Obtainment of Lignocellulose Degradation Microbial Community: The Effect of Acid-Base Combination After Restrictive Enrichment

Obtainment of Lignocellulose Degradation Microbial Community: The Effect of Acid-Base Combination After Restrictive Enrichment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Obtainment of Lignocellulose Degradation Microbial Community: The Effect of Acid-Base Combination After Restrictive Enrichment Binbin Hua, Xiaofen Wang, Zongjun Cui This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1074700/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Acid-base combination is used in some cases expecially after restricted enrichment, and has created many lignocellulose-degrading communities. While how it worked is not well understood. In this study, compost was used as inoculum source. Induced community structure changes were analyzed with high throughput sequencing to elucidate the formation processes and determine the mechanisms of acid-base combination. We found that after restricted enrichment, retaining primarily bacteria not only included that could decompose and utilize lignocellulose, such as Clostridium and Pseudomonas , but also synergistic microbiota such as Pseudoxanomonas and Alkalobacillaceae. When the proportion of these two types of bacteria was not balanced, the degradation ability of the microbial community was low or pH changes of it did not compound regular changes , which maybe lead to the failure of restricted enrichment. Microbial communities were re-constituted by acid-base combination, whereby the degrading and synergistic strains were adjusted to a more appropriate proportion. Acid-base combination fixed the instability of microbial communities caused by randomness of restrictive screening enrichment. In this study, the mechanism of acid-base combination was analyzed, which enriched the theoretical system of restricted culture, and provided an effective and controllable technical method for obtaining high-quality lignocellulose-degrading microbial community resources. Molecular Biology General Microbiology acid-base combination restrictive enrichment lignocellulose microbial community Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction In nature, lignocellulose degradation often requires various microorganisms to cooperate as a microbial consortium (Tesfaw and Assefa, 2014 ; Wang et al., 2020 ). Inspired by natural microbial consortia, artificial microbial consortia can be created to address questions in scientific research and problems in industrial production (Xu and Yu, 2021 ). Nevertheless, simple artificial consortia suffer from the shortage of characterized strains for synthetic consortia construction and the inability to co-culture microorganisms stably (Weiland, 2010 ). In contrast, undefined natural consortia originate from environmental microbial communities with an unknown number of constituents, often with outstanding self-stability and low operation requirements (Jawed et al., 2019 ). Given the advantages of restrictive screen enrichment, such as reduced metabolic burden and robustness to environment disturbances, many lignocellulose-degrading microbial communities are enriched by this process (Cui et al., 2002 ; Hui et al., 2013 ; S. et al., 2002; Wang et al., 2011 ). Despite these benefits, however, the uncontrollability of enrichment culturing requires many parallel experiments in order to obtain microbial communities with high lignocellulose degradability. Further, often the degradability of initially suitable communities can decrease in subsequent successive transferring. To overcome these challenges, acid-base combination of microbial community was used in the restrictive screen process (S. et al., 2002). For acid-base variety, one microbial community with low pH was combined with another microbial community with high pH to obtain a novel community whose pH fluctuated within a specific range. With the method of restricted culture and acid-base combination, many lignocellulose-degrading microbial communities, such as MC1 (S. et al., 2002), XDC-2(Guo et al., 2010 ), and WCS-6 (Wang et al., 2011 ), were obtained. MC1, for example, was constructed from compost and is capable of effectively degrading various cellulosic materials, including rice straw (Cui et al., 2002 ; Hua et al., 2014 ), filter paper (Kato et al., 2004 ), cardboard (Yuan et al., 2012 ). In addition, it has been used for hydrolysis and acidification of agricultural waste (Yu et al., 2017 ), to enhance anaerobic digestion (Hua et al., 2016 ; Yuan et al., 2016 ), for concurrent saccharification and anaerobic digestion of Napier grass (Wen et al., 2015 ). After more than 40 successive generations of culture (S. et al., 2002) and approximately 20 years of research and application (Wang et al., 2021 ), the microbial community has remained stable with a high degradation efficiency. Therefore, in terms of practical application, restrictive screen enrichment and acid-base combination have achieved good results. At the same time, however, little research has been conducted into understanding the mechanisms of acid-base combinations. It is currently unknown how these communities form, particularly how acid-base combinations influence the microbial communities. With recent advances in sequencing, high throughput approaches have made it possible to quantify the composition of microbial communities during this process (Estrela et al., 2021 ) . This study used compost as the inoculum source for the restrictive screen lignocellulosic degradation microbial community. The community structure changes were analyzed by high throughput sequencing to understand the formation process and reveal the mechanisms of acid-base combinations after long-time enrichment cultures. Materials And Methods Inoculation and enrichment culturing Our inoculum source was compost, sourced from the Zhuozhou Experimental Station, China Agricultural University. The pile was turned over once every two days during the thermophilic period (average 60 ℃) and once every five days after the temperature dropped to 35℃. Overall, the experiment lasted for 60 days. Inoculum sources (5g in fresh weight) were sampled during the ripening period (D1) and thermophilic period (D2). These were then inoculated into PCS medium with three replicates (A1, A2, A3 and B1, B2, B3, respectively). Cultures were maintained in 100 mL triangular bottles for 15 days in each generation. The PCS medium was 0.5% peptone, 1 % lignocellulose, 0.5% NaCl, 0.2% CaCO 3 , 0.1% yeast powder, pH natural (about 7.3) (S. et al., 2002). Rice straw was utilised as a carbon source, and small filter paper strips were added to the cultures. Those with decomposed filter paper strips were regarded as degradable and were chosen for the succession cultures. The inoculation was performed statically at 50 ℃ at a volume ratio of 5% (v/v). Pretreatment of lignocellulosic materials and analysis of weight loss The rice straw (harvested from Shangzhuang Experimental Station of China Agricultural University) was cut into approximately 10 cm length, soaked in 1% NaOH for 24 h, and washed with tap water until the pH value was between 7.0 and 8.0. Pieces were then dried at 50℃ and stored for future use. The weight loss of rice straw was tested as previously reported (Hua et al., 2014). Acid-base combination of microbial community Dynamic changes in pH were measured, communities with a slight pH decrease and a slow pH increase were selected. 5 mL liquid of each community was extracted, taken, and combined to create a novel PCS medium. After merging, these new communities were named with the original group numbers (A1A3, A1B3, A3, and B2B3), as shown in Fig 1. Extraction of genomic DNA After five months of restrictive screening, samples were extracted from D1, D2, A1, A2, A3, B1, B2, B3, respectively, and after acid-base combination, samples were also extracted from A1A3, A1B3, A3B2, and B2B3, respectively. Genomic DNA was extracted using the CTAB method (Murray and Thompson, 1980). High-throughput sequencing analysis Genomic DNA was sent off for high throughput sequencing (Allwegene Tech., Co., Ltd, Beijing). The V3+V4 region was amplified using the following universal primer sets: 336 F and 806 R (5′-GTACTCCTACGGG AGGCAGCA-3′and 5′-GTGGACTACHVGG GTWTCTAAT-3′). The amplicon libraries were sequenced using the Illumina Miseq and a 2×300 bp paired-end protocol (Bartram et al., 2011). Sequences were filtered out with a quality value <20. For each sample, the qualified reads were defined as a library and then down-sampled to the smallest library size, which was determined to compare the alpha diversity metrics, including Chao and Shannon (mothur v.1.30.1). After demultiplexing, the reads were assigned species-equivalent operational taxonomic units (OTUs) at 97% sequence similarity. The remaining OTUs were denominated at different classification levels according to the Silva database release 115 (http://www.arb-silva.de). Results Lignocellulosic degradation ability of the obtained microbial communities. The microbial community A1 had the lowest weight loss of rice straw (13.91±4.83%), followed by community B2 (38.28±1.77%). Community B1, A2, B3 and A3 increased successively (42.77±2.86%, 46.03±6.96%, 46.28±3.43%, and 47.08±0.40%) (Fig. 2 A). These values corresponded to changes in pH. Decreases in pH reflected rapid decomposition of cellulose, while increases in pH occurred with decreasing rates or cessation of cellulose degradation (Liu et al., 2006 ). Solutions' pH decreases were a prominent feature of the cellulosic materials degraded microbial community (Liu et al., 2006 ). The pH of all six microbial communities decreased on the third day Fig. 2 C. The pH of A1 and B2 decreased to 6.6 and then began to rise rapidly to alkaline conditions, while A2 and B1 decreased to approximately 6.3. On the third day, A3 and B3 decreased to 6.1 and 6.3, respectively, and remained acidic until the 12th day. On the 15th day, the pH of A3 returned to neutral, while the pH of B3 remained acidic. Microbial degradation of straw resulted in pH decreases in all treatments before rising to slightly alkaline conditions. Although A3 and B3 displayed faster degradation rates, the recovery time to slightly alkaline was longer than 12 days. Therefore, we selected B1 and A2 for further research and utilization. For A3 and B3, as the acidic products were not rapidly utilized, accumulation of these products may inhibit the degrading strains, thus inducing instability of the bacterial communities. To study the acid-base combinations, A1 and B3 were pairwise combined with A3 and B3, respectively (Fig. 1 ). The new microbial communities A1A3, A1B3, A3B2, and B2B3 were created. The rice straw weight losses of A1A3, A1B3, A3B2, and B2B3 were 43.24±6.73%, 32.18±1.19%, 44.01±2.93%, and 38.56±2.83%, respectively, which were significantly higher than those of A1 and B2, but lower than those of A3 and B3, respectively (Fig. 2 B). On the third day, the pH of A1A3, A1B3, A3B2, and B2B3 was 6,6.5, 6.3, and 6.1, and increased to 7.1, 7.4, 8.2, and 7, on the 9th day, respectively (Fig. 2 D). All returned to neutral and then became weakly alkaline. The combined microbial communities all followed the rule of pH fluctuating within 6-9 range, similar to other stable and efficient lignocellulose-decomposing microbial communities in previous reports (Liu et al., 2006 ). Acid-base combinations did not obtain a more efficient lignocellulose-decomposing microbial community, while the pH was more similar to a stable community. Effect of inoculum source on the enrichment of microbial communities Based on the T-test results, group A and group B’s degradation ratio was not significantly different. However, the parallelity of group B was much higher than A. Therefore, both D1 and D2 had potential, while D2 was more suitable to screen high-efficiency microbial communities (Fig. 3 ). Bacterial biodiversity was higher in the thermophilic period than in the ripening period. The bacteria composition of both compost samples was significantly different, which resulted in group A and group B also exhibiting significant differences. The inoculum source had a greater impact on restrictive screen results. In this study, composting during the thermophilic period was a better choice of inoculum source than during the ripening period. Composting in the thermophilic period is often used as an inoculum source to screen cellulose-degrading bacteria (Tesfaw and Assefa, 2014 ). For D2, the temperature of the compost body in the thermophilic period was higher, and organic matter decomposed rapidly. Cellulose materials decomposed more rapidly during this period, and bacteria with cellulose degradation abilities were also the most active. For D1, in the ripening period, the rapid decomposition of the organic matter ended. The structure of the abtained microbial communities after restrictive screen culture A significant number of OTUs in D1 and D2 were eliminated (Fig. 4 A and B). The number of retained OTUs in A1, A2, and A3 were 337, 310, and 418, respectively, with B1, B2, and B3 retaining 408, 392, and 353, respectively. In addition, some OTUs that were not detected in D1 and D2 were detected in our microbial communities. A1, A2, and B2 had 827, 807, and 713 OTUs that were not found in D1, while B1, B2, and B3 had 1128, 862, and 663 OTUs that were not identified in D2. The retained bacteria (amount ratio more than 0.1%) are shown in Table 1 . Bacteria such as Cellulosilyticum have the ability to degrade cellulose. In the community, EMSD5, Cellulosilyticum , and other bacteria, and fungi synergistically degrade whole corn with cobs removed (Zhu et al., 2016 ). In the community SV79, which decomposes various lignocellulosic substrates to produce ethanol, Cellulosilyticum and Acetivibrio, Clostridium, Ruminococcus , and Sporomusa are the dominant bacteria (Zhao et al., 2014 ). Treponema primitia in the intestine of termites can degrade lignin (Lucey and Leadbetter, 2014 ). Pseudomonas, Comamonas , and Lachnoclostridium can also decompose cellulose in a composite community (Liu et al., 2011 ; Xue et al., 2020 ; Zagrodnik et al., 2021 ). Table 1 The bacteria microbiome covered all samples(quantitative proportion above 0.1%) Taxonomy Quantitative proportion (%) A1 A2 A3 B1 B2 B3 D1 D2 A1A3 A1B3 A3B2 B2B3 Alcaligenes 67.1 59.0 10.7 2.2 3.4 1.4 1.7 1.1 2.6 6.1 6.8 4.2 Lachnoclostridium 6.0 0.8 1.4 0.8 1.2 4.1 1.0 0.6 0.5 0.3 0.8 1.8 Cellulosilyticum 3.6 2.5 41.1 1.2 0.6 2.6 0.3 0.2 0.3 0.3 0.3 0.6 Proteiniphilum 3.6 5.1 20.4 2.4 0.7 2.1 0.3 0.2 4.9 3.5 0.7 2.5 unidentified 3.0 2.0 1.0 2.1 5.0 1.0 0.2 1.0 0.4 0.4 1.9 0.6 Comamonas 0.7 0.7 2.1 2.3 31.8 36.5 1.0 1.2 51.3 18.3 4.1 27.0 Pseudomonas 0.7 3.2 2.8 12.4 11.7 11.6 0.5 0.7 10.5 11.5 0.8 2.4 Advenella 0.6 0.3 0.5 47.8 4.1 1.7 0.3 0.2 0.6 3.1 4.4 6.3 Treponema 0.2 0.3 0.5 0.7 4.6 0.8 0.3 0.3 0.9 29.9 18.8 27.8 Clostridium 0.1 0.2 0.6 0.2 0.1 0.3 2.6 0.7 0.1 0.5 0.8 0.7 Some retained bacteria could not use cellulose directly, however, they can exist in communities that decomposed lignocellulose, such as Alcaligenes. They can utilize organic acids and amino acids to produce ammonia and carbon dioxide. Alcaligenes faecalis have been reported in plants, soil, and other environments (Rosenberg, 2014), and Alcaligenes sp TB is an aerobic denitrifying bacterium (Chen et al., 2016 ). Proteiniphilum is widely reported in anaerobic fermentation sludge, as it cannot decompose lignocellulose and cellobiose (Maspolim et al., 2015 ). It can, however, use peptone, arabinose, etc., and its metabolites include small molecular organic acids, CO 2, and H 2 (Langer et al., 2016 ). Treponema caldarium is a strictly anaerobic hyperthermia spirochete, which uses glucose, lactose, and other sugars as substrates, and has been reported in a complex community that degraded naphthalene (Koelschbach et al., 2017 ). These bacteria may assist in the elimination of acidic byproduct that inhibits cellulose-degrade strains. Some bacteria, like Sporomusa , Proteiniclasticum , Dethiosulfovibrionaceae, Desulfotomaculum , Desulfosporosinus , Pseudoxanthomonas , Nocardia , Alteromonadaceae , Erysipelotrichaceae , Novosphingobium , Propionigenium , Nonomuraea , Balneimonas , and Arcobacter which were found in all the six enriched communities, but not in D1 or D2. These newcomers may play specific roles in the microbial community and may have originated from the compost. Their abundance in compost may be extremely low and difficult to detect with high throughput sequencing. After the growth environment changed, their abundance amount increased, allowing for detection with high-throughput sequencing. Additionally, they may also come from the air as the PCS medium was not sterilized. Generally, the enriched microbial community needs to have a high anti-pollution capacity. It was not hortative to be operated in a sterile environment such as an ultra-clean workbench, and thus other bacteria may contaminate them (Himanshu et al., 2017 ). The abundance and composition of fungal OTUs from our communities were similar, which were different from that of the compost (Fig. 4 C). While many fungi were eliminated as a result of the restricted culture, some survived, though their OTUs were less abundant. Some may be involved in straw catabolism, such as Penicillium (Ogunyewo et al., 2020 ), while most fungi had little connection to straw decomposition, like Guehomyces pullulans (Nakagawa et al., 2006 ) and Hyphopichia burtonii (Groenewald and Smith, 2010 ). Effect of acid-base combination The change of microbial community structure by an acid-base combination Among the obtained communities by acid-base combination, A1A3 had the lowest pH (6.0) on the 3rd day and had a greater degradation ratio (43.24±6.73%). In contrast, A1B2 had the highest pH (6.5), and the degradation ratio was only 32.18±1.19%. Therefore, analysis was focused on these two communities to elucidate the mechanism of acid-base combination. A1A3 was combined within group A, and A1B3 was combined between group A and B. Before mixing, the dominant bacteria in A1 was Alcaligenes , with a ratio of 67% (Table 1 , Fig. 5 ), which cannot decompose cellulose. Alcaligenes can use peptone and yeast extract powder in PCS medium and produce alkaline substances, inducing a pH increase. The relative abundance of Cellulosilyticum was less than 3.6%, other bacteria producing acid were much lower than Alcaligenes . The dominant bacteria of A3 was Cellulosilyticum , followed by Proteiniphilum , while the relative abundance of Alcaligenes was 11%. The dominant bacteria of B3 was Commonas , followed by Pseudomonas . The relative abundance of Lachnoclostridium , which may have the ability to decompose cellulose, was only 4%. The total proportion of Advenella , Alcaligenes , and Bordetella , which belong to Alkalobacillaceae, was 4.8%. After the combination, the dominant bacterium of A1A3 was Comamonas , which was different from A1 and A3, and the proportion of Hydrogenispora increased to 12%. The database information may be hydrogenase poraethanolica , a strictly anaerobic bacteria producing hydrogen and ethanol (Liu et al., 2014 ). The relative abundance of Alcaligenes was 3%. After the combination, the dominant strain of A1B3 was Treponema , followed by Comamonas , Pseudomonas , Anaerophaga , and Alcaligenes . Whether it was the combination within one group or between two groups, the dominant bacteria varied greatly. After combination, the ratio of Cellulosilyticum decreased, the Pseudomonas ratio increased, and Clostridium ratio was low. Those three genera all have the potential ability to decompose lignocellulose. Their total proportions in A1, A3, and B3 were 4.4%, 44.5%, and 14.5%, respectively. The relative abundance of them in A1A3 and A1B3 was 10.9%, 12.3%, respectively. The numerical relationship was A3>A1A3>A1, B3>A1B3>A1. A1A3 and A1B3 were significantly higher than A1 before the combination but lower than A3 and B3. The results were consistent with straw degradation. The pH of A1 was higher than 6.6 (Fig. 2 A), and the proportion of Alcaligenes in A1 was the highest corresponding. After the combination, the proportion of Alcaligenes in A1A3 and A1B3 is lower than that in A1, and their pH decreased below 6.5. Change of fungi in the process of acid-base combination Generally, all obtained fungal communities spatially overlapped, indicating a similar species diversity (Fig. 4 C). Take A3B2, for example; the proportion of dominant fungi (1%) in the communities is shown in Fig. 6 . Before the combination, the dominant fungi in A1 were Scedosporium, Candida, Machimura, Penicillium , etc. The dominant fungi in B2 were Candida, Machimura, Acremonium, Penicillium, Scedosporium, IsSatchenkia . After combination, the dominant fungi in A3B2 included Candida, Machimura, Scedosporium, Acremonium, Penicillium, and Clavulina . For fungi, the acid-base combination was just a simple mixing process. The dominant fungi were gathered into the new community, while the proportions likely changed as a result of competition. Discussion The screened bacteria can be divided into three groups. The first group exhibited lignocellulose decomposing functions, such as Clostridium , Cellulosilyticum, Pseudomonas , etc. The second group was likely to have auxiliary functions, such as Alcaligenes, Pseudomonas (decomposing bacteria or auxiliary bacteria), etc. The third group was not directly related to the decomposition of lignocellulose by current reports. It may have unknown functions or may be eliminated in the subculture in the future. While in the screening process, the randomness was apparent. The obtained microbial communities were significantly different with the same inoculum source and under the same culture conditions. For example, the community structure of A1 was significantly different from that of parallel A2 and A3, and the straw degradation rate was much lower than that of A2 and A3. The acid-base combination aimed to obtain a microbial community with a stable and robust ability to decompose straw. The goal is to optimize the variety of different microbiota-producing acids and alkaline without damaging the synergistic relationship between microorganisms in nature. The pH of the obtained microbiota decreased rapidly with the decomposition of straw before returning to a slightly alkaline level. The regular change of pH was closely related to the structure of the community. The composition of dominant bacteria varied greatly, whether within or between groups. The cellulose-degrading strains represented by Clostridium can metabolize straw to produce small molecular organic acids, reducing the pH of the solution. Helper strains represented by Alkalobacillus can metabolize organic acids and amino acids to produce ammonia and carbon dioxide, increasing the solution's pH. When the auxiliary bacteria were dominant in the microbiota, their abundance was very high, such as A1, while the abundance of other strains was low. The decomposition ability of microbial bacteria was insufficient, and the pH was difficult to decline. When the number of auxiliary bacteria in the community was low, such as A3, B3, pH only slowly rose back to slightly alkaline. After the acid-base combination, the microbial community was reconstituted. The degrading and auxiliary strains were adjusted to a more appropriate quantity and proportion, and unrelated strains were knocked out simultaneously, which changed the pH regularly. The acid-base combination was conducive to obtaining an efficient and stable microbial community and correcting microbial communities' reduced decomposition ability caused by a random process in the restricted culture process. Composting in the thermophilic period was a better choice of inoculum source than that of the ripening period. Many bacteria and most fungi were eliminated after the restrictive screening. Degrading strains, such as Clostridium and Pseudomonas , synergistic microbial such as Pseudoxanomonas and Alkalobacillaceae were obtained. Microbial communities were re-constituted by acid-base combination. The degrading and synergistic strains were adjusted to a more appropriate proportion, which changed the pH regularly. Acid-base combination fixed the instability of microbial communities caused by randomness of restrictive screening enrichment. The combination of the two techniques is high conducive to obtain efficient lignocellulose degradation microbial resources. Declarations Acknowledgements This research was financially supported by the National Natural Science Foundation of China (NO. 31900107) . Authors’ Contributions Binbin Hua: Investigation, Data curation, Formal analysis, Validation, Writing - original draft, Writing - review & editing. Xiaofen Wang: Conceptualization, Methodology, Supervision, Writing-review & editing. Zongjun Cui: Supervision,Conceptualization. Conflict of Interest The authors declare that they have no conflicts of interest. Funding Information This research was financially supported by the National Natural Science Foundation of China (NO. 31900107) . References Bartram AK, Lynch MDJ, Stearns JC, Moreno-Hagelsieb G, Neufeld JD (2011) Generation of Multimillion-Sequence 16S rRNA Gene Libraries from Complex Microbial Communities by Assembling Paired-End Illumina Reads. Appl Environ Microb 77(15):5569–5569 DOI Chen J, Gu S, Hao H, Chen J (2016) Characteristics and metabolic pathway of Alcaligenes sp. TB for simultaneous heterotrophic nitrification-aerobic denitrification. Appl Microbiol Biot 100(22):9787–9794 DOI Cui Z, Li M, Piao Z, Huang Z, Ishii M, Igarashi Y (2002) Selection of a composite microbial system MC1 with efcient and stability cellulose degradation bacteria and its function. Environ Sci 23:36–39 DOI Estrela S, Sánchez Á, Rebolleda-Gómez M (2021) Multi-Replicated Enrichment Communities as a Model System in Microbial Ecology. Front Microbiol 12:657467 DOI Groenewald M, Smith MT (2010) Re-examination of strains formerly assigned to Hyphopichia burtonii, the phylogeny of the genus Hyphopichia, and the description of Hyphopichia pseudoburtonii sp. nov. Int J Syst Evol Micr 60(11):2675–2680 DOI Guo P, Zhu W, Wang H, Lu Y, Wang X, Zheng D, Cui Z (2010) Functional characteristics and diversity of a novel lignocelluloses degrading composite microbial system with high xylanase activity. J Microbiol Biotechnol 20(2):254–264 DOI Himanshu H, Voelklein MA, Murphy JD, Grant J, O'Kiely P (2017) Factors controlling headspace pressure in a manual manometric BMP method can be used to produce a methane output comparable to AMPTS. Bioresour Technol 238:633–642 Hua B, Dai J, Liu B, Zhang H, Yuan X, Wang X, Cui Z (2016) Pretreatment of non-sterile, rotted silage maize straw by the microbial community MC1 increases biogas production. Bioresour Technol 216:699–705 Hua B, Lu Y, Wang J, Wen B, Cao Y, Wang X, Cui Z (2014) Dynamic Changes in the Composite Microbial System MC1 During and Following its Rapid Degradation of Lignocellulose. Appl Biochem Biotech 172(2):951–962 DOI Hui W, Jiajia L, Yucai L, Peng G, Xiaofen W, Kazuhiro M, Zongjun C (2013) Bioconversion of un-pretreated lignocellulosic materials by a microbial consortium XDC-2. Bioresour Technol 136:481–487 DOI Jawed K, Yazdani SS, Koffas MA (2019) Advances in the development and application of microbial consortia for metabolic engineering.Metabolic Engineering Communications,9. https://doi.org/10.1016/j.mec.2019.e00095 Kato S, Haruta S, Cui ZJ, Ishii M, Igarashi Y (2004) Effective cellulose degradation by a mixed-culture system composed of a cellulolytic Clostridium and aerobic non-cellulolytic bacteria. FEMS Microbiol Ecol 51(1):133–142 Koelschbach JS, Mouttaki H, Pickl C, Heipieper HJ, Meckenstock RU (2017) Rectinema cohabitans gen. nov., sp. nov., a rod-shaped spirochete isolated from an anaerobic naphthalene-degrading enrichment culture. Int J Syst Evol Microbiol 67(5):1288–1295 DOI Langer T, Hahnke S, Klocke M, Koeck DE (2016) Description of Proteiniphilum saccharofermentans sp. nov., Petrimonas mucosa sp. nov. and Fermentimonas caenicola gen. nov., sp. nov. isolated from mesophilic lab-scale biogas reactors and emended description of the genus Proteiniphilum.Int. J. Syst. Evol. Microbiol., 66(3). DOI : 10.1099/ijsem.0.000902 Liu J, Wan L, XiuHong X, HongTao L (2011) Effect of Cellulose-decomposing Strain on Microbial Community of Cow Manure Compost. Environ Sci 32(10):3073 DOI Liu JB, Wang WD, Yang HY, Wang XF, Gao LJ, Cui ZJ (2006) Process of rice straw degradation and dynamic trend of pH by the microbial community MC1. J Environ Sci (China) 18(6):1142–6 DOI Liu Y, Qiao JT, Yuan XZ, Guo RB, Qiu YL (2014) Hydrogenispora ethanolica gen. nov., sp nov., an anaerobic carbohydrate-fermenting bacterium from anaerobic sludge. Int J Syst Evol Micr 64(5):1756–1762 DOI Lucey KS, Leadbetter JR (2014) Catechol 2,3-dioxygenase and other meta‐cleavage catabolic pathway genes in the 'anaerobic' termite gut spirochete Treponema primitia. Mol Ecol 23(6):1531–1543 DOI Maspolim Y, Zhou Y, Guo C, Xiao K, Ng WJ (2015) The effect of pH on solubilization of organic matter and microbial community structures in sludge fermentation.Bioresource Technol. DOI : 10.1016/j.biortech.2015.04.087 Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8(19):4321–5 DOI Nakagawa T, Ikehata R, Uchino M, Miyaji T, Takano K, Tomizuka N (2006) Cold-active acid beta-galactosidase activity of isolated psychrophilic-basidiomycetous yeast Guehomyces pullulans. Microbiol Res 161(1):75–79 DOI Ogunyewo OA, Randhawa A, Gupta M, Kaladhar VC, Yazdani SS (2020) Synergistic Action of a Lytic Polysaccharide Monooxygenase and a Cellobiohydrolase from Penicillium funiculosum in Cellulose Saccharification Under High Substrate Loading.Appl. Environ. Microb., 86(23). DOI : 10.1128/AEM.01769-20 Rosenberg E, Delong EF, Lory S (2014) et al The Prokaryotes:The Family Methylobacteriaceae[J] Chapter 256313–340. 10.1007/978-3-642-30197-1 S ., H ., Z ., C ., Z ., H ., M ., L ., M ., I ., Y ., I ., Construction of a stable microbial community with high cellulose-degradation ability .Appl. Microbiol. Biot., 59 ( 4-5 ), 529-534 . DOI : 10.1007/s00253-002-1026-4 Tesfaw A, Assefa F (2014) Co-culture: A great promising method in single cell protein production. Biotechnology and Molecular Biology Reviews 9(2):12–20 Wang J, Zhu D, Zhao S, Xu S, Yang R, Zhao W, Zhang X, Huang Z (2021) Effect of liquid volume and microflora source on degradation rate and microbial community in corn stover degradation.AMB Express, 11(1). DOI : 10.1186/s13568-021-01233-5 Wang R, Zhao S, Wang Z, Koffas MA (2020) Recent advances in modular co-culture engineering for synthesis of natural products. Curr Opin Biotech 62:65–71 Wang W, Yan L, Cui Z, Gao Y, Wang Y, Jing R (2011) Characterization of a microbial consortium capable of degrading lignocellulose. Bioresour Technol 102(19):9321–9324 Weiland P (2010) Biogas production: current state and perspectives. Appl Microbiol Biot 85(4):849–860 DOI Wen B, Yuan X, Li QX, Liu J, Ren J, Wang X, Cui Z (2015) Comparison and evaluation of concurrent saccharification and anaerobic digestion of Napier grass after pretreatment by three microbial consortia. Bioresour Technol 175:102–111 DOI Xu C, Yu H (2021) Insights into constructing a stable and efficient microbial consortium. Chinese J Chem Eng 30:112–120 Xue C, Zhang Q, Owens G, Chen Z (2020) A cellulose degrading bacterial strain used to modify rice straw can enhance Cu(II) removal from aqueous solution. Chemosphere 256:127142 DOI Yu J, Zhao Y, Zhang H, Hua B, Yuan X, Zhu W, Wang X, Cui Z (2017) Hydrolysis and acidification of agricultural waste in a non-airtight system: Effect of solid content, temperature, and mixing mode. Waste Manage 59:487–497 DOI Yuan X, Cao Y, Li J, Wen B, Zhu W, Wang X, Cui Z (2012) Effect of pretreatment by a microbial consortium on methane production of waste paper and cardboard. Bioresour Technol 118:281–288 DOI Yuan X, Ma L, Wen B, Zhou D, Kuang M, Yang W, Cui Z (2016) Enhancing anaerobic digestion of cotton stalk by pretreatment with a microbial consortium (MC1). Bioresour Technol 207:293–301 DOI Zagrodnik R, Duber A, Seifert K (2021) Hydrogen production during direct cellulose fermentation by mixed bacterial culture: The relationship between the key process parameters using response surface methodology. J Clean Prod 314:127971 DOI Zhao C, Deng Y, Wang X, Li Q, Huang Y, Liu B (2014) Identification and characterization of an anaerobic ethanol-producing cellulolytic bacterial consortium from Great Basin hot springs with agricultural residues and energy crops. J Microbiol Biotechnol 24(9):1280–1290 DOI Zhu N, Yang J, Ji L, Liu J, Yang Y, Yuan H (2016) Metagenomic and metaproteomic analyses of a corn stover-adapted microbial consortium EMSD5 reveal its taxonomic and enzymatic basis for degrading lignocellulose. Biotechnol Biofuels 9(1):243 DOI Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 22 Nov, 2021 Reviewers invited by journal 22 Nov, 2021 Editor invited by journal 15 Nov, 2021 Editor assigned by journal 13 Nov, 2021 First submitted to journal 12 Nov, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1074700","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":65080439,"identity":"1e66627f-47bb-4f89-ae45-b72463c2a47e","order_by":0,"name":"Binbin Hua","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYBACAwYGNmaGCiCLh4HhAAMDM7FazpCshbENooWBKC3mEsnPHhfOuyNvznP44QGGCuvEBvazB/BqsZyRZm48c9szw529bQYHGM6kJzbw5CXgd9iNBDNp3m2HGTecZzA4wNh2OLFBgseAgJb0b9K8cw7bbzjP/uEA4z+itOQAbWk4nLjhbA/QlgZitJx5U24849jh5A1nzhQcSDiWbtzGk0NAy/H0bY8Lag7bbjiTvvnDhxpr2X72M/i1oIIEIGYjQf0oGAWjYBSMAhwAAGjrSzgMuJpaAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-5781-5900","institution":"Nankai University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Binbin","middleName":"","lastName":"Hua","suffix":""},{"id":65080440,"identity":"eecc4b39-6563-4bde-a63e-4a317e80af15","order_by":1,"name":"Xiaofen Wang","email":"","orcid":"https://orcid.org/0000-0003-3543-1073","institution":"China Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaofen","middleName":"","lastName":"Wang","suffix":""},{"id":65080441,"identity":"8e20df9e-0c00-40f9-ba77-e8b23b95378d","order_by":2,"name":"Zongjun Cui","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zongjun","middleName":"","lastName":"Cui","suffix":""}],"badges":[],"createdAt":"2021-11-12 14:16:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1074700/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1074700/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":15987663,"identity":"bc26e5b7-1719-4661-95f2-71272e5ca7ba","added_by":"auto","created_at":"2021-11-29 23:03:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":234239,"visible":true,"origin":"","legend":"Schematic diagram of experimental design process","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-1074700/v1/04b11c76383869a902a6b21c.png"},{"id":15987668,"identity":"f137396a-7232-4df7-87e7-ab595fdfbbf4","added_by":"auto","created_at":"2021-11-29 23:03:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":52275,"visible":true,"origin":"","legend":"The weight loss ratio of rice straw dynamic change of pH in microbial communities. A represents six directly enriched microbial communities and B represents four acid and alkaline mixed microbial communities. C represents six directly enriched microbial communities and D represents four acid and alkaline mixed microbial communities.","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-1074700/v1/4ca45dee6db9e4323a9fed5d.png"},{"id":15987820,"identity":"7e0e4f2b-e8d2-43cd-a811-de3489ca0cd8","added_by":"auto","created_at":"2021-11-29 23:06:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":281658,"visible":true,"origin":"","legend":"Bacteria community structure of each group (genus level). “g” represents genus, after “_” are genus names. Other means reference sequence is not found in the database.","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-1074700/v1/c35c95bb6bc7278bbd7dfd35.png"},{"id":15987664,"identity":"86c7550c-4382-4dd1-821f-cbc8770d501b","added_by":"auto","created_at":"2021-11-29 23:03:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":69921,"visible":true,"origin":"","legend":"OTUs venn analysis of microbial community A1, A2, A3 and D1(A); and OTUs venn analysis of microbial community B1, B2, B3 and D2(B); PCA analysis of community diversity based on OTU abundant; (C) is the PCA analysis of bacteria , and (D) is the PCA ananlysis of fungi.","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-1074700/v1/3e9ac3e804c1757952de819e.png"},{"id":15987821,"identity":"b948d69c-9f80-4681-94ce-0a5faa362e48","added_by":"auto","created_at":"2021-11-29 23:06:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":95710,"visible":true,"origin":"","legend":"The dominant bacteria in microbial community A1, A3, B3, A1A3 and A1B3 (genus level).The quantitative proportion lower than 1% are combined as “low proportion”","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-1074700/v1/e8d3a69214ea307970819dd1.png"},{"id":15987667,"identity":"963583b4-715a-450b-a044-ef55a029429d","added_by":"auto","created_at":"2021-11-29 23:03:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":116684,"visible":true,"origin":"","legend":"The preponderant fungus (quantitative proportion above 1%, genus level) in microbial community A3, B2 and A3B2. Unidentified means lack of identified genus level name in the database.Other means no reference sequnance in the database.","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-1074700/v1/9cc555d5a39cbc08c02b8928.png"},{"id":15987822,"identity":"cd61c822-ad9c-4216-94d0-55d75d5e7708","added_by":"auto","created_at":"2021-11-29 23:07:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1296743,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1074700/v1/3fea0dea-57df-4aa2-aceb-3e4235b8c537.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eObtainment of Lignocellulose Degradation Microbial Community: The Effect of Acid-Base Combination After Restrictive Enrichment\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn nature, lignocellulose degradation often requires various microorganisms to cooperate as a microbial consortium (Tesfaw and Assefa, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Inspired by natural microbial consortia, artificial microbial consortia can be created to address questions in scientific research and problems in industrial production (Xu and Yu, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Nevertheless, simple artificial consortia suffer from the shortage of characterized strains for synthetic consortia construction and the inability to co-culture microorganisms stably (Weiland, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In contrast, undefined natural consortia originate from environmental microbial communities with an unknown number of constituents, often with outstanding self-stability and low operation requirements (Jawed et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Given the advantages of restrictive screen enrichment, such as reduced metabolic burden and robustness to environment disturbances, many lignocellulose-degrading microbial communities are enriched by this process (Cui et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Hui et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; S. et al., 2002; Wang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite these benefits, however, the uncontrollability of enrichment culturing requires many parallel experiments in order to obtain microbial communities with high lignocellulose degradability. Further, often the degradability of initially suitable communities can decrease in subsequent successive transferring. To overcome these challenges, acid-base combination of microbial community was used in the restrictive screen process (S. et al., 2002). For acid-base variety, one microbial community with low pH was combined with another microbial community with high pH to obtain a novel community whose pH fluctuated within a specific range. With the method of restricted culture and acid-base combination, many lignocellulose-degrading microbial communities, such as MC1 (S. et al., 2002), XDC-2(Guo et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), and WCS-6 (Wang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), were obtained. MC1, for example, was constructed from compost and is capable of effectively degrading various cellulosic materials, including rice straw (Cui et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Hua et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), filter paper (Kato et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), cardboard (Yuan et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In addition, it has been used for hydrolysis and acidification of agricultural waste (Yu et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), to enhance anaerobic digestion (Hua et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yuan et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), for concurrent saccharification and anaerobic digestion of Napier grass (Wen et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). After more than 40 successive generations of culture (S. et al., 2002) and approximately 20 years of research and application (Wang et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), the microbial community has remained stable with a high degradation efficiency. Therefore, in terms of practical application, restrictive screen enrichment and acid-base combination have achieved good results. At the same time, however, little research has been conducted into understanding the mechanisms of acid-base combinations. It is currently unknown how these communities form, particularly how acid-base combinations influence the microbial communities. With recent advances in sequencing, high throughput approaches have made it possible to quantify the composition of microbial communities during this process (Estrela et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) .\u003c/p\u003e \u003cp\u003eThis study used compost as the inoculum source for the restrictive screen lignocellulosic degradation microbial community. The community structure changes were analyzed by high throughput sequencing to understand the formation process and reveal the mechanisms of acid-base combinations after long-time enrichment cultures.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003ch3\u003e\u003cem\u003eInoculation and enrichment culturing\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eOur inoculum source was compost, sourced from the Zhuozhou Experimental Station, China Agricultural University. The pile was turned over once every two days during the thermophilic period (average 60 ℃) and once every five days after the temperature dropped to 35℃. Overall, the experiment lasted for 60 days.\u003c/p\u003e\n\u003cp\u003eInoculum sources (5g in fresh weight) were sampled during the ripening period (D1) and thermophilic period (D2). These were then inoculated into PCS medium with three replicates (A1, A2, A3 and B1, B2, B3, respectively). Cultures were maintained in 100 mL triangular bottles for 15 days in each generation.\u003c/p\u003e\n\u003cp\u003eThe PCS medium was 0.5% peptone, 1 % lignocellulose, 0.5% NaCl, 0.2% CaCO\u003csub\u003e3\u003c/sub\u003e, 0.1% yeast powder, pH natural (about 7.3) (S. et al., 2002). Rice straw was utilised as a carbon source, and small filter paper strips were added to the cultures. Those with decomposed filter paper strips were regarded as degradable and were chosen for the succession cultures. The inoculation was performed statically at 50 ℃ at a volume ratio of 5% (v/v).\u003c/p\u003e\n\u003ch3\u003e\u003cem\u003ePretreatment of lignocellulosic materials and analysis of weight loss\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eThe rice straw (harvested from Shangzhuang Experimental Station of China Agricultural University) was cut into approximately 10 cm length, soaked in 1% NaOH for 24 h, and washed with tap water until the pH value was between 7.0 and 8.0. Pieces were then dried at 50℃ and stored for future use. The weight loss of rice straw was tested as previously reported (Hua et al., 2014).\u003c/p\u003e\n\u003ch3\u003e\u003cem\u003eAcid-base combination of microbial community\u0026nbsp;\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eDynamic changes in pH were measured, communities with a slight pH decrease and a slow pH increase were selected. 5 mL liquid of each community was extracted, taken,\u0026nbsp;and\u0026nbsp;combined to create a novel PCS\u0026nbsp;medium.\u0026nbsp;After merging, these new communities were named with the original group numbers (A1A3, A1B3, A3, and B2B3), as shown in Fig 1.\u003c/p\u003e\n\u003ch3\u003e\u003cem\u003eExtraction of genomic DNA\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eAfter five months of restrictive screening, samples were extracted from D1, D2, A1, A2, A3, B1, B2, B3, respectively, and after acid-base combination, samples were also extracted from A1A3, A1B3, A3B2, and B2B3, respectively.\u0026nbsp;Genomic DNA was extracted using the CTAB method (Murray and Thompson, 1980).\u003c/p\u003e\n\u003ch3\u003e\u003cem\u003eHigh-throughput sequencing analysis\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was sent off for high throughput sequencing (Allwegene Tech., Co., Ltd, Beijing). The V3+V4 region was amplified using the following universal primer sets: 336 F and 806 R (5\u0026prime;-GTACTCCTACGGG AGGCAGCA-3\u0026prime;and 5\u0026prime;-GTGGACTACHVGG GTWTCTAAT-3\u0026prime;). The amplicon libraries were sequenced using the Illumina Miseq and a 2\u0026times;300 bp paired-end protocol (Bartram et al., 2011).\u003c/p\u003e\n\u003cp\u003eSequences were filtered out with a quality value \u0026lt;20. For each sample, the qualified reads were defined as a library and then down-sampled to the smallest library size, which was determined to compare the alpha diversity metrics, including Chao and Shannon (mothur v.1.30.1). After demultiplexing, the reads were assigned species-equivalent operational taxonomic units (OTUs) at 97% sequence similarity. The remaining OTUs were denominated at different classification levels according to the Silva database release 115 (http://www.arb-silva.de).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eLignocellulosic degradation ability of the obtained microbial communities.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eThe microbial community A1 had the lowest weight loss of rice straw (13.91\u0026plusmn;4.83%), followed by community B2 (38.28\u0026plusmn;1.77%). Community B1, A2, B3 and A3 increased successively (42.77\u0026plusmn;2.86%, 46.03\u0026plusmn;6.96%, 46.28\u0026plusmn;3.43%, and 47.08\u0026plusmn;0.40%) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). These values corresponded to changes in pH.\u003c/p\u003e\n\u003cp\u003eDecreases in pH reflected rapid decomposition of cellulose, while increases in pH occurred with decreasing rates or cessation of cellulose degradation (Liu et al., \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e). Solutions\u0026apos; pH decreases were a prominent feature of the cellulosic materials degraded microbial community (Liu et al., \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e). The pH of all six microbial communities decreased on the third day Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC. The pH of A1 and B2 decreased to 6.6 and then began to rise rapidly to alkaline conditions, while A2 and B1 decreased to approximately 6.3. On the third day, A3 and B3 decreased to 6.1 and 6.3, respectively, and remained acidic until the 12th day. On the 15th day, the pH of A3 returned to neutral, while the pH of B3 remained acidic.\u003c/p\u003e\n\u003cp\u003eMicrobial degradation of straw resulted in pH decreases in all treatments before rising to slightly alkaline conditions. Although A3 and B3 displayed faster degradation rates, the recovery time to slightly alkaline was longer than 12 days. Therefore, we selected B1 and A2 for further research and utilization. For A3 and B3, as the acidic products were not rapidly utilized, accumulation of these products may inhibit the degrading strains, thus inducing instability of the bacterial communities.\u003c/p\u003e\n\u003cp\u003eTo study the acid-base combinations, A1 and B3 were pairwise combined with A3 and B3, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The new microbial communities A1A3, A1B3, A3B2, and B2B3 were created. The rice straw weight losses of A1A3, A1B3, A3B2, and B2B3 were 43.24\u0026plusmn;6.73%, 32.18\u0026plusmn;1.19%, 44.01\u0026plusmn;2.93%, and 38.56\u0026plusmn;2.83%, respectively, which were significantly higher than those of A1 and B2, but lower than those of A3 and B3, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). On the third day, the pH of A1A3, A1B3, A3B2, and B2B3 was 6,6.5, 6.3, and 6.1, and increased to 7.1, 7.4, 8.2, and 7, on the 9th day, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). All returned to neutral and then became weakly alkaline. The combined microbial communities all followed the rule of pH fluctuating within 6-9 range, similar to other stable and efficient lignocellulose-decomposing microbial communities in previous reports (Liu et al., \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e). Acid-base combinations did not obtain a more efficient lignocellulose-decomposing microbial community, while the pH was more similar to a stable community.\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eEffect of inoculum source on the enrichment of microbial communities\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eBased on the T-test results, group A and group B\u0026rsquo;s degradation ratio was not significantly different. However, the parallelity of group B was much higher than A. Therefore, both D1 and D2 had potential, while D2 was more suitable to screen high-efficiency microbial communities (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Bacterial biodiversity was higher in the thermophilic period than in the ripening period. The bacteria composition of both compost samples was significantly different, which resulted in group A and group B also exhibiting significant differences. The inoculum source had a greater impact on restrictive screen results. In this study, composting during the thermophilic period was a better choice of inoculum source than during the ripening period.\u003c/p\u003e\n\u003cp\u003eComposting in the thermophilic period is often used as an inoculum source to screen cellulose-degrading bacteria (Tesfaw and Assefa, \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). For D2, the temperature of the compost body in the thermophilic period was higher, and organic matter decomposed rapidly. Cellulose materials decomposed more rapidly during this period, and bacteria with cellulose degradation abilities were also the most active. For D1, in the ripening period, the rapid decomposition of the organic matter ended.\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eThe structure of the abtained microbial communities after restrictive screen culture\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eA significant number of OTUs in D1 and D2 were eliminated (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). The number of retained OTUs in A1, A2, and A3 were 337, 310, and 418, respectively, with B1, B2, and B3 retaining 408, 392, and 353, respectively. In addition, some OTUs that were not detected in D1 and D2 were detected in our microbial communities. A1, A2, and B2 had 827, 807, and 713 OTUs that were not found in D1, while B1, B2, and B3 had 1128, 862, and 663 OTUs that were not identified in D2.\u003c/p\u003e\n\u003cp\u003eThe retained bacteria (amount ratio more than 0.1%) are shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Bacteria such as \u003cem\u003eCellulosilyticum\u003c/em\u003e have the ability to degrade cellulose. In the community, EMSD5, \u003cem\u003eCellulosilyticum\u003c/em\u003e, and other bacteria, and fungi synergistically degrade whole corn with cobs removed (Zhu et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). In the community SV79, which decomposes various lignocellulosic substrates to produce ethanol, \u003cem\u003eCellulosilyticum\u003c/em\u003e and \u003cem\u003eAcetivibrio, Clostridium, Ruminococcus\u003c/em\u003e, and \u003cem\u003eSporomusa\u003c/em\u003e are the dominant bacteria (Zhao et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). \u003cem\u003eTreponema primitia\u003c/em\u003e in the intestine of termites can degrade lignin (Lucey and Leadbetter, \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). \u003cem\u003ePseudomonas, Comamonas\u003c/em\u003e, and \u003cem\u003eLachnoclostridium\u003c/em\u003e can also decompose cellulose in a composite community (Liu et al., \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e; Xue et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zagrodnik et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003ctable border=\"1\" id=\"Tab1\" style=\"margin-right: calc(19%); width: 81%;\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe bacteria microbiome covered all samples(quantitative proportion above 0.1%)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\" style=\"width: 23.5633%;\"\u003e\n \u003cp\u003eTaxonomy\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"12\" style=\"width: 76.2451%;\"\u003e\n \u003cp\u003eQuantitative proportion (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" style=\"width: 5.9386%;\"\u003e\n \u003cp\u003eA1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eA2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eA3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eB1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eB2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eB3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eD1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eD2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eA1A3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eA1B3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eA3B2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eB2B3\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 23.5633%;\"\u003e\n \u003cp\u003e\u003cem\u003eAlcaligenes\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 5.9386%;\"\u003e\n \u003cp\u003e67.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e59.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 23.5633%;\"\u003e\n \u003cp\u003e\u003cem\u003eLachnoclostridium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 5.9386%;\"\u003e\n \u003cp\u003e6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 23.5633%;\"\u003e\n \u003cp\u003e\u003cem\u003eCellulosilyticum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 5.9386%;\"\u003e\n \u003cp\u003e3.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e41.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 23.5633%;\"\u003e\n \u003cp\u003e\u003cem\u003eProteiniphilum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 5.9386%;\"\u003e\n \u003cp\u003e3.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 23.5633%;\"\u003e\n \u003cp\u003eunidentified\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 5.9386%;\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 23.5633%;\"\u003e\n \u003cp\u003e\u003cem\u003eComamonas\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 5.9386%;\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e51.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 23.5633%;\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 5.9386%;\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 23.5633%;\"\u003e\n \u003cp\u003e\u003cem\u003eAdvenella\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 5.9386%;\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e47.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 23.5633%;\"\u003e\n \u003cp\u003e\u003cem\u003eTreponema\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 5.9386%;\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 23.5633%;\"\u003e\n \u003cp\u003e\u003cem\u003eClostridium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" style=\"width: 5.9386%;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eSome retained bacteria could not use cellulose directly, however, they can exist in communities that decomposed lignocellulose, such as \u003cem\u003eAlcaligenes.\u003c/em\u003e They can utilize organic acids and amino acids to produce ammonia and carbon dioxide. \u003cem\u003eAlcaligenes faecalis\u003c/em\u003e have been reported in plants, soil, and other environments (Rosenberg, 2014), and \u003cem\u003eAlcaligenes sp\u003c/em\u003e TB is an aerobic denitrifying bacterium (Chen et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). \u003cem\u003eProteiniphilum\u003c/em\u003e is widely reported in anaerobic fermentation sludge, as it cannot decompose lignocellulose and cellobiose (Maspolim et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). It can, however, use peptone, arabinose, etc., and its metabolites include small molecular organic acids, CO\u003csub\u003e2,\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003e (Langer et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). \u003cem\u003eTreponema caldarium\u003c/em\u003e is a strictly anaerobic hyperthermia spirochete, which uses glucose, lactose, and other sugars as substrates, and has been reported in a complex community that degraded naphthalene (Koelschbach et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). These bacteria may assist in the elimination of acidic byproduct that inhibits cellulose-degrade strains.\u003c/p\u003e\n\u003cp\u003eSome bacteria, like \u003cem\u003eSporomusa\u003c/em\u003e, \u003cem\u003eProteiniclasticum\u003c/em\u003e, Dethiosulfovibrionaceae, \u003cem\u003eDesulfotomaculum\u003c/em\u003e, \u003cem\u003eDesulfosporosinus\u003c/em\u003e, \u003cem\u003ePseudoxanthomonas\u003c/em\u003e, \u003cem\u003eNocardia\u003c/em\u003e, \u003cem\u003eAlteromonadaceae\u003c/em\u003e, \u003cem\u003eErysipelotrichaceae\u003c/em\u003e, \u003cem\u003eNovosphingobium\u003c/em\u003e, \u003cem\u003ePropionigenium\u003c/em\u003e, \u003cem\u003eNonomuraea\u003c/em\u003e, \u003cem\u003eBalneimonas\u003c/em\u003e, and \u003cem\u003eArcobacter\u003c/em\u003e which were found in all the six enriched communities, but not in D1 or D2. These newcomers may play specific roles in the microbial community and may have originated from the compost. Their abundance in compost may be extremely low and difficult to detect with high throughput sequencing. After the growth environment changed, their abundance amount increased, allowing for detection with high-throughput sequencing. Additionally, they may also come from the air as the PCS medium was not sterilized. Generally, the enriched microbial community needs to have a high anti-pollution capacity. It was not hortative to be operated in a sterile environment such as an ultra-clean workbench, and thus other bacteria may contaminate them (Himanshu et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe abundance and composition of fungal OTUs from our communities were similar, which were different from that of the compost (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). While many fungi were eliminated as a result of the restricted culture, some survived, though their OTUs were less abundant. Some may be involved in straw catabolism, such as \u003cem\u003ePenicillium\u003c/em\u003e (Ogunyewo et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e), while most fungi had little connection to straw decomposition, like \u003cem\u003eGuehomyces pullulans\u003c/em\u003e (Nakagawa et al., \u003cspan class=\"CitationRef\"\u003e2006\u003c/span\u003e) and \u003cem\u003eHyphopichia burtonii\u003c/em\u003e (Groenewald and Smith, \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003e\u003cem\u003eEffect of acid-base combination\u003c/em\u003e\u003c/h3\u003e\n\u003cdiv class=\"Section2\" id=\"Sec10\"\u003e\n \u003ch2\u003eThe change of microbial community structure by an acid-base combination\u003c/h2\u003e\n \u003cp\u003eAmong the obtained communities by acid-base combination, A1A3 had the lowest pH (6.0) on the 3rd day and had a greater degradation ratio (43.24\u0026plusmn;6.73%). In contrast, A1B2 had the highest pH (6.5), and the degradation ratio was only 32.18\u0026plusmn;1.19%. Therefore, analysis was focused on these two communities to elucidate the mechanism of acid-base combination.\u003c/p\u003e\n \u003cp\u003eA1A3 was combined within group A, and A1B3 was combined between group A and B. Before mixing, the dominant bacteria in A1 was \u003cem\u003eAlcaligenes\u003c/em\u003e, with a ratio of 67% (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e), which cannot decompose cellulose. \u003cem\u003eAlcaligenes\u003c/em\u003e can use peptone and yeast extract powder in PCS medium and produce alkaline substances, inducing a pH increase. The relative abundance of \u003cem\u003eCellulosilyticum\u003c/em\u003e was less than 3.6%, other bacteria producing acid were much lower than \u003cem\u003eAlcaligenes\u003c/em\u003e. The dominant bacteria of A3 was \u003cem\u003eCellulosilyticum\u003c/em\u003e, followed by \u003cem\u003eProteiniphilum\u003c/em\u003e, while the relative abundance of \u003cem\u003eAlcaligenes\u003c/em\u003e was 11%. The dominant bacteria of B3 was \u003cem\u003eCommonas\u003c/em\u003e, followed by \u003cem\u003ePseudomonas\u003c/em\u003e. The relative abundance of \u003cem\u003eLachnoclostridium\u003c/em\u003e, which may have the ability to decompose cellulose, was only 4%. The total proportion of \u003cem\u003eAdvenella\u003c/em\u003e, \u003cem\u003eAlcaligenes\u003c/em\u003e, and \u003cem\u003eBordetella\u003c/em\u003e, which belong to Alkalobacillaceae, was 4.8%.\u003c/p\u003e\n \u003cp\u003eAfter the combination, the dominant bacterium of A1A3 was \u003cem\u003eComamonas\u003c/em\u003e, which was different from A1 and A3, and the proportion of \u003cem\u003eHydrogenispora\u003c/em\u003e increased to 12%. The database information may be \u003cem\u003ehydrogenase poraethanolica\u003c/em\u003e, a strictly anaerobic bacteria producing hydrogen and ethanol (Liu et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). The relative abundance of Alcaligenes was 3%. After the combination, the dominant strain of A1B3 was \u003cem\u003eTreponema\u003c/em\u003e, followed by \u003cem\u003eComamonas\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eAnaerophaga\u003c/em\u003e, and \u003cem\u003eAlcaligenes\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eWhether it was the combination within one group or between two groups, the dominant bacteria varied greatly. After combination, the ratio of \u003cem\u003eCellulosilyticum\u003c/em\u003e decreased, the \u003cem\u003ePseudomonas\u003c/em\u003e ratio increased, and \u003cem\u003eClostridium\u003c/em\u003e ratio was low. Those three genera all have the potential ability to decompose lignocellulose. Their total proportions in A1, A3, and B3 were 4.4%, 44.5%, and 14.5%, respectively. The relative abundance of them in A1A3 and A1B3 was 10.9%, 12.3%, respectively. The numerical relationship was A3\u0026gt;A1A3\u0026gt;A1, B3\u0026gt;A1B3\u0026gt;A1. A1A3 and A1B3 were significantly higher than A1 before the combination but lower than A3 and B3. The results were consistent with straw degradation. The pH of A1 was higher than 6.6 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA), and the proportion of \u003cem\u003eAlcaligenes\u003c/em\u003e in A1 was the highest corresponding. After the combination, the proportion of \u003cem\u003eAlcaligenes\u003c/em\u003e in A1A3 and A1B3 is lower than that in A1, and their pH decreased below 6.5.\u003c/p\u003e\n \u003ch2\u003e\u003cem\u003eChange of fungi in the process of acid-base combination\u0026nbsp;\u003c/em\u003e\u003c/h2\u003e\n\u003c/div\u003e\n\u003cp\u003eGenerally, all obtained fungal communities spatially overlapped, indicating a similar species diversity (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). Take A3B2, for example; the proportion of dominant fungi (1%) in the communities is shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e. Before the combination, the dominant fungi in A1 were \u003cem\u003eScedosporium, Candida, Machimura, Penicillium\u003c/em\u003e, etc. The dominant fungi in B2 were \u003cem\u003eCandida, Machimura, Acremonium, Penicillium, Scedosporium, IsSatchenkia\u003c/em\u003e. After combination, the dominant fungi in A3B2 included \u003cem\u003eCandida, Machimura, Scedosporium, Acremonium, Penicillium, and Clavulina\u003c/em\u003e. For fungi, the acid-base combination was just a simple mixing process. The dominant fungi were gathered into the new community, while the proportions likely changed as a result of competition.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe screened bacteria can be divided into three groups. The first group exhibited lignocellulose decomposing functions, such as \u003cem\u003eClostridium\u003c/em\u003e, \u003cem\u003eCellulosilyticum, Pseudomonas\u003c/em\u003e, etc. The second group was likely to have auxiliary functions, such as \u003cem\u003eAlcaligenes, Pseudomonas\u003c/em\u003e (decomposing bacteria or auxiliary bacteria), etc. The third group was not directly related to the decomposition of lignocellulose by current reports. It may have unknown functions or may be eliminated in the subculture in the future.\u003c/p\u003e \u003cp\u003eWhile in the screening process, the randomness was apparent. The obtained microbial communities were significantly different with the same inoculum source and under the same culture conditions. For example, the community structure of A1 was significantly different from that of parallel A2 and A3, and the straw degradation rate was much lower than that of A2 and A3.\u003c/p\u003e \u003cp\u003eThe acid-base combination aimed to obtain a microbial community with a stable and robust ability to decompose straw. The goal is to optimize the variety of different microbiota-producing acids and alkaline without damaging the synergistic relationship between microorganisms in nature. The pH of the obtained microbiota decreased rapidly with the decomposition of straw before returning to a slightly alkaline level.\u003c/p\u003e \u003cp\u003eThe regular change of pH was closely related to the structure of the community. The composition of dominant bacteria varied greatly, whether within or between groups. The cellulose-degrading strains represented by \u003cem\u003eClostridium\u003c/em\u003e can metabolize straw to produce small molecular organic acids, reducing the pH of the solution. Helper strains represented by \u003cem\u003eAlkalobacillus\u003c/em\u003e can metabolize organic acids and amino acids to produce ammonia and carbon dioxide, increasing the solution's pH. When the auxiliary bacteria were dominant in the microbiota, their abundance was very high, such as A1, while the abundance of other strains was low. The decomposition ability of microbial bacteria was insufficient, and the pH was difficult to decline. When the number of auxiliary bacteria in the community was low, such as A3, B3, pH only slowly rose back to slightly alkaline. After the acid-base combination, the microbial community was reconstituted. The degrading and auxiliary strains were adjusted to a more appropriate quantity and proportion, and unrelated strains were knocked out simultaneously, which changed the pH regularly. The acid-base combination was conducive to obtaining an efficient and stable microbial community and correcting microbial communities' reduced decomposition ability caused by a random process in the restricted culture process.\u003c/p\u003e \u003cp\u003eComposting in the thermophilic period was a better choice of inoculum source than that of the ripening period. Many bacteria and most fungi were eliminated after the restrictive screening. Degrading strains, such as \u003cem\u003eClostridium\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e, synergistic microbial such as \u003cem\u003ePseudoxanomonas\u003c/em\u003e and \u003cem\u003eAlkalobacillaceae\u003c/em\u003e were obtained. Microbial communities were re-constituted by acid-base combination. The degrading and synergistic strains were adjusted to a more appropriate proportion, which changed the pH regularly. Acid-base combination fixed the instability of microbial communities caused by randomness of restrictive screening enrichment. The combination of the two techniques is high conducive to obtain efficient lignocellulose degradation microbial resources.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThis research was financially supported by the National Natural Science Foundation of China (NO. 31900107) .\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026rsquo; Contributions\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eBinbin Hua: Investigation, Data curation, Formal analysis, Validation, Writing - original draft, Writing - review \u0026amp; editing. Xiaofen Wang: Conceptualization, Methodology, Supervision, Writing-review \u0026amp; editing. Zongjun Cui: Supervision,Conceptualization.\u003c/p\u003e\n\u003ch2\u003eConflict of Interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFunding Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was financially supported by the National Natural Science Foundation of China (NO. 31900107) .\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBartram AK, Lynch MDJ, Stearns JC, Moreno-Hagelsieb G, Neufeld JD (2011) Generation of Multimillion-Sequence 16S rRNA Gene Libraries from Complex Microbial Communities by Assembling Paired-End Illumina Reads. Appl Environ Microb 77(15):5569\u0026ndash;5569\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen J, Gu S, Hao H, Chen J (2016) Characteristics and metabolic pathway of Alcaligenes sp. TB for simultaneous heterotrophic nitrification-aerobic denitrification. Appl Microbiol Biot 100(22):9787\u0026ndash;9794\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui Z, Li M, Piao Z, Huang Z, Ishii M, Igarashi Y (2002) Selection of a composite microbial system MC1 with efcient and stability cellulose degradation bacteria and its function. Environ Sci 23:36\u0026ndash;39\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEstrela S, S\u0026aacute;nchez \u0026Aacute;, Rebolleda-G\u0026oacute;mez M (2021) Multi-Replicated Enrichment Communities as a Model System in Microbial Ecology. Front Microbiol 12:657467\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGroenewald M, Smith MT (2010) Re-examination of strains formerly assigned to Hyphopichia burtonii, the phylogeny of the genus Hyphopichia, and the description of Hyphopichia pseudoburtonii sp. nov. Int J Syst Evol Micr 60(11):2675\u0026ndash;2680\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo P, Zhu W, Wang H, Lu Y, Wang X, Zheng D, Cui Z (2010) Functional characteristics and diversity of a novel lignocelluloses degrading composite microbial system with high xylanase activity. J Microbiol Biotechnol 20(2):254\u0026ndash;264\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHimanshu H, Voelklein MA, Murphy JD, Grant J, O'Kiely P (2017) Factors controlling headspace pressure in a manual manometric BMP method can be used to produce a methane output comparable to AMPTS. Bioresour Technol 238:633\u0026ndash;642\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHua B, Dai J, Liu B, Zhang H, Yuan X, Wang X, Cui Z (2016) Pretreatment of non-sterile, rotted silage maize straw by the microbial community MC1 increases biogas production. Bioresour Technol 216:699\u0026ndash;705\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHua B, Lu Y, Wang J, Wen B, Cao Y, Wang X, Cui Z (2014) Dynamic Changes in the Composite Microbial System MC1 During and Following its Rapid Degradation of Lignocellulose. Appl Biochem Biotech 172(2):951\u0026ndash;962\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHui W, Jiajia L, Yucai L, Peng G, Xiaofen W, Kazuhiro M, Zongjun C (2013) Bioconversion of un-pretreated lignocellulosic materials by a microbial consortium XDC-2. Bioresour Technol 136:481\u0026ndash;487\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJawed K, Yazdani SS, Koffas MA (2019) Advances in the development and application of microbial consortia for metabolic engineering.Metabolic Engineering Communications,9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.mec.2019.e00095\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKato S, Haruta S, Cui ZJ, Ishii M, Igarashi Y (2004) Effective cellulose degradation by a mixed-culture system composed of a cellulolytic Clostridium and aerobic non-cellulolytic bacteria. FEMS Microbiol Ecol 51(1):133\u0026ndash;142\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoelschbach JS, Mouttaki H, Pickl C, Heipieper HJ, Meckenstock RU (2017) Rectinema cohabitans gen. nov., sp. nov., a rod-shaped spirochete isolated from an anaerobic naphthalene-degrading enrichment culture. Int J Syst Evol Microbiol 67(5):1288\u0026ndash;1295\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLanger T, Hahnke S, Klocke M, Koeck DE (2016) Description of Proteiniphilum saccharofermentans sp. nov., Petrimonas mucosa sp. nov. and Fermentimonas caenicola gen. nov., sp. nov. isolated from mesophilic lab-scale biogas reactors and emended description of the genus Proteiniphilum.Int. J. Syst. Evol. Microbiol., 66(3). \u003cb\u003eDOI\u003c/b\u003e: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1099/ijsem.0.000902\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Wan L, XiuHong X, HongTao L (2011) Effect of Cellulose-decomposing Strain on Microbial Community of Cow Manure Compost. Environ Sci 32(10):3073\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu JB, Wang WD, Yang HY, Wang XF, Gao LJ, Cui ZJ (2006) Process of rice straw degradation and dynamic trend of pH by the microbial community MC1. J Environ Sci (China) 18(6):1142\u0026ndash;6\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Qiao JT, Yuan XZ, Guo RB, Qiu YL (2014) Hydrogenispora ethanolica gen. nov., sp nov., an anaerobic carbohydrate-fermenting bacterium from anaerobic sludge. Int J Syst Evol Micr 64(5):1756\u0026ndash;1762\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLucey KS, Leadbetter JR (2014) Catechol 2,3-dioxygenase and other meta‐cleavage catabolic pathway genes in the 'anaerobic' termite gut spirochete Treponema primitia. Mol Ecol 23(6):1531\u0026ndash;1543\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaspolim Y, Zhou Y, Guo C, Xiao K, Ng WJ (2015) The effect of pH on solubilization of organic matter and microbial community structures in sludge fermentation.Bioresource Technol. \u003cb\u003eDOI\u003c/b\u003e: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.biortech.2015.04.087\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8(19):4321\u0026ndash;5\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakagawa T, Ikehata R, Uchino M, Miyaji T, Takano K, Tomizuka N (2006) Cold-active acid beta-galactosidase activity of isolated psychrophilic-basidiomycetous yeast Guehomyces pullulans. Microbiol Res 161(1):75\u0026ndash;79\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOgunyewo OA, Randhawa A, Gupta M, Kaladhar VC, Yazdani SS (2020) Synergistic Action of a Lytic Polysaccharide Monooxygenase and a Cellobiohydrolase from Penicillium funiculosum in Cellulose Saccharification Under High Substrate Loading.Appl. Environ. Microb., 86(23). \u003cb\u003eDOI\u003c/b\u003e: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/AEM.01769-20\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosenberg E, Delong EF, Lory S (2014) et al The Prokaryotes:The Family Methylobacteriaceae[J] Chapter 256313\u0026ndash;340. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-3-642-30197-1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cb\u003eS\u003c/b\u003e., \u003cb\u003eH\u003c/b\u003e., \u003cb\u003eZ\u003c/b\u003e., \u003cb\u003eC\u003c/b\u003e., \u003cb\u003eZ\u003c/b\u003e., \u003cb\u003eH\u003c/b\u003e., \u003cb\u003eM\u003c/b\u003e., \u003cb\u003eL\u003c/b\u003e., \u003cb\u003eM\u003c/b\u003e., \u003cb\u003eI\u003c/b\u003e., \u003cb\u003eY\u003c/b\u003e., \u003cb\u003eI\u003c/b\u003e., \u003cb\u003eConstruction of a stable microbial community with high cellulose-degradation ability\u003c/b\u003e.Appl. Microbiol. Biot., \u003cb\u003e59\u003c/b\u003e(\u003cb\u003e4-5\u003c/b\u003e),\u003cb\u003e529-534\u003c/b\u003e. \u003cb\u003eDOI\u003c/b\u003e: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00253-002-1026-4\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTesfaw A, Assefa F (2014) Co-culture: A great promising method in single cell protein production. Biotechnology and Molecular Biology Reviews 9(2):12\u0026ndash;20\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Zhu D, Zhao S, Xu S, Yang R, Zhao W, Zhang X, Huang Z (2021) Effect of liquid volume and microflora source on degradation rate and microbial community in corn stover degradation.AMB Express, 11(1). \u003cb\u003eDOI\u003c/b\u003e: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13568-021-01233-5\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang R, Zhao S, Wang Z, Koffas MA (2020) Recent advances in modular co-culture engineering for synthesis of natural products. Curr Opin Biotech 62:65\u0026ndash;71\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang W, Yan L, Cui Z, Gao Y, Wang Y, Jing R (2011) Characterization of a microbial consortium capable of degrading lignocellulose. Bioresour Technol 102(19):9321\u0026ndash;9324\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeiland P (2010) Biogas production: current state and perspectives. Appl Microbiol Biot 85(4):849\u0026ndash;860\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWen B, Yuan X, Li QX, Liu J, Ren J, Wang X, Cui Z (2015) Comparison and evaluation of concurrent saccharification and anaerobic digestion of Napier grass after pretreatment by three microbial consortia. Bioresour Technol 175:102\u0026ndash;111\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu C, Yu H (2021) Insights into constructing a stable and efficient microbial consortium. Chinese J Chem Eng 30:112\u0026ndash;120\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXue C, Zhang Q, Owens G, Chen Z (2020) A cellulose degrading bacterial strain used to modify rice straw can enhance Cu(II) removal from aqueous solution. Chemosphere 256:127142\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu J, Zhao Y, Zhang H, Hua B, Yuan X, Zhu W, Wang X, Cui Z (2017) Hydrolysis and acidification of agricultural waste in a non-airtight system: Effect of solid content, temperature, and mixing mode. Waste Manage 59:487\u0026ndash;497\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan X, Cao Y, Li J, Wen B, Zhu W, Wang X, Cui Z (2012) Effect of pretreatment by a microbial consortium on methane production of waste paper and cardboard. Bioresour Technol 118:281\u0026ndash;288\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan X, Ma L, Wen B, Zhou D, Kuang M, Yang W, Cui Z (2016) Enhancing anaerobic digestion of cotton stalk by pretreatment with a microbial consortium (MC1). Bioresour Technol 207:293\u0026ndash;301\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZagrodnik R, Duber A, Seifert K (2021) Hydrogen production during direct cellulose fermentation by mixed bacterial culture: The relationship between the key process parameters using response surface methodology. J Clean Prod 314:127971\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao C, Deng Y, Wang X, Li Q, Huang Y, Liu B (2014) Identification and characterization of an anaerobic ethanol-producing cellulolytic bacterial consortium from Great Basin hot springs with agricultural residues and energy crops. J Microbiol Biotechnol 24(9):1280\u0026ndash;1290\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu N, Yang J, Ji L, Liu J, Yang Y, Yuan H (2016) Metagenomic and metaproteomic analyses of a corn stover-adapted microbial consortium EMSD5 reveal its taxonomic and enzymatic basis for degrading lignocellulose. Biotechnol Biofuels 9(1):243\u003cb\u003eDOI\u003c/b\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aomi","sideBox":"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)","snPcode":"203","submissionUrl":"https://submission.nature.com/new-submission/203/3","title":"Archives of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"acid-base combination, restrictive enrichment, lignocellulose, microbial community","lastPublishedDoi":"10.21203/rs.3.rs-1074700/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1074700/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAcid-base combination is used in some cases expecially after restricted enrichment, and has created many lignocellulose-degrading communities. While how it worked is not well understood. In this study, compost was used as inoculum source. Induced community structure changes were analyzed with high throughput sequencing to elucidate the formation processes and determine the mechanisms of acid-base combination. We found that after restricted enrichment, retaining primarily bacteria not only included that could decompose and utilize lignocellulose, such as \u003cem\u003eClostridium\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e, but also synergistic microbiota such as \u003cem\u003ePseudoxanomonas \u003c/em\u003eand \u003cem\u003eAlkalobacillaceae.\u003c/em\u003e When the proportion of these two types of bacteria was not balanced, the degradation ability of the microbial community was low or pH changes of it did not compound regular changes\u003cem\u003e , \u003c/em\u003ewhich maybe lead to the failure of restricted enrichment. Microbial communities were re-constituted by acid-base combination, whereby the degrading and synergistic strains were adjusted to a more appropriate proportion. Acid-base combination fixed the instability of microbial communities caused by randomness of restrictive screening enrichment. In this study, the mechanism of acid-base combination was analyzed, which enriched the theoretical system of restricted culture, and provided an effective and controllable technical method for obtaining high-quality lignocellulose-degrading microbial community resources.\u003c/p\u003e","manuscriptTitle":"Obtainment of Lignocellulose Degradation Microbial Community: The Effect of Acid-Base Combination After Restrictive Enrichment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-11-29 23:03:57","doi":"10.21203/rs.3.rs-1074700/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-11-22T11:20:13+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-11-22T11:15:23+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Archives of Microbiology","date":"2021-11-15T12:31:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-11-13T16:43:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Microbiology","date":"2021-11-12T09:15:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aomi","sideBox":"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)","snPcode":"203","submissionUrl":"https://submission.nature.com/new-submission/203/3","title":"Archives of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"47b2905e-f584-4685-b00d-f61bede82e3d","owner":[],"postedDate":"November 29th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":8740365,"name":"Molecular Biology"},{"id":8740366,"name":"General Microbiology"}],"tags":[],"updatedAt":"2022-08-17T05:36:58+00:00","versionOfRecord":[],"versionCreatedAt":"2021-11-29 23:03:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1074700","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1074700","identity":"rs-1074700","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}