weed seed survival during anaerobic digestion in biogas plants

Weed Seed Survival during Anaerobic Digestionin Biogas Plants

Paula R. Westerman1,2 & Bärbel Gerowitt1

1 Group Crop Health, Faculty of Agricultural and Environmental Sciences, University of Rostock,Satower Str. 48, 18059 Rostock, Germany

2 Author for Correspondence; e-mail: [email protected]

Published online: 7 August 2013# The New York Botanical Garden 2013

Abstract Anaerobic digestion using animal manure and crop biomass is increasinglybeing used to produce biogas as a durable alternative to fossil fuel. The sludge, theleftover after processing, is returned to the field as a crop fertilizer. If weed seeds surviveanaerobic digestion, the use of contaminated sludge poses a phytosanitary risk. Theconditions that seeds are likely to encounter in biogas plants, and the effect of these, inparticular temperature, on seed viability were reviewed. Knowledge on seed defencemechanisms and how these might protect seeds from inactivation in biogas reactors wassummarized. Mechanisms of seed inactivation can be classified as thermal, biologicaland chemical. Weed species with hard seeds (physical dormant), high thermoresistance,a thick seed coat or adapted to endozoochory were identified as high-risk species.Specific seed traits could be used in future tests to circumvent extensive testing of seedsin biogas reactors.

Keywords Anaerobic digestion . Biogas reactors . High-risk species .

Physical dormancy . Thermoresistance .Weed seed survival

Introduction

General

Anaerobic digestion of animal manure, organic waste and crop biomass is used forbiogas production as a sustainable alternative to fossil fuels, as a more efficient wayof disposing of waste products, as a sludge reducing procedure, and as an odour-lessalternative to composting. Various European governments provide financial andlegislative incentives to biogas production. This has resulted in Germany, for exam-ple, in some 7,000 biogas plants, processing tons of manure, and some five mega tonsof maize biomass per year (http://www.biogasportal.info/). The biogas produced issufficient to satisfy 5.5 % of the current demand for electricity in Germany (http://www.erneuerbare-energien.de/). These numbers are expected to rise in the future.

The sludge or digestate, the semi-solid leftover after anaerobic digestion, has severaladvantages as a crop fertilizer compared to animal slurry; a lower C/N ratio, higher

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concentrations of K, P and N, and a better availability of the latter, as NH4, increased pH,improved fluidity, and reduced odour emissions (Arthurson, 2009). However, the ingre-dients for biogas production, namely manure, organic wastes and crop biomass, can becontaminated with pests and diseases, and, if they survive the process of anaerobicdigestion, the use of contaminated sludge as a crop fertilizer could constitute a threat tothe health of humans, animals and plants alike. Known human and veterinary pathogensand parasites commonly found in animal manures, biological and household wastes, andsewage and sewage sludge are listed, for example, in Bendixen (1994); Déportes et al.(1995); Colleran (2000), or Martens and Böhm (2001). Little is known with regard to thephytosanitary risks associated with the presence and survival of weed seeds.

In Germany, most anaerobic digesters are located on-farm and operated by the samefarmers that produce the manure or biomass. In contrast, in Denmark, centralisedanaerobic digesters exist, which process manure and other feedstocks from 60 to 80farms per plant (Colleran, 2000). The digestate can be used either on-farm or sold andused elsewhere. In the latter case, the sludge can serve as a vehicle for the spread of pestsand diseases. The likelihood of spread will be highest for common organisms, as thesehave the highest probability to enter the anaerobic digestion cycle, and organismsresistant to the adverse conditions in anaerobic digesters.

Because it is impossible to test the fate of all (micro-)organisms in anaerobicdigesters, testing is usually restricted to a limited sets of organisms, the so-calledindicator organisms (Colleran, 2000; Sahlström, 2003). These include organisms thatare prescribed by national authorities for the sanitation of biosolids or biowaste, e.g.,Ascaris suum and poliovirus 1 (PVS-1)(USA; U.S. Environmental Protection Agency,2003), tobacco mosaic virus, Plasmodiophora brassicae, and tomato seeds (Germany;Bundesministerium für Umwelt, Naturschutz und Reaktorsicherheit, 1998, BioAbfV),Streptococcus feacalis (Denmark; Bendixen, 1994),Clostridium perfringens, Salmonellaspp., and amember of the Enterobacteriaceae, e.g., Escherichia coli (EU; The Europeanparliament and council, 2002. Regulation (EC) No. 1774/2002 and supplement No.208/2006). The emphasis in legislation is on limiting veterinary and human health risks;phytosanitary risks have been less of a priority, except in Germany, where indicators forplant pathogens are explicitly included in the BioAbfV regulation. The use of anindicator species is based on the assumption that “it can be reliably used to evaluatethe hygienisation efficiency of the anaerobic treatment process” (Colleran, 2000),meaning that other organisms will be inactivated as efficiently as or more efficientlythan the indicator species. The advantage is that the hygienisation efficiency of instal-lations and processes can be compared. A disadvantage is that organisms that are moreresistant to anaerobic digestion than the indicator species may survive and spread.

The speed of reduction in biogas reactors is usually expressed as the decimalreduction time, Dx (Lewis, 1956), which is the time required at temperature x to kill90 % (one log-unit) of the organisms being studied. In general, bacterial spores (e.g.,D35=D53≥28 day, Olsen & Larsen, 1987) tend to bemore resistant to anaerobic digestionthan fungi (e.g., D37=0.8–10 day, D55=0.02−>30 day, Schnürer & Schnürer, 2006),which, in turn, tend to be more resistant than viruses (e.g., D35=0.2–2.3 day, D55=0.01–1.5 day; Lund et al., 1996), vegetative bacteria (e.g., D35=0.9–7.1 day, D53=0.3–1.2 h;Olsen& Larsen, 1987), helminth cysts (D35=0.4 day; Turner et al., 1983), ova and larvae(e.g., full inactivation at 35 °C in <1 day and at 53 °C in 0.04–0.2 day, Olsen et al.,1985). However, there are always some that are more resistant than others, such as, for

282 P.R. Westerman, B. Gerowitt

example, thermoresistant fungi (Schnürer & Schnürer, 2006). Decimal reduction timesfor weed seeds tend to be comparable to those of fungi (e.g., D41=0.8–19.7; Westermanet al., 2012b).

Risks Associated with Sludge Contaminated with Weed Seeds

In the case of weeds, no indicator species are routinely included in sanitation procedures,except tomato seeds, which are prescribed in the BioAbfVregulation as a proxy for weedseeds (Germany; Bundesministerium für Umwelt, Naturschutz und Reaktorsicherheit,1998, BioAbfV). Seeds are a logical choice, because they are the most likely structure tosurvive anaerobic digestion. Seeds tend to be well-protected by seed coat and otherprotective structures, they usually have a low metabolic rate and dormancy mechanismsthat prevent germination, and they are known to be able to endure and survive adverseenvironmental conditions. However, weed species differ considerably in seed charac-teristics, and are, therefore, expected to differ in survival probability during anaerobicdigestion. Systematic research on the ability of weeds from different taxonomic andfunctional groups to survive anaerobic digestion is lacking, and it is, therefore, difficultto say how well tomato represents weeds.

Weed seeds can enter the biogas chain either via crop biomass or via animalmanure. In maize, the most common feedstock in Germany, 180 species of weedshave been recorded (Mehrtens et al., 2005). The most frequently encountered weedspecies were: Chenopodium spp. (79.7 % of the fields), Stellaria media (61.0 %),Fallopia convolvulus (55.7 %), Echinochloa crus-galli (53.0 %), Matricaria spp.(50.3 %), Viola arvensis (47.8 %), Polygonum aviculare (45.8 %), Lamium spp.(41.6 %), Galium aparine (39.7 %), Elytrigia repens (39.4 %), Solanum nigrum(36.3 %), Thlaspi arvense (34.3 %), Capsella bursa-pastoris (33.8 %), Veronica spp.(31.1 %), Poa spp (27.7 %), Cirsium arvense (25.9 %), Polygonum persicaria(24.8 %), Atriplex patula (21.6 %), and Polygonum lapathifolium (20.8 %). Theabove percentages were related to incidences, not weed abundances. Figures for weedidentity and intensity in other energy crops are lacking.

The presence of weeds in a field does not necessarily mean that the harvestedbiomass will contain their seeds. For example, the density of a weed could be so lowthat the risk of contamination of biomass during harvest is negligible. Some weedplants are physically so small that they escape the combine-harvester, such as forexample S. media, Veronica spp, Vi. arvensis, Myosotis arvensis, Anagallis arvensis,or Argentina anserine (Westerman & Gerowitt, 2012). Most farmers cut maize at aheight of 10 to 30 cm (Wu & Roth, 2004). Many weeds remain vegetative (e.g.,Mertens, 1998) due to competition for light. Some weeds produce seeds either soearly that they are all shed prior to harvest, or so late that most seeds will be immatureand non-viable at harvest (e.g., Harker et al., 2003). Compared to maize grown forgrain, maize for silage and biogas production is usually harvested early, giving fewweeds the possibility to reproduce. In maize, the number of weed seeds in flowerheads above the cutting height varied between fields from 0 to 157 000 seed m−2

(Westerman & Gerowitt, 2012).With regard to the probability that manure is contaminated with seeds, research on

cattle and sheep manure shows that large numbers of viable seeds from different weedspecies can survive and be present in the manure, but density and identity vary with

Weed Seed Survival during anaerobic Digestion in Biogas Plants 283

farm, depending on what animals had been fed (e.g., Dastgheib, 1987; Cudney et al.,1992; Mt. Pleasant & Schlather, 1994).

Risks associated with spread and infestation of weeds will be highest for invasive,quarantine and troublesome weeds that do not have a widespread distribution yet. Inaddition to the infestation risk associated with the use of contaminated sludge, lowlevels of weed survival could select for ‘anaerobic-digestion-resistant’ biotypes,provided that the sludge is repeatedly recycled on-farm and provided that seedsurvival has a genetic basis.

Scope and Aim of this Review

The purpose of this review was 1) to provide an overview of what is known withregard to weed seed survival after anaerobic digestion in biogas plants (ChaptersIII and IV) and 2) identify high-risk weed species, i.e., species that have aparticularly high probability of surviving the conditions encountered in biogasplants.

Unfortunately, literature on these subjects was scarce and fragmentary. The liter-ature search was furthermore hampered by a large amount of ‘grey’ literature, i.e.,papers that are not peer-reviewed, not easily accessible, and often published in alanguage other than English. Despite an extensive search, the only literature describ-ing empirical data on seed survival in anaerobic digesters consists of seven peer-reviewed publications (Jeyanayagam & Collins, 1984; Engeli et al., 1993; Šarapatkaet al., 1993; Ryckeboer et al., 2002; Strauß et al., 2012 (in German); Westerman et al.,2012a, b), one article in a popular scientific journal (Schrade et al., 2003 (inGerman)), and six project reports (Hansen & Hansen, 1983 (in Danish); Böhmet al., 2000 (in German); Lorenz et al., 2001 (in German); Katovich et al., 2004;Marcinisyn et al., 2004 (in German); Westerik & Kleizen, 2006 (in Dutch); Leonhardtet al., 2010 (in German)). Three of these only report on the survival of tomato seeds(Böhm et al., 2000; Lorenz et al., 2001; Ryckeboer et al., 2002), and one only on thesurvival of crop seeds (Strauß et al., 2012).

To achieve the second goal, i.e., identify high-risk weed species, survival probabilitiesfor a large range of weed species, involving different taxonomic or functional groups orcontrasting seed characteristics, would be required, such that generalizations are possi-ble. However, systematic research onweed seeds is lacking andmost studies included nomore than 5–6 weed species, which is insufficient for the purposes of this review.Mechanisms of seed inactivation can be classified as thermal, biological or chemical.However, evidence of chemical inactivation is completely lacking and is, therefore,excluded from this review. We here provided a brief background on the conditions thatseeds are likely to encounter in biogas plants (Chapter III) and summarized what isknown with regard to the effect of these conditions, in particular temperature andduration of exposure (Chapter V), and microbial activity (Chapter VI), on seed viability.We borrowed knowledge from related fields, such as endozoochory, weed seed banksand survival of microorganisms in biogas reactors, to complement the deficient litera-ture. In addition, we tried to identify seed traits that might be linked to seed survival inbiogas reactors (Chapter VII), such that in the future extensive testing of seeds inbiogas reactors can be omitted and replaced by simple, predictive tests, basedon specific seed traits.


Anaerobic Digestion

Reactor Types

Continuous Flow-Through, Stirred Tank Reactor. The conditions to which patho-gens, pests and weed seeds are exposed depend on the type of reactor used and theoperational settings chosen. The most commonly used type of anaerobic digester forbiogas production in Germany, the country with the highest number of biogas plantsin the world, is the so called single-stage, continuous flow-through, stirred tankreactor (CSTR) that uses high solids as a feedstock in wet fermentation and isoperated at the mesophilic temperature range. ‘Single-stage’ refers to the fact thatall four microbiological steps involved in digestion, namely hydrolysis, acidogenesis,acetogonesis and methanogenesis, occur simultaneously in the same reactor. Eachprocess is conducted by a specific group of microorganisms, with its own set ofrequirements with regard to pH, temperature, etc. The fact that all four processes takeplace in the same reactor means that the prevailing conditions are a compromise thatis suboptimal for all groups involved. ‘Continuous flow-through’ refers to the factthat the reactor is fed at the inlet with new biomass and slurry, and relieved fromsludge at the outlet, continuously and simultaneously. The content of the tank isstirred continuously, using various designs of agitators (e.g., Deublein & Steinhauser,2011), such that new feedstock is mixed in with the partially digested feedstockalready present. ‘Mesophilic’ refers to the temperature range at which anaerobicdigestion takes place, namely 20–45 °C (92 % of the biogas plants in Germany), asopposed to psychrophylic (< 20 °C) or thermophilic (>45 °C). ‘High solid, wetfermentation’ refers to the fact that the concentration of total suspended solids inthe liquid is >15 %; the slurry in the reactor is thick and viscous, but can still bepumped around. This type of reactor is typically fed a mixture of agricultural waste,such as animal slurry or mist (swine or cattle), in combination with agricultural rawproducts, such as maize, small-grain cereals, or potato- or sugar beet residues (co-digestion), but the exact composition of the feed varies from reactor to reactor.

The residence time, i.e., the time that the substrate, including weed seeds, isexposed to the conditions inside anaerobic digesters, is variable. The average resi-dence time, or hydraulic retention time (HRT), is calculated as the volume of the tankdivided by the flow rate. Depending on the operational temperature, the HRT ofmesophilic CSTRs is usually between 20 and 40 days, that of thermophilic CSTRsbetween 8 and 15 days (Deublein & Steinhauser, 2011). However, there is consider-able variation in the actual residence time inside a reactor, which can be studied withthe help of tracers (e.g., Monteith & Stephenson, 1981; Smith et al., 1993; Teefy,1996). In an ideal CSTR, where mixing is perfect and instantaneous, a sudden pulseof a tracer at the inlet would cause an immediate peak followed by an exponentialdecline of the tracer material at the outlet. An important consequence is that a certainproportion of the tracer material will be exposed for (much) shorter or (much) longerperiods of time than the HRT. However, no CSTR is perfect because of short-circuiting in the reactor, imperfect mixing, or dead space in the vessel. Deviationsfrom the ideal CSTR affect the shape and width of the tracer curve, causing higher orlower variance around the HRT. Tracer studies, assisted by modelling, can help tocharacterize real reactors to optimize biogas production (e.g., Capela et al., 2009) and


to predict the level of disinfection that can be achieved in water treatment facilities(e.g., Teefy, 1996). The shape and width of the tracer curve will be variable betweenreactors used in biogas production, as most are custom-build and unique to somedegree.

Batch Reactor. Another type of reactor is the so-called ‘batch’ reactor. The differencewith the CSTR is that it is fully loaded with organic materials, sealed, the contents aredigested and the reactor unloaded. The reason that this type of reactor is mentionedhere is not because it is important in commercial biogas production (e.g., approx. 5 %of the biogas plants in Germany), but because it is often used in controlled laboratoryconditions as a substitute for CSTRs and easier to handle. Batch reactors differ fromCSTRs in that the four steps in the microbiological conversion of organic material tobiogas occur sequential, not simultaneously, causing fluctuations in pH and theconcentration of intermediate substances, and that the residence time is fixed andwithout variance. The relationships between results obtained under laboratory con-ditions vs. full-scale commercial conditions, or batch vs. continuous conditions areambiguous. For example, pathogenic bacteria were more easily reduced under labo-ratory conditions than in full-scale, continuous digesters (Carrington et al., 1982).Similarly, batch reactors were more effective at destroying seeds and pathogenicbacteria than CSTRs, operated at the same temperature and HRT (Jeyanayagam &Collins, 1984; Kearney et al., 1993). In contrast, Olsen and Larsen (1987) found thatbatch and continuous digesters were equally effective at reducing bacterial pathogens,and Westerman et al. (2012a, b) found that the ranking of weed species differedbetween experimental batch reactors and commercial CSTRs.

Plug-Flow Reactor. Three publications on seed survival in biogas reactors(Ryckeboer et al., 2002; Katovich et al., 2004; Marcinisyn et al., 2004) deal with aplug-flow reactor (PFR), which is a horizontal pipe or tunnel, to which substrate isadded at one end and digestate is removed from the other end. A main difference withthe above-mentioned types of reactors is that the substrate is stackable (dry fermen-tation), not liquid, and that the substrate is pushed through the PFR and not mixed.The retention time is, therefore, fixed and without variance. In this sense, processing isas in a batch loaded reactor, although the actual loading and unloading is continuous.

Gastrointestinal Tract of Animals. Because of interest in plant dispersal by animals(endozoochory), a large amount of literature is available on seed survival in thealimentary tract of animals, in particular ruminants (for overview see, for example;Bonn & Poschlod, 1998; Hogan & Phillips, 2011). The processes that take place inthe gastrointestinal tract of animals bear resemblance to those in anaerobic reactors,although temperature is fixed, dependent on the body temperature of the animalspecies. Insights gained in one system could be used in the other. However, thereare a number of differences in process characteristics and research methodology.

1) The digestion of lignocellulosics in the digestive tract of ruminants is much moreeffective than in anaerobic digesters (Bayane & Guiot, 2011). Lignocellulosicsare the plant’s main structural components, they occur in cell walls, and they areone of the main energy carriers in biomass. The biodegradability of cellulose and


hemicellulose is affected by the degree of lignification (Van Soest, 1982, 1988).Lignin cannot be cleaved by hydrolytic enzymes, and thus protects cellulose andhemicellulose from hydrolysis (Hofrichter, 2002). Only a few specialized fungiand bacteria can effectively digest lignin via lignin-modifying enzymes, such asperoxidises and phenol-oxidases. Some of these occur in the digestive tract ofruminants (Trinci et al., 1994) and some other animals. Because the degradationof lignocellulose in bioreactors is slow and limited, lignin degrading enzymesand microorganisms have attracted attention because of their potential to improvebiogas and biofuel production (e.g., Bayane & Guiot, 2011; Jin et al., 2011;Sanderson, 2011).

2) The residence time in ruminants is much shorter than in commercial reactorvessels. The mean retention time (MRT) ranges from 25 h in deer (Mouissieet al., 2005) to 50–63 h in cattle and sheep (Glendening & Paulsen, 1950; SimaoNeto et al., 1987; Cosyns et al., 2005).

3) Anaerobic digestion in the reticulorumen (1st and 2nd chamber of the stomach)and intestine of ruminants is interrupted by acidic hydrolysis in the abomasum(4th chamber or true stomach) with a pH of around 2. In cattle, normally only 2–4 h are spent in the strongly acidic abomasums and duodenum (first section of thesmall intestine) (Gardener et al., 1993b). Some found that the duration of stayand the conditions encountered in the rumen were decisive for seed survival(Simao Neto & Jones, 1987; Carpanelli et al., 2005) and that additional passagethrough the true stomach and intestines had little effect on seed degradation andgermination. However, using a simulated (in vitro) digestive process, othersfound that both the rumen and the stomach negatively affected seed survival(e.g., Edwards & Younger, 2006). Pepsin incubation, as in the stomach, was verydamaging to seeds and made prior rumen incubation largely irrelevant.

4) The residence time in the alimentary tract of ruminants is influenced by particlesize and density. The reasons for this are a) selective filtering by the mat, a thickmass of partially degraded, fibrous material that is formed in the rumen, incombination with ruminal contractions, and b) smaller particles are better ableto pass through the orifice, the opening between the reticulorumen and theabomasums, than larger particles (Poppi et al., 1985). For example, the meanretention time (MRT) was found to be approx. 20 h longer for 10 mm particlesthan for 1 mm particles (Kaske & van Engelhardt, 1990). The MRTwas minimalfor particles of 6.4 mm and longer for particles that were either shorter (3.2 mm)or longer (12.7 mm) (Ehle & Stern, 1986). The MRT in the reticulorumen waslongest for particles with a density around 1.2–1.4 g.ml−1, and shorter for bothlighter and heavier particles (DesBordes & Welch, 1984; Ehle & Stern, 1986;Kaske & van Engelhardt, 1990). Differences in the fate of ingested seeds can,therefore, be caused by differences in sensitivity of weed species to anaerobicdigestion as such, or by differences in the duration that particles remain in thedigestive tract. Often, it is impossible to distinguish between the two causes. InCSTRs, the residence time cannot be influenced by the size, shape or density ofthe particles that inhabit them, because solids are carried along with liquids andthe retention time of solids (SRT) is equal to that of liquids (HRT). In some typeof CSTRs, however, the solid fraction is separated from the liquid fraction afteroutflow and re-circulated into the reactor, resulting in SRT > HRT. In that case,


seeds could be processed either as the liquid or as the solid fraction. We are notaware of any literature investigating the fate of differently sized particles in suchreactors.

5) Usually, only germination after passage through the gastrointestinal tract wasscored and not seed viability. This means that in many studies on zoochorymortality is confounded with dormancy. Seeds that were fully digested were oftenignored, making it impossible to get a pure estimate of seed mortality. In the mostextreme cases, the number of seeds recovered of a particular species was notexpressed as a proportion of the number of seeds that entered the animal, but as aproportion of the total number of seeds recovered from the faeces (e.g., Pakemanet al., 2002). As a consequence, differences in the availability of seeds to grazersand the probability of ingestion are ignored (for discussion see Bruun & Poschlod,2006). The latter category of papers was, therefore, omitted from the review.

Despite the differences and recognized problems, literature on seed survival afterpassage through animals was included in this review, to supplement the scarceinformation available on seed survival in anaerobic digesters. It is clear that theoutcome of animal feeding experiments should be interpreted cautiously.

Conditions During Anaerobic Digestion

In mesophilic CSTRs, weed seeds will be exposed to dark, moist, anaerobic conditions,constant temperatures that can differ between reactors from approx. 20 to 40 °C, and apH that is ideally between 6.8 and 8. Most environmental conditions will be kept withintight bounds, because deviations could disrupt biogas production. In addition to water,methane and CO2, a wide range of substances can occur in bioreactors, includinghydrolases, lipases, proteases, and other enzymes involved in decomposition, aminoand organic acids, including long chain fatty acids, H2S, HS

-, S-, alcohols, NH4+/NH3,

and cyanides. Particularly when sewage and organic household waste are used as biogasfeedstock, light and heavy metal ions may be present as well as a large range of organic(micro) pollutants, including polycyclic aromatic hydrocarbons, N-substituted aromatics(e.g., nitrobenzenes, nitrophenols), chlorophenols, halogenated carbohydrates (e.g.,chloroform), phytohormones and analogues, and their degradation products (e.g.,Déportes et al., 1995; McGrath, 1999; Langenkamp et al., 2001). These substanceshave been studied because they are important intermediates in the process of biogasproduction, because of negative side-effects, such as disruption of biogas production(Chen et al., 2008) and odour nuisance (Hansen et al., 2004), or because of a reduction inthe quality of the digestate as a crop fertilizer (Lukehurst et al., 2010).

As soon as seeds and other biomass enter the liquid medium of CSTRs, theirsurfaces will be colonized by microorganisms, such as bacteria, archaea, and protists.They form a biofilm, a coordinated functional community that is much more efficientthan a mixed population of floating organisms (Costerton et al., 1995). Biofilmmicroorganisms produce and maintain spatial and temporal gradients of pH, metab-olites, etc., which allow the coexistence with specialized and fastidious species, suchas methanogenic bacteria (Costerton et al., 1995 and references therein). Onceestablished, associations between microorganisms and surfaces are often difficult todisrupt physically or chemically.


The conversion of biomass to biogas develops via four microbiological steps, eachinvolving a specific group ofmicroorganisms. During hydrolysis, microorganisms breakdown the insoluble organic polymers, such as proteins, lipids and carbohydrates, intoamino acids, fatty acids and sugars. The acidogenics, in particular members of thegenera Clostridium, Paenibacillus, and Ruminococcus, convert the amino acids, fattyacids and sugars into carbonic acids, alcohols, hydrogen, carbon dioxide and ammonia(acidogenesis). Next, acetogenics and sulphate-reducing bacteria convert the organicacids into acetic acid, hydrogen, and carbon dioxide (acetogenesis), and, finally,methanogenics, in particular those belonging to the Methanosaeta, the generaMethanobacterium and Methanosarcina, and the species Methanospirillum hungatiiconvert the hydrogen, acetic acid and carbon dioxide into methane and carbon dioxide.For a short overview of the chemical pathways and the microorganisms involved thevarious steps in anaerobic digestion, we refer to Deublein and Steinhauser (2011).

Up- and Downstream Processes

Anaerobic digestion is not a stand-alone process. Biomass, including weed seeds, willrun through a series of events before (upstream processes) and after (downstreamprocesses) anaerobic digestion. These processes will affect weed seeds as well.

Upstream. Weed seeds will enter biogas plants either via animal manure or via biomass.In the former case, seeds must pass through the gastrointestinal tract of animals, usuallypigs or cattle. At one time, these seeds had been fed to the animals either as fresh(grazing), dried (hay), or ensiled material. In the latter case, seeds were freshly harvestedtogether with maize or other feedstock, or they had been stored in silage. Both thealimentary tract of animals and silage are hostile environments to seeds (Blackshaw &Rode, 1991; Westerman et al., 2012a). Anaerobic digesters are often preceded bygrinders or cutters for reducing the size of biomass particles andmixers for homogenizingwater, manure and biomass. Mechanical damage to seeds prior to exposure to anaerobicdigestion is possible. In several countries, anaerobic digestion has to be preceded by apasteurization step, which usually involves treatment of animal manure and other organicwastes in a (aerobic) sanitation tank at 70 °C for at least 1 h (Bendixen, 1994; Colleran,2000; Sahlström, 2003). Chemical sanitation is also possible and involves enhancing thepH to above 8 by the addition of large quantities of calcium hydroxide (lime) (Haas et al.,1995; Deublein & Steinhauser, 2011). High pH and the presence of ammonia (NH3),which is made available at pH>8, are effective biocides, particularly against viruses. Hightemperatures, high pH and ammonia are likely to influence seed viability. Consequently,the probability that undamaged, highly vital and vigorous weed seeds will enter thebiogas reactors directly is low. Most experiments in anaerobic digesters, however, usenewly produced or laboratory stored seeds and single out anaerobic digestion as such.This is likely to cause overestimation of seed survival over the entire chain.

Downstream. Usually, the output from anaerobic digesters is stored in a basin untilfurther use. Either aerobic or anaerobic digestion will continue, causing additionalseed mortality (Strauß et al., 2012). In addition, post-sanitation may take place, as analternative to pre-sanitation, involving thermal hygienisation, exposing seeds to thesame kind of stress as described for upstream hygienisation (Strauß et al., 2012).


Seed Viability as Affected by Anaerobic Digestion

In Biogas Plants

In anaerobic digesters, the viability of seed declines exponentially over time (Jeyanayagam& Collins, 1984; Schrade et al., 2003; Westerik & Kleizen, 2006; Leonhardt et al., 2010;Strauß et al., 2012;Westerman et al., 2012b), in a similar way as the exponential inactivationof bacterial, fungal and viral pathogens. The exponential inactivation of seeds tends to bepreceded by a lag phase, indicating that seeds are initially unaffected by anaerobic digestion.Temperature was found to be the most important factor influencing seed survival; lag phasewas shorter and decline faster with increasing temperatures. For example, in experimentalCSTRs at approximately 54 °C, eight species of seeds were killed within 24 h, while 3 dayswere required to inactive four out of eight species at 36 °C (Schrade et al., 2003; Table 1).Similarly, ten species of weed seeds were inactivated within 1 day in a batch loaded reactorat 50 °C, whereas at least 3 day were required to inactivate seven out of ten weed species at35 °C (Leonhardt et al., 2010; Table 1). Temperaturewas also found to be themost importantfactor influencing survival of pathogenic bacteria (Olsen & Larsen, 1987; Dumontet et al.,1999), fungi (Schnürer & Schnürer, 2006), and viruses (Lund et al., 1996). Not only seedviability was affected, but also seed vigour. The speed of germination of surviving seedsdecreasedwith increasing exposure time to anaerobic digestion in experimental batch loadedreactors (Westerik & Kleizen, 2006). By physically separating hydrolysis from the otherthree steps in the anaerobic digestion process (two stage digester), Engeli et al. (1993)showed that most seeds were inactivated during hydrolysis, the first step in anaerobicdigestion.

Some species survived for appreciably longer periods inside biogas reactors thanothers, in particular under mesophilic conditions (Table 1). For example, tomato seedsproved to be more resistant to mesophilic digestion than most other species (Westerik &Kleizen, 2006; Strauß et al., 2012; Table 1), leading to the opinion that tomatowouldmakean appropriate indicator for sanitation. However, Schrade et al. (2003) and Westermanet al. (2012a, b) found that some species survivedmuch better than did tomato. Frequently,the same species were included in studies and, therefore, the same species tend to surfaceas being resistant to mesophilic anaerobic digestion; Abutilon theophrasti, C. album andAmaranthus retroflexus at approximately 37 °C (Katovich et al., 2004); C. album, A.retroflexus, At. patula, E. crus-galli, P. lapathifolium, and Rumex obtusifolius at 35 °C(Leonhardt et al., 2010); Amaranthus retroflexus, C. album, E. crus-galli and R.obtusifolius at approximately 30 °C (Šarapatka et al., 1993); C. album and R. obtusifoliusat approximately 36 °C (Schrade et al., 2003); flax, mustard and oilseed rape at 38 °C(Strauß et al., 2012), and A. theophrasti,Datura stramonium, Erodium cicuratium,Malvaneglecta and Vicia tetrasperma at 37 °C (Westerman et al., 2012a).

Differences between species were also found under thermophilic conditions.Polygonum lapathifolium (59 %), C. album (57 %), Amaranthus sp. (23 %) and E.crus-galli (1 %) survived 3 day at 45 °C, in contrast to six other species (Leonhardtet al., 2010). Tomato and Urtica urens required 24 h at 51 °C to be fully inactivated,in contrast to four other species that were inactivate much faster (Westerik & Kleizen,2006; Table 1).

Ranking of species differed between studies. For example, survival of P.lapathifolium was lowest in the study by Šarapatka et al. (1993) and one of the


Table 1 Inactivation of seeds during anaerobic digestion, either expressed as survival (%) given a certainexposure time, or as the length of time required until full inactivation, given specific reactor types andoperational temperatures

Reference Species Reactor type Temperature[°C]

Exposure[d]

Survival(%)

Inactivation[d]

Böhm et al.,2000

tomato Exp. CSTR 33 21

Engeli et al.,1993

Rumex obtusifolius Exp. batch 35 14 0

Engeli et al.,1993

tomato Exp. batch 35 14 56a

Jeyanayagam &Collins, 1984

Panicumdichotomoflora

Exp. CSTR 35 28 55–63b

Jeyanayagam &Collins, 1984

Sorghumhalepense

Exp. CSTR 35 28 75–80b

Katovich et al.,2004

Abutilontheophrasti

PFR 37 20 16


Amaranthusretroflexus

PFR 37 20 1


Chenopodiumalbum

PFR 37 20 12


Panicum miliaceum PFR 37 20 0


Polygonumpersicaria

PFR 37 20 0


Setaria faberi PFR 37 20 0

Leonhardtet al., 2010

Amaranthus sp. Exp. batch 35/50 7/1


Atriplex patula Exp. batch 35/50 7/1


Avena fatua Exp. batch 35/50 3/1


Bromus sp. Exp. batch 35/50 1/1


Chenopodiumalbum

Exp. batch 35/50 21/1


Echinocloacrus-galli

Exp. batch 35/50 7/1


Elytrigia repens Exp. batch 35/50 1/1


Galium aparine Exp. batch 35/50 1/1


Polygonumlapathifolium

Exp. batch 35/50 7/1


Rumex obtusifolius Exp. batch 35/50 7/1


Amaranthus sp. Comm. CSTR 42/45/45 3/7/3


Table 1 (continued)


Exposure[d]

Survival(%)

Inactivation[d]


Atriplex patula Comm. CSTR 42/45/45 3/3/3


Avena fatua Comm. CSTR 42/45/45 3/3/3


Bromus sp. Comm. CSTR 42/45/45 3/3/3


Chenopodiumalbum

Comm. CSTR 42/45/45 7/7/3


Echinocloa crus-galli

Comm. CSTR 42/45/45 3/7/3


Elytrigia repens Comm. CSTR 42/45/45 3/3/3


Galium aparine Comm. CSTR 42/45/45 3/3/3


Rumex obtusifolius Comm. CSTR 42/45/45 3/3/3



Comm. CSTR 42/45/45 3/7/3


Ambrosiaartemesiifolia

Exp. batch 35 3


Capsella bursa-pastoris

Exp. batch 35 0.42


maize Exp. batch 35 3


red clover Exp. batch 35 3


Stellaria media Exp. batch 35 0.42

Leonhardtet al. 2010

Trifolium aestivum Exp. batch 35 1

Lorenzet al., 2001


Lorenzet al., 2001


Marcinisynet al., 2004

Rumex obtusifolius Comm. CSTR 39–47 20 0.09


tomato Comm. CSTR 38–48 21/42 0/0


tomato Comm. PFR 49 14 0


tomato Comm. CSTR 55 14/28 0/0

Ryckeboeret al., 2002

tomato Exp. PFR 52 0.8–1.0

Šarapatkaet al., 1993

Agropyron repens Comm. batch 50→30c 30 0


Table 1 (continued)


Exposure[d]

Survival(%)

Inactivation[d]



Comm. batch 50→30c 30 4


Avena fatua Comm. batch 50→30c 30 0


Chenopodiumalbum



Chenopodiumstrictum



Echinochloa crus-galli



Plantago major Comm. batch 50→30c 30 0





Rumex obtusifolius Comm. batch 50→30c 30 19


Thlaspi arvense Comm. batch 50→30c 30 0


Tripleurospermummaritimum


Schrade et al.,2003

Alopecurusmyosuroides

Exp. CSTR 36/54 1/1

Schrade et al.,2003

Chenopodiumalbum

Exp. CSTR 36/54 21/1

Schrade et al.,2003

oilseed rape Exp. CSTR 36/54 1/1

Schrade et al.,2003

Rumex obtusifolius Exp. CSTR 36/54 7/1

Schrade et al.,2003

Sinapis arvensis Exp. CSTR 36/54 1/1

Schrade et al.,2003

Thlaspi arvense Exp. CSTR 36/54 3/1

Schrade et al.,2003

tomato Exp. CSTR 36/54 7/1

Schrade et al.,2003

wheat Exp. CSTR 36/54 1/1

Strauß et al.,2012

flax Exp. batch 38 6

Strauß et al.,2012

maize Exp. batch 38 2

Strauß et al.,2012

mustard Exp. batch 38 6

Strauß et al.,2012

oilseed rape Exp. batch 38 6

Strauß et al.,2012

soyabean Exp. batch 38 2


Table 1 (continued)


Exposure[d]

Survival(%)

Inactivation[d]

Strauß et al.,2012

tomato Exp. batch 38 30

Strauß et al.,2012

wheat Exp. batch 38 3

Westerik &Kleizen, 2006

Agrostemmagithago

Exp. batch 38/51 0.25/0.25


Jacobaea vulgaris Exp. batch 38/51 1/0.1


Rumex acetosella Exp. batch 38/51 1/0.1


Taraxacumofficinale

Exp. batch 38/51 0.25/0.04


tomato Exp. batch 38/51 5/1


Urtica urens Exp. batch 38/51 2/1

Westermanet al., 2012a

Abutilontheophrasti

Exp. batch 37 30 36.5



Exp. batch 37 30 0


Anchusa arvensis Exp. batch 37 30 <1


Bromus secalinus Exp. batch 37 30 0


Capsellabursa-pastoris

Exp. batch 37 30 0


Chenopodium album Exp. batch 37 30 <1


Datura stramonium Exp. batch 37 30 6.7


Echinochloacrusrgalli

Exp. batch 37 30 0


Erodium cicuratium Exp. batch 37 30 21.1


Fallopiaconcolvulus

Exp. batch 37 30 <1


Galium aparine Exp. batch 37 30 0


Geranium pusillum Exp. batch 37 30 0


Lithospermumarvense

Exp. batch 37 30 0


Malva neglecta Exp. batch 37 30 30.4


Rumex obtusifolius Exp. batch 37 30 0


highest in the study by Leonhardt et al. (2010); survival of E. crus-galli was higherthan that of C. album in the study by Šarapatka et al. (1993), but lower in the study byLeonhardt et al. (2010). Either the initial seed quality or the conditions inside reactorsmust have varied between studies. Furthermore, ranking of species may change withexposure time due to differences in the shape of the seed survival curves(Jeyanayagam & Collins, 1984; Leonhardt et al., 2010; Strauß et al., 2012;Westerman et al., 2012b).

In the Digestive Tract of Animals

As in biogas reactors, seed viability decreased exponentially over time after an initiallag phase, in the digestive tract of ruminants (Alomar et al., 1994; Alomar & Ulloa,1994; Fredrickson et al., 1997; Gökbulak, 2002). The same was observed when seedswere exposed to the (simulated) rumen alone (Simao Neto & Jones, 1987; Blackshaw& Rode, 1991; Alomar et al., 1992; Fredrickson et al., 1997; Edwards & Younger,2006), or (simulated) stomach alone (Edwards & Younger, 2006). Both the length ofthe lag phase and the rate of decrease differ between species (Blackshaw & Rode,

Table 1 (continued)


Exposure[d]

Survival(%)

Inactivation[d]


Solanum nigrum Exp. batch 37 30 0


Stachus arvensis Exp. batch 37 30 0


Stellaria media Exp. batch 37 30 0


tomato Exp. batch 37 30 2.8


Tripleurospermummaritimum

Exp. batch 37 30 <1


Vicia tetrasperma Exp. batch 37 30 12.6

Westermanet al., 2012b

Abutilon theophrasti Comm. CSTR 41 1.5–2.0d


Chenopodium album Comm. CSTR 41 4.7–19.7d


Fallopia convolvulus Comm. CSTR 41 1.2–9.1d


Malva neglecta Comm. CSTR 41 17–23.6d


tomato Comm. CSTR 41 0.8–8.1d

a Nylon bags not in close contact with digestateb Seeds first subjected to simulated rumen treatmentc Cooling from 50 to 30 °C during operationd Estimate of decimal reduction time


1991). For example, Bromus tectorum remained fully viable for up to approx. 10 h inthe rumen of cattle, after which viability decreased rapidly to almost zero. In contrast,viability of F. convolvulus decreased gradually over time, with no noticeable lag time(Blackshaw & Rode, 1991). Ranking of species, therefore, depends on the exposuretime, similar to the situation in biogas reactors. Seed vigour, estimated as the speed ofgermination, is also affected by (simulated) digestion in animals (e.g., Peco et al.,2006), as in anaerobic digesters.

D’hondt and Hoffmann (2011) compared the germination success of seeds of 48grassland species after passage through cattle. Included were 12 species that mayoccur as weeds in maize in Germany (Mehrtens et al., 2005). The best germinatingweeds were Trifolium pratense (100 %), Juncus bufonis (83 %), Agrostis stolonifera(35 %), Plantago lanceolata (32 %), Poa annua (25 %), R. obtusifolius and C. album(both 21 %). Similarly, Cosyns et al. (2005) compared the germination success ofseeds of 19 grassland species after passage through rabbit, cattle, sheep, donkey andhorse. Included were six species that may occur as weeds in maize in Germany.Ranking of species in order of decreasing germination success was as follows;Trifolium arvensis, P. lanceolata, Prunella vulgaris, V. arvensis, T. repens, and T.pratense.

Studies in which seeds were exposed to ruminal conditions under more controlledconditions and for known periods of time, i.e., via fistulated animals or an artificialrumen (e.g., Rusitec; Czerkawski & Breckenridge, 1977), may yield relationshipsthat are more valuable for predicting seed survival in biogas reactors. Although suchstudies do exist, they tend to investigate the process of ruminal digestion, such asspeed of inactivation and mortality curves of individual species (e.g., Fredricksonet al., 1997; Alomar et al., 1992), and do not compare the survival probabilities of arange of weed species, with one exception. In a study with 12 weed species, seedsthat survived exposure to fistulated cows for 24 h particularly included; T. arvense(68 %), Malva pusilla (57 %), F. convolvulus (56 %), C. album (52 %) and A.retroflexus (45 %) (Blackshaw & Rode, 1991).

Thermoresistance

Temperature was found to be the most important factor influencing seed survival inbiogas reactors. With increasing temperatures, the lag phase of the survival curvedecreased and the rate of decline increased. Thermoresistance depends very much onthe moisture contents (mc) of the seeds. First, the available knowledge with regard tothe response of seeds to temperature as a function of moisture contents will besummarized, before proceeding to thermoresistance in biogas reactors. Knowledgeon this subject is fundamental in understanding the mechanism of inactivation ofseeds in the hot and moist environments of biogas reactors, and in understandingdeviations from the expected patterns.

Response to Temperature Depending on Seed Moisture Content

The thermoresistance of seeds can be divided into four categories, based on thesurvival characteristics as influenced by seed moisture contents and temperature; 1)


low seed mc, low and medium T, 2) medium seed mc, high T, 3) high seed mc,medium T, and 4) high seed mc, high T.

Ad 1. Dry Seeds at Low and Medium Temperatures. There is a lot of knowledgeavailable regarding the longevity of very dry (mc<<15 %), orthodox seeds, at low andmedium temperatures (T<30 °C). Almost all arable weed species belong to thecategory of orthodox (=desiccation tolerant) seeds. Seed mortality decreases expo-nentially with decreasing temperatures and moisture contents. This means that seedscan survive for extended periods of time at low T and low mc. Research on thesurvival of dry seeds has been motivated by the need to define optimal conditions formaintaining seed viability for a long period of time for seeding purposes and storageof genetic material in gene banks. This knowledge has culminated in the developmentof the so-called seed viability equation (Ellis & Roberts, 1980; Pritchard & Dickie,2003), whose parameters are constant for a given seed species at a given temperatureand moisture content, making seed longevity predictable. The seed viability equationfails completely in fully imbibed (mc>20 %), but not in partially imbibed seeds(mc=15–20 %, see ad 2).

Ad 2. Moist Seeds at High Temperatures. Seeds with medium seed moisture contents(mc=15–20 %) age very quickly when exposed to high temperatures (T=40–45 °C).This fact has found application in three areas, namely procedures to estimate theparameters of the seed viability equation to predict storage behaviour at low mc andlow temperatures (accelerated ageing test), procedures to predict and compare ‘vig-our’ or ‘quality’ of seed lots (controlled deterioration test (CDT)(Powell & Matthews,1981) or controlled ageing test (CAT)(Delouche & Baskin, 1973)), and research withregard to mechanisms involved in the deterioration of seeds, i.e., the loss of mem-brane integrity, impairment of RNA and protein synthesis and DNA degradation,caused by the accumulation of reactive oxygen species (ROS) and lipid peroxidation(e.g., Walters, 1998; Bailly et al., 2008; Lehner et al., 2008).

Ad 3. Fully Imbibed Seeds at Medium Temperatures. In contrast to partially imbibedseeds, fully imbibed seeds (mc>20 %) can survive for extended periods of time aslong as temperatures are not too high (T=20–35 °C), and provided that they do notgerminate or rot (e.g., Villiers, 1974; Murdoch & Ellis, 2000). Under anaerobicconditions, the deterioration of seeds is merely halted (Ibrahim & Roberts, 1983),but under aerobic conditions, the process of seed deterioration is reversed andlongevity is restored to some extent (Ibrahim et al., 1983). This improvement seemsto be caused by metabolic repair of previously sustained damage (Powell et al., 2000).It is not necessary for seeds to be continuously imbibed; even a short period ofimbibition can improve seed longevity (Villiers & Edgecumbe, 1975). This principlehas found application in seed priming and other seed invigoration treatments toimprove, synchronize and speed up germination and emergence (e.g., Powell et al.,2000).

Ad 4. Fully Imbibed Seeds at High Temperatures. At higher temperatures (T>35 °C)the viability of fully imbibed seeds declines exponentially over time (Economouet al., 1998, Dahlquist et al., 2007). Thermal death models have been fitted, using


exponential or other non-linear models, to describe mortality of imbibed seeds overtime. These models allow the prediction of the duration of exposure at a giventemperature to cause 100 % mortality, or, alternatively, the temperature required tocause 100 % mortality at a given exposure time (thermal death point), and are used toassist weed control via soil solarisation and steaming (Horowitz & Taylorson, 1983;Egley, 1990; Economou et al., 1998; Thompson et al., 1997a; Dahlquist et al., 2007).In general, the higher the temperature the shorter the period of time required to reachthe thermal death point.

At the thermophilic range, loss of viability of imbibed seeds is quick. Depending onthe species, 15–312 h at 46 °C, 4–113 h at 50 °C, 0.25–3 h at 60 °C and 0.17–0.67 h at70 °Cwere sufficient to kill 100% (Dahlquist et al., 2007). About 800 h were required toinactivate Avena sterilis at 38 °C, 450 h at 39 °C, 300 h at 40 °C and less than 50 h at45 °C (Economou et al., 1998). One day at 55 or 65 °C was sufficient to kill all seeds ofsix weed species; 2 day were required to inactivate two out of six species at 45 °C and5 day at 45 °C for another species; three species were not inactivated within 9 day at45 °C (Nishida et al., 2002). Loss of viability is slower at the mesophilic range. Seedviability was unaffected at 39 °C, and that of three of the six species tested wasunaffected at 42 °C (up to 28 day exposure). For the remaining three species, 4–16 day was required to cause 100 % mortality (Dahlquist et al., 2007). Similarly, seedviability was unaffected for five out of six species at 35 °C (Nishida et al., 2002).

Differences in Thermoresistance between Weed Species

The imbibed seeds of some species are more sensitive to exposure to hot water or hotmoist soil than others. Thermal death models indicate that both the lag phase and thespeed of decline may vary between species (Economou et al., 1998; Dahlquist et al.,2007). Summarizing seven studies on the effects of soil solarisation, Elmore (1991)concluded that, in general, summer annuals tended to be more thermoresistant thanwinter annuals. Senecio vulgaris was among the least, and Melilotus sp. and Medicagosp. among the most thermoresistant species (Elmore, 1991).

Ranking of six weed species in order of decreasing thermoresistance in the studyby Nishida et al. (2002) was as follows; Amaranthus spinosus, Solanum carolinense,A. patula, S. americanum, Phytolacca americana, A. theophrasti. Based on the periodrequired to kill 100 % of the seeds at 50 °C ranking of six species was as follows;Amaranthus albus (113 h), S. nigrum (71 h), Portulaca oleracea (56 h), E. crus-galli(9 h), Sisymbrium irio (6 h), Sonchus oleraceus (4 h) (Dahlquist et al., 2007). Allseeds of R. obtusifolius were inactivated by ten minutes in water at 70 °C, but 15 % ofthe seeds of A. fatue were still viable at this temperature; tomato and P. persicariawere inactivated by 10 min at 65 °C; T. maritima and Anthemis arvensis wereinactivated by 10 min at 60 °C (Lorenz et al., 2001). Xanthium strumarium seedswere fully inactivated after 6 h in moist soil at 60 °C, or 3 day at 50 °C, Sida spinosarequired 1 day to be inactivated at 60 °C and 6 h at 70 h, A. theophrasti required 1 dayat 70 °C, Sorghum halepense 2 day, Anoda cristata 5 day and P. oleracea 7 day at70 °C to be fully inactivated (Egley, 1990). Seeds of A. retroflexus needed eithertemperatures >70 °C or exposure periods >7 day to be fully inactivated. Seven daysexposure to moist soil at 40 °C had no effect on any of the eight species (Egley, 1990).


In anaerobic digesters, the temperature is 20ºC or higher and seeds should be fullyimbibed, corresponding to the situation as described under ad 4. Exposure of seeds tohot water baths could, therefore, be used as a first screen to determine the minimumpercentage of mortality that can be expected and to identify potentially thermoresistantweed species, i.e., species whose seeds might be able towithstand the thermal conditionsin anaerobic digestion for a longer period of time. This methodwas employed by Lorenzet al. (2001) and Böhm et al. (2000), but unfortunately not verified in biogas reactors.Results by Dahlquist et al. (2007) and others make clear that, at least within themesophilic temperature range, the inactivation of seeds in biogas reactors cannot solelybe due to high temperatures. This means that exposure of seeds to hot water or moist soilcould (strongly) underestimate seed mortality in biogas reactors. For example, seedviability was unaffected by exposure to 39 °C in water baths (Dahlquist et al., 2007),while most seeds in anaerobic digesters died within 3 day at 35 °C. The fact that hightemperatures alone cannot fully explain the demise of seeds during anaerobic digestionalso means that additional mortality factors have to be involved.

Mechanisms and Compounds that modify the Effects of High Temperatures

Similar as with seeds, high temperatures could not fully explain the demise ofpathogenic bacteria, viruses or helminths during anaerobic digestion (Berg &Berman, 1980; Olsen & Larsen, 1987; Sahlström, 2003; Popat et al., 2010). As anexplanation for the higher than expected decimation rates, the involvement of sub-stances with bactericidal or virucidal activity, such as ammonia (Ward, 1978) or longchain fatty acids, was put forward (for review see: Sahlström, 2003). In the case ofbacteria, competition for limiting supplies of nutrients could be involved.

Interestingly, certain substances and mechanisms occurring in biogas reactorsseem to be able to protect bacteria and viruses from thermal inactivation. Theresulting decimation rates are then lower than expected on the basis of thermalinactivation alone. Substances and mechanisms involved include, for example, cer-tain anionic detergents, food additives and amino acids (Popat et al., 2010), adhesionto suspended solids (Bar-Or, 1990) or embedding of virus particles and clumps ofbacterial cells in viscous envelops (Rollins & Colwell, 1986; Lund et al., 1996).

It is unknown if substances with herbicidal activity or seed protective propertiescould occur in biogas reactors, and if these could be responsible for the higher orlower than expected decimation rates based on thermal inactivation alone. Certainamounts of chemical herbicides may enter via the biomass feedstock, allelochemicalsmay be present in crop residues, such as in wheat straw, bacterial phytotoxins may bepresent in anaerobic digesters, as well as phytohormones, such as ethylene, auxinesand cytokinin analogues (e.g., Belay & Daniels, 1987; Marchaim et al., 1997).Furthermore, a wide range of ‘other’ substances may be present or produced inbiogas plants. Whether the concentrations in biogas reactors are high enough toaffect seed viability is unknown.

Seed damaging or seed protecting mechanisms may exist in biogas reactors too. Forexample, if seeds in biogas reactors would not fully imbibe (mc<20 %), they wouldrespond as if exposed to the accelerated ageing test and die much more rapidly.Imbibition is a strictly physical process, and depends on the difference in water potentialbetween the seed and its environment, the protein, lipid and starch composition of the


seed, and the permeability of the seed coat (Nelson, 2004). Water will be absorbedregardless of whether seeds are dormant (except physically dormant) or non-dormantseeds, viable or nonviable (Bewley & Black, 1994). The water potential in seeds mayrange from −350 MPa to −50 MPa (Nelson, 2004; Bewley & Black, 1994). However,the water potential of mixtures inside biogas reactors is unknown. If the difference isinsufficient, seeds will not or only partially imbibe. Furthermore, heavy metals areknown to block water uptake in seeds (Kranner & Colville, 2011). If seeds are indeedonly partially imbibed in biogas reactors, the accelerated aging test could be used topredict seed resistance to anaerobic digestion. A special case involves seeds with awater-impermeable layer (see VII.A.) that do not imbibe at all when exposed to water,rendering them much more thermoresistant than partially or fully imbibed seeds. Thissituation will be discussed in more detail in section VII.A.

Seed Defence Mechanisms

Because thermosensitivity can explain only part of the inactivation of seeds in biogasreactors, in particular under mesophilic conditions, chemical and biological processesoccurring during anaerobic digestion have to play a role as well. Understanding the variousways that seeds defend themselves against microbial attack and toxins may help to identifyweak spots in the defence, and, identify groups of seed species that might be either unusuallysusceptible or unusually resistant to inactivation during anaerobic digestion.

The seed coat is an effective barrier against microbial attack and toxic compounds(Mohamed-Yasseen et al., 1994). Halloin (1983) described it as the most importantcomponent of resistance against microbial attack. Seeds with deliberately damagedseed coats have a much lower survival probability both in the soil (Davis et al., 2008)and in the rumen (e.g., Michael et al., 2006), and reduced longevity during storage(Mohamed-Yasseen et al., 1994 and references therein). To gain access to the embryo,microorganisms have to breach the protection offered by the seed coat and otherprotective layers. Various reviews summarize the available knowledge with regard tothe structure and functioning of the seed coat and other layers in seeds in the defenceagainst microbial infections (e.g., Halloin, 1983; Mohamed-Yasseen et al., 1994;Dalling et al., 2011). Here, the most important findings will be summarized with anemphasis on those aspects that are most relevant to the issue at hand.

Halloin (1983) eloquently summarized the functioning of seed coat as; ‘a chemicalbarrier of inhibitory polyphenolic compounds, as a mechanical barrier, and as a barrier tothe availability of nutrients to fungi’. This division will be used to structure the followingsub-sections.

Seed Coat Composition, Thickness and Weak Spots

The first barrier is a physical one. The seed coat is presumed to be the initial point ofaccess for microorganisms (e.g., Chee-Sanford et al., 2006). Seed coat compositionand thickness may determine to a large extent the sensitivity to microbial degradation.

Unfortunately, very little research has focussed on the composition of seed coats(Graven et al., 1996). Consequently, it is largely unknown if and how seeds of variousweed species differ in seed coat composition. The general assumption is that the seed


coat consists mainly of the usual cell wall components, including cellulose, hemicel-lulose, pectin and lignin. Because hydrolytic bacteria produce cellulases andhemicellulases profusely, seed coats could be digested. There is only a limitedamount of direct evidence for this to happen. Observations by scanning electronmicroscope (Simao Neto et al., 1987) and light microscope (Michael et al., 2006)indicate that a thin outer layer of the seed coat of legume and Malva parviflora seedswas partly or wholly removed during passage through the digestive tract of rumi-nants. Legume seeds that survived passage through animals were often blackened andpartially swollen, indicating that they had been acted upon by the digestive processes(Glendening & Paulsen, 1950). Fermentation in the rumen was accompanied by lossof dry matter, which increased with MRT (Alomar et al., 1992; Fredrickson et al.,1997). The weight of C. album and oilseed rape seeds decreased when exposed toselected bacteria (Streptomyces spp.) and fungi (Phanerochaete chrysosprium) inliquid medium or soil (Einhorn & Brandau, 2006). The weight loss suggests thateither part of the seed coat or entire seeds were digested. Weight loss was alsoobserved when only the seed coats of oilseed rape seed were exposed, despite thefact that these contained a large percentage of lignin (Einhorn & Brandau, 2006).Lignin cannot easily be digested by hydrolytic bacteria (Hofrichter, 2002). However,the bacteria and fungi used in the study by Einhorn and Brandau (2006) had beenselected on the basis of known or suspected production of high amounts of cellulaseor lignin-modifying enzymes.

Chemical Defence

A second function of the seed coat as a defence against microbial attack, as definedby Halloin (1983), is a chemical barrier. In addition to lignin, the seed coat maycontain all kinds of alkaloids, terpenoids, biogenic silica, peptides, suberin, proteaseinhibitors, lectins and (poly)phenolic compounds, such as flavanols, catechins, tan-nins and other pigments, etc. (Rolston, 1978, Halloin, 1983; Broekaert et al., 1995).These substances can have multiple functions, such as waterproofing the seed coat,inhibit germination, maintain dormancy, inhibit or kill microbes and deter granivores(e.g., Rolston, 1978; Mohamed-Yasseen et al., 1994; Dalling et al., 2011). Anindication of the involvement of biogenic silica in seed resistance against anaerobicdigestion was found in Jeyanayagam and Collins (1984), who claimed that the higherash content of S. halepense (9.4 %) in comparison to Panicum dichotomoflora(2.6 %) was responsible for the lower digestibility of S. halepense in anaerobicdigesters. Unfortunately, the conclusion was based on only two species and requiresconfirmation.

With the exception of A. theophrasti (Kremer, 1993 and references therein), almostnothing is known with regard to the identity or effectiveness of the chemicalcompounds involved in chemical defence in weed seeds. It is, therefore, unknownhow and how much species of weed seeds differ in this respect. Tests to determine theidentity and concentrations of antimicrobial compounds require specialized laborato-ries and cannot be determined quickly and easily. However, there is no doubt thatsecondary plant metabolites affect microorganisms. Some secondary plant metabo-lites affect digestibility of plant materials in both ruminants and biogas reactors (VanSoest, 1982, 1988; Deublein & Steinhauser, 2011). Digestibility is sometimes more


limited by substances such as tannins and silica than by lignin (Jackson, 1977; VanSoest, 1988). Biogenic silica, for example, reduces the digestibility of cellulose andhemicellulose in a manner that is additive to lignin (Van Soest & Jones, 1968).

Nutrient Availability and Microorganisms

The third function of the seed coat is described by Halloin (1983) as ‘a barrier to theavailability of nutrients to fungi’. Based on insights gained over the last four decades, thethird function needs to be slightly revised, because the seed coat naturally harbours andnurtures microorganisms that are generally considered beneficial to the seed (Nelson,2004; Chee-Sanford et al., 2006). These mutualistically associated microorganismsprovide protection against pathogenic microorganisms, via competition for nutrientsand space, the production of inhibitors and antimicrobial compounds (Broekaert et al.,1995), and the decomposition of chemical cues that could attract deleterious microor-ganisms in the spermophere (Nelson, 2004; and references therein). In turn, the seed coatfunctions a source of carbon and nitrogen, and it provides structure, binding sites formicrobial attachment, and protection from predators and adverse environmental condi-tions. It seems that if microorganisms cannot be kept at bay, it is better to team up withthe beneficial ones, rather than to be exposed to the pathogenic ones.

Beneficial seed-microbial associations can be formed during seed set, or after seeddispersal in the soil. In the latter case, community composition depends to a largeextent on the microorganisms present in the substrate surrounding the seed (Nelson,2004; Chee-Sanford et al., 2006). Seeds influence their relationship with microor-ganisms; different weed species and different genotypes harbour different microbialassemblages (Nelson, 2004; Chee-Sanford et al., 2006). Also, the number of micro-organisms that colonize a seed and their spatial distribution on the seed surface differsbetween species and genotypes (Nelson, 2004 and references therein). The selectivitymay be based on nutritional selection, selection by antimicrobial compounds, or byproviding distinctive opportunities for nutrition and surface attachment (Chee-San-ford et al., 2006).

Initially, biofilm formation will not be extensive, given the relatively dry andoligotrophic conditions on seed surface and in the spermosphere. However, when theconditions are right for germination, in particular with regard to water availability andtemperature, seeds will start to imbibe water; a process that is immediately accom-panied by the leaking of cellular and vacuolar constituents from the seed (Nelson,2004). The substances released are usually low-molecular-weight molecules andinclude carbohydrates and amino acids. Leaking stops after approximately 12 h whencellular membranes are fully hydrated and functional (Nelson, 2004 and referencestherein). During this short period of time, the attached microorganisms will grow,such that a more extensive biofilm is present when some time later the radicle extendsand protrudes through the seed coat; the most vulnerable stage in the life of a seed.

The interaction with microorganisms is a double-edged sword. Exogenous soilmicroorganisms will be stimulated to germinate or attracted by the exudates, andcompete with the resident microorganism for the available resources. Pathogenic mi-croorganisms can outcompete the beneficial ones, colonize the seed coat, and induceseed coat decomposition. Studies that search for microorganisms that can either provideprotection against plant diseases to crop seeds (Nelson, 2004 and references therein,


Dalling et al., 2011) or help to control weeds by decomposing seeds in the weed seedbank (Kremer, 1993, Kennedy, 1999; Chee-Sanford et al., 2006) reflect this dualism.Compared to the conditions in the soil, conditions in biogas reactors may be favourablefor prolific growth of microorganisms (moist, warm, anaerobic and eutrophic). It isuncertain how much protection the associated, beneficial microorganisms, if any, mayprovide under these conditions, but most likely they will be outcompeted by the activehydrolytic assembly in the slurry.

Seeds can also harbour microorganisms that have colonized internal tissues(endophytes). These microorganisms may confer various benefits to seedling and plant,including enhanced nutrient uptake, greater stress tolerance, and protection from her-bivory, plant pathogens and pests (Clay & Schardl, 2002 and references therein).However, it is unknown if and how they help protect the seed itself. Furthermore, thefate of endophytes in anaerobic digesters is unexplored.

Species Resistant to Anaerobic Digestion

Based on the previous sections, four groups of plant species could be identified thatmight have a higher than usual probability of surviving anaerobic digestion, namelyweed species with hard seeds (see VII.A.), thermoresistance not based on a water-impermeable layer in the seed coat (see VII.B.), seeds with a thick seed coat (seeVII.C.), and seeds adapted to endozoochory (see VII.D.).

Seed with a Water-Impermeable Layer (Hard Seeds)

Mechanism and Occurrence

Seeds with water-impermeable layers in the seed coat or fruit form a special category,because they do not imbibe water. Seeds that do not imbibe water are less sensitive to heatstress than partially or fully imbibed seeds (see V.A. Ad 1). Ergo, seeds with a water-impermeable seed coat are expected to be able to survive high temperatures, such as inanaerobic digesters or the intestinal tract of animals, much better than imbibed seeds. Theyare termed ‘hardseeded’ or ‘hard seed’, because they remain hard compared to imbibedseeds, which swell and soften during imbibition (Rolston, 1978). Hardseededness is one ofthe mechanisms responsible for keeping seeds in a state of dormancy (called ‘physicaldormant’ by Baskin & Baskin, 1998 or ‘intrinsically quiescent’ by Murdoch & Ellis,2000). This term is not to be confused with the thickness or hardness of the material of theseed coat itself.

Usually, the water-impermeable barrier consists of one or more palisade layers oflignified cells in the seed coat, waterproofed by wax, lignin, tannin, suberin, pectin,quinine derivates, or phenolic compounds (e.g., Rolston, 1978; Baskin et al., 2000;Ma et al., 2004). Impermeability develops during seed dehydration, meaning thatnewly formed seeds are not immediately water-impermeable. Initially, water is lostvia the seed coat, but once water impermeability develops, the hilum (the mark on theseed coat indicating the former attachment site to the ovary wall) acts as a hygro-scopic valve that prevents water uptake but allows water loss (Rolston, 1978). Thepercentage of water-impermeable seeds in a seed lot increases with decreasing


moisture content of the seeds and is influenced by genetics and by environmentalfactors during maturation. If dehydration is insufficient (> 10 % mc), water imper-meability can be reversed. The status of a seed lot can, therefore, be described interms of both degree and percentage of water impermeability (Rolston, 1978).

Hardseededness is usually associated with members of the Fabaceae, but can also befound in members of many other families, including Convolvulacea, Geraniaceae,Malvaceae, and Solanaceae (Rolston, 1978; Baskin et al., 2000; Murdoch & Ellis,2000). Not all members of these families exhibit hardseededness. For many species itis unknown if they have a water-impermeable layer. Arable weed species with knownhardseededness include A. theophrasti, An. cristata, M. parviflora, M. pusilla, Sidahermaphrodita and S. spinosa (Malvaceae),Datura ferox (Solanaceae), Erodium botrys,E. cicutarium, Geranium carolinianum, G. dissectum, G. molle, G. pusillum and G.robertianum (Geraniaceae), Convolvulus arvensis, C. sepium, Ipomoea purpurea, I.hederaceae and Cuscuta campestris (Convolvulaceae), Lespedeza capitata, Melilotusalba, M. officinalis and Vicia sp. (Fabaceae), (Horowitz & Taylorson, 1984, Marowski& Morrison, 1989, Baskin & Baskin, 1998; and references therein, Meisert, 2002,Michael et al., 2006, Dorado et al., 2009). A large number of legume crop species arehardseeded, including lucerne, clovers, fetch, lupin, soybean, and pea, although forseveral crops varieties have been bred without hardseededness.

Survival of Hard Seeds during Anaerobic Digestion

There is little information on the survival of water-impermeable seeds in biogasreactors. Red clover (Fabaceae) was among the best survival species in simulatedbatch reactors (Leonhardt et al., 2010) and A. theophrasti (Malvacaea) survived bestin a PFR (Katovich et al., 2004). A. theophrasti and M. neglecta (Malvacaea), D.stramonium (Solanaceae), E. cicuratium (Geraniaceae), and V. tetrasperma(Fabaceae) were the best surviving out of 21 species in simulated batch reactors(Westerman et al., 2012a); M. neglecta was also the best surviving species incommercial biogas plants, but A. theophrasti was not (Westerman et al., 2012b).Soyabean (Fabaceae) was among the worst surviving species in simulated batchreactors (Strauß et al., 2012; but see also VII.A.3.). In soil solarisation studies,Melilotus sp. and Medicago sp. (Fabaceae; Elmore, 1991), and A. theophrasti andAn. cristata (Malvaceae; Egley, 1990) were among the more thermoresistant species.

More information is available on the survival of water-impermeable seeds in thedigestive tract of animals. Legumes and other species with physical dormancyinvariable stand out as being among the most resistant to anaerobic digestion in thealimentary tract of animals. For example, of the four most resistant species in thestudy by D’hondt and Hoffmann (2011), one belonged to the Malvaceae(Helianthemum nummularum), two to the Fabaceae (T. campestre, T. pratense), andone to the Juncaceae (J. bufonius), whereby the latter species was recognized forhaving highly water-impermeable seeds (Peco et al., 2006). Of the five most resistantspecies in the study by Cosyns et al. (2005), one belonged to the Malvaceae (H.nummularum), two to the Fabaceae (T. arvense, T. pratense), one to the Cyperaceae(Carex arenaria), and one to the Poaceae (Agrostis capillaries). Of the five mostresistant species in the study by Peco et al. (2006), two belonged to the Fabaceae(=Leguminosae) (Astragalus pelecinus, Ornithopus compressus), one to the


Campanulaceae (Jasione montana), one to the Lamiaceae (Lavandula stoechas), andone to the Plantaginaceae (P. lanceolata).

For a range of legume species, the percentage of seed survival in the rumen wasdirectly related to the percentage of hard seeds in the original material (Gardeneret al., 1993a, b). Soft seeds die, while most of the remaining hard seeds survivepassage through the tract (Gardener et al., 1993a, Glendening & Paulsen, 1950).Similar results were obtained for M. parviflora; seeds that were made water perme-able through mechanical scarification were completely digested within 24 h in therumen, while hard seeds remained largely intact until the end of the experiment (48 h;Michael et al., 2006).

Controlled and prolonged exposure to the rumen via fistulas suggest the existenceof two seed survival curves; one for water-permeable soft seed with a short lag time isshort and a rapid rate of decline, and one for water-impermeable hard seeds with along lag time and slow rate of decline. In several legume species, the time required tokill hard seeds exceeded the natural MRT in the digestive tract of cattle (35–51 h;Gardener et al., 1993a, b).

Restoring Water Permeability

When water permeability is restored, seeds will imbibe water and thermoresistancewill be lost. It is unknown how well conditions inside biogas reactors fulfil therequirements for restoring water permeability. Water permeability can naturally berestored by high temperatures, low winter temperatures, temperature fluctuations,fire, passage through the digestive tract of animals, and, possibly, by microbialactivity (Rolston, 1978, Baskin & Baskin., 1998). Methods of artificially restoringwater permeability include acid scarification (concentrated sulphuric acid), mechan-ical scarification, organic solvents (ethanol, acetone), wet heat (60–100ºC), dry heat(50–150ºC), prolonged storage, soaking, high pressure, percussion, freezing, heating,and radiation or ultrasound treatments (Rolston, 1978, Baskin & Baskin, 1998).Conditions in biogas reactors are such that dormancy is likely to be broken in atleast some hardseeded species.

All treatments act via one of the build-in areas of weakness in the seed coat, i.e.,hilum, strophiole (a crestlike excrescence about the hilum), micropyle (openingthrough which the pollen tube enters), or chalaza (the region opposite the micropyle,where the integuments and nucellus (central part in which the embryo sac develops)are joined) that either softens, cracks, ruptures, or collapses during treatment(Rolston, 1978, Baskin et al., 2000). However, in soybean cultivars that have beenbred for reduced hardseededness, cracks develop outside these pre-designed areas(Ma et al., 2004), which may have been the reason for the low survival of soyabean inthe study by Strauß et al. (2012). Not all mechanisms work for all species. Forexample, Gardener et al. (1993b) found large differences between legume species inresistance to breakdown in the digestive system of ruminants.

Once water permeability is restored, seeds respond to anaerobic digestion as anyother species without a water-impermeable layer. However, water uptake can cause adoubling in seed size or weight (Leopold, 1983, Mullin & Xu, 2001, Geneve et al.,2007), which results in an increase in the pressure within the seed coat. The straincauses a decrease in thickness and hardness of the seed coat, making it more


vulnerable to mechanical damage and infection (Fraczek et al., 2005). When thedifference in water potential between seed and environment is large, water uptake isfast and both seed coat and embryonic tissues can weaken or rupture (soaking injury;Yasue & Hibino, 1984, Bewley & Black, 1994, Fraczek et al., 2005).

Other Thermoresistant Species

Based on studies comparing the thermoresistance of seeds in hot water or hot, moistsoil, seeds of, for example, Melilotus sp., Medicago sp., S. spinosa, A. theophrasti,An. cristata, A. retroflexus, A. spinosus, A. albus, S. nigrum, S. carolinense, tomato,R. obtusifolius, P. oleracea, Po. persicaria, A. fatue, and S. halepense would qualifyas relatively thermoresistant. The first five are hardseeded but the other species arenot. The mechanism involved in thermoresistance of the latter 11 species is unknownand, therefore, testing in hot water baths will be needed to identify such species.

Seeds with a Thick Seed Coat

It is reasonable to assume that the thicker the seed coat, the longer it will take to bedigested. There is very little direct evidence that the thickness of the seed coat isrelated to seed survival in anaerobic digesters. Only a few species of weed seeds havebeen tested in biogas plants and for most of these the thickness of the seed coat isunknown (but see: Davis et al., 2008, Gardarin et al., 2010). Nevertheless, differencesin seed coat thickness were consistent with the longer period required to inactivate A.artemisiifolia (159 μm; Gardarin et al., 2010) than C. bursa-pastoris (18 μm) or S.media (27 μm) in simulated batch reactors (Leonhardt et al., 2010).

There is indirect evidence that the thickness of the seed coat may aid in seedsurvival during anaerobic digestion. A significant relationship was found betweenseed coat thickness and seed persistence in the soil (Davis et al., 2008, Gardarin et al.,2010). This relationship was not linear, but seed mortality declined exponentiallywith increasing thickness (Gardarin et al., 2010), meaning that for thin-coated seedsan increase in thickness causes a relatively large reduction in seed mortality, while forthick-coated species a similar increase causes only a small reduction in seed mortality.Seed longevity in the soil, in turn, has been related to seed survival during anaerobicdigestion in ruminants, suggesting that factors responsible for seed survival in theseed bank also protect seeds during anaerobic digestion (Cosyns et al., 2005,Mouissie et al., 2005). The latter relationships tend to be significant, but weak (slopeof the regression line=0.15, Cosyns et al., 2005; R=0.4, Mouissie et al., 2005),suggesting the involvement of modifying factors. Further research is required on thissubject. Techniques for measuring the thickness of the seed coat, based on light orelectron microscopy or X-rays in combination with image analysis, are available(e.g., Fraczek et al., 2005, Davis et al., 2008, Gardarin et al., 2010).

Microbes could bypass the tedious process of decomposing the seed coat if theycould gain access directly via pre-designed weak spots, such as hilum, strophiole,chalaza, or micropyle, or via entrances created by cracks, nicks or wrinkles in theseed coat. There is some evidence that suggest that this may be happening. Forexample, Halloin (1975) observed that certain fungi entered cotton seeds predomi-nantly via the chalaza. Decomposed seeds with entirely intact seed coats were found


by Chee-Sanford et al. (2006). It is unknown how common this phenomenon is. Mostseed lots contain at least some seeds with cracked or damaged seed coats. The degreeof cracking, in terms of the percentage of the population affected and severity ofcracking, is largely influenced by the conditions during seed filling and imbibition. Itcan also have a hereditary basis, for instance, via genes that code for seed coatcomposition (e.g., Moïse et al., 2005; and references therein). The same applies towrinkling and shrinking of seeds (Halloin, 1983 and Mohamed-Yasseen et al., 1994and references therein). Consequently, analysing seed coat thickness as a predictor forresistance against anaerobic digestion is only useful for a certain category of seedspecies.

If seeds would actually germinate during anaerobic digestion they would mostlikely be doomed. There are some indications for this to happen. Janzen (1981) foundpartly digested, newly germinated seed of Enterolobium cyclocarpum in the dung ofhorses. Some species of seeds can germinate under anaerobic conditions, i.e., weedsin rice (Kennedy et al., 1980, Yamasue, 2001). The presence of phytohormones inbiogas plants, such as ethylene, auxines and cytokinin analogues (e.g., Belay &Daniels, 1987, Marchaim et al., 1997) may help breaking dormancy and initiategermination. It is completely unknown if germination of seeds inside anaerobicdigesters occurs or not.

Openings in the seed coat, due to germination, cracking, or other causes do notalways have to lead to seed mortality. Resistance mechanisms exist within the embryoand other tissues inside the seed coat (Halloin, 1983 and reference therein). Forexample, when seeds of the grass Pennisetum clandestinum were deliberately dam-aged by clipping the tip of the seed, mortality in the ruminal fluid was enhanced(48 % vs. 37 %), but not 100 %, as expected. A similar result was obtained withdamaged grass seeds in comparison with damaged legume seeds (Simao Neto et al.,1987). The need for additional protective layers may be higher in seeds that normallygerminate hypogeally vs. epigeally. Monocotyledons germinate hypogeally, whiledicotyledons germinate predominantly epigeally. In the case of epigeal germination,the cotyledons and seed coat are pushed out of the soil during germination, such thatfurther colonization by soil microorganisms is prevented (Nelson, 2004). In the caseof hypogeal germination, only the epicotyl with the meristematic tissue or thecoleoptile that covers the shoot reach the soil surface, while all other structures, suchas endosperm and cotyledons, remain underground requiring further protection.

Seeds Selected for Endozoochory

Usually, seed defence mechanisms have developed in response to adverse conditionsand microbial pressure associated with the soil environment. However, Janzen (1984)suggested that seed attributes could have developed in response to selection exerted bygrazers. In analogy with selection of specific seed traits that accommodate dispersal byants, birds or wind, Janzen (1984) proposed that grazers could exert a selective force toaccommodate endozoochory by herbivorous mammals, such that seeds would becomemore resistant to anaerobic digestion. The ideal zoochorical seed as anticipated byJanzen would be small, tough, hard and inconspicuous, and its seed coat would haveto be able to resist digestion for a period of days or months, and would have to containtoxins to protect against seed predators, without affecting the large herbivores that eat


them. Unfortunately, there is little empirical evidence to support Janzen’s theory.Nevertheless, the idea that grazers can at least in some cases exert selection has beenaccepted (e.g., D’hondt & Hoffmann, 2011). This would suggest that seed specieswhose mode of dispersal is mainly through endozoochory could prove to be moreresistant to anaerobic digestion in biogas reactors. The identification of seeds predom-inantly dispersed via endozoochory will be difficult, because seed traits associated withthis means of dispersal are very general. Besides, most could have originated throughselection by other forces as well.

Several studies compared the survival (or germination) of a range of seed speciesafter passage through the digestive tract of ruminants and other animals. All studiesindicated clear differences in survival probability between seed species. Attemptswere made to correlate seed survival to certain seed characteristics, such as seed size,weight, and shape (ecological correlates); if successful, dispersal distance and spatialdistribution patterns of species due to endozoochory could be simulated (e.g., Will &Tackenberg, 2008). However, results were inconsistent. For example, some foundthat round seeds survived or germinated better than elongated seeds (Simao Netoet al., 1987, Mouissie et al., 2005). In contrast, others found that ovate-lanceolateseeds survived or germinated better than round seeds (Cosyns et al., 2005). Somefound that seed survival and germination were positively influenced by seed mass(Cosyns et al., 2005, Peco et al., 2006); others that seed survival was negativelyinfluenced by seed mass (Mouissie et al., 2005).

The only seed characteristic that was more or less consistently positively related toseed survival after passage through the alimentary tract of animals was the seedlongevity index (LI), i.e., the proportion of records in a database that report a speciesas having a persistent seed bank (Thompson et al., 1997b). This suggests that thesame factors responsible for seed persistence in the seed bank also protect seeds in thegastrointestinal tract. Because seed persistence is strongly correlated with seed coatthickness (Davis et al., 2008, Gardarin et al., 2010), it would be worthwhile to includeseed coat thickness in future studies searching for ecological correlates.

Various explanations for the observed inconsistencies have been put forward, suchas a) the fact that large-seeded species are more prone to mechanical damage bychewing (e.g., Mouissie et al., 2005), b) the residence time in the alimentary tract ofruminants is influenced by particle size and density, and c) statistical problems relatedto small sample size and taxonomic interdependencies (D’hondt & Hoffmann, 2011).

The latter authors pointed out that the high survival rate of seeds with water-impermeable seed coats (physical dormancy), which do not imbibe water, overruledsimple seed traits, such as size, weight and shape. Omission of seeds with physicaldormancy from the data sets may elicit correlations with other seed traits. Theyfurthermore suggested that the water-impermeable seed coat itself could be the mostimportant ecological correlate and that it may have evolved through frequent inges-tion by grazers. This is corroborated by data by Gardener et al. (1993a, b).

Gardener et al. (1993b) furthermore found that the seeds of dense, low-growing,rhizomatous or stoloniferous grasses have a higher survival probability during passagethrough the gastrointestinal tract of cattle than seeds of tall, tussock grasses. The formercategory of seeds has a higher probability of being ingested during cattle grazing thanthe latter. It would also mean that the selection of ‘anaerobic-digestion-resistant’ bio-types in repeatedly recycled sludge from biomass to crop field and vice versa is likely.


Prospects

Potential Test Procedures

In summary, the available, scarce information gathered in this review suggests thatseeds die during anaerobic digestion due to a combination of thermal inactivation andinactivation due to microbial activity. Evidence for inactivation by toxic compoundsis lacking, but may exist too. Given these mechanisms, tests could be selected to helpto screen a large range of seed species for tentative resistance to anaerobic digestionin biogas reactors.

Sensitivity to thermal inactivation could be determined relatively easily using expo-sure of seeds to hot water baths (e.g., Dahlquist et al., 2007). Such tests would estimateminimum seed mortality to be expected inside biogas reactors; the actual mortality willbe higher, in particular in reactors operated in the mesophilic temperature range.

Procedures to screen seed resistance to microbial inactivation are much more spec-ulative, because the mechanisms and processes involved are not well understood andmay differ between seed species. Systematic empirical data is lacking. There are someindications that seed coat thickness may be related to sensitivity to microbial inactiva-tion; the thicker the seed coat, the longer it will take to decompose. Various methods areavailable for estimating seed coat thickness. However, the relationship may be disruptedby seeds that have cracks, wrinkles, or pre-designed ‘weak spots’ in the seed coat thatmicroorganisms could use as an easy point of entry. It is not clear how to identify these.

There is a positive correlation between the degree of persistence of seeds in the soilseed bank and seed survival during anaerobic digestion. This would make sense, giventhat microbial processes are involved in the degradation of seeds in both systems,although the one proceeds much faster than the other. The seed longevity index, whichis usually used to quantify seed persistence and which is defined as the proportion ofrecords in a database that report a species as having a persistent seed bank (Thompsonet al., 1997b), may not necessarily be the most appropriate measure for seed persistence(e.g., Bekker et al.,1998). Seed persistence, in turn, is positively correlated with seedcoat thickness, and it is possible that the two are based on the same principle.

It is likely that the seed coat composition, in particular the degree of lignificationand the presence and concentration of secondary metabolites with antimicrobialactivity, influences the speed of decomposition by microorganisms. However, empir-ical evidence is largely lacking. Methods to determine and compare the chemicalcomposition are generally complicated and expensive, and the routine use of these isunlikely. One exception involves ash content as an estimate of biogenic silica, whichstrongly limits digestibility.

Testing of seeds via ingestion by animals, in particular ruminants, could help todetermine the sensitivity of seed species to anaerobic digestion in biogas reactors,because the two systems bear close resemblance. However, interpretation of the datasets derived from such studies is obscured by several factors that are irrelevant to thesituation in biogas plants, such as species-specific residence times and damage due tochewing. Studies in which seeds are exposed to ruminal conditions under morecontrolled conditions and for known periods of time, i.e., via fistulated animals orin artificial rumen (e.g., Rusitec), may yield relationships that are much morevaluable for predicting seed survival in biogas reactors.


Groups of Seed Species Resistant to Anaerobic Digestion

Two groups of seeds stood out with regard to their tentative potential to withstandanaerobic digestion; those with a water-impermeable seed coat and those adapted todispersal via endozoochory. The two groups may partially overlap.

Seeds with a water-impermeable layer in the seed coat (hardseededness or phys-ically dormant) do not absorb water when imbibed, and are, therefore, protected fromthermal inactivation. However, once the water-impermeable layer is breached, theseeds imbibe water and thermoresistance is lost. Several physical cues necessary forbreaking physical dormancy are present in biogas reactors. Hardseeded species andbiotypes within species may differ, genetically or phenotypically, in the percentage ofhardseededness within a population.

Seeds adapted to dispersal via ingestion by grazers (endozoochory) are expected tobe more resistant to anaerobic digestion than those that rely on other modes ofdispersal. Unfortunately, the anticipated physical characteristics of endozoochoricallydispersed seeds, i.e., small, tough, hard, and inconspicuous (Janzen, 1984), cannot bedistinguished from other seeds, and such seeds will, therefore, be hard to identify. Ithas been proposed that hardseededness has evolved in response to frequent ingestionby grazers (D’hondt & Hoffmann, 2011), meaning that hard seeded species are boththermoresistant and resistant to anaerobic digestion. Other groups of seed speciesprone to frequent ingestion by grazers, in particular small grasses, may have devel-oped resistance to anaerobic digestion, which is neither based on hardseededness noron seed coat thickness.

Acknowledgment This study was made possible with the financial support of the German Agency forRenewable Resources (Fachagentur für Nachwachsende Rohrstoffe e.V.; Bundesministerium fürErnährung, Landwirtschaft und Verbraucherschutz (= German Federal Ministry of Food, Agriculture andConsumer Protection)), project No. 22028408‚ ‘Untersuchungen zum phytosanitären Risiko durch dieanaerobe Vergärung von pflanzlichen Biomassen in Biogasanlagen; Teilvorhaben 2.’

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http://www.fao.org/wairdocs/ILRI/x5495E/x5495e06.htm

http://www.epa.gov/nrmrl/pubs/625r92013/625R92013.pdf

http://www.vergisting.nl/wp/wp-content/uploads/2007/11/1492rap01-rev-02-22-10-07.pdf

http://www.vergisting.nl/wp/wp-content/uploads/2007/11/1492rap01-rev-02-22-10-07.pdf

http://www.das.psu.edu/research-extension/dairy/nutrition/pdf/cscutheight.pdf

weed seed survival during anaerobic digestion in biogas plants

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