Microbial Community Succession is Associated With Corn Straw Degradation in Microbial Consortium M44 During Subculture

To systematically analyze the succession of functional microbiota that plays an important role during culture of microbial consortia M44 and its relationship with straw degradation characteristics, we determined the straw degradation ratio and activities of cellulose, hemicellulose, lignin enzyme, and VFA content of M44 in different culture periods. We also used 16S rRNA gene sequencing to analyze the change in microbial community structure in M44 and explore the differences in microbial composition in the original sample. The results showed that at 15 ℃ for 21 days, the straw degradation rate, endoglucanase activity, and lter paper enzyme activity of M44 generally decreased with increasing culture age, reaching their highest values at F1. The activities of xylanase, laccase, and lignin peroxidase, as well as VFA content, were the highest at F5, showing a single-peak curve change with rst an increase and then decrease. At the phylum level, Proteobacteria, Bacteroidetes, and Firmicutes were dominant in the original samples and in different culture stages. At the genus level, Devosia and Bacillus were dominant in the original sample. During subculture, the dominant bacteria in the rst generation (F1) were Pseudomonas, Flavobacterium, Brevundimonas, Achromobacter, Chryseobacterium, and Devosia. The dominant genera in the last generation (F11) were Trichococcus, Acinetobacter, Dyssgonomonas, and Rhizobium. In conclusion, we identied changes in microbial community structure occurring in M44 during subculture, as well as similarities and differences in microbial communities from the original sample.


Introduction
Lignocellulose is one of the most abundant renewable carbon sources in the biosphere, and its resource utilization e ciency has potential signi cance for sustainable development and environmental protection 1,2 . However, ine cient lignocellulose deconstruction is a primary bottleneck for its economic conversion and further utilization (i.e., of hemicellulose and cellulose, which are enclosed by lignin) 3,4 , especially under low-temperature conditions. Biodegradation, accomplished through coordination of various microorganisms, is currently considered a highly e cient method for lignocellulosic degradation 5,6 . Studies have shown that original samples determine the species composition and functional characteristics of microbial consortia. For example, Zheng et al. 7 obtained the lignocellulosedegrading bacterium LTF-27 from cold perennial forest soil and found that the degradation rates of cellulose, hemicellulose, and lignin in rice straw were 71.7%, 65.6%, and 12.5% at 15°C for 20 days, respectively. Di, F. R. et al. 8 found that the degradation rate of alkanes and aromatic hydrocarbons reached 98% and 88% with the highly e cient petroleum hydrocarbon-degrading strain Tust-DM21, which was identi ed in a mixture of water and oil samples collected from a Bohai Bay oil pollution area and incubated at 28°C for 10 days. Previous studies have shown that complete degradation of lignocellulose requires the synergistic action of various microorganisms in the natural environment [9][10][11] . Su, X. et al. 12 found that the species and abundance of microorganisms were constantly changing during degradation of corn straw by microbial consortia LDC, with Pseudomonas, Pannonibacter, Thauera, Rumino libacter, and Anaerocolumna identi ed as the dominant bacteria. Yan, L. et al. 13 screened the mesophytic lignocellulolytic microbial consortium BYND-5, which is mainly composed of Bacteroidetes, Clostridium, Lentisphaerae, Deltaproteobacteria, and other major groups. Alessi, A. M. et al. 14 showed that as wheat straw degraded, the complexity and diversity of the microbial consortia gradually decreased, while the relative abundance of Asticcacaulis, Leadbetterella, and Truepera remained relatively high.
In an early study, a microbial consortium designated M44 with degrading corn straw was identi ed from air-dried sheep dung by restricted subculture that degraded the cellulose, hemicellulose, and lignin of corn stalks by 35.12%, 30.34%, and 17.44%, respectively, after incubation at 15°C for 21 days 15 . However, the bacterial source sample (air-dried sheep dung) and changes in microbial community composition of microbial consortia M44 in different culture stages were not well de ned. Therefore, in this study, the dynamics of species composition and differences in straw degradation e ciency during subculture were explored by measuring changes in straw degradation characteristics and M44 microbial composition. With these results, we aimed to provide a reference and theoretical basis for understanding microbial diversity and discovering and screening new degrading bacterial strains.
Corn straw was taken from the experimental eld of the Corn Center of Inner Mongolia Agricultural University (110°28' E, 40°32' N). The maize variety planted in the experimental eld is Xianyu696 that is sold in the Chinese market and allowed to be purchased as test materials. After corn harvest, the corn straw with no disease and insect pests, complete surface without serious damage, uniform size and uniform thickness was selected. After being brought back to the laboratory, washed and dried (80°C), and cut into small pieces of 2-3 cm for use. This study complies with relevant institutional, national, and international guidelines and legislation.

Medium and culture conditions
Mandels medium (M medium) was composed of K 2 HPO 4 (3.0 g), NaNO 3 (3.0 g), CaCl 2 (0.5 g), CaCl 2 (0.02 g), NaCl (0.2 g), and distilled water (1 L). Then, 40 mL M medium or EP medium and 1.0 g corn straw were added to a 100-mL triangular ask for subsequent subculture, and the corn straw degradation ratio and enzyme activity were measured. Following this, the mixtures were sterilized at 121°C for 20 min and set aside.

Cultivation of microbial consortia M44
Two grams of dried sheep dung was put into a triangular bottle lled with 40 mL sterile distilled water and glass beads and placed on a shaker at 15°C for 2 hours. Then, 5% (V/V) supernatant was absorbed in 40 mL M medium, corn straw was used as the substrate carbon source, and the cells were cultured at 15°C for 21 days. After culturing for 21 days, the fermentation liquid at an inoculation rate of 5% (V/V) was transferred to new M medium and cultured successively until the 11th generation (F11). The F1, F5, F8, and F11 generations were stored at -80°C for later use.

Determination of straw degradation characteristics
The fermentation broth of microbial consortium M44 at F1, F5, F8, and F11 generations was inoculated into 40 mL M medium or EP medium at 5% (V/V) and cultured at 15°C for 21 days. Then, the corn straw degradation ratio was determined using the weight loss method, and 5 mL of fermentation material was centrifuged at 12,000 rpm at 4°C for 10 min. The supernatants were used as extracellular crude enzyme samples to analyze the enzyme activities and volatile fatty acid (VFA) content in different stages. Filter paper enzyme activity and endonuclease 1,4-β-glucanase activity were assessed using the DNS method 16 , and xylanase activity was established by DNS method 16 . The activity of laccase was assessed using the ABTS method, and that of lignin peroxidase was examined according to the resveratrol method 17 . One milliliter supernatant was integrated into a 1.5-mL centrifuge tube, and an acid adsorbent was added to an Agilent GC-6890N meteorological chromatographic column was an Agilent l9091F-112 (length 30 m, inner diameter 0.32 mm, lm thickness 0.5 µm). The column temperature was raised gradually. The gasi cation chamber temperature was 220°C, the detector temperature was 240°C, the carrier gas was hydrogen, the ow rate was 30.0 mL•min −1 , and the tail was blowing at 30 mL•min −1 injection volumes 1 µL.

Analysis of microbial consortium composition stability during successive subcultures
Under aseptic conditions, genomic DNA from the source sample and consortium M44 at different culture periods (F1, F5, F8, F11) was extracted using a bacterial genomic DNA extraction kit (China, Tiengen Biochemical Technology Co., Ltd.), and a 1% agarose gel was used for electrophoresis. A NanoDrop 2000 UV-V spectrophotometer (Thermo Fisher Scienti c, Waltham, MA, USA) was used to determine the concentration and purity of DNA. The hypervariable region V3-V5 of the bacterial 16S rRNA gene was ampli ed using primer pair 338F (5'-ACTCCTACGGGAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') with an ABI Gene Amp® 9700 PCR thermonuclear system (Applied Biosystems, Thermo Fischer Scienti c). PCR ampli cation products were sent to Shanghai Meiji Biomedical Technology Co., Ltd. for sequencing.

Dynamics of M44 straw degradation across culture stages
The corn straw degradation rates at different culture stages is shown in Fig. 1. Corn straw weight loss in M44 at F1 reached 35.90% at 15 ℃ for 21 days, which was greater than that at F5, F8, and F11 by 2.33%, 3.01%, and 3.35%, respectively. There were no signi cant differences between F8 and F11. Therefore, the straw degradation e ciency in the early period (F1) was signi cantly higher than that in the late period (F8 and F11), indicating that with increasing subculture time, the species composition of the microbial consortium changed.

Dynamics of M44 enzyme activities across culture stages
The enzyme activities at each culture stage are shown in Fig. 2. The highest endoglucanase activity was 2.01 U•mL −1 in F1, which was signi cantly higher than that of other stages. Filter paper enzyme activities and xylanase activities were the highest in F1 and F5, with enzyme activities of 2.16 and 21.50 U•mL −1 , respectively, and there were no signi cant differences between the different stages. Laccase activity reached 101.02 U•L −1 at F5, which was signi cantly different from that in F8 and F11. The activity of lignin peroxidase in F5 was 80.37 U•L −1 , which was considerably higher than that in F11.

Dynamics of M44 VFA content across culture stages
Changes in VFA content in the microbial consortia M44 at different culture stages are shown in Fig. 3. Acetic acid, prophetic acid, and butyric acid contents were all the highest at F5. Acetic acid and propionic acid contents were 143.91 mmol•L −1 and 6.70 mmol•L −1 at F5, respectively, which were signi cantly different from those at F1 and F8. Butyric acid was 3.80 mmol•L −1 in F5, which was signi cantly different from that in F1 but not from that in F8 and F11.

Alpha diversity of microorganisms in M44 across culture stages
Alpha diversity was used as a measure of microbial community diversity within the sample. As shown in Fig.4, the Ace, Shannon, and Sobs indexes for the bacterial source samples were 1,358.22, 5.17, and 1,101.33, respectively, which were signi cantly higher than those for other stages.
3.5 Beta diversity of microorganisms in M44 across culture stages A principal component analysis (PCA) was conducted on the bacterial community in each group of samples, and the results are shown in Fig. 5. The contribution rates of PC1 and PC2 were 42.64% and 23.3% of the total, respectively. Samples of S and F1, F8, and F11 clustered together, indicating that the composition of microbial communities in these two groups was similar. On the other hand, samples from F5 clustered far from each other and into a single cluster, indicating that the microbial community compositions of the F5 samples were signi cantly different from those of other periods. To further de ne the differences, ANOSIM and PERMANOVA were performed at the OTU level based on the Bray-Curtis distance algorithm. The results showed that there were signi cant differences between different stages (p < 0.05; N = 999 permutations).
3.6 Diversity analyses of microbial consortia in M44 across culture stages 3.6.1 Phylum level The relative abundance of bacterial groups according to classi cation level is shown in Fig. 6. At the phylum level, microbial consortium M44 was mainly composed of Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, and Verrucomicrobia. Among these, Proteobacteria was dominant in the microbial consortia, with abundances of 56.84%, 87.09%, 61.64%, and 53.94% at F1, F5, F8, and F11, respectively. Its abundance in the original sample was signi cantly lower than that in the subcultured microbial consortia by 21.87%. The abundance of Bacteroidota in F1 was 32.11%, which was signi cantly higher than that of other stages, and Bacteroidota accounted for 21.87% of the total bacterial content in the original sample. The relative abundance of Firmicutes increased steadily, accounting for 26.80% of the total in F11, which was considerably higher than that at F1 (5.83%), F5 (7.68%), and F8 (17.37%). The relative abundance of Actinobacteriota in the initial sample was 41.19%, but decreased with subculture of the microbial consortia. The relative abundance of Verrucomicrobiota uctuated with increasing culture period, increasing to its maximum of 3.03% in F11.

Genus level
At the genus level (Fig.7), the relative abundance of Pseudomonas was 0.46% in the original sample, with its abundance increasing with increasing culture time, reaching 8.75% in F11. The relative abundance of Brevundimonas was highest (0.53%) in the original sample (F1), accounting for 10.79% of the total bacteria, and was signi cantly different from that in F5, F8 and F11. The relative abundance of Flavobacteria in the original sample was 0.53%, which rst increased and then decreased across culture ages. The relative abundance of Devosia was 3.09%, with its highest abundance at 1.71% in the F1 generation, after which time it decreased. The relative abundances of Achromobacter and Ochrobactrum in F1 were 5.60% and 6.56%, respectively, but bacteria in these genera were rarely found in the source samples. Their relative abundances decreased with increasing culture time. Trichococcus, Acinetobacter, and Azospirillum were found in F5. The relative abundances of F11 were 19.65%, 13.01%, and 2.96%, respectively.

Correlation analyses of physicochemical characteristics and dominant genera of the M44 microbial consortium.
Correlation analysis between the TOP20 genera in M44 and straw degradation characteristics (Fig. 8) showed that endoglucanase activity was positively correlated with Brevundimonas, Achromobacter, Hydrogenophaga, Chryseobacterium, Sphingobacterium, and some bacteria that degrade lignocellulosic or intermediate products in the colony. Dysgonomonas had a signi cant negative correlation with lter paper enzyme activity, Acinetobacter had a signi cant negative correlation with xylanase activity, and Pseudomonas and Enterobacter had a signi cant positive correlation with laccase and lignin peroxidase activity. Rhizobium and Proteiniphilum were positively correlated with acetic acid, prophetic acid, and butyric acid contents.

Discussion
In nature, degradation of lignocellulose is coordinated by various active enzymes secreted by various microorganisms. Pure-cultured single bacterial strains produce fewer enzymes types or unbalanced ratios of enzymes. Therefore, using single lignocellulose-degrading bacteria in combination with composite oraos an effective way to degrade lignocellulose 18 . Many studies have shown that by simulating the decomposition process of lignocellulose under natural conditions (i.e., taking the original environmental samples as the inoculum and adopting restrictive culture techniques), composite ora that can e ciently degrade lter paper, rice straw, and pulp waste can be identi ed 19,20,21 . However, there are limited reports on composite ora that can e ciently degrade lignocellulose under low temperature conditions. In this study, microbial consortium M44 was cultured at 15°C for 21 days, and the rate at which corn straw was degraded was found to be 35.90%.
The microbial consortium M44 is a complex microbial mix composed of aerobic bacteria, anaerobic bacteria, and strict anaerobic bacteria, and its microbiome structure changed considerably during degradation of straw. Through an analysis of alpha and beta diversity of complex microorganisms in different culture periods, we found that although the dominant microorganisms were similar in different culture periods, the overall community structure was still different, and the microbiome of the original sample was the richest, probably because the microbial composition of the original sample itself was rich. However, during the culture process, carbon and other nutrients were limited in the culture medium, and some bacteria that mainly used the straw degradation products did not grow and reproduce su ciently. At the phylum level, the abundance of Proteobacteria and Firmicutes in the F11 generation was signi cantly higher than that in the F1 generation. These types of bacteria are common in rice straw compost 22 , decaying wood 23 , and rumen 24, which suggests the importance of these types of bacteria for the degradation of lignocellulolytic materials. For example, among Firmicutes, most bacilli have been shown to degrade lignin. Bacillus atrophaeus and Bacillus pumilus isolated from tropical rainforest soil in Peru were shown to produce laccase and degrade Kraft lignin 25 . Among Proteobacteria, Sphingobium paucimobiliz SYK-6 and Pseudomonas putida are capable of degrading lignin monoaryls, biaryls, and phenolic intermediates using extracellular laccases and peroxidases 26,27 . Based on our genus-level analysis, in the process of subculturing, because the oxygen in the medium was su cient at an initial stage of culture, aerobic microorganisms Pseudomonas, Devosia,and Azospirillum used the nutrients in the medium for their own growth, and oxygen was consumed rapidly, which created a good growth environment for facultative anaerobic and anaerobic bacteria such as Trichococcus, Acinetobacter, Rihizobium, and Dysgonomonas. In later stages of culture, their abundance increased, organic acids and some intermediate metabolites were consumed, and they became the main microorganisms degrading the lignocellulose. Across the subculture process, the species and abundance of microorganisms change with changes in the environment, so microbial consortium M44 is able to e ciently and stably degrade straw at a low temperature.
Acinetobacter, Azospirillum, Pseudomonas, Brevundimonas, Devosia, Achromobacter, and Chryseobacterium play major roles in this process. Acinetobacter is found in cellulose-containing agricultural waste as the only carbon source, and e ciently secretes extracellular cellulase and hemicellulose enzymes 28, 29 ; Azospirillum has been shown to produce hydrogen peroxide enzymes, oxidase, methyl cellulase, and produce acetic acid, butyric acid, and lactic acid, and to participate in straw degradation metabolism 30,31 ; Dye-decolonizing peroxidases (DYPs) secreted by Pseudomonas have the ability to degrade lignin and lignin model compounds 32,33 and have high laccase and lignin peroxidase activities 34 . They are believed to be important functional bacteria for degradation of straw lignin. In this study, lignin enzyme activity and VFA content of the microbial consortium fermentation broth at generation F5 were considerably increased, which may be the result of massive growth of the above bacteria. Studies have shown that Brevundimonas secretes oxidase and catalase to promote the decomposition of cellulose 35 ; Devosia decomposes catalase and utilizes xylose, glyceraldehyde, cellulose, etc 36 ; and Achromobacter can oxidize xylose, secrete oxidase and xylanase, which effectively degrade cellulose and hemicellulose 37 . These may be the basic functional bacteria for degrading straw cellulose. Our correlation analysis showed that the activities of endoglucanase and lter paper enzyme were signi cantly positively correlated with the abundance of Brevundimonas, Devosia, and Achromobacter. With subculture, their relative abundance decreased, resulting in a reduction in straw degradation by the microbial consortium. The degradation characteristics stabilized at the F11 generation and were not signi cantly different from those in the F8 generation. Chryseobacterium decomposes cellulase and protease, degrading cell walls, and can cooperate with Pseudomonas to degrade cellulose and hemicellulose 38,39 , which correlates with xylanase activity. This is speculated to be a functional bacterium for straw hemicellulose degradation. This microbial consortium is rich in microbial diversity that continuously changes through subculture. The microorganisms in M44 cooperate for e cient straw degradation.

Conclusion
With increasing subculture times of M44, the straw degradation rate, endoglucanase activity, and lter paper enzyme activity showed a decreasing trend, whereas xylanase activity, laccase activity, lignin peroxidase activity, and volatile fatty acid content rst increased and then decreased in a single-peak curve. Throughout subculture, the species and abundance of microorganisms in M44 changed. Pseudomonas, Devosia, Azospirillum, Trichococcus, Acinetobacter, Rihizobium, Achromobacter, Brevundimonas, and Chryseobacterium were found to be the key bacteria for straw degradation, playing a synergistic role in breaking down lignocellulose to e ciently degrade straw.  Figure 1 Corn straw degradation rate of M44 at different stages of culture Note: The same small letter means there was no signi cant difference, and different small letters indicate signi cant differences at P = 0.05.