Survival of human and plant pathogens during anaerobic mesophilic digestion of vegetable, fruit, and garden waste

European Journal of Soil Biology 39 (2003) 165–171 www.elsevier.com/locate/ejsobi

Survival of human and plant pathogens during anaerobic mesophilic digestion of vegetable, fruit, and garden waste A.J. Termorshuizen a,*, D. Volker a, W.J. Blok a, E. ten Brummeler b, B.J. Hartog d, J.D. Janse c, W. Knol d, M. Wenneker c a

Biological Farming Systems, Marijkeweg 22, 6709 PD Wageningen, The Netherlands b Arcadis, P.O. Box 660, 5140 AR Waalwijk, The Netherlands c Plant Protection Service, P.O. Box 9102, 6700 HC Wageningen, The Netherlands d TNO Nutrition and Food Research Institute, P.O. Box 360, 3700 AJ Zeist, The Netherlands Accepted 6 May 2003

Abstract Five pathogens were added to vegetable, fruit and garden waste and their survival was studied during mesophilic (maximum temperature 40 °C) anaerobic digestion. Digestion during 6 weeks took place with a 50/50% (v/v) ratio of digested and fresh, undigested material, respectively. Survival of the plant pathogens Fusarium oxysporum f.sp. asparagi and Ralstonia solanacearum, and of the human pathogen Salmonella typhimurium was below the detection levels, and survival of the plant pathogen Plasmodiophora brassicae was low in one experiment and below the detection level in two replicates. In addition, numbers of Enterobacteriaceae originally present in the waste decreased significantly during digestion. However, sclerotia of the plant pathogen Sclerotium cepivorum recovered from the digestion vessel were, at least in part, viable. It is concluded that many pathogens may be inactivated readily, but that anaerobically digested compost may involve some significant phytohygienic problems. From this study, it can be concluded that vegetable, fruit and garden waste containing onions infected with S. cepivorum should be avoided. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Enterobacteriaceae; Fusarium oxysporum f.sp asparagi; Ralstonia solanacearum; Plasmodiophora brassicae; Salmonella typhimurium; Sclerotium cepivorum

1. Introduction In the Netherlands, an increasing amount of compost is produced since the separate collection of organic household waste (vegetable, fruit and garden waste) became compulsory. Since the compost is used primarily in agriculture as a soil conditioner, it needs to be free of pathogens. In aerobic composting, temperatures are reached that are lethal to the majority of plant [4] and human [12] pathogens. An alternative to aerobic composting is anaerobic digestion. In this * Corresponding author. E-mail address: [email protected] (A.J. Termorshuizen).

process, methane can be recovered from the waste. With this technology, however, inactivation of plant pathogens by heat does not occur since temperatures do not pass 40 °C [5]. Information on the fate of pathogens during mesophilic anaerobic digestion is scant. Turner et al. [27] reported the complete inactivation of the plant pathogens F. oxysporum f.sp. dianthi, Corynebacterium michiganense, and Globodera pallida in an anaerobic digester filled with tomato material, run at 35 °C, within 4, 7, and 10 d, respectively. Bollen [4] reported the complete inactivation of Sclerotium cepivorum in an anaerobic digester filled with onion material, run also at 32 °C, within 7 d. The formation of volatile organic acids during anaerobic digestion has been suggested to play a determinative role in the inactivation process of

© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. doi:10.1016/S1164-5563(03)00032-3

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F. oxysporum [20] and the cyst nematode G. rostochiensis [26]. The strong inactivation of various bacterial human pathogens during anaerobic digestion has been reported for animal waste [13,15,21], but their fate in more solid biowaste products is unknown. Jones [13] reported a positive correlation between survival of Salmonella dublin and the solids content of cattle slurry, suggesting that the bacterial pathogens may be more persistent in vegetable, fruit and garden waste which has a much higher solids content than cattle slurry. The goal of the present research was to evaluate the survival of six pathogens during anaerobic composting. We studied three plant pathogens that form persistent survival structures: F. oxysporum (Fungi) causing wilts and root rots in various hosts and forming chlamydospores, S. cepivorum (Fungi) causing white rot in onion and forming sclerotia, and Plasmodiophora brassicae (Protoctista) causing clubroot in brassicas and forming resting spores. In addition, we studied Ralstonia solanacearum (syn. Pseudomonas solanacearum) (Prokaryota), the causal agent of potato brown rot. Although this bacterium does not form persistent structures, the phytohygienic implications of its recent introduction in the Netherlands are important [24]. The human pathogen S. typhimurium (Prokaryota), which causes a gastro-intestinal form of paratyphus and is a notorious cause of food infections, was also included in the study. Finally, the presence of Enterobacteriaceae (Prokaryota) in digested samples was determined. This family of bacteria has pathogenic members such as Salmonella spp., and total counts of Enterobacteriaceae is generally considered as an indicator for hygiene and sanitation [19]. We also aimed to relate volatile organic acid compositions in percolate tapped from the digester at various times to inactivation rate of F. oxysporum, R. solanacearum, S. typhimurium, and Enterobacteriaceae.

2. Materials and methods 2.1. Digestion system An experimental 300-l- Biocel-type reactor (diameter 60 cm, height 106 cm) was used as described by ten Brummeler et al. [5]. The reactor was filled with a mix (50/50, v/v) of (unsieved) vegetable, fruit, and garden waste (64 kg fw) and similar waste that had been anaerobically composted in a commercial digester at Lelystad serving as methanogenic seed (82 kg fw). After adding 40 l of water, the dry matter content was 39% and the organic matter content was 35% (determined by loss on ignition according to Allison et al. [1]). During filling of the reactor, samples of the pathogens were added at different depths in the digester. Percolate was recirculated at a rate of 4 l h–1. The temperature in the digester was recorded continuously using 3 Pt-100 sensors, placed at 0.5, 0.75, and 1 m height in the middle of the digester and CH4 and CO2 concentrations were recorded every 3 min using an automated gas detection system that includes an IR-based CO2-sensor (Dräger, Hagen, Germany) and a catalytic combustion based CH4 sensor (GIG Gesellschaft für Gerätebau, Bonn, Germany). Both temperature data and gas detection data were recorded with a datalogger (AXIOM, Andorra). At the end of the composting process (21 d), the pathogens (see below) were collected and their survival was determined as described below. In addition, the quantity of F. oxysporum, R. solanacearum, S. typhimurium and Enterobacteriaceae in the percolate from the digestion system during anaerobic digestion, was determined. The experiments and the different pathogens involved are listed in Table 1. 2.2. Pathogens Field-collected potato tubers (Solanum tuberosum cv. Karnico) found to be infected with R. solanacearum were selected and used in survival experiments. The presence of

Table 1 Overview of the six experiments performed in this study and pathogens and number of samples involved Experiment number Date

Survival of pathogens tested in anaerobic digester a

Additional survival tests

1

18 September 1997

E-3, Foa-3 b , Sc-3, Pb-3, St-3

Pathogen E, St

2 3

19 October 1997 13 January 1998

Foa-3, Sc-3, Pb-3 Pb-9, Rs-5,7 c , St-5, E-5

– Foa, Rs

4

4 February 1998

Rs-0,11 c

Rs Foa

5 6

13 October 1998 3 November 1998

Sc-9 Sc-9

Foa Foa

Test Presence in percolate samples – Survival in percolate, harvested at different dates As in experiment 3 Effect of centrifuging the percolate before plating Foa d As in experiment 3 As in experiment 3

a E, Enterobacteriaceae spp.; Foa, Fusarium oxysporum f.sp. asparagi; Rs, R. solanacearum; Pb, P. brassicae; Sc, S. cepivorum; St, S. typhimurium. b Number of samples tested. c Number of whole and halved potato tubers tested, respectively. d Described in Section 2.

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living cells of R. solanacearum was confirmed by plating onto SMSA medium (modified by Elphinstone et al. [11]). On this medium, R. solanacearum produces a typical morphology. The identity was regularly confirmed by immunofluorescence microscopy [2,3] or PCR [25]. The survival of the pathogen in percolate obtained from the anaerobic digester was studied using field-collected potato tubers (described above) or a pure culture of R. solanacearum biovar 2 race 3, strain PD2762. Tuber material was macerated in phosphate buffer (pH 7; 4.26 g Na2HPO4 and 2.72 g KH2PO4 l–1 distilled water) to obtain a suspension containing 107–108 cfu ml–1. The pure culture was maintained on Yeast Peptone Glucose medium [18]. To study the survival in percolate, three samples of 200 µl of bacterial suspension were put into the tubes closed with a rubber stopper and flushed with N2 to obtain anaerobic conditions. The tubes were transported to the anaerobic digester and 5 ml of the percolate was injected into the tubes. Incubation took place at room temperature. Survival was determined by plating 100 µl of 10-fold dilutions of the inoculum onto each of two plates containing SMSA medium and incubating at 28 °C for 5–6 d. F. oxysporum (Schlechtend.) emend. W.C. Snyder & H.N. Hans. f.sp. asparagi (S.I. Cohen & Heald) strain CWB1 was originally isolated from asparagus roots in the Netherlands. For the preparation of inoculum, the strain was grown in malt extract broth for 10 d at 25 °C. After comminuting the culture in a blender, the slurry was centrifuged at 3400 × g for 30 min, and the precipitate was resuspended in sterile distilled water and mixed with silver sand (1:2, w/w) that had been washed two times with hot tap water. This mixture was dried with forced air for 21 d, after which the silver sand was passed through a 0.36 mm sieve. This inoculum contained primarily microchlamydospores. The samples of F. oxysporum consisted of ca. 100 g of sand inoculum in a nylon bag. Survival was tested by plating 10-fold dilutions of the inoculum in 0.1% water agar on Komada’s medium [16] with 10 and five replicates 0.25-ml aliquots per dilution for experiments 1 and 2, respectively. To study the survival in percolate, three 2.0-g samples of sand inoculum were put into the tubes covered with a rubber cap, N2-flushed to obtain anaerobic conditions, transported to the anaerobic digester. Percolate (5 ml) was injected into the tubes which were then incubated at 35 °C (experiment 3) or 30 °C (experiments 4 and 5). Three milliliters of the percolate were injected into the tube as described above after 1, 3, 7, 14, and 21 d. The remaining 3 ml percolate was acidified by adding 0.5 ml of 10% formic acid. The concentration of volatile fatty acids (C2–C5) was determined on a gas chromatograph equipped with a packed column (2000 × 6 × 6 mm) with a 10% Fluorad 431 Supelco-port 100–120 mesh, and a flame ionisation detector. The carrier gas was nitrogen saturated with formic acid (40 l min–1), and the oven temperature was 130 °C. In experiment 3, it was observed that removing the percolate before diluting and plating by centrifugation for 3 min at 1600 × g had no effect on the recovery of F. oxysporum.

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Undiluted samples showed no recovery of F. oxysporum. A 100-fold dilution of the percolate with water lead to recoveries not significantly different from those of control samples (water only). S. cepivorum Berk. strain 0900 was obtained from infected onions grown in a commercial field in the Netherlands. To obtain large quantities of sclerotia, the strain was incubated in fresh onions for 7 weeks at 15 °C. The sclerotia were scraped from the onions, air-dried, and 1.2 × 106 sclerotia (approximately 30 g) was mixed with 50 g sand (pHKCl 7.0, organic matter 0.3%) that had been steamed and allowed to be recolonised for at least 4 weeks, and put in a nylon bag (total volume approximately 50 cm3). Testing the survival by plating onto semiselective medium (inulinquintozene agar, [22]) failed due to the presence of Trichoderma sp. Concurrently, survival was tested following a method described by Coley-Smith [6] that involved placing 1.0 g of the inoculum (equalling approximately 1.5 × 104 sclerotia) in a small hole made in a fresh onion. After placing the inoculum in the hole, this was closed with onion tissue and taped to prevent drying out. For each sample, the viability was tested for four replicate 1.0-g samples. The onions were incubated for 7 weeks at 15 °C and inspected for formation of new sclerotia in the onion tissue under the dissecting microscope (Zeiss, Jena, Germany) at 15× magnification. P. brassicae Wor. was obtained from infected brassica roots from a naturally infested site at Wageningen, The Netherlands. The roots were cut into final sizes of 2 cm and 22, 30, and 70 g was put in nylon bags in experiments 1, 2 and 3, respectively. Survival of inoculum was determined by chopping the contents of the nylon bags into 5-mm pieces followed by mixing with 1000 ml potting soil. One Chinese cabbage plant (Brassica oleracea cv. Granaat) was planted in 1 l pots containing the test soil and after 5 week plants were uprooted and observed for clubroot symptoms. S. typhimurium, strain ATCC13311, originally obtained from the American Type Culture Collection, was grown on Tryptone Soya Broth (Oxoid) for 1 d at 37 °C and the culture was diluted 50-fold with a sterile 0.85% NaCl to obtain 1.7 × 108 cfu ml–1. Five hundred milliliters of this suspension were equally distributed over the to-be digested material by spraying aliquots of 100 ml on the surface of the material five times during filling of the digester. The survival of S. typhimurium and other representatives of the family of the Enterobacteriaceae, eventually present in the starting material, was determined in three composite samples of (1) the uninoculated material, (2) the material after inoculation with S. typhimurium, and (3) after completion of the fermentation when the anaerobic digester was opened. Each sample (250 g) was suspended in 1 l sterile peptone-physiological salt solution (0.1% peptone, Oxoid, in 0.85% NaCl). In order to investigate the survival during the digestion period counts of Enterobacteriaceae and S. typhimurium were determined in percolate samples taken from the digester at various time intervals. Ten-fold dilutions of the suspended samples and of the percolate samples were plated onto Brilliant Green Agar

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(BGA; Oxoid) and Violet Red Bile Glucose Agar (Oxoid) for quantifying S. typhimurium and Enterobacteriaceae, respectively. Plates were incubated for 1 d at 37 °C. For quantifying S. typhimurium in the digested compost, the suspension of the samples was cultivated in Rappaport-Vassiliadis medium (Oxoid) for 1 d at 42 °C and subsequently subcultured on BGA plates for 1 d at 37 °C. In cases of doubtful identity colonies were isolated and identified using various biochemical characteristics with the Api20E System (BioMérieux, Lyon).

3. Results After 21 d of anaerobic digestion, dry matter content had decreased from 39% to 29% and organic matter content had increased from 35% to 41%. The temperature in all experiments was 35–37 °C during digestion. Typically, volatile fatty acids peaked shortly after onset of the digestion, with acetic acid reaching the highest concentration at 5.1 g l–1 percolate, followed by a sharp decline to 0.79 g l–1 at the end of the incubation time (day 21; Fig. 1). A concomitant increase in pH of the percolate from 6.0 to 8.0 was observed. The decline in concentration of volatile fatty acids is caused by the increased activity of methanogenic bacteria. In experiments 3 and 5, no decline in concentrations of volatile fatty acids occurred and this coincided with no increase in pH, indicating that composting partially failed in these experiments. F. oxysporum, R. solanacearum, and S. typhimurium decreased dramatically during digestion and were undetectable with at least three, six, and sevenfold reductions, respectively (Table 2). For P. brassicae, in experiments 1 and 3, no clubroot was observed in the bioassay plants. However, in experiment 2, one very small infection was observed for one of the three samples that had been incubated in the anaerobic

digester, while in the control samples all three bioassay plants were heavily infected. Enterobacteriaceae originally present in the waste were detectable, showing a threefold reduction after 3 weeks of digestion down to 1.2 × 103 cfu g–1. Samples of S. cepivorum that had been incubated in the anaerobic digester were all viable. The toxicity of percolate collected from the anaerobic digester after various times was tested by incubating R. solanacearum and F. oxysporum in percolate samples for various times under anaerobic conditions at room temperature and 30–35 °C. At all harvests the survival was very low (<0.1%) within 1 d of incubation irrespective of the time the percolate was taken. The survival was not correlated with the concentration of any of the volatile fatty acids determined. S. typhimurium could not be detected in any of the percolate samples taken at various time intervals during anaerobic digestion of the inoculated compost, while the number of total Enterobacteriaceae decreased rapidly (Table 3). R. solanacearum was practically absent in samples that were incubated in percolate for 2 h (Table 4) and not detected when incubated for 18 h. The survival of R. solanacearum increased only slightly when macerated infected potato tubers were used as inoculum instead of cells from a pure culture. There was a difference in the toxicity of percolate, with percolate taken 7 (experiment 3) or 9 and 13 d (experiment 4) after onset of the digestion causing not any survival even when incubated for only 15 min, while at the other sampling times there was at least some survival at incubation for 15 min. Persistence of F. oxysporum in the percolate was slightly higher than for the bacterial pathogens (Table 5), with only slight differences between different percolate samples. The survival rate was very low (down to 0.0095%) after 3 d of incubation and after 7 d the pathogen was not detected at all. In experiment 3, no survival was found in any of the percolate incubations.

Fig. 1. Evolution in time of six volatile fatty acids occurring in the percolate of anaerobically digesting vegetable, fruit and garden waste (experiment 1).

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Table 2 Percent survival of six pathogens during anaerobic digestion of vegetable, fruit, and garden waste a for 21 d at 35–40 °C Pathogen

Experiment number 1 2 <1.2 × 10 –3 <1.2 × 10 –3 0/3 1/3 100 100 n.d. n.d. n.d. n.d. n.d. n.d.

F . oxysporum a P . brassicae c S . cepivorum d R . solanacearum a,e S . typhimurium a Enterobacteriaceae a

3 n.d. b 0/9 n.d. <4.0 × 10 –6 <2.1 × 10 –5 7.0 × 10 –3

4 n.d. n.d. n.d. <4.0 × 10 –6 n.d. n.d.

5 n.d. n.d. 100 n.d. n.d. n.d.

6 n.d. n.d. 100 n.d. n.d. n.d.

a

Data preceded by “<” indicate lower limit of detection. n.d., not determined. c a/b, No. of bioassay cabbage plants with clubroot in samples incubated in anaerobic digester/total no. of plants. d Per cent of bioassay onions showing newly formed sclerotia (%). e Experiment 3, no differences between survival of R. solanacearum in whole and half potato tubers. f At day 1 of the composting process, vegetable, fruit, and garden waste was mixed with anaerobically composted vegetable, fruit, and garden waste (50/50,v/v). b

Table 3 Colony-forming units of S. typhimurium and total Enterobacteriaceae per ml percolate taken from the anaerobic digester 1, 5, 7, and 21 d after onset of the fermentation Sampling time (d) of percolate after onset of the fermentation 1 5 7 21

4. Discussion The results show that three pathogens are quickly inactivated during mesophilic anaerobic digestion of vegetable, fruit and garden waste at 35–37 °C. Inactivation of F. oxysporum during anaerobic digestion of biowaste of tomato plants at 35 °C was also reported by Turner et al. [27] although these authors did not mention the type of structures that were tested which may well have consisted of mycelium and conidia. Our study shows that also the persistent structures of F. oxysporum (Chlamydospores) are sensitive to conditions during anaerobic digestion. S. typhimurium and R. solanacearum were also undetectable after digestion of the biowaste. Comparing the counts of S. typhimurium in the digester contents directly after inoculation (1.4 × 107 g–1; data not shown) with the counts in percolate water (below detection limit), it is

Pathogen S . typhimurium a

Enterobacteriaceae

<10 5 <10 5 <10 2 <1

4.7 × 10 6 8.6 × 10 3 1.3 × 10 2 1.1 × 10 2

a Competitive growth of the accompanying microflora occurred, which caused the high detection levels of S. typhimurium. Since the determination of Enterobacteriaceae include S. typhimurium, levels of the latter species are likely lower or equal to the levels measured for Enterobacteriaceae.

Table 4 Percent survival of R. solanacearum applied in pure culture or in macerated infected potato tubers incubated for various times at room temperature in percolate taken from the anaerobic digester various days after onset of the fermentation Sampling time (d) of percolate after onset of the fermentation Experiment 3

7 21

Time (h) the samples were incubated in the percolate Pure culture

Macerated infected potato tubers

0.25

1

2

18

0.25

<4.0 × 10 –6 ∞

<4.0 × 10 –6 <4.0 × 10 –6 <6.0 × 10 –5 <1.6 × 10 –6

0.25

2

4

0.25

2

4

∞ ∞ ∞ <1.2 × 10 –6 <1.2 × 10 –6 ∞

1.2 × 10 –5 <1.2 × 10 –6 1.5 × 10 –4 <1.2 × 10 –6 <1.2 × 10 –6 2.8 × 10 –6

2.8 × 10 –6 <1.2 × 10 –6 2.2 × 10 –5 <1.2 × 10 –6 <1.2 × 10 –6 1.7 × 10 –6

∞ ∞ ∞ ∞ ∞ ∞

1.5 × 10 –2 <6.3 × 10 –4 1.3 × 10 –2 <6.3 × 10 –4 <6.3 × 10 –4 8.4 × 10 –2

8.5 × 10 –4 <6.3 × 10 –4 6.4 × 10 –4 <6.3 × 10 –4 <6.3 × 10 –4 2.0 × 10 –2

<4.0 × 10 –6 <3.1 × 10 –3 <1.6 × 10 –6 ∞

1

2

<3.1 × 10 –3 <3.1 × 10 –3 <3.1 × 10 –3 <4.3 × 10 –1 <1.6 × 10 –3 <1.6 × 10 –3

Experiment 4

1 2 6 9 13 21

18

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Table 5 Survival (%) of F. oxysporum incubated for various times at 35 (experiment 3) and 30 °C (experiments 5 and 6) in percolate taken from the anaerobic digester various days after onset of the fermentation; experiment numbers coincide with those mentioned in Table 1 Sampling time (d) of percolate after onset of the fermentation 1 3 7 14 21

Time (d) the samples were incubated in the percolate Experiment 3 1

Experiment 5 3

–2

<1.4 × 10 <1.4 × 10 –2 <1.4 × 10 –2 <1.4 × 10 –2 <1.4 × 10 –2

7 –2

<5.1 × 10 <5.1 × 10 –2 <5.1 × 10 –2 <5.1 × 10 –2 <5.1 × 10 –2

1 –1

<5.5 × 10 <5.5 × 10 –1 <5.5 × 10 –1 <5.5 × 10 –1 <5.5 × 10 –1

–3

5.8 × 10 7.2 × 10 –1 5.8 × 10 –3 <1.5 × 10 –4 <1.5 × 10 –5

obvious that the inactivation of this pathogen was also very strong. Within 1 d most of the inoculum (>99%) was already killed. The order of magnitude of inactivation is even higher than the 90%-inactivation rate of 0.7–2.5 d reported by Kearney et al. [14] for survival of S. typhimurium in cattle slurry anaerobically digested at 35 °C. Apparently the solid content itself is not a predominant factor predicting survival, as suggested by Jones [13] for cattle slurry. It may be questioned whether the pathogens studied have really been killed or whether they have entered a nonculturable state. For fungi, dormancy of young survival structures does exist [9], but entering from a culturable into a (reversible) nonculturable state is currently an unknown phenomenon [8]. For Enterobaceriaceae, including Salmonella spp., reversible nonculturability has been reported for cells that were incubated in a nutrient-poor water suspension and then exposed to nutrients [23]. In our experiments, it is not likely that nonculturability due to shortage of nutrients has occurred in the nutrient-rich biowaste. Turpin et al. [28] found a 105-fold decline of S. typhimurium after incubation for 23 d in nonsterile soil while no decrease in direct fluorescent antibody counts were noticed. Cells that had been killed by UV-radiation became very quickly undetectable by fluorescent antibody counts after incorporation in soil [28]. The difference between cultivated cells and direcly counted cells was interpreted as viable but nonculturable. However, it was not tested whether the nonculturable cells were able to cause disease or whether they could revert to a culturable state. The percolate appears to be very toxic to F. oxysporum and R. solanacearum with complete or nearly complete (>99.9%) inactivation within 2 d or less. Incubation of inoculum of F. oxysporum in percolate sampled at different times consistently resulted in complete inactivation, although concentration of organic acids varied considerably (Fig. 1). In addition, at the prevailing high pH (8.0) in the percolate sample at 21 d, the majority of volatile fatty acids occurs in nontoxic, undissociated form. Thus, organic acids seem not to be the predominant factor causing inactivation of chlamydospores of F. oxysporum, which was suggested by Okazaki and Nose [20]. Similarly, the complete or nearly complete inactivation of cells of R. solanacearum incubated in percolate that was sampled at 1–21 d after onset of the experiment invariably showed high levels of inactivation.

Experiment 6 3

7 –4

<1.6 × 10 <1.6 × 10 –4 <1.6 × 10 –4 <1.6 × 10 –4 <1.6 × 10 –4

1 –3

<1.2 × 10 <1.2 × 10 –3 <1.2 × 10 –3 <1.2 × 10 –3 <1.2 × 10 –3

3 –1

1.7 × 10 7.0 × 10 –2 2.9 × 10 –3 5.3 × 10 –1 2.1 × 10 –1

7 –4

<1.6 × 10 <1.6 × 10 –4 <1.6 × 10 –4 9.5 × 10 –3 1.1 × 10 –3

<1.2 × 10 –3 <1.2 × 10 –3 <1.2 × 10 –3 <1.2 × 10 –3 <1.2 × 10 –3

Temperature during anaerobic digestion has a great effect on survival of pathogens. Kearney et al. [15] assessed that 90%-inactivation of S. typhimurium during anaerobic digestion at 28 °C was reached after 34.5 d. Kumar et al. [17] reported a reduction of the survival time of Escherichia coli, Streptococcus faecalis, S. typhi, and Shigella dysenteriae by about 50% when the temperature during anaerobic digestion of cattle dung slurry was increased from room temperature (18–25 °C) to 35 °C. The reason for this large effect of temperature, which is in the nonlethal range, may be temperature-dependent dynamics of decomposition. Thus, our results are valid only for anaerobic digestion at 35 °C and probably also for higher, sublethal temperatures, but not for lower temperatures. There appears to be a phytohygienic risk of presence of sclerotia of S. cepivorum in the biowaste. Our data do not permit precise quantitative interpretation since the germination assay on agar failed due to presence of Trichoderma sp. It would be interesting to study the ability of this well-known hyperparasite of various plant pathogens to survive anaerobic mesophilic digestion. As S. cepivorum can survive anaerobic digestion and levels as low as 1 sclerotium kg–1 soil can cause significant disease [10], efforts to avoid white rot infected material in the anaerobic digestion of vegetable, fruit and garden waste need to be undertaken. In contrast to our results, Bollen [4] reported total inactivation of sclerotia of S. cepivorum in an anaerobic digester fed with onion juice at 32 °C. The germination of sclerotia on agar after anaerobic exposure to percolate decreased from 62% to 0% [4]. It is likely that during anaerobic digestion of material consisting only of onions, sulphur-containing compounds are produced at concentrations that are lethal to the sclerotia of S. cepivorum, or that induce germination followed by lysis, as volatile degradation products of the alkyl and alkenyl sulphoxides are responsible for triggering the germination of sclerotia of S. cepivorum [7]. Alternatively, the sclerotia of S. cepivorum may have been exposed to more toxic substances in the onion juice than to those in the much more solid vegetable, fruit and garden waste. Host tissue in which inoculum is embedded may provide protection if the host tissue is resistant to decomposition, but it may contribute to inactivation of the pathogen if the host tissue is readily decomposed. For R. solanacearum, there

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appeared to be no significant protection when it was incubated in the form of macerated potato tissue. On the other hand, it may explain the low survival of P. brassicae in one experiment as it was incorporated in the form of infected root pieces. It would be worthwhile to study the inactivation rate of human pathogens such as Salmonella spp. during anaerobic composting of pieces of contaminated meat. The minimum retention time of biowaste in Biocel reactors is 21 d. The majority of inoculum is already killed within a few days and therefore no danger exists for the survival of F. oxysporum, R. solanacearum, and S. typhimurium given that the recirculating percolate is distributed well in the reactor. To further explore the mechanism of inactivation, it would be interesting to incubate these pathogens in anaerobic digesters while being protected from direct exposure to the percolate.

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