3.1 Optimization of anaerobic co digestion of PW and ES
Mixing ratio is the key parameter of anaerobic co-digestion, which affects the performance of co-digestion (Haider et al. 2015). The effect of co-digestion ratio of PW and ES on the methane yield was presented in in Fig. 1. It can be clearly found that the methane production in each reactor showed a sharp rise first, then a steady increase, and finally a basically stable trend. However, different proportions of PW and ES have a great impact on the cumulative methane production. In R1 or R5, PW or ES were digested alone, and the maximum cumulative methane production was 103.2 mL/g and 128.6 mL/g, respectively, which was similar to the previous reports (Wang et al. 2019). When PW and ES were co-digested, the cumulative methane production was significantly increased (Table S1), and when the ratio of PW to ES reduced from 2/1 to 1/1, the maximum cumulative methane production increased from 146.5 mL/g to 172.6 mL/g. However, when the ratio of PW to ES further reduced to 1/2, the cumulative maximum methane production decreased to 136.4 mL/g, but still higher than that of PW or ES digestion alone. The above experimental results clearly show that co-digestion of PW and ES improved methane production.
Figure 2 further showed the daily methane production during the co-digestion of PW and ES. As shown in Fig. 2, the daily methane production rate in each group showed two peaks, about on 2 d and 20 d, respectively. The first peak was due to the high content of digestive substrate and the low concentration of ammonia nitrogen, which led to the high activity of methanogens. The second peak appeared mainly because methanogenic microorganisms adapted to the digestion and operation conditions. The mixing ratio of PW and ES also had a great impact on the daily methane production. In R1, the daily methane production rate was low during the first 15 d, which was mainly due to the high content of refractory organic matter (lignin, cellulose, etc.) in PW (Croce et al. 2016). When the ratio of PW to ES reduced to 1/1, the daily methane production rate reached the maximum value of the experimental group, indicating that the daily methane production in R3 was the best. However, when the ratio of PW to ES further reduced, the daily methane production decreased slightly.
The mixing of PW and ES mainly affected the C/N ratio in the fermentation substrate, because PW was a kind of organic matter with high carbon content, while ES was a kind of organic waste with high proportion of nitrogen. Table 1 showed the variation of C/N ratio of each reactor. It can be seen from Table 1 that the mixed ratio of PW and ES reduced from 1/0 to 1/1, the C/N in the fermentation substrate reduced from 34.1/1 to 23.5/1, and the ratio of PW and ES further reduced to 0/1, and the C/N in the fermentation substrate also reduced to 7.2/1. C/N is one of the key factors affecting the activity of anaerobic digestion microorganisms, which has an important impact on the transformation of organic matter and the expression of key enzyme activities (Dai et al. 2016; Paul and Dutta 2018). Previous studies have shown that the optimal C/N for anaerobic digestion was in the range of 20–30/1(Zahan et al. 2018; Parkin and Owen 1986). When the ratio of PW and ES was 1/1, the C/N ratio of fermentation substrate was 23.5/1, which was in the range of optimal digestion ratio. Although the C/N ratio in R2 was also in the optimal range, the PW content in R2 was higher and the refractory components (lignin, hemicellulose, etc.) were more, which were difficult to be hydrolyzed. Therefore, the optimal mixture ratio of PW and ES to produce methane was 1/1.
Table 1
Changes of C/N ratio of digestive matrix with PW and ES mixture ratio
Reactor
|
R1
|
R2
|
R3
|
R4
|
R5
|
C/N
|
34.1 ± 2.3/1
|
28.6 ± 1.3/1
|
23.5 ± 1.6/1
|
16.5 ± 1.1/1
|
7.2 ± 0.8/1
|
3.2 CP enhanced the co-digestion of PW and ES
Figure 3 presented the effect of CP content on the co- digestion of PW and ES to produce methane. It can be clearly seen that the change trend of methane in each reactor showed a sharp increase at first and then a slow change trend. However, when CP was present in the co-digestion system, the methane production potential of the mixed matrix changed greatly. When the content of CP increased from 0 to 0.2 g/g, the maximum cumulative methane production increased from 172.6 mL/g to 234.8 mL/g. However, further increasing the content of CP to 0.3 g/g, the maximum cumulative methane production decreased slightly and decreased to 209.7 mL/g, but it was still about 1.2 times of the blank. CP has the characteristics of strong oxidation, and the presence of excess CP can inhibit the activity of methanogenic archaea, thereby reducing methane production. It can be clearly found that CP promoted the co-digestion of PW and ES to produce methane.
Methane is the end product of anaerobic digestion of organic matter. In the blank, the maximum cumulative methane production time could be considered as 21 d (Fig. 2). The maximum cumulative methane production time was slightly delayed with the ES digestion time alone, which may be related to the presence of refractory hemicellulose and ligneous acid in the digestion matrix in this work (Mustafa et al. 2016; Mamimin et al. 2020). When CP was present in the digestive system, the maximum cumulative methane production time was shortened to 19 d, 2 days earlier than the blank. The reduction of the optimal digestion time is of great significance in practical engineering, which can improve the treatment efficiency of organic matter (Qu et al. 2020). Therefore, the results obtained in this work clearly showed CP improved the co digestion efficiency of PW and ES, and shortened the optimal digestion time. The mechanism of CP enhancing co digestion of PW and ES would be described in detail in the following chapters.
It is considered that anaerobic digestion of organic matter must go through disintegration stage, and the change of SCOD in fermentation system can reflect the degree of disintegration (Liu et al. 2019; Kuang et al.2020a; Dong et al. 2020). As shown in Fig. 4, the concentration of SCOD in each group increased first and then decreased slowly. However, the content of CP greatly changed the concentration of SCOD in the fermentation broth. It can be clearly seen that the higher the content of CP, the concentration of SCOD in fermentation broth. For example, in the blank, the maximum content of SCOD was 1584 mg/L, and when the content of CP was 0.05 g/g, the maximum concentration of SCOD increased to 1826 mg/L. The concentration of CP further increased to 0.3 g/g, the maximum concentration of SCOD also increased to 2793 mg/L, which was about 1.7 times of the blank. When CP was dissolved in the fermentation system, calcium hydroxide and hydrogen peroxide were released slowly, which increased the pH value of fermentation (Wang et al. 2018). Previous studies have shown that alkaline pH was conducive to the dissolution of cellulose and hemifiber in the straw, which leaded to the increase of degradable organic matter content (Mancini et al. 2018; Bolado-Rodríguez et al. 2016). PW is rich in carbon sources, most of which are cellulose, lignocellulose and so on. The degradation of lignocellulose can be enhanced by CP (Wang et al. 2019), which improved the anaerobic digestion performance of PW. In addition, alkaline environment was conducive to the decomposition of extracellular polymer in ES, releasing extracellular organic matter and increasing the content of dissolved organic matter in fermentation broth (Chi et al. 2011; Kim et al. 2013; Kuang et al.2020b). The hydrogen peroxide released by CP can also promote the transformation of refractory organic matter from PW or ES, thus increasing the concentration of SCOD (Ping et al. 2018).
Protein and polysaccharide are the main organic compounds in ES. This work also explored the effect of CP on the dissolution of the two organic compounds. As shown in Fig. 5, the change trend of soluble protein and soluble polysaccharide was similar to that of SCOD. The higher the content of CP, the greater the release of soluble protein and polysaccharide. For example, when the content of CP was 0.3 g/g, the contents of soluble protein and polysaccharide on 8 d were 535 mg/L and 442 mg/L (calculated by COD concentration), respectively, which were 1.9 and 2.2 times of those in the blank. In this study, the concentration of polysaccharides was high, accounting for about 36% of the total SCOD, which was significantly higher than the results of ES anaerobic digestion alone reported before (Wang et al. 2019). The reason for this phenomenon may be due to the large amount of PW in the digestive matrix, and there was a certain amount of polysaccharide in the PW. The strong oxidation of CP increased the release of organic matter from fermentation substrate, which provided sufficient materials for subsequent acidification and methanation (Liu et al. 2019).
3.3 Effect of CP on yield of VFA during the co-digestion of PW and ES
VFA is a key intermediate in organic anaerobic fermentation process, which has an important impact on the utilization and activity of methanogens. As shown in Fig. 6, CP can affect VFA accumulation during the co- digestion of PW and ES, and the maximum accumulation of VFA was closely related to the CP content. When the content of CP increased from 0 to 0.2 g/g, the maximum accumulation of VFA increased from 361 mg/L to 751 mg/L (calculated by COD concentration), which indicated that increasing CP content promoted VFA accumulation. It should be noted that when the content of CP increased to 0.3 g/g, the accumulation of VFA was inhibited to a certain extent, and the maximum accumulation of VFA was 503 mg/L, lower than that of 0.2 g/g CP group, but still higher than that of blank. The conversion of dissolved organic matter to VFA requires the metabolism of microorganisms, which is a biochemical reaction. The change of pH in this study is shown in Fig.S1. The results showed that the higher the CP content, the greater the increase of pH. 0.3 g/g CP reduced the content of VFA, which may be due to the fact that the maximum pH was beyond the tolerance of acidifying microorganisms (Zhao et al. 2010; Lin et al. 2013). The high pH produced by high dose of CP had a negative effect on microorganisms, and the VFA content was not the highest in the presence of 0.3 g/g CP.
VFA is the main substrate in the process of methane production, and the components of VFA can also affect the accumulation of methane. As shown in the appendix, CP could affect the proportion of VFA components and promote the relative percentage of acetate. When the content of CP increased from 0 to 0.1 g/g, the proportion of acetate increased from 42.3–49.5%, indicating that proper increase of CP content was beneficial to the biogenesis of acetate. Further increasing the content of CP had little effect on the increase of acetate percentage. In the blank, propionate was also a major VFA component, accounting for about 26.5%, while the increase of CP content reduced the proportion of propionate, especially when the content of CP was 0.2 g/g, the proportion of propionate decreased to 24.3%. Previous studies have shown that methanogens were more likely to consume acetate and convert to methane, while more difficult to consume propionate (Zhao et al. 2019; Zhang et al. 2021). In this work, the increase of CP content increased the proportion of acetic acid and decreased the proportion of propionic acid, which also provided the basis for improving the activity of methanogens.
3.4 Effect of CP on VSS reduction during co-digestion of PW and ES
One of the main functions of anaerobic digestion is to reduce organic matter (Li et al. 2011; Zhao et al.2020b; Zhu et al.2020b). The effect of CP dosage on VSS reduction was also evaluated. As shown in Fig. 7, the presence of CP improved the reduction rate of VSS, and the higher the content of CP, the greater the reduction rate of VSS. In the blank, the reduction rate of VSS was about 26.5%, while when the content of CP was 0.05 g/g, the reduction rate of VSS was slightly increased to 26.9%, indicating that the reduction rate of low content CP was not obvious (p>0.05). When the content of CP was further increased to 0.2 g/g and 0.3 g/g, the reduction rate of VSS increased to 32.6% and 33.9%, which were 1.23 and 1.27 times of the blank, respectively, indicating that high concentration of CP significantly increased the content of organic matter in the PW and ES co-digestion system. Previous studies have shown that the removal rate of VSS was consistent with the gas production rate, that was, the higher the removal efficiency of VSS, the higher the gas production (Rafique et al. 2010; Li et al. 2019b). However, in this work, 0.3 g/g CP led to the highest removal rate of VSS, but the content of CP in the group with the highest gas production rate was 0.2 g/g. This inconsistency may be due to the strong oxidation effect of 0.3 g/g CP on anaerobes, thus inhibiting the activity of methanogens.
3.5 Effect of CP on the activities of key enzymes in the co-digestion of PW and ES
Anaerobic digestion is mainly carried out under the control of anaerobic microorganisms and key enzymes. The activities of key enzymes can reflect the activities of microorganisms (Zhao et al.2020c; Zheng et al.2021a; Zhu et al.2020c). Protease and amylase are the key enzymes to decompose protein and polysaccharide in ES, while filter paper enzyme and CMC enzyme are responsible for the degradation of total lignocellulose and cellulose in PW (Romero-Güiza et al. 2016; Gu et al. 2014). As shown in Fig. 8, the presence of CP has a great impact on the activity of hydrolysis and acidification enzymes in anaerobic digestion process. For example, when the content of CP was 0.2 g/g, the relative activities of protease and amylase were 115% and 109%, respectively, which were significantly higher than those of blank group. Similar experimental results were also found in filter paper enzyme and CMC enzyme. The presence of CP promoted the dissolution and release of refractory organic compounds in PW and ES, thus providing the favorable conditions for the hydrolysis of key enzymes. In addition, high dose of CP inhibited the above key enzymes, especially filter paper enzyme and CMC enzyme, which may be related to the strong oxidation of CP. Coenzyme F420 is unique to methanogens and is sensitive to the external environment (Zheng et al. 2020b). The presence of CP decreased the activity of F420, and the higher the content of CP, the greater the inhibition of F420. This phenomenon may be due to the increase of oxidative stress by CP, and the optimum pH range of methanogens was 7.0–8.0 (Lay et al. 1997). CP can strengthen PW and ES anaerobic digestion to produce methane mainly because CP promoted the release of organic matter and provides sufficient material guarantee for the digestion process. Although it has a little inhibition on F420 enzyme activity, it promotes methane production as a whole.