Running title: Bacteria-enzyme synergy modulates microbiome and metabolome of wheat straw silage
标题： 细菌-酶协同作用调节小麦秸秆青贮的微生物组和代谢组

Novel strategy to understand the bacteria-enzyme synergy action regulates the ensiling performance of wheat straw silage by multi-omics analysis
通过多组学分析了解细菌-酶协同作用调节小麦秸秆青贮青贮性能的新策略

Shuai Du^{1, *}, Lijun Xu², Chao Jiang³, Yanzi Xiao^{3, *}
杜帅^{1， *}徐丽军²， 江超³， 肖燕子 ^{3， *}

¹ Key Laboratory of Grassland Resources, Ministry of Education, Key Laboratory of Forage Cultivation, Processing and High Efficient Utilization, Ministry of Agriculture, College of Grassland, Resources and Environment, Inner Mongolia Agricultural University, Hohhot Inner Mongolia, China.
¹ 内蒙古农业大学草原资源与环境学院，农业部草原资源与高效利用教育部重点实验室，内蒙古呼和浩特农业大学，草原资源与环境学院。

² Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Science, Hulunber Grassland Ecosystem Observation and Research Station, Beijing, China.
²中国农业科学院农业资源与区域规划研究所，呼伦贝尔草原生态系统观测研究站，中国北京。

³ Grass Industry Collaborative Innovation Research Center, Hulunbuir University, Hulunber, China
³ 呼伦贝尔大学草产业协同创新研究中心，呼伦贝尔

* Corresponding author at:
* 通讯作者：

E-mail address: dushuai_nm@sina.com, xiaoyz1113@126.com
电子邮件地址dushuai_nm@sina.com， xiaoyz1113@126.com

Graphical Abstract
图形摘要

Abstract
A小区

Background: Ensiling technology holds promise for preserving and providing high-quality forage. Nevertheless, the high polymeric contents and compact properties of fiber results in a low biodigestibility. The study aimed to evaluate the ensiling technology in storing wheat straw. Changes of fermentation related product, chemical component, bacterial community and metabolite profiles of wheat straw ensiled without or with cellulase or Lactiplantibacillus plantarum (L. plantarum) were also analyzed.
背景： 青贮技术有望保存和提供高质量的牧草。然而，纤维的高聚合物含量和致密性导致生物消化率低。该研究旨在评估贮藏小麦秸秆的青贮技术。 还分析了无纤维素酶 或 植物乳植杆菌 （L. plantarum） 青贮小麦秸秆发酵相关产物、化学成分、细菌群落和 m代谢物谱的变化。

Results: Results showed that the inoculated with L. plantarum alone or with cellulase produced abundant organic acids, degraded the fiber, suppressed most microbes and increased some metabolites in wheat straw silage. The wheat straw inoculated with L. plantarum alone or with cellulase exhibited significantly lower neutral detergent fiber, and acid detergent fiber contents compared to that in the control treatment. Similarly, the higher lactic acid and acetic acid contents were also found in these treatments. The microbiome revealed that the Lactobacillus was dominance and Kosakonia was suppressed. The metabolic analysis remarkably increased the amino acids, peptides, and analogues, and organic acids and derivatives related metabolites.
结果表明：单独接种植物乳杆菌或纤维素酶接种后，小麦秸秆青贮饲料中产生丰富的有机酸，降解纤维，抑制大多数微生物，增加一些代谢物。 与对照处理相比，单独接种植物乳杆菌或加纤维素酶的 wheat 秸秆表现出显著降低的中性洗涤纤维和酸性洗涤纤维含量。同样，在这些处理中也发现了较高的乳酸和乙酸含量。微生物组显示 乳酸菌 占主导地位，Kosakonia 受到抑制。代谢分析显着增加了氨基酸、肽和类似物以及有机酸和衍生物相关的代谢物。

Conclusions: Overall, wheat straw inoculated with L. plantarum alone or with cellulase can produce well-preserved silage, which offers a new insight into recycling and utilizing the wheat straw by the bacteria-enzyme synergy.
C结论： 总体而言，单独接种植物乳杆菌或纤维素酶接种的小麦秸秆可以产生保存完好的青贮饲料，这为通过 b acteria-酶协同作用回收和利用小麦秸秆提供了新的见解。

Keywords: Cellulase; Lactiplantibacillus plantarum; Microbiome; Metabolome; Wheat straw silage
关键词： 纤维素酶; 植物乳植杆菌; Microbiome; M依他泊肟; W加热秸秆青贮

1. Introduction
1. 引言

Approximately thousands of million tons of crop straw are produced after the crop collection, processing and production every year, and only 30% of these processing leftovers can be utilized, and the crop straw is receiving an increasing interest to be used as animal feed (Yan et al., 2022; Jairath et al., 2023). The wheat straw is considered as an important renewable bioresource for producing ruminant feed because of its high yield and carbohydrate content (Niu et al., 2020). However, the high and complex polymeric contents can prevent glycoside hydrolases from contacting their substrates results in a low biodigestibility (Fu et al., 2023). Chemical and thermal pretreatments have proved effective methods to improve the digestibility, but these methods often produce some by-products related lignocellulose that inhibit the microorganisms and enzymatic biocatalysts (Jonsson & Martin 2016). Ensiling is an effective, economically viable method to transform low-value agricultural by-product efficiently and produce high value-added by bioaugmentation for ruminants’ feed (Su et al., 2022; Luo et al., 2023), including the apple pomace silage (Yang et al., 2021), corn straw silage (Okoye et al., 2023), mulberry silage (Du et al., 2023) and others (Javourez et al., 2021).
每年在作物收集、加工和生产后生产约数千万吨农作物秸秆，只有 30% 的加工剩余物可以利用，农作物秸秆被用作动物饲料的兴趣越来越大（Yan et al.， 2022;Jairath等人，2023 年）。 wheat 秸秆因其高产和高碳水化合物含量而被认为是生产反刍动物饲料的重要可再生生物资源（Niu et al.， 2020）。然而，高而复杂的聚合物含量会阻止糖苷水解酶接触其底物，从而导致生物消化率低（Fu et al.， 2023）。Chemical和thermal预处理已被证明 可以提高消化率的有效方法，但这些方法经常产生一些与抑制微生物和酶生物催化剂相关的副产物木质纤维素 （Jonsson & Martin 2016）。青贮是一种有效、经济可行的方法，可以有效地转化低价值的农业副产品，并通过生物强化为反刍动物饲料生产高附加值 （Su et al.， 2022;Luoet t al.， 2023），包括苹果渣青贮饲料（Yang et al.， 2021）、玉米秸秆青贮饲料（Okoye et al.， 2023）、桑树青贮饲料（Du et al.， 2023）和其他（Javourez et al.， 2021）。

Silage is produced under anaerobic conditions, this process depends on the substrate, the water-soluble carbohydrates (WSC) are fermented mostly by lactic acid bacteria (LAB) into lactic acid (LA), causing a pH drop, inhibiting the growth of spoilage microorganisms and storage (Vlierberghe et al., 2022). Generally, the wheat straw has a low WSC and epiphytic (LAB, which it is difficult to produce high-quality silage by the natural anaerobic fermentation. Several previously published studies have recommended using silage additives can effectively improve the fermentation process (Okoye et al., 2023). The microbial and enzymes additives, such as LAB and cellulase inoculants, play a critical role in enhancing the fermentation process and are widely used in the production of silage (Cai et al., 2020). Previous report indicated that the LAB not only improve the fermentation quality, but can increase the in vitro dry matter digestibility of wheat straw (Nyang’au et al., 2023). Nevertheless, some studies indicated that the crystallinity of the wheat straw’s cellulose and lignin content limits the fermentation process, as the decomposable components are protected, subsequently influences the microbial and enzymatic attack during the hydrolysis stage (Mirmohamadsadeghi et al., 2021). Previously reported the enzyme produced by the microorganisms have positive effect on the cellulose content degradation in crop straw silage (Chen et al., 2012; Nazar et al., 2023). The cellulase can degrade and transfer the cellulose and hemicellulose into monosaccharides, and these monosaccharides can be used by LAB to accelerate the fermentation process (Du et al., 2023).
Silage 是在厌氧条件下产生的，这个过程取决于底物，水溶性碳水化合物 （WSC） 主要由乳酸菌 （LAB） 发酵成乳酸 （LA），导致 pH 值下降，抑制腐败微生物的生长和储存 （Vlierberghe等人，2022 年）。一般来说，小麦秸秆的 WSC 和附生 （LAB） 较低， 很难通过自然厌氧发酵生产出高质量的青贮饲料。 先前发表的几项研究建议使用青贮添加剂可以有效改善发酵过程（Okoye et al.， 2023）。微生物和酶添加剂，如 LAB 和纤维素酶接种剂，在增强发酵过程中起着关键作用，并广泛用于青贮饲料的生产（Cai et al.， 2020）。以前的报告表明，LAB 不仅可以提高发酵质量，还可以提高 小麦秸秆的体外干物质消化率（Nyang'au et al.， 2023）。 然而，一些研究表明，小麦秸秆的纤维素和木质素含量的结晶度限制了发酵过程，因为可分解成分受到保护，随后影响 水解阶段的微生物和酶攻击（Mirmohamadsadeghi et al.， 2021）的 以前报道的微生物产生的酶对作物秸秆青贮中纤维素含量的降解有积极影响（Chen等人，2012 年;Nazar et al.， 2023）的纤维素酶可以降解纤维素和半纤维素并将其转化为单糖，这些单糖可以被 LAB 用来加速发酵过程（Du et al.， 2023）。

The inoculated with cellulase in conjunction with LAB have been investigated on some crops. Previously published studies have indicated that the fermentation quality of silage is closely related to the relationship between the microorganisms and metabolites, and the fermentation quality is determined by the microorganisms and metabolites (Xu et al., 2021). Recently, the metabolomics has been applied in the ensiling ecosystems (Guo et al., 2018; Guan et al., 2020). But ensiling wheat straw still poses challenges due to the characteristics of the raw materials, and the comprehensive and detailed characterization of wheat straw silage by microbiome and metabolome analysis has been limited. Therefore, the effects of cellulase or Lactiplantibacillus plantarum (L. plantarum) on the chemical composition, bacterial community and metabolite profiles of wheat straw silage were investigated. This study aimed to offers new insights into the role of bacteria-enzyme synergy in enhancing the quality of rice straw silage, from both microbiological and metabolomic perspectives.
T 他接种纤维素酶与 LAB 一起已在一些作物上进行了研究。先前发表的研究表明 ，青贮饲料的发酵质量与微生物和代谢物之间的关系密切相关，发酵质量由微生物和代谢物决定（Xu et al.， 2021）。最近，代谢组学已应用于青贮生态系统（Guo等人，2018 年;Guan等人，2020 年）。但由于原料的特性，柩化小麦秸秆仍然面临挑战，通过微生物组和代谢组分析对小麦秸秆青贮进行全面和详细的表征受到限制。 因此，纤维素酶或植物乳植杆菌 （L. 植物）对小麦秸秆青贮的化学成分、细菌共性Y和代谢物谱进行了研究。本研究旨在从微生物学和代谢组学的角度为细菌-酶协同作用在提高水稻秸秆青贮质量中的作用 提供新的见解。

2. Materials and methods
2. 材料和方法

2.1. Substrate and silage
2.1. 基质和青贮饲料

Wheat (
小麦 （Triticum aestivum
小麦L.) straw was from the   Hulunber  Grassland Ecosystem National Observation and Research Station of the Chinese Academy of Agricultural Sciences in   Hulunber  , Inner Mongolia, China (E 119°55′, N49°19′). The   experimental  zone belongs to the temperate semi humid zone, with an average annual precipitation of 380~400  mm, an average annual temperature of -2 ℃ ~ -1 ℃ and a humidity level of 0.49~0.50.  The wheat straw was harvested at the late   maturity stage  on 15 Aug. 2022  , then chopped and immediately   taken to the laboratory for  silage making  when the   wheat straw  samples   were picked  . Inoculants  , Cellulase  (CE  , (total cellulase activity of   50,000 U/g)) purchased from Hefei Bomei Biotechnology Co., Ltd, Hefei, China; L. plantarum (MTD-1) purchased from Jiangsu Lvke Biotechnology Company, Gaoyou, China. The treatments were as follows: control (CK), CE, Lp, and CELp. The inoculants were distinctively diluted in distilled water and added according to the manufacturer’s guidelines with the amount of CE was 50,000 U/g on a fresh matter (FM) basis, after which the number of LAB was equivalent to 1.0 × 10⁵ colony-forming unit per gram (cfu/g), the CK treatment was also treated with the same volume of distilled water. Both wheat straw and wheat straw silage were stored at a small-scale fermentation system (260 cm × 380 cm; Hiryu KN type; Asahi kasei, Tokyo, Japan). A 200 gram of the samples were packed into the polyethylene plastic bag, and removing air with a vacuum sealer (N-14886, Deli Group Co., Ltd., Zhejiang, China). A total of 24 bags (4 treatments × 6 replicates) of wheat straw were stored at room temperature (23 ± 2 °C). After 60 days of fermentation process, these bags were opened and the ensiling performance, bacterial community and metabolites profiles were analyzed.

2.2. Ensiling performance and nutritive values analyses

Clean containers were used to collect FM and wheat straw silage after being uniformly blended for ensiling performance and nutritive values analyses. The dry matter (DM) content of the FM and silage samples were measured after drying the sample for 72 h at 65 °C with an oven (AOAC 2005). The dried samples were ground and through a 1 mm screen for the nutritive values analysis. The crude protein (CP) and acid detergent lignin (ADL) contents were analyzed according to the method of the Association of Official Analytical Chemists (AOAC, 2005). The neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents were determined by a ANKOM A200i Fiber Analyzer (ANKOM Technology, Macedon, NY, USA) with the report (Van Soest et al., 1991). The fraction of cell wall constituents, including cellulose, hemicellulose and holocellulose was calculated using methods briefed by Nyang’au (2023). The anthrone method was selected to evaluate the WSC content (Thomas, 1977). A 20 gram of the wheat straw silage samples were mixed with 180 mL sterile water and stored for 24 h at 4 °C fridge for the extractions, then the extracts were filtered through four layers of cheesecloth. A glass-electrode pH meter was used to measure the pH value of the filtrate. The organic acids concentrations in the filtrate, mainly LA, acetic acid (AA), propionic acid and butyric acid, were measured by the high-performance liquid chromatography methods (You et al., 2021). The plate count method was used to analysis the microbial population of the wheat straw silage according to the previously published methods (You et al., 2021).

2.3. Bacterial community analysis

The genomic DNA of bacterial community was extracted from the FM and wheat straw silage samples by the CTAB method (Bao et al., 2023). The Nano Drop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the concentrations and qualities of the extracted genomic DNA. The V3–V4 regions of 16S rDNA gene was targeted with the universal primer pair 341F and 806R. The Illumina NovaSeq 6000 platform (IlluminaInc., San Diego, CA, USA) was used to sequence. The Raw pair-end reads were analyzed by the Qiime2 platform (https:// qiime2. org/). Amplicon sequence variants (ASVs) were obtained by eliminating low-quality data using DADA2 (Callahan et al., 2016). Subsequently, the ASVs were taxonomically annotated against the SILVA database (https://www. arb-silva.de/, Release 138) using mothur (Schloss et al., 2020). The raw data were uploaded to the sequence read archive database with the accession number PRJNA1145488.

2.4. Metabolites profiles analyses

The metabolites in the wheat straw silage samples were extracted according to the previous methods (Du et al., 2023). The raw data files of the 24 wheat straw silage samples generated by the liquid chromatography-mass spectrometry (LC–MS) platform (Thermo Fisher, Ultimate 3000LC, Q Exactive) using the compound Discover 3.1 (CD 3.1 Thermo Fisher) to perform peak picking, peak alignment and quantitation for each metabolite. After that, peak intensities were normalized to the total spectral intensity (Want et al., 2012). The normalized data was used to predict the molecular formula based on additive ions, molecular ion peaks and fragment ions (Yuan et al., 20212). Then peaks were matched with the mzCloud (https://www.mzcloud.org/) mz Vault and Mass List database to obtain the accurate qualitative and relative quantitative results. After mean centering and unit variance scaling, the principle component analysis (PCA) and (orthogonal) partial least squares discriminant analysis (O)PLS-DA were selected to show the differences of the metabolites among the treatments by R package (Warnes et al., 2007; Worley et al., 2013). The variable importance in the projection (VIP) ranks and VIP > 2.0 were considered as the relevant for treatment discrimination, and the results were displayed by the (O)PLS-DA plots. Significant differences in metabolites among the treatments were analyzed involved a combination of (O)PLS-DA and p < 0.05 as statistical significance (Yoon et al., 2020). The gplots package in R was used for significant metabolites for expression pattern clustering using. Hierarchical clustering method were used for distance calculation algorithms (Du et al., 2023). The metabolic pathways and metabolites set enrichment was analyzed with the Stats package in R and the SciPy package in Python using the MetaboAnalyst 6.0 (https://www.metaboanalyst.ca).

2.5. Statistical assessments

This experiment was conducted independently with six biological replicates. All experimental data were analyzed using the one-way analysis of variance with the general linear models of SAS (version 9.2, SAS Institute Inc., Cary, NC). Multiple comparisons among the treatments were conducted using the Tukey's method, and significant differences were considered at p < 0.05 level.

3. Results

3.1 Characteristics of fresh materials wheat silage

The chemical and microbial characteristics of the FM before ensiling are presented in Table 1. The raw materials contained 50.30% of DM, and the contents of WSC, CP, ADF, and NDF were 3.42%, 12.90%, 29.50%, and 37.10% of DM, respectively. The counts of LAB, yeasts, aerobic bacteria, and coliform bacteria were 4.27, 8.47, 8.35, 8.40 and 8.32 log₁₀ cfu/g of FM, respectively.

3.2 Ensiling performance of wheat straw silage

The parameters of the wheat straw ensiling characteristics are displayed in Table 2. The results showed that the inoculants significantly (p < 0.05) influenced the pH value and organic acids contents of the wheat straw silage. The inoculated silages showed markedly (p < 0.05) lower pH values than that in the control treatment. The lowest pH values were observed in the Lp and CELp treatments and reached at 4.05. The LA and AA contents, which are directly responsible for the decreasing in pH, were significantly (p < 0.05) increased in the inoculants treated wheat straw silages, especially in the Lp and CELp treatments. The composition of structural carbohydrates (NDF, ADF, ADL, cellulose, hemicellulose and holocellulose) and nutritional compositions of wheat straw silages are reported in Table 2. No (p > 0.05) significant differences were observed on the DM contents among the treatments, however, the DM content in the additives treated silages was lower than that in the CK treatment. The CP content in the CE treatment was markedly higher (p < 0.05) than that in the CK treatment, followed by the CELp and Lp treatments. Moreover, the concentrations of ADF, NDF and ADL of wheat straw silage were significantly (p < 0.05) affected by the additives treatments. Compared with the CK treatment, the additives clearly lowered the ADF, NDF and ADL contents. There were no (p > 0.05) obvious changes in the cellulose, hemicellulose and holocellulose contents among the treatments.

3.3 Bacterial diversity of wheat straw silage

The bacterial community of all the wheat straw silages was analyzed by 16S rRNA sequencing (Table 3). Overall, 3,201,925 raw reads were obtained for the 6 fresh materials wheat straw and 24 wheat straw silages from the four treatments. And, 3,092,643 valid reads were also obtained for these samples, with the average sequences of 54,463–117,832 for sample from these treatments, these valid reads were clustered into 15,515 ASVs with the confidence threshold value of 80%. No significant (p < 0.05) differences were observed among these samples. The Goods’ coverage of the fresh materials and wheat straw silages exceeded 0.99, indicating the sequencing depth perfectly meet the demand for the diversity analysis of these samples. The changes of the Chao1 value among the fresh materials and silage samples were similar with the ASV numbers. After 60 days of ensiling, the Shannon index in the wheat straw silage samples was markedly (p < 0.05) decreased compared to that in the fresh materials, and the lowest Shannon index was found in the Lp treatments.

To probe the influence of inoculants on the bacterial communities of wheat straw silage, the Venn and principal coordinate analysis (PCoA) plots were used to characterized the bacterial compositions of wheat straw silage treated with cellulase or LAB. The Venn diagram showed 187, 660, 148, 259 and 317 unique ASVs in the FM, control, CE, Lp and CELp treatments, respectively, and all treatments shared 97 ASVs. The PCoA plot of the FM and wheat straw silages unveiled discernible differences among the treatments was estimated by the Bray-Curtis distance metrics, wherein the wheat straw silage samples were distinctly partitioned into three groups: FM, control and CE treatments, Lp and CELp treatments (Fig. 1B). The result indicated that 54.94% of the variation was captured by axis 1, while axis 2 represents 11.51% of the variation. On the other hand, there markedly distinct separation and the permutational multivariate analysis of variance (PERMANOVA) shows significant difference (p = 0.001) among the three groups.

The taxonomic bacterial communities of the wheat silage treated without or with inoculants were subsequently analyzed at both the phylum and genus levels. In the absence of L. plantarum inoculation, the Proteobacteria stood as the most abundant phylum with significant difference (Fig. 1D), accounting over 60% of the entire community (Fig. 1C). Additionally, the relative abundance of Firmicutes in the L. plantarum- treated wheat straw silages were markedly higher than that in the FM and other treatments (Fig. 1D). Further the distribution of the bacterial compositions in the wheat silage treated without or with inoculants were analyzed at the genus level, yielding insights into the changes of the bacterial compositions. The dominant relative abundance shifted in the FM from Kosakonia and Xanthomonas into Lactobacillus in the wheat straw silages (Fig. 1E), with significant difference (Fig. 1F). Silage samples lacking L. plantarum exhibited elevated bacterial diversity, mirroring the higher Shannon indices observed (Table 3). The genus Lactobacillus dominated the entirety of the bacterial communities in L. plantarum- treated silage samples (Fig. 1E).

3.4. Metabolite profiles of wheat straw silage

The metabolomic method was applied to delve deeper into the metabolic consequences of the wheat straw treated without or with inoculants. As expected, the principal component analysis (PCA) of the metabolite profiles on the positive and negative mode ionization distinctly grouped into L. plantarum-inoculated and non-inoculated samples, indicating distinctly separated metabolite profiles due to the L. plantarum inoculation (Fig. 2A and 2B). The differentially accumulated metabolites in these treatments were displayed in the Fig. 2. by the orthogonal partial least squares discriminant analysis [(O)PLS-DA] and supervise the multivariate analysis. The (O)PLS-DA provides valuable insights into treatments relationships from all wheat straw silage samples with simple visual inspection of scores-space clustering patterns. All the wheat straw silage samples in the score plots were within the 95% Hotelling T2 ellipse. For the positive ionization analysis, the (O)PLS-DA fitted model (Fig. 2C) resulted in one predictive and two orthogonal components. Furthermore, 42% of the total explained variation in the data set (R²X = 0.42) was used to account for 99.9% of the variance in the class separation (R²Y = 0.46), and the cross-validated predictive ability of the model was 0.29 (Q² cum). As shown in Fig. 2E, the permutation test (R²Y = 0.26, Q²Y = -0.32) indicated that the model was adequate for its efficacy. The results of the (O)PLS-DA results and permutation tests following negative mode ionization are shown in Fig. 2D and 2F. Both positive and negative data revealed clear separation and discrimination among the four treatments, illustrating the effectiveness of the (O)PLS-DA model can be used to identify different metabolites among the four treatments.

Overall, a total of 2, 559 metabolites were identified in the wheat straw silage samples. According to the t-test and variable importance in projection (VIP) filtering for the relative contents of wheat straw silage samples, 30 metabolites were significantly different between the two groups (p < 0.05 and VIP > 2.0), 15 of these metabolites were positively ionized metabolites (Fig. 3A and 3B) and 15 of these metabolites were negatively ionized metabolites (Fig. 3C and 3D), including carboxylic acids and derivatives, amino acids, peptides, and analogues, and other metabolites. Compared to the CK treatment, the CE-treated wheat straw silages significantly improved the concentration of carboxylic acids and derivatives, such as valylproline, L-arginine, proline and valine. Similarly, the L. plantarum-treated wheat straw silages significantly improved the concentration of carboxylic acids and derivatives than these in the CK and CE treatments, such as isocitric acid, L-pyroglutamic acid and tyrosine.

To understand the possible metabolic pathways of differential metabolites, the identified metabolites were conducted by a KEGG pathway enrichment analysis. Results revealed the differential metabolites between CK versus CE treatments, involving “vancomycin resistance”, “glycerophospholipid metabolism” and “pantothenate and CoA biosynthesis” (p < 0.05) (Fig. 4A). Compared to the CK treatment, the main metabolic pathways encompassed in the Lp treatment were “Lysine degradation”, “β-Alanine metabolism” and “aminoacyl-tRNA biosynthesis” (p < 0.05) (Fig. 4B). Between the CK and Lp treatments, the main metabolic pathways concentrated in the Lp treatments were “tropane, piperidine and pyridine alkaloid biosynthesis”, “lysine degradation” and “pyrimidine metabolism” (p < 0.05) (Fig. 4C). Results also revealed the differential metabolites between CE versus Lp treatments, involving “lysine degradation”, “cyanoamino acid metabolism” and “linoleic acid metabolism” (p < 0.05) (Fig. 4D). Results displayed the differential metabolites between CE versus CELp treatments, involving “Tropane, piperidine and pyridine alkaloid biosynthesis”, “Lysine degradation” and “cyanoamino acid metabolism” (p < 0.05) (Fig. 4E). Between the Lp and CELp treatments, the main metabolic pathways concentrated in the CELp treatments were “antineoplastics-agents from natural products”, “Glycine, serine and threonine metabolism” and “Degradation of aromatic compounds” (p < 0.05) (Fig. 4F).

To delve the specific bacteria and differential metabolites for plastisphere microbial variation, the Mantel test between the composition of wheat silage microbiota and the differential metabolites in wheat straw silages were performed (Fig. 5). The Mantel analysis revealed that the genus Lactobacillus exhibited the most significant correlations with the identified metabolites, the genus Lactobacillus was positive correlations with succinic acid (r = 0.740, p < 0.01), purine (r = 0.700, p < 0.01), isocitric acid (r = 0.428, p < 0.01), tyrosine (r = 0.437, p < 0.01), hypoxanthine (r = 0.694, p < 0.01) and xanthine (r = 0.676, p < 0.01). The Kosakonia was another primary genus, also positive correlations with succinic acid (r = 0.471, p < 0.01), purine (r = 0.644, p < 0.01), glutaric acid (r = 0.454, p < 0.01), hypoxanthine (r = 0.321, p < 0.01) and xanthine (r = 0.357, p < 0.01). Furthermore, the mental analysis also reflects the relationships among the microbial members. The genus Lactobacillus was negative correlations with the Xanthomonas (r = -0.702), Kosakonia (r = -0.794), Allorhizobium (r = -0.791), Pseudomonas (r = -0.006), Paenibacillus (r = -0.183), Pantoea (r = -0.667), Sphingomonas (r = -0.078) and Flavobacterium (r = -0.266).

4. Discussion

Ensiling is considered as an effective method to extend the availability of feed for ruminants. Globally, the inoculants are commonly employed for improving the ensiling characteristics during the process, including LAB, acid and enzyme. The application of additives has been extensively proved on improve the ensiling performance (da Silva et al., 2018). Accordingly, certain metabolites produced by the microorganisms could also directly impact the fermentation quality of the silage (Guo et al., 2018; Li et al., 2021). Unfortunately, there is limited information available regarding the changes of wheat straw inoculated without or with L. plantarum or cellulase. In the current study, a combination of multiple physicochemical analyses, microbiome and metabolome analysis was employed to investigate the changes of fermentation quality, microbial community and metabolites of wheat straw treated without or with L. plantarum or cellulase. This is the first study that integrates microbiome and metabolome in response to anaerobic bioaugmentation of wheat straw ensiling without or with L. plantarum or cellulase. In the current study, the author found that the wheat straw could be well-preserved after the bacteria-enzyme synergy, which could provide a new method for recycling and utilizing the wheat straw.

The pH and contents of LA and AA in the inoculants treated wheat straw silages was markedly dropped and elevated (p < 0.05) after 60 days of ensiling, respectively. These results were proven by the poor fermentation characteristics observed in the CK treatment. The lowest pH, highest concentrations of LA and AA were detected in the CELp treatment, which is in accordance with the previous report that the silage treated with L. plantarum and cellulase showed the lowest pH and maximum LA concentration as compared to the control or L. plantarum- or cellulase- treated silage alone (Ebrahimi et al., 2014). The hydrolysis and release of fermentable sugar were enhanced by the activities of the cellulase enzymes and could provide sufficient fermentable subtract for L. plantarum and acceleration the accumulation of LA and AA might be the main reason (Nazar et al., 2022). The CP is considered as the main factor because of it is linked with animal performance (Okoye et al., 2023). In the current study, the highest CP contents were found in the CE treatment, followed by the CELp and Lp treatments, and the lowest CP contents were found in the CK treatments, and the lowest CP contents were found in the CK treatments, which could be explained by the mixed action of cellulase and L. plantarum on decomposing hemicellulose and utilizing WSC contents (Okoye et al., 2023; Du et al., 2023). There were significantly decreased ADF, NDF and ADL contents in all cellulase- or L. plantarum- treated wheat straw silages than these in the CK treatment, which could be leads to by the chemical and enzymatic hydrolysis. As previous report, the accumulated LA and AA can degrade the lignocellulose (Alengebawy et al., 2023). Furthermore, the cellulase or other enzymes (such as feruloyl esterase) that produced by the microbes also could decompose the fiber components (Alengebawy et al., 2023; Sun et al., 2023; Xiao et al., 2023).

The fermentation quality of silage is mainly determined by the compositions of the microbiota, and the bacterial compositions varies with the different treatments (Khota et al., 2016). The alpha diversity indices, including the Chao1 value and Shannon index, reflect the richness and diversity of microbiota in the FM and wheat straw silage. As shown in Table 1, the Shannon index dropped after 60 days of fermentation process, especially in the Lp and CELp treatments. This might be because of some aerobic microorganisms and gram-negative bacteria could not survived in the anaerobic and acidic environment (Du et al., 2023). The influences of different inoculants on the wheat straw silage bacterial compositions were also explored, the principal coordinate analysis (PCoA) was conducted to reflect the bacterial compositions of wheat straw silage. The PCoA plot unveiled distinguishable changes between the FM and silages, wherein the wheat straw silage samples were distinctly separated into two groups: without or with L. plantarum (Fig. 1B). This phenomenon could be contributed to the growth of diverse bacterial species under various pH conditions (Avila et al., 2020; Du et al., 2023). Compared to the FM, the relative abundance of Proteobacteria decreased after ensiling, especially in the Lp and CELp treatments, while Firmicutes became the predominant phylum. The shift from Proteobacteria to Firmicutes in the bacterial community in wheat straw silage was consistent with the previous reports on rice straw and corn straw silage (Sun et al., 2023; Okoye et al., 2023), which could be contributed to the Firmicutes could thrive at a low pH and anaerobic conditions might be the main reason (Xiao et al., 2023), resulting in the changes in wheat straw silage bacterial community after fermentation process. Further granularity emerged at the genus level distribution of bacterial community, yielding insights into community changes. As expected, a markedly higher relative abundance of Kosakonia and Lactobacillus were found in the CK and CE treatments, and Lp and CELp treatments after ensiling, respectively. Kosakonia, a new genus of aerobic Gram-negative bacteria, belongs to Enterobacteriaceae. Nevertheless, there were few studies involved in the activity and mechanisms in silage fermentation of Kosakonia (Wang et al., 2023). The abundance of Lactobacillus markedly increased in the Lp and CELp treatments than that in the CK and CE treatments, which could lead to by the inoculated with the L. plantarum. Interestingly, the inoculated with the cellulase did not increase the abundance of Lactobacillus, however, it increased the growth of Xanthomonas after fermentation process. This result is in accordance with the previous report that the degradation of lignocellulose could be the main reason (Liu et al., 2024).

To delve deeper into the metabolic consequences of various additives, the LC-MS based metabolomic analysis was used to further understand the effects of various additives on the metabolites of wheat straw silage. Interestingly, similarly to the minor fluctuations of the bacterial compositions of wheat straw silage observed above, the various inoculations have large effects on the metabolites of wheat straw silage. A total of 2, 559 metabolites were identified in the wheat straw silage samples, which far exceeded the amounts of metabolites in rice straw silage, corn silage and paper mulberry leaf silage (Sun et al., 2023; He et al., 2021; Li et al., 2021). The differences of the compositions and numbers metabolites could be determined by the raw materials (Xia et al., 2023). The bacteria-enzyme synergy alternated the substrate and fermentation processes, which the numerous metabolites were produced in the current study.

The PCA and (O)PLS-DA scatter plots displayed significant differences in the metabolites of wheat straw silage among the treatments, and the metabolites were separated into two groups, the CK and CE treatments versus the Lp and CELp treatments (Fig. 2). These significantly different metabolites belonged to amino acids, peptides, and analogues, pyrimidines and pyrimidine derivatives, purines and purine derivatives and others. Certainly, the amino acids, peptides, and analogues, pyrimidines and pyrimidine derivatives, purines and purine derivatives not only directly affected by the additives, but are also critical parameters for wheat straw silage.

Compared to the CK and CE treatments, the concentrations of amino acids and carbohydrates significantly increased in the Lp and CELp treatments (Fig. 3), which focused on the amino acids, peptides, and analogues and fatty acids and conjugates. The ADF, NDF and ADL contents were broken-down by the enzyme and could provide additional substrate to improve the fermenting processes, especially the increasing on LA that produced by the Lactobacillus (Cubas-Cano et al., 2018). Furthermore, the higher amino acid and carbohydrate contents in wheat straw silage are more beneficial for the ruminants (Desta et al., 2016). Amino acids, peptides, and analogues related metabolites, valine, L-phenylalanine, proline, L-pyroglutamic acid, L-arginine, pipecolic acid, N6-Acetyl-L-lysine, asparagine, L-aspartic acid and tyrosine consisting of cyanoamino acid metabolism, arginine biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, alanine, aspartate and glutamate metabolism, glucosinolate biosynthesis and the other pathways (Fig. 3; Fig. 6). The prior report found that the amino acids, peptides, and analogues were related to the nutrient metabolism and the synthesis of other bioactive compounds (Li et al., 2018; Liu et al., 2019). In the present study, the higher amino acids, peptides, and analogues related metabolites were detected in the CK and CE treatments than that in the Lp and CELp treatments, which indicating the protein degradation process produced by the undesirable microorganisms during ensiling (Fig. 4). As described, the wheat straw was surrounded by a lower acidic environment in the Lp and CELp treatments compared to that in the CK and CE treatments. Many spoilage and harmful microorganisms belong to Proteobacteria were sensitive to the acidic environment, the enzymatic activity of these microorganisms was depressed and the growth of the bacteria was also inhibited (Wang et al., 2020; Xia et al., 2023). Therefore, the lower CP content was found in the CK and CE treatments. Organic acids and derivatives related metabolites, including succinic acid, isocitric acid, trans-aconitic acid, glutaric acid and pipecolic acid, are participating in citrate cycle, phenylalanine, tyrosine and tryptophan biosynthesis, glucosinolate biosynthesis, arginine and proline metabolism, and the other pathways (Fig. 3; Fig. 6). Majority of these metabolites were enriched in the Lp and CELp treatments compared to the CK and CE treatments (Fig. 4), which is similar with the results found in the rice straw silage and Italian ryegrass silage that the inoculated with the L. plantarum could increase the organic acids and derivatives related metabolites (Xia et al., 2023; Sun et al., 2023). These results can be explained by the LAB promoted the depolymerization of complex compounds breakdown into simple compounds (He et al., 2021). Therefore, the above metabolic investigation suggested that inoculated L. plantarum alone or with cellulase is an effective way to improve the multifunctional profiles of wheat straw silage.

The Mantel analysis was selected to characterize the intricate interplay between the specific bacteria and metabolites. These results revealed that the genus Lactobacillus exhibited the most significant relationships with the identified metabolites (Fig. 5), indicating the Lactobacillus dominate the fermentation processes by modulating the metabolite profiles. Organic acids and derivatives related metabolites, succinic acid, purine and glutaric acid demonstrated positive correlations with Lactobacillus, due to the Lactobacillus stimulate the generation of organic acid and improve the ensiling performance (Sun et al., 2023).

5. Conclusion

In conclusion, this study investigated the effect of L. plantarum- or with cellulase-mediated anaerobic fermentation on the fermentation quality, bacterial community and metabolites profiles of wheat straw silage. Thes effects of cellulase and L. plantarum had positives of the pH value and organic acids contents of the wheat straw silage. The LA and AA contents, which are directly responsible for the decreasing in pH, were significantly increased in in the Lp and CELp treatments. The PCoA plot showed a distinct partition of bacterial community in these treatments. The genus Lactobacillus dominated the entirety of the bacterial communities, especially in the L. plantarum- treated silage samples. The cellulase and LAB synergistic effects during the wheat straw anaerobic fermentation and most of the spoilage microbes in wheat straw could be suppressed. Augmenting the authors comprehension, the metabolomic analysis illuminates the mechanisms underlying the quality enhancement of wheat straw silage achieved through the bacteria-enzyme synergy. Overall, the wheat straw inoculated with L. plantarum alone or with cellulase can produce well-preserved silage, and the bacteria-enzyme synergy is a novel strategy to improve the fermentation quality of wheat straw and enhanced the feed resource for ruminants. However, the economic analysis and the available feed resource for ruminants of the wheat straw silage should be evaluated in the future.

Acknowledgements

This work was supported by the Research Foundation for Advanced Talents (RK2400001896), Scientific and Technological Projects (NC2023022).

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Table 1. Chemical and microbial characteristics of substrates before ensiling

DM, dry matter; FM, fresh matter; cfu, colony-forming units.

Table 2. Fermentation quality and chemical composition in wheat straw silage

DM, dry matter; CP, crude protein; ADF, acid detergent fiber; NDF, neutral detergent fiber; ADL, acid detergent lignin. Propionic acid and butyric acid were not detected in all wheat straw silages. Control, control treatment; CE, wheat straw inoculated with cellulase treatment; Lp, wheat straw inoculated with L. plantarum treatment; CELp, wheat straw inoculated with cellulase and L. plantarum treatment.

Table 3. Diversity indices of wheat straw inoculated without or with cellulose or L. plantarum

FM, fresh matter; ASV, amplicon sequence variants; No, number; Control, control treatment; CE, wheat straw inoculated with cellulase treatment; Lp, wheat straw inoculated with L. plantarum treatment; CELp, wheat straw inoculated with cellulase and L. plantarum treatment.

Fig. 1. Analysis of bacterial communities in wheat straw silage with various inoculations. (A) Venn diagram representing the common and unique ASVs found at different treatments. (B) Principal coordinate analysis of different treatments based on Bray-Curtis distance metrics. (C) The relative abundance of bacterial phyla of the raw materials and wheat straw silage. (D) Extended error bar plot showing the bacteria at the phylum level that had significant differences among the raw materials and wheat straw silage. (E) The relative abundance of bacterial genus of the raw materials and wheat straw silage. (F) Extended error bar plot showing the bacteria at the genus level that had significant differences among the wheat straw materials and wheat straw silage. Control, control treatment; CE, wheat straw inoculated with cellulase treatment; Lp, wheat straw inoculated with L. plantarum treatment; CELp, wheat straw inoculated with cellulase and L. plantarum treatment.

Fig. 2. Total principal component analysis of the wheat straw silage samples corresponding to different treatments following (A) positive and (B) negative mode ionization. Orthogonal partial least squares discriminant analysis [(O)PLS-DA] plot of wheat straw silage metabolites in comparisons of the four treatments following (C, E) positive and (D, F) negative mode ionization. Control, control treatment; CE, wheat straw inoculated with cellulase treatment; Lp, wheat straw inoculated with L. plantarum treatment; CELp, wheat straw inoculated with cellulase and L. plantarum treatment.

Fig. 3. Hierarchical clustering analysis for identification of different metabolites in wheat straw silage by comparison of the four treatments following (A) positive and (B) negative mode ionization. Bar plot with significantly differential metabolites among the wheat straw silage inoculated without or with additive. (C) Positive mode ionization for different metabolites by comparison of the four treatments. (D). Negative mode ionization for different metabolites by comparison of the four treatments. Each column in the figure represents a sample, each row represents a metabolite, and the color indicates the relative abundance of metabolites expressed in the group; Red indicates that the metabolite is expressed at high levels, and green indicates that the metabolite is expressed at high levels. Control, control treatment; CE, wheat straw inoculated with cellulase treatment; Lp, wheat straw inoculated with L. plantarum treatment; CELp, wheat straw inoculated with cellulase and L. plantarum treatment.

Fig. 4. KEGG pathway enrichment analysis of differential metabolites between wheat straw silage inoculated without or with additive. (A). Differential abundance scores of KEGG pathways between the CK and CE treatments. (B). Differential abundance scores of KEGG pathways between the CK and Lp treatments. (C). Differential abundance scores of KEGG pathways between the CK and CELp treatments. (D). Differential abundance scores of KEGG pathways between the CE and Lp treatments. (E). Differential abundance scores of KEGG pathways between the CE and CELp treatments. (F). Differential abundance scores of KEGG pathways between the Lp and CELp treatments. Pathway size corresponds to differential metabolite counts. CK, control treatment; CE, wheat straw inoculated with cellulase treatment; Lp, wheat straw inoculated with L. plantarum treatment; CELp, wheat straw inoculated with cellulase and L. plantarum treatment.

Fig. 5. Mantel analysis between the composition of wheat straw silage and differential metabolites. Red, positive correlation; blue, negative correlation. The square chart indicates the magnitude of the correlation coefficients, and the shade of color indicates the significance of the correlation coefficients.

Fig. 6 Metabolic pathways associated with differential metabolites variations in the ensiling process of wheat straw silage. Red indicates that the metabolite is expressed at high levels, and blue indicates that the metabolite is expressed at high levels. CK, control treatment; CE, wheat straw inoculated with cellulase treatment; Lp, wheat straw inoculated with L. plantarum treatment; CELp, wheat straw inoculated with cellulase and L. plantarum treatment.