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Anaerobic digestion of nitrogen-rich substrate often causes process inhibition due to the susceptibility of the microbial community facing ammonia accumulation. However, the precise response of the microbial community has remained largely unknown. To explore the reasons, bacterial communities in ammonia-stressed reactors and control reactors were studied by amplicon pyrosequencing of 16S rRNA genes and the active methanogens were followed by terminal restriction fragment length polymorphism (T-RFLP) analyses of mcrA/mrtA gene transcripts. The results showed that the diversity of bacterial communities decreased in two parallel ammonia-inhibited reactors compared with two control reactors, but different levels of inhibitions coinciding with different community shifts were observed. In one reactor, the process was completely inhibited, which was preceded by a decreasing relative abundance of the phylum Firmicutes. Despite the same operating conditions, the process was stabilized in the parallel, partially inhibited reactor, in which the relative abundance of Firmicutes greatly increased. In particular, both ammonia-inhibited reactors lacked taxa assumed to be syntrophic bacteria (Thermoanaerobacteraceae, Syntrophomonadaceae, and Synergistaceae). Besides the predominance of the hydrogenotrophic methanogens Methanoculleus and Methanobacterium, activity of Methanosarcina and even of the strictly aceticlastic genus Methanosaeta were found to contribute at very high ammonia levels (> 9 g NH4-N L−1) in the stabilized reactor (partial inhibition). In contrast, the lack of aceticlastic activity in the parallel reactor might have led to acetate accumulation and thus process failure (complete inhibition). Collectively, ammonia was found to be a general inhibitor while accumulating acetate and thus acidification might be the key factor of complete process failure. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop an effective bioaugmentation concept for anaerobic digesters treating lignocellulosic biomass such as straw. For that purpose, lignocellulose-degrading methanogenic communities were enriched on wheat straw from cow and goat rumen fluid as well as from a biogas reactor acclimated to lignocellulosic biomass (sorghum as mono-substrate). The bacterial communities of the enriched cultures and the different inocula were examined by 454 amplicon sequencing of 16S rRNA genes while the methanogenic archaeal communities were analyzed by terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of the mcrA gene. Bacteroidetes was the most abundant phylum in all samples. Within the Bacteroidetes phylum, Bacteroidaceae was the most abundant family in the rumen-derived enrichment cultures, whereas Porphyromonadaceae was the predominant one in the reactor-derived culture. Additionally, the enrichment procedure increased the relative abundance of Ruminococcaceae (phylum: Firmicutes) in all cultures. T-RFLP profiles of the mcrA gene amplicons highlighted that the ruminal methanogenic communities were composed of hydrogenotrophic methanogens dominated by the order Methanobacteriales regardless of the host species. The methanogenic communities changed significantly during the enrichment procedure, but still the strict hydrogenotrophic Methanobacteriales and Methanomicrobiales were the predominant orders in the enrichment cultures. The bioaugmentation potential of the enriched methanogenic cultures will be evaluated in further studies. [ABSTRACT FROM AUTHOR]

The effect of pig manure mixed with rice straw on methane yield and the microbial community involved in a thermophilic (55°C) anaerobic digestion process was investigated. Three substrates composed of mixed pig manure and rice straw at different ratios (95:5; 78:22 and 65:35 w/w, which resulted in C/N ratios of 10:1, 20:1 and 30:1) were used for the experiment. The substrate type had a major influence on the total bacterial community, while the methanogens were less affected. The members of the class Clostridia (phylum Firmicutes ) were predominant regardless of mixture ratio (C/N ratio), but at species level there was a major difference between the low and high C/N ratio samples. The hydrogenotrophic methanogenic genus of Methanothermobacter was predominant in all samples but higher C/N ratio sequences affiliated to the genus Methanosarcina were also detected. The appearance of Methanosarcina sp. is most likely due to the less inhibition of ammonia during the anaerobic digestion. [ABSTRACT FROM AUTHOR]

Background: During strategic planning of a biogas plant, the local availability of resources for start-up and operation should be taken into consideration for a cost-efficient process. Because most bioethanol/sugar industries in Brazil are located in remote areas, the use of fresh cattle manure from local farms could be a solution for the inoculation of the biogas process. This study investigated the diversity and dynamics of bacterial and archaeal communities and the performance of biogas reactors inoculated with manure and a mixed inoculum from different biogas reactors as for a controlled start-up until steady state. Results: Laboratory-scale biogas reactors were fed semi-continuously with sugarcane filter cake alone (mono-digestion) or together with bagasse (co-digestion). At the initial start-up, the reactors inoculated with the mixed inoculum displayed a less diverse taxonomic composition, but with higher presence of significant abundances compared to reactors inoculated with manure. However, in the final steady state, the communities of the differently inoculated reactors were very similarly characterized by predominance of the methanogenic genera Methanosarcina and Methanobacterium, the bacterial families Bacteroidaceae, Prevotellaceae and Porphyromonadaceae (phylum Bacteroidetes) and Synergistaceae (phylum Synergistetes). In the mono-digestion reactors, the methanogenic communities varied greater than in the co-digestion reactors independently of the inoculation strategy. Conclusion: The microbial communities involved in the biogas production from waste products of the Brazilian bioethanol/sugar industry were relatively similar and stable at the reactor's steady phase independently of the inoculum source (manure or mixed inoculum). Therefore, the locally available manure can be used as inoculum for start-up of the biogas process, since it also contains the microbial resources needed. The strong fluctuation of methanogenic communities in mono-digestion reactors indicates higher risk of process instability than in co-digestion reactors. [ABSTRACT FROM AUTHOR]

The anaerobic digestion of filter cake and its co-digestion with bagasse, and the effect of gradual increase of the organic loading rate (OLR) from start-up to overload were investigated. Understanding the influence of environmental and technical parameters on the development of particular methanogenic pathway in the biogas process was an important aim for the prediction and prevention of process failure. The rapid accumulation of volatile organic acids at high OLR of 3.0 to 4.0 gvs.L-1.day-1 indicated strong process inhibition. Methanogenic community dynamics of the reactors was monitored by stable isotope composition of biogas and molecular biological analysis. A potential shift toward the aceticlastic methanogenesis was observed along with the OLR increase under stable reactor operating conditions. Reactor overloading and process failure were indicated by the tendency to return to a predominance of hydrogenotrophic methanogenesis with rising abundances of the orders Methanobacteriales and Methanomicrobiales and drop of the genus Methanosarcina abundance. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We present a simple protocol for the cost- and time-efficient profiling of methanogens based on T-RFLP fingerprinting of mcrA amplicons. Sequence data were compiled from mesophilic lab-scale and full-scale biogas reactors operated under various conditions and fed with various substrates. The database facilitates the rapid identification of methanogens, thus reducing the need of cloning and sequencing. [ABSTRACT FROM AUTHOR]

In this study, the effects of nitrogen, phosphate and trace elements supplementation were investigated in a semi-continuously operated upflow anaerobic sludge blanket system to enhance process stability and biogas production from sugarcane vinasse. Phosphate in form of KH 2 PO 4 induced volatile fatty acids accumulation possibly due to potassium inhibition of the methanogenesis. Although nitrogen in form of urea increased the reactor’s alkalinity, the process was overloaded with an organic loading rate of 6.1 g COD L −1 d −1 and a hydraulic retention time of 3.6 days. However, by supplementing urea and trace elements a stable operation even at an organic loading rate of 9.6 g COD L −1 d −1 and a hydraulic retention time of 2.5 days was possible, resulting in 79% higher methane production rate with a stable specific methane production of 239 mL g COD −1 . [ABSTRACT FROM AUTHOR]

The effects of hydraulic retention time (HRT) reduction at constant high organic loading rate on the activity of hydrogen-producing bacteria and methanogens were investigated in reactors digesting thin stillage. Stable isotope fingerprinting was additionally applied to assess methanogenic pathways. Based on hydA gene transcripts, Clostridiales was the most active hydrogen-producing order in continuous stirred tank reactor (CSTR), fixed-bed reactor (FBR) and anaerobic sequencing batch reactor (ASBR), but shorter HRT stimulated the activity of Spirochaetales . Further decreasing HRT diminished Spirochaetales activity in systems with biomass retention. Based on mcrA gene transcripts, Methanoculleus and Methanosarcina were the predominantly active in CSTR and ASBR, whereas Methanosaeta and Methanospirillum activity was more significant in stably performing FBR. Isotope values indicated the predominance of aceticlastic pathway in FBR. Interestingly, an increased activity of Methanosaeta was observed during shortening HRT in CSTR and ASBR despite high organic acids concentrations, what was supported by stable isotope data. [ABSTRACT FROM AUTHOR]

• Lignosulfonic acid counteracts urea induced ammonia inhibition of anaerobic digestion. • Tannic acid with fluoride inhibits methanogenesis, particularly acetoclastic methanogenesis. • Swine and cattle manures were dominated by hydrogenotrophic methanogens. • Tannic acid was degraded over time eventually resulting in methane production. Livestock waste is stored and used as soil fertilizer or directly as substrate for biogas production. Methane emissions from manure storages and ammonia inhibition of anaerobic digesters fed with manure, are well-known problems related to manure management. This study examines the effect of adding tannic acid with fluoride (TA-NaF) and lignosulfonic acid (LS) on methanogenic activity in batch reactors with ammonia inhibited maize silage digestate and in batch reactors with manure. Lignosulfonic acid counteracted urea induced ammonia inhibition of methanogenesis, whereas TA-NaF inhibited methanogenesis itself. Stable carbon isotope ratio analysis and methanogen community analysis suggested that TA-NaF affected acetoclastic methanogens the most. The combined findings suggest that TA-NaF could be used to reduce methane emissions from stored manure. Conversely, LS could be used as supplement in anaerobic digesters prone to urea induced ammonia inhibition. [ABSTRACT FROM AUTHOR]