Facilitation of interspecies electron transfer in anaerobic processes through pine needle biochar

In the Himalayan region of India, pine forest generates lignocellulosic residue in the form of pine needles (PN) with a productivity of about 1.9 million tons/year (Bisht et al. 2018). Due to its non-biodegradable characteristics, PN biodegrades very slowly and accumulates over the forest floor. Accumulation of PN leads to several environmental problems such as forest fires and consequent air pollution, inhibition to groundwater recharge by blocking the soil pores, hindrance to the growth of plants and shrubs as well as soil acidification. PN primarily contains cellulose 22–27%, hemicellulose 24–28% and higher lignin content of 38–40% (Mandal et al. 2018; Mohan & Annachhatre 2022). Lignin comprises phenyl propane as the basic unit with cross-linking and has an amorphous structure. On the other hand, cellulose is made up of glucopyranose sugar molecules with linear chain, whereas hemicellulose is comprised of different sugar molecules attached in a branched structure. Since lignin content in PN is high, it creates several environmental problems due to its nondegradable nature.

Researchers have used biochar as support material for attachment and growth of biofilm in AD processes (Park et al. 2018). Biochar is a carbon-rich solid material which is produced through pyrolysis of biomass such as forest/agricultural residue or animal waste in the absence of oxygen at the temperature of 300–1,000 °C (Rosales et al. 2017). Biochar is a highly porous material with pore size ranging from 0.004 to 150 μm (Leng et al. 2021) and high specific surface area (SSA) up to 500 m²/g (Yargicoglu et al. 2015). Several structural components of biochar such as graphite and surface functional groups are known to induce redox activity. Furthermore, accumulation of active cations such as Na, K, Mg, Ca, etc. also helps in promoting buffering characteristics of biochar (Yuan et al. 2017; Liu et al. 2019). These properties of biochar promote attachment and growth of methanogenic archaea under anaerobic conditions and lead to increased methane production (Qin et al. 2020; Ren et al. 2020).

Accordingly, this research seeks to explore the effect of application of pine needle biochar (PNB) on the process of AD. The PNB is used as a support material in different types of anaerobic reactors and their performances are compared to understand the role of biochar in anaerobic processes.

PN forest residue was collected from the floor of the Himalayan pine forest, pre-treated and stored for biochar production according to the procedure presented elsewhere (Bashir et al. 2022). The stored PN was analysed for its cellulose, hemicellulose and lignin content.

The stored PN was then converted into PNB through pyrolysis at 650 °C as per the procedure reported elsewhere (Bashir et al. 2022). The PNB was ground with pestle and mortar and sieved to a particle size of 500–710 μm for use as support in continuously stirred tank reactor (CSTR) and to 1,000–1,200 μm for use in fixed bed reactor (FBR) in the subsequent experiments.

Initially, a separate experiment was conducted to check the effect of direct addition of biochar into CSTR containing tap water. 10 g/L of PNB with particle size of 500–710 μm was directly added to the CSTR and the reactor contents were continuously stirred by magnetic stirrer at 325 RPM. However, within span of about 2 weeks of CSTR operation, the biochar particles started disintegrating into finer particles possibly due to collisions on to the reactor wall as well as magnetic stirrer. Furthermore, finer biochar particles would have a tendency to wash-out of the reactor since they had poor settling characteristics. Considering that CSTR anaerobic reactor was to be operated for about 9–10 months, it was decided to use a permeable bag to avoid the breakage of biochar particles.

5 g/L and 10 g/L of PNB with particle size of 500–710 μm were filled in two separate non-degradable and permeable plastic bags having mesh size of about 300 μm. Porous bags containing 5 g/L and 10 g/L of PNB were kept hanging in R1 and R2, respectively, at the height of 3 cm from the bottom of the reactor so that the bags always remained immersed inside the liquid phase of the reactor. On the other hand, reactor R0 did not contain any PNB. A magnetic stirrer with the hot plate was used for maintaining the reactor contents under suspension at RPM of 325 ± 2 and at the temperature of 35–37 °C. All three reactors were operated in a fed-batch mode, as this mode of operation was preferred since it allowed monitoring of diurnal variation in concentrations of various species.

The inoculum used in this study was a mixture of cow dung and laboratory-scale anaerobic CSTR effluent. The ratio of cow dung:effluent in the inoculated sludge was 1:5 (v/v) for CSTR as well as FBR. The mixture of cow dung and CSTR were mixed properly using the magnetic stirrer and was allowed to settle overnight while the supernatant was used as an inoculum.

Synthetic wastewater (SWW) which simulates spent wash, was prepared by dissolving jaggery (chemical oxygen demand (COD) 5,000–10,700 mg/L), salts of nitrogen (NH₄Cl), and phosphorus (KH₂PO₄) in deionized water to obtain the final COD:N:P as 200:5:1. pH of the influent was adjusted to a value of 7.0–8.0 using 1 N NaOH solution.

CSTR and FBR were initially filled up to 75% of the working volume by a mixture of inoculum and SWW in the ratio of 70:30 (v/v) (Mohan et al. 2020). The CSTR and FBR content was kept for 48 h for the initial acclimatization of the inoculum. During the start-up, the COD of the influent was maintained in the range of 5,000–5,600 mg/L to keep the low loading rate in the range of 0.4–3.2 kg-COD/(m³d) for FBR and 0.12–0.5 kg-COD/(m³.d) for CSTR.

The elemental composition of the PNB was determined by its C, H, N, O content using the CHNO analyzer (UNICUBE plus). Physical properties of PNB such as SSA, pore size and pore volume were determined using the Brunauer–Emmett–Teller (BET) analyser (Quanta chrome, Autosorb iQ3). For BET analysis, all the samples were degassed at 150 °C under vacuum. pH and electrical conductivity were measured according to methods listed elsewhere (Singh et al. 2017). Functional groups on the biochar surface were determined using Fourier transform infrared (FTIR) spectroscopy (FTIR L1600312, Agilent Technologies). Samples of feed and effluents from the reactors were collected daily and analyzed for pH, volatile fatty acids (VFA), COD, and total and volatile solids (TS and VS). The pH was measured using a benchtop pH meter (EI, Deluxe pH meter model-101). COD (closed reflux method), VFA (distillation method), TS, and VS were analysed using standard methods 5220C, 5560C, 2540B and 2540E, respectively, listed in American Public Health Association (APHA) (Baird & Bridgewater 1995). Conductivity was measured using Eutech CON700 conductivity meter. Sulphate analysis was carried out using the spectrophotometer (HACH DR6000). Biogas samples were analysed using a gas chromatograph (Agilent 7820A) equipped with a thermal conductivity detector (TCD).

The lignocellulosic composition of pine residue including PN is presented in Table S1. As this data reveals, PN has a higher lignin content of 40%. It is reported in the literature that higher lignin content in the agricultural residue also leads to higher carbon content in the biochar (Sohi et al. 2010). The elemental composition of the biochar produced at temperatures ranging from 450 to 950 °C is presented in Table S2. From this data it can be concluded that the PNB produced at 650 °C has superior properties as compared to other pyrolysis temperatures, such as higher carbon content, higher specific surface area and lower ash content.

Biochar primarily consists of two predominant structural fractions, namely, stacked crystalline graphene sheets and randomly ordered amorphous aromatic structures (Bashir et al. 2022). A variety of functional groups such as quinone (CO), phenol (OH) and carboxyl (COOH) etc. (Figure S1) (Bashir et al. 2022) are present on the surface of biochar, which are formed due to molecular rearrangement during pyrolysis (Azlina et al. 2013; Mia et al. 2016). Functional groups present on the biochar surface can accept or donate electrons due to the coexistence of basic to acidic and hydrophobic to hydrophilic properties of different functional groups (Uchimiya et al. 2011; Ahmad et al. 2014). The presence of phenolic and quinone groups imparts redox properties to the biochar.

The physicochemical properties of the PNB prepared at 650 °C are presented in Table 1. Its higher specific surface area facilitates microbial colonization during start-up, while its alkaline nature improves its buffering capacity. On the other hand, its higher electrical conductivity facilitates CM-IET among VFA degrading bacteria and methane-producing archaea for effective and efficient operation of AD processes.

During the steady state operation, the FBR was operated for more than 20 days at each OLR value and its performance was recorded as shown in Figure 5(a) and 5(b). Data in Figure 5(b) reveals that as the OLR was increased beyond 66 days of reactor start-up the COD removal remained at a high value of about 80% till OLR of 10.4 kgCOD/(m³.d) was achieved in 246 days of reactor operation. COD removal rate (CRR) also increased concomitantly to a value of 8.2 kgCOD/(m³.d) in 246 days of reactor operation. As the OLR was further increased to a value of 13.8 kgCOD/(m³.d) the COD removal showed a marginal reduction to 68–70% (Figure 5(b)). Interestingly, in spite of this marginal reduction in COD removal, the CRR further increased to a value of 9.5 kgCOD/(m³.d).

Initially, up to 40 days of FBR start-up, the VFA concentration remained in the range 1,400–2,300 mg/L (Figure 5(c)) while pH fluctuated in the range 5.9–7.4 (Figure 5(d)). However, as microbial attachment and subsequent biofilm growth occurred on PNB surface, electron transfer through CM-IET was initiated with PNB as the conductive material. As a result, VFA concentration showed a rapid drop beyond 40 days of reactor start-up and eventually stabilized to a value of 1,000 mg/L (Figure 5(c)) while the pH was stabilized to a value of 6.8–7.0. Subsequent increase in OLR from 1.4 kgCOD/(m³.d) to 13.8 kgCOD/(m³.d) resulted only in marginal increase in VFA concentration at each intermediate steady state OLR value. Finally, at OLR of 13.8 kgCOD/(m³.d), the VFA stabilized in the range of 1,400–1,500 mg/L (Figure 5(c)) while pH value remained in the range 6.6–6.8 (Figure 5(d)). Alkaline nature of biochar helped in maintaining the pH in FBR in the range 6.6–6.8. Furthermore, alkaline nature of the functional groups (C = C, CO, COOH and OH) present on the surface of biochar also assisted in maintaining suitable pH for methanogenesis (Ryue et al. 2020).

The methane yield of 0.34 L/(g.COD removed) is obtained from the experimental data. The high methane yield achieved was due to CM-IET facilitation by the conductive nature of PNB as it utilizes electrons at faster rate for conversion of CO₂ and H₂ to methane.

In general, IIET employs hydrogen as a carrier for electron transfer and is found in the conventional AD processes such as CSTR, which generally fails at higher loading rate due to accumulation of VFA. In biological DIET, electron transfer takes place via conductive pili or through EPS, and this mechanism is found in reactors like up-flow anaerobic sludge blanket reactors (UASB) (Morita et al. 2011). These reactors can be operated at higher OLR values of about 10 kgCOD/(m³.d) as compared to CSTR, which typically operates at the OLR of 1.5–2 kgCOD/(m³.d). In these investigations as well, the reactor R0, which represented IIET, could be satisfactorily operated at an OLR of 1.75 kgCOD/(m³.d), beyond which reactor failure occurred. On the other hand, reactors R1 (5 g/L of PNB) and R2 (10 g/L of PNB) showed satisfactory operation up to OLR of 2.5 kgCOD/(m³.d). From R1 and R2, reactor R2 continued to give more stable operation with lower VFA accumulation and higher pH value in the reactor. These investigations also revealed that the FBR which represents CM-IET through PNB as conductive material continued to operate satisfactorily up to OLR of 13.8 kgCOD/(m³.d) with methane productivity of about 3.4 L/(L.d) (Figure 6).

The increased rate of electron transfer is because biochar as conductive material bypasses the H₂ production and creates direct connection between electron producing bacteria and electron consuming methanogenic archaea. It is reported in the literature that electron transfer rate per cell pair through CM-IET (44.9 ×10³ e⁻/(cp.s)) is substantially higher than the IHT (5.24×10³ e⁻/(cp.s)) (Storck et al. 2016). Accordingly, in the present study CM-IET mode of electron transfer through conductive PNB plays an important role in accomplishing higher electron transfer rate, thereby reducing the VFA accumulation in the reactor. Table 2 presents the comparison of CSTRs, namely R0, R1 and R2, with FBR.

As the data in Table 2 reveals, the FBR could achieve higher OLR of 13.8 kgCOD/(m³.d) and COD removal rate of 9.38 kgCOD/(L.d), primarily due to highest biochar concentration of 80 g/L, thus achieving higher electron transfer rate. On the other hand, the COD removal rates decline in R1, R2 and R0 as the biochar maintained in these reactors declines from 10 to 5 and 0 g/L, respectively. Similar trend was observed in gas productivity as well, with FBR achieving the highest value of 3.32 ± 0.024 L/(L.d). The VFA concentration also brings out interesting comparison amongst the reactors. The lowest VFA of 1,442.22 ± 49.93 mg/L was achieved in FBR, signifying rapid consumption of VFA as a result of higher electron transfer rate through CM-IET due to higher biochar concentration in FBR. The VFA concentrations in R2, R1 and R0 in Table 2 show an increasing trend as the biochar concentration in these reactors reduces. The highest VFA of 6,313.97 ± 45.19 mg/L is recorded in R0, in which the biochar concentration is 0 g/L, meaning thereby the electron transfer in R0 is only through IIET. At such a high concentration of VFA the COD removal efficiency reduced to less than 40%, indicating reactor failure.

Table 3 compares the findings of the present study with the literature values. Data presented on FBR in Table 3 reveals that the methane yield of 0.34 L CH₄/(g.COD) obtained in this research is comparable to 0.33 and 0.35 L CH₄/(g.COD) as reported in literature. COD removal efficiency of 80% in FBR in current research compares well with COD removal of 93% (Shanmugam et al. 2017) and 77% (Wambugu et al. 2019) in biochar reactors as reported in the literature. PNB-supplemented FBR in the current research shows less VFA accumulation of about 1,442 mg/L due to CM-IET mechanism, which maintains the syntrophic balance among the VFA degrading bacteria and methanogenic archaea.

The main objective of this investigation was to demonstrate the beneficial role of biochar in promoting CM-IET and thereby achieving improved stability of anaerobic process and its performance. Three anaerobic CSTRs with varying biochar concentrations and one FBR were established and operated and their performance was compared.

The study revealed that CSTRs having higher concentration of biochar were more stable, had lower VFA concentration and were better buffered against pH drop. On the other hand, the FBR performance was the most superior with lowest VFA concentrations even at higher OLR of 13.8 kgCOD/(m³.d). These investigations clearly bring out that several properties of PNB, such as its surface characteristics, functional groups as well as its redox and buffering characteristics, play a beneficial role in promoting methanogenesis, as discussed below.

The surface properties of PNB help in colonization and growth of microorganisms on the surface of biochar. PNB, when in contact with near neutral aqueous medium, acquires negative charge, since PNB has point of zero charge of 5.75 pH (Bashir et al. 2022). During the entire span of 291 days of this investigation, the pH in the anaerobic reactors was in the range of 6.6–7.4. The negative surface charge acquired by PNB in this pH range results in the accumulation of cations, which are major micronutrients for the methanogenic archaea. This, in turn, initiates initial microbial attachment and growth. Porous structure of PNB also offers abundant sites for microbial attachment and growth. Once the initial microbial attachment occurs on the surface of biochar, the functional groups such as OH, COOH and CO present on the biochar surface, which have excellent redox properties, play an important role in biofilm formation and growth (Qin et al. 2020). A stable carbon pool comprising graphene sheets, aromatic carbon and various functional groups is an essential part of any biochar structure, which also plays an important role in promoting CM-IET in anaerobic processes (Zhou et al. 2014; Kobayashi et al. 2015).

Likewise, the alkali metals such as Na⁺, K⁺, Mg ⁺², Ca ⁺² present in biochar ash also neutralize the accumulated VFA and prevent pH drop in the acidic range (Shen et al. 2015).

Biochar addition helps in reducing VFA concentration and maintaining pH necessary for methanogens. Firstly, the redox properties of biochar facilitate CM-IET. As a result, the methanogens are no more dependent on H₂-mediated IET. Secondly, CM-IET between VFA degrading bacteria and methanogenic archaea substantially improves IET. This increment in electron transfer leads to much improved methanogenic activity, which results in a substantial reduction in VFA concentration. This fact is verified in this research, since the CSTR with no biochar resulted in the highest VFA concentration. On the other hand, CSTR with highest biochar concentration resulted in the lowest VFA concentration. In comparison to CSTR, FBR could perform satisfactorily at OLR value 10 times higher than CSTR and still could maintain much lower VFA concentration. These results clearly indicate the importance of biochar addition in anaerobic processes for maintaining low VFA concentrations, which is necessary for the smooth functioning of AD.

Since these CSTR investigations involved biochar in a porous bag, a discussion on their limitations is called for. The biochar was filled loosely in the non-degradable but permeable plastic bag. Although biochar was filled inside the bag, the bag was highly porous and hence, liquid as well as suspended microorganisms could easily pass through the pores. This was mainly done to avoid formation of biochar fines due to collision of biochar particles on reactor wall and magnetic stirrer. However, in reality, addition of biochar in porous bag may impact the performance of CSTR adversely due to lack of possible contact between the biomass and the biochar particles. As a result, addition of biochar directly into the UASB type of reactor which employs granular sludge would be more beneficial, since biochar particles could promote initial microbial attachment and growth, yielding eventual granular sludge formation. Accordingly, further experiments can be planned for assessing the effectivity of the PNB in the AD processes which employ granular sludge.

Role of biochar in promoting methanogenesis in anaerobic processes is investigated in this research. Biochar produced from Himalayan pine needle forest residue has been used as a conducting material for electron transfer amongst the methanogenic species. Three CSTRs, namely R0, R1 and R2, were operated at steady state with varying biochar concentration of 0, 5 and 10 g/L, respectively. Failure of R0 (reactor with no biochar addition), which represented indirect interspecies electron transfer (IIET), occurred at OLR of 2.0 kgCOD/(m³.d), while R1 (with 5 g/L biochar) and R2 (with 10 g/L biochar) could be operated satisfactorily at steady state OLR of 2.5 kgCOD/(m³.d). On the other hand, fixed bed reactor (FBR) operated with pine needle biochar as a support material could operate satisfactorily at OLR of 13.8 kgCOD/(m³.d). Addition of biochar in CSTRs and FBR resulted in much improved performance due to their superior buffering capacity and also led to lower VFA accumulation, mainly due to the conductive nature of biochar, which facilitated CM-IET amongst the microbial species. These investigations confirm the beneficial role of biochar in anaerobic processes by promoting CM-IET amongst the VFA degrading bacteria and methane producing archaea.

These investigations were carried out when Mr Chander Mohan was a doctoral student at Indian Institute of Technology, Mandi in Himachal Pradesh, India. These investigations were led through cooperation under Ministry of Human Resource and Development (MHRD), Government of India (GoI). This help is appreciatively recognized.

Chander Mohan: Conceptualization, Methodology, Investigation, Data curation, Writing – Original draft, Review and Editing. Ajit Annachhatre: Conceptualization, Supervision.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.