), Li et al. (2016) and Li et al. (2021) and grazing yaks by Wei et al. (2015, 2017), in this study, 3 new types of natural anaerobic fungus–methanogen co-culture combinations were first obtained from the rumen of yaks, namely: N. frontalis + M. gottschalkii, C. communis + M. ruminantium, and C. communis + M. millerae. These three types of fungus-methanogen co-cultures all included one fungus and one methanogen, and each methanogen coexisting with each fungus belonged to Methanobrevibacter sp., consistent with the natural fungus–methanogen co-cultures isolated from the rumen or faeces of ruminants and non-ruminants previously reported by Jin et al. (2011), Leis et al. (2014), Sun et al. (2014), Li et al. (2016), Li et al. (2021) and Wei et al. (2015, 2017).

Our study revealed that different combinations of natural fungus–methanogen co-cultures in the rumen of grazing yaks in different regions, probably because of the different types of wild herbages eaten by the grazing yaks in different areas, and these different types of natural fungus–methanogen co-cultures differed in their ability to degrade lignocelluloses. This suggests that there are new and abundant microbial resources for efficiently degrading lignocelluloses in the rumen of yaks grazing on the Qinghai-Tibet Plateau, which have not yet been fully explored.

Roughage degradation by fungus–methanogen co-cultures from the rumen of grazing yaks

During the 5-day incubation, the N. frontalis + M. gottschalkii co-culture YakQH5 degraded the 5 kinds of roughages and showed degradation potential, including high lignocellulose-degrading enzyme activities, IVDMD 59.0%-68.1% (from oat straw to rice straw), NDFD 49.5%-59.7% (from wheat straw to corn stalk) and large amounts of FA and PCA releases, which are described in Sect. 3. Accordingly, we found that the degradation degrees of roughages were different. Ranked in terms of highest to lowest decomposition, the substrates were rice straw, corn stalk, sorghum straw, wheat straw, and oat straw, while in terms of highest to lowest degradation, they were corn stalk, rice straw, sorghum straw, oat straw, and wheat straw. The N. frontalis + M. gottschalkii co-culture YakQH5 degraded lignocelluloses by secreting main-chain degrading polysaccharide hydrolases (CMCase, FPase and xylanase) and side-chain degrading esterases (FAE, AE and CAE) with high activities, which could be key lignin-degrading enzymes in enhancing plant cell wall degradation. All these enzymes acted synergistically to effectively decompose lignocelluloses. The N. frontalis + M. gottschalkii YakQH5 degraded sorghum straw to release PCA 11.7 mg/g DM (70.1 μg/mL) as a result of the high PCA content in sorghum straw, consistent with the high activity of CAE when using sorghum stalk as a substrate, implying that the N. frontalis + M. gottschalkii YakQH5 from the rumen of Qinghai yaks can decompose lignin efficiently. Meanwhile, the N. frontalis + M. gottschalkii co-culture YakQH5 degraded wheat straw, corn stalk, rice straw, oat straw and sorghum straw to release very small amounts of VA and PA. The yields of VA and PA releases appeared unrelated to their contents in the roughages. Further study is needed to clarify this finding.

Among the 31 fungus–methanogen co-cultures, the N. frontalis + M. gottschalkii co-culture YakQH5 was screened out with only wheat straw as substrate by measuring gas production. In this case, the lignocellulose degradation and gas production are generally positive correlation.

The N. frontalis + M. gottschalkii co-culture YakQH5 degraded different roughages as substrates, the order of the lowest to highest IVDMD was: oat straw, wheat straw, sorghum straw, corn stalk, and rice straw; the order of the lowest to highest NDFD was: wheat straw, oat straw, sorghum straw, rice straw and corn stalk; and the order of the lowest to highest gas production was: corn stalk, sorghum straw, oat straw, rice straw and wheat straw. When the 5 kinds of roughages with different lignocellulose contents were used as substrates, there was not always a linear correlation between IVDMD, NDFD and gas production. This phenomenon may have been related to the different compositions of the five roughages. The lignocellulose degradation mechanism of the anaerobic fungi needs to be further studied to reveal the reason behind this phenomenon.

In 2015 and 2017, we first reported the natural fungus–methanogen co-culture N. frontalis + M. ruminantium Yaktz1 and Piromyces + M. ruminantium Yak-G18 that degraded straws with remarkable efficiency were isolated from the rumen of yaks grazing in Tianzhu Tibetan Autonomous County in Gansu Province of China (Wei et al. 2015, 2016, 2017). During the 7-day incubation, the N. frontalis + M. ruminantium co-culture Yaktz1 degraded 61.7% of wheat straw, 68.8% of corn stalk, and 71.9% of rice straw, with NDFD values of 56.0% on wheat straw, 61.7% on corn stalk, and 55.6% on rice straw, while exhibiting the highest enzyme activity values as follows: xylanase 12,500 mU/mL on wheat straw; FPase 430.3 mU/mL, FAE 11.4 mU/mL, AE 199.3 mU/mL and CAE 5.0 mU/mL on corn stalk, and FA release 24.1 μg/mL and PCA release 50.3 μg/mL on corn stalk as the peak values. Across the 7-day incubation, the Piromyces + M. ruminantium co-culture Yak-G18 degraded 60.5% of wheat straw, 65.0% of corn stalk, 65.9% of rice straw, 66.0% of Chinese wildrye, and 75.0% of alfalfa, with NDFD values of 40.8%–47.5% on the five substrates, showing peak values of xylanase activity ranging from 2750 to 5023 mU/mL (from alfalfa to Chinese wildrye), FPase ranging from 71.9 to 123.5 mU/mL(from rice straw to Chinese wildrye), and AE 66.3–118.1 mU/mL (from alfalfa to Chinese wildrye), releasing little FA and PCA. To date, 3 types of extremely effective fungus–methanogen co-cultures for straw degradation have been obtained from the rumen of yaks: the N. frontalis + M. ruminantium co-culture Yaktz1, the Piromyces + M. ruminantium co-culture Yak-G18, and the N. frontalis + M. gottschalkii co-culture YakQH5. According to degradation capability, the N. frontalis + M. ruminantium co-culture Yaktz1 and N. frontalis + M. gottschalkii co-culture YakQH5 showed the most prominent ability to degrade straws. The N. frontalis + M. gottschalkii co-culture YakQH5 from Qinghai yaks decomposed wheat straw, corn stalk, rice straw, oat straw, and sorghum straw to produce higher xylanase, FPase, and CAE activities than the N. frontalis + M. ruminantium co-culture Yaktz1 from Tianzhu yaks, other natural fungus-methanogen co-cultures (from rumen or faeces of ruminants and non-ruminants), and artificially mixed anaerobic fungus–methanogen co-cultures previously reported, with all kinds of roughages or fiter paper as substrates (Jin et al. 2011; Teunissen et al. 1992a, b; Wei et al. 2015, 2016, 2017). Specifically, the N. frontalis + M. gottschalkii co-culture YakQH5 showed FPase activity on corn stalk that was approximately 2.7 times higher than that of the N. frontalis + M. ruminantium co-culture Yaktz1 on corn stalk. The xylanase produced by N. frontalis + M. gottschalkii YakQH5 has good prospects for industrial application. Concurrently, the N. frontalis + M. gottschalkii co-culture YakQH5 could effectively degrade wheat straw, corn stalk and rice straw with IVDMD and NDFD values analogous to those of the N. frontalis + M. ruminantium co-culture Yaktz1 (Wei et al. 2016) but obviously higher IVDMD values than for other fungus–methanogen co-cultures from the rumen or faeces of ruminants and non-ruminants previously reported, with roughages as substrates (Jin et al. 2011). Values of 33.6%–53.1% IVDMD for wheat straw, corn stalk, bagasse, distiller’s dried grains with solubles (DDGS), wheat bran and rice straw by the Piromyces + M. thaueri CW co-culture from the rumen of goats; 26.8%–57.0% IVDMD for wheat straw, corn stalk, bagasse, DDGS, wheat bran, and rice straw by the Piromyces + Methanobrevibacter sp. Z8 co-culture from the rumen of goats; and 33.5%–48.3% IVDMD for rice straw by the Anaeromyces + M. gottschalkii strain PG co-culture from faeces of mules, the Piromyces + M. gottschalkii strain PG co-culture from faeces of mules, the Piromyces + Methanobrevibacter sp. Z8 co-culture from faeces of camel, the Neocallimastix + Methanobrevibacter sp. Z8 co-culture from feces of camel, and the Piromyces + Methanobrevibacter sp. 1Y co-culture from feces of buffalo, all after a 5-day incubation. The N. frontalis + M. ruminantium co-culture Yaktz1 degraded wheat straw, corn stalk, and rice straw to release the maximum values of FA 24.1 μg/mL and PCA 50.3 μg/mL on corn stalk, slightly lower than those of the N. frontalis + M. gottschalkii co-culture YakQH5 with corn stalk as substrate (Wei et al. 2016).

Our study showed that the N. frontalis + M. gottschalkii co-culture YakQH5 and the N. frontalis + M. ruminantium co-culture Yaktz1 from the rumen of grazing yaks degraded roughages more effectively than previously reported fungus-methanogen co-cultures from the digestive tract of herbivores, including ruminants and non-ruminants, and even some current industrial strains. This study also highlighted that a new-type fungus–methanogen combination, the N. frontalis + M. gottschalkii YakQH5, has been obtained from the rumen of Qinghai yaks, and it can superiorly degrade lignocellulosic materials. The N. frontalis + M. ruminantium co-culture Yaktz1 was isolated from yaks grazing in a Wushaoling pasture located in Tianzhu Tibetan Autonomous Prefecture in Gansu Province of China, an alpine meadow pasture with Kobresia myosuroides (Villars) Foiri as the main species, while the N. frontalis + M. gottschalkii co-culture YakQH5 was isolated from yaks grazing in Xinghai County located in Hainan Tibetan Autonomous Prefecture in Qinghai Province of China, where the pasture was alpine meadow with Festuca Ovina L. as the main species. It can be concluded that the combinations of fungus–methanogen co-cultures from the grazing yaks in different regions may vary, and these natural fungus–methanogen co-cultures had different characteristics and abilities to degrade lignocelluloses. Further studies on the host specificity or substrate specificity of anaerobic fungi are needed.

Meanwhile, the N. frontalis + M. gottschalkii co-culture YakQH5 degraded wheat straw straw, corn stalk, rice straw, oat straw, and sorghum straw to produce the highest yields of CH4 4.6 mmol/g DM on wheat straw and acetate 8.6 mmol/g DM (55.7 mM) on rice straw. These are slightly higher yields than the CH4 and acetate yields produced by the N. frontalis + M. ruminantium co-culture Yaktz1 with wheat straw, corn stalk, and rice straw as substrates during the 7-day incubation; markedly higher than those produced by the Piromyces + M. ruminantium co-culture Yak-G18 on wheat straw, corn stalk, rice straw, Chinese wildrye, and alfalfa during the 7-day incubation; and higher than those produced by most of natural fungus–methanogen co-cultures from the rumen or faeces of ruminants and non-ruminants, and artificially mixed anaerobic fungus–methanogen co-cultures, with roughages, fiter paper, cellulose or glucose as substrates (Jin et al. 2011; Teunissen et al. 1992a, b; Li et al. 2016; Nakashimada et al. 2000; Wei et al. 2015, 2016, 2017).

After methanogen inhibition, the pure fungus N. frontalis YakQH5 degraded wheat straw, corn stalk, rice straw, oat straw and sorghum straw to produce the highest yields of H2 3.9 mmol/g DM and ethanol 45.8 mmol/g DM (260.1 mM) on wheat straw, formate 2.5 mmol/g DM (15.0 mM) on sorghum straw, and lactate 2.5 mmol/g DM (15.0 mM) on sorghum straw. The yields of these end-products were generally higher than those produced by anaerobic fungi from the digestive tract of common ruminants and non-ruminants. The most interesting finding was that its ethanol yield was more greater than that produced by all reported anaerobic fungi with many kinds of roughages as substrates, even exceeding those of some industrial strains that produced ethanol (Jin et al. 2011; Sirohi et al 2013; Nagpal et al. 2011; Paul et al. 2010; Teunissen et al. 1992a, b; Sijtsma and Tan 1993; Thareja et al. 2006; Wei et al. 2016, 2017; Saye et al. 2021). Thus, the fungus N. frontalis YakQH5 is promising for use in the development of ethanol production.

Prospects for the fungus–methanogen co-cultures from the rumen of grazing yaks

Anaerobic fungi have been studied for more than 40 years and are considered to play a crucial role in degrading lignocelluloses. Some methanogens can further enhance the anaerobic fungi's ability to degrade lignocelluloses. Anaerobic fungus–methanogen co-cultures degrade lignocellulosic materials to produce CH4, acetate and lignocellulose degradation enzymes with high activities, even exceeding those of some industrial strains (Teunissen et al. 1993; Cheng et al. 2018; Mountfort and Asher 1989; Solomon et al. 2016; Yang and Xie 2010; Chang and Park 2020). However, until now, anaerobic fungi have not been used for widespread industrial applications due to their strict anaerobic growth requirements, limited preservation methods, difficulty in scale-up, and genetic intractability. Recently, with the improvement of culture media, more species of anaerobic fungi have been discovered and a large number of their nucleotides and proteins have been sequenced (Chang et al. 2020; Hess et al. 2020).

In the present study, the 31 fungus–methanogen co-cultures were first obtained from the rumen of yaks grazing in Qinghai Province of China. These co-cultures included 5 combination types. Among them, during the 5-day incubation, the new-type combination N. frontalis + M. gottschalkii co-culture YakQH5 degraded 59.0%–68.1% of the DM and 49.5%–59.7% of the NDF of wheat straw, corn stalk, rice straw, oat straw and sorghum straw to produce CH4 (3.0–4.6 mmol/g DM) and acetate (7.3–8.6 mmol/g DM) as end-products and released the most FA (4.8 mg/g DM) on corn stalk, PCA (11.7 mg/g DM) on sorghum straw. The peak values of enzyme activitie were as follows: xylanase 12,910 mU/mL on wheat straw, FAE 10.5 mU/mL on corn stalk and CAE 20.5 mU/mL on sorghum straw. The N. frontalis + M. gottschalkii co-culture YakQH5 degraded roughages to produce higher xylanase, CMCase, FAE, AE activities, IVDMD, NDFD, and more CH4 and acetate yields without any pretreatment, than reported for natural fungus–methanogen co-cultures isolated from the digestive tract of ruminants and non-ruminants. This study convincingly proved our original hypothesis that Yak-derived ruminal fungus–methanogen co-cultures have evolved to possess high efficiency to degrade plant lignocellulose.

In follow-up studies, it will be useful strategy to further construct a large-scale continuous culture facility to produce natural complex lignocellulose-degrading enzymes, CH4 and acetate by the N. frontalis + M. gottschalkii co-culture YakQH5, or express its xylanase, CMCase, FEA, AE and CAE genes under aerobic conditions using molecular biology techniques to realize large-scale industrial production and application. The study on combined multiple omics analysis including genomics, transcriptomics, proteomics and metabolomics of the N. frontalis + M. gottschalkii co-culture YakQH5 will help to reveal its lignocellulose degradation mechanism. The fermentation process of the fungus–methanogen co-cultures is mainly carried out in the cytoplasm and hydrogenosomes, and the detailed fermentation mechanism also needs to be further studied and optimized. The fungus–methanogen co-cultures can efficiently degrade and change lignocelluloses into CH4 and acetate, representing potential for biological energy production that cannot be ignored. Therefore, screening the dominant combination of fungus–methanogen co-cultures from grazing yaks for the production of high-quality cellulase, hemicellulase, esterase and CH4 will have broad application prospects. Improving the preservation methods of anaerobic fungi with high activity will further promote their practical application in industrial and agricultural production. In the future, the study of the fungus–methanogen co-cultures from the rumen of Qinghai yaks will potentially be highly important to address feed shortages, agricultural wastes utilization, environmental pollution and energy crises.

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