Changes in the Taxonomic and Functional Structure of Microbial Communities During Vegetable Waste Silage Fermentation

Juntao Zhang Sichuan Agricultural University Xiying Huang Sichuan Agricultural University Menggen Ma Sichuan Agricultural University Quanju Xiang Sichuan Agricultural University Ke Zhao Sichuan Agricultural University Petri Penttinen Sichuan Agricultural University Yunfu Gu (  [email protected] ) Department of Microbiology, College of Resources, Sichuan Agricultural University, Chengdu 611130, China https://orcid.org/0000-0001-8238- 9360

Research article

Keywords: Vegetable waste, Silage, Bacterial communities, Functional prediction

Posted Date: October 19th, 2020

DOI: https://doi.org/10.21203/rs.3.rs-92855/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Page 1/13 Abstract

Background：Silage fermentation, a sustainable way to use vegetable waste resources, is a complex process driven by a variety of microorganisms. We used lettuce waste as the raw material for silage, analyzed changes in the physico-chemical characteristics and bacterial community composition of silage during a 60 day fermentation, identifed differentially abundant taxa, predicted the functional profles of bacterial communities, and determined the associated effects on the quality of silage.

Results: The biggest changes occurred in the early stage of silage fermentation. Changes in the physico-chemical characteristics included a decrease in pH and increases in ammonia nitrogen to total nitrogen ratio and lactic acid content. The numbers of lactic acid increased and those of molds, yeasts and aerobic bacteria decreased. The bacterial communities and their predicted functions on day 0 were clearly different from those on day 7 to day 60. The relative abundances of phylum Firmicutes and genus Lactobacillus increased. Nitrite ammonifcation and nitrate ammonifcation were more prevalent after day 0. The differences in the predicted functions were associated with differences in pH and amino acid, protein, carbohydrate, NH3-N, ether extract and crude ash contents.

Conclusion: Firmicutes and Lactobacillus were the dominant taxa during vegetable waste silage fermentation. The microbial communities and the predicted functions changed in different stages of silage fermentation, and the changes were accompanied with changes in the physico-chemical characteristics, especially with a decrease in pH and increases in ammonia nitrogen to total nitrogen ratio and lactic acid content.

Background

Vegetable waste refers to withered, residual leaves of vegetables after harvest, derived from the production, transportation, storage, distribution and consumption of vegetables [1]. With the rapid economic development and structural adjustment of agriculture in China, the vegetable industry has expanded quickly, and needs to dispose of large amounts of vegetable waste. In China, approximately 1.3 million tons of vegetable waste is produced per day [2]. Vegetable waste is mostly randomly discarded, causing a huge waste of resources and serious environmental problems. In the felds, vegetable waste with high moisture and organic matter content will easily rot, attract mosquitoes and fies, and cause the spread of plant diseases and pests [3, 4]. Dumping vegetable waste into water bodies pollutes rivers, lakes and groundwater systems, and incineration decreases air quality; all these affect the environment and human health [5].

Making silage, ensiling, is a sustainable way to use vegetable waste resources [6]. Silage fermentation relies on lactic acid bacteria (LAB) that ferment water soluble carbohydrates (WSC) and lignocellulolytic material anaerobically to produce lactic acid (LA); LA lowers pH that further inhibits the growth of molds, aerobic bacteria, yeasts and other unfavorable microorganisms, thus improving the quality and storage stability of silage [7, 8]. Silage has been mainly made from corn and alfalfa [9, 10]. Vegetable waste is also suitable raw material for silage due to its abundant nutrient content including vitamins, minerals and vegetable fber [6, 11]. However, silage fermentation using vegetable waste as raw material has received limited attention, and more knowledge is needed to develop vegetable waste fermentation practices.

Silage fermentation depends on the microbial communities. LAB has been found the dominant group in the fermentation of silage and to affect the quality of silage. [12] studied microbial communities during the fermentation of soybean by high-throughput sequencing, and showed that Lactobacillus was the dominant genus in the fermentation process. [13] found that Lactobacillus, Weissella and Pediococcus were the predominant genera in alfalfa silage. Ensiling was accompanied with a change in the taxonomic composition of the silage microbiomes [14]. Changes in the taxonomic composition do not necessarily result in changes in the functional structure of the microbiomes [15]. However, the functions of LAB and other microorganisms in silage have not received attention. Studying the functional variation, for example using Functional Annotation of Prokaryotic Taxa (FAPROTAX) that has been used to predict microbial functions in ecosystems like soil and wastewater [16–18], could elucidate how microbial communities affect the whole silage ecosystem.

Studies on the composition and variation of microbial communities in fermentation of vegetable waste and on the predicted functions are still limited. In this study, we used lettuce waste as the raw material for silage, analyzed changes in the physico-chemical characteristics and bacterial community composition of silage during a 60 day fermentation, identifed differentially abundant taxa, predicted the functional profles of bacterial communities, and determined the associated effects on the quality of silage.

Results Changes in silage characteristics and microbial population during ensiling

The pH of silage decreased from 5.93 to 3.98 from day 0 to day 45 (Table 1). Ammonia nitrogen (AN) to total nitrogen (TN) ratio and lactic acid (LA) and butyric acid (BA) contents increased from 1.87 to 5.03%, 1.24 to 2.46% and 0.04 to 0.27%, respectively, during the 60 day fermentation (p < 0.05). Acetic acid (AA), propionic acid (PA) and total amino acid (TAA) contents decreased from 0.25 to 0.15%, 0.52 to 0.22% and 8.95 to 6.41%, respectively (p < 0.05). Dry matter (DM), crude protein (CP), acid detergent fber (ADF), neutral detergent fber (NDF), and water soluble carbohydrate (WSC) contents decreased during ensiling, and ether extract (EE) and crude ash (CA) contents increased (p < 0.05). For most of the characteristics, the biggest changes were detected after 7 or 15 days of fermentation.

Page 2/13 Table 1 Physico-chemical characteristics of vegetable waste silage during the 60-day fermentation Time DM pH AN/TN LA AA PA BA TAA CP EE ADF NDF WSC CA (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%)

T0 28.9 ± 5.9 ± 1.9 ± 1.2 ± 0.23 0.5 ± 0.04 ± 8.9 ± 13.5 3.0 ± 18.7 ± 34.3 19.9 9.3 ± 0.03b 0.03d 0.04a 0.02a ± 0.01d 0.00b 0.21d ± 0.08a 0.09d ± ± 0.15a 0.01c 0.12f 0.05c 0.11a

T1 28.5 ± 4.5 ± 4.4 ± 1.6 ± 0.2 ± 0.4 ± 0.02 ± 7.7 ± 12.1 3.4 ± 18.5 ± 34.5 13.3 10.1 0.21ab 0.00c 0.08b 0.02b 0.01b 0.00c 0.00ab 0.06c ± 0.01b 0.20 cd ± ± ± 0.18e 0.07c 0.05b 0.14b

T2 28.1 ± 4.1 ± 4.8 ± 1.7 ± 0.2 ± 0.3 ± 0.02 ± 7.5 ± 11.6 3.5 ± 18.2 ± 34.1 12.3 11.4 0.16a 0.01b 0.34c 0.01c 0.00b 0.01b 0.00a 0.06c ± 0.08bc 0.07abc ± ± ± 0.15d 0.03b 0.05b 0.24c

T3 28.2 ± 4.0 ± 4.7 ± 2.1 ± 0.2 ± 0.2 ± 0.13 ± 7.3 ± 11.2 3.7 ± 18.4 ± 35.2 10.7 11.6 0.14a 0.01ab 0.04c 0.15d 0.01b 0.00a 0.01c 0.10bc ± 0.07 cd 0.05bc ± ± ± 0.05c 0.01d 0.13c 0.13c

T4 28.0 ± 4.0 ± 4.9 ± 2.5 ± 0.2 ± 0.2 ± 0.20 ± 7.1 ± 10.5 3.8 ± 18.1 ± 34.4 10.5 11.7 0.11a 0.01a 0.07c 0.02e 0.01b 0.00a 0.00d 0.08b ± 0.06d 0.24ab ± ± ± 0.09b 0.04c 0.06d 0.10c

T5 28.15 3.99 ± 5.03 ± 2.14 0.15 0.22 0.27 ± 6.41 ± 10.06 3.59 ± 17.87 ± 33.90 10.22 10.16 ± 0.01a 0.04d ± ± ± 0.01e 0.18a ± 0.08bc 0.11a ± ± ± 0.18a 0.00d 0.00a 0.01a 0.07a 0.01a 0.05e 0.04b

Data are average ± SEM (n = 3). Different letters in a column denote statistically signifcant differences (p < 0.05). Nutrient contents are percentages of the dry matter. DM: dry matter; AN: ammonia nitrogen; TN: total nitrogen; LA: lactic acid; AA: acetic acid; PA: propionic acid; BA: butyric acid; TAA: total amino acid; CP: crude protein; EE: ether extract; NDF: neutral detergent fber; ADF: acid detergent fber; WSC: water soluble carbohydrate; CA: crude ash. T0: day 0, T1: day 7, T2: day 15, T3: day 30, T4: day 45, T5: day 60.

The number of LAB increased after the frst 7 days (p < 0.05) and remained relatively stable later on (Table 2). The number of molds decreased from day 0 to day 7 (p < 0.05) and was below detection level afterwards. The number of yeasts and aerobic bacteria decreased from day 0 to day 7 (p < 0.05) and remained relatively stable later on.

Table 2 Culturable microbial population in vegetable waste silage during the 60-day fermentation Time LAB Mold Yeast Aerobic bacteria (lg CFU/g FM) (lg CFU/g FM) (lg CFU/g FM) (lg CFU/g FM)

T0 4.2 ± 0.49a 2.45 ± 0.33b 5.8 ± 0.38c 7.3 ± 0.16c

T1 8.2 ± 0.24bc 1.49 ± 0.49a 4.0 ± 0.32b 4.2 ± 2.13b

T2 8.4 ± 0.27bc ND 3.7 ± 0.22ab 3.6 ± 0.29ab

T3 8.9 ± 0.13c ND 3.2 ± 0.13ab 3.2 ± 0.27a

T4 8.2 ± 0.20bc ND 3.2 ± 0.66ab 3.0 ± 0.46a

T5 7.8 ± 0.11b ND 2.9 ± 0.16a 3.1 ± 0.23a

Data are expressed as the average ± SEM. Different letters in a column denote statistically signifcant differences (p < 0.05). LAB: lactic acid bacteria; FM: fresh material. T0: day 0, T1: day 7, T2: day 15, T3: day 30, T4: day 45, T5: day Bacterial diversity during ensiling

The 614151 reads from the 18 samples that passed the quality criteria, with an average of 34119 reads per sample, were assigned to 3311 OTUs at 97% similarity level. The rarefaction curves (Fig. S1) indicated that the OTUs represented well the sampled populations. The Shannon index decreased slightly from day 0 to day 45 (p < 0.05). The Chao1 index was lowest on day 0 (p < 0.05) (Table 3).

Based on relative abundances, (96.25%) was the dominant phyla on day 0 (Fig. 1A). The relative abundance of Firmicutes was highest on day 7 (81.86%), after which the relative abundance of Firmicutes decreased gradually and that of Proteobacteria increased. At the genus level, the relative abundances of Pseudomonas (59.27%) and Pantoea (24.93%) were highest on day 0 (Fig. 1B). From day 7 to day 60, Lactobacillus (58.38–73.70%) and Kluyvera (5.83–18.08%) dominated the populations. The relative abundance of Pediococcus was high on day 15 (9.28%).

In the NMDS based on the unweighted UniFrac distances, the bacterial communities on day 0 were clearly separated from those on day 7 to day 60 (Fig. 2). Besides, communities on day 7 and day 15 (the early stage of silage fermentation) and day 30 to day 60 (the middle and late stages of silage fermentation) grouped separately. Distance decay analysis based on Bray-Curtis similarity confrmed that the communities on day 0 were

Page 3/13 clearly different from those on later sampling days (R = -0.45, p < 0.001), and the within sampling day similarities were approximately on the same level (R = 0.33, p > 0.05) (Fig. 3). Differential abundance analysis

Altogether 1164 (35.16%) differentially abundant OTUs were detected between day 0 and the early stage of silage fermentation (day 7 and day 15), 1290 (38.96%) between day 0 and the middle and late stages of silage fermentation (day 30 to day 60), and 645 (19.48%) between the early stage of silage fermentation and the middle and late stages of silage fermentation (Fig. 4). At the phylum level (Fig. S2A), the relative abundances of Firmicutes were higher and those of Actinobacteria lower in the early, middle and late stages of silage fermentation than on day 0. Compared to the early stage of fermentation, the relative abundances of Bacteroidetes were higher on day 0 and in the middle and late stages, and those of Proteobacteria and Actinobacteria were higher in the middle and late stages. Thirteen genera were more abundant and 36 genera less abundant in the early stage of silage fermentation than at day 0 (Fig. S2B): the more abundant genera included Lactobacillus, Weissella, unidentifed Lactobacillaceae, Pediococcus and Kluyvera, and the less abundant genera included Pseudomonas, Samsonia, Franconibacter, Sphingobacterium and Curtobacterium. Fourteen genera were more abundant and 38 genera less abundant in the middle and late stages of silage fermentation than on day 0, including Kluyvera, Lactobacillus, Citrobacter, Pediococcus, Leuconostoc and Pseudomonas, and Samsonia, Curtobacterium, Franconibacter and Pantoea, respectively. Eight genera were more abundant and four less abundant in the middle and late stages than in the early stage, including Allorhizobium, Sphingobacterium, Brachybacterium and Ochrobactrum, and Pediococcus, unidentifed Lactobacillacea, Weissella and Timonella, respectively (Table S1 and S2). Functional prediction

In line with the NMDS (Fig. 2), the predicted functions on day 0 were separated from those on day 7 to day 60. The differences in the predicted functions were associated with differences in pH, organic acid contents, TAA and AN contents, and DM, WSC, ADF and CA contents (p < 0.001) (Fig. 5). Among the predicted functions, functional groups related to nitrite ammonifcation and nitrate ammonifcation were more prevalent after day 0 (Fig. 6A), and groups related to denitrifcation, invertebrate parasites, cellulolysis, aromatic hydrocarbon degradation, hydrocarbon degradation, aromatic compound degradation, plastic degradation, dark thiosulfate oxidation and dark oxidation of sulfur compounds were less prevalent after day 0. Chemoheterotrophy was prevalent during the whole fermentation period.

The contribution of taxa to the predicted functions was estimated based on relative abundances. Five taxa contributed 100% to a single function: Curtobacterium to aerobic chemoheterotrophy, and Erwinia, Leuconostoc, Pediococcus and Weissella to fermentation (Fig. 6B). The 100% contribution of Bromus tectorum to chloroplasts was probably an artefact created by the FAPROTAX function prediction program. Eight genera contributed to at least fve functions.

Discussion

Silage fermentation is a complex process where various bacteria affect the chemical composition and nutritional quality of the silage [19]. Elucidating the community composition and functional structure of the silage microbiome will increase our understanding of vegetable waste silage fermentation. Previous studies have mainly focused on the diversity, abundance and composition of the bacterial communities in alfalfa and corn silage [9, 13], and the bacterial communities and their functional structure in vegetable waste silage have not been addressed.

Fermentation products and physicochemical characteristics, such as pH and lactic acid (LA), acetic acid (AA) and ammonium nitrogen (AN) contents, indicate the nutritional quality of silage [20, 21]. In our study, the biggest changes in fermentation products and physicochemical characteristics occurred in the early stage of silage fermentation. Possibly the rapid increases in lactic acid bacteria (LAB) in the early stage of the fermentation allowed them to convert a large amount of water soluble carbohydrates (WSC) into organic acids, which led to a decrease in pH and made the silage a stable, anaerobic and acidic environment [8, 22]. At the same time, crude protein (CP) decomposion by microorganisms led to an increase in AN content. During the middle and late stages of silage fermentation, the physicochemical characteristics and the numbers and community composition of microorganisms remained relatively stable, indicating that the early stage of silage fermentation is the key stage in determining the fnal quality of silage.

Silage is a complex microbial system. A variety of microorganisms participates in the silage fermentation, including microorganisms attached to the raw materials, fermenting microorganisms and spoilage microorganisms [23]. [24] studied the fermentation process and concluded that in the frst stage carbon dioxide is released by respiration of the plant ensiled in the silage. In the second, relatively short stage, coli-type bacteria and fungi produce acetic acid. In the third stage after approximately three days of fermentation, LAB produce lactic acid and the lactic fermentation begins. In the fourth stage that lasts until approximately 17–21 days, lactic acid content reaches a peak and pH decreases to 4.2 or less. In the ffth stage water-soluble carbohydrates are exhausted and pH does not decrease, and the butyric acid bacteria produce butyric acid. Based on the changes in microbial communities, [25] divided the microbial communities in maize silage fermentation into an early-ensiling cluster, a midterm-ensiling cluster, a late-ensiling cluster and an aerobic exposure cluster. In our study, the communities were divided into an initial community at day 0 dominated by Proteobacteria, early stage communities and middle and late stage communities.

Page 4/13 Similar to [26], the fermentation stages were dominated by Firmicutes. Firmicutes and Proteobacteria have been found as the predominant phyla in grass [26] and alfalfa silages [27]. Firmicutes that include LAB, Bacillus and Clostridium are capable to degrade macromolecular compounds such as cellulose, protein and starch [28]. LAB have an important role of in silage fermentation [26, 29]. Clostridium include strains capable to convert sugar or lactic acid to butyric acid and to degrade amino acids to form ammonia or amines. These metabolic activities result in a decrease in DM, and the metabolites reduce the quality of silage [30, 31]. Bacillus compete with LAB for WSC at the beginning of fermentation, and produce lignocellulose degrading enzymes that break down plant material and release carbohydrates [32, 33]. Clostridium and Bacillus favor neutral pH, and their growth will gradually decrease with the decreasing pH during the silage fermentation. The proteobacterial Pseudomonas and Enterobacter have been frequently detected in the silage fermentation. Enterobacter strains are among the principal competitors of LAB for WSC in silage and produce acetic acid, succinic acid, 2,3-butanediol and endotoxins, which can decrease the nutritional quality of silage [34]. However, [35] concluded that Enterobacter might account for the rapid decrease in undesirable microorganisms during the ensiling process and increase the storage stability of silage. [13] found a negative correlation between Pseudomonas and AN concentration and yeast count in alfalfa silage, indicating that Pseudomonas might contribute to protein preservation and yeast inhibition. In wastewater treatment Pseudomonas species were more abundant toward the onset of nitrifcation where ammonia is oxidized to nitrite and then to nitrate [36], which may occur in silage fermentation as well.

Lactococcus, Leuconostoc, Pediococcus and Weissella are often detected in the early stage of silage fermentation [14, 37]. In agreement, in our study LAB, including Lactobacillus, Weissella and Pediococcus, were abundant in all except the initial community. Generally, Pediococcus are the dominant LAB during the early stage of ensiling and initiate lactic acid fermentation; Lactobacillus become dominant as the pH decreases [20, 38]. In our study, the relative abundance of Pediococcus was higher in the early stage of the silage fermentation, but Lactobacillus was the dominant genus during the whole process, possibly due to the Lactobacillus plantarum inoculant. The abundance of enterobacterial Kluyvera that have been rarely found in silage increased as the fermentation proceeded; Kluyvera may be found in silages made from crops that had been fertilized with manure [39]. Since Kluyvera have a biochemical profle similar to that of other [40], the increase in the abundance of Kluyvera may have resulted in the increase of BA content in the middle and late stages of silage.

The functional structures of microbiomes can be predicted with FAPROTAX that provides insights into the functions based on functional data on cultured bacteria [16, 41]. In our study, changes in the taxonomic composition were accompanied with changes in the functional structure of the microbiomes. Possibly the abundant taxa, including Lactobacillus, Leuconostoc, Pediococcus, Weissella, Buttiauxella, Citrobacter, Enterobacter and Klebsiella, that participated in the fermentation process decomposed organic matter and produced primary or secondary metabolites, e.g. LA and BA, contents of which increased during fermentation. Functional groups related to nitrite ammonifcation and nitrate ammonifcation increased after the silage fermentation started. The increase in ammonifcation was accompanied with increases in AN and a decrease in pH, which may improve the quality of silage. FAPROTAX has its limitations, thus the sugar metabolism in fermentation requires further study.

Conclusion

Firmicutes and especially Lactobacillus were the dominant taxa during vegetable waste silage fermentation. The microbial communities and the predicted functions changed in different stages of silage fermentation, and the changes were accompanied with changes in the physico-chemical characteristics, especially with a decrease in pH and increases in AN to TN ratio and LA content. Abbreviations

LA: lactic acid LAB: lactic acid bacteria

WSC: water soluble carbohydrates FAPROTAX: Functional Annotation of Prokaryotic Taxa

BA: butyric acid DM: Dry matter

AA: Acetic acid CP: crude protein

PA: propionic acid ADF: acid detergent fber

TAA: total amino acid NDF: neutral detergent fber

EE: ether extract WSC: water soluble carbohydrate

CA: crude ash AN: ammonium nitrogen

Methods

Ensiling material and silage preparation Lactuca sativa var. asparagina Bailey (lettuce) waste [42], dry rice straw, wheat bran and molasses were collected from local farms in Chengdu, Sichuan, China, and their nutrient compositions were analyzed (Table S3). To prepare silage, the lettuce waste and rice straw were chopped into 1– 2 cm pieces, and the molasses was diluted with water in a ratio of 1:2. A mixture of lettuce waste, 15% dry rice straw, 15% wheat bran and 20% molasses was made, an inoculant with 108 colony forming units (cfu) per ml of Lactobacillus plantarum C20, isolated from vegetable waste in our laboratory, was prepared, and the mixture was inoculated with 1% (w/w) inoculant. Silage was packed into plastic bags, the bags were sealed using Page 5/13 a vacuum sealer and kept at room temperature, approximately 26 °C to 28 °C. At 0, 7, 15, 30, 45 and 60 days after ensiling, three bags were opened for analyses. Chemical and nutritional composition analysis Ten g of silage was suspended in 90 mL of sterilized distilled water and let stand 24 h at 4 °C. The pH was measured using a glass electrode pH meter (FiveGo, Mettler Toledo, Greifensee, Switzerland). Ammonia nitrogen (AN) content was determined using a spectrophotometer (UV/VIS Spectrometer, PG Instruments Ltd., London, UK) [43]. The organic acid (lactic acid, LA; acetic acid, AA; propionic acid, PA; butyric acid, BA) and water soluble carbohydrate (WSC) contents were measured using HPLC with a 8.0 mm × 300 mm SC1011 column (Shoko, Tokyo, Japan) and a RI-1530 detector (Jasco Corp., Tokyo, Japan) at 80 °C oven temperature and water as the mobile phase at 1.0 mL min− 1 as described by [44]. The dry matter (DM), crude protein (CP), ether extract (EE) and crude ash (CA) were analyzed according to the standard procedures by the Association of Ofcial Analytical Chemists [45], while total nitrogen (TN) content was calculated by dividing CP by 6.25. The neutral detergent fber (NDF) and acid detergent fber (ADF) were analyzed as described by [46]. Total amino acid (TAA) content was analyzed using an automatic amino acid analyzer (L- 8800, Hitachi, Japan). Microbial population analysis The numbers of colony forming units (cfu) in fresh silage were determined by mixing 10 g of silage and 90 mL of sterilized water, followed by serial dilution from 10− 1 to 10− 9 in sterilized water. To quantify LAB, serial dilutions were inoculated on de Man, Rogosa, and Sharpe (MRS) agar and incubated at 37 °C for 48 h under anaerobic conditions. Yeasts and molds were quantifed on Potato Dextrose Agar incubated at 28 °C for 24 h. The yeasts were distinguished from molds and bacteria by colony appearance and cell morphology. Aerobic bacteria were quantifed on Nutrient Agar incubated at 30 °C for 24 h under aerobic conditions. DNA extraction, amplifcation and sequencing Genomic DNA was extracted from 10 g of silage mixed with 90 mL sterile 0.85% NaCl. The suspension was shaken at 120 rpm for 2 h, fltered with carcass, centrifuged at 10,000 rpm for 10 min at 4 °C, the pellet was suspended in 3 mL sterile 0.85% NaCl solution, centrifuged at 10,000 rpm for 10 min at 4 °C, and the supernatant was discarded. DNA was extracted from the pellet using a Fast DNA™ SPIN Kit (MP Biomedicals, Solon, OH, USA) and eluted in a fnal volume of 70 µL. The quantity and quality of DNA was assessed by agarose gel electrophoresis. DNA samples were stored at -20 °C.

The 16S rRNA gene V4 hypervariable region was amplifed with primers 515F (5′-GTGCC-AGCMGCCGCGGTAA-3′) and 806R (5′- GGACTACVSGGGTATCTAAT-3′) with adapter and barcode sequences [47]. Amplifcation was done in a 50 µL reaction mixture with 3 U of TaKaRa Ex

Taq HS (TaKARA Shuzo Co., Shiga, Japan), 5 mM dNTP mixture (TaKARA), 2.0 mM MgCl2, 5 µL of 10 × Ex Taq Buffer (TaKARA), 0.6 µM of each primer, and 4.0 ng of DNA. The PCR procedure included initial denaturation at 94 °C for 4 min, and 30 cycles of 15 s at 94 °C, 15 s at 55 °C and 30 s at 72 °C, and a fnal extension at 72 °C for 10 min. PCR products were purifed using PCR Clean-up Purifcation Kit (MP Biomedicals, Solon, OH, USA) and quantifed using Qubit 2.0 fuorometer (Invitrogen, Carlsbad, CA, USA). Purifed amplicons were pooled in equimolar concentrations and sequenced using MiSeq Reagent Kit V2 on an Illumina MiSeq platform [48]. The sequence data were submitted to NCBI Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra/) with accession number PRJNA637532. Bioinformatics analysis The sequencing reads were assembled using FLASH V1.2.7 [49]. Low quality reads were removed according to the QIIME quality control process (V1.7.0) [50]. Chimeric sequences were removed by using both UCHIME denovo and UCHIME reference [51]. Sequences were assigned into operational taxonomic units (OTUs) at 97% similarity cutoff using Uparse v7.0.1001 [51]. The representative sequences of the OTUs were aligned to the Greengene Database [52] based on the RDP classifer algorithm V2.2 to annotate taxonomic information. Statistical analysis

Alpha diversity indices (Chao1, Shannon, Simpson and ACE) were calculated with QIIME software (Version 1.7.0) using the Statistical Package for the Social Sciences (SPSS Version 19.0, SPSS Inc., Chicago, IL, USA). Differences in the nutrient composition, microbial population and alpha diversity were tested using two-way ANOVA. Differences were considered statistically signifcant at p < 0.05. Relative abundances of taxa at the phylum and genus levels were visualized using the multtest package in R v3.4 [53]. Distance decay analysis was applied to test the rate of decay of microbial community similarity (Bray-Curtis) along the silage fermentation process. Non-metric multidimensional scaling (NMDS) based on the unweighted UniFrac distance was done using the vegan package in R [53, 54]. Differentially abundant OTUs were defned using DESeq2 as described previously [48, 55] by comparing the relative abundances of OTUs in three stages: day 0, day 7 and day 15 (the early stage of silage fermentation) and day 30 to day 60 (the middle and late stages of silage fermentation). OTUs with twofold higher or lower relative abundance (p < 0.05) were considered enriched OTUs (eOTUs) and depleted OTUs (dOTUs), respectively. Functional prediction

The functional profles of the microbial communities were predicted using FAPROTAX [16]. Correlation between the nutrient compositions and predicted functions were analyzed using distance based redundancy analysis (dbRDA). The relative importance of each variable in the dbRDA model was estimated using envft function in R package vegan [53, 54]. Differential functional profles were detected using Kruskal-Wallis rank sum test (p < 0.05) and visualized in a heatmap. The contributions of taxa to differences in functional profles were estimated based on relative abundances using Python and R scripts.

Page 6/13 Declarations

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

The sequence data were submitted to NCBI Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra/) with accession number PRJNA637532.

Competing interests

Juntao Zhang declares that he has no competing interests. Xiying Huang declares that she has no competing interests. Quanju Xiang declares that she has no competing interests. Ke Zhao declares that she has no competing interests. Menggen Ma declares that he has no competing interests. Petri Penttinen declares that he has no competing interests. Yunfu Gu declares that he has no competing interests.

Funding

This study was funded by the grant from the Ministry of Education of China (grant no. Z2016116). Yunfu Gu was the person in charge of this project.

Authors' contributions

JT Zhang and YF Gu designed the experiments. JT Zhang and XY Huang performed the experiments. JT Zhang, P Penttinen and YF Gu wrote and revised the manuscript. MG Ma, QJ Xiang and K Zhao approved the fnal version of the manuscript. All authors read and approved the fnal manuscript.

Acknowledgements

Not applicable

Statement

We obtained the relevant fermentation raw materials from the farm owner through legal purchase.

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Figures

Page 9/13 Figure 1

Bacterial community composition at phylum (A) and genus (B) level in the vegetable waste silage during the 60-day fermentation. T0: day 0, T1: day 7, T2: day 15, T3: day 30, T4: day 45, T5: day 60.

Page 10/13 Figure 2

Non-metric multidimensional scaling (NMDS) plot based on the Bray-Curtis dissimilarity between bacterial communities in the vegetable waste silage during the 60-day fermentation. T0: day 0, T1: day 7, T2: day 15, T3: day 30, T4: day 45, T5: day 60.

Figure 3

Distance decay analysis of the bacterial communities in the vegetable waste silage during the 60-day fermentation. T0: day 0, T1: day 7, T2: day 15, T3: day 30, T4: day 45, T5: day 60.

Page 11/13 Figure 4

Volcano plots illustrating OTUs that were signifcantly enriched (green) and depleted (red) in the bacterial communities in the vegetable waste silage during the 60-day fermentation. T0: day 0, T1: day 7, T2: day 15, T3: day 30, T4: day 45, T5: day 60.

Figure 5

Redundancy analysis (RDA) of predicted functional profles and physico-chemical factors in the vegetable waste silage during the 60-day fermentation. * p < 0.05; ** p < 0.01; *** p < 0.001. DM: dry matter; NH4: ammonia nitrogen; TN: total nitrogen; LA: lactic acid; AA: acetic acid; PA:

Page 12/13 propionic acid; BA: butyric acid; TAA: total amino acid; CP: crude protein; NDF: neutral detergent fber; ADF: acid detergent fber; WSC: water soluble carbohydrate; CA: crude ash. T0: day 0, T1: day 7, T2: day 15, T3: day 30, T4: day 45, T5: day 60.

Figure 6

The differential functions among treatments (A), and the estimated bacterial contributions to these functions based on the relative abundance at the genus level (B) in the vegetable waste silage during the 60-day fermentation. T0: day 0, T1: day 7, T2: day 15, T3: day 30, T4: day 45, T5: day 60.

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