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 .

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.

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.

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.

Adaptation of continuous biogas reactors operating under wet fermentation conditions to dry conditions with corn stover as substrate.

Corn stover (CS) is the agricultural by-product of maize cultivation. Due to its high abundance and high energy content it is a promising substrate for the bioenergy sector. However, it is currently neglected in industrial scale biogas plants, because of its slow decomposition and hydrophobic character. To assess the maximum biomethane potential of CS, long-term batch fermentations were carried out with various substrate concentrations and particle sizes for 72 days. In separate experiments we adapted the biogas producing microbial community in wet fermentation arrangement first to the lignocellulosic substrate, in Continuous Stirred Tank Reactor (CSTR), then subsequently, by continuously elevating the feed-in concentration, to dry conditions in solid state fermenters (SS-AD). In the batch tests, the <10 mm fraction of the grinded and sieved CS was amenable for biogasification, but it required 10% more time to produce 90% of the total biomethane yield than the <2 mm sized fraction, although in the total yields there was no significant difference between the two size ranges. We also observed that increasing amount of substrate added to the fermentation lowered the specific methane yield. In the CSTR experiment, the daily substrate loading was gradually increased from 1 to 2 gvs/L/day until the system produced signs of overloading. Then the biomass was transferred to SS-AD reactors and the adaptation process was studied. Although the specific methane yields were lower in the SS-AD arrangement (177 mL CH4/gvs in CSTR vs. 105 mL in SS-AD), the benefits of process operational parameters, i.e. lower energy consumption, smaller reactor volume, digestate amount generated and simpler configuration, may compensate the somewhat lower yield.

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.

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.

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.

HupO, a Novel Regulator Involved in Thiosulfate-Responsive Control of HupSL [NiFe]-Hydrogenase Synthesis in Thiocapsa roseopersicina.

[NiFe]-hydrogenases are regulated by various factors to fulfill their physiological functions in bacterial cells. The photosynthetic purple sulfur bacterium Thiocapsa roseopersicina harbors four functional [NiFe]-hydrogenases: HynSL, HupSL, Hox1, and Hox2. Most of these hydrogenases are functionally linked to sulfur metabolism, and thiosulfate has a central role in this organism. The membrane-associated Hup hydrogenases have been shown to play a role in energy conservation through hydrogen recycling. The expression of Hup-type hydrogenases is regulated by H2 in Rhodobacter capsulatus and Cupriavidus necator; however, it has been shown that the corresponding hydrogen-sensing system is nonfunctional in T. roseopersicina and that thiosulfate is a regulating factor of hup expression. Here, we describe the discovery and analysis of mutants of a putative regulator (HupO) of the Hup hydrogenase in T. roseopersicina. HupO appears to mediate the transcriptional repression of Hup enzyme synthesis under low-thiosulfate conditions. We also demonstrate that the presence of the Hox1 hydrogenase strongly influences Hup enzyme synthesis in that hup expression was decreased significantly in the hox1 mutant. This reduction in Hup synthesis could be reversed by mutation of hupO, which resulted in strongly elevated hup expression, as well as Hup protein levels, and concomitant in vivo hydrogen uptake activity in the hox1 mutant. However, this regulatory control was observed only at low thiosulfate concentrations. Additionally, weak hydrogen-dependent hup expression was shown in the hupO mutant strain lacking the Hox1 hydrogenase. HupO-mediated Hup regulation therefore appears to link thiosulfate metabolism and the hydrogenase network in T. roseopersicina.

Connection between the membrane electron transport system and Hyn hydrogenase in the purple sulfur bacterium, Thiocapsa roseopersicina BBS.

Thiocapsa. roseopersicina BBS has four active [NiFe] hydrogenases, providing an excellent opportunity to examine their metabolic linkages to the cellular redox processes. Hyn is a periplasmic membrane-associated hydrogenase harboring two additional electron transfer subunits: Isp1 is a transmembrane protein, while Isp2 is located on the cytoplasmic side of the membrane. In this work, the connection of HynSL to various electron transport pathways is studied. During photoautotrophic growth, electrons, generated from the oxidation of thiosulfate and sulfur, are donated to the photosynthetic electron transport chain via cytochromes. Electrons formed from thiosulfate and sulfur oxidation might also be also used for Hyn-dependent hydrogen evolution which was shown to be light and proton motive force driven. Hyn-linked hydrogen uptake can be promoted by both sulfur and nitrate. The electron flow from/to HynSL requires the presence of Isp2 in both directions. Hydrogenase-linked sulfur reduction could be inhibited by a QB site competitive inhibitor, terbutryne, suggesting a redox coupling between the Hyn hydrogenase and the photosynthetic electron transport chain. Based on these findings, redox linkages of Hyn hydrogenase are modeled.

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.

Pretreatment of lignocellulosic biogas substrates by filamentous fungi.

Decomposition of lignocellulosic plant biomass by four filamentous fungi was carried out to facilitate subsequent anaerobic degradation and biogas formation. Agricultural side products, wheat straw and corn stover and forestry energy plant willow chips were selected as plant biomass sources. The substrates were confronted by pure cultures of Penicillium aurantiogriseum (new isolate from rumen), Trichoderma reesei (DSM768), Gilbertella persicaria (SZMC11086) and Rhizomucor miehei (SZMC11005). In addition to total cellulolytic filter paper degradation activity, the production of endoglucanase, cellobiohydrolase, ß-glucosidase enzymes were followed during the pretreatment period, which lasted for 10 days at 37 °C. The products of pretreatments were subsequently tested for mesophilic biogas production in batch reactors. All 4 strains effectively pretreated the lignocellulosic substrates albeit in varying degrees, which was related to the level of the tested hydrolytic enzyme activities. Penicillium aurantiogriseum showed outstanding hydrolytic enzyme production and highest biogas yield from the partially degraded substrates. Corn stover was the best substrate for biomass decomposition and biogas production. Scanning electron microscopy confirmed the deep penetration of fungal hyphae into the lignocellulosic substrate in all cases.

Triploid Hybrid Vigor in Above-Ground Growth and Methane Fermentation Efficiency of Energy Willow.

Hybrid vigor and polyploidy are genetic events widely utilized to increase the productivity of crops. Given that bioenergy usage needs to be expanded, we investigated triploid hybrid vigor in terms of the biology of biomass-related willow traits and their relevance to the control of biomethane production. To produce triploid hybrid genotypes, we crossed two female diploid Swedish cultivars (Inger, Tordis) with two male autotetraploid willow (Salix viminalis) variants (PP-E7, PP-E15). Field studies at two locations and in two successive years recorded considerable midparent heterosis (MPH%) in early shoot length that ranged between 11.14 and 68.85% and in the growth rate between 34.12 and 97.18%. The three triploid hybrids (THs) developed larger leaves than their parental cultivars, and the MPH% for their CO2 assimilation rate varied between 0.84 and 25.30%. The impact of hybrid vigor on the concentrations of plant hormones in these TH genotypes reflected essentially different hormonal statuses that depended preferentially on maternal parents. Hybrid vigor was evinced by an elevated concentration of jasmonic acid in shoot meristems of all the three THs (MPH:29.73; 67.08; 91.91%). Heterosis in auxin-type hormones, such as indole-3-acetic acid (MPH:207.49%), phenylacetic acid (MPH:223.51%), and salicylic acid (MPH:27.72%) and benzoic acid (MPH:85.75%), was detectable in the shoots of TH21/2 plants. These hormones also accumulated in their maternal Inger plants. Heterosis in cytokinin-type hormones characterized the shoots of TH3/12 and TH17/17 genotypes having Tordis as their maternal parent. Unexpectedly, we detected abscisic acid as a positive factor in the growth of TH17/17 plants with negative MPH percentages in stomatal conductance and a lower CO2 assimilation rate. During anaerobic digestion, wood raw materials from the triploid willow hybrids that provided positive MPH% in biomethane yield (6.38 and 27.87%) showed negative MPH in their acid detergent lignin contents (from -8.01 to -14.36%). Altogether, these insights into controlling factors of above-ground growth parameters of willow genotypes support the utilization of triploid hybrid vigor in willow breeding to expand the cultivation of short rotation energy trees for renewable energy production.

Comparison of transcriptional and translational changes caused by long-term menadione exposure in Aspergillus nidulans.

Under long-term oxidative stress caused by menadione sodium bisulfite, genome-wide transcriptional and proteome-wide translational changes were compared in Aspergillus nidulans vegetative cells. The comparison of proteomic and DNA microarray expression data demonstrated that global gene expression changes recorded with either flip-flop or dendrimer cDNA labeling techniques supported proteome changes moderately with 40% and 34% coincidence coefficients, respectively. Enzyme levels in the glycolytic pathway were alternating, which was a direct consequence of fluctuating gene expression patterns. Surprisingly, enzymes in the vitamin B2 and B6 biosynthetic pathways were repressed concomitantly with the repression of some protein folding chaperones and nuclear transport elements. Under long-term oxidative stress, the peroxide-detoxifying peroxiredoxins and cytochrome c peroxidase were replaced by thioredoxin reductase, a nitroreductase and a flavohemoprotein, and protein degradation became predominant to eliminate damaged proteins.

Use of ensiled green willow biomass in biogas fermentation.

The biggest challenges of our era include climate change and the global fossil energy problem. Extensive utilization of renewable energy sources should be a part of the solution for both these problems. Biogas is a versatile renewable energy carrier that has the potential to substitute fossil fuels. The most frequently utilized substrates for the anaerobic digestion (AD) process include maize silage today, but there is an increasing demand for second-generation biomass sources, which are cheaper and do not interfere with the cultivation of food production. Green biomass from short rotation coppice willow (GWB) may be a promising alternative. However, to ensure feedstock quantity and quality all year round, a preservation method has to be developed. We attempted to ensilage the biomass and subsequently utilized the resulting willow-silage in batch fermenters. Various mixtures of lactic acid bacteria were employed to facilitate ensiling by inoculation of the substrate in anaerobic jars for 60 days. During the ensiling analytical investigations, (HPLC, pH, oTS/TS%) were carried out in order to follow the build-up of fermentation products. AD fermentations were assembled from the ensilaged biomass and the methane production was measured for 56 days. The total methane yields of the ensilaged biomass were 8-15% higher than that of the fresh biomass and methane production rates were also improved. Our findings suggest that ensiling is not only an excellent preservation method for willow biomass, but also stimulates its AD.

Enhancing biogas production from agroindustrial waste pre-treated with filamentous fungi.

Biogas is the product of anaerobic digestion (AD) of organic waste and is considered to be one of the most valuable natural renewable energy carriers. Plant biomass represents the most abundant eco-friendly energy reservoir on Earth. However, the tenacious and heterogeneous structure of the lignocellulose-rich elements makes it difficult for the involved microbes to digest the recalcitrant substrates. Both the degradation process and the biogas production yield can be enhanced by appropriate pre-treatment of lignocellulosic materials. Filamentous fungi have been known as proficient colonizers of lignocellulosic plant tissues and have been recognized as producers of exceptionally rich and diverse hydrolytic enzymes. We tested Aspergillus nidulans, Trichoderma reesei, Rhizomucor miehei and Gilbertella persicaria filamentous fungal strains for pre-treatment of various agricultural lignocellulosic wastes. During the pre-treatment phase, the ß-glucosidase and endoglucanase activity was measured spectrophotometrically. In the AD step, methane production was monitored by gas chromatography. The preliminary results showed that all the applied strains (Aspergillus nidulans, Trichoderma reesei, Rhizomucor miehei and Gilbertella persicaria) were highly effective in producing both ß-glucosidase and endo-(1,4)-ß-D-glucanase enzymes, which might explain the greatly improved AD results. Pre-treatment with the above-mentioned filamentous fungi positively affected the biogas production, although the effect strongly depended on the selection of the fungal partner for any given biomass substrate. Depending on the used substrate and the pre-treatment strain, overall methane yields were elevated two-fold relative to the controls.

Interactions between probiotic and oral pathogenic strains.

More than 6 billion bacteria and other microorganisms live in the adult oral cavity. As a result of any deleterious effect on this community, some microorganisms will survive better than others, which may trigger pathogenic processes like caries, halitosis, gingivitis or periodontitis. Oral dysbiosis is among the most frequent human health hazards globally. Quality of life of patients deteriorates notably, while treatments are often unpleasant, expensive and irreversible, e.g. tooth loss. In the experiments reported here, we investigated the individual interactions between 8 pathogenic and 8 probiotic strains and a commercially available probiotic product. Almost all pathogens, namely Fusobacterium nucleatum, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Streptococcus mutans, Streptococcus oralis, Streptococcus gordonii, Enterococcus faecalis and Prevotella buccae are pathogens frequently occurring in the oral cavity. The used probiotic strains were Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus delbrueckii, Bifidobacterium thermophilum and two Streptococcus dentisani isolates. Using a modified agar diffusion method, we investigated capability of the probiotic bacteria to prevent the growth of the pathogenic ones in order to identify candidates for future therapeutic treatments. The results indicated successful bacteriocin production, i.e. growth inhibition, against every pathogenic bacterium by at least 5 probiotic strains.

Methane production from green and woody biomass using short rotation willow genotypes for bioenergy generation.

Short rotation plantations of willow genotypes, harvested in vegetative growth phases, were tested as an alternative biomass for methane production. The substrate characteristics, maximal methane yields (K) and highest methane production rates (µmax) were determined. Leaves and stems from diploid Energo (EN) and tetraploid (PP) plants, harvested in June were superior methane sources to woody tissue. This could be related to the lower lignin contents in green willow. Fermentation of pooled biomasses from tetraploid genotypes harvested in June-August was more efficient than methane production from diploid tissues. Microbial community analyses by 16S rRNA genes showed a dominance of the order Clostridiales. In field study, based on Energo plantation, the maximum in green biomass accumulation was in early month 9 of the vegetation period. A theoretical calculation showed similar or better energy potential per unit area for willow than in the case of maize silage. This study encourages the use of green willow biomass as feedstock in biomethanation processes due to its relatively low production costs and uncomplicated agricultural practice.