ARTICLE IN PRESS

Anaerobe 14 (2008) 181– 183

Contents lists available at ScienceDirect

Anaerobe

1075-99

doi:10.1

� Corr

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journal homepage: www.elsevier.com/locate/anaerobe

Ecology/environmental microbiology

Survival of bacterial pathogens during the thermophilic anaerobic digestionof biowaste: Laboratory experiments and in situ validation

Andreas Otto Wagner a,�, Gudrun Gstraunthaler b, Paul Illmer a

a Institute of Microbiology, University of Innsbruck, Technikerstr. 25, A-6020 Innsbruck, Austriab Abfallbeseitigungsverband Westtirol, Breite Mure, A-6426 Roppen, Austria

a r t i c l e i n f o

Article history:

Received 16 November 2006

Received in revised form

1 February 2008

Accepted 20 March 2008Available online 28 March 2008

Keywords:

Anaerobic digestion

Pathogen carrier

Salmonella

Listeria

Campylobacter

Sanitation

64/$ - see front matter & 2008 Elsevier Ltd. A

016/j.anaerobe.2008.03.004

esponding author. Tel.: +43 512 5076006; fax

ail address: Andreas.Wagner@uibk.ac.at (A.O.

a b s t r a c t

Anaerobic digestion is continually gaining importance for the processing of the organic fraction of

municipal solid wastes. Although methods for studying the survival of pathogen exist, these methods

often need adaptations, are expensive, time consuming or generally not well suited for the harsh

conditions within an anaerobic digestion system. In the present study we investigated the applicability

of commercially available, mechanically stable and inexpensive pathogen carriers to validate in situ

pathogen inhibition within a 750,000 l thermophilic, bio-waste treating anaerobic digester. None of the

pathogens investigated (Listeria monocytogenes, Salmonella enterica, Escherichia coli, and Campylobacter

jejuni) was capable of survival under the conditions of the biogas reactor for more than 24 h indicating

that the temperature and physico-chemical properties of the sludge of the fermenter were effective in

inhibiting the survival of these microorganisms.

& 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Anaerobic digestion is a well-established technique for treatingvarious organic substrates, including the organic fraction ofmunicipal solid wastes [1]. Hygiene problems with the endproduct of these fermentation processes mainly occur at meso-philic temperatures [2] and can increase as digesters operate on asemi-continuous feed-draw basis, since digesters are potentiallyvulnerable to by-pass flow of pathogens through the process [2].

Most anaerobic digestion processes are operated at tempera-tures of more than 50 1C. Together with a suitable exposure time,the temperature is considered to be the most important factor formicrobial growth inhibition in an anaerobic digestion environ-ment [3]. The use of appropriate time and temperature conditionsin addition should allow the safe spreading of sludges in theenvironment, thus posing no threat to humans or animals health[2,4]. However, some pathogenic microorganisms, includingspecies of Salmonella, Campylobacter, and spore forming bacteriaare known to be resistant to high temperature [5]. Thermal deathcurves of a number of enteric bacteria have been determined[3,6–8] in order to get minimum requirements for digestion plantoperation, so as to ensure that resultant sludges are pathogen-free[9]. Nevertheless, an in situ evaluation should additionally be

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Wagner).

performed to confirm pathogen-inhibiting conditions within thebiogas plant [10].

Several methods for studying the survival of pathogens inanaerobic digestion plants have been developed [11–14]. How-ever, such methods often need to be adopted for the use within aspecific fermentation process. In addition, the containers are oftenexpensive, some are constructed for the use in water or for greatertest organisms, or the vessels are mechanically instable. Althoughin some countries (e.g. Germany) the use of diffusion chambershas been proposed in the official guidelines, there are stillproblems with recovering the exposed vessels after a specificexposure time and with mechanical destruction of the containersthrough the harsh process conditions [10].

In the present study we introduced a variable, low cost andmechanically stable microbial carrier system in the form of filterholders which are usually used for sterile-filtration of liquids.Results from the lab were confirmed through an in situ experi-ment, which was carried out in a 750,000 l fermenter.

2. Material and methods

Twenty-four-hour-old cultures of the following organismswere used: Listeria monocytogenes (DSM 15675; DSM-DeutscheSammlung von Mikroorganismen und Zellkulturen), Salmonella

enterica subsp. enterica serovar Senftenberg (DSM 10062), Escher-

ichia coli K12 (DSM 613), and Campylobacter jejuni subsp. jejuni

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A.O. Wagner et al. / Anaerobe 14 (2008) 181–183182

(DSM 4688). Pure cultures of L. monocytogenes were grown inDSMZ medium 92, C. jejuni, E. coli, and S. enterica in DSMZmedium 220 following supplier’s protocols. L. monocytogenes, E.

coli, and S. enterica were cultured aerobically, whereas C. jejuni

was cultured under microaerophilic conditions in a candle jar. Thefollowing selective media and corresponding supplement solu-tions were used: PALCAM-Listeria-Selektivagar (Merck 1.11755.and supplement Merck 1.12122.), Salmonella Shigella Agar (OXOID

CM0099), TBX Chromocult Agar (Merck 1.16122.) for E. coli, and forC. jejuni CCDA Medium (OXOID CM0739 and supplement SR0174).Cell numbers of L. monocytogenes, S. enterica, E. coli, and C. jejuni

were evaluated by a Thoma-chamber. Suspensions of each culturecontaining 2�108 cells ml�1 were prepared using 0.85% NaClsolution. An aliquot of 0.5 ml of this suspension (so consequently1�108 cells of each organism) was placed on a 1�1 cm piece ofrubber foam (Fig. 1), this piece was enclosed in a pathogen carrier,which was comprised of a polypropylene filter-holder (e.g.Millipores Swinnex-25; Millipore Catalogue no.: SX00 025 00)equipped with a silicone gasket and a 0.2 mm polyethersulfon filter

Fig. 1. Setup of the pathogen carrier consisting of in- and outlet part of the filter

holder, silicone gasket, female luer-lock, and a 1�1 cm piece of rubber foam (A).

After assembling (B and C) the filter holder was daubed with silicone (not shown).

In D a simplified pattern of the carrier is shown.

(Pall Supors-200). The inlet of the filter holder was closed by afemale luer-lock and daubed with silicone. On the outlet side ofthe carrier an exchange with the outer matrix was possible,guaranteeing the same conditions (pressure, concentrations ofVFA, temperature, etc.) as the sludge itself. This setup resulted inmicroorganisms being trapped inside the filter holder and notreaching the surrounding sludge.

For verifying the tightness of the system, rubber foams filledwith E. coli cells were put into the carriers and assembled asdescribed above. Several filter holders were placed in sterile E. coli

cultivation medium and the bottle was closed under sterileconditions. It was important not to contaminate the outer parts ofthe filter holder neither with E. coli from the rubber foam nor withany other organism. The whole carrier unit was incubated at 37 1Cat a rotation speed of 150 rpm for 2 weeks and checked regularlyfor perturbation of the medium.

For the lab scale experiment, 15 filter holders containing therubber foam with the pathogen suspension (108 cfu per organism)were placed in a 5 l bottle filled with 1:3 diluted sludge of thedigester and heated at 50 1C. It was necessary to dilute the sludgefor practicability reasons (e.g. liquid handling). Sampling (threereplicate each) was conducted after 0, 3, and 24 h and after 7 and14 days. After collecting samples, the silicone was removed, thefilter holder opened, and the piece of rubber foam was taken outand cut into four pieces under sterile conditions. Pieces wereplaced into 20 ml of enrichment medium for the respectiveorganism prepared using an Austrian standard guideline [15].After 48 h of incubation, the pieces of rubber foam were taken outof the enrichment medium and the liquid (100 ml) was spread onthe respective selective agar plates described above.

For the in situ experiment, 15 filter holders were placed in ahigh-grade steel basket fixed to a 3 m pole and plugged into thereactor using a sampling port. Filter holders were taken out after0, 3, and 24 h and after 7 and 14 days (three replicates each) andthe recultivation procedure was conducted in same way as for thelab scale experiment.

The sludge used, originated from a 750,000 l fermenter(Bioheizkraftwerk Roppen, Austria) and the in situ experimentwas carried out within this biogas plant. The digester follows theKOMPOGAS-principle, is run as an anaerobic solid phase plug-flow fermenter treating bio-waste with a mean operationtemperature of 5370.3 1C, a retention time of 29.474.5 days,and a dry matter content of 26.272.0% (averages of the year 2004(7S.D.)). Additional information about the fermenter is given at /www.thoeni.comS and /www.kompogas.chS.

3. Results and discussion

The tightness test for the pathogen carriers was performedprior to the pathogen experiments, and the filter holders werefound to be tight. Bacterial cells were not found to escape from theinside of the filter holder and the filter membrane stayed in onepiece and did not distort. The intactness of the containers was alsofound in the in situ experiment. Comparable studies have reporteddamaged diffusion chambers following the passage through afermenter, this being connected with the liberation of test strainsand invasion of surrounding microorganisms [10]. With ourmechanically stable pathogen carrier, there should be nearly nolimitations regarding the investigated process or the choice ofmicroorganisms. With variable sized filter holders (filter dia-meters between 13 and 47 mm) different volumes and thereforedifferent numbers of cells can be investigated. It was veryimportant to fix the pathogen carriers within the fermenter toimmerse them into the sludge and to enable a sampling afterspecific exposure times.

ARTICLE IN PRESS

Table 1Recultivation of L. monocytogenes, S. enterica, E. coli, and C. jejuni from pathogen

carriers after heat treatment in digester sludge

Time of heat treatment L. monocytogenes S. enterica E. coli C. jejuni

Lab In situ Lab In situ Lab In situ Lab In situ

0 h +++ +++ +++ +++ +++ +++ +++ +++

3 h +++ +++ +++ +++ +++ +++ +++ +++

24 h +++ ��� +++ ��� +++ ��� +++ ��+

7 days ��� ��� ��� ��� ��� ��� ��� ���

14 days ��� ��� ��� ��� ��� ��� ��� ���

‘‘+’’: Positive recultivation; ‘‘�’’: no viable cells detected; each character indicating

a single replicate. For experimental details of lab and in situ experiment refer to the

text.

A.O. Wagner et al. / Anaerobe 14 (2008) 181–183 183

In the present study, bacteria codified in a national guidelinefor compost quality monitoring [15] were investigated. Thequestion arises, however, as to whether these organisms derivedfrom aerobic process control are also well suited to monitoranaerobic digestion. In the lab scale experiment growth of allinvestigated microorganisms could be verified for up to 24 h ofheat treatment (positive for 0, 3, and 24 h). No viable cells were,however, detected in the 7 and 14 days samples (Table 1).

The in situ experiment confirmed the results obtained in thelab scale experiments, except that viable cells of L. monocytogenes,S. enterica, and E. coli could only be detected in samples taken after0 and 3 h of exposure in the digester but not in samples collectedafter 24 h or subsequent (Table 1), and that only one replicate of C.

jejuni was positive after 24 h in the digester. Also in previousinvestigations, carried out by employees of the biogas plant, C.

jejuni was detected as the sole test strain after the aerobiccomposting of the fermenter sludge. However, based on ourresults, a survival of the digestion process should not be possibleeven for C. jejuni. The most probable explanation for the detectionwithin the compost would be a recontamination within the plant,as was also found to be a serious problem by [5]. For mesophilicanaerobic digestion processes a high resistance of C. jejuni todecay was observed compared to other enteric bacteria [11]. Thelonger survival of the strains in the lab scale experimentscompared with the in situ test might be due to the dilution ofthe sludge in the lab scale experiment.

In conclusion this study has shown that:

(i)

the pathogen carriers consisting of polypropylene filterholders and a polyethersulfon filter are well suited toinvestigate the survival of pathogens in lab scale experimentsand full scale digester experiments,

(ii)

the time–temperature conditions applied at the digestionplant investigated were suitable to inhibit the survival of thepathogens studied.

Acknowledgments

The study (Project no. 1405) was supported by the AustrianFederal Ministry of Agriculture, Forestry, Environment and WaterManagement (BMLFUW) and the County of Tyrol.

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