Microbial decomposition of rice straw: alkaline pretreatment, genome analysis and fermentation broth on seed growth

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It is difficult to degrade cellulose only by biodegradation, chemical or physical degradation. The weight loss rate of rice straw reached 38.86% by the inoculation of Bacillus amyloliquefaciens N5 combined with alkali pretreatment. The fermentation broth of strain N5 and straw could promote the rice seed’s germination and the growth of rice seedlings. Moreover, the addition of peptone as a nutrient source further amplified the cellulose degradation. The whole genome analysis revealed genes associated with cellulose degradation in the genome of strain N5, including glycoside hydrolase family 1 (GH1), GH13, GH43, and three polysaccharide lyases (PLs), eleven carbohydrate esterases (CEs), twenty-six glycosyltransferases (GTs), and twenty-five carbohydrate-binding modules (CBMs). cellulose rice straw alkali pretreatment microbial degradation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction As agricultural production advances, many crop straws with high cellulose content are produced annually [1, 2] . The surplus accumulation of these straws in agricultural areas is becoming a big issue. Farmers often consider these straws as waste, which, if burned directly or left piled up in the fields, can severely damage the ecological environment and disrupt the agricultural cycle [3–5] . This, in turn, can restrict the sustainable growth of agriculture [6] . The proper management of crop straw is essential for promoting sustainable agricultural growth, addressing the energy crisis, and fostering the development of green agriculture [7] . Approximately 70% of biomass is composed of cellulosic and hemicellulosic polymers, which are bound to the lignin component by covalent bonds, providing the lignocellulosic biomass with great robustness and resistance to (bio-)chemical or physical treatments. In agricultural residues, paddy straw is notable for its composition, which includes 43% cellulose, 23% hemicellulose, and 6.27% lignin [8, 9] . The complex network structure formed by these three components in crop straw presents a challenge to the degradation and utilization of the material [10, 11] . Consequently, cellulose requires treatment to increase the utilization rate of straw. The benefits of microbial degradation over alternative techniques are unparalleled. Bacteria capable of decomposing cellulose are plentiful [12–14] . Microorganisms in nature may release enzymes that synergistically affect cellulose degradation, which is produced by several microorganisms, especially bacteria and fungi [15–17] . Enzyme proteins must work in concert with enzyme systems and can be categorized into three groups based on their characteristics and roles: β-glucosidase, endoglucanase, and exoglucanase [12, 18] . Cellulase functions by hydrolyzing cellulose into glucose, utilizing the combined action of multiple enzymes to break down cellulose effectively [19] . In this context, fungi and bacteria have garnered significant interest due to their capacity to generate diverse cellulases. While fungi are the primary sources of enzymes that break down lignocellulose, bacteria typically exhibit the advantages of being environmentally friendly, faster growth rates, cheap, convenient, and not causing secondary pollution compared to fungi [3, 20] . Finding a more efficient degradation approach is essential because the microbial method alone may not fully degrade straw due to its complex structure. To maximize the combination, combined therapy can overcome the limitations of physical, chemical, and biological treatments. Currently, the most widely used treatment technology combines chemical and physical methods. To overcome the resistance of lignocellulose, TANG et al. [21] pretreated corn stover using organic solutions, resulting in high-yield fermentable sugars and superior, salt-free lignin. The overall sugar yield was 83.2%, and the delignification rate was 81.7%. Combining chemical techniques with microbial treatment can also achieve a better result in straw decomposition. WU et al. [22] subjected rice straw to a bacterial breakdown in conjunction with Fenton treatment. The findings demonstrated that bacterial inoculation and Fenton treatments altered the fungal community's composition, promoted fungal diversity, altered the genes responsible for cellulose breakdown, and accelerated the rate of rice straw degradation. Chen et al. [23] used dilute acid, lime, ammonia/dilute acid, and alkali, four different chemical reagents, to pretreat corn straw. The results showed that dilute alkali pretreatment was the most effective in improving the enzymatic hydrolysis efficiency of fibre residue. Therefore, this study selected dilute alkali to treat straw and studied the degradation of Bacillus amyloliquefaciens N5 on pretreated straw. This study investigated the preliminary effects of chemical pretreatment and degrading bacteria on straw degradation, verifying the modifications resulting from pretreatment on straw following biodegradation. Through the study on the growth promoting effect of straw biological fermentation broth on rice seeds, the effect of fermentation broth on plants after microbial degradation of straw was explored. This study also provides new ideas for recycling straw. The strain genes were investigated based on whole-genome sequencing analysis to overcome the barrier of poor adaptability and low enzyme production in the future application of cellulose resources. This allowed for excellent strains and technical support, providing valuable resources for later synthetic biology applications. 2. Materials and methods 2.1 Degradation of rice straw The Bacillus amyloliquefaciens N5 was screened in cow manure compost, and the strain was inoculated into LB liquid medium (yeast extract 5.0 g/L, peptone 10.0 g/L, sodium chloride 10.0 g/L, pH 7.0 ~ 7.2, sterilized at 121°C for 30 minutes), and cultured at 35°C and 160 rpm. The strain N5 was inoculated into the straw culture medium (rice straw was dried and cut into 2–3 cm segmental straw 2 g, inorganic salt culture medium (contained NH 4 Cl 1 g/L, KH 2 PO 4 0.5 g/L, K 2 HPO 4 1.5 g/L, MgSO 4 0.2 g/L, NaCl g/L, and 1 mL/L Hunter’s trace elements solution) 100 mL, natural pH, 121°C sterilization for 30 minutes) at inoculation amount 5% (V/V). The liquid fermentation was carried out at 35°C and 160 rpm for 7 days. After the degradation of liquid fermentation, the residue after straw degradation was filtered and rinsed with a large amount of water to remove the bacteria and other soluble substances attached to the straw and dried to constant weight at 105°C. At the same time, the dry weight of straw residue was weighed to calculate the weight loss rate of straw [24] . $$\:\text{weight loss rate of straw}\text{}\text{(}\text{%}\text{)}\text{=}\raisebox{1ex}{$（{\text{M}}_{\text{0}}\text{-}{\text{M}}_{\text{1}}）$}\!\left/\:\!\raisebox{-1ex}{${\text{M}}_{\text{0}}$}\right.\text{×100}$$ In the formula: M 0 is the dry weight of straw residue in the control group (g); M 1 is the dry weight (g) of straw residues in the treatment group. 2.2 Effect of chemically combined treatment on rice straw degradation ability 2.2.1 Alkali treatment combined with strain N5 on the degradation of rice straw Straw alkali treatment: Rice straw was soaked in a 1.5% sodium hydroxide (NaOH) solution for 24 h, rinsed with water to neutrality, and dried in an oven at 80°C for later use. The treated rice straw was added to the inorganic salt medium (not sterilized). The experiment set up four treatments: 1)untreated straw, 2༉untreated straw + N5 bacterial solution, 3༉pretreated straw, and 4༉pretreated straw + N5 bacterial solution. The strain N5 was inoculated into the pretreated straw medium at 1% (V/V) (pretreated straw 2 g, inorganic salt medium 100 mL, pH 7.0, medium not sterilized). After 7 days of degradation at 35°C and 160 rpm under natural conditions, the rice straw degradation residues were filtered and washed. Drying to constant weight at 105°C, each treatment had three replicates, and the weight loss rate of the straw was calculated. The residue was weighed to calculate the weight loss rate of the straw 2.2.2 The effect of nutrient addition on the combined degradation of rice straw The strain N5 was inoculated into the pretreated straw medium at a 1% (V/V) ratio, and nutrients were added for bioaugmentation. To the pretreated straw medium, yeast (0.5 g), peptone (0.5 g), urea (0.5 g), and glucose (0.5 g) were added as nutrients to provide sustenance for both natural microorganisms and strain N5. Liquid fermentation was conducted at 35°C with a rotation speed of 160 rpm for 7 days, with three replicates for each treatment, during which the straw degradation rate of the strain was determined. The variations in pH, CMCase activity, OD600, and microbial quantity during the straw degradation process were measured, and the changes in natural degradation and biodegradable straw residues were observed. Each treatment included three replicates. 2.3 The growth-promoting effect of biological fermentation broth on rice Pretreatment of rice seeds: Rice seeds with intact surfaces and no black spots were soaked in sterile water for 24 hours, then sterilized with 75% alcohol for 5 minutes and washed with sterile water [25] . The strain was introduced into a nutrient straw medium at a 1% (volume/volume) inoculation rate. Following a 7-day biodegradation period, the fermentation supernatant was centrifugated for 10 minutes at 8000 rpm and 4°C. The resulting pellet from the centrifuged biological fermentation broth was then diluted to a 10 − 6 concentration, the standard dilution factor. A disposable petri dish was prepared by laying a filter paper for water retention at the bottom. Each dish was then populated with 50 sterilized rice grains. Three experimental groups were established: sterile water, nutrient medium, and biological fermentation broth. 10 mL of the 10 − 6 diluted biological fermentation broth was added to each culture dish. The sterile water and nutrient medium were diluted similarly to negative controls. The petri dishes were then placed in an artificial climate chamber for incubation. The incubation conditions were set at a temperature of 25°C with a light/dark cycle of 16 hours light and 8 hours darkness. After a 7-day cultivation period, the growth of the rice seedlings was assessed by measuring both the height of the seedlings and the length of the rice roots. Each treatment was conducted in triplicate for consistency. The sterile water, nutrient medium, and biological fermentation broth were subjected to varying levels of salt stress using NaCl solutions at concentrations of 100 and 200 mmol/L. Each petri dish was inoculated with 50 grains, and this process was repeated three times. Germination counts were recorded daily. Under the same culture temperature and photoperiod conditions, the germination rate of rice seeds was recorded daily in an artificial climate incubator until the seventh day. Three replicates were set for each treatment, and the calculation formula of seed germination rate is as follows [26] : $$\:\text{Germination rate (%)}\text{=}\raisebox{1ex}{$\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{s}\text{e}\text{e}\text{d}\:\text{g}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{i}\text{o}\text{n}$}\!\left/\:\!\raisebox{-1ex}{$\text{t}\text{o}\text{t}\text{a}\text{l}\:\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{s}\text{e}\text{e}\text{d}\text{s}$}\right.\text{×100}$$ 2.4 Scanning electron microscope analysis of rice straw The degradation of rice straw after different treatments was observed and analyzed by scanning electron microscope. After the fermentation is completed, the medium is filtered, and the treated rice straw is air-dried. After processing by slicing, fixing, washing, dehydration, and replacement, the samples were adhered to the scanning electron microscope sample table with conductive tape and vacuum gold plating, and the surface morphology and structure of rice straw were observed by scanning electron microscope. 2.5 Whole genome sequencing The strain N5 was inoculated into LB medium and cultured for 24 h. The logarithmic phase bacteria were collected, and the bacterial solution was centrifuged for 5 minutes (rotation speed 10000 rpm, temperature 4°C). The supernatant was discarded to retain the bacteria, placed in liquid nitrogen for quick freezing, and then stored in a refrigerator at -80°C. PacBio RS II and Illumina HiSeq 4000 platforms were used to sequence and assemble the whole genome of strain N5. The Pacbio platform uses SMRT to generate a sub-reading set and deletes Pacbio sub-readings (length < 1kb). The Canu program is used for self-correction, drafting genomic units, and assembling a corrected cyclic common sequence sub-reading set using high-quality Canu. In order to improve the accuracy of the genome sequence, GATK is used for single base correction, and the Blast alignment tool is used for the best matching of functional annotations. 3 Results 3.1 Rice straw degradation by strain N5 The results are depicted in Fig. 1 c. After 7 days, the weight loss rate of rice straw in the control experiment without strain N5 was 4.05%. In contrast, the addition of N5 degrading bacteria increased the weight loss rate to 23.40%, indicating that the presence of N5 degrading bacteria significantly enhances the degradation of rice straw, thereby achieving effective treatment. In summary, the strain N5 identified in this study possesses excellent degradation capabilities for rice straw, elevating the weight loss rate by 19.35% within 7 days. The silicon structure within the rice straw, as highlighted in the electron microscope image shown in Fig. 1 a, is characterized by a surface layer of wax. The primary resistance to degradation in rice straw is attributed to its silicon structure and the protective wax layer [27, 28] . The untreated straw, which did not fully dissolve its surface silicon structure and wax layer, displayed a smooth surface, intact integrity, and a regular, dense morphological structure compared to the straw treated with N5 (Fig. 1 a). After 7 days of treatment with N5, the straw's surface exhibited large, irregular fractures and pores (Fig. 1 b). This observation suggests that strain N5 may cause the wax layer on the straw's surface to detach, exposing the underlying cellulose and silicon structure. This exposure would facilitate the microbes' more efficient utilization of the substrate, thereby enhancing the breakdown of the straw [29] . 3.2 Effect of combined treatment on rice straw degradation Figure 2 d displays the microstructure of rice straw following alkali pretreatment. After NaOH treatment, the straw surface's convex structure is loose, and the gap widens. Chemical pretreatment appears to expand the cellulose's exposed area in straw, making it more favorable for the adherence and uptake of strains [30] . The straw's appearance following NaOH treatment is depicted in Fig. 2 a. Rice straw has a rough surface and a whole structure. The pretreated straw after seven days of incipient deterioration is shown in Fig. 2 b. The straw sample is not finely crushed, and the straw's edge has fine hair edges. After being subjected to N5 degradation for seven days, the pretreatment rice straw in Fig. 2 c lost much of its structural integrity and fractured into pieces of varying sizes. As shown in Fig. 2 e, the weight loss rate of rice straw was 6.3% for untreated rice straw and 22.58% for rice straw that had been treated with NaOH, suggesting that treating rice straw with NaOH can accelerate its natural degradation rate. When rice straw was treated with 1.5% NaOH, its weight loss rate increased to 38.86% from 16.3% when left untreated. The outcomes demonstrated that the complicated structure of straw might be broken down by strain N5 in conjunction with a chemical pretreatment, increasing the bioconversion efficiency of cellulose biomass [31] . 3.3 Effects of nutrients on straw degradation by alkali treatment Strain N5 demonstrated good adaptation to various nutrients, as seen in Fig. 3 . Peptone, yeast, glucose, and urea were the ideal nutrient sequences for the fermentation medium, and the corresponding weight loss rates were 65.28%, 62.18%, 57.97%, and 44.45%. By adding peptone to the fermentation medium, strain N5 reached the maximum breakdown rate (65.28%). Straw under natural conditions degraded at a rate ranging from 24.16–30.2%, consistently lower than that of the biological treatment with strain N5. For further culture, peptone was added to the pretreated straw media. 3.4 Changes in physicochemical properties of alkali-treated straw during biodegradation As the fermentation period was extended during the natural degradation, the enzyme activity decreased, and on the second day, the CMC enzyme activity achieved its maximum value of 124.28 U/mL (Fig. 4 a). The CMC enzyme activity of N5 biodegradation fluctuated sporadically throughout the entire degradation process, but overall, there was a high and, subsequently, a low trend. The additional strain N5's biodegradation could break down the substrate more quickly on the second day of fermentation and hasten the breakdown of the straw, as evidenced by the greatest enzyme activity appearing on the second day and the CMC enzyme activity of 224 U/mL, 206.01 U/mL, 134.32 U/mL, 179.54 U/mL, 166.21 U/mL, and 156.93 U/mL were the CMC enzyme activity, in that order. This might be because early fermentation had enough nutrients, which improved the strain's ability to create enzymes through metabolism. As time passes and the nutrients from mixed biological fermentation minimize the drop in dissolved oxygen, intracellular enzymes are released, and enzyme activity becomes unstable [32, 33] . The OD600 value of natural degradation peaked at 1.55, while the OD600 value of biodegradation was 2.38 on the third day, as illustrated in Fig. 4 b. The biodegradation's OD600 value peaked at 2.67 on the fifth day, but the natural degradation value was just 1.17. It demonstrates that the biodegradation of rice straw by strain N5 has a comparatively high bacterial concentration and robust bacterial growth and metabolism. The pH was stable for the first four days of the fermentation process, which could be attributed to other microorganisms in the medium. Following inoculation, strain N5 did not cause pH change during the lag phase. On the fourth day, the pH increased significantly from 6.45 to 7.56, and on the sixth day of fermentation, the pH value stayed at 7.53 (Fig. 4 c). Straw naturally degrades over a prolonged period, which is not favorable for the growth of microorganisms. After four days of bio-pretreatment, the medium could recover to its neutral state. However, the average pH of the natural deterioration followed a pattern of initially lowering and gradually increasing. These outcomes demonstrated the strain N5's potent capacity to regulate pH during the biodegradation of straw. After biodegradation, the straw is more suitable for subsequent utilization regarding pH value. As shown in Fig. 4 d, between day 1 and day 4, the natural degradation gradually increased (from 0.29×10 6 CFU/mL to 0.90×10 6 CFU/mL), and the number of biodegradable microorganisms introduced with strain N5 increased initially before declining. Consistent with the OD results from the previous investigation, it rose rapidly on days two and three (0.88×10 6 CFU/mL to 2.72×10 6 CFU/mL) and reached its maximal biological growth (3.01×10 6 CFU/mL) on day four. As the fermentation progressed, the bacteria quantity started to decline on day five (2.53×10 5 CFU/mL). 3.5 The growth-promoting effect of biological fermentation broth on plants Studies have shown that rice straw contains trace elements that will be released and transferred after gasification [34] . After the straw decomposes through fermentation, these trace elements can be released and utilized. The outcomes are displayed in Fig. 5 . When rice seedlings of strain N5 biological fermentation broth was added, their height increased relative to the control group. These outcomes demonstrated that strain N5's fermentation broth was comparatively safe and had no negative effects on the growth of rice seedlings during the biodegradation process. As shown in Fig. 5 a, While the height range of seedlings treated with biological fermentation broth was 2.51 ~ 4.06 cm, the height range of seedlings treated with sterile water and nutrient medium diluent in the two control groups was 1.70 ~ 2.98 cm and 2.14 ~ 3.23 cm, respectively. These results indicated that the biological fermentation broth of strain N5 had a certain growth-promoting effect on rice seedlings. Similarly, When Penicillium sp. strain JiTF01 biological fermentation broth is applied to rice seeds, it has an obvious growth-promoting effect [26] . In a study, Bacillus sp . PG-8 fermentation broth was applied to peanut seeds and showed a good growth-promoting effect [35] . The results indicated that the biological fermentation broth of strain N5 had a certain promoting effect on the growth and root of rice seedlings. Figure 5 b shows the effect of fermentation broth on rice root length. The average root length of rice cultured in sterile water and blank medium is 5.30 cm and 5.25 cm, respectively. The average root length of rice cultured in the biological fermentation broth of strain N5 is 7.43 cm. 3.6 Effects of biological fermentation broth on the germination rate of rice seeds under salt stress Rice is a sensitive crop to salt content [36] . This study further evaluated fermentation broth's influence on rice seeds' germination rate under varying salt concentrations. The image illustrates how similar circumstances were shown for the germination of rice seedlings treated with sterile water and nutritional medium at the same concentration of salt stress. In the presence of a 100 mmol/L salt stress, the diluted biological fermentation broth encouraged rice seedlings to germinate. At 200 mmol/L of salt stress, there was no significant difference between the treatments. This may be because the inhibition of seed growth at high concentrations of salt stress was more severe than that under low concentrations. The rice seeds diluted by biological fermentation broth under 100 mmol/L salt stress had a higher second day than the other groups, as Fig. 6 illustrates. On the third day of culture, the germination rate was 70% under 100 mmol/L salt stress, and on the sixth day, the germination rate reached its maximum. The germination rates of seedlings treated with sterile water and nutrient medium were 52% and 54% on the third day, respectively, and the germination index was completed on the seventh day. Furthermore, under salt stress of 200 mmol/L, there was no discernible variation in the rate of germination of seeds among the various treatments. The third day showed comparable germination rates of 47%, 53%, and 53% for various treatments of nutritional medium, sterile water, and biological fermentation broth. On the seventh day, 99% of the seeds treated with biological fermentation broth germinated, compared to 92% and 97% of the seeds treated with sterile water and nutritional medium. 3.7 Genome-wide analysis of cellulose-degrading strain N5 The N5 strain was found to contain a circular chromosome but no plasmids (Fig. 7 a). Its genome size was determined to be 3,492,876 base pairs, with a GC content of 47.33%. The non-coding RNA included 86 tRNAs, 27 rRNAs (with nine copies each of 5S, 16S, and 23S rRNAs), and 31 sRNAs. Gene annotation based on the COG database revealed that the most abundant functional categories were amino acid transport and metabolism (305 sequences, 10.25%), transcription (286, 9.62%), translation and ribosomal structure (259, 8.71%), general function prediction (256, 8.61%), cell cycle control (213, 7.16%), and signal transduction (210, 7.06%). This suggests that strain N5 has an active synthesis pathway and a robust capacity to utilize amino acids and carbohydrates for substrate transport. The presence of cellulose-coding genes indicates their involvement in carbohydrate transport and metabolism, including 6 α-amylases, 4 β-xylosidases, 3 β-glucosidases, and 3 glycoside hydrolases, which are crucial for cellulose hydrolysis [37, 38] . The synergistic action of these multiple hydrolases is believed to facilitate cellulose degradation. Additionally, 140 genes of unknown function were identified, offering potential for further research. The GO database annotated 2338 genes (Fig. 7 c), representing 59.02% of the total coding genes, with the majority involved in biological pathways, followed by molecular function and cellular components. The top three biological pathways were cell processes (1280), metabolic processes (1247), and biological regulation (304). In molecular functions, the majority of genes were involved in catalytic activity (1398) and binding (927), while the most annotated cellular component was cell structure (621). This indicates that strain N5's metabolism and regulatory network are complex, with a focus on catalytic activity, suggesting that enzyme regulation plays a dominant role in its metabolic processes. Functional annotation of strain N5's genome via the KEGG database identified 2536 genes (Fig. 7 d). Among the six major metabolic pathways, 1956 genes were related to metabolic pathways and at least 59 to tissue systems. Carbohydrate metabolism (259), amino acid metabolism (214), and vitamin and cofactor metabolism (176) were the most represented. In the biosynthetic pathways of secondary metabolites, genes related to potassium uptake proteins (K03498, K03499), proline transport systems (K11928, K03762), and proline biosynthesis (proA, proB, proC) were identified. These genes enhance strain N5's activity under high salt stress by synthesizing and accumulating small molecule solutes to counteract osmotic pressure imbalances. The synergistic action of multiple carbohydrate-active enzymes (CAZymes) is required to degrade straw cellulose polymers [12] . The genome of strain N5, when compared to the CAZy database, was found to contain 115 CAZy genes across six categories: auxiliary active enzymes (AAs), carbohydrate-binding modules (CBMs), carbohydrate esterases (CEs), glycoside hydrolases (GHs), glycosyltransferases (GTs), and polysaccharide lyases (PLs). The GH family predominates, with 52 annotated genes spread across 27 families (Fig. 8 ). Notably, GH1 (5 genes) is associated with cellulase, GH13 (5 genes) with hemicellulose degradation, and GH43 (4 genes) with xylan breakdown, all of which are crucial for cellulose degradation. GH23 (3 genes) encodes peptidoglycan lyase, also implicated in this process. The presence of these genes in strain N5 suggests a genetic predisposition for carbohydrate degradation, including cellulose and glycosides. In addition, the function of carbohydrate-binding modules (CBMs) is to help immobilize cellulases to the surface structure of cellulose molecules, enabling enzymes to catalyze the hydrolysis of cellulose to the greatest extent and to maintain the hydrolysis state throughout the subsequent degradation process [39, 40] . Carbohydrate-binding components (CBMs) are annotated to 25 gene quantities, and glycosyltransferase families (GTs) are enzyme systems that catalyze glycosylation reactions [41] . Strain N5 possesses 26 glycosyltransferase (GT) genes, among which GT2 (5) is related to carboxyltransferases. There are 11 carbohydrate esterase (CE) genes, as well as 3 polysaccharide lyase (PL) genes and 2 auxiliary activity (AA) genes, which can assist other enzyme systems in degrading cellulose. Among them, the glycoside hydrolase family is a crucial enzyme in bacterial polysaccharide degradation and promotes bacterial growth and catalytic efficiency alongside CBM glycohydrolase-active enzymes [42] . 4 Conclusion This study introduced the degrading bacterium N5 to the straw medium. The findings revealed that adding bacterium N5 could potentially increase the weight loss rate of straw by 19.35%. When the straw was subjected to additional alkali treatment, it resulted in a biodegradation rate of 38.86%. Moreover, the microbial fermentation broth generated from the straw exhibited a stimulatory effect on the germination and growth of rice seeds, with the incorporation of peptone as a nutrient significantly amplifying the degradation. A comprehensive analysis of the molecular biological characteristics of the degrading bacterium N5 identified a total of 52 glycoside hydrolase (GH) genes and other relative genes; the presence of these genes confirms that strain N5 is capable of hydrolyzing lignin, hemicellulose, and cellulose. Declarations Declaration of competing interest The authors have no conflicts of interest to declare. Funding This research was supported by the Zhejiang Province Key Research and Development Plan (2021C03190), the Zhejiang Province "Sannong Jiufang" (2022SNJF077), and the 111 Project (D18008). Author contributions Tao Zhang: Implemented the experiments, analysed the data and wrote the manuscript; Wen-fan Wang: Implemented the experiments, analysed the data; Zhuo-qun Zhao: Guided experiment and analysed the data; Hua-bao Zheng: Conceived and designed experiment. Data availability The authors declare that all data and materials are available to be shared upon a formal request. References Yao X, Liu Q, Li D., 2024. Mechanism underlying effects of cellulose-degrading microbial inoculation on amino acid degradation and biosynthesis during composting[J]. Bioresource Technology. 403, 130899. Wei Y, Wu D, Wei D, et al.,2019. Improved lignocellulose-degrading performance during straw composting from diverse sources with actinomycetes inoculation by regulating the key enzyme activities[J]. Bioresource technology. 271, 66–74. Li Y, Kuramae E E, Nasir F, et al.,2023. Addition of cellulose degrading bacterial agents promoting keystone fungal-mediated cellulose degradation during aerobic composting: Construction the complex co-degradation system[J]. Bioresource Technology. 381, 129132. Wang X, Tian L, Li Y, et al., 2022. Effects of exogenous cellulose-degrading bacteria on humus formation and bacterial community stability during composting[J]. Bioresource Technology. 359, 127458. Shi ZuLiang S Z L, Wang Fei W F, Wang JiuChen W J C, et al., 2019. Utilization characteristics, technical model and development suggestion on crop straw in China[J]. 2019. Kumar V, Mitra D, Rani A, et al.,2022. Bio-inoculants for Biodegradation and Bioconversion of Agrowaste: Status and Prospects[J]. Bioremediation of Environmental Pollutants: Emerging Trends and Strategies. 2022, 351–367. Liu H, Sun S, Han Y., 2021. The current situation and development strategy of corn straw returning in Heilongjiang Province [J]. China Seed Industry. 11, 20–22. Padhan K, Patra R K, Sethi D, et al., 2024. Exploitation of cellulose degrading bacteria in bioconversion of agro-wastes[J]. Chemosphere. 347, 140654. Pandit L, Sethi D, Pattanayak S K, et al., 2020. Bioconversion of lignocellulosic organic wastes into nutrient rich vermicompost by Eudrilus eugeniae[J]. Bioresource Technology Reports. 12, 100580. Zhang G, Dong Y., 2022. Design and application of an efficient cellulose-degrading microbial consortium and carboxymethyl cellulase production optimization[J]. Frontiers in Microbiology. 13, 957444. Kaur P, Sharma N, Munagala M, et al., 2021. Nanocellulose: Resources, physio-chemical properties, current uses and future applications[J]. Frontiers in Nanotechnology. 3, 747329. He Z, Ding B, Ali Q, et al., 2023. Screening and isolation of cold-adapted cellulose degrading bacterium: A candidate for straw degradation and De novo genome sequencing analysis[J]. Frontiers in Microbiology. 13, 1098723. Wang L, Wang T, Xing Z, et al., 2023. Enhanced lignocellulose degradation and composts fertility of cattle manure and wheat straw composting by Bacillus inoculation[J]. Journal of Environmental Chemical Engineering. 11(3), 109940. Wang S, Long H, Hu X, et al., 2024. The co-inoculation of Trichoderma viridis and Bacillus subtilis improved the aerobic composting efficiency and degradation of lignocellulose[J]. Bioresource Technology. 394, 130285. Meneses C, Silva B, Medeiros B, et al., 2016. A metagenomic advance for the cloning and characterization of a cellulase from red rice crop residues[J]. Molecules. 21(7), 831. Karthika A, Seenivasagan R, Kasimani R, et al., 2020. Cellulolytic bacteria isolation, screening and optimization of enzyme production from vermicompost of paper cup waste[J]. Waste Management. 116, 58–65. Hemati A, Aliasgharzad N, Khakvar R, et al., 2021. Role of lignin and thermophilic lignocellulolytic bacteria in the evolution of humification indices and enzymatic activities during compost production[J]. Waste Management. 119, 122–134. Haruta S, Cui Z, Huang Z, et al., 2022. Construction of a stable microbial community with high cellulose-degradation ability[J]. Applied microbiology and biotechnology. 59, 529–534. Bajpai P. Paper Bioprocessing., 2018. Biermann’s handbook of pulp and paper[J]. 2018. Li H, Zhang M, Zhang Y, et al., 2023. Characterization of Cellulose-Degrading Bacteria Isolated from Silkworm Excrement and Optimization of Its Cellulase Production[J]. Polymers. 15(20), 4142. Tang C, Shan J, Chen Y, et al., 2017. Organic amine catalytic organosolv pretreatment of corn stover for enzymatic saccharification and high-quality lignin[J]. Bioresource technology. 232, 222–228. Wu D, Qu F, Li D, et al., 2022. Effect of Fenton pretreatment and bacterial inoculation on cellulose-degrading genes and fungal communities during rice straw composting[J]. Science of The Total Environment. 806, 151376. Chen M, Zhao J, Xia L., 2009. Comparison of four different chemical pretreatments of corn stover for enhancing enzymatic digestibility[J]. Biomass and Bioenergy. 33(10), 1381–1385. Jain D, Ravina, Bhojiya A A, et al., 2021. Polyphasic characterization of plant growth promoting cellulose degrading bacteria isolated from organic manures[J]. Current Microbiology. 78, 739–748. Ji J, Yuan D, Jin C, et al., 2020. Enhancement of growth and salt tolerance of rice seedlings (Oryza sativa L.) by regulating ethylene production with a novel halotolerant PGPR strain Glutamicibacter sp. YD01 containing ACC deaminase activity[J]. Acta Physiologiae Plantarum. 42, 1–17. Dong X, Ji J, Zhang S, et al., 2022. Study on a Low-temperature Cellulose-degrading Strain: Fermentation Optimization, Straw Degradation, and the Effect of Fermentation Broth on Seed Growth[J]. Biotechnology and Bioprocess Engineering. 27(4), 652–667. Tamilselvan R, Selwynraj A I., 2024. A novel g-C3N4 photocatalytic pretreatment for reducing silica and modifying the structure of rice straw for sustainable biofuel production[J]. Fuel. 357, 129901. Han X, Luo Q, Bao A, et al., 2019. Research on the mechanism of improving rice straw self-bonding via NaOH solution pretreatment[J]. BioResources. 14(4), 9352–9363. Guo H, Chang J, Yin Q, et al., 2013. Effect of the combined physical and chemical treatments with microbial fermentation on corn straw degradation[J]. Bioresource technology. 148, 361–365. Guan R, Li X, Wachemo A C, et al., 2018. Enhancing anaerobic digestion performance and degradation of lignocellulosic components of rice straw by combined biological and chemical pretreatment[J]. Science of the Total Environment. 637, 9–17. Samar W, Arora A, Sharma A, et al., 2021. Material flow of cellulose in rice straw to ethanol and lignin recovery by NaOH pretreatment coupled with acid washing[J]. Biomass Conversion and Biorefinery. 2021, 1–10. Vogel H C, Todaro C M., 1996. Fermentation and biochemical engineering handbook: principles, process design and equipment[M]. William Andrew, 1996. Gutiérrez-Correa M, Villena G K., 2010. Characteristics and techniques of fermentation systems[J]. Comprehensive Food Fermentation and Biotechnology. 2010, 1. Thao N T N L, Chiang K Y., 2020. The migration, transformation and control of trace metals during the gasification of rice straw[J]. Chemosphere. 260, 127540. Gohil R B, Raval V H, Panchal R R, et al., 2022. Plant growth-promoting activity of Bacillus sp. PG-8 isolated from fermented panchagavya and its effect on the growth of Arachis hypogea[J]. Frontiers in Agronomy. 4, 805454. Grattan S R, Zeng L, Shannon M C, et al., 2002. Rice is more sensitive to salinity than previously thought[J]. California agriculture. 2002, 56(6). Chukwuma O B, Rafatullah M, Tajarudin H A, et al., 2020. Lignocellulolytic enzymes in biotechnological and industrial processes: a review[J]. Sustainability. 12(18), 7282. Raheja Y, Kaur B, Falco M, et al., 2020. Secretome analysis of Talaromyces emersonii reveals distinct CAZymes profile and enhanced cellulase production through response surface methodology[J]. Industrial crops and products. 152, 112554. Várnai A, Siika-Aho M, Viikari L., 2013. Carbohydrate-binding modules (CBMs) revisited: reduced amount of water counterbalances the need for CBMs[J]. Biotechnology for biofuels. 6, 1–12. Aïssa K., 2020. The potential of using carbohydrate-binding modules (CBMs) for the characterization and modification of cellulose surfaces[D]. University of British Columbia. 2020. Ortiz-Soto, M.E. and J. Seibel., 2024. An overview on glycoside hydrolases and glycosyltransferases [J]. Zeitschrift für Naturforschung C. 2024(0). Ma D, Chen H, Liu D, et al., 2024. Soil-derived cellulose-degrading bacteria: screening, identification, the optimization of fermentation conditions, and their whole genome sequencing[J]. Frontiers in Microbiology. 15, 1409697. Supplementary Files 05SupplementaryInformation.docx Cite Share Download PDF Status: Posted Version 1 posted 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-6325052","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":444099545,"identity":"15a142fc-d5e3-4b32-852e-d979279e028b","order_by":0,"name":"Tao Zhang","email":"","orcid":"","institution":"GuiZhou Zhuxin Water Environmental Industry Co.,Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Zhang","suffix":""},{"id":444099546,"identity":"72d084ab-b5b7-413e-8deb-df99c8e70c17","order_by":1,"name":"Wen-fan Wang","email":"","orcid":"","institution":"Zhejiang Agriculture and Forestry University: Zhejiang A and F University","correspondingAuthor":false,"prefix":"","firstName":"Wen-fan","middleName":"","lastName":"Wang","suffix":""},{"id":444099547,"identity":"1c9c9a59-8778-4c27-8cc1-1267fceb1243","order_by":2,"name":"Zhuo-qun Zhao","email":"","orcid":"","institution":"Zhejiang Agriculture and Forestry University: Zhejiang A and F University","correspondingAuthor":false,"prefix":"","firstName":"Zhuo-qun","middleName":"","lastName":"Zhao","suffix":""},{"id":444099548,"identity":"b0171d32-f8db-4d76-bd00-c753df35be95","order_by":3,"name":"Hua-bao Zheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYDACCSBmbGBgMABSDxgY5EBiBkRrYQZiY9K0sEkQpUV+dvOxh193HJY3Zz98rPJHjUFiA3vzNgmGmjs4tTDOOZZuLHvmsOHOnrS0GxLHgFp4jpVJMBx7hlMLs0SOmbRk22HGDQdyzG4YsP1JbACKSDA2HMaphU0i/xtIi/2G82/MChL+AW2Rf4NfC49EDpvkx7bDiRtu5JgxHGwDapHgwa9FQiLNTJqxLT1554xnyZKNfQbGbTxpxRYJx3BrkZ+R/EzyZ5u17Xb+5IMff3wzkO1nP7zxxoca3FrAQcCD4jsQkYBXAzCgfxBQMApGwSgYBSMcAACPN1UCy6LNcgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-3509-7634","institution":"Zhejiang Agriculture and Forestry University: Zhejiang A and F University","correspondingAuthor":true,"prefix":"","firstName":"Hua-bao","middleName":"","lastName":"Zheng","suffix":""}],"badges":[],"createdAt":"2025-03-28 05:36:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6325052/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6325052/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81399589,"identity":"f46a8c93-16c1-4b6d-9bc2-ca8e34ccda40","added_by":"auto","created_at":"2025-04-25 16:17:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11830809,"visible":true,"origin":"","legend":"\u003cp\u003eDegradation effect of N5 on rice straw\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6325052/v1/51f1f2d23abc1977f9fd69e8.png"},{"id":81400167,"identity":"e38b66da-9702-4e33-9829-ea79bf68f8c9","added_by":"auto","created_at":"2025-04-25 16:25:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2059775,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of chemical pretreatment on biodegradation of rice straw\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6325052/v1/e127e1707033ed6c6da03eb0.png"},{"id":81399584,"identity":"404ce3fe-0792-4696-9228-64384af279ca","added_by":"auto","created_at":"2025-04-25 16:17:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":81563,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of nutrients on the biodegradation of rice straw\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6325052/v1/f2b32c36711786c80233c056.png"},{"id":81399588,"identity":"53db9d3a-343c-43eb-8d39-bc3649cc3d40","added_by":"auto","created_at":"2025-04-25 16:17:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":612890,"visible":true,"origin":"","legend":"\u003cp\u003eThe changes of CMC enzyme activity, pH and microbial quantity during the biodegradation of rice straw\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6325052/v1/dead17da0347a12c30dc3b60.png"},{"id":81400170,"identity":"776a4104-0a1d-464f-8cb5-a2b24ce22462","added_by":"auto","created_at":"2025-04-25 16:25:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":17897180,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of rice straw fermentation broth on the growth of rice seedlings\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6325052/v1/41718095c25358ca2106de39.png"},{"id":81399592,"identity":"bfc89000-07ba-4948-84fb-161ff0605cb4","added_by":"auto","created_at":"2025-04-25 16:17:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":800956,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of rice straw fermentation broth on seed germination\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6325052/v1/0e70c29fb2ae21ca050c08b6.png"},{"id":81399593,"identity":"95f6a228-7d96-42f6-9cdd-05e1c3368a8b","added_by":"auto","created_at":"2025-04-25 16:17:41","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":231153,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic information of the strain N5. (a) Genome circle map. (b) COG annotation results. (c) GO annotation results. (c) KEGG annotation results\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6325052/v1/3bfc2cc7a24075e0c344a4de.png"},{"id":81399597,"identity":"5e8aa124-f7ab-4ed6-8ae9-b8ef1deb4415","added_by":"auto","created_at":"2025-04-25 16:17:41","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":225358,"visible":true,"origin":"","legend":"\u003cp\u003eStrain N5 gene CAZy annotation classification\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6325052/v1/a3b24799e55f3c441fc257de.png"},{"id":85335188,"identity":"25238c2d-d3e0-428c-87c2-ac919be79c8a","added_by":"auto","created_at":"2025-06-24 19:52:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7907833,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6325052/v1/a03e62d9-dae4-45c5-9ccf-ba926904ba7a.pdf"},{"id":81399585,"identity":"abe67a78-55f0-4aec-a5b1-48948a6e1c7b","added_by":"auto","created_at":"2025-04-25 16:17:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17107,"visible":true,"origin":"","legend":"","description":"","filename":"05SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6325052/v1/220b49fd9c2096cc85ba0428.docx"}],"financialInterests":"","formattedTitle":"Microbial decomposition of rice straw: alkaline pretreatment, genome analysis and fermentation broth on seed growth","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAs agricultural production advances, many crop straws with high cellulose content are produced annually \u003csup\u003e[1, 2]\u003c/sup\u003e. The surplus accumulation of these straws in agricultural areas is becoming a big issue. Farmers often consider these straws as waste, which, if burned directly or left piled up in the fields, can severely damage the ecological environment and disrupt the agricultural cycle \u003csup\u003e[3\u0026ndash;5]\u003c/sup\u003e. This, in turn, can restrict the sustainable growth of agriculture \u003csup\u003e[6]\u003c/sup\u003e. The proper management of crop straw is essential for promoting sustainable agricultural growth, addressing the energy crisis, and fostering the development of green agriculture \u003csup\u003e[7]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eApproximately 70% of biomass is composed of cellulosic and hemicellulosic polymers, which are bound to the lignin component by covalent bonds, providing the lignocellulosic biomass with great robustness and resistance to (bio-)chemical or physical treatments. In agricultural residues, paddy straw is notable for its composition, which includes 43% cellulose, 23% hemicellulose, and 6.27% lignin \u003csup\u003e[8, 9]\u003c/sup\u003e. The complex network structure formed by these three components in crop straw presents a challenge to the degradation and utilization of the material \u003csup\u003e[10, 11]\u003c/sup\u003e. Consequently, cellulose requires treatment to increase the utilization rate of straw.\u003c/p\u003e \u003cp\u003eThe benefits of microbial degradation over alternative techniques are unparalleled. Bacteria capable of decomposing cellulose are plentiful \u003csup\u003e[12\u0026ndash;14]\u003c/sup\u003e. Microorganisms in nature may release enzymes that synergistically affect cellulose degradation, which is produced by several microorganisms, especially bacteria and fungi \u003csup\u003e[15\u0026ndash;17]\u003c/sup\u003e. Enzyme proteins must work in concert with enzyme systems and can be categorized into three groups based on their characteristics and roles: β-glucosidase, endoglucanase, and exoglucanase \u003csup\u003e[12, 18]\u003c/sup\u003e. Cellulase functions by hydrolyzing cellulose into glucose, utilizing the combined action of multiple enzymes to break down cellulose effectively \u003csup\u003e[19]\u003c/sup\u003e. In this context, fungi and bacteria have garnered significant interest due to their capacity to generate diverse cellulases. While fungi are the primary sources of enzymes that break down lignocellulose, bacteria typically exhibit the advantages of being environmentally friendly, faster growth rates, cheap, convenient, and not causing secondary pollution compared to fungi \u003csup\u003e[3, 20]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFinding a more efficient degradation approach is essential because the microbial method alone may not fully degrade straw due to its complex structure. To maximize the combination, combined therapy can overcome the limitations of physical, chemical, and biological treatments. Currently, the most widely used treatment technology combines chemical and physical methods. To overcome the resistance of lignocellulose, TANG et al. \u003csup\u003e[21]\u003c/sup\u003e pretreated corn stover using organic solutions, resulting in high-yield fermentable sugars and superior, salt-free lignin. The overall sugar yield was 83.2%, and the delignification rate was 81.7%. Combining chemical techniques with microbial treatment can also achieve a better result in straw decomposition. WU et al. \u003csup\u003e[22]\u003c/sup\u003e subjected rice straw to a bacterial breakdown in conjunction with Fenton treatment. The findings demonstrated that bacterial inoculation and Fenton treatments altered the fungal community's composition, promoted fungal diversity, altered the genes responsible for cellulose breakdown, and accelerated the rate of rice straw degradation. Chen et al. \u003csup\u003e[23]\u003c/sup\u003e used dilute acid, lime, ammonia/dilute acid, and alkali, four different chemical reagents, to pretreat corn straw. The results showed that dilute alkali pretreatment was the most effective in improving the enzymatic hydrolysis efficiency of fibre residue. Therefore, this study selected dilute alkali to treat straw and studied the degradation of \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e N5 on pretreated straw.\u003c/p\u003e \u003cp\u003eThis study investigated the preliminary effects of chemical pretreatment and degrading bacteria on straw degradation, verifying the modifications resulting from pretreatment on straw following biodegradation. Through the study on the growth promoting effect of straw biological fermentation broth on rice seeds, the effect of fermentation broth on plants after microbial degradation of straw was explored. This study also provides new ideas for recycling straw. The strain genes were investigated based on whole-genome sequencing analysis to overcome the barrier of poor adaptability and low enzyme production in the future application of cellulose resources. This allowed for excellent strains and technical support, providing valuable resources for later synthetic biology applications.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Degradation of rice straw\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e N5 was screened in cow manure compost, and the strain was inoculated into LB liquid medium (yeast extract 5.0 g/L, peptone 10.0 g/L, sodium chloride 10.0 g/L, pH 7.0\u0026thinsp;~\u0026thinsp;7.2, sterilized at 121\u0026deg;C for 30 minutes), and cultured at 35\u0026deg;C and 160 rpm.\u003c/p\u003e \u003cp\u003eThe strain N5 was inoculated into the straw culture medium (rice straw was dried and cut into 2\u0026ndash;3 cm segmental straw 2 g, inorganic salt culture medium (contained NH\u003csub\u003e4\u003c/sub\u003eCl 1 g/L, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 0.5 g/L, K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 1.5 g/L, MgSO\u003csub\u003e4\u003c/sub\u003e 0.2 g/L, NaCl g/L, and 1 mL/L Hunter\u0026rsquo;s trace elements solution) 100 mL, natural pH, 121\u0026deg;C sterilization for 30 minutes) at inoculation amount 5% (V/V). The liquid fermentation was carried out at 35\u0026deg;C and 160 rpm for 7 days. After the degradation of liquid fermentation, the residue after straw degradation was filtered and rinsed with a large amount of water to remove the bacteria and other soluble substances attached to the straw and dried to constant weight at 105\u0026deg;C. At the same time, the dry weight of straw residue was weighed to calculate the weight loss rate of straw \u003csup\u003e[24]\u003c/sup\u003e.\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{weight loss rate of straw}\\text{}\\text{(}\\text{%}\\text{)}\\text{=}\\raisebox{1ex}{$（{\\text{M}}_{\\text{0}}\\text{-}{\\text{M}}_{\\text{1}}）$}\\!\\left/\\:\\!\\raisebox{-1ex}{${\\text{M}}_{\\text{0}}$}\\right.\\text{\u0026times;100}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eIn the formula: M\u003csub\u003e0\u003c/sub\u003e is the dry weight of straw residue in the control group (g); M\u003csub\u003e1\u003c/sub\u003e is the dry weight (g) of straw residues in the treatment group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Effect of chemically combined treatment on rice straw degradation ability\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Alkali treatment combined with strain N5 on the degradation of rice straw\u003c/h2\u003e \u003cp\u003eStraw alkali treatment: Rice straw was soaked in a 1.5% sodium hydroxide (NaOH) solution for 24 h, rinsed with water to neutrality, and dried in an oven at 80\u0026deg;C for later use.\u003c/p\u003e \u003cp\u003eThe treated rice straw was added to the inorganic salt medium (not sterilized). The experiment set up four treatments: 1)untreated straw, 2༉untreated straw\u0026thinsp;+\u0026thinsp;N5 bacterial solution, 3༉pretreated straw, and 4༉pretreated straw\u0026thinsp;+\u0026thinsp;N5 bacterial solution. The strain N5 was inoculated into the pretreated straw medium at 1% (V/V) (pretreated straw 2 g, inorganic salt medium 100 mL, pH 7.0, medium not sterilized). After 7 days of degradation at 35\u0026deg;C and 160 rpm under natural conditions, the rice straw degradation residues were filtered and washed. Drying to constant weight at 105\u0026deg;C, each treatment had three replicates, and the weight loss rate of the straw was calculated. The residue was weighed to calculate the weight loss rate of the straw\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 The effect of nutrient addition on the combined degradation of rice straw\u003c/h2\u003e \u003cp\u003eThe strain N5 was inoculated into the pretreated straw medium at a 1% (V/V) ratio, and nutrients were added for bioaugmentation. To the pretreated straw medium, yeast (0.5 g), peptone (0.5 g), urea (0.5 g), and glucose (0.5 g) were added as nutrients to provide sustenance for both natural microorganisms and strain N5. Liquid fermentation was conducted at 35\u0026deg;C with a rotation speed of 160 rpm for 7 days, with three replicates for each treatment, during which the straw degradation rate of the strain was determined. The variations in pH, CMCase activity, OD600, and microbial quantity during the straw degradation process were measured, and the changes in natural degradation and biodegradable straw residues were observed. Each treatment included three replicates.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3 The growth-promoting effect of biological fermentation broth on rice\u003c/h2\u003e \u003cp\u003ePretreatment of rice seeds: Rice seeds with intact surfaces and no black spots were soaked in sterile water for 24 hours, then sterilized with 75% alcohol for 5 minutes and washed with sterile water \u003csup\u003e[25]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe strain was introduced into a nutrient straw medium at a 1% (volume/volume) inoculation rate. Following a 7-day biodegradation period, the fermentation supernatant was centrifugated for 10 minutes at 8000 rpm and 4\u0026deg;C. The resulting pellet from the centrifuged biological fermentation broth was then diluted to a 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e concentration, the standard dilution factor. A disposable petri dish was prepared by laying a filter paper for water retention at the bottom. Each dish was then populated with 50 sterilized rice grains. Three experimental groups were established: sterile water, nutrient medium, and biological fermentation broth. 10 mL of the 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e diluted biological fermentation broth was added to each culture dish. The sterile water and nutrient medium were diluted similarly to negative controls. The petri dishes were then placed in an artificial climate chamber for incubation. The incubation conditions were set at a temperature of 25\u0026deg;C with a light/dark cycle of 16 hours light and 8 hours darkness. After a 7-day cultivation period, the growth of the rice seedlings was assessed by measuring both the height of the seedlings and the length of the rice roots. Each treatment was conducted in triplicate for consistency. The sterile water, nutrient medium, and biological fermentation broth were subjected to varying levels of salt stress using NaCl solutions at concentrations of 100 and 200 mmol/L. Each petri dish was inoculated with 50 grains, and this process was repeated three times. Germination counts were recorded daily. Under the same culture temperature and photoperiod conditions, the germination rate of rice seeds was recorded daily in an artificial climate incubator until the seventh day. Three replicates were set for each treatment, and the calculation formula of seed germination rate is as follows \u003csup\u003e[26]\u003c/sup\u003e:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\text{Germination rate (%)}\\text{=}\\raisebox{1ex}{$\\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r}\\:\\text{o}\\text{f}\\:\\text{s}\\text{e}\\text{e}\\text{d}\\:\\text{g}\\text{e}\\text{r}\\text{m}\\text{i}\\text{n}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}$}\\!\\left/\\:\\!\\raisebox{-1ex}{$\\text{t}\\text{o}\\text{t}\\text{a}\\text{l}\\:\\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r}\\:\\text{o}\\text{f}\\:\\text{s}\\text{e}\\text{e}\\text{d}\\text{s}$}\\right.\\text{\u0026times;100}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Scanning electron microscope analysis of rice straw\u003c/h2\u003e \u003cp\u003eThe degradation of rice straw after different treatments was observed and analyzed by scanning electron microscope. After the fermentation is completed, the medium is filtered, and the treated rice straw is air-dried. After processing by slicing, fixing, washing, dehydration, and replacement, the samples were adhered to the scanning electron microscope sample table with conductive tape and vacuum gold plating, and the surface morphology and structure of rice straw were observed by scanning electron microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Whole genome sequencing\u003c/h2\u003e \u003cp\u003eThe strain N5 was inoculated into LB medium and cultured for 24 h. The logarithmic phase bacteria were collected, and the bacterial solution was centrifuged for 5 minutes (rotation speed 10000 rpm, temperature 4\u0026deg;C). The supernatant was discarded to retain the bacteria, placed in liquid nitrogen for quick freezing, and then stored in a refrigerator at -80\u0026deg;C.\u003c/p\u003e \u003cp\u003ePacBio RS II and Illumina HiSeq 4000 platforms were used to sequence and assemble the whole genome of strain N5. The Pacbio platform uses SMRT to generate a sub-reading set and deletes Pacbio sub-readings (length\u0026thinsp;\u0026lt;\u0026thinsp;1kb). The Canu program is used for self-correction, drafting genomic units, and assembling a corrected cyclic common sequence sub-reading set using high-quality Canu. In order to improve the accuracy of the genome sequence, GATK is used for single base correction, and the Blast alignment tool is used for the best matching of functional annotations.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Rice straw degradation by strain N5\u003c/h2\u003e \u003cp\u003eThe results are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec. After 7 days, the weight loss rate of rice straw in the control experiment without strain N5 was 4.05%. In contrast, the addition of N5 degrading bacteria increased the weight loss rate to 23.40%, indicating that the presence of N5 degrading bacteria significantly enhances the degradation of rice straw, thereby achieving effective treatment. In summary, the strain N5 identified in this study possesses excellent degradation capabilities for rice straw, elevating the weight loss rate by 19.35% within 7 days.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe silicon structure within the rice straw, as highlighted in the electron microscope image shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, is characterized by a surface layer of wax. The primary resistance to degradation in rice straw is attributed to its silicon structure and the protective wax layer \u003csup\u003e[27, 28]\u003c/sup\u003e. The untreated straw, which did not fully dissolve its surface silicon structure and wax layer, displayed a smooth surface, intact integrity, and a regular, dense morphological structure compared to the straw treated with N5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). After 7 days of treatment with N5, the straw's surface exhibited large, irregular fractures and pores (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). This observation suggests that strain N5 may cause the wax layer on the straw's surface to detach, exposing the underlying cellulose and silicon structure. This exposure would facilitate the microbes' more efficient utilization of the substrate, thereby enhancing the breakdown of the straw \u003csup\u003e[29]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effect of combined treatment on rice straw degradation\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed displays the microstructure of rice straw following alkali pretreatment. After NaOH treatment, the straw surface's convex structure is loose, and the gap widens. Chemical pretreatment appears to expand the cellulose's exposed area in straw, making it more favorable for the adherence and uptake of strains \u003csup\u003e[30]\u003c/sup\u003e. The straw's appearance following NaOH treatment is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. Rice straw has a rough surface and a whole structure. The pretreated straw after seven days of incipient deterioration is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb. The straw sample is not finely crushed, and the straw's edge has fine hair edges. After being subjected to N5 degradation for seven days, the pretreatment rice straw in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec lost much of its structural integrity and fractured into pieces of varying sizes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, the weight loss rate of rice straw was 6.3% for untreated rice straw and 22.58% for rice straw that had been treated with NaOH, suggesting that treating rice straw with NaOH can accelerate its natural degradation rate. When rice straw was treated with 1.5% NaOH, its weight loss rate increased to 38.86% from 16.3% when left untreated. The outcomes demonstrated that the complicated structure of straw might be broken down by strain N5 in conjunction with a chemical pretreatment, increasing the bioconversion efficiency of cellulose biomass \u003csup\u003e[31]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effects of nutrients on straw degradation by alkali treatment\u003c/h2\u003e \u003cp\u003eStrain N5 demonstrated good adaptation to various nutrients, as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Peptone, yeast, glucose, and urea were the ideal nutrient sequences for the fermentation medium, and the corresponding weight loss rates were 65.28%, 62.18%, 57.97%, and 44.45%. By adding peptone to the fermentation medium, strain N5 reached the maximum breakdown rate (65.28%). Straw under natural conditions degraded at a rate ranging from 24.16\u0026ndash;30.2%, consistently lower than that of the biological treatment with strain N5. For further culture, peptone was added to the pretreated straw media.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Changes in physicochemical properties of alkali-treated straw during biodegradation\u003c/h2\u003e \u003cp\u003eAs the fermentation period was extended during the natural degradation, the enzyme activity decreased, and on the second day, the CMC enzyme activity achieved its maximum value of 124.28 U/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The CMC enzyme activity of N5 biodegradation fluctuated sporadically throughout the entire degradation process, but overall, there was a high and, subsequently, a low trend. The additional strain N5's biodegradation could break down the substrate more quickly on the second day of fermentation and hasten the breakdown of the straw, as evidenced by the greatest enzyme activity appearing on the second day and the CMC enzyme activity of 224 U/mL, 206.01 U/mL, 134.32 U/mL, 179.54 U/mL, 166.21 U/mL, and 156.93 U/mL were the CMC enzyme activity, in that order. This might be because early fermentation had enough nutrients, which improved the strain's ability to create enzymes through metabolism. As time passes and the nutrients from mixed biological fermentation minimize the drop in dissolved oxygen, intracellular enzymes are released, and enzyme activity becomes unstable \u003csup\u003e[32, 33]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe OD600 value of natural degradation peaked at 1.55, while the OD600 value of biodegradation was 2.38 on the third day, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb. The biodegradation's OD600 value peaked at 2.67 on the fifth day, but the natural degradation value was just 1.17. It demonstrates that the biodegradation of rice straw by strain N5 has a comparatively high bacterial concentration and robust bacterial growth and metabolism.\u003c/p\u003e \u003cp\u003eThe pH was stable for the first four days of the fermentation process, which could be attributed to other microorganisms in the medium. Following inoculation, strain N5 did not cause pH change during the lag phase. On the fourth day, the pH increased significantly from 6.45 to 7.56, and on the sixth day of fermentation, the pH value stayed at 7.53 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Straw naturally degrades over a prolonged period, which is not favorable for the growth of microorganisms. After four days of bio-pretreatment, the medium could recover to its neutral state. However, the average pH of the natural deterioration followed a pattern of initially lowering and gradually increasing. These outcomes demonstrated the strain N5's potent capacity to regulate pH during the biodegradation of straw. After biodegradation, the straw is more suitable for subsequent utilization regarding pH value.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, between day 1 and day 4, the natural degradation gradually increased (from 0.29\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mL to 0.90\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mL), and the number of biodegradable microorganisms introduced with strain N5 increased initially before declining. Consistent with the OD results from the previous investigation, it rose rapidly on days two and three (0.88\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mL to 2.72\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mL) and reached its maximal biological growth (3.01\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/mL) on day four. As the fermentation progressed, the bacteria quantity started to decline on day five (2.53\u0026times;10\u003csup\u003e5\u003c/sup\u003e CFU/mL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5 The growth-promoting effect of biological fermentation broth on plants\u003c/h2\u003e \u003cp\u003eStudies have shown that rice straw contains trace elements that will be released and transferred after gasification \u003csup\u003e[34]\u003c/sup\u003e. After the straw decomposes through fermentation, these trace elements can be released and utilized. The outcomes are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. When rice seedlings of strain N5 biological fermentation broth was added, their height increased relative to the control group. These outcomes demonstrated that strain N5's fermentation broth was comparatively safe and had no negative effects on the growth of rice seedlings during the biodegradation process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, While the height range of seedlings treated with biological fermentation broth was 2.51\u0026thinsp;~\u0026thinsp;4.06 cm, the height range of seedlings treated with sterile water and nutrient medium diluent in the two control groups was 1.70\u0026thinsp;~\u0026thinsp;2.98 cm and 2.14\u0026thinsp;~\u0026thinsp;3.23 cm, respectively. These results indicated that the biological fermentation broth of strain N5 had a certain growth-promoting effect on rice seedlings. Similarly, When Penicillium sp. strain JiTF01 biological fermentation broth is applied to rice seeds, it has an obvious growth-promoting effect \u003csup\u003e[26]\u003c/sup\u003e. In a study, \u003cem\u003eBacillus sp\u003c/em\u003e. PG-8 fermentation broth was applied to peanut seeds and showed a good growth-promoting effect \u003csup\u003e[35]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe results indicated that the biological fermentation broth of strain N5 had a certain promoting effect on the growth and root of rice seedlings. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb shows the effect of fermentation broth on rice root length. The average root length of rice cultured in sterile water and blank medium is 5.30 cm and 5.25 cm, respectively. The average root length of rice cultured in the biological fermentation broth of strain N5 is 7.43 cm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Effects of biological fermentation broth on the germination rate of rice seeds under salt stress\u003c/h2\u003e \u003cp\u003eRice is a sensitive crop to salt content \u003csup\u003e[36]\u003c/sup\u003e. This study further evaluated fermentation broth's influence on rice seeds' germination rate under varying salt concentrations. The image illustrates how similar circumstances were shown for the germination of rice seedlings treated with sterile water and nutritional medium at the same concentration of salt stress. In the presence of a 100 mmol/L salt stress, the diluted biological fermentation broth encouraged rice seedlings to germinate. At 200 mmol/L of salt stress, there was no significant difference between the treatments. This may be because the inhibition of seed growth at high concentrations of salt stress was more severe than that under low concentrations.\u003c/p\u003e \u003cp\u003eThe rice seeds diluted by biological fermentation broth under 100 mmol/L salt stress had a higher second day than the other groups, as Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e illustrates. On the third day of culture, the germination rate was 70% under 100 mmol/L salt stress, and on the sixth day, the germination rate reached its maximum. The germination rates of seedlings treated with sterile water and nutrient medium were 52% and 54% on the third day, respectively, and the germination index was completed on the seventh day. Furthermore, under salt stress of 200 mmol/L, there was no discernible variation in the rate of germination of seeds among the various treatments. The third day showed comparable germination rates of 47%, 53%, and 53% for various treatments of nutritional medium, sterile water, and biological fermentation broth. On the seventh day, 99% of the seeds treated with biological fermentation broth germinated, compared to 92% and 97% of the seeds treated with sterile water and nutritional medium.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Genome-wide analysis of cellulose-degrading strain N5\u003c/h2\u003e \u003cp\u003eThe N5 strain was found to contain a circular chromosome but no plasmids (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Its genome size was determined to be 3,492,876 base pairs, with a GC content of 47.33%. The non-coding RNA included 86 tRNAs, 27 rRNAs (with nine copies each of 5S, 16S, and 23S rRNAs), and 31 sRNAs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGene annotation based on the COG database revealed that the most abundant functional categories were amino acid transport and metabolism (305 sequences, 10.25%), transcription (286, 9.62%), translation and ribosomal structure (259, 8.71%), general function prediction (256, 8.61%), cell cycle control (213, 7.16%), and signal transduction (210, 7.06%). This suggests that strain N5 has an active synthesis pathway and a robust capacity to utilize amino acids and carbohydrates for substrate transport. The presence of cellulose-coding genes indicates their involvement in carbohydrate transport and metabolism, including 6 α-amylases, 4 β-xylosidases, 3 β-glucosidases, and 3 glycoside hydrolases, which are crucial for cellulose hydrolysis \u003csup\u003e[37, 38]\u003c/sup\u003e. The synergistic action of these multiple hydrolases is believed to facilitate cellulose degradation. Additionally, 140 genes of unknown function were identified, offering potential for further research.\u003c/p\u003e \u003cp\u003eThe GO database annotated 2338 genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec), representing 59.02% of the total coding genes, with the majority involved in biological pathways, followed by molecular function and cellular components. The top three biological pathways were cell processes (1280), metabolic processes (1247), and biological regulation (304). In molecular functions, the majority of genes were involved in catalytic activity (1398) and binding (927), while the most annotated cellular component was cell structure (621). This indicates that strain N5's metabolism and regulatory network are complex, with a focus on catalytic activity, suggesting that enzyme regulation plays a dominant role in its metabolic processes.\u003c/p\u003e \u003cp\u003eFunctional annotation of strain N5's genome via the KEGG database identified 2536 genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). Among the six major metabolic pathways, 1956 genes were related to metabolic pathways and at least 59 to tissue systems. Carbohydrate metabolism (259), amino acid metabolism (214), and vitamin and cofactor metabolism (176) were the most represented. In the biosynthetic pathways of secondary metabolites, genes related to potassium uptake proteins (K03498, K03499), proline transport systems (K11928, K03762), and proline biosynthesis (proA, proB, proC) were identified. These genes enhance strain N5's activity under high salt stress by synthesizing and accumulating small molecule solutes to counteract osmotic pressure imbalances.\u003c/p\u003e \u003cp\u003eThe synergistic action of multiple carbohydrate-active enzymes (CAZymes) is required to degrade straw cellulose polymers \u003csup\u003e[12]\u003c/sup\u003e. The genome of strain N5, when compared to the CAZy database, was found to contain 115 CAZy genes across six categories: auxiliary active enzymes (AAs), carbohydrate-binding modules (CBMs), carbohydrate esterases (CEs), glycoside hydrolases (GHs), glycosyltransferases (GTs), and polysaccharide lyases (PLs). The GH family predominates, with 52 annotated genes spread across 27 families (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Notably, GH1 (5 genes) is associated with cellulase, GH13 (5 genes) with hemicellulose degradation, and GH43 (4 genes) with xylan breakdown, all of which are crucial for cellulose degradation. GH23 (3 genes) encodes peptidoglycan lyase, also implicated in this process. The presence of these genes in strain N5 suggests a genetic predisposition for carbohydrate degradation, including cellulose and glycosides.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, the function of carbohydrate-binding modules (CBMs) is to help immobilize cellulases to the surface structure of cellulose molecules, enabling enzymes to catalyze the hydrolysis of cellulose to the greatest extent and to maintain the hydrolysis state throughout the subsequent degradation process \u003csup\u003e[39, 40]\u003c/sup\u003e. Carbohydrate-binding components (CBMs) are annotated to 25 gene quantities, and glycosyltransferase families (GTs) are enzyme systems that catalyze glycosylation reactions \u003csup\u003e[41]\u003c/sup\u003e. Strain N5 possesses 26 glycosyltransferase (GT) genes, among which GT2 (5) is related to carboxyltransferases. There are 11 carbohydrate esterase (CE) genes, as well as 3 polysaccharide lyase (PL) genes and 2 auxiliary activity (AA) genes, which can assist other enzyme systems in degrading cellulose.\u003c/p\u003e \u003cp\u003eAmong them, the glycoside hydrolase family is a crucial enzyme in bacterial polysaccharide degradation and promotes bacterial growth and catalytic efficiency alongside CBM glycohydrolase-active enzymes \u003csup\u003e[42]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eThis study introduced the degrading bacterium N5 to the straw medium. The findings revealed that adding bacterium N5 could potentially increase the weight loss rate of straw by 19.35%. When the straw was subjected to additional alkali treatment, it resulted in a biodegradation rate of 38.86%. Moreover, the microbial fermentation broth generated from the straw exhibited a stimulatory effect on the germination and growth of rice seeds, with the incorporation of peptone as a nutrient significantly amplifying the degradation. A comprehensive analysis of the molecular biological characteristics of the degrading bacterium N5 identified a total of 52 glycoside hydrolase (GH) genes and other relative genes; the presence of these genes confirms that strain N5 is capable of hydrolyzing lignin, hemicellulose, and cellulose.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by the Zhejiang Province Key Research and Development Plan (2021C03190), the Zhejiang Province \"Sannong Jiufang\" (2022SNJF077), and the 111 Project (D18008).\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eTao Zhang: Implemented the experiments, analysed the data and wrote the manuscript; Wen-fan Wang: Implemented the experiments, analysed the data; Zhuo-qun Zhao: Guided experiment and analysed the data; Hua-bao Zheng: Conceived and designed experiment.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe authors declare that all data and materials are available to be shared upon a formal request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYao X, Liu Q, Li D., 2024. Mechanism underlying effects of cellulose-degrading microbial inoculation on amino acid degradation and biosynthesis during composting[J]. Bioresource Technology. 403, 130899.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei Y, Wu D, Wei D, et al.,2019. Improved lignocellulose-degrading performance during straw composting from diverse sources with actinomycetes inoculation by regulating the key enzyme activities[J]. Bioresource technology. 271, 66\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Kuramae E E, Nasir F, et al.,2023. Addition of cellulose degrading bacterial agents promoting keystone fungal-mediated cellulose degradation during aerobic composting: Construction the complex co-degradation system[J]. Bioresource Technology. 381, 129132.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Tian L, Li Y, et al., 2022. Effects of exogenous cellulose-degrading bacteria on humus formation and bacterial community stability during composting[J]. Bioresource Technology. 359, 127458.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi ZuLiang S Z L, Wang Fei W F, Wang JiuChen W J C, et al., 2019. Utilization characteristics, technical model and development suggestion on crop straw in China[J]. 2019.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar V, Mitra D, Rani A, et al.,2022. Bio-inoculants for Biodegradation and Bioconversion of Agrowaste: Status and Prospects[J]. Bioremediation of Environmental Pollutants: Emerging Trends and Strategies. 2022, 351\u0026ndash;367.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu H, Sun S, Han Y., 2021. The current situation and development strategy of corn straw returning in Heilongjiang Province [J]. China Seed Industry. 11, 20\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePadhan K, Patra R K, Sethi D, et al., 2024. Exploitation of cellulose degrading bacteria in bioconversion of agro-wastes[J]. Chemosphere. 347, 140654.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePandit L, Sethi D, Pattanayak S K, et al., 2020. Bioconversion of lignocellulosic organic wastes into nutrient rich vermicompost by Eudrilus eugeniae[J]. Bioresource Technology Reports. 12, 100580.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang G, Dong Y., 2022. Design and application of an efficient cellulose-degrading microbial consortium and carboxymethyl cellulase production optimization[J]. Frontiers in Microbiology. 13, 957444.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaur P, Sharma N, Munagala M, et al., 2021. Nanocellulose: Resources, physio-chemical properties, current uses and future applications[J]. Frontiers in Nanotechnology. 3, 747329.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe Z, Ding B, Ali Q, et al., 2023. Screening and isolation of cold-adapted cellulose degrading bacterium: A candidate for straw degradation and De novo genome sequencing analysis[J]. Frontiers in Microbiology. 13, 1098723.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Wang T, Xing Z, et al., 2023. Enhanced lignocellulose degradation and composts fertility of cattle manure and wheat straw composting by Bacillus inoculation[J]. Journal of Environmental Chemical Engineering. 11(3), 109940.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S, Long H, Hu X, et al., 2024. The co-inoculation of Trichoderma viridis and Bacillus subtilis improved the aerobic composting efficiency and degradation of lignocellulose[J]. Bioresource Technology. 394, 130285.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeneses C, Silva B, Medeiros B, et al., 2016. A metagenomic advance for the cloning and characterization of a cellulase from red rice crop residues[J]. Molecules. 21(7), 831.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarthika A, Seenivasagan R, Kasimani R, et al., 2020. Cellulolytic bacteria isolation, screening and optimization of enzyme production from vermicompost of paper cup waste[J]. Waste Management. 116, 58\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHemati A, Aliasgharzad N, Khakvar R, et al., 2021. Role of lignin and thermophilic lignocellulolytic bacteria in the evolution of humification indices and enzymatic activities during compost production[J]. Waste Management. 119, 122\u0026ndash;134.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaruta S, Cui Z, Huang Z, et al., 2022. Construction of a stable microbial community with high cellulose-degradation ability[J]. Applied microbiology and biotechnology. 59, 529\u0026ndash;534.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBajpai P. Paper Bioprocessing., 2018. Biermann\u0026rsquo;s handbook of pulp and paper[J]. 2018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, Zhang M, Zhang Y, et al., 2023. Characterization of Cellulose-Degrading Bacteria Isolated from Silkworm Excrement and Optimization of Its Cellulase Production[J]. Polymers. 15(20), 4142.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang C, Shan J, Chen Y, et al., 2017. Organic amine catalytic organosolv pretreatment of corn stover for enzymatic saccharification and high-quality lignin[J]. Bioresource technology. 232, 222\u0026ndash;228.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu D, Qu F, Li D, et al., 2022. Effect of Fenton pretreatment and bacterial inoculation on cellulose-degrading genes and fungal communities during rice straw composting[J]. Science of The Total Environment. 806, 151376.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen M, Zhao J, Xia L., 2009. Comparison of four different chemical pretreatments of corn stover for enhancing enzymatic digestibility[J]. Biomass and Bioenergy. 33(10), 1381\u0026ndash;1385.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJain D, Ravina, Bhojiya A A, et al., 2021. Polyphasic characterization of plant growth promoting cellulose degrading bacteria isolated from organic manures[J]. Current Microbiology. 78, 739\u0026ndash;748.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJi J, Yuan D, Jin C, et al., 2020. Enhancement of growth and salt tolerance of rice seedlings (Oryza sativa L.) by regulating ethylene production with a novel halotolerant PGPR strain Glutamicibacter sp. YD01 containing ACC deaminase activity[J]. Acta Physiologiae Plantarum. 42, 1\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong X, Ji J, Zhang S, et al., 2022. Study on a Low-temperature Cellulose-degrading Strain: Fermentation Optimization, Straw Degradation, and the Effect of Fermentation Broth on Seed Growth[J]. Biotechnology and Bioprocess Engineering. 27(4), 652\u0026ndash;667.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTamilselvan R, Selwynraj A I., 2024. A novel g-C3N4 photocatalytic pretreatment for reducing silica and modifying the structure of rice straw for sustainable biofuel production[J]. Fuel. 357, 129901.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan X, Luo Q, Bao A, et al., 2019. Research on the mechanism of improving rice straw self-bonding via NaOH solution pretreatment[J]. BioResources. 14(4), 9352\u0026ndash;9363.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo H, Chang J, Yin Q, et al., 2013. Effect of the combined physical and chemical treatments with microbial fermentation on corn straw degradation[J]. Bioresource technology. 148, 361\u0026ndash;365.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuan R, Li X, Wachemo A C, et al., 2018. Enhancing anaerobic digestion performance and degradation of lignocellulosic components of rice straw by combined biological and chemical pretreatment[J]. Science of the Total Environment. 637, 9\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSamar W, Arora A, Sharma A, et al., 2021. Material flow of cellulose in rice straw to ethanol and lignin recovery by NaOH pretreatment coupled with acid washing[J]. Biomass Conversion and Biorefinery. 2021, 1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVogel H C, Todaro C M., 1996. Fermentation and biochemical engineering handbook: principles, process design and equipment[M]. William Andrew, 1996.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuti\u0026eacute;rrez-Correa M, Villena G K., 2010. Characteristics and techniques of fermentation systems[J]. Comprehensive Food Fermentation and Biotechnology. 2010, 1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThao N T N L, Chiang K Y., 2020. The migration, transformation and control of trace metals during the gasification of rice straw[J]. Chemosphere. 260, 127540.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGohil R B, Raval V H, Panchal R R, et al., 2022. Plant growth-promoting activity of Bacillus sp. PG-8 isolated from fermented panchagavya and its effect on the growth of Arachis hypogea[J]. Frontiers in Agronomy. 4, 805454.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrattan S R, Zeng L, Shannon M C, et al., 2002. Rice is more sensitive to salinity than previously thought[J]. California agriculture. 2002, 56(6).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChukwuma O B, Rafatullah M, Tajarudin H A, et al., 2020. Lignocellulolytic enzymes in biotechnological and industrial processes: a review[J]. Sustainability. 12(18), 7282.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaheja Y, Kaur B, Falco M, et al., 2020. Secretome analysis of Talaromyces emersonii reveals distinct CAZymes profile and enhanced cellulase production through response surface methodology[J]. Industrial crops and products. 152, 112554.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eV\u0026aacute;rnai A, Siika-Aho M, Viikari L., 2013. Carbohydrate-binding modules (CBMs) revisited: reduced amount of water counterbalances the need for CBMs[J]. Biotechnology for biofuels. 6, 1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA\u0026iuml;ssa K., 2020. The potential of using carbohydrate-binding modules (CBMs) for the characterization and modification of cellulose surfaces[D]. University of British Columbia. 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrtiz-Soto, M.E. and J. Seibel., 2024. An overview on glycoside hydrolases and glycosyltransferases [J]. Zeitschrift f\u0026uuml;r Naturforschung C. 2024(0).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa D, Chen H, Liu D, et al., 2024. Soil-derived cellulose-degrading bacteria: screening, identification, the optimization of fermentation conditions, and their whole genome sequencing[J]. Frontiers in Microbiology. 15, 1409697.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"cellulose, rice straw, alkali pretreatment, microbial degradation","lastPublishedDoi":"10.21203/rs.3.rs-6325052/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6325052/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe rich cellulose in rice straw is an obstacle to the resource utilization of rice straw. It is difficult to degrade cellulose only by biodegradation, chemical or physical degradation. The weight loss rate of rice straw reached 38.86% by the inoculation of \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e N5 combined with alkali pretreatment. The fermentation broth of strain N5 and straw could promote the rice seed\u0026rsquo;s germination and the growth of rice seedlings. Moreover, the addition of peptone as a nutrient source further amplified the cellulose degradation. The whole genome analysis revealed genes associated with cellulose degradation in the genome of strain N5, including glycoside hydrolase family 1 (GH1), GH13, GH43, and three polysaccharide lyases (PLs), eleven carbohydrate esterases (CEs), twenty-six glycosyltransferases (GTs), and twenty-five carbohydrate-binding modules (CBMs).\u003c/p\u003e","manuscriptTitle":"Microbial decomposition of rice straw: alkaline pretreatment, genome analysis and fermentation broth on seed growth","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-25 16:17:36","doi":"10.21203/rs.3.rs-6325052/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1c161fcf-64bf-4a9d-b29e-ad226ef8f19e","owner":[],"postedDate":"April 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-24T19:44:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-25 16:17:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6325052","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6325052","identity":"rs-6325052","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}