*3.1. Substrate and Biochar Properties*

The liquid digestate used for BMP had 7.86 of pH, 68.8 <sup>μ</sup><sup>S</sup> × cm−<sup>1</sup> of EC, 94.7% of MC, 5.3% of TS, 59.3% of VS, and 40.7% of AC, while the FW mixture (substrate) used for BMP tests had 5.6% of MC, 94.4% of TS, 95.8 of VS, and 4.2% of AC (Table 1). The elemental analysis showed that FW mixture was characterized by 44–47.8%, 5.7–6.2%, 39.9–44.4%, 1.45–1.58%, 0.24–0.26% of C, H, O, N, S, respectively (by dry mass base). In addition, the FW mixture was characterized by a pH of 5.62 and EC of 3.6 mS × cm<sup>−</sup>1.

The five types of biochars were used depending on the production conditions as follows: temperature/time/pressure; however, the HTC280 means a hydrothermal carbonization process at 280 ◦C in 60 min. The biochars were characterized by MY ranging from 34.3% to 56.4% for 400/60/15 and HTC280, respectively (Table 3). The highest MY was noted in the case of HTC and the 300/60/15 process (Table 3). As result, for biochars with high MY, less substrate and energy are needed for their production in comparison to biochars with low MY. Nevertheless, in such a scenario, the substrate is less converted, and biochar may not have the desired properties [31]. Produced biochars had a relatively low volatile solid content compared to FW used for biochar production. On the other hand, biochars had a much higher ash content than the FW mixture. The ash content in biochar varied from 10.4% to 39.1%, while the FW mixture had only 4.2% of ash. The produced biochar was also analyzed for specific surfaces area (SSA) according to BET theory, total pore volume <50 nm (Vt), and average pore size <50 nm (L). Moreover, produced biochars had a value of SSA ranging from 0.26 to 0.64 g × <sup>m</sup>−2, and pore size ranging from 5.2 to 7.1 nm (Table 3). The total pore volume ranged from 3.3 × <sup>10</sup>−<sup>4</sup> cm3 × <sup>g</sup>−<sup>1</sup> to 8.2 × <sup>10</sup>−<sup>4</sup> cm<sup>3</sup> × <sup>g</sup><sup>−</sup>1, excluding 400/60/15 biochar that had Vt of 11.3 × <sup>10</sup>−<sup>4</sup> cm<sup>3</sup> × <sup>g</sup>−<sup>1</sup> (Table 3). The pyrolysis results in biochars' pH increase from 5.62 to 8.61–10.75, except HTC280, for which pH decreased to 5.59. Except for biochar produced at 300 ◦C, all biochars had higher EC in comparison to the FW (Table 3).

**Table 3.** Low-temperature biochar properties.


\* as-received base, \*\* dry base, \*\*\* measured in solution: 1 g BC to 10 mL deionized water, after 30 min.

The pore volume, pore size, specific surface area, pH, elemental composition, surface functional groups, electrical conductivity (EC), and cation exchange capacity (CEC) are considered as key biochar physicochemical properties which affect the AD and biogas production [32]. Porosity is considered a key factor to recognize the plausible relations with microbes in AD. The porosity is characterized in terms of the average diameter [33] and is described by three main pore type: micropores (<2 nm), mesopores (2–50 nm), and macropores (>50 nm). For activated carbon, a specific surface area of micropores may constitute up to 95% of the total SSA of activated carbon. As result, micropores decide about the adsorption capacity. On the other hand, mesopores significantly contribute to the adsorption of larger particles, such as dye or humic acids [34]. Generally, pores with a radius over 25 nm are considered transport pores, while pores smaller than 25 nm are considered adsorbing ones [35]. Besides absorption, pores provide a microorganism habitat for proliferating since the typical size of bacteria is 0.3 μm to 13 μm. The higher the SSA, the more effective biochar is in the interaction with the surrounding species [33]. The SSA of biochar varied significantly depending on substrate and process conditions. The SSA in activated carbons varies from 419 to 3102 m<sup>2</sup> × <sup>g</sup>−<sup>1</sup> [36], while for low-temperatures and not activated biochar (350–500 ◦C), it varies from 0.36 to 5.31 g × <sup>m</sup><sup>−</sup>2. Moreover, pore volume and average pore size in such biochars vary from 10 × <sup>10</sup>−<sup>4</sup> to 80 10−<sup>4</sup> cm<sup>3</sup> × <sup>g</sup><sup>−</sup>1, and 2.39 to 14.60 nm, respectively [37]. It means that biochars produced in the current study do not differ significantly in comparison with other biochars produced at similar temperatures but have incomparably smaller SSA in comparison to activated carbon.

Since electrically conductive materials (i.e., mineral particles, carbon materials) added to AD show a reduction in lag phase and increased methane production rates, electrically conductive materials found more attention. Conductive materials (i.e., biochar, graphite, activated carbon) added to AD can promote direct interspecies electron transfer (DIET) between syntrophic partners [38]. The DIET is an alternative to interspecies H2/formate transfer for syntrophic electron exchange between microbial species. In AD, some methanogens can receive electrons from other microorganisms by molecular electric connections or by conductive materials [39]. For that reason, materials with good electrical conductivity properties are assumed to help enhance methane fermentation. The biochar electrical conductivity can be measured in solid-state [40], as powder [41], or in water solution, like soil EC is measured [42]. The EC varies depending on the method, and therefore caution is needed when data are compared between studies. Nevertheless, results from the same method show that an increase in pyrolysis temperature increases EC value. In addition, this is due to higher carbonization and an increase in ash content [41]. Biochar EC values may vary from 0.04 mS × cm–1 to 54.2 mS × cm–1, and besides pyrolysis temperature, the feedstock affects EC as well [42]. These show that biochar produced in this study had relatively low EC (3.04–7.69 mS × cm–1) in comparison to biochars found in the literature.

The pH is an important factor affecting the BMP test results and will be described in more detail later. It is worth noting here that all biochars except HTC280 were alkaline, and their pH increased with process temperature, while HTC280 become more acidic. In addition, it is worth noting that pH did not change when pressure was applied, while EC increased, 3.04 vs. 3.57 mS × cm–1 for biochars made at 300 ◦C, and 4.53 vs. 7.69 mS × cm–1 for biochars made at 400 ◦C. This suggests that pressure may potentially be a parameter that can be used to modify EC. This finding should be further investigated.

#### *3.2. Biochemical Methane Potential—Theoretical and Experimental*

The effect of low-temperature biochar addition on the cumulative biomethane production process for 21 days was investigated (Figure 3). The result shows that the highest methane production was obtained for biochar from hydrothermal carbonization (HTC280) and biochar produced at 400/60/0. The control reactors obtained 347.9 mlCH4 × gVS−1, while reactors with biochars 400/60/0 and HTC280 had 360.1 mlCH4 × gVS−<sup>1</sup> and 365.2 mlCH4 × gVS<sup>−</sup>1, respectively (Figure 3). The lowest value of BMP was obtained for reactors where biochar 400/60/15 was added (331.7 mlCH4 × gVS<sup>−</sup>1).

**Figure 3.** The biomethane production from food waste (*n* = 4). The results show CH4 production in ml per gram of food waste volatile solids, and the CH4 produced by inoculum (digestate) was subtracted.

The theoretical biochemical methane potential of the food waste mixture was 460 mlCH4 × gVS−<sup>1</sup> (Equation (3)). In addition, theoretical calculations showed that, for complete substrate conversion into biogas, 437 mlCO2 × gVS−1, 25 mlNH3 × gVS−1, and 2 mlH2S × gVS−<sup>1</sup> will be produced. The experimental BMP test for control samples after 21 days obtained 347.9 mlCH4 × gVS−<sup>1</sup> (Figure 3) reaching 75.5% substrate biodegradation.

Experimental BMP values obtained in this study are lower than the BMP value for source-separated domestic FW collected in the EU, for which BMP ranges from 420 to 470 mlCH4 × gVS−<sup>1</sup> [43]. Nevertheless, the theoretical potential is in this range, and most reactors reached BD over 75%, which suggests that BMP was done properly, especially since the processing time was only 21 days.

The CH4 production effect shows a difference between the value obtained from the control (D + FW) and the reactor with biochar (Table 4). When the value is greater than 0, biochar increased the methane production, while when the value is lower than 0, biochar decreased methane production in comparison to control. The biochar addition had a positive effect on methane production from FW. Only biochar 400/60/15 showed a decrease in methane production. For this biochar, all reactors produced less methane than control. For other biochars, mean value from the repetitions was generally positive, and more methane was produced than by control. Nevertheless, biochars produced at 300 ◦C led to a decrease in methane production in some repetitions. The highest methane production was obtained from reactors where 400/60/0 and HTC280 were added, 3.5%, and 3.6% respectively (Table 4). Among literature, various effects of biochar addition on methane production effect can be found. Results differ from total process inhibition to a several-fold increase in methane production. The effect is highly dependent on factors such as initial conditions of the batch test, used inoculum and substrate, the substrate to inoculum ratio, biochar dose, biochar type, and conditions of its production) [22,44–47]. Kaur et al. [47] added biochars produced at 550 ◦C and 700 ◦C from wood, oilseed rape, and wheat straw at a dose of 10 gBC·L−<sup>1</sup> to co-fermentation of food waste and sewage sludge under a high SIR level of 11.5 by VS. As a result, cumulative methane production increased from 4.5% to 24%. In addition, the highest increase was observed for biochar made from wheat straw at 550 ◦C, and the lowest for oilseed rape produced at 700 ◦C [47]. On the other hand, Sunyoto et al. [22] added biochar made from pine sawdust at 650 ◦C to anaerobic digestion of food waste. Biochar doses of 8.3, 16.6, 25.1, and 33.3 gBC·L−<sup>1</sup> were studied, and results showed that only a dose of 8.3 increased methane production by 6.2%, while others decreased methane production up to 12.9%. It is also worth noting that biochar doses that increased methane potential did not do it significantly, while biochar doses higher than 25.1 gBC·L−<sup>1</sup> significantly decreased methane production (at the *<sup>p</sup>*-value of 0.002) [22]. Furthermore, the results of Zhang et al. [45] that conducted methane fermentation of FW at thermophilic conditions showed that the lowest of tested biochar doses (6 gBC·L−1) gave the highest cumulative methane production [45]. Because, in the current study, only one dosage of 0.65 gBC·L−<sup>1</sup> was tested, and other research proved that a biochar dose of up to 10 gBC·L−<sup>1</sup> can improve methane production, higher doses of 400/60/0 and HTC280 should be tested in the future.

The initial pH in all reactors with FW and biochar differed from 7.62 to 7.91, while EC differed from 56.1 to 67.9 <sup>μ</sup><sup>S</sup> × cm−1. After 21 days of the process, pH differed from 7.92 to 8.03, and EC differed from 68.7 to 77.7 <sup>μ</sup><sup>S</sup> × cm−<sup>1</sup> (Table 4). For comparison, digestate alone had an initial pH and EC of 7.86, and 66.8 <sup>μ</sup><sup>S</sup> × cm−1, respectively, while, after 21 days, these parameters were 8 and 71.8 <sup>μ</sup><sup>S</sup> × cm−1, respectively (Table 4). The initial pH is an important parameter affecting methane yield in batch experiments, but no one value would show the correctness of the process [48]. The initial pH and then its changes during the process affect product yield, as optimal pH was reported value from 6.8 to 7.4 [49]. Anaerobic digestion is a four-stage process consisting of hydrolysis, acidogenesis, acetogenesis, and methanogenesis. The pH is crucial in each stage, and each of them required a different value. A positive correlation was found between the hydrolysis rate and pH [49]. The optimal pH for acidogenesis is 5.5–6.5 [50], while methanogenesis is effective

when pH is around 6.5–8.2 (with optimum pH of 7.0) [51]. Even though methanogenesis is effective at 6.5, the methanogens' growth rate is reduced significantly at a pH lower than 6.6 [52]. Therefore, the best result of AD can be obtained by a division process into two-stage hydrolysis with acidogenesis, and acetogenesis with methanogenesis [49]. The pH also affects the decomposition of total solids, and volatile solids in the reactor, as well as volatile fatty acid composition [53,54]. Nevertheless, in this study, biochar addition did not significantly change pH (*p* < 0.05), and as result, all reactors had similar conditions. Here it is worth noting that, for some reason, biochars with completely different pH, 10.19 vs. 5.59 for 400/60/0 and HTC280, respectively, showed the best methane production enhancement. The reason for that may be some other biochar properties that were not considered in this study. Maybe these biochars enhanced buffer capacity in the highest way despite different pH, and, as a result, provided better conditions for microorganism growth.

**Table 4.** The biochar addition effect on the process residues and methane production, after 21 days.


The EC shows the number of dissolved salts in solutions and is proportional to the quantity of these salts. The solutions with higher salt concentration have a greater ability to conduct an electrical current [42]. In the methane fermentation process, this parameter alone is rather useless. Nevertheless, EC can be used in online monitoring of biogas plants for prediction in advanced methane production of up to two days [55], or alkalinity [56]. As mentioned previously, conductive materials can enhance methane production by DIET. Nevertheless, in this study, biochar addition did not change the electrical conductivity of the solution significantly (*p* < 0.05); therefore, it is highly probable that DIET had no effect here.

Generally, biochar addition did not lead to significant (*p* < 0.05) changes in pH, and EC obtained biodegradability, substrate mass reduction, and amount of produced CH4. However, even though no statistically significant differences were found, results of biochar made at 400/60/0 and HTC280 showed to always have higher methane production than control, on average by 3.5% (Table 4). At first sight, it looks small; however, when the 1 MWe FW biogas plant working for 8000 h per year is considered, after the addition of BC, the additional 280 MWh of electricity may be produced. It is worth noting that usually

biogas plants have problems with the utilization of heat, which in this case may be used for BC production.
