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Waste Management 26 (2006) 1205–1211

Fungal survival during anaerobic digestion of organic household waste

Anna Schnurer *, Johan Schnurer

Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, SE-750 07 Uppsala, Sweden

Accepted 15 September 2005Available online 15 November 2005

Abstract

Anaerobic digestion of organic waste yields energy rich biogas and retains nutrients (N, P, K, S, etc.) in a stabilised residue. For theresidue to be used as a soil fertiliser, it must be free from pollutants and harmful microorganisms. Fungal survival during sanitation andanaerobic treatment of source-separated organic household waste and during aerobic storage of the residue obtained was investigated.Decimal reduction times were determined for inoculated fungi (Aspergillus flavus and Aspergillus fumigatus, Penicillium roqueforti, Rhi-

zomucor pusillus, Thermoascus crustaceus and Thermomyces lanuginosus). Several different fungal species were found after waste sanita-tion treatment (70 �C, 1 h), with Aspergillus species dominating in non-inoculated waste. Anaerobic waste degradation decreased thediversity of fungal species for processes run at both 37 and 55 �C, but not total fungal colony forming units. Fungi surviving the mes-ophilic anaerobic digestion were mainly thermotolerant Talaromyces and Paecilomyces species. T. crustaceus and T. lanuginosus were theonly inoculated fungi to survive the thermophilic anaerobic degradation process. Aerobic storage of both types of anaerobic residues forone month significantly decreased fungal counts.� 2005 Elsevier Ltd. All rights reserved.

1. Introduction

Application of anaerobic digestion residues to agricul-tural land reduces the need for artificial fertilisers, whileat the same time improving the physical and chemicalproperties of the soil (Richert Stintzig, 2000). However,to be used as a fertiliser, such residues should be free fromheavy metals, organic pollutants and harmful microorgan-isms. Chemical and biological contaminants can constitutehealth hazards to people handling the waste or the residue,as well as causing problems related to the production offood and feed when added to soil. Organic wastes can con-tain many different types of biological contaminants,including bacteria, viruses, fungi and parasites (Deporteset al., 1995; Weinrich et al., 1999; Rundberget et al.,2004). In order to kill pathogenic microorganisms, thewaste can be heated before the anaerobic treatment pro-cess. In Sweden the recommended process is pre-heating

0956-053X/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.wasman.2005.09.007

* Corresponding author. Tel.: +46 (0) 18 673209/671000; fax: +46 (0) 18673392.

E-mail address: Anna.Schnurer@mikrob.slu.se (A. Schnurer).

of the waste at 70 �C for 1 h, a procedure originallydescribed by the Danish Ministry of Agriculture (Bendixenand Ammendrup, 1992). Heating at 70 �C for 1 h is suffi-cient to kill faecal streptococci, which are used as indicatororganisms, as well as different viruses, plant pathogens,parasites and human pathogens such as Salmonellatyphimurium and Listeria monocytes (Bendixen andAmmendrup, 1992; Engeli et al., 1993; Larsen et al.,1994; Lund et al., 1996; Burtscher et al., 1998; Dumontetet al., 1999). However, some spore-forming microorgan-isms that are resistant to high temperatures might survivethe heating procedure and eventually end up in the diges-tion residue (Palop et al., 1999; Schnurer et al., 1999).Spore-forming organisms likely to be present in the wasteinclude different fungi, as well as Bacillus and Clostridium

species.Fungi are known to cause problems during aerobic

treatment (composting) of different types of organic wastes.To date, most of the concern about fungi and waste hasfocused on the composting processes and a possiblebuild-up of potentially pathogenic Aspergillus species. Var-ious members of the genus Aspergillus, including A. fumig-

1206 A. Schnurer, J. Schnurer / Waste Management 26 (2006) 1205–1211

atus and A. flavus, have been isolated from different com-posting systems, and high levels of fungal spores have beendetected in the air close to compost systems (Millner et al.,1980; Beffa et al., 1998; Fischer et al., 1998; Haas et al.,1999). Furthermore, several investigations report inci-dences of fungal infections and allergic responses to aero-sols of fungal spores in compost process workers (Clarket al., 1984; Fischer et al., 1998; Bunger et al., 2000; Kitsan-tas et al., 2000).

The aim of the present study was to investigate the sur-vival of fungi during anaerobic digestion of organic house-hold waste. We examined the presence of fungi in sanitised,source-separated, organic household waste, and in residuesproduced by mesophilic (37 �C) and thermophilic (55 �C)anaerobic treatment of the waste. We also studied the sur-vival of six external fungal species. The species added arecommonly found in different types of compost processes,or are extremely thermotolerant, �microaerophilic�, knownto produce mycotoxins and to cause allergic reactions, orare pathogens: Aspergillus flavus (mycotoxins, allergenic),Aspergillus fumigatus (allergenic, pathogen), Penicillium

roqueforti (�microaerophilic�, mycotoxins), Rhizomucor pus-illus (pathogen), Thermoascus crustaceus (thermotolerant)and Thermomyces lanuginosus (thermotolerant). Thesefungi were inoculated into the waste during: (1) heating(70 �C, 1 h) of the waste; (2) anaerobic digestion of thewaste in batch cultures at mesophilic (37 �C) or thermo-philic (55 �C) temperature; and (3) aerobic storage at differ-ent temperatures (+2, +10, +20 �C) of residues producedduring the anaerobic treatment of the waste. In this way,both the natural occurrence of fungi and the survival ofintroduced model fungi could be followed throughout theanaerobic waste-to-residue process.

2. Materials and methods

2.1. Anaerobic reactors

The residues used in this study were taken from two45 L anaerobic reactors, operated semi-continuously (fedonce a day) on source-separated organic household waste(kitchen waste) at either mesophilic (37 �C) or thermophilic(55 �C) temperature. The waste was collected from a muni-cipal waste handling plant in Uppsala, Sweden. Afterremoval of visible impurities, such as glass, metals andplastics, the waste was sieved, mixed and finally frozen(�20 �C) in 24-kg portions (Eklind et al., 1997). The chem-ical composition of the waste was thoroughly investigatedas described by Eklind et al. (1997) and Nilsson (2000).Some chemical parameters of the waste were: pH, 4.9;dry matter, 342 g kg�1 fresh weight (FW); ash, 252 g kg�1

dry weight (DW); carbon, 368 g kg�1 DW; C/N, 16.9; cel-lulose, hemicellulose, lignin, starch, sugar and crude fat at156, 32, 99, 132, 16 and 150 g kg�1 ash free DM, respec-tively; and lactic acid, acetic acid and ethanol, 0.39, 0.14and 0.13 (% of FW). Before being used as a substrate inthe reactors, the waste was diluted with water to a total sol-

ids (TS) concentration of 17% and heated to 70 �C for 1 hin order to kill pathogenic organisms. Both reactors beganoperating in 1995 and since then they have been running onthe same feed. The organic loading rate and the hydraulicretention time are 3 g VS L�1 day�1 (g volatile solids perL reactor volume and day) and 30 days for the mesophilicprocess and 5 g VS L�1 day�1 and 19 days for the thermo-philic process. The gas yield, methane content and degreeof volatile solid reduction are 0.75 L g �1 VS, 60% and70%, respectively, in both reactors.

2.2. Isolation of fungi

Samples (1 g) from the sanitised and diluted organichousehold waste and the residue obtained from the anaer-obic reactors were serially diluted in peptone water. Theresidues were collected just before feeding of the reactors.From each dilution, 1 g portions were placed into petridishes (90 mm) and sterile malt extract agar (Oxoid, Eng-land), supplemented with chloramphenicol (0.1 g L�1;Sigma) and cycloheximide (10 ppm; Sigma), was added.Chloramphenicol is a broad-spectrum bacteriostatic antibi-otic, while cycloheximide at the concentration used in thisstudy inhibits growth of many yeast species without affect-ing mould growth (Bjornberg and Schnurer, 1993). Eachsample was analysed in duplicate and the plates were incu-bated at 25 or 37 �C. As described by Deportes et al.(1997), the fungi were transferred to new agar plates assoon as they appeared. Isolated fungi detected on morethan one plate were identified using morphological criteriaat our laboratory or at CBS (Centraalbureau voor Schim-melcultures, Baarn, The Netherlands). The numbers of col-ony forming units (CFU) were approximated as the highestdilution at which a certain fungus could be detected. Theexact number occurring after plating of each dilution wasnot determined.

2.3. Source of organisms

The fungal strains A. fumigatus (J9) and A. flavus (J7)came from the Department of Microbiology culture collec-tion. The strain P. roqueforti (A 432188) was providedcourtesy of Dr. P. Haggblom from the culture collectionof the National Veterinary Institute, Uppsala, Sweden.The strains R. pusillus (CBS. 294.63), T. crustaceus (CBS348.92) and T. lanuginosus (CBS 224.63) were obtainedfrom the Centraalbureau voor Schimmelcultures (CBS),Delft, The Netherlands.

2.4. Growth and collection of fungal spores

A. flavus, A. fumigatus and P. roqueforti were cultivatedon malt extract agar (MEA) (2%, Oxoid, Hampshire, Eng-land) at 25 �C. R. pusillus was cultivated on MEA (4%;Oxoid) at 30 �C. T. crustaceus and T. lanuginosus were cul-tivated on oatmeal agar (Difco, Michigan England) at37 �C. Spore suspensions of the moulds were prepared by

A. Schnurer, J. Schnurer / Waste Management 26 (2006) 1205–1211 1207

collecting spores from 7-day-old colonies in sterile water,supplemented with peptone (0.02 g L�1 distilled water,BBL, Becton Dickson and Co., Cockeysville, USA) andTween 80 (0.05 g L�1, KEBO, Stockholm; Sweden). Toobtain homogeneous suspensions without aggregates, thespores were shaken with sterile glass beads, filtered throughsterile glass wool, centrifuged (15 min, 6100g), and re-sus-pended in peptone water supplemented with Tween 80(0.05 g L�1, KEBO, Stockholm; Sweden). To further purifythe spore suspension, the centrifugation procedure wasrepeated twice. The spore concentration was then deter-mined using a haemocytometer.

2.5. Survival of inoculated fungi during sanitation of thewaste

Spores of either A. flavus, A. fumigatus, P. roqueforti, R.

pusillus, T. crustaceus or T. lanuginosus were added individ-ually to the diluted organic waste used as a substrate in theanaerobic reactors, to a final concentration of 105 colonyforming units (CFU) per g wet waste. Then 50 ml aliquotsof each treatment of the inoculated waste were transferredto two beakers. The beakers were placed in boiling waterand the waste was quickly heated to 70 �C while stirring.After heating, the beakers were placed in a water bathand incubated for 1 h at 70 �C. Waste inoculated witheither T. crustaceus or T. lanuginosus was also heated to47, 52, 57, 62, 67 or 70 �C and incubated for 1 h at the sametemperature. After heating, the waste material was allowedto cool at room temperature for 10 min, serially diluted inpeptone water and then 0.1 ml from each dilution was sur-face spread on oatmeal agar plates. Fungal CFU weredetermined after incubation of the plates at 37 �C for 5days. All experiments were repeated at least twice withduplicates.

2.6. Survival of inoculated fungi during anaerobic digestion

of the waste

Residues (10 g wet weight) from both the anaerobicreactors, taken before feeding, were transferred to separateserum vials (118 ml) during flushing with N2/CO2 (80/20%). The bottles were closed with butyl-rubber stoppersand sealed with aluminium caps. During the anaerobic deg-radation of the waste in the reactors, the organic materialwas only partly converted to biogas, leaving approximately30% not degraded. After transfer of the residues to thesmall serum vials, the degradation process could continueand these batch cultures were used in order to imitate theenvironment in the large mother reactors. Such batch cul-tures are commonly used in anaerobic biodegradationstudies. Gas chromatography determination of methaneproduction in these cultures showed that the microbialpopulations originating from the laboratory scale reactorswere still active after the transfer of residue to the smallserum vials. However, the exact methane production rateswere not determined. Spore suspensions of the different

fungi were added to the vials with syringes to a final con-centration of 106–108 CFU per g wet residue. The bottleswere incubated at 37 or 55 �C and for each fungus and tem-perature two bottles were withdrawn on each samplingoccasion. The sampling intervals were different for the dif-ferent fungal species, as treatment sensitivity varied sub-stantially among species (determined in preliminaryexperiments). The contents of the bottles were diluted 10-fold with peptone water and homogenised for 2 min at nor-mal speed in a Stomacher 400 (Colworth, UK). After serialdilution of the samples in peptone water, 0.1 ml from eachsample was surface plated. Samples with A. flavus, A.

fumigatus and P. roqueforti were spread on malt extractagar (MEA) (2%, Oxoid, Hampshire, England) and incu-bated at 25 �C. Samples with R. pusillus was cultivatedon MEA (4%, Oxoid) at 30 �C. Samples with T. crustaceus

and T. lanuginosus were cultivated on oatmeal agar (Difco,Michigan England) at 37 �C. The CFU were determinedafter incubation of the plates for 5 days.

2.7. Survival of fungi during aerobic incubation of residue

sludge

Non-inoculated residues (20 ml aliquots) straight fromboth the thermophilic and the mesophilic reactor weretransferred to petri dishes and incubated at +2 or +20 �Cfor 1 or 4 weeks (two petri dishes per temperature and sam-pling occasion). Furthermore, residue from the thermo-philic reactor was inoculated with spores (106 spores g�1

residue) from either A. flavus, A. fumigatus, P. roqueforti,R. pusillus, T. crustaceus or T. lanuginosus, transferred(20 ml aliquots) to petri dishes and incubated at 2, 10 or20 �C for 1, 3, 7 or 30 days (two petri dishes per fungal spe-cies, temperature and sampling occasion). The incubationtemperature interval chosen represents typical autumnand spring temperatures during storage in south-centralSweden. The petri dishes were incubated in plastic bagsin the presence of a piece of wet cotton wool, in order toavoid evaporation of water from the inoculated residues.After incubation, the samples were diluted 10-fold withpeptone water and homogenised for 2 min at normal speedin a Stomacher 400 (Colworth, UK). The samples werethen further diluted in peptone water and finally plated(0.1 ml per plate). The plating, incubation and CFU deter-mination were performed as described in Sections 2.2 and2.6.

3. Results

3.1. Isolation of fungi from waste

Nine different genera of fungi were identified from thesanitised waste used as a substrate for the anaerobic reac-tors (Table 1). The genera present in the highest numbersin the waste were Aspergillus, Talaromyces and Byssochla-

mys. Several different species of Aspergillus and Talaromy-

ces were identified and the species occurring in the highest

0

1

2

3

4

5

6

45 50 55 60 65 70 75 80

Temperature (oC)

)etsaw g(

UFC gol

1-

T.crustaceus

T.lanuginosus

Detection limit

Fig. 1. Survival curves for T. lanuginosus and T. crustaceus during heatingof household waste for 1 h at different temperatures (47, 52, 57, 62, 67 or70 �C).

Table 1Fungal genera identifieda in household waste sanitised by heating at+70 �C for 1 h and in residue from mesophilic (37 �C) and thermophilic(55 �C) anaerobic treatment of the waste

Fungal genera Sanitised waste(CFU g�1)b

Residue, 37 �C(CFU g�1)b

Residue,55 �C(CFU g�1)b

Cladosporiumc 101 – –Chromelosporiumd 101 – –Aspergilluse 103 –Talaromycesf 102 103 102

Byssochlamys 102 – –Mucor 101 – –Paecilomycesg 101 101 102

Botrytis 101 – –Penicillium 101 – –Trichocladiumh – – 101

Arachniotus – 101 –

The CFU data from both sanitised waste and residue are given on a wetweight basis.

a Fungal isolates of Cladosporium, Chromelosporium, Talaromyces,Paecilomyces, Trichocladium and Arachniotus were identified at CBS(Centralbureau voor schimmelcultures), Baarn, The Netherlands. Strainsof Mucor, Aspergillus, Byssochlamys, Botrytis and Penicillium were iden-tified at the Department of Microbiology, Swedish University of Agri-cultural Sciences, Uppsala, Sweden.

b Isolation and counting of fungi were performed by plating of serialdiluted residues followed by transfer of all visible colonies to new agarplates as soon as they appeared. CFU number refers to dilution at whichgrowth occurred.

c The isolate was identified as Cladosporium herbarum.d The isolate was identified as Chromelosporium fulvum.e In total, five different isolates were identified, one which was clearly

identified as Aspergillus fumigatus.f Talaromyces leycettanus (waste, 37 �C residue and 55 �C residue, 102),

T. bacillisporus (55 �C residue), T. byssochlamydoides (waste, 37 �C resi-due) and T. emersonii (waste, 37 �C residue).

g Several different strains were identified in the waste but only twostrains were identified after anaerobic treatment of the waste; a Paecilo-

myces state of Byssochlamys nivea and an unidentified species.h Trichocladium minimum.

1208 A. Schnurer, J. Schnurer / Waste Management 26 (2006) 1205–1211

numbers were A. fumigatus (103 CFU g�1 waste), Talar-

omyces leycettanus (102 CFU g�1 waste) and Talaromyces

byssochlamydoides (102 CFU g�1 waste). Isolation of fungifrom non-sanitised waste showed that the dominant speciesbelonged to the genus Penicillium (103 CFU g�1 waste,data not shown).

3.2. Survival of inoculated fungi during sanitation of thewaste

After heating at +70 �C for 1 h, all fungi except T.

lanuginosus were reduced to numbers below the detectionlimit (102 CFU g�1 waste). T. lanuginosus was present at103 CFU g�1 waste after the sanitation. Heating of thewaste to + 47, +52, +57, +62, +67 or 70 �C for 1 h(Fig. 1) showed that T. crustaceus was reduced to belowthe detection limit at temperatures between +52 and+57 �C. The numbers of T. lanuginosus were reduced attemperatures above +55 �C, but did not decline as fast asT. crustaceus.

3.3. Survival of fungi during anaerobic digestion of the waste

Anaerobic digestion of the waste at 37 �C did not reducethe total number of fungal CFU (103 CFU g�1) originallypresent in the waste, while digestion at 55 �C caused onlya slight decrease (102 CFU g�1). However, the number ofgenera was significantly reduced during anaerobic digestionat both mesophilic and thermophilic temperatures (Table1). In residues from both temperatures, only three differentgenera of fungi were found (Table 1). The dominant generain both residues were Talaromyces and Paecilomyces. Twodifferent species of Paecilomyces were observed; Paecilomy-ces state of Byssochlamys nivea (103, 37 �C residue, 102,55 �C residue) and an unknown (according to CBS) speciesof Paecilomyces (103, 37 �C residue). Four different speciesof Talaromyces were identified in the residues: Talaromyces

emersonii (103, 37 �C residue), T. leycettanus (102, 37 �Cand 55 �C residue), T. byssochlamydoides (102, 37 �C resi-due) and Talaromyces bacillisporus (101, 37 �C residue,102 55 �C residue).

The decimal reduction times (D-value), the timerequired to kill 90% of the fungal population (i.e., onelog-unit decrease in CFU) at a given condition, were calcu-lated for the six external fungi A. flavus, A. fumigatus, P.

roqueforti, R. pusillus, T. crustaceus and T. lanuginosus

added to anaerobic batch systems and incubated at meso-philic and thermophilic temperatures (Table 2). Duringanaerobic digestion at 37 �C, the D-values varied between0.83 and 10 days. During anaerobic digestion at 55 �C,the numbers of A. flavus, A. fumigatus, P. roqueforti, R.

pusillus were reduced more quickly than at the mesophilictemperature, with D-values for these fungi varying between0.02 and 0.05 days. However, T. crustaceus and T. lanugi-

nosus survived for a much longer period of time in the ther-mophilic process and had D-values above 30 days. Thedecimal reduction curves observed for T. crustaceus andT. lanuginosus were highly similar at both temperatures,as were the reduction curves for A. flavus, A. fumigatus,P. roqueforti and R. pusillus. To exemplify, the representa-tive decimal reduction curves for T. lanuginosus and A. fla-

Table 2Decimal reduction time (D-value) for different fungi during anaerobicdigestion of organic household waste at +37 or +55 �C and during aerobicstorage of residue produced by these anaerobic digestion processes

Fungi D-value (days)

Anaerobic digestiona Aerobic storageb

37 �C 55 �C

A. flavus 0.83–1.2 0.05 1.9–4.0A. fumigatus 1.7–2.8 0.02 2.6–4.4P. roqueforti 1.6–1.7 0.02 4.8–5.1T. crustaceus 4.6–8.3 >30 32T. lanuginosus 4.0–10 >30 7.2–11R. pusillus 2.2–3.6 0.05 3.3–5.2

a The D-values given are the span of results obtained after repeatedexperiments (P2) with two replicates on each occasion. When only onevalue is given, identical D-values were obtained in the differentexperiments.

b The D-values given are the span of results obtained after aerobicincubation of two replicate samples at different temperatures, +2, +10 or+20 �C.

A. Schnurer, J. Schnurer / Waste Management 26 (2006) 1205–1211 1209

vus for both mesophilic and thermophilic conditions areshown in Fig. 2.

3.4. Survival of fungi during aerobic incubation of digestion

residue

Isolation of fungi after aerobic storage of non-inocu-lated residues showed a significant decrease in the totalnumber of fungi from 103 to 1–10 CFU g�1. The incuba-tion time did not influence the total number of fungi. Inter-estingly, the fungal species present in the waste afteraerobic incubation did not only belong to the genera Talar-

omyces and Paecilomyces, since some Penicillium specieswere also identified (data not shown). Before the aerobicstorage, these fungi were not detected, possibly insteadbeing outcompeted on the agar plates by higher numbersof Talaromyces spp. and Paecilomyces spp.

Aerobic storage of residue inoculated with A. flavus, A.

fumigatus, P. roqueforti, R. pusillus, T. crustaceus and T.

lanuginosus at different temperatures showed that all fungi,

0

2

4

6

8

0 5 10 15 20 25 30 35Days

)eudiser g( UF

C gol1-

Detection limit

Fig. 2. Survival curves of fungal spores incubated under conditions ofmesophilic (37 �C) or thermophilic (55 �C) anaerobic degradation oforganic household waste. T. lanuginosus incubated at 55 �C (–j–) and at37 �C (–h–). A. flavus incubated at 55 �C (–d–) and at 37 �C (–s–).

except T. lanuginosus and T. crustaceus, had D-valuesbetween 2 and 5 days (Table 2). T. crustaceus had a D-valueof 11 days, while T. lanuginosus survived for an even longerperiod of time and had a D-value of 32 days. Furthermore,the D-values were not to any great extent influenced by thestorage temperature.

4. Discussion

4.1. Survival of fungi during sanitation of the waste

The presence of fungi in the sanitised waste suggests thatseveral different fungi can survive the recommended heat-ing procedure of +70 �C for 1 h. Alternatively the presenceof these fungi is a consequence of an insufficient heatingprocedure, i.e., that it was not possible to keep the temper-ature constant at 70 �C in the waste material during a com-plete sanitation process. For the anaerobic reactors used inthis study and also for large-scale reactors, large quantitiesof waste are sanitised in each batch. It is therefore difficultto obtain full control of the heating process for the entiremass, and the waste material might not always be com-pletely disinfected by this method. However, our resultsshow that one of the external fungi inoculated into thewaste, the thermotolerant species T. lanuginosus, survivedeven a careful and controlled heating procedure. Therefore,it is most likely that other thermotolerant fungal speciescan also survive the sanitation process and enter the anaer-obic reactor.

4.2. Survival of fungi during anaerobic digestion of the waste

Isolation of fungi from the residues obtained afteranaerobic digestion of household waste at both thermo-philic and mesophilic temperatures showed that fungi canalso survive the environment prevailing in the reactors.These fungi survived in an environment with very low oxy-gen concentration and at rather high temperatures. Thedominant fungi were identified as four different species ofthe genera Talaromyces and two different species of Paeci-

lomyces (one was identified as Paecilomyces state of B.

nivea). Both Talaromyces and Paecilomyces consist of spe-cies that mostly are soil dwelling and species belonging tothese genera of fungi are known for their ability to produceheat-resistant ascospores (Scholte et al., 2000). Further-more, species from both genera are also able to grow underreduced oxygen conditions, a possible explanation for theirability to survive in the reactors. These fungi have previ-ously been reported to cause spoilage in pasteurised foodproducts such as fruit juices, canned fruit and vegetables(Tournas, 1994; Kotzeikidou, 1997; Pitt and Hocking,1999; Pieckova and Samson, 2000). The reported produc-tion of different toxic secondary metabolites, includingthe carcinogen patulin, by B. nivea (Tournas, 1994; Pittand Hocking, 1999) is also noteworthy.

The small batch cultures carried out in order to imitatethe environment in the large mother reactors also demon-

1210 A. Schnurer, J. Schnurer / Waste Management 26 (2006) 1205–1211

strated survival of thermotolerant fungi. Both T. crustaceus

and T. lanuginosus had D-values of more than 30 dayswhen added to batch cultures incubated at 55 �C. That is,both fungi survived for a period of time longer than themean retention times (19 days at 55 �C and 30 days at37 �C) of the anaerobic mother processes. If present inthe waste, these thermotolerant fungi would thus probablyend up in an anaerobic digestion residue. Both T. crustac-

eus and T. lanuginosus survived for a shorter period of timeat the mesophilic temperature compared to the thermopilictemperature, suggesting that they are more likely to bepresent in residues produced at thermophilic temperature.These results are not surprising, since thermotolerant fungiin general have their growth optimum at or above 50 �C(Maheshwari et al., 2000). All other fungi added to thebatch cultures, A. flavus, A. fumigatus, P. roqueforti, andR. pusillus, had D-values much shorter than the retentiontimes of 19 and 30 days for the thermophilic and meso-philic mother process, respectively. Furthermore, thereduction of these fungi was faster at the higher processtemperature. Therefore, it does not seem likely that thesefungi will be found in anaerobic digestion residues. Thisresult is also in accordance with data obtained after isola-tion of fungi from the waste and residues produced fromthe laboratory scale reactors. Here it was possible to isolateand identify different species of Aspergillus, including A.

fumigatus, in the sanitised waste but not in the residueobtained after anaerobic degradation of the waste. Mostfungi present in the sanitised waste are thus not likely tosurvive an anaerobic biogas process. However, some ther-motolerant species are likely to survive the process andeventually end up in the residue.

4.3. Survival of fungi during aerobic incubation of digestion

residue

The plant-growing season is short in Sweden and inother parts of northern Europe, so the residues from biogasprocesses cannot be used as a fertiliser during most of theyear. During the period of plant non-growth, the residuescommonly are stored in tanks where anaerobic conditionsare not maintained and oxygen may enter. The presentstudy demonstrated that the amount of fungi originallypresent in the residues was significantly reduced by aerobicincubation of process residues for 1–4 weeks. Furthermore,A. flavus, A. fumigatus, P. roqueforti, R. pusillus, T. crustac-eus and T. lanuginosus, externally added to the residues,had D-values ranging from 1.9 to 32 days during aerobicincubation, with T. crustaceus surviving for the longest per-iod of time. These results suggest that the environment pre-vailing in the process residues was not suitable for growthor survival of fungi even under aerobic conditions and thataerobic storage of residues could further decrease theamount of fungi present. The residue used in this studyhad a pH of 7.3 and low contents of shorter (<C7) organicacids (0.1 g L�1) and salts, so it is not likely that the fungiwere inhibited by general environmental conditions. Possi-

bly, the fungi instead experienced limited access to carbonsources. During the anaerobic degradation process most ofthe easily available carbon in the organic waste is convertedto biogas, while other components are retained. The C/Nratio changed during the process from 17 to approximately5. However, there could also be other explanations for thereduction of fungi during aerobic storage; both the wasteand the residues are known to contain different types offungicides (Nilsson, 2000). These fungicides are present atlow concentrations, but in combination with a low contentof carbon and energy sources they could have negativelyinfluenced the fungi.

4.4. Concluding remarks

Our findings show that sanitation of organic householdwaste at 70 �C for 1 h is not sufficient to kill all fungalspores. Anaerobic degradation of the waste significantlydecreased the diversity of fungi present. However, somethermotolerant species survived the anaerobic processand would eventually end up in the sludge. Since certainthermotolerant fungi can be pathogenic, mycotoxigenicor form spores causing allergic reactions in humans, theymay be a potential hazard for the people handling thesludge and possibly also during food and feed productionfrom soil amended with residues as organic fertilisers.One way to decrease the fungal hazard associated withthe handling and use of the residues can be aerobic storageof the anaerobic residues for at least 1 month, shown hereto significantly decrease the level of fungal CFU.

Acknowledgements

The authors thank Mrs. Inger Ohlsson, Mrs. MarihJohnsson and Ms. Anne-Marie Somberg for technicalassistance. This research was funded by SLUs research pro-gramme �Biological Waste in Circulation Between UrbanAnd Rural Areas� and the Alice and Knut WallenbergFoundation.

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