Kakuk et al. Microb Cell Fact (2021) 20:127 https://doi.org/10.1186/s12934-021-01618-y Microbial Cell Factories

RESEARCH Open Access Early response of methanogenic archaea to ­H2 as evaluated by metagenomics and metatranscriptomics Balázs Kakuk1,2, Roland Wirth2,3, Gergely Maróti3, Márk Szuhaj2, Gábor Rakhely2,4, Krisztián Laczi2, Kornél L. Kovács2,5* and Zoltán Bagi2*

Abstract Background: The molecular machinery of the complex microbiological cell factory of biomethane production is not fully understood. One of the process control elements is the regulatory role of hydrogen (H­ 2). Reduction of carbon dioxide ­(CO2) by ­H2 is rate limiting factor in methanogenesis, but the community intends to keep H­ 2 concentration low in order to maintain the redox balance of the overall system. ­H2 metabolism in methanogens becomes increas- ingly important in the Power-to-Gas renewable energy conversion and storage technologies.

Results: The early response of the mixed mesophilic microbial community to ­H2 gas injection was investigated with the goal of uncovering the frst responses of the microbial community in the CH­ 4 formation and ­CO2 mitiga- tion Power-to-Gas process. The overall microbial composition changes, following a 10 min excessive bubbling of H­ 2 through the reactor, was investigated via metagenome and metatranscriptome sequencing. The overall composi- tion and taxonomic abundance of the biogas producing anaerobic community did not change appreciably 2 hours after the ­H2 treatment, indicating that this time period was too short to display diferences in the proliferation of the members of the microbial community. There was, however, a substantial increase in the expression of genes related to hydrogenotrophic methanogenesis of certain groups of Archaea. As an early response to H­ 2 exposure the activity of the hydrogenotrophic methanogenesis in the genus Methanoculleus was upregulated but the hydrogenotrophic pathway in genus Methanosarcina was downregulated. The RT-qPCR data corroborated the metatranscriptomic

Results: H2 injection also altered the metabolism of a number of microbes belonging in the kingdom Bacteria. Many Bacteria possess the enzyme sets for the Wood-Ljungdahl pathway. These and the homoacetogens are partners for syntrophic community interactions between the distinct kingdoms of Archaea and Bacteria.

Conclusions: External ­H2 regulates the functional activity of certain Bacteria and Archaea. The syntrophic cross-king- dom interactions in ­H2 metabolism are important for the efcient operation of the Power-to-Gas process. Therefore, mixed communities are recommended for the large scale Power-to-Gas process rather than single hydrogenotrophic methanogen strains. Fast and reproducible response from the microbial community can be exploited in turn-of and turn-on of the Power-to-Gas microbial cell factories. Keywords: Hydrogen, Biomethane, Anaerobic digestion, Methanogenesis, Hydrogenotrophic methanogens, Metagenome, Metatranscriptome, Renewable energy, Power-to-Gas

*Correspondence: [email protected]; [email protected] 2 Department of Biotechnology, University of Szeged, Szeged, Hungary Full list of author information is available at the end of the article

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Background and ­CO2 [10]. Te electrons from H­ 2 are frst taken up Anaerobic digestion (AD) of organic wastes and by- by coenzyme F­420, which is embedded in the enzyme products by specialized microbial communities and ­F420-dependent hydrogenase. Te reduced coenzyme F­ 420 the concomitant biogas production is an environmen- is the central electron carrier in methanogenic archaea. tally attractive bioenergy production technology. In the Other hydrogenases from methanogens cannot reduce context of climate change, the generation of biogas as a ­F420 [11]. Methanogenesis from formate involves oxi- renewable energy form has become popular and inten- dation of the substrate to produce CO­ 2 and a reduced sively examined over the last few decades [1]. electron carrier. Te reaction is catalyzed by a formate Biogas provides environmental benefts with regard dehydrogenase [12]. A novel hydrogenase (Ech) was dis- to waste treatment, pollution reduction, production of covered in acetate-grown cells of Methanosarcina bark- CO­ 2-neutral renewable energy and the improvement of eri, which shows sequence homologies to hydrogenases 3 economy of agricultural practices through the recycling and 4 of Escherichia coli and to the CO-induced hydroge- of plant nutrients and replacing artifcial fertilizers [2]. nase from Rhodospirillum rubrum. Te purifed enzyme Biogas can be burnt to produce heat or combusted in from Ms. barkeri catalyzed the ­H2-dependent reduction gas engines for electricity generation and, after purifca- of a 2[4Fe-4S] ferredoxin and is also able to perform the tion, it can be used in any application for which fossil fuel reverse reaction, namely, H­ 2 formation from reduced natural gas is utilized today [3]. AD is applicable to a wide ferredoxin [13]. Some hydrogenases are components of range of waste streams derived from the agro-food indus- the ­H+-translocating system in methanogens [14]. Te try, which is a source of vast amounts of readily degra- efect of ­H2 on the expression of genes coding for hydro- dable organic material composed mainly of complex genases and other methanogenesis genes has not been organic molecules, as well as in liquid or solid communal systematically examined yet. It is astonishing to note the waste treatments. complexity of the molecular machinery, which handles While the main microorganisms and mechanisms the simplest molecule, ­H2. Te exploration of the molec- involved in the methane producing anaerobic micro- ular networks, which afect the expression of these genes bial cell factories are fairly well-known, the regulation could improve our extended knowledge concerning and management of the overall process is far from being molecular redox mechanisms in microbial cell factories. fully understood [4, 5]. Despite the industrial-economic AD is one of the most promising among the various importance of the underlying microbiological events, lit- biomass conversion processes. Te regulatory roles of tle is known about the roles, networking interactions of the ­H2 levels have been recognized as a signifcant ele- the microorganisms and the regulatory mechanisms of ment in the concerted action of the complex microbial the methane production. Terefore, the microbiological community [14, 15]. We demonstrated earlier that by events representing the bottlenecks of the process are the introduction of H­ 2-producing bacteria into a natu- difcult to manage. AD demands the concerted action ral biogas-generating consortium appreciably increased of a complex community of microbes, each member per- the efcacy of biogas production both in batch fermen- forming their special role in the overall degradation pro- tations and in scaled-up continuous AD [14]. One of the cess [6, 7]. In the absence of terminal electron acceptors rate-limiting factors of AD is the actual level of H­ 2 in the such as nitrate, oxygen or sulfate, the methanogenic con- system [16]. Te presence of excessive amounts of ­H2 version of organic matter is an essential feature of many inhibits the activity of the acetogenic bacteria that gener- ecosystems [8]. ate ­H2 in the system, whereas limiting ­H2 levels have an H2 metabolism is one of the most important rate-lim- adverse efect on an important group of methane pro- iting processes in methanogenesis. ­H2-coupled electron ducing Archaea, the hydrogenotrophic methanogens. In transfer has been verifed as an important extracellu- natural ecosystems, a very low partial pressure of ­H2 is lar pathway of sharing reducing equivalents within the maintained, which may be a limiting factor for the meth- anaerobic environment, especially in microbial elec- anogenesis [8, 17]. Te relationship between the ace- trosynthesis systems [9]. togens and methanogens is syntrophic, supported by a H2 conversion is performed at molecular level by the process called interspecies hydrogen transfer or interspe- class of enzymes called hydrogenases. Several hydroge- cies electron fow [18]. We have only incomplete infor- nases have been identifed in methanogenic archaea their mation about the detailed mechanism of interspecies brief overview is appropriate here. Te series of reac- hydrogen transfer [19]. Te actual ­H2 concentration has tions involved in methane ­(CH4) formation from ­H2 and been shown to determine the composition of the metha- carbon-dioxide ­(CO2) are initiated by the formylmetha- nogenic community [20–22]. Te expression of up to 10% nofuran dehydrogenase. Tis enzyme catalyzes the for- of the total proteins in a hydrogenotrophic methanogen mation of N-carboxymethanofuran from methanofuran were reported to change in response to ­H2 limitation Kakuk et al. Microb Cell Fact (2021) 20:127 Page 3 of 18

[23], indicating that the H­ 2 availability is sensed by the most sensitive group of microbes in the AD process [34]. + −1 methanogens and this gas has a major efect on their Te ­NH4 concentration was below 1000 mg L­ during physiology. the whole fermentation process. Te biogas productivity Metagenomic analyses ofer a new toolbox for the of the digesters was also stable: 650 mL­ N biogas alpha- −1 −1 investigation of the complex microbial cell factories. Te cellulose ­g day­ were produced with 53% of CH­ 4 con- reconstruction of the genomes (metagenome assembled tent. Te frst samples for DNA and RNA analysis were genomes: MAGs) of the individual members of a com- taken on day 20 from the stabilized reactors. After sam- plex microenvironment and their subsequent functional pling the digesters were fushed with ­H2 gas from a gas and phylogenetic analysis is termed genome-centric cylinder for 10 min and 2 h later the second sampling was metagenomics [24, 25]. Genome-centric metagenomics carried out. Tis protocol was repeated after 2 months of (referred to as MG hereafter) already yielded valuable reactor operation. insights into the functional organization of biogas reac- Te reactors displayed stable operation during the tors and the microbial cell factories operating within [26, course of the experiment. Te daily biomethane produc- 27]. Additionally, its combination with metatranscrip- tion varied by < 10%. Te H­ 2 injection took place on days tomics (the analysis of the whole microbial community 15 and 71 (blue dotted arrows in Additional fle 1: Figure mRNA in a microenvironment), i.e., genome-centric S1). metatranscriptomics (referred to as MTR, hereafter) Te reactors responded with a sudden increase in daily enables the examination of the gene expression of each ­CH4 evolution by 20–25% at both time points, which individual MAG, has been used for the in-depth analysis lasted for 1–2 days (Additional fle 1: Figure S1). Te CH­ 4 of the process control, regulation and interactions among content of the biogas was 53% throughout the experi- the members of these cell factories. mental period. Afterwards the reactors returned to their In previous approaches the consequences long-term previous biomethane production levels. It is worth not- and/or steady H­ 2 exposure have been investigated [28– ing that the microbial community responded exactly the 31]. Tis study is dedicated to unveil the early response same manner to the H­ 2 spike 2 months apart, which indi- of the anaerobic mixed microbial consortium, with spe- cates the robustness, reproducibility and quick response cial emphasis on methanogens to the presence of ­H2 dis- time by the microbial community. Assuming ­H2 satura- tress. Tis is a realistic scenario in large scale AD plants tion of the liquid phase by the 10 min long ­H2 bubbling, due to local concentration gradients as well as in natural we estimated that more than 95% of the injected ­H2 was environments, e.g. in swamps or rice felds. More impor- converted to CH­ 4 by the community within 16–24 h, tantly, a quick turn-on and turn-of of H­ 2 supply can be although the amount of available dissolved ­H2 decreased expected in the Power-to-Gas technologies, where the rapidly during the second half of the ­H2 consumption fuctuating production of renewable electricity, e.g. by phase. Tis was in line with the observations of Szuhaj photovoltaic or wind power, is coupled with its biological et al. [35], who found in fed-batch ­H2 feeding experi- conversion to biomethane [17, 21]. Te central challenge ments at much lower scale that the injected H­ 2 was com- to be understood is the regulatory role of H­ 2 in CH­ 4 for- pletely consumed in 16–24 h. Te H­ 2 injection apparently mation and the early response by the methanogens and did not alter markedly the cumulative biomethane pro- other ­H2-metabolizing microbes, which regulates and duction curve, which showed a straight line throughout balances the fragile bioenergetic processes in AD. the experiment.

Results Fermentation Genom‑centric metagenome and metatranscriptome analyses A constant value of VOA/TIC is a reliable indicator of a stable mesophilic fermentation process [32]. Each experi- In the early response of the residing microbial consor- ment started with a 20 days long start-up period in order tium to the sudden ­H2 burst at transcriptome level of to adapt the microbial community to the alpha-cellulose metagenome-assembled genomes (MAGs) [36] it was substrate. During this period the average VOA and TIC anticipated that the microbial composition and the rela- −1 tive abundances of species did not change substantially values stabilized at VOA = 1.1 g ­L and the TIC = 14 g −1 within 2 h, i.e., sampling before and after H­ exposure. ­CaCO3 ­L . Because of the relatively low substrate load- 2 ing rate, the VOA/TIC ratios were moderate, which An extensive binning procedure became possible as the + number of metagenomic samples elevated. Terefore allowed balanced operations. Te amount of ­NH4 is also an important indicator of AD process stability [33]. the ­H2 triggered diferences in the gene expression lev- + −1 els could be precisely assessed together with associated Teoretically, levels above 3000 mg NH­ 4 ­L may have a negative efect on the methanogenic archaea, which is the alterations in cell physiology. Kakuk et al. Microb Cell Fact (2021) 20:127 Page 4 of 18

Te extensive binning procedure on the co-assembled previous studies [8, 26]. Tis can be attributed to their contigs and read mapping, employing the three metagen- diverse capability in polysaccharide and oligosaccharide omic binners and the DAS tool, yielded 84 bins. Out of degradation, which is the frst step in the AD of complex these, 16 were high, 49 were medium and 19 were low organic substrates [37]. quality, according to the MIMAG initiative [36]. 73 bins Te second well-represented phylum was Bacterioi- harbored enough single copy marker genes (SCG) for detes (12 bins), all of them belonged in the order Bac- the phylogenetic tree building (center part in Fig. 1)—the teroidales. Most Bacterioidetes produce succinic acid, phylogenetic relationship of the remaining 11 bins could acetic acid, and in some cases propionic acid, these mol- not be determined probably because of the low quality of ecules fuel the acetotrophic methanogenesis. In addition, the metagenomes. representatives of the phyla Synergistota, Spirochaetes, Te taxonomic assignment of the 84 bins (or MAGs as Verrucomicrobia, Cloacimonadota, Fibrobacterota, Cal- both of these synonymous expressions will be used in this datribacteria, and Chlorofexota were identifed (Addi- discussion) resulted in seven Archaea, 61 Bacteria and 16 tional fle 5: Table S1). Te overall microbial landscape unclassifed bins (details are compiled in Additional fle 5: is in line with previous studies [8, 26]. A typical micro- Table S1). Archaea represented about 10% of the micro- bial community fourished in our biogas digesters, which biome. Within the domain Bacteria, most bins (34) were indicated that the synthetic medium containing only associated with the phylum Firmicutes. Te dominance cellulose as a carbon source proved to be a good model of Firmicutes in biogas reactors is in accordance with system for the metatranscriptomic investigations [38].

Fig. 1 A Anvi’o plot of binning results (from innermost to outermost): phylogenetic relationship of bins according to phylophlyan3; completion and redundancy of the bins, according to single-copy marker gene (SCG) content; taxonomic Class and Genus assignment for the bins and relative abundance of bins in samples. The list of Classes at the bottom part indicates the color code and the number of bins in the Classes. B depicts the relative abundance of Archaeal Classes (the summary of bins in the Classes) Kakuk et al. Microb Cell Fact (2021) 20:127 Page 5 of 18

Tis has been corroborated in a comparison of our 84 addition). Tis demonstrates a rapid response to the bins with the MAGs library compiled in Bio-Gas Micro- appearance of excess ­H2. biome database (https://​micro​bial-​genom​es.​org) (Addi- Te elevation of the total number of transcribed tional fle 5: Table S1) [39]. Te comparison of the coding Archaea genes (H2_MTR samples) was mainly attrib- sequences, i.e., fasta fles, revealed the counterparts of 70 uted to representatives of the genus Methanobacterium of our 84 MAGs in the Bio-Gas Microbiome database, 56 (bins 35 and 51), which increased from 4.33 to 17.39% of the MAGs had more than 95% similarity. Te remain- ­(log2FC = 2.84) of all bins’ abundance. Methanobacte- ing bins contained medium or low quality metagenomes, ria are hydrogenotrophic methanogens. Te second which could be the main reason for not fnding more major contributor to Archaea transcripts was the order matches. Two high quality bins (bin_1—Herbivorax sac- Methanomicrobiales, from 2.69 to 7.03% (log­ 2FC = 2.16). cincola and bin_35—Methanobacterium sp.) was appar- Te genera Methanoculleus and Methanosarcina both ently not represented among the nearly 1600 species belong in this order. Te three bins of the genus Metha- identifed in Bio-Gas Microbiome. noculleus showed elevated overall abundance, the ­log2FC A comparison of the DNA-based omics data clearly values of bin_6, bin_60 and bin_66 were 3.64, 2.37 and indicated that the community compositions were very 2.18, respectively. Te increase upon ­H2 exposure was similar in all four samples (Fig. 2), respectively (Addi- the most apparent in the case of bin_6. Methanocul- tional fle 5: Table S1). Te overall Archaea gene abun- leus_bourgensis, whose proportion increased from 1.65 dance, i.e., sum of read counts, was 18.49 ± 2.04% in to 10.66%. Remarkably, the genus Methanosarcina efec- N2-MG or H2-MG samples (each DNA-based). Tis tively ceased to express genes to near zero upon ­H2 dis- observation corroborates that (i) all reactors that worked pensation. Methanosarcina are known to possess genes under the same conditions maintained the same micro- coding for all three methanogenic pathways, i.e., hydrog- bial community; (ii) as expected, the microbial communi- enotrophic, acetotrophic and methylotrophic methano- ties did not change perceptibly within 2 h; and (iii) the genesis [8, 16]. Members of the genus Methanoculleus observations were highly reproducible after 2 months. are solely hydrogenotrophic methanogens. H­ 2 exposure In contrast, the mRNA-based metatranscriptome analy- apparently turns on the activity of the hydrogenotrophic sis showed striking changes in the transcriptome-based methanogenesis in both Methanoculleus and Methano- community composition when ­H2 was ofered to the bacterium but turns of the hydrogenotrophic pathway in reactors’ microbial community. Te N2_MTR sam- Methanosarcina. ples (RNA-based, before H­ 2 addition) showed a simi- lar total Archaea abundance to that of the MG samples: Metatranscriptomic pathways analysis 18.99 ± 11.64%, but this was elevated to 36.53 ± 3.74% A community-level pathway enrichment analysis was in the case of H2_MTR samples (RNA-based, after H­ 2 performed to examine the overall metatranscriptomic

Fig. 2 PCA biplot of the rlog-transformed (regularized-logarithm transformation) total gene expressions, i.e., copy number in the MG sample, of each MAG in each sample Kakuk et al. Microb Cell Fact (2021) 20:127 Page 6 of 18

changes that occurred as a result of the ­H2-addition. therefore we combined them as a group of “unbinned” Te contig assembly and ORF prediction/annotation genes. workfow yielded 219,353 KEGG Orthology (KO) anno- tated ORFs. Out of these 98,791 ORFs were binned in Changes in the expression levels in methanogenesis genes the refned MAGs. Te remaining 120,562 ORFs were Te enrichment analysis revealed that the CH­ 4 metabo- used for the community-level pathway analysis. Te lism was the most afected, hence the contribution of changes in the expression levels of the genes involved each individual MAGs was examined next to gain a in the various methanogenesis related metabolic path- deeper insight to the molecular mechanism. An over- ways and modules were examined according to KEGG all of 103 genes of the 8 Archaea MAGs from this path- annotation. Te results indicated that the methano- way were down-regulated ­(log2FC lower than − 2), and genesis pathway was primarily afected as the result of 37 that were up-regulated ­(log2FC higher than 2), but of ­H2 injection (Fig. 3). Te upregulation of diferentially these only 61 were found to be signifcantly diferentially expressed (DE) genes was the highest in this pathway expressed based on the p-value threshold of 0.05. MAGs (48) and in the associated modules. It is noteworthy harboring more than fve KEGG map00680 pathway that some other carbon metabolism associated path- genes were plotted in Fig. 4. ways were also afected, such as Glycolysis/Gluconeo- Te two MAGs identifed as belonging in the genus genesis and Propanoate metabolism, which suggest that Methanobacterium (bin_35 and bin_51) and Methanoc- acetogenic and acetate utilizing microbes were also ulleus (bin_6, bin_60 and bin_66) showed a very similar afected by the specifcally altered environment. ­H2 is response (Fig. 4), many of their map00680 genes were known to inhibit acetogenic microbes [40], thus their expressed at log­ 2FC higher than 2, i.e., four-times higher response to the H­ 2 addition is not surprising. Te RNA expression. Two additional Methanoculleus MAGs polymerase pathway also changed signifcantly, this was (bin_60 and bin_66), a low and a medium quality MAG due to triggered transcription machinery as a response according to CheckM, were identifed but not presented to the altered environment. in Fig. 4. Tis implies that several Methanoculleus strains Despite the binning eforts, many KEGG annotated actively participate in the Power-to-Gas (P2G) reaction. genes remained unbinned (Additional fle 2: Figure S2). Te expression level of numerous genes increased Omitting these from the downstream analysis would shortly after ­H2 injection in the hydrogenotrophic have distorted the pathway and statistical analyses, strains, which indicated that several metabolic path- ways responded to the increased H­ 2 concentrations.

Fig. 3 A Results of KEGG Module enrichment analysis (left), and B KEGG Pathway (right). The pathways, which were signifcantly diferent between N2_MTR and H2_MTR samples are presented. X-axis indicates the number of KEGG IDs found as signifcantly diferent in the given pathway (listed along the Y axis). P-adjust stands for corrected p-values Kakuk et al. Microb Cell Fact (2021) 20:127 Page 7 of 18

Fig. 4 Violin plot of genes (small dots) involved in the methanogenesis KEGG pathway (map00680) in each bin (arranged on the X-axis) and the unbinned gene collection. Only bins, which contain at least are plotted. Filling colors indicate taxonomy at Class level. Each dot represents a KEGG 5

methanogenesis genes orthologue (KO) in the respective bin. Colors of the dots indicate the p-value of the log­ 2FC diference between N2_MTR and H2_MTR samples. Horizontal dashed red lines mark the ­log2FC thresholds for signifcantly diferent KOs (respective p-value < 0.05)

Te ­log2FC values of the genes ENO (phosphopyruvate decarboxylase, EC 4.1.1.25), ADC (aspartate 1-decar- hydratase, EC 4.2.1.11), COF (7,8-didemethyl-8-hydroxy- boxylase, EC 4.1.1.11), FMD/FWD (formylmethanofuran 5-deazaribofavin synthase, EC 4.3.1.32), and COM (sul- dehydrogenase, EC 1.2.99.5), AKS (methanogen homoci- fopyruvate decarboxylase, EC 4.1.1.79) were the largest trate synthase, EC 2.3.3.14 2.3.3), COM increased. Tese in M. bourgensis i.e., 5.06, 4.2 and 1.25, respectively. Te enzymes also play an important role in the hydrogeno- ENO enzyme takes part in the biosynthesis of the Coen- troph methanogenesis pathway (Additional fle 3: Figure zyme B, which is an essential molecule in the fnal step S3). Te MFN and ADC enzymes are normally involved of the methanogenesis. Te COF enzymes are responsi- in the methanofuran biosynthesis pathway, when they ble for the synthesis of the other important coenzyme, catalyze the l-tyrosine to tyramine reaction. Te FMD/ Coenzyme ­F420. Te COM enzymes catalyze the 3-sul- FWD redox enzyme complex contains a molybdopterin fopyruvate to 2-sulfoacetaldehyde reaction, which is an cofactor and numerous [4Fe-4S] clusters in order to cata- intermediate step in the synthesis of the third impor- lyze the reversible reaction the formyl-methanofuran tant coenzyme, Coenzyme-M [38]. Tese results clearly synthesis from methanofuran, which is an important suggested that the cells increased the synthesis of all methanogenesis step in ­CO2 conversion and the oxida- coenzymes, which were involved in methanogenesis to tion of coenzyme-M to ­CO2. Te reaction is endergonic support the quick conversion of ­H2 and ­CO2 to ­CH4. and is driven by coupling the soluble CoB-CoM het- In the MAGs belonging Methanobacterium strains, erodisulfde reductase via electron bifurcation. Te AKS the expression level of the enzymes MFN (tyrosine enzyme also takes part in the synthesis of Coenzyme-B. Kakuk et al. Microb Cell Fact (2021) 20:127 Page 8 of 18

Overall, the results signifed that the hydrogeno- EC 4.2.1.147) signifcantly decreased. Te ACS enzyme trophic methanogenic cells activated a large number is responsible for the conversion of acetate to acetyl- of the key enzymes in the methanogenesis pathway to CoA, which is a typical step in the acetotrophic metha- consume more efectively the ­H2 from the environment. nogenesis pathway. Te next enzyme, FAE generates It is noteworthy that the genes of the MCR enzymes 5,10-methylene tetrahydromethanopterin (5,10-Methyl- (methyl-coenzyme M reductase, EC 2.48.4.1.) showed ene-THMPT) from formaldehyde, an important interme- lower expression in all hydrogenotrophic bins. Te MCR diate of methanogenesis. enzymes (methyl-coenzyme M reductase) catalyze the Te substantial decrease in the transcriptional response fnal step of the methanogenesis (Additional fle 3: Fig- of M. honorobensis to H­ 2 injection corroborated that this ure S3). One of the possible considerations explaining strain is unable to utilize H­ 2 and signaled an active inhibi- this observation could have been that 2 h was not enough tory role of H­ 2 on acetotrophic methanogenesis. Tis for redirecting this section of methanogenesis pathways. implicates a hitherto unrecognized tight regulatory role If the local substrate availability did not increase signif- of ­H2 on diverse pathways coupled to methanogenesis cantly, the cells did not need to increase the transcrip- (Fig. 4). tional activity of the MCR enzymes (Additional fle 4: Figure S4). qPCR validation of the transcriptomic data Almost all genes in Methanosarcina honoroben- Eleven genes were selected for testing the metatranscrip- sis showed decreased expression in the presence of H­ 2 tomic data by Real-Time quantitative polymerase chain (Fig. 4). Tis strain has been described as acetotrophic, reaction (RT-qPCR). Te genes were selected to cover a which also grew on methanol, dimethylamine, trimeth- broad range of genes displaying various gene expression ylamine, dimethylsulfde and acetate but not on mono- levels and signifcant (p < 0.05) expression change accord- methylamine, ­H2/CO2, formate, 2-propanol, 2-butanol ing to the metatranscriptomic data. Genes participat- or cyclopentanol [41]. Te expression levels of MCR, ing in methanogenesis as well as others involved in cell ACS (acetyl-CoA decarbonylase/synthase, EC 3.1.2.1) metabolism were included. Based on the ­log2FC values and FAE (5,6,7,8-tetrahydromethanopterin hydro-lyase, (Fig. 5) most of the examined genes showed consistent

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-14 cdhC mcrB frhA mcrC ppdK oppA gapA enorplJmfnFcogF

Fig. 5 Comparison of metatranscriptomic and qPCR results of selected genes afected by early H­ 2 treatment. The threshold value of signifcant gene expression was set to fold change 2 in gene expression ­(log FC 1). The selected genes are from bin_1: ppdK (pyruvate, phosphate dikinase); 2 = bin_6: mfnF ((4-{4-[2-(gamma-L-glutamylamino)ethyl]phenoxymethyl}furan-2-yl)methanamine synthase), cofG (7,8-didemethyl-8-hydroxy-5-deazari bofavin synthase), rplJ (ribosomal protein L10), eno (enolase); bin_27: cdhC (acetyl-CoA decarboxylase/synthase), mcrB (methyl-CoM reductase beta

subunit), frhA (Coenzyme F­ 420 hydrogenase subunit alpha); bin_35: mcrC (methyl-CoM reductase gamma subunit); bin_59: gapA (glyceraldehyde 3-phosphate dehydrogenase), oppA (peptide nickel transport system substrate binding protein). Blue columns: metatranscriptomic expression, red columns: qPCR results Kakuk et al. Microb Cell Fact (2021) 20:127 Page 9 of 18

results with the metatranscriptomic data, although in Teir overall gene expression did increase (log­ 2FC of several cases their fold change was slightly lower than 2.19 and 2.37, respectively), thus the observed diferences derived from the metatranscriptomic analysis. Te might as well indicate a slower response by bin_60 and slight deviation could have been the result of the difer- bin_66 and perhaps further ­H2 addition would have trig- ences between the distinct evaluation methods. Te fold gered a response more similar to that of the abundant change (FC) calculation in the qPCR experiments is done M bourgensis (bin_6). If future experiments corroborate on the traditional way ­(log2FC = ­log2(treated/control). this situation, than the observation may indicate the time Te DESeq2 program employed in metatranscriptomics resolution limit of ­H2 triggered transcription and meta- uses a series of mathematical transformations to normal- bolic changes. It seems that the whole RNA machinery ize the log­ 2FC values [71]. Despite the minor diferences, must be altered for responding to a signifcant change the RT-qPCR data clearly corroborated the MTR results. in the environment. Indeed, almost all genes (including Despite the minor diferences, the RT-qPCR data clearly the subunits of RNA polymerase for instance) from these corroborated the MTR results. pathways were highly expressed in the Methanomicro- Tree genes may deserve special attention. Te cofG bia and Methanobacteria bins, and 64% of them with a gene had a more than 4 times smaller gene expression ­log2FC of 2 or higher (p-value of 0.05 or lower). Te early fold change in the qPCR experiment and it apparently response to H­ 2 injection by Methanosarcina horonoben- diminished into the “unchanged”. category. Similarly, sis (bin_27) was quite the opposite as the expression of the FC of mfnF also droppped threefold although it still all investigated KEGG orthologs and metabolic pathways remained “upregulated” according to qPCR. It should be were hindered signifcantly, i.e., up to 33% (Fig. 6). noted that DESeq2 attempts to flter the biologically rele- Other carbon metabolism-related pathways that vant changes from the background noise thus in the case showed an overall signifcant diference in the pathway of cofG and mfnF the DESeq2 algorithm overestimated enrichment analysis were “carbon fxation” pathways the FC values. Te oppA decreased from “unchanged” to in prokaryotes and “glycolysis/gluconeogenesis”, which “downregulated”. Tis gene is a substrate-binding protein, showed a similar pattern. For example the folD gene of responsible for the transport of various oligopeptides the reductive acetyl-CoA pathway (Wood-Ljungdahl across the cell membrane [42]. pathway) was transcribed vigorously in bin_6 (M. bour- gensis) ­(log2FC = 3.7). Te relative enrichment of Metha- Interactions between methanogenesis and other nogenesis, acetate to methane was overall the highest in metabolic processes this bin (mean log­ 2FC = 3.55), this can be linked to the In addition to the methanogenesis pathways in the elevated acetotrophic methanogenesis, as there were no Archeal bins, we identifed nine additional pathways that other major diference between the expression change in were expressed diferently as the early response of the these pathways. Interestingly though, the Methanogen- microbiota to H­ 2 injection (Fig. 3B). Figure 6 presents the esis, CO2 to methane module did not increase drastically Archaea and Bacteria bins that indicate substantial up- or (nor did the methylotrophic module), with the excep- down regulation of several KEGG pathways. It is clear tion of a handful of genes showing ­log2FC higher than that ­H2 addition rapidly caused gene expression changes 2, including methenyltetrahydromethanopterin cyclo- in the Archaea, i.e., bin_6, bin_27, bin_35 and bin_51, hydrolase gene in bin_6 and bin_35 (log­ 2FC = 2.56 and since the Ribosome, RNA polymerase and Methanogen- 3.49, respectively), and some others with smaller but still esis pathways were altered mainly in these bins. signifcant diferences, including the ­F420-non-reducing In the case of Archaea, one Methanoculleus bin (bin_6) hydrogenase iron-sulfur subunit gene of bin_6 and the two Methanobacteria bins (bin_35 and bin_51) ­(log2FC = 1.32, p-value = 0.04). responded with elevated gene expression in all pathways, while the Methanosarcina (bin_27) and Iainarchaeia Changes in gene expression levels in bacterial bins (bin_18) responded with a substantial and general loss of Some genes involved in, or related to elements of the transcripts, i.e., biological activity, in them. methanogenesis pathway could be found in bacterial Interestingly, the three Methanoculleus bins responded bins as well, e.g. Herbivorax saccincola, Ruminiclostrid- diferently to the H­ 2 injection. Apparently, the entire met- ium sp001512505, two unknown Limnochorida and a abolic activity, including all KEGG orthologs, were tuned Mahellia MAG. However, when inspecting the change up in bin_6 (classifed as M. bourgensis), whereas only of the methanogenesis-related KEGG orthologs in the Ribosomal activity, RNA transport and Lysine biosyn- MAGs, it became clear that these genes showed sig- thesis was strongly upregulated in bin_60 and hardly any nifcant diference only in a few cases, i.e., their ­log2FC change in metabolic activity took place in bin_66 repre- values were spread between the threshold lines that indi- senting presumably a separate strain of M. bourgensis. cated signifcance. Consequently, they were involved in Kakuk et al. Microb Cell Fact (2021) 20:127 Page 10 of 18

Fig. 6 Heatmap of signifcantly various KEGG Pathways in bins that harbor a total of at least 10 genes in any of these pathways or modules. Top

panel shows Archaea, while the bottom panel shows Bacteria bins. Filling colors are according to the log­ 2FC of all the genes in that pathway/ module in the given bin. Violin plots represent ­log2FC values of every gene participating in the given pathway/module Kakuk et al. Microb Cell Fact (2021) 20:127 Page 11 of 18

the overall methanogenesis, and closely related metabolic microbes tended to increase their activity in a ­H2-fed sys- pathways (which are included in the KEGG map00680 tems [45]. pathway), but they did not respond to the H­ 2 provision change. Tis was substantially diferent from the behav- Discussion ior of the Archaea MAGs, which clearly expressed their Te interest in converting the fossil fuel based energy genes diferently as a respond to ­H2 injection. market to renewable energy carriers is growing world- In the case of Bacteria, the RNA-machinery pathways wide. Tis is a very positive trend to avoid threatening (ko03010) showed an overall decrease in gene expression, global climate change and associated environmental with the exception of bin_40 (Treponema brennaborense), catastrophes. Te overwhelming majority of renewable bin_8 (Fermentimonas massiliensis), bin_11 (UBA3941_ energy production approaches employ photovoltaics and sp002385665) and bin_7 (Unknown Fermentimonas). wind power today. Both of these technologies gener- Tese MAGs had low abundance, though they showed an ate electricity in an intermittent fashion. Te power dis- increase in the MTR samples. Te related pathways seem tribution grids are designed to harmonize electricity to be up-regulated in bin_40 and in bin_11 (mapped in production and consumption continuously, these grids class Mahellia, order Caldicoprobacterales). Most of the can operate in a fuctuating mode only with substantial small and large ribosomal subunits showed log­ 2FC of 2 or energy loss. Hence, technologies to balance the fuctua- higher. Another member of the family Treponemataceae tions are urgently needed. A very promising solution to (bin_28 Spiro-10 sp001604405) showed a clear downreg- this problem is ofered by the fexible biogas technol- ulation in all discussed pathways. ogy [46]. Biogas plants have controllable energy output In AD, Treponema behave like homoacetogenes, they to bufer the fuctuations in renewable electricity pro- consume ­H2 and CO­ 2 to produce acetate, hence they duction. Moreover, with a coupled technology called may compete with hydrogenotrophic methanogens [43], Power-to-Gas (P2G), Power-to-Methane (P2CH­ 4) or although not very efciently [17]. We identifed only Power-to-Biomethane ­(P2bioCH4), biogas reactors two methanogenesis related genes in bin_28 and bin_40 can efciently convert the temporarily surplus renew- (formate-tetrahydrofolate ligase and methylenetetrahy- able electricity to biomethane ­(bioCH4). Clean ­bioCH4 is drofolate reductase NADPH), bin_40 showed an overall chemically indistinguishable from the fossil natural gas, activity increase ­(log2FC = 2.216), indicating either that therefore it can be stored and transported efciently and this pathway would become more active at a later time- inexpensively in the natural gas grids. Te biotechnologi- point, or these bacteria utilize alternative catabolic activi- cal route to P2bioCH­ 4 requires specifc microbes capa- ties. In a relevant observation Treponema abundance ble of converting ­H2 + ­CO2 to ­CH4 in a carbon neutral increased in digesters spiked with H­ 2 [44], although after or negative carbon footprint process. Te key potential 90 h the signs of H­ 2 stress were noted in the digester. player microbes are methanogenic Archaea, a group of Essential genes of the Wood-Ljungdahl (WL) path- rare and obscure obligate anaerobic microbes. Te pre- way were apparently not expressed in bacterial bins in cise biochemical events leading to ­CH4 formation are a recent study [30]. In contrast, in the present work we only understood in a broad sense today. identifed several bins harboring these genes, includ- Understanding of the molecular regulation and control ing bin_7 (Unknown Fermentimonas), bin_8 (Fermenti- of the highly complex cell factory pathways of micro- monas massiliensis) and bin_20 (DTU074 sp002385885, bial communities carrying out AD, is a challenge for although all of them showed low abundance (~ 0.3–1%). both basic and applied research. In this study we aimed Interestingly, bin_20 exhibited an overall decrease, but at mapping the early response of the entire community, the expression of its WL pathway genes increased. Tis with particular attention to methanogens, a scenario fre- can be attributed to the elevation of the transcriptional quently envisaged and expected in the P2bioCH­ 4 indus- activity of only two genes, the fhs gene (formate-tetrahy- try [35]. drofolate ligase) and the folD gene (methylene-tetrahy- In a recent study thermophilic biogas reactors were drofolate oxidase), which are important in WL pathway fed with ­H2, and after 18 h and 36 days MTR analyses ­(log2FC = 6.31 and 3.14, respectively). Tis response to were carried out to unveil the involvement of the indi- ­H2 is thus the opposite to that of bin_40, suggesting that vidual MAGs in the global microbiome functions [30]. as acetogenic methanogenesis increased, it might have Te results revealed a multi-trophic role to Methano- tried to compete with the Archaea for acetate. Te other sarcina thermophila, although the hydrogenotrophic two potential homoacetogens, which increased their Methanoculleus thermophilus prevailed as the dominant transcriptional activity ­(log2FC = 1.40 and 2.56, respec- Archaea species in terms of relative gene expressions, at tively), apparently included the fhs and folD genes as well. the expense of M. thermophila. Some community mem- It was also demonstrated earlier that homoacetogenic bers emerged in the later stages of methanogenesis were Kakuk et al. Microb Cell Fact (2021) 20:127 Page 12 of 18

below the detection limit in the starting sample, i.e., community with alpha-cellulose substrate continued Methanobacteriaceae spp. as before. We interpret that this behavior indicated the Te changes in the metatranscriptome of an AD com- presence of sufcient hydrogenotrophic methanogenesis munity triggered by H­ 2 addition were studied before in activity in the “raw” biogas community, i.e., in the large thermophilic reactors, but the short-term response at scale biogas plant efuent, to perform the P2bioCH­ 4 con- mRNA-level to ­H2 was not Mapping the early response of version at full speed. In other words, the diverse, “raw” the microbial community via genome-centric metatran- anaerobic communities can be used in switching on scriptomics is therefore important for understanding ­P2bioCH4 without a lengthy adaptation and enrichment and managing the turn-on and turn-of steps of the P2bi- period. Tis allows a quick and efcient turn-on and oCH4 process. Genome-centric MG linked MTR inves- turn-of response by the mixed methanogenic commu- tigations enables the distinction of the activity of each nity. Te microbial community composition rearranges individual MAG and the identifcation of the key and upon long-term exposure to ­H2 (and ­CO2), particularly most sensitive members of the community. when no other organic substrate is available for the com- In the examination of the initial response of the com- munity [49]. Te vigorous ­P2bioCH4 activity returned plex AD microbial community and assessment of the frst to normal biogas production as soon as the dissolved up- or down-regulated genes by the ­H2 injection a custom ­H2 diminished, but the community was ready to adjust bioinformatics workfow was employed for the down- its biochemistry to instant H­ 2 conversion and P2bioCH­ 4 stream analysis of the genes and pathways of each MAGs. repeatedly. Tis involved primarily the SqueezeMeta [47] pipeline, Te metatranscriptomic responses to the ­H2 treat- which can jointly analyze MG and MTR sequencing data. ments separated 2 months apart were very similar to In addition a more extensive binning procedure, a subse- each other indicating that the metabolic pathways were quent pathway enrichment analysis and statistical evalu- fexibly restored after switching on and of the ­P2bioCH4 ation of the log2FC of the gene expressions of the MAGs operational mode. A thorough analysis of the difer- between the H­ 2 and N­ 2 MTR samples were carried out. In ences between the ­H2-treated metatranscriptomes and order to gain higher statistical confdence in the results, corresponding controls identifed the early events in the we used biological duplicates separated by a 2-month microbial communities brought about by ­H2. interval in CSTR AD reactors. Te following important H2 (and dissolved CO­ 2) is readily converted to CH­ 4 by considerations were also adopted: (1) Te metagenomes both direct (hydrogenotrophic) and indirect (homoace- of the samples separated by just a 2-h time-window, i.e., togenesis and subsequent acetotrophic) methanogenesis. before ­H2 addition and 2 h later, to make sure an unal- Our results suggest that the second route is unlikely the tered microbial community. (2) qPCR tests of a handful predominant one in the early response of the microbial of selected genes validated the results from the metatran- community to ­H2 addition at least under mesophilic scriptomics pipeline. conditions, since the acetotrophic pathways reacted First we established that the composition of the micro- sluggishly, while the gene transcription of the hydrogen- bial community did not change signifcantly (Fig. 2), otrophic route increased dramatically after a very short therefore the diferent reproduction rates of the vari- period of extensive H­ 2 feeding (Figs. 4, 5). Tis predicts ous taxa did not disturb the picture of early functional that under the P2bioCH­ 4 operation conditions the physi- response. Up-to-date metagenomic and metatran- ological readiness of the hydrogenotrophic methanogen scriptomic methods were employed to determine the members of the community will determine the reactor biochemical events taking place as the result of ­H2 response rate upon switch-on of the ­H2 addition. administration. Te reproducibility of the system was Interestingly, this study revealed an extensive reac- tested by repeated ­H2 injections 2 months apart. Practi- tion to the transient ­H2 stress within the Bacteria cally identical results were obtained (Fig. 2). community as well although Bacteria cannot directly Four metagenome (MG) sequencing datasets were generate ­CH4 from ­H2 as many Archaea can. Some of combined to assemble a fairly large number of bins (84 these Bacteria possess the complete or partial enzyme bins: seven Archaea, 61 Bacteria and 16 unclassifed sets for the Wood-Ljungdahl pathway. Tese and the bins). Te non-H2-adapted, “raw” biogas forming micro- homoacetogens are probably the best candidates for bial community was essentially the same in structure and syntrophic community interactions between members composition as the ones sampled previously from the of the distinct kingdoms of Archaea and Bacteria. Te same industrial biogas plant fed with manure and maize details of these interactions in the complex anaero- silage [48, 49]. Tis community switched to ­H2 consump- bic environment and consequences to stabilize robust tion and biomethane production almost immediately and vigorous P2bioCH­ 4 microbial communities dur- following ­H2 injection, although feeding of the entire ing long term P2G operation should be the subjects of Kakuk et al. Microb Cell Fact (2021) 20:127 Page 13 of 18

future studies. Nevertheless, the transcriptional activity and ­CO2 depletion in the selection of hydrogenotrophic of the primary potential syntrophic bacterial partners methanogens predominating the P2bioCH­ 4 community. (bin_1 (Herbivorax saccincola), bin_68 (Ruminococcus In a thorough in situ syngas bioconversion study running sp.), and unidentifed bins_59, _61, _63, see Fig. 4) did two UASB reactors in sequence at mesophilic temperature not change substantially upon ­H2 exposure. Tis may [54], observed the predominance of the genus Methanotrix mean that either there is enough syntrophic capacity (formerly Methanosaeta). Te reactors were continuously already in the non-adapted, “raw” community to sup- fed with varying glucose loads. Methanotrix species appar- port increased hydrogenotrophic methanogen activity ently cannot carry out hydrogenotrophic methanogenesis, or the syntrophic partners respond slowly to the sud- therefore their predominance under these conditions can den ­H2 burst appearing in the microbial environment. be rationalized by the combined efects of glucose and Te development of a stable P2CH4 community CO-rich syngas addition via carboxydotrophic methano- strongly depends on environmental conditions and on genesis [55]. In addition, the recently recognized capability the starter microbial community composition. Various of Methanotrix species to carry out direct electron transfer reactor designs, operational parameters and inocula are (DIET) to drive ­CO2 reduction could facilitate the Metha- being tested making rigorous comparison of the results notrix predominance [56, 57]. difcult. Taking into account the recent results and considera- In a brief review to summarize in situ biogas upgrad- tions, the development of a stable P2bioCH4 mixed AD ing Zhang et al. [50], pointed out the predominant roles community depends on a number of important param- of the genus Methanoculleus under mesophilic condi- eters, such as the origin of inoculum, ­H2 supply and its tions and the thermophilic genus Methanothermobac- fuctuation, composition of added growth supporting sub- − ter at elevated temperatures. Te species M. bourgensis strates, the dissolved ­CO2/HCO3 concentration, temper- (bin_6) was identifed to play an important role in vari- ature and reactor confguration. In the future the extension ous biogas reactor systems. Methanoculleus species of these studies should be carried out, i.e., mapping the grow on CO­ 2 and H­ 2 and hence perform the hydrog- molecular events after longer exposure of the microbial enotrophic pathway for ­CH4 synthesis [51]. In line with cell factory and linking the metagenomic approach to these conclusions, the mesophilic AD methanogenic more detailed transcriptomic and proteomic studies. community of palm oil mill efuent with eventual addi- tion of formate was predominated by members of the Conclusion genus Methanoculleus [52]. Various inocula were com- In this study the early response of the mixed biogas pared for biomethane production at mesophilic condi- microbial community to the presence of saturat- tions in batch fermentations. It was concluded that the ing amount of ­H2 was examined. Metagenomic and abundance and activity of the genera Methanosarcina metatranscriptomic analyses have been carried out to and Methanoculleus played key roles in methanogen- determine the changes of the expression levels of the esis of added ­H2 [52], while the authors also noted the various genes related to methanogenesis. Te results regulatory role of the available ­CO2/bicarbonate in the indicated that the microbial community responded production of CH­ 4 and/or volatile fatty acids. instantaneously to the presence of ­H2. Te activity of In a recent work [53] the microbial community acetotrophs reduced signifcantly. In addition, the meta- changes were followed under various operational con- bolic activity of numerous bacterial strains changed ditions starting from two distinct inocula, i.e., waste- substantially as a response to ­H2. Clearly, the excess ­H2 water (WW) sludge and plug-fow reactor operated does not only afect the methanogenesis pathways in with agricultural waste (PF). Te study pointed out the Archaea, rather the microbial community respond with importance of the history of the inoculum communi- a multifarious gene expression profle change, which ties. In the WW inoculated batch reactors the metha- seems to be rather selective. Tis indicates a more global nogenic genus Methanobacterium and Methanothrix regulatory role of ­H2 in the life of anaerobic communi- predominated and upon ­H2 feeding the genus Methano- ties than assumed earlier. Te syntrophic interactions bacterium took over. In the plug-fow reactor, supplied contribute to the stability and metabolic activity of the with animal manure and ensilaged plant biomass, the hydrogenotrophic methanogens. Tis, together with the initial abundance of genus Methanothrix diminished non-sterile operation conditions and continuous supply and the methanogenic gap was flled in by members of inexpensive catalyst, underlines the benefts of using of the genera Methanobacterium and Methanoculleus. mixed communities in the P2bioCH­ 4 process instead of Tis study corroborated the previous observations [19, pure hydrogenotrophic cultures [35, 58, 59]. 30] concerning the regulatory role of ­H2 concentration Kakuk et al. Microb Cell Fact (2021) 20:127 Page 14 of 18

Materials and methods acid/bufer capacity ratio were constant. 2 mL of reac- Anaerobic fermentation tor content was withdrawn and total RNA for transcrip- Anaerobic digestions (AD) were carried out in continu- tome analysis (sample names: N2-MTR) and DNA for ously stirred tank reactors [60]. Te fermentation vol- metagenome analysis (sample names: N2_MG) were ume was 5000 mL, leaving a headspace of 2000 mL. Te isolated immediately after sampling. Tan the digesters apparatus can be fed continuously or intermittently via a were fushed with pure ­H2 gas for 10 min on day 15 and piston type delivery system, the fermentation efuent is 71. ­H2 was injected directly from a pure ­H2 gas cylinder removed through an air-tight overfow. Te reactors are through custom made nozzles (10 pieces) having 0.2 mm equipped with a spiral strip mixing device driven by an holes. Te applied gas pressure was 2 bar, the gas purity electronic engine. An electronically heated jacket sur- was 99.999%. 2 h after fushing the reactors with ­H2 sam- rounds the cylindrical stainless steel body, electrodes for ples were also taken for RNA (sample names: H2_MTR) the measurement of pH and redox potential are inserted and DNA (sample names: H2_MG) isolation. Te head- through the reactor wall, in sealed sockets. Te device space was then replaced with N­ 2 and the reactors were can be drained at the bottom. Te evolved gas leaves the run under the same conditions as before. After 2 months reactor through the top plate, where ports for gas sam- the whole ­H2 treatment procedure was repeated in order pling and the delivery of liquids by means of syringes to test the reproducibility of the set-up. At the sampling through silicone rubber septa are also installed. Gas time points two biological parallels were withdrawn. volumes are measured with thermal mass fow devices (DMFC SLA5860S, Brooks). A fresh sample from an RNA isolation and cDNA synthesis industrial scale mesophilic biogas plant, fed with pig For RNA isolation 2 mL of reactor liquid samples were slurry and maize silage mix (Zöldforrás Biogas Plant, used. Te samples were centrifuged at 12,000 rpm for Szeged, Hungary) was used as an inoculum, i.e., the 10 min. RNA extractions were carried out with the Zymo microbial community adopted to heterogeneous sub- Research Soil/Fecal RNA kit (R2040, Zymo Research, strate degradation. Te reactors were fushed with N­ 2 to Irvine, CA, United States). After lysis (bead beating), ensure anaerobic conditions and were closed air tight. the Zymo Research kit protocol was followed. Te DNA During the experiment the digesters were fed twice a day contamination was removed by Termo Scientifc Rapi- with synthetic medium in which only alpha-cellulose was dout™ DNA removal kit (K2981, Termo Fisher Scien- added as a carbon source at a loading rate of 1 g oDM tifc, Waltham, MA, United States). Ribosomal RNA was L-1 day-1. Te reactors were operated under mesophilic depleted using the Ribo-Zero™ rRNA Removal Kit for conditions, at 37 °C. Bacteria (Illumina, Madison, USA) according to the man- ufacturer’s instructions. Te rRNA depleted samples were Determination of fermentation parameters purifed via the RNA Clean and Concentrator Columns Organic dry matter (ODM): Te dry matter content was from Zymo Research (Irvine, USA). During this step, an determined by drying the biomass at 105 °C overnight additional in-column DNase I treatment was included and weighing the residue. Further, heating of this residue to ensure complete removal of DNA. Subsequently, syn- at 550 °C provided the organic total solids content. thesis of double-stranded cDNA was conducted using + + NH4 –N: For the determination of ­NH4 –N con- the Maxima H Minus Double-Stranded cDNA Synthesis tent, the Spectroquant Ammonium test (1.00683.0001 Kit from TermoScientifc (Waltham, USA). In the frst- test, Merck, Kenilworth, N.J, USA) was used in a Nova strand cDNA synthesis reaction, 2 μL of random hexamer 60 spectrophotometer according to the manufacturer’s primer were used. Final purifcation of the blunt-end instructions. double-stranded cDNA was carried out using SureClean VOA/TIC (Volatile organic acids/Total inorganic car- Plus solution from Bioline (Luckenwalde, Germany). Te bon): 5 g of each AD samples were taken for analysis and cDNA was sequenced in the same way as the total DNA. diluted appropriately with distilled water. Te subsequent Te quality of the RNA preparation was checked by aga- measurement was carried out with a Pronova FOS/TAC rose gel electrophoresis (data not shown). 2000 Version 812-09.2008 automatic titrator (Pronova, Germany). DNA isolation DNA extractions were carried out from 2 mL reac- Sampling tor liquid using a slightly modifed version of the Zymo Te frst set of samples were taken when the reactor Research Fecal DNA kit (D6010, Zymo Research, Irvine, USA). Te lysis mixture contained 100 µL of 10% CTAB operation was stabilized under ­N2 in the headspace, the (cetyltrimethylammonium bromide) to improve the daily biogas production, CH­ 4 content and total organic Kakuk et al. Microb Cell Fact (2021) 20:127 Page 15 of 18

efciency [61]. After lysis (bead beating was performed was calculated based on the size and concentration of the by Vortex Genie 2, bead size: 0.1 mm; beating time: particular gene fragment. Dilution series were created 9 15 min, beating speed: max) and subsequently the Zymo from the PCR products with a factor of 10 from 1 × ­10 1 −1 Research kit protocol was followed. to 1 × ­10 copies µL . Te dilution series were measured Te quantity of DNA was determined in a NanoDrop on the same plate with their corresponding cDNA sam- ND-1000 spectrophotometer (NanoDrop Technolo- ples in the RT-qPCR experiments. Te RT-qPCR runs gies, Wilmington, DE, United States) and a Qubit 2.0 were evaluated with CFX Maestro version: 4.1.2433.1219 Fluorometer (Life Technologies, Carlsbad, CA, United (BioRad). ­log2FC of the gene expression was calculated as States). DNA purity was tested by agarose gel electro- for the transcriptomics data. phoresis and on an Agilent 2200 TapeStation instrument (Agilent Technologies, Santa Clara, CA, United States). Bioinformatics Quality fltering and trimming of the raw reads were car- Sequencing ried out with FastQC. Assembly with MegaHIT, ORF Paired-end libraries were prepared for the metagenome prediction with prodigal and predicted gene functional and metatranscriptome samples using the NEBNext­ ® annotation was carried out within the SqueezeMeta Ultra™ II DNA Library Prep Kit for Illumina (Cat.Num.: workfow [47]. For the KEGG KO annotation EggNOG E7645L). Paired-end fragment reads were generated database (v. 5) was used [62]. Binning of the contigs on an Illumina NextSeq sequencer using TG NextSeq­ ® was carried out with four diferent binning algorithms: 500/550 High Output Kit v2 (300 cycles). Raw read Metabat2 [25], Maxbin2 [63], Concoct [24] and Binsan- sequences (.fastq fles) are available on NCBI-SRA under ity [64]. Te result of each binning procedure was further the following BioProject ID: PRJNA 698464. improved with DAS tool [65]. Bin qualities were esti- mated with CheckM [36] and bin taxonomy was deter- Reverse transcription coupled quantitative PCR mined using the GTDB database. A phylogenomic tree Elevenmpared with the Bio-Gas Microbiome dat genes from the protein genomes of the MAGs were built with were selected for reverse transcription coupled quanti- the phylophlan3 program [66]: phylophlan –diversity tative PCR (RT-qPCR) based on the metatranscriptomic high –fast -f phylophlan_confgs/supermatrix_aa.cfg -t a data. From every sample, 500 ng of RNA was transcribed –min_num_markers 75. Te assembly, annotation, bin- into cDNA with the High Capacity cDNA Reverse Tran- ning and phylogenomic results were imported into and scription Kit (Termo Fisher Scientifc, Waltham, MA, subsequently visualized with the Anvi’o [67] platform. USA) according to the instructions of the manufacturer. Te results were compared with the Bio-Gas Microbiome Te PCR primer oligonucleotides were synthesized by database (Additional fle 5: Table S1). Eurofns Genomics (Eurofns Genomics, Ebersberg, Te fltered reads from each sample were mapped back Germany). Te primers are listed in Additional fle 6: onto each bins with bowtie2 [68] and FeatureCounts Table S2. Te reactions were prepared in a fnal vol- [69] was used to calculate the gene count table by using ume of 25 µL with Kapa SYBR Fast Universal qPCR kit the ORF predictions of the bins. Since we were primar- (Roche, Basel, Switzerland). Te qPCR experiments were ily interested in pathway analysis, genes that could be carried out on a BioRad CFX96 Touch Real-Time PCR annotated with a KEGG Orthology (KO) were kept [70]. Detection System (BioRad, Hercules, CA, USA) with the For the assessment of ­log2 fold changes ­(log2FC) between following parameters: initial denaturation was done at the samples the DESeq2 package was used [71], which 95 °C for 3 min then 40 cycles of 95 °C for 10 s and 60 °C was proven to be an appropriate method to infer difer- for 30 s. For quantifcation of the gene copies, standards ences between metagenomic and metatranscriptomic were prepared with every primer pair from the genomic gene counts [72]. Te following parameters were set: DNA. Te standards were amplifed with DreamTaq test = "Wald", ftType = "parametric", flterFun = ihw. DNA Polymerase in a BioRad T100 Termal Cycler with For the assessment of signifcance, the Benjamini-Hoch- the following parameters: initial denaturation was at berg-adjusted p-values were used (termed ‘padj’), with a 95 °C for 3 min, then 35 cycles of 95 °C for 30 s, 60 °C for threshold of 0.05. 30 s, 72 °C for 20 s. After amplifcation, the PCR products Diferentially expressed KOs and pathways were assessed were purifed with Viogene PCR Advanced PCR Clean at two levels: First, counts of genes with the same KO anno- Up Miniprep System (Viogene Biotek Corp., New Tai- tation were grouped together and summed in each sam- pei City, Taiwan) following the manufacturer’s instruc- ple. Diferentially expressed KOs between the two MTR tions. Te PCR product concentration was determined samples were then determined with DESeq2 as described on a Qubit4 fuorimeter (Termo Fisher Scientifc) with a above. Te resulting DE KO list was the input for Cluster- Broad Range Assay Kit. Te molarity of the PCR products profler R package [73] to detect diferentially expressed Kakuk et al. Microb Cell Fact (2021) 20:127 Page 16 of 18

pathways. Ten counts of genes with the same KO annota- Availability of data and materials All the R scripts that were used to analyze the data are available upon request. tion were grouped together in each sample and in each bin, Raw read sequences (.fastq fles) are available on NCBI-SRA under the follow- since our main focus was to assess which pathways changed ing BioProject ID: PRJNA698464 (https://​www.​ncbi.​nlm.​nih.​gov/​biopr​oject/​ in the individual genome bins. Genes that did not belong to PRJNA​698464). any bin were grouped together as unbinned. Diferentially expressed KOs of every bin between the two MTR samples Declarations were then determined with DESeq2, based on log­ 2FC and Ethics approval and consent to participate p-values. Tis bin-KEGGKO-sample table was also rlog- No human or animal participants, data or tissue has been involved in the transformed (regularized logarithm transformation) with study, therefore no need for ethics approval is needed. the rlog function of the DESeq2 package and results were Consent for publication subjected to a Principial Component Analysis (PCA) using The manuscript contains no personal information data, therefore no consent the FactoMineR package. is required. Competing interest Supplementary Information The authors declare that they have no competing interest. The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s12934-​021-​01618-y. Author details 1 Institute of Medical Biology, University of Szeged, Szeged, Hungary. 2 Depart- ment of Biotechnology, University of Szeged, Szeged, Hungary. 3 Institute Additional fle 1: Figure S1. Daily biogas productions (green spots) and of Plant Biology, Biological Research Center, Szeged, Hungary. 4 Institute 5 their average (red line) during the experimental period. H­ 2 injection took of Biophysics, Biological Research Center, Szeged, Hungary. Department place at time points marked with dotted blue arrows. Increment CH­ 4 of Oral Biology and Experimental Dental Research, University of Szeged, production is highlighted with yellow curves ftted to the data points. The Szeged, Hungary. areas under these curves were used for CH­ 4 conversion estimation. Additional fle 2: Figure S2. Relative abundances of Archaea and Bacteria Received: 27 March 2021 Accepted: 24 June 2021 bins.

Additional fle 3: Figure S3. Methanogenesis enzymes afected by H­ 2 addition. Additional fle 4: Figure S4. KEGG heatmap. References 1. Maurya R, Tirkey SR, Rajapitamahuni S, Ghosh A, Mishra S. Recent Additional fle 5: Table S1. Comparison of bins identifed using the GTDB advances and future prospective of biogas production. In: Advances in database with Bio-Gas Microbiome database. The two bins containing feedstock conversion technologies for alternative fuels and bioproducts. high quality metagenomes, which did not fnd their corresponding MAGs Sawston: Woodhead Publishing, Elsevier; 2019;159–78. are highlighted. Percent AAI stands for percent amino acid identity. # 2. Scarlat N, Dallemand JF, Fahl F. Biogas: Developments and perspectives in Indicates the bins, which were not identifed by the one or both of the Europe. Renew Energy. 2018;129:457–72. databases. 3. Bagi Z, Ács N, Böjti T, Kakuk B, Rákhely G, Strang O, et al. Biomethane: the Additional fle 6: Table S2. Genes and qPCR primers used in this study. energy storage, platform chemical and greenhouse gas mitigation target. Anaerobe. 2017;46:13–22. 4. Goswami R, Chattopadhyay P, Shome A, Banerjee SN, Chakraborty AK, Acknowledgements Mathew AK, et al. An overview of physico-chemical mechanisms of ZB acknowledges the funding of Hungarian NKFIH/OTKA program, project biogas production by microbial communities: a step towards sustainable number FK 123902. RW and GM received partial support from the Hungar- waste management. 3 Biotech. 2016;6:1–12. ian NKFIH fund projects PD132145 and FK123899. This study has been also 5. Liu CM, Wachemo AC, Tong H, Shi SH, Zhang L, Yuan HR, et al. Biogas supported by the Hungarian National Research, Development and Innova- production and microbial community properties during anaerobic tion Fund projects GINOP- 2.2.1-15-2017-00081, EFOP- 3.6.2-16-2017-00010, digestion of corn stover at diferent temperatures. Bioresour Technol. 2020-3.1.2.-ZFR-KVG-2020-00009 and 2020-1.1.2-PIACI-KFI-2020-00117. GM 2018;261:93–103. was also supported by the Lendület-Programme of the Hungarian Academy 6. Maus I, Koeck DE, Cibis KG, Hahnke S, Kim YS, Langer T, et al. Unraveling of Sciences (LP2020-5/2020). the microbiome of a thermophilic biogas plant by metagenome and metatranscriptome analysis complemented by characterization of bacte- Authors’ contributions rial and archaeal isolates. Biotechnol Biofuels. 2016;9:171. BK and RW performed the bioinformatic analyses. GM did the sequencing 7. Hassa J, Maus I, Of S, Pühler A, Scherer P, Klocke M, et al. Metagenome, and primary evaluation of data. MSz and ZB developed the interpretation and metatranscriptome, and metaproteome approaches unraveled composi- rationalization of the data. KL contributed with the qPCR experiments. GR tions and functional relationships of microbial communities residing in critically read the manuscript. KLK and BZ designed and conceived the experi- biogas plants. Appl Microbiol Biotechnol. 2018;102:5045–63. mental protocol and compiled the manuscript together with BK. All authors 8. Wirth R, Kovacs E, Martín G, Bagi Z, Rakhely G, Kovacs KL. Characterization read and approved the fnal manuscript. of a biogas producing microbial community by short read next genera- tion DNA sequencing. Biotechnol Biofuels. 2012;5:1–16. Funding 9. Cai W, Liu W, Wang B, Yao H, Guadie A, Wang A. Semiquantitative detec- ZB has been awarded by funding of Hungarian NKFIH/OTKA program, project tion of hydrogen-associated or hydrogen-free electron transfer within number FK 123902. RW and GM received partial support from the Hungar- methanogenic bioflm of microbial electrosynthesis. Appl Environ Micro- ian NKFIH fund projects PD132145 and FK123899. This study has been also biol. 2020. https://​doi.​org/​10.​1128/​AEM.​01056-​20. supported by the Hungarian National Research, Development and Innova- 10. Thauer RK, Kaster AK, Goenrich M, Schick M, Hiromoto T, Shima S. Hydro- tion Fund projects GINOP- 2.2.1-15-2017-00081, EFOP- 3.6.2-16-2017-00010, genases from methanogenic archaea, nickel, a novel cofactor, and ­H2 2020-3.1.2.-ZFR-KVG-2020-00009 and 2020-1.1.2-PIACI-KFI-2020-00117. GM storage. Annu Rev Biochem. 2010;79:507–36. was also supported by the Lendület-Programme of the Hungarian Academy of Sciences (LP2020-5/2020). Kakuk et al. Microb Cell Fact (2021) 20:127 Page 17 of 18

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