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The aim of this study was to investigate the production of succinic acid from lactose concentrate, a by-product of cheese-making, using Actinobacillus succinogenes and Basfia succiniciproducens. Although the ability of these strains to metabolize different sugars is already known, their application in the conversion of lactose bears high potential for optimization. With regard to B. succiniciproducens, this approach is completely novel. In particular, the effect of the medium’s sugar concentration as well as the ability of its supplementation with yeast extract to prevent a lack of essential nutrient proteins and vitamins was examined. Lactose-based media containing sugar concentrations of between 20 and 65 g L−1 and 5 g L−1 of yeast extract were fermented, with both strains showing comparable performances. The best results in terms of succinic acid yield and acid concentration—0.57 g g−1 initial sugar and 23 g L−1—were achieved at an initial sugar concentration of 43 g L−1. The necessity of yeast extract was demonstrated using the sugar-optimized medium without supplementation. As a result, the yield and concentration of succinic acid dropped to 0.34 g g−1 and 13 g L−1 and the sugar consumption decreased from more than 99 to less than 55%. Therefore, the supplementation amount of 5 g L−1 of yeast extract can be regarded as well-balanced.

The aim of this study was to investigate succinic acid production from lactose concentrate, a by-product of cheese-making, using Actinobacillus succinogenes and Basfia succiniciproducens. Although the capability of these strains to metabolize different sugars is already known, their application in the conversion of lactose bears high potential for optimization. With regard to B. succiniciproducens this approach is completely novel. In particular the influence of the mediums sugar concentration and its supplementation with yeast extract to prevent a lack of proteins and vitamins were examined. Lactose based media containing sugar concentrations between 20 and 65 g L-1 and 5 g L-1 yeast extract were fermented, whereby both strains showed comparable performances. The best results in succinic acid yield and acid concentration, 0.57 g g-1 initial sugar and 23 g L1, were achieved at an initial sugar concentration of 43 g L-1. The necessity of yeast extract was demonstrated using the sugar optimized medium without supplementation. As a result, yield and concentration of succinic acid dropped to 0.34 g g-1 and 13 g L-1, the sugar consumption decreased from more than 99 to less than 55 %. Therefore the supplementation amount of 5 g L-1 yeast extract can be regarded as well-balanced.

This work demonstrates the suitability of nitrogen removal during anaerobic acidification in batch configuration for a more efficient pre-treatment of chicken manure prior to anaerobic digestion. High loading rates corresponding to a total nitrogen input between 6.3 and 9.5 g L−1 allowed successful suppression of methanogenic archaea. To eliminate nitrogen, NH3-stripping and MAP (magnesium ammonium phosphate hexahydrate) precipitation were compared. In spite of decreased cell quantities detected using qPCR, removal of nitrogen caused an increase in volatile fatty acid (VFA) formation from 13 to 19%. The highest nitrogen removal during acidification (up to 29%) was achieved with three consecutives MAP precipitation steps, however, conductivity values were affected too, reaching 53.3 and 53.1 mS cm−1 after the three consecutive MAP precipitations. Additionally, MAP-precipitation reduced the concentration of important trace elements and 16S-rRNA amplicon sequencing revealed an altered taxonomic pattern, in which especially the bacterial families Marinilabiliaceae, Bacteroidales UCG-001, M2PB4-65 termite group and Idiomarinaceae were impaired. However, in spite of these inhibitory effects, nitrogen removal proved able to prevent unwanted methanogenesis and to enhance the yield of VFAs, and this strategy thus holds great potential for the optimized production of biogas in a two-phase system., We are thankful for the funding received from the German Ministry of Economic Affairs and Energy (grant no. 16KN041338, 16KN041342, 16KN041339 and 16KN041341).

We report here the impact of heat-shock treatments (55 and 70 °C) on the biogas production within the acidification stage of a two-stage reactor system for anaerobic digestion and biomethanation of grass. The microbiome proved both taxonomically and functionally very robust, since heat shocks caused minor community shifts compared to the controls, and biogas yield was not decreased. The strongest impact on the microbial profile was observed with a combination of heat shock and low pH. Since no transient reduction of microbial diversity occured after the shock, biogas keyplayers, but also potential pathogens, survived the treatment. All along the experiment, the heat-resistant bacterial profile consisted mainly of Firmicutes, Bacteroidetes and Proteobacteria. Bacteroides and Acholeplasma were reduced after heat shocks. An increase was observed for Aminobacterium. Our results prove the stability to thermal stresses of the microbial communities involved in acidification, and the resilience in biogas production irrespectively of the thermal treatment.

Background Separating acidification and methanogenic steps in anaerobic digestion processes can help to optimize the process and contribute to producing valuable sub-products such as methane, hydrogen and organic acids. However, the full potential of this technology has not been fully explored yet. To assess the underlying fermentation process in more detail, a combination of high-throughput sequencing and proteomics on the acidification step of plant material (grass) at both mesophilic and thermophilic temperatures (37 and 55 °C, respectively) was applied for the first time. Results High-strength liquor from acidified grass biomass exhibited a low biodiversity, which differed greatly depending on temperature. It was dominated by Bacteroidetes and Firmicutes at 37 °C, and by Firmicutes and Proteobacteria at 55 °C. At the methane stage, Methanosaeta, Methanomicrobium and Methanosarcina proved to be highly sensitive to environmental changes as their abundance in the seed sludges dropped dramatically after transferring the seed sludges from the respective reactors into the experimental setup. Further, an increase in Actinobacteria coincided with reduced biogas production at the end of the experiment. Over 1700 proteins were quantified from the first cycle of acidification samples using label-free quantitative proteome analysis and searching protein databases. The most abundant proteins included an almost complete set of glycolytic enzymes indicating that the microbial population is basically engaged in the degradation and catabolism of sugars. Differences in protein abundances clearly separated samples into two clusters corresponding to culture temperature. More differentially expressed proteins were found under mesophilic (120) than thermophilic (5) conditions. Conclusion Our results are the first multi-omics characterisation of a two-stage biogas production system with separated acidification and suggest that screening approaches targeting specific taxa such as Methanosaeta, Methanomicrobium and Methanosarcina could be useful diagnostic tools as indicators of environmental changes such as temperature or oxidative stress or, as in the case of Actinobacteria, they could be used as a proxy of the gas production potential of anaerobic digesters. Metaproteome analyses only detected significant expression differences in mesophilic samples, whereas thermophilic samples showed more stable protein composition with an abundance of chaperones suggesting a role in protein stability under thermal stress. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0859-0) contains supplementary material, which is available to authorized users.

Here, we present the genome sequence and annotation of HV4-6-C5C, a bacterial strain isolated from a mesophilic two-stage laboratory-scale leach bed biogas reactor system. Strain HV4-6-C5C may represent a new genus of the family Bacteroidaceae and may have a key role in acidogenesis and acetogenesis steps during anaerobic biomass digestion.

Anaerobic digestion is a technology known for its potential in terms of methane production. During the digestion process, multiple metabolites of high value are synthesized. However, recent works have demonstrated the high robustness and resilience of the involved microbiomes; these attributes make it difficult to manipulate them in such a way that a specific metabolite is predominantly produced. Therefore, an exact understanding of the manipulability of anaerobic microbiomes may open up a treasure box for bio-based industries. In the present work, the effect of nalidixic acid, γ-aminobutyric acid (GABA), and sodium phosphate on the microbiome of digested sewage sludge from a water treatment plant fed with glucose was investigated. Despite of the induced process perturbations, high stability was observed at the phylum level. However, strong variations were observed at the genus level, especially for the genera Trichococcus, Candidatus Caldatribacterium, and Phascolarctobacterium. Ecological interactions were analyzed based on the Lotka–Volterra model for Trichococcus, Rikenellaceae DMER64, Sedimentibacter, Candidatus Cloacimonas, Smithella, Cloacimonadaceae W5 and Longilinea. These genera dynamically shifted among positive, negative or no correlation, depending on the applied stressor, which indicates a surprisingly dynamic behavior. Globally, the presented work suggests a massive resilience and stability of the methanogenic communities coupled with a surprising flexibility of the particular microbial key players involved in the process.

Conventional anaerobic digesters intended for the production of biogas usually operate in complete darkness. Therefore, little is known about the effect of light on microbial communities operating in anaerobic digesters. In the present work, we have studied through 16S rRNA gene amplicon Nanopore sequencing and shotgun metagenomic sequencing the taxonomic and functional structure of the microbial community forming a biofilm on the inner wall of a lab-scale transparent anaerobic biodigester illuminated with natural sunlight. The biofilm was composed of microorganisms involved in the four metabolic processes needed for biogas production. The biofilm proved surprisingly rich in Rhodopseudomonas faecalis, a versatile bacterium able to carry out a photoautotroph metabolism when grown under anaerobic conditions. Our results suggest that this bacterium, able to fix carbon dioxide, could be considered for its use in transparent biogas fermenters in order to contribute to the production of optimized biogas with a higher CH4:CO2 ratio than the biogas produced in regular, opaque digesters. To the best of our knowledge, this is the first study supporting illuminated bioreactors as a new bioprocess for the obtention of biogas enriched in methane.

Here, we present the genome sequence and annotation of HV4-6-C5C, a bacterial strain isolated from a mesophilic two-stage laboratory-scale leach bed biogas reactor system. Strain HV4-6-C5C may represent a new genus of the family Bacteroidaceae and may have a key role in acidogenesis and acetogenesis steps during anaerobic biomass digestion., We acknowledge funding by the European Union through the BioRoboost project (H2020-NMBP-TR-IND-2018-2020/BIOTEC-01-2018 [Coordination and Support Action], project identifier 210491758). Moreover, this study was funded by the German Federal Ministry of Economic Affairs and Energy (grant numbers KF 2050830SA4, KF 3400701SA4, and KF 2112205SA4).

A new Ruminococcaceae bacterium, strain HV4-5-B5C, participating in the anaerobic digestion of grass, was isolated from a mesophilic two-stage laboratory-scale leach bed biogas system. The draft annotated genome sequence presented in this study and 16S rRNA gene sequence analysis indicated the affiliation of HV4-5-B5C with the family Ruminococcaceae outside recently described genera.

In this work, liquid and solid fractions of grass biomass were used as co-substrates for anaerobic co-digestion of sewage sludge. The input of grass biomass was increased gradually, and the underlying methanogenic microbiome was assessed by means of microscopy-based cell counting and full-length 16S rRNA gene high-throughput sequencing, proving for the first time the suitability of nanopore-based portable sequencers as a monitoring tool for anaerobic digestion systems. In both cases co-fermentation resulted in an increased number of bacteria and methanogenic archaea. Interestingly, the microbial communities were highly different between solid and liquid-fed batches. Liquid-fed batches developed a more stable microbiome, enriched in Methanosarcina spp., and resulted in higher methanogenic yield. In contrast, solid-fed batches were highly unstable at higher substrate concentrations, and kept Methanosaeta spp. – typically associated to sewage sludge – as the majoritary methanogenic archaea.

A range of parameters are known to shape the methanogenic communities of biogas-producing digesters and to strongly influence the amount of biogas produced. In this work, liquid and solid fractions of grass biomass were used separately for semicontinuous batch methanation using sewage sludge as seed sludge. During 6 months of incubation, the amount of input COD was increased gradually, and the underlying methanogenic microbiome was assessed by means of microscopy-based automated cell counting and full-length 16S rRNA high-throughput sequencing. In this sense, we prove for the first time the suitability of the ONT™MinION platform as a monitoring tool for anaerobic digestion systems. According to our results, solid-fed batches were highly unstable at higher COD input concentrations, and kept Methanosaeta spp. typically associated to sewage sludge-as the majoritary methanogenic archaea. In contrast, liquid-fed batches developed a more stable microbiome, proved enriched in Methanosarcina spp, and resulted in higher methanogenic yield. This work demonstrates the high repowering potential of microbiomes from sewage sludge digesters, and highlight the effectiveness of liquefied substrates for increasing biogas productivity in anaerobic digestions.

The kinetochore proteins assemble onto centromeric chromatin and regulate DNA segregation during cell division. The inner kinetochore proteins bind centromeres while most outer kinetochore proteins assemble at centromeres during mitosis, connecting the complex to microtubules. The centromere-kinetochore complex contains specific nucleosomes and nucleosomal particles. CENP-A replaces canonical H3 in centromeric nucleosomes, defining centromeric chromatin. Next to CENP-A, the CCAN multi-protein complex settles which contains CENP-T/W/S/X. These four proteins are described to form a nucleosomal particle at centromeres. We had found the CENP-T C-terminus and the CENP-S termini next to histone H3.1 but not to CENP-A, suggesting that the Constitutive Centromere-Associated Network (CCAN) bridges a CENP-A- and a H3-containing nucleosome. Here, we show by in vivo FRET that this proximity between CENP-T and H3 is specific for H3.1 but neither for the H3.1 mutants H3.1(C96A) and H3.1(C110A) nor for H3.2 or H3.3. We also found CENP-M next to H3.1 but not to these H3.1 mutants. Consistently, we detected CENP-M next to CENP-S. These data elucidate the local molecular neighborhood of CCAN proteins next to a H3.1-containing centromeric nucleosome. They also indicate an exclusive position of H3.1 clearly distinct from H3.2, thus documenting a local, and potentially also functional, difference between H3.1 and H3.2.

A new Firmicutes isolate, strain HV4-6-A5C, was obtained from the hydrolysis stage of a mesophilic and anaerobic two-stage lab-scale leach-bed system for biomethanation of fresh grass. It is assumed that the bacterial isolate contributes to plant biomass degradation. Here, we report a draft annotated genome sequence of this organism.

In the present work we investigated how the state of a biogas reactor impacts the enumeration of prokaryotic cells by fluorescencein situhybridisation (FISH). Therefore, the correlation between gas production and FISH hybridisation rates was analysed in different anaerobic digester sludges. High gasification activity coincided with high hybridisation rates. Low hybridisation rates were especially achieved with reactor samples subjected to long starvation periods showing low biogas production.Based on our findings we conclude that samples for FISH analysis should be fixed as soon as possible to prevent a loss of microbial activity resulting in lower FISH signals. Furthermore, the location of sampling is of importance, since samples from different fermenters within the same biogas plant also varied strongly in their FISH hybridisation rate. Our results indicate that FISH could be a useful method for assessing the metabolic state of microorganisms in anaerobic digester plants.

Background Only a fraction of the microbial species used for anaerobic digestion in biogas production plants are methanogenic archaea. We have analyzed the taxonomic profiles of eubacteria and archaea, a set of chemical key parameters, and biogas production in samples from nine production plants in seven facilities in Thuringia, Germany, including co-digesters, leach-bed, and sewage sludge treatment plants. Reactors were sampled twice, at a 1-week interval, and three biological replicates were taken in each case. Results A complex taxonomic composition was found for both eubacteria and archaea, both of which strongly correlated with digester type. Plant-degrading Firmicutes as well as Bacteroidetes dominated eubacteria profiles in high biogas-producing co-digesters; whereas Bacteroidetes and Spirochaetes were the major phyla in leach-bed and sewage sludge digesters. Methanoculleus was the dominant archaea genus in co-digesters, whereas Methanosarcina and Methanosaeta were the most abundant methanogens in leachate from leach-bed and sewage sludge digesters, respectively. Conclusions This is one of the most comprehensive characterizations of the microbial communities of biogas-producing facilities. Bacterial profiles exhibited very low variation within replicates, including those of semi-solid samples; and, in general, low variation in time. However, facility type correlated closely with the bacterial profile: each of the three reactor types exhibited a characteristic eubacteria and archaea profile. Digesters operated with solid feedstock, and high biogas production correlated with abundance of plant degraders (Firmicutes) and biofilm-forming methanogens (Methanoculleus spp.). By contrast, low biogas-producing sewage sludge treatment digesters correlated with high titers of volatile fatty acid-adapted Methanosaeta spp. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0271-6) contains supplementary material, which is available to authorized users.

This report describes the results from anaerobic batch acidification of chicken manure as a mono-substrate studied under mesophilic conditions. The manure was diluted with tap water to prevent methane formation during acidification and to improve mixing conditions by reducing fluid viscosity; no anaerobic digester sludge has been added as an inoculum. Highest acidification rates were measured at concentrations of 10 gVS L−1 and 20 gVS L−1; the pH value remained high (pH 6.9–7.9) throughout the test duration and unexpected fast methane formation was observed in every single batch. At substrate concentrations of 10 gVS L−1 there was a remarkable methane formation representing a value of 82% of the respective biochemical methane potential of chicken manure. Increasing substrate concentrations did not supress methane formation but impaired acid production. Consequently, the liquor cannot be stored over longer periods but should immediately be used in a digestion process.