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The Fate of Plant Pathogens and SeedsDuring Anaerobic Digestion and AerobicComposting of Source SeparatedHousehold WastesJaak Ryckeboera, Stef Copsa & Jozef Coosemansa

a Laboratory of Phytopathology and Plant Protection, KatholiekeUniversiteit Leuven, BelgiumPublished online: 23 Jul 2013.

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204 Compost Science & Utilization Summer 2002

Compost Science & Utilization, (2002), Vol. 10, No. 3, 204-216

The Fate of Plant Pathogens and Seeds During Anaerobic Digestion and Aerobic Composting of

Source Separated Household Wastes

Jaak Ryckeboer, Stef Cops and Jozef CoosemansLaboratory of Phytopathology and Plant Protection,

Katholieke Universiteit Leuven, Belgium

Anaerobic digestion is becoming a more common method for treatment of organicwastes. Little is known, however, about the effects of this process on the fate of plantpathogens and seeds. Therefore, the fates of the plant pathogens Plasmodiophora bras-sicae, Heterodera schachtii, Meloidogyne incognita, Ralstonia solanacearum, tobacco mosa-ic virus (TMV) and tomato seeds were followed during anaerobic digestion of sourceseparated household wastes. With the exception of TMV, all test organisms were de-stroyed to below detectable limits within one day of anaerobic digestion at 52°C. Twodays of anaerobic digestion did not reduce the concentration of TMV. However, twodays of anaerobic digestion followed by 19 days of high temperature composting at58°C reduced the concentration of infectious TMV particles by a factor of almost threeorders. Anaerobic digestion followed by 12 days of composting at 68°C was evenmore effective. Although TMV concentrations were not eliminated entirely, we con-clude that short-term high temperature anaerobic digestion followed by high tem-perature composting is a highly efficient process for the eradication of detrimentalagents from solid wastes.

Introduction

Anaerobic digestion is a common method for treatment of organic wastes. Howev-er, little is known about the effects of anaerobic digestion on survival of plant pathogens(Williams 1979; Bollen and Volker 1996). Whereas heat is thought to be the main inacti-vating factor of pathogenic agents during high temperature composting, which is a pre-dominantly aerobic process, toxic agents seem to play the major role under anaerobicconditions in compost piles, especially at lower temperatures. Unfortunately, data con-cerning the identity of the toxic agents are lacking (Bollen 1993). According to Bollen andVolker (1996), pathogens may be more sensitive to toxic agents under low redox poten-tials. These authors also illustrated the effects of anaerobiosis on the destruction ofpathogens during farm-scale composting of flower residues. They determined that theoxygen content of the air at depths of 30 and 70 cm inside a large, compact and poorlyaerated heap at the end of a 32-week composting period, was 14 and 1%, respectively.Surprisingly, even at sites within the heap where temperatures did not exceed 35°C, rel-atively heat-resistant pathogens such as formae speciales of Fusarium oxysporum weredestroyed. Two pathogens known for their resistance to adverse conditions in soil, i.e.F. oxysporum f. sp. dianthi and Sclerotium cepivorum, were eradicated within a few daysof treatment under these low oxygen tensions at temperatures of 32-35°C (Turner et al.1983; Bollen 1993; Bollen and Volker 1996). Furthermore, Horiuchi et al. (1983) observedthat infectivity of Plasmodiophora brassicae in galls on turnip (Brassica rapa) was lost afterone day of exposure to 45°C under (semi)anaerobic conditions. The foregoing suggeststhat some fungi may be highly sensitive to anaerobic processes.

It has been determined that plant pathogenic bacteria are highly sensitive to anaer-obic digestion. For example, Clavibacter michiganense is destroyed by fermentation at35°C in less than seven days (Turner et al. 1983).

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Furthermore, plant pathogenic nematodes such as cysts of Globodera rostochiensisand G. pallida are also sensitive to this process (Williams 1979; Turner et al. 1983;Heinicke 1989). Turner et al. (1983) showed that the population of viable juveniles ofGlobodera rostochiensis decreased 1,000-fold during 26 h of anaerobic digestion at 35°C.Viability of cysts was completely lost within seven days.

Data concerning the fate of plant pathogenic viruses are lacking. Furthermore, lit-tle is known about inactivation of seeds through anaerobic digestion. Jeyanayagamand Collins (1984) reported that survival of seeds of Sorghum halepense and of Panicumdichotomiflorum in the temperature range of 35°C ± 1°C is influenced by digestion time,the species and dormancy of the seeds. Dormant seeds were less sensitive and seed ofP. dichotomiflorum was more sensitive than that of S. halepense.

In conclusion, thermal death is assumed to be the most important cause of eradi-cation of plant pathogens during composting (Hoitink et al. 1976; Ylimäki et al. 1983;Yuen and Raabe 1984; Bollen 1985; Lopez-Real and Foster 1985; Herrmann et al. 1994;Bollen and Volker 1996), while in contrast, pathogen kill during anaerobic digestion atmesophilic temperatures is often attributed to toxic conversion products or to directbreakdown of pathogen structures by microorganisms (Lopez-Real and Foster 1985;Bollen et al. 1989; Bollen and Volker 1996). Unfortunately, the contribution of factorsother than heat in sanitation during composting or anaerobic digestion is difficult toquantify. Therefore, temperature and time should be considered as the most reliableparameters for prediction of inactivation of pathogens and seeds (Bollen et al. 1989).

Anaerobic digesters that are operated continuously pose potential problems whenthe feedstock contains infected plant materials. Turner et al. (1983) reported that a smallpercentage of the fresh material leaving such reactors has not been sanitized. For exam-ple, in a continuously fed digester system running with a hydraulic retention time of tendays, statistically 10% of the influent will pass straight through the system. Therefore,pretreatment and/or follow-up treatment with chemicals or heat treatment should beperformed. The obvious solution to this problem is to treat the digested material furtherthrough high temperature composting. This, to our knowledge, has not been tested.

The objective of this research was to investigate the fate of TMV, Plasmodiophorabrassicae, Heterodera schachtii, Meloidogyne incognita, Ralstonia solanacearum and tomato(Lycopersicon esculentum L.) seeds during anaerobic digestion of source separatedhousehold wastes (also called vegetable, fruit and garden wastes, referred hereafter asbiowastes) in small-scale digesters. Modified German BioAbfV-norms (LAGA-Merk-blatt 1994; BioAbfV 1998; Ryckeboer 2001) were used as the basis for this research, asdescribed further.

Materials and Methods

DRANCO Process

The anaerobic digestion process used in this work was based on the DRANCOprocess (De Baere et al. 1986; Baeten and Verstraete 1993; Gellens et al. 1995; De Baere2000). The DRANCO process is a solid phase (25-35% solids) fermentation process. Thefull-scale reactor is designed as a vertical plug flow reactor that operates at 52°C. In thesereactors, organic loading rates of 20 kg Chemical Oxygen Demand (COD) per m3 reactorper day are obtained. Fresh biowastes blended with a small amount of digested residue(inoculum) enters the top and leaves the bottom of the reactor with retention times of 16to 21 days. The digestion phase is followed by an aerobic composting phase of one tothree weeks (De Baere et al. 1986; Baeten and Verstraete 1993; Gellens et al. 1995; De Baere

The Fate of Plant Pathogens and Seeds During Anaerobic Digestion and Aerobic Composting of Source Separated Household Wastes

Compost Science & Utilization Summer 2002 205

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2000). Due to this continuous feeding process with fresh wastes, a small amount of freshwaste necessarily leaves the digester after having been treated as little as two days.

In this research small-scale digesters with a content of 2 to 6 liters were filled withDRANCO digester inoculum (= predigested biowastes) and incubated in a hot air ovenat 52°C. Inocula of plant pathogens and tomato seeds were prepared and incorporat-ed into these digesters according to the German BioAbfV-norms (LAGA-MerkblattM10 1994; BioAbfV 1998). In all experiments, a controlled quantity of pathogen inocu-lum or tomato seeds was added to an excess quantity of DRANCO digester inoculum(weight ratio pathogen inoculum/digester inoculum between 1:10 and 1:100) to es-tablish optimum digestion conditions (C/N ratio of feedstock: 15-40; pH in reactor: 7.5-

9; concentration of NH4-N inreactor: 300-2000 mg/kg etc.)in the small-scale reactors.The methane gas, convertedto normal conditions of pres-sure and temperature, wasquantified with a gas meter orwater column (Figure 1). TheDRANCO digester inoculumoriginated from a 40-literanaerobic digestion-unit thathad been fed in batch-mode(= discontinue) with bio-wastes and diapers and di-gested for two to three weeks(Ryckeboer 2001).

BioAbfV-norms

The German BioAbfV-norms describe how the hygienic safety of composting anddigestion processes must be evaluated. German regulations also specify that facilitieswhich process biowastes under controlled aerobic (composting) or anaerobic condi-tions (fermentation) must meet the BioAbfV-norms before the end product is com-mercialized. Direct process validation must be performed within twelve months aftera new biowaste treatment facility has been started up and the tests must be performedon a regular basis. The norms prescribe the use of TMV, P. brassicae and tomato seedsas indicator organisms for evaluation of phytohygienic safety. These test organismsare rather resistant to conditions that prevail during composting. Based on the resultsof test bioassays the phytohygienic safety of the compost is then predicted (LAGA-Merkblatt M10 1994; BioAbfV 1998).

Experimental Design

Several preliminary experiments were performed to determine the time requiredfor eradication of P. brassicae, H. schachtii, R. solanacearum or of tomato seeds duringanaerobic digestion at 52°C. In a second series of experiments, destruction of theseorganisms as well as that of TMV was investigated. The effect of anaerobic digestionon survival was examined in several experiments. In follow-up experiments, how-ever, the effect of anaerobic digestion followed by forced aerated composting wasdetermined as well, which is how the full-scale DRANCO process operates.

Jaak Ryckeboer, Stef Cops and Jozef Coosemans


Figure 1. Schematic of lab-scale DRANCO digester.

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Tobacco Mosaic Virus

Inactivation of TMV was tested by mixing tobacco leaves harvested from Nicotianatabacum cv. ‘Samsun’ plants infected with TMV with DRANCO digester inoculum ata weight ratio of 1:10. The mixture was then loaded into a digester. Control samplesconsisting of a 110-g mixture of TMV-infected tobacco leaves and DRANCO digesterinoculum (1:10; w/w) were stored at the beginning of each experiment at –21°C, ex-cept for in one short-term experiment where samples were stored at 4°C. Three di-gesters were used per treatment and several types of anaerobic digestions were per-formed as described below.

TMV-infected tobacco leaves used in these experiments were excised at the six-leafstage from Nicotiana tabacum cv. ‘Samsun’ plants grown in a heated greenhouse withphotosynthetic illumination (SON-T; 16-h photoperiod) at 21 to 22°C. Two or threelower leaves were thinly dusted with carborundum powder (500 mesh; Sigma AldrichChemie GmbH, Steinheim, Germany). The extract of pressed TMV-infected tobaccoleaves mixed with 0.05 mol/l phosphate buffer (pH 7), was carefully applied to car-borundum-dusted leaves using a glass spatula. TMV-infected leaves displaying mo-saic symptoms were selected three weeks after inoculation for use in this work(BioAbfV 1998; Ryckeboer 2001).

In a first short-term experiment, TMV-infested samples were digested for 72 h. Forpractical reasons controls were stored at 4°C. In later experiments, a minimal digesterretention time of two days was simulated followed by an aerobic composting periodof 19 days. During the normal transition from anaerobic to aerobic composting, the ma-terial leaving the DRANCO digester is dewatered with a screw press. The press waterfrom our experiments, therefore, was collected also and analyzed for TMV-titer. Thepressed solids were incubated under aerobic conditions in an oven for 19 days at 58°Cwhere it was subjected to humidified forced-aeration. Triplicate 110-g samples wereremoved after 0, 1, 2, 5, 10 and 19 days of aerobic composting. Control samples werestored at -21°C during the course of the experiment.

In a follow-up experiment, the material was digested for two days and then com-posted at 58°C or 68°C for 12 days. Triplicate 110-g samples were removed after 0, 1,2, 3, 5, 6, 7, 8 and 12 days of composting. In these experiments, all samples were storedat –21°C. At the end of the experiment, samples were thawed for 24 h at 21°C and thensubjected to a bioassay to determine TMV-titer. In additional experiments, the mater-ial was either digested for five weeks or digested for two weeks followed by threeweeks aerobic composting at 58°C.

Plasmodiophora brassicae

Survival of P. brassicae was tested by mixing 30 g cauliflower roots infected withP. brassicae and 430 g of a sandy loam soil infested with P. brassicae with 200 g DRAN-CO digester inoculum. This mixture had a composition (on a weight basis) of approx-imately 5% tuberous roots, 65% soil and 30% digester inoculum. Non-infested controlsamples were placed in separate digesters.

Details for all other test substances were as those described above with the fol-lowing exceptions.

Ralstonia solanacearum

Survival of R. solanacearum was examined by incorporating a mixture of 6 g groundpotato pieces severely infested with the pathogen with 200 g noninfested potato peels



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and 1 kg DRANCO digester inoculum. This preparation was then placed in a digester.Non-infested control samples were placed in separate digesters.

Plant Parasitic Nematodes

Survival of H. schachtii was examined by placing fine-mesh nylon nets (3 cm x 3cm, mesh 100 µm) each containing 200 cysts of H. schachtii in 4 kg DRANCO digesterinoculum. The preparation was then placed into a 6-liter digester.

Survival of M. incognita was determined by incorporating a mixture of 450 g ofshredded tomato roots severely infected with M. incognita into 3 kg DRANCO digesterinoculum. The mixture was then placed into a 6-liter digester. Non-infested controlsamples were placed in a separate digester.

Tomato Seeds

Destruction of tomato seeds was examined by placing fine-mesh nylon nets (3 cmx 3 cm, mesh 100 µm) each containing 400 tomato seeds (Lycopersicon esculentum L. cv.‘Moneymaker’, germination rate: 98%) into 4 kg DRANCO digester inoculum in a 6-liter digester.

For all experiments, three replicates were used per treatment for each agent. Fi-nally, each experiment was repeated once. In all experiments except one with TMV,survival of TMV, pathogens or seeds was determined immediately after samples wereremoved from the digester (Ryckeboer 2001).

Evaluation of Pathogen and Seed Survival Tobacco Mosaic Virus

A modified tobacco (Nicotiana tabacum cv. ‘Samsun NN’) half-leaf method was usedto detect infectious virus particles (Walkey 1991). This cultivar expresses a TMV infec-tion as local lesions (small, round spots with a necrotic center) within six days after in-oculation. Contrary to the BioAbfV-norms (LAGA-Merkblatt M10 1994; BioAbfV 1998)which prescribe the use of entire plants, we used detached leaves in this work. The de-tached leaves were the second and third leaves from plants in the 6-8 leaf stage grownin a heated greenhouse with photosynthetic illumination (SON-T; 16-h photoperiod) at21 to 22°C. Digester samples (3 replicates of 110 g each) were cut up into 1-cm2 pieceswith sterilized scissors. The material was then ground with a pestle in 30 ml of phosphatebuffer (0.05 mol, pH 7.0) and pressed through a nylon net (mesh 1 mm). The extract wasthen applied to one half of each of two tobacco leaves that had been dusted with car-borundum powder (600 mesh), while the opposite leaf halves were treated with extractof the frozen control. It was spread on the leaves with rotating movements using a glassspatula, also according to Walkey (1991). After 30 s, the leaves were rinsed for ten withrunning tap water. The inoculated leaves were then placed on two moistened filter pa-pers in a Petri dish and incubated in a growth chamber under controlled conditions (22-24°C; 16 h 210 µE m-2 s-1). Six days later, the number of local lesions was determined(Ryckeboer 2001). The BioAbfV-norms prescribe that the sum of TMV lesions of two leafhalves should be ≤ 8 for each replicate (LAGA-Merkblatt M10 1994; BioAbfV 1998).


Survival of P. brassicae was evaluated with a Brassica juncea bioassay (Ryckeboer2001). According to BioAbfV-norms, a potting mix (pH(CaCl2) of 6.0) was prepared

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from the digested sample by amending each with dry white sand and light sphagnumpeat. To accomplish this, 325 ml of the digested three replicate samples was mixed with82.5 ml sand and 192.5 ml peat and then placed into a 600-ml pot. For each samplingdate, 325-ml control samples were taken from non-infested separate digesters andmixed with 82.5 ml of sand and 192.5 ml of peat and processed in the same manner asdescribed for the infested samples. To allow for evaporation of phytotoxic compounds,fifteen-day-old Brassica juncea plants were planted ten days after filling of the pots (4plants/pot; 3 pots/treatment). The plants were incubated during 5 weeks in a heatedgreenhouse with photosynthetic illumination (SON-T; 16-h photoperiod) at 21 to 22°C.A completely randomized design was used in this bioassay. After this growth period,pathogen survival was determined by counting the total number of plants per treat-ment. Roots were rated for clubroot disease severity according to the following scale:0 = no visible symptoms; 1 = little gall development on lateral roots; 2 = moderate galldevelopment on lateral and main roots; and 3 = severe gall development on lateral andmain roots (Buczacki et al. 1975; LAGA-Merkblatt M10 1994; BioAbfV 1998). A diseaseseverity index was then calculated for each replicate with the following equation:

∑(Number of infected plants � disease severity rating)Disease severity index =

Total number of plants

According to the BioAbfV-norms, the mean clubroot disease severity index should be≤ 0.5 for each replicate.


Survival of R. solanacearum was determined by removing three replicate 10-g sam-ples of digested material from the digesters at each sampling time (Ryckeboer 2001).The sample was diluted with 50 mM phosphate buffer (pH 7.45; 7.75 g Na2HPO4 and1.65 g KH2PO4/l distilled water) and a serial triplicate dilution series was then made.Non-infested control samples were treated as the infested samples. From each dilu-tion, a 50 µl suspension was plated on SMSA selective medium (Engelbrecht 1994). TheSMSA-medium contained 1 g casamino acids, 10 g bacto-peptone, 5 ml glycerol, 15 gagar and 1 liter distilled water. The mixture was autoclaved for 15 min at 121°C. Aftercooling to 40°C, 5 mg crystal violet, 100 mg polymixin B sulfate, 25 mg bacitracin, 5 mgchloramphenicol, 0.5 mg penicillin, 20 mg 2,3,5-triphenyltetrazolium and 100 mg cy-cloheximide dissolved in 70% ethanol were added to the mixture. After three days ofincubation at 28°C, the number of colony forming units was enumerated. The coloniesof R. solanacearum were easily recognizable as milky white, liquid, irregular colonieswith a separated red to purple center. The identity of the colonies was confirmed withan ELISA-test (Adgen Ltd, Ayr, UK) and a PCR-test (Seal et al. 1993).


Survival of H. schachtii was determined by soaking fine-mesh nylon nets filled with200 cysts of H. schachtii in a funnel containing a ZnCl2-solution (620 µg/ml) accordingto Clarke and Perry (1977). After three weeks, this solution was collected and the num-ber of juveniles hatched from the cysts was counted under a light microscope.

A plant bioassay was used to determine viability of M. incognita. At each samplingdate, three 200-ml samples of the digested mixture were removed from the digester.



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Each sample was blended with 400 ml potting soil and then placed into a 600-ml pot.Two-week-old tomato plants cv. ‘Trend F1’ were planted ten days after filling of thepots (4 plants/pot; 3 pots/treatment). This process avoided symptoms of phytotoxici-ty caused by partially digested samples. The plants were grown in a heated greenhousewith photosynthetic illumination (SON-T; 16-h photoperiod) at 21 to 22°C. Four weeksafter planting, survival of M. incognita was evaluated by counting the number of rootknots on the entire root system (an approximately 27-cm3 clump of roots). For each sam-pling date, a non-infested control sample was treated as described for the infested sam-ple. A completely randomized design was used in this bioassay (Ryckeboer 2001).

Tomato Seeds

Survival of tomato seeds was evaluated by removing three fine-mesh nylon netsfrom the digester at each harvest date (Ryckeboer 2001). The nets were rinsed with tapwater and 200 tomato seeds were removed per sample replicate and placed on twomoistened filter papers in each of four Petri dishes (50 seeds/dish). The dishes werethen incubated in a growth chamber (24/22.5°C; 16-h photoperiod) and at intervals ofseven days the number of germinated seeds was counted. A seed was considered tohave germinated when the root and/or shoot was visible. After three weeks of incu-bation, the total number of germinated seeds was recorded and a germination indexwas calculated as follows:

Germination (%) of digested seedsGermination index = � 100 (%)

Germination (%) of untreated seeds

The remaining 200 seeds per treatment were air-dried and stored in an air-tightcontainer at room temperature for repeated germination capacity tests that might be-come necessary at a later stage. According to the BioAbfV-norms (LAGA-MerkblattM10 1994; BioAbfV 1998) the germination index of the treated tomato should be lessthan 2% seeds for each replicate.

Statistical Analysis

Data, unless otherwise indicated, were analyzed using one-way analysis of vari-ance (ANOVA) with SPSS (version 6.1, SPSS Inc., Chicago, IL). If a significant (P ≤ 0.05)F-test was observed, separation of means was performed in SPSS by using Fisher’s LeastSignificant Difference (LSD) test for multiple comparisons. Where necessary, data werelog10 transformed to improve homogeneity of variance. To determine significant (P ≤0.05) differences among mean clubroot severity values, the CATMOD procedure wasused in SAS (version 6.12, SAS Institute Inc., Cary, NC). CATMOD is a procedure formodeling of categorical data. Separation of means was performed in SAS by a weight-ed-least-square-estimation method that is based on a Wald chi square-test (Agresti 1990,1996). For each sample and control, a standard deviation was calculated.

Results

Tobacco Mosaic Virus

Infectivity of TMV-infested material was affected only slightly after 72 h of anaero-bic digestion at 52°C (Figure 2). After this short-digestion period, the number of lesions



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on test leaves caused by di-gested samples was signifi-cantly higher than thatcaused by control samplesstored at 4°C. None of the in-dividual sample replicatesmet the BioAbfV-norms. In asecond experiment (Figure3), almost complete inactiva-tion of TMV was achieved.In this experiment, two daysof anaerobic digestion wasfollowed by 19 days of aer-obic composting at 58°C.The BioAbfV-norms hadbeen met within two daysof anaerobic digestion fol-lowed by ten days of com-posting in this procedure.The mean number of lesionscaused by TMV-infestedsamples after 21 days oftreatment was 0.3 on twoleaf halves (Figure 3). Theaverage sum of lesionscaused by TMV-infestedpress water harvested after21 days was 237 (data notshown). In a final experi-ment, two days of anaero-bic digestion of TMV-in-fested samples at 52°C wasfollowed by 12 days of com-posting at 58 or 68°C.BioAbfV-norms were metwithin 2 days of anaerobicdigestion followed by eightand five days of compostingat 58 or 68°C, respectively(Figure 4). Infectivity de-creased sharply during fivedays of aerobic compostingat 68°C and it remained lowthereafter. To achieve thesame degree of TMV inacti-vation during composting at58°C, three more days wererequired. After 12 days ofcomposting at 58 or 68°C,the mean number of lesions



Figure 2. Fate of TMV during the initial 72 hours of anaerobic digestion ofbiowastes. Means (n = 3) followed by the same letter do not differ signifi-cantly (P ≤ 0.05). Bars represent standard deviation, while + and – indicatewhether the BioAbfV-norms were met.

Figure 3. Fate of TMV during anaerobic digestion followed by composting ofbiowastes. Samples were digested for two days and then composted there-after for 19 days at 58°C. Arrow indicates time of placement of sample fromdigester into composter. Means (n = 3) followed by the same letter do notdiffer significantly (P ≤ 0.05). Bars represent standard deviation, while + and– indicate whether the BioAbfV-norms were met.

Figure 4. Fate of TMV after two days of anaerobic digestion followed bytwelve days of composting at 58 or 68°C. Means followed by the same letterdo not differ significantly (P ≤ 0.05). Bars represent standard deviation,while + and – indicate whether the BioAbfV-norms were met.

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caused by the TMV-infested samples was 4 and 1 lesion(s) per two leaf halves, respec-tively. In additional experiments for which the details are not presented here, infectivi-ty was highly reduced after five weeks of anaerobic digestion at 52°C. Finally, completeinactivation was achieved if three weeks of anaerobic digestion was followed by twoweeks of aerobic composting at 58°C (Ryckeboer 2001).


In a preliminary experiment,destruction of P. brassicaeseemed complete within 12 hof anaerobic digestion at 52°C(data not shown). In a secondexperiment, therefore, sam-ples were removed after 6, 7, 8,9 and 10 h of anaerobic diges-tion (Figure 5). Temperaturesin the center of the reactor af-ter 2 h slowly increased from34 to 48°C after 2 and 10 h ofanaerobic digestion at 52°C.Survival of P. brassicae de-creased significantly within 9h of anaerobic digestion ofbiowastes. Complete destruc-tion was achieved within 10 hof anaerobic digestion (Rycke-boer 2001).


Eradication of R. solanacearum during anaerobic digestion depended on theconcentration of the pathogen in the source material. When the concentration of R.solanacearum was 108 CFU per gram feed material (experiment 1), the pathogen was

eradicated within 12 h ofanaerobic digestion at52°C, while a drastic re-duction in viability wasobserved after only 6 h of anaerobic digestion(Ryckeboer 2001). Howev-er, when the concentrationof R. solanacearum in feedmaterial was approxi-mately two orders lower(experiment 2), eradica-tion of the pathogen wasachieved within 6 h ofanaerobic digestion at52°C (Figure 6).



Figure 5. Fate of Plasmodiophora brassicae during anaerobic digestion ofbiowastes. Brassicae juncea plants (4 plants/pot; n = 3) were rated fiveweeks after planting with a disease severity index: 0 = no visible symp-toms; 1 = little gall development on lateral roots; 2 = moderate gall devel-opment on lateral and main roots; and 3 = severe gall development on lat-eral and main roots. Control plants were planted in compost not infestedwith P. brassicae. Means followed by the same letter do not differ signifi-cantly (P ≤ 0.05). Bars represent standard deviation, while + and – indicatewhether the BioAbfV-norms were met.

Figure 6. Fate of Ralstonia solanacearum during anaerobic digestion ofbiowastes. Non-infested control samples were taken from separated di-gesters. Means for each of two experiments followed by the same letter donot differ significantly (P ≤ 0.05); bars represent standard deviation.

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Cysts of H. schachtiiwere inactivated at firstsampling, i.e. within 30 minof anaerobic digestion at52°C, suggesting that thisnematode is highly sensi-tive to anaerobic digestion(data not shown).

M. incognita apparentlywas eradicated within 12 hof anaerobic digestion at52°C (Figure 7). The meannumber of root knots enu-merated on the entire rootsystem (root clump of ap-prox. 27-cm3) of tomatoplants grown in the infest-ed samples declined from18 in the control to 4, 2 and 0within 3, 6, and 12 h ofanaerobic digestion, respec-tively (Ryckeboer 2001).

Tomato Seeds

Percent germination oftomato seed was reduced to0.2% of untreated seeds after24 h of anaerobic digestionat 52°C in one experiment(Ryckeboer 2001). Seedswere completely inactivat-ed within 20 h of anaerobicdigestion in a second exper-iment (Figure 8).

Discussion

The results of this work show that propagules of the plant pathogens P. brassi-cae, R. solanacearum, H. schachtii, M. incognita as well as tomato seeds were rapidlydestroyed during treatment of biowastes in a thermophilic anaerobic digester basedon the DRANCO process. Thus, further high temperature composting should not berequired for the destruction of these pathogens. Because they were destroyed with-in 24 h, it may be assumed that they will be destroyed also in the full-scale systemeven though part of the materials in this continuous flow reactor may exit in twodays. In contrast, TMV could cause problems where anaerobic digestion is not fol-lowed by the high temperature composting process. Two days of anaerobic diges-tion did not significantly reduce the infectivity of the digested preparations. During



Figure 7. Fate of Meloidogyne incognita during anaerobic digestion ofbiowastes. Four weeks after planting of Lycopersicon esculentum plants thenumber of root knots in a 27-cm3 clump of roots was determined. Controlswere planted in samples not infested with M. incognita. Means followed bythe same letter do not differ significantly (P ≤ 0.05); bars represent stan-dard deviation.

Figure 8. Impact of anaerobic digestion of biowastes on the viability oftomato (Lycopersicon esculentum) seeds. Means for each of two experimentsfollowed by the same letter do not differ significantly (P ≤ 0.05). Bars repre-sent standard deviation, while + and – indicate whether the BioAbfV-normswere met.D

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this short-term experiment, the number of lesions caused by the digested samples ontest leaves actually was significantly higher than that of control samples (stored at4°C). This suggests that release of virus particles from decaying plant cells increasedfurther during anaerobic digestion of the infected plant material. It is interesting,however, that anaerobic digestion followed by 19 days of aerobic composting at 58°Csignificantly reduced the concentration of infectious virus particles, while anaerobicdigestion followed by 12 days of aerobic composting at 68°C was even more effec-tive. Even so, the virus was not entirely inactivated revealing its tolerance to heattreatment as well as anaerobic digestion. In some experiments, complete inactivationof TMV apparently was achieved after three weeks of thermophilic anaerobic diges-tion followed by two weeks of composting at 58°C. At least, the concentration of par-ticles had been reduced to below detectable limits for the bioassay. It seems unlike-ly, therefore, that TMV would pass through the DRANCO process as infectiveparticles under full-scale conditions in concentrations high enough to cause diseasebecause the retention time in the anaerobic phase for this process is 16-21 days andthe material typically is composted at high temperatures for several weeks there-after. Finally, the quantity of virus particles that might remain in the compost underfull-scale conditions should be infinitely small because most plant materials inbiowastes typically should not be infected with TMV. Nevertheless, TMV could posea threat, in theory at least, under conditions when crop residues from severely in-fested plants are treated. For heat-resistant pathogens, such pathogen populationscould be minimized by heat treatment of the feed material (Demuynck et al. 1985;Turner et al. 1983; Baeten and Verstraete 1993; Pagilla et al. 1996; Ward et al. 1998;Schieder et al. 2000). This should not be necessary, however, except for under themost extreme conditions such as those occurring at vegetable processing plants.Even here, however, experiments should be performed to determine the risk of dis-semination of such pathogens.

Several reports indicate that eradication of plant pathogenic fungi, bacteria andnematodes is achieved within a few days of anaerobic digestion at mesophilic tem-peratures (Williams 1979; Horiuchi et al. 1983; Turner et al. 1983; Heinicke 1989;Bollen 1993; Bollen and Volker 1996). Weed seeds on the other hand are inactivatedwithin a few weeks (Jeyanayagam and Collins 1984). Unfortunately, data concern-ing the fate of plant pathogens and seeds during thermophilic anaerobic digestionare lacking but this should occur readily because most plant pathogens are heat sen-sitive (Bollen 1993).

We conclude that thermophilic anaerobic digestion offers a suitable system fortreatment of organic waste typically delivered to treatment plants when the popula-tions of plant pathogens, especially detrimental agents such as TMV, and seeds areminimized or when anaerobic digestion is followed by thermophilic aerobic compost-ing. Field scale testing will need to be performed on full-scale plants to verify this con-clusion (Ryckeboer 2001).

Acknowledgements

J. Ryckeboer is indebted to the Flemish Institute for scientific-technologic research(IWT) in Belgium. The authors want to thank Organic Waste Systems n.v. (Ghent, Bel-gium) for technical assistance and providing the schematic of the lab-scale DRANCOdigester and Dr. H.A.J. Hoitink for critical review of the manuscript. We also thank J.Van Vaerenbergh (CLO, Merelbeke, Belgium) for providing potatoes severely infest-ed with R. solanacearum.



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