3.1 Variations of methane production, specific methane yield, and VS removal rate
Methane yield is an important indicator in reflecting the efficiency of AD. Figure 2 shows the daily and cumulative methane yields of the control, RI, and BA groups. All groups started normally, and similar daily methane variation was detected during the first phase (0–10 days). The first methanogenesis peak for each reactor was observed during the third day, resulting from the assimilation of readily degradable and dissolved substances into methane. Subsequently, the occurrence, duration, and methane production values differed among all reactors after re-inoculating the RI reactor and adding biochar to the BA reactor. From phase 2, it was difficult to observe obvious methane production peaks in the control reactors. Strong daily peaks were observed in the RI reactors on day 23 (0.12 NL) and day 42 (0.07 NL), but these peaks were significantly less than those observed in the BA reactors, which showed stable daily peaks (0.10–0.22 NL) during the first 6 phases.
A kinetic study of the cumulative methane production for all reactors during the entire AD process (100 days) was performed using a modified Gompertz model (see Table 2). The maximum methane yield (Rm) for the control reactor was 0.10 NL d− 1. For RI and BA reactors, Rm increased to 0.07 and 0.12 NL d− 1, respectively. The values of Rm and P showed that re-inoculation and biochar addition improved the rate and total volume of methane production. In particular, the methane potential of the BA reactor reached 5.48 NL, which was nearly three times that of the RI reactor.
Table 2
Gompertz kinetics data for methane production in different experimental reactors.
| Gompertz kinetics for biogas production |
P (NL) | Rm (NL d− 1) | λ (d) | R2 |
Control | 0.66 ± 0.01 | 0.10 ± 0.40×10− 2 | 2.61 ± 0.08 | 0.98 |
RI | 2.11 ± 0.05 | 0.07 ± 0.20×10− 2 | 2.05 ± 0.38 | 0.98 |
BA | 5.48 ± 0.07 | 0.12 ± 0.20×10− 2 | 7.72 ± 0.29 | 0.99 |
In the absence of precise process control and recovery strategies, the high protein and lipid contents in FW, impurities in CG, and propionic acid derived from CG led to inhibitory levels of ammonia, hydrogen sulfide, and LCFAs. These harmful intermediate compounds can be easily generated to cause reduced system stability, low methane yield, or foaming. Therefore, under continuous feeding conditions, the methane production performance of the control reactor declines greatly, and almost no methane can be produced from day 16.
As shown in Fig. 1, the RI reactors were re-inoculated every 10 days to alleviate VFA accumulation and overcome inhibition periods. However, although the re-inoculation strategy was effective until day 50, its long-term effect (50–100 days) was weak. After the fourth re-inoculation, the daily methane production was significantly lower than that in the first three phases. During the 50- to 100-day period, the RI reactor was no longer able to produce methane, even if re-inoculation was performed. Over the entire AD process, the BA reactor was more effective than the RI reactor because of the consistently higher methane production over the first five phases (0–50 days). In the later stages of AD, the control and RI reactors stopped producing biogas, whereas the BA reactor kept working up to day 100, even though its methane production rate decreased slightly. Higher rates of VS removal generally means that more organic material can be converted to biogas. In the control reactor, the VS removal rate reached 53.21 ± 5.09% in the first phase, but remained at lower levels (27.81–47.14%) in the subsequent phases, indicating accumulation and poor degradation of input material. Compared with RI treatment, the addition of biochar resulted in higher VS removal efficiency in AD treatment of FW and CG, which remained above 50% throughout the trial period. The specific methane yield (SMY), defined as the amount of methane produced for a given quantity of removed VS, is the result of the activity of anaerobic flora. It is constant under steady-state conditions for a given carbon substrate under anaerobic respiration conditions (i.e., catabolism) and depends on the proportion of biodegradable substances [20] and the nature of the compound. The SMYs of the different reactors were determined for each phase (Table 3). The SMY data for the BA group (0.12–0.56 NL g− 1 VS) indicates that the unique electrochemical properties of biochar contributed to methanogenic performance to varying degrees. Yin et al. [21] also found that biochar enhanced VS removal efficiency and methane production by 18% and 25%, respectively, through promoted direct interspecies electron transfer by enriching methanogens and substituting exoelectrogens. Conversely, although the RI group also showed higher VS removal rate than the control group, the consumed VS was not well converted to methane based on SMY values (0.002–0.09 NL g− 1 VS) after phase 5.
Table 3
Specific methane yield, electrical conductivity, and VS removal rate of digestate from different experimental reactors.
Phase | Control | RI | BA |
| VS removal rate (%) | SMY (NL g− 1 VS) | VS removal rate (%) | SMY (NL g− 1 VS) | VS removal rate (%) | SMY (NL g− 1 VS) |
1 | 53.21 ± 5.09 | 0.53 ± 1.10×10− 2 | 49.45 ± 8.17 | 0.65 ± 0.07 | 51.05 ± 3.53 | 0.56 ± 0.01 |
2 | 34.09 ± 4.79 | 0.21 ± 5.10×10− 2 | 58.29 ± 3.02 | 0.31 ± 0.02 | 64.66 ± 4.61 | 0.49 ± 0.04 |
3 | 35.03 ± 8.01 | 0.03 ± 0.70×10− 2 | 65.93 ± 2.55 | 0.28 ± 0.01 | 70.58 ± 4.74 | 0.56 ± 0.04 |
4 | 41.54 ± 3.42 | 0.02 ± 1.10×10− 2 | 63.41 ± 1.94 | 0.13 ± 0.01 | 77.70 ± 0.38 | 0.27 ± 0.01 |
5 | 47.14 ± 5.65 | 0.02 ± 0.30×10− 2 | 65.19 ± 2.73 | 0.09 ± 0.01 | 67.57 ± 7.74 | 0.44 ± 0.05 |
6 | 45.77 ± 1.05 | 0.02 ± 0.60×10− 2 | 62.40 ± 1.05 | 1.40×10− 2±0.50×10− 2 | 68.26 ± 1.68 | 0.24 ± 0.20×10− 2 |
7 | 45.49 ± 6.56 | 0.90×10− 2±5.92×10− 2 | 57.43 ± 3.72 | 0.01 ± 0.20×10− 2 | 66.21 ± 3.40 | 0.24 ± 0.03 |
8 | 27.81 ± 3.66 | 0.70×10− 2±0.20×10− 2 | 49.38 ± 0.66 | 0.70×10− 2±0.20×10− 2 | 61.42 ± 1.48 | 0.13 ± 0.01 |
9 | 41.13 ± 4.51 | 0.01 ± 0.20×10− 2 | 35.13 ± 8.07 | 0.40×10− 2±0.20×10− 2 | 61.27 ± 3.14 | 0.12 ± 0.80×10− 2 |
10 | 41.56 ± 2.85 | 0.20×10− 2±6.92×10− 4 | 52.23 ± 5.12 | 0.20×10− 2±3.63×10− 4 | 55.84 ± 7.59 | 0.17 ± 0.02 |
It is also worth noting that the observed results can be attributed to the accumulation of biochar decreasing methane production because of the adsorption of VFAs before their conversion to biogas [22]. In a study by Shen et al. [23], no significant difference was observed between biochar-modified digesters and controls in terms of reaction rate at thermophilic temperature because the high dosage of biochar likely inhibited microbial activity and kinetics. Therefore, excessive biochar accumulation over time in the reactors may cause a decline in biomethane production because of the nonselective adsorption behavior of biochar.
3.2 Effects of RI and BA strategies on reactor chemical conditions
One of the core objectives of this study was to determine the effects of RI and BA treatments on AD performance using easily acidified substrates. This approach required the monitoring of pH, VFA, alkalinity, TAN, and EC as process performance parameters.
3.2.1 Variation of VFAs in AD reactors
Co-digestive systems exhibit high hydrolysis and acid production because of the high biodegradability of the FW and CG present in such systems. Figure 3 shows the accumulation of VFA and individual acid components in the effluent of the control, RI, and BA reactors. The volatile fatty acids (acetic acid, propionic acid, butyric acid, valeric acid) were the core products of acidogenesis in this study. The predominant accumulated VFAs were acetic acid and propionic acid in all three treatments. Similar VFA concentrations were observed in the initial start-up phase with subsequent increased degradation of organic matter from FW and CG as reflected by increasing VFA levels up to day 10. For the control treatment without any recovery strategy, the soluble products continued to accumulate in the reactors during the continuous feeding period and reached its first peak (16,250.11 ± 120.90 mg L− 1) on day 33, thereby resulting in significantly higher VFAs concentration than the other two reactors. High levels of propionic acid accumulation, reaching 5.39 and 4.54 g L− 1 on days 30 and 40, respectively, were also observed in the control reactor, which reduced the buffering capacity and lowered the pH, thereby weakening the efficiency of the biomethanation process. At the end of AD on day 100, the propionic acid concentration in the control reactor was 5.68 g L− 1 and seemed to be difficult to consume. Propionic and butyric acids are generally considered difficult to oxidize because the process is thermodynamically unfavorable in the absence of hydrogen consumption by methanogens [24]. Re-inoculation and biochar treatments helped to alleviate the accumulation of VFAs, with the BA reactor having lower VFAs concentrations and more effective conversion of propionic acid. In previous research by Xu et al. [25], the secondary accumulation of VFA was also reduced in the AD treatment of sulfate wastewater when mediated by biochar. Generally, in the presence of biochar, the enhanced acetic acid and butyric acid degradation can provide abundant hydrogen for more efficient degradation of propionic acid [26].
3.2.2 Variation of total alkalinity concentration and total ammonium nitrogen in AD reactors
The total alkalinity concentrations in the reactors were determined daily (see Fig. 4). The addition of biochar maintained the CaCO3 alkalinity in the BA reactor at a high level of 5000–9000 mg L− 1, thus providing buffering capacity for the AD treatment of FW with CG. These results demonstrated the potential benefits of using biochar as an additive to help maintain alkalinity and counter the effects of acidification. Buffering capacity is necessary to alleviate AD reactor instability while the biochar elemental composition, soluble ash, fixed carbon, and volatile matter contents will contribute to total alkalinity [27]. It has been reported that the alkalinity of biochar can increase the alkalinity in AD and increase the methane content through the reaction of CO2 and H2S [28]. However, biochar alkalinity cannot be based directly on its composition of N, P, and K and more research is needed to establish correlations between biochar production parameters and its elemental composition. Cations of Na, K, Ca, Mg, Fe, organic functional groups, and inorganic basic species in biochar will help the AD system to equilibrate in the BA reactor. Biochar character and composition also contribute to AD performance in terms of biomethanation productivity. This can also be attributed to the microspore surface area of biochar and its relative adsorption properties and pyrolysis temperature to alleviate inhibition and attract microorganisms to its surface.
From day 30, the TAN concentration in the control reactor was noticeably higher than the TAN levels in the RI and BA reactors, which may indicate that the TAN concentration (2797.26–4495.51 mg L− 1) in the digesters was the main driver of total alkalinity. During the AD process, the accumulation of higher ammonium concentrations affected microbial growth rate and metabolic performance and resulted in low methane production. Although TAN ions (NH3 or NH4+) may be toxic to the growth and development of methanogens, biochar reactors maintained favorable pH values (7.5–8.5) at higher TAN concentrations above 2000 mg L− 1 with no significant inhibitory effect. This may have been caused by the adsorption capacity of individual biochars, which are capable of adsorbing ammonium ions and promoting electron transfer of NH4+ with cations on the biochar surface [29]. The BA reactor tolerated TAN concentrations above the recommended ammonia suppression threshold of 1700–1800 mg L− 1 [30]. This study demonstrates the efficacy of biochar in reducing TAN inhibition and retaining robust reactor performance at higher concentrations. Furthermore, biochar can be used as a suitable additive to control high TAN content while maintaining reactor stability for enhanced energy recovery without increasing environmental risk.
3.2.3 Variation of pH in AD reactors
The initial pH values of the control, RI and BA groups were 8.42, 8.29, and 8.18, respectively. Figure 5 shows the daily variation in pH for the control, RI, and BA treatments. For each treatment, pH was observed to fluctuate in accordance with feed degradation. In the initial treatment phase (the first 10 days), similar trends of pH fluctuation were observed for the three groups, which was expected in the absence of any difference between the three groups up until day 10. The pH of the control group showed a brief recovery on day 23, but afterward presented a mainly downward trend. During the AD process, high VFA concentration (5536.90–19270.51 mg L− 1) in the control reactor caused the pH to drop to 5.34, which ultimately led to lower methane production.
The pH trend of the control reactor suggested that intervention was necessary to prevent acidification of thermophilic anaerobic co-digestion reactors. To ease the acidification trend, 50% of the working volume of the RI reactor was replaced with fresh digestate. The positive effect of re-inoculation was immediately observed. The methane yield and pH were obviously stabilized after the external intervention. From day 10, the pH of the RI and BA reactors recovered to between pH 7.0 and 8.0. The ability of AD to adapt to changes in pH to a stable working range is known as self-buffering. There were regular fluctuations in pH in the RI and BA reactors from day 20 to day 40, which were attributed to the acclimatization and adaption of the reactors. However, during the period from day 50 to day 100, the pH of the RI reactor showed a sustained drop after re-inoculation until the end of each phase. This means that simple re-inoculation of acidified digestate was unable to moderate the destabilized reactors. It was expected that the pH in BA reactors would increase with biochar addition because of the alkaline nature of biochar (pH = 8.85), which was significantly higher than that of the control. This is mainly attributable to the release of alkali cations and alkaline earth metals (K, Ca, and Mg) in the biochar and the generation of ammonia during AD, which can consume CO2 to generate HCO3−/CO32− buffer [23]. Therefore, even with daily feeding with easily acidified substrates, the BA reactors could tolerate high VFA accumulation in the presence of biochar.
3.2.4 Electrical conductivity of digestate
The electrical conductivity (EC) reflects the number of free ions in the solution and can be used as a measure of the salinity level of the liquid digestate. At the same temperature, EC is positively correlated with the soluble salt content of the anaerobic digestate [31]. Shen et al. [32] quantified the correlation between the increase in methane production from digesters amended with biochar and a positive linear correlation was noted between methane production and the electron-donating capacity of biochar.
Figure 6 shows the variation of EC with digestion time. The EC of all reactors increased in the first 5 days, probably because of an increase in soluble components generated by degradation of FW and CG. Because of continuous feeding, the EC value of the control group showed an increasing trend from 18.64 to 38.53 mS cm− 1. The partial degradation of organic matter into small soluble organic anions is also expected to contribute to EC. The EC value of the RI reactor was at a moderate level for the three groups because it was reinoculated with fresh inoculant with the EC value of 19.83 mS cm− 1 every 10 days throughout the whole process. Decreases in EC on some days was observed in all three reactors, which might be relevant to the reduced soluble salt levels caused by increased nutrient consumption from methanogenic activities. For the BA reactor, the addition of biochar reduced the EC value throughout the AD process. Biochar produced under superheated conditions has a relatively high adsorption capacity and promotes methanogenesis, which leads to a reduction in the soluble salt content of the digestate. However, the EC increased significantly as the amount of biochar increased from day 60 (24.81–36.08 mS cm− 1), which corresponds to the decline in methane production from phase 6. Higher concentrations of soluble salts in anaerobic digesters will generate reverse osmosis pressure, which displaces water within microbial cells and leads to dehydration and inactivation of methanogenic archaea. Moreover, soluble salts could dissociate and block the material flows during AD process, resulting in the inhibition of nutrient and metabolite transport. The EC should be kept at a moderate level even though remarkably high VFA bioconversion was achieved with EC up to 90 mS cm− 1 [33]. EC of anaerobic digestates should be considered when using them as fertilizers because their use might directly affect the electrical properties of soil. In future work, the clean treatment of digestate after AD and its use in horticulture should be investigated with a view to controlling high conductivity in the reactor caused by continuous accumulation of biochar.
3.2.5 Characteristics of biochars before and after AD
Fagbohungbe et al. [34] proposed that the addition of biochar to AD processes can alleviate substrate-induced instability in three main ways: (1) the adsorption of inhibitors; (2) the increased buffering capacity of AD; and (3) the immobilization of bacterial cells. SEM was used to examine the solid particle morphology of biochar and to provide information on microstructural changes (Fig. 7). Sponge-, honeycomb-, and fence-like porous structures, which originated from the tissue structure in the precursor plants, were observed in the biochar particles. The existence of pores on biochar is important for microbial activities, and the presence of macropores on biochar provides suitable habitats for microbial communities [35].
Table 4 summarizes the pH, EC, ash content, total carbon and nitrogen, nitrate nitrogen and ammonia nitrogen, and elemental composition of biochar before and after AD. The pH of biochar was alkaline before (pH 8.85) and after AD (pH 8.59) because of the gradual loss of acidic surface groups and volatile matter at high pyrolysis temperatures [36]. Fe, Ni, Co, Mg, K, and Ca are necessary as supplements to avoid nutritional deficiencies in the AD process [37]. The importance of Fe depends on its redox properties and its role in energy metabolism, where Fe reacts as an electron acceptor and donor in the transport system of methanogenic bacteria to convert CO2 to CH4 [38]. Ni and Co are also vital cofactors for carbon monoxide dehydrogenase, acetyl coenzyme-A decarboxylase, and other enzymes involved in the methanogenic pathway of acetate fragmentation [39]. Likewise, K, Ca, and Mg are essential for the growth and development of some methanogenic bacteria and are crucial for the formation of microbial aggregates [40]. Therefore, the presence of these elements in biochar may explain the enhanced effect of biochar addition on the performance of AD processes. After use in AD processing, the concentration of ammonia nitrogen in biochar increased significantly, indicating that biochar addition had the effect of capturing ammonia nitrogen.
Table 4
The characteristics of biochar before and after AD.
Parameter | Biochar before AD | Biochar after AD |
pH | 8.85 | 8.59 |
Electrical conductivity (mS cm− 1) | 0.62 | 3.67 |
Ash (%) | 19.64 | 12.57 |
C (%) | 76.93 | 78.02 |
N (%) | 0.52 | 0.96 |
Nitrate nitrogen (ppm) | 4.24 | 5.17 |
Ammonia nitrogen (ppm) | 15.52 | 184.26 |
P (%) | 0.09 | 0.32 |
Ca (%) | 1.22 | 1.10 |
Mg (%) | 0.22 | 0.34 |
K (%) | 0.47 | 0.74 |
Mn (ppm) | 328.60 | 246.09 |
Zn (ppm) | 73.74 | 89.07 |
Cu (ppm) | 14.35 | 13.95 |
Fe (%) | 0.34 | 0.31 |
Ni (ppm) | 20.51 | 31.48 |
Co (ppm) | 8.67 | 10.85 |
Data given on dry matter basis |