Casting Light on the Micro-Organisms in Digestate: Diversity and Untapped Potential

Abstract

Anaerobic digestion (AD) is an established process for waste conversion to bioenergy. However, for the AD process to be viable, it is imperative that all products be adequately valorized to maximize the benefits associated with the technology and in turn promote economic feasibility and technology uptake. Digestate is a byproduct of the AD process that is oftentimes overshadowed by the primary product, biogas, however the potential of digestate is vast. Digestate is composed of undigested organic matter, inorganic matter, and microorganisms. Whilst digestate has frequently been utilized as a soil amendment due to its abundance of readily available plant nutrients, the microbial content of digestate is oftentimes neglected or undermined. The array of microbes prevalent in digestate may contribute to expanding its potential applications. This microbial composition is shaped by several factors including resident microbial communities in inoculum and feedstock, feedstock composition, temperature of the AD system, AD additives and augmenting agents as well as post-treatment strategies, amongst others. Hence, it is hypothesized that digestate microbial content can be manipulated to target particular downstream applications by altering the above-mentioned factors. In so doing, the value of the produced digestate may be improved, which may even lead to digestate becoming the most lucrative product of the AD process. This review provides a holistic overview of the factors influencing the microbial community structure of digestate, the microorganisms in digestate from diverse AD systems and the associated microbial functionality as well as the potential applications of the digestate from a perspective of the resident microflora. The aim of the paper is to highlight the vast potential of microorganisms in digestate so as to broaden its applicability and value.

1. Introduction

Anaerobic digestion (AD) has long been recognized as a promising technology for organic waste management with the generation of bioenergy in the form of biogas [1,2]. In recent years, climate and energy policies as well as schemes for the promotion of renewable energy resources have been major drivers promoting household and industrial applications of AD [3]. As such, renewed interest has been diverted to unravelling the AD process in terms of factors influencing biogas yield and process stability [4], the microbial communities driving the process [5] and strategies to enhance methane yield [6,7]. However, the management of the effluent from the AD process has received much less attention.

The effluent of the AD process, i.e., digestate, is composed of a mixture of partially degraded organic matter, inorganic substances and microbial biomass, with an elevated moisture content [8,9]. Whilst direct digestate discharge is an option, such management strategies require substantial digestate treatment prior to discharge in order to minimize environmental impacts such as greenhouse gas (GHG) emissions and the contamination of receiving water and soil. Such treatment methods are cost intensive and may negate the economic benefits associated with biogas production. Moreover, digestate disposal is considered wasteful in a modern society where circular economy concepts are receiving more attention [10]. A more attractive alternative is the recovery and valorization of the constituents of digestate, thereby modifying the outlook of the digestate from waste to a valuable renewable resource [9].

Conventional digestate valorization strategies include utilization of the digestate as a fertilizer or as a substrate for thermo-chemical conversion to value-added products such as biochar [11,12]. Oftentimes, the nutrient content of the digestate takes precedence when establishing methods of digestate valorization. For example, the chemical composition of digestate, in terms of the presence of plant-essential macro- and micro-nutrients often receives most attention when considering digestate as a fertilizer. However, digestate is a complex substance consisting of chemical compounds and an array of microbial entities. The often-neglected native microbial community associated with the digestate may even broaden its applicability.

It has been well established that digestate contains a wide diversity of microorganisms which is oftentimes related to the operational conditions utilized for biogas production and the choice of the feedstock and inocula used [13,14,15,16]. When evaluating this microbial community, focus is frequently directed to the biosecurity of the digestate in terms of the prevalence of pathogens and post-treatment to remove such microbial entities [17]. Recent studies have however revealed that the AD process has the potential to reduce/eliminate selected pathogens in digestate [18,19,20] and focus has been redirected to digestate microbes of agronomic and environmental importance [21]. It has been established that digestate is rich in agronomically important microbes such as plant growth-promoting bacteria, arbuscular mycorrhizal fungi, denitrifying and nitrifying bacteria, nitrogen-fixing bacteria, saprophytic fungi as well as soil methanogens [21,22]. Other studies have reported on the potential of microbes obtained from digestate to degrade aliphatic hydrocarbons in petroleum-contaminated soil [23]. A recent study by Bankston and Higgins, reported on the influence of microbial communities in digestate on algal growth [24].

Whilst reviews and chapters have been published on the potential applications of digestate [25,26], oftentimes these applications are purely based on the nutrient content of the digestate and neglect the microbial potential. Therefore, this review aims to collate data obtained from studies related to micro-organisms in digestate to provide a holistic view of the current status of such microbes from the point of view of potential applications. In order to provide a thorough overview of the topic, the factors that shape the microbial community of digestate will first be unraveled followed by the functionality of microbes in digestate, which dictates the eventual applications. In so doing, this review reveals strategies for manipulating the AD process in order to achieve the desired microbial constituents in the digestate for particular applications that may be the most economically rewarding. This paper is of particular value to investors in biogas technology, researchers, policy makers, non-government organizations and anyone interested in a circular economy and maximizing the output of the AD process.

2. Factors Shaping Microbial Composition of Digestate

The microbial composition of digestate is largely determined by many interacting factors which could be categorized into pre-process, in-process and post-process parameters (Figure 1). Pre-process factors are related to external parameters that exist prior to AD and are introduced when starting materials such as feedstock and inoculum are fed into digesters. Specifically, the feedstock and inoculum harbor diverse resident microflora that find their way into AD systems together with incoming material, and these allochthonous microorganisms have the ability to modulate the AD microbiome and consequently the microbial constituents of the corresponding digestate. The in-process factors, on the other hand, are primarily operating conditions such as temperature and supplementation with additives, both of which have the potential to influence the digestate microbiome. Post-treatment methods such as digestate drying and composting could also modulate its microbial composition. The following subsections outline how these factors influence the type of microorganisms that colonize digestate.

2.1. Resident Microbial Communities in Inoculum and Feedstock

Traditionally, the inoculum is introduced into the digester with the primary objective of providing microbial consortia in order to initiate and stimulate the AD process. Therefore, their important roles in facilitating organic matter degradation through the supply of seed microbes and modulation of the microbial community in anaerobic digesters are well documented [27,28]. Although the effects of the inoculated seed on the AD microbiome during the process have long been recognized, the fate of the inoculum microbiota post-AD remains a debatable question that ongoing research seeks to clarify. Current research has revealed contrasting results for inoculum effects, which in some studies appear to influence digestate microbiota, but in others, no correlation was found between these parameters. For instance, an investigation of various inocula obtained from different sources and the microbial community assembly in anaerobic digesters treating cellulose supported the inoculum effects [29]. In their study, dendrograms from Bray–Curtis similarity matrices revealed that the microbial community of the sample from each digester and its corresponding inoculum clustered together. This indicated that inoculum played a more important role in shaping microbial communities than niche-based ecology theory, which accentuates the role of environmental factors in the distribution of microbial species [29]. On the other hand, other researchers have disputed the effects of inoculum, as no correlations were found between microbial profiles of inoculum and digestate microbiome [30,31,32]. In these studies, the impact of the sludge used as inoculum and as feed was obvious at the start-up phase, ensuring a smooth start-up for the AD process. As the process progressed and reached a steady phase or during long-term operation, marked bacterial and archaeal successional dynamics were observed, and a shift between the inoculum and digestate microbiota was attributed to acclimation and other factors [33].

Similarly, conflicting evidence was published on the influence of the initial microbiota in feedstock on digestate microbial composition. In an earlier study of abundant and growing organisms in full-scale anaerobic digester systems that received waste-activated sludge, 16S rRNA gene amplicon sequencing revealed that the microbial composition of the influent and the digestate was similar [34] indicating the influence of the feedstock. Likewise, a feedstock-induced microbial shift was found in a more recent study comparing the metagenome of AD digestate and a variety of feedstock, with the predominant phyla in feedstock (Firmicutes, Bacteroidetes and Proteobacteria) being similar to that of AD digestate [35]. However, the microbial diversity of feedstock samples in the same study was higher than that of AD digestate samples. It is possible that the conditions imposed by AD promote the growth of particular microbial groups but also impair or kill off others, leading to the loss of microbial diversity in the digestate [36]. In addition, the relative abundance of most genes involved in methanogenesis increased from feedstock to AD digestate, which is consistent with the higher content of methanogens in digestate. Furthermore, evidence for the contribution of the raw organic waste microbiome to the product, including digestate and compost bacterial content, was provided by Aigle et al. [37]. In this study, the raw organic waste content was found to be more of a key factor for predicting bacterial constituents in digestate compared to compost. On the contrary, Wang and co-workers described differences in the bacterial community between digested sludge and feeding sludge whereby archaeal, bacterial, and fungal communities of the digested sludge system remained relatively stable but varied from the microbial constituents in the feeding sludge [38]. The authors suggested that under different feedstock conditions, dominant archaeal, bacterial, and fungal compositions in an AD reactor are likely to remain stable regardless of the type of feedstock. Although the exact cause of the discrepancies among these studies is unknown, variations in inoculum and the composition of the substrates, operational parameters, analytical tools and sample sizes could be possible contributing factors.

2.2. Feedstock Composition

It is well known that variation in organic matter input resulting from differences in feedstock nutrient content can influence the microbial composition of AD and the resulting digestate. For example, one study investigating the impact that inoculum source, substrate type and operating parameters exert on AD and digestate microbiota showed that substrate and selected operating parameters and not the inoculum source are important factors modulating digestate-associated microbiota after AD of lignocellulose-rich material (manure and grass mixture) [32]. Many recent studies have also described the feedstock effects on AD and the resulting digestate microbiome when digesting a variety of organic waste including cattle and swine manure [39], rice straw [40], food waste and sludge [41], animal manure [35], wastewater and bio waste [42] as well as food waste alone [31]. These studies have identified the high variability of feedstock characteristics, digestibility and nutrient contents as important factors shaping the structure of the prokaryotic community in digestate.

Among the different nutrient components in feedstock, the carbon to nitrogen (C/N) ratio of the influent substrates significantly affects the entire AD process, with a substantial impact on the digestate microbial content [43]. Carbon and Nitrogen are essential nutrients for microbial metabolism and their concentration in feedstock governs the enrichment of AD microbes with diverse nutritional needs and metabolic activity [44]. Increased C/N ratio is associated with an increase of Clostridiales, which are known lignocellulose-degrading microorganisms, as well as the enrichment of genes encoding lignocellulolytic activity, conferring higher degradation of monosaccharides and polysaccharides. Conversely, a lowered C/N ratio generated solid digestates richer in family Thermotogaceae and genus Caldicoprobacter and in genes encoding proteins with the capacity to protect cells directly from reactive nitrogen intermediates [43]. Members of the Thermotogaceae family possess unique metabolic and genomic features that support growth and degradation of complex substrates at high temperatures; as such, they are associated with thermophilic AD of nitrogen-rich substrates [45,46]. In addition, the Thermotogaceae family is identified as one of the key players involved in phosphorus (P) mineralization in artificial soil mixtures [47]. Therefore, the application of digestate containing such P-solubilizing microorganisms could transfer beneficial microbes and thereby influence nutrient turnover and support more P recycling in soil. As a driver of digestate microbiota variation, the C/N ratio could be regulated by co-digestion of different feedstock in order to ensure a ratio that favors desirable microorganisms both for the AD process and the quality of the finished product.

Alternatively, the effects of feedstock on digestate microbial community could be related not only to their nutritional content but also to the deleterious effects of the by-products formed during the AD process. The decomposition of organic feedstock with high concentrations of nitrogenous matter in the form proteins and urea often results in ammonia (NH₃) production. During AD, high levels of ammonia have regularly been implicated in digester failure due to the inhibition of microbial activity, which could alter AD microbiota as well as influence digestate microbial diversity [48]. Studies on the impact of an increasing amount of NH₃ on digestate microbiome consistently described a shift in microbial community in support of the high relative abundance of Clostridiales with high total ammonia [49,50]. The Clostridiales order was composed of several NH₃-tolerant species that participate in syntrophic acetate oxidation, which is the key pathway for acetate removal at high NH₃ concentration [49,51]. In another study, the anaerobic genera Longilinea (fam. Chloroflexi) and Alloprevotella (fam. Bacteroidetes) were rather prevalent in digesters operated under a high NH₃ content (7 mg/L) while Nitratalea sp. was the predominant bacterium under low ammonia (1.9 mg/L) [52]. Likewise, pretreatment of lignocellulosic feedstock as a way to enhance biogas production lead to the generation of toxic by-products such phenolics and furan derivatives [53,54]. At high concentrations, phenolic compounds can hinder the degradation of organic matter through their negative effects on AD microorganisms and consequently influence digestate microbes [55]. Increasing the concentrations of phenol from 0.01 g/L has been shown to induce level-dependent structural modification in the digestate microbial community [56]. Furthermore, increased phenol levels (0.2 g/L) were related to an increase in the relative abundance of families belonging to the Bacteroidales order and a lower relative abundance of families affiliated with the Clostridiales order [57]. Increased levels of toxic by-products in AD contribute to modification in the microbial community due to the selective pressure imposed, which reorganizes the initial microbial population. Finally, several studies indicate that feedstock composition also affects the proportion and diversity of specific microbial groups in digestate. With regard to the archaea community across different digesters, the feedstock effects are minimal, since different digesters demonstrated community similarity, with Methanosaeta and Methanosarcina reported as the dominant phyla in most digestate [40,43,58]. This could be due to the fact that archaea are fewer than bacteria, as they typically make up less than 30% of digester microbes [36]. However, significant differences in the bacteria community have been reported according to substrate type. For instance, the microbiota associated with lignocellulose-derived digestate is characterized by less bacterial diversity, with the predominant bacteria belonging to phyla Bacteriodetes and Firmicutes, whereas the sludge bacteria population is more diverse [41]. In food waste digestate, the phyla Synegistota, Cloacimonadota, Thermotogota and Chloroflexi show an upward trend in their relative abundance when compared to lignocellulose digestate. In addition to other factors, microbial activity and survival in anaerobic digesters are dependent on the input of resources, but different organisms have different nutritional requirements. Thus, the variation in feedstock input has the potential to cause variation in AD and digestate microbial composition with implications for future applications.

2.3. Temperature

Temperature has been identified (together with nutrient availability) as one of the primary operating parameters affecting the bacterial and methanogenic community structures of digesters, and previous studies have described significant variations in the diversity of digestate microbiota based on the temperature of digestion. For example, a recent microbiome population study using data from 18 full-scale biogas reactors investigated factors influencing the microbial community composition in food waste digestate. The study revealed that the microbial communities were shaped by operational parameters such as the primary substrate type and operational temperature [31]. Similarly, Jiang et al. studied the impact of food waste source and operating temperature on the evolution of AD and digestate microbes, observing that temperature influenced digestate-associated microbiota more than the source of the substrate [59].

Most studies examining the effect of temperature have revealed differences in microbial diversity and composition between psychrophilic (<20 °C), mesophilic (30–40 °C) and thermophilic (50–60 °C) digesters. The microbiota associated with psychrophilic AD and its digestate is characterized by low bacterial diversity and species richness with predominant bacteria belonging to Bacteroides, Clostridium, Geobacter and Syntrophomans, Methanosaeta as well as Methanobacterium [60,61,62,63,64]. However, mesophilic digesters reportedly produce digestate with a high microbial diversity and richness comprising bacterial phyla Bacteriodetes, Firmicutes and Proteobacteria, and enriched methanogens Methanosphaera and Methanobacterium with a hydrogenotrophic methanogenic pathway [31,65]. In thermophilic digesters, heat-tolerant microbes such as Defluviitoga and Methanothermobacter were selectively enriched [66]. Thermophilic conditions have also been found to modulate the digestate resistome profile and reduce the abundance of several antibiotic resistance genes (ARGs) as well as show greater ARG removal than other temperatures [67,68,69,70]. The decrease in ARGs with thermophilic AD could be attributed to the reduction of genes encoding antibiotic efflux pump, rapid biomass decomposition, loss of microbial diversity, and the inactivation of multidrug-resistant bacteria under high temperatures [70]. Generally, the temperature of AD affects both the growth and the persistence of microorganisms in the system, since different microbial groups exhibit variable temperature tolerance. Thus, process temperature is an important factor that imposes a strong selective pressure on digestate microbial composition.

2.4. Use of Additives

Process intensification techniques, such as the use of additives, is common during AD either as a way to improve process stability or increase the range of AD substrates [71,72,73]. Many researchers have demonstrated that the use of additives such as non-biological conductive materials e.g., biochar, inorganic additives and biological additives, have significant effects on several AD processes as well as digestate microbial parameters. This was demonstrated by Johnravindar et al., who described an increase in the level of different microbial species and their diversity in the digestate from biochar-supplemented AD [58]. In addition, previous studies have reported a higher relative abundance of Clostridia and Methanosarcina in the digestate obtained from thermophilic AD of sewage sludge amended with corn stover biochar [74]. Changes in digestate microbial composition triggered by inorganic or micronutrient supplementation have also been reported. A study on the optimization of AD through trace element supplementation showed that digestate from supplemented digesters was dominated by Bacteroides, Clostridium, Ruminococcus, and Methanosarcina [40]. In contrast, digestate recovered from non-supplemented controls was dominated by the bacterial phyla Proteobacteria and the methanogen Methanosaeta [40]. Besides inorganic additives, the introduction of biological additives (bioaugmentation) including microbial inocula for the enhancement of organic matter degradation can also modulate digestate microbial composition. This was demonstrated by Yan et al. who showed changes in microbiota of digestate in response to bioaugmentation with ammonia-tolerant methanogen [75]. Previous studies also reported an increased proportion of propionate-degrading microorganisms in the resulting digestate of AD bioaugmented with propionate-degrading culture [76,77]. Furthermore, Methanothrix, Methanobacterium, and Methanomassiliicoccus predominated in bioaugmented digesters, while Methanoculleus and Methanobrevibacter were the dominant methanogens in non-bioaugmented digesters [78]. Taken together, these findings suggest that supplementation of AD with additives affects microbial growth and alters the digestate microbial structure. Optimization techniques supporting diverse beneficial microbes, particularly those with plant growth-promoting traits, offer the best opportunity for harnessing digestate microbiomes to enrich soil nutrients and boost agricultural production.

2.5. Post-Treatment Strategies

Post-treatment approaches including liquid–solid separation, drying and composting are relevant contributing factors to a more sanitized AD product, which sets the basis for digestate microbiota variations. In order to promote the agricultural application of digestate while reducing the risks of environmental contamination with pathogenic microbes or antibiotic resistance genes, post-treatment techniques such as liquid–solid separation, drying and composting are suggested [79]. Since these management practices are established as a means of sanitizing AD products, their impact on digestate microbial composition is not unexpected. A link between digestate microbiota and post-digestate composting has also been demonstrated in studies using 16S rRNA analysis and qPCR assays to evaluate microbial composition and antimicrobial resistance genes (ARGs). Bacterial phyla Bacteroidetes, Firmicutes, and Proteobacteria dominated fresh digestate, whereas the emergence of Actinobacteria, Planctomycetes and Verrucomicrobia phyla during composting shifted the microbial composition [79]. Moreover, compared with the microbiome of composted digestate, that of fresh digestate was correlated with higher levels of antibiotic-resistant microbes and enriched in ARGs, indicating the negative impact of composting on ARGs. Thus, further treatment of digestate through composting is likely to modulate the digestate microbiome and reduce the risks related to ARG spread with digestate land application.

In summary, numerous factors influence digestate microbiota, such as the resident microbial community in the inoculum and feedstock, feedstock composition, the AD temperature, the use of additives and the post-treatment of digestate. However, the function of this microbial component of digestate is unknown. A functional approach to elucidating the role of each microbial component in digestate would not only help inform the choice of its possible end use but also help develop optimal post-AD intervention strategies to minimize the risk of environmental contamination.

3. Digestate Microbial Communities and Associated Functions

Digestate arising from AD contains microbial communities which change based on several factors, including the operational parameters of the digester such as temperature, digester type, feedstock and duration of digestion. Bacterial, archaeal and fungal taxa reported in studies, which investigated microbial communities in digestate primarily utilized for downstream applications, are presented in Table 1. It was observed that more studies investigated the bacterial and archaeal communities of digestate compared to fungal communities. Hanseniaspora, Cryptococcus, Acaulospora, Guehomyces, Kazachstania, Penicillium, Saccharomyces, Scedosporium, Cyllamyces and Mucor, which were fungal taxa present in digestate investigated by Coelho et al., are primarily associated with fermenting and decomposing functions [21,80,81,82,83,84,85]. Moreover, the arbuscular mycorrhizal fungi, Acaulospora, plays an important role in bioremediation and soil management due to its resilience to abiotic stresses and ability to provide plants with improved access to nutrients [86]. Although taxonomic attributes and potential metabolic capabilities of various digestate fungal communities have not been investigated extensively, their predominant role in deconstructing lignocellulosic biomass and recalcitrant fibers into soluble sugars, metabolites and nutrients has been well established in AD processes [87,88,89,90]. Furthermore, fungal communities present potential for alleviating fatty acid accumulation and were shown to have higher resistance to ammonia inhibition, which oftentimes impact bacterial and archaeal communities during AD [91]. Research has also revealed that anaerobic fungi can stimulate the growth of methanogens, and in turn, methanogens stimulated fungal metabolic activity which indicates potential to improve AD systems [92,93,94,95]. Overall, fungi have great potential to enhance biofuel production, however further development and investigation is required [96]. In addition to their role in AD, fungi from the Ascomycota phylum group were found to play a key role in organic composting processes [97,98]. Future studies should explore the metabolic potential of digestate fungal communities. Focusing research to fill this knowledge gap will pave the way for further development in downstream applications of digestate.

The predominant bacterial taxa occurring in Table 1 (>2%) is shown in Figure 2a. Clostridium, Lactobacillus, Sedimentibacter, Bacteroides and Leuconostoc are associated with fermentative functions, whereas Syntrophomonas, Treponema and Clostridium are associated with acetogenic functions [60,99,100,101]. Fermentative functions include the metabolism of biopolymers to oligomers or monomers and/or monomers to volatile fatty acids (VFAs), alcohols or carbonic acids. On the other hand, acetogenic functions involve the conversion of VFAs, alcohols, and carbonic acids to acetate/acetic acid, hydrogen, and/or carbon dioxide. Several studies have shown that the accumulation of VFAs during AD impacts the digestate microbial community [91,102,103]. This suggests that the digestate microbial composition and the metabolic capabilities in digestate may shift depending on the type and concentration of VFAs present during AD. Further investigation into the effect of VFAs on the metabolic dynamics of digestate microbial communities is required. Such studies may aid in the development of biofuel and VFA production systems. The predominant archaeal taxa occurring in Table 1 are shown in Figure 2b. The highest frequency of archaeal taxa in these digestate communities were Methanobrevibacter, Methanosarcina and Methanobacterium. These methanogenic archaeal genera play a role in converting carbon dioxide and hydrogen to methane through hydrogenotrophic methanogenesis. Furthermore, Methanosarcina converts methylated compounds and acetate to methane through methylotrophic and acetoclastic methanogenesis, respectively [60]. The majority of the digestate archaeal communities were associated with hydrogenotrophic methanogenesis, while 22% and 19% were associated with methylotrophic and acetoclastic methanogenesis, respectively (Figure 2b, inset figure). Based on the type of functions associated with the digestate microbial communities, digestate may be valorized for various applications such as bioremediation, soil amendment, composting, biofertilization, microalga cultivation, VFA production, recirculation into operating anaerobic digestion plants, aquaculture and hydroponics.

Table 1. Notable microbial communities in digestate arising from various anaerobic digesters.

Feedstock	Treatment	Digester	Duration	Temperature	Bacterial Taxa	Archaeal Taxa	Fungal Taxa	References
Pig manure and plant silage		Industrial mesophilic biogas plant	10 days	Mesophilic (37 °C)	Streptococcus, Clostridium, Syntrophaceticus, Candidatus cloacimonas, Chitinispirillum, Lactobacillus, Treponema, Bacteroides, Ruminiclostridium, Nitrosomonas, Sedimentibacter, Prevotella, Paenibacillus, Bacillus, Arcobacter, Herbinix, Pseudomonas, Candidatus riflebacteria	Methanobacterium, Methanosaeta, Methanosarcina, Methanospirillum, Methanoculleus, Methanobrevibacter, Methanothermobacter		[104]
Pig manure and plant silage digestate	H2	Batch—bio power to methane reactor	4 weeks	Mesophilic (37 °C)	Streptococcus, Clostridium, Syntrophaceticus, Candidatus cloacimonas, Lactobacillus, Treponema, Bacteroides, Ruminiclostridium, Nitrosomonas, Sedimentibacter, Prevotella	Methanobacterium, Methanosaeta, Methanosarcina, Methanospirillum, Methanoculleus, Methanobrevibacter		[104]
Pig manure and plant silage digestate + α-Cellulose	H2	Batch—bio power to methane reactor	4 weeks	Mesophilic (37 °C)	Streptococcus, Clostridium, Syntrophaceticus, Candidatus cloacimonas, Lactobacillus, Treponema, Bacteroides, Ruminiclostridium, Nitrosomonas, Sedimentibacter, Prevotella, Paenibacillus, Herbinix	Methanobacterium, Methanosaeta, Methanosarcina, Methanospirillum, Methanoculleus, Methanobrevibacter		[104]
Pig manure and plant silage digestate	N2	Batch—bio power to methane reactor	4 weeks	Mesophilic (37 °C)	Streptococcus, Clostridium, Syntrophaceticus, Lactobacillus, Treponema, Bacteroides, Ruminiclostridium, Nitrosomonas, Prevotella, Pseudomonas	Methanobacterium, Methanosaeta, Methanosarcina, Methanospirillum, Methanoculleus, Methanobrevibacter		[104]
Pig manure and plant silage digestate + α-Cellulose	N2	Batch—bio power to methane reactor	4 weeks	Mesophilic (37 °C)	Streptococcus, Clostridium, Syntrophaceticus, Chitinispirillum, Lactobacillus, Treponema, Bacteroides, Ruminiclostridium, Nitrosomonas, Sedimentibacter, Prevotella, Pseudomonas	Methanobacterium, Methanosaeta, Methanosarcina, Methanospirillum, Methanoculleus, Methanobrevibacter		[104]
Pig manure and plant silage digestate	H2 and CO2	Batch—bio power to methane reactor	12 weeks	Mesophilic (37 °C)	Streptococcus, Clostridium, Syntrophaceticus, Ruminiclostridium	Methanobacterium, Methanoculleus, Methanosarcina, Methanobrevibacter		[104]
Pig manure and plant silage digestate	N2	Batch—bio power to methane reactor	12 weeks	Mesophilic (37 °C)	Streptococcus, Clostridium, Syntrophaceticus, Candidatus cloacimonas, Ruminiclostridium, Herbinix	Methanobacterium, Methanosaeta, Methanosarcina, Methanobrevibacter		[104]
Dairy cow manure containing wheat straw and woodchip		Commercially operating biogas plant		Unknown	Peptostreptococcaceae, Family XI, Ruminococcaceae, Bacteroidales UCG-001, Rikenellaceae, Porphyromonadaceae, Helicobacteraceae, Clostridiaceae 1, Carnobacteriaceae, Lachnospiraceae, Syntrophomonadaceae, Erysipelotrichaceae	Methanobacteriaceae, Methanosarcinaceae		[105]
Food waste from the dairy industry	Pasteurization pre-digestion	Commercial biogas plant	70 days (HRT)	Mesophilic	Clostridium, Cloacimonas, Bacteroides, Acetivibrio, Rikenella, Eubacterium, Bacillus, Proteiniphilum, Symbiobacterium, Petrotoga, Sporobacter, Syntrophomonas, Cytophaga, Acidaminococcus, Bellilinea	Methanobacterium, Methanobrevibacter, Methanoculleus	Hanseniaspora, Cryptococcus, Guehomyces	[21]
Food waste and pig slurry		Commercial biogas plant	90 days (HRT)	Mesophilic (40 °C)	Clostridium, Cloacimonas, Bacteroides, Acetivibrio, Rikenella, Pseudoalteromonas, Eubacterium, Bacillus, Proteiniphilum, Symbiobacterium, Syntrophomonas, Bellilinea, Verrucomicrobium	Methanobacterium, Methanobrevibacter, Methanosarcina, Methanoculleus, Methanothermobacter	Acaulospora, Kazachstania, Penicillium, Saccharomyces	[21]
Farm and food industry wastes		Commercial biogas plant	54 days (HRT)	Mesophilic (38 °C)	Clostridium, Cloacimonas, Bacteroides, Acetivibrio, Rikenella, Pseudoalteromonas, Eubacterium, Bacillus, Proteiniphilum, Symbiobacterium, Natronoanaerobium, Cytophaga, Syntrophomonas, Bellilinea, Verrucomicrobium	Methanobacterium, Methanobrevibacter, Methanosaeta, Methanospirillum, Methanosarcina, Methanoculleus	Acaulospora, Scedosporium, Cyllamyces	[21]
Food waste and municipal wastes		Commercial biogas plant	60 days (HRT)	Mesophilic (37–42 °C)	Clostridium, Cloacimonas, Bacteroides, Acetivibrio, Pseudoalteromonas, Eubacterium, Bacillus, Proteiniphilum, Symbiobacterium, Natronoanaerobium, Cytophaga, Syntrophomonas, Bellilinea, Verrucomicrobium, Acidaminococcus	Methanobacterium, Methanosarcina, Methanobrevibacter, Methanoculleus, Methanothermobacter	Acaulospora, Penicillium, Paramicro-sporidium, Mucor	[21]
Green waste (leaves, grass, prunings and trimmings, branches and stumps) and food waste (dog food) mixture with digestate sludge inoculum	C/N ratio of 17	Batch anaerobic digester	14–15 days	Thermophilic (55 °C)	Thermotogaceae, Halanaerobiaceae, Lachnospiraceae, Caldicoprobacteraceae, Tissierellaceae, Clostridiaceae, Ruminococcaceae, Porphyromonadaceae, Anaerobaculaceae	Methanosarcinaceae		[43]
Green waste (leaves, grass, prunings and trimmings, branches and stumps) and food waste (dog food) mixture with digestate sludge inoculum	C/N ratio of 20	Batch anaerobic digester	14–15 days	Thermophilic (55 °C)	Thermotogaceae, Halanaerobiaceae, Lachnospiraceae, Caldicoprobacteraceae, Tissierellaceae, Clostridiaceae, Ruminococcaceae, Porphyromonadaceae, Anaerobaculaceae	Methanosarcinaceae		[43]
Green waste (leaves, grass, prunings and trimmings, branches and stumps) and food waste (dog food) mixture with digestate sludge inoculum	C/N ratio of 23	Batch anaerobic digester	14–15 days	Thermophilic (55 °C)	Thermotogaceae, Halanaerobiaceae, Lachnospiraceae, Clostridiaceae, Ruminococcaceae, Tissierellaceae, Syntrophomonadaceae	Methanosarcinaceae		[43]
Green waste (leaves, grass, prunings and trimmings, branches and stumps) and food waste (dog food) mixture with digestate sludge inoculum	C/N ratio of 27	Batch anaerobic digester	14–15 days	Thermophilic (55 °C)	Thermotogaceae, Halanaerobiaceae, Lachnospiraceae, Clostridiaceae, Ruminococcaceae, Caldicoprobacteracea, Tissierellaceae, Porphyromonadaceae, Anaerobaculaceae, Syntrophomonadaceae	Methanosarcinaceae		[43]
Green waste (leaves, grass, prunings and trimmings, branches and stumps) and food waste (dog food) mixture with digestate sludge inoculum	C/N ratio of 34	Batch anaerobic digester	14–15 days	Thermophilic (55 °C)	Thermotogaceae, Lachnospiraceae, Clostridiaceae, Ruminococcaceae, Halanaerobiaceae, Porphyromonadaceae, Anaerobaculaceae	Methanosarcinaceae		[43]
Unknown	Unknown	Biogas plant	Unknown	Unknown	Psychrobacter, Mycobacterium, Acinetobacter, Microbacterium			[23]
Corn stover	NaOH pretreatment	Continuously stirred tank reactor	1–60 days	Mesophilic (35 °C)	Bacteroidetes, Firmicutes, WS6, Spirochaetae, Verrucomicrobia, Synergistetes, Proteobacteria, Cloacimonetes, Saccharibacteria, Acidobacteria, Chloroflexi	Methanosarcina, Methanobacterium		[106]
Livestock effluent and agricultural waste	Unknown	Unknown	Unknown	Unknown	Bacillales, Bacteroidales, Clostridiales, Acholeplasmatales	Methanomicrobiales		[107]
Mixture of sewage sludge (53% total solids [TS]) and municipal solid waste (47% TS)		Industrial-scale plant	19 days (HRT)	Mesophilic (37 °C)	Holophagae, Actinobacteria, Anaerolineae, Bacteroidetes_vadinHA17, Bacteroidia, Sphingobacteriia, Cloacimonetes, Fibrobacteria, Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Clostridia, Synergistia	Methanobacterium, Methanobrevibacter, Methanosaeta, Methanomassiliicoccus		[108]
50% digestate and 50% mixture of sewage sludge (53% total solids [TS]) and municipal solid waste (47% TS)		Industrial-scale plant	10 days (HRT)	Mesophilic (37 °C)	Holophagae, Anaerolineae, Bacteroidetes_vadinHA17, Bacteroidia, Sphingobacteriia, Fibrobacteria, Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Clostridia, Synergistia	Methanobacterium, Methanobrevibacter, Methanosaeta, Methanomassiliicoccus, Methanospirillum		[108]
Mixture of sewage sludge (53% total solids [TS]) and municipal solid waste (47% TS)	Thermal hydrolysis processing	Industrial-scale plant	23 days (HRT)	Mesophilic (40–42 °C)	Anaerolineae, Bacteroidetes_vadinHA17, Bacteroidia, Sphingobacteriia, W27, Clostridia, Thermotogae, LD1-PB3, Synergistia	Methanobacterium, Methanosaeta, Methanomassiliicoccus		[108]
50% digestate and 50% mixture of sewage sludge (53% total solids [TS]) and municipal solid waste (47% TS)	Thermal hydrolysis processing	Industrial-scale plant	20 days (HRT)	Mesophilic (39 °C)	Anaerolineae, Bacteroidetes_vadinHA17, Bacteroidia, Sphingobacteriia, W27, W5, Clostridia, Thermotogae, LD1-PB3, Synergistia	Methanobacterium, Methanosaeta, Methanomassiliicoccus		[108]
Organic household waste 37%, manure 31%, slaughter residues 19%, other organic food waste 13% and iron chloride 0.03%		Biogas plant		Unknown	Pseudomonas, Sporosarcina, Leuconostoc, Romboutsia			[109]
Organic household waste 37%, manure 31%, slaughter residues 19%, other organic food waste 13% and iron chloride 0.03%	Nitrification	Biogas plant		Unknown	Lactobacillus, Ralstonia, Pseudomonas, Mycobacterium, Chujaibacter, Romboutsia			[109]
Sewage sludge produced by wastewater treatment plants from urban sites		Organic waste treatment plants	34–38 days	Mesophilic (32–42 °C)	Acidobacteria, Actinobacteria, Atribacteria, Bacteroidetes, Chlorobi, Chloroflexi, Deferribacteres, Firmicutes, Lentisphaerae, Nitrospirae, Planctomycetes, Proteobacteria, Spirochaetae, Verrucomicrobia			[37]
Animal manure or slurry from agricultural sites and a mix of food industry waste, harvest residues, livestock effluents or sewage sludge from central sites		Organic waste treatment plants	30–90 days	Mesophilic (32–42 °C)	Actinobacteria, Atribacteria, Bacteroidetes, Cloacimonetes, Firmicutes, Planctomycetes, Proteobacteria, Spirochaetae, Synergistetes, Tenericutes			[37]
Mixed sludge		Anaerobic digestion plant	Unknown	Mesophilic (36 °C)	Clostridia, Bacillus, Bacteroidia, Sphingobacteriia, Cytophagia, Flavobacteriia, Alphaproteobacteria, Gammaproteobacteria, Betaproteobacteria			[110]
Cattle manure digestate inoculum, pig manure (85%) and food waste (15%), volatile solids basis		Anaerobic digestion reactor (semi-continous)	41 days (HRT)	Mesophilic (39 °C)	Acholeplasma, Treponema, Sphaerochaeta, Succinivibrio, RFN20, ML1228J-1, Sedimentibacter, Syntrophomonas, Coprococcus, Clostridium, Alkaliphilus, Caldicoprobacter, Leuconostoc, Lactobacillus, Corynebacterium	Methanosarcina, Methanoculleus, Methanosphaera, Methanobrevibacter, Methanobacterium		[111]
Cattle manure digestate inoculum, pig manure (62.5%) and food waste (37.5%), volatile solids basis		Anaerobic digestion reactor (semi-continous)	41 days (HRT)	Mesophilic (39 °C)	Acholeplasma, Treponema, Sphaerochaeta, Succinivibrio, RFN20, ML1228J-1, Sedimentibacter, Syntrophomonas, Coprococcus, Clostridium, Alkaliphilus, Caldicoprobacter, Leuconostoc, Lactobacillus, Corynebacterium	Methanosarcina, Methanoculleus, Methanosphaera, Methanobrevibacter, Methanobacterium		[111]
Cattle manure digestate inoculum, pig manure (40%) and food waste (60%), volatile solids basis		Anaerobic digestion reactor (semi-continous)	41 days (HRT)	Mesophilic (39 °C)	Acholeplasma, Treponema, Sphaerochaeta, Succinivibrio, RFN20, ML1228J-1, Sedimentibacter, Syntrophomonas, Coprococcus, Clostridium, Alkaliphilus, Caldicoprobacter, Leuconostoc, Lactobacillus, Corynebacterium	Methanosarcina, Methanoculleus, Methanosphaera, Methanobrevibacter, Methanobacterium		[111]
Cattle manure digestate inoculum, pig manure (85%) and food waste (15%), volatile solids basis		Anaerobic digestion reactor (semi-continous)	29 days (HRT)	Mesophilic (39 °C)	Acholeplasma, Treponema, Sphaerochaeta, RFN20, ML1228J-1, Sedimentibacter, Syntrophomonas, Coprococcus, Clostridium, Alkaliphilus, Caldicoprobacter, Leuconostoc, Lactobacillus, Propionicimonas, Corynebacterium	Methanomassiliicoccus, Methanosarcina, Methanospirillum, Methanogenium, Methanoculleus, Methanosphaera, Methanobrevibacter, Methanobacterium		[111]
Cattle manure digestate inoculum, pig manure (62.5%) and food waste (37.5%), volatile solids basis		Anaerobic digestion reactor (semi-continous)	29 days (HRT)	Mesophilic (39 °C)	Acholeplasma, Treponema, Sphaerochaeta, RFN20, ML1228J-1, Sedimentibacter, Syntrophomonas, Coprococcus, Clostridium, Alkaliphilus, Caldicoprobacter, Leuconostoc, Lactobacillus, Propionicimonas, Corynebacterium	Methanomassiliicoccus, Methanosarcina, Methanogenium, Methanoculleus, Methanosphaera, Methanobrevibacter		[111]
Cattle manure digestate inoculum, pig manure (40%) and food waste (60%), volatile solids basis		Anaerobic digestion reactor (semi-continous)	29 days (HRT)	Mesophilic (39 °C)	Acholeplasma, Treponema, Sphaerochaeta, RFN20, ML1228J-1, Sedimentibacter, Syntrophomonas, Coprococcus, Clostridium, Alkaliphilus, Caldicoprobacter, Leuconostoc, Lactobacillus, Propionicimonas, Corynebacterium	Methanomassiliicoccus, Methanosarcina, Methanosaeta, Methanoculleus, Methanosphaera, Methanobrevibacter		[111]
Cattle manure digestate inoculum, pig manure (85%) and food waste (15%), volatile solids basis		Anaerobic digestion reactor (semi-continous)	21 days (HRT)	Mesophilic (39 °C)	Acholeplasma, Treponema, Sphaerochaeta, RFN20, ML1228J-1, Sedimentibacter, Syntrophomonas, Coprococcus, Clostridium, Alkaliphilus, Caldicoprobacter, Leuconostoc, Lactobacillus, Propionicimonas, Corynebacterium	Methanomassiliicoccus, Methanosarcina, Methanospirillum, Methanogenium, Methanoculleus, Methanosphaera, Methanobrevibacter, Methanobacterium		[111]
Cattle manure digestate inoculum, pig manure (62.5%) and food waste (37.5%), volatile solids basis		Anaerobic digestion reactor (semi-continous)	21 days (HRT)	Mesophilic (39 °C)	Acholeplasma, Treponema, Sphaerochaeta, RFN20, ML1228J-1, Sedimentibacter, Syntrophomonas, Coprococcus, Clostridium, Alkaliphilus, Caldicoprobacter, Leuconostoc, Lactobacillus, Corynebacterium	Methanomassiliicoccus, Methanosarcina, Methanogenium, Methanoculleus, Methanosphaera, Methanobrevibacter		[111]
Cattle manure digestate inoculum, pig manure (40%) and food waste (60%), volatile solids basis		Anaerobic digestion reactor (semi-continous)	21 days (HRT)	Mesophilic (39 °C)	Acholeplasma, Treponema, Sphaerochaeta, RFN20, ML1228J-1, Sedimentibacter, Syntrophomonas, Coprococcus, Clostridium, Alkaliphilus, Caldicoprobacter, Leuconostoc, Lactobacillus, Corynebacterium	Methanomassiliicoccus, Methanosarcina, Methanosaeta, Methanoculleus, Methanosphaera, Methanobrevibacter		[111]
Cow manure, wood straw and wood chip		Biogas plant	Unknown	Unknown	Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Plantomycetes, Proteobacteria, OP9, Synergistetes	Crenarchaeota, Euryarchaeota		[112]
Mixture of dairy cattle manure (70%), energy crops (maize silage up to 30%) and waste from agro-food industries		Biogas plant		Mesophilic	Acidimicrobiia, Actinobacteria, Alphaproteobacteria, Bacilli, Bacteroidia, Clostridia, Deltaproteobacteria, Gammaproteobacteria, Gemmatimonadetes, Mollicutes, Phycisphaerae, Saccharomonadia, Verrucomicrobiae	Methanosarcina	Ascomycota, Basidiomycota	[113]
Anaerobically digested sludge inoculum, food waste and waste activated sludge (TS: 5%)		Continuous stirred tank reactors	30 days	Mesophilic (35 °C)	Mycobacterium, Thermovirga, Mesotoga, Erysipelatoclostridium, Eubacterium, Aminobacterium, Bacillus, Clostridium	Methanobrevibacter, Methanofollis		[58]
Anaerobically digested sludge inoculum, food waste and waste activated sludge (TS: 5%)	Wheat straw pellet biochar	Continuous stirred tank reactors	30 days	Mesophilic (35 °C)	Mycobacterium, Thermovirga, Mesotoga, Erysipelatoclostridium, Eubacterium, Bacteroides, Aminobacterium, Clostridium	Methanosarcina, Methanomicrobiales, Methanofollis, Methanoculleus, Methanolinea		[58]
Anaerobically digested sludge inoculum, food waste and waste activated sludge (TS: 7.5%)		Continuous stirred tank reactors	30 days	Mesophilic (35 °C)	Thermovirga, Mesotoga, Erysipelatoclostridium, Syntrophomonas, Bacteroides, Aminobacterium	Methanocorpusculum, Methanobrevibacter, Methanosarcina		[58]
Anaerobically digested sludge inoculum, food waste and waste activated sludge (TS: 7.5%)	Wheat straw pellet biochar	Continuous stirred tank reactors	30 days	Mesophilic (35 °C)	Thermovirga, Mesotoga, Erysipelatoclostridium, Eubacterium, Aminobacterium, Bacillus, Clostridium	Methanobrevibacter, Methanosarcina		[58]

Table 1. Notable microbial communities in digestate arising from various anaerobic digesters.

Feedstock	Treatment	Digester	Duration	Temperature	Bacterial Taxa	Archaeal Taxa	Fungal Taxa	References
Pig manure and plant silage		Industrial mesophilic biogas plant	10 days	Mesophilic (37 °C)	Streptococcus, Clostridium, Syntrophaceticus, Candidatus cloacimonas, Chitinispirillum, Lactobacillus, Treponema, Bacteroides, Ruminiclostridium, Nitrosomonas, Sedimentibacter, Prevotella, Paenibacillus, Bacillus, Arcobacter, Herbinix, Pseudomonas, Candidatus riflebacteria	Methanobacterium, Methanosaeta, Methanosarcina, Methanospirillum, Methanoculleus, Methanobrevibacter, Methanothermobacter		[104]
Pig manure and plant silage digestate	H2	Batch—bio power to methane reactor	4 weeks	Mesophilic (37 °C)	Streptococcus, Clostridium, Syntrophaceticus, Candidatus cloacimonas, Lactobacillus, Treponema, Bacteroides, Ruminiclostridium, Nitrosomonas, Sedimentibacter, Prevotella	Methanobacterium, Methanosaeta, Methanosarcina, Methanospirillum, Methanoculleus, Methanobrevibacter		[104]
Pig manure and plant silage digestate + α-Cellulose	H2	Batch—bio power to methane reactor	4 weeks	Mesophilic (37 °C)	Streptococcus, Clostridium, Syntrophaceticus, Candidatus cloacimonas, Lactobacillus, Treponema, Bacteroides, Ruminiclostridium, Nitrosomonas, Sedimentibacter, Prevotella, Paenibacillus, Herbinix	Methanobacterium, Methanosaeta, Methanosarcina, Methanospirillum, Methanoculleus, Methanobrevibacter		[104]
Pig manure and plant silage digestate	N2	Batch—bio power to methane reactor	4 weeks	Mesophilic (37 °C)	Streptococcus, Clostridium, Syntrophaceticus, Lactobacillus, Treponema, Bacteroides, Ruminiclostridium, Nitrosomonas, Prevotella, Pseudomonas	Methanobacterium, Methanosaeta, Methanosarcina, Methanospirillum, Methanoculleus, Methanobrevibacter		[104]
Pig manure and plant silage digestate + α-Cellulose	N2	Batch—bio power to methane reactor	4 weeks	Mesophilic (37 °C)	Streptococcus, Clostridium, Syntrophaceticus, Chitinispirillum, Lactobacillus, Treponema, Bacteroides, Ruminiclostridium, Nitrosomonas, Sedimentibacter, Prevotella, Pseudomonas	Methanobacterium, Methanosaeta, Methanosarcina, Methanospirillum, Methanoculleus, Methanobrevibacter		[104]
Pig manure and plant silage digestate	H2 and CO2	Batch—bio power to methane reactor	12 weeks	Mesophilic (37 °C)	Streptococcus, Clostridium, Syntrophaceticus, Ruminiclostridium	Methanobacterium, Methanoculleus, Methanosarcina, Methanobrevibacter		[104]
Pig manure and plant silage digestate	N2	Batch—bio power to methane reactor	12 weeks	Mesophilic (37 °C)	Streptococcus, Clostridium, Syntrophaceticus, Candidatus cloacimonas, Ruminiclostridium, Herbinix	Methanobacterium, Methanosaeta, Methanosarcina, Methanobrevibacter		[104]
Dairy cow manure containing wheat straw and woodchip		Commercially operating biogas plant		Unknown	Peptostreptococcaceae, Family XI, Ruminococcaceae, Bacteroidales UCG-001, Rikenellaceae, Porphyromonadaceae, Helicobacteraceae, Clostridiaceae 1, Carnobacteriaceae, Lachnospiraceae, Syntrophomonadaceae, Erysipelotrichaceae	Methanobacteriaceae, Methanosarcinaceae		[105]
Food waste from the dairy industry	Pasteurization pre-digestion	Commercial biogas plant	70 days (HRT)	Mesophilic	Clostridium, Cloacimonas, Bacteroides, Acetivibrio, Rikenella, Eubacterium, Bacillus, Proteiniphilum, Symbiobacterium, Petrotoga, Sporobacter, Syntrophomonas, Cytophaga, Acidaminococcus, Bellilinea	Methanobacterium, Methanobrevibacter, Methanoculleus	Hanseniaspora, Cryptococcus, Guehomyces	[21]
Food waste and pig slurry		Commercial biogas plant	90 days (HRT)	Mesophilic (40 °C)	Clostridium, Cloacimonas, Bacteroides, Acetivibrio, Rikenella, Pseudoalteromonas, Eubacterium, Bacillus, Proteiniphilum, Symbiobacterium, Syntrophomonas, Bellilinea, Verrucomicrobium	Methanobacterium, Methanobrevibacter, Methanosarcina, Methanoculleus, Methanothermobacter	Acaulospora, Kazachstania, Penicillium, Saccharomyces	[21]
Farm and food industry wastes		Commercial biogas plant	54 days (HRT)	Mesophilic (38 °C)	Clostridium, Cloacimonas, Bacteroides, Acetivibrio, Rikenella, Pseudoalteromonas, Eubacterium, Bacillus, Proteiniphilum, Symbiobacterium, Natronoanaerobium, Cytophaga, Syntrophomonas, Bellilinea, Verrucomicrobium	Methanobacterium, Methanobrevibacter, Methanosaeta, Methanospirillum, Methanosarcina, Methanoculleus	Acaulospora, Scedosporium, Cyllamyces	[21]
Food waste and municipal wastes		Commercial biogas plant	60 days (HRT)	Mesophilic (37–42 °C)	Clostridium, Cloacimonas, Bacteroides, Acetivibrio, Pseudoalteromonas, Eubacterium, Bacillus, Proteiniphilum, Symbiobacterium, Natronoanaerobium, Cytophaga, Syntrophomonas, Bellilinea, Verrucomicrobium, Acidaminococcus	Methanobacterium, Methanosarcina, Methanobrevibacter, Methanoculleus, Methanothermobacter	Acaulospora, Penicillium, Paramicro-sporidium, Mucor	[21]
Green waste (leaves, grass, prunings and trimmings, branches and stumps) and food waste (dog food) mixture with digestate sludge inoculum	C/N ratio of 17	Batch anaerobic digester	14–15 days	Thermophilic (55 °C)	Thermotogaceae, Halanaerobiaceae, Lachnospiraceae, Caldicoprobacteraceae, Tissierellaceae, Clostridiaceae, Ruminococcaceae, Porphyromonadaceae, Anaerobaculaceae	Methanosarcinaceae		[43]
Green waste (leaves, grass, prunings and trimmings, branches and stumps) and food waste (dog food) mixture with digestate sludge inoculum	C/N ratio of 20	Batch anaerobic digester	14–15 days	Thermophilic (55 °C)	Thermotogaceae, Halanaerobiaceae, Lachnospiraceae, Caldicoprobacteraceae, Tissierellaceae, Clostridiaceae, Ruminococcaceae, Porphyromonadaceae, Anaerobaculaceae	Methanosarcinaceae		[43]
Green waste (leaves, grass, prunings and trimmings, branches and stumps) and food waste (dog food) mixture with digestate sludge inoculum	C/N ratio of 23	Batch anaerobic digester	14–15 days	Thermophilic (55 °C)	Thermotogaceae, Halanaerobiaceae, Lachnospiraceae, Clostridiaceae, Ruminococcaceae, Tissierellaceae, Syntrophomonadaceae	Methanosarcinaceae		[43]
Green waste (leaves, grass, prunings and trimmings, branches and stumps) and food waste (dog food) mixture with digestate sludge inoculum	C/N ratio of 27	Batch anaerobic digester	14–15 days	Thermophilic (55 °C)	Thermotogaceae, Halanaerobiaceae, Lachnospiraceae, Clostridiaceae, Ruminococcaceae, Caldicoprobacteracea, Tissierellaceae, Porphyromonadaceae, Anaerobaculaceae, Syntrophomonadaceae	Methanosarcinaceae		[43]
Green waste (leaves, grass, prunings and trimmings, branches and stumps) and food waste (dog food) mixture with digestate sludge inoculum	C/N ratio of 34	Batch anaerobic digester	14–15 days	Thermophilic (55 °C)	Thermotogaceae, Lachnospiraceae, Clostridiaceae, Ruminococcaceae, Halanaerobiaceae, Porphyromonadaceae, Anaerobaculaceae	Methanosarcinaceae		[43]
Unknown	Unknown	Biogas plant	Unknown	Unknown	Psychrobacter, Mycobacterium, Acinetobacter, Microbacterium			[23]
Corn stover	NaOH pretreatment	Continuously stirred tank reactor	1–60 days	Mesophilic (35 °C)	Bacteroidetes, Firmicutes, WS6, Spirochaetae, Verrucomicrobia, Synergistetes, Proteobacteria, Cloacimonetes, Saccharibacteria, Acidobacteria, Chloroflexi	Methanosarcina, Methanobacterium		[106]
Livestock effluent and agricultural waste	Unknown	Unknown	Unknown	Unknown	Bacillales, Bacteroidales, Clostridiales, Acholeplasmatales	Methanomicrobiales		[107]
Mixture of sewage sludge (53% total solids [TS]) and municipal solid waste (47% TS)		Industrial-scale plant	19 days (HRT)	Mesophilic (37 °C)	Holophagae, Actinobacteria, Anaerolineae, Bacteroidetes_vadinHA17, Bacteroidia, Sphingobacteriia, Cloacimonetes, Fibrobacteria, Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Clostridia, Synergistia	Methanobacterium, Methanobrevibacter, Methanosaeta, Methanomassiliicoccus		[108]
50% digestate and 50% mixture of sewage sludge (53% total solids [TS]) and municipal solid waste (47% TS)		Industrial-scale plant	10 days (HRT)	Mesophilic (37 °C)	Holophagae, Anaerolineae, Bacteroidetes_vadinHA17, Bacteroidia, Sphingobacteriia, Fibrobacteria, Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Clostridia, Synergistia	Methanobacterium, Methanobrevibacter, Methanosaeta, Methanomassiliicoccus, Methanospirillum		[108]
Mixture of sewage sludge (53% total solids [TS]) and municipal solid waste (47% TS)	Thermal hydrolysis processing	Industrial-scale plant	23 days (HRT)	Mesophilic (40–42 °C)	Anaerolineae, Bacteroidetes_vadinHA17, Bacteroidia, Sphingobacteriia, W27, Clostridia, Thermotogae, LD1-PB3, Synergistia	Methanobacterium, Methanosaeta, Methanomassiliicoccus		[108]
50% digestate and 50% mixture of sewage sludge (53% total solids [TS]) and municipal solid waste (47% TS)	Thermal hydrolysis processing	Industrial-scale plant	20 days (HRT)	Mesophilic (39 °C)	Anaerolineae, Bacteroidetes_vadinHA17, Bacteroidia, Sphingobacteriia, W27, W5, Clostridia, Thermotogae, LD1-PB3, Synergistia	Methanobacterium, Methanosaeta, Methanomassiliicoccus		[108]
Organic household waste 37%, manure 31%, slaughter residues 19%, other organic food waste 13% and iron chloride 0.03%		Biogas plant		Unknown	Pseudomonas, Sporosarcina, Leuconostoc, Romboutsia			[109]
Organic household waste 37%, manure 31%, slaughter residues 19%, other organic food waste 13% and iron chloride 0.03%	Nitrification	Biogas plant		Unknown	Lactobacillus, Ralstonia, Pseudomonas, Mycobacterium, Chujaibacter, Romboutsia			[109]
Sewage sludge produced by wastewater treatment plants from urban sites		Organic waste treatment plants	34–38 days	Mesophilic (32–42 °C)	Acidobacteria, Actinobacteria, Atribacteria, Bacteroidetes, Chlorobi, Chloroflexi, Deferribacteres, Firmicutes, Lentisphaerae, Nitrospirae, Planctomycetes, Proteobacteria, Spirochaetae, Verrucomicrobia			[37]
Animal manure or slurry from agricultural sites and a mix of food industry waste, harvest residues, livestock effluents or sewage sludge from central sites		Organic waste treatment plants	30–90 days	Mesophilic (32–42 °C)	Actinobacteria, Atribacteria, Bacteroidetes, Cloacimonetes, Firmicutes, Planctomycetes, Proteobacteria, Spirochaetae, Synergistetes, Tenericutes			[37]
Mixed sludge		Anaerobic digestion plant	Unknown	Mesophilic (36 °C)	Clostridia, Bacillus, Bacteroidia, Sphingobacteriia, Cytophagia, Flavobacteriia, Alphaproteobacteria, Gammaproteobacteria, Betaproteobacteria			[110]
Cattle manure digestate inoculum, pig manure (85%) and food waste (15%), volatile solids basis		Anaerobic digestion reactor (semi-continous)	41 days (HRT)	Mesophilic (39 °C)	Acholeplasma, Treponema, Sphaerochaeta, Succinivibrio, RFN20, ML1228J-1, Sedimentibacter, Syntrophomonas, Coprococcus, Clostridium, Alkaliphilus, Caldicoprobacter, Leuconostoc, Lactobacillus, Corynebacterium	Methanosarcina, Methanoculleus, Methanosphaera, Methanobrevibacter, Methanobacterium		[111]
Cattle manure digestate inoculum, pig manure (62.5%) and food waste (37.5%), volatile solids basis		Anaerobic digestion reactor (semi-continous)	41 days (HRT)	Mesophilic (39 °C)	Acholeplasma, Treponema, Sphaerochaeta, Succinivibrio, RFN20, ML1228J-1, Sedimentibacter, Syntrophomonas, Coprococcus, Clostridium, Alkaliphilus, Caldicoprobacter, Leuconostoc, Lactobacillus, Corynebacterium	Methanosarcina, Methanoculleus, Methanosphaera, Methanobrevibacter, Methanobacterium		[111]
Cattle manure digestate inoculum, pig manure (40%) and food waste (60%), volatile solids basis		Anaerobic digestion reactor (semi-continous)	41 days (HRT)	Mesophilic (39 °C)	Acholeplasma, Treponema, Sphaerochaeta, Succinivibrio, RFN20, ML1228J-1, Sedimentibacter, Syntrophomonas, Coprococcus, Clostridium, Alkaliphilus, Caldicoprobacter, Leuconostoc, Lactobacillus, Corynebacterium	Methanosarcina, Methanoculleus, Methanosphaera, Methanobrevibacter, Methanobacterium		[111]
Cattle manure digestate inoculum, pig manure (85%) and food waste (15%), volatile solids basis		Anaerobic digestion reactor (semi-continous)	29 days (HRT)	Mesophilic (39 °C)	Acholeplasma, Treponema, Sphaerochaeta, RFN20, ML1228J-1, Sedimentibacter, Syntrophomonas, Coprococcus, Clostridium, Alkaliphilus, Caldicoprobacter, Leuconostoc, Lactobacillus, Propionicimonas, Corynebacterium	Methanomassiliicoccus, Methanosarcina, Methanospirillum, Methanogenium, Methanoculleus, Methanosphaera, Methanobrevibacter, Methanobacterium		[111]
Cattle manure digestate inoculum, pig manure (62.5%) and food waste (37.5%), volatile solids basis		Anaerobic digestion reactor (semi-continous)	29 days (HRT)	Mesophilic (39 °C)	Acholeplasma, Treponema, Sphaerochaeta, RFN20, ML1228J-1, Sedimentibacter, Syntrophomonas, Coprococcus, Clostridium, Alkaliphilus, Caldicoprobacter, Leuconostoc, Lactobacillus, Propionicimonas, Corynebacterium	Methanomassiliicoccus, Methanosarcina, Methanogenium, Methanoculleus, Methanosphaera, Methanobrevibacter		[111]
Cattle manure digestate inoculum, pig manure (40%) and food waste (60%), volatile solids basis		Anaerobic digestion reactor (semi-continous)	29 days (HRT)	Mesophilic (39 °C)	Acholeplasma, Treponema, Sphaerochaeta, RFN20, ML1228J-1, Sedimentibacter, Syntrophomonas, Coprococcus, Clostridium, Alkaliphilus, Caldicoprobacter, Leuconostoc, Lactobacillus, Propionicimonas, Corynebacterium	Methanomassiliicoccus, Methanosarcina, Methanosaeta, Methanoculleus, Methanosphaera, Methanobrevibacter		[111]
Cattle manure digestate inoculum, pig manure (85%) and food waste (15%), volatile solids basis		Anaerobic digestion reactor (semi-continous)	21 days (HRT)	Mesophilic (39 °C)	Acholeplasma, Treponema, Sphaerochaeta, RFN20, ML1228J-1, Sedimentibacter, Syntrophomonas, Coprococcus, Clostridium, Alkaliphilus, Caldicoprobacter, Leuconostoc, Lactobacillus, Propionicimonas, Corynebacterium	Methanomassiliicoccus, Methanosarcina, Methanospirillum, Methanogenium, Methanoculleus, Methanosphaera, Methanobrevibacter, Methanobacterium		[111]
Cattle manure digestate inoculum, pig manure (62.5%) and food waste (37.5%), volatile solids basis		Anaerobic digestion reactor (semi-continous)	21 days (HRT)	Mesophilic (39 °C)	Acholeplasma, Treponema, Sphaerochaeta, RFN20, ML1228J-1, Sedimentibacter, Syntrophomonas, Coprococcus, Clostridium, Alkaliphilus, Caldicoprobacter, Leuconostoc, Lactobacillus, Corynebacterium	Methanomassiliicoccus, Methanosarcina, Methanogenium, Methanoculleus, Methanosphaera, Methanobrevibacter		[111]
Cattle manure digestate inoculum, pig manure (40%) and food waste (60%), volatile solids basis		Anaerobic digestion reactor (semi-continous)	21 days (HRT)	Mesophilic (39 °C)	Acholeplasma, Treponema, Sphaerochaeta, RFN20, ML1228J-1, Sedimentibacter, Syntrophomonas, Coprococcus, Clostridium, Alkaliphilus, Caldicoprobacter, Leuconostoc, Lactobacillus, Corynebacterium	Methanomassiliicoccus, Methanosarcina, Methanosaeta, Methanoculleus, Methanosphaera, Methanobrevibacter		[111]
Cow manure, wood straw and wood chip		Biogas plant	Unknown	Unknown	Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Plantomycetes, Proteobacteria, OP9, Synergistetes	Crenarchaeota, Euryarchaeota		[112]
Mixture of dairy cattle manure (70%), energy crops (maize silage up to 30%) and waste from agro-food industries		Biogas plant		Mesophilic	Acidimicrobiia, Actinobacteria, Alphaproteobacteria, Bacilli, Bacteroidia, Clostridia, Deltaproteobacteria, Gammaproteobacteria, Gemmatimonadetes, Mollicutes, Phycisphaerae, Saccharomonadia, Verrucomicrobiae	Methanosarcina	Ascomycota, Basidiomycota	[113]
Anaerobically digested sludge inoculum, food waste and waste activated sludge (TS: 5%)		Continuous stirred tank reactors	30 days	Mesophilic (35 °C)	Mycobacterium, Thermovirga, Mesotoga, Erysipelatoclostridium, Eubacterium, Aminobacterium, Bacillus, Clostridium	Methanobrevibacter, Methanofollis		[58]
Anaerobically digested sludge inoculum, food waste and waste activated sludge (TS: 5%)	Wheat straw pellet biochar	Continuous stirred tank reactors	30 days	Mesophilic (35 °C)	Mycobacterium, Thermovirga, Mesotoga, Erysipelatoclostridium, Eubacterium, Bacteroides, Aminobacterium, Clostridium	Methanosarcina, Methanomicrobiales, Methanofollis, Methanoculleus, Methanolinea		[58]
Anaerobically digested sludge inoculum, food waste and waste activated sludge (TS: 7.5%)		Continuous stirred tank reactors	30 days	Mesophilic (35 °C)	Thermovirga, Mesotoga, Erysipelatoclostridium, Syntrophomonas, Bacteroides, Aminobacterium	Methanocorpusculum, Methanobrevibacter, Methanosarcina		[58]
Anaerobically digested sludge inoculum, food waste and waste activated sludge (TS: 7.5%)	Wheat straw pellet biochar	Continuous stirred tank reactors	30 days	Mesophilic (35 °C)	Thermovirga, Mesotoga, Erysipelatoclostridium, Eubacterium, Aminobacterium, Bacillus, Clostridium	Methanobrevibacter, Methanosarcina		[58]

4. Applications of Digestate in Relation to Microbial Composition

Anaerobic digestion not only generates methane, but also produces a digestate that can be used for various applications. These potential applications include soil remediation, soil amendment, biofuel production and aquaculture (Figure 3). Significant environmental changes can result from the use of digestate, and soil ecology can be impacted by these modifications. This might influence microbial competitiveness and antagonistic interactions, which in turn may help prevent plant diseases or facilitate soil remediation [43,114].

Understanding the behavior and dynamics of microbial communities is crucial for AD optimization, where microorganisms play an integral role, and this later determines digestate microbes. The presence of various microbes in the digestate can have either beneficial or detrimental effects on the subsequent application. The microbiology of anaerobic digestion processes has been studied extensively in recent years, using both physical and molecular methods [60,115,116]. The potential uses of digestate and the microbial populations contained in the digestate are currently not extensively studied. As such, it is important to understand the digestate’s microbial functions in light of various applications.

4.1. Digestate Use as a Biofertilizer

Ultimately, a digestate containing nutrients and organic molecules that were not broken down during the process is produced. Digestate can be re-used as a fertilizer, contributing to the goal of creating a sustainable food system. By reusing the plant nutrients in the organic waste, the amount of chemical fertilizers needed to grow crops may be significantly reduced, making the use of digestate as a fertilizer environmentally friendly. Furthermore, due to the mineralization of organic nutrients during the AD process, digestate contains a significantly higher quantity of plant nutrients in plant-available forms compared to undigested organic wastes [114].

The use of digestate as an organic fertilizer has also been shown to improve nutrient uptake, soil structure and soil microbial activity [117]. Digestate contains a wide range of beneficial microbes, a unique chemical profile, and a wealth of macro and micronutrients for plant growth [21]. In general, the variety of microorganisms present in biogas production facilities is proportional to the variety of feedstock and the specifics of the production process [13]. In most cases, the microbial population in digestates is not given enough credit or research for the value it could potentially bring to biofertilizers. Direct interactions with the plant rhizosphere (such as nitrogen fixers and mycorrhizae) and/or indirect solubilization of plant nutrients in the soil are two ways in which the active microbial community in biofertilizers can significantly affect crop nutrition, as reported by Mohammadi and Sohrabi [118]. Although AD is well-known for lowering microbial pathogens in organic wastes, biosecurity concerns connected to the land spreading of microorganisms from digestate, such as the presence of pathogenic microbes, remains a primary issue [21]. However, in addition to pathogenic organisms, digestate contains numerous other microorganisms with potential for agronomical benefit or environmental impact, such as nitrogen-fixing bacteria (e.g., Bradyrhizobium) [119] as well as denitrifying and nitrifying bacteria (e.g., Achromobacter denitrificans, Thiobacillus denitrificans and Nitrosomonas) [38]. The majority of microorganisms previously listed were from studies focusing on biogas production. Hence, numerous questions remain unanswered, such as do microorganisms in the digestate exist in sufficient amounts to have an effect on the soil and plant systems?

Some research suggests that applying digestate to soils can boost microbial biomass and activity even if the digestate itself does not contain sufficient levels of microorganisms [120], whereas others reported little effects [21]. These results may have been influenced by experimental factors such as sampling time after digestate application and the duration over which applications were performed. This can also be modified by the feedstock composition and AD operation parameters. The AD process can be undertaken at different temperatures, such as mesophilic and thermophilic, and as a result, the digestate microbial community can be affected by these temperatures [60].

Soil bacterial growth was positively affected by digestate treatments, although fungus population changes were minimal, as reported by Walsh et al. [121]. Although, the focus of this paper is on the microbes in the digestate and not the soil microbes, it is still mandatory to understand the soil dynamics post digestate application, as digestate microbial content will influence the soil microbial content. For instance, plant growth-promoting bacteria (PGPB) in the digestate are representative of a large bacterial community that is found in the rhizosphere of many plant species and directly stimulates host plant growth through the solubilization of minerals like phosphorus, the production of siderophores that chelate iron, and the production of phytohormones [22].

4.2. Digestate Use in Aquaculture

The AD process converts complex organic molecules in the presence of microorganisms into biogas while digestate, which is rich in nitrogen and phosphorus, is also formed. Algal biomass has been proven to thrive on anaerobic digestate, with the added benefit of sequestering nitrogen and phosphorus in a biologically fixed form [24]. Fernandes et al. examined the usage of digestate from various feedstock (pig manure, potato peel, and agricultural waste) for microalgae production [122]. Ammonium was the most important nutrient in the digestate for microalgal cultivation, and enhancing its availability and consequent uptake is essential for optimal growth. Both Scenedesmus obliquus and Chlorella vulgaris had higher growth yields as a result of ammonium in the digestate media. However, the findings also revealed that digestates from different feedstock could result in different growth yields and biomass composition.

Higgins et al. conducted an experiment in which algae were grown in anaerobic digestate from chicken sludge [123]. Algae reduced nitrogen and phosphorus nutrient concentrations in the digestate proportionally to algal growth, and the microbes in the digestate helped lower the chemical oxygen demand (COD), including photosynthate secreted by algae, thereby improving water quality. Tawfik et al. considered mixotrophic algal cultivation on anaerobic digestate as a potential alternative for nutrient recovery, pollution removal, and biofuel production [124]. They concluded that anaerobic liquid digestate is rich in macro- and micronutrients, both of which contribute to the growth of algae. However, high concentrations of digestate inhibits algal development and diminishes their biodegradability. Therefore, pretreatments, such as dilution, are recommended as a means to reduce the effect of contaminants on algal growth. Uggetti et al. investigated the viability of using the liquid phase of anaerobic digestate effluent as a substrate for microalgal development [125]. In a batch experiment conducted in the lab, the impact of the inoculum/substrate ratio on microalgal growth was investigated. Based on their findings, digestate showed promise as a substrate for the growth of microalgae, which also has a potential for biofuel production. Whilst much focus has been directed to the influence of inherent digestate nutrient content on algal growth, limited research on the influence of the resident microbes is available, and this highlights a potential future research scope.

4.3. Digestate Use in Biofuel Production—Digestate Recirculation

Digestate from lignocellulose-fed anaerobic digesters is a rich source of soluble nutrients and lignocellulose-degrading bacteria [106]. The performance of anaerobic systems can be enhanced by recycling the digestate within the system for reuse, which in turn reduces its outflow. The buffering capacity, system stability, and hydrolytic performance of biodegradable materials are all enhanced by recirculating effluent from the methanogenic phase to the acidogenic phase digester, as reported by Hao et al. [126]. In addition, the evenness, diversity, and richness of the microbial population affects the performance of the reactor, which is dependent on the operating parameters, in terms of both anaerobic efficiency and system stability.

Maria et al. report on the study of the microbial community structure in the AD system both before and after digestate recirculation [127]. In the anaerobic system using recycled digestate, the two most common archaeal groups were Methanosarcina (acetoclastic and hydrogenotrophic) and Methanoculleus (hydrogenotrophic). A study by Lin et al. investigated the effect of using digestate recirculation as inoculum on the microbial communities during thermophilic solid-state anaerobic digestion (SS-AD) of grass clippings [128]. Up to the third run, the results showed a clear progression of microbes toward higher variety. The presence of VFAs seemed to correspond with the occurrence of Clostridia and Thermotogae. They found that reusing digestate could be an effective ‘vaccine delivery mechanism’. Moreover, this frequent exposure allowed these microbes to thrive under harsh conditions.

4.4. Digestate Use for Bioremediation

Soil contaminants such as petroleum hydrocarbons (PHCs) can be effectively treated through bioremediation [129]. Specialized microbial biomass capable of metabolizing the pollutants are essential for successful bioremediation treatment. During bioremediation, the oxidation of alkanes is catalyzed by alkane monooxygenases, which are encoded by the alkB gene family [130]. During bioremediation, bioaugmentation has also been found to be a powerful method for increasing the number of contaminant-degrading microbes in the soil and, in turn, the quantity of the alkB genes present in that soil [131].

Soil bioremediation research has previously focused on organic fertilizers, but primarily in their nutrient-providing capacities. Here, we also review digestates’ advantageous properties for bioremediation. Research on the microorganisms involved in the degradation of contaminants has typically been limited to either broad surveys of microbial community composition (such as DGGE) or the isolation and identification of culturable bacterial species [130].

A study by Gielnik et al. investigated the effect of sewage sludge digestate that can be used as soil inoculum in small-scale aerated bioreactors in the laboratory [23]. The aim was to determine if and how the concentration of alkB genes and the rate of contaminant removal may be improved by adding sewage sludge digestate to soil contaminated with polychlorinated biphenyls (PCBs). They found that monooxygenases produced by alkB genes have the potential to breakdown organic materials in digestate. Interestingly, the same enzymes that are important for the metabolism of alkanes and other TPH components may also play a significant part in this process. It was then postulated that the alkB gene content of soil may be increased by the bacteria found in digestate, which would boost the metabolic capacity of the soil by introducing new contaminant-degrading taxa. Provided that the injected microorganisms are already acclimated to treatment conditions, bioaugmentation could further boost process efficiency.

5. Conclusions and Future Prospects

Overall, this paper provides a holistic overview of the current state of knowledge on resident microorganisms in digestate and applications thereof. A multitude of factors contributing to the composition of the microbial community are elaborated on, and it was postulated that these factors may be manipulated in order to obtain a microbial community in the digestate that is conducive to the proposed digestate application. For example, thermophilic AD and post-treatments such as composting may be suggested when considering digestate for application in the field or as an animal feed, since such approaches aid in modulating the resistome profile of the resident microbes in the digestate by reducing the abundance of ARGs [132,133,134].

A thorough literature survey was conducted in order to report on the microorganisms detected in the digestate from several studies, and the functionality of the microbes in the AD process was eluded. Such data are valuable to researchers in the AD field as they will guide future studies on the engineering of the digestate microbiome. Overall, it is well known that for any process to be environmentally sustainable and economically viable, the optimization of all aspects of the process needs to be achieved. Focus needs to be diverted to the microbiome associated with digestate in order to maximize the value of the product. In so doing it is anticipated that value addition from the digestate in AD may even exceed that of the generated biogas.

Future studies are proposed related to the evaluation of microorganisms in digestate obtained from several digesters under varying conditions. Such studies are anticipated to aid in the modelling of targeted microbiome composition of digestate. Studies are also proposed for assessing the potential of isolates from digestate in several spheres, including plant growth promotion, bioremediation, etc. Overall, the current state of knowledge on the potential of microbes in digestate is still in its infancy. Future research may lead to unlocking the vast potential of this valuable resource and aid in closing the loop in the circular economy of the AD process.