ENVIRONMENTAL MICROBIOLOGY

Screening Escherichia coli, Enterococcus faecalis,and Clostridium perfringens as Indicator Organismsin Evaluating Pathogen-Reducing Capacity in Biogas Plants

Montira Watcharasukarn & Prasad Kaparaju &

Jean-Philippe Steyer & Karen A. Krogfelt &Irini Angelidaki

Received: 21 July 2008 /Accepted: 2 February 2009 /Published online: 5 March 2009# Springer Science + Business Media, LLC 2009

Abstract This study was conducted to identify an indicatororganism(s) in evaluating the pathogen-reducing capacityof biogas plants. Fresh cow manure containing 104 to 105

colony forming unit (CFU) per milliliter of Escherichia coliand Enterococcus faecalis along with an inoculatedClostridium perfringens strain were exposed to 37°C for15 days, 55°C for 48 h, and 70°C for 24 h. C. perfringenswas the most heat-resistant organism followed by E.faecalis, while E. coli was the most heat-sensitive organ-ism. E. coli was reduced below detection limit at alltemperatures with log10 reductions of 4.94 (10 s), 4.37(40 min), and 2.6 (5 days) at 70°C, 55°C, and 37°C,respectively. Maximum log10 reductions for E. faecalis

were 1.77 at 70°C (1 day), 1.7 at 55°C (2 days) and 3.13 at37°C (15 days). For C. perfringens, maximum log10reduction at 37°C was 1.35 log10 units (15 days) comparedto less than 1 unit at 55 and 70°C. Modeling results showedthat E. faecalis and C. perfringens had higher amount ofheat-resistant fraction than E. coli. Thus, E. faecalis and C.perfringens can be used as indicator organisms to evaluatepathogen-reducing capacity in biogas plants at high temper-atures of 55°C and 70°C while at 37°C E. coli could also beincluded as indicator organism.

Introduction

Livestock manures contain a number of pathogens and othermicrobial indicators of fecal pollution. According to AnimalBy-Products (ABP) Regulation (EC No 1774/2002), any useof by-products of animal origin, animals parts accepted forhuman consumption (Category 3), and household wastes assubstrates for biogas plants should be treated in a separatesanitation tank at 70°C for 1 h (particles of max. 12 mm) [3].Pasteurization is usually carried out by heating the substratein a separate step prior to anaerobic digestion (AD) [2, 12].In Denmark, controlled sanitation is usually performed aspasteurization (70°C for 1 h) before a mesophilic AD (30–38°C) or at different time–temperature regulations withthermophilic AD (50–55°C) [6].

AD is generally performed either under mesophilic orthermophilic conditions. At mesophilic temperatures, ADprovides not more than 1–2 log10 units of pathogenreduction within treatment times of 24 h or less (timeinterval between withdrawal and feeding), whereas thermo-philic conditions (about 55°C) can achieve a pathogendecay of 3–4 log10 units within hours [15]. Inactivation

Microb Ecol (2009) 58:221–230DOI 10.1007/s00248-009-9497-9

M. Watcharasukarn : P. Kaparaju : J.-P. Steyer : I. Angelidaki (*)Department of Environmental Engineering,Technical University of Denmark,Building 115,2800 Kgs. Lyngby, Denmarke-mail: ria@env.dtu.dk

K. A. KrogfeltDepartment of Bacteriology, Mycology and Parasitology,Statens Serum Institute,Artilerivej 5,2300 S Copenhagen, Denmark

Present address:M. WatcharasukarnDepartment of Mechanical Engineering, University of Canterbury,Private Bag 4800 Christchurch, New Zealand

Present address:J.-P. SteyerINRA, UR050, Laboratoire de Biotechnologie del’Environnement,Avenue des Etangs,Narbonne 11100, France

rates are faster in thermophilic than in mesophilic AD [27].Although thermophilic AD provides sufficient pathogen-reducing capacity, recontamination of pasteurized anddigested material has been shown to occur in full-scalebiogas plants [7]. Some spore-forming bacteria, certainviruses, and (oo)cysts of endoparasites are quite heat-resistant. For instance, Cryptosporidia in sentinel chamberswere not safely eliminated by anaerobic treatment of semi-liquid cattle manure in a single continuously stirred tankreactor within 24 h at 55°C [8]. Contamination is believed tooccur when the digested materials are transported. Pathogenssuch as Salmonella, Escherichia, Campylobacter, Mycobac-teria, Clostridia, and Brucella found in manures are zoonoticand may cause infections in both animals and humans [8].Destruction of these pathogens is a major concern for anysubsequent use of manure, e.g., land application, recycle, ordisposal. For instance, silage, which is proved to be thesource of the Clostridium spores in the cows and conse-quently their milk, was reported to be contaminated bydigested materials used as fertilizer [39]. Similarly, Yersiniaenterocolitica was isolated from a slaughter swine in Europe[18] and may be a potential risk factor when slaughterhousewaste is used as a substrate in biogas plants.

Temperature is considered as the dominant inactivatingfactor [28]. Pathogens such as Salmonella and Mycobacteriaparatuberculosis were reported to be inactivated within 24 hin thermophilic AD compared to weeks and even months inmesophilic AD [31]. However, spores of Bacillus cereus andClostridium perfringens were not inactivated at mesophilicor thermophilic AD. Nevertheless, high concentrations oforganic acids at low pH value (4.5–5.5) during thermophilicAD were reported to cause C. perfringens decay [32]. Thus,factors other than temperature such as ammonia, organicacids, alcohols, ketones, aldehydes, duration of heat treat-ment, and/or presence of other microorganisms were alsoshown to inactivate bacterial pathogens [32].

Fecal coliform (FC) is the most commonly usedindicator for public health monitoring. Although E. coli isthe most common FC, it is regarded as an unreliableindicator as it has low thermostability [12]. In Denmark,Fecal streptococci (FS) are suggested as suitable indicatororganisms [14]. However, the FS method has limitations asFS are quickly reduced and are impossible to quantify whenthe treatment temperature exceeds 55°C [7]. Alternately,Enterococcus faecalis, Enterococcus faecium, Enterococcusgallinarum, and Enterococcus avium which belong to thegenus Enterococcus are suggested as indicator bacteria [9].FS species were known to display longer survival times instorage tanks and greater thermostability in digesters thanthe majority of bacteria tested excluding spore-formingbacteria, such as C. perfringens and B. cereus [5, 12].Finally, highly heat-resistant C. perfringens, common inraw sludges, is suggested as ideal indicator organism as its

destruction should ensure a higher reduction of spore-bearing bacteria, e.g., Bacillus spp. [22]. In the presentstudy, E. faecalis (Group D), C. perfringens, and E. coliwere used as target organisms to examine their suitability asindicator organism(s) in evaluating the pathogen-reducingcapacity in full-scale biogas plants. The criteria for the useof E. coli, E. faecalis, and C. perfringens as indicatororganisms was to represent a broad spectrum of humanpathogens that have different heat tolerances or resistance[6]. The rates of inactivation were measured at differenttime–temperature relevant to biogas process, i.e., meso-philic (37°C), thermophilic (55°C), and pasteurization step(70°C). In addition, role of pH, ammonia, and organic acidson pathogen decay was also investigated.

Materials and Methods

Experimental Setup

Two sets of experiments were conducted in parallel. In oneset, the effect of time–temperature on rates of inactivationof indicator organisms was conducted while the changes inchemical composition of manure during the same time–temperature conditions was studied in another set ofexperiment.

Cow Manure

Fresh cow manure collected directly from a delivery lorry(Snertinge biogas plant, Denmark) was used as experimen-tal material within 4 h of arrival at the laboratory.

Target Indicator Organisms

E. coli, E. faecalis, and C. perfringens were used as targetorganisms. A cultured strain of C. perfringens, obtainedfrom National Serum Institute (Denmark), was used in thestudy as C. perfringens content in manure was low(101 CFU mL−1). Prior to the experiment, manure sampleswere spiked with 103–104 CFU mL−1 of the cultured C.perfringens strain. On the other hand, no cultured strainswere used for E. coli and E. faecalis as fresh cow manurecontained 104 to 105 CFU mL−1 of indigenous strains of E.coli and E. faecalis.

Inactivation Experiments

Two milliliters of manure (inoculated manure in case of C.perfringens) were transferred to 10-mL glass test tubes andsealed with cotton plugs. Assays were then incubatedstatically at 37°C, 55°C, and 70°C by complete submer-gence in a thermostatically controlled water bath. Well-

222 M. Watcharasukarn et al.

homogenized samples (50 µL) were withdrawn periodicallyat time–temperature conditions mentioned below. Eachtime–temperature treatment combination was replicatedthrice (pseudoreplicate). Samples were collected at intervalsof 0, 10, and 30 s, after 2, 5, 10, and 30 min, and after 1, 3,5, and 24 h at 70°C; 0, 2, 10, 30, 40, and 60 min and after3, 5, 10, 24, and 48 h at 55°C; and 0, 1, 3, 5, 7, 10, and15 days at 37°C. Sampling was carried out once the desiredtemperatures of 37°C, 55°C, and 70°C were attained (t=0).The obtained data were then used for both experimental andmodeling study. On the other hand, samples taken prior toimmersion of test tubes in the water bath gave the initialcounts of the selected microorganisms in the raw manure.Upon completion of thermal exposure, treated manure wasdiluted with 0.9% sodium chloride. Later, the dilutedsample was spread on the selective medium agar platesfor enumeration. The enumeration was performed bycounting the colonies on each plate.

Isolation and Enumeration Methods

C. perfringens

Fifty microliters of the sample were spread on triplicatedtryptose sulfite cycloserine (TSC) plates and incubated at44°C for 24 h [16]. Black colonies of C. perfringens werethen enumerated on TSC agar as described elsewhere [16].D-cycloserine was added to TSC medium to improve theselectivity of the medium for isolation and enumeration ofthe vegetative and spore forms of C. perfringens [17].Further, black colonies of C. perfringens were confirmedby testing double-zone beta hemolysin on blood agar plate(5% defibrinated horse blood; SSI catalog No. 677) and eggyolk agar plate (SSI catalog No. 699) for detection oflecithinase, lipase, and proteolytic activity of Clostridium.

E. coli

M-lauryl sulfate (MLS) agar was used for detection andenumeration of E. coli. After incubation at 44°C for 21 h,the appeared colonies were confirmed in Lactose TryptoneLaurylsulphate Bouillon recommended by ISO Committeeunder the specification ISO 9308-1:1997 [20].

E. faecalis

E. faecalis in 50-µL manure samples was enumerated anddetected using Enterococcus selective agar medium [35]. E.faecalis colonies appeared in pink to dark red after 24 h ofincubation at 37°C. However, the contaminants were foundas pink colonies, gram positive, and rod-shaped. Therefore,only pink or dark red colonies, the form confirmed ascoccus, were counted.

Chemical Composition of Manure During Inactivation

Chemical composition of the manure during inactivationperiod was investigated in batch assays. To each bottle(118-mL), 50 mL of homogenized fresh manure (from thesame batch used for inactivation experiments) was trans-ferred. Prepared assays were incubated statically at 37°C,55°C, and 70°C. Bottles were mixed before and after eachsampling. Total ammonia and volatile fatty acid (VFA)concentrations along with pH during inactivation periodwere determined. Well-homogenized samples were collect-ed as mentioned in the time–temperature conditions(previous section). Each treatment combination was con-ducted in triplicate.

Chemical Analyses

Total solids (TS), volatile solids (VS), and total ammonia(both ionized and unionized ammonia) were determinedaccording to Standard Methods [4]. VFAs were determinedusing a gas chromatograph as described elsewhere [36].

Data Analyses

Data from inactivation experiments were analyzed assum-ing that there was a heat-sensitive population and a heat-resistant population of each studied pathogen [1]. It wasinitially assumed that each population decayed exponen-tially but at different rates, such that the total (heat-sensitiveplus heat-resistant strains) concentration C of a givenpathogen at any time t would correspond to:

C=C0 ¼ fe�k1t þ 1� fð Þe�k2t ð1ÞWhere C0 is the concentration at initial time, f is thefraction of the total concentration that appears to be heat-resistant, (1−f) is the fraction of the total concentrationthat appears to be heat-sensitive, and k1 and k2 are thefirst-order inactivation rate coefficients for the heat-resistant and heat-sensitive fractions, respectively. Equa-tion 1 was solved for the three parameters f, k1, and k2using Windows Excel spread sheet.

Calculations

The unionized fraction of the total ammonia (determined asdescribed in Standard Methods, [4]) was calculated by thefollowing equation:

FNH3 ¼ 1þ 10 pKw� pKb� pHð Þ� ��1

ð2Þ

The values of the dissociation constant of water (Kw) andthe ionization constant of NH4

+ (Kb) obtained from

Evaluating Pathogen-Reducing Capacity in Biogas Plants 223

literature [24] at 25°C were pKb=4.76 and pKw=13.995,respectively. The calculated pKb values for ammonia were4.731 at 37°C, 4.721 at 55°C, and 3.87 at 70°C. Similarly,the calculated pKw values were 13.62, 13.15, and 12.81 at37°C, 55°C, and 70°C, respectively.

The fraction of unionized VFAs was calculated with thefollowing equation:

FVFA ¼ 10 pKa�pHð Þ

1þ 10 pKa�pHð Þ ð3Þ

From the values of the dissociation constants (Ka), pKa

values obtained from the literature [24], valid only at 25°C,were 4.757 for acetate, 4.874 for propionate, 4.812 forbutyrate, 4.840 for iso-butyrate, 4.835 for valerate, and4.767 for iso-valerate. As enthalpy of the dissociationreaction of organic acids is independent of temperature overthe temperature range of interest (37°C to 70°C), pKa

values valid at 25°C were used to calculate the unionizedVFAs values at 37°C, 55°C, and 70°C.

Results

Chemical and Microbial Characteristics of Cow Manure

Fresh cow manure had TS content of 6.6%, a VS/TS ratioof 0.8, and pH of 7.5. VFA and total ammonia content was

6.2 g L−1 and 1.5 g NL−1, respectively. Microbial analysesshowed that fresh cow manure contained 6×104 CFUmL−1

of E. coli, 6.6×105 CFU mL−1 of E. faecalis, and 101

CFUmL−1 of C. perfringens.

Effect of Time–Temperature Regime on Inactivationof Indicator Organisms

Temperature profiles during inactivation period (data notshown) showed that it took 2.91±0.04, 3.17±0.07, and 3.35±0.9min to attain 37°C, 55°C, and 70°C, respectively. The decaypatterns plotted on a log10 scale are shown in Fig. 1. Resultsshowed that C. perfringens was the most heat-resistantorganism followed by E. faecalis while E. coli was the mostsensitive organism at all tested time–temperatures. E. coli wasreduced to below detection limit within 2 min at 70°C, after40 min at 55°C, and after 6 d at 37°C. The correspondinglog10 reduction values for E. coli were 4.94, 4.37, and 2.6 at37°C, 55°C, and 70°C, respectively. The maximum log10reductions obtained for E. faecalis were 1.77 at 70°C (1 day),1.7 at 55°C (2 days), and 3.13 at 37°C (15 days). Decaypattern of C. perfringens was similar to that of E. faecalis, butthe inactivation rates were slower for C. perfringens than E.faecalis at all tested temperatures. For C. perfringens,maximum log10 reduction of 1.35 log10 units was achievedat 37°C (after 15 days) while less than 1 log10 reductions wereobtained at 55°C and 70°C.

-6

-4

-2

055oC

-4

-3

-2

-1

0

-2.0

-1.5

-1.0

-0.5

0.0

0.00 0.05 0.10 0.15 0.20Time (d)

-4

-3

-2

-1

0

-6

-4

-2

070oC

-2.0

-1.5

-1.0

-0.5

0.0

0.00 0.01 0.02 0.03 0.04Time (d)

-6

-4

-2

037oC

log1

0C/C

o

-4

-3

-2

-1

0

-2.0

-1.5

-1.0

-0.5

0.0

0 5 10 15Time (d)

log

10C

/Co

log

10C

/Co

Figure 1 Inactivation patterns of E. coli (filled diamonds), E. faecalis(filled triangles), and C. perfringens (filled squares) in cow manureincubated in batches at 37°C, 55°C, and 70°C: experimental data

(broken line) and simulated data (unbroken line). X-axis has beenshortened to show pathogen decay during the initial period

224 M. Watcharasukarn et al.

Regression Analysis and T90 values

Results from nonlinear regression analyses are summarizedin Table 1 and illustrated in Fig. 1. Results showed that thetwo-population exponential decay model fitted well withthe experimental data. In general, the rates of inactivationfor all target organisms were an order of magnitude higherat 70°C and 55°C than at 37°C. Inactivation rates forindividual organisms were shown to be dependent on theapparent heat-resistant and heat-sensitive fractions of eachspecies and their interaction with the exposed temperature.One can first notice that because of the dynamicsexperimentally observed for E. coli, the value found forits heat-resistant fraction is very small compared to theother organisms. Temperature had a strong effect on theinactivation rate of the heat-sensitive fraction but not onthe heat-resistant fraction of E. coli. Opposite was true forE. faecalis and C. perfringens. For instance, the estimatedinactivation rate coefficients for heat-resistant (k1) and heat-sensitive (k2) fractions of E. coli were 0.001 to 0.04 and 2.5to 1×105, respectively, at 37–70°C (Table 1). Conversely,the values for E. faecalis and C. perfringens ranged from 1to 800 for heat-sensitive fractions (k2) compared to a valueof 0.005 to 2.5 for the heat-resistant fractions (k1).Temperature had only a little influence on the heat-sensitive fractions (1−f) of E. faecalis and C. perfringenswhile the effect on heat-resistant fractions (f) increasedexponentially for E. faecalis and reached maximum for C.perfringens at 55°C. C. perfringens and E. faecalis in

general had an order of higher magnitude of heat-resistantfraction (0.1 to 10–3) than E. coli (10–4 to 10–5).

T90 values are listed in Table 2. These values wereobtained using the mathematical model since, in somecases, not enough experimental data were available toprecisely determine T90. T90 results indicated that decimer-ation times for E. coli were always short and decreased withinactivation temperature (22.2 h at 37°C, 3.3 min at 55°Cand less than 1 min at 70°C). An optimum T90 values for E.faecalis was obtained only at 55°C (T90 was around 1.5 h at55°C) while the values at 37°C and 70°C were 27.7 and6.6 h, respectively. Further, a T90 value of 4 days wasobtained for C. perfringens at 37°C whereas no T90 valueswere attained at 55°C and 70°C.

The effect of temperature (K) on the relative rate ofinactivation (1/T90) exhibited a reasonable fit to theArrhenius relationships for the actual E. coli data at alltemperatures but not for C. perfringens and E. faecalis (datanot shown). The slope of the curve indicated that C.perfringens by far the most thermotolerant organism testedand would need more than 20 days to notice total log10reduction at 70°C.

Chemical Composition of the Manure During Inactivationof Indicator Organisms

The pH and ammonia and VFA levels in manure during theinactivation period were followed (Table 3). Incubation athigher temperatures generally resulted in increased concen-trations of VFA and slightly higher pH’s while ammonialevels remained between 1.6 and 1.7 g L−1. The calculatedunionized VFA and free-ammonia profiles along withpathogen reductions are shown in Fig. 2. The calculatedunionized VFA levels when T90 values were attained were56–73 mg L−1 at 37°C, 19–23 mg L−1 at 55°C, and 13 mgL−1 at 70°C. Conversely, free-ammonia levels at T90 were12–33 mg L−1 at 37°C compared to 64–67 at 55°C and456–491 mg L−1 at 70°C.

Discussion

In the present study, E. coli, E. faecalis, and C. perfringenswere selected as indicator organisms in order to represent awide spectrum of human pathogens that have different heattolerances or resistance [6]. C. perfringens was particularlyincluded as it is an important human pathogen and habitatof the gastrointestinal tract of mammals [39]. Inactivationof these indicator organisms, as noticed in the present study,would guarantee a more hygiene of the treated materials.The present study clearly showed that both C. perfringensand E. faecalis were relatively resistant to high temperature(55°C to 70°C) induced inactivation and could be used as

Table 1 Numerical values of the parameters of the nonlinearregression model

Model parameter Pathogen Temperature

37°C 55°C 70°C

Heat resistantfraction(f, cfu/cfu)

E. coli 1×10–5 5×10–5 2.5×10–3

E. faecalis 0.002 0.02 0.2

C. perfringens 0.1 0.4 0.15

Inactivation ratecoefficientfor heat-resistantfraction(k1, day

−1)

E. coli 0.001 0.04 0.001

E. faecalis 0.1 0.01 2.5

C. perfringens 0.04 0.1 0.005

Inactivation ratecoefficientfor heat-sensitivefraction(k2, day

−1)

E. coli 2.5 1×103 1×105

E. faecalis 2 100 800

C. perfringens 1 100 400

r2 E. coli 0.961 0.767 0.569

E. faecalis 0.949 0.903 0.897

C. perfringens 0.869 0.846 0.594

Evaluating Pathogen-Reducing Capacity in Biogas Plants 225

indicator organisms for future studies in evaluatingpathogen-reducing capacity in full-scale biogas plants.Previously, use of C. perfringens [22, 32] and E. faecalis[9, 15] as indicator organisms in evaluating the pathogen-reducing capacity in biogas plants was also reported.However, use of C. perfringens, usually present in higherconcentrations in the municipal sludges than in farmmanures, as indicator organism would depend on its initial

population, waste type, operating temperature, and expo-sure time. On the other hand, the low thermostability of E.coli at high temperatures of 55°C and 70°C suggests that E.coli cannot be a reliable indicator organism at these processconditions, and pathogens belonging to this group consti-tute a low risk.

The high thermo-resistance of C. perfringens and E.faecalis than E. coli at all tested time–temperatures could be

Pathogen T90 (in days) at 37°C T90 (in days) at 55°C T90 (in days) at 70°C

E. coli 0.92±0.024 2.3±0.151×10–3 4.6±0.151×10–4

E. faecalis 1.15±0.081 0.062±0.026 0.277±0.060

C perfringens 4.05±0.054 n.a. n.a.

Table 2 T90 values derivedfrom the nonlinear regressionmodel for E. coli, E. faecalis,and C. perfringens inactivated inbatches at 37°C, 55°C, and 70°C

n.a. not attained

Table 3 pH, ammonia, volatile fatty acids (VFA) and T90 values obtained during inactivation of E. coli, E. faecalis, and C. perfringens in manureat 37°C, 55°C, and 70°C

Time pH NH4-N (g/L) Free-NH3-N (mg/L) VFA (g/L) Unionized-VFA (mg/L) T90 (% change in CFU)

E. coli E. faecalis C. perfringens

37°C

0 day 7.49 1.67 63.6 5022 10.8 0.0 0.0 0.0

1 day 6.90 1.60 33.6 7167 55.8 30.8 94.4 64.9

3 days 6.80 1.59 12.7 7955 77.8 99.8 98.8 69.2

5 days 6.85 1.66 14.9 8307 72.4 100 99.9 90.8

7 days 6.86 1.66 15.7 8432 64.0 100 99.9 91.5

10 days 6.88 1.67 16.7 8624 59.0 100 99.9 95.5

15 days 6.91 1.66 16.6 8712 10.8 100 99.9 95.7

55°C

0 min 7.36 1.58 8.1 6342 17.2 0.0 0.0 0.0

2 min 7.31 1.68 7.3 6382 19.4 60.3 10.0 28.5

10 min 7.26 1.69 6.6 6390 21.8 96.1 76.2 46.2

30 min 7.24 1.56 6.3 6437 22.9 99.1 80.3 51.5

40 min 7.21 1.65 5.9 6234 23.8 100 88.2 58.5

1 h 7.16 1.67 5.3 6483 27.8 100 94.2 58.5

3 h 7.09 1.60 4.5 6695 33.7 100 96.1 60.8

5 h 7.04 1.65 4.1 7551 42.6 100 96.0 60.8

10 h 7.00 1.73 3.7 9026 55.4 100 97.8 60.8

24 h 6.99 1.75 1.2 9298 57.2 100 97.8 63.1

70°C

0 s 7.52 1.66 22.8 7194 13.5 0 0.0 0.0

10 s 7.56 1.56 24.5 7540 12.9 0 42.9 42.9

30 s 7.56 1.66 24.5 7489 12.8 25 57.1 53.6

120 s 7.55 1.74 24.1 7459 13.0 100 57.1 58.9

300 s 7.58 1.62 25.3 7499 12.2 100 66.7 67.0

0.17 h 7.60 1.55 26.2 7181 11.2 100 64.3 69.6

0.5 h 7.60 1.57 26.2 7764 12.0 100 64.3 72.3

1 h 7.62 1.58 27.1 7764 11.5 100 76.2 80.4

3 h 7.67 1.55 29.5 7700 10.1 100 81.0 82.1

5 h 7.71 1.59 30.9 8085 9.9 100 86.2 86.6

226 M. Watcharasukarn et al.

attributed to the differences in the cell wall structure,physiology, and amount of heat-resistant fraction of eachspecies. For instance, the vegetative cells of C. perfringensare usually destroyed at temperatures of 60°C and abovewhile the spores are considerably heat-resistant and cansurvive at temperatures of 95–100°C for periods of up to1 h [21]. E. faecalis can survive at 60°C for at least 30 min[34] while E. coli can be rapidly inactivated by heating at71°C [30].

The higher rates of inactivation at 55°C and 70°C than at37°C indicate that temperature was the principal factorresponsible for inactivation [28]. These results are inagreement to Aitken et al. [1] who have also reported asharp raise in inactivation rates with increasing temper-atures. The underlying mechanism for bacterial inactivationat high temperatures could be attributed to an increasedfluidity of the cellular membrane [25], thereby facilitatingmore rapid diffusion of inhibitory compounds, e.g.,ammonia or organic acids into the cytoplasm [13]. Therelatively small inactivation rates noticed at higher temper-ature (55°C and 70°C) than at 37°C was probably due tothe fact that inactivation could have occurred before thedesigned temperature was reached. For instance, the timerequired reaching 55°C and 70°C in the present study wasmuch longer than the time required to reach 37°C. Most ofthe heat-sensitive population must have been killed beforethe designed temperatures were attained.

The higher log10 reduction noticed for C. perfringens at37°C than at 55°C or 70°C indicate that factors other than

temperature such as VFA, through formation of unionizedVFA, and exposure time could have also affected theinactivation rates especially at lower temperature. The VFAlevels in the present study were approximately 6 g L−1 at alltested temperatures. However, assays incubated at 37°C had3–6 times higher unionized VFA levels (56–73 mg L−1)than those incubated at 55°C or 70°C (Fig. 2). In a similarstudy, no significant reduction in C. perfringens wasreported at 35°C when the VFA levels in the digester werebetween 0.7–1.5 g L−1; but at 6 g L−1, a decrease of 18% at35°C, 20% at 42°C, 60% at 49°C, and completely inhibitedprocess at 55°C was reported [32]. The decreased concen-trations of VFAs and slightly higher pH’s with increase ininactivation temperature were probably due to release ofammonia at these temperature. A similar observation wasalso reported by Scanlan et al. [33]. However, the pH in thepresent study was never higher than 8, indicating that thefree-ammonia levels (12–33 mg NL−1) at 37°C neverreached concentrations that would induce any death. Thesefree-ammonia values were 3–7 times lower than theminimum value of 85 mg L−1 required to induce anybacterial death [29]. Nevertheless, in full-scale systemsoperated with higher solids concentration in the feed and/orat higher residence times, free-ammonia concentrationscould be two or three times higher than the concentrationsnoticed in the present study.

The difference in heat resistance, evident from the two-population exponential decay model, could be attributed to theheterogeneous distribution of resistance among the cells of

55oC

0

100

200

300

400

500

25

50

75

100

125

0.00 0.03 0.05 0.08 0.10

Time (day)

37oC

0

100

200

300

400

500

Fre

e N

H3-N

(mg/L

)

0

25

50

75

100

125

0 2 4 6 8

Time (day)

T9

0 (%

change in C

FU

)

0

25

50

75

100

unio

nis

ed-V

FA

(mg/L

)

0

25

50

75

100

70oC

0

100

200

300

400

500

0

25

50

75

100

125

0.0 0.1 0.2 0.3

Time (day)

0

25

50

75

100

Figure 2 Free-ammonia(NH3-N) and unionized VFAconcentrations in the manurealong with the T90 valuesobtained during inactivation ofE. coli (filled triangle),E. faecalis, (filled square), andC. perfringens (filled diamonds)incubated in batches for 15 daysat 37°C and for 5 h at 55°C and70°C

Evaluating Pathogen-Reducing Capacity in Biogas Plants 227

indicator organisms or due to changes in environmentalconditions [19]. A similar two-population exponential decaywas reported for fecal coliforms [38] and for two differentstrains of E. coli [1] while a first-order decay was reported forC. perfringens [10]. The increase in heat-resistant fractionwith increase in inactivation temperature for E. faecalis and C.perfringens and not for E. coli indicate that temperature doesnot affect the fractionation, i.e., percentage of heat-resistantversus heat-sensitive fractions for E. coli. These results inpractice would suggest that temperature had to be more than55°C to affect the inactivation rates of C. perfringens and E.faecalis, while temperature cannot improve the inactivationrate of the heat-resistant fraction of E. coli. The oppositewould be true for the heat-sensitive fraction of the three targetorganisms. It should be noted that the decay curves for E. coliat 70°C should not be considered as an absolute values asthese were based on only two data points due to the virtue ofsampling interval. However, the values of the modelparameters for E. coli at 70°C were very low compared tothe other strains, and so, they should be accounted for as orderof magnitude for E. coli. Nevertheless, model data showedthat no matter what the inactivation temperature is, the heat-resistant population of E. coli (i.e., the f value in Table 1) isvery low compared to the other strains and so, E. coli couldbe considered as a pure heat-sensitive strain.

The higher maximum log10 reduction obtained at 37°Cthan at 55°C or 70°C indicate that inactivation of E. coli attemperatures of 55°C and above would result not only insmall T90 values but also in low log10 reductions indepen-dent of exposure time (Table 2). However, to obtainmaximum log10 reductions, a lower temperature, i.e., 37°C should be applied and for a longer period. For C.perfringens, inactivation rates at temperature equal to orlower than 55°C will not affect the final log10 reductions asinactivation rates seem to be independent of exposuretimes. For instance, exposure time for C. perfringensshould be over 20 days to notice any increase of totallog10 reduction at 70°C (data not shown). This greatthermostability of C. perfringens is attributed to theirspore-bearing nature [37]. Similarly, Enterococcus spp.were also shown to be more resistant to different kinds ofwaste treatments [23] and a thermal exposure either at 37°Cor 70°C (and 70°C is better) should be adopted for E.faecalis as at 55°C; the total log reduction was low.

Several researchers have suggested that a 4 log10reduction of pathogenic or indicator microorganisms [11]or a 5 log10 reduction of E. faecalis or Salmonellasenftenberg (775W, H2S negative) must be demonstratedto validate the intended process is hygienically safe [3]. Inthe present study, maximum log10 reductions of 1.35 (for C.perfringens) and 4.94 (for E. coli) were obtained at 37°Cbut only after 15 and 5 days of inactivation, respectively.The relatively low log10 reductions noticed in the present

study was probably due to fact that inactivation could haveoccurred before the designed temperatures were attained.However, previous studies showed a complete inactivationof E. coli over an 8 log10 range at 37°C but at retentiontimes between 2 and 9 days [19]. Both these results are inagreement that at mesophilic temperatures, AD cannotprovide more than 1–2 log10 units of pathogen reductionwithin treatment times of 24 h or less [15]. On the otherhand, 1–4.4 log10 reductions obtained within 40 min ofinactivation at 55°C in the present study are in accord toearlier studies that under thermophilic conditions, apathogen decay of 3–4 log10 unit reduction can be achievedwithin hours [1, 15]. Nevertheless, a minimum guaranteeretention time of 2 h at not less than 55°C, or at least 4 h atnot less than 50°C, or similar conditions with a retentiontime of at least 48–72 h, is generally applied in biogasplants treating manure in combination with wastes belong-ing to categories A and B [5]. These results, thus, reaffirmthat AD of manure in a controlled environment isdependent on treatment time and temperature. For full-scale application, a separate sanitation step must be used,and thermophilic temperatures must be achieved in theentire material. Hence, the temperature has to be monitoredfrequently and at different positions in the material.

Finally, selection of a relatively heat-resistant indicatororganism(s) is important and should depend on the targetorganism of economic interest. Moreover, a good indicatororganism of pathogen destruction should behave in a waysimilar to relevant pathogens. For instance, the mainconcern of fecal contamination through manure is Salmo-nella and E. coli O157, especially in farms with lowsanitary standards. Salmonella spp. and E. coli serotypesare common zoonotic pathogens in manure and can survivefor more than 1 year under favorable conditions [26].However, those bacteria were shown to be inactivatedwithin 24 h in thermophilic AD and may not have a longersurvival in manure [31]. In order to show their destruction,E. coli can be used as indicator organisms as bothorganisms have similar die-off pattern. Similarly, ABPregulation (EC No. 1774/2002) recommends the use of S.senftenberg 775W or E. faecalis as a suitable test organismfor the validation of the hygienization in biogas plants [3].The successful application of E. faecalis in processvalidation with the indigenous flora has been also reported[6]. The rapid inactivation of E. coli, a Gram-negativebacterium, in the present study suggests that pathogensbelonging to this group constitute a low risk. On the otherhand, the Gram-positive E. faecalis had similar inactivationrate to that of C. perfringens but other Gram positiveswould, if pathogenic, result in a lower risk than for C.perfringens, since infectious doses for these bacteria aregenerally higher than those for other gram-negative bacteriasuch as Campylobacter, Enterobacter, Shigella, or Yersinia

228 M. Watcharasukarn et al.

sp. This study shows the value of including more than oneindicator pathogen in order to represent a wide spectrum ofpotentially pathogen bacteria.

Acknowledgements The authors thank Dr. Dimitar Karakashev,Hector Garcia, A. Dilokpimol, V. Siriwongrungson, and Dawei Liufrom the Technical University of Denmark and I. B. Jensen fromStatens Serum Institute, Denmark for their technical support to thisresearch.

References

1. Aitken MD, Sobsey MD, Van Abel NA, Blauth KE, Crunk PL,Melsch ME, Nichols CC (2005) Inactivation of Escherichia coliO157:H7 during thermophilic anaerobic digestion of manure fromdairy cattle. Proc 10th World Congress—Anaerobic Digestion2:377–382

2. Albihn A, Eriksson E, Wallen C, Aspán A (2002) Verotoxino-genic E. coli (VTEC) O157:H7—A nation-wide Swedish surveyof bovine faeces. Acta Vet Scand 44:43–52

3. Animal by-Products Regulation (2002) Impacts on composting andAnaerobic Digestion. The European Parliament and the Council ofthe European Union, Brussels, Regulation (EC) No 1774/2002

4. APHA (1998) In: Lenore SC, Greenberg AE, Eaton AD (eds)Standard Methods for the Examination of Water and Wastewater,20th edn. American Public Health Association, Washington DC

5. Bendixen HJ (1994) Safeguards against pathogens in DanishBiogas Plants. Wat Sci Tech 30:171–180

6. Bendixen HJ (1999) Hygienic safety—results of scientific investiga-tions in Denmark Sanitation requirements in Danish Biogas Plants. In:Bohn R, Wellinger A (eds) Hygienic and Environmental Aspects ofAnaerobic Digestion: Legislation and Experiences in Europe,Stuttgart-Hohenheim. Universität Hohenheim, Stuttgart, pp 27–47

7. Bendixen HJ, Ammendrup S (1992) Safeguards against pathogensin biogas plants. Practical measure to prevent dissemination ofpathogens and requirements for sanitation. Danish Vet. Service,Copenhagen, 32 pp

8. Bicudo JR, Goyal SM, Zhu J (2000) Animal production, manuremanagement and pathogens: A review. In: Animal, Agriculturaland Food Processing Wastes. Moore JA (ed) Proc of the 8thInternational Symposium, 09–11.10.2000, Des Moines, Iowa,USA. American Society of Agricultural Engineers, St. Joseph,Michigan, USA. pp. 507–521

9. Bitton G (2005) Microbial indicators of fecal contamination:Application to microbial source tracking, report submitted toFlorida Stormwater Association

10. Byrne B, Dunne G, Bolton DJ (2006) Thermal inactivation ofBacillus cereus and Clostridium perfringens vegetative cells andspores in port luncheon roll. Food Microbiol 23:803–808

11. Carrington EG (2001) Evaluation of sludge treatments forpathogen reduction-Final report. Study contract no. B4–3040/2001/322179/MAR/A2, For the European Commissiondirectorate-General Environment. ISBN 92–894–1734-X. Avail-able at: http://europa.eu.int/comm/enivronment/waste/sludge/sludge_eval.pdf. (visited on July 2006)

12. Colleran E (2000) Hygienic and sanitation requirements in biogasplants treating animal manure or mixtures of manure and otherorganic waste. In: Ortenblad H (Ed.) Anaerobic Digestion:Making Energy and Solving Modern Waste Problem 77–86

13. Demeyer DJ, Henderickx HK (1967) The effects of unsaturatedfatty acids on methane production in vitro by mixed rumenbacteria. Biochem Biophys Acta 137:484–497

14. Espensen B (1996) Praktiske forsög med smitstofreducerendebahandling af husholdingaffald. Dansk Vet Tidsskr 79(14):615–622, (in Danish)

15. Gibbs RA, Hu CJ, Ho GE, Phillips PA, Unkovich I (1995) Pathogendie-off in stored wastewater sludge. Wat Sci Tech 31:93–95

16. Harmon SM, Kautter DA, Peeler JT (1997a) Comparison of mediafor the enumeration of Clostridium perfringens. Appl Microbiol21(5):922–927

17. Harmon SM, Kautter DA, Peeler JT (1997b) Improved mediumfor enumeration of Clostridium perfringens. Appl Microbiol 22(4):688–692

18. Hartung M, Gerigk K (1991) Yersinia in effluents from the food-processing industry. Rev Sci Tech 10:799–811

19. Himathongkham S, Bahari S, Riemann H, Cliver D (1999)Survival of Escherichia coli O157:H7 and Salmonella typhimu-rium in cow manure and cow manure slurry. FEMS Microbiol Lett178:251–257

20. ISO (1997) Water Quality—Detection and Enumeration ofEscherichia coli and Coliform Bacteria—Part 1: MembraneFiltration Method. International Organization for Standardization,Geneva, ISO/DIS 9308–1

21. Labbe RG (2001) Clostridium perfringens. In: Downs FP, Ito K(eds) Compendium of Methods for the Microbiological Examina-tion of Foods. American Public Health Association, Washington,DC, pp 325–330

22. Langeland G, Paulsrud B (1985) Aerobic thermophilic stabilisa-tion. In: Strauch D, Havelaar A, Hermite P (eds) Inactivation ofmicroorganims in sewage sludge by stabilisation process. Elsevier,Amsterdam, pp 38–47

23. Larsen HE, Munch B, Schlundt J (1994) Use of indicators formonitoring the reduction of pathogens in animal waste treated inbiogas plants. Zentralbl Hyg Umveltmed 195:544–555

24. Lide RD (1997) Handbook of Chemistry and Physics, 77th edn.CRC, Boca Raton

25. Logue CM, Sheridan JJ, McDowell DA, Blair IS, Hegarty T(2000) The effect of temperature and selective agents on thegrowth of Yerwina enterocolitica serotype O:3 in pure culture. JAppl Microbiol 88:1001–1008

26. Mitscherlich E, Marth EH (1984) Microbial Survival in theEnvironment. Springer, Berlin

27. Olsen JE, Larsen HE (1987) Bacterial decimation times inanaerobic digestions of animal slurries. Biol Wastes 21:153–168

28. Oropeza MR, Cabirol N, Ortega S, Ortiz LPC, Noyola A (2001)Removal of fecal indicator organisms and parasites (fecal coliformsand helminth eggs) from municipal biologic sludge by anaerobicmesophilic and thermophilic digestion. Wat Sci Tech 44:97–101

29. Park GW, Diez-gonzalez F (2003) Utilization of carbonate andammonia-based treatments to eliminate Escherichia coli O157:H7and Salmonella Typhimurium DT104 from cattle manure. J ApplMicrobiol 94(4):675–685

30. Park S, Worobo RW, Durst RA (1999) Escherichia coli O157:H7as an emerging food borne pathogen. A literature review. CriticalRev Food Sci Nut 39:481–502

31. Plym-Forshell L (1995) Survival of salmonellas and Ascaris suumeggs in a thermophilic biogas plant. Acta vet Scandina 36:79–85

32. Salsali HR, Parker WJ, Sattar SA (2005) The effect of volatilefatty acids on the inactivation of Clostridium perfringens inadvanced anaerobic digestion. Proc. 10th World Congress—Anaerobic digestion 2:486–490

33. Scanlan P, Santha H, Fronek S (2005) Advanced anaerobicdigestion for Class A pathogen removal. Proc 10th WorldCongress—Anaerobic Digestion 2:491–497

34. Sinton LW, Donnison AM, Hastie CM (1993) Faecal streptococcias faecal pollution indicators: a review. Part I: Taxonomy andenumeration. N Z J Mar Freshw Res 27:101–115

Evaluating Pathogen-Reducing Capacity in Biogas Plants 229

35. Slanetz LW, Bartley CH (1957) Numbers of enterococci in water,sewage, and faeces determined by the membrane filter techniquewith an improved medium. J Bact 74:591–595

36. Sorensen AH, Winther-Nielsen M, Ahring BK (1991) Kinetics oflactate, acetate and propionate in unadapted and lactate-adaptedthermophilic, anaerobic sewage sludge: the influence of sludgeadaptation for start-up of thermophilic UASB-reactors. ApplMicrobiol Biotechnol 34:823–827

37. Wery N, Pourcher A-M, Stan V, Delgenes J-P, Picard-Bonnaud F, Godon J-J (2006) Survival of Listeria mono-

cytogenes and Enterococcus faecium in sludge evaluated byreal-time PCR and culture methods. Lett Appl Microbiol 43(2):131–136

38. Zhai Q, Coyne MS, Barnhisel RI (1995) Mortality rates of fecalbacteria in subsoil amended with poultry manure. BioresourTechnol 54:165–169

39. Van Immerseel F, De Buck J, Pasmans F, Huyghebaert G,Haesebrouck F, Ducatelle R (2004) Clostridium perfringens inpoultry: an emerging threat for animal and public health. AvianPathol 33(6):537–549, Review

230 M. Watcharasukarn et al.