Meso- and thermophilic posttreatment of press water coming from a thermophilic municipal solid waste digester.

An efficient biogas production out of organic (waste) materials is important to contribute to a carbon-neutral future. In this study, thermophilic press water (PW) coming from an organic fraction of the municipal solid waste digester was further digested in a thermo- and mesophilic posttreatment approach using two semicontinuous 14 L digesters. The results showed that the PW can still have considerable high biogas potential-at least during the touristic high season in central Europe. The change in temperature led to an increase in volatile fatty acid concentrations and a decrease in biogas production in the mesophilic approach in the first days. However, the losses in biogas production at the beginning could be compensated thus there were no considerable differences in biogas production between thermo- and mesophilic posttreatment at the end of incubation. This can most probably be contributed to a change in the microbial community, and potentially problematic intermediates like valerate could be better degraded in the mesophilic reactor. Especially the abundance of representatives of the phylum Bacteroidota, like Fermentimonas spp., increased during mesophilic anaerobic digestion.

Effects of phenyl acids on different degradation phases during thermophilic anaerobic digestion.

Aromatic compounds like phenyl acids (PA) can accumulate during anaerobic digestion (AD) of organic wastes due to an increased entry of lignocellulose, secondary plant metabolites or proteins, and thermodynamic challenges in degrading the benzene ring. The effects of aromatic compounds can be various - from being highly toxic to be stimulating for methanogenesis - depending on many parameters like inoculum or molecular characteristics of the aromatic compound. To contribute to a better understanding of the consequences of PA exposure during AD, the aim was to evaluate the effects of 10 mM PA on microbial communities degrading different, degradation phase-specific substrates in thermophilic batch reactors within 28 days: Microcrystalline cellulose (MCC, promoting hydrolytic to methanogenic microorganisms), butyrate or propionate (promoting syntrophic volatile fatty acid (VFA) oxidisers to methanogens), or acetate (promoting syntrophic acetate oxidisers to methanogens). Methane production, VFA concentrations and pH were evaluated, and microbial communities and extracellular polymeric substances (EPS) were assessed. The toxicity of PA depended on the type of substrate which in turn determined the (i) microbial diversity and composition and (ii) EPS quantity and quality. Compared with the respective controls, methane production in MCC reactors was less impaired by PA than in butyrate, propionate and acetate reactors which showed reductions in methane production of up to 93%. In contrast to the controls, acetate concentrations were high in all PA reactors at the end of incubation thus acetate was a bottle-neck intermediate in those reactors. Considerable differences in EPS quantity and quality could be found among substrates but not among PA variants of each substrate. Methanosarcina spp. was the dominant methanogen in VFA reactors without PA exposure and was inhibited when PA were present. VFA oxidisers and Methanothermobacter spp. were abundant in VFA assays with PA exposure as well as in all MCC reactors. As MCC assays showed higher methane yields, a higher microbial diversity and a higher EPS quantity and quality than VFA reactors when exposed to PA, we conclude that EPS in MCC reactors might have been beneficial for absorbing/neutralising phenyl acids and keeping (more susceptible) microorganisms shielded in granules or biofilms.

Prediction of hospitalisations based on wastewater-based SARS-CoV-2 epidemiology.

Wastewater-based epidemiology is widely applied in Austria since April 2020 to monitor the SARS-CoV-2 pandemic. With a steadily increasing number of monitored wastewater facilities, 123 plants covering roughly 70 % of the 9 million population were monitored as of August 2022. In this study, the SARS-CoV-2 viral concentrations in raw sewage were analysed to infer short-term hospitalisation occupancy. The temporal lead of wastewater-based epidemiological time series over hospitalisation occupancy levels facilitates the construction of forecast models. Data pre-processing techniques are presented, including the approach of comparing multiple decentralised wastewater signals with aggregated and centralised clinical data. Time­lead quantification was performed using cross-correlation analysis and coefficient of determination optimisation approaches. Multivariate regression models were successfully applied to infer hospitalisation bed occupancy. The results show a predictive potential of viral loads in sewage towards Covid-19 hospitalisation occupancy, with an average lead time towards ICU and non-ICU bed occupancy between 14.8-17.7 days and 8.6-11.6 days, respectively. The presented procedure provides access to the trend and tipping point behaviour of pandemic dynamics and allows the prediction of short-term demand for public health services. The results showed an increase in forecast accuracy with an increase in the number of monitored wastewater treatment plants. Trained models are sensitive to changing variant types and require recalibration of model parameters, likely caused by immunity by vaccination and/or infection. The utilised approach displays a practical and rapidly implementable application of wastewater-based epidemiology to infer hospitalisation occupancy.

Expanding the Pathogen Panel in Wastewater Epidemiology to Influenza and Norovirus.

Since the start of the 2019 pandemic, wastewater-based epidemiology (WBE) has proven to be a valuable tool for monitoring the prevalence of SARS-CoV-2. With methods and infrastructure being settled, it is time to expand the potential of this tool to a wider range of pathogens. We used over 500 archived RNA extracts from a WBE program for SARS-CoV-2 surveillance to monitor wastewater from 11 treatment plants for the presence of influenza and norovirus twice a week during the winter season of 2021/2022. Extracts were analyzed via digital PCR for influenza A, influenza B, norovirus GI, and norovirus GII. Resulting viral loads were normalized on the basis of NH4-N. Our results show a good applicability of ammonia-normalization to compare different wastewater treatment plants. Extracts originally prepared for SARS-CoV-2 surveillance contained sufficient genomic material to monitor influenza A, norovirus GI, and GII. Viral loads of influenza A and norovirus GII in wastewater correlated with numbers from infected inpatients. Further, SARS-CoV-2 related non-pharmaceutical interventions affected subsequent changes in viral loads of both pathogens. In conclusion, the expansion of existing WBE surveillance programs to include additional pathogens besides SARS-CoV-2 offers a valuable and cost-efficient possibility to gain public health information.

Transcriptome profiling of Paraburkholderia aromaticivorans AR20-38 during ferulic acid bioconversion.

The importance and need of renewable-based, sustainable feedstocks increased in recent years. Lignin-derived monomers have high potential, energetic and economic value in the microbial bioconversion to valuable biomolecules. The bacterium Paraburkholderia aromaticivorans AR20-38 produces a remarkable yield of vanillic acid from ferulic acid at moderate and low temperatures and is therefore a good candidate for biotechnological applications. To understand this bioconversion process on a molecular level, a transcriptomic study during the bioconversion process was conducted to elucidate gene expression patterns. Differentially expressed genes, cellular transporters as well as transcriptional factors involved in the bioconversion process could be described. Additional enzymes known for xenobiotic degradation were differentially expressed and a potential membrane vesicle mechanism was detected. The bioconversion mechanism on a transcriptional level of P. aromaticivorans could be elucidated and results can be used for strain optimization. Additionally, the transcriptome study showed the high potential of the strain for other degradation applications.

Viral variant-resolved wastewater surveillance of SARS-CoV-2 at national scale.

SARS-CoV-2 surveillance by wastewater-based epidemiology is poised to provide a complementary approach to sequencing individual cases. However, robust quantification of variants and de novo detection of emerging variants remains challenging for existing strategies. We deep sequenced 3,413 wastewater samples representing 94 municipal catchments, covering >59% of the population of Austria, from December 2020 to February 2022. Our system of variant quantification in sewage pipeline designed for robustness (termed VaQuERo) enabled us to deduce the spatiotemporal abundance of predefined variants from complex wastewater samples. These results were validated against epidemiological records of >311,000 individual cases. Furthermore, we describe elevated viral genetic diversity during the Delta variant period, provide a framework to predict emerging variants and measure the reproductive advantage of variants of concern by calculating variant-specific reproduction numbers from wastewater. Together, this study demonstrates the power of national-scale WBE to support public health and promises particular value for countries without extensive individual monitoring.

Low-Temperature Biodegradation of Lignin-Derived Aromatic Model Monomers by the Cold-Adapted Yeast Rhodosporidiobolus colostri Isolated from Alpine Forest Soil.

The contribution of cold-adapted yeasts to the emerging field of lignin biovalorization has not yet been studied. The red-pigmented basidiomycetous yeast strain Rhodosporidiobolus colostri DBVPG 10655 was examined for its potential to degrade five selected lignin-derived aromatic monomers (syringic acid, p-coumaric acid, 4-hydroxybenzoic acid, ferulic acid, and vanillic acid). The strain utilized p-coumaric acid, 4-hydroxybenzoic acid, and ferulic acid not only as the sole carbon source; full biodegradation occurred also in mixtures of multiple monomers. Vanillic acid was not utilized as the sole carbon source, but was degraded in the presence of p-coumaric acid, 4-hydroxybenzoic acid, and ferulic acid. Syringic acid was utilized neither as the sole carbon source nor in mixtures of compounds. Biodegradation of lignin-derived aromatic monomers was detected over a broad temperature range (1-25 °C), which is of ecological significance and of biotechnological relevance.

Extraction of Cofactor F420 for Analysis of Polyglutamate Tail Length from Methanogenic Pure Cultures and Environmental Samples.

The cofactor F420 plays a central role as a hydride carrier in the primary and secondary metabolism of many bacterial and archaeal taxa. The cofactor is best known for its role in methanogenesis, where it facilitates thermodynamically difficult reactions. As the polyglutamate tail varies in length between different organisms, length profile analyses might be a powerful tool for distinguishing and characterizing different groups and pathways in various habitats. Here, the protocol describes the extraction and optimization of cofactor F420 detection by applying solid-phase extraction combined with high-performance liquid chromatography analysis independent of cultural or molecular biological approaches. The method was applied to gain additional information on the expression of cofactor F420 from microbial communities in soils, anaerobic sludge, and pure cultures and was evaluated by spiking experiments. Thereby, the study succeeded in generating different F420 tail-length profiles for hydrogenotrophic and acetoclastic methanogens in controlled methanogenic pure cultures as well as from environmental samples such as anaerobic digester sludge and soils.

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.

Microbial community dynamics in mesophilic and thermophilic batch reactors under methanogenic, phenyl acid-forming conditions.

BACKGROUND: Proteinaceous wastes exhibit high theoretical methane yields and their residues are considered valuable fertilisers. The routine anaerobic degradation of proteins often raises problems like high aromatic compound concentrations caused by the entry of aromatic amino acids into the system. A profound investigation of the consequences of aromatic compound exposure on various microorganisms, which cascade-like and interdependently degrade complex molecules to biogas, is still pending. RESULTS: In mesophilic samples, methane was predominantly produced via acetoclastic methanogenesis. The highest positive correlation was observed between phenylacetate (PAA) and Psychrobacter spp. and between phenylpropionate (PPA) and Haloimpatiens spp. Moreover, Syntrophus spp. negatively correlated with PAA (Spearman's rank correlations coefficient (rs) = - 0.46, p < 0.05) and PPA concentrations (rs = - 0.44, p < 0.05) and was also associated with anaerobic benzene ring cleavage. In thermophilic samples, acetate was predominantly oxidised by Tepidanaerobacter spp. or Syntrophaceticus spp. in syntrophic association with a hydrogenotrophic methanogen. The genera Sedimentibacter and Syntrophaceticus correlated positively with both PAA and PPA concentrations. Moreover, Sedimentibacter spp., Tepidanaerobacter spp., Acetomicrobium spp., and Sporanaerobacter spp. were significant LEfSe (linear discriminant analysis effect size) biomarkers for high meso- as well as thermophilic phenyl acid concentrations. Direct negative effects of phenyl acids on methanogenic properties could not be proven. CONCLUSIONS: Anaerobic phenyl acid formation is not restricted to specific microbial taxa, but rather done by various meso- and thermophilic bacteria. The cleavage of the highly inert benzene ring is possible in methanogenic batch reactors-at least in mesophilic fermentation processes. The results indicated that phenyl acids rather affect microorganisms engaged in preceding degradation steps than the ones involved in methanogenesis.

Biomethanation at 45 °C offers high process efficiency and supports hygienisation.

The aim of this work was to prove a process temperature of 45 °C as a practical alternative to commonly applied mesophilic (37 °C) and thermophilic (55 °C) anaerobic digestion (AD). Regarding methane production, no differences were found between the three temperature regimes. However, the maximum possible loading rate at 45 °C exceeded that at 37 °C and 55 °C. Pathogen inactivation at 45 °C was higher than at 37 °C and similarly efficient as at 55 °C. At each process temperature, a unique microbial community established. In addition, the archaeome at 55 °C was dominated by hydrogenotrophs, while at 37 °C and 45 °C it was dominated by acetotrophs. For the investigated substrate mixture, liquid cattle manure with wheat straw as co-substrate, 45 °C turned out to be preferable for AD. For other substrates, these findings still need to be confirmed.

Microbial and Phenyl Acid Dynamics during the Start-up Phase of Anaerobic Straw Degradation in Meso- and Thermophilic Batch Reactors.

Aromatic compounds like phenyl acids derived from lignocellulose degradation have been suspected to negatively influence biogas production processes. However, results on this topic are still inconclusive. To study phenyl acid formation in batch reactors during the start-up phase of anaerobic degradation, different amounts of straw from grain were mixed with mesophilic and thermophilic sludge, respectively. Molecular biological parameters were assessed using next-generation sequencing and qPCR analyses. Metagenomic predictions were done via the program, piphillin. Methane production, concentrations of phenylacetate, phenylpropionate, phenylbutyrate, and volatile fatty acids were monitored chromatographically. Methanosarcina spp. was the dominant methanogen when high straw loads were effectively degraded, and thus confirmed its robustness towards overload conditions. Several microorganisms correlated negatively with phenyl acids; however, a negative effect, specifically on methanogens, could not be proven. A cascade-like increase/decrease from phenylacetate to phenylpropionate, and then to phenylbutyrate could be observed when methanogenesis was highly active. Due to these results, phenylacetate was shown to be an early sign for overload conditions, whereas an increase in phenylbutyrate possibly indicated a switch from degradation of easily available to more complex substrates. These dynamics during the start-up phase might be relevant for biogas plant operators using complex organic wastes for energy exploitation.

Formation of phenylacetic acid and phenylpropionic acid under different overload conditions during mesophilic and thermophilic anaerobic digestion.

BACKGROUND: Substrate spectra for anaerobic digestion have been broadened in the past decade, inter alia, due to the application of different pretreatment strategies and now include materials rich in lignocellulose, protein, and/or fat. The application of these substrates, however, also entails risks regarding the formation of undesired by-products, among which phenolic compounds are known to accumulate under unfavorable digestion conditions. METHODS: Different states of overload were simulated in batch experiments while reviewing the generation of phenyl acids out of different lab-use substrates in order to evaluate the impact on biogas and methane production as well as some additional process performance parameters under defined laboratory conditions. Investigations were conducted under both mesophilic and thermophilic conditions. RESULTS: It could be shown that the tested input materials led to the formation of phenyl acids in a substrate-dependent manner with the formation itself being less temperature driven. Once formed, the formation of phenyl acids turned out to be a reversible process. CONCLUSIONS: Although a mandatory negative impact of phenyl acids per se on the anaerobic digestion process in general and the methanogenesis process in particular could not be proven, phenyl acids, however, seem to play an important role in the microbial response to overloaded biogas systems.

Temperature shapes the microbiota in anaerobic digestion and drives efficiency to a maximum at 45 °C.

Throwing longstanding habits over the pile may be necessary to improve biogas production, in particular when it comes to the process temperature. Its effect on biogas production was investigated with lab-scale reactors operated in fed-batch mode (cattle slurry and maize straw) at 10-55 °C over six months. Biochemical and microbial changes were comprehensively investigated. Production was highest and most efficient at 45 °C with an average methane yield of 166 NL kg-1 VS, and thus 12.8% and 9.6% higher than at 37 and 55 °C. Temperature significantly affected the microbiota and higher temperature provoked a shift from Bacteroidetes/Proteobacteria to Firmicutes. A transition from hydrogenotrophic to acetoclastic methanogenesis was observed from 10 to 45 °C, while the trend was reversed at 55 °C. The results contest the textbook notion of preferred and most efficient temperatures for AD and suggest reconsideration of the temperature range around 45 °C for efficient manure-based co-fermentation.

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.

Biological Pretreatment Strategies for Second-Generation Lignocellulosic Resources to Enhance Biogas Production.

With regard to social and environmental sustainability, second-generation biofuel and biogas production from lignocellulosic material provides considerable potential, since lignocellulose represents an inexhaustible, ubiquitous natural resource, and is therefore one important step towards independence from fossil fuel combustion. However, the highly heterogeneous structure and recalcitrant nature of lignocellulose restricts its commercial utilization in biogas plants. Improvements therefore rely on effective pretreatment methods to overcome structural impediments, thus facilitating the accessibility and digestibility of (ligno)cellulosic substrates during anaerobic digestion. While chemical and physical pretreatment strategies exhibit inherent drawbacks including the formation of inhibitory products, biological pretreatment is increasingly being advocated as an environmentally friendly process with low energy input, low disposal costs, and milder operating conditions. Nevertheless, the promising potential of biological pretreatment techniques is not yet fully exploited. Hence, we intended to provide a detailed insight into currently applied pretreatment techniques, with a special focus on biological ones for downstream processing of lignocellulosic biomass in anaerobic digestion.

Biological pre-treatment: Enhancing biogas production using the highly cellulolytic fungus Trichoderma viride.

With regard to renewable sources of energy, bioconversion of lignocellulosic biomass has long been recognized as a desirable endeavor. However, the highly heterogeneous structure of lignocellulose restricts the exploitation of its promising potential in biogas plants. Hence, effective pre-treatment methods are decisive prerequisites to overcome these challenges in order to improve the utilization ratio of (ligno) cellulosic substrates during fermentation. In the present study, the application of Trichoderma viride in an aerobic upstream process prior to anaerobic digestion led up to a threefold increase in the yield of methane and total gas in a lab-scale investigation. Due to its highly efficient cellulolytic activities, T. viride seemed to be responsible for an improved nutrient availability that positively influenced the anaerobic microbiocenosis. Aerobic pre-treatment of organic matter with T. viride is therefore a promising solution to achieve higher methane yields and degradation performances without any additional energy demand, nor undesired by-product inhibition.

Lactic acid fermentation within a cascading approach for biowaste treatment.

Limited availability of resources and increased amounts of waste coupled with an ever-increasing demand for raw materials are typical characteristics of our times. As such, there is an urgent need to accordingly update waste treatment technology. The aim of this study was to determine whether a separate treatment of the liquid and the solid fraction of biowaste could enhance overall efficiency. Liquid fractions obtained from two different separation procedures were fermented at a pH of 5 and uncontrolled pH conditions for 72 h. The fermentation conditions leading to highest lactic acid productivity and yield were evaluated. The substrates gained by both separation procedures showed efficient lactic acid production up to <25 g L(-1). The pH control increased lactic acid concentration by about 27 %. Furthermore, quantitative real-time PCR analyses revealed stronger Lactobacilli growth in these fermentations. As identified via Illumina sequencing Lactobacillus delbrueckii and its closest relatives seemed to drive the fermentation independent of the substrate. These results could help to improve today's resourcing concept by providing a separate treatment of the liquid and solid biowaste fraction.