fungal survival during anaerobic digestion of organic household waste
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zomucor pusillus, Thermoascus crustaceus and Thermomyces lanuginosus). Several dierent 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 the
to be used as a fertiliser, such residues should be free fromheavy metals, organic pollutants and harmful microorgan-
waste can be heated before the anaerobic treatment pro-cess. In Sweden the recommended process is pre-heating
typhimurium and Listeria monocytes (Bendixen andAmmendrup, 1992; Engeli et al., 1993; Larsen et al.,
Fungi are known to cause problems during aerobictreatment (composting) of dierent 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-
* Corresponding author. Tel.: +46 (0) 18 673209/671000; fax: +46 (0) 18673392.
E-mail address: Anna.Schnurer@mikrob.slu.se (A. Schnurer).
Waste Management 26isms. 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 dierent 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, the
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 dierent fungi, as well as Bacillus and Clostridiumspecies.diversity 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 signicantly decreased fungal counts. 2005 Elsevier Ltd. All rights reserved.
Application of anaerobic digestion residues to agricul-tural land reduces the need for articial fertilisers, whileat the same time improving the physical and chemicalproperties of the soil (Richert Stintzig, 2000). However,
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 su-cient to kill faecal streptococci, which are used as indicatororganisms, as well as dierent viruses, plant pathogens,parasites and human pathogens such as SalmonellaFungal survival during anaerobic d
Department of Microbiology, Swedish University of Ag
Accepted 15Available online
Anaerobic digestion of organic waste yields energy rich biogasresidue to be used as a soil fertiliser, it must be free from pollutantsanaerobic treatment of source-separated organic household wasteDecimal reduction times were determined for inoculated fungi (As0956-053X/$ - see front matter 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.wasman.2005.09.007estion of organic household waste
ltural Sciences, Box 7025, SE-750 07 Uppsala, Sweden
tember 2005November 2005
retains nutrients (N, P, K, S, etc.) in a stabilised residue. For thed harmful microorganisms. Fungal survival during sanitation andduring aerobic storage of the residue obtained was investigated.
gillus avus and Aspergillus fumigatus, Penicillium roqueforti, Rhi-
Maatus and A. avus, have been isolated from dierent com-posting systems, and high levels of fungal spores have beendetected in the air close to compost systems (Millner et al.,1980; Bea 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 dierent types of compost processes,or are extremely thermotolerant, microaerophilic, knownto produce mycotoxins and to cause allergic reactions, orare pathogens: Aspergillus avus (mycotoxins, allergenic),Aspergillus fumigatus (allergenic, pathogen), Penicilliumroqueforti (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 dier-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 nally 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 kg1 fresh weight (FW); ash, 252 g kg1
dry weight (DW); carbon, 368 g kg1 DW; C/N, 16.9; cel-lulose, hemicellulose, lignin, starch, sugar and crude fat at156, 32, 99, 132, 16 and 150 g kg1 ash free DM, respec-tively; and lactic acid, acetic acid and ethanol, 0.39, 0.14
1206 A. Schnurer, J. Schnurer / Wasteand 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 L1 day1 (g volatile solids perL reactor volume and day) and 30 days for the mesophilicprocess and 5 g VS L1 day1 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 L1;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 aect-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 identied 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. avus (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. avus, 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-
nagement 26 (2006) 12051211tivated on oatmeal agar (Difco, Michigan England) at37 C. Spore suspensions of the moulds were prepared by
Macollecting spores from 7-day-old colonies in sterile water,supplemented with peptone (0.02 g