Biodegradation of lignin monomers and bioconversion of ferulic acid to vanillic acid by Paraburkholderia aromaticivorans AR20-38 isolated from Alpine forest soil.

Lignin bio-valorization is an emerging field of applied biotechnology and has not yet been studied at low temperatures. Paraburkholderia aromaticivorans AR20-38 was examined for its potential to degrade six selected lignin monomers (syringic acid, p-coumaric acid, 4-hydroxybenzoic acid, ferulic acid, vanillic acid, benzoic acid) from different upper funneling aromatic pathways. The strain degraded four of these compounds at 10°C, 20°C, and 30°C; syringic acid and vanillic acid were not utilized as sole carbon source. The degradation of 5 mM and 10 mM ferulic acid was accompanied by the stable accumulation of high amounts of the value-added product vanillic acid (85-89% molar yield; 760 and 1540 mg l-1, respectively) over the whole temperature range tested. The presence of essential genes required for reactions in the upper funneling pathways was confirmed in the genome. This is the first report on biodegradation of lignin monomers and the stable vanillic acid production at low and moderate temperatures by P. aromaticivorans. KEY POINTS: • Paraburkholderia aromaticivorans AR20-38 successfully degrades four lignin monomers. • Successful degradation study at low (10°C) and moderate temperatures (20-30°C). • Biotechnological value: high yield of vanillic acid produced from ferulic acid.

Proposal of Thermoactinomyces mirandus sp. nov., a filamentous, anaerobic bacterium isolated from a biogas plant.

We isolated a filamentous, thermophilic, and first anaerobic representative of the genus Thermoactinomyces, designated strain AMNI-1T, from a biogas plant in Tyrol, Austria and report the results of a phenotypic, genetic, and phylogenetic investigation. Strain AMNI-1T was observed to form a white branching mycelium that aggregates into pellets when grown in liquid medium. Cells could primarily utilize lactose, glucose, and mannose as carbon and energy sources, with acetate accelerating and yeast extract being mandatory for growth. The optimum growth temperature and pH turned out to be 55 °C and pH 7.0, respectively, with an optimum NaCl concentration of 0-2% (w/v). 16S rRNA gene sequence comparison indicated that the genetic relatedness between strain AMNI-1T and Thermoactinomyces intermedius, Thermoactinomyces khenchelensis, and Thermoactinomyces vulgaris was less than 97%. The G + C content of the genomic DNA was 44.7 mol%. The data obtained suggest that the isolate represents a novel and first anaerobic species of the genus Thermoactinomyces, for which the name Thermoactinomyces mirandus is proposed. The type strain is AMNI-1T (= DSM 110094T = LMG 31503T). The description of the genus Thermoactinomyces is emended accordingly.

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.

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.

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.

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.

The glutamyl tail length of the cofactor F420 in the methanogenic Archaea Methanosarcina thermophila and Methanoculleus thermophilus.

The cofactor F420 is synthesized by many different organisms and as a redox cofactor, it plays a crucial role in the redox reactions of catabolic and biosynthetic metabolic pathways. It consists of a deazaflavin structure, which is linked via lactate to an oligoglutamate chain, that can vary in length. In the present study, the methanogenic Archaea Methanosarcina thermophila and Methanoculleus thermophilus were cultivated on different carbon sources and their coenzyme F420 composition has been assayed by reversed-phase ion-pair high-performance liquid chromatography with fluorometric detection regarding both, overall cofactor F420 production and distribution of F420 glutamyl tail length. In Methanosarcina thermophila cultivated on methanol, acetate, and a mixture of acetate and methanol, the most abundant cofactors were F420-5 and F420-4, whereby the last digit refers to the number of expressed glutamyl rests. By contrast, in the obligate CO2 reducing Methanoculleus thermophilus the most abundant cofactors were F420-3 and F420-4. In Methanosarcina thermophila, the relative proportions of the expressed F420 tail length changed during batch growth on all three carbon sources. Over time F420-3 and F420-4 decreased while F420-5 and F420-6 increased in their relative proportion in comparison to total F420 content. In contrast, in Methanoculleus thermophilus the relative abundance of the different F420 cofactors remained stable. It was also possible to differentiate the two methanogenic Archaea based on the glutamyl tail length of the cofactor F420. The cofactor F420-5 in concentrations >2% could only be assigned to Methanosarcina thermophila. In all four variants a trend for a positive correlation between the DNA concentration and the total concentration of the cofactor could be shown. Except for the variant Methanosarcinathermophila with acetate as sole carbon source the same could be shown between the concentration of the mcrA gene copy number and the total concentration of the cofactor.

Detection and Stability of SARS-CoV-2 Fragments in Wastewater: Impact of Storage Temperature.

SARS-CoV-2 wastewater epidemiology suffers from uncertainties concerning sample storage. We show the effect of the storage of wastewater on the detectable SARS-CoV-2 load. Storage at 4 °C for up to 9 days had no significant effect, while storage at -20 °C led to a significant reduction in gene copy numbers.

pH and Phosphate Induced Shifts in Carbon Flow and Microbial Community during Thermophilic Anaerobic Digestion.

pH is a central environmental factor influencing CH4 production from organic substrates, as every member of the complex microbial community has specific pH requirements. Here, we show how varying pH conditions (5.0-8.5, phosphate buffered) and the application of a phosphate buffer per se induce shifts in the microbial community composition and the carbon flow during nine weeks of thermophilic batch digestion. Beside monitoring the methane production as well as volatile fatty acid concentrations, amplicon sequencing of the 16S rRNA gene was conducted. The presence of 100 mM phosphate resulted in reduced CH4 production during the initial phase of the incubation, which was characterized by a shift in the dominant methanogenic genera from a mixed Methanosarcina and Methanoculleus to a pure Methanoculleus system. In buffered samples, acetate strongly accumulated in the beginning of the batch digestion and subsequently served as a substrate for methanogens. Methanogenesis was permanently inhibited at pH values ≤5.5, with the maximum CH4 production occurring at pH 7.5. Adaptations of the microbial community to the pH variations included shifts in the archaeal and bacterial composition, as less competitive organisms with a broad pH range were able to occupy metabolic niches at unfavorable pH conditions.

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions.

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.

Removal of free extracellular DNA from environmental samples by ethidium monoazide and propidium monoazide.

Recently, new DNA extraction techniques (using ethidium monoazide and propidium monoazide) have been developed to discriminate between alive and dead bacterial cells. Nevertheless, for complex environmental samples, no data are available yet. In the present study, these new methods were applied to anaerobic-fermentor sludge and the results were compared to a conventional microbiological approach.

Sample preparation, preservation, and storage for volatile fatty acid quantification in biogas plants.

Volatile fatty acids (VFA) represent short-chain fatty acids consisting of six or fewer carbon atoms that can be distilled at atmospheric pressure. In anaerobic digestion processes VFAs are of central importance for maintaining stable reactor performance and biogas production, are used as indicators for arising problems and are important process monitoring parameters. In the present study, sludge derived form a full-scale anaerobic digester of a wastewater treatment plant was spiked with formate, acetate, propionate, and butyrate in order to evaluate various commonly used techniques for VFA extraction, preservation, and storage. It was shown that VFA extraction after centrifugation warranted the highest recovery rates for spiked VFAs. Moreover, experiments clearly indicated the importance of a fast sample handling, including the necessity of immediate cooling of the samples. Chemical sample preservation within a narrow time frame or deep freezing emerged as an alternative to instant VFA extraction. Short-time storage of extracted VFA samples at + 4°C is an option for up to 7 days, for longer periods storage at -20°C was found to be applicable.

Abundance and potential metabolic activity of methanogens in well-aerated forest and grassland soils of an alpine region.

Although methanogens were recently discovered to occur in aerated soils, alpine regions have not been extensively studied for their presence so far. Here, the abundance of archaea and the methanogenic guilds Methanosarcinales, Methanococcales, Methanobacteriales, Methanomicrobiales and Methanocella spp. was studied at 16 coniferous forest and 14 grassland sites located at the montane and subalpine belts of the Northern Limestone Alps (calcareous) and the Austrian Central Alps (siliceous) using quantitative real-time PCR. Abundance of archaea, methanogens and the methanogenic potentials were significantly higher in grasslands than in forests. Furthermore, methanogenic potentials of calcareous soils were higher due to pH. Methanococcales, Methanomicrobiales and Methanocella spp. were detected in all collected samples, which indicates that they are autochthonous, while Methanobacteriales were absent from 4 out of 16 forest soils. Methanosarcinales were absent from 10 out of 16 forest soils and 2 out of 14 grassland soils. Nevertheless, together with Methanococcales they represented the majority of the 16S rRNA gene copies quantified from the grassland soils. Contrarily, forest soils were clearly dominated by Methanococcales. Our results indicate a higher diversity of methanogens in well-aerated soils than previously believed and that pH mainly influences their abundances and activities.

Methane-cycling microorganisms in soils of a high-alpine altitudinal gradient.

Methanogens and methanotrophs play unique roles as producers and consumers of the greenhouse gas methane (CH4) in soils, respectively. Here, we aimed to reveal whether and to which extent methane-cyclers occur in high-alpine soils, and to assess their spatial distribution along an altitudinal gradient (2700-3500 m) in the Austrian Alps at sites located within the alpine (2700-2900 m), the alpine-nival (3000-3100 m) and the nival belts (3200-3500 m). Methanococcales and Methanocella spp. were most abundant among all quantified methanogenic guilds, whereas Methanosarcinales were not detected in the studied soil. The detected methanogens seem to be capable of persisting despite a highly oxic low-temperature environment. Methanogenic and methanotrophic activities and abundances of methanotrophs, Methanococcales and Methanocella spp. declined with altitude. Methanogenic and methanotrophic abundances were best explained by mean annual soil temperature and dissolved organic carbon, respectively. Alpine belt soils harbored significantly more methane-cyclers than those of the nival belt, indicating some influence of plant cover. Our results show that methanogens are capable of persisting in high-alpine cold soils and might help to understand future changes of these environments caused by climate warming.

Methane yields and methanogenic community changes during co-fermentation of cattle slurry with empty fruit bunches of oil palm.

The biomethane potential and structural changes of the methanogenic community in a solid-state anaerobic digestion process co-digesting cattle slurry and empty fruit bunches were investigated under mesophilic (37°C) and thermophilic (55°C) conditions. Phylogenetic microarrays revealed the presence of two hydrogenotrophic genera (Methanoculleus and Methanobrevibacter) and one acetoclastic genus (Methanosarcina). Methanosarcina numbers were found to increase in both mesophilic and thermophilic treatments of empty fruit bunches. Methanobrevibacter, which dominated in the cattle slurry, remained constant during anaerobic digestion (AD) at 37°C and decreased in numbers during digestion at 55°C. Numbers of Methanoculleus remained constant at 37°C and increased during the thermophilic digestion. Physicochemical data revealed non-critical concentrations for important monitoring parameters such as total ammonia nitrogen, free ammonia nitrogen and volatile fatty acids in all treatments after AD. The biomethane potential of empty fruit bunches was higher under thermophilic conditions than under mesophilic conditions.

Methanogenic activities in alpine soils.

Uncontrolled microbial methane production is playing an important role in global warming. In the present study, we showed that water content and incubation temperature increase the potential for methane formation in the two alpine soils under investigation. Beside these factors, the grazing of cows and thus the amendment of methanogenic microorganisms by cattle dung is the most important factor determining the potential of methane production in those soils.

Effects of various fatty acid amendments on a microbial digester community in batch culture.

Since biogas production is becoming increasingly important the understanding of anaerobic digestion processes is fundamental. However, large-scale digesters often lack online sensor equipment to monitor key parameters. Furthermore the possibility to selectively change fermenting parameter settings in order to investigate methane output or microbial changes is limited. In the present study we examined the possibility to investigate the microbial community of a large scale (750,000 L) digester within a laboratory small-scale approach. We studied the short-term response of the downscaled communities on various fatty acids and its effects on gas production and compared it with data from the original digester sludge. Even high loads of formic acid led to distinct methane formation, whereas high concentrations of other acids (acetic, butyric, propionic acid) caused a marked inhibition of methanogenesis coupled with an increase in hydrogen concentration. Molecular microbial techniques (DGGE/quantitative real-time-PCR) were used to monitor the microbial community changes which were related to data from GC and HPLC analysis. DGGE band patterns showed that the same microorganisms which were already dominant in the original digester re-established again in the lab-scale experiment. Very few microorganisms dominated the whole fermenting process and species diversity was not easily influenced by moderate varying fatty acid amendments--Methanoculleus thermophilus being the most abundant species throughout the variants. MCR-copy number determined via quantitative real-time-PCR--turned out to be a reliable parameter for quantification of methanogens, even in a very complex matrix like fermenter sludge. Generally the downscaled batch approach was shown to be appropriate to investigate microbial communities from large-scale digesters.

Microbial community related to volatile organic compound (VOC) emission in household biowaste.

Malodorous emissions and potentially pathogenic microorganisms which develop during domestic organic waste collection are not only a nuisance but may also pose health risks. The aim of the present study was to determine whether the presence of specific microorganisms in biowastes is directly related to the composition of the emitted volatile organic compounds (VOCs). The succession of microbial communities during 16 days of storage in organic waste collection bins was studied by denaturing gradient gel electrophoresis (DGGE) of amplified 16S ribosomal DNA in parallel with a classical cultivation and isolation approach. Approximately 60 different bacterial species and 20 different fungal species were isolated. Additionally, some bacterial species were identified through sequencing of excised DGGE bands. Proton transfer reaction mass spectrometry (PTR-MS) was used to detect VOCs over the sampling periods, and co-inertia analyses of VOC concentrations with DGGE band intensities were conducted. Positive correlations, indicating production of the respective VOC or enhancement of microbial growth, and negative correlations, indicating the use of, or microbial inhibition by the respective compound, were found for the different VOCs. Measurement of the VOC emission pattern from a pure culture of Lactococcus lactis confirmed the positive correlations for the protonated masses 89 (tentatively identified as butyric acid), 63 (tentatively identified as dimethylsulfide), 69 (likely isoprene) and 73 (likely butanone).