Effect of Alfalfa-Derived Biochar on Anaerobic Digestion of Dairy Manure

: Biochemical methane potential (BMP) tests were conducted for investigating the effects of alfalfa-derived biochar (AF-BC) on anaerobic digestion (AD) of dairy manure under various loading of AF-BC (0–10 g/L). BMP tests were performed at mesophilic temperature (37 ◦ C) with the addition of AF-BC. Biogas and methane volumes and concentrations, water quality parameters (i.e., COD (chemical oxygen demand)), and volatile fatty acids (VFAs) were measured during the AD process. The addition of 1 and 5 g/L of AF-BC increased the biogas yields by 15.51% and 26.09% and methane yields by 14.61% and 26.88% compared with the control without addition of AF-BC. Additionally, the addition of AF-BC (1–10 g/L) decreased the lag phase by 7.14–22.45% and the CO 2 content of biogas by 13.60–32.48%, while increasing the COD removal efﬁciency by 19.19–35.94% in the AD of dairy manure. Moreover, the addition of AF-BC also decreased total VFAs and acetic acid concentrations in the AD process. The increase in AD performance was mainly owing to the improvement of buffering ability of the AD system and direct interspecies electron transfer (DIET) among AD microorganisms resulting from the addition of AF-BC. In contrast, the addition of 10 g/L AF-BC did not show any obvious improvement in biogas and methane yields in the AD of dairy manure, possibly because of toxic effects from excessive addition of AF-BC toward the AD microorganisms. Therefore, this study supported practical feasibility of AF-BC-enhanced AD of dairy manure.


Introduction
Anaerobic digestion (AD) has been widely used as a sustainable route to convert various waste to biogas and digestate as fuel and biofertilizer while mitigating environmental problems and greenhouse gas emission [1][2][3][4]. A large quantity of animal manures (about 132 million metric tons of dried manure per year in United States) has been used as the major feedstock for AD [5,6]. Dairy manure, among the animal manures, is generated by up to 9.4 million dairy cows each year [7] and has caused increasing pollution in soil, water and air when it has been applied to farms as a fertilizer [8]. Therefore, effective AD of dairy manure has been considered as a sustainable remediation process for production of renewable energy (either cleaned biogas or renewable natural gas), effective disposal of manure, and reduction of manure-derived greenhouse gas emission [9]. To date, a substantial number of works about AD of dairy manure have been reported. Li et al. [5] conducted AD tests with diary manure under various substrate concentrations and achieved a methane yield up to 270 mL/g VS. Zheng et al. [10] evaluated the impact of co-digestion ratios on the performance of AD in mixtures of dairy manure and switchgrass, and found that the optimal methane production (155.1 mL/g VS) was observed under a 2:2 mixture ratio of manure to grass. In addition, Zeng et al. [11] applied mechanical refining pretreatment to

Preparation and Characterization of BC
The grass alfalfa (AF), obtained from a local hay vendor (Stephenville, TX, USA), was used as the feedstock for BC. After being dried, ground, and sieved (<500 µm), 10 g of dry AF was placed into a quartz-tube furnace (MTI corporation, Richmond, VA, USA) for pyrolysis. In order to lower the production cost of BC, AF was pyrolyzed at a low pyrolysis temperature. The pyrolysis conditions were set at 350 • C with the heating rate of 10 • C/min and heating time of 120 min under continuous flow of nitrogen gas (2 L/min). The resulting BC was named as "AF-BC". Then, AF-BC was milled and sieved until the particle size was less than 106 µm. The analysis of elemental and mineral compositions of AF and AF-BC was conducted at Robert Microlit Lab (Ledgewood, NJ, USA) and the Soil, Forage and Water Testing Lab at Texas A&M AgriLife (College Station, TX, USA), respectively. Proximate analysis, including fixed carbon, volatile carbon, and ash, was determined according to ASTM D7582-12 [33]. The surface functional groups of AF-BC were determined via a FTIR Spectrometer (Bruker Optik GmbH, Ettlingen, Germany). The surface area of AF-BC was evaluated by a surface area analyzer (Particle Technology Lab, IL, USA).

Anaerobic Digestion Experiments
BMP (Biochemical Methane Potential) tests were performed for evaluating the impacts of AF-BC on the AD of dairy manure. Briefly, 130 mL of inoculum and dry dairy manure with a ratio of 1 (TS basis) was loaded into a 280 mL serum bottle. The AF-BC was added into the serum bottle with four loading rates (0, 1, 5, and 10 g of BC/L of manure). After inoculation, each serum bottle was sealed with a rubber plug and a screw cap. Then, N 2 gas was flushed into each bottle for removing oxygen and ensuring anaerobic condition in each bottle. All bottles, in replicate, were incubated at mesophilic temperature (37 • C) and manually mixed every day. In this study, the BMP tests were carried out for 36 d. The experiment sets are referred to as control, A1, A5, and A10. The control represents the experiment group without addition of AF-BC, while A1, A5, and A10 represent the addition of 1, 5, and 10 g of AF-BC/L of manure. The experiment setup is shown in Figure S1.

Analytical Methods
TS, VS, pH, TCOD, SCOD, NH 3 -N, PO 4 3− , and TA were analyzed via APHA standard methods [34] and commercial test kits (Hach Company, Loveland, CO, USA). The biotoxicity tests of leaching solution from the addition of various concentrations of AF-BC (0-10 g/L) into DI water was conducted using a Toxi-ChromoTest TM kit (Environmental Bio-Detection Products Inc., Mississauga, ON, Canada) [35]. The biogas volume was evaluated using a 60 mL syringe, and methane and carbon dioxide contents of biogas were determined through the gas chromatograph (GC) (GC-2014, Shimaza Corp., Kyoto, Japan) connected with a packed column, a thermal conductivity detector (TCD) and a flame ionization detector (FID). The concentrations of VFAs were also determined by the GC as previously described [25]. The FID temperature was 250 • C, and the carrier gas was helium.

Modified Gompertz Model
For examining the impact of AF-BC addition on lag phase, maximum production potential and production rate from AD of dairy manure in the current work, the methane production data from the AD tests were fitted to a modified Gompertz model shown in the following equation [4,25]: where M(t) represents the methane yield in a time t (mL/g VS removed ), P represents the maximum methane potential (mL/g VS removed ), R max represents the maximum methane production rate (mL/g VS removed ·d), λ represents the lag phase (d), and e is the Euler's constant (2.7183). In the present study, the fitting precision of the model was determined through the determination coefficient (R 2 ): where M e , M c , and M mean represent the experimental value, calculated value, and mean of experimental value, respectively.

Effects of AF-BC Addition on Methane and Biogas Production
Biogas and methane production from the AD of dairy manure with various loading rates of AF-BC are displayed in Figure 1. The highest cumulative biogas and methane production in the AD of dairy manure was achieved with the addition of 5 g/L (A5) of AF-BC (Figure 1a,b). However, compared to the control, the addition of 10 g/L (A10) of AF-BC to the AD of dairy manure showed negative effects on cumulative biogas and methane volumes, probably due to the potential biotoxicity from AF-BC addition, which is described in Section 3.2. Conversely, the biogas yields in A1 and A5 were improved by 15.51% and 26.09%, respectively, compared to the control (625.99 mL/g VS removed ) ( Figure 1c). Similarly, the methane yields in A1 and A5 increased by 14.61% and 26.88%, respectively, compared to the control (300.01 mL/g VS removed ) ( Figure 1d). Therefore, compared to the control, the addition of AF-BC (1-5 g/L) to the AD of dairy manure resulted in effectively enhancing biogas and methane production. As shown in Table S1, these results were remarkably consistent with the positive impacts of BC on methane and biogas production reported by previous studies [4,36]. Wei et al. [37] evaluated the impacts of corn stover-derived BC on the AD of primary sludge and found that BC significantly improved the methane yield by 8.6-17.8%. Pan et al. [4] conducted the AD of chicken manure with the addition of nine different kinds of BCs, and the results showed that all BCs significantly improved methane yields, and the addition of fruitwood-derived BC achieved a methane yield of 294 mL/g VS. Moreover, the AD of sewage sludge and orange peels carried out by Martínez et al. [36] indicated that 10 g/L of vineyard pruning-derived BC improved the methane yield by 33% in the batch digestion system. However, the addition of 10 g/L of AF-BC (A10) showed no obvious improvement of methane and biogas yields compared with the control. The biogas and methane yields of A10 were 641.70 mL/g VSremoved and 301.68 mL/g VSremoved (Figure 1), which were lower than those of A1 and A5, with almost no difference from the control, indicating that a high loading rate of AF-BC was not favorable for the AD of dairy manure. Similar results have also been observed by Shen et al. [2], Shen et al. [38], and Sunyoto et al. [39]. Shen et al. [2] found that CH4 yield from the AD of mixtures of straw and cattle manure increased from 267.55 to 281.48 mL/g VS with the addition of coconut shell-derived BC at a 2% loading rate. However, the CH4 yield was reduced to 271.5 mL/g VS with 4% loading of the BC, mainly because of the accumulation of higher concentrations of acidic intermediates resulting in the imbalance of the AD system. Shen et al. [38] also reported that the high loading rates of wood-derived BC to the AD of primary sludge showed no significant difference for methane production compared to the control due to possible inhibition of microbial activities among AD microorganisms. Moreover, Sunyoto et al. [39] investigated the two-phase AD process using food waste with the addition of sawdust-derived BC. The results indicated that increasing loading rates of the BC ranging from 8.3-33.3 g/L caused a decrease in methane production. It was found that the addition of BC at high However, the addition of 10 g/L of AF-BC (A10) showed no obvious improvement of methane and biogas yields compared with the control. The biogas and methane yields of A10 were 641.70 mL/g VS removed and 301.68 mL/g VS removed (Figure 1), which were lower than those of A1 and A5, with almost no difference from the control, indicating that a high loading rate of AF-BC was not favorable for the AD of dairy manure. Similar results have also been observed by Shen et al. [2], Shen et al. [38], and Sunyoto et al. [39]. Shen et al. [2] found that CH 4 yield from the AD of mixtures of straw and cattle manure increased from 267.55 to 281.48 mL/g VS with the addition of coconut shell-derived BC at a 2% loading rate. However, the CH 4 yield was reduced to 271.5 mL/g VS with 4% loading of the BC, mainly because of the accumulation of higher concentrations of acidic intermediates resulting in the imbalance of the AD system. Shen et al. [38] also reported that the high loading rates of wood-derived BC to the AD of primary sludge showed no significant difference for methane production compared to the control due to possible inhibition of microbial activities among AD microorganisms. Moreover, Sunyoto et al. [39] investigated the two-phase AD process using food waste with the addition of sawdust-derived BC. The results indicated that increasing loading rates of the BC ranging from 8.3-33.3 g/L caused a decrease in methane production. It was found that the addition of BC at high loading rates caused a significant accumulation of propionic acid, which could inhibit methane production [39].
As displayed in Figure 2, the first daily methane yield peak appeared on the fourth day of the AD process for all experiment groups, which might have been caused by the consumption of easily degradable organic matter by anaerobic microorganisms [4]. The maximum daily methane yields in A1 (26.84 mL/g VS removed ), A5 (34.35 mL/g VS removed ), and A10 (27.45 mL/g VS removed ) were obviously higher than that in the control (23.29 mL/g VS removed ), which means that AF-BC could enhance utilization efficiency of organic substrates in the initial stage of AD. In addition, the second and third peaks in A5 (17th and 24th day) appeared earlier than those in the control (18th and 26th day), revealing that the addition of AF-BC to the AD process can accelerate the degradation of complex intermediates in the middle and later stages, thus increasing methane production overall [4]. Pan et al. [4] also observed three daily methane yield peaks during the AD of chicken manure and indicated that the addition of BC produced methane earlier than the control without the addition of BC.
Agronomy 2022, 12, x FOR PEER REVIEW 6 of 14 loading rates caused a significant accumulation of propionic acid, which could inhibit methane production [39]. As displayed in Figure 2, the first daily methane yield peak appeared on the fourth day of the AD process for all experiment groups, which might have been caused by the consumption of easily degradable organic matter by anaerobic microorganisms [4]. The maximum daily methane yields in A1 (26.84 mL/g VSremoved), A5 (34.35 mL/g VSremoved), and A10 (27.45 mL/g VSremoved) were obviously higher than that in the control (23.29 mL/g VSremoved), which means that AF-BC could enhance utilization efficiency of organic substrates in the initial stage of AD. In addition, the second and third peaks in A5 (17th and 24th day) appeared earlier than those in the control (18th and 26th day), revealing that the addition of AF-BC to the AD process can accelerate the degradation of complex intermediates in the middle and later stages, thus increasing methane production overall [4]. Pan et al. [4] also observed three daily methane yield peaks during the AD of chicken manure and indicated that the addition of BC produced methane earlier than the control without the addition of BC. In the present study, the experimental data were also fitted to the Gompertz model to develop a microbial kinetic model for the AD of dairy manure with the addition of AF-BC. Table 1 shows that A1 and A5 achieved significantly higher Rmax (mL CH4/g VSremoved·d) and P (mL CH4/g VSremoved) than the control. Both Rmax and P increased by 17.70% and 13.89% in A1, and 30.86% and 25.60% in A5, respectively, compared with the control. The results highly agree with positive impacts of BC addition on the methane production rate and yield (Rmax and P) during the AD process [1,25,40]. For example, Wei et al. [37] showed that both Rmax and P were improved by 53.79% and 13.72% when the corn stover-derived BC was applied to the AD of primary sludge obtained from the WWTP. However, there were no significant differences between the control and A10 for Rmax (15.20 vs. 15.51 mL/g VSremoved·d) and P (295.78 vs. 297.47 mL/g VSremoved). Shen et al. [38] indicated that a high concentration of wood-derived BC (4.97 g BC/g dry sludge) caused the lower Rmax and P than a low concentration of BC (2.49 g BC/g dry sludge). Moreover, Wang et al. [1] also reported that Rmax and P significantly increased during the AD of sludge and food waste In the present study, the experimental data were also fitted to the Gompertz model to develop a microbial kinetic model for the AD of dairy manure with the addition of AF-BC. Table 1 shows that A1 and A5 achieved significantly higher R max (mL CH 4 /g VS removed ·d) and P (mL CH 4 /g VS removed ) than the control. Both R max and P increased by 17.70% and 13.89% in A1, and 30.86% and 25.60% in A5, respectively, compared with the control. The results highly agree with positive impacts of BC addition on the methane production rate and yield (R max and P) during the AD process [1,25,40]. For example, Wei et al. [37] showed that both R max and P were improved by 53.79% and 13.72% when the corn stover-derived BC was applied to the AD of primary sludge obtained from the WWTP. However, there were no significant differences between the control and A10 for R max (15.20 vs. 15.51 mL/g VS removed ·d) and P (295.78 vs. 297.47 mL/g VS removed ). Shen et al. [38] indicated that a high concentration of wood-derived BC (4.97 g BC/g dry sludge) caused the lower R max and P than a low concentration of BC (2.49 g BC/g dry sludge). Moreover, Wang et al. [1] also reported that R max and P significantly increased during the AD of sludge and food waste with increasing loading of the sawdust-derived BC up to 6 g/L. However, the R max and P markedly decreased with high loading of BC (more than 6 g/L). In addition, as noted in Table 1, the lag phase (λ) in AD was shortened after the addition of AF-BC. Compared to the control, λ was reduced by 7.14%, 22.45%, and 12.24% under the addition of 1, 5, and 10 g/L of AF-BC, possibly owing to faster adaptation and communication of microorganisms with the addition of AF-BC. Similarly, Fagbohungbe et al. [41] applied three kinds of BCs (coconut shell BC, rice husk BC, and wood BC) in the process of AD of citrus peel and indicated that all BCs could lower the lag phase of AD. Wang et al. [42] also found that Douglas fir-derived BCs pyrolyzed at 500 and 600 • C led to the decrease in lag phase by 14.29% and 9.52% during AD of wastewater sludge. The impacts of AF-BC addition on COD, ammonia, and phosphate removal in the AD process are presented in Figure 3. A1, A5 and A10 achieved COD removal efficiencies of 56.64%, 64.60%, and 62.39% (Figure 3a), which were higher than that of the control (47.52%). This supported that the addition of AF-BC improved COD reduction from the AD of dairy manure. It was thought that microbial activity of AD can increase with the addition of AF-BC to AD, resulting in a higher consumption of organic compounds in the AD of dairy manure. The result was consistent with that reported by Shanmugam et al. [43], showing that switchgrass-derived BC could significantly improve the efficiency of COD removal by 16% during the AD of glucose. Moreover, Choe et al. [44] added the bambooderived hydrochar into the AD of fish processing waste while significantly enhancing COD reduction. However, the COD removal efficiency in A10 (Figure 3a) was comparable to that in A5, revealing that high loading of AF-BC was not favorable for further COD reduction. One assumption could be that AF-BC might contain some toxic compounds, which were produced during the pyrolysis process, resulting in the negative effects on AD microorganism activity due to the high loading of AF-BC. Lyu et al. [45] also reported that toxic compounds such as polycyclic aromatic hydrocarbons (PAHs) and dioxin-compounds were generated during the BC production by pyrolyzing the sawdust at 300 to 700 • C, with a higher toxicity of BC at lower temperatures. In addition, Smith et al. [46] indicated that toxicity of pine wood-derived BCs was mainly from lignin-derived phenolic compounds in BCs, and the toxicity of BCs was higher at a low pyrolysis temperature (300-400 • C). Moreover, in this study, the biotoxicity of leaching solution of AF-BC showed an increasing trend with an increase in AF-BC concentration from 1 to 10 g/L (Table S2). Therefore, high loading of AF-BC, produced from pyrolysis of grass alfalfa (containing 13% lignin) at low temperature (350 • C), in this study, might contain possible toxic compounds such as PAHs and phenolic compounds, which would be detrimental to AD microorganisms and negatively affect the AD process. Thus, this could explain the decrease in methane production with high loading of AF-BC (10 g BC/L, A10 in Figure 1).  As shown in Figure 3b, after AD, ammonia concentrations significantly increased in all experiment groups, from 96.2-102.4 to 773-803 mg/L NH3-N, which resulted from effective degradation of nitrogen-containing organic matters by AD microorganisms. Moreover, the final ammonia concentrations in A1 and A5 showed no significant difference with that in the control. However, compared to the control, a high dosage of AF-BC (A10) increased the final ammonia concentration from 777 to 803 mg/L. Similarly, Figure 3c indicates that the AD with AF-BC also enhanced phosphate concentration due to the enhanced hydrolysis of organic phosphate into inorganic phosphate. The high ammonium and increased phosphate concentrations, after the AD of manure, could be valuable sources of liquid biofertilizer when properly irrigated to agricultural farms.

CO2 Content
Biogas, produced from the AD process, largely consists of methane, carbon dioxide, and other impurities. CO2, considered as a greenhouse gas, can cause climate change, while higher CO2 concentrations result in the lower energy value of biogas [47]. Therefore, possibly low amounts of CO2 in biogas are beneficial for high energy values of biogas and have a low impact on climate change. Figure 4 shows that, after 36 days of AD, the CO2 As shown in Figure 3b, after AD, ammonia concentrations significantly increased in all experiment groups, from 96.2-102.4 to 773-803 mg/L NH 3 -N, which resulted from effective degradation of nitrogen-containing organic matters by AD microorganisms. Moreover, the final ammonia concentrations in A1 and A5 showed no significant difference with that in the control. However, compared to the control, a high dosage of AF-BC (A10) increased the final ammonia concentration from 777 to 803 mg/L. Similarly, Figure 3c indicates that the AD with AF-BC also enhanced phosphate concentration due to the enhanced hydrolysis of organic phosphate into inorganic phosphate. The high ammonium and increased phosphate concentrations, after the AD of manure, could be valuable sources of liquid biofertilizer when properly irrigated to agricultural farms.

CO 2 Content
Biogas, produced from the AD process, largely consists of methane, carbon dioxide, and other impurities. CO 2 , considered as a greenhouse gas, can cause climate change, while higher CO 2 concentrations result in the lower energy value of biogas [47]. Therefore, possibly low amounts of CO 2 in biogas are beneficial for high energy values of biogas and have a low impact on climate change. Figure 4 shows that, after 36 days of AD, the CO 2 content of biogas in A1, A5, and A10 decreased from 48.59% to 41.98%, 38.57%, and 32.81% compared to the control, respectively, implying that AF-BC addition was favorable for the reduction of the CO 2 content in biogas. In addition, it is obvious that the CO 2 content in biogas decreased with an increased loading rate of AF-BC. It is well known that BC has Agronomy 2022, 12, 911 9 of 14 the adsorption capacity for CO 2 in biogas via physisorption (e.g., Van der Waals' force), which could be enhanced by chemical interactions between acidic CO 2 and basic nitrogen functional groups of BC [48]. Creamer et al. [49] also mentioned that nitrous groups in the BC played an important role in CO 2 adsorption by the BC. As seen in Figure S2, AF-BC contained some nitrous functional groups (C-N and N-O), which was beneficial for CO 2 capture in the AD of dairy manure. Similarly, Baltrėnas et al. [47] conducted the AD of chicken manure with the addition of wood BC and indicated that the addition of wood BC did not result in enhancement for biogas production, but CO 2 content decreased from 47.5% to 33.1%.
Agronomy 2022, 12, x FOR PEER REVIEW 9 of 14 content of biogas in A1, A5, and A10 decreased from 48.59% to 41.98%, 38.57%, and 32.81% compared to the control, respectively, implying that AF-BC addition was favorable for the reduction of the CO2 content in biogas. In addition, it is obvious that the CO2 content in biogas decreased with an increased loading rate of AF-BC. It is well known that BC has the adsorption capacity for CO2 in biogas via physisorption (e.g., Van der Waals' force), which could be enhanced by chemical interactions between acidic CO2 and basic nitrogen functional groups of BC [48]. Creamer et al. [49] also mentioned that nitrous groups in the BC played an important role in CO2 adsorption by the BC. As seen in Figure S2, AF-BC contained some nitrous functional groups (C-N and N-O), which was beneficial for CO2 capture in the AD of dairy manure. Similarly, Baltrėnas et al. [47] conducted the AD of chicken manure with the addition of wood BC and indicated that the addition of wood BC did not result in enhancement for biogas production, but CO2 content decreased from 47.5% to 33.1%.

VFAs Analysis
VFAs accumulation during AD processes often causes drastic pH drops and inhibition of microbial activity [19]. Figure 5 indicates that all experiments in the control, A1, A5 and A10 exhibited a similar trend of total VFAs concentration variation. Total VFAs concentration at 7 d was the highest and then rapidly decreased in all experiments. This is due to the rapid hydrolysis of the easily degradable organic matter in the manure by AD microorganisms at the initial stage. Compared to the control, total VFAs concentration was significantly reduced with the addition of AF-BC. For instance, in the seventh day, total VFAs concentrations in A1, A5, and A10 (124.89, 94.64, and 153.69 mg/L) were lower than the control (214.66 mg/L). Wang et al. [24] and Sunyoto et al. [39] found that the addition of vermicompost BC and sawdust BC to the AD of chicken manure and white bread can significantly reduce VFAs accumulation and lead to an increase in methane production. In contrast, Pan et al. [23] reported that excess BC could cause an excessive acceleration of hydrolysis and acidogenesis-acetogenesis to an unbearable extent, resulting in an imbalance to the AD system and the accumulation of intermediates, which fi-

VFAs Analysis
VFAs accumulation during AD processes often causes drastic pH drops and inhibition of microbial activity [19]. Figure 5 indicates that all experiments in the control, A1, A5 and A10 exhibited a similar trend of total VFAs concentration variation. Total VFAs concentration at 7 d was the highest and then rapidly decreased in all experiments. This is due to the rapid hydrolysis of the easily degradable organic matter in the manure by AD microorganisms at the initial stage. Compared to the control, total VFAs concentration was significantly reduced with the addition of AF-BC. For instance, in the seventh day, total VFAs concentrations in A1, A5, and A10 (124.89, 94.64, and 153.69 mg/L) were lower than the control (214.66 mg/L). Wang et al. [24] and Sunyoto et al. [39] found that the addition of vermicompost BC and sawdust BC to the AD of chicken manure and white bread can significantly reduce VFAs accumulation and lead to an increase in methane production. In contrast, Pan et al. [23] reported that excess BC could cause an excessive acceleration of hydrolysis and acidogenesis-acetogenesis to an unbearable extent, resulting in an imbalance to the AD system and the accumulation of intermediates, which finally reduced methane production. Therefore, in the present study, total VFAs concentration in A5 was less than that in A10, which supported the result that high concentrations of AF-BC were not favorable for methane production.
nally reduced methane production. Therefore, in the present study, total VFAs concentration in A5 was less than that in A10, which supported the result that high concentrations of AF-BC were not favorable for methane production. As seen in Figure 5, acetic acid was the primary VFA from the AD of dairy manure under all experimental conditions. Acetic acid is one of the main precursors for methane production and could be directly utilized by acetotrophic methanogens for methane production [19]. Figure 5 also indicates that the addition of AF-BC led to the decrease in the concentration of acetic acid, implying that AF-BC could facilitate the transformation of acetic acid to methane and finally improve the methane yield. Lately, DIET has been considered as an effective pathway for enhancing methane production in the AD process [19,23]. Figure S2 shows that AF-BC possessed various functional groups, including C-H, C-O, C-N, N-O, and C=C, which are usually related with quinone, phenazine, and hydroquinone moieties [50,51]. In addition, these moieties can act as electron shuttles, accepting electrons from microorganisms and donating electrons to microorganisms, which are favorable for the DIET [50,51]. Thus, it can be inferred that AF-BC can enhance the metabolism of acetic acid via the DIET between acetogens and methanogens on the surface of AF-BC, which improved the efficiency of conversion of acetate to methane. As seen in Figure 5, acetic acid was the primary VFA from the AD of dairy manure under all experimental conditions. Acetic acid is one of the main precursors for methane production and could be directly utilized by acetotrophic methanogens for methane production [19]. Figure 5 also indicates that the addition of AF-BC led to the decrease in the concentration of acetic acid, implying that AF-BC could facilitate the transformation of acetic acid to methane and finally improve the methane yield. Lately, DIET has been considered as an effective pathway for enhancing methane production in the AD process [19,23]. Figure S2 shows that AF-BC possessed various functional groups, including C-H, C-O, C-N, N-O, and C=C, which are usually related with quinone, phenazine, and hydroquinone moieties [50,51]. In addition, these moieties can act as electron shuttles, accepting electrons from microorganisms and donating electrons to microorganisms, which are favorable for the DIET [50,51]. Thus, it can be inferred that AF-BC can enhance the metabolism of acetic acid via the DIET between acetogens and methanogens on the surface of AF-BC, which improved the efficiency of conversion of acetate to methane. Figure 6a indicates that TA in A1 was comparable to that in the control; however, TAs in A5 and A10 were much higher than that in the control during the AD process. A higher TA concentration with the addition of AF-BC was thought to exist because AF-BC contained various alkali and alkaline-earth metals such as K (1 g/kg BC), Ca (14 g/kg BC), and Mg (3 g/kg BC) (Table S3), which endowed the AF-BC with alkalinity properties and buffering abilities in the AD system [4,24]. Moreover, macronutrients such as N (49 g/kg) and P (2 g/kg) in AF-BC (Table S3) also played an important role as buffering agents in the AD system [3,4]. Therefore, Figure 6b also shows that the pH values in the AD of dairy manure with the addition of AF-BC increased compared to the control. Wang et al. [24] also mentioned that vermicompost BC increased the buffering capacity to avoid drastic reduction of pH owing to a high accumulation of VFAs and maintaining high activity of AD microorganisms during the AD of kitchen wastes and chicken manure, while resulting in an increase in AD performance and methane production. In this study, the increased TA concentration resulted in higher pH and OH − ions, which reacted with CO 2 to produce CO 3 2− or HCO 3 − . Both CO 2 and CO 3 2− /HCO 3 − could act as the electron acceptors and were reduced to methane by hydrogenotrophic methanogens [52,53]. Therefore, the increase in TA and pH values due to the addition of AF-BC could facilitate the maintenance of CO 2 in the form of CO 3 2− /HCO 3 − in aqueous solution, which enhanced the CO 2 utilization for methane production [25,52]. Thus, based on the results from this study, the addition of AF-BC at a moderate loading rate (or appropriate loading rate) could significantly improve the performance of AD of dairy manure. Future works will focus on analyzing microbial communities and the understanding of possible mechanisms associated with the AD of dairy manure with the addition of AF-BC. Figure 6a indicates that TA in A1 was comparable to that in the control; however, TAs in A5 and A10 were much higher than that in the control during the AD process. A higher TA concentration with the addition of AF-BC was thought to exist because AF-BC contained various alkali and alkaline-earth metals such as K (1 g/kg BC), Ca (14 g/kg BC), and Mg (3 g/kg BC) (Table S3), which endowed the AF-BC with alkalinity properties and buffering abilities in the AD system [4,24]. Moreover, macronutrients such as N (49 g/kg) and P (2 g/kg) in AF-BC (Table S3) also played an important role as buffering agents in the AD system [3,4]. Therefore, Figure 6b also shows that the pH values in the AD of dairy manure with the addition of AF-BC increased compared to the control. Wang et al. [24] also mentioned that vermicompost BC increased the buffering capacity to avoid drastic reduction of pH owing to a high accumulation of VFAs and maintaining high activity of AD microorganisms during the AD of kitchen wastes and chicken manure, while resulting in an increase in AD performance and methane production. In this study, the increased TA concentration resulted in higher pH and OH − ions, which reacted with CO2 to produce CO3 2− or HCO3 − . Both CO2 and CO3 2− /HCO3 − could act as the electron acceptors and were reduced to methane by hydrogenotrophic methanogens [52,53]. Therefore, the increase in TA and pH values due to the addition of AF-BC could facilitate the maintenance of CO2 in the form of CO3 2− /HCO3 − in aqueous solution, which enhanced the CO2 utilization for methane production [25,52]. Thus, based on the results from this study, the addition of AF-BC at a moderate loading rate (or appropriate loading rate) could significantly improve the performance of AD of dairy manure. Future works will focus on analyzing microbial communities and the understanding of possible mechanisms associated with the AD of dairy manure with the addition of AF-BC.

Conclusions
The effects of AF-BC on the AD of dairy manure were examined in the present study. The AD of dairy manure with the addition of AF-BC at 1-5 g/L showed a significant enhancement of biogas and methane production compared to the control. The biogas and methane yields during the AD of dairy manure increased by 15.51% and 14.61%, with the addition of 1 g/L AF-BC, and 26.09% and 26.88% with the addition of 5 g/L AF-BC. The main reasons for the increase in AD performance were that the addition of AF-BC improved the buffering ability of the AD system and DIET between AD microorganisms. However, the addition of AF-BC at 10 g/L was not favorable for methane production, possibly owing to toxicity of AF-BC at high loading. Besides, the addition of AF-BC also reduced the lag phase and VFAs concentrations while increasing total alkalinity in the AD process. Overall, this study provides valuable information for application of AF-BC in the enhancement of AD of dairy manure. This sustainable reuse of various wastes (waste hay, manure) at dairy farms through interaction of biochar and AD could significantly enhance environmental and agricultural sustainability at dairy and other animal farms.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/agronomy12040911/s1, Table S1: Summary of effects of various biochars on methane production during anaerobic digestion [54,55]; Table S2: The biotoxicity of leaching solutions of AF-BC at various concentrations; Table S3: The physicochemical characteristics of AF and AF-BC; Figure S1: Experiment set-up for anaerobic digestion of dairy manure with the addition of AF-BC; Figure S2: FT-IR spectrum of AF-BC.