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Chemical and Biological Technologies in Agriculture
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Effects of pyroligneous acid as silage additive on fermentation quality and bacterial community structure of waste sugarcane tops

Chemical and Biological Technologies in Agriculture volume 9, Article number: 67 (2022) Cite this article

Abstract

This article intends to improve the recycling of waste sugarcane (Saccharum officinarum) tops and the value-added utilization of pyroligneous acid. Fresh sugarcane tops can be used by ruminants, but they are prone to dehydration and mildew during storage, reducing their feeding value. Pyroligneous acid, a by-product in the process of making biochar, has good antibacterial effects. Adding pyroligneous acid to sugarcane tops for silage fermentation may be an effective way to promote the recycling of sugarcane tops. Thus, the fermentation quality and bacterial community of sugarcane tops ensiled with or without 1–2% pyroligneous acid for 5, 10, 20, or 100 days were investigated. Results showed that pyroligneous acid increased the acetic acid content and reduced ammonia-N concentration, and numbers of coliform bacteria and molds in sugarcane tops silages. On the other hand, the addition of pyroligneous acid decreased the diversity of bacteria in sugarcane-top silage. Pyroligneous acid decreased Firmicutes and Leuconostoc relative abundances while increasing Lactobacillus relative abundances. Fermentation was also limited by the addition of pyroligneous acid, which reduced metabolic activities during ensiling.

Graphical abstract

Introduction

Sugarcane (Saccharum officinarum) is a significant crop for sugar production and is largely planted in tropical and subtropical regions, such as China, Thailand and Brazil [43]. Sugarcane tops, a major by-product accounting for 15–25% of the aerial part of the plant, are inexpensive and abundant. It is estimated that 79.4 and 36.9 million tons of sugarcane tops are produced every year in India and China, respectively [20, 21]. Sugarcane tops are usually burned or wasted in the field after the sugarcane harvest. This is not only a waste of resources, but also produces large amounts of greenhouse gases, resulting in environmental pollution. Using sugarcane tops as an alternative forage for ruminants might be an effective approach for reducing the environmental burden and alleviating shortages of animal feed. Although fresh sugarcane tops can be fed directly to ruminants, it is easy to dehydrate and mildew during storage. Ensiling is a traditional conservation method for fresh forages, which can also be used for the storage of fresh sugarcane tops. However, it is reported that the natural fermentation of sugarcane silage always produces useless fermentation, resulting in dry matter loss, due to a large number of epiphytic yeasts and spoilage microorganisms [4, 8]. Therefore, it is necessary to identify additives with bacteriostatic effect to improve the fermentation quality of sugarcane tops silage.

In recent years, biochar produced from thermal degradation of agricultural and forestry residues has been increasingly used in agricultural, environmental, and biorefinery activities [25]. Pyroligneous acid (PA), a coproduct during the process of making biochar, is a complex mixture containing a variety of chemical components, such as phenols, organic acids, furan and pyran derivatives [22, 24]. These compounds have promoted its application in many areas. For example, PA can be used as food or feed additive to prevent lipid peroxidation because it contains a variety of phenolic compounds with high in vitro antioxidant activity [34]. Oramahi et al. (2018) found that the termiticidal performance of PA was consistent with the concentration of total acid in PA. In addition, owing to the existence of phenolics and acids, PA also has strong antimicrobial capacity against Escherichia, Staphylococcus, and Pseudomonas (De Souza Araujo et al. 2018; [33], which are all abundant in silages [16, 28]. And De Souza Araujo et al. (2018) also found that PA also has a significant inhibitory effect on yeasts which are abundant in sugarcane tops. Given the prominent antibacterial properties of PA, we therefore hypothesized that it could improve the fermentation quality of sugarcane tops silage by way of altering the bacterial community during ensiling.

To the best of our knowledge, few studies have investigated the bacterial community and fermentation quality of sugarcane tops silage supplemented with PA. Therefore, the purpose of this study is to investigate the effect of PA on the fermentation quality of sugarcane tops, focusing on the effect of PA on the bacterial community of sugarcane top fermentation.

Materials and methods

Ensiling processes

Sugarcane tops were manually collected from an experimental plot at South China Agricultural University (Guangzhou, China) on 28 December 2019. Samples were immediately cut into pieces approximately 2 cm long without wilting. Then the chemical compositions and microbial populations of sugarcane tops were determined from three homogenized samples. PA was obtained from blended wood wastes and then filtered through a 0.45 μm cellulose acetate membrane. Approximately 500 g sugarcane tops were treated with 1% PA (5 ml PA and 5 ml distilled water), 2% PA (10 ml PA), or without PA (10 ml distilled water as control) on a fresh-matter basis. After mixing evenly, the sugarcane tops with or without PA were equally packed into three bags with a vacuum sealer, respectively. Finally, 18 silage bags were made for each treatment and stored at room temperature (around 28 °C). After 5, 10, 20, and 100 days of fermentation, respectively, three bags were randomly selected for each treatment to analyze fermentation parameters and bacterial communities. The remaining six bags for each treatment were opened at day 100 for aerobic stability investigation.

Chemical composition and microbial population analysis

The number of microorganisms in silage was detected according to the method of Wang et al. [38]. Briefly, 20 g materials were mixed with 180 ml of sterile saline water and diluted after 1 h of shaking at 120 rpm. Then the population of LAB, coliform bacteria, and fungi (yeasts and molds) were obtained by using the plate counting method. Another 20 g samples were mixed with 180 ml distilled water and incubated overnight at 4 °C, and the filtrate was used to measure pH value, ammonia-N and organic acids content. [5, 19]. Sufficient silage samples were stoved for dry matter determination and chemical composition analysis. Crude protein (CP) and true protein (TP) were analyzed using a Kjeldahl nitrogen analyzer (Kjeltec 2300 Auto Analyzer, FOSS Analytical AB, Hoganas, Sweden) according to the methods of the AOAC [2]. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were analyzed according to the method of Van Soest et al. [35]. The content of water-soluble carbohydrates (WSC) was detected using the anthrone method [27].

Microbial community analysis

For bacterial community analysis, total DNA in sugarcane tops silage was extracted with the E.Z.N.A. stool DNA Kit (Omega Biotek, Norcross, GA, US) following the manufacture’s protocols. The V3–V4 regions of 16S rDNA were amplified, sequenced and analyzed according to He et al. [16]. Sequence information for the bacterial community was deposited in the National Center of Biotechnology Information (NCBI) with accession number PRJNA735080.

Statistical analysis

All data were analyzed using the IBM SPSS 20.0. And results were evaluated using two-way analysis of variance (ANOVA) with Bonferroni multiple range tests. Statistical significance was determined at the P < 0.05 level. All figures were downloaded from the Omicsmart online platform and further embellished using the software Adobe Illustrator CS 6.0.

Results

Characteristics of fresh sugarcane tops prior to ensiling

The chemical compositions and microbial populations of fresh sugarcane tops prior to ensiling are shown in Table 1. The DM content of sugarcane tops was 337 g/kg FM. Nutrition parameters including CP, TP, non-protein nitrogen (NPN), NDF, and ADF were 71.6 g/kg DM, 899 g/kg total nitrogen (TN), 101 g/kg TN, 745 g/kg DM, and 396 g/kg DM, respectively. The WSC content of sugarcane tops was 55.2 g/kg DM. Moreover, the epiphytic LAB count of fresh sugarcane tops in this trial was 5.66 log10 CFU/g FM. The count for yeasts was 5.02 log10 CFU/g FM, while that for molds was below the level of detection (< 3.00 log10 CFU/g FM). Unexpectedly, the number of coliform bacteria was also at a relatively lower level (< 4.00 log10 CFU/g FM).

Table 1 Chemical composition and microbial population of fresh sugarcane top prior to ensiling (± SD, n = 3)
Full size table

Fermentation quality and microbial population of sugarcane tops silage

The fermentation quality and microbial populations of sugarcane tops dynamically ensiled with or without 1–2% PA are presented in Table 2. DM content was not influenced by PA addition or ensiling days. Ensiling days had a highly significant effect (P < 0.01) on pH decline, and PA addition significantly increased (P < 0.01) pH at the early stage of ensiling (day 5 and day 10). Furthermore, on day 100, all treatments of sugarcane-top silage showed relatively low pH values (< pH 4.20). Meanwhile, lactic acid (LA) and acetic acid (AA) were the dominant fermentation products detected, and their contents were significantly increased (P < 0.01) with prolonged ensiling. PA additive significantly reduced (P < 0.01) LA content, but it significantly improved (P < 0.01) AA content. Butyric acid (BA) was not detected in the current study. The number of LAB in the control showed a trend with decreased distances, while that in the PA-treated sugarcane-top silages showed a trend of first rise and then decline from day 5 to day 100. But, anyway, LAB were the predominant microorganisms throughout the ensiling process, and no significant differences were observed between all treatments after a 100-day ensiling. Pleasingly, the yeast numbers were significantly decreased (P < 0.01) with prolonged ensiling, and yeasts were not detected on day 100. Molds and coliform bacteria were only detected in the control at the early ensiling stage, and PA had obvious inhibitory effect on them. Moreover, the addition of PA significantly decreased the ammonia-N (P < 0.01) content of sugarcane-top silage; however, ammonia-N content was significantly improved with increased ensiling days (P < 0.01).

Table 2 Organic acid contents, pH and microbial population of ensiled sugarcane tops (n = 3)
Full size table

Bacterial diversity and abundance in sugarcane-top silage

The alpha diversity of bacterial communities in dynamically ensiled sugarcane tops is shown in Table 3. The addition of PA decreased the Shannon and Simpson indexes compared with control. However, the indexes of Sobs, Shannon, Simpson, Chao, and Ace were all significantly increased (P < 0.05) with prolonged ensiling. Good-coverage values for all treatments were above 0.99. Results of unweighted principal coordinate analysis (PCoA) are shown in Fig. 1; PCoA 1 and PCoA 2 for sugarcane-top silage were 43.92% and 28.70%, respectively. Moreover, the bacterial community of sugarcane tops ensiled alone showed a clear separation from those of the PA-treated samples.

Table 3 Alpha diversity of bacterial community of ensiled sugarcane tops (n = 3)
Full size table
Fig. 1
figure 1

Principal coordinate analysis of bacterial communities for sugarcane-top silage treated without or with 1–2% pyroligneous acid after 5, 10, 20, and 100 days of ensiling, respectively

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The relative abundances of different taxa in bacterial communities of sugarcane-top silages at the phylum and genus levels are presented in Fig. 2. Overall, Firmicutes, Proteobacteria, and Cyanobacteria were the three dominant phyla, while Lactobacillus, Leuconostoc, and Acinetobacter were the dominant genera among all sugarcane-top silages. With prolonged ensiling, the relative abundances of Proteobacteria and Cyanobacteria decreased, while the proportion of Firmicutes increased rapidly to become the dominant phylum. Furthermore, with more days of ensiling, the relative abundance of Lactobacillus increased while that of Leuconostoc and Acinetobacter was reduced. However, at each ensiling stage, the relative abundance of Lactobacillus was highest in the group treated with 1% PA. As shown in Fig. 3, the relative abundances of Leuconostoc, Lactococcus and Enterococcus in the control increased, while those of the PA-treated silages remained low during ensiling. In addition, the relative abundance of Pseudomonas increased in 1% PA-treated silage after 100 days of ensiling. Predicted functional profiles of silage bacteria based on 16S rRNA gene sequences in sugarcane-top silages are shown in Fig. 4. The results showed that PA could reduce microbial metabolism, especially at the initial stage of fermentation.

Fig. 2
figure 2

Relative abundance of different taxa in bacterial communities of sugarcane-top silage treated without or with 1–2% pyroligneous acid after 5, 10, 20, and 100 days of ensiling, respectively

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Fig. 3
figure 3

Heatmap of bacterial communities in sugarcane-top silage treated without or with 1–2% pyroligneous acid after 5, 10, 20, and 100 days of ensiling, respectively

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Fig. 4
figure 4

Predicted functional profiles of silage bacteria based on 16S rRNA gene sequences in sugarcane-top silage treated without or with 1–2% pyroligneous acid after 5, 10, 20, and 100 days of ensiling, respectively

Full size image

Characteristics of fresh sugarcane tops prior to ensiling

The characteristics of fresh material have a crucial effect on silage quality. In this study, the DM of sugarcane tops was 33.7%, approximating the ideal DM (30–35%) for satisfactory silage [14]. And excessive moisture in raw materials may increase the number of undesirable microorganisms, causing nutrition loss to effluent and mildew in the course of ensiling process [26]. The CP content of sugarcane tops is poor compared with other forages, such as stylo (CP content:120 g/kg DM) or mulberry leaf (CP content: 196 g/kg DM, [36]). However, sugarcane tops contain a high proportion of TP, which should be utilized more efficiently by livestock than NPN [15]. The fiber content of sugarcane tops is relatively high, which is not conducive to the digestion and absorption of ruminants. An appropriate processing strategy is therefore required for development of this agricultural resource. It is well known that appropriate WSC content is a prerequisite for harvesting high-quality silage. Insufficient WSC will limit the fermentation of LAB, cause the metabolism of harmful microorganisms and reduce the silage quality. In this study, the WSC content of sugarcane tops was slightly lower than 60–70 g/kg DM, the theoretical requirement for obtaining well-preserved silage [32]. But compared to other common forages, such as alfalfa and stylo, the WSC of sugarcane tops is relatively high. Moreover, Cai et al. [6] believed that high-quality silage can only be obtained when the epiphytic LAB count of fresh material reached 5 log10 CFU/g FM at least. The epiphytic LAB count of sugarcane tops, in this study, was higher than 5 log10 CFU/g FM. In summary, ensiling is a suitable approach for storing sugarcane tops.

Fermentation quality, microbial population, and temperature dynamics of sugarcane-top silage

Ensiling is a conservation method for fresh forages worldwide [3]. Epiphytic microbes (mostly LAB) produce organic acids (such as LA and AA) during ensiling, which cause the pH to decrease accordingly. Acidic and anaerobic conditions inhibit detrimental anaerobes and preserve the nutrients in forage [39]. We could easily observe an increase in concentrations of LA and AA, while the pH and counts of undesirable microorganisms such as coliform bacteria, yeasts, and molds decreased during the sugarcane-top ensiling process. Organic acids, especially AA, are the main components of PA [40]. This partially explains the reason for the increase in AA concentration in silages with added PA compared to the control. Plant cell respiration and activities of microorganisms always result in nutrient losses during ensiling, especially at the early stage. PA decreased these losses, possibly because the direct acidification inhibited respiration of plant cells and microorganism activity. LA is the product of fermentation by LAB and results in pH decline during the initial stage of ensiling [37]. Addition of PA decreased the number of LAB at the initial stage of the sugarcane-top fermentation process. This is consistent with the lower concentration of LA and higher pH in the PA-treated silages, especially the 2% PA treatment. The accumulation of NPN in silage is the result of protein degradation. However, it is undesirable for NPN to accumulate in silage because ruminants have a low usage efficiency of NPN as compared to protein [15]. Furthermore, animals that digest NPN emit plenty of waste gas, which pollutes the environment. The ammonia-N, a crucial indicator of protein breakdown during ensiling [29], is usually produced by the decomposition of proteins by undesirable microorganisms. In our study, the addition of PA could reduce the content of ammonia-N significantly. This may be due to the effective inhibition of coliform bacteria by PA. In addition, the number of molds in sugarcane-top silage was obviously reduced by PA. The result was generally consistent with the observation of Jung [18] and Suresh et al. [33], who noted a growth inhibition effect of PA on fungi such as Aspergillus. The reason why PA can inhibit coliform bacteria and molds may be that organic acids and phenols destroy cell membrane, inhibit protein synthesis and enzyme activity [1, 7, 11]. Aerobic deterioration is a major concern for sugarcane-top silage [40]. And AA helps to maintain the aerobic stability of silage [9]. From Table 2, we could observe that the addition of PA contributed to the accumulation of AA during sugarcane tops fermentation, which may be beneficial to the aerobic stability of sugarcane tops silage.

Bacterial diversity and abundance of sugarcane-top silage

The distinction of bacterial communities among treatments was highlighted by PCoA. Control groups were separated from PA treatment groups, as shown in Fig. 1. This suggests that PA influenced the bacterial community during ensiling. Moreover, PA decreased the α-diversity of bacteria in sugarcane-top silage, as evidenced by reduced Shannon and Simpson indexes, as shown in Fig. 2. At the phylum level, Proteobacteria and Firmicutes were the main phyla in sugarcane-top silages. These results corresponded with those of Liu et al. [23], who found that Firmicutes and Proteobacteria were the dominant phyla (over 99% of the total relative abundance) in barley silages. And Proteobacteria became less abundant and Firmicutes became more abundant with prolonged ensiling. Similar results were demonstrated by Wang et al. [37] in Moringa oleifera leaf silage; Proteobacteria and Firmicutes were the dominant phyla at early and late stages of ensiling, respectively. Members of the genus Lactobacillus were the main LAB in the sugarcane-top silage. This result was consistent with that of Ren et al. [31], who also found that Lactobacillus became a dominant genus in sugarcane-top silage after 90 days of fermentation. Lactobacillus is a rigorous homofermentative bacterium that can decompose one mole of glucose to produce two moles of LA. Thus, members of this genus can rapidly decrease silage pH in the early stage of ensiling, inhibiting undesirable bacteria such as Clostridium [12]. Silages treated with 1% PA showed higher abundance of Lactobacillus than control. However, lower Lactobacillus abundance was observed with 2% PA treatment. Addition of 2% PA might be too high for some species of Lactobacillus to tolerate. Strains of Leuconostoc, Enterococcus, and Lactococcus are widely used as inoculants during silage making as they produce LA that acidifies the environment, especially during the initial stage of fermentation [28, 37]. Addition of PA decreased the relative abundance of Leuconostoc, Enterococcus, and Lactococcus in sugarcane-top silage (Fig. 3), potentially due to the relatively low acid tolerance of these genera [30]. Acinetobacter is mainly associated with the aerobic stability of silage; it can utilize acetate as a substrate and survive in anaerobic environments [13]. Accordingly, the energy for Acinetobacter growth is supplied by carbohydrate degradation and thus causes DM loss of silages. Gluconobacter is an obligately aerobic bacterium that usually uses oxygen as a final electron acceptor for generating oxidation reactions [17]. However, it is reported infrequently in silages. Acinetobacter and Gluconobacter were abundant only at the early stage of ensiling of sugarcane tops, indicating that the bacterial community improved with prolonged enisling.

Predicted functional profiles of silage bacteria showed that PA could reduce microbial metabolism, especially at the initial stage of fermentation (Fig. 4). The direct acidification caused by PA addition might inhibit the activities of bacteria in silage. Furthermore, PA has strong antimicrobial and antiviral activities attributed to the presence of compounds such as phenolic derivatives and carbonyls (De Souza Araújo et al. 2018; [22, 33]. On the other hand, we know antibiotics are responsible for the spread of multi-antibiotic-resistant bacteria. It has been reported that PA could mitigate dissemination of antibiotic resistance genes [41]. Therefore, using PA as silage additive can not only increase its value, but also promote the healthy development of animal husbandry.

Conclusions

We demonstrated that ammonia-N content and numbers of coliform bacteria and molds can be decreased during the sugarcane-top ensiling process by PA addition. Both ensiling days and PA addition could influence bacterial community composition in sugarcane tops silage. The relative abundance of the dominant genus, Lactobacillus, increased with prolonged ensiling. Addition of PA decreased the bacterial diversity and the abundance of Firmicutes and Leuconostoc during the ensiling process of sugarcane-top. In conclusion, the application of PA could make an improvement in the fermentation quality of sugarcane-top silage.

Availability of data and materials

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

This work was financially supported by Guangdong Science Forestry Technology and Innovation Commission (grant nos. 2018KJCX001, 2019KJCX001) and National Key R&D Projects (Grant no. 2017YFD0502102-02).

Funding

Guangdong Science Forestry Technology and Innovation Commission (grant nos. 2018KJCX001, 2019KJCX001) and National Key R&D Projects (Grant No. 2017YFD0502102-02).

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Author notes

  1. Shuo Wu and Cheng Wang have contributed equally to this article

Authors and Affiliations

  1. College of Forestry and Landscape Architecture, Guangdong Province Research Center of Woody Forage Engineering Technology, Guangdong Research and Development Center of Modern Agriculture (Woody Forage) Industrial Technology, Guangdong Key Laboratory for Innovative Development and Utilization of Forest Plant Germplasm, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agricultural University, Guangzhou, China

    Shuo Wu, Cheng Wang, Dandan Chen, Wei Zhou, Xiaoyang Chen & Qing Zhang

  2. College of Animal Science and Technology, Heibei Agricultural University, Baoding, China

    Mingya Wang

Authors
  1. Shuo Wu
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  2. Cheng Wang
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  3. Dandan Chen
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  4. Wei Zhou
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  5. Xiaoyang Chen
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  6. Mingya Wang
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  7. Qing Zhang
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Contributions

SW: investigation, data curation, writing original draft. CW: investigation, data curation. DC: software. WZ: project administration, resources, validation. XC: supervision, validation. MW: resources, validation. QZ: methodology, project administration. All authors read and approved the final manuscript

Corresponding authors

Correspondence to Wei Zhou, Xiaoyang Chen, Mingya Wang or Qing Zhang.

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Wu, S., Wang, C., Chen, D. et al. Effects of pyroligneous acid as silage additive on fermentation quality and bacterial community structure of waste sugarcane tops. Chem. Biol. Technol. Agric. 9, 67 (2022). https://doi.org/10.1186/s40538-022-00335-x

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