Six novel species of the genus Methanoculleus isolated from various environments in Taiwan.

Taiwan is situated in the subtropical region and its geographical location and topographical features contribute to a rich ecological diversity and scenic landscapes. We investigated the diversity of methanogens in different environments of Taiwan using a culture-dependent method. This report presents the characterization and taxonomy of six hydrogenotrophic methanogens obtained from cold seep sediments (strain FWC-SCC1T and FWC-SCC3T), marine sediments (strain CWC-02T and YWC-01T), estuarine sediments (strain Afa-1T), and a hot spring well (strain Wushi-C6T) in Taiwan. The proposed names of the six novel species are Methanoculleus frigidifontis (type strain FWC-SCC1T=BCRC AR10056T=NBRC 113993T), Methanoculleus oceani (CWC-02T=BCRC AR10055T=NBRC 113992T), Methanoculleus methanifontis (FWC-SCC3T=BCRC AR10057T=NBRC 113994T), Methanoculleus nereidis (YWC-01T=BCRC AR10060T=NBRC 114597T), Methanoculleus formosensis (Afa-1T=BCRC AR10054T=NBRC 113995T), and Methanoculleus caldifontis (Wushi-06T=BCRC AR10059T= NBRC 114596T).

Patterns of syntrophic interactions in methanogenic conversion of propionate.

Methanogenesis is central to anaerobic digestion processes. The conversion of propionate as a key intermediate for methanogenesis requires syntrophic interactions between bacterial and archaeal partners. In this study, a series of methanogenic enrichments with propionate as the sole substrate were developed to identify microbial populations specifically involved in syntrophic propionate conversion. These rigorously controlled propionate enrichments exhibited functional stability with consistent propionate conversion and methane production; yet, the methanogenic microbial communities experienced substantial temporal dynamics, which has important implications on the understanding of mechanisms involved in microbial community assembly in anaerobic digestion. Syntrophobacter was identified as the most abundant and consistent bacterial partner in syntrophic propionate conversion regardless of the origin of the source culture, the concentration of propionate, or the temporal dynamics of the culture. In contrast, the methanogen partners involved in syntrophic propionate conversion lacked consistency, as the dominant methanogens varied as a function of process condition and temporal dynamics. Methanoculleus populations were specifically enriched as the syntrophic partner at inhibitory levels of propionate, likely due to the ability to function under unfavorable environmental conditions. Syntrophic propionate conversion was carried out exclusively via transformation of propionate into acetate and hydrogen in enrichments established in this study. Microbial populations highly tolerant of elevated propionate, represented by Syntrophobacter and Methanoculleus, are of great significance in understanding methanogenic activities during process perturbations when propionate accumulation is frequently encountered. Key points • Syntrophobacter was the most consistent bacterial partner in propionate metabolism. • Diverse hydrogenotrophic methanogen populations could serve as syntrophic partners. • Methanoculleus emerged as a methanogen partner tolerant of elevated propionate.

A snapshot of microbial community structures in 20 different field-scale anaerobic bioreactors treating food waste.

This study aimed to identify significant factors shaping the microbial populations in biogas plants treating food waste (FW). Twenty full-scale anaerobic acidogenic/methanogenic bioreactors, located at 11 FW treatment facilities, were compared to find patterns in their microbial community structures and potential interactions with the process parameters. Temperature, hydraulic retention time, and organic loading rate were design parameters that systematically influenced the microbial communities. The latter two clearly separated the acidogenic and methanogenic bioreactors. Lactobacillus was the dominant (69.7 ±â€¯19.8%) bacteria in the acidogenic reactors, while hydrogen-utilizing methanogens, such as Methanoculleus (65.1 ±â€¯33.5%), were the dominant archaea in most methanogenic digesters. Defluiviitoga was the dominant (82.7 ±â€¯1.4%) bacteria in the thermophilic digesters, but was also the most abundant (33.1-33.6%) bacteria in dry mesophilic digesters. The two bioreactor categories had lower bacterial diversities, and also higher propionate concentrations (>5 g/L in 4 out of 5 cases), which may impose potential risks for the management of such digesters. The current 'snapshot' of the microbial communities suggests several bacterial and archaeal taxa as potential indicators of bioreactor categories and/or process variables.

Isolation and characterization of a new Methanoculleus bourgensis strain KOR-2 from the rumen of Holstein steers.

OBJECTIVE: To isolate and identify new methanogens from the rumen of Holstein steers in Korea. METHODS: Representative rumen contents were obtained from three ruminally cannulated Holstein steers (793±8 kg). Pre-reduced media were used for the growth and isolation of methanogens. Optimum growth temperature, pH, and sodium chloride (NaCl) concentration as well as substrate utilization and antibiotic tolerance were investigated to determine the physiological characteristics of the isolated strain. Furthermore, the isolate was microscopically studied for its morphology. Polymerase chain reaction of 16S rRNA and mcrA gene-based amplicons was used for identification. RESULTS: One strain designated as KOR-2 was isolated and found to be a non-motile irregular coccus with a diameter of 0.2 to 0.5 µm. KOR-2 utilized H2+CO2 and formate but was unable to metabolize acetate, methanol, trimethylamine, 2-propanol, and isobutanol for growth and methane production. The optimum temperature and pH for the growth of KOR-2 were 38°C and 6.8 to 7.0, respectively, while the optimum NaCl concentration essential for KOR-2 growth was 1.0% (w/v). KOR-2 tolerated ampicillin, penicillin G, kanamycin, spectromycin, and tetracycline. In contrast, the cell growth was inhibited by chloramphenicol. Phylogenetic analysis of 16S rRNA and mcrA genes revealed the relatedness between KOR-2 and Methanoculleus bourgensis. CONCLUSION: Based on the physiological and phylogenetic characteristics, KOR-2 was thought to be a new strain within the genus Methanoculleus and named Methanoculleus bourgensis KOR-2.

The time response of anaerobic digestion microbiome during an organic loading rate shock.

Knowledge of connections between operational conditions, process stability, and microbial community dynamics is essential to enhance anaerobic digestion (AD) process efficiency and management. In this study, the detailed temporal effects of a sudden glycerol-based organic overloading on the AD microbial community and process imbalance were investigated in two replicate anaerobic digesters by a time-intensive sampling scheme. The microbial community time response to the overloading event was shorter than the shifts of reactor performance parameters. An increase in bacterial community dynamics and in the abundances of several microbial taxa, mainly within the Firmicutes, Tenericutes, and Chloroflexi phyla and Methanoculleus genera, could be detected prior to any shift on the reactor operational parameters. Reactor acidification already started within the first 24 h of the shock and headed the AD process to total inhibition in 72 h alongside with the largest shifts on microbiome, mostly the increase of Anaerosinus sp. and hydrogenotrophic methanogenic Archaea. In sum, this work proved that AD microbial community reacts very quickly to an organic overloading and some shifts occur prior to alterations on the performance parameters. The latter is very interesting as it can be used to improve AD process management protocols.

Long-term influence of trace element deficiency on anaerobic mono-digestion of chicken manure.

Recent findings showed that some trace elements essential for anaerobic digestion might be deficient in chicken (laying hens) manure. In this study, the long-term influence of trace element deficiency on anaerobic mono-digestion of chicken manure was investigated. Three bench-scale anaerobic reactors were operated with or without trace element supplementation. As trace element, only Se or a mix containing Co, Mo, Ni, Se, and W was added to the reactors. The results revealed that in anaerobic digestion of chicken manure at total ammonium nitrogen concentrations over 6000 mg L-1, Se supplementation was critical but not sufficient alone for long-term stable CH4 production. Addition of a mix consisting of Co, Mo, Ni, Se and W resulted in a more stable digestion performance. Daily trace element mix supplementation promoted the hydrogenotrophic Methanoculleus bourgensis, which is an ammonia tolerant methanogen. The decrease in the relative abundance of Methanoculleus detected after termination of trace element addition and resulted in accumulation of acetate and propionate that followed by a significant decrease in CH4 production.

Toward the structure of presenilin/γ-secretase and presenilin homologs.

Presenilin is the catalytic component of the γ-secretase complex, a membrane-embedded aspartyl protease that plays a central role in biology and in the pathogenesis of Alzheimer's disease. Upon assembly with its three protein cofactors (nicastrin, Aph-1 and Pen-2), presenilin undergoes autoproteolysis into two subunits, each of which contributes one of the catalytic aspartates to the active site. A family of presenilin homologs, including signal peptide peptidase, possess proteolytic activity without the need for other protein factors, and these simpler intramembrane aspartyl proteases have given insight into the action of presenilin within the γ-secretase complex. Cellular and molecular studies support a nine-transmembrane topology for presenilins and their homologs, and small-molecule inhibitors and cysteine scanning with crosslinking have suggested certain presenilin residues and regions that contribute to substrate recognition and handling. Identification of partial complexes has also offered clues to protein-protein interactions within the γ-secretase complex. Biophysical methods have allowed 3D views of the γ-secretase complex and presenilins. Most recently, the crystal structure of a microbial presenilin homolog has confirmed a nine-transmembrane topology and intramembranous location and proximity of the two conserved and essential aspartates. The crystal structure also provides a platform for the formulation of specific hypotheses regarding substrate interaction and catalysis as well as the pathogenic mechanism of Alzheimer-causing presenilin mutations. This article is part of a Special Issue entitled: Intramembrane Proteases.

Development of Methanoculleus-specific real-time quantitative PCR assay for assessing methanogen communities in anaerobic digestion.

AIM: To develop a Methanoculleus-specific real-time quantitative PCR (RT-qPCR) assay with high coverage and specificity for the analysis of methanogenic populations in anaerobic digestion. METHODS AND RESULTS: A Methanoculleus-specific primer/probe set for RT-qPCR was designed in this study based on all Methanoculleus 16S rRNA gene sequences in Ribosomal Database Project (RDP) according to TaqMan chemistry. The newly designed primer/probe set was shown to have high coverage and specificity by both in silico and experimental analyses. Amplification efficiency of the Methanoculleus-specific primer/probe set was determined to be ideal for RT-qPCR applications. Subsequent field testing on anaerobic digesters showed that results from RT-qPCR were consistent with those from clone library analysis, validating the accuracy of the RT-qPCR assay. CONCLUSIONS: The Methanoculleus-specific RT-qPCR assay designed in this study can serve as a rapid and effective tool for the quantification of Methanoculleus populations in anaerobic digestion. SIGNIFICANCE AND IMPACT OF THE STUDY: Methanoculleus populations represent important members of archaeal communities in methanogenic processes, necessitating the need to develop effective tools to monitor Methanoculleus population abundance. The RT-qPCR developed in this study provides an essential tool for the quantification of Methanoculleus populations in anaerobic digestion and for the understanding of the functions of these methanogens in anaerobic biotransformation.

Metataxonomic characterization of an autochthonous and allochthonous microbial consortium involved in a two-stage anaerobic batch reactor applied to hydrogen and methane production from sugarcane bagasse.

Enzymatically pre-treated sugarcane bagasse (SCB) was used as substrate for sequential production of hydrogen (H2) and methane (CH4). In stage I, SCB was used by autochthonous bacteria, such as Enterococcus (42.4 % relative abundance) and Paraclostridium (16.8 %) for H2 production (166.8 mL H2/L), while the metabolites accumulated at the end of this stage (9140.5 mg HAc/L) were used by allochthonous inoculum for CH4 production (870.8 mL CH4/L) in stage II. In stage II, hydrogenotrophic (Methanoculleus, 49.1 %) and acetoclastic (Methanosaeta, 15.5 %) archaeal genera were identified and considered important to maintain low H2 pressure in the system. According to gene inference, the hydrolysis of the SCB fiber was performed in both stages, as potential ß-glucosidase and 1,4-ß-xylosidase encoding genes were predicted. However, the energy metabolism of microbial populations differed, as potential genes involved in CH4 metabolism were predominant in phase II (39.0 %).

Inhibitory mechanisms on dry anaerobic digestion: Ammonia, hydrogen and propionic acid relationship.

Inhibitory pathways in dry anaerobic digestion are still understudied and current knowledge on wet processes cannot be easily transferred. This study forced instability in pilot-scale digesters by operating at short retention times (40 and 33 days) in order to understand inhibition pathways over long term operation (145 days). The first sign of inhibition at elevated total ammonia concentrations (8 g/l) was a headspace hydrogen level over the thermodynamic limit for propionic degradation, causing propionic accumulation. The combined inhibitory effect of propionic and ammonia accumulation resulted in further increased hydrogen partial pressures and n-butyric accumulation. The relative abundance of Methanosarcina increased while that of Methanoculleus decreased as digestion deteriorated. It was hypothesized that high ammonia, total solids and organic loading rate inhibited syntrophic acetate oxidisers, increasing their doubling time and resulting in its wash out, which in turn inhibited hydrogenotrophic methanogenesis and shifted the predominant methanogenic pathway towards acetoclastic methanogenesis at free ammonia over 1.5 g/l. C/N increases to 25 and 29 reduced inhibitors accumulation but did not avoid inhibition or the washout of syntrophic acetate oxidising bacteria.

Novel bioaugmentation strategy boosted with biochar to alleviate ammonia toxicity in continuous biomethanation.

This study investigated for the first time if ammonia tolerant methanogenic consortia can be stored in gel (biogel) and used in a later time on-demand as bioaugmentation inocula, to efficiently relieve ammonia inhibition in continuous biomethanation systems. Moreover, wood biochar was assessed as a potential enhancer of the novel biogel bioaugmentation process. Three thermophilic (55 °C), continuous stirred-tank reactors (RBgel, RChar and RMix), operated at 4.5 g NH4+-N L-1 were exposed to biogel, biochar and mixture of biogel and biochar, respectively, while a fourth reactor (RCtrl) was used as control. The results showed that the methane production yields of RMix, RChar and RBgel increased by 28.6%, 20.2% and 10.7%, respectively compared to RCtrl. The highest methane yield was achieved by the synergistic interaction between biogel and biochar. Additionally, biogel stimulated a rapid recovery of Methanoculleus thermophilus sp. and syntrophic acetate oxidising bacteria populations.

Osmoprotectants boost adaptation and protect methanogenic microbiome during ammonia toxicity events in continuous processes.

Different osmoprotectants were used to counteract ammonia toxicity in continuous anaerobic reactors. The anaerobic microbiome osmoadaptation process and its role to the methanogenic recovery are also assessed. Three osmoprotectants (i.e., glycine betaine, MgCl2 and KCl) were respectively introduced in continuous reactors at high ammonia levels, namely RGB, RMg, RK, while a control reactor (RCtrl) was also used. After ammonia was introduced, the RGB, RMg, RK and RCtrl suffered 39.0%, 36.6%, 39.9% and 36.2% methane production loss, respectively. Osmoprotectants addition recovered significantly methane production by up to 68.9%, 54.3% and 32.2% for RGB, RMg and RK, respectively contrary to RCtrl, where production increased only by 13.6%. The recovered methane production was maintained in RGB and RMg for at least four HRTs, even after the addition of osmoprotectants was stopped, due to the formed methanogenic microbiota by osmoadaptation process, with Methanoculleus sp. as the dominant species.

Characterization of Blf4, an Archaeal Lytic Virus Targeting a Member of the Methanomicrobiales.

Today, the number of known viruses infecting methanogenic archaea is limited. Here, we report on a novel lytic virus, designated Blf4, and its host strain Methanoculleus bourgensis E02.3, a methanogenic archaeon belonging to the Methanomicrobiales, both isolated from a commercial biogas plant in Germany. The virus consists of an icosahedral head 60 nm in diameter and a long non-contractile tail of 125 nm in length, which is consistent with the new isolate belonging to the Siphoviridae family. Electron microscopy revealed that Blf4 attaches to the vegetative cells of M. bourgensis E02.3 as well as to cellular appendages. Apart from M. bourgensis E02.3, none of the tested Methanoculleus strains were lysed by Blf4, indicating a narrow host range. The complete 37 kb dsDNA genome of Blf4 contains 63 open reading frames (ORFs), all organized in the same transcriptional direction. For most of the ORFs, potential functions were predicted. In addition, the genome of the host M. bourgensis E02.3 was sequenced and assembled, resulting in a 2.6 Mbp draft genome consisting of nine contigs. All genes required for a hydrogenotrophic lifestyle were predicted. A CRISPR/Cas system (type I-U) was identified with six spacers directed against Blf4, indicating that this defense system might not be very efficient in fending off invading Blf4 virus.

Similar Methanogenic Shift but Divergent Syntrophic Partners in Anaerobic Digesters Exposed to Direct versus Successive Ammonium Additions.

During anaerobic digestion (AD) of protein-rich wastewater, ammonium (NH4+) is released by amino acid degradation. High NH4+ concentrations disturb the AD microbiome balance, leading to process impairments. The sensitivity of the AD microbiome to NH4+ and the inhibition threshold depend on multiple parameters, especially the previous microbial acclimation to ammonium stress. However, little is known about the effect of different NH4+ acclimation strategies on the differential expression of key active microbial taxa. Here, we applied NH4+ inputs of increasing intensity (from 1.7 to 15.2 g N-NH4+ liters-1) in batch assays fed with synthetic wastewater, according to two different strategies: (i) direct independent inputs at a unique target concentration and (ii) successive inputs in a stepwise manner. In both strategies, along the NH4+ gradient, the active methanogens shifted from acetoclastic Methanosaeta to Methanosarcina and eventually hydrogenotrophic Methanoculleus. Despite shorter latency times, the successive input modality led to lower methane production rate, lower soluble chemical oxygen demand (sCOD) removal efficiency, and lower half maximal inhibitory concentration, together with higher volatile fatty acid (VFA) accumulation, compared to the independent input modality. These differential performances were associated with a drastically distinct succession pattern of the active bacterial partners in both experiments. In particular, the direct exposure modality was characterized by a progressive enrichment of VFA producers (mainly Tepidimicrobium) and syntrophic VFA oxidizers (mainly Syntrophaceticus) with increasing NH4+ concentration, while the successive exposure modality was characterized by a more dynamic succession of VFA producers (mainly Clostridium, Sporanaerobacter, Terrisporobacter) and syntrophic VFA oxidizers (mainly Tepidanaerobacter, Syntrophomonas). These results bring relevant insights for improved process management through inoculum adaptation, bioaugmentation, or community-driven optimization. IMPORTANCE Anaerobic digestion (AD) is an attractive biotechnological process for wastewater bioremediation and bioenergy production in the form of methane-rich biogas. However, AD can be inhibited by ammonium generated by protein-rich effluent, commonly found in agro-industrial activities. Insights in the microbial community composition and identification of AD key players are crucial for anticipating process impairments in response to ammonium stress. They can also help in defining an optimal microbiome adapted to high ammonium levels. Here, we compared two strategies for acclimation of AD microbiome to increasing ammonium concentration to better understand the effect of this stress on the methanogens and their bacterial partners. Our results suggest that long-term cumulative exposure to ammonia disrupted the AD microbiome more strongly than direct (independent) ammonium additions. We identified bioindicators with different NH4+ tolerance capacity among VFA producers and syntrophic VFA oxidizers.

Microbial Resource Management for Ex Situ Biomethanation of Hydrogen at Alkaline pH.

Biomethanation is a promising solution to convert H2 (produced from surplus electricity) and CO2 to CH4 by using hydrogenotrophic methanogens. In ex situ biomethanation with mixed cultures, homoacetogens and methanogens compete for H2/CO2. We enriched a hydrogenotrophic microbiota on CO2 and H2 as sole carbon and energy sources, respectively, to investigate these competing reactions. The microbial community structure and dynamics of bacteria and methanogenic archaea were evaluated through 16S rRNA and mcrA gene amplicon sequencing, respectively. Hydrogenotrophic methanogens and homoacetogens were enriched, as acetate was concomitantly produced alongside CH4. By controlling the media composition, especially changing the reducing agent, the formation of acetate was lowered and grid quality CH4 (≥97%) was obtained. Formate was identified as an intermediate that was produced and consumed during the bioprocess. Stirring intensities ≥ 1000 rpm were detrimental, probably due to shear force stress. The predominating methanogens belonged to the genera Methanobacterium and Methanoculleus. The bacterial community was dominated by Lutispora. The methanogenic community was stable, whereas the bacterial community was more dynamic. Our results suggest that hydrogenotrophic communities can be steered towards the selective production of CH4 from H2/CO2 by adapting the media composition, the reducing agent and the stirring intensity.

Comparative genomic analyses reveal trehalose synthase genes as the signature in genus Methanoculleus.

To date, the only methanoarchaea isolated directly from methane hydrate bearing sediments were Methanoculleus submarinus Nankai-1T and Methanoculleus sp. MH98A. Here, we provide the genome of Methanoculleus taiwanensis CYW4T isolated from the deep-sea subseafloor sediment at the Deformation Front offshore southwestern Taiwan, where methane hydrate deposits are likely located. Through comparative genomics analyses of nine Methanoculleus strains from various habitats, 2-3 coding genes for trehalose synthases were found in all nine Methanoculleus genomes, which were not detected in other methanogens and are therefore suggested as a signature of genus Methanoculleus among methane-producing archaea. In addition, the structural genes adjacent to trehalose synthase genes are comprised of the signaling module of Per-Arnt-Sim (PAS) domain-containing proteins, Hsp20 family proteins, arabinose efflux permeases and multiple surface proteins with fasciclin-like (FAS) repeat. This indicates that trehalose synthase gene clusters in Methanoculleus might play roles in the response to various stresses and regulate carbon storage and modification of surface proteins through accumulation of trehalose. The non-gas hydrate-associated Methanoculleus strains harbor carbon-monoxide dehydrogenase (cooS/acsA) genes, which are important for the conversion of acetate to methane at the step of CO oxidation/CO2 reduction in acetoclastic methanogens and further implies that these strains may be able to utilize CO for methanogenesis in their natural habitats. In addition, both genomes of M. bourgensis strains MS2T and MAB1 harbor highly abundant transposase genes, which may be disseminated from microbial communities in their habitats, sewage treatment plants and biogas reactors, which are breeding grounds for antibiotic resistance. Through comparative genomic analyses, we gained insight into understanding the life of strictly anaerobic methane-producing archaea in various habitats, especially in methane-based deep-sea ecosystems.

Hydrogenotrophic methanogens are the key for a successful bioaugmentation to alleviate ammonia inhibition in thermophilic anaerobic digesters.

Bioaugmentation to alleviate ammonia inhibition under thermophilic anaerobic digestion has never been reported, as well as the working mechanism that allows a fast and successful bioaugmentation. Thus two bioaugmentation inocula (an enriched culture, and a mixed culture composed 50/50 by Methanoculleus thermophilus and the enriched culture) on the recovery of ammonia-inhibited thermophilic continuous reactors was assessed. The results showed that bioaugmentation improved methane yield by 11-13% and decreased the volatile fatty acids (VFA) by 45-52% compared to the control reactor (abiotic augmentation). Moreover, the importance of hydrogenotrophic methanogens to a fast and successful bioaugmentation was recognized. Specifically, the instant hydrogen partial pressure reduction by the bioaugmented hydrogenotroph created thermodynamically favourable conditions for the acetate oxidation process and consequently, the catabolism of other VFA. High-throughput sequencing results strengthened this explanation by showing that the bioaugmented M. thermophilus stimulated the growth of syntrophic acetate oxidising bacterium Thermacetogenium phaeum, immediately after bioaugmentation.

Bioaugmentation strategy for overcoming ammonia inhibition during biomethanation of a protein-rich substrate.

High ammonia levels inhibit anaerobic digestion (AD) process and bioaugmentation with ammonia tolerant methanogenic culture is proposed to alleviate ammonia inhibition. In the current study, hydrogenotrophic Methanoculleus bourgensis was bioaugmented in an ammonia-inhibited continuous reactor fed mainly with microalgae (a protein-rich biomass), at extreme ammonia levels (i.e. 11 g NH4+-N L-1). The results showed 28% increase in methane production immediately after bioaugmentation. Moreover, volatile fatty acids decreased rapidly from more than 5 g L-1 to around 1 g L-1, with a fast reduction in propionate concentration. High throughput 16s rRNA gene sequencing demonstrated that the bioaugmented M. bourgensis doubled its relative abundance after bioaugmentation. "Microbiological domino effect", triggered by the bioaugmented M. bourgensis establishing a newly efficient community, was proposed as the working mechanism of the successful bioaugmentation. Additionally, a strong aceticlastic methanogenesis was found at the end of the experiment evidenced by the dominant presence of Methanosarcina soligelidi and the low abundance of syntrophic acetate oxidising bacteria at the final period. Overall, for the first time, this study proved the positive effect of bioaugmentation on ammonia inhibition alleviation of the microalgae-dominating fed reactor, paving the way of efficient utilization of other protein-rich substrates in the future.

Metabolic Adaptation of Methanogens in Anaerobic Digesters Upon Trace Element Limitation.

Anaerobic digestion (AD) is a complex multi-stage process relying on the activity of highly diverse microbial communities including hydrolytic, acidogenic and syntrophic acetogenic bacteria as well as methanogenic archaea. The lower diversity of methanogenic archaea compared to the bacterial groups involved in AD and the corresponding lack of functional redundancy cause a stronger susceptibility of methanogenesis to unfavorable process conditions such as trace element (TE) deprivation, thus controlling the stability of the overall process. Here, we investigated the effects of a slowly increasing TE deficit on the methanogenic community function in a semi-continuous biogas process. The aim of the study was to understand how methanogens in digester communities cope with TE limitation and sustain their growth and metabolic activity. Two lab-scale biogas reactors fed with distillers grains and supplemented with TEs were operated in parallel for 76 weeks before one of the reactors was subjected to TE deprivation, leading to a decline of cobalt and molybdenum concentrations from 0.9 to 0.2 mg/L, nickel concentrations from 2.9 to 0.8 mg/L, manganese concentrations from 38 to 18 mg/L, and tungsten concentrations from 1.4 to 0.2 mg/L. Amplicon sequencing of mcrA genes revealed Methanosarcina (72%) and Methanoculleus (23%) as the predominant methanogens in the undisturbed reactors. With increasing TE limitation, the relative abundance of Methanosarcina dropped to 67% and a slight decrease of acetoclastic methanogenic activity was observed in batch tests with 13C-methyl-labeled acetate, suggesting a shift toward syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis. Metaproteome analysis revealed abundance shifts of the enzymes involved in methanogenic pathways. Proteins involved in methylotrophic and acetoclastic methanogenesis decreased in abundance while formylmethanofuran dehydrogenase from Methanosarcinaceae increased, confirming our hypothesis of a shift from acetoclastic to hydrogenotrophic methanogenesis by Methanosarcina. Both Methanosarcina and Methanoculleus increased the abundance of N5-methyltetrahydromethanopterin-coenzyme M methyltransferase and methyl-coenzyme M reductase. However, these efforts to preserve the ion motive force for energy conservation were seemingly more successful in Methanoculleus. We conclude that both methanogenic genera use different strategies to stabilize their energy balance under TE limitation. Methanosarcina switched from TE expensive pathways (methylotrophic and acetoclastic methanogenesis) to hydrogenotrophic methanogenesis. Methanoculleus showed a higher robustness and was favored over the more fastidious Methanosarcina, thus stabilizing reactor performance under TE limitation.

Anaerobic Process for Bioenergy Recovery From Dairy Waste: Meta-Analysis and Enumeration of Microbial Community Related to Intermediates Production.

Dairy wastes are widely studied for the hydrogen and methane production, otherwise the changes in microbial communities related to intermediate valuable products was not deeply investigated. Culture independent techniques are useful tools for exploring microbial communities in engineered system having new insights into their structure and function as well as potential industrial application. The deep knowledge of the microbiota involved in the anaerobic process of specific waste and by-products represents an essential step to better understand the entire process and the relation of each microbial population with biochemical intermediates and final products. Therefore, this study investigated the microbial communities involved in the laboratory-scale anaerobic digestion of a mixture of mozzarella cheese whey and buttermilk amended with 5% w/v of industrial animal manure pellets. Culture-independent methods by employing high-throughput sequencing and microbial enumerations highlighted that lactic acid bacteria, such as Lactobacillaceae and Streptococcaceae dominated the beginning of the process until about day 14 when a relevant increase in hydrogen production (more than 10 ml H2 gVS-1 from days 13 to 14) was observed. Furthermore, during incubation a gradual decrease of lactic acid bacteria was detected with a simultaneous increase of Clostridia, such as Clostridiaceae and Tissierellaceae families. Moreover, archaeal populations in the biosystem were strongly related to inoculum since the non-inoculated samples of the dairy waste mixture had a relative abundance of archaea less than 0.1%; whereas, in the inoculated samples of the same mixture several archaeal genera were identified. Among methanogenic archaea, Methanoculleus was the dominant genus during all the process especially when the methane production occurred, and its relative abundance increased up to 99% at the end of the incubation time highlighting that methane was formed from dairy wastes primarily by the hydrogenotrophic pathway in the reactors.