Effect of sulfate addition on carbon flow and microbial community composition during thermophilic digestion of cellulose.

Substrates with high sulfate levels pose problems for biogas production as they allow sulfate reducing bacteria to compete with syntrophic and methanogenic members of the community. In addition, the end product of sulfate reduction, hydrogen sulfide, is toxic and corrosive. Here we show how sulfate addition affects physiological processes in a thermophilic methanogenic system by analyzing the carbon flow and the microbial community with quantitative PCR and amplicon sequencing of the 16s rRNA gene. A sulfate addition of 0.5 to 3 g/L caused a decline in methane production by 73-92%, while higher sulfate concentrations had no additional inhibitory effect. Generally, sulfate addition induced a shift in the composition of the microbial community towards a higher dominance of Firmicutes and decreasing abundances of Bacteroidetes and Euryarchaeota. The abundance of methanogens (e.g., Methanoculleus and Methanosarcina) was reduced, while sulfate reducing bacteria (especially Candidatus Desulforudis and Desulfotomaculum) increased significantly in presence of sulfate. The sulfate addition had a significant impact on the carbon flow within the system, shifting the end product from methane and carbon dioxide to acetate and carbon dioxide. Interestingly, methane production quickly resumed, when sulfate was no longer present in the system. Despite the strong impact of sulfate addition on the carbon flow and the microbial community structure during thermophilic biogas production, short-term process disturbances caused by unexpected introduction of sulfate may be overcome due to the high resilience of the engaged microorganisms.

Spiking a Silty-Sand Reference Soil with Bacterial DNA: Limits and Pitfalls in the Discrimination of Live and Dead Cells When Applying Ethidium Monoazide (EMA) Treatment.

In the present study, EMA (ethidium monoazide) treatment was applied to a silty-sand reference soil prior to DNA extraction to enable a differentiation between dead and living cells. For this purpose, a reference soil was spiked with Listeria monocytogenes cells or cell equivalents, respectively. With the purpose of evaluating optimum treatment conditions, different EMA concentrations have been tested. However, the results remained largely inconclusive. Furthermore, varied dark incubation periods allowing EMA to penetrate dead cells did not allow the selective removal of DNA from membrane-compromised cells in downstream analyses. In contrast to undiluted soil, an effect of EMA treatment during DNA extraction could be observed when using a 1:10 dilution of the reference soil; however, the effect has not been sufficiently selective to act on heat-treated cells only. Although the application of EMA to soil requires further evaluation, the procedure harbors future potential for improving DNA-based approaches in microbial ecology studies.

Hydrogenotrophic Methanogenesis and Autotrophic Growth of Methanosarcina thermophila.

Although Methanosarcinales are versatile concerning their methanogenic substrates, the ability of Methanosarcina thermophila to use carbon dioxide (CO2) for catabolic and anabolic metabolism was not proven until now. Here, we show that M. thermophila used CO2 to perform hydrogenotrophic methanogenesis in the presence as well as in the absence of methanol. During incubation with hydrogen, the methanogen utilized the substrates methanol and CO2 consecutively, resulting in a biphasic methane production. Growth exclusively from CO2 occurred slowly but reproducibly with concomitant production of biomass, verified by DNA quantification. Besides verification through multiple transfers into fresh medium, the identity of the culture was confirmed by 16s RNA sequencing, and the incorporation of carbon atoms from 13CO2 into 13CH4 molecules was measured to validate the obtained data. New insights into the physiology of M. thermophila can serve as reference for genomic analyses to link genes with metabolic features in uncultured organisms.

Distribution of Prokaryotic Abundance and Microbial Nutrient Cycling Across a High-Alpine Altitudinal Gradient in the Austrian Central Alps is Affected by Vegetation, Temperature, and Soil Nutrients.

Studies of the altitudinal distributions of soil microorganisms are rare or have led to contradictory results. Therefore, we studied archaeal and bacterial abundance and microbial-mediated activities across an altitudinal gradient (2700 to 3500 m) on the southwestern slope of Mt. Schrankogel (Central Alps, Austria). Sampling sites distributed over the alpine (2700 to 2900 m), the alpine-nival (3000 to 3100 m), and the nival altitudinal belts (3200 to 3500 m), which are populated by characteristic plant assemblages. Bacterial and archaeal abundances were measured via quantitative real-time PCR (qPCR). Moreover, microbial biomass C, microbial activity (dehydrogenase), and enzymes involved in carbon (CM-cellulase), nitrogen (protease), phosphorus (alkaline phosphatase), and sulfur (arylsulfatase) cycling were determined. Abundances, microbial biomass C, and activities almost linearly decreased along the gradient. Archaeal abundance experienced a sharper decrease, thus pointing to pronounced sensitivity toward environmental harshness. Additionally, abundance and activities were significantly higher in soils of the alpine belt compared with those of the nival belt, whereas the alpine-nival ecotone represented a transitional area with intermediate values, thus highlighting the importance of vegetation. Archaeal abundance along the gradient was significantly related to soil temperature only, whereas bacterial abundance was significantly related to temperature and dissolved organic carbon (DOC). Soil carbon and nitrogen concentrations explained most of the variance in enzyme activities involved in the cycling of C, N, P, and S. Increasing temperature could therefore increase the abundances and activities of microorganisms either directly or indirectly via expansion of alpine vegetation to higher altitudes and increased plant cover.

Archaeal Distribution in Moonmilk Deposits from Alpine Caves and Their Ecophysiological Potential.

(Alpine) caves are, in general, windows into the Earth's subsurface. Frequently occurring structures in caves such as moonmilk (secondary calcite deposits) offer the opportunity to study intraterrestrial microbial communities, adapted to oligotrophic and cold conditions. This is an important research field regarding the dimensions of subsurface systems and cold regions on Earth. On a methodological level, moonmilk deposits from 11 caves in the Austrian Alps were collected aseptically and investigated using a molecular (qPCR and DGGE sequencing-based) methodology in order to study the occurrence, abundance, and diversity of the prevailing native Archaea community. Furthermore, these Archaea were enriched in complex media and studied regarding their physiology, with a media selection targeting different physiological requirements, e.g. methanogenesis and ammonia oxidation. The investigation of the environmental samples showed that all moonmilk deposits were characterized by the presence of the same few habitat-specific archaeal species, showing high abundances and constituting about 50 % of the total microbial communities. The largest fraction of these Archaea was ammonia-oxidizing Thaumarchaeota, while another abundant group was very distantly related to extremophilic Euryarchaeota (Moonmilk Archaea). The archaeal community showed a depth- and oxygen-dependent stratification. Archaea were much more abundant (around 80 %), compared to bacteria, in the actively forming surface part of moonmilk deposits, decreasing to about 5 % down to the bedrock. Via extensive cultivation efforts, it was possible to enrich the enigmatic Moonmilk Archaea and also AOA significantly above the level of bacteria. The most expedient prerequisites for cultivating Moonmilk Archaea were a cold temperature, oligotrophic conditions, short incubation times, a moonmilk surface inoculum, the application of erythromycin, and anaerobic (microaerophilic) conditions. On a physiological level, it seems that methanogenesis is of marginal importance, while ammonia oxidation and a still undiscovered metabolic pathway are vital elements in the (archaeal) moonmilk biome.

Abundances, diversity and seasonality of (non-extremophilic) Archaea in Alpine freshwaters.

The objectives of this study were to assess abundances and community compositions of Archaea within a heterogeneous set of freshwater systems in the Austrian Alps. Seasonal changes and geographical differences within Archaea, considering abiotic and biotic factors (e.g. temperature, pH, total organic carbon (TOC), NH4 (+), bacteria, fungi), were analysed in this context. Water samples were collected from 8 lakes, 10 creeks and the river Inn in 2014. Qualitative-quantitative data were derived via a comprehensive set of (quantitative) PCR assays and PCR-DGGE (denaturing gradient gel electrophoresis) based methodology, which was evaluated concerning specificity and reliability either previously or in this study. QPCR-derived archaeal abundances reached values of 10(3) copies mL(-1) on average, with a peak in winter-spring ('Cold Peak'), and covered 0-15 % (average: 1 %) of the microbial populations. This peak correlated with significantly raised TOC and low NH4 (+) levels during the cold seasons. Stagnant waters showed significantly higher archaeal abundances and diversities than flowing ones. Among methanogens, Methanosarcinales were the most common order. PCR-DGGE data showed that the archaeal communities were site-specific and could function as an ecological marker, in contrast to the more heterogeneous and unsteady bacterial and fungal community. This is attributable to the highly heterogeneous community of methanogenic Archaea (MA, Euryarchaeota), while only two species, Nitrosopumilus maritimus and Ca. Nitrososphaera gargensis, were found to be the ubiquitous representatives of ammonia-oxidizing Archaea (AOA, Thaumarchaeota) in Alpine freshwaters. This work emphasises the diversity, distribution and seasonality of non-extremophilic Archaea in Alpine freshwaters, with a first insight into their ecophysiological potential.

Temporal patterns of prokaryotic abundance, community structure and microbial activity in glacier foreland soils.

To reveal temporal variability of archaeal and bacterial abundance, community structure, as well as microbial biomass and activity, soils of different ages (young, intermediate, mature) were sampled along a glacier foreland in the Austrian Central Alps, at the beginning (summer) and at the end (autumn) of the plant growing season. No significant changes of 16S rRNA gene copy numbers of bacteria or archaea occurred in the young, recently de-glaciated soil. However, in intermediate and mature soils, bacteria were more abundant in autumn than in summer, whereas archaea decreased at the end of the growing season. Bacterial and archaeal community structures were differentially affected by the sampling date. Both soil microbial biomass and microbial activities in intermediate and mature soils significantly increased in autumn. On the contrary, the overall abiotic parameters did not undergo changes during summer. Bacterial communities at young, intermediate, and mature sites formed different clusters, thus reflecting changes according to soil age. Archaeal communities, on the other hand, were mainly influenced by the growing season, at least in young and intermediate soils. Our results indicate that temporal variations of microbial activities, biomass, and abundance in alpine glacier foreland soils distinctly increased along with the age of the soils and highlight the importance of sampling date for ecological studies.

Effect of DNA extraction procedure, repeated extraction and ethidium monoazide (EMA)/propidium monoazide (PMA) treatment on overall DNA yield and impact on microbial fingerprints for bacteria, fungi and archaea in a reference soil.

Different DNA extraction protocols were evaluated on a reference soil. A wide difference was found in the total extractable DNA as derived from different extraction protocols. Concerning the DNA yield phenol-chloroform-isomyl alcohol extraction resulted in high DNA yield but also in a remarkable co-extraction of contaminants making PCR from undiluted DNA extracts impossible. By comparison of two different extraction kits, the Macherey&Nagel SoilExtract II kit resulted in the highest DNA yields when buffer SL1 and the enhancer solution were applied. The enhancer solution not only significantly increased the DNA yield but also the amount of co-extracted contaminates, whereas additional disintegration strategies did not. Although a three times repeated DNA extraction increased the total amount of extracted DNA, microbial fingerprints were merely affected. However, with the 5th extraction this changed. A reduction of total DGGE band numbers was observed for archaea and fungi, whereas for bacteria the diversity increased. The application of ethidium monoazide (EMA) or propidium monoazide (PMA) treatment aiming on the selective removal of soil DNA derived from cells lacking cell wall integrity resulted in a significant reduction of total extracted DNA, however, the hypothesized effect on microbial fingerprints failed to appear indicating the need for further investigations.

Cultivation of moonmilk-born non-extremophilic Thaum and Euryarchaeota in mixed culture.

PCR-DGGE, qPCR and sequencing highlighted a quite homogenous archaeal community prevailing in secondary calcite deposits, so-called moonmilk, within the cold alpine Hundalm cave in Tyrol (Austria). Furthermore, the depth profile of this moonmilk could prove that the Archaea are located in oxygen-rich near- and oxygen-depleted sub-surface layers. To gather these communities we therefore applied an aerobic and anaerobic cultivation approach in oligotrophic and methanotrophic media. The mixed moonmilk community was analyzed with a combination of molecular methods using qPCR, PCR-DGGE and sequencing. Anaerobic and aerobic cultures were additionally investigated with GC and HPLC analyses. It was possible to initially cultivate and enrich the supposed aerobic/microaerophilic and anaerobic archaeal fraction, representing the natural archaeal community. While the naturally less abundant near-surface Archaea are closely related to members of the Thaumarchaeota (Nitrosopumilus maritimus), the highly abundant anaerobic Archaea are more distantly related to members within the Euryarchaeota. It is possible that these cultivable moonmilk-born Archaea represent new ecotypes or are so far undescribed. Based on the sequencing results and the production of very low amounts of methane, a corresponding methanogenic community is thought to represent only a minor abundant archaeal fraction. On a physiological level the cultivated moonmilk community is cold-adapted and basically of oligotrophic and organotrophic character.

Reactor performance of a 750 m(3) anaerobic digestion plant: varied substrate input conditions impacting methanogenic community.

A 750 m(3) anaerobic digester was studied over a half year period including a shift from good reactor performance to a reduced one. Various abiotic parameters like volatile fatty acids (VFA) (formic-, acetic-, propionic-, (iso-)butyric-, (iso-)valeric-, lactic acid), total C, total N, NH4 -N, and total proteins, as well as the organic matter content and dry mass were determined. In addition several process parameters such as temperature, pH, retention time and input of substrate and the concentrations of CH4, H2, CO2 and H2S within the reactor were monitored continuously. The present study aimed at the investigation of the abundance of acetogens and total cell numbers and the microbial methanogenic community as derived from PCR-dHPLC analysis in order to put it into context with the determined abiotic parameters. An influence of substrate quantity on the efficiency of the anaerobic digestion process was found as well as a shift from a hydrogenotrophic in times of good reactor performance towards an acetoclastic dominated methanogenic community in times of reduced reactor performance. After the change in substrate conditions it took the methano-archaeal community about 5-6 weeks to be affected but then changes occurred quickly.

Primer evaluation and adaption for cost-efficient SYBR Green-based qPCR and its applicability for specific quantification of methanogens.

In the present study nine promising primer sets, targeting Archaea and methanogenic Archaea in particular, were evaluated in silico, in vitro and in situ concerning specificity, accuracy and applicability in end-point (ep-) and especially quantitative (q-)PCR research. The main goal was to adapt and evaluate already adapted primer sets, which were partially designed in combination with TaqMan probes, in substantially cheaper SYBR Green-based qPCR applications. An initial 16S rRNA gene bank-based in silico evaluation revealed high coverage potentials for all primers within targeted groups, ranging from 71 to 90%, except the Methanosaeta specific set showing a low potential of 37%. Mentionable cross-reacting potentials could be detected for the Methanothermobacter, Methanomicrobiales and Methanoculleus sets. The in vitro evaluation with selected reference organisms revealed a specific behavior for most primer sets, while the Methanosarcina and Methanothermobacter sets showed most problematic cross-reactions in epPCR application. We were able to show that primers for detecting the total archaeal community, methanogenic orders Methanosarcinales, Methanobacteriales, Methanococcales and the genus Methanoculleus performed in a highly specific way and allowed an accurate quantification of targeted organisms without the use of expensive TaqMan probes. However, primer pairs designed for detecting Methanomicrobiales, Methanothermobacter, Methanosarcina and Methanosaeta are not suitable for SYBR Green applications. The reliability of in situ quantifications was assessed for a typical methanogenic community, derived from a thermophilic fermenter, and confirmed via denaturing gradient gel band quantification and sequencing. Thereby, we revealed high abundances of methanogenic Archaea, mainly comprising Methanoculleus and Methanosarcinales, while Methanobacteriales only formed a minor fraction.

Microbial perspective of inhibited carbon turnover in Tangel humus of the Northern Limestone Alps.

Tangel humus primarily occurs in montane and subalpine zones of the calcareous Alps that exhibit low temperatures and high precipitation sums. This humus form is characterized by inhibited carbon turnover and accumulated organic matter, leading to the typical thick organic layers. However, the reason for this accumulation of organic matter is still unclear, and knowledge about the microbial community within Tangel humus is lacking. Therefore, we investigated the prokaryotic and fungal communities along with the physical and chemical properties within a depth gradient (0-10, 10-20, 20-30, 30-40, 40-50 cm) of a Tangel humus located in the Northern Limestone Alps. We hypothesized that humus properties and microbial activity, biomass, and diversity differ along the depth gradient and that microbial key players refer to certain humus depths. Our results give the first comprehensive information about microbiota within the Tangel humus and establish a microbial zonation of the humus. Microbial activity, biomass, as well as microbial alpha diversity significantly decreased with increasing depths. We identified microbial biomarkers for both, the top and the deepest depth, indicating different, microbial habitats. The microbial characterization together with the established nutrient deficiencies in the deeper depths might explain reduced C-turnover and Tangel humus formation.

Pre-treatment with Trichoderma viride: Towards a better understanding of its consequences for anaerobic digestion.

Understanding and optimising biological pre-treatment strategies for enhanced bio-methane production is a central aspect in second-generation biofuel research. In this regard, the application of fungi for pre-treatment seems highly promising; however, understanding the mode of action is crucial. Here, we show how aerobic pre-treatment of crystalline cellulose with the cellulolytic Trichoderma viride affects substrate degradability during mesophilic, anaerobic digestion. It could be demonstrated that fungal pre-treatment resulted in a slightly reduced substrate mass. Nevertheless, no significant impact on the overall methane yield was found during batch fermentation. Short chain organic acids accumulation, thus, overall degradation dynamics including methane production kinetics were affected by the pre-treatment as shown by Gompertz modelling. Finally, 16S rRNA amplicon sequencing followed by ANCOM-BC resulted in up to 53 operative taxonomic units including fermentative, syntrophic and methanogenic taxa, whereby their relative abundances were significantly affected by fungal pre-treatment depending on the duration of the pre-treatment. The results demonstrated the impact of soft rot fungal pre-treatment of cellulose on subsequent anaerobic cellulose hydrolysis as well as on methanogenic activity. To the best of our knowledge, this is the first study to investigate the direct causal effects of pre-treatment with T. viride on basic but crucial anaerobic digestion parameters in a highly standardised approach.

Comparison of DNA extraction methods on different sample matrices within the same terrestrial ecosystem.

Metataxonomic studies of ecosystem microbiotas require the simultaneous processing of samples with contrasting physical and biochemical traits. However, there are no published studies of comparisons of different DNA extraction kits to characterize the microbiotas of the main components of terrestrial ecosystems. Here, and to our knowledge for the first time, five DNA extraction kits were used to investigate the composition and diversity of the microbiota of a subset of samples typically studied in terrestrial ecosystems such as bulk soil, rhizosphere soil, invertebrate taxa and mammalian feces. DNA extraction kit was associated with changes in the relative abundance of hundreds of ASVs, in the same samples, resulting in significant differences in alpha and beta diversity estimates of their microbiotas. Importantly, the impact of DNA extraction kit on sample diversity varies according to sample type, with mammalian feces and soil samples showing the most and least consistent diversity estimates across DNA extraction kits, respectively. We show that the MACHEREY-NAGEL NucleoSpin® Soil kit was associated with the highest alpha diversity estimates, providing the highest contribution to the overall sample diversity, as indicated by comparisons with computationally assembled reference communities, and is recommended to be used for any large-scale microbiota study of terrestrial ecosystems.

Improvement of methane generation capacity by aerobic pre-treatment of organic waste with a cellulolytic Trichoderma viride culture.

Trichoderma viride is known as a potent cellulose decomposer and was successfully used to improve and accelerate the decomposition process of aerobic composting. In contrast, the role of fungi as pre-treatment organisms for anaerobic digestion is not clear, since the fast aerobic decomposition is thought to be responsible for a rapid depletion of easily available nutrients, leading to a lack of these for the anaerobic community. In the present study carried out in lab-scale, the application of T. viride for the aerobic pre-incubation of organic matter derived from the inlet port of a 750,000 L anaerobic digester led to an increase in total gas and methane production in a subsequent anaerobic digestion step. A high cellulase activity caused by the addition of T. viride seemed to be responsible for a better nutrient availability for anaerobic microorganisms. Therefore, aerobic pre-incubation of organic residues with T. viride for subsequent anaerobic digestion is a promising approach in order to increase methane yields.

Mock community as an in situ positive control for amplicon sequencing of microbiotas from the same ecosystem.

Metataxonomy has become the standard for characterizing the diversity and composition of microbial communities associated with multicellular organisms and their environment. Currently available protocols for metataxonomy assume a uniform DNA extraction, amplification and sequencing efficiency for all sample types and taxa. It has been suggested that the addition of a mock community (MC) to biological samples before the DNA extraction step could aid identification of technical biases during processing and support direct comparisons of microbiota composition, but the impact of MC on diversity estimates of samples is unknown. Here, large and small aliquots of pulverized bovine fecal samples were extracted with no, low or high doses of MC, characterized using standard Illumina technology for metataxonomics, and analysed with custom bioinformatic pipelines. We demonstrated that sample diversity estimates were distorted only if MC dose was high compared to sample mass (i.e. when MC > 10% of sample reads). We also showed that MC was an informative in situ positive control, permitting an estimation of the sample 16S copy number, and detecting sample outliers. We tested this approach on a range of sample types from a terrestrial ecosystem, including rhizosphere soil, whole invertebrates, and wild vertebrate fecal samples, and discuss possible clinical applications.

The use of FAME analyses to discriminate between different strains of Geotrichum klebahnii with different viabilities.

A considerable decline in viability of spray dried cells of Geotrichum klebahnii was observed and was attributed to an undefined alteration of the used strain. As common techniques were not able to distinguish the altered from the still viable strains, we used the fatty acid methyl ester (FAME) analysis. On the basis of FAME data we were able to discriminate the three strains under investigation. Especially the ratios of cis/trans fatty acid ratios and of saturated/unsaturated fatty acid were significantly reduced in the less viable strain, pointing to an increased stress level in this strain. These findings clearly show the applicability of the FAME analysis to detect strain alterations and that this method is therefore a suitable, fast and feasible tool for quality assurance.

Methanogenic potential of formate in thermophilic anaerobic digestion.

In the present study the methanogenic potential of formate (HCOO(-)) during thermophilic anaerobic digestion was investigated. After appropriate conditions for methanogenesis (HCOO(-) and inoculum concentration, pH and duration of incubation) were assessed, an experiment with initial 31 replicates was run. Diluted fermenter sludge was used as inoculum, and process parameters including the pH, quality and quantity of the produced biogas and the concentrations of volatile fatty acids and HCO(3) (-) were determined. Remarkably, after 5 days of incubation the highest CH(4) production was calculated for a HCOO(-) concentration of 200 mmol L(-1), a concentration, however, which might not occur in situ. During the phase of high CH(4) production HCOO(-) was degraded with a rate of 1.5 mmol L(-1) h(-1), and distinct changes of Gibbs free energy for several reactions were observed. Based on denaturing high-performance liquid chromatography, denaturing gradient gel electrophoresis, and additional subsequent sequencing approaches the hydrogenotrophic Methanothermobacter wolfeii was the dominant methanogen responsible for CH(4) production. Further confirmation was achieved due to the detection of autofluorescing rods with a size of up to ~3 µm, which were often arranged in pairs and chains. It was shown that even high concentrations of HCOO(-) are readily degraded, which might lead to an underestimation of both, the concentration and thus, the importance of HCOO(-) in anaerobic digestion.

Carbon-dependent growth, community structure and methane oxidation performance of a soil-derived methanotrophic mixed culture.

Soil-borne methane-oxidizing microorganisms act as a terrestrial methane (CH4) sink and are potentially useful in decreasing global CH4 emissions. Understanding the ecophysiology of methanotrophs is crucial for a thorough description of global carbon cycling. Here, we report the in situ balance of soils from abandoned landfills, meadows and wetlands, their capacities to produce and oxidize CH4 at laboratory-scale and the isolation of a soil-borne methanotrophic-heterotrophic mixed culture that was used for carbon (C1 and C2) feeding experiments. We showed that even with similar soil properties, the in situ CH4 balance depends on land-use. Different soils had different potentials to adapt to increased CH4 availability, leading to the highest CH4 oxidation capacities for landfill and wetland soils. The most efficient mixed culture isolated from the landfill was dominated by the methanotrophs Methylobacter sp. and Methylosinus sp., which were accompanied by Variovorax sp. and Pseudomonas sp. and remained active in oxidizing CH4 when supplied with additional C-sources. The ratios between type I and type II methanotrophs and between methanotrophic and heterotrophic bacteria changed when C-sources were altered. A significant effect of the application of the mixed culture on the CH4 oxidation of soils was established but the extent varied depending on soil type.

Lignin intermediates lead to phenyl acid formation and microbial community shifts in meso- and thermophilic batch reactors.

BACKGROUND: Lignin intermediates resulting from lignocellulose degradation have been suspected to hinder anaerobic mineralisation of organic materials to biogas. Phenyl acids like phenylacetate (PAA) are early detectable intermediates during anaerobic digestion (AD) of aromatic compounds. Studying the phenyl acid formation dynamics and concomitant microbial community shifts can help to understand the microbial interdependencies during AD of aromatic compounds and may be beneficial to counteract disturbances. RESULTS: The length of the aliphatic side chain and chemical structure of the benzene side group(s) had an influence on the methanogenic system. PAA, phenylpropionate (PPA), and phenylbutyrate (PBA) accumulations showed that the respective lignin intermediate was degraded but that there were metabolic restrictions as the phenyl acids were not effectively processed. Metagenomic analyses confirmed that mesophilic genera like Fastidiosipila or Syntrophomonas and thermophilic genera like Lactobacillus, Bacillus, Geobacillus, and Tissierella are associated with phenyl acid formation. Acetoclastic methanogenesis was prevalent in mesophilic samples at low and medium overload conditions, whereas Methanoculleus spp. dominated at high overload conditions when methane production was restricted. In medium carbon load reactors under thermophilic conditions, syntrophic acetate oxidation (SAO)-induced hydrogenotrophic methanogenesis was the most important process despite the fact that acetoclastic methanogenesis would thermodynamically be more favourable. As acetoclastic methanogens were restricted at medium and high overload conditions, syntrophic acetate oxidising bacteria and their hydrogenotrophic partners could step in for acetate consumption. CONCLUSIONS: PAA, PPA, and PBA were early indicators for upcoming process failures. Acetoclastic methanogens were one of the first microorganisms to be impaired by aromatic compounds, and shifts to syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis occurred in thermophilic reactors. Previously assumed associations of specific meso- and thermophilic genera with anaerobic phenyl acid formation could be confirmed.