**3. Discussion**

RS and WW belonging to lignocellulosic biomass are normally composed of three major components, namely polysaccharide cellulose and hemicellulose and the aromatic non-polysaccharide lignin. The higher the lignin content in WW, the tighter the physical structure of WW than RS (Table 1) [8]. Thus, the affinity of internal cellulose and hemicellulose of WW to hydrolase was relative weak. Maintaining the pH close to neutral (6.8–7.2) is preferable for methanogenesis, whereas the optimal pH for hydrolysis and acidogenesis is within the range of 5.5–6.5 [17,18]. At the initial stage of digestion, the accumulation of organic acids produced by hydrolysis and acidogenesis was difficult due to the low affinity of wood chips to hydrolase, thus the system pH cannot be lowered (Figure 1c). The high pH value in turn suppressed the activities of hydrolysis and acidogenesis, as well as methanogenesis of the single digestion of WW. Therefore, the methane production of RS was higher than WW during the single-substrate digestion process (Figure 1b).


**Table 1.** Characteristics of WW, RS, PM and inoculum.

Notes: NA: not analyzed; PM: pig manure, WW: wood waste, RS: rice straw. WW + NaOH: WW pretreated with NaOH, RS + NaOH: RS pretreated with NaOH.

Salehian et al. [7] found that the yield of methane produced from pine wood pretreated with 8% NaOH (100 ◦C for 10 min) was increased from 65 mL CH4/g VS to 178.2 mL CH4/g VS after 45 days of incubation. Similarly, Mirahmadi et al. [15] observed a 50% enhancement in methane production after the 7% NaOH pretreatment (100 ◦C for 2 h) of birch. Though under different conditions of alkaline pretreatment, the yield of methane in the current work of eucalyptus was also remarkably elevated from 175.81 to 243.53 mL CH4/g VS after NaOH pretreatment (Figure 2b). The complex structures of cellulose, hemicellulose, and lignin are difficult for microorganisms to degrade [19]. The daily and cumulative methane production of the single-digestions of WW and RS was evidently increased by NaOH pretreatment (Figure 2). This result was because alkali can break down the ether bonds between lignin and saponified the ester bonds between hemicellulose and lignin to weaken the internal hydrogen bonds within cellulose and hemicellulose [14], thus making cellulose and hemicelluloses accessible to hydrolytic enzymes. The NaOH pretreatment decreased the C/N ratio of WW and RS to a low level from 54.5 and 47 to 27.2 and 29.1, respectively, which was more acceptable for biodegradation over the first several days (Table 1) [20]. Notably, the improvement of methane production of the single digestion of WW (increased by 38.5%) by NaOH pretreatment was significantly higher (*p* < 0.05) than that of RS (increased by 12.2%) (Figure 4). This result might be occasioned by the following reasons. First, the NaOH pretreatment could increase the effective contact area between anaerobic microorganisms and substrates via reducing lignin content or breaking down lignin-hemicellulose complexes [14,21]. Therefore, WW, which contained higher amounts of lignin content than RS (Table 1) [8], had higher potential in increasing effective contact area when pretreated with NaOH. Second, the amounts of cellulose and hemicellulose that can be used to produce methane by methanogens were more contained in WW than in RS (Table 1). Thus, the improvement of the methane yield of WW could be visible when the effective contact area between anaerobic microorganisms and cellulose and hemicellulose was increased. Finally, after NaOH pretreatment, the pH value of the single digestion of WW was decreased to neutral, which was preferable for methanogenesis. However, the pH of the single-digestion of RS was almost unchanged (Figure 2e).

Compared with the single-substrate anaerobic digestions of PM and RS, the biomethane yield of the co-digestion of PM and RS was significantly (*p* < 0.05) increased (Figure 3). The methane yield of RS and PM co-digestion had increased by over 30% than both of the PM and RS single-digestions (Figures 3 and 4). High C/N ration and rich NH4–N content are the dominant factors limiting the methane production rates of RS and PM (Table 1), respectively [22]. Mixing PM and RS could balance the C/N ratio (Table 2) and nutrition, as well as toxic compounds generated during the digestion [23]. The AcoD with different substrates could also stimulate the synergistic effects of microorganisms for achieving improved biogas production [24].


**Table 2.** Experimental design.

Notes: PM/WW indicates the AcoD of PM and WW. PM/RS indicates the AcoD of PM and RS. PM/WW(NaOH) indicates the AcoD of PM and WW (pretreated with NaOH), PM/RS(NaOH) indicates the AcoD of PM and RS (pretreated with NaOH).

Co-digestion with WW had no beneficial effect on the methane production of PM (Figure 3b), which may be ascribed to the rigid and recalcitrant lignocellulosic structure of WW. On the other hand, the methane production of the co-digestion of WW and PM had nonsignificant difference with that of the single-digestion of PM (Figure 3b), suggesting WW can be utilized as a supplementary during the PM anaerobic digestion without affecting its methane production efficiency. We showed that the WW pretreated with NaOH had a satisfactory methane production performance in anaerobic digestion (Figure 2a,b). The methane production of WW treated with AcoD (pretreated with NaOH) was increased by 75.8% compared with the untreated WW. Furthermore, the growth rates of methane production of WW treated with NaOH and AcoD (pretreated with NaOH) were signally higher than the RS that under the same optimizing strategies (Figure 4). WW is widely distributed in vast rural areas and has huge reserves. Therefore, when treated with targeted approaches, WW has a considerable potential transforming from the worthless organic waste to a promising fermentation substrate.
