# Addition of biochar and trace elements in the form of industrial FeCl3 to stabilize anaerobic digestion of food waste: dosage optimization and long-term study

Gabriel Capson-Tojo, Chloé Girard, Maxime Rouez, Marion Crest, Jean-Philippe Steyer, Nicolas Bernet, Jean-Philippe Delgenes, Renaud Escudié

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## To cite this version:

Gabriel Capson-Tojo, Chloé Girard, Maxime Rouez, Marion Crest, Jean-Philippe Steyer, et al. Addition of biochar and trace elements in the form of industrial FeCl3 to stabilize anaerobic digestion of food waste: dosage optimization and long-term study. Journal of Chemical Technology and Biotechnology, 2019, 94 (12), pp.505-515. 10.1002/jctb.5797. hal-03685985

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Submitted on 2 Jun 2022

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Biochar and industrial FeCl3 as additives to favor the
consumption of volatile fatty acids during anaerobic
digestion of food waste

<table><tr><td>Journal:</td><td>Journal of Chemical Technology &amp; Biotechnology</td></tr><tr><td>Manuscript ID</td><td>JCTB-18-0700</td></tr><tr><td>Wiley - Manuscript type:</td><td>Research Article</td></tr><tr><td>Date Submitted by the Author:</td><td>10-Jun-2018</td></tr><tr><td>Complete List of Authors:</td><td>Capson-Tojo, Gabriel; LBE, INRA, Univ. Montpellier; Suez, CIRSEE<br>Girard, Chloe; Suez, CIRSEE<br>Rouez, Maxime; Suez, CIRSEE<br>Crest, Marion; Suez, CIRSEE<br>Steyer, Jean-Philippe; LBE, INRA, Univ. Montpellier<br>Bernet, Nicolas; LBE, INRA, Univ. Montpellier<br>Delgenès, Jean-Philippe; LBE, INRA, Univ. Montpellier<br>Escudié, Renaud; LBE, INRA, Univ. Montpellier</td></tr><tr><td>Key Words:</td><td>Biogas, Anaerobic Digestion, Char, Design of Experiment (DOE)</td></tr></table>

SCHOLARONE™
Manuscripts
1 Biochar and industrial $FeCl_3$ as additives to favor the consumption of volatile fatty acids

2 during anaerobic digestion of food waste

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4 Short title: Biochar and industrial $FeCl_3$ to stabilize the anaerobic digestion of food waste

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6

7 Gabriel Capson-Tojo<sup>a,b</sup>, Chloé Girard<sup>b</sup>, Maxime Rouez<sup>b</sup>, Marion Crest<sup>b</sup>, Jean-Philippe Steyer<sup>a</sup>

8 Nicolas Bernet<sup>a</sup>, Jean-Philippe Delgenès<sup>a</sup>, Renaud Escudié<sup>a,*</sup>

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10 <sup>a</sup> LBE, INRA, Univ. Montpellier, 102 avenue des Etangs, 11100 Narbonne, France

11 <sup>b</sup> Suez, CIRSEE, 38 rue du Président Wilson, 78230 Le Pecq, France

12 * Corresponding author: tel. +33 (0) 468.425.173, e-mail: renaud.escudie@inra.fr

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14 Abstract

15 BACKGROUND

16 Although anaerobic digestion is a promising alternative for the valorization of complex substrates such as food waste, this process is yet to be optimized and options for its stabilization must be developed. The goal of this study was to assess for the first time if the addition of biochar, together with trace elements in the form of an industrial $FeCl_3$ solution, could serve as method for stabilizing anaerobic digestion of food waste.

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21 RESULTS

22 Results from batch reactors demonstrated that the supplementation of both biochar and industrial $FeCl_3$ favored the digestion kinetics. Their addition improved the maximum methane production rates (from 886 up to 1498 ml·g VS<sup>-1</sup>·d<sup>-1</sup>) and the average daily methane production rates (from 280 up to 376 ml·d<sup>-1</sup>), related to acetate and propionate consumption,

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26 respectively. Continuous reactors confirmed the batch results, with higher methane production

27 rates (up to 1.75 l·l⁻¹·d⁻¹) and lower concentrations of both acetate and propionate when

28 biochar and FeCl3 were added.

29 **CONCLUSION**

30 Addition of biochar and industrial FeCl3 favored the digestion kinetics, improving volatile

31 fatty acid consumption and the methane production rates. Although more research is needed,

32 these materials appear as an economically-feasible alternative for stabilizing the valorization

33 of food waste at industrial scale.

34

35 **Introduction**

36 The increasing production of food waste (FW) and novel international regulations call for the

37 development of novel sustainable technologies for its treatment and valorization (1). Among

38 the existing options, anaerobic digestion (AD) is an environmental-friendly process that

39 provides an efficient waste treatment, producing at the same time biogas and digestate.

40 Nevertheless, FW AD is a complex biological process that often leads to inefficient results or

41 reactor acidification. The first complication occurring during FW AD is related to the fast

42 biodegradability of this substrate, which is mainly composed of easily degradable

43 carbohydrates. Therefore, the reactors can be easily overloaded, especially in batch systems

44 (2), causing an unbalance between the acidogenesis/acetogenesis and the methanogenesis

45 steps, which results in an initial accumulation of volatile fatty acids (VFAs) and a consequent

46 pH drop (1,3–6). The second issue to be dealt with is related to the high protein content of this

47 substrate and its low water content (~20 % total solids; TS). During AD, organic nitrogen

48 (usually in the form of proteins) is reduced into ammonia nitrogen (total ammonia nitrogen;

49 TAN), which, in its free form (free ammonia nitrogen; FAN) is toxic to microorganisms,

50 especially to methanogenic archaea, eventually resulting also in an accumulation of VFAs.
51 Thus, different FW AD studies have reported inefficient performances due to high concentrations of FAN (7-9).

53 The main alternative that has been applied to favor the consumption of VFAs in FW AD is the supplementation of trace elements (TEs) (10-14). TEs have been found to favor VFA consumption in both mesophilic and thermophilic AD of FW, mainly due to a favored synthesis of critical enzymes in the process of syntrophic hydrogenotrophic methanogenesis (HM), which is known to be the predominant methane-producing pathway during FW AD (2,11,15-17). TEs addition has been successfully applied to avoid accumulation of VFAs at high organic loading rates (OLRs) (10,18,19) and to recover acidified reactors (20,21).

60 Another strategy to favor VFA consumption during AD that has gained attention in the last few years is the addition of conductive materials (22,23). This approach is based on the capability of these materials for improving microbial interactions, by allowing the formation of biofilm onto their surfaces and by facilitating the occurrence of direct interspecies electron transfer (DIET), a mechanism in which the transfer of electrons between species occurs through shared physical connections. DIET is a faster and more efficient route for electron transfer than mediated interspecies electron transfer (23) and, as molecular hydrogen is no longer formed, VFA oxidation is thermodynamically independent of its concentration (22,24).

68 Moreover, recent studies have suggested that acetic acid (HAc), butyric acid (HBu) and propionic acid (HPr) can be metabolized through DIET (22,25-27). Materials such as granular activated carbon (GAC), carbon cloth or magnetite have been found to favor VFA consumption and methane production during AD (26,28,29). Among them, GAC appears as a particularly performant alternative that has been found to promote the consumption of HAc, HBu and HPr during AD of dog-food waste and FW (22,28,30).

74 However, both TEs and GAC are expensive and their application at an industrial scale is far from being feasible. Thus, cheaper alternatives must be found. A simple substitute for the TEs
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76 solution may be industrial $FeCl_3$, which consists on an acid solution, highly concentrated in different metals (mainly Fe) that is commonly applied in wastewater treatment plants and anaerobic digesters for pH control worldwide. Concerning GAC, an affordable substitute may be biochar, which is also a carbon-conductive material with the capacity of accepting and donating electrons (31). In addition, biochar may also improve the AD process by providing buffering capacity and by sorption of inhibitors (32). It also favors nutrient retention in the digestates, facilitating nutrient uptake if the digestate is spread on land for plant cultivation (32,33). Moreover, as biochar can be produced from digestate (34) or directly from FW (35), its addition for AD improvement clearly fits within the approach of environmental biorefinery.
80 Biochar has been previously used as amendment for AD: (i) for biogas purification (removal of $H_2S$ or $CO_2$) (36–38), (ii) as reactor packing for biofilm support (39), (iii) as AD substrate (40), (iv) for nutrient supplementation (41), (v) as matrix for sorption of inhibitors (42,43) or (vi) as means to increase the buffer capacity of the system (33). However, only three studies have been carried out dealing with low-dosage of biochar for improving the AD kinetics. Luo et al. (44) studied the addition of biochar for improving the kinetics of methane production and VFA degradation ($HAc$ and $HBu$) with glucose as substrate, concluding that a positive effect existed. Zhao et al. (27) investigated the effect of biochar and ethanol supplementation in reactors degrading $HPr$ and $HBu$, observing that the degradation of both VFAs was improved by adding biochar and concluding that bacteria known to participate in DIET (i.e. $Geobacter$ species) were attached onto the biochar surface. Finally, Sunyoto et al. (45) assessed the influence of biochar addition in a two-phase AD reactor treating aqueous carbohydrates FW, concluding that biochar supplementation increased the hydrogen and methane production rates. They attributed this improvement to a promotion of the biofilm formation.
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101 To the knowledge of the authors, no study has been carried out so far to assess if addition of biochar can favor VFA consumption during AD of complex substrates such as FW. Moreover, the stabilizing effect of jointly adding industrial $FeCl_3$ (to provide TEs) and biochar on the performance of FW AD has been investigated for the first time. Therefore, the goal of this study was to: (i) optimize the concentrations of both biochar and industrial $FeCl_3$ using a batch experimental design to improve the kinetics of FW AD and (ii) evaluate the effect of industrial $FeCl_3$ and biochar supplementation on the AD performance of semi-continuous pilot reactors treating commercial FW.

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## 110 Experimental

### 111 *Inoculum, substrate and AD additives*

112 The reactors were inoculated with digestate from an industrial plant treating different organic streams at high TAN/FAN concentrations (5.04 g TAN·l⁻¹; 615 mg FAN·l⁻¹). Thus, it was assumed that the microorganisms would be adapted to high the high TAN/FAN levels associated with FW AD (9). The TS content of the inoculum was 5.81 ± 0.02 %, with a proportion of volatile solids (VS) of 59.13 ± 0.08 %. The activity of the inoculum was verified prior its utilization using ethanol as substrate. The commercial FW was collected from five major producers from the region of the Grand Narbonne, in the south of France. A proportional mixture (wet weight) was used as substrate. The main characteristics of the FWs, as well as those of the mixture used as substrate and the inoculum are presented in Table 1.
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121 The characteristics of the FW mixture were in agreement with typical values presented in the literature (1), which indicated that it could be considered as a representative sample of a general FW. It had a TS contents of 21 % (90.3 % VS), it was mainly composed of carbohydrates (618 g·kg TS⁻¹) and it had relatively low C/N ratios (16.1). No sulfur was detected. A more extensive characterization and a deeper discussion of the results can be
126 found in Capson-Tojo et al. (9).

127 The industrial $FeCl_3$ was provided by an industrial AD plant, where it is still used for pH

128 controlling purposes. The characteristics and composition of this industrial solution, rich in

129 TEs, are shown in Table 2.

130 The biochar was natural slow-pyrolyzed wood charcoal, commonly found in the market for

131 several applications. Before utilization, the biochar was grinded and sieved at 600 µm.

132 *Batch experimental design for dosage optimization*

133 A multilevel factorial design was used to optimize the dosage of biochar and industrial $FeCl_3$

134 and to evaluate their individual effect on the methane production and the VFA production-

135 consumption kinetics. Digestate from continuous reactors digesting FW was used as inoculum

136 (after consumption of the remaining VFAs). This sludge had a TS content of 5.17 %, with

137 60.2 % corresponding to VS and had a TAN content of 7.27 g·l⁻¹. Sixty g of FW were added

138 as substrate in all the reactors. A substrate to inoculum (S/X) ratio of 1 g VS·g VS⁻¹ was

139 applied, leading to initial FW concentrations of approximately 27 g VS·FW·l⁻¹. The reactors

140 were incubated at 37 °C. The working volumes ranged from 487 ml to 529 ml. Two different

141 concentrations of the $FeCl_3$ solution (0.1 and 0.2 g Fe·l⁻¹) and three of biochar (10, 55 and 100

142 g·l⁻¹) were tested. As a result, an experimental design with 12 conditions was defined (6

143 conditions in duplicate; Table 3). Two consecutive batch feeding were carried out. The first

144 feeding served for inoculum adaptation (results not presented), and the results of the second

145 one were used for modelling purposes. It must be mentioned that, based on previous results, it

146 was decided not to include into the experimental design reactors containing only biochar and

147 $FeCl_3$. As it was demonstrated in Capson-Tojo et al. (30), although the separate addition of

148 GAC and TEs also enhanced the VFAs degradation kinetics during FW AD, the best

149 performances were achieved when both of these reagents were added simultaneously. With

150 these results as starting point, the current experimental designed was defined. The same study
151 was also used to define the levels of industrial $FeCl_3$ and biochar to be tested (30).

152 This experimental design was chosen because it allows analyzing the effects of the selected factors (biochar and $FeCl_3$ concentrations) on a chosen output through the entire experimental region covered. This implied that it was possible to distinguish between the effect of biochar and $FeCl_3$ without the need of single additive treatments. It allows so by predicting the responses using a quadratic model (Eq. 1):

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$$y = a_0 + \sum_{i=1}^{k} a_i \cdot x_i + \sum_{i=1}^{k} a_{ii} \cdot x_i^2 + \sum_{i<j} a_{ij} \cdot x_i \cdot x_j \quad \text{Equation 1}$$

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160 Where y is the response to be predicted, $x_i$ are the studied factors and $a_i$, $a_{ii}$ and $a_{ij}$ are the parameters corresponding to each factor. These parameters represent the linear effects, the quadratic effects and the interactional effects, respectively. The first coefficient $a_0$ is required for fitting the mathematical model. The p-values from F-tests (95 % confidence) and the coefficient of determination $R^2$ were used to evaluate the fitness of the model. The experiment was designed and evaluated using the software STATGRAPHICS Centurion XVI Version 16.1.03 (©StatPoint Technologies Inc.).

167 The reactors were incubated in an Automated Methane Potential Testing System (AMPTSII)

168 (Bioprocess Control, Sweden). Twelve reactors from the AMPTSII system, with a total volume of 600 ml, were used. According to the manufacturer instructions, they were connected to $CO_2$ traps (NaOH solutions) and to gas flow meters to determine continuously the methane flow rates. These reactors also allowed the follow-up of the dynamics of VFA production-consumption. A hole present in each vessel was used as sampling port, facilitating an easy sampling of the digestate. The reactors were agitated during one minute every 10 minutes at 40 rpm.

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175 The criterion followed to decide when the batch had finished was the total consumption of the
176 HPr in the reactors (26 days). Blank reactors (without substrate addition) were carried out to account for the endogenous respiration of the inoculum.

178 *Continuous pilot scale reactors*

179 In parallel to the batch reactors, two different pilot scale reactors were run: a control system simply digesting FW and a doped reactor supplemented with biochar and the FeCl₃ solution.

180 Both reactors were incubated at 37 °C and had a working volume of 12 l. The reactors were fed once per day, initially with an OLR of 1.4 g VS·l⁻¹·d⁻¹, corresponding to a hydraulic retention time (HRT) of 110 days. This value was increased to 2.8 g VS·l⁻¹·d⁻¹ (HRT of 55 days) after 77 days of operation.

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The volume of the reactors constant. The FeCl₃ solution was diluted with water (x20 vol:vol) and dosed into the supplemented reactor to keep a constant concentration of 100 mg Fe·l⁻¹ (value calculated from optimal results reported in the literature (10–12)). This corresponded to concentrations of 0.38 mg Mn·l⁻¹, 0.19 mg Zn·l⁻¹, 0.03 mg Ni·l⁻¹, 0.01 mg Co·l⁻¹ and 0.01 mg Mg·l⁻¹ in the reactor. The initial concentration of biochar in the supplemented reactor was 10 g·l⁻¹. As it will be further explained, this concentration was increased up to 50 g·l⁻¹ to favor the consumption of the accumulated VFAs, according to the results obtained from the batch experimental design.

The pilot reactors consisted of jacketed cylindrical vessels made of stainless steel that had inner stirring blades to provide continuous agitation. A more detailed description of the reactors can be found elsewhere (9). The experiments lasted for 196 days.
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201 solvent extraction (46), respectively. The protein content was calculated using the total

202 Kjeldahl nitrogen (TKN) contents (6.25 g protein·g N⁻¹ (48)). The TKN and NH₄⁺

203 concentrations were determined using an AutoKjeldahl Unit K-370, BUCHI. The contents of

204 organic (TOC) and inorganic carbon (IC) were measured with a Shimadzu (Kyoto, Japan)

205 TOC-VCSN Total Organic Carbon Analyzer coupled to a Shimadzu ASI-V tube rack. The C/N

206 ratio was calculated as TOC/TKN. A WTW (London, United Kingdom) pHmeter series

207 inoLab pH720 was used for pH measurement. Finally, the biochemical methane potentials

208 (BMPs) of the substrates were measured according to Motte et al. (49).

209 *Gas quantification and analysis*

210 The amount of methane produced was automatically measured in the AMPTSII system and

211 the volume of biogas produced in the pilot reactors was continuously measured using Ritter

212 MilliGascounters MGC-1 V3.0. The composition of the biogas was determined by gas

213 chromatography as described in Cazier et al. (50).

214 *Analysis of metabolites and final products of the digestion*

215 A sample of digestate from the pilot reactors was taken once per week for measurement of the

216 concentrations of VFAs and ionic species in the reactors. Concerning the batch study, a

217 plastic tube submerged in the reaction media served for digestate sampling when required.

218 Before sampling, a clip was used for blocking the gas output and the equivalent volume of

219 digestate to be removed was injected as nitrogen gas, avoiding an overestimation of the

220 produced gas. The concentrations of VFAs and ionic species in the digestates were measured

221 as described in Motte et al. (51), by gas and ion chromatography, respectively.

222 *Data analysis*

223 The concentration of FAN was calculated according to Chen et al. (52) as a function of

224 temperature, pH, and TAN concentration.

225 The methane yields were calculated by dividing the total volume of methane produced by the
226 initial mass of VS of substrates. They were corrected to take into account the digestate

227 removed when sampling. The concentrations of FAN in the reactors were determined

228 according to Rajagopal et al. (8), as a function of temperature, pH, ionic strength and TAN

229 concentration. The concentrations of the main ions present in the reactors (Cl⁻, Na⁺, NH₄⁺, K⁺,

230 Mg²⁺, H⁺, VFAs and Ca²⁺) were taken into account in this calculation.

231

232 **Results and discussion**

233 *Optimization of biochar and FeCl₃ dosing in batch reactors*

234 As aforementioned, the first objective of this study was to evaluate the effect of different

235 concentrations of both biochar and industrial FeCl₃, aiming at optimizing their dosage. The

236 results of the experimental design (using the second consecutive batch) are shown in Table 4.

237 All the reactors produced methane efficiently, with yields ranging from 456 to 505 ml CH₄·g

238 VS⁻¹ and final pH values between 8.15 and 8.42. These high pH values were caused by the

239 high TAN concentrations, up to 9.75 g·l⁻¹. As it can be observed, the main differences were

240 related to the AD kinetics. The maximum methane production rates (consequence of the

241 initial consumption of HAc) varied widely, from 907 to 1498 ml·g·VS⁻¹·d⁻¹. The total time for

242 consumption of HPr (most recalcitrant VFA to be degraded) ranged from 18.9 to 24.4 days.

243 The average daily methane production was calculated by dividing the total volume of methane

244 produced by the batch duration (i.e. time to completely degrade HPr), according to Capson-

245 Tojo et al. (30). It varied from 302 to 376 ml CH₄·d⁻¹. The values of the methane yields, the

246 maximum methane rates and the average daily methane production rates were used as inputs

247 for the quadratic model (Equation 1), obtaining the results presented in Table 5. The raw

248 kinetic curves of the methane yields and production rates, the pH values and the

249 concentrations of the different VFAs are presented in Figure S1 and Figure S2 (supplementary

250 material).
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251 Starting with the methane yields, the modeled results indicated that both linear coefficients

252 from the biochar and the FeCl₃ (i.e. a<sub>biochar</sub> and a<sub>FeCl3</sub>) had a significant effect on this variable

253 (p-values linear coefficients < 0.05). However, the quadratic parameter a<sub>biochar-biochar</sub> was

254 statistically different than zero with a 99 % of certainty (p-value < 0.01) and the high value of

255 this parameter indicated that the experimental design was not properly centered for predicting

256 the methane yield. The relatively low R² value (86.7 %) also suggests the lack of fit of the

257 model. In addition, the Durbin-Wilson test (with a p-value < 0.05) could not exclude that

258 correlations existed due to the order in which the data were used as input. All these results

259 suggest that the model was not able to predict any direct effect of the biochar or the FeCl₃ on

260 the final methane yields obtained, indicating that no significant differences existed between

261 the obtained results. Considering that the biodegradable matter content of these two additives

262 should be negligible, this is a logical outcome.

263 Nevertheless, the model was able to reproduce precisely the maximum methane production

264 rates (R² of 94.2 % and p-value Durbin-Wilson 0.20) and the average daily methane

265 production rates (R² of 100 %), indicating that highly significant differences existed between

266 the reactors. The model responses for these two variables are presented in Figure 1.

267 The maximum methane production rate was mainly affected by the biochar concentration,

268 with a negligible influence of the industrial FeCl₃ concentration. This can be verified by the p-

269 values of the parameters in Table 5, where it can be observed that the only parameter with a p-

270 value < 0.5 was a<sub>biochar</sub>. This indicates that addition of biochar clearly improved the

271 degradation of the HAc that accumulated at the beginning of the AD process, when the

272 maximum methane production rates were registered (Figure S1 and Figure S2). This is in

273 agreement with previous results, where GAC addition enhanced the HAc consumption during

274 batch FW AD (30). Regarding the average daily methane production rates (estimated by

275 dividing the total volume of methane produced by the time to completely degrade the HPr),
276 the value of this variable also increased greatly at higher biochar concentrations. However, in

277 this case the effect of the $FeCl_3$ was significant, with both $a_{biochar}$ and $a_{FeCl_3}$ showing p-values

278 lower than 0.5. This variable was mainly affected by the batch duration, which was

279 determined by the time required for HPr consumption. Thus, it can be concluded that both

280 biochar and industrial $FeCl_3$ addition favored the degradation of the HPr accumulated during

281 the first days of the batch process. This can be related to favored syntrophic interactions and

282 to the occurrence of DIET in the case of biochar and to the synthesis of enzymes for the

283 industrial $FeCl_3$ (i.e., TEs contained in this solution). Again, this is in agreement with

284 previous results presented in the literature. Adding GAC and TEs into batch reactors, a

285 reduction of the time required to completely degraded the accumulated HPr of around 40 %

286 was observed during batch FW AD (30).

287 Although optimal values could be extrapolated ($162 \ g \cdot l^{-1}$ of biochar at $0.1 \ g \ Fe \cdot l^{-1}$ for

288 maximizing the methane production rate and $111 \ g \cdot l^{-1}$ of biochar at $0.2 \ g \ Fe \cdot l^{-1}$ for

289 maximizing the average methane production rates), it is clear that the experimental design

290 was no properly centered and therefore further experiments must be performed to precisely

291 optimize the dosing of these reagents.

292 Performance of the continuous pilot reactors

293 Two continuous pilot reactors were run in parallel to the batch reactors during 196 days: a

294 control reactor fed only with FW and a reactor supplemented with biochar and industrial

295 $FeCl_3$. The main operational parameters and the obtained results are presented in Figure 2.

296 After starting the reactors at an OLR of $1.4 \ g \ VS \cdot l^{-1} \cdot d^{-1}$, it was required to wait around 30-40

297 days to achieve a stable methane production of $10 \ l \cdot d^{-1}$ in both systems. Interestingly,

298 differences between both reactors could already be appreciated in this start-up period. After

299 15 days, the reactor supplemented with biochar and industrial $FeCl_3$ showed higher methane

300 production rates than the control (Figure 2B), which were associated with higher initial
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301 methane yields, lower concentrations of HAc and HPr (i.e. peaks of HAc of 13 and 16 g·l⁻¹,

302 respectively) and higher pH values. This suggests that the added AD enhancers favored the

303 consumption of the VFAs accumulated during the start-up period, even at a biochar

304 concentration of 10 g·l⁻¹. This is in agreement with the batch results, confirming the positive

305 effect of adding these reagents on the initial HAc consumption (leading to a lower extent of

306 HPr accumulation) and on the HPr degradation.

307 Moving forwards, after 60 days both reactors achieved efficient methane productions at the

308 first OLR applied, with yields around 400 ml CH₄·g VS⁻¹ (95 % of the BMP), with relatively

309 low concentrations of VFAs in the reactors (2 g·l⁻¹ of HAc and 0.3 g·l⁻¹ for HPr) and with high

310 pH values (around 8.1). It must be mentioned that high TAN concentrations were already

311 present in the reactors at this point, with values around 8 g·l⁻¹ in both reactors at day 68. It

312 must also be commented that the average TAN concentrations in the reactors were 8513 ± 362

313 mg·l⁻¹ in control reactor and 8432 ± 777 mg·l⁻¹ in the biochar-supplemented reactor

314 (corresponding to 398 and 550 mg FAN·l⁻¹, respectively). This indicates that no significant

315 differences existed and that the observed improvement of the AD performance was no caused

316 by a reduction in the TAN/FAN concentrations related to its adsorption on the biochar

317 particles.

318 However, due to the low OLR applied, relatively low methane production rates (around 10 l

319 CH₄·d⁻¹) were obtained. Therefore, the OLR was doubled in day 77 to reach 2.8 g VS·l⁻¹·d⁻¹.

320 This caused a sudden drop in the methane yields to 160 ml CH₄·g VS⁻¹, which was associated

321 with an increase in the HAc and HPr concentrations in the reactors. In agreement with the

322 previous results, the levels of both VFAs were always lower in the supplemented reactor.

323 In an attempt to reduce the intensity of VFA accumulation in the reactor containing biochar

324 and FeCl₃, the biochar concentrations were increased to 20 g·l⁻¹ on day 105 and to 50 g·l⁻¹ on

325 day 146 (based on the results from the batch optimization experiment described above). While
326 the first increase did not have significant effects, the second one (to 50 g·l⁻¹) caused a drop in the HAc concentration from 13 to 7 g·l⁻¹, raising the methane yields up to 350 ml CH₄·g VS⁻¹.

327 A consequent decrease in the HPr concentration was observed (from 3.1 to 1.8 g·l⁻¹). Sadly, on day 167 a problem occurred with the heating of the biochar-supplemented reactor. This caused a temperature drop, which led to a sudden decrease of the methane yields obtained and, again, an accumulation of HAc at the end of the operational period (days 167 to 196).

332 Besides the aforementioned complication, Figure 2 clearly shows that addition of biochar and industrial FeCl₃ decreased the HPr concentrations in reactors, with considerable differences between the supplemented reactor and the control. This discrepancy was particularly important after increasing the biochar concentrations, with HPr levels of 7.2 g·l⁻¹ in the control reactor and of 1.8 g·l⁻¹ in the reactor containing biochar and industrial FeCl₃ at the end of the operational period.

335 Although the obtained results further suggest that addition of biochar and industrial FeCl₃ can improve the AD kinetics and favor VFA consumption during FW AD, it is also clear that further continuous experiments must be carried out, allowing longer operational periods.

341 These experiments should aim to reach an operational steady-state, with results that can be extrapolated to a potential industrial-scale facility.

343 *Biochar as feasible option for enhancing FW AD*

344 The results obtained both in batch and continuous reactors are in agreement with different studies carried out to study the effect of biochar on AD. The kinetics of consumption of HAc and HBu were reported to be faster when adding biochar using glucose as substrate (44). Also the direct degradation of HPr and HBu has been improved by supplementing biochar and ethanol (27). Sunyoto et al. (45) observed that biochar supplementation increased the methane production rates and enhanced the consumption of HAc and HBu in the second stage of a 2-phase AD reactor treating aqueous FW.
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351 Using GAC as carbon-based AD enhancer, different studies have suggested that HAc, HBu

352 and HPr can be directly metabolized through DIET, improving the kinetics of consumption of

353 these VFAs (22,25–27). In addition, biochar has also been found to promote the growth of

354 bacteria known to participate in DIET (i.e. Geobacter species) onto its surface (27).

355 Therefore, it can be hypothesized that the improved VFA degradation kinetics could be

356 related to an enhancement of the syntrophic interactions between microorganisms via biofilm

357 formation and to the occurrence of DIET. The degradation of HAc through DIET has already

358 been proposed in the literature and, although being more limited thermodynamically (9),

359 DIET may have also played an important role in the oxidation of HPr. Besides, even if direct

360 DIET of HPr might not have occurred extensively, its degradation would be favored anyway

361 due to lower HAc and hydrogen/formate concentrations. Further studies analyzing the

362 microbial communities attached on the biochar, as well as the properties of the biochar used,

363 should be performed to verify this hypothesis. Concerning the industrial $FeCl_3$ addition, this

364 additive favored the HPr degradation due to the supplementation of TEs, critical for enzyme

365 synthesis (11).

366 These experiments proved that a regular biochar (natural slow-pyrolyzed wood charcoal)

367 could also improve greatly the AD performance using complex FW as substrate. It must also

368 be considered that, other than the concentration applied, many parameters and variables which

369 have not been considered in this study have a huge potential for optimization when

370 considering biochar as AD enhancer. It is clear that the textural characteristics of the biochar

371 (e.g. specific surface, pore volume, pore size or pore distribution) as well as its surface

372 chemistry (e.g. hydrophobicity) or its particle size play a major role on biofilm formation. In

373 addition, also its resistivity (conductivity) might have a huge impact on its capability for

374 favoring DIET. All these characteristics are dependent on different variables that clearly

375 deserve further study, such as the raw material used for biochar production (41), the
376 temperature and pressure applied during pyrolysis (i.e. slow or fast pyrolysis) or the

377 pretreatment applied to the biochar before its addition into the AD reactor (i.e. mechanical

378 grinding) (32).

379 Although deep techno-economic analyses must be carried out before considering its

380 application at industrial scale (particularly at the high concentrations of biochar applied,

381 which would require recirculation of the solid fraction of the digestate for biochar

382 reutilization), the obtained results suggest for the first time that biochar and industrial $FeCl_3$

383 can be a feasible alternative for stabilizing AD of FW, favoring the consumption of VFAs and

384 improving the methane productivities. When compared to substrate dilution (1:1 vol:vol),

385 which is the most commonly applied stabilization method in industrial facilities (increasing

386 greatly the HRTs and the volumes of digestate produced and requiring an input of clean

387 water), the proposed technology is clearly a more environmental-friendly option that can be

388 coupled with other waste treatment processes (i.e. green waste pyrolysis).

389

390 Acknowledgement

391 Suez is gratefully acknowledged for financing this research under the CIFRE convention N°

392 2014/1146. The authors also want to express their gratitude to the Communauté

393 d'Agglomération du Grand Narbonne (CAGN) for the financial support.

394

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551 Figure and Table captions

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<figure><img src="image_4.png" /><figcaption>**Figure 1.** Surface responses (left) and average individual effects (right) of (up) the maximum methane rates and (down) the average daily methane production rates</figcaption></figure>

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<figure><img src="image_5.png" /><figcaption>**Figure 2.** Evolution of the (A) average weekly methane yield, (B) methane production rate, (C) acetic acid concentration, (D) propionic acid concentration and (E) pH in the pilot reactors. The days in which an operational parameter (i.e. OLR or biochar concentration) was modified are also indicated (vertical lines)</figcaption></figure>

<caption>**Table 1.** Characteristics of the food waste mixture and the inoculum (9)</caption>

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<table><thead><tr><th>Parameter</th><th>Food waste mixture</th><th>Inoculum</th></tr></thead><tbody><tr><td>TS (%)</td><td>21.0</td><td>5.81</td></tr><tr><td>VS/TS (%)</td><td>90.3</td><td>59.1</td></tr><tr><td>Carbohydrates (g·kg TS<sup>-1</sup>)</td><td>618</td><td>n.m.<sup>1</sup></td></tr><tr><td>Proteins (g·kg TS<sup>-1</sup>)</td><td>187</td><td>n.m.<sup>1</sup></td></tr><tr><td>Lipids (g·kg TS<sup>-1</sup>)</td><td>121</td><td>n.m.<sup>1</sup></td></tr><tr><td>BMPs (ml CH<sub>4</sub>·g VS<sup>-1</sup>)</td><td>420</td><td>n.m.<sup>1</sup></td></tr><tr><td>pH</td><td>5.02</td><td>8.10</td></tr><tr><td>TAN (g·kg TS<sup>-1</sup>)</td><td>0.90</td><td>5.04</td></tr><tr><td>TKN (g·kg TS<sup>-1</sup>)</td><td>30.0</td><td>93.0</td></tr><tr><td>C/N</td><td>16.1</td><td>3.04</td></tr></tbody></table>

1. n.m. stands for “not measured”

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<caption>**Table 2.** Characteristics and composition of the industrial $FeCl_3$ solution</caption>

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<table><thead><tr><th>Parameter</th><th>Unit</th><th>Value</th></tr></thead><tbody><tr><td>Density at 20 °C</td><td>(g·cm<sup>-3</sup>)</td><td>1.45</td></tr><tr><td>FeCl<sub>3</sub></td><td>(%)</td><td>41.1</td></tr><tr><td>Cl total</td><td>(g·l<sup>-1</sup>)</td><td>397</td></tr><tr><td>Fe total</td><td>(g·l<sup>-1</sup>)</td><td>206</td></tr><tr><td>HCl</td><td>(g·l<sup>-1</sup>)</td><td>2.2</td></tr><tr><td>Mn</td><td>(mg·l<sup>-1</sup>)</td><td>780</td></tr><tr><td>Zn</td><td>(mg·l<sup>-1</sup>)</td><td>390</td></tr><tr><td>Pb</td><td>(mg·l<sup>-1</sup>)</td><td>220</td></tr><tr><td>Ni</td><td>(mg·l<sup>-1</sup>)</td><td>67</td></tr><tr><td>Co</td><td>(mg·l<sup>-1</sup>)</td><td>28</td></tr><tr><td>Cu</td><td>(mg·l<sup>-1</sup>)</td><td>65</td></tr><tr><td>Cr</td><td>(mg·l<sup>-1</sup>)</td><td>45</td></tr><tr><td>Ca</td><td>(mg·l<sup>-1</sup>)</td><td>540</td></tr><tr><td>Na</td><td>(mg·l<sup>-1</sup>)</td><td>110</td></tr><tr><td>Al</td><td>(mg·l<sup>-1</sup>)</td><td>100</td></tr><tr><td>Mg</td><td>(mg·l<sup>-1</sup>)</td><td>15</td></tr></tbody></table>

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<caption>**Table 3.** Experimental design of the batch experiment. All the reactors were fed with 60 g of</caption>

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<table><caption>625 FW at an S/X ratio of 1 g VS·g VS⁻¹ and incubated at 37 °C</caption><thead><tr><th rowspan="2">Reactor</th><th colspan="2">Normalized level</th><th colspan="2">Real concentrations (g·l⁻¹)</th></tr><tr><th>Biochar</th><th>FeCl<sub>3</sub></th><th>Biochar</th><th>FeCl<sub>3</sub></th></tr></thead><tbody><tr><td>1</td><td>0</td><td>-1</td><td>55</td><td>0.1</td></tr><tr><td>2</td><td>-1</td><td>1</td><td>10</td><td>0.2</td></tr><tr><td>3</td><td>1</td><td>1</td><td>100</td><td>0.2</td></tr><tr><td>4</td><td>-1</td><td>-1</td><td>10</td><td>0.1</td></tr><tr><td>5</td><td>1</td><td>-1</td><td>100</td><td>0.1</td></tr><tr><td>6</td><td>0</td><td>1</td><td>55</td><td>0.2</td></tr><tr><td>7</td><td>0</td><td>-1</td><td>55</td><td>0.1</td></tr><tr><td>8</td><td>-1</td><td>-1</td><td>10</td><td>0.1</td></tr><tr><td>9</td><td>0</td><td>1</td><td>55</td><td>0.2</td></tr><tr><td>10</td><td>1</td><td>1</td><td>100</td><td>0.2</td></tr><tr><td>11</td><td>-1</td><td>1</td><td>10</td><td>0.2</td></tr><tr><td>12</td><td>1</td><td>-1</td><td>100</td><td>0.1</td></tr></tbody></table>

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640 **Table 4.** Results of the batch experimental design

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<table><thead><tr><th>Reactor</th><th>Methane yield (ml·g VS<sup>-1</sup>)</th><th>Maximum methane rate (ml·g VS<sup>-1</sup>·d<sup>-1</sup>)</th><th>Time for HPr consumption (d)</th><th>Average daily methane production rates (ml·d<sup>-1</sup>)<sup>1</sup></th><th>Final pH</th></tr></thead><tbody><tr><td>1</td><td>483</td><td>1327</td><td>18.9</td><td>363</td><td>8.15</td></tr><tr><td>2</td><td>505</td><td>948</td><td>23.0</td><td>302</td><td>8.22</td></tr><tr><td>3</td><td>484</td><td>1498</td><td>18.9</td><td>376</td><td>8.19</td></tr><tr><td>4</td><td>509</td><td>886</td><td>24.4</td><td>280</td><td>8.21</td></tr><tr><td>5</td><td>489</td><td>1436</td><td>18.9</td><td>374</td><td>8.18</td></tr><tr><td>6</td><td>459</td><td>1249</td><td>18.9</td><td>356</td><td>8.29</td></tr><tr><td>7</td><td>461</td><td>1281</td><td>20.1</td><td>329</td><td>8.29</td></tr><tr><td>8</td><td>501</td><td>907</td><td>21.5</td><td>316</td><td>8.17</td></tr><tr><td>9</td><td>456</td><td>1142</td><td>20.1</td><td>323</td><td>8.29</td></tr><tr><td>10</td><td>466</td><td>1489</td><td>18.9</td><td>361</td><td>8.27</td></tr><tr><td>11</td><td>496</td><td>913</td><td>21.5</td><td>326</td><td>8.42</td></tr><tr><td>12</td><td>496</td><td>1457</td><td>20.1</td><td>360</td><td>8.19</td></tr></tbody></table>

1. Calculated as final methane yield divided by the time required for HPr consumption (batch duration)

641

642

643

644

645

646

647

648

649

650

651

652

653

654

655

656

657

**Table 5.** Coefficients of the quadratic model for the main responses of the experimental

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60
658 design

<table><thead><tr><th>Parameter/coefficient</th><th>Methane yield (ml·g VS<sup>-1</sup>)</th><th>Maximum methane rate (ml·g VS<sup>-1</sup>·d<sup>-1</sup>)</th><th>Average daily methane production rates (ml·d<sup>-1</sup>)<sup>1</sup></th></tr></thead><tbody><tr><td>a<sub>0</sub></td><td>465</td><td>1250</td><td>343</td></tr><tr><td>a<sub>biochar</sub></td><td>-9.54*</td><td>278.2**</td><td>37.3***</td></tr><tr><td>a<sub>FeCl3</sub></td><td>-9.04*</td><td>-4.71</td><td>4.02***</td></tr><tr><td>a<sub>biochar-biochar</sub></td><td>28.3**</td><td>-57.9</td><td>-12.2***</td></tr><tr><td>a<sub>biochar-FeCl3</sub></td><td>-8.03</td><td>3.25</td><td>-6.94***</td></tr><tr><td>R<sup>2</sup></td><td>86.7 %</td><td>94.2 %</td><td>100 %</td></tr><tr><td>p-value Durbin-Wilson</td><td>0.025</td><td>0.20</td><td>-</td></tr></tbody></table>

659 1. Calculated as final methane yield divided by the time required for HPr consumption (batch duration)

660 * p-value < 0.05

661 ** p-value < 0.001

662 *** p-value < 0.0001

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

678
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>**Figure S1.** Evolution of (A) the methane yields, (B) the methane production rates and (C) the pH in the reactors. The legend indicates the reactor number and the normalized levels of each factor (B stands for biochar and Fe for FeCl₃ solution)</figcaption></figure>
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>**Figure S2.** Evolution of the concentrations of (A) acetic acid, (B) propionic acid, (C) butyric acid and (D) valeric acid in the reactors. The legend indicates the reactor number and the normalized levels of each factor (B stands for biochar and Fe for FeCl₃ solution)</figcaption></figure>