Diversity of Microbial Communities in Biogas Reactors

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Diversity of Microbial Communities in Biogas Reactors Bernadett Papa and Gergely Marótib,* b Seqomics Biotechnology Ltd., H-6782 Mórahalom, Vállalkozók útja 7., Hungary; Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, H-6726 Szeged, Temesvári krt. 62., Hungary

a

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Abstract: Biomethane has gained increasing attention in the recent years as an alternative, local energy source option. Biogas is generated during the anaerobic digestion of organic materials via a multistep process catalyzed by complex microbial communities. This review aims at providing a concise summary of recent studies on the microbial communities in various biogas reactors. The effects of acid composition, C/N ratio, mixing and the geometry of the anaerobic digester on the microbial ecosystem is discussed. Recent studies demonstrated the extensive fluctuations in the biogas microbial communities in response to changes in temperature, substrate type, pH, type of volatile fatty acids, organic loading rate, etc. The goals to ensure the stability of the anaerobic degradation process and to maximize the biogas production require the better understanding of these metabolic changes, since functional stability strongly correlates with the state and composition of microbial communities. The safe and controlled intensification of biogas production would be an important step to make biogas a real competitor of fossil fuels.

Keywords: Anaerobic digestion, biogas, metagenomics, methane, microbial community, syntrophic interactions. Received: May 26, 2015

Revised: July 15, 2015

1. INTRODUCTION Beside the extensive use of fossil fuels increasing attention is paid for the research and implementation of alternative methods to provide sustainable, environmentally friendly energy sources. The use of renewable energy sources can contribute to the reduction of greenhouse gas emissions thereby mitigating the proposed climate change effects. A range of wastes such as agricultural, industrial, household and municipal wastes are available in excess and remain untapped as energy source. Furthermore, different untreated waste materials are often hazardous for the environment since these slowly degrade and the produced various gases escape into the atmosphere increasing the greenhouse gas effect. However, controlled biogas technology allows us to transform these waste materials easily and advantageously into a unique alternative energy source by anaerobic digestion. Biogas technology offers economic, health, social, and environmental benefits [1]. This energy source is suitable to run generators to produce electricity by burning the biogas, and the generated heat also can be utilized locally. In addition, the residual digestate can replace or complete artificial fertilizers in agricultural utilization. *Address correspondence to this author at the Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, H-6726 Szeged, Temesvári krt. 62., Hungary; Tel: +36308270455; E-mail: [email protected] 2212-7119/16 $58.00+.00

Accepted: July 18, 2015

2. ANAEROBIC DIGESTION/FERMENTATIVE METHANE PRODUCTION Methane-rich biogas is one of the most widely used renewable energy source, it is produced through anaerobic digestion (AD) of various organic-rich materials. AD is a conversion of organic material into a mixture of mainly CO4 and CO2. Coordinated interactions between the microbial consortia in the digested material are responsible of this biodegradation process (Fig. 1). There are four key steps in the AD process: hydrolysis, acidogenesis, acetogenesis and methanogenesis. In the first step of AD the complex organic matter is transformed into soluble organic material, mainly carbohydrates, proteins and lipids are broken down by hydrolytic bacteria. The generated sugars, amino acids, fatty acids are appropriate substrates for fermentative bacteria involved in the acidogenesis step. Thereafter the previously formed organic acids and alcohols are transformed into acetate and hydrogen via acetogenesis. In the last step the methane production takes place via aceticlastic or hydrogenotrophic methanogenesis depending on the operating parameters and substrates [2]. The hydrogenotrophic methanogenesis is usually connected to syntrophic bacteria which are able to convert the previously formed short-chain fatty acids into H2 and CO2 through endergonic reactions. The maintenance of the equilibrium between acetogenesis and methanogenesis is a crucial factor for continuous methane production. Disturbances can initiate restricted VFA (volatile fatty acids) utilization leading to pH decrease © 2016 Bentham Science Publishers

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Pap and Maróti

Fig. (1). Generalized pathway of the anaerobic digestion process.

which can have a strong negative effect on the methanogenesis, since the pH optimum of the methanogenic Archaea is between 6.5-8.0 (somewhat higher than the optimum for acetogen bacteria). Furthermore, too high levels of hydrogen and acetate (end products of acetogenesis) inhibit the consumption of hydrogen and acetate. A number of studies already reviewed the microbial participants of biogas generation processes. Heyer et al. discussed studies of proteomics results from full-scale biogas plants [3]. Other studies examined the methanogenic cellulolytic communities in bioreactors using various substrates [4] or operating temperatures [5], or observed the microbial structure influenced by other operation conditions [6]. Syntrophic associations in AD were also discussed by Kouzuma et al. in a review [7]. Close interactions are needed between the syntrophs and methanogens for hydrogen-transfer which is frequently strengthened by co-aggregation or biofilm production especially in the presence of poor, energetically unfavorable substrates [7]. In an earlier work the dynamics of the continuously stirred tank reactors (CSTRs) was examined. Microbial communities of acetate, propionate, butyrate, long-chain fatty acids, glycerol, protein, glucose and starch substrate-degrading biogas reactors were summarized [8]. 3. APPROACHES FOR THE ANALYSIS OF MICROBIAL COMMUNITIES A number of cultivation-independent techniques have been developed in the past, for instance fluorescence in situ hybridization (FISH), real-time PCR, terminal restriction fragment length polymorphism (T-RFLP), denaturing gradient gel electrophoresis (DGGE) and clone library approaches were demonstrated and shown to be suitable for the identification of complex microbial communities or at least certain groups of them [9, 10]. However, according to a study revealed that DGGE and clone library approaches missed important taxonomic groups [11]. Thus, new techniques permitting more precise and representative community data were investigated. Nowadays, the high-throughput genomic tech-

nologies are becoming a benchmark approach for the detailed analysis of microbial communities. Accordingly, highthroughput metagenomics, metatranscriptomics, metaproteomics [3] and metabolomics approaches supplemented with advanced visualization and isotope labeling techniques provide realistic opportunities for process engineering of anaerobic digestion through the understanding of the taxonomic and metabolic complexities [12]. Metagenomics techniques such as 454-pyrosequencig, SOLiD™ short-read DNA sequencing, Ion Torrent Personal Genome Machine™ sequencing have been successfully used to describe microbial communities of AD [13-22]. 4. DOMINANT BACTERIAL AND ARCHAEAL MEMBERS OF THE BIOGAS MICROBIAL COMMUNITIES The Bacteroidetes and Firmicutes phyla were observed as highly stable and dominant bacterial groups in laboratory scale biogas reactors fed with various substrates [23, 24]. Other studies also confirmed the highest abundance of the Firmicutes phylum in co-digesting reactors, however, under thermophilic conditions the Thermotogae phylum was also highly dominant [25]. Regueiro et al. identified Firmicutes, Bacteroidetes and Proteobacteria as the most dominant phyla in six full-scale and one laboratory-scale co-digesters [26]. The predominance of the Firmicutes phylum is mostly explained by the capability of these bacteria to produce diverse enzymes performing hydrolysis, acidogenesis and acetogenesis. Concerning the methanogenic Archaea, the predominance of the aceticlastic Methanosaeta genus was described principally at low acetate level in stable anaerobic digesters [27, 28]. Information is accumulating on the possible distinct roles of acetotrophic and hydrogenotrophic methanogens. It is to note that in numerous cases dramatic decrease in the relative abundance of the Methanosaeta genus was described in AD under perturbated conditions which may indicate the high sensitivity of the members of this aceticlastic methanogenic genus. Under various disturbed cir-

Diversity of Microbial Communities in Biogas Reactors

cumstances (elevated temperature, decreased pH, high VFA level and high ammonia level) a clear transition was observed from aceticlastic to hydrogenotrophic methanogenesis [19, 20, 29-35]. This takeover of hydrogenotrophic methanogens is mostly linked to the increased relative abundance of syntrophic acetate oxidizing bacteria (SAOB) in the biogas fermentors. Furthermore Lerm et al. revealed that the high organic loading rate (OLR) also had similar effect on methanogenesis, the enrichment of hydrogenotrophic methanogens was observed, more specifically two hydrogenotrophic species, Methanospirillum hungatei and Methanoculleus receptaculi was shown to be present with strongly increased relative abundance [36]. Further studies also revealed the Methanoculleus dominance over other hydrogenotrophic methanogenic genera, this phenomenon is ascribed to the possible high general stress tolerance of the members of this genus [16, 37]. It was specifically shown that certain Methanoculleus strains showed increased tolerance to high salt concentration, to elevated ammonium level and even against aeration in the bioreactors [16, 37-39]. The high differences of the various AD systems is depicted by the results of Li et al., their observations might sound to be in contradiction with the general above-mentioned picture since in this system the Methanosaeta genus dominated the reactors in different examined reactor states regardless of the stability level of the digestion process [40]. A similar observation was explained in another study where the robustness and high stability of the Methanosaeta genus was observed in the reactor even in the presence of high acetate level [41]. Syntrophic associations in AD are gaining more and more attention, the acetate-, propionate- or butyrateoxidizing bacteria interact with H2-scavenging methanogens. Syntrophic acetate oxidation (SAO) was described in stressed (particularly high ammonia-level) biogas reactors [42, 43]. Westerholm et al. reported a study about quantification of syntrophic acetate-oxidizing bacteria (SAOB), the putative roles of Thermacetogenium phaeum, Clostridium ultunense, Syntrophaceticus schinkii and Tepidanaerobacter acetatoxydans were discussed [44]. The presence of syntrophic fatty acid degrading bacteria (SFAB) in biogas reactors was also shown, members of the Syntrophobacter, Smithella, Pelotomaculum and Syntrophomonas bacterial genera were identified, their relative abundances were observed to vary according to reactor configuration and substrate characteristics [45]. 4.1. Effects of Operational Parameters on Microbial Community 4.1.1. Effects of Temperature On the Microbial Community Structure Temperature is one of the crucial factors in shaping the microbial community structure during the anaerobic digestion (beside substrate type, OLR, VFA composition, ammonium concentration, pH of the digested sludge, alkalinity, mixing and the geometry of the anaerobic digester) [20, 46]. Elevated operation temperature enhances the efficacy of the enzymatic processes and initiates faster growth rate of the methanogens, thereby ensure that besides lower HRT (hydraulic retention time) microbes remain in optimal concentration within the fermentor [47, 48]. Moreover, thermophilic

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anaerobic fermentation minimizes the numbers of pathogens, viruses, fungi, and parasites, which is an important requirement for the agricultural utilization of the residual digested sludge as fertilizer [47, 49]. Detailed analyses revealed the fundamental effects of the operation temperature both on the biogas and VFA yields as well as on the microbial diversity (Fig. 2. and Fig. 3). In general the mesophilic bioreactors maintain higher general biodiversity than the thermophilic reactors [46, 50, 51]. Under mesophilic conditions the major bacterial components of the fermentation ecosystem are the Bacteroidetes (in some cases up to 50% of the bacterial community), the Firmicutes and the Proteobacteria phyla according to various studies [19, 20]. However, in the thermophilic anaerobic digestion process Firmicutes phylum was identified as the most abundant bacterial group in the ecosystem, while the relative abundance of the Bacteroidetes and Proteobacteria phyla were very low indicating that these organisms play only marginal role in the decomposition of the organic materials at elevated temperature [19, 52] (Fig. 3A). In sugar beet pressed pulp-degrading biogas reactors the Thermotogae phylum was also enriched during an adaptation from mesophilic to thermophilic operation, Petrotoga mobilis became the most abundant species in the system [20]. In another temperature adaptation study using maize-silage as substrate the most striking expansions were detected for the hydrogen-producing Clostridium and Caldicellulosiruptor genera within the Firmicutes phylum [19, 37]. Hollister et al. compared mesophilic and thermophilic microbial communities in bioreactors fed by lignocellulosic feedstock [50]. The mesophilic anaerobic digester was dominated by Firmicutes, Proteobacteria, Actinobacteria phyla and Bacteroidia class within Bacteriodetes phylum, while under thermophilic operation two classes of Firmicutes phylum Clostridia and Bacilli and Thermoanaerobacterium genus within Clostridia class were identified as the most abundant taxons. In the mesophilic reactor the majority of the genes could be assigned to biodegradation of hemicellulose derivatives (especially for a five-carbon sugar arabinose degradation). The ability for degradation of arabinose and other hemicellulose derivatives by Bacteriodetes phylum and arabinose transformation into propionate by members of the Bacteroides genus were shown [53]. Thus, the elevated propionate concentration is likely the result of the dominant Bacteroidetes phylum metabolism (The presence of higher concentration of propionate under mesophilic conditions (compared to the thermophilic reactor) confirmed this observation). However, the thermophilic reactor was rather enriched in genes related to cellobiose uptake. Clostridia and Bacilli classes were identified also by Ritari et al. as the most prevalent groups in thermophilic reactors, moreover members of the Thermotogae phylum could be exclusively identified in the thermophilic reactor confirming the results of other studies [54]. Thus, in general Firmicutes and Thermotogae are the most abundant bacterial phyla under thermophilic operation of the anaerobic bioreactor. In response to temperature drop (switch from mesophilic to psychrophylic operation) an increase was observed in the Bacteroides genus and significantly decreased relative abundances were found for Syntrophomonas and Clostridium genera [55]. These observations indicate a generally increasing importance of Clostridia by


Pap and Maróti

Fig. (2). Effects of temperature adaptation on daily biogas production and VFA level.

Fig. (3). An example for the microbial diversity in biogas reactors. (A) Phylogenetic distribution of the bacterial communities (at phylum level) at mesophilic and thermophilic temperatures in a reactor fed with maize silage. (B) Structure of the archaeal communities (at genus level) at mesophilic and thermophilic temperatures in a reactor run on maize silage.

elevating the temperature and vice versa. At the same time the members of phyla Proteobacteria and Bacteroidetes are more characteristic for mesophilic systems. 4.1.2. Roles of Ammonia, VFA Level and pH in Shaping the Microbial Ecosystem The free ammonia diffusing across cell membranes is considered the main form of ammonium toxicity [56, 57]. Methanogens in biogas reactors exhibit different reactions to ammonia stress. In general, the aceticlastic methanogens are

more sensitive than the hydrogenotrophic ones [57, 58]. It has been shown that the relative abundance of the aceticlastic groups declined whereas that of the hydrogenotrophic methanogens increased when total ammonium concentration reached 3 g L1 in anaerobic digesters [44, 59]. For example, the extreme reduction of the Methanosaeta genus and a clear shift from acetate to hydrogen utilization was observed in the anaerobic digesters at increased ammonium concentration and VFA level [34, 60]. Also, the increasing ammonium level favor the development of syntrophic acetate oxidation


Table 1.


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Effects of operational parameters on microbial community.

Operational Parameters

Dominant Microbial Community

References

Temperature mesophilic temperature

Firmicutes, Bacteriodetes, Proteobacteria phyla

[19, 20]

thermophilic temperature

Firmicutes, Thermotogae phlya

[20, 25, 46]


Firmicutes phylum, Clostridium genus

[19, 52]


Methanosarcina, Methanothermobacter genera

[19, 46, 52, 95]


Methanosarcina genus

[96]


Methanoculleus genus

[29, 37, 39]


Methanosarcina, Methanoculleus genera

[97]

VFA low acetate

Methanosaeta genus

[27, 28, 34]

high acetate


[34]

Ammonia low ammonia

Methanosaeta genus

[33, 34]

high ammonia


[34]

high ammonia


[16, 37, 38]

Substrate low C/N ratio

Bacilli class, Thermotogae phylum

[17, 18]

low C/N ratio


[74, 75]

Other parameters high salt


[16, 37, 38]

aeration


[16, 37-39]

(SAO)[61]. Certain syntrophic acetate oxidizers were found to be tolerant up to 8 g L1 ammonium level at neutral pH [44, 62]. Under high ammonium conditions CH4 production from acetate is probably shifted from aceticlastic methanogenesis to SAO combined with hydrogenotrophic methanogenesis [44, 58, 63]. However, the sensitivity of individual methanogens can be highly different and is influenced by further reactor parameters like pH and temperature. Methanosarcina spp. were found to be either sensitive [44, 59] or tolerant to ammonia stress [64] in different systems. Hydrogenotrophic methanogens also showed differential responses in different setups [63-65]. Interestingly, the species Methanoculleus bourgensis was identified to play a significant role in different biogas reactor systems especially under high ammonium concentration. Comparative genome analysis of M. bourgensis MS2 and Methanoculleus marisnigri JR1 revealed significant similarities and differences between the two Methanoculleus species. The absence of genes for a putative ammonium uptake system in M. bourgensis MS2 may indicate that this species is specifically adapted to environments with high levels of ammonium/ammonia [66]. Although the dominance of the acetoclastic Methanosaeta spp.

was generally observed in anaerobic digesters operating under low ammonia/ammonium level [33, 34], some exceptions can be found in the literature again [40, 41]. The accumulation of VFA also represents stress for the microbial communities, especially for the methanogenic Archaea fraction. Methanosarcina the Methanoculleus genera showed the most robust tolerance to rapid VFA increase in biogas reactors, where VFA increase was induced by gradual temperature elevation causing a perturbation in the stable mesophilic community. The relative abundance of Methanosarcina the Methanoculleus genera significantly increased in response to temperature adaptation and replaced the members of the Methanosaeta genus being dominant in the mesophilic reactor with low VFA level [19] (Fig. 3B). The pH and alkalinity are also among the most important influencing parameters since sufficient methanogenesis requires pH value between 6.5-8.0, the methanogens are especially sensitive for the acidic environment. As acidogenic bacteria are capable to lower the pH below the optimal range it is essential to continuously monitor the AD process to maintain constant and safe methane production.


4.1.3. The Substrate Dependence of the AD Microbial Communities It is evident, that different substrates are metabolized through different pathways either by single microbes or by complex microbial communities as well. A large number of studies examined the microbial composition dynamics in response to changes in feedstock composition and feeding rates. For example, propionic acid accumulates in reactors overloaded with carbohydrates, in such cases members of the Lactobacillus genus were found to increase significantly. Acetate accumulation was observed in reactors supplemented with lipids, which was accompanied by the occurrence of Dialister and Kyrpidia genera. Desulfotomaculum was the only bacterial genus exhibiting significant increase in response to addition of proteins, it can be justified by the appearing need for H2S elimination as a consequence of protein degradation [22]. According to many scientists the efficient biodegradation of the excessively available cellulosic materials could be a competitive solution for biomass-based energy generation. However, the anaerobic degradation of cellulosic biomass is not a straightforward approach, in most cases the hydrolysis of such substrates is a major ratelimiting factor in AD, consequently pretreatment strategies are required [67]. During anaerobic digestion of grass silage which consists mainly of polymers of cellulose, hemicellulose and lignin mostly various members of Firmicutes (dominated by Clostridia) and Bacteroidetes bacterial phyla were identified by clone library analysis, while the methanogenic Archaea were represented by the hydrogenotrophic Methanobacterium genus as the most dominant group [68]. Maize-silage is a common substrate for biogas generation, similar community structures were observed in different mesophilic biogas digesters with the clear dominance of the Firmicutes and Bacteroidetes phyla [19, 69]. Within the thermophilic cellulolytic community fermenting microcrystalline cellulose as substrate and glucose as co-substrate the Anaerolineales, Clostridiales, Bacteroidales and Thermotogales orders belonging to Chloroflexi, Firmicutes, Bacteriodetes and Thermotogae phyla, respectively, were dominant. The dominance of Methanobacteriales and Methanosarcinales archaeal orders were observed in this system. Interestingly, metatranscriptomic analysis showed stronger transcriptional activities of genes and pathways involved in aceticlastic methanogenesis of Methanosarcinales compared to the hydrogenotrophic pathways of Methanobacteriales suggesting that aceticlastic methanogens were more active than the hydrogenotrophic methanogens [70]. This finding also highlights the importance of the combination of various approaches to describe the microbial communities. Application of algal biomass for biogas production is another promising possibility due to its low lignin content, certain algal species are described to have advantageous cell wall characteristics with easily degradable materials. A single physical pretreatment of the algal biomass might be sufficient to use them as substrate [71]. During co-fermentation of mixed algal-bacterial biomass with maize silage a microbial community dominated by the Proteobacteria phylum was developed, which showed clear differences compared to the anaerobic digestion based on maize silage alone where Firmicutes and Bacteroidetes were the most abundant phyla [72].

Pap and Maróti

Byproducts of various food processing industries, predominantly dairy and meat industries are often used as substrates for biogas generation. The anaerobic digestion of such protein-rich waste materials containing high concentration of nitrogen (resulting in elevated ammonia/ammonium level) has an intrinsic risk for inhibition issues in the biogas fermentors. However, these materials are valuable and suitable for biogas production under well-controlled conditions and with careful operation, the bacterial and archaeal communities can be successfully adapted to the protein-rich substrate [18, 73]. Firmicutes was shown to be the most abundant phylum in the biogas reactor fed by protein-rich substrates and within the Firmicutes phylum a decreasing relative abundance of the Bacilli class was observed over time [17, 18]. However, the Thermotogae phylum also showed constant increase due to its adaptability for protein-rich substrates [17, 18]. The highest methane yield was described in proteindegrading batch reactors compared to batch systems running on cellulose-rich material or using high lipid content. The most explicit dominance of the Methanoculleus genus was described in the batch reactors fed with protein-rich substrate suggesting that this group was responsible for the surplus methane production [74]. The archaeal community was examined by terminal restriction fragment length polymorphism (T-RFLP) method when the biogas fermentor was fed with protein-rich substrate and the overwhelming dominance of the hydrogenotrophic Methanoculleus genus was observed [75]. Industrial wastewaters, sewage sludges, swine manure and biofermentors digesting different organic solids such as slaughter-house waste, animal manure and food wastes are often sources of toxic phenolic compounds which can negatively affect the microorganisms involved in anaerobic digestion. Inhibited degradation of phenol was observed at thermophilic operation temperature while these phenols and derivatives were degraded rapidly into methane in mesophilic reactors [20, 76]. This can be explained by the different metabolic capability and enzyme sortiment (harbored by the strongly different microbial communities) under mesophilic or thermophilic operation. The members of Syntrophorhabdaceae (Proteobacteria phylum) and the Desulfotomaculum genus (Firmicutes phylum) were identified as community members with potential phenol-degrading capability [76, 77]. Handling of sewage sludge waste is difficult because of the floc structure of the sludge. Therefore, usually thermal, chemical, mechanical or biological pretreatment methods [78, 79] were used to treat sludge waste materials in order to enhance their biodegradability by solubilization of the organic matters [80]. Coprothermobacter has been identified as a dominant bacterial genus in sewage sludge digesters operating under thermophilic temperature [81-84]. Sewage sludge has low C/N ratio between 6 and 16 [85], therefore co-digestion of this material with high carbohydrate containing substrates such as food wastes can be advantageous [86]. Coprothermobacter has also been found dominant by Lee et al. [87] in thermophilic two-stage sewage sludge and kitchen garbage co-digesters. Using other co-substrates such as grease waste [88-90] or organic fraction from the municipal solid waste


[91] can also be beneficial in intensification of methane production. Silvestre et al. found that the methane yield was doubled using grease waste and the aceticlastic way of methanogenesis was favored [88]. Nakakihara et al. observed changes in the microbial community of sewage sludge co-digestation with rice straw, mostly Methanosaeta and Methanoculleus genera were observed among the methanogenic Archaea when only sewage sludge was used as substrate, while Methanosarcina and Methanoculleus were the dominant genera when the sewage sludge digester was supplemented with rice straw [92]. The co-digestion of sewage sludge and crude glycerol increased the abundance of the Thermotogae bacterial phylum while the methanogenic population remained unaltered and was mainly represented by the Methanosaeta genus [93]. Karakashev et al. examined the methanogenic population of sewage sludge and animal manure digesters. In the sewage sludge digesters low organic acid and ammonia concentrations were observed with the dominance of the Methanosaeta genus, while manure digesters have been shown to accumulate higher organic acid and ammonia concentrations, and these digesters were dominated by the Methanosarcina genus [34]. The dominance of the Methanosarcina genus at high acetate level can be explained by the lower acetate and ammonia sensitivity of this methanogenic genus [57, 58, 94] compared to the Methanosaeta genus. 5. MICROBES USED FOR BIOAUGMENTATION AND PRETREATMENT Biological pretreatments of various substrates might provide more improvements in the anaerobic biodegradation processes than thermochemical pretreatments [98]. Efficiency of the lignocellulosic biomass degradation is a key factor to provide appropriate substrate for sufficient methane production via anaerobic digestion. However, lignocellulose is a barely degradable material, its breakdown into lignin, cellulose and hemicellulose might be enhanced by various biological pretreatment approaches. The controlled addition of selected hydrogen evolving bacteria (e.g. Enterobacter cloacae and/or Caldicellulosiruptor saccharolyticus) into the biogas fermentor may enhance the biogas production rate by promoting hydrogenotroph methanogenesis through mitigating the hydrogen limitation [99, 100]. Biogas production using protein-rich meat extract as substrate was improved by bioaugmentation, a mixture of three bacterial strains naturally displaying high affinity for protein degradation (Pseudomonas fluorescens, Bacillus coagulans and B. subtilis) was applied to enhance hydrolysis in the anaerobic digestor [18]. It was suggested and demonstrated that specific fungi able to survive anoxic conditions at mesophilic and thermophilic temperature may contribute to feeding hydrolytic bacteria and methanogenic Archaea during the biogas generation process [54]. Thus, beside bacteria some fungi are also able to help the better utilization of biomass and to speed up the anaerobic degradation of fiber-rich substrates in biogas fermentors [101]. Another study described experiments where biogas production was enhanced by 4-22% in pig slurry fermenting reactors supplemented with cellulose-rich energy crop substrate by the addition of a mixture of rumen anaerobic fungi, the Anaeromyces, Piromyces and Orpinomyces strains possess fibrolytic activity [102]. Similarly, improved


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biogas production was observed when paddy straw was pretreated by the Thermoascus aurantiacus MTCC 375 fungus [103]. 6. FURTHER POSSIBILITIES FOR PRODUCTION OF VFA AND HYDROGEN During the AD of organic materials a mixture of acids, gases and other molecules are synthesized. Together with the increasing demand for environmentally friendly energy sources, the processes involved in the biogas production are to be improved to increase efficiency. The versatility of the AD processes offers a number of possibilities for intervention. There are methods for directed engineering of complex communities in order to influence the end-products of the fermentation. To facilitate the shift from biogas production towards biohydrogen development different kinds of pretreatment approaches were developed. The goal is the selective elimination or restriction of hydrogen-consuming microbes, thus achieve efficient inhibition of methanogenesis [104-107]. Some enrichment techniques were introduced for selection of hydrogen-producing bacteria such as heat-shock, acid and base treatment, aeration, freezing and thawing, chloroform, sodium-2-bromoethanesulfonate or 2bromoethanesulfonic acid and iodopropane treatments [105, 106, 108]. Boboescu et al. investigated a combined hydrogen evolving and wastewater treating system based on the selective enrichment of hydrogen-producing consortia by heat pretreatment, acid pretreatment, ultrasonication and a combination of these pretreatments. The successful treatments where significantly increased biohydrogen production was observed - favored the Firmicutes phylum while Proteobacteria phylum was restricted. In addition butyric and acetic acid concentrations were shown to directly correlate with the hydrogen level [109], thus this phenomenon could be also exploited. Another study also confirmed the importance of the members of the Firmicutes phylum in hydrogen production, the highest hydrogen level was achieved by applying heat pretreatment on the complex sludge community, which resulted in the enrichment of the Clostridium genus [110]. Additionally, a complex, statistics-based optimization strategy was developed for biohydrogen production using defined microbial consortium as the starting inoculum and beer brewery wastewater as the fermentation substrate [110]. A further possibility for exploitation of AD is represented by a two-stage system, where algal-bacterial hydrogen evolution is connected with algal-bacterial biomass based biogas production [72]. Another example for the dual utilization of microalgae biomass is the combination of enzymatic pretreatment of microalgae for hydrogen production and fermentation of the microalgal residue for methane generation [111]. Lipid-depleted microalgal residue was used for VFA production, both VFA and methane productions were enhanced by 30-40% by the appropriate pretreatment of microalgal residue [112]. CONCLUSION Biogas production can be coupled to the recycling of various agricultural, municipal, food, animal and forestry wastes. The anaerobic digestion process is relying on a well-


concerted microbial network, the stability of this complex and sensitive community depends on the process parameters including substrate-type and feeding rate (C/N ratio, specific inorganic compounds), operation temperature, mixing and the geometry of the digester. A more detailed insight into the microbial community and especially the differential tolerance levels of community members to various stress factors can help us in developing and maintaining efficient and safe biogas production, thereby contribute to the competitiveness of the production and utilization of biomass-based energy sources. CONFLICT OF INTEREST The author(s) confirm that this article content has no conflict of interest.

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ACKNOWLEDGEMENTS This work was supported by the grants TÁMOP-4.2.2.A11/1/KONV-2012-0007 (supported by the European Union and co-financed by the European Social Fund), and PIAC_13-1-2013-0145 supported by the Hungarian Government, and financed by the Research and Technology Innovation Fund.

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