Qiming Cheng1,2,3†
Dianpeng Liu4†
Yao Lei2†
Maoya Li2Yulian Chen2Jiachuhan Wang2Xiangjiang He2Yuanyuan Zhao2Chao Chen2*
Xiaoqing Zhang1,3*- 1Institute of Grassland Research of Chinese Academy of Agricultural Sciences, Hohhot, China
- 2College of Animal Science, Guizhou University, Guiyang, China
- 3Northern Agriculture and Livestock Husbandry Technology Innovation Center, CAAS, Hohhot, China
- 4Tongliao Institute of Agriculture and Animal Husbandry, Tongliao, China
Introduction: Carbon dioxide (CO2) generated during the ensiling process is a source of greenhouse gas emissions and a reason for the loss of nutrients during ensiling.
Methods: Broussonetia papyrifera (B), Tritriale (T) and their mixtures (B7T3, B5T5 and B3T7) were ensiled with sour soup (S) and Lactobacillus acidophilus (LAB) to investigate the effects of additives on silage quality, CO2 production and bacterial communities.
Results: After 45 days of fermentation, the B3T7 treatment resulted in the lowest CO2 production, a relatively high lactic acid content (pH < 4.2), and relatively high relative abundances of Lactiplantibacillus and Weissella after fermentation; the quality of silage in all the treatments with additives was greater than that in the CK treatment, and the CO2 content was significantly lower than that in the CK treatment (P < 0.05). In addition, the overall silage quality was better than that of CK after the addition of additives, the CO2 content was significantly lower (P < 0.05), and adding sour soup resulted in a greater effect than adding LAB. CO2 production was positively correlated with the relative abundances of Lactococcus, Enterococcus, and Neoscardovia and negatively correlated with the relative abundances of Lactiplantibacillus, Weissella, and Sphingomonas.
Conclusions: In summary, selecting an appropriate proportion of different forages for mixed silage and adding sour soup may be effective ways to improve silage quality and reduce CO2 production during ensiling.
1 Introduction
Intensification of the greenhouse effect in the atmosphere has led to global warming, precipitating a series of global climate problems that cannot be readily predicted by our current level of scientific understanding. The International Report on the Economics of Climate Change shows that if humans continue to maintain the lifestyles they have today, there is a 50 percent chance that the average global temperature will rise by 4 degrees Celsius by the year 2,100 (Masson-Delmotte et al., 2022). In 2016, many countries worldwide signed the Paris Agreement to combat climate change. China, being among the world's largest emitters of carbon dioxide (CO2), has established a goal to curtail CO2 emissions by 60–65% relative to the levels in 2005 by the year 2030 (Chen et al., 2019). CO2, as one of the principal greenhouse gases, has drawn increasing attention. Approximately 15% of greenhouse gas emissions caused by human activities stem from the livestock industry, according to Adegbeye et al. (2019). Ensiling is a way to preserve the nutritive value of forage over time to not only solve problems related to waste associated with seasonal forage but also provide a source of forage for livestock over the winter (Van Pamel et al., 2011). China produces more than 280 million tons of silage annually, and the global production volume is even greater (Liu et al., 2020). Cai et al. reported that 6 liters of gas can be produced per kg of sorghum silage fermented for 60 days (Cai et al., 1997). Mceniry et al. studied the gases produced during Teagasc ensiling, and they reported CO2 levels of up to 90% (McEniry et al., 2011). In recent years, the main concern in silage production has been the quality of the silage, and little attention has been given to CO2 emissions during silage production.
Broussonetia papyrifera (B) is a tall or shrub-like plant of the genus Broussonetia in the family Moraceae. It is widely planted, has a high crude protein content and high biological yield, is rich in nutrients, and contains a variety of bioactive ingredients, which increase the immunity and quality of livestock and poultry and are widely used in animal production (Peng et al., 2019). Although the crude protein content of B leaves is high, their amino acid composition is not balanced, the crude fiber content of the branches and the whole plant is high, the materials are not easy for ruminants to digest when directly fed, and palatability is low; thus, processing is needed to make B an unconventional feed resource with high utilization value (Sun et al., 2022). Common feed processing methods include green hay preparation and silage preparation; however, the biomass of B is high, and the harvest season is typically the rainy season, so green hay is not easy to prepare and preserve (Du et al., 2021). In addition, the high buffer energy value of B, low number of attached lactic acid bacteria, and low water-soluble carbohydrate (WSC) content make traditional ensiling of this material difficult (Cheng et al., 2021). According to previous research reports, the mixing of different forage grasses and the use of silage additives are two effective methods for improving the fermentation quality of silage, and the addition of appropriate silage additives can effectively reduce the production of CO2 during the silage process (Ni et al., 2018; Muck et al., 2018). Triticale (T) is a new annual grass and forage crop that was formed by the intergeneric hybridization of Triticum and Secale; it strong disease resistance, wide adaptability, high yield and high nutritional value. After alfalfa (Medicago sativa L.) and corn (Zea mays L.), it is the industrialized feed crop that is most worthy of promotion and utilization (Nascimento et al., 2015). T has a high WSC content (Li et al., 2023). When B and T are combined for silage, T can provide additional fermentation substrates, promoting silage fermentation, and we predict that a mixed silage consisting of B and T can effectively improve overall silage quality.
Sour soup (S) is traditional food that consists of fermented vegetables, mainly tomato, which is rich in a variety of nutritional and functional ingredients (Lin et al., 2020). At present, the production of S in China has reached 500,000 tons. Due to improper operation, pathogenic bacteria and sulfur-containing compounds are produced during the fermentation process of S, resulting in the waste of one fifth of the S and causing environmental pollution (Lin et al., 2022), whereas ruminants preferred the odor of sour soup due to their unique intestines which are more resistant than those of humans (Thacharodi et al., 2024). S contains many beneficial lactic acid bacteria, including Lactobacillus namurensis, Pediococcus pentosaceus, Lentilactobacillus buchneri, Lacticaseibacillus casei and Lacticaseibacillus rhamnosus (Xiong et al., 2021). Silage fermentation is driven by lactic acid bacteria under anaerobic conditions, and the dominant genus of S is Lactiplantibacillus. We hypothesized that S could be used as a new type of silage additive to improve silage quality and reduce the production of CO2 during the ensiling process. To date, the application of S in silage has not been reported.
The development of sour soup additives for silage applications can result in effectively resource utilization. At present, research on the addition of acid soup to silage is limited. Therefore, the nutritional quality, fermentation quality and microbial community structure of silage with different proportions of B and T were studied and analyzed in this study to reduce the production of CO2 and improve the quality of silage.
2 Materials and methods
2.1 Raw material preparation
The sour soup used in this study is Guizhou red sour soup, primarily composed of fresh cherry tomatoes and small red chili peppers. These ingredients are washed, stemmed, and chopped in specific proportions before being mixed with ginger, garlic, table salt, glutinous rice flour, and distilled liquor. The mixture is then placed in jars and fermented for ~10–20 days in cool, ventilated conditions. The main fermentation products include lactic acid, free amino acids, and functional compounds such as polyphenols and flavonoids (Li, 2022; Liu et al., 2023). The predominant microorganisms involved are lactic acid bacteria and yeast, with key bacterial genera comprising Lentilactobacillus, Lactiplantibacillus, and Levilactobacillus, while the primary fungal genera are Kazachstania and Pichia (Liu et al., 2025; Du et al., 2021).
B and T were taken from Jinbi Town, Bijie City, Guizhou Province (25° 73′n, 101° 32′e, Guizhou, China) on June 14, 2022. The study selected the leaf-expansion stage as the harvesting period for Broussonetia papyrifera, with the stems and leaves being the primary research focus for cutting. For rye, the harvesting was conducted at the heading stage, with the entire plant being cut. For Broussonetia papyrifera (B), dry matter (DM) content accounted for 41.28% of the fresh weight (FW). In terms of nutrient composition, the crude protein (CP) content was 15.56% DM, the neutral detergent fiber (NDF) content was 47.39% DM, the acid detergent fiber (ADF) content was 33.29% DM, and the WSC content was 4.31% DM. The DM content of Tritriale (T) constituted 34.21% of the FM. The CP content was 7.25% DM, the NDF content was 57.92% DM, the ADF content was 37.27% DM, and the WSC content was 6.48% DM. B10T0, B7T3, B5T5, B3T7 and B0T1 were obtained by mixing chopped Broussonetia papyrifera and Tritriale at ratios of 100:0, 70:30, 50:50, 30:70 and 0:100, respectively. Approximately 300 grams of the chopped feed was carefully mixed in a set proportion until a uniform mixture was achieved. Next, the homogeneously mixed feed was carefully hand-packed into polyethylene bags, each measuring 25 cm in width and 30 cm in length. Following this step, a vacuum packaging machine (model SJ-400, manufactured by Shanghai Precision Machinery Manufacturing Co., Ltd.) was employed to remove the air from the bags and seal them. The experimental treatments were established as follows. (1) Control treatment (CK): In this case, 6 milliliters per kilogram of FW of pure distilled water, devoid of any additives, was utilized. (2) Treatment: This treatment included the application of 6 milliliters per kilogram FW of sour soup. (3) LAB treatment: This treatment included the addition of 1 × 106 colony-forming units per gram FW of high-quality Lactobacillus acidophilus, sourced from the Zhongke Jiayi Bioengineering Co., Ltd., in Shandong, China. To guarantee the reliability and reproducibility of the experimental findings, three replicates were established for each of these treatments. The total gas (TG) and CO2 production were measured after 45 days of ensiling at room temperature (20–25°C), and the relevant indices were determined by opening the bags.
2.2 Gas production analysis
Gas production from silage was determined using the drainage method reported by Zheng et al. (2022). Before and after the ensiling process, the silage bags were carefully positioned inside a 5 L beaker. The volume of these bags was precisely measured in a constant-temperature water bath maintained at 25°C. The difference in the bag volume observed before and after ensiling served as an indicator of gas production. To determine the concentration of CO2, gas chromatography was employed (Shimadzu GC-20A).
2.3 Chemical composition analysis
After 45 days of ensiling, open the silage bags, take ~200 grams of sample from each bag, and dry it in a 105°C oven until a constant weight is achieved to determine the DM content. The dried sample was ground and passed through a 1 mm screen for the analyses of other indices. The CP content was determined via the Dumas combustion method (Gerhardt DUMATHERM; AOAC, 1990). The NDF and ADF contents were determined via an Ankom 200 fiber analyzer system (Ankom Technology Corp.; Zhao et al., 2017). The WSC content was determined via the Thrace-non-sulfuric acid method (Arthur Thomas, 1977).
2.4 Analysis of fermentation characteristics
After 45 days of silage fermentation, the bags were opened. A 10-gram sample from each bag was mixed with 90 ml of distilled water, stored at 4°C for 24 h and filtered through four layers of cheesecloth for fermentation index tests. The pH was measured with a portable pH meter. The concentrations of lactic acid (LA), acetic acid (AA), propionic acid (PA), and butyric acid (BA) were measured by HPLC (He et al., 2020), and the ammonia nitrogen (AN) concentration was determined via the phenol-hypochlorite reaction method (Li et al., 2019). This process allowed for a full analysis of silage quality after fermentation.
2.5 Microbial community analysis
The 3 samples from each silage treatment were evenly mixed together. The total microbiome DNA in the silage was extracted using a QIamp Fast DNA Stool Mini Kit. A preprimer (5′-ACTCCTACGGGAGGCAGCA-3′) and postprimer (5′-GGACTACHVHHHTWTCTAAAT-3′) were used to obtain the highly variable V3-V4 region of the bacterial 16SrRNA gene. The amplified products were recovered and purified, the sequencing library was prepared using Illunina's TruSeq NanoDNA Library Prep Kit, and the original sequences were analyzed according to a procedure proposed by Wang et al. (2018).
2.6 Data analysis
To analyze the impacts of the mixing ratio and additives on fermentation quality as well as the chemical composition of the silages, IBM SPSS 25.0 statistical software was used. The results were evaluated through two-way analysis of variance (ANOVA) and Duncan's multiple range test, with the threshold for statistical significance set to P < 0.05. Additionally, regression analysis was used to explore the correlation between gas production and the bacterial community. All graphics presented in this article were generated using GraphPad Prism 9 software.
3 Results and discussion
3.1 Effects of different additives on the chemical composition of silages
The chemical compositions of B and T after ensiling are shown in Table 1. There was a significant effect of the additives, mixing ratios and their interaction on the DM, CP, ADF and WSC contents of the silages (P < 0.001). With increasing proportion of B in the silage, the CP content significantly increased (P < 0.05), whereas the dry matter (DM), neutral detergent fiber (NDF), and acid detergent fiber (ADF) contents significantly decreased (P < 0.05). This may be related mainly to the properties of the material under study. This may have occurred because the T samples selected for this study were harvested at the late tasseling stage, and the fresh T samples presented relatively high NDF (57.92% DM) and ADF (37.27% DM) contents, whereas the fresh B samples presented relatively high CP (15.56% DM) contents. This is in line with the results of Ni et al. (2018), who reported that with increasing amounts of raw materials with high CP and fiber contents, the CP and fiber contents of mixed silage increases. The WSC content of all the treatments decreased with decreasing proportion of B (P < 0.05). This may have occurred due to the high content of antimicrobial substances such as polyphenols and flavonoids in B, which can inhibit microbial activity and reduce microbial utilization of WSCs (He et al., 2020). At the same ratio, the contents of DM, CP and WSCs were greater in the S treatment than in the LAB and CK treatments. This may have occurred due to the high content of lactic acid bacteria and organic acids in sour soup, which can facilitate the rapid formation of an acidic environment and inhibit the utilization of DM, CP and WSCs by undesirable microorganisms (Li et al., 2021). At the same ratio of silage materials, the NDF and ADF contents in the S treatment were lower than those in the LAB and CK treatments. Zhao et al. (2018) reported that Lactiplantibacillus plantarum can reduce the fiber content of silage. Additionally, Liao et al. (2022) reported that Saccharomyces cerevisiae and Lactobacillus brevis can reduce the fiber content of silage. Sour soup contains many microorganisms (lactic acid bacteria and yeasts), which may lead to a reduction in the NDF and ADF content of silage due to the ability of certain yeasts and lactic acid bacteria to degrade cellulose (Lin et al., 2020). The above results revealed that the mixing ratio of silage materials significantly affected the chemical composition of the silage and that the addition of sour soup effectively preserved nutrients and reduced the fiber content of the silage.
Table 1. Analysis of the chemical composition of mixed B and T silage with different additives.
3.2 Effects of different additives on the fermentation quality of silages
As shown in Table 2, the additives, mixing ratios and their interactions had significant effects on the silage pH and fermentation products (P < 0.05). The pH of the silage was greater (P < 0.05) when the proportion of B was greater. Due to the low WSC content and high buffer energy value of B, the pH of this material is difficult to reduce, even at a high LA content (Si et al., 2018). Numerous studies have shown that forage crops that are difficult to ferment during ensiling, such as legumes and woody crops, exhibit good results in mixed silage. Similar results were obtained by Li et al. (2022) for a mixed silage of broad bean and oats, who reported that the best overall silage quality was obtained from a mixture of 30% broad bean and 70% oats. Similar results were obtained by Zong et al. (2023) for a mixed silage of whole-plant corn and hairy vetch, who reported that the silage quality was better when the proportion of maize was 60–80%. LA is a direct product of DM and WSC fermentation by lactic acid bacteria, which influence the whole silage process and play an important role in determining silage quality and silage preservation time (Ni et al., 2017). The LA content of the B3T7 treatment was significantly greater (P < 0.05) than that of the other treatments under the same additives, indicating that silage fermentation is favored under a B to T ratio of 3:7. Keshri et al. (2019) reported that aerobic spoilage was inhibited under an AA content of < 3% DM. The AA content of all the treatments in this study ranged from 0.85 to 2.75% DM, which ensured aerobic stabilization of the silage. The formation of BA is usually undesirable during silage, as it implies nutrient loss and energy depletion (Muck, 2010). BA was not detected in any of the treatments in this study, which implies that the treatments were not subject to secondary fermentation. AN production usually implies CP degradation (Pahlow, 2003). The AN content decreased with decreasing percentage of B. This may have been influenced mainly by the material properties of B and T. Due to the higher CP content of B (15.56% DM) than T, the AN content increased following B degradation. In addition, this may be related to the higher buffer energy value of B, as a result of which B is less likely to successfully ferment during ensiling, nutrients are less likely to be preserved, and protein degradation is likely to occur; these are in line with the findings of Sun et al. (2022). Numerous studies have shown that additives are effective in improving silage quality (Dunière et al., 2013). The same results were observed in the present study, where the S-added treatments had lower pH values; lower AA, PA and AN contents; and higher LA contents than did the CK treatment. This may be related to the presence of organic acids and numerous microorganisms in the sour soup, whose presence led to the rapid formation of an acidic environment, lowering the pH of the silage and inhibiting the activity of undesirable microorganisms (Xiong et al., 2021). These findings suggest that the addition of sour soup can effectively inhibit CP degradation, reduce PA production and lower silage nutrient losses, which is consistent with the observation that the S treatment resulted in a higher CP content, as shown in Table 1. The above results indicated that among the treatments with different mixing ratios, the fermentation quality of the B3T7 treatment was better than that of the other treatments (pH < 4.2, higher LA content, and lower AA, PA and AN contents), and the addition of sour soup effectively improved the fermentation quality of the silage.
Table 2. Analysis of silage quality under mixed B and T silage with different additives.
3.3 Effects of different additives on the bacterial diversity of silages
The α diversity of the silage samples obtained from each treatment is shown in Table 3. The average coverage of all the samples was >0.998, indicating that the sequencing process was able to adequately represent the bacterial diversity of the silage samples (Yang et al., 2019). The Chao1, Shannon and Simpson indices of the treatments decreased with a decreasing proportion of B, and those of B3T7 were the lowest (P < 0.05). In addition, the additives had significant effects on the microbial diversity of the mixed B and T silage (P < 0.05). At the same mixing ratio, the Chao1, Shannon and Simpson indices were lowest in the S treatment (P < 0.05). These findings indicate that lactic acid bacteria were dominant and that the addition of sour soup reduced the relative abundance of other bacteria and promoted the reproduction of lactic acid bacteria.
Table 3. Analysis of α bacterial diversity in silage.
The relative abundance of microorganisms at the phylum level is shown in Figure 1A. The phyla Firmicutes and Proteobacteria were the main phyla after 45 days of mixed B and T ensiling, which is in agreement with the results of Wang et al. (2022). In this study, the relative abundance of Firmicutes was lower when the proportion of B was greater. Studies have reported that due to the high buffer energy values of legumes and some woody feeds with high protein contents, the pH of these materials is difficult to reduce, and studies have shown that acidic environments are more suitable for the growth of Firmicutes, so the relative abundance of Firmicutes is low when the proportion of B is high (He et al., 2021). The relative abundance of genus-level microorganisms is shown in Figure 1B. In this study, Lactiplantibacillus, Weissella and Enterococcus were the main genera in the silage samples. Enterococci are silage probiotics that act mainly in the early stages of ensiling, as they do not tolerate acidic environments and are usually replaced by lactic acid bacteria in the later stages of ensiling (Delpech et al., 2019). In this study, a greater abundance of Enterococcus was detected when the ratio of B was greater. This may have occurred because an acidic environment was not formed when the ratio of B was high, which allowed Enterococcus strains to survive. The presence of Enterobacter and Clostridia during silage fermentation is considered unfavorable, as these bacteria compete with lactic acid bacteria for fermentation substrates, slowing lactic acid production and increasing protein degradation (Dunière et al., 2013). In this study, the abundances of Enterobacter and Clostridia were below the detection level, and BA was not detected (Table 2), which indicated that butyric acid was not formed in the silage. Ensiling is a Lactobacillus-driven fermentation process, and Lactobacilli gradually take over as the ensiling process progresses (Muck, 2013). Lactiplantibacillus are lactic acid-producing microorganisms in the ensiling process, and there is a significant correlation between the abundance of these microorganisms and lactic acid content (Guan et al., 2018). Compared with those in the B silage alone, the relative abundances of Lactobacilli (Figure 1C) and Weissella (Figure 1D) gradually increased with increasing proportion of T in the silage, and the relative abundances of lactic acid bacteria and Weissella in the B3T7 mixture were greater than those in the other treatments, which indicated that the optimum fermentation environment was formed under this proportion. This finding is in line with the results of Wang et al. (2019), who reported that mixed alfalfa and maize silage was best ensiled at ratios of 4:6 and 6:4. Under the same mixing ratio, the addition of sour soup effectively improved the fermentation environment of the silage, and the relative abundance of lactic acid bacteria in the S treatment was greater than that in the CK and LAB treatments, which may be attributed to the various nutrients (organic acids, vitamins, minerals, etc.) and the large number of microorganisms (mainly lactobacilli) that were present in the silage fermentation environment (Lin et al., 2020; Choi et al., 2019). Moreover, this study revealed that the additives did not have a good effect on the ensiling of B and T alone, which may be related to the properties of the materials. B has few lactic acid bacteria attached to the material, a low WSC content, and abundant bioactive compounds, including flavonoids and terpenes with antioxidant and anti-inflammatory properties, which are not conducive to silage fermentation (Chen et al., 2023). Many studies have shown that T, which is rich in fermentation substrates, can ferment quickly without additives and that the effects of additives are not obvious. The above results revealed that the mixing ratio affected the bacterial community composition of the B and T silages, that the optimal B proportion was 30%, and that the addition of sour soup effectively promoted the growth of Lactiplantibacillus and Weissella.
Figure 1. Microbial communities in silages after 60 days of ensiling. Bacterial communities are reported at the phylum level (A), the genus level (B) and the relative abundances of Lactiplantibacillus (C) and Weissella (D). CK, Control treatment (devoid of any additives); S, included the application of 6 milliliters per kilogram FW of sour soup; LAB, included the addition of 1 × 106 colony-forming units per gram FW of high-quality Lactobacillus acidophilus; B10T0, obtained by mixing chopped Broussonetia papyrifera and Tritriale at ratios of 100:0; B7T3, obtained by mixing chopped Broussonetia papyrifera and Tritriale at ratios of 70:30; B5T5, obtained by mixing chopped Broussonetia papyrifera and Tritriale at ratios of 50:50; B3T7, obtained by mixing chopped Broussonetia papyrifera and Tritriale at ratios of 30:70; B0T10, obtained by mixing chopped Broussonetia papyrifera and Tritriale at ratios of 0:100.
3.4 Effects of different additives on CO2 production and TG volume of silages
Silage fermentation is a complex process that generally includes four stages: an aerobic respiration period, an anaerobic microbial competition period, a lactic acid accumulation period and a relatively stable period (Murdoch, 1961). In the aerobic fermentation stage, due to plant cell and aerobic microbial respiration, nutrients are consumed, which results in the production of many gases. The production of these gases not only results in the loss of nutrients but also may cause greenhouse gas emissions (Guo et al., 2021). The effects of different additives on CO2 and TG production in the mixed B and T silage are shown in Table 4. The additives and their blending proportions significantly influenced CO2 and TG yields in silage (P < 0.001). With changing B-to-T ratio, the CO2 and TG production exhibited the same trend. The carbon dioxide and TG production tended to decrease and then increased as the proportion of T increased, with the B3T7 treatment resulting in the lowest carbon dioxide content and TG production. This may have occurred because the mixing ratio for B3T7 resulted in a silage environment that facilitated microbial fermentation, and the microorganisms in the additives utilized the substrates to produce more LA, which led to a lower-pH silage environment, inhibited the growth of harmful bacteria, and reduced the nutrient loss, thus lowering gas production. At the same mixing ratio, the LAB treatment significantly (P < 0.05) reduced TG production and CO2 production compared with those under CK. This may have occurred because the addition of lactic acid bacteria facilities the rapid formation of an acidic environment, inhibiting the aerobic respiration phase of the ensiling and creating a new microbial community structure, which in turn reduces CO2 production. This finding is consistent with the findings of Cai et al. (1997), who also showed that the addition of lactic acid bacteria was effective in reducing CO2 production during ensiling. In this study, the S treatment reduced TG production and CO2 production more effectively than the LAB treatment (P < 0.05). There are two reasons why S treatment is more effective than LAB treatment: one is that S itself is more acidic, which is conducive to the rapid formation of an acidic environment, and the other reason is that sour soup contains a mixed flora of various beneficial lactic acid bacteria, whereas the LAB used in this study only consisted of a single strain, which was not as effective as the sour soup. As mentioned above, both the mixing ratio and additives significantly affected CO2 production and TG production, and the addition of sour soup to B3T7 resulted in the lowest CO2 production and TG production. The primary sources of CO2 are plant respiration and microbial metabolic activities, while the reduction of CO2 emissions mainly involves either decreasing CO2 production or consuming the already produced CO2. The results of this study indicate that adding sour soup accelerates the silage fermentation process, promoting the formation of an acidic anaerobic environment, thereby inhibiting plant respiration and microbial metabolic activities. Additionally, mixed silage with different raw materials creates a survival environment for CO2-fixing microorganisms, further reducing CO2 production. According to the results of this study, under the mixing ratio employed in B3T7, CO2 production was reduced by 130.56 ml per 100 g of S-treated silage compared with that in the CK treatment, and CO2 production was reduced by 53.97 ml per 100 g of S-treated silage compared with that in the LAB treatment. China's annual silage production volume is ~280 million tons, and the global silage production volume is even greater. Therefore, selecting an optimal proportion of mixed silage consisting of different crops and adding sour soup may be effective ways to reduce CO2 emissions during ensiling.
Table 4. Analysis of CO2 and TG production.
3.5 Analysis of correlations of bacterial relative abundance with fermentation products and gas production
The results of the correlation analysis of the relative abundance of bacteria with fermentation products and gas production are shown in Figure 2. Regression analysis revealed that Lactococcus and Enterococcus were significantly positively correlated with pH, AA, PA, AN, and CO2 and significantly negatively correlated with LA; Lactiplantibacillus and Weissella were negatively correlated with pH, AA, PA, AN, and CO2 and significantly positively correlated with LA. Considering bacterial diversity (Figure 1) and gas production (Table 4), the results revealed an inextricable relationship between silage quality, CO2 production and silage bacterial community composition. Silage is a fermentation process driven by microorganisms, and in the presilage period, the process is carried out mainly by microorganisms such as Lactococcus and Enterococcus. A relatively high pH (>5) provides a suitable environment for the survival of microorganisms such as Lactococcus and Enterococcus (Ni et al., 2018; Andersen et al., 2005), and the presence of many Lactococcus and Enterococcus bacteria results in incomplete fermentation or poor fermentation quality, which explains the high CO2 production when the mixing ratios in CK treatment and B are high. Lactiplantibacillus and Weissella are the main microorganisms that produce lactic acid, and high levels of lactic acid are produced by Lactiplantibacillus and Weissella when they are present at relatively high relative abundances, decreasing pH and creating an acidic environment, which inhibits the activity of many aerobic microorganisms. This may be an important reason for the negative correlation between Lactiplantibacillus and Weissella and CO2 production. This finding is consistent with the results of Chen et al. (2021), who also reported that the relative abundances of Lactococcus and Leuconostoc were positively correlated with CO2 yield. Regression analysis revealed that Neoscardovia was significantly positively correlated with pH, AA, PA, AN and CO2 and significantly negatively correlated with LA. Neoscardovia is a specialized anaerobic bacterium in Bifidobacteriaceae in the Actinobacteria family that cannot tolerate strongly acidic environments (pH > 5; García-Aljaro et al., 2012; Mekadim et al., 2019), which may be one reason it was positively correlated with pH and CO2 production and negatively correlated with LA. Aerobic bacteria are the main microorganisms that produce CO2, and inhibiting the growth and reproduction of aerobic bacteria can effectively reduce CO2 production (Pahlow, 2003). Komagatales are gram-negative, specialized aerobic bacteria of the Acetobacteriaceae family that grow optimally at pH values ranging from 5.0 to 6.5, and they may contribute to the production of CO2 (Bishop et al., 2022). In addition, CO2 production may be related to some functions of bacteria. Streptococcus produces H2O2, which inhibits the growth of certain fungi in the phylum Ascomycetes, and these bacteria may contribute to the inhibition of aerobic bacterial growth. Sphingomonas may be useful in carbon sequestration and in reducing carbon dioxide emissions, and Wang et al. (2020) reported that rice fields with relatively high relative abundances of Sphingomonas presented relatively low CO2 emissions. The above results show that many microorganisms are involved in the fermentation of silage, which affect the quality of silage fermentation and CO2 production, and the bacterial community structure of silage can be changed by choosing an appropriate ratio of mixed silage substrates consisting of different types of forage materials. Appropriate additives further reduce the production of CO2 during ensiling.
Figure 2. Pearson analysis between nutrient indices, fermentation indices and CO2 production and the bacterial community after ensiling. LA, lactic acid; AA, acetic acid; TG, total gas; PA, propionic acid; AN, ammonia nitrogen. This study utilized Pearson correlation coefficients for the heatmap analysis. Red squares indicate a positive correlation (values approaching 1) between taxa and production parameters, while blue squares represent a negative correlation (values approaching−1). Significance is denoted by *P < 0.05, **P < 0.01, and ***P < 0.001, with all P-values adjusted using the FDR method.
4 Conclusions
In summary, silage nutrients are better balanced and silage fermentation is promoted when a mixed silage consisting of different forage crops is used, additives can further promote silage fermentation and improve silage quality, and the combined use of the two methods may be an effective measure to solve problems related to the low nutritive value of forage and poor silage fermentation. Mixing B and T can improve silage quality and reduce CO2 production during ensiling, and the best effect is achieved when the mixing ratio of B and T is 3:7. Additives can further improve silage quality when mixed B and T silage is used, and the addition of sour soup contributes the most to an improvement in fermentation quality and nutrient preservation, as evidenced by a relatively high LA content, low pH, low AN content and low CO2 production under sour soup treatment. Sour soup provides a sufficiently acidic environment for silage microorganisms and increases the relative abundance of Lactiplantibacillus and Weissella. A considerable amount of silage is produced in China annually, and the selection of suitable proportions of different forages for mixed silage and the addition of sour soup may be effective ways to improve silage quality and reduce greenhouse gas emissions during the ensiling process.
Data availability statement
The original contributions presented in the study are publicly available. This data can be found here: https://pan.baidu.com/s/1P7VkNLGhWuaGykGhmAWfZw with extraction code 1234.
Author contributions
QC: Writing – original draft, Writing – review & editing. DL: Formal analysis, Methodology, Writing – review & editing. YL: Data curation, Formal analysis, Writing – original draft. ML: Data curation, Formal analysis, Methodology, Writing – review & editing. YC: Data curation, Methodology, Writing – review & editing. JW: Data curation, Writing – review & editing. XH: Resources, Writing – review & editing. YZ: Data curation, Writing – review & editing. CC: Project administration, Writing – review & editing. XZ: Project administration, Writing – review & editing.
Funding
The author(s) declare that financial support was received for the research and/or publication of this article. This project was supported by the supported by the National Key Research and Development Subject (2021YFD1300302), Youth Science Fund Projects of National Natural Science Foundation (32401494), Guizhou Provincial Science and Technology Project (Qiankehe Chengguo [2022]005), Guizhou Provincial Science and Technology Project (Qiankehe Jichu-ZK [2023]120), Guizhou Provincial Science and Technology Projects (QKHPTRC-GCC[2023]012), Guizhou Provincial Science and Technology Project (QKHPTRC-CXTD [2022]011), “Development of Inner Mongolia Through Science and Technology” Action (Grant No. NMKJXM202401-05), and Hohhot Science and Technology Innovation Project (grant No. 2023RC-Industry-Research-Institite-7).
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declare that no Gen AI was used in the creation of this manuscript.
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References
Adegbeye, M. J., Elghandour, M. M., Monroy, J. C., Abegunde, T. O., Salem, A. Z., Barbabosa-Pliego, A., et al. (2019). Potential influence of Yucca extract as feed additive on greenhouse gases emission for a cleaner livestock and aquaculture farming-a review. J. Clean Prod. 239:118074. doi: 10.1016/j.jclepro.2019.118074
Andersen, A. Z., Lauritsen, F. R., and Olsen, L. F. (2005). On-line monitoring of CO2 production in Lactococcus lactis during physiological pH decrease using membrane inlet mass spectrometry with dynamic pH calibration. Biotechnol. Bioeng. 92, 740–747. doi: 10.1002/bit.20641
Arthur Thomas, T. (1977). An automated procedure for the determination of soluble carbohydrates in herbage. J. Sci. Food Agric. 28, 639–642. doi: 10.1002/jsfa.2740280711
Bishop, P., Pitts, E. R., Budner, D., and Thompson-Witrick, K. A. (2022). Kombucha: Biochemical and microbiological impacts on the chemical and flavor profile. Food Chem. Adv. 1:100025. doi: 10.1016/j.focha.2022.100025
Cai, Y., Ohmomo, S., Ogawa, M., and Kumai, S. (1997). Effect of NaCl-tolerant lactic acid bacteria and NaCl on the fermentation characteristics and aerobic stability of silage. J. Appl. Microbiol. 83, 307–313. doi: 10.1046/j.1365-2672.1997.00229.x
Chen, D., Zheng, M., Guo, X., Chen, X., and Zhang, Q. (2021). Altering bacterial community: a possible way of lactic acid bacteria inoculants reducing CO2 production and nutrient loss during fermentation. Bioresour. Technol. 329:124915. doi: 10.1016/j.biortech.2021.124915
Chen, J., Shen, L., Shi, Q., Hong, J., and Ochoa, J. J. (2019). The effect of production structure on the total CO2 emissions intensity in the Chinese construction industry. J. Clean Prod. 213, 1087–1095. doi: 10.1016/j.jclepro.2018.12.203
Chen, S., Xi, M., Gao, F., Li, M., Dong, T., Geng, Z., et al. (2023). Evaluation of mulberry leaves' hypoglycemic properties and hypoglycemic mechanisms. Front. Pharmacol. 14:1045309. doi: 10.3389/fphar.2023.1045309
Cheng, Q., Chen, Y., Bai, S., Chen, L., You, M., Zhang, K., et al. (2021). Study on the bacterial community structure and fermentation characteristics of fresh and ensiled paper mulberry. Anim. Sci. J. 92:e13656. doi: 10.1111/asj.13656
Choi, Y., Yong, S., Lee, M. J., Park, S. J., Yun, Y., Park, S., et al. (2019). Changes in volatile and non-volatile compounds of model kimchi through fermentation by lactic acid bacteria. LWT-Food Sci. Technol. 105, 118–126. doi: 10.1016/j.lwt.2019.02.001
Delpech, G., Hebert, E. M., Sparo, M., and Saavedra, L. (2019). Draft genome sequence of Enterococcus faecalis strain CECT7121, a corn silage isolate with antibacterial activity against Gram-positive pathogens. Microbiol. Resour. Ann. 8, 10–1128. doi: 10.1128/MRA.00245-19
Du, Z., Sun, L., Chen, C., Lin, J., Yang, F., and Cai, Y. (2021). Exploring microbial community structure and metabolic gene clusters during silage fermentation of paper mulberry, a high-protein woody plant. Anim. Feed Sci. Technol. 275:114766. doi: 10.1016/j.anifeedsci.2020.114766
Dunière, L., Sindou, J., Chaucheyras-Durand, F., Chevallier, I., and Thévenot-Sergentet, D. (2013). Silage processing and strategies to prevent persistence of undesirable microorganisms. Anim. Feed Sci. Technol. 182, 1–15. doi: 10.1016/j.anifeedsci.2013.04.006
García-Aljaro, C., Ballesté, E., Rosselló-Móra, R., Cifuentes, A., Richter, M., and Blanch, A. R. (2012). Neoscardovia arbecensis gen. nov., sp. nov., isolated from porcine slurries. Syst. Appl. Microbiol. 35, 374–379. doi: 10.1016/j.syapm.2012.06.007
Guan, H., Yan, Y., Li, X., Li, X., Shuai, Y., Feng, G., et al. (2018). Microbial communities and natural fermentation of corn silages prepared with farm bunker-silo in Southwest China. Bioresour. Technol. 265, 282–290. doi: 10.1016/j.biortech.2018.06.018
Guo, X., Zheng, P., Zou, X., Chen, X., and Zhang, Q. (2021). Influence of pyroligneous acid on fermentation parameters, CO2 production and bacterial communities of rice straw and stylo silage. Front. Microbiol. 12:701434. doi: 10.3389/fmicb.2021.701434
He, L., Zhou, W., Xing, Y., Pian, R., Chen, X., and Zhang, Q. (2020). Improving the quality of rice straw silage with Moringa oleifera leaves and propionic acid: Fermentation, nutrition, aerobic stability and microbial communities. Bioresour. Technol. 299:122579. doi: 10.1016/j.biortech.2019.122579
He, Q., Zhou, W., Chen, X., and Zhang, Q. (2021). Chemical and bacterial composition of Broussonetia papyrifera leaves ensiled at two ensiling densities with or without Lactobacillus plantarum. J. Clean Prod. 329:129792. doi: 10.1016/j.jclepro.2021.129792
Keshri, J., Chen, Y., Pinto, R., Kroupitski, Y., Weinberg, Z. G., and Sela Saldinger, S. (2019). Bacterial dynamics of wheat silage. Front. Microbiol. 10:1532. doi: 10.3389/fmicb.2019.01532
Li, D., Duan, F., Tian, Q., Zhong, D., Wang, X., and Jia, L. (2021). Physiochemical, microbiological and flavor characteristics of traditional Chinese fermented food kaili red sour soup. LWT-Food Sci. Technol. 142:110933. doi: 10.1016/j.lwt.2021.110933
Li, H., Zeng, T., Du, Z., Dong, X., Xin, Y., Wu, Y., et al. (2022). Assessment on the fermentation quality and bacterial community of mixed silage of faba bean with forage wheat or oat. Front. Microbiol. 13:875819. doi: 10.3389/fmicb.2022.875819
Li, J. (2022). Study on the Correlation Between Dominant Bacterial Groups and Characteristic Flavors of Guizhou Red Sour Soup. Guiyang: Guizhou University.
Li, P., Zhang, Y., Gou, W., Cheng, Q., Bai, S., and Cai, Y. (2019). Silage fermentation and bacterial community of bur clover, annual ryegrass and their mixtures prepared with microbial inoculant and chemical additive. Anim. Feed Sci. Technol. 247, 285–293. doi: 10.1016/j.anifeedsci.2018.11.009
Li, S., Ke, W., Zhang, G., Zhang, Q., and Undersander, D. (2023). Effects of Bacillus coagulans and Lactobacillus plantarum on the fermentation quality, aerobic stability and microbial community of triticale silage. Chem. Biol. Technol. Agric. 10:79. doi: 10.1186/s40538-023-00446-z
Liao, C., Tang, X., Li, M., Lu, G., Huang, X., Li, L., et al. (2022). Effect of lactic acid bacteria, yeast, and their mixture on the chemical composition, fermentation quality, and bacterial community of cellulase-treated Pennisetum sinese silage. Front. Microbiol. 13:1047072. doi: 10.3389/fmicb.2022.1047072
Lin, L. J., Du, F. M., Zeng, J., Liang, Z. J., Zhang, X. Y., and Gao, X. Y. (2020). Deep insights into fungal diversity in traditional Chinese sour soup by Illumina MiSeq sequencing. Food Res. Int. 137:109439. doi: 10.1016/j.foodres.2020.109439
Lin, L. J., Zeng, J., Tian, Q. M., Ding, X. Q., Zhang, X. Y., and Gao, X. Y. (2022). Effect of the bacterial community on the volatile flavour profile of a Chinese fermented condiment–red sour soup–during fermentation. Food Res. Int. 155:111059. doi: 10.1016/j.foodres.2022.111059
Liu, N., Hu, Y., Qin, L., Bao, A., Qin, W., and Miao, S. (2023). Flavor and quality characteristics of Guizhou red sour soup prepared by different artificially fortified fermentation methods. LWT 186:115247. doi: 10.1016/j.lwt.2023.115247
Liu, N., Zhang, B., Wu, M., Hou, Y., Qin, L., Bao, A., et al. (2025). Effects of different fermentation methods on the microflora composition and flavor quality of mixed red sour soup. LWT-Food Sci. Technol. 222:117653. doi: 10.1016/j.lwt.2025.117653
Liu, Z., Liu, Z., Li, J., Xie, N., Qin, W., Feng, W., et al. (2020). Analysis on the development status of Chinese silage feed industry. J. Grassl. Forage Sci. 251, 70–75.
Masson-Delmotte, V., Zhai, P., Pörtner, H. O., et al. (2022). Global Warming of 1.5 C: IPCC Special Report on Impacts of Global Warming of 1.5 C Above Pre-industrial Levels in Context of Strengthening Response to Climate Change, Sustainable Development, and Efforts to Eradicate Poverty. London: Cambridge University Press.
McEniry, J., Forristal, P. D., and O'Kiely, P. (2011). Gas composition of baled grass silage as influenced by the amount, stretch, colour and type of plastic stretch-film used to wrap the bales, and by the frequency of bale handling. Grass Forage Sci. 66, 277–289. doi: 10.1111/j.1365-2494.2011.00788.x
Mekadim, C., Bunešová, V., Vlková, E., Hroncová, Z., and Killer, J. (2019). Genetic marker-based multi-locus sequence analysis for classification, genotying, and phylogenetics of the family Bifidobacteriaceae as an alternative approach to phylogenomics. Antonie Van Leeuwenhoek 112, 1785–1800. doi: 10.1007/s10482-019-01307-2
Muck, R. (2013). Recent advances in silage microbiology. Agr. Food Sci. 22, 3–15. doi: 10.23986/afsci.6718
Muck, R. E. (2010). Silage microbiology and its control through additives. Rev. Bras. Zootecn. 39, 183–191. doi: 10.1590/S1516-35982010001300021
Muck, R. E., Nadeau, E. M. G., McAllister, T. A., Contreras-Govea, F. E., Santos, M. C., Kung, L.et al. (2018). Silage review: recent advances and future uses of silage additives. J. Dairy Sci. 101, 3980–4000. doi: 10.3168/jds.2017-13839
Murdoch, J. C. (1961). A review of silage-making techniques. Grass Forage Sci. 16, 253–259. doi: 10.1111/j.1365-2494.1961.tb00248.x
Nascimento, A. D., Bassoi, M. C., Silva, M. S. E., et al. (2015). BRS harmonia-triticale cultivar. Crop. Breed. Appl. Biotechnol. 15, 40–42. doi: 10.1590/1984-70332015v15n1c6
Ni, K., Wang, F., Zhu, B., Yang, J., Zhou, G., Pan, Y., et al. (2017). Effects of lactic acid bacteria and molasses additives on the microbial community and fermentation quality of soybean silage. Bioresour. Technol. 238, 706–715. doi: 10.1016/j.biortech.2017.04.055
Ni, K., Zhao, J., Zhu, B., Su, R., Pan, Y., Ma, J., et al. (2018). Assessing the fermentation quality and microbial community of the mixed silage of forage soybean with crop corn or sorghum. Bioresour. Technol. 265, 563–567. doi: 10.1016/j.biortech.2018.05.097
Peng, X., Liu, H., Chen, P., Tang, F., Hu, Y., Wang, F., et al. (2019). A chromosome-scale genome assembly of paper mulberry (Broussonetia papyrifera) provides new insights into its forage and papermaking usage. Mol. Plant. 12, 661–677. doi: 10.1016/j.molp.2019.01.021
Si, B., Tao, H., Zhang, X., Guo, J., Cui, K., Tu, Y., et al. (2018). Effect of Broussonetia papyrifera L.(paper mulberry) silage on dry matter intake, milk composition, antioxidant capacity and milk fatty acid profile in dairy cows. Asian Australas. J. Anim. Sci. 31:1259. doi: 10.5713/ajas.17.0847
Sun, W. T., Huang, Y., Wu, C. R., Peng, C., Zheng, Y. L., Chen, C., et al. (2022). Addition of lactic acid bacteria can promote the quality and feeding value of Broussonetia papyrifera (paper mulberry) silage. Fermentation 8:25. doi: 10.3390/fermentation8010025
Thacharodi, A., Hassan, S., Ahmed, Z. H. T., Singh, P., Maqbool, M., Meenatchi, R., et al. (2024). The ruminant gut microbiome vs enteric methane emission: The essential microbes may help to mitigate the global methane crisis. Environ. Res. 261:119661. doi: 10.1016/j.envres.2024.119661
Van Pamel, E., Verbeken, A., Vlaemynck, G., De Boever, J., and Daeseleire, E. (2011). Ultrahigh-performance liquid chromatographic–tandem mass spectrometric multimycotoxin method for quantitating 26 mycotoxins in maize silage. J. Agric. Food Chem. 59, 9747–9755. doi: 10.1021/jf202614h
Wang, M., Lan, X., Xu, X., et al. (2020). Steel slag and biochar amendments decreased CO2 emissions by altering soil chemical properties and bacterial community structure over two-year in a subtropical paddy field. Sci. Total Environ. 740:140403. doi: 10.1016/j.scitotenv.2020.140403
Wang, M., Wang, L., and Yu, Z. (2019). Fermentation dynamics and bacterial diversity of mixed lucerne and sweet corn stalk silage ensiled at six ratios. Grass Forage Sci. 74, 264–273. doi: 10.1111/gfs.12431
Wang, S., Li, J., Zhao, J., Dong, Z., Dong, D., and Shao, T. (2022). Dynamics of the bacterial communities and predicted functional profiles in wilted alfalfa silage. J. Appl. Microbiol. 132, 2613–2624. doi: 10.1111/jam.15417
Wang, Y., Wang, C., Zhou, W., Yang, F. Y., Chen, X. Y., and Zhang, Q. (2018). Effects of wilting and Lactobacillus plantarum addition on the fermentation quality and microbial community of Moringa oleifera leaf silage. Front. Microbiol. 9:1817. doi: 10.3389/fmicb.2018.01817
Xiong, K., Han, F., Wang, Z., Du, M., Chen, Y., Tang, Y., et al. (2021). Screening of dominant strains in red sour soup from Miao nationality and the optimization of inoculating fermentation conditions. Food Sci. Nutr. 9, 261–271. doi: 10.1002/fsn3.1992
Yang, L., Yuan, X., Li, J., Dong, Z., and Shao, T. (2019). Dynamics of microbial community and fermentation quality during ensiling of sterile and nonsterile alfalfa with or without Lactobacillus plantarum inoculant. Bioresour. Technol. 275, 280–287. doi: 10.1016/j.biortech.2018.12.067
Zhao, J., Dong, Z., Li, J., Chen, L., Bai, Y., Jia, Y., et al. (2018). Ensiling as pretreatment of rice straw: The effect of hemicellulase and Lactobacillus plantarum on hemicellulose degradation and cellulose conversion. Bioresour. Technol. 266, 158–165. doi: 10.1016/j.biortech.2018.06.058
Zhao, X., Liu, J., Liu, J., Yang, F., Zhu, W., Yuan, X., et al. (2017). Effect of ensiling and silage additives on biogas production and microbial community dynamics during anaerobic digestion of switchgrass. Bioresour. Technol. 241, 349–359. doi: 10.1016/j.biortech.2017.03.183
Zheng, M., Zhou, W., Zou, X., Wu, S., Chen, X., and Zhang, Q. (2022). Fermentation quality and bacterial diversity of broussonetia papyrifera leaves ensiled with Lactobacillus plantarum and stored at different temperatures. Agronomy-Basel 12:986. doi: 10.3390/agronomy12050986
Keywords: sour soup, silage, carbon dioxide, bacterial communities, fermentation
Citation: Cheng Q, Liu D, Lei Y, Li M, Chen Y, Wang J, He X, Zhao Y, Chen C and Zhang X (2025) Altering the bacterial community: sour soup decreased CO2 production and improved the fermentation quality of Broussonetia papyrifera, Tritriale and mixed silages. Front. Microbiol. 16:1606628. doi: 10.3389/fmicb.2025.1606628
Received: 07 April 2025; Accepted: 03 June 2025;
Published: 25 June 2025.
Edited by:
Alicja Wegrzyn, University of Gdansk, PolandReviewed by:
Atte Johannes Von Wright, University of Eastern Finland, FinlandJie Zhang, Southwest University, China
Copyright © 2025 Cheng, Liu, Lei, Li, Chen, Wang, He, Zhao, Chen and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Chao Chen, Y2hlbmNAZ3p1LmVkdS5jbg==; Xiaoqing Zhang, emhhbmd4aWFvcWluZ0BjYWFzLmNu
†These authors have contributed equally to this work