Potential risks when spreading anaerobic digestion residues on grass silage crops – survival of bacteria, moulds and viruses M. Johansson*, E. Emmoth†, A.-C. Salomonsson‡ and A. Albihn* *Department of Disease Control and Biosecurity, †Department of Virology, and ‡Department of Feed, National Veterinary Institute, Uppsala, Sweden
Abstract
Introduction
The survival of pathogenic and spoilage micro-organisms in soil and on grass fertilized with spiked anaerobic digestion residue (ADR) was investigated in a climate chamber during periods of up to 56 d. In addition, the survival of these organisms over time was investigated during ensiling of grass at 390 g dry matter (DM) kg)1 or 610 g DM kg)1. Micro-organisms included in these studies were: Clostridium tyrobutyricum, Salmonella serotype Typhimurium, Listeria monocytogenes, Campylobacter jejuni, Escherichia coli, Talaromyces emersonii, Byssochlamys nivea, Porcine parvovirus and Swine vesicular disease virus. Soil and grass still contained high numbers of E. coli, Cl. tyrobutyricum and T. emersonii (1Æ9–5Æ4 log10) 49 and 56 d after fertilization with spiked ADR. Listeria monocytogenes and S. Typhimurium were generally found in the samples. This indicates that, within this time span, there is a risk of silage contamination by bacteria, moulds and viruses present in ADR spread on grassland. An increase in DM content of the crop decreased its ensilability as measured by pH and short-chain fatty acid content. However, no clear differences were found in survival of pathogenic and spoilage micro-organisms between the two silages with different DM contents, regardless of storage time. The lack of moisture and oxygen was probably sufficient to cause the reduction in vegetative bacteria in the 610 g DM kg)1 silage. However, bacterial and fungal spores and the viruses studied were not significantly inactivated by ensiling at these high DM contents and could, therefore, pose a health risk to farm animals fed on the silage if present in ADR applied to crops prior to ensilage.
Biowaste can be a useful resource in agriculture as it contains plant nutrients and organic material, both of which are needed in, for example, organic farming. An increased recycling of nutrients from biowaste to feed and food production is of importance in developing sustainable agriculture systems. Production of biogas from anaerobic digestion is increasing in European countries. Mineralization of organic material through anaerobic digestion represents a complex pathway in which organic matter is progressively degraded by several microbial communities, ultimately resulting in the release of most of the carbon as methane and bicarbonate or carbon dioxide (Gujer and Zehnder, 1983). In addition, anaerobic digestion of biowaste produces a nutrient-rich residue that can be used as a fertilizer. The use of biowaste, such as anaerobic digestion residue (ADR), in agriculture is an alternative to incineration. It is possible for undesirable micro-organisms to contaminate grass crops and silage via several routes, such as soil, wild animals and the use of manure (Fenlon, 1985; Rammer and Lingvall, 1997; Fenlon et al., 2000). These undesirable micro-organisms can grow during storage of silage and lead to decreased nutritional value, spoilage or a risk for the spread of infectious diseases to animals (McDonald et al., 1991; Driehuis and Elferink, 2000). It is a common practice to fertilize silage crops with farmyard manure, although there can be an increased risk of undesirable micro-organisms in the silage. Several studies have shown high levels of Enterobacteriaceae, Bacillus and Clostridium spores in manure ¨ stling and Lindgren, 1991; Rammer et al., 1994) (O and manure may therefore have an adverse effect on the nutritional and hygiene quality of silage (Rammer and Lingvall, 1997). Biowaste often contains many different bacteria, viruses, moulds and parasites (Deportes et al., 1995) and could contribute to an increased hygiene risk if used as a fertilizer in agriculture. In order to obtain a safe product, the material therefore needs to be treated.
Keywords: biowaste, hygiene, silage, high dry-matter content, micro-organism
Correspondence to: E. Emmoth, National Veterinary Institute, SE-751 89 Uppsala, Sweden. E-mail:
[email protected] Received 10 May 2004; revised 5 February 2005
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176 M. Johansson et al.
If anaerobic digestion is used as a biological treatment, the recommended treatment process in Sweden is preheating at 70C for 1 h, which is sufficient to kill vegetative bacteria, for example faecal streptococci, Salmonella and Listeria, different viruses and non-cystic parasites (Engeli et al., 1993; Larsen et al., 1994; Lund et al., 1996; Burtscher et al., 1998; Dumontet et al., 1999). However, it should be noted that, when the residue is stored and transported after processing, there is a risk of recontamination, for example by equipment not properly cleaned or by animals. Some microorganisms and parasites form spores and cysts, which are resistant to high temperatures, and hence may survive the treatment process, and eventually may end up in the residue (Palop et al., 1999; Schnu¨rer et al., 1999). Examples of spore-forming micro-organisms, likely to be present in organic waste, are heat-resistant moulds and Bacillus and Clostridium species. Clostridium tyrobutyricum is a spoilage organism and interesting from the point of view of silage nutritional quality but also as the causative agent of the phenomena called ‘late blowing’ of hard cheese. The ability of some of these heat-resistant moulds to grow at body temperature makes them potential pathogens, although far from all are considered pathogenic under normal circumstances. Many of these heat-resistant moulds can also produce toxic metabolites such as gliotoxin (Wenehed et al., 2003) and patulin. Mycotoxin-producing moulds and Listeria monocytogenes have been identified as a serious risk to animal health in aerobically deteriorated silage (Driehuis and Elferink, 2000). The practice of making silage with highly wilted forage is often used to reduce the risk of clostridial growth. However, it is also well-known that increased dry matter (DM) content can reduce the extent and rate of fermentation, resulting in higher pH and decreased formation of short-chain fatty acids (Driehuis et al., 1997). Slow fermentation (Woolford, 1984) and wilting (Rammer et al., 1994) favour the growth of enterobacteria. Most research on undesirable micro-organisms in silage has focused on bacteria and fungi (Wilkinson, 1999) and virus survival in silage has received little attention to date. Porcine parvovirus (PPV) is a very resistant virus and has been used as a viral indicator in other studies (Lund et al., 1996). The PPV is a potential health risk for pigs. Swine vesicular disease virus (SVDV) can also be used as a model for other enteroviruses, such as bovine and porcine enteroviruses, as it belongs to this genus (Van Regenmortel, 2000). There is limited knowledge regarding the risk of transferring undesired micro-organisms from biowaste to silage. The increased use of highly wilted crops for silage production also deserves further attention. Thus, the objective of the present study was to evaluate the survival of pathogenic and spoilage micro-organisms in
soil and on grass fertilized with ADR, and to study the survival of these organisms in silage with two different high DM contents.
Materials and methods Experimental design In the first study, survival of bacteria, moulds and viruses in soil and on grass was evaluated. The first experiments included studies of the survival of bacteria/ moulds (Experiment 1) and PPV (Experiment 2) in soil. Survival on grass was evaluated in Experiment 3 (bacteria/moulds) and Experiment 4 (PPV). In the second study, the survival of bacteria/moulds (Experiment 5) and viruses (Experiments 6a; PPV, and 6b; PPV and SVDV) in silage was evaluated. In addition, survival of PPV and SVDV in aqueous extracts of the forages was investigated (Experiment 7). Unless otherwise stated, the National Veterinary Institute (SVA), Uppsala, Sweden supplied the material and media used in the experiments.
Bacterial, fungal and viral strains The bacteria used in Experiments 1, 3 and 5 were: Cl. tyrobutyricum (Ct 209), originally isolated from silage (Jonsson, 1990), Salmonella enterica ssp. enterica serotype Typhimurium (CCUG 31969), L. monocytogenes (CCUG 15527), Campylobacter jejuni (CCUG 11284) and Escherichia coli (ATCC 352189). Two thermo-tolerant moulds were used in the experiments above: Talaromyces emersonii and Byssochlamys nivea, (isolated from biogas digesters by Dr A. Schnu¨rer, Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden). For Experiments 2, 4, 6 and 7, the PPV strain 893/76, originally isolated at the Danish Institute for Food and Veterinary Research, Lindholm, Denmark, and obtained from the Department of Vaccine Research, SVA, was grown in PK-15 (pig kidney) cells (ATCC CCL33), using Eagle’s minimal essential medium (MEM) (Eagle, 1959) supplemented with non-essential amino acids (L -alanine 8Æ9 mg L)1, L -asparagine monohydrate 15 mg L)1, L -aspartic acid 13Æ3 mg L)1, )1 )1 L -glutamic acid 14Æ7 mg L , L -proline 11Æ5 mg L , )1 L -serine 10Æ5 mg L ) and containing 4% foetal calf serum (FCS). The virus suspension was clarified by compressed air filtration, using two sets of filters: 3Æ0 and 0Æ45 lm (Millipore, Billerica, MA, USA), aliquoted and stored at )70C. The SVDV strain 27/72 used in Experiments 6b and 7 was grown in IB-RS-2 (pig kidney) cells, both obtained from Pirbright Laboratory, Pirbright, UK, using Eagle’s MEM containing 2% FCS. After full cytopathogenic effect, the virus-infected cells and supernatant were
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frozen and thawed twice, and clarified by low-speed centrifugation (2500 g for 20 min). The virus suspension was aliquoted and stored at )70C.
Survival of micro-organisms in soil and on the crop (Experiments 1–4) The soil was collected from an organic farm 20 km north of Uppsala, Sweden. Soil sampling was performed in the autumn of 2001 to a depth of 20 cm, shortly before ploughing. The soil was directly transported to the laboratory, sieved to pass a 4-mm sieve and stored frozen at )20C until the start of experiments. Soil texture, classified according to the International Union of Soil Science, was 45% clay, 40% silt and 15% sand. Soil organic carbon content was 5Æ0%, total nitrogen 0Æ49% and pH 7Æ8. Soil analysis was performed at the Department of Soil Science, SLU, Uppsala, Sweden. The ADR used in this study was taken from an anaerobic semi-continuous mesophilic (37C) digester (45 L), degrading source separated organic household waste (Department of Microbiology, SLU, Uppsala, Sweden). The waste (12 tonnes) was collected in 1995 from households in Uppsala, Sweden. It was sieved, mixed and finally frozen ()20C) in 24-kg portions (Eklind et al., 1997). The chemical composition of the waste has been described by Eklind et al. (1997) and Nilsson (2000). Before it was used as a substrate for the digester, the waste was treated (70C, 1 h) in order to kill pathogens. The digester was started in 1995 and has been running satisfactorily since then. The organic loading rate of the digester is 3-g volatile solids L)1, the hydraulic retention time is 30 d, the gas yield is 0Æ75 L g)1 volatile solids, the methane content is 0.6 L L)1 and the degree of solids reduction is 0.7 g g)1.
In Experiment 1, ADR was spiked with bacteria/ moulds and in Experiment 2 with PPV. Clostridium tyrobutyricum was prepared and applied to the ADR in the form of spores, while the other bacteria were prepared from overnight cultures. The ADR was then mixed with the soil to a total rate of 10 g ADR kg)1 DM soil. The spiked soil was transferred to 0Æ5 L pots. The starting numbers of bacteria (except for S. Typhimurium, L. monocytogenes and Ca. jejuni) and moulds in the spiked soil are presented in Table 1 (day 0). The calculated numbers of S. Typhimurium, L. monocytogenes and Ca. jejuni in the spiked ADR were 108, 2 · 107 and 5 · 107 g)1 respectively. In Experiment 2, the PPV titre in the spiked soil was approximately 106 tissue-culture infectious dose (TCID50) g)1. The pots were incubated in a climate chamber with a 16-h light period (430–450 lmol photons m)2s)1) and a temperature (range) of 18 (0Æ5)C, followed by 8 h of darkness and a temperature of 15 (0Æ5)C . The light did not emit a UV component. Relative humidity was 80 (2). Soil water content was set to )7 kPa in all pots. The pots were weighed at the start of the experiment and during the experiment and adjusted with deionized water three times a week. The pots for Experiments 3 and 4 were filled with non-spiked soil. Italian ryegrass (Lolium multiflorum) was sown in all pots. Approximately 30–40 seeds per pot were used. In Experiments 3 and 4, contamination with spiked ADR was performed when the grass had reached a height of approximately 10 cm. Concentrations of bacteria, moulds and viruses in the ADR were similar to those used in Experiments 1 and 2. Anaerobic digestion residue was applied using gloved hands for mixing, in order to get an even distribution of ADR on the grass surface. As a result of this procedure, the amount of ADR applied to each pot therefore varied
Table 1 Quantitative and qualitative microbiological results over time after addition of spiked anaerobic digestion residue to soil (Experiment 1) or grass (Experiment 3) (Lolium multiflorum). Mean log10 cfu g)1 dry matter (s.d.)
Time (d) Soil Soil Soil Soil Soil Grass Grass Grass Grass
0 7 14 28 49 0 14 35 56
E. coli 5Æ2 5Æ9 4Æ9 4Æ3 4Æ1 2Æ6 3Æ1
