The appropriate amount of winter forest soil samples from northeast China were collected in natural environments below 10°C, loaded in sterile self-sealing bags, brought back to the laboratory, and refrigerated at 4°C. 10.0 g of the above samples were placed in a triangular bottle with 90 ml of sterile water for 20 min, followed by fully absorbing 1 ml of supernatant into 100 ml of enrichment medium at 10°C, observation, and shaking once a day. When the straw section became loose and soft and the culture medium became darker, it was transferred to the fresh enrichment medium with 5% inoculum and then repeated four times.

Hutchinson enrichment liquid medium containing: KH₂PO₄ 1 g/l,NaCl 0.1 g/l,NaNO₃ 2.5 g/l, MgSO₄·7H₂O 0.3 g/l, FeCl₃·6H₂O 0.01 g/l, CaCl₂ ·6H₂O 0.01 g/l, and corn straw 5 g/l, pH = 7.0 and the Luria-Bertani (LB) liquid and solid media containing: tryptone 10 g/l, yeast extract 5 g/l, and NaCl 10 g/l, pH = 7.0; Carboxymethylcellulose sodium culture (CMC) medium: sodium CMC 5 g/l, NaNO₃ 3 g/l, KH₂PO₄ 1 g/l, MgSO₄·7H₂O 0.5 g/l, KCl 0.5 g/l, FeSO₄ 0.01 g/l, and agar 15 g/l, pH = 7.0 were prepared and used in this study. The activities of different cellulases in the bacterial solution were detected by 3,5-dinitrosalicylic acid (DNS) method (Lu et al., 2015). Briefly, the degradation ability was detected using cellulose substances CMC (Somogyi, 1926), absorbent cotton, and salicin as substrates, in which CMC was the substrate for endo-1,4-β-glucanase (CMCase), absorbent cotton was the substrate for exo-1,4-β-glucanase (C₁), and salicin was the substrate for BG (Somogyi, 1952).

The enriched bacterial solutions with dilution ratios of 10⁻⁴, 10⁻⁵, and 10⁻⁶ were coated on the above isolation medium, and cultured at 10°C for 3 d to obtain pure culture. Then the purified strains were inoculated into the cellulose Congo red medium and cultured at 10°C for 4 d, followed by preliminary screening based on the ratio of the diameter of the hydrolysis circle (D) to the diameter of the colony (d; D/d). Finally, 1 ml of the suspension (1 × 10⁹ bacteria) of the cellulose-degrading strains screened was inoculated into the liquid fermentation medium (50 ml in a 250 ml conical flask) and cultured by constant temperature shaking at 10°C and 160 r/min for 6 d, and its enzyme activity was measured (Liu et al., 2014).

Polymerase chain reaction (PCR) amplification was conducted with 16S ribosomal ribonucleic acid (rRNA) V3-V4 as the amplification region, and 27F (5’-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5’-TACGGYTACCTTGTACGACTT-3′) were used as forward and reverse primers. Amplicons were examined by 1% agarose gel electrophoresis (Chuanjiao et al., 2021). A phylogenetic tree of the sequences was retrieved from National Center for Biotechnology Information (NCBI) GenBank and the reference sequences were constructed by the neighbor-joining method using MEGA 7. The PCR was conducted with following conditions: pre-denaturation at 94°C for 2 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 100 s, and repair and extension at 72°C for 2 min.

A single colony was inoculated into the LB liquid medium, cultured in a shaker at 30°C and 170 r/min for 2 d, and collected by centrifugation at 8,000 rpm for 3 min. The genomic DNA was extracted from the strains through CTAB method (Motham et al., 2014), and the total microbial DNA was extracted using Power Soil^® DNA Isolation Kit. The purity and concentration of the total DNA were measured by virtue of a NanoDrop ND-1000 nucleic acid quantitation instrument. The purified DNA was sent to Wuhan OneMore Technology Co., Ltd. for whole genome sequencing. The genome was filtered and assembled by HGAP 2.0 (Westbrook et al., 2015).

The cultured bacteria were collected at the initial logarithmic stage and were sent to Wuhan Guao Technology Co Ltd. for genome extraction and sequencing. Illumina PCR adapter reads and low-quality reads from the paired-end were filtered using the readfq (Version 10) remove reads with less than a certain percentage of low-quality bases (mass value B 38 or default is 40 bp), a certain percentage of reads with N bases (default is 10 bp), overlap exceeds a certain threshold (default is 15 bp) and the possibility reads originating from the host (Chuanjiao et al., 2021). Coding regions of coding genes were predicted by Glimmer 3.0. The predicted genes were aligned to NCBI non-redundant protein sequence database (NR), clusters of orthologous groups of proteins (COGs) database, Swiss-Pro, Genome Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database by BLAST for gene functional annotation.

Bacillus subtilis K1 and the pre-screened composite strains were selected for subsequent straw composting experiment. Corn straws were the composting materials, containing 11.9% water, 0.62% total N, 0.09% total P, 0.28% total K, C/N 37.42, and urea (46% N). Three treatments were set up in the composting experiment:

Bio-composting (M): About 1,000 kg of corn straws were composted and added with 5 l (5 × 10⁷ CFU/l) of composite strain (included yeast, and Trichoderma viride, obtained from The Liaoning Academy of Agricultural Sciences Institute of Plant Nutrition and Environmental Resources Microbiology Laboratory) and 10 kg of urea. (2) Bio-composting (MC): About 1,000 kg of corn straws were composted, with 2.5 l each of Bacillus subtilis K1 and composite strain fermentation broth and 10 kg of urea. (3) Control (CK): About 1,000 kg of corn straws were composted without adding microbial agents but added with 10 kg of urea. The experiment was repeated 3 times.

Specifically, a flat head land was selected and stacked with crushed corn straws, and a little microbial agents and urea were sprinkled on the surface for every 20 cm rise. Meanwhile, the straws were compacted by treading. The composting height was kept at 1.5–1.8 m, and the outer surface was covered with an agricultural plastic film. The experiment was from March 13, 2019, and the composting lasted for 45 d in total. Test site: Shenyang Xidi Testing Base (123°55′43.16″ E, 41°82′17.74” N).

During the composting process, temperature changes were detected. At the same time, the content of N, P₂O₅, K₂O and active organic matters and the pH of straws were measured in accordance with China’s organic fertilizer standard (NY 525–2012). The humus components were determined according to Liu Xiaojing’s method Sodium pyrophosphate-sodium hydroxide extraction, potassium dichromate oxidation capacity method (Liu et al., 2017). The content of cellulose and hemicellulose was measured through Van Soest method (Zhang et al., 2017). The morphological and physicochemical indexes of the strains were observed, and the OD₆₀₀ of the strains under different carbon sources, pH, and temperature conditions were determined (Liu et al., 2017) with some modifications.

Every experiment was conducted using a completely random design. The results of three replicates (n = 3) were expressed as SD (standard deviations). Following an Analysis of variance (ANOVA), the means were determined using Tukey’s Honestly Significant Difference (HSD) test at p ≤ 0.05. The data were statistically analyzed using IBM SPSS Statistics 22.0. Origin’s graphics and analysis software was used to create the graphical representations (Version 2022, OriginLab Corporation, Northamptom, MA, United States).

The bacteria were cultured using CMC selection medium, and the isolated strains were subjected to the Congo red plate test, forming transparent circles as shown in (Figure 1). Therefore, K1 was identified as B. subtilis and named B. subtilis K1. The B. subtilis K1 is a regular single cell, 2–5.5 μm long and ~ 0.3–0.4 μm wide, with flagellum around its body as shown in its SEM micrograph (Figure 1B), and it is a small, round, moist, smooth, viscous, and easy to pick (Figure 1A). It has G+ rod, milk-white round colony with the growing conditions of 5–40°C (optimal temperature 25°C), pH 3.3–8.5 (optimal pH 6.5), optimal carbon source is sodium CMC. It entered the logarithmic period at 8 h, with the highest OD₆₀₀ value at 30 h, and then entered the stabilization period (Figure 2A). The ratio of the diameter of the transparent circle to the diameter of the colony (D/d) was calculated to preliminarily determine the strains with strong cellulose-degrading ability. As shown in Figure 3B, the mean D/d was the highest in K, followed by W, V, and Z. A total of 12 strains with larger transparent circles were screened for further screening. The K1 was higher, among which CMCase had the highest activity (24.69 U/ml), followed by endoglucanase (1.72 U/ml) and exoglycanase (1.14 U/ml). Considering the growth rate and the cellulose-degrading ability, K1 was further studied (Figure 3A).

Degradation of crop straw is a bottleneck in natural agricultural settings, there has been a recent increase in the exploration of cold-adapted microorganism, as it can solve the problem of difficult degradation of corn straws under low temperature offering new alternatives for the sustainable development of agriculture. In this study, a cold-adapted cellulose-degrading strain, B. subtilis K1, enrichment of culture by cold domestication was utilized. The cellulose activity of B. subtilis K1 was higher, among which β-glucanase (24.69 U/ml), exo-β-glucanase (1.72 U/ml) and β-glucosaccharase (1.14 U/ml) were found to have the highest degradation activity. B. subtilis K1 is a regular single cell, 2–5.5 μm long and ~ 0.3–0.4 μm wide, with flagellum around its body as shown in its SEM micrograph. In addition, it is a small, round, moist, smooth, viscous, easy to pick, having G+ rod, milk-white round colony with the growing conditions of 5–40°C (optimal temperature 25°C), pH 3.3–8.5 (optimal pH 6.5), optimal carbon source of sodium CMC-Na. It entered the logarithmic period at 8 h, with the highest OD₆₀₀ value at 30 h, and then entered the stabilization period. Despite a strong cellulose-degrading ability, fungi grow more slowly than bacteria, and their cellulase has poor alkali resistance (Ruijssenaars and Hartmans, 2004). Smith et al. (2018) found that Sphingobacteria and Flavobacteria were the major microbes in different environments of Dry Valley of Antarctica (Huang et al., 2021), proving that such genera are capable of adapting to the low temperature. Cho et al. (2021) isolated Geobacter from the Antarctic soil (Smith et al., 2018), and found that Geobacter is still able to degrade arabinose and hemicellulose in the Antarctic environment. Shang et al. (2012) screened a strain of low temperature-resistant cellulose-degrading bacterium from compost (Cho et al., 2021). The above results demonstrate that B. subtilis K1 is expected to be developed into commercially available cellulose biodegrading microbial agents for the sake of comprehensive utilization of straws in Northeastern China.

Composting is effective in degrading organic matters, during which carbohydrate metabolism can produce various compounds and degrade lignin and hemicellulose and cellulose under aerobic conditions. As lignin and cellulose and hemicellulose are degraded, the microbes preferentially degrade available degradable substances (Zhang et al., 2016). It can be speculated that carbohydrate metabolism is crucial for the degradation of hemicellulose and cellulose in the process of corn straw composting. In the present experiment, the physicochemical parameters of the cold-adapted cellulose-degrading strain B. subtilis K1 applied to the process of low-temperature corn straw composting were different. Specifically, under an average outside temperature of 6°C, after the cold-adapted corn straw decomposing bacteria were added. It was found that through adding cold-adapted cellulose-degrading B. subtilis K1 in the corn straw composting under 6°C (ambient temperature), the average temperature of straw composting was 58.7°C higher 86.7% than the control. The HA/FA was higher 94.02% than the control and the lignocellulose degradation rate was lower 18.01–41.39% than the control. Correspondingly, different treatment C/N ratio dropped from 37.25 to 21.40%、21.92% throughout the process. Given the low temperature in Northeastern China, the microbial diversity is high in the straw incorporated soil and the rotten straws, and many types of microbes may degrade the main parts of plants and release nutrients into the environment, thereby participating in nutrient cycling (Li and Jin, 2011). Therefore, it is of great significance to effectively develop a catalytic pathway for microbial degradation of cellulose and prepare an efficient low-temperature straw decomposing microbial agent for solving the problem of straw burning and protecting the black soil in Northeastern China (Yao et al., 2009).

Whole genome sequencing is an effective means for studying bacterial genetic and molecular mechanisms. In this experiment, a cold-adapted cellulose-degrading strain, B. subtilis K1, was screened and subjected to bio-informatics analysis. The results showed that the strain K1 was 4,060,823 bp in genome size and composed of 4,213 genes, among which 3,665, 3,656, 2,755, 3,240, 1,261, 3,336 and 4,003 genes were annotated in the NR, Pfam, COG, GO, KEGG and Annotation databases, respectively, and a large number of lignocellulose degradation-related genes were annotated in the genome. Furthermore, the strain K1 had genes encoding important enzymes (such as GH, GT, PL, CE, and auxiliary oxidoreductase) in the cellulose degradation pathway, and these genes were different in the expression, resulting in different expressions of CMCase, C1, and BG in the strain. These three enzymes act synergistically to completely hydrolyze cellulose GHS family cellulases function as endocellulases and/or exocellulases and play a key role in the early stage of cellulose degradation (Merlin et al., 2014).