3.1 Analysis of the difference in gas production via AD under different proportioning conditions
The results of stagewise and cumulative methane production experiments on the AD of coal and fishmeal under single-substrate conditions (coal or fishmeal) and various ratios are illustrated in Fig. 1. As depicted in Fig. 1(a), the single coal group (1:0) peaked in gas production on day 12, reaching 135 mL/day. The single fishmeal group (0:1) peaked on day 3, with a production of 203.5 mL/day. The CY-2:1 group reached its highest gas production on day 12, recording the highest among all proportional stages at 480 mL/day, with a cumulative gas production of 1758 mL. As illustrated in Fig. 1(b), the single coal group peaked in methane production on day 9, with a production of 65.75 mL/day. The single fishmeal group reached its peak methane production on day 6, with a production of 29.49 mL/day. The 2:1 group achieved the highest stagewise methane production of 353.42 mL/day on day 12, with the highest cumulative methane production recorded at 1121.49 mL. The comparison revealed a significant difference in cumulative methane production among combinations with different ratios. Various combinations of fishmeal displayed distinct peaks in methane production, with the 2:1 group peaking on day 12, the single coal group on day 9, and the 1:1 group on day 18. Thus, the addition of an appropriate amount of fishmeal can shorten the gas production cycle.
3.2 COD phase change
The COD can be utilized to characterize the concentration of substances that undergo oxidation during anaerobic fermentation. A higher COD value indicates a greater concentration of organic matter in the fermentation broth (Soomro et al.2020). Table 3 presents the variations in COD values during the AD of fishmeal mixed with lignite at different ratios. As shown in Table 3, the initial COD value of the bacterial broth before the experiment was 15.3 mg/L. At the peak of gas production, there was an increase in organic matter generation compared to hydrolysis rate, resulting in an elevated COD value. Subsequently, through anaerobic digestion reaction, organic matter gradually underwent hydrolysis and was utilized by microorganisms for methane production, leading to a decrease in COD value. In the 2:1 group, at the peak of gas production, the COD value reached 1197.5 mg/L and after degradation it decreased to 95.2 mg/L, indicating superior degradation efficiency. The blank group had COD values of 561.4 mg/L and 774.7 mg/L respectively, which were approximately 3.67 and5 .06 times higher than those observed before experimentation (pre-experiment). Combined with Fig. 1 illustrating gas production results, it can be inferred that microorganisms within the fermentation system continuously engage in various metabolic activities throughout hydrolysis phase, acidification phase, and acetic acid production phase. The microorganisms in the system ultimately metabolize the organic matter present in the solution to generate methane, resulting in a reduction of the COD value. In Fig. 1, group 2:1 exhibited the most favorable gas production outcomes, with maximum daily gas production observed during its peak period. This suggests that microorganisms can effectively utilize high concentrations of soluble organic matter within the system for methane conversion.
Table 3
Changes in liquid-phase COD during AD
samples | B (mg/L) | M (mg/L) | E(mg/L) |
CY-5: 1 | 15.3 15.3 15.3 15.3 15.3 15.3 | 385 | 125.94 |
CY-2: 1 | 1197.5 | 95.2 |
CY-1: 1 | 498.6 | 885.6 |
CY-1: 2 | 1692.5 | 957.15 |
CY-1: 0 | 561.4 | 349.93 |
CY-0: 1 | 774.7 | 276.55 |
Note: B, before the experiment; M, the peak of gas production; E, after degradation |
3.3 Dissolved organic matter
The fermentation broths from the CY-5-1, CY-2-1, CY-1-1, CY-2-1, CY-1-0, and CY-0-1 groups during the peak gas production period were collected to analyze the changes in soluble organic matter using a fluorescence spectrophotometer. According to the findings of relevant studies on fluorescence spectral partitioning, the fluorescence region can be categorized into five parts (Huang et al.2021;Xiong et al.2022) (Fig. 2). The fermentation broth of the anaerobically digested samples exhibited two fluorescence peaks, with consistent positions but differing intensities. These peaks corresponded to complex amino acids, tryptophan-like proteins, fulvic acid-like substances, dissolved microbial metabolites, and humic acid-like substances, according to the coloration of the respective regions. As shown in Fig. 2(b), the fluorescence peaks of the 2:1 group were primarily concentrated in the II and IV regions, indicating tryptophan-like and dissolved microbial products. During the peak gas production period, microbial activity was enhanced, leading to a greater production rate of tryptophan-like microbial metabolic protein substances than their degradation rate. This resulted in intensified fluorescence peak intensity, explaining the highest gas production observed in the 2:1 group. As depicted in Fig. 2(e), the fluorescence peaks resolved in the single coal group were predominantly distributed in zone III, with stronger peaks of fulvic acid-like humic substances. In the spectra of the single fishmeal group, the fluorescence peaks of tryptophan-like protein substances and microbial metabolism protein substances were resolved, with the spectra displaying enhanced fluorescence peaks (Fig. 2f). In summary, the 2:1 group displayed enriched peaks concentrated in zones II and IV and higher levels of dissolved microbial products compared with the other groups.
3.4 GC-MS
The fermentation broths from groups CY-5:1, CY-2-1, CY-1-1, CY-1-2, CY-1-0, and CY-0-1 at the peak of gas production were collected for GC–MS analysis. As depicted in Fig. 3, the single coal group contained fewer types of compounds, primarily 2-bromododecane, tetradecane, and a small amount of amines and alcohols. In contrast, the single fishmeal group was mainly characterized by 1,2-diphenoxyethane, n-eicosane, n-octadecane, and n-octacosane compounds. The 2:1 group exhibited the largest number of compounds, comprising 61 species, among which n-octadecane and n-octadecane hydrocarbons were more common, while these compounds were less abundant in the single coal group 1:0. Fishmeal addition resulted in a significantly greater number of compound species compared with the blank group (Fig. 3). The higher content of n-oicosane in the single coal group 1:0 and the single fishmeal group 0:1 and the lower content in the other mixed ratios can be attributed to reactions occurring between the two substrates during the co-digestion process. The single coal group was predominated by benzene ring analogs, which are difficult to be degraded and digested by microorganisms, and the generation of benzene ring analogs is unfavorable for methane production through AD (Shimoyama et al.2019). In summary, fishmeal addition increased the variety of compounds in the fermentation system and provided the basis for further AD for methane production.
3.5 Analysis of ammonia, total phosphorus, pH, and VFA
Ammonia nitrogen, one of the common inhibitory substances during the AD reaction, primarily exists in the forms of NH4+-N and free NH3. The ammonia nitrogen content serves as a crucial indicator of AD environment stability and influences the activity of methanogenic bacteria (Peng et al.2019). A high concentration of ammonia nitrogen can inhibit the digestive system. The fermentation broth collected at the gas production peak was tested, and the results are illustrated in Fig. 4. The ammonia nitrogen content of the 2:1 group was 76.97 mg/L, which was 3.19, 1.39, and 3.88 times those of the 5:1, 1:0, and 0:1 groups, respectively: 245.6 mg/L, 107.3 mg/L, and 298.6 mg/L (Fig. 4). Ouyang et al. investigated the effect of phytoplankton-added phosphorus on the functioning of bacterial colonies in terms of methanogenicity. They demonstrated that phosphorus inhibited the relative abundance of methanogenic bacteria, thereby affecting methane production [Ouyang et al.2024]. The total phosphorus content in the 2:1 group was 73 mg/L, and those in the single coal and fishmeal groups were 5.6 mg/L and 79 mg/L, respectively. Hess, Sbarciog, and Shen et al. discovered that the accumulation of VFA produced during AD inhibited the growth of methanogenic bacteria, leading to the acidification of the reaction system, consequently resulting in the failure of the AD experiments (Hess et al.2008; Sbarciog et al.2010; Shen et al.2007). Figure 4(d) illustrates that the VFA content of the 2:1 group was 4.8 mmol/L, higher than the 0.25 mmol/L for the single coal group and the 4.7 mmol/L for the single fishmeal group. This comparison indicates that fishmeal addition helped to maintain the levels of ammonia nitrogen, pH, and VFA necessary for the AD reaction, thereby enhancing the stability of the AD system and improving gas production. In conclusion, the ammonia nitrogen, total phosphorus, and VFA contents were inversely related to methane production.
3.6 Law of change of enzyme activity
The fermentation broth collected during peak gas production was tested for coenzyme F420, cellulase, and hydrogenase contents, and results are displayed in Fig. 5. Overall, activity levels showed an initial rise and then a decline as the proportion of fishmeal increased. Cellulose was converted into fermentable sugars by cellulase, facilitating the production of various valuable products during AD, ultimately enhancing gas production (Terranova et al.2024). As depicted in Fig. 5, the cellulase activities of the single coal and single fishmeal groups were 16 U/g and 134 U/g, respectively, while the cellulase activity of the 2:1 group was 326 U/g. Cheng investigated the changes in coenzyme F420 activity and methane production in the simulation of organic waste landfills. A positive correlation existed between the activity of coenzyme F420 and both methane production and methane concentration (p < 0.05) (Cheng et al.2007). In another study, hydrogenase, a biological enzyme present in microorganisms, promoted AD by catalyzing the oxidation of H2 in methanogenic bacteria (Seigo et al.2011). As shown in Fig. 5, the 2:1 group exhibited the highest hydrogenase activity at 0.38 ml/(mg·min), while the single coal group displayed the lowest hydrogenase activity at 0.0124 ml/(mg·min). This pattern of change corresponds to the enzyme promotion during methane production digestion, consistent with the stagewise methane production and cumulative methane production depicted in Fig. 1.
3.7 Changes in soluble sugars, proteins, and lipids
The peak fermentation broth was collected for testing soluble sugars, proteins, and lipids, and the results are presented in Table 4. Fishmeal is high in soluble organic matter, containing dissolved sugars, proteins, and polyester compounds, which promote anaerobic digestion for methane production during and coal and synergistic process. Soluble sugars primarily comprise sucrose, glucose, and fructose (Cho and Yoo et al.2011). As indicated in Table 4, the concentrations of soluble sugars in the single coal and fishmeal groups were 21.2 µg/mL and 52.3 µg/mL, respectively. The experimental group 2:1 exhibited a concentration of 137.3 µg/mL, which was 6.48 and 2.63 times those of the single coal and fishmeal groups, respectively. Microorganisms do not absorb proteins directly but metabolize them to VFA and sulfur compounds under acid-producing conditions and finally to methane under AD conditions (Mirko et al.2021). The protein content was 13.3 µg/mL in the single coal group, 27.9 µg/mL in the single fishmeal group, and 58.2 µg/mL in the 2:1 group. Lipid addition enhanced the expression of functional metabolism enzyme genes in the AD process and extended the reaction cycle of the AD system. As presented in Table 4, the 2:1 group exhibited the highest content of lipid compounds, 21.7 mg/mL, which was 6.78 and 2.65 times those of the single coal and single fishmeal groups, respectively. In summary, with the increase in the proportion of fishmeal, the dissolved sugar, protein, and lipid compound contents first increased and then decreased the 1:2 group exhibited the highest contents of dissolved sugars, proteins, and lipid compounds. Moreover, the 1:2 group had the highest fishmeal content; consequently, toxic substances, including ammonia nitrogen and VFA accumulated within the AD system, which results in gas production and methane production below the 2:1 group.
Table 4
Changes in soluble sugars, proteins, and lipids
Samples | S ug/mL | P ug/mL | L g/mL |
CY-5: 1 | 107.3 137.7 164.2 213.8 21.2 52.3 | 38.8 | 13.2 |
CY-2: 1 | 58.2 | 21.7 |
CY-1: 1 | 72.4 | 33.9 |
CY-1: 2 | 93.3 | 42.5 |
CY-1: 0 | 13.3 | 3.2 |
CY-0: 1 | 27.9 | 8.2 |
Note: S, soluble sugars;P, soluble proteins ༛L, soluble lipids |
According to the results of the AD methane production experiment with different ratios, the CY-2:1 group exhibited the highest stagewise gas production and methane production: 480 mL/d and 353.42 mL/d, respectively; thus, the addition of an appropriate amount of fishmeal can promote the AD of methane production, which can shorten the gas production cycle and increase the amount of methane production. As the proportion of fishmeal increased, COD, F420, the hydrogenase content, and the cellulase content first increased and then decreased, and the Highest enzyme activity in 2:1 group. Consistent with the trend of producing the most gas and methane. Among the groups, the 2:1 group exhibited the lowest levels of ammonia nitrogen, phosphorus, and fatty acids, which were not high enough to inhibit the reaction. Consequently, the 2:1 group had the highest gas and methane production. The GC–MS results revealed that the 2:1 group exhibited the highest number of compounds, totaling 61, which was conducive to increasing gas and methane production. The dissolved sugar, protein, and lipid contents first increased and then decreased with increasing fishmeal proportion, peaking in the 1:2 group. High contents of sugars, proteins, and lipids inhibited the systemic reactions in the AD system, which in turn affected gas and methane production. Overall, the results showed that the 2:1 group exhibited the highest gas and methane production.