Vigor, Sex and Woody Substrates: Lessons from the Cultivation of Pleurotus Ostreatus

UNIVERSITY OF WISCONSIN SYSTEM SOLID WASTE RESEARCH PROGRAM Undergraduate Project Report

May 2010

Student Investigator: Kymberly R. Draeger

Faculty Advisor: Dr. Amy Wolf

University of Wisconsin-Green Bay

Vigor, Sex, and Woody Substrates: Lessons from the cultivation of Pleurotus ostreatus

SUMMARY

The purpose of this project was to comparatively assess high lignocellulosic waste substrates for

Pleurotus ostreatus cultivation and to understand the lifecycle governing the fruiting production of oyster mushrooms. Using short-fiber paper sludge, office paper, newspaper, and Phragmites australis

(invasive common reed) as substrates, I wanted to transform waste, and was adamant about not creating waste. Throughout the study, I increased my appreciation for the complexity regarding fungal systems, genetics, sexual recombination and their adaptive responses to environmental factors. With further manipulation, it was shown that strains could be cultivated to grow within sub-optimal environmental conditions, in an effort to be cognizant of human safety. With knowledge about the fungal recyclers of the world, we can move toward a better understanding of waste and it’s utility.

INTRODUCTION

The purpose of this project was to assess the bioconversion of lignocellulosic paper wastes and residues through the cultivation of Oyster mushrooms, Pleurotus spp., as an opportunity to utilize renewable-waste resources in the value-added production of an edible, medicinal, and protein rich food source. What began as a simple experiment of growth became a seminar in fungal genetics, sexual recombination, substrate suitability, and human environmental factors. The following is an account of the lessons learned.

I applied for this grant in the middle of Dr. John F. Katers’s “Waste Management & Resource

Recovery” course; and by the completion of the class, my perspectives on what waste is had been

2 completely transformed. According to Katers (personal communication), waste is any material perceived to have no value. However, since the world works in cycles and food chains, it’s likely that waste is simply misplaced nutrients, unavailable because decomposition is not adequately taking place within established cycles and its value unseen. With a more complete understanding of waste and suggestions from the UW- Solid Waste board, I felt it necessary to re-evaluate the wastes I was going to both utilize and create.

Substrate Selection

I initially proposed using five different waste materials: shredded office paper, shredded cardboard, shredded newspaper, short fiber sludge waste from recycling paper, and white sawdust.

Knowing that wood and paper waste came from many sources: municipal, commercial, industrial, process, and agricultural (Shah 2000), I thought these five waste materials would be good representatives of the types of waste materials that might provide suitable substrate for mushroom cultivation. Processed lignocellulosic wastes in the form of paper and cardboard constituted 40% of the typical composition of domestic waste in America (Shah 2000). The unclassified and unprocessed garden, yard, and wood waste comprised 21.5% (Shah 2000). The utilization of these wood and paper wastes could counter the important problem of solid waste disposal.

Looking closer to home, the Wisconsin Department of Natural Resources (WDNR) commissioned a Statewide Waste Characterization Study in 2002 to assess how much waste Wisconsin generated and where it came from. The purpose of the study was to estimate the composition and quantities of: (1) instate waste disposed in Wisconsin’s municipal solid waste (MSW) landfills; (2) waste disposed by residents, industrial, commercial, and institutional (ICI) generators, and construction and demolition

(C&D) activities within the state; and (3) establish a baseline for measuring the impact of future waste reduction and recycling programs (Cascadia Consulting Group, Inc. 2003). Construction & demolition

(28.7%), paper (20.8%), and organic materials (18.0%) accounted for the largest portions of Wisconsin’s disposed waste. These combined accounted for nearly 70% of the total instate waste disposed in

Wisconsin’s MSW landfills; the equivalent of about 3.2 million tons per year (Cascadia Consulting Group,

Inc. 2003).

However, Wisconsin is very proactive in its waste reduction. The recycling law of 1990 (WDNR,

2010) made the disposal of paper and cardboard wastes illegal and mandated recycling of paper and cardboard through the Wisconsin Administrative Codes: Chapters 150, 502, 544, 546 and Wisconsin

State Statute: Chapter 287 (WDNR, 2010). Because programs are already in place to eliminate unnecessary waste of these products, I removed cardboard waste from my list of substrates for cultivation of Oyster mushrooms.

I maintained to use of both recycled newspaper waste and recycled office paper because both were readily available and disposed of near the UW- Green Bay hallway garbage and recycling centers.

I was also able to obtain white sawdust waste from the UW-Green Bay wood shop dust collector, where residues from several table saws and planers was being disposed of. I wanted to use only wastes that were not already being used elsewhere, and that were readily available on the UWGB campus.

I added the entire vegetative body of Phragmites australis, the invasive, common reed grass to my design. Phragmites australis is a wetland pest species throughout the United States due to its rapid colonization and domination of marsh plant communities. High plant heights, stem densities, and detrital accumulation reduce light to the marsh surface and affect air temperatures (Meyerson et al

2000). With reported aboveground biomasses that are higher than all other native plant species occurring in the same marsh systems, the problem is exacerbated with the slow decomposition rates of the organic material (Meyerson et al 2000). This troublesome grass is truly the definition of a waste material because it has invaded important wetland environments, displaced native vegetation, wildlife,

4 and nutrient cycles, and affects several ecological factors of aboveground productivity, plant species diversity, and sediment biochemistry (Meyerson et al. 2000).

Mushroom Species Selection Saprophytic fungi have evolved to exploit every possible habitat on earth, wherever degradable organic matter exists, and are by definition the premier natural recyclers in the world (Dix & Webster 1995). All ecosystems depend on the fungal decomposition of organic plant material yielding carbon, hydrogen, nitrogen, and various other compounds available to plants, insects, and other organisms (Stamets 2000). Waste is naturally converted into soil nutrients by fungi weaving their filamentous mycelia network through cell walls of plants. Through the secretion of enzymes and acids, large molecular complexes of lignin and cellulose are broken down into smaller, simpler compounds (Sinsabaugh 2005). Most gourmet mushrooms are saprophytic.

One of the most widely distributed edible and medicinal mushrooms is the oyster mushroom from the genus Pleurotus, belonging to a phylum of fungi called Basidiomycetes (referring to the morphology of fungi with a stalk, cap, and gills) (Kendrick 2000). Commonly referred to as white rot fungi, Pleurotus are naturally found decaying wood materials and lignocelluloses, based on their ability to synthesize relevant hydrolytic and oxidative extracellular enzymes. The fungi oxidize carbon in organic compounds for every energy gain, while any nitrogen that is contained within these compounds is often released because nitrogen is bound directly to carbon (Treseder 2005). An adaptive feature the wood-decay basidiomycetes possess is the ability to grow at very low nitrogen concentrations, necessary when the undecayed wood contains a maximum of 0.2-0.3% by weight. Cellulase (enzyme to dissolve cellulose) activity is maintained with C: N ratios of 2000:1, while the C: N ratio of wood ranges between 300-1000:1 (Dix & Webster 1995). Whereas the fungi could potentially receive carbon, nitrogen, and phosphorus from only one source, the soil, this experiment proposed to utilize only the inoculated grains for nitrogen and phosphorus additions.

Humans have been ingesting mushrooms for their medicinal properties since before the birth of

Christ (Gregori 2007). Various species of Pleurotus have been shown to possess a number of medicinal, antimicrobial, and antiviral properties. Of particular interest, the polysaccharides appear to be potent anti-tumor and immune-enhancing substances (Gregori 2007). While research is continually being conducted regarding the medicinal benefits, Americans continue to consume the oyster mushroom for its taste. United States farmers received an average of $4.40(US) per kg for fresh oyster mushrooms, while growers of Agaricus bisporus (the most commonly grown white button mushroom) received an average of $2.29 per kg in the 2001-2002 growing season (Royse 2003). Oyster mushrooms are favored for their favorable organoleptic and medicinal properties, vigorous growth, and undemanding cultivation conditions (Gregori 2007).

Several studies reported cultivation of oyster mushrooms on various types of lignocellulosic wastes. For example, Baysal (2003) used waste paper and husk rice as substrates for growing oyster mushrooms in Turkey. Chopped office paper, cardboard, sawdust, and plant fibers substrates were compared through the cultivation of three types of oyster mushroom with similar results despite using three different fruiting containers (Mandeel 2005). Additional substrates including corncobs (Naralan

2008), switchgrass, cottonseed hulls, and wheat straw (Royse 2003), leaves of hazelnut, tilia, aspen, wheat straw, sawdust, and waste paper (Yildez 2001), wheat straw, cotton waste, peanut shells

(Philippoussis 2001) have been used to grow oyster mushrooms with various degrees of success. In fact,

Poppe (2004) reported 200 different types of wastes that were useful in the cultivation of Oyster mushrooms. Pleurotus species are one of the most efficient lignocellulose decomposing types of white rot fungi and are particularly useful because of their ability to colonize a wide range of agricultural and industrial wastes.

I initially chose to culture three species of Pleurotus. These included Italian Oyster Pleurotus cornucopiae Pers. var. pulmonarius Fr., Golden Oyster Pleurotus cornucopiae var. citrinopileatus (Singer),

6 and the Grey Dove Oyster Pleurotus ostreatus (Jacquin ex Fries) Kummer. The species were obtained from Field and Forest Products, Peshtigo, WI in November of 2009. Pure culture lines were maintained on potato dextrose agar, PDA.

Within one week of inoculating the PDA plates, I noticed a difference in the mycelia colonization of the plates. Whereas one species, Pleurotus ostreatus (Jacquin ex Fries) Kummer (herein referred to as the Grey Dove Oyster), exhibited healthy mycelial spreading and cottony white growth, the other two species were not as vigorous. In fact, some plates did not have any growth. According to the growth parameters listed by Stamets (2000), the lab temperature where the plates were located was well within range for growth.

I looked to primary literature for more specific growth parameters related to my slow growing

Pleurotus cornucopiae Pers. Var. pulmonarius Fr. (herein referred to as the Italian Oyster) and Pleurotus cornucopiae var. citrinopileatus (Singer) (herein referred to as the Golden Oyster). Vilgalys et al. (1993) published that the Italian Oyster is virtually indistinguishable from the Grey Dove Oyster. The differences were largely due to habitat preference, altitude differences, and seasonal variety. Singer

(1986) described the Italian Oyster as having a tawny brown cap, whereas the Golden Oyster had a brilliant yellow pileus; however both are so closely allied that they are often considered varieties of each other. Stamets (2000) own experiences with Golden Oyster resulted in a ‘culturing out’ of the characteristic color, resulting in a grayish brown mushroom conforming macroscopically to the Italian

Oyster. It appeared that all strains were genetically one species, yet the conditions surrounding fruiting lead to differential phenotypes.

Developmental plasticity is the inborn property of an organism to adapt to the fluctuating environment and is enabled by the differential expression of the genome according to the immediate needs of the organism. Spontaneous fruitbody plasticity is widely observed during mushroom cultivation (Chiu 1993). Old school taxonomists stressed morphological features or phenotypic

7 variations for classification purposes. Contemporary developmental biologists regard phenotypes as the product of interactions between environment and genotype, essentially “the nature vs. nurture” debate with fungi. Therefore, because I essentially had only one species, I utilized only the vigorous Grey Dove

Oyster for my studies.

METHODS AND MATERIALS

Induction of

Spawn Substrate Spawn Primordial Fruitbody Production Pasteurization Run Formation development (~25 days) (12 hours) (~25 days) (3 days) (~5-10 days)

Cropping Containers

I had initially decided to fruit my crop from disposable, heat-tolerant polypropylene bags.

However, in assessing the purpose for this project, waste reduction and transformation, I decided to replace the disposable bags with reusable Mason jars. Both Stamets (2000) and Oei (1991) state that the utilization of jar culture is acceptable for the cultivation of the Grey Dove Oyster. Methods for mushroom production were taken from P. Oei’s Manual on Mushroom Cultivation (1991) and P. Stamets

Growing Gourmet and Medicinal Mushrooms (2000).

Spawn Run

The Grey Dove Oyster spawn was inoculated on PDA plates and allowed to incubate at ~24°C for

1-2 weeks or until the mycelial growth had covered the entire surface of the PDA plate without reaching the outer edges.

First Generation G1 Grain Spawn Production

Organic rye grains were obtained from Streu’s Pharmacy & Bay Natural of Green Bay, Wisconsin.

Two hundred grams of rye were added to quart sized Mason jars with 220 mL of distilled water and 1 gram of gypsum. Jars were autoclaved for 50 minutes at 15 psi and allowed to cool to room temperature.

After successful colonization of the mycelium upon the PDA plate, mycelial cuttings were aseptically transferred from the PDA growth plates onto sterilized organic rye grains.

The spawn was allowed to run for ~ 25 days in the dark at ~ 24⁰C (75⁰F). Fresh air exchange was facilitated by three- ½ inch holes drilled into the Mason jar lids, and micro porous filters were used to prevent contamination.

Substrate Preparation

Heat-treatment of the substrates was performed as a means of sterilizing competitive microorganisms to allow the successful colonization of the Grey Dove Oyster spawn. Using a modified hot water bath method of submerged pasteurization described by Stamets (2000), each of four substrates (100 grams)were added to clean, numbered Mason jars, with ten replicates each. The substrates were wetted with distilled water to about 85% moisture and incubated at 105⁰C for 12 hours.

After 12 hours of incubation, the moisture contents were raised back up to 60%. Given that the substrates had similar initial moisture contents of 18-22% moisture, the moisture levels during incubation never dropped below 50% for any substrates.

After cooling, the pasteurized bulk substrates were aseptically inoculated with 10% grain spawn

(by mass).

The spawn was allowed to run for ~ 25 days in the dark at ~ 24⁰C (75⁰F). Fresh air exchange was

9 again facilitated by three - ½ inch holes drilled into the Mason Jar lids, and micro porous filters were used to prevent contamination.

Primordia Formation

Environmental initiation of primordial formation was stimulated by decreasing the temperatures to 50-60 ⁰F. I moved the jars from the dark cupboard spawning environment into the UW-Green Bay greenhouse for three days. The lids with microporous filters were left on the Mason jars to avoid contaminating the moist greenhouse with mycelial spores. The temperature within the greenhouse never topped 65⁰F during the daytime and never dropped below 50⁰F at night. Hyphal knots and primordia were visualized, signaling the development of the fruiting body.

Fruitbody Development

Following primordial formation, the opened jars were moved into the fume hood for fruiting.

Because of allergic reactions to the basidiospores released during fruitbody formation (Horner et al.

1993) and for the purposes of human safety, I initially felt it necessary to control the generated spores.

Several weeks prior, I had fruiting successes (with 80% biological efficiency) using other paper-derived by-product substrates in polyethelene bags within the same fumehood. The relative humidity within the fume hood was measured at 20%, and Stamets (2000) reports fruitbody development requires relative humidities of 85-90%.

After two days under the fume hood, it was obvious I had dried out the tops of my myceliated substrates. I had been misting the jars with water from a spray bottle, and positioned microporous filters in clear garbage bags that I draped atop the jars. I scratched the top of the myceliated substrate to stimulate fruit body development through mechanical wounding (Stamets 2000). Wounding causes the outgrowth of fresh hyphae and only fresh hyphae can be induced into fruitbody production (Kues &

Liu 2000). The substrate composed of short fiber paper mulch was revived and mushrooms began to fruit. The fungi grown on other substrates did not revive.

I then decided to move the jars from the fume hood into the lab and reasoned that because I had microporous filters within the garbage bags, I would not have to worry about human health issues.

The garbage bags were removed twice a day to allow for abundant fresh air exchange, as well as misted twice a day. The relative humidity measured within the bagged environment was 60%.

RESULTS

The generation of grain spawn was 100% effective. The grains were fully colonized with white fluffy mycelium. The mycelium descended throughout the substrates and proliferated.

Table 1 Comparison of growth of Pleurotus ostreatus on different substrates______Substrate Spawn Run: Colonization Period: Mean B.E. % Fresh Weight Mycelia density Spawn run (days) of Mushroom (g)

Sawdust + 50+ n.a. n.a. Office Paper + + + 22 n.a. n.a. Newspaper + + + 25 n.a. n.a. Phragmites + + + 21 n.a. n.a. Short-fiber sludge + + + 27 7.32 +/- 5.8 2.06+/- 1.6 Degree of mycelia colonization: + poor running growth, + + mycelium grows throughout but is not uniformly white, + + + mycelium growth throughout and is uniformly white.

The spawn run was 75% effective. The paper, newspaper, and Phragmites substrates were fully colonized prior to environmental initiation of primordial formation. The sawdust substrate did not fully colonize during the 25 day spawn run. I attribute this to inadequate moisture. Kues & Liu (2000) state that for wooden substrates adequate moisture is between 35-60%. Croan (2003) reported success with

60% moisture content for alder wood chips; however I believe the increased surface area and overall density of the sawdust required higher moisture content for adequate spawn run. Additionally, because

I took the sawdust from the wood shop of UW-Green Bay, the attendant suggested that some treated, stained and finished woods had been run through various machines throughout the semester and he couldn’t assure the purity of the dust.

Despite my efforts to revive my myceliated substrates after being in the fume hood, the conditions were not such to allow for a successful crop. The short fiber paper sludge substrate was the only substrate to actually fruit; however the biological efficiency (B.E.) of fruiting averaged at 2%, as opposed to reports of >100% efficiency (Stamets 2000 & Oei 1991). The fruiting bodies were malformed with elongated stipes and reduced pilei. This was due to the high CO2 concentrations (Kues & Liu 2000) achieved once the garbage bags were utilized to reduce moisture loss.

One recycled paper substrate did have one primordial formation, but it did not produce an adequate fruit.

It was obvious that the low humidity within the fume hood was the deterrent to healthy growth; however the positive news comes from the sexual recombination success of the fruited paper sludge substrate. Once the hyphae are initiated to produce primordia, only a few of those come into maturation. The relocation of nutrients is thought to occur throughout the mycelium to favor one specific or a few selected primordia, though what determines that preference is still unknown (Kues &

Liu 2000). Because the Grey Dove Oyster has heterothallic fertility mechanisms (meaning it needs two separate sex types found in separate individuals), crosses between compatible mycelia derived from single spores can provide a practical method for hybridization and strain improvement (Peberdy et al

1993). Therefore, the fruited sexual recombination favored high concentrations of carbon dioxide and low humidity and can be used to create strains capable of producing higher yields under such conditions. Also, because of these sexual recombination effects, it is necessary to receive primary cultures directly from a type culture collection to ensure genetics with the strain selected.

I would repeat this study with the polyethylene bags instead of the Mason jars. Though

12 deemed an acceptable fruiting container, neither Baysal (2003), Mandeel (2005), Naralan (2008), Royse

(2003) nor Yildez (2001) used jars for their studies. While it was determined that the size of the fructification surface had no significant interaction between substrate productivity (Lelley & Jan Ben

1993), the jars only allowed for fruiting from the top. Had they been allowed to fruit from the sides, the malformations may not have been so pronounced.

Lastly, because of my concern with creating wastes during a waste-transformation study, I am very pleased to note that the degraded substrate post mushroom production has tremendous potentials for improved ruminant feed, production of biogas, garden compost, recycling for Agaricus cultivation, production of saccharification enzymes (Yildez 2002), and bioremediation (Eggen 1999; Stamets 2000) through the degradation of Anthracenes, Poly-Aromatic Hydrocarbons(PAHs), Pentachlorophenol (PCP),

Dioxins, pesticides, petroleum hydrocarbons (Stamets 2005) and Polychlorinated Biphenyls (PCBs)

(Moeder et al. 2004). Therefore, the spent compost will not be a waste but beneficial additions to the surrounding ecosystems.

The purpose of this project was to comparatively assess high lignocellulosic waste substrates for

Pleurotus ostreatus cultivation and to understand the lifecycle governing the fruiting production of oyster mushrooms. I wanted to transform waste, and was adamant about not creating waste.

Throughout the study, I increased my appreciation for the complexity regarding fungal systems and their adaptive responses to environmental factors. With knowledge about the fungal recyclers of the world, we can move toward a better understanding of waste and it’s utility.

ACKNOWLEDGEMENTS

I would like to acknowledge my late professor Dr. V.M.G. Nair for his introduction and contribution to my love of fungi and learning what is the best. I would also like to acknowledge Dr. Amy

Wolf for her self-less character which has molded many great minds into communicable scientists.

Lastly, I would like to thank the UW – Solid Waste Research Program for this waste transformation grant and their commitment to a more thorough understanding of waste and waste resources.

LITERATURE CITED

Baysal, E. 2002. Cultivation of oyster mushroom on waste paper with some added supplementary materials. Bioresource Technology 89: 95-97.

Cascadia Consulting Group, Inc. 2003. Wisconsin Statewide Waste Characterization Study. Prepared for Wisconsin DNR. http://dnr.wi.gov/org/aw/wm/publications/recycle/wrws-finalrpt.pdf

Croan, Suki C. 2003. Utilization of treated conifer wood chips by Pleurotus (Fr.) P. Karst. Species for cultivating mushrooms. Mushrooms International Newsletter: 91: 4-7.

Chiu, Siu Wai. 1993. Physiology, Cytology and Genetics of Mushrooms. . Mushroom Biology and Mushroom Products. Proceedings of the First International Conference on Mushroom Biology and Mushroom Products 23-26 August 1993, The Chinese University of Hong Kong. Chinese University Press.

Dix, N.J. & Webster, J. (1995) Fungal Ecology. Chapman & Hall: University Press, Cambridge.

Eggen, T. 1999. Application of fungal substrate from commercial mushroom production– Pleuorotus ostreatus – for bioremediation of creosote contaminated soil. International Biodeterioration & Biodegradation 44: 117-126.

Gregori, A. et al. 2007. Cultivation Techniques and Medicinal Properties of Pleurotus spp. Food Technology and Biotechnology 45: 238-249.

Horner et al. 1993. Basidiospore allergen release: elution from intact spores. Journal of Allergy and Clinical Immunology 92(2): 306-312.

Kendrick, Bryce. 2000. The Fifth Kingdom 3rd edition. Mycologue Publications. Focus Publishing, Newburyport, MA.

Kues U. & Liu Y. 2000. Fruiting body production in basidiomycetes. Appl. Microbiol. Biotechnol 54:141- 152.

Lelley, J. I. and JanBen, A. 1993. Interactions between supplementation, fructification-surface and productivity of the substrate of Pleurotus spp. Proceedings of the First International Conference on Mushroom Biology and Mushroom Products 23-26 August 1993, The Chinese University of Hong Kong. Chinese University Press.

Mandeel, Q.A. et al. 2005. Cultivation of oyster mushrooms (Pleurotus spp.) on various lignocellulosic wastes. World Journal of Microbiology & Biotechnology 21: 601-607.

Meyerson et al. 2000. A comparison of Phragmites australis in freshwater and brackish marsh environments in North America. Wetlands Ecology and Management 8: 89-103.

Moeder et al. 2005. Structure selectivity in degradation and translocation of polychlorinated biphenyls (Delor 103) with a Pleurotus ostreatus (oyster mushroom) culture. Chemosphere 61: 1370- 1378.

Naraian, R. et al. 2008. Influence of different nitrogen rich supplements during cultivation of Pleurotus florida on corn cob substrate. Environmentalist 29: 1-7.

Oei, P. 1991 Manual on mushroom cultivation. Transfer of Technology for Development: Amsterdam & Technical Centre for Agricultural and Rural Co-operation: Wageningen.

Peberdy et al. 1993. New Perspectives on the Genetics of Pleurotus. Mushroom Biology and Mushroom Products. Proceedings of the First International Conference on Mushroom Biology and Mushroom Products 23-26 August 1993, the Chinese University of Hong Kong. Chinese University Press.

Philippoussis, A. et al. 2001. Bioconversion of agricultural lignocellulosic wastes through the cultivation of the edible mushrooms Agrocybe aegerita, Volvariella volvacea and Pleurotus spp. World Journal of Microbiology & Biotechnology 17:191-200.

Poppe, J. 2004. Oyster Mushroom Cultivation. The Mushroom Growers Handbook. MushWorld. http://www.fungifun.org/mushworld/Oyster-Mushroom-Cultivation/mushroom-growers- handbook-1-mushworld-com-preface.pdf

Poppe, J. 2000. Use of agricultural waste materials in the cultivation of mushrooms. Mushroom science: Proceedings of the International Conference on Scientific Aspects of Mushroom Growing, 15: 3- 23.

Royse, D.J. et al. 2003. Yield, mushroom size and time to production of Pleurotus cornucopiae (oyster mushroom) grown on switch grass substrate spawned and supplemented at various rates. Bioresource Technology 91: 85-91.

Sinsabaugh, R.L. 2005. Fungal enzymes at the community scale. Published in The Fungal Community; Its organization and role in the ecosystem edited by J. Dighton, J. White, and P. Oudemans. Taylor & Francis; New York.

Shah, K.L. 2000. Basics of solid and hazardous waste management technology. Prentice Hall, pgs 130- 155.

Singer, R. 1986. The Agaricales in Modern Taxonomy (4th Edition) Koeltz. Scientific Books. Germany. Wisconsin Department of Natural Resources. Waste & Materials Management: Recycling Program. http://www.dnr.state.wi.us/org/aw/wm/information/wiacssr.htm

Stamets, Paul. 2000. Growing gourmet and medicinal mushrooms, 3rd edition. Ten Speed Press, Berkeley.

Stamets. Paul. 2005. Mycelium Running: How mushrooms can help save the world. Ten Speed Press, Berkeley.

Treseder, K.K. 2005. Nutrient acquisition strategies of fungi and their relation to elevated atmospheric CO2. The fungal community: it’s organization and role in the ecosystem 3rd edition. Edited by Dighton et al. Taylor & Francis Group, New York.

Thomas et al. 1999. Mycoremediation: a method for test-to pilot-scale application. Phytoremediation and innovative strategies for specialized remedial applications. Edited by Leeson & Alleman. Battelle.

Tuininga, A.R. 2005. Interspecific Interaction Terminology: From Mycology to General Ecology. The fungal community: its organization and role in the ecosystem 3rd edition. Edited by Dighton et al. Taylor & Francis Group, New York.

Vilgalys et al. 1993. Intersterility groups in the Pleurotus ostreatus complex from the continental United States and adjacent Canada. Canadian Journal of Botany 71: 113-128.

WDNR. Recycling Program: Waste and Materials Management. Obtained 30 April 2008. http://dnr.wi.gov/org/aw/wm/information/wiacssr.htm

Yildiz, S. et al. 2001. Some lignocellulosic wastes used as raw material in cultivation of the Pleurotus ostreatus culture mushroom. Process Biochemistry 38: 301-306.