Lignocellulolytic microbial decomposer

The microbial consortia M44 has been previously reported and was screened by restricted subculture and low-temperature domestication. The main microbial composition was Pseudomonas, Flavobacteria, Azospirillum, Trichococcus, Acinetobacter, and Brevundimonas [13]. The microbial decomposer (1.0×10⁸ CFU·g^-1) was obtained by freeze-drying, in which the ratio of filler carrier (straw husk powder: soluble starch: and bran = 2: 1.5: 4.5) and the fermentation liquid broth was 4: 1.

Microbial decomposer M44 (2.0 g) was precisely weighed and added into 20 mL aseptic water containing 7–10 glass beads. This mixture was placed in a 15°C constant-temperature oscillator and fully oscillated at 200 r·min-1 for 5–10 min The prepared agent suspension (5% (v/v)) was inoculated into 40 mL Mandels medium with 1.0 g straw and then cultured at 15°C under static conditions and repeated three times. Samples were taken on the 1st, 7th, 14th, 21st, and 28th days to determine the degradation characteristics of straw and the microbial diversity of the microbial decomposer M44.

Medium and culture conditions

Mandels medium (M medium) was composed of K₂HPO₄ (3.0 g), NaNO₃ (3.0 g), CaCl₂ (0.5 g), MgSO₄·7H₂O (0.5 g), Fe₂SO₄·7H₂O (7.5 mg), MnSO₄·H₂O (2.5 mg), ZnSO₄ (2.0 mg), CoCl₂ (3.0 mg), and distilled water (1 L). M medium (40 mL) and 1.0 g straw were added to a 100-mL triangular flask for subsequent subculturing, and then the straw degradation ratio and enzyme activity were measured. Following this, the mixtures were sterilized at 121°C for 20 min and set aside. Harvested straw (the maize variety was Xianyu696) was taken from the experimental field of the corn center of Inner Mongolia Agricultural University (110°28’ E, 40°32’ N). The selected corn straw had no diseases or insect pests, a complete surface without serious damage, uniform size, and uniform thickness. It was washed and dried (60°C) and cut into small pieces of 2–3 cm for use.

Enzyme activity and straw degradation rate measurement

The suspension of the microbial agent M44 was inoculated in the M medium, and 5 mL of the fermentation liquid and degraded straw was taken on the 1st, 7th, 14th, 21st, and 28th days. The fermentation liquid was centrifuged at 4°C and 5000 r·min^-1 for 10 min. The filter paper activity (FPase), endonuclease 1,4-β-glucanase activity (Cx), and xylanase activity were determined by the DNS method [14]. The activity of laccase (Lac) was determined by the ABTS method, and the activity of lignin peroxidase (LiP) was determined by the resveratrol method [15]. Straw was taken out and washed with distilled water. Then, the corn straw degradation ratio was determined using the weight loss method. The lignin, cellulose, and hemicellulose contents were determined by a cellulose analyzer (ANKOM A200i). The degradation rate of lignocellulose was calculated in triplicate.

High-throughput sequencing

The fermentation liquid of decomposer M44 was taken on the 1st, 7th, 14th, 21st, and 28th days after inoculation and centrifuged at 4°C for 10 min at 5000 r·min^-1. The supernatant was discarded, and the precipitate remained. This was repeated several times to ensure complete precipitation. Under aseptic conditions, a bacterial genomic DNA extraction kit (China Tiangen Biochemical Technology Co., Ltd.) was used to extract genomic DNA from samples at different culture times, and 1% agarose gel was used for electrophoresis. PCR amplification was performed with the primers 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) of standard bacteria 16S V3-V5 region, and the product was purified and quantified. An Illumina MiSeq sequencer was used to construct qualified libraries for sequencing, and sequencing was completed at Shanghai Meiji Biomedical Technology Co., Ltd.

Statistical analysis

Operational taxonomic units (OTUs) with a 97% similarity cutoff were clustered using UPARSE (version 7.1, http://drive5.com/uparse/), and chimeric sequences were identified and removed. The taxonomy of each OTU representative sequence was analyzed using RDP Classifier (http://rdp.cme.msu.edu/) against the 16S rRNA database (Silva SSU128) with a confidence threshold of 0.7. Data of enzyme activities and straw degradation characteristics were analyzed by IBM SPSS Statistics 25.0 (IBM Inc., Armonk, NY, USA, https://www.ibm.com/cn-zh/analytics/spss-statistics-software), and R (V3.6.1) (https://www.r-project.org/)and Origin 2018(https://www.Originlab.com) was used to create figures.

Straw and lignocellulose degradation rates of microbial decomposer M44

The straw and lignocellulose degradation rates increased upon extending the culture time and tended to be stable in the late stage (S1 Fig). The straw degradation rate increased the most at 1–7 days of culture. The straw degradation rate after culturing for 21 days was 30.51%, accounting for 97.07% of the total degradation rate. 31.43% of straw was degraded at the end of the culture. Hua et al. also showed that rice straw degraded rapidly at 0–9 days of culture and tended to be stable at the later stage [16]. Changes in the straw components’ degradation rate were generally consistent with the straw decomposition trend. Among them, the degradation rates of hemicelluloses and cellulose were significantly higher than those of lignin, and the maximum degradation rates were respectively 21.69% and 25.04% at the end of the culture. The degradation rate of lignin was the highest from day 14 to 21 and reached the highest value of 13.67% on day 28, which was significantly higher than the value at the early stage of culture.

Previous research on the screening of straw degradation using microbial consortia has mainly focused on normal or high-temperature conditions, but the in situ decomposition of returned corn straw in autumn at low temperatures is often poor. Previous studies have indicated that the degradation rate of straw was about 50% at 30–40°C [7,17]. In our study, the corn straw degradation rate using microbial decomposer M44 reached 30.51% at 15°C on day 21. Although this is lower than what is suitable for medium and high-temperature microbial consortia studied by other researchers, it can be adapted to corn straw returning in cold and arid regions.

Enzyme activities of microbial decomposer M44

In the early stage of straw degradation by M44, the FPase and Cx activities increased rapidly and peaked on day 14 with 2.55 U·mL^-1 and 2.34 U·mL^-1, respectively, which were significantly higher than on the 1st, 21st, and 28th days (S2A Fig). The xylanase activity increased slowly after the 7th day, reaching 9.86 U·mL^-1 at day 28 (S2B Fig). This was mainly because polysaccharides, amino acids, organic acids, proteins, and other soluble organic matters in straw were readily used by microorganisms in the early stage of degradation, and microorganisms secreted more cellulase and hemicellulase, which degraded straw. Therefore, the FPase, Cx, and xylanase activity increased rapidly in the early stage of culture. After culturing for 21 days, the Lac and LiP enzyme activity increased and reached 134.43 U·L^-1 and 86.88 U·L^-1, and then remained stable until day 28 (S2C Fig). Upon extending the degradation time, the easily-degradable components of straw gradually decreased, and highly refractory substances (such as lignin, waxy, and tannin) gradually increased, so the lignin activity increased rapidly in the later stage of culture. This result is consistent with changes in the straw lignocellulose degradation rate.

Alpha diversity of microbial decomposer M44

Alpha diversity analysis was performed on the microbial communities of samples at different degradation stages (S3 Fig). The Ace index fluctuated slightly and reached the highest of 127.82 on day 21. The Shannon and Sobs indices increased from 1.29 and 58.67 on day 1 to 2.72 and 109.33 on day 28. The Simpson index decreased significantly from 0.41 on day 1 to 0.16 at the end of the culture. The alpha diversity indices showed no significant differences between days 21 and 28 of culturing, indicating that the diversity and richness of the microbial composition were most stable at the later stage of degradation.

Beta diversity of microbial decomposer M44

Principal coordinate analysis (PCoA) based on weighted UniFrac distance was conducted for bacterial communities of samples at different culture periods (S4 Fig). There was a certain distance between each sample, indicating that there were differences in the species composition, relative abundance, and phylogenetic relationship of compound bacteria in different culture periods. To further clarify the differences, we performed ANOSIM and PERMANOVA analyses at the OTU level based on the Bray-Curtis distance algorithm. The results showed remarkable differences between samples at different culture stages (p < 0.05; N = 999 permutations).

PCoA showed that 21 and 28-day treatments clustered together, and there was no significant difference between the alpha diversity indices, indicating that the species composition abundance and structure were similar in the later stage of culture.

Microbial composition dynamics using microbial decomposer M44

At the phylum level (others < 1%), the microbial decomposer M44 mainly contained Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria. Most of the species related to the biomass cycle had relatively complete metabolic mechanisms, similar to the results of Wongwilaiwalin et al. [18], Zhu et al. [19], and Guo et al. [20]. In previous reports, Proteobacteria and Firmicutes were found in straw compost [21], rotten wood [22], and rumen [23]. These can secrete cellular laccase and peroxidase, which degrade the monoaryl, bisaryl, and phenolic intermediates of lignin and sulfate lignin [24–26]. Many studies have documented that Proteobacteria, Firmicutes, and Bacteroidetes are the main species involved in cellulose hydrolysis [27,28]. Among them, Proteobacteria accounted for the highest proportion at different culture stages and reached the maximum of 92.89% culture on day 14. Contrary to Proteobacteria, the abundance of Firmicutes decreased from 11.33% on day 1 to 4.80% on day 14 and then increased to 7.18% at the end of the culture. The richness of Bacteroidetes increased significantly from 0.17% on day 1 of culture to 10.24% at the end of the culture. There was a significant difference between samples at different culture stages (p < 0.05). There were no significant changes in Actinobacteria throughout the culture process (S5 Fig).

At the genus level (others < 1%), there were significant differences in the abundance of dominant genera at different culture periods (p < 0.05) (S6 Fig). Pseudomonas was the dominant bacterium in the samples at different culture stages, accounting for 55.25% and 54.82% of the total bacteria on days 7 and 14. Pseudomonas possesses a high capacity to utilize and degrade petroleum hydrocarbons and is classified as a hydrocarbonoclastic microorganism [29,30]. Guo et al. [31] and Wang et al. [32] detected Pseudomonas in the composite microbial XDC-2 and LDC, which efficiently degraded lignin and aromatic compounds. The relative abundance of Enterococcus was the highest on culture day 1 (10.44%), which was much higher than in other culture periods. Delftia and Ochrobactrum accounted for the highest proportions on day 14, which were 5.83% and 1.26%, respectively. In addition, the relative abundance of Kaistia, Novosphingobium, and Trichococcus were the highest on day 21, with 3.58%, 1.90%, and 3.89%, respectively, which were remarkably higher than that on days 1, 7, and 14. It has been reported that Trichococcus produces lactic acid, formic acid, and ethanol and participates in the decomposition of organic matter at temperatures as low as -5°C [33,34]. The relative abundance of Chryseobacterium, Sphingobacterium, and Rhizorhapis was 2.90%, 3.09%, and 2.43%, respectively, on day 28, which was remarkably higher than that in the early stage of degradation. Among them, Chryseobacterium has been found to produce cellulase and protease, which degrade cell walls [35,36]; Sphingobacterium can utilize and degrade lignocellulosic material for growth and reproduction, resulting in efficient straw degradation, and are classified as hydrocarbonoclastic microorganisms [11]. During straw decomposition, Paenibacillus remained stable at 1.28–1.65%, and there was no significant difference among samples at different degradation stages. Paenibacillus sp. was reported to produce xylanases that specifically decompose xylans [37]. It has a high thermal stability and wide pH adaptability and has been widely used for industrial enzyme production [38].

Comprehensive analysis of straw degradation characteristics and species composition changes shows that in the initial stage of straw degradation (1, 7, and 14 days), the structure of straw was relatively complete, and there was sufficient oxygen. Pseudomonas, Paenibacillusm, and Delftia mainly used nutrients in the medium to proliferate in large numbers. This was accompanied by the secretion of degrading enzymes (FPase, Cx, and xylanase), while the cellulose and hemicelluloses in the system were decomposed into small-molecule acids and esters that provided nutrients for the growth of other microorganisms. It is speculated that the above bacteria may be functional microorganisms during cellulose and hemicellulose degradation by compound bacteria. At the last stage of cultivation (21–28 d), the contents of Kaistia, Novosphingobium, Brevundimonas, Chryseobacterium and Sphingobacterium continued to increase, and the cellulase activities (FPase and Cx) declined. The Lip, Lac activity, and lignin degradation ratio rapidly increased and stabilized at relatively high values. It is speculated that the above bacteria are functional microorganisms related to lignin degradation.

There were also other strains (Kaistia and Rhizorhapis) without lignocellulosic degradation activity in M44, and non-functional aerobic bacteria may play an important role in providing an anaerobic environment, as well as growth promotion and pH neutralization [39,40]. Overall, interactions among these bacteria in the microbial decomposer M44 might be responsible for the high straw degradation activity and stable maintenance of the community structure.

Correlation between the microbial composition of M44 and straw degradation characteristics

Correlation analysis of the vital genera of M44 and straw degradation characteristics (S7 Fig) showed that the Cx and FPase activity were positively correlated with Delftia, Pseudomonas, Paenibacillus, and some bacteria that degraded straw or intermediate products in the colony. Chryseobacterium, Brevundimonas, Rhizobium, Delftia, and Devosia were positively correlated with the xylanase, Lac, and LiP activities. The above results showed that Pseudomonas, Delftia and Paenibacillus mainly participated in the degradation of straw in the early stage, which increased the activities of cellulase and hemicellulase, and degraded cellulose and hemicellulose components efficiently. Rhizobium, Chryseobacterium, Sphingobacterium, Brevundimonas and Devosia are mainly involved in the degradation of straw at the later stage, which improved the activities of laccase and lignin peroxidase and efficiently degraded lignin components.