Letters in Applied Microbiology ISSN 0266-8254 ORIGINAL ARTICLE Fungi from koala (Phascolarctos cinereus) faeces exhibit a broad range of enzyme activities against recalcitrant substrates R.A. Peterson, J.R. Bradner, T.H. Roberts and K.M.H. Nevalainen Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia Keywords enzyme activity, faeces, fungi, hemicellulase, lignin, rDNA internal transcribed spacer Correspondence K.M.H. Nevalainen, Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, 2109, Australia. E-mail: hnevalai@els.mq.edu.au 2008 ⁄ 0932: received 2 June 2008, revised 10 October 2008 and accepted 11 October 2008 doi:10.1111/j.1472-765X.2008.02513.x Abstract Aims: Identification of fungi isolated from koala faeces and screening for their enzyme activities of biotechnological interest. Methods and Results: Thirty-seven fungal strains were isolated from koala faeces and identified by the amplification and direct sequencing of the internal transcribed spacer (ITS) region of the ribosomal DNA. The fungi were screened for selected enzyme activities using agar plates containing a single substrate for each target class of enzyme. For xylanase, endoglucanase, ligninase (ligninolytic phenoloxidase) and protease over two-thirds of the isolates produced a clearing halo at 25C, indicating the secretion of active enzyme by the fungus, and onethird produced a halo indicating amylase, mannanase and tannase activity. Some isolates were also able to degrade crystalline cellulose and others displayed lipase activity. Many of the fungal isolates also produced active enzymes at 15C and some at 39C. Conclusions: Koala faeces, consisting of highly lignified fibre, undigested cellulose and phenolics, are a novel source of fungi with high and diverse enzyme activities capable of breaking down recalcitrant substrates. Significance and Impact of the Study: To our knowledge, this is the first time fungi from koala faeces have been identified using ITS sequencing and screened for their enzyme activities. Introduction The koala, Phascolarctos cinereus, has a unique diet consisting entirely of Eucalyptus leaves, which are very high in lignin, cellulose, tannin and essential oils (Tyndale-Biscoe 2005). Although Eucalyptus leaves are extremely toxic to most animals, koalas are able to extract enough nutrition from the fermentation and enzymatic degradation of the leaves in their hindgut to maintain metabolism. The material remaining consists of highly lignified fibre, undigested cellulose and phenolics, and is excreted in the faeces. Fungi colonize and degrade koala faeces (Cribb 1997) and therefore must produce enzymes capable of utilizing this unique and recalcitrant substrate. Most research on coprophilous fungi (fungi that grow on faeces) has been concerned with fungal identification, usually based on morphology, and the succession in which different species appear over time (Krug et al. 2004). These fungi have been found to produce metabolites with valuable antibacterial and antifungal properties (Wang et al. 1997; Soman et al. 1999) and enzymes with the potential to break down complex substrates (Magnelli and Forchiassin 1999; Anh et al. 2007). Despite this, there has been surprisingly little research into enzyme production of coprophilous fungi in general and, to our knowledge, none concerning the enzyme activity of fungi isolated from koala faeces or any other Australian animal. The types of enzymes produced by fungi growing on koala faeces are likely to be useful for biotechnological applications. Enzymes that break down complex substances such as hemicellulose, cellulose and lignin are of great interest to the pulp and paper industry, for textile ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 1 Enzymes from fungi on koala faeces R.A. Peterson et al. and detergent manufacture, and for the production of animal feed. In addition, enzymes with lignin-degrading ability have bioremediation potential because their nonspecific enzyme action can also break down a variety of organopollutants such as chlorophenols (Leontievsky et al. 2000), polyaromatic hydrocarbons (Juhasz and Naidu 2000), polychlorobiphenyls (Beaudette et al. 1998) and synthetic dyes (Kirby et al. 2000). The well-studied white rot fungus Phanerochaete chrysosporium has been the focus of much of bioremediation research; however, the performance of this species under laboratory conditions has not transferred well to the natural environment. This failure has led to a renewed interest in screening for new species of ligninolytic fungi with bioremediation potential (Rigas et al. 2003; Dritsa et al. 2007). Apart from cellulose, hemicellulose and lignin, koala faeces also contain a high level of tannin, as well as some protein and lipids (Tyndale-Biscoe 2005); thus fungi colonizing the faeces may additionally produce tannases, proteases and lipases. Tannases are of industrial interest for tea and wine manufacture, in the feed industry and in the production of gallic acid. Fungal proteases are used in detergents, dairy products and in the leather industry, while lipases are particularly valuable in cheese manufacture, for detergents and cosmetics, and for bioremediation (Saxena et al. 2004). Industrial enzymes often need to perform at temperatures higher or lower than ambient, depending on the requirements of the process in which they are involved. For example, thermostable enzymes are necessary in pulp and paper manufacture and for baking and brewing (Haki and Rakshit 2003), whereas cold-tolerant enzymes are utilized for the production of dairy products, cosmetics and for inclusion in detergents (Gerday et al. 2000). Enzymes used in bioremediation, particularly of waterways, can also be required to act at low temperatures. Koala faeces provide a previously untapped source of fungi with the potential for a diverse range of enzyme activities. Here, we identify fungi isolated from koala faeces using internal transcribed spacer (ITS) sequencing, and screen the fungi for their enzyme activities at 15, 25 and 39C. Materials and methods Isolation of fungi from faeces Koala faeces were obtained from Koala Park Sanctuary, West Pennant Hills, Sydney, Australia. Within 10 min of falling to the ground, the faeces were collected by hand, using clean disposable gloves, and placed into a clean plastic bag. The faecal pellets were lightly brushed to remove soil or any other material from the surface, and 2 then dried indoors in a clean cardboard box for seven weeks. The koala faeces were lightly brushed again, and soaked in bleach (NaOCl, 0Æ01% v ⁄ v) for 1 min to surface sterilize, rinsed twice in MilliQ water to remove the bleach, and dried by blotting on sterile filter paper (Bradner et al. 2000). A variety of methods was used to incubate the faeces to maximize the number of fungi to screen for enzymatic activity. Moist-chambers were prepared, each consisting of a faecal pellet on sterile filter paper in a sterile Petri dish. The chamber was kept moist with sterile MilliQ water applied in 1-ml drops to the filter paper as necessary (Krug 2004). Faeces-in-agar plates were prepared by pouring cooled molten potato dextrose agar (PDA) around a faecal pellet in the centre of a sterile Petri dish. Chloramphenicol (100 mg l)1) and ampicillin (100 mg l)1) were added to the cooled PDA to suppress bacterial growth. To eliminate some rapidly growing fungi and favour the Ascomycota species (Warcup 1950; Krug et al. 2004) ethanol-faeces-agar plates were prepared. For each plate, a faecal pellet was macerated manually with 60% ethanol to form a suspension. After soaking for 4 min (Bills and Polishook 1993), 1 ml of the suspension was added to a sterile Petri dish. Semi-cooled PDA containing chloramphenicol and ampicillin (as above) was then poured into the Petri dish, and the dish rotated carefully to distribute the suspension evenly. All the plates and moist chambers were incubated at room temperature (daily temperatures fluctuating between 18 and 28C). Plates were kept until there was a period of 20 consecutive days in which it was not possible to isolate any new fungi. The fungi were isolated as they appeared by touching a colony or individual fruiting body with a sterile inoculating loop and transferring to a separate PDA plate for each isolate. The isolates were tentatively identified by their morphology using macroscopic features such as colony shape, size, colour and texture and microscopic features such as conidiophore structure (von Arx 1981). DNA extraction from the fungi For each isolate, spores were collected from a pure culture on PDA and spread onto a sterile cellophane disc placed over a fresh PDA plate. After 5–7 days of growth at 25C, the mycelia were scraped off, freeze dried for 3 days and then ground to a fine powder with a sterile mortar and pestle. Genomic DNA was isolated using phenol extraction and isopropanol precipitation (Romero et al. 2007). The DNA quantity and quality were checked by 1% (w ⁄ v) agarose gel electrophoresis. ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology R.A. Peterson et al. PCR amplification and direct sequencing of fungal ITS regions The ITS1, 5Æ8S and ITS2 regions of the fungal DNA were amplified by PCR using 10 ng of each ITS5 (5¢-GGAAGTAAAAGTCGTAACAAGG-3¢) and ITS4 (5¢-TCCTCCGCTTATTGATATGC-3¢) (White et al. 1990), 1 U TripleMaster enzyme mix (Eppendorf, Germany), 1 · high fidelity buffer, 50 lmol l)1 dNTPs, approx. 100 ng genomic DNA and H2O to 50 ll. The thermal cycle consisted of 1 · (94C, 12 min), 35 · (94C, 1 min; 58C, 1 min; 72C, 1 min) and 1 · (72C, 5 min). The PCR products were analysed by 1% (w ⁄ v) agarose gel electrophoresis and purified using the QIAquick PCR purification kit (Qiagen, Germany) in accordance with the manufacturer’s protocol. The DNA samples were sequenced directly using BigDye Terminator chemistry and an ABI Prism 377 DNA sequencer (Applied Biosystems, USA). Blast searches of the GenBank nucleotide database (http:// www.ncbi.nlm.nih.gov/blast/Blast.cgi) were conducted to identify the isolate or reveal the closest known analogue. Screening for enzyme activity on solid media All isolates were screened for production of each enzyme on 2% (w ⁄ v) agar (Difco Bacto, USA) plates containing one of the following substrates (supplied by SigmaAldrich, USA, unless otherwise stated): birch xylan (0Æ5% w ⁄ v) for xylanases (Bradner et al. 1999); locust bean gum (0Æ5% w ⁄ v) for mannanases (Rättö and Poutanen 1988); carboxymethylcellulose (0Æ5% w ⁄ v) for endoglucanases (Maijala et al. 1991); Avicel cellulose (0Æ5% w ⁄ v, Fluka, Ireland) for cellobiohydrolases (Teather and Wood 1982); lignin (0Æ25% w ⁄ v) for ligninases (ligninolytic phenoloxidases); tannin (1% w ⁄ v, BDH, UK) for tannases (Pinto et al. 2001); commercial skim milk powder (1% w ⁄ v) for proteases (Saran et al. 2007); Remazol Brilliant Bluestarch (1% w ⁄ v) for amylases (Akpan et al. 1999) and Tween 20 (1% v ⁄ v, Amresco, USA) for lipases and esterases (Hankin and Anagnostakis 1975). Isolates exhibiting enzyme activity on the Tween 20 plates were also tested on plates containing commercial olive oil (2Æ5% w ⁄ v) and Rhodamine B (0Æ001% w ⁄ v) in order to determine true lipase activity (Kouker and Jaeger 1987). All plates contained minimal medium salts consisting of KH2PO4 (1Æ5% w ⁄ v), (NH4)2SO4 (0Æ5% w ⁄ v), MgSO4.7H2O (0Æ06% w ⁄ v) and CaCl2.2H2O (0Æ06% w ⁄ v) (Nevalainen 1981), slightly modified for the tannin plates to enable solidification (Murugan et al. 2007) and with the addition of ammonium tartrate (0Æ5% w ⁄ v, Univar, Australia) in the lignin plates (Mswaka and Magan 1998). Triton X100 (0Æ01% v ⁄ v) was added to all the media to prevent the colonies spreading too extensively and the pH was Enzymes from fungi on koala faeces adjusted to pH 5Æ5 with KOH (1 mol l)1) (Nevalainen and Palva 1978). Each fungal isolate was streaked onto test plates containing the substrate for the detection of the target enzyme class. This process was replicated four times for each isolate at each temperature of incubation: 15, 25 and 39C. The mesophilic, saprophytic fungus, Trichoderma reesei Rut-C30 (ATCC 56765) (Montenecourt and Eveleigh 1979), known to produce a broad range of hydrolytic enzymes, was used as a reference strain. Enzyme activity was detected by the presence of a clearing zone (halo) around the fungal colony, indicating the degradation of the substrate as a result of the production and secretion of enzyme. Visualization of the mannanase, xylanase and endoglucanase halos was facilitated by flooding the plates with Congo Red (1% w ⁄ v) for 5 min, and destaining with NaCl (1 mol l)1) for 15 min (Teather and Wood 1982). Protease plates were flooded with tannic acid (10% w ⁄ v) (Saran et al. 2007) and lignin plates were flooded with equal parts of 1% (w ⁄ v) aqueous solutions of FeCl3 and K3[Fe(CN)6], mixed immediately before use (Mswaka and Magan 1998). Assessments were made on day 5 (25 and 39C) or day 7 (15C) after inoculation. An index of relative enzyme activity was determined by dividing the total area of activity (the area of the clearing zone less the area of the colony) by the area of the colony (Bradner 2003). Results Isolation and identification of the fungi on the koala faeces Thirty-seven fungal strains were isolated from the koala faeces: eight from the moist chambers, 10 from the faeces-in-agar and 19 from the ethanol-faeces-agar. The ITS regions of the ribosomal DNA from each of the 37 isolates were amplified by PCR and the sequences have been deposited in GenBank (see Table 1). Morphological analyses supported identification obtained from the GenBank sequence data. Twenty-seven isolates had matches with a percentage identity of at least 98% (E = 0) with fungal species listed in the NCBI database. Of the remaining 10 isolates, four had less than 90% identity with database species. Enzyme activity of fungi isolated from koala faeces An index of relative activity was used to provide a broad measure of the enzyme production of the isolates (Bradner et al. 1999). Maximum enzyme activity for all isolates occurred at 25C. For xylanase, endoglucanase, ligninase and protease, more than 25 isolates formed clearing halos ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 3 Enzymes from fungi on koala faeces R.A. Peterson et al. Table 1 Identification of fungi isolated from koala faeces in moist chambers (M), faeces-in-agar (A) and ethanol-faeces-agar (E) NCBI Accession number EU551178 EU551179 EU551180 EU551181 EU551182 EU551183 EU551184 EU551185 EU551186 EU551187 EU551188 EU551189 EU551190 EU551191 EU551192 EU551193 EU551194 EU551195 EU551196 EU551197 EU551198 EU551199 EU551200 EU551201 EU551202 EU551203 EU551204 EU551205 EU551206 EU551207 EU551208 EU551209 EU551210 EU551211 EU551212 EU551213 EU551214 Closest species by ITS sequence match in NCBI nucleotide database* Culture method M A E Species Accession number Similarity (%) to known species Aspergillus crystallinus Penicillium concentricum Penicillium concentricum Sporobolomyces lactosus Sordaria alcina Fusarium oxysporum Sporormiella isomera Doratomyces stemonitis Mucoraceae sp. Mucoraceae sp. Sordaria superba Neurospora cerealis Trichosporon sp. Asordaria tenerifae Cylindrocladiella peruviana Pseudallescheria boydii Sordaria alcina Preussia africana Trichosporon asahii Trichoderma atroviride Penicillium concentricum Neosartorya fisheri Eurotium amstelodami Eurotium amstelodami Penicillium concentricum Penicillium commune Penicillium lanosum Gelasinospora cratophora Mariannaea camptospora Preussia australis Preussia africana Phoma sp. Phoma sp. Trichosporon faecale Preussia minima Microdiplodia sp. Coprinellus micaceus AF033486 DQ339561 DQ681333 AB038132 AY681198 AY462580 AY943053 EF029213 EF060714 EF060714 AY681173 AY681187 AF444397 AY681172 AY793467 AJ888404 AY681198 DQ865095 AB369919 EF417482 DQ339561 AB369900 EF151446 EF652084 DQ339561 DQ132843 DQ681336 AY681197 AB112029 AY943052 DQ865095 AJ972797 AJ972797 EF153624 AY510425 EF432267 AY461832 99 99 98 99 98 99 98 100 87 89 99 98 98 99 99 98 98 92 99 100 99 97 100 95 97 94 98 96 98 99 99 87 87 98 99 100 98 Sequence length obtained (bp) 550 571 565 541 568 538 502 388 595 593 552 549 504 549 519 598 547 508 510 571 550 577 520 452 573 522 478 522 536 525 495 521 536 474 519 579 659 *Some isolates were found to have closest matches to the same species in the database but are listed separately due to sustained differences in enzyme production and slight but consistent differences in morphology that suggest that they are separate strains or species. Complete ITS1 sequence was not obtained for this isolate due to difficulties in extracting genomic DNA; however, 100% match (E = 0) for the obtained sequence length and strong morphological features support the identification as Doratomyces stemonitis. on agar plates containing the substrate for the target enzyme. For mannanase, tannase and amylase, more than 13 isolates formed halos. Many of these isolates also produced the enzyme at 15C and ⁄ or at 39C (Table 2). The relative activity of top performing isolates on selected substrates is illustrated in Fig. 1. Isolates that exhibited enzyme activity on Tween 20 plates also exhibited enzyme activity on Rhodamine B plates, confirming lipase production. The enzyme activity of T. reesei Rut-C30 (ATCC 4 56765) was as expected on each substrate (unpublished laboratory data), providing support for the validity of the tests. Discussion We used ITS sequencing to identify fungi isolated from koala faeces, and consequently provided the first such information to NCBI GenBank. Twenty-seven of the 37 ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology R.A. Peterson et al. Enzymes from fungi on koala faeces Table 2 Number of fungal isolates from koala faeces that produced halos on agar plate tests containing substrate for each target enzyme. Incubation was for 5 days (25 and 39C) or 7 days (15C) Number of isolates with enzyme activity Target enzyme 15C 25C 39C Xylanase Mannanase Endoglucanase Cellobiohydrolase Ligninase Tannase Protease Amylase Lipase 13 15 12 5 14 10 21 6 3 26 19 25 6 25 14 26 19 5 6 4 8 5 9 4 11 2 0 All isolates shown were active at 25C; however, the specific isolates exhibiting activity at 15 and 39C sometimes differed. isolates could be identified with sequence matches of at least 98% with known fungal species in GenBank. Morphological features of the isolates were consistent with the identifications. Of these species, Aspergillus, Penicillium, Sporormiella and Sordaria have previously been identified on koala faeces on the basis of their morphology (Cribb 1997). Most of the other genera isolated and identified in the project, Mucor, Preussia, Doratomyces, Trichosporon, Fusarium, Pseudallescheria and Gelasinospora, are known to occur as coprophilous fungi on the faeces of other herbivores (Krug et al. 2004). To our knowledge, Mariannaea and Cylindrocladiella species have not been reported to occur on the faeces of any animal. Four of the isolates, tentatively identified as Mucor and Phoma species, had ITS sequence data with less than a 90% match to known species in the NCBI database, suggesting that they might be new strains or species yet to be described by molecular means. The fungi isolated followed a commonly reported succession over time from mucoraceous species to species from the phylum Ascomycota and then Basidiomycota (Krug et al. 2004). The early successional mucoraceous species were isolated only using the faeces-in-agar method, whilst mid to late successional species were isolated using all incubation methods. The ethanol-faecesagar method was most successful in eliminating early rapidly growing fungi and resulted in the isolation of late successional species, such as Coprinellus micaceus from the phylum Basidiomycota. Enzyme activities of the isolated fungi were shown to be diverse and substantial. The high content of hemicellulose, cellulose and lignin in the faeces was reflected in the large proportion of isolates (more than twothirds) that exhibited xylanase, endoglucanase and ligninolytic phenoloxidase activity, as well as protease activity. The proportion of isolates showing amylase, mannanase and tannase activity was almost as high in each case. The early successional mucoraceous species produced only proteases and amylases. Mid to late successional species were able to break down more complex molecules such as hemicellulose, cellulose and lignin. Many of the fungi produced active enzymes at 15 and ⁄ or 39C, thereby increasing the potential of the enzymes for biotechnological applications. Trichoderma, Aspergillus, Penicillium and Fusarium species are known for their hemicellulase, cellulase, tannase and ligninase production (Gerhartz 1990; Monkemann et al. 1997; Abdel-Sater and El-Said 2001; Panagiotou et al. 2003; Demain et al. 2005; Murugan et al. 2007); however, very little data concerning enzyme production is available for the other species isolated. The enzyme activity of the fungi at 15C was particularly notable. Fourteen of the isolates had ligninase (ligninolytic phenoloxidase) activity at this temperature. Cold active phenoloxidases are rare (Martins et al. 2002) but are in high demand for washing detergents and the in situ bioremediation of soil and waterways. The isolate exhibiting the highest ligninase, xylanase, tannase and protease activity at 15C was identified as Fusarium oxysporum (Fig. 1). Strains of F. oxysporum are known to have huge genetic variation worldwide, and Australian strains are thought to be indigenous and genetically distinct from overseas strains (Wang et al. 2006). The enzyme activity at 15C of the strain isolated from koala faeces appears worthy of further investigation, particularly as the temperature is generally suboptimal for the growth of most Fusarium species (Bakshi et al. 2001). Also of interest is the isolate identified as Mariannaea camptospora, which exhibited high lipase, protease, ligninase and mannanase activities at 15C (Fig. 1). Cold-active lipases are highly valued for use in the pharmaceutical, food and brewing industries, for detergents for cold water washing and for oil degradation and bioremediation (Saxena et al. 1999, 2004). Continued investigation of the enzyme production of this isolate also appears warranted. The index of relative activity (Bradner et al. 1999) used in this study compares the enzyme activity of the isolates (in terms of halo size) in relation to colony size, providing a preliminary gauge of enzyme production. Isolates found to have particularly high relative enzyme activity in this study (Fig. 1) will be the focus of our future work. We plan to extend the study into liquid culture for the production and characterization of enzymes showing potential for industrial or bioremedial applications. The enzyme activity of fungi isolated from the faeces of other Australian herbivores is also an untapped area that we soon plan to investigate. ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 5 Doratomyces stemonitis Sordaria superba Cylindrocladiella peruviana Cylindrocladiella peruviana Sordaria superba Sordaria superba Cylindrocladiella peruviana Trichoderma atroviride Trichoderma atroviride Cylindrocladiella peruviana Trichoderma atroviride Cylindrocladiella peruviana Trichoderma atroviride Trichoderma atroviride Mariannaea camptospora Mariannaea camptospora Microdiplodia sp· Mariannaea camptospora Mariannaea camptospora Mariannaea camptospora Trichoderma reesei Rut-C30 Trichoderma reesei Rut-C30 Trichoderma reesei Rut-C30 Trichoderma reesei Rut-C30 Trichoderma reesei Rut-C30 Penicillium concentricum Relative activity 16·0 14·0 12·0 10·0 8·0 6·0 4·0 2·0 0·0 14·0 12·0 10·0 8·0 6·0 4·0 2·0 0·0 16·0 14·0 12·0 10·0 8·0 6·0 4·0 2·0 0·0 Fusarium oxysporum Fusarium oxysporum Fusarium oxysporum Doratomyces stemonitis Doratomyces stemonitis Doratomyces stemonitis Doratomyces stemonitis Cylindrocladiella peruviana Cylindrocladiella peruviana Sordaria superba Cylindrocladiella peruviana Trichoderma atroviride Penicillium concentricum Penicillium concentricum Trichoderma atroviride Penicillium concentricum Mariannaea camptospora Mariannaea camptospora Mariannaea camptospora Mariannaea camptospora Trichoderma reesei Rut-C30 Trichoderma reesei Rut-C30 Trichoderma reesei Rut-C30 Trichoderma reesei Rut-C30 Tannase Cylindrocladiella peruviana Protease Lipase Trichoderma atroviride Amylase Fusarium oxysporum Trichoderma atroviride (f) Relative activity 14·0 12·0 10·0 8·0 6·0 4·0 2·0 0·0 Relative activity (g) (h) (i) Relative activity Xylanase Doratomyces stemonitis 14·0 Doratomyces stemonitis 8·0 Doratomyces stemonitis 12·0 10·0 Doratomyces stemonitis Mannanase Fusarium oxysporum Endoglucanase Fusarium oxysporum Cellobiohydrolase Fusarium oxysporum 6·0 4·0 2·0 0·0 18·0 16·0 14·0 12·0 10·0 8·0 6·0 4·0 2·0 0·0 12·0 10·0 8·0 6·0 4·0 2·0 0·0 12·0 10·0 8·0 6·0 4·0 2·0 0·0 Fusarium oxysporum Ligninase Fusarium oxysporum Enzymes from fungi on koala faeces Relative activity (a) Relative activity (b) Relative activity (c) Relative activity (d) (e) 12·0 10·0 8·0 6·0 4·0 2·0 0·0 R.A. Peterson et al. ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology Figure 1 Relative enzyme activity displayed by the best enzyme-producing fungi isolated from koala faeces, incubated at the indicated temperatures (h15C; 25C; n39C) on agar plates containing the substrate for the target enzyme. (a) birch xylan (0Æ5% w ⁄ v) for xylanase; (b) locust bean gum (0Æ5% w ⁄ v) for mannanase; (c) carboxymethylcellulose (0Æ5% w ⁄ v) for endoglucanase; (d) Avicel cellulose (0Æ5% w ⁄ v) for cellobiohydrolase; (e) lignin (0Æ25% w ⁄ v) for ligninase; (f) tannin (1% w ⁄ v) for tannase; (g) skim milk powder (1% w ⁄ v) for protease; (h) Remazol Brilliant Blue-starch (1% w ⁄ v) for amylase and (i) Tween 20 (1% v ⁄ v) for lipase. Error bars indicate one standard deviation above and below the mean (n = 4). Fungal strains are in order of Accession number (see Table 1). The Penicillium concentricum strain indicated is EU551198. 6 Relative activity R.A. Peterson et al. References Abdel-Sater, M.A. and El-Said, A.H.M. (2001) Xylan-decomposing fungi and xylanolytic activity in agricultural and industrial wastes. Int Biodeterior Biodegradation 47, 15–21. Akpan, I., Bankole, M.O. and Adesemowo, A.M. (1999) A rapid plate culture method for screening of amylase producing micro-organisms. Biotechnol Tech 13, 411–413. Anh, D.H., Ullrich, R., Benndorf, D., Svatos, A., Muck, A. and Hofrichter, M. (2007) The coprophilous mushroom Coprinus radians secretes a haloperoxidase that catalyzes aromatic peroxygenation. Appl Environ Microbiol 73, 5477–5485. von Arx, J.A. (1981) The Genera of Fungi Sporulating in Pure Culture, 3rd edn. Vaduz: J. Cramer. Bakshi, S., Sztejnberg, A. and Yarden, O. (2001) Isolation and characterisation of a cold-tolerant strain of Fusarium proliferatum, a biocontrol agent of grape downy mildew. Phytopathology 91, 1062–1068. Beaudette, L.A., Davies, S., Fedorak, P.M., Ward, O.P. and Pickard, M.A. (1998) Comparison of gas chromatography and mineralization experiments for measuring loss of selected polychlorinated biphenyl congeners in cultures of white rot fungi. Appl Environ Microbiol 64, 2020–2025. Bills, G.F. and Polishook, J.D. (1993) Selective isolation of fungi from dung of Odocoileus hemionus (mule deer). Nova Hedwigia 57, 195–206. Bradner, J.R. (2003) Antarctic microfungi as a potential bioresource. PhD Thesis, Macquarie University, Sydney. Bradner, J.R., Gillings, M. and Nevalainen, K.M.H. (1999) Qualitative assessment of hydrolytic activities in antarctic microfungi grown at different temperatures on solid media. World J Microbiol Biotechnol 15, 131–132. Bradner, J.R., Sidhu, R.K., Yee, B., Skotnicki, M.L., Selkirk, P.M. and Nevalainen, K.M.H. (2000) A new microfungal isolate, Embellisia sp., associated with the Antarctic moss Bryum argenteum. Polar Biol 23, 730–732. Cribb, A.B. (1997) Two coprophilous fungi on koala dung. Queensl Nat 35, 4–6. Demain, A.L., Velasco, J. and Adrio, J.L. (2005) Industrial mycology: past, present and future. In Handbook of Industrial Mycology ed. An, Z. pp. 1–26 New York, NY: CRC Press. Dritsa, V., Rigas, F., Natsis, K. and Marchant, R. (2007) Characterization of a fungal strain isolated from a polyphenol polluted site. Bioresour Technol 98, 1741–1747. Gerday, C.C., Aittaleb, M.M., Bentahir, M.M., Chessa, J.P., Claverie, P.P., Collins, T.T., D’Amico, S.S., Dumont, J.J. et al. (2000) Cold adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol 18, 103–107. Gerhartz, W. (1990) Enzymes in Industry: Production and Applications. Weinheim: VCH. Haki, G.D. and Rakshit, S.K. (2003) Developments in industrially important thermostable enzymes: a review. Bioresour Technol 89, 17–34. Enzymes from fungi on koala faeces Hankin, L. and Anagnostakis, S.L. (1975) The use of solid media for detection of enzyme production by fungi. Mycologia 67, 597–607. Juhasz, A.L. and Naidu, R. (2000) Bioremediation of high molecular weight polycyclic aromatic hydrocarbons: a review of the microbial degradation of benzo[a]pyrene. Int Biodeterior Biodegradation 45, 57–88. Kirby, N., Marchant, R. and McMullan, G. (2000) Decolourisation of synthetic textile dyes by Phlebia tremellosa. FEMS Microbiol Lett 188, 93–96. Kouker, G. and Jaeger, K. (1987) Specific and sensitive plate assay for bacterial lipases. Appl Environ Microbiol 53, 211–213. Krug, J.C. (2004) Moist chambers for the development of fungi. In Biodiversity of Fungi: Inventory and Monitoring Methods ed. Mueller, G.M., Bills, G.F. and Foster, M.S. pp. 589–593 San Diego, CA: Elsevier. Krug, J.C., Benny, G.L. and Keller, H.W. (2004) Coprophilous fungi. In Biodiversity of Fungi: Inventory and Monitoring Methods ed. Mueller, G.M., Bills, G.F. and Foster, M.S. pp. 467–500 San Diego, CA: Elsevier. Leontievsky, A.A., Myasoedova, N.M., Baskunov, B.P., Evans, C.S. and Golovleva, L.A. (2000) Transformation of 2,4,6trichlorophenol by the white rot fungi Panus tigrinus and Coriolus versicolor. Biodegradation 11, 331–340. Magnelli, P. and Forchiassin, F. (1999) Regulation of the cellulase complex production by Saccobolus saccoboloides: induction and repression by carbohydrates. Mycologia 91, 359–364. Maijala, P., Fagerstedt, K.V. and Raudaskoski, M. (1991) Detection of extracellular cellulolytic and proteolytic activity in ectomycorrhizal fungi and Heterobasidion annosum (Fr.) Bref. New Phytol 117, 643–648. Martins, L.O., Soares, C.M., Pereira, M.M., Teixeira, M., Costa, T., Jones, G.H. and Henriques, A.O. (2002) Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat. J Biol Chem 277, 18849–18859. Monkemann, H., Holker, U. and Hofer, M. (1997) Components of the ligninolytic system of Fusarium oxysporum and Trichoderma atroviride. Fuel Process Technol 52, 73–77. Montenecourt, B.S. and Eveleigh, D.E. (1979) Selective screening methods for the isolation of high yielding cellulase mutants of Trichoderma reesei. Adv Chem Ser 181, 289–301. Mswaka, A.Y. and Magan, N. (1998) Wood degradation, and cellulose and ligninase production by Trametes and other wood-inhabiting basidiomycetes from indigenous forests of Zimbabwe. Mycol Res 102, 1399–1404. Murugan, K., Saravanababu, S. and Arunchalam, M. (2007) Screening of tannin acyl hydrolase (E.C.3.1.1.20) producing tannery effluent fungal isolates using simple agar plate and SmF process. Bioresour Technol 98, 946–949. Nevalainen, K.M.H. (1981) Induction, isolation, and characterization of Aspergillus niger mutant strains producing elevated levels of beta-galactosidase. Appl Environ Microbiol 41, 593–596. ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 7 Enzymes from fungi on koala faeces R.A. Peterson et al. Nevalainen, K.M.H. and Palva, E.T. (1978) Production of extracellular enzymes in mutants isolated from Trichoderma viride unable to hydrolyze cellulose. Appl Environ Microbiol 35, 11–16. Panagiotou, G., Kekos, D., Macris, B.J. and Christakopoulos, P. (2003) Production of cellulolytic and xylanolytic enzymes by Fusarium oxysporum grown on corn stover in solid state fermentation. Ind Crop Prod 18, 37–45. Pinto, G., Leite, S., Terzi, S. and Couri, S. (2001) Selection of tannase-producing Aspergillus niger strains. Braz J Microbiol 32, 24–26. Rättö, M. and Poutanen, K. (1988) Production of extracellular enzymes in mutants isolated from Trichoderma reesei unable to hydrolyze cellulose. Appl Environ Microbiol 35, 11–16. Rigas, F., Marchant, R., Dritsa, V., Kapsanaki-Gotsi, E. and Avramides, L. (2003) Screening of wood rotting fungi potentially useful for the degradation of organic pollutants. Water Air Soil Pollut Focus 3, 201–210. Romero, E., Speranza, M., Garcia-Guinea, J., Martinez, A.T. and Martinez, M.J. (2007) An anamorph of the white-rot fungus Bjerkandera adusta capable of colonizing and degrading compact disc components. FEMS Microbiol Lett 275, 122–129. Saran, S., Isar, J. and Saxena, R.K. (2007) A modified method for the detection of microbial proteases on agar plates using tannic acid. J Biochem Biophys Methods 70, 697–699. Saxena, R.K., Ghosh, P.K., Gupta, R., Davidson, W.S., Bradoo, S. and Gulati, R. (1999) Microbial lipases: potential biocatalysts for the future industry. Curr Sci 77, 101–115. 8 Saxena, R.K., Malhotra, B. and Batra, A. (2004) Commercial importance of some fungal enzymes. In Handbook of Fungal Biotechnology, Mycology, Vol. 20 ed. Arora, D.K. pp. 287–298. New York, NY: Marcel Dekker. Soman, A.G., Gloer, J.B., Koster, B. and Malloch, D. (1999) Sporovexins a-c and a new preussomerin analog: antibacterial and antifungal metabolites from the coprophilous fungus Sporormiella vexans. J Nat Prod 62, 659–661. Teather, R.M. and Wood, P.J. (1982) Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol 43, 777–780. Tyndale-Biscoe, H. (2005) Life of Marsupials. Collingwood, VIC: CSIRO Publishing. Wang, H., Gloer, K.B., Gloer, J.B., Scott, J.A. and Malloch, D. (1997) Anserinones a and b: new antifungal and antibacterial benzoquinones from the coprophilous fungus Podospora anserina. J Nat Prod 60, 629–631. Wang, B., Brubaker, L., Tate, W., Woods, J., Matheson, B.A. and Burdon, J.J. (2006) Genetic variation and population structure of Fusarium oxysporum f.sp. vasinfectum in Australia. Plant Pathol 55, 746–755. Warcup, J.H. (1950) The soil-plate method for isolation of fungi from soil. Nature 166, 117–118. White, T., Burns, T., Lee, S. and Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications ed. Innis, M.A., Gelfand, D.H., Sninsky, J.J. and White, T.J. pp. 315–322 San Diego, CA: Academic Press. ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology