Cultivation of olive trees and oil production are vital activities, mainly in Mediterranean countries. However, uncontrolled olive mill wastewater (OMW) discharge into the environment may beget serious problems owing to its high pollution degree, acidic pH and polyphenols compounds generating antimicrobial effects which involve the inhibition of natural biodegradability of organic load in natural water bodies (Sayadi et al., 2000). Among the numerous processes proposed for the effluent detoxification, biological treatments were considered less expensive and environmental friendly (McNamara et al., 2008). It has been reported that anaerobic digestion (AD) is more efficient than aerobic processes referring basically to the plausibility to treat effluents with high organic load and energy potential like OMW (Gelegenis et al., 2007). In addition, it displays several merits like operational economy, reduction in energy consumption, generation of biogaz and use of the stabilized digestate as a fertilizer (Dareioti et al., 2009).

However, AD effectiveness is limited by the slow metabolism between syntrophs and archaea (Zhao et al., 2016). In fact, some reactor operational modifications may cause volatile fatty acids (VFAs) or hydrogen (H₂) accumulation that might be toxic to methanogens and acetogenic bacteria respectively, which triggers souring of anaerobic reactors and leads as a matter of fact to the process failure.

Particularly, for OMW, AD was affected chiefly by inhibitory substances such as phenolic compounds as well as long chain fatty acids (Beccari et al., 1999) as they inhibit anaerobic microorganisms. For these reasons, many researchers have been particularly oriented towards improving OMW AD efficiency through dilution, physico-chemical pretreatments, co-digestion or integrated treatments (Khoufi et al., 2015; Vavouraki et al., 2020). Khoufi et al. (2007) proved that OMW pretreatment with electrocoagulation followed by sedimentation led to removal of 76.2% of phenolic compounds and a chemical oxygen demand COD reduction of 43%. After this pretreatment, anaerobic biomethanization was conducted with high methane yield at a loading rate of 6 g COD L⁻¹ day⁻¹ compared to raw OMW which was toxic to microorganisms. The working mode for syntrophic metabolism during anaerobic methanogenesis was commonly reported as interspecies hydrogen transfer (IHT) (Boone et al., 1999), where H₂ acts as a diffusive electron shuttle to mediate electron transfer from secondary fermenting bacteria to methanogens. However, the production of hydrogen catalysed by secondary fermenting bacteria is thermodynamically feasible (i.e., ΔG < 0) uniquely if hydrogen concentrations are quite low (H₂ < 10^–4 atm (Logan et al., 2002)). Since this condition is accomplished through the consumption of hydrogen by hydrogenotrophic methanogens, the syntrophic metabolism between oxidizing bacteria and archaea is crucial (Mcinerney et al., 2010). Yet, H₂ diffusion between H₂ producers and H₂-consuming methanogens is slow (Stams et al., 2006), which reduces the methane formation rate during AD. Therefore, this syntrophic metabolism network has been confirmed to be metabolically low-efficient.

Over the last years, a new electron transfer pathway, referred to direct interspecies electron transfer (DIET), has been set forward as an alternative network which is more efficient than IHT (Xu et al., 2019). DIET may occur through biological electrical connections such as pili and outer surface c-type cytochromes (Xu et al., 2019). Moreover, several electrically conductive materials, such as graphene (Zhang and Tremblay, 2020) and carbon cloth (Zhao et al., 2016), served as additives for DIET enhancement between syntrophic microorganisms. However, excessive costs as well as the environmental risks of these materials like graphene might limit their use (Kang et al., 2007). Thus, recently, various researchers have investigated biochar as an effective additive to enhance AD. Biochar is an amorphous and a porous carbon-rich material produced by pyrolysis of biomass varieties in the absence or presence of a little amount of oxygen (Cantrell et al., 2012). It is reported that during AD, biochar increases buffering capacity (Zhang et al., 2014), immobilizes microbial cell and improves the methane production rate (Cai et al., 2016). Recently, Wang et al. (2020) have asserted that biochar derived from biowaste promoted DIET to accelerate syntrophic phenol oxidation during AD. The authors suggested a probable shift of syntrophic phenol metabolism from indirect transfer via H₂ to direct interspecies electron transfer.

This work corresponds to a pioneering research that focuses on the impact of the biochar supplementation on AD of OMW. The chief objective of this research work was: Firstly, to examine the impact of different biochar concentrations on biochemical methane potential assays of OMW at different increasing COD loads: 10 g/L, 15 g/L and 20 g/L. Secondly, the prokaryotic community structure, both in the suspended solution and those integrated with biochar surface, were investigated by Illumina to explore their potential implication in DIET.

This work would provide new findings on the effect of biochar supplementation upon the production of methane from OMW which has an important implication for potential application at large scales.

In the current work, we attempt to assess the influence of biochar concentrations (5 g/L and 10 g/L) on anaerobic digestion of OMW with different increasing COD loads (10 g/L, 15 g/L and 20 g/L) over an incubation period of 56 days. Statistical differences in methane production and methane yields per Gram of COD_introduced were determined. When examining the final CH₄ yield (mL/g COD _introduced) for all the treatments, ANOVA analysis revealed a significant difference among the various COD loads treatments (Table 1). However, the Tukey’s post hoc test highlighted these points: Firstly, a significant lower yield was reached in raw OMW cultures with 20 g/L COD compared to cultures with 10 g/L COD. However, after methane production enhancement with 5 g/L biochar addition, final yield became significantly higher than that achieved in batch with 10 g/L COD load. Hence, we deduce that biochar supplementation during AD of OMW may entail promising results in terms of the recovery of metabolic activity when operating at high organic load rate at large scale applications. Furthemore, for all the treatments, 5 g/L biochar addition improved significantly methane yield compared to the control and treatments with 10 g/L biochar. These results suggested that, for all initial loads of COD, the addition of 5 g/L of biochar was more effective than the 10 g/L dose in increasing yield methane production.

Biochar supplementation may enhance methane production by adsorbing polyphenols which inhibit methanogens. This likelihood may be excluded since a higher concentration of biochar is needed for polyphenols elimination (Abid et al., 2022). It would be highly useful to analyze the microbial communities (bacteria and archaea) at the end of operation (56 days) in the cultures with initial COD load of 20 g/L to get insights into the microbial community response during AD with biochar supplementation. Microbial communities of biochar free group (R0) and 5 g/L biochar supplemented group (R1), involving suspended fraction of R0 (S0) and R1 (S1), loosly cells (L1) and tightly ones bound to biochar (T1) were analyzed. Results from high-throughput sequencing revealed that biochar supplementation improved the abundance of hydrolytic bacteria, mainly in the suspended fraction, such as Bacteroidaceae, as well as acidogenic bacteria such as Clostridiacea, Proteinivoraceae and fermentative genus group such as Erysipelatoclostridium. Bacteroidaceae play a key role in organic matter depolymerisation during acidogenesis phase thanks to several hydrolyzing enzymes (Wang et al., 2017). They seemed to be part of bacterial key players during AD exposed to high concentrations of phenol reaching 2 g/L (Poirier et al., 2016). Consequently, they were involved in the hydrolysis step to enhance polyphenols degradation. Our results go in good consistency with those of Pytlak et al. (2020) who asserted that biochar addition to a fermentation sludge containing sugar beet pulp leads to an enrichment of Bacteroidales. On the other side, it was emphasized that Bacteroides are probably able for the extracellular electron transfer since some species were enriched on the anode of a bio-electrolysis cell system and were able to transfer electrons directly to ferric iron (Wang et al., 2010). Clostridiaceae were known not only as acidogenic bacteria (Yin et al., 2018) but also as exoelectrogens. In particular, Natronincola peptidivorans was reported as an interesting new potential electro-synthesizing bacterium in the Clostridiaceae family capable of direct electron transfer in a microbial electrolysis cell (Quéméner et al., 2019). Erysipelatoclostridium were known for their ability to ferment several carbohydrates such as acetate, propionate and butyrate (Yutin and Galperin, 2013).

Stimulated CH₄ after 5 g/L biochar addition to OMW with initial COD load of 20 g/L may be explained by the bacterial and archaeal communities shift with a significant enrichment of microbes involved in DIET. In this line, our results demonstrated the enrichement of Anaerolineaceae which were known for their capacities for extracellular transfer of electrons using fulvic acids as electron acceptors (Dang et al., 2016). These results proved to be consistent with the findings of Wang et al. (2018) who reported that biochar supplementation during AD of complex organic wastes entailed the enrichment of Anaerolineaceae. In this respect, Cloacibacillus species which were enriched in the suspended fraction of biochar supplemented group proved to be syntrophic amino-acid-oxydizing bacteria (Zhao et al., 2017). Recently, Yang et al. (2021) have argued that biochar addition during anaerobic digestion of swine manure enriched Cloacibacillus which might participate in DIET with Methanothrix.

More importantly, our results revealed that Rikenellaceae and Parabacteroides were distinctly more enriched in the fixed biomass of biochar. Recently, Rikenellaceae were identified as potential syntrophic bacteria capable of establishing magnetite-mediated direct electron transfer with methanogens to accelerate VFAs degradation (Lee et al., 2019). Parabacteroides are fermentative species. However, many researchers have recently reported the abundant growth of Parabacteroides under DIET-simulated conditions (Baek et al., 2019), which is suggestive that this group may potentially be involved in electro-syntrophic interactions during anaerobic digestion. Consequently, our results go in good conformity with previous researchers’ findings indicating that these groups may possibly be involved in electro-syntrophic interactions during anaerobic digestion.

Lower relative abundance of hydrogenetrophic methanogens such as Methanoculleus and Methanomassiliicoccus in S1 compared to S0 proved that IHT wan’t enhanced by biochar. Methanoculleus emergence has been reported as an early indicator which portends phenol inhibition towards microbial community during AD exposed to phenol concentrations between 1 and 2 g/L (Poirier et al., 2016). Hence, the rigorous hydrogenotrophic Methanoculleus seemed to play a key role to maintain AD via IHT in R0. However, Methanobacterium seemed to be enriched in the suspended fraction for the biochar treatment. Methanobacterium are hydrogenotrophic methanogens, which had been broadly detected in the AD of phenol culture (Na et al., 2016; Poirier et al., 2016). Consequently, positive effects of biochar addition on polyphenols alleviation can be attributed to Methanobacterium enrichement. They were recognized also as syntrophic microbes which participated to DIET and proved to be enriched in anaerobic digestion of sewage sludge process with biochar addition (Wu et al., 2019). Recently, they have been reported to be able to change the primary working mode of the syntrophic metabolism from IHT to DIET during anareobic digestion of swine manure with biochar addition (Yang et al., 2021).

Additionally, the enrichment of genera of archaea like Methanothrix and Methanosarcina is suggestive that DIET would be accelerated in the biochar treatment samples. Methanosarcina barkeri is known as a methanogen that is able to participate to DIET (Rotaru et al., 2014). Methanothrix can use both acetoclastic methanogenesis and DIET-CO₂ reduction during AD with the addition of biochar (Wang C et al., 2018). Thus, the stimulation of methane production rate seems to be associated with the increase in abundance of these genera with independence of the syntrophy between bacteria and archaea via IHT. To sum up, several putative exoelectrogenic microbes were enriched in biochar treatment samples and particularly in suspended fractions such as Bacteroides, Natronincola and Cloacibacillus. More importantly, since the potential exoelectrogenic bacteria like Parabacteroides Rikenellaceae and Anaerolineaceae were enriched exclusively on the biochar, they could be directly interacting with archaea of Methanothrix and Methanosarcina via DIET. These archaea species which were enriched on the biochar, proved to be responsible for DIET. A Conceptual illustration of DIET possible mechanism in anaerobic digestion of OMW with biochar is described in Figure 7.

As a conductive material, biochar can serve as an electron acceptor as well as a donor and allows DIET to take place (Park et al., 2018). When compared to IHT, DIET has been suggested as faster and energetically more effective than IHT (Xu et al., 2019) since it does not require energy to produce H₂. As electrical conductivity plays an important role in terms of promoting DIET (Zhao et al., 2017), the used biochar in our study, which was derived from olive mill waste water sludge, would stand for a good candidate as it possesses high electrical conductivity (11 m/cm).

The inhibition of OMW polypenols towards methanogens begets the imbalance in the relationship between bacteria and methanogens, which results in significant VFAs accumulation and AD inhibition in the control cultures with the highest COD load (20 g/L). Furthermore, biochar supplementation at 5 g/L to OMW with initial COD load of 20 g/L mitigated VFAs accumulation. Our findings go in good correlation with the results of Wang G et al. (2018). These authors disclosed that biochar derived from sawdust alleviated VFAs accumulation during AD of complex organic wastes. Positive effects of biochar addition on the VFAs alleviation can be interpreted as follows: Our study revealed that biochar enhanced the growth of bacteria known for their ability to ferment several carbohydrates like acetate, propionate and butyrate (Yutin and Galperin, 2013) such as Erysipelatoclostridium. Consequently, enhanced degradation of VFAs was probably ascribed to the improved solubilization of OMW following the bacteria enrichement. Besides, our study demonstrated that biochar addition suppressed IHT and enabled DIET to take place. This pathway enhances the electron transport rates when compared to IHT. Hence, the H₂ and formate concentrations are lower than during IHT. Therfore, syntrophic VFAs oxydation becomes thermodynamically feasible (Capson-Tojo et al., 2018) .

This study addresses the impact of biochar addition during AD of OMW in batch at different COD increasing loads. AD of OMW with initial COD load of 20 g/L led to process unbalance owing to VFAs accumulation. However, biochar addition at 5 g/L increased methane yield by 97.8%, mitigated VFAs accumulation and enriched prokaryotic as well as methanogenic communities.

Results indicated that biochar displayed a great potential in terms of enhancing VFAs degradation referring to the improved solubilization of OMW through microbial hydrolysis with increased abundance of hydrolytic bacteria such as Bacteroidaceae, as well as acidogenic bacteria such as Clostridiaceae, Proteinivoraceae. In addition, the enrichment of genera like Methanothrix and Methanosarcina may indicate the establishment of DIET in the biochar treatment samples.

This work provides new findings on the effect of biochar supplementation upon the production of methane from OMW which has an important implication for potential applications at large scales.