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IntroductionPlant seeds from weeds and energy crops have the potential to survive anaerobic digestion (AD). Species able to form physically dormant, i.e., hardseeded (HS) seeds seem particularly resistant. However, it is not yet known to what extent the storage of the digestate after AD affects seed viability.MethodsSeed survival of five HS and six non-HS (NHS) species was investigated in three combinations of digestate storage (DS) and AD. First, untreated seeds were exposed to DS for maximum 12 weeks. To simulate short-circuited AD (ADshort) in biogas reactors, seeds were second subjected to lab-scale AD for 1 day before DS. Third, seeds of six species were exposed to full-scale AD (ADfull) followed by DS. Seed viability was determined using a combination of germination tests and tetrazolium staining. Viability was modeled as a function of exposure time.Results and discussionSeed viability was affected by DS, AD and AD + DS, but responses varied greatly between species and treatments. With increasing exposure time, viability decreased after a lag-phase, remained stable or even increased. The NHS species Cichorium intybus, Daucus carota, Echium vulgare, and Verbascum thapsus were most susceptible, with seed-killing close to 100% if DS was involved. The HS species Malva sylvestris, Melilotus albus and Melilotus officinalis were most resistant. They survived all treatments and were alive after 35 days of ADfull plus 3 months of DS. The resistance potential of the HS species Abutilon theophrasti and Malva alcea and of the NHS species Chenopodium album and two tomato varieties was intermediate. None of them survived ADfull + DS, but except A. theophrasti they were viable after ADshort + DS. With few exceptions, seed-killing by AD + DS was higher than that by AD alone. In conclusion, DS and AD + DS have the potential to reduce seed viability, but do not completely inactivate all species. Therefore, digestate can be contaminated with viable seeds and may lead to the spread of weeds, especially after a short-circuited AD. In order to ensure the sustainable use of digestates in terms of weeds, we recommend to investigate the factors contributing to seed inactivation and the quantity of seed introduced to AD.

When plant biomass is anaerobically digested, seeds may survive the energy production process and contaminate the digestate. Hard-seeded (HS), i.e., physically dormant, species were found to be difficult to inactivate. Here, we aimed to verify this finding from lab-scale experimental reactors (ERs) in a full-scale commercial reactor (CR). In addition, we tested seed survival in a pH-buffered water bath (WB). Seeds were exposed to CR, ER and WB treatments at 42 °C for a maximum of 36 days. The viability of seeds was checked by measuring germination and response to tetrazolium staining and modeled as a function of exposure time using a dose–response approach. CR killed seeds more effectively than ER and WB treatments. The non-HS reference species, Chenopodium album, was completely inactivated by all treatments. Responses of the HS species ranged from complete inactivation to complete insensitivity. The most resistant was Malva sylvestris. The least resistant species were inactivated mainly by temperature, while additional mortality factors were effective in the more resistant species. We concluded that mesophilic AD in CRs can reduce the risk of seed contamination in the digestate for non-HS but not for HS species. Moreover, WB treatments seem suitable to estimate the minimum mortality of non-HS species in CR.

The use of wildflower species as biogas feedstock carries the risk that their seeds survive anaerobic digestion (AD) and cause weed problems if spread with the digestate. Risk factors for seed survival in AD include low temperature, short exposure and hardseededness (HS). However, it is not possible to predict how AD will affect seed viability of previously unstudied species. In laboratory-scale reactors, we exposed seeds of eight species from a mixture of flowering wild plants intended as biogas feedstock and three reference species to AD at two mesophilic temperatures. Half of the species were HS, the other was non-HS (NHS). Viability was determined using a combination of tetrazolium and germination tests. Viability and germinability were modeled as functions of exposure time using a dose-response approach. Responses to AD varied considerably among species, and none of the considered influencing factors (time, temperature, HS) had a consistent effect. Seed lots of a species differed in inactivation times and seed-killing efficacy. The HS species Melilotus officinalis, Melilotus albus, and Malva sylvestris were particularly AD-resistant. They were the only ones that exhibited biphasic viability curves and tended to survive and germinate more at 42°C than at 35°C. Viability of the remaining species declined in a sigmoidal curve. Most NHS species were inactivated within a few days (Cichorium intybus, Daucus carota, Echium vulgare, and Verbascum thapsus), while HS species survived longer (Malva alcea). AD stimulated germination in the HS species A. theophrasti and its AD-resistance overlapped with that of the most resistant NHS species, C. album and tomato. In all seed lots, germinability was lost faster than viability, implying that mainly dormant seeds survived. After the maximum exposure time of 36 days, seeds of HS species and Chenopodium album were still viable. We concluded that viability responses to mesophilic AD were determined by the interplay of AD-conditions and species- and seed-lot-specific traits, of which HS was an important but only one factor. For the use of wildflowers as biogas feedstock, we recommended long retention times and special care with regard to HS species.

Ensiling, a lactic acid fermentation process, is mainly used to preserve biomass. In addition, it has been shown to affect seed viability of some plant species. The extent to which this makes ensiling suitable as a weed control measure, however, has not yet been determined. Both the range of controllable species and the parameters of an ensiling process that safely kills seeds are still undefined. We aimed to determine the effect of varying substrate and ensiling conditions on the seed viability of 10 species selected to represent a wide range of different seed traits. Five different types of silages were made from maize or mixtures of wildflower and maize biomass and ensiled in lab-scale silos for 8 months. The pure maize silages were prepared under conditions either ideal or suboptimal for ensiling forage. Seeds of important weeds (Chenopodium album, Abutilon theophrasti) and of species from a wildflower mixture suitable for ensiling and biogas production (Cichorium intybus, Daucus carota, Echium vulgare, Malva alcea, Malva sylvestris, Melilotus albus, Melilotus officinalis) were tested. Seed viability was determined using a combination of tetrazolium and germination tests. Ensiling reduced seed viability across all 10 species significantly. Seed-killing efficacies of ensiling, however, differed widely among the species studied, largely related to whether the species could produce hard (physically dormant) seeds. Seeds from species without hardseededness were completely inactivated by ensiling, while the seed-killing efficacies for hardseeded species ranged from 5 to 60%. Variation in ensiled substrate and ensiling conditions had no consistent effect on seed survival. We concluded that ensiling has the potential to sustainably reduce seed viability of a wide range of species and therefore should be adopted as a component of integrated weed management in organic agriculture.

Flower strips are grown to an increasing degree in order to enhance the ecological value of agricultural landscapes. Depending on their profitable life span and the crop sequence, the strips’ biomass must be mulched after flowering to enable repeated tillage. A promising alternative is the use of the flower strips’ biomass as a co-substrate for biomethanisation – thereby contributing to the climate-friendly generation of energy. This potential bioenergy substrate occurs only seasonally and is commonly produced only in limited quantities at a farm scale. To realize the additional benefit of flower strips as energy suppliers, stock piling of the strips’ biomass is required. However, information about the ensilability of flower strip biomass is still rare. We conducted a 2-year study to analyze the ensilability of pure biomass from effloresced flower strips and mixtures of flower strip biomass with 33 and 67% whole crop maize, respectively. Ensiling took place in 3 l model silos at laboratory scale after chopping the substrate. Before ensiling several chemical characteristics of the biomass stock were determined to assess the substrate’s biochemical ensilability potential (dry matter content, water-soluble carbohydrates, buffering capacity, nitrate content). The process-engineered ensiling success after 90 days was determined based on fermentation patterns. The ensilability potential of the pure flower strip substrates reached modest levels (fermentability coefficients according to Weißbach vary around the threshold of 45). Nevertheless, acceptable silage qualities were achieved under the laboratory conditions (pH ranging from 4.2 to 4.7). Compared to pure flower strip biomass, the addition of maize noticeably improved both the substrate’s biochemical ensilability potential and the quality of real fermented silage. We conclude that a mixture of 33% biomass from flower strips with 67% whole crop maize can be regarded as a recommendable ratio if proper ensiling technology is applied.

Wenn Pflanzen-Samen in eine Biogas-Anlage gelangen, besteht das Risiko, dass sie die anaerobe Vergärung überleben und mit Ausbringung des Gärrestes verbreitet werden. Neuerdings kommt durch den Einsatz von Blühmischungen eine Vielzahl von Wildpflanzen-Samen mit dem Biogas-Prozess in Kontakt. Das von diesen Arten ausgehende Kontaminationsrisiko lässt sich derzeit nicht verlässlich abschätzen, da systematische Forschung zur Überlebensfähigkeit von Pflanzensamen fast vollständig fehlt. Um dem abzuhelfen bietet sich die Überprüfung der Thermoresistenz der Arten an, da Temperatur und Verweildauer die wichtigsten Parameter sind, von denen das Überleben der Samen in Biogas-Anlagen abhängt. Wir haben das Überleben von 11 Wildpflanzen-Arten aus verschiedenen Familien unter den Temperatur-Bedingungen einer mesophilen Biogas-Anlage mit einem Screening im Wasserbad untersucht. Die Samen wurden bei 42 °C und pH 7 in einer wässrigen Lösung inkubiert und ihre Lebensfähigkeit über einen Zeitraum von bis zu 18 Tagen bestimmt. Zur Validierung der Ergebnisse wurden Samen ausgewählter Arten einer anaeroben Vergärung bei 42 °C im Labormaßstab ausgesetzt. Die mittlere Inaktivierungszeit unterschied sich bei 8 der 11 untersuchten Wildpflanzen-Arten signifikant, wobei 6 Arten nur sehr langsam bzw. nicht vollständig inaktiviert wurden. Bei diesen Arten besteht das Risiko, dass sie mit dem Gärrest verbreitet werden. Mit Hilfe des Wasserbad-Screenings ließ sich das Überlebensrisikos der Samen in der mesophilen, anaeroben Vergärung verlässlich abschätzen.

Peatland restoration can have several objectives, for example re-establishing the natural habitat, supporting unique biodiversity attributes or re-initiating key biogeochemical processes, which can ultimately lead to a reduction in greenhouse gas (GHG) emissions. Every restoration measure, however, is itself a disturbance to the ecosystem.Here, we examine an ecosystem shift in a coastal fen at the southern Baltic Sea which was rewetted by flooding. The analyses are based on one year of bi-weekly closed chamber measurements of methane fluxes gathered at spots located in different vegetation stands. During measurement campaigns, we recorded data on water levels, peat temperatures, and chemical properties of peat water. In addition we analyzed the first 20 cm of peat before and after flooding for dry bulk density (DBD), content of organic matter and total amounts of carbon (C), nitrogen (N), sulfur (S), and other nutrients.Rewetting turned the site from a summer dry fen into a shallow lake with water levels up to 0.60 m. We observed a substantial die-back of vegetation, especially in stands of sedges (Carex acutiformis Ehrh). Concentrations of total organic carbon and nitrogen in the peat water, as well as dry bulk density and concentrations of C, N and S in the peat increased. In the first year after rewetting, the average annual exchange of methane amounted to 0.26 ± 0.06 kg m-2. This is equivalent to a 190-times increase in methane compared to pre-flooding conditions. Highest methane fluxes occurred in sedge stands which suffered from the heaviest die-back. None of the recorded environmental variables showed consistent relationships with the amounts of methane exchanged.Our results suggest that rewetting projects should be monitored not only with regard to vegetation development but also with respect to biogeochemical conditions. Further, high methane emissions that likely occur directly after rewetting by flooding should be considered when forecasting the overall effect of rewetting on GHG exchange.

Giardiasis, a gastrointestinal disease caused by Giardia duodenalis, is currently treated mainly with nitroimidazoles, primarily metronidazole (MTZ). Treatment failure rates of up to 20 percent reflect the compelling need for alternative treatment options. Here, we investigated whether orlistat, a drug approved to treat obesity, represents a potential therapeutic agent against giardiasis. We compared the growth inhibitory effects of orlistat and MTZ on a long-term in vitro culture adapted G. duodenalis strain, WB-C6, and on a new isolate, 14-03/F7, from a patient refractory to MTZ treatment using a resazurin assay. The giardiacidal concentration of the drugs and their combined in vitro efficacy was determined by median-effect analysis. Morphological changes after treatment were analysed by light and electron microscopy. Orlistat inhibited the in vitro growth of G. duodenalis at low micromolar concentrations, with isolate 14-03/F7 (IC50(24h) = 2.8 µM) being more sensitive than WB-C6 (IC50(24h) = 6.2 µM). The effect was significantly more potent compared to MTZ (IC50(24h) = 4.3 µM and 11.0 µM, respectively) and led to specific undulated morphological alterations on the parasite surface. The giardiacidal concentration of orlistat was >14 µM for 14-03/F7 and >43 µM for WB-C6, respectively. Importantly, the combination of both drugs revealed no interaction on their inhibitory effects. We demonstrate that orlistat is a potent inhibitor of G. duodenalis growth in vitro and kills parasites at concentrations achievable in the gut by approved treatment regimens for obesity. We therefore propose to investigate orlistat in controlled clinical studies as a new drug in giardiasis.