Fungal survival during anaerobic digestion of organic household waste

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<ul><li><p>ig</p><p>*,</p><p>ricu</p><p>Sep15</p><p>andanandper</p><p>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</p><p>to be used as a fertiliser, such residues should be free fromheavy metals, organic pollutants and harmful microorgan-</p><p>waste can be heated before the anaerobic treatment pro-cess. In Sweden the recommended process is pre-heating</p><p>typhimurium and Listeria monocytes (Bendixen andAmmendrup, 1992; Engeli et al., 1993; Larsen et al.,</p><p>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-</p><p>* Corresponding author. Tel.: +46 (0) 18 673209/671000; fax: +46 (0) 18673392.</p><p>E-mail address: Anna.Schnurer@mikrob.slu.se (A. Schnurer).</p><p>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</p><p>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.</p><p>1. Introduction</p><p>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,</p><p>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</p><p>Anna Schnurer</p><p>Department of Microbiology, Swedish University of Ag</p><p>Accepted 15Available online</p><p>Abstract</p><p>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</p><p>Johan Schnurer</p><p>ltural Sciences, Box 7025, SE-750 07 Uppsala, Sweden</p><p>tember 2005November 2005</p><p>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.</p><p>gillus avus and Aspergillus fumigatus, Penicillium roqueforti, Rhi-</p><p>www.elsevier.com/locate/wasman</p><p>(2006) 12051211</p></li><li><p>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).</p><p>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.</p><p>2. Materials and methods</p><p>2.1. Anaerobic reactors</p><p>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</p><p>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</p><p>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.</p><p>2.2. Isolation of fungi</p><p>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.</p><p>2.3. Source of organisms</p><p>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.</p><p>2.4. Growth and collection of fungal spores</p><p>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-</p><p>nagement 26 (2006) 12051211tivated on oatmeal agar (Difco, Michigan England) at37 C. Spore suspensions of the moulds were prepared by</p></li><li><p>Macollecting spores from 7-day-old colonies in sterile water,supplemented with peptone (0.02 g L1 distilled water,BBL, Becton Dickson and Co., Cockeysville, USA) andTween 80 (0.05 g L1, KEBO, Stockholm; Sweden). Toobtain homogeneous suspensions without aggregates, thespores were shaken with sterile glass beads, ltered throughsterile glass wool, centrifuged (15 min, 6100g), and re-sus-pended in peptone water supplemented with Tween 80(0.05 g L1, KEBO, Stockholm; Sweden). To further purifythe spore suspension, the centrifugation procedure wasrepeated twice. The spore concentration was then deter-mined using a haemocytometer.</p><p>2.5. Survival of inoculated fungi during sanitation of thewaste</p><p>Spores of either A. avus, 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 nal 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.</p><p>2.6. Survival of inoculated fungi during anaerobic digestion</p><p>of the waste</p><p>Residues (10 g wet weight) from both the anaerobicreactors, taken before feeding, were transferred to separateserum vials (118 ml) during ushing 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 small</p><p>A. Schnurer, J. Schnurer / Wasteserum vials. However, the exact methane production rateswere not determined. Spore suspensions of the dierentfungi were added to the vials with syringes to a nal con-centration of 106108 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 dierent 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. avus, 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. crustaceusand T. lanuginosus were cultivated on oatmeal agar (Difco,Michigan England) at 37 C. The CFU were determinedafter incubation of the plates for 5 days.</p><p>2.7. Survival of fungi during aerobic incubation of residue</p><p>sludge</p><p>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 g1</p><p>residue) from either A. avus, 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 nally plated(0.1 ml per plate). The plating, incubation and CFU deter-mination were performed as described in Sections 2.2 and2.6.</p><p>3. Results</p><p>3.1. Isolation of fungi from waste</p><p>Nine dierent genera of fungi were identied from thesanitised waste used as a substrate for the anaerobic reac-tors (Table 1). The genera present in the highest numb...</p></li></ul>