Detection and abundance of SARS-CoV-2 in wastewater in Liechtenstein, and the estimation of prevalence and impact of the B.1.1.7 variant.

The new coronavirus 2 (SARS-CoV-2) is known to be also shed through feces, which makes wastewater-based surveillance possible, independent of symptomatic cases and unbiased by any testing strategies and frequencies. We investigated the entire population of the Principality of Liechtenstein with samples from the wastewater treatment plant Bendern (serving all 39,000 inhabitants). Twenty-four-hour composite samples were taken once or twice a week over a period of 6 months from September 2020 to March 2021. Viral RNA was concentrated using the PEG centrifugation method followed by reverse transcription quantitative PCR. The aim of this research was to assess the suitability of SARS-CoV-2 fragments to relate the viral wastewater signal to the incidences and assess the impact of the emerging B.1.1.7. variant. The viral load in the wastewater peaked at almost 9 × 108 viral fragments per person equivalent (PE) and day on October 25, and showed a second peak on December 22 reaching a viral load of approximately 2 × 108 PE-1d-1. Individual testing showed a lag of 4 days and a distinct underestimation of cases at the first peak when testing frequency was low. The wastewater signal showed an immediate response to the implementation of non-pharmaceutical interventions. The new virus variant B.1.1.7. was first detected in wastewater on December 23, while it was first observed with individual testing on January 13, 2021. Further, our data indicate that the emergence of new virus variant may change the wastewater signal, probably due to different shedding patterns, which should be considered in future models.

Biogas purification with biomass ash.

The aim of the study was to investigate the option to purify biogas from small-scale biogas plants by entrapping CO2 and H2S with regionally available biomass ash. Connected to the existing biogas plant Neustift (Tyrol) wood ash placed in a 1 m3 container was used as a trap for CO2 and H2S in the biogas. With the process conditions chosen, for a period of a few hours CO2 was trapped resulting in pure methane. The removal of H2S was much longer-lasting (up to 34 d). The cumulative H2S uptake by the biomass ash ranged from 0.56 to 1.25 kg H2S per ton of ash. The pH of the ash and the leachability of Lead and Barium were reduced by the flushing with biogas, however toxicity towards plants was increased thus reducing the potential of ash use in agriculture. It can be concluded that biomass ash may be used for removal of hydrogen sulphide from biogas in small and medium biogas plants. The economic evaluation, however, indicated that the application of this system is limited by transport distances for the ash and its potential use afterwards.

Lactic acid and methane: improved exploitation of biowaste potential.

This feasibility study investigated a two-step biorefining approach to increase the value gained by recycling of organic municipal solid waste. Firstly, lactic acid was produced via batch fermentation at 37°C using the indigenous microbiome. Experiments revealed an optimal fermentation period of 24h resulting in high yields of lactic acid (up to 37gkg(-1)). The lactic acid proportion of total volatile fatty acid content reached up to 83%. Lactobacilli were selectively enriched to up to 75% of the bacterial community. Additionally conversion of organic matter to lactic acid was increased from 22% to 30% through counteracting end product inhibition by continuous lactic acid extraction. Secondly, fermentation residues were used as co-substrate in biomethane production yielding up to 618±41Nmlbiomethaneg(-1) volatile solids. Digestate, the only end product of this process can be used as organic fertilizer.

Merging two waste streams, wood ash and biowaste, results in improved composting process and end products.

A trial was carried out to evaluate the influence of wood ash admixture on biowaste composting. The aim was to find the optimal dosage of ash addition to enhance the composting process without endangering the final compost characteristics and use. Six treatments including an unamended control (K0) and composts with additions of 3% (K3), 6% (K6), 9% (K9), 12% (K12) and 15% (K15) of wood ash (w/w) were studied. The composting process was monitored in situ for 49days, by measuring temperature, CO2, O2, and CH4 in the piles and pH, electric conductivity (EC), and inorganic N in the laboratory. At the end of the process, the products were tested for Reifegrad (maturity), toxicity and quality. The addition of up to 15% of wood ash to biowaste did not negatively affect the composting process, and the initial differences found between both the low and high ash-treated composts were attenuated with the ongoing process development. Nevertheless, and mainly due to Cd level, composts with higher ash amendment did not comply with the highest quality standards established by the Austrian Compost Ordinance. The failure of obtaining class A+ quality after ash amendment emphasizes the need for a rigid quality selection of (bottom) ashes and thus reducing environmental risks related to high pollutant loads originating from the ashes.

Archaeal community dynamics and abiotic characteristics in a mesophilic anaerobic co-digestion process treating fruit and vegetable processing waste sludge with chopped fresh artichoke waste.

This study evaluated the feasibility of obtaining methane in anaerobic digestion (AD) from the waste products generated by the processing of fruit and vegetables. During the first phase (0-55 d) of the AD using sludge from fruit and vegetable processing, an average value of 244±88 L kg(-1) dry matter d(-1)of biogas production was obtained, and methane content reached 65% of the biogas. Co-digestion with chopped fresh artichoke wastes in a second phase (55-71 d) enhanced biogas production, and resulted in an average value of 354±68 L kg(-1) dry matter d(-1), with higher methane content (more than 70%). The archaeal community involved in methane production was studied using the ANAEROCHIP microarray and real-time PCR. Results indicated that species of Methanosaeta and Methanosarcina were important during the AD process. Methanosarcina numbers increased after the addition of chopped fresh artichoke, while Methanosaeta numbers decreased.

Microbiological community analysis of vermicompost tea and its influence on the growth of vegetables and cereals.

Vermicompost, the digestion product of organic material by earthworms, has been widely reported to have a more positive effect on plant growth and plant health than conventional compost. A study was conducted to investigate the effects of different vermicompost elutriates (aerated compost teas) on soils and plant growth. The teas were analyzed by chemical, microbiological, and molecular methods accompanied by plant growth tests at laboratory and field scale. The number of microorganisms in the teas increased during the extraction process and was affected by substrate addition. The vermicompost tea found to increase plant growth best under laboratory tests was applied to cereals (wheat and barley) and vegetables (Raphanus sativus, Rucola selvatica, and Pisum sativum) in a field study. The results revealed no effects of tea application on plant yield; however, sensoric tests indicated an improvement in crop quality. The soils from laboratory and field studies were investigated to detect possible microbial or chemical changes. The results indicated that minor changes to the soil microbial community occurred following tea application by foliar spray in both the laboratory-scale and field-scale experiments.

The effect of maize silage as co-substrate for swine manure on the bacterial community structure in biogas plants.

The qualitative and quantitative changes in the bacterial community composition in two mesophilic, commercially used biogas plants were monitored by denaturing gradient gel electrophoresis (DGGE) and real-time PCR. The main objective was to evaluate the influence of the co-substrate maize silage on total bacteria and some selected bacterial groups by comparing full-scale reactors fed solely with pig manure or additionally with maize silage. DGGE fingerprints reflected shifts in the bacterial community structure associated with maize silage as co-substrate and the real-time PCR results showed clear changes in the quantitative composition of the bacterial consortia of each fermenter. A clear dominance of Clostridia in all surveyed fermenters and considerably lower abundance of Bacteroidetes in the biogas plant fed with maize silage was shown.

Burning fire-prone Mediterranean shrublands: immediate changes in soil microbial community structure and ecosystem functions.

Wildfires subject soil microbes to extreme temperatures and modify their physical and chemical habitat. This might immediately alter their community structure and ecosystem functions. We burned a fire-prone shrubland under controlled conditions to investigate (1) the fire-induced changes in the community structure of soil archaea, bacteria and fungi by analysing 16S or 18S rRNA gene amplicons separated through denaturing gradient gel electrophoresis; (2) the physical and chemical variables determining the immediate shifts in the microbial community structure; and (3) the microbial drivers of the change in ecosystem functions related to biogeochemical cycling. Prokaryotes and eukaryotes were structured by the local environment in pre-fire soils. Fire caused a significant shift in the microbial community structure, biomass C, respiration and soil hydrolases. One-day changes in bacterial and fungal community structure correlated to the rise in total organic C and NO(3)(-)-N caused by the combustion of plant residues. In the following week, bacterial communities shifted further forced by desiccation and increasing concentrations of macronutrients. Shifts in archaeal community structure were unrelated to any of the 18 environmental variables measured. Fire-induced changes in the community structure of bacteria, rather than archaea or fungi, were correlated to the enhanced microbial biomass, CO(2) production and hydrolysis of C and P organics. This is the first report on the combined effects of fire on the three biological domains in soils. We concluded that immediately after fire the biogeochemical cycling in Mediterranean shrublands becomes less conservative through the increased microbial biomass, activity and changes in the bacterial community structure.

Bacterial community composition of the gut microbiota of Cylindroiulus fulviceps (diplopoda) as revealed by molecular fingerprinting and cloning.

Bacterial clone libraries of the gut microbiota of nurtured and starved Cylindroiulus fulviceps specimens displayed the predominance of the phyla Bacteroidetes (55 and 37 %, respectively) and Proteobacteria (40 and 35 %, respectively) and a high similarity to bacteria previously detected in the intestinal tract of termites and beetles, which are known to harbor symbiotic bacteria essential for digestive activity. Bacterial isolates were dominated by Proteobacteria (74 %), followed by members of the phyla Actinobacteria, Firmicutes and Bacteroidetes. PCR-DGGE fingerprints of the gut samples showed that intestinal bacteria were affected by starvation, although the change was not significant.

Quantitative and qualitative effects of bioaugmentation on ammonia oxidisers at a two-step WWTP.

Large waste water treatment plants (WWTP) often operate nitrification in two different process environments: the cold-dilute sewage is treated in the mainstream nitrification/denitrification system, while the high strength ammonia liquors from sludge dewatering are treated in a separate high temperature reactor (SBR). This study investigates transfer from nitrifier biomass into a two-stage WWTP, commonly referred to as bioaugmentation. Besides the quantitation of ammonia oxidising bacteria (AOB), community differences were analysed with two techniques, denaturing gradient gel electrophoresis and real-time PCR melt curve analysis. It was shown that, without bioaugmentation, two distinct AOB communities establish in the mainstream and in the SBR, respectively. A gradual shift of the two AOB communities with increasing pump rates between the systems could be demonstrated. These molecular findings support process engineering experience, that cycling of waste activated sludge improves the ability of AOB to adapt to different process environments.

Effect of biowaste sludge maturation on the diversity of thermophilic bacteria and archaea in an anaerobic reactor.

Prokaryotic diversity was investigated near the inlet and outlet of a plug-flow reactor. After analyzing 800 clones, 50 bacterial and 3 archaeal phylogenetic groups were defined. Clostridia (>92%) dominated among bacteria and Methanoculleus (>90%) among archaea. Significant changes in pH and volatile fatty acids did not invoke a major shift in the phylogenetic groups. We suggest that the environmental filter imposed by the saline conditions (20 g liter(-1)) selected a stable community of halotolerant and halophilic prokaryotes.

Application of denaturing gradient gel electrophoresis for analysing the gut microflora of Lumbricus rubellus Hoffmeister under different feeding conditions.

The earthworm, Lumbricus rubellus, plays an essential role in soil ecosystems as it affects organic matter decomposition and nutrient cycling. By ingesting a mixture of organic and mineral material, a variety of bacteria and fungi are carried to the intestinal tract of the earthworm. To get a better understanding of the interactions between L. rubellus and the microorganisms ingested, this study tried to reveal if the diet affects the composition of the gut microflora of L. rubellus or if its intestinal tract hosts an indigenous, species-specific microbiota. A feeding experiment with L. rubellus was set up; individuals were collected in the field, transferred to a climate chamber and fed with food sources of different quality (dwarf shrub litter, grass litter or horse dung) for six weeks. DNA was extracted from the guts of the earthworms, as well as from the food sources and the surrounding soil, and further analysed by a molecular fingerprinting method, PCR-DGGE (Polymerase Chain Reaction -- Denaturing Gradient Gel Electrophoresis). We were able to demonstrate that the gut microbiota was strongly influenced by the food source ingested and was considerably different to that of the surrounding soil. Sequencing of dominant bands of the bacterial DGGE fingerprints revealed a strong occurrence of y-Proteobacteria in all gut samples, independent of the food source. A specific microflora in the intestinal tract of L. rubellus, robust against diet changes, could not be found.

Control of GHG emission at the microbial community level.

All organic material eventually is decomposed by microorganisms, and considerable amounts of C and N end up as gaseous metabolites. The emissions of greenhouse relevant gases like carbon dioxide, methane and nitrous oxides largely depend on physico-chemical conditions like substrate quality or the redox potential of the habitat. Manipulating these conditions has a great potential for reducing greenhouse gas emissions. Such options are known from farm and waste management, as well as from wastewater treatment. In this paper examples are given how greenhouse gas production might be reduced by regulating microbial processes. Biogas production from manure, organic wastes, and landfills are given as examples how methanisation may be used to save fossil fuel. Methane oxidation, on the other hand, might alleviate the problem of methane already produced, or the conversion of aerobic wastewater treatment to anaerobic nitrogen elimination through the anaerobic ammonium oxidation process might reduce N2O release to the atmosphere. Changing the diet of ruminants, altering soil water potentials or a change of waste collection systems are other measures that affect microbial activities and that might contribute to a reduction of carbon dioxide equivalents being emitted to the atmosphere.

Model-based design of an agricultural biogas plant: application of anaerobic digestion model no.1 for an improved four chamber scheme.

Different digestion technologies for various substrates are addressed by the generic process description of Anaerobic Digestion Model No. 1. In the case of manure or agricultural wastes a priori knowledge about the substrate in terms of ADM1 compounds is lacking and influent characterisation becomes a major issue. The actual project has been initiated for promotion of biogas technology in agriculture and for expansion of profitability also to rather small capacity systems. In order to avoid costly individual planning and installation of each facility a standardised design approach needs to be elaborated. This intention pleads for bio kinetic modelling as a systematic tool for process design and optimisation. Cofermentation under field conditions was observed, quality data and flow data were recorded and mass flow balances were calculated. In the laboratory different substrates have been digested separately in parallel under specified conditions. A configuration of four ADM1 model reactors was set up. Model calibration identified disintegration rate, decay rates for sugar degraders and half saturation constant for sugar as the three most sensitive parameters showing values (except the latter) about one order of magnitude higher than default parameters. Finally, the model is applied to the comparison of different reactor configurations and volume partitions. Another optimisation objective is robustness and load flexibility, i.e. the same configuration should be adaptive to different load situations only by a simple recycle control in order to establish a standardised design.

Microbial community structure at different depths in disturbed and undisturbed semiarid Mediterranean forest soils.

Metabolic abilities and micrfiobial community structure were investigated through three semiarid Mediterranean soils of SE Spain. The soils were (1) a Typic Calcixerept under an adult pine plantation (PP), growing on abandoned agricultural terraces; (2) a Typic Calcixeroll under a native pinewood (NP); and (3) a Typic Haploxerept covered with a grass steppe (GS). PP and NP were similar as regards their genesis, but the former used to be tilled. NP and GS were undisturbed and supported natural and seminatural vegetation, respectively. Seven samples in 10-cm depth increments were taken in triplicate along each soil profile. Community-level physiological profiles based on sole-C-source use were determined to characterize the metabolic abilities. A 16S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis analysis was performed to investigate the microbial genetic structure. Plant cover and land-use history were major determinants of microbial community structure. Microbial communities residing in soils under a native pinewood, the most diverse and stable plant cover, were the most complex both metabolically and genetically. The microbial community structure distinctly changed with depth, related to the quantity and quality of total organic carbon. Both undisturbed soils showed falling gradients of metabolic and genetic complexity, which were invariably of a greater magnitude in the mature woodland than in the grass steppe. In the planted pinewood, however, the substrate-use diversity increased with depth, apparently a response to the depleted metabolic abilities within its upper layer (0-30 cm). Tilling and plant cover removal might be responsible for such a perturbation. In the same profile, molecular fingerprint patterns of the topsoil layer (0-10 cm) indicated a disturbed genetic structure that might underlie the loss of metabolic abilities. However, the genetic structure of the deeper layers of the planted and native pinewoods was not dissimilar, revealing that equivalent genetic resources perform different environmental functions under changing soil scenarios.

Elevated CO2 alters community-level physiological profiles and enzyme activities in alpine grassland.

Plots of an alpine grassland in the Swiss Alps were treated with elevated (680 microl l(-1)) and ambient CO2 (355 microl l(-1)) in open top chambers (OTC). Several plots were also treated with NPK-fertilizer. Community level physiological profiles (CLPPs) of the soil bacteria were examined by Biolog GN microplates and enzyme activities were determined through the release of methylumbelliferyl (MUF) and methylcoumarin (MC) from MUF- or MC-labelled substrates. A canonical discriminant analysis (CDA) followed by multivariate analysis of variance showed a significant effect of elevated CO2 on the CLPPs both under fertilized and unfertilized conditions. Further, the installation of the OTCs caused significant shifts in the CLPPs (chamber effect). Of the four enzyme activities tested, the beta-D-cellobiohydrolase (CELase) and N-acetyl-beta-D-glucosaminidase (NAGase) activity were enhanced under elevated CO2. L-Leucin-7-aminopeptidase (APEase) activity decreased, when the plots received fertilizer. Beta-D-glucosidase (GLUase) remained unaffected. The results suggest effects of elevated CO2 on specific microbial activities even under low mineral nutrient conditions and when bulk parameters like microbial biomass or respiration, which have been investigated on the same site, remain unaffected. The observed medium-term changes point at possible long-term consequences for the ecosystem that may not be specified yet.

Responses of the soil microbiota to elevated CO2 in an artificial tropical ecosystem.

Plants in artificial tropical ecosystems were grown under ambient (340 microl l(-1)) and elevated (610 microl l(-1)) atmospheric CO2 for 530 d under low-nutrient conditions on a substrate free of organic C. At the end of the experiment a number of soil chemical and microbiological variables were determined. Although we found no changes in total soil organic matter under elevated CO2, we did find that after physical fractionation the amount of organic C in the supernatant (< 0.2 microm) and the amount of water extractable organic C (WEOC) was lower under elevated CO2. The extractable optical density (OD) indicated a higher degree of humification for the elevated than for the ambient CO2 samples (P = 0.032). Microbial biomass C was not significantly altered under high CO2, but total bacterial counts were significantly higher. The microbial biomass C-to-N ratio was also higher at elevated (15.0) than at ambient CO2 (10.0). The number of mycorrhizal spores was lower at high CO2, but ergosterol contents and fungal hyphal lengths were not significantly affected. Changes were found neither in community level physiological profiles (CLPPs) nor in the structural attributes (phospholipid fatty acids, PLFAs) of the microbial community. Overall, the effects on the soil microbiota were small, perhaps as a result of the low nutrient supply and low organic matter content of the soil used in our study. The few significant results showing changes in specific, though relatively minor, organic matter pools may point to possible long-term changes of the more major pools. Furthermore, the data suggest increased competition between plants and microbes for N at high CO2.

Changes in functional abilities of the microbial community during composting of manure.

The objective of this study was (a) to detect changes of the functional abilities of the microflora during composting of manure as a result of windrow turning frequency and (b) to detect differences between distinct zones within the windrows. Biolog GN microtiter plates containing 95 different carbon sources were inoculated with diluted suspensions of compost material containing 15,000 microorganisms per well (120 µl). We found a dramatic shift in functional microbial community structure during the 8-week composting process. The shift was more rapid when the compost windrows were turned. The substrate use pattern in the outer, well-aerated zone of the unturned windrow was similar to that of the turned windrows. Microbial biomass and respiration decreased more rapidly in the turned than in the unturned windrows, indicating a different pace of compost maturation. The data suggest that the Biolog assay may be a suitable approach to determine compost maturity.

Metabolic quotient of the soil microflora in relation to plant succession.

In this study we propose the hypothesis that ecosystem succession is accompanied by a decrease in the metabolic quotient qCO2 (respiration-to-biomass ratio) of the soil microflora. The qCO2 is calculated from basal respiration (CO2-C·h-1) per unit microbial biomass carbon (Cmier). The hypothesis was tested by studying two primary successions on recessional moraines of the Rotmoos Ferner (Austria) and the Athabasca Glacier (Canada). For both soil seres (0->200 years) it was shown that the qCO2 decreased with time, which corroborated the hypothesis. In addition, the short term development of the qCO2 was demonstrated with a revegetation trial. We observed a rise in qCO2 for the first two years after reclamation, followed by a subsequent decrease.