Thermophilic Anaerobic Digestion: An Advancement towards Enhanced Biogas Production from Lignocellulosic Biomass

Abstract

Thermophilic anaerobic digestion (TAD) technology has been adopted worldwide mainly due to it being a pathogen-free process in addition to the enhanced biogas yield and short hydraulic retention time (HRT). Taking the high metabolic rate of the thermophilic microbial community with highly efficient enzymatic systems into consideration, thermophiles are being widely explored as efficient inocula for lignocellulosic biomass (LCB) degradation and improved biomethane production. The advantages of TAD over mesophilic anaerobic digestion (MAD), including improved kinetics, efficient degradation of organic matter, and economic and environmental sustainability, make it one of the best strategies to be operated at moderately high temperatures. This review sheds light on the relevant role of thermophilic microorganisms as inocula in the anaerobic digestion of organic matter and factors affecting the overall process stability at high temperatures. Further, the discussion explains the strategies for enhancing the efficiency of thermophilic anaerobic digestion.

1. Introduction

The continuous depletion of environmental conditions due to the consistent and long-term use of the conventional and non-renewable energy resources has compelled the search for cleaner and relatively environmentally safe alternative resources. Biomass-derived energy is an emerging renewable alternative energy source to meet the interminable requirements for livelihood with a lower environmental impact [1]. Recent reports of the Intergovernmental Panel on Climate Change (IPCC) have reported the projected production of bioenergy to 100 EJ by 2050, which will account for almost 20% of the total energy needs [2,3,4,5]. Correspondingly, biogas is one of the sources of bioenergy produced through the anaerobic digestion (AD) of an organic matter in a cost-effective manner using a group of microorganisms [6,7,8]. It has gained attention in the past few decades to meet the demands for heat, electricity, and transportation fuel whilst reducing the environmental impact. Lignocellulosic biomass (LCB) is the most abundant biomass on Earth, with an estimated annual production of 181.5 billion tonnes, and is a potential feedstock for AD and also fulfills the demand for a renewable and “carbon-neutral” energy resource [9]. LCB mainly includes agricultural residues, energy crops, forestry residues, and yard trimmings without compromising global food or feed security [8]. LCB is a fibrous material, made up of polysaccharides (cellulose and hemicellulose) and embedded covalently or non-covalently with aromatic polymer (lignin) along with extractive and inorganic compounds [10]. One of the major limitations to LCB valorization into bioenergy through AD is its recalcitrant structure, strongly interconnected and difficult to dissociate. The pretreatment of LCB with physical, chemical, biological, or a combination of methods has been widely adopted to disintegrate the recalcitrant structure of LCB, but this is expensive and, to an extent, unfriendly to the environment [11].

Recently, the focus has shifted from expensive pretreatment methods to the search for an efficient source of microorganisms or inoculum sources which could be highly efficient in degrading LCB in a sustainable, economical, and eco-friendly manner [12]. As an important process parameter, temperature exerts a direct impact on the growth and activity of the microbial community inside an anaerobic digester, which in turn affects the process kinetics. The characteristics and nutritional requirements of microorganisms along with the metabolites during AD are influenced by the temperature. The enzymatic activity of the microbial cells doubles with the rise in temperature by 1 °C until reaching the threshold value [6]. Any deviation from the optimum temperature range denatures or inactivates the enzyme protein, which in turn lowers its activity. Among the various temperature ranges of AD, thermophilic anaerobic digestion (TAD), wherein the digester is operated at 50–60 °C [13] using microbial community functioning under thermophilic conditions, has gained interest over the common and convenient process, mesophilic anaerobic digestion (MAD) at 25–45 °C [14,15], due to the enhanced organic loading rate (OLR) and substrate degradation [16]. Higher degradation of the organic matter is due to the improved solubility of the substrates (composed of polysaccharides, proteins, lipids, etc.) in a high temperature range, which shortens the start-up time, improves the kinetics and stability of the process, and lowers the risk of contamination [17,18,19,20]. Therefore, TAD is an advanced strategy which has been adopted by various countries such as Brazil, the United States of America, Norway, Sweden, Denmark, Germany, and the Czech Republic at a large scale to avoid contamination and enhanced methane yield as well as to shorten the hydraulic retention time [21,22,23,24]. Thus, the present review focuses on the various perspectives of TAD, including strategies such as the development of a resilient, robust, and acclimatized microbiome to conquer the key challenges associated with the process, and also some of the emerging advanced technologies to enhance biomethane/biogas production along with future perspectives for bridging the technical gaps and potential research directions for further improvisation at a larger scale.

2. Lignocellulosic Biomass (LCB)

LCB generally comprises cellulose (38–50%), hemicelluloses (23–32%), and lignin (15–25%) [7,8]. Cellulosic and hemicellulosic fractions are polymeric sugars, which need to be hydrolyzed into monomeric fermentable sugars. On the other hand, lignin is a polyphenolic heteropolymer, which shields cellulose and hemicellulose from pathogenic attacks in nature. Readily available LCBs include agricultural, industrial, and urban wastes, as well as forestry residues and energy crops. Due to enriched cellulosic and hemicellulosic fractions in LCB, they exhibit potential to be explored for biofuel production by using their monomeric hexose (glucose, galactose, mannose) and pentose (xylose, arabinose) sugars. Various LCBs have been extensively studied by researchers for their potential in biogas production. Agricultural wastes include paddy and wheat straw, alfalfa fiber, groundnut shell, cotton stalks, corn stover, sunflower stalks, sugarcane bagasse, rice husks, corn cobs, palm bagasse, wheat barn and barley, and sunflower hulls [6,8]. Generally, wastes from industries and urban areas include food processing waste, processing papers, vegetables and fruit processing waste, cotton linters, pulps, and household waste [25,26]. Forestry wastes include branches from dead trees, wood chips, hardwood, softwood, slashes, and prunings [27]. The compositions of several LCBs and their methane yields are listed in

Table 1. Composition and methane yield of different lignocellulosic residues.

Residue	C:H:L	TS%	VS%	Methane Yield (m3/kg-VS)	References
Silage maize	16:11:38	30.8	94.1	0.259	[28]
Grass silage	31:29:10	50	92	0.344–0.383	[29,30]
Paddy straw	38:23:13	93	80	0.202	[7]
Wheat straw	33:22:19	93.1	76.8	0.282	[31,32]
Corn stover	39:26.6:19	86	94.3	0.296	[33,34]
Sugarcane bagasse	42:22:18	94	97	0.122–0.236	[35,36]
Coffee pulp	31:11:23	55	91	0.131	[37,38]
Pulp and paper sludge	na	24.2	77	0.432	[39]
Forestry residues	42:na:44	50	64	0.214	[40]
Banana stalks (sun-dried)	56:8:18	92	83	0.236	[39]
Chicken manure	12:20:2	40	75	0.309 *	[41,42]
Cattle manure	27:12:13	25	76	0.236 *	[41,42]

C:H:L = cellulose:hemicellulose:lignin; TS = total solids; VS = volatile solids; FM = fresh matter; na = data not available. Methane is primarily estimated as 55% of the reported biogas yield values; * indicates that methane is estimated as 60% of reported biogas yield values.

.

3. Thermophilic Anaerobic Digestion

AD is an attractive option to recover biomethane from organic waste with the help of various microbes under anaerobic conditions [43]. AD can be carried out at diverse temperature ranges, including psychrophilic (<20 °C) [14], mesophilic (25–45 °C), thermophilic (50–60 °C), and hyperthermophilic (70–80 °C) [44,45]. Apparently, elevated temperature leads to an increased hydrolysis rate due to an acceleration of the biochemical processes, which determines the rate of degradation. In addition, the growth rate of thermophilic methanogens is 2 to 3 times higher than that of the mesophilic methanogens with reduced hydraulic retention time (HRT) [46]. Other related advantages of TAD are increased biogas yield, shortened HRT, enhanced digestate quality, and higher rates of pathogen removal as compared to MAD [47,48,49], as highlighted in

Table 2. Comparison of TAD and MAD [50,51,52,53].

Parameters	MAD	TAD
Start-up period	Long	Short
Hydrolysis rate	Low	High
Biogas production	Low	High
Methane content	Low	High
Retention time	Long	Short
Pathogen reduction	Low	High
Reactor volume	Large	Small
Process stability	High	Low
Energy—consumption and recovery	Low; Low	High; High
Digestate quality	Low	High

.

Despite these advantages, TAD is often considered as an energy-exhaustive and unstable process as compared to MAD. Mostly, the microbiomes in thermophilic digesters are more sensitive to changes in the environmental conditions, such as pH, temperature, etc. [54]. Therefore, it is important to understand and control the process stability and conduct in-depth study of the thermophilic microbiome in the digester. There are some traditional methods to identify the microorganisms present in the AD consortium by the construction of 16S rRNA gene clone libraries followed by Sanger sequencing [55], which recently has been replaced by high-throughput next-generation sequencing of 16S rRNA gene amplicons [56]. However, the metabolic pathways of the microorganisms involved in the process can be studied indirectly by the analysis of metabolic products or intermediate products formed [57]. Figure 1 shows the metabolic pathway followed, along with all the major intermediate products formed during the AD process, such as formate, butyrate, acetate, ethanol, lactate, propionate, and methane, marked by letters (a) to (h).

For example, Gomez-Quiroga et al. [58] reported the thermophilic (55 °C) anaerobic co-digestion of sugar beet pulp and cow manure in semi-continuous reactors with anaerobic sludge as inoculum obtained from a wastewater treatment plant (WWTP), tested at ten different HRTs ranging from 3 to 30 days and OLR ranges of 2–24 gVS/L_reactor∙d. The results showed that the best performance (regarding stability, biogas production, and organic matter removal) was achieved at an HRT as short as 5 days (OLR of 12.47 gVS/L_reactor∙d) with a methane yield of 315 mL/gVS_added. However, similar AD conditions at mesophilic (35 °C) temperature yielded 178.6 mL CH₄/gVS_added in 30 days of HRT. Fang et al. (2011) obtained the methane yields of 260 and 280 mL CH₄/gVS_added for co-digestion of fresh beet pulp and cow manure operating at OLRs of 2.95 and 6.75 gVS/L_reactor⋅d and mixing ratios of 15:85 and 50:50 (% w/w), respectively, at 55 °C. In a study by Li et al. (2014), mesophilic anaerobic co-digestion of chicken manure and corn stover was carried out in stirred tank reactors operating with a concentration of 12% in TS. They reported a long HRT (22.5-day) for an OLR of 4 gVS/_Lreactor∙d and obtained a methane yield of 223 ± 7 mL/gVS_added. These results of methane yields were found to be about 30% smaller and the OLR 3 times shorter than those obtained and reported by Gomez-Quiroga et al.

Figure 1. Pathway mapped for major AD steps (hydrolysis, acidogenesis, acetogenesis, and methanogenesis) with major intermediates formed and labeled with alphabetic letters (a–h) [59]. Abbreviations: CoA—coenzyme A; P—phosphate; TCA—tricarboxylic acid; CO₂—carbon dioxide; H₂—hydrogen; MFR—methanofuran; CoM—coenzyme M; THMPT—tetrahydromethanopterin.

Figure 1. Pathway mapped for major AD steps (hydrolysis, acidogenesis, acetogenesis, and methanogenesis) with major intermediates formed and labeled with alphabetic letters (a–h) [59]. Abbreviations: CoA—coenzyme A; P—phosphate; TCA—tricarboxylic acid; CO₂—carbon dioxide; H₂—hydrogen; MFR—methanofuran; CoM—coenzyme M; THMPT—tetrahydromethanopterin.

Hence, based on the reported studies, it is shown that the shortened start-up period and enhanced organic loading capacity directly affect digester volume size at elevated temperatures (thermophilic) compared to the conventional, i.e., mesophilic, method, which also depends on the type of microbial flora present and feedstock composition in the digester [59]. Further in-depth insight is given in the following sections of this review to explain the significance of thermophilic inoculum in an anaerobic digester for better performance.

There is limited or negligible research on digestate quality obtained from TAD of LCB. However, the most-discussed biomass for TAD is sewage sludge, whose digestate quality has been examined from the final disposal point of view. Lanko et al. [13] reported the digestate quality of single-stage mesophilic (SMAD) and thermophilic AD (STAD) and double-stage thermophilic AD (DTAD) systems for treating waste-activated sludge, in terms of the dewaterability, pathogenic safety, and lower calorific value (LCV). The experimental results showed that the DTAD system is the most beneficial in terms of organic matter degradation efficiency (32.4% against 27.2% for STAD and 26.0% for SMAD), producing a digestate with high dewaterability (8.1–9.8% worse than for STAD and 6.2–12.0% better than for SMAD) and pathogenic safety (coliforms and Escherichia coli were not detected, and Clostridium perfringens were counted up to 4.8–4.9 × 10³ CFU/g, when it was 1.4–2.5 × 10³ CFU/g only for STAD, and 1.3–1.8 × 10⁴ CFU/g for SMAD with the lowest LCV (19.2% against 15.4% and 15.8% under STAD and SMAD, respectively)). Regarding the final disposal, the digested sludge after STAD can be applied directly in agriculture; after DTAD, it can be used as a fertilizer only in the case where the fermenter’s HRT assures the pathogenic safety. The SMAD digestate is the best for being used as a fuel (briquettes), preserving a higher portion of organic matter and not transforming into biogas during AD. The aforementioned example proved the benefits of a high degradation rate of organic matter under TAD, which could result in a better quality of digestate in terms of dewaterability, pathogenic safety, and LCV, as discussed.

4. Impact of Thermophilic Inoculum on AD

The multi-stage AD process is driven by the complex microbial consortium that plays a central role in degrading or solubilizing the organic substrate. Therefore, to make a robust and effective consortium, the selection of the source of microbial community is considered as one of the important factors. Over the past few decades, extensive research has uncovered the potential of thermophilic microbes to produce robust and effective carbohydrate-degrading enzymes with survival under harsh bioprocessing conditions that reflect their natural biotopes [60]. These microbes are often isolated from wastewater discharge, bio-waste streams, acid-mine effluents, as well as geothermal and volcanic areas, terrestrial hot springs, submarine hydrothermal vents, geothermally heated oil reserves and oil wells, sun-heated litter, soils/sediments, etc. [15,21]. Various microbial species involved in TAD are summarized in Table 3.

AD of LCB requires pre-processing or pretreatment of feedstocks to overcome the effect of recalcitrant nature, although this causes a substantial cost implication. On the other hand, microorganisms such as thermophiles are capable of hydrolyzing the carbohydrates of LCB, which is considered as the rate-limiting step in AD. Therefore, in the presence of thermophiles, the possibility is that mild or no pretreatment would be required [60,61]. Shikata et al. [62] isolated the thermophilic, alkaliphilic, and non-pretreated LCB-degrading consortium, ISHI-3, from bovine manure compost, employed for TAD of non-pretreated cornstalk (CS) (32.2% ± 1.9% of cellulose, 29.1% ± 2.7% of xylan, 7.5% ± 2.1% of arabinan) and rice straw (RS) feedstocks (40.4% ± 2.8% of cellulose, 18.7% ± 3.0% of xylan, 4.6% ± 1.1% of arabinan) at 55 °C. The residue weights decreased by 64.1% (w/v) and 65.3% (w/v) compared with the total initial dry weights of CS and RS after inoculation with ISHI-3, respectively. Reportedly, the isolated ISHI-3 consortium comprised one major cellulolytic–xylanolytic anaerobe (Herbivorax saccincola) and three non-cellulolytic anaerobes (belonging to Pelotomaculum, Tepidimicrobium, and Tepidanaerobacter), as determined by DGGE and metagenomics analysis, based on the 16S rRNA genes. The characterization of the microbiome in combination with all possible techniques facilitates a multi-dimensional view of the performance, species composition, carbohydrate-active enzymes (CAZymes), and metabolic functions at varying residence times during the overall digestion period. Dai et al. [63] reported the identification of a CAZyme family of cellulolytic bacteria, glycoside hydrolase (GH), classified mainly as GH 5, GH 9, GH 45, and GH 48. Likewise, Kabaivanova et al. [47] and Maus et al. [64] identified that in general, the most abundant phyla, Firmicutes, Bacteroidetes, and Proteobacteria, are found in the thermophilic microbial consortium with the ability to solubilize and metabolize the carbohydrates (cellulose and hemicelluloses) of lignocelluloses in the TAD process. Liang et al. [65] reported the development of stable switchgrass-fermenting enrichment cultures maintained at various residence times (20, 10, 5, and 3.3 days) and thermophilic (55 °C) temperature. The results revealed stable, time-invariant cellulolytic methanogenic cultures with minimal accumulation of organic acids which solubilized carbohydrates (cellulose and hemicellulose) by about 71.1% at 20 days of RT. However, the coexistence of a thermophilic cellulose degrader with methanogens had been commonly observed in the genus of Clostridium (phylum Firmicutes), also known as a stable cellulose-utilizing thermophilic methanogenic community [66].

The substrate to inoculum (S/I) ratio plays an important role in the proper functioning of the AD process, and depends on the composition of the feedstock. Lin et al. [67] worked on the optimization of the S/I ratio for yard trimmings as substrate and dewatered effluent from a mesophilic liquid anaerobic digester as the initial inoculum in the subsequent sequential batch thermophilic solid-state AD (SS-AD). To speed up the acclimatization of mesophilic inoculum to thermophilic conditions, recirculation of solid digestate (~51%) was performed. At an S/I ratio of 1, the sequential batch SS-AD gradually reached a steady state after three runs (30 days/run), with an increase in both methane yields (up to 11.5%) and cellulose degradation (up to 55%), indicating that recirculated digestate could be a feasible inoculum, acclimated to the substrates and operating conditions.

Table 3. Various microorganisms involved in the TAD process.

Stages of TAD	Microbes Reported	Function
Hydrolysis (bacteria and fungi) Microbial domain (genera -> species)	1Defluviitoga tunisiensis L3 [64] 2 Caldicellulosiruptor bescii [68] 3 Gracilibacter thermotolerans JW/YJL-S1 [69]	1,2 Cellulolytic, 3 polysaccharides
	Proteiniborus indolifex [70]	Protein-utilizing
	Acidaminococcus intestini	Amino acid-utilizing, mainly glutamic acid
	Thermoanaerobacterium thermosaccharolyticum [71], Caldanaerobacter subterraneus [71], Thermoanaerobacter pseudethanolicus [71], Clostridium cellulolyticum [71]	Thermophilic cellulolytic
Acidogenesis (fermentation bacteria)	G. thermotolerans [69]	Glucose-degrading
Acetogenesis (bacteria)	1Syntrophaceticus sp. [54] 2 Thermogymnomonas acidicola [54] 3 Gelria glutamica [54]	1 Syntrophic acetate-oxidizing bacterium 2 Thermophilic acetogen 3 Syntrophic glutamate-degrading
Methanogenesis (archaea)	Methanoculleus, Methanobacterium Thermogymnomonas, Thermoplasmata, Methanospirillum, Thermoprotei Methanobrevibacter, Methanolinea, Methanosaeta, Methanimicrococcus [54]	Methane-forming archaea

Note: The superscript prefix numbering in the “Microbes Reported” column represents the corresponding function of it as mentioned in the adjacent column “Function”.

Table 3. Various microorganisms involved in the TAD process.

Stages of TAD	Microbes Reported	Function
Hydrolysis (bacteria and fungi) Microbial domain (genera -> species)	1Defluviitoga tunisiensis L3 [64] 2 Caldicellulosiruptor bescii [68] 3 Gracilibacter thermotolerans JW/YJL-S1 [69]	1,2 Cellulolytic, 3 polysaccharides
	Proteiniborus indolifex [70]	Protein-utilizing
	Acidaminococcus intestini	Amino acid-utilizing, mainly glutamic acid
	Thermoanaerobacterium thermosaccharolyticum [71], Caldanaerobacter subterraneus [71], Thermoanaerobacter pseudethanolicus [71], Clostridium cellulolyticum [71]	Thermophilic cellulolytic
Acidogenesis (fermentation bacteria)	G. thermotolerans [69]	Glucose-degrading
Acetogenesis (bacteria)	1Syntrophaceticus sp. [54] 2 Thermogymnomonas acidicola [54] 3 Gelria glutamica [54]	1 Syntrophic acetate-oxidizing bacterium 2 Thermophilic acetogen 3 Syntrophic glutamate-degrading
Methanogenesis (archaea)	Methanoculleus, Methanobacterium Thermogymnomonas, Thermoplasmata, Methanospirillum, Thermoprotei Methanobrevibacter, Methanolinea, Methanosaeta, Methanimicrococcus [54]	Methane-forming archaea

Note: The superscript prefix numbering in the “Microbes Reported” column represents the corresponding function of it as mentioned in the adjacent column “Function”.

5. Factors Affecting the Performance of Thermophilic Inoculum

5.1. Ammonia Inhibition

Ammonia is one of the common hydrolysis products formed during the degradation of organic nitrogen present in biomass in the form of urea and proteins [72]. Karkat et al. [73] reported that only a proportionate fraction of organic nitrogen available in feedstock is converted biologically into ammonia (${\mathrm{NH}}_4^{+}-\mathrm{N}/{ \mathrm{NH}}_3-\mathrm{N})$. Subsequently, Gallert and Winter [72] evidenced that during TAD, the ammonia formation is high due to the higher protein degradation rate than MAD. On the other hand, few process parameters such as pH and temperature have been considered as crucial influences on ammonia formation and stability of TAD. To an extent, the carbon dioxide dissolving capacity is reduced at a higher temperature, which causes the elevation of pH and leads to high-impact ammonia [74].

However, ammonia concentrations up to 200 mg/L are considered beneficial for metabolic activities of the thermophilic microbiome [74], and the rest of the ammonia serves as a natural buffer to resist acidification from the accumulation of organic acids in the digesters [75]. However, the total ammonia nitrogen (TAN) concentration exceeding the threshold limit for TAD, i.e., 1500–11,000 mg-N/L, results in the strong inhibition of the digestion process, usually indicated by the decrease in methane production rates [76,77] (

Table 4. Ammonia inhibition reported for TAD.

Substrate	Temperature (°C)	TAN Conc.	FAN Critical Conc.	Major Findings/Impacts	References
Pig manure	51	4.6–11.0 g-N/L	1450 mg-N/L	50% inhibition of methanogenesis at 11.0 g NH4-N/L	[81]
Paper and yard waste	54–60	0.75–2.5 g-N/kg	-	50% reduction in CH4 at 1.5 g-N/kg Complete failure of digester at 2.5 g-N/kg	[82]
OFMSW	55	1.75–3 g-N/L	660 mg-N/L	Methane yield decreased by 63% at 432 mg-N/L of FAN	[83]
OFMSW	55	1.7–5.6 g/L	468 mg/L	50% methane inhibition at specified TAN and FAN levels	[84]

OFMSW = organic fraction of municipal solid waste; TAN = total ammonia nitrogen; FAN = free ammonia nitrogen.

). There are some digesters which can tolerate higher TAN concentrations due to various factors such as acclimatization of inoculum, inoculum source, robust microbial communities, etc. [78,79]. TAN exists in the digester in the form of ammonium ions $\left({\mathrm{NH}}_4^{+}-\mathrm{N}\right)$ and free ammonia nitrogen (NH₃-N) (FAN). Among these two species, FAN is the main concern due to its ability to diffuse passively into the microbial cell membrane, cause proton imbalance, and create potassium deficiencies [80].

Methanogens are highly sensitive to high TAN levels, more than other microbes in AD. The inhibitory concentration of TAN is reported as 3500 mg/L ${\mathrm{NH}}_4^{+}-\mathrm{N}$ and 250 mg/L NH₃-N for acetoclastic methanogens and 7000 mg/L ${\mathrm{NH}}_4^{+}-\mathrm{N}$ and 500 mg/L NH₃-N for hydrogenotrophic methanogens at pH of 7.2–7.3 under thermophilic conditions [72,85,86]. However, Tian et al. [79] performed a step-wise acclimatization strategy to tolerate high TAN level such as 10,000 mg/L on methanogen communities and reported results indicating the robustness of acetoclastic methanogens to the highly inhibitory conditions. Furthermore, it has been found that the choice of feedstock can also influence the ammonia inhibition potential due to an imbalanced C/N ratio. The optimal range of C/N ratio is between 20 and 30, and outside this range, nutrient imbalance usually takes place, which causes low performance. To mitigate ammonia inhibition caused by an imbalanced C/N ratio, the co-digestion of carbon and nitrogen-rich feedstocks could be a solution to balance the C/N to an appropriate value [7].

5.2. Organic Acid Accumulation

AD is a biochemical process involving hydrolysis, acidogenesis, acetogenesis, and methanogenesis in a sequence (Figure 1). In hydrolysis and acidogenesis, the complex organic matter is converted into short-chain volatile fatty acids (VFAs) such as acetic acid, propionic acid, butyric acid, lactic acid, and valeric acid by acidogenic or fermentative bacteria [87]. Acetogens further utilize these organic acids to break them into the simplest acetic acid along with hydrogen (H₂), which in turn forms a precursor of methane to be utilized by methanogens. Acetate can be utilized directly by methanogens [88]. It has been estimated that about 75% of methane is produced from the degradation of acetate. The accumulation of VFAs, i.e., acidification, creates an imbalance between acidogenic and methanogenic stages. In the case of TAD, the high hydrolysis rate of organic matter may cause process inhibition due to the accumulation of VFAs, which causes the pH to drop. The common VFAs accumulated in TAD are reported to be acetate and propionate, giving an indication of the system instability. Kim et al. [89] observed that the threshold concentration of acetate in TAD of rice straw was about 5000 mg/L, which led to a decrease in pH from 7.2 to 4.9, causing the complete failure of the system. Hori et al. [90] reported the dynamic transition of the methanogenic population in a TAD digester (at 55 °C) mainly due to propionate and acetate accumulation, which was investigated using a combination approach of the process analyses and molecular ecological techniques, i.e., PCR-mediated single-strand conformation polymorphism (SSCP) based on the 16S rRNA gene, quantitative PCR, and fluorescence in situ hybridization (FISH). They observed a dynamic transition of predominantly presented hydrogenotrophic methanogens from Methanoculleus sp. to Methanothermobacter sp. at a propionate accumulation of >1.4 mmol/L along with the associated bacteria (analyzed by FISH). The VFA concentration has been reported to be closely related to the dissolved hydrogen concentration [91] and it is suggested that the dissolved hydrogen concentration plays a direct role in the predominance of the hydrogenotrophic methanogens. Thus, VFAs could be considered as a useful tool to identify the microbial presence. The organic loading rate (OLR) is a key factor which influences the accumulation of VFAs. Enhanced OLR and subsequent hydrolysis lead to the higher VFA concentration, which causes pH to drop, inactivating the metabolic pathways for methane production [92]. Therefore, it can be concluded that the optimization of OLR according to the target feedstock may lead to overcoming organic acid accumulation and provide stability to the TAD process.

6. Strategies to Enhance the Efficiency of TAD

6.1. Development of Robust Microbiome: Selection, Acclimatization, and Bioaugmentation of Inoculum

A robust inoculum in AD refers to one which provides stable and abundant microbiota to degrade the substrate efficiently into a useable product such as methane. The source of origin plays a vital role in shaping a robust inoculum characteristic, such as its adaptation to the substrate and/or the operating conditions. In the case of LCB, the hydrolysis step requires longer time periods because the inoculum employed is not adapted to lignocelluloses and/or the thermophilic conditions. TAD performs well with the inocula that are already acclimated to thermophilic temperature due to the presence of active thermophiles, which can shorten the start-up period considerably [93]. However, the limited availability of thermophilic inocula compelled the incubation of mesophilic inocula at a high temperature to allow the microbiota to adapt to thermophilic conditions. Similarly, many strategies have been reported in the literature for the shifting of inoculum from mesophilic to thermophilic conditions, such as the single-step increase in temperature from 35 to 55 °C along with an initial drop in OLR and then a gradual increase, which improved the digester performance [94,95]. Palatsi et al. [96] performed a similar temperature transition study where a single-step increase in temperature from 35 to 55 °C was adapted, and the system recovered after 20 days in terms of methane yield, with 0.371 L-CH₄/g-VS, although the temperature transition caused higher VFA accumulation and propionate/acetate ratio. On the other hand, the slow and gradual increase in the temperature at a constant OLR requires longer transition times to adapt and stabilize the process. Tian et al. [97] also applied the one-step strategy to directly increase the temperature of their mesophilic CSTR to thermophilic conditions (55 °C) within a day; the effect was observed on the methanogenic communities in ~11 days, with the start-up period shortened to 20 days. However, it is possible that the optimum inoculum source may vary with the composition of the substrate as well as the operating conditions of digesters.

Bioaugmentation is used as an approach to provide a boost in a variety of microbes in the reactor by co-inoculation from different sources. It helps in reducing the start-up period of the process by reducing the time of rate-limiting steps (i.e., hydrolysis and methanogenesis) in TAD. The consistent increment in the biogas and methane yields could be considered for analyzing the adequate adaptation of co-inoculated consortia. Strang et al. [71] worked on two stable thermophilic mixed cellulolytic consortia marked as AD1 and AD2, enriched from an industrial-scale biogas fermenter and used for bioaugmentation. The corn stover was used as a substrate in the laboratory-scale batch reactors, and reported an enhanced methane yield by 22–24%. Next-generation sequencing revealed the predominant microbial strains as Thermoanaerobacterium thermosaccharolyticum, Caldanaerobacter subterraneus, Thermoanaerobacter pseudethanolicus, and Clostridium cellulolyticum. The effect of these strains, cultivated in the pure cultures, was investigated with the aim of reconstructing the defined cellulolytic consortium. The addition of the four bacterial strains and their mixture to the biogas digesters enhanced the methane yield by 10–11%, but it was not as efficient as the original communities, indicating the significant contribution by the members of the enriched communities present in low abundance. Therefore, mixing inocula from different sources can be selected as another strategy to promote a diverse and robust microbial consortium in TAD with enhanced buffering capacity and lower propionate accumulation [48,98] (Figure 2).

6.2. Adoption of Emerging Technologies

6.2.1. Bioelectrochemical Technologies

The bioelectrochemical system refers to an electrochemical interaction involved with at least one electrode, i.e., an anode or cathode, with the electroactive bacteria or microorganism. The presence of microorganisms on an anode yields the electrons from an organic substrate [91]. The reported bioelectrochemical technologies are microbial electrolysis cells (MECs) and microbial fuel cells (MFCs) coupled with a thermophilic anaerobic digester in a single- or double-chambered manner or in series [99,100,101,102]. These technologies have been recently adopted to promote the methane yield enhancement, and to overcome the organic as well as ammonia inhibitions (Figure 2).

Cerrillo et al. [99] investigated the effects of doubling OLR and the ammonia loading in TAD of pig slurry by hydraulically connecting an MEC with an AD reactor to demonstrate how the MEC could be used to alleviate ammonia inhibition. The independent TAD reactor failed when the OLR was doubled from 3.02 to 6.25 kg COD/(m³-d) due to severe inhibition by ammonia accumulation. However, when the recirculation loop was established to recirculate effluent from the dual-chamber MEC back to the AD reactor, recovery of the AD process was observed. The benefits of the recirculation loop were due to the contribution of MEC to decrease ammonia loading by in situ ammonia recovery to the cathode, as well as decreasing total organic load due to the removal of 20–30% of COD occurring within the MEC. In a dual-chamber MEC having a cation exchange membrane (CEM), ammonium ions can transport from the anode chamber to the cathode chamber to maintain charge neutrality in the system [103,104]. Zhang and Angelidaki [105] developed another strategy to lower the ammonia levels in a continuously stirred tank digester and recover ammonia by submerging a bioelectrochemical cell inside a thermophilic digester. The system, named the submersible microbial desalination cell (SMDC), was designed to carry out in situ ammonia recovery and electricity production. The anode chamber is continuously fed with an acetate-modified nutrient solution and generates a supply of electron flow to the cathode; the aerated cathodic chamber takes in ammonium ions (NH₄⁺) through a CEM from the surrounding ammonia-rich wastewater, which is then recovered as ammonia (NH₃). They achieved an average recovery rate of 80 g-N/(m²-d) over 30 days by decreasing the TAN concentration from 6 to 0.7 g/L. However, the current status of SMDC applications is limited to synthetic waste streams, and further efforts need to be made to test it on real ammonia-rich substrates such as livestock manures. Compared to conventional technologies such as air-stripping or electrodialysis, the SMDC is highly advantageous because of the lower energy costs and because there is no need for chemical additives. Thus, the integration of MEC is promising for increasing the robustness of the TAD process. However, this application is still in its early stages, especially regarding scale-up issues associated with bioelectrochemical technologies [106,107].

Zhang et al. [108] studied single-chambered MECs combined with thermophilic anaerobic digesters for the digestion of ammonia-rich synthetic substrates and sludge under high FAN concentrations. The results showed that the highest methane yields from both the substrates were obtained at an applied voltage of 0.7 V as 25 and 97.8 mL g⁻¹ VSS_added for synthetic substrates and sludge, respectively. Methanosarcina and Methanothermobacter were the predominant archaea, but coupling of MECs with a thermophilic digester indicated that it could increase the number and mass of microbial species in the digesters under ammonia inhibition. A similar study was conducted by Cerrillo et al. [99] for TAD of pig slurry as substrate combined with a two-chambered MEC reactor, which was coupled to an ammonia stripping unit as a post-treatment of effluent and a loop configuration recirculation. The MEC allowed the system to achieve chemical oxygen demand (COD) removal of 46 ± 5% after doubling the overload of inhibitors and recovered 40 ± 3% of ammonia. However, the AD-MEC system in loop configuration recovered the system with a more stable and robust operation (55% increase in the methane productivity). The microbial population assessment revealed an enhancement of AD methanogenic archaea numbers and a shift in the eubacterial population. Tremouli et al. [100] explored the usage of MFC technology (in four air cathode MFC units) for processing digestate post-AD from thermophilic and mesophilic anaerobic digesters fed with fermentable food waste. This approach helped facilitate COD removal from the digestate by 80–90% within the first 24 to 48 h of each cycle of operation to convert it into power as the product. The power obtained from mesophilic fed cells (~0.24 mW) was lower than the thermophilic fed cells (~0.42 mW).

Therefore, it can be concluded from the above studies that the bioelectrochemical technologies, viz., MEC-TAD or MFC-TAD, could be proven as the best paths forward due to their high energy efficiency combined with their overcoming of inhibitions and power output from post-AD digestate treatment, but they are not commercialized at a large scale.

6.2.2. Addition of Conductive Materials

The addition of conductive materials (CMs), such as granular activated carbon (GAC), biochar, carbon cloth, carbon-based nanomaterials, magnetite (Fe₃O₄), stainless steel, and conductive polymeric material, is considered as one of the emerging strategies to enhance the efficiency of the TAD process. It has been shown that the growth of microorganisms involved in the AD process depends on the electron transfer among the syntrophic community through an intermediate such as hydrogen or formic acid. This direct interspecies electron transfer (DIET) can be stimulated by adding conductive materials into the digester which act as conduits for electron exchange between syntrophic partners [107]. DIET is one of the alternative ways of electron transfer without hydrogen involvement and is reported to be 10⁶ times faster than interspecies hydrogen transfer (IHT) [109,110]. This advantage of DIET allows a much more rapid conversion of organic waste into methane, which ultimately can be an effective solution to accelerate and stabilize the TAD start-up process. Recently, to create a DIET environment in the TAD system, CMs, i.e., activated carbon, carbon nano-tubes, biochar, magnetite particles, graphene, carbon fibers, etc., were reported to be capable of serving as conduits for electron transfer, enriching electro-active bacteria and accelerating the methanogenesis process [111]. Yan et al. [112] reported in their study that the methane production rate in CM-supplemented reactors was almost two times higher than the control reactors. Caloramator sp., a candidate of electro-active bacteria, was significantly enriched in the carbon nano-tube (CNT)-supplemented groups (12.89%) as compared to control groups (only 1.26%). Together with the doubled abundance of Methanosaeta and Methanosarcina methanogens in CM groups, it is highly possible that Caloramator sp. and Methanosaeta/Methanosarcina have established syntrophic DIET by adopting CMs as electron conduits. Microbial community analysis indicates that DIET was likely to be an unstable condition and trigger the syntrophic process. This study demonstrated that conductive materials could promote microbial activity and shorten the start-up period for the thermophilic anaerobic system. There are few studies which report the advantages of adding CMs, such as magnetic particles which triggered the fast and robust syntrophic pathway of methanogenic propionate degradation [109].

Tiwari et al. [113] studied the TAD of recalcitrant agro-waste (wheat husk and sewage sludge) co-digestion supplemented with CMs such as granular activated carbon (GAC) and granular biochar (GBC) to enhance the biogas yield. The results revealed that samples amended with GAC and GBC at 20 g/L dosage of CM yielded the maximum biogas production of 263 and 273 mL/g VS_added, respectively, with an additional advantage of a shorter lag phase than the control. However, these studies have shown that supplementing thermophilic digesters with various CMs facilitate the ability to control acidification and control the accumulation of common inhibitors, such as ammonia [58,59]. The purpose of CM addition is to accelerate and stabilize the TAD process start-up and methane production [109,114,115,116].

7. Future Perspectives and Conclusions

LCBs are considered as suitable feedstock for the production of biofuels, especially biogas, through the TAD process, contributing to a cleaner environment and carbon neutrality. TAD seems to be a promising technology to be implemented for the efficient digestion of feedstocks such as LCB. However, most of these technologies have been tested on the lab scale, and therefore, techno-economic data, mass and energy balance, and other social issues are not known. Moreover, the TAD process suffers from various technological gaps which could be mitigated by cost-effective re-engineering of digesters’ design with scale-up approaches. The progress in design may include the application of emerging technologies along with the provision of mixing, heat transfer, and processing of the digested slurry from the bench scale to the commercial scale.

The present review summarizes the advantages of TAD of recalcitrant LCB as one of the cleaner energy production processes, which not only reduces GHG emissions but also facilitates waste management relatively more efficiently over the MAD process in terms of the improved kinetics, better degradation of substrate, and reduced chances of contamination, and it is economical as well as eco-friendly. Selecting suitable thermophilic inocula is a more effective strategy instead of expensive pre-treatment approaches for AD of LCB. The adoption of emerging technologies could also help in enhancing the process stability and efficiency. However, the overall techno-economic analysis of TAD process must be explored further for scaling up.