The oral microbiome of a family including Papillon-Lefèvre-syndrome patients and clinically healthy members.

AIMS: The oral microbiota composition of patients diagnosed with Papillon-Lefèvre-syndrome and treated for several years were compared to those existing in the oral cavity of the clinically healthy family members and a cohort of patients having various stages of chronic periodontitis. MATERIALS AND METHODS: A family with two sisters affected with severe periodontitis and with the typical skin symptoms of Papillon-Lefèvre-syndrome, and symptomless parents and third sibling were investigated. The Patients received periodontal treatment for several years and their oral microbiome was analysed by amplicon sequencing. Data were evaluated by microbial cluster analysis. RESULTS: The microbiome of the patients with Papillon-Lefèvre-syndrome was predominated with Aggregatibacter actinomycetemcomitans and associated oral periodontopathogens. Although the clinically healthy family members showed no oral disorder, their microbiome resembled that of subjects having mild periodontitis. CONCLUSIONS: Predominance of A. actinomycetemcomitans in the subgingival microbiome of patients with Papillon-Lefèvre-syndrome suggests that specific treatment strategies directed against this pathobiont may improve the oral health status of the affected individuals. TRIAL REGISTRATION: The study was conducted in accordance with the Declaration of Helsinki and the ethical permission has been issued by the Human Investigation Review Board of the University of Szeged, Albert Szent-Györgyi Clinical Centre (Permission No. 63/2017-SZTE). September 19, 2017.  https://u-szeged.hu/klinikaikutatas/rkeb-altal-jovahagyott/rkeb-2017 .

Regulation of the methanogenesis pathways by hydrogen at transcriptomic level in time.

The biomethane formation from 4 H2 + CO2 by pure cultures of two methanogens, Methanocaldococcus fervens and Methanobacterium thermophilum, has been studied. The goal of the study was to understand the regulation of the enzymatic steps associated with biomethane biosynthesis by H2, using metagenomic, pan-genomic, and transcriptomic approaches. Methanogenesis in the autotrophic methanogen M. fervens could be easily "switched off" and "switched on" by H2/CO2 within about an hour. In contrast, the heterotrophic methanogen M. thermophilum was practically insensitive to the addition of the H2/CO2 trigger although this methanogen also converted H2/CO2 to CH4. From practical points of view, the regulatory function of H2/CO2 suggests that in the power-to-gas (P2G) renewable excess electricity conversion and storage systems, the composition of the biomethane-generating methanogenic community is essential for sustainable operation. In addition to managing the specific hydrogenotrophic methanogenesis biochemistry, H2/CO2 affected several, apparently unrelated, metabolic pathways. The redox-regulated overall biochemistry and symbiotic relationships in the methanogenic communities should be explored in order to make the P2G technology more efficient. KEY POINTS : • Hydrogenotrophic methanogens may respond distinctly to H2/CO2 in bio-CH4 formation. • H2/CO2 can also activate metabolic routes, which are apparently unrelated to methanogenesis. • Sustainable conversion of the fluctuating renewable electricity to bio-CH4 is an option.

Inter-kingdom interactions and stability of methanogens revealed by machine-learning guided multi-omics analysis of industrial-scale biogas plants.

Multi-omics analysis is a powerful tool for the detection and study of inter-kingdom interactions, such as those between bacterial and archaeal members of complex biogas-producing microbial communities. In the present study, the microbiomes of three industrial-scale biogas digesters, each fed with different substrates, were analysed using a machine-learning guided genome-centric metagenomics framework complemented with metatranscriptome data. This data permitted us to elucidate the relationship between abundant core methanogenic communities and their syntrophic bacterial partners. In total, we detected 297 high-quality, non-redundant metagenome-assembled genomes (nrMAGs). Moreover, the assembled 16 S rRNA gene profiles of these nrMAGs showed that the phylum Firmicutes possessed the highest copy number, while the representatives of the archaeal domain had the lowest. Further investigation of the three anaerobic microbial communities showed characteristic alterations over time but remained specific to each industrial-scale biogas plant. The relative abundance of various microorganisms as revealed by metagenome data was independent from corresponding metatranscriptome activity data. Archaea showed considerably higher activity than was expected from their abundance. We detected 51 nrMAGs that were present in all three biogas plant microbiomes with different abundances. The core microbiome correlated with the main chemical fermentation parameters, and no individual parameter emerged as a predominant shaper of community composition. Various interspecies H2/electron transfer mechanisms were assigned to hydrogenotrophic methanogens in the biogas plants that ran on agricultural biomass and wastewater. Analysis of metatranscriptome data revealed that methanogenesis pathways were the most active of all main metabolic pathways.

Nocardioides carbamazepini sp. nov., an ibuprofen degrader isolated from a biofilm bacterial community enriched on carbamazepine.

From the metagenome of a carbamazepine amended selective enrichment culture the genome of a new to science bacterial species affiliating with the genus Nocardioides was reconstructed. From the same enrichment an aerobic actinobacterium, strain CBZ_1T, sharing 99.4% whole-genome sequence similarity with the reconstructed Nocardioides sp. bin genome was isolated. On the basis of 16S rRNA gene sequence similarity the novel isolate affiliated to the genus Nocardioides, with the closest relatives Nocardioides kongjuensis DSM19082T (98.4%), Nocardioides daeguensis JCM17460T (98.4%) and Nocardioides nitrophenolicus DSM15529T (98.2%). Using a polyphasic approach it was confirmed that the isolate CBZ_1T represents a new phyletic lineage within the genus Nocardioides. According to metagenomic, metatranscriptomic studies and metabolic analyses strain CZB_1T was abundant in both carbamazepine and ibuprofen enrichments, and harbors biodegradative genes involved in the biodegradation of pharmaceutical compounds. Biodegradation studies supported that the new species was capable of ibuprofen biodegradation. After 7 weeks of incubation, in mineral salts solution supplemented with glucose (3 g l-1) as co-substrate, 70% of ibuprofen was eliminated by strain CBZ_1T at an initial conc. of 1.5 mg l-1. The phylogenetic, phenotypic and chemotaxonomic data supported the classification of strain CBZ_1T to the genus Nocardioides, for which the name Nocardioides carbamazepini sp. nov. (CBZ_1T = NCAIM B.0.2663 = LMG 32395) is proposed. To the best of our knowledge, this is the first study that reports simultaneous genome reconstruction of a new to science bacterial species using metagenome binning and at the same time the isolation of the same novel bacterial species.

Microbiomes in supragingival biofilms and saliva of adolescents with gingivitis and gingival health.

BACKGROUND: Important alterations exist in the microbiomes of supragingival biofilm and saliva samples from adolescent patients developing induced or spontaneous gingivitis relative to healthy controls. These and the relationships to dental health are not fully understood yet. SUBJECTS AND METHODS: Supragingival biofilm samples (n = 36) were collected from the teeth of 9 adolescents with gingivitis induced by orthodontic appliances, as well as dental plaques (n = 40) from 10 adolescents with spontaneous gingivitis, in addition to similar samples (n = 36) from 9 healthy controls. The bacterial metagenomes were analyzed by 16S rRNA gene amplicon sequencing. Salivary microbiomes of the same persons were characterized by shotgun metagenome sequencing. The data sets were examined using advanced bioinformatics workflows and two reference databases. RESULTS: The composition and diversity of bacterial communities did not differ extensively among the three study groups. Nevertheless, the relative abundances of the genera Fusobacterium, Akkermansia, Treponema, and Campylobacter were prominently higher in gingivitis patients versus controls. In contrast, the genera Lautropia, Kingella, Neisseria, Actinomyces, and Rothia were significantly more abundant in controls than in either of the two gingivitis groups. CONCLUSIONS: The abundance pattern of certain taxa rather than individual strains shows characteristic features of potential diagnostic value. Stringent bioinformatics treatment of the sequencing data is mandatory to avoid unintentional misinterpretations.

A comparison of the antimicrobial resistance of fecal Bacteroides isolates and assessment of the composition of the intestinal microbiotas of carbapenem-treated and non-treated persons from Belgium and Hungary.

The antimicrobial susceptibilities of Bacteroides strains isolated from the feces of imipenem-treated patients from Belgium and Hungary were compared with those isolated from the normal microbiota from these two and five other European countries and assessed. Of the 10 antibiotics tested, highly significant differences were found with cefoxitin (decrease for Belgium and for this two and the five countries from the previous study), clindamycin (decrease for Belgium and for this two and the five countries from the previous study) and moxifloxacin (increase for Belgium and for this two and the five countries from the previous study) relative to normal microbiota strains reported earlier. Imipenem treatment brought about modest, but notable differences in the compositions of the microbiomes where there was less diversity in the treated group relative to the non-treated group.

Genome-centric investigation of anaerobic digestion using sustainable second and third generation substrates.

Biogas production through co-digestion of second and third generation substrates is an environmentally sustainable approach. Green willow biomass, chicken manure waste and microalgae biomass substrates were combined in the anaerobic digestion experiments. Biochemical methane potential test showed that biogas yields of co-digestions were significantly higher compared to the yield when energy willow was the sole substrate. To scale up the experiment continuous stirred-tank reactors (CSRTs) are employed, digestion parameters are monitored. Furthermore, genome-centric metagenomics approach was employed to gain functional insight into the complex anaerobic decomposing process. This revealed the importance of Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes phyla as major bacterial participants, while Methanomicrobia and Methanobacteria represented the archaeal constituents of the communities. The bacterial phyla were shown to perform the carbohydrate hydrolysis. Among the representatives of long-chain carbohydrate hydrolysing microbes Bin_61: Clostridia is newly identified metagenome assembled genome (MAG) and Bin_13: DTU010 sp900018335 is common and abundant in all CSTRs. Methanogenesis was linked to the slow-growing members of the community, where hydrogenotrophic methanogen species Methanoculleus (Bin_10) and Methanobacterium (Bin_4) predominate. A sensitive balance between H2 producers and consumers was shown to be critical for stable biomethane production and efficient waste biodegradation.

Early response of methanogenic archaea to H2 as evaluated by metagenomics and metatranscriptomics.

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 (H2). Reduction of carbon dioxide (CO2) by H2 is rate limiting factor in methanogenesis, but the community intends to keep H2 concentration low in order to maintain the redox balance of the overall system. H2 metabolism in methanogens becomes increasingly 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 first responses of the microbial community in the CH4 formation and CO2 mitigation Power-to-Gas process. The overall microbial composition changes, following a 10 min excessive bubbling of H2 through the reactor, was investigated via metagenome and metatranscriptome sequencing. The overall composition 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 differences 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 H2 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-kingdom interactions in H2 metabolism are important for the efficient 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-off and turn-on of the Power-to-Gas microbial cell factories.

A comparative analysis of biogas production from tomato bio-waste in mesophilic batch and continuous anaerobic digestion systems.

Annually, agricultural activity produces an enormous amount of plant biomass by-product. Many studies have reported the biomethane potential of agro-industrial wastes, but only a few studies have investigated applying the substrates in both batch and continuous mode. Tomato is one of the most popular vegetables globally; its processing releases a substantial amount of by-product, such as stems and leaves. This study examined the BMP of tomato plant (Solanum lycopersicum Mill. L. cv. Alfred) waste. A comparative test revealed that the BMPs of corn stover, tomato waste,and their combination were approximately the same, around 280 mL methane/g Volatile Solid. In contrast, the relative biogas production decreased in the presence of tomato waste in a continuous mesophilic anaerobic digestion system; the daily biogas productions were 860 ± 80, 290 ± 50, and 570 ± 70 mL biogas/gVolatile Solid/day in the case of corn stover, tomato waste, and their mixture, respectively. The methane content of biogas was around 46-48%. The fermentation parameters of the continuous AD experiments were optimal in all cases; thus, TW might have an inhibitory effect on the microbial community. Tomato plant materials contain e.g. flavonoids, glycoalkaloids (such as tomatine and tomatidine), etc. known as antimicrobial and antifungal agents. The negative effect of tomatine on the biogas yield was confirmed in batch fermentation experiments. Metagenomic analysis revealed that the tomato plant waste caused significant rearrangements in the microbial communities in the continuously operated reactors. The results demonstrated that tomato waste could be a good mono-substrate in batch fermentations or a co-substrate with corn stover in a proper ratio in continuous anaerobic fermentations for biogas production. These results also point to the importance of running long-term continuous fermentations to test the suitability of a novel biomass substrate for industrial biogas production.

Toward Personalized Oral Diagnosis: Distinct Microbiome Clusters in Periodontitis Biofilms.

Periodontitis is caused by pathogenic subgingival microbial biofilm development and dysbiotic interactions between host and hosted microbes. A thorough characterization of the subgingival biofilms by deep amplicon sequencing of 121 individual periodontitis pockets of nine patients and whole metagenomic analysis of the saliva microbial community of the same subjects were carried out. Two biofilm sampling methods yielded similar microbial compositions. Taxonomic mapping of all biofilms revealed three distinct microbial clusters. Two clinical diagnostic parameters, probing pocket depth (PPD) and clinical attachment level (CAL), correlated with the cluster mapping. The dysbiotic microbiomes were less diverse than the apparently healthy ones of the same subjects. The most abundant periodontal pathogens were also present in the saliva, although in different representations. The single abundant species Tannerella forsythia was found in the diseased pockets in about 16-17-fold in excess relative to the clinically healthy sulcus, making it suitable as an indicator of periodontitis biofilms. The discrete microbial communities indicate strong selection by the host immune system and allow the design of targeted antibiotic treatment selective against the main periodontal pathogen(s) in the individual patients.

Chlorella vulgaris and Its Phycosphere in Wastewater: Microalgae-Bacteria Interactions During Nutrient Removal.

Microalgae-based bioenergy production is a promising field with regard to the wide variety of algal species and metabolic potential. The use of liquid wastes as nutrient clearly improves the sustainability of microalgal biofuel production. Microalgae and bacteria have an ecological inter-kingdom relationship. This microenvironment called phycosphere has a major role in the ecosystem productivity and can be utilized both in bioremediation and biomass production. However, knowledge on the effects of indigenous bacteria on microalgal growth and the characteristics of bacterial communities associated with microalgae are limited. In this study municipal, industrial and agricultural liquid waste derivatives were used as cultivation media. Chlorella vulgaris green microalgae and its bacterial partners efficiently metabolized the carbon, nitrogen and phosphorous content available in these wastes. The read-based metagenomics approach revealed a diverse microbial composition at the start point of cultivations in the different types of liquid wastes. The relative abundance of the observed taxa significantly changed over the cultivation period. The genome-centric reconstruction of phycospheric bacteria further explained the observed correlations between the taxonomic composition and biomass yield of the various waste-based biodegradation systems. Functional profile investigation of the reconstructed microbes revealed a variety of relevant biological processes like organic acid oxidation and vitamin B synthesis. Thus, liquid wastes were shown to serve as valuable resources of nutrients as well as of growth promoting bacteria enabling increased microalgal biomass production.

A case study of salivary microbiome in smokers and non-smokers in Hungary: analysis by shotgun metagenome sequencing.

OBJECTIVE: To investigate the role of cigarette smoking in disease-development through altering the composition of the oral microbial community. Periodontitis and oral cancer are highly prevalent in Hungary; therefore, the salivary microbiome of smoker and non-smoker Hungarian adults was characterized. METHODS: Shotgun metagenome sequencing of salivary DNA samples from 22 individuals (11 non-smokers and 11 current smokers) was performed using the Ion Torrent PGMTM platform. Quality-filtered reads were analysed by both alignment-based sequence similarity searches and genome-centric binning. RESULTS: Prevotella, Veillonella and Streptococcus were the predominant genera in the saliva of both groups. Although the overall composition and diversity of the microbiota were similar, Prevotella was significantly more abundant in salivary samples of current smokers compared to non-smokers. Members of the genus Prevotella were implicated in the development of inflammatory diseases and oral cancer. The abundance of the genus Megasphaera also increased in current smokers, whereas the genera Neisseria, Oribacterium, Capnocytophaga and Porphyromonas were significantly reduced. The data generated by read-based taxonomic classification and genome-centric binning mutually validated the two distinct metagenomic approaches. CONCLUSION: Smoking-associated dysbiosis of the salivary microbiome in current cigarette smokers, especially increased abundance of Prevotella and Megasphaera genera, may facilitate disease development.

Integrated evolutionary analysis reveals antimicrobial peptides with limited resistance.

Antimicrobial peptides (AMPs) are promising antimicrobials, however, the potential of bacterial resistance is a major concern. Here we systematically study the evolution of resistance to 14 chemically diverse AMPs and 12 antibiotics in Escherichia coli. Our work indicates that evolution of resistance against certain AMPs, such as tachyplesin II and cecropin P1, is limited. Resistance level provided by point mutations and gene amplification is very low and antibiotic-resistant bacteria display no cross-resistance to these AMPs. Moreover, genomic fragments derived from a wide range of soil bacteria confer no detectable resistance against these AMPs when introduced into native host bacteria on plasmids. We have found that simple physicochemical features dictate bacterial propensity to evolve resistance against AMPs. Our work could serve as a promising source for the development of new AMP-based therapeutics less prone to resistance, a feature necessary to avoid any possible interference with our innate immune system.

The Planktonic Core Microbiome and Core Functions in the Cattle Rumen by Next Generation Sequencing.

The cow rumen harbors a great variety of diverse microbes, which form a complex, organized community. Understanding the behavior of this multifarious network is crucial in improving ruminant nutrient use efficiency. The aim of this study was to expand our knowledge by examining 10 Holstein dairy cow rumen fluid fraction whole metagenome and transcriptome datasets. DNA and mRNA sequence data, generated by Ion Torrent, was subjected to quality control and filtering before analysis for core elements. The taxonomic core microbiome consisted of 48 genera belonging to Bacteria (47) and Archaea (1). The genus Prevotella predominated the planktonic core community. Core functional groups were identified using co-occurrence analysis and resulted in 587 genes, from which 62 could be assigned to metabolic functions. Although this was a minimal functional core, it revealed key enzymes participating in various metabolic processes. A diverse and rich collection of enzymes involved in carbohydrate metabolism and other functions were identified. Transcripts coding for enzymes active in methanogenesis made up 1% of the core functions. The genera associated with the core enzyme functions were also identified. Linking genera to functions showed that the main metabolic pathways are primarily provided by Bacteria and several genera may serve as a "back-up" team for the central functions. The key actors in most essential metabolic routes belong to the genus Prevotella. Confirming earlier studies, the genus Methanobrevibacter carries out the overwhelming majority of rumen methanogenesis and therefore methane emission mitigation seems conceivable via targeting the hydrogenotrophic methanogenesis.

Anaerobic gaseous biofuel production using microalgal biomass - A review.

Most photosynthetic organisms store and convert solar energy in an aerobic process and produce biomass for various uses. Utilization of biomass for the production of renewable energy carriers employs anaerobic conditions. This review focuses on microalgal biomass and its use for biological hydrogen and methane production. Microalgae offer several advantages compared to terrestrial plants. Strategies to maintain anaerobic environment for biohydrogen production are summarized. Efficient biogas production via anaerobic digestion is significantly affected by the biomass composition, pretreatment strategies and the parameters of the digestion process. Coupled biohydrogen and biogas production increases the efficiency and sustainability of renewable energy production.

Bioaugmentation of the thermophilic anaerobic biodegradation of cellulose and corn stover.

Two stable, thermophilic mixed cellulolytic consortia were enriched from an industrial scale biogas fermenter. The two consortia, marked as AD1 and AD2, were used for bioaugmentation in laboratory scale batch reactors. They enhanced the methane yield by 22-24%. Next generation sequencing method revealed the main orders being Thermoanaerobacterales and Clostridiales and the predominant strains were Thermoanaerobacterium thermosaccharolyticum, Caldanaerobacter subterraneus, Thermoanaerobacter pseudethanolicus and Clostridium cellulolyticum. The effect of these strains, cultivated in pure cultures, was investigated with the aim of reconstructing the defined cellulolytic consortium. The addition of the four bacterial strains and their mixture to the biogas fermenters enhanced the methane yield by 10-11% but it was not as efficient as the original communities indicating the significant contribution by members of the enriched communities present in low abundance.

Biomethane: The energy storage, platform chemical and greenhouse gas mitigation target.

Results in three areas of anaerobic microbiology in which methane formation and utilization plays central part are reviewed. a.) Bio-methane formation by reduction of carbon dioxide in the power-to-gas process and the various possibilities of improvement of the process is a very intensively studied topic recently. From the numerous potential methods of exploiting methane of biological origin two aspects are discussed in detail. b.) Methane can serve as a platform chemical in various chemical and biochemical synthetic processes. Particular emphasis is put on the biochemical conversion pathways involving methanotrophs and their methane monooxygenase-catalyzed reactions leading to various small molecules and polymeric materials such as extracellular polysaccharides, polyhydroxyalkanoates and proteins. c.) The third area covered concerns methane-consuming reactions and methane emission mitigation. These investigations comprise the anaerobic microbiology of ruminants and approaches to diminishing methane emissions from ruminant animals.

Pretreatment of poultry manure for efficient biogas production as monosubstrate or co-fermentation with maize silage and corn stover.

Water extraction of raw chicken manure elevated the carbon-to-nitrogen ratio 2.7-fold, i.e. from 7.48 to 19.81. The treated chicken manure (T-CM) thus became suitable for biogas fermentation as monosubstrate. Improved methane production was achieved in co-fermentations with either maize silage (24% more methane) or corn stover (19% more methane) relative to T-CM monosubstrate. The standardized biogas potential assay indicated that the methane yields varied with the organic loading rate between 160 and 250 mL CH4/g organic total solid (oTS). Co-fermentation with maize silage was sustainable in continuous anaerobic digestion for at least 4 months.

Induced Hyperon-Nucleon-Nucleon Interactions and the Hyperon Puzzle.

We present the first ab initio calculations for p-shell hypernuclei including hyperon-nucleon-nucleon (YNN) contributions induced by a similarity renormalization group transformation of the initial hyperon-nucleon interaction. The transformation including the YNN terms conserves the spectrum of the Hamiltonian while drastically improving model-space convergence of the importance-truncated no-core model, allowing a precise extraction of binding and excitation energies. Results using a hyperon-nucleon interaction at leading order in chiral effective field theory for lower- to mid-p-shell hypernuclei show a good reproduction of experimental excitation energies while hyperon separation energies are typically overestimated. The induced YNN contributions are strongly repulsive and we show that they are related to a decoupling of the Σ hyperons from the hypernuclear system, i.e., a suppression of the Λ-Σ conversion terms in the Hamiltonian. This is linked to the so-called hyperon puzzle in neutron-star physics and provides a basic mechanism for the explanation of strong ΛNN three-baryon forces.

Metagenome changes in the mesophilic biogas-producing community during fermentation of the green alga Scenedesmus obliquus.

A microalgal biomass offers a potential alternative to the maize silage commonly used in biogas technology. In this study, photoautotrophically grown Scenedesmus obliquus was used as biogas substrate. This microalga has a low C/N ratio of 8.5 relative to the optimum 20-30. A significant increase in the ammonium ion content was not observed. The methane content of the biogas generated from Sc. obliquus proved to be higher than that from maize silage, but the specific biogas yield was lower. Semi-continuous steady biogas production lasted for 2 months. Because of the thick cell wall of Sc. obliquus, the biomass-degrading microorganisms require additional time to digest its biomass. The methane concentration in the biogas was also high, in co-digestion (i.e., 52-56%) as in alga-fed anaerobic digestion (i.e., 55-62%). These results may be related to the relative predominance of the order Clostridiales in co-digestion and to the more balanced C/N ratio of the mixed algal-maize biomass. Predominance of the order Methanosarcinales was observed in the domain Archaea, which supported the diversity of metabolic pathways in the process.