Bioresource Technology 87 (2003) 161166

Review paper

A review of survival of pathogenic bacteria in organic waste used in biogas plants

m Leena Sahlstro
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National Veterinary Institute, SE-75189 Uppsala, Sweden

Abstract Anaerobic digestion is one way of handling biowaste and generating energy in the form of methane (biogas). The digested residue may be used as fertiliser on agricultural land. Biowaste is known to contain pathogenic bacteria such as Salmonella and other microorganisms that may be a health risk for both people and animals. The biosecurity risk associated with using digested residue as fertiliser is hard to assess, but this risk cannot be neglected. It is of greatest importance that the treatment in the biogas plants (BGPs) minimise the survival of pathogens. Temperature is the most important factor when considering the reduction of pathogens in BGP, but there are also other factors involved. Dierent indicator bacteria are used to evaluate the hygienic treatment, but an indicator that is good enough to give an overall picture has not yet been found. 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Anaerobic digestion; Biowaste; Pasteurisation; Indicator bacteria; Pathogenic bacteria; Fertiliser; Agriculture use

1. Introduction Anaerobic digestion in biogas plants (BGPs) is an alternative way to handle biowaste, which includes animal and human waste. In Europe, increasing numbers of BGPs use food waste and manure as energy sources. In Denmark there are 19 BGPs, in Germany 11, and in Sweden there are 10 large scale BGPs operating today rtenblad, 2000) and additional (Albihn et al., 1999; O plants are under construction. Anaerobic digestion produces methane (biogas), reduces odour, and the digested residues may be used as fertiliser in agriculture. The main suppliers of biowaste are slaughterhouses, households, restaurants, food and beverage industries as well as sewage treatment plants (STPs) and animal farms. In the Swedish BGPs, animal wastes from slaughterhouses are used with other biowaste, mainly manure and food industrial waste. In some countries, BGPs use sewage sludge with other biowaste, but in Sweden sewage sludge is treated separately in STPs. Because biowaste is known to contain pathogens, the digested residues must be proven hygienically safe for both people and animals in order to be recycled. Otherwise, new ways of transmission of pathogens between
*

people and animals could be established. There are no EU regulations concerning the hygienic standard of the BGP residue. In Denmark, Austria, Germany, and Sweden dierent regulations exist for the residues and the treatment in BGPs (Colleran, 2000). The growing interest in BGPs in Europe makes it important to consider and regulate biosecurity aspects of recycling residues. This paper reviews the literature about pathogenic bacteria in biowaste and the hygienic eect of anaerobic (mesophilic and thermophilic) digestion and pasteurisation on pathogenic bacteria in BGPs. Because anaerobic digestion is also common in STPs, this review refers to several relevant studies about STPs.

2. Pathogenic bacteria in biowaste The potential health risk with digested residues from BGPs is partly dictated by the substrate that is treated in the plant. It is well known that biowastes contain pathogenic bacteria. They originate from tissues of diseased animals and people and from healthy carriers who excrete bacteria in faeces, urine, and exudates. Therefore, biowaste may contain pathogenic bacteria of dierent species such as Salmonella, Listeria, Escherichia coli, Campylobacter, Mycobacteria, Clostridia, and Yersinia (Dudley et al., 1980; Larsen, 1995; Larsen and

Fax: +46-18-674445. m). E-mail address: leena.sahlstrom@sva.se (L. Sahlstro

0960-8524/03/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 0 2 ) 0 0 1 6 8 - 2

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Munch, 1986, 1981; Strauch, 1991). Many of these bacteria are zoonotic pathogens. They may cause infections in both animals and peoplethis is a public health issue. Furthermore, several of the bacteria are very persistent and may even multiply in the BGP environment. Salmonella is one of the most likely pathogens to be spread in the environment by animal slurry and sewage sludge (Jones, 1980). All serovars of Salmonella are potentially pathogenic to both animals and people. Food borne enteritis (food poisoning) is the most common infection caused by Salmonella. Salmonella may survive in slurry for more than 77 days and grow in temperatures ranging from 6 to 47 C (Mitscherlich and Marth, 1984). A recent survey concludes that Salmonella is detected in more than 50% of the treated sewage sludge from Swedish sewage treatment plants (Sahlm et al., 2001). A similar survey in Norway reveals stro 10% Salmonella positive samples from sewage sludge (Rosef, 1999). In Denmark, sewage sludge is considered Salmonella positive if the STP serves more than 4000 persons (Larsen, 1995). Listeria monocytogenes is found in soil, silage, faeces, and excudates. For people, L. monocytogenes is primarily considered a food borne pathogen. It is a zoonosis and the bacteria may cause dierent clinical manifestations including abortion in both ruminants and people. It may survive and even grow at 145 C (Junttila et al., 1988). De Luca et al. (1998) notes that sewage sludge contains L. monocytogenes and can cause listerosis. This makes L. monocytogenes a pathogen that also should be included as a potential health risk as the result of digested residue from BGPs. Verotoxin producing E. coli O157 is an important food borne pathogen that causes colitis or haemolytic uremic syndrome in people. Cattle are the main reservoir of E. coli O157 (Dorn, 1993; Kudva et al., 1998). The bacteria are found in bovine manure, which is a common substrate in BGPs and hence a possible source of contamination of the environment. Wang et al. (1996) report that E. coli O157:H7 survives for up to 10 weeks and can produce verotoxins for up to 10 weeks. They even report that E. coli O157:H7 is able to multiply in bovine faeces at 22 and 37 C. Mycobacterium paratuberculosis is found worldwide and causes severe chronic enteritis in ruminants. The bacteria are excreted in faeces of infected animals and mainly spread to other animals by contaminated water or feed. M. paratuberculosis is highly resistant to various environmental conditions (Hirsh and Zee, 1999). In a few countries such as Australia, Norway, and Sweden there are national eradication plans for M. paratuberculosis. In these countries, animals infected with M. paratuberculosis are slaughtered and herds are certied if they are M. paratuberculosis free.

In many countries, Campylobacter is one of the major bacterial reasons of gastro-enteritis in people. It is mainly a food borne pathogen and it is often associated eating chicken (Berndtson, 1996). Campylobacter jejuni has been found in raw sewage sludge (Stampi et al., 1998/1999; Steltzer et al., 1991). Campylobacter was quite sensitive to anaerobic digestion and was not found in digested sludge treated more than 90 days in the sewage treatment plants studied. Consequently, Campylobacter is not regarded as a high risk when spread in the environment as digested sludge (Stampi et al., 1998/ 1999; Steltzer et al., 1991). On the contrary, Kearney et al. (1993a) showed in an investigation of mesophilic anaerobic digestion under laboratory conditions that viable organisms of C. jejuni could still be detected when stored for 112 days after digestion. Due to their spore forming capacity, Clostridium spp. as well as other spore forming bacteria are very resistant. Spores can survive for many years in the environment. Several severe diseases are caused by Clostridium spp, such as tetanus (Clostridium tetani), botulism, (Clostridium botulinum) and black leg (Clostridium chauvoie) (Hirsh and Zee, 1999). Clostridium tyrobutyricum causes economic losses in the dairy industry especially in the cheese industry (Dasgupta and Hull, 1989). Silage, which is proved the source of the spores into the cows and consequently their milk, could be contaminated by digested residues used as fertiliser. Yersinia enterocolitica causes acute enteritis mainly in children. Yersinia spp. is commonly associated with water and is of primary concern in sludge (Bitton, 1999). Yersinia may grow in temperatures approaching 0 C (Holt et al., 1994). Y. enterocolitica is isolated from slaughter swine in Europe (Hartung and Gerigk, 1991) and may be a potential risk factor when slaughterhouse waste is used as a substrate in BGPs. There are few reported cases with evidence that infections in animals or people have originated from environmental sources such as contaminated water. Probably one reason for this is that retrospective epidemiological studies are complicated. However, Reilly et al. (1981) reported 26 episodes of Salmonella in Scotland that is believed to originate from contaminated water and sewage sludge spread on farmland. Jack and Hepper (1969) reported that one outbreak of Salmonella was caused by cattle grazing on slurry irrigated pasture. For more references on outbreaks, see the review by Larsen and Munch (1986).

3. Parameters inuencing the reduction of pathogens in BGPs The decay rate of viable bacteria is dependent on many factorstemperature, treatment time, pH, volatile fatty acids (VFA), batch or continuous digestion,

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bacterial species, and available nutrients (Farrah and Bitton, 1983; Kearney et al., 1993a; Larsen, 1995). Inactivation of pathogens is also dependent on the initial amount of pathogens in the biowaste (Strauch, 1991). In BGPs, the temperature and time factors are taken into account using several checkpoints that control such things as the temperature and the length of treatment. The anaerobic digestion is performed either mesophilically or thermophilically in a batchwise or continuous way. In the batchwise system, all the substrate is replaced at the same time but approximately 10% of the fresh substrate contains inoculated digested material (Wellinger, 2000). A continuous system lls and removes material continuously. In Austria, Denmark, Germany, and Sweden BGPs may also have a separate pasteurisation step where the substrate is heated to 70 C for 30 or 60 min (Albihn et al., 1999; Colleran, 2000) before digestion. Many studies on anaerobic digestion are done under laboratory conditions. Laboratory conditions are relatively easy to maintain; however, to maintain pathogen kill and to prevent recontamination in full-scale digesters is dicult. In addition, more resistant indigenous bacteria are found in a full-scale digester than in a laboratory digester (Carrington et al., 1982). However, Olsen and Larsen (1987) produced similar results in a laboratory digester and in a full-scale digester. 3.1. Temperature and time The temperature is the most important factor concerning survival of pathogenic bacteria during anaerobic digestion (Dumontet et al., 1999). Anaerobic digestion can be performed either mesophilic at 3038 C or thermophilic at 5055 C. Bacterial inactivation due to temperature is related to time (Olsen and Larsen, 1987). The time required for a 90% reduction of viable counts of a population of microorganisms or a decrease by one logarithmic unit (log 10) is called the decimation reduction time (T90 ). T90 for many bacteria can be counted in hours in thermophilic digestion and in days in mesophilic digestion, compared to weeks and months in conventional treatment (storage) (Gibbs et al., 1995; Larsen et al., 1989). Digestion at higher temperatures needs less time for bacterial inactivation and this causes bacteria to die much faster in thermophilic than in mesophilic digestion. This was demonstrated by Olsen and Larsen (1987) in a study including Salmonella typhimurium, Salmonella dublin, E. coli, Staphylococcus aureus, Streptococcus faecalis, Erysopelotrix rhusiopathiae, Bacillus cereus, and Clostridium perfringens. Salmonella and M. paratuberculosis are inactivated within 24 h in thermophilic anaerobic digestion compared to weeks and even months in mesophilic anaerobic digestion (Plym-Forshell, 1995; Olsen et al., 1985). However, spores of B. cereus and Cl.

perfringens were not inactivated in either mesophilic or thermophilic digestion. Olsen and Larsen (1987) also showed that a rise of 5 C (from 30 to 35 C) in mesophilic digestion signicantly shortens the time for bacterial inactivation. In a study by Gadre et al. (1986), survival of S. typhimurium was studied during mesophilic anaerobic digestion of cattle dung. After an incubation period of 10 days at 37 C, all Salmonella were inactivated. PlymForshell (1995) investigated the survival of S. typhimurium and S. Senftenberg in cattle manure in a continuous thermophilic digester at a constant 55 C and revealed that no Salmonella could be detected after 24 h in the digester chamber. Olsen et al. (1985) studied the reduction of M. paratuberculosis in batchwise mesophilic and thermophilic anaerobic digestion in bovine slurry. In this investigation, M. paratuberculosis could not be detected after 3 h in thermophilic digestion under laboratory conditions. De Luca et al. (1998) concluded that L. monocytogenes is reduced by mesophilic anaerobic digestion of sewage sludge. However, a slight increase in the number of L. monocytogenes in the following dewatering phase could indicate that L. monocytogenes is only stressed during digestion (De Luca et al., 1998; Steltzer et al., 1991). In a full-scale experiment, Y. enterocolitica was sensitive to the mesophilic anaerobic digestion of cattle slurry and other organic waste (Kearney et al., 1993b). The decimation reduction time was 18 days compared to S. typhimurium in the same experiment, which had twice as long T90 values. 3.2. Batch versus continuous system Anaerobic digestion in BGPs is run either batchwise or continuously. However, due to economic and practical reasons most of the digesters are run continuously. Kearney et al. (1993a) reported a greater decline of viable counts of E. coli, S. typhimurium, L. monocytogenes, and Y. enterocolitica in batch versus continuous anaerobic digestion. Based on another laboratory study, continuous mesophilic digestion with 12 days maximum intervals between additions and removal would not ensure elimination of viable M. paratuberculosis whereas batchwise mesophilic digestion for about a month or 3 h in a thermophilically run digester would (Olsen et al., 1985). 3.3. Pasteurisation Pasteurisation is an eective way of heat-treatment in order to reduce pathogens. For example, Salmonella will not survive more than 5 min in sludge heated to 70 C (Mitscherlich and Marth, 1984). However, bacterial spores are not reduced by pasteurisation. Pasteurisation

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can be run either before or after AD and can be run batchwise or continuously. The batchwise method is preferred because it is more easily controlled in terms of temperature and time. In Swedish BGPs, undigested substrate is heated to 70 C in 60 min in a separate batchwise step prior to anaerobic digestion (Albihn et al., 1999). This is in accordance to the Swedish regulations concerning low risk animal waste (Swedish Board of Agriculture, SJVFS 1998.34, K14). In Denmark, pasteurisation (70 C in 60 min) may be replaced with a similar method, which means a longer time at a lower temperature. This is combined with thermophilic or mesophilic digestion with regulated temperature and time (Bendixen, 1999). In Germany and Austria, a presanitation treatment at 70 C for 60 min before mesophilic or for 30 min before thermophilic anaerobic digestion is recommended (Colleran, 2000). In economic terms, pasteurisation after digestion would be more eective than pre-pasteurisation. However, in the early 1980s there were problems in Switzerland with re-growth of pathogens in sludge that were treated with post-pasteurisation (Keller, 1983; Clements, 1983). Sludge pasteurised after digestion is particularly prone to recontamination. This led to abandoning pasteurisation after digestion and incorporating pasteurisation before anaerobic digestion. In 1999, Ward et al. studied the growth of Salmonella and Enterobacteriaceae in pasteurised digested residues. They looked for conditions that would favour or prevent growth of faecal coliforms and Salmonella in digested residues after pasteurisation. In a laboratory study, they showed that Salmonella and faecal coliforms will not re-grow if the substrate is eciently pasteurised (70 C in 30 min). However, recontamination of pasteurised and digested substrate has been shown to occur in full-scale BGPs (Bagge, 2001). Contamination is believed to occur when the digested residues are transported. 3.4. Volatile fatty acids and pH The amount of VFA and the pH in the substrate correlate to bacterial survival during anaerobic digestion (Abdul and Lloyd, 1985b; Farrah and Bitton, 1983; Fay and Farias, 1975; Henry et al., 1983). Toxicity of VFA is dependent on the pH. Henry et al. (1983) note that the toxicity of VFA to S. typhimurium is much greater at pH 4 than at pH 5 and higher. Kunte et al. (1998) investigated the in vitro eect of VFA on the survival of S. typhi in cattle dung undergoing anaerobic digestion. They also found that high levels of VFA in combination with acidic pH cause greater inactivation of Salmonella than in the control. Abdul and Lloyd (1985a) also noticed a toxic eect of VFA on the survival of E. coli strains in laboratory anaerobic digesters. In contrast, Kearney et al. (1993b)

could not see any correlation between VFA and a decline in the number of dierent pathogens. 3.5. Variation in survival between bacterial species The survival of bacteria is dependent on the characteristics of dierent bacterial species. Kearney et al. (1994) showed a signicantly greater mean T90 value of C. jejuni in a continuous anaerobic digester compared to these for S. typhimurium, L. monocytogenes and Y. enterocolitica. They suggested that the decimation time could dier because of the bacterial species and the supply of nutrients (Kearney et al., 1993b). C. jejuni uses amino acids and vitamins that are not used by other bacteria and hence the competition is lower whereas S. typhimurium, L. monocytogenes, and Y. enterocolitica must compete with other non-methanogenic bacteria for carbohydrates. In a continuous system, the availability of nutrients is dependent on how fast the digester is fed. Kearney et al. (1993b) reported a rapid decline in viable cells for S. typhimurium, L. monocytogenes, and Y. enterocolitica shortly after the inoculation at the beginning of the digestion. They suggested that this was due to an insucient supply of available nutrients for the number of bacteria. The uctuation of nutrients in continuous systems may inuence bacteria to exist in a transient stage between viable culturable states and viable non-culturable states (Kearney et al., 1993b). Ward et al. (1999) also studied the eect of substrate addition and the eect of other active anaerobic digester bacteria on pathogen growth. They showed that some anaerobic bacteria in the digester prevented Salmonella growth. When other active anaerobic bacteria were present (such as in a full-scale digester) Salmonella dieo did occur.

4. Indicator bacteria Many pathogens are present in low concentrations and they may be dicult or time consuming to detect. Therefore, indicator bacteria are used to detect the possible presence of faecal pathogens and to indicate the eect of hygienic treatment of biowaste. Indicator bacteria are preferably non-pathogenic bacteria naturally occurring in large numbers in human and animal intestinal tracts. When choosing indicator bacteria, at least two conditions should be fullled: The bacteria should be excreted in large numbers in the stool and they should be easily detected and counted (Bitton, 1999). Enterococci are suggested as the most suitable indicator bacteria to validate the hygienic treatment of biowaste in BGPs (Larsen et al., 1994). The genus Enterococcus, also called faecal streptococci (FS), consists of Enterococcus faecalis, Enterococcus faecium, Enterococcus avium, and Enterococcus galinarium (Holt et al.,

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1994). In Denmark, the so-called FS-method is used for quality assurance of digested residues for commonly existing pathogens (Salmonellae, Listeria, Campylobacter, and Yersinia) (Espensen, 1996). However, the FS-method has limitations when the temperature in the treatment process exceeds 55 C because FS are then quickly reduced and are impossible to quantify above this temperature according to Bendixen and Ammendrup (1992). De Luca et al. (1998) found FS to be the only indicator bacteria with a statistically signicant correlation to L. monocytogenes in a STP. Other indicator bacteria included in their study were total coliforms and faecal coliforms. Berg and Berman (1980) studied the ratios of indicator bacteria to enteroviruses in mesophilic and thermophilic anaerobic digestion. Indicator bacteria that were tried included faecal coliforms, total coliforms, and FS. Berg and Berman showed that FS closely reected the number of viruses destroyed in mesophilic and thermophilic digestion. They suggested FS as the indicator bacteria of choice because this bacterium may serve as the most useful indicator of faecal viruses. However, this study also shows that using FS as indicator bacteria for viruses has a disadvantage: it is more sensitive than the viruses to both mesophilic and thermophilic anaerobic digestion. To control the hygienic standard, FS have shown to be the best indicator bacteria studied even though limitations exist when the pasteurisation temperature exceeds 55 C. For this reason, there is a need to discover new indicators or a need to actively check for pathogens. Acknowledgements The author thanks Professor Marie-Louise Danielsson-Tham and Assistant Professor Ann Albihn for their critical reading of this manuscript. References
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