Evaluation of brewers' spent grain as low cost substrate for the cultivation of pleurotus eryngii (k by Oswald Gekko Kierkegaard

Evaluation of Brewers ' Spent Grain as Low-cost Substrate for the Cultivation of Pleurotus eryngii (King Oyster Mushroom)

by Biswas Palikhey A Research Paper Submitted in Partial Fulfillment of the Requirements for the Master of Science Degree III

Food and Nutritional Sciences Approved: 6 Semester Credits

C Vf~

Naveen Chikthimmah, hD, ~rch Adviser

'I<itlillacaIlS, PhD The Graduate School University of Wisconsin-Stout May, 2011

The Graduate School University of Wisconsin-Stout Menomonie, WI

Author:

Palikhey, B.

Title: Evaluation of Brewers' Spent Grain as Low-cost Substrate for the Cultivation of Pleurotus eryngii (King Oyster Mushroom) Graduate Degree/ Major: MS Food and Nutritional Sciences Research Adviser:

Naveen Chikthimmah, Ph.D.

MonthrYear:

May, 2011

Number of Pages:

Style Manual Used: American Psychological Association, 6 th edition

Abstract

Brewers' spent grain (BSG) is the major waste of brewery operation accounting for around 85% of total solid wastes generated. Although BSG is composed of rich lignocellulosic components that include cellulose (17%), non-cellulosic polysaccharides including arabinoxylans (28%), and lignin (28%), it has drawn little attention for the recycling, apart from its use as animal feed. The efficacy of BSG (65% moisture content) as basal substrate for the cultivation of Pleurotus eryngii was evaluated at 0%,25% and 50% wheat bran (WB) supplementation. There was significant increase in the apical extension rate, Ilr of P. eryngii mycelia in the agar media plates prepared from BSG substrate at 0%, 25%, and 50% WB supplementation (p < 0.05). Contrastingly, Ilr decreased significantly with the dilution level of substrate (gram of substrate/milliliter water) at 1:20, 1: 10, and 1:5 respectively. Bottle cultivation of P. eryngii with the above substrate combinations showed complete colonization of all three substrate treatments including 100% BSG at 22Âą1 DC for 30 days.

3 Fruiting body formation was initiated at 18Â°C and 95% relative humidity for 35 days. There was no mushroom production from BSG without WB supplementation. The biological efficiency of P. eryngii mushroom from 25% and 50% WB supplemented BSG were 9.37% and 66.97% respectively. Results from this research showed that BSG upon WB supplementation might be used successfully as basal substrate to grow P. eryngii.

4 The Graduate School University of Wisconsin-Stout Menomonie, WI

Acknowledgement

Firstly and importantly I would like to offer my deep and sincere gratitude to Dr. Naveen Chikthimmah for his tremendous contribution to my thesis work. He was instrumental in providing me direction and encouragement that helped me in every aspect of the thesis work from planning, material collections, experimental work, writing, and presenting. His proactiveness and logical way of thinking were great help that boosted the study. I would like to thank Dr. Alma Rodriguez for bringing her expertise and key guidance that were important to this study. Her positive attitude and willingness to give was highly appreciated. I also owe my deep gratitude to Dr. Carolyn Barnhart for her constant encouragement and important support throughout the work. My hemifelt thanks to Dr. Kitrina Carlson who added zest for the completion of this thesis. I am also thankful to the departments of Food and Nutrition and Biology at the University of Wisconsin-Stout for providing laboratory facilities and resources needed for my work. I would also like to offer my high regards to Connie Galep, Josiah Ray, and Frank Dogbatsey who helped me in the laboratory logistics during conduction of the experiments. One very special person I have to appreciate is Sharmila Thapa for being very understanding to facilitate my focus towards the thesis work. Her support in the form of love and encouragement were the major players in the success of this work.

5 Table of Contents Abstract ................................................................................................................................................. 2 Chapter I: Introduction .......................................................................................................................... 8 Statement of the Problem ................................................................................................................ 11 Purpose of the Study ....................................................................................................................... 12 Objectives of the Study ................................................................................................................... 12 Definition of Terms ......................................................................................................................... 13 Assumption of the Study ................................................................................................................. 13 Chapter II: Literature Review ............................................................................................................. 15 Introduction ..................................................................................................................................... 15 Lignocellulosic Materials ................................................................................................................ 15 Enzymatic Hydrolysis of Lignocellulosic Materials ....................................................................... 17 Brewers' Spent Grain: A Byproduct of the Brewing Industry ....................................................... 18 Generation of BSG in Brewing Operations. . ............................................................................ 19 Composition ofBSG ................................................................................................................. 20 Preservation technique of BSG. ................................................................................................ 21 Applications of BSG. . ............................................................................................................... 22 Chapter III: Methodology ................................................................................................................... 34 Materials ......................................................................................................................................... 34 Fungal Growth Studies in Undefined Media in Petri Plates ........................................................... 34

Fungal Growth Studies under Solid State Conditions .................................................................... 35 Preparation of spawn. ................................................................................................................ 35 Substrate preparation and inoculation ....................................................................................... 35 Cultivation of P. eryngii mushrooms .......................................................................................... 36 Biological efficiency. ................................................................................................................ 36 Statistical Analysis .......................................................................................................................... 36 Chapter IV: Results and Discussion ................................................................................................... 38 Fungal Growth Studies in Undefined Media in Petri Plates ........................................................... 38 Apical Extension Rate ........................................................................................ 41 Cultivation of P. eryngii mushrooms .............................................................................................. 45 Chapter V: Conclusion ........................................................................................................................ 54 Recommendations for future: ......................................................................................................... 55 References ........................................................................................................................................... 57 Appendix A: Apical Extension Rate of P. eryngii colony ................................................... 67

7 List of Figures

Figure 1: Structures of a) cellulose; b) arabinoxylan (an example of hemicellulose) and c) ferulic linkages between xylans and lignin .............. " ........................................................ 16 Figure 2: Structure of Pleurotus eryngii mushroom ..................................................... 29 Figure 3: Mechanism of compound degradation by laccase (Lac), versatile peroxidase (VP) and aryl-alcohol Oxidase (AAO) of P. eryngii ....................................................................... 31 Figure 4: Growth of P. eryngii colony on BSG-WB media plates at different substrate combination and dilution levels ............................................................................ 38 Figure 5: Pleurotus eryngii WC888 mycelia radial growth on BSG/WB substrate combination and varying dilution levels (1:20, 1: 10 and 1:5) at 23±2°C. ........................................... 39 Figure 6: Pleurotus eryngii WC943 mycelia radial growth on BSG/WB substrate combination and varying dilution levels (1 :20, 1: 10 and 1:5) at 23±2°C ........................................... .40 Figure 7: Mean apical extension rate of P. eryngii WC888 at different substrate combinations and dilution levels ............................................................................................ 41 Figure 8: Mean apical extension rate of P. eryngii WC943 at different substrate combination and dilution levels ................................................................................................. 42 Figure 9: Pleurotus eryngii (WC888 and WC943) mycelial growth on BSG/WB combination substrate ........................................................................................................ 45 Figure 10: Pleurotus eryngii WC888 cultivation on BSG and WB substrates a) Rye spawn b) Bottles inside environmental chamber c) Mushroom growing in the bottles d) & e) Fruiting bodies produced from 50% BSG + 50% WB f) Fruiting body produced from 75% BSG + 25% WB ............................................................................................................. 46

Chapter I: Introduction A major output of most agricultural and food processing operations is unusable lignocellulosic (LC) waste material. The predominant components of LC materials are cellulose (35-50%), followed by hemicelluloses (20-30%), and lignin (l 0-25%), in addition to minor components such as proteins, oils and ash making up the remaining fraction (Rashad et aI., 2009). LC materials are also one ofthe most abundant naturally available and renewable complex organic carbons. Annual production of LC materials is over 150 billion tons worldwide (Saber et aI., 2010). Only a very small amount of the total lignocellulosic waste produced is utilized for useful applications, while most are disposed causing environmental problems and incur to disposal cost to agricultural and food producers and processors. Nature is abound with microorganisms having the ability to produce cell wall degrading enzymes that solubilize complex carbon chains in lignocellulosic materials to smaller molecules (Gao et aI., 2007). The use of such microorganisms in a solid state fermentation (SSF) of LC materials (agricultural polymeric bypro ducts) such as wheat straw, rice bran, corn cops, and spent brewers grain can produce a variety of useful products with high economic value. Microbial solid-state fermentations are attractive techniques for the utilization of LC materials because this fermentation does not require costly inputs such as air (aeration), water and energy (Hong et aI., 2011). Operation costs are low due to the use of simple bioreactors requiring no energy input. SSF also do not create any significant amounts of waste water that require treatment prior to disposal. Utilizing lignocellulosic material via SSF has been successful in the production of fermentable sugars (Hong et aI., 2011) and organic acids (Saber et aI., 2010). Lignocellulosic materials serve as good SSF substrates for the growth of saprophytic microorganisms including fungi used for commercial edible mushroom cultivation. Presently,

9 the most extensively used lignocellulosic materials for the production of edible fungi are wheat straw, rice straw, sawdust, hard wood chips, sugarcane bagasse, cotton seed hulls, corn cobs, rice bran, and wheat bran (Kirbag & Akvuz, 2008, Saber et aI., 2010; Orts et aI., 2008). Other green materials, such as cotton stalk and soybean straw (Panjabrao et aI., 2007), coffee pulp (MartinezCarrera, 1989) etc. have also been used or tried for growing edible mushrooms in some countries. Brewers' spent grain (BSG) is the most abundant lignocellulosic waste in brewing operations. BSG is the solid material that is remaining after the grains have been mashed. It accounts for about 85% of the total wastes generated in a brewery (Mussato et aI., 2007). Currently the main application of BSG is limited to ruminant animal feed purpose only (Bartolome et aI., 2002). It is available at low or no cost throughout the year. In the United States (U.S.), 34 million metric tons of distillers' spent grains including BSG were generated in 2010 (USDA, 2010). While a very small amount ofBSG is used as animal feed and agricultural amendments, the bulk of BSG generated in the United States is considered a solid waste and requires proper disposal thereby incurring costs to the brewing industry. In brewing process, the mashing operation results in the extraction of soluble sugars, proteins, and minerals from the malted barley grains into a liquid fraction known as wort leaving behind water insoluble proteins and the cell wall residues of the husk, pericarp, and seed coat in the spent grain known as the Brewer's spent grain (BSG). BSG is rich in cellulose and noncellulosic polysaccharides and lignin, and small amounts of proteins, lipids, and minerals (Mussatto et aI., 2006). Owing to its residual nutrient content of cellulose, protein and fiber, BSG can be an attractive substrate ingredient for the cultivation of edible mushrooms (Gregori et

10 aI.,2008), production ofbioethanol (Xiros & Christakopoulos, 2009), enzymes (Mandalari, 2008), organic acids (Mussato et aI., 2007), and sugars (Mussotto & Roberto, 2008). Mushroom along with other fungi are special because they are neither plants nor animals. The increase in consumption and appreciation of mushrooms over the years is due to their flavor, economical and ecological values, and medicinal properties (Sanchez, 2004). In the U.S., the cultivation of edible oyster mushroom (Pleuratus spp.) started since early 1980's (Rodriguez & Royse, 2008). Pleuratus spp. is third in world production volume after Agaricus bispraus and Lentinula edades. The sales volume of oyster mushroom in the U.S. market has increased from 1.2 million pounds in 1988-89 to around 5.05 million pounds in the 2008-09 season (USDA, 2009). Pleuratus spp. is well acknowledged as an economically important genus among higher fungi due to its world-wide availability, broad adaptability to various growing conditions and preferable nutritional propeliies (Hassan et aI., 2010). Among the genus Pleuratus, Pleuratus elyngii is generally considered as the best tasting species. The commercial production of P. eryngii in the U.S. began in 2000 and by 2004,85 tons were produced (Rodriguez & Royse, 2008). Pleuratus eryngii, commonly known as the "king oyster" mushroom has great nutritional and medicinal value. This mushroom fully justifies its name with its large fruiting body, fleshy meat-like texture, longer shelf-life, superior nutritional profile, and desirable nutraceutical properties. BSG favors the growth of mushrooms not only due to high protein content, but also due to high moisture content and physical properties such as particle size, volume weight, specific density, porosity, and water holding capacity (Gregori et aI., 2008). The cultivation of P. eryngii ob BSG can be done using three basis systems - bag, bottle, or outdoor cultivation. The bag system is mainly used in Europe and China (Rodriguez & Royse, 2008). Polypropylene or

polyethylene bags (0.5-3kg substrate/bag) are used as cultivation containers. The bottle system is used by many growers in Japan, South Korea, and China and in some modern production facilities in the U.S. and Canada. The bottle system is advantageous since it is highly mechanized in addition to the bottles being autoclavable, re-usable, and easy to handle (Rodriguez & Royse, 2005). Outdoor cultivation is practiced in the farms that do not have controlled environment. In this system the initial colonization of substrate is done in bags which are then trans felTed onto the ground and then cased with soil to prevent desiccation for promotion of the fruiting body formation. Statement of the Problem The use of expensive carbon source such as glucose, sucrose or starch is not economical in the large production of biotechnological products. Instead, the exploitation of less expensive sources would be beneficial. This has lead, in recent year, an increasing trend in utilizing byproducts and wastes as raw material in the production of value added products. BSG which is low-cost and high volume byproduct of brewery is rich in cellulose and non-cellulosic polysaccharides. Fungi through the action of extracellular enzymes are able to degrade these complex materials present in BSG to produce various products of impOliance. BSG thus holds a strong potential to be recycled. At present, feed use and dumping are the major means of elimination ofBSG from brewery. However dumping is not sustainable because of the environmental pollution associated with the disposal method. The waste management problems for BSG thus require developing new ways to utilize the spent grains taking into account the adverse impact on environment and health. Apart from the current general use as an animal feed, other alternative uses of BSG like in mushroom production can be examined.

12 Out of numerous types of edible mushrooms, P. eryngii is a highly valuable edible mushroom with superior nutritional and economic benefits. Cultivation of P. eryngii on readily available inexpensive BSG will be prized in the context of effective bioconversion of locally available substrate and for mushroom crop diversification. Producing nutritious food at a profit, while using materials that would otherwise be considered "waste," also contributes to sustaining agricultural practices. There are about 72 small and large breweries in Wisconsin producing abundant amounts of BSG annually. The research work on understanding the potential use of BSG on P. eryngii mycelia growth and mushroom production can contribute to supporting a knowledge-based entrepreneurial business 0ppOliunity in Wisconsin. Purpose of the Study BSG is generated in bulk quantities and can easily deteriorate due to its high moisture content, which makes the transportation, storage and preservation of BSG a major challenge for the brewing industries. Therefore, the use of BSG can solve disposal cost and environmental problems associated in the brewery process. At the same time it can provide cheaper alternative substrate for the production of high value product. Since, the cultivation of P. elyngii needs a moderate initial investment; even local farmers can get an opportunity to start small scale entrepreneurial business to generate a potential profit. Objectives of the Study The main objective of this work was to evaluate and optimize brewers' spent grain (BSG) as a substrate for the cultivation of Pleurofus eryngii. The specific objectives include: 1. Determine the effect of BSG-WB substrate combinations and dilution levels on P. eryngii mycelia growth.

13 2. Examine biological efficiency of P. eryngii mushroom cultivated at various BSG-WB substrate combinations

Definition of Terms Lignocellulose. Complex non-edible plant cell wall material primarily composed of cellulose, hemicellulose, and lignin.

Brewers' Spent Grain (BSG). Waste product of the brewery industry consisting of the solid material remaining after the mashing process.

Oyster mushrooms. Specialty edible mushrooms with particular taste, medicinal, and nutritional properties.

Spawn. Grain colonized with fungal mycelium used to inoculate substrates for the cultivation of mushroom.

Mycelium. Vegetative fungus growth appearing as cotton like networks of filaments. Fruiting body. Fleshy structure of the fungus (sexual stage) generally called mushroom. It is produced by reproductive stage of the fungal growth.

Biological efficiency. Ability of the fungus to utilize the dry substrate expressed as percentage of yield of mushroom in dry or wet basis produced from dry wet of the substrate.

Assumption of the Study Wet BSG coming out from brewing process has high moisture and nutrient content and is liable to rapid microbial and chemical deterioration. In this study the wet BSG obtained from the brewery was dried to a stable moisture content level in order to be stored for longer period of time. But since mushroom production requires higher moisture content of around 65% in the substrate, BSG was later added with water to reach to this optimum level. In larger scale BSG

14 utilization, the drying operation incurs huge amount of additional cost thereby the use of wet BSG becomes more economical and practicable. But since dried BSG had to be used in the study, it was assumed that dried BSG when added with water for moisture optimization will behave characteristically and functionally similar to wet BSG which is recommended in large scale utilization in mushroom cultivation.

Chapter II: Literature Review Introduction

This chapter will cover background oflignocellulosic materials and brewers' spent grain (BSG). It would be followed by oyster mushrooms, and Pleurotus eryngii, its characteristics, enzymatic system, and will conclude with the cultivation of P. eryngii. Lignocellulosic Materials

Lignocellulosic (LC) materials are components of plant or wood cell walls in which cellulose is intimately associated with hemicelluloses and lignin (Saber et aI., 2010). LC materials are inexpensive, readily available in nature as forest or agricultural wastes, easily processed to high value products with high yield and are suitable for both hydrolysis and production of enzymes. Development of technological advances for optimal utilization of these materials requires an understanding of the morphological arrangement, and efficient and economical ways of degrading their complex structures (Orts et aI., 2008). Cellulose is the main molecular entity in lignocellulosic materials. It is a glucose-based polymer consisting of two fJ-l, 4 linked glucose residues (cellobiose) in their repeating units (Figure la). The fJ acetal linkage plays an important role in structural properties and strength of cellulose fiber. Unlike starch that is processed by a number of organisms, cellulose cannot be easily utilized by many organisms due to the presence of fJ acetal linkage in cellulose that requires unique enzymes for digestion (Wertz et aI., 2010). The insolubility of cellulose in normal aqueous solutions is due to its tendency to form crystals utilizing extensive intra- and intermolecular hydrogen bonding. Part of the cellulose preparation is amorphous between the crystalline sections (Orts et aI., 2008).

~-O

~O~O

HO~~q

HO~"'~

HO~OHO-L--z:::rf OH

S--O~O\

c.)

(0"-J~O--~

o)~ OH

Ugnln"--.

R:-H

(p~coumaric

Hdd)

or R

R=OCII 3 (ferulic acid)

(Orts, Holtman, & Seiber, 2008) Figure 1. Structures of a) cellulose; b) arabinoxylan (an example of hemicellulose) and c) ferulic linkages between xylans and lignin

Hemicelluloses are branched polymers of relatively low molecular weight. They bind tightly to the surface ofthe cellulose micro fibrils and to each other by hydrogen bonding, and can be referred to as

cross~linking

glycans (Weliz et aI., 2010). It contains a variety

of5~

and 6-

carbon sugars such as glucose, mannose, xylose, arabinose, galactose, and 4-0-methyl glucuronic acid (Figure 1b). Hemicelluloses are cross-linked via ester linkages to predominately

17 cinnamic (ferulic) acids, forming covalent linkages with lignin, making plant cell wall hydrolysis even more difficult (Saha, 2003). Lignin is a highly branched aromatic polymer of largely guaiacyl and syringyl monomers with connecting three-carbon aliphatic sidechains (Figure 1c) (Orts et aI., 2008). It is a cementing material in lignocellulosic fibers providing structural rigidity to the cell wall. Enzymatic Hydrolysis of Lignocellulosic Materials

Utilization of lignocellulosic materials is important for the bioconversion of inexpensive and readily available raw agricultural products and wastes. One of the main approaches for the utilization of LC wastes is their microbiological and/or enzymatic conversion to value added and high-value food, pharmaceutical, nutraceutical, cosmeceutical, and specialty chemical products. The conversion ofLCs to value-added products includes two sub-processes: hydrolysis ofLC materials to fermentable sugars and fermentation of sugars to target products (Saber et aI., 2010). Efficient cellulose degradation requires multi-enzyme systems produced by variety of cellulolytic bacteria and fungi. The fungal kingdom constitutes a promising group of microorganisms that are capable of hydrolyzing lignocellulosic components. The hydrolysis of cellulose is primarily accomplished by cellulase enzyme. Cellulase constitutes endoglucanases that cleave the internal fJ-l, 4-glucosidic bonds and exoglucanases that act in a processive manner on the reducing or non-reducing ends of cellulose polysaccharide chains, thus liberating either glucose or cellobiose as major products (Saber et aI., 2010). The action of cellulase system on insoluble cellulose results in three processes simultaneously: physical and chemical changes in the solid-phase cellulose; primary hydrolysis; and secondary hydrolysis (Wertz et aI., 2010). The endoglucanases largely causes chemical changes in solid-phase cellulose while exoglucanase causes solubilization.

18 Hemicellulose is not easily nor economically degraded due to presence of xylose which takes longer time and higher cost for fermentation (Orts et aI., 2008). Studies have shown a positive correlation between hemicellulose removal and cellulose digestion. Ideally, hemicelluloses hydrolysis is obtained by pretreatment with sulphuric acid in order to facilitate subsequent enzymatic hydrolysis of cellulose. Lignin is a complex polymer that physically surrounds and protects cellulose and hemicelluloses from enzymatic hydrolysis and is also resistant to microbial degradation (Yamac & Tamer, 2008). In this case, effective uses of lignocelluloses depend on delignification of plant

material. The removal of lignin not only increases cellulose accessibility and availability. Only some microorganisms such as white rot fungi and actinobacteria are capable of degrading lignin by an oxidative process. This oxidative process involves enzymes such as lignin peroxidases (LiP), manganese peroxidases (MnP), and laccases (Kerem & Hadar, 1998). Brewers' Spent Grain: A Byproduct of the Brewing Industry Beer is a popular beverage with world production volume increasing annually (F AO, 2009). The brewing process generates a significant amount of bypro ducts and wastes. The most common bypro ducts of a brewery are brewers' spent grain (BSG), spent hops, and surplus yeast. Brewers who do their own malting also produce malt sprouts. The major challenge in a brewery is not only these wastes produced but also their bulk. A typical small brewery brewing 1500 litres three times a week will produce around two tons ofBSG in a week (Thomas & Rahman, 2006). Large regional breweries producing 1,OOOHL beer per day will have 40 tons per day to remove. With the emergence of global concerns on climate change, and environmental pollution, regulations call for reduction in waste generation, and the transition towards sustainable and green production methods.

19 BSG accounts for approximately 85% of the total bypro ducts generated in a brewery (Tang, et aI., 2009). It is the solid material left after the malt has been mashed. BSG is produced in large quantities and is readily available at low or no cost throughout the year. It accounts, on average, for 31 % of the original malt weight, representing approximately 20 kg per 100 L of beer produced (Mussatto et aI., 2006). In 2008, 1.8 billion hI of beer was produced worldwide, and this represents the generation of36 million tons ofBSG (Robertson et aI., 2010). While BSG contains significant energy resources from its organic contents, there are major difficulties in a sustainable energy balance because of its high moisture and nutrient content and handling difficulties (Thomas & Rahman, 2006). Since BSG is prone to chemical and microbial deterioration mechanisms, the urgency to remove BSG from brewery area is of prime importance to brewing operations. Large breweries have mechanized system to collect, process, and distribute the BSG for further use. However, in small breweries, BSG in most cases is given away for little or no economic value for use in animal feeds. In other cases, BSG is disposed off into landfills (Aliyu & Bala, 2011). Therefore, there is a need of technologies for sustainable use of BSG as a method of waste management.

Generation ofBSG in Brewing Operations. Barley is the world's most important cereal grain after wheat, maize, and rice (Mussatto et aI., 2006). It is used commercially for animal feed, malt production for beer and for human consumption. About 13% of barley produced worldwide is processed into malt (F AO, 2009). Barley grain is rich in starch and proteins and consists of three main pmis: the endosperm (starchy material), the germ (proteins) and the grain covering (fiber). The malting of barley activates enzymes that modify the structure of the starchy endosperm. In the brewery, the malted barley is milled, and then mixed with water in a mash

20 tun. The enzymes act on barley substrate to convert starch into fermentable sugars (mainly maltose, maltotriose, glucose) and non-fermentable sugars (dextrins). Also proteins are partially degraded to polypeptides and amino acids. These soluble fractions are extracted into the water which forms sweet liquid known as wort. The wort is filtered off for the fermentation step to make beer while the insoluble, undegraded solid part of the malted barley is left behind which constitutes the brewers' spent grain. Thus, the brewing process only removes certain nutrients from the malt necessary to produce the wOli, but leaves behind washed, water insoluble proteins and the cell wall residues of the husk, pericarp and seed coat within the spent grain. Composition of BSG. BSG basically consists of the husk-pericarp-seed coat layers that

covered the original barley grain. Wide variability in the composition of BSG can be found due to variations in grain used, harvest time, malting and mashing condition, and quality and type of adjuncts used in the brewing process. BSG is considered as a lignocellulosic material rich in protein and fiber (Mussatto et aI., 2006). BSG contains cellulose and non-cellulosic polysaccharides and lignin, and may contain some lipids. Compositionally BSG has about 17% cellulose, 28% non-cellulosic polysaccharides, mainly arabinoxylans, and 28% lignin. The protein and fiber content is very high around 20% and 70% dry basis, respectively. Xylose, glucose, and arabinose are the most abundant monosaccharides found in BSG (Aliyu & Bala, 2011). BSG also constitutes some minerals, vitamins, and amino acids. The minerals found in BSG are calcium, cobalt, copper, iron, magnesium, manganese, phosphorus, potassium, selenium, sodium and sulphur. The vitamins include (ppm): biotin (0.1), choline (1800), folic acid (0.2), niacin (44), pantothenic acid (8.5), riboflavin (1.5), thiamine (0.7) and pyridoxine (0.7). Some essential amino acids have also been found in BSG.

21 The crude fiber content (including lignin and carbohydrates mostly cellulose) in brewers' spent grain is about 14-15% on a dry solids basis (Keilbach, 2009). Other fiber components include hemicelluose, pectins, gums, mucilages, and maillard products. Total dietary fiber in BSG in about 56% of which 2.5% is soluble dietary fiber and 53.5% is insoluble. Preservation technique of BSG. Due to high moisture content (70-75% in wet BSG)

and some residual fermentable sugars, BSG can be rapidly deteriorated due to chemical and microbiological reactions. Therefore, BSG imposes significant handling, storage and transportation challenges to brewing operations. It has been proposed that the moisture content ofBSG has to be reduced to below 10% (wet basis, wb) to prolong its storage time (Tang et aI., 2005). Drying is an appropriate technique for the preservation of BSG as it reduces product volume, and consequently reduces transportation and storage costs (Aliyu & Bala, 2011). However, breweries typically do not dry the BSG generated due to high energy costs involved. Instead, most breweries send it for cattle feed, composting, soil amendments in farmlands or disposed in landfills. Some breweries apply a two stage preservation techniques: first, BSG is pressed to reduce moisture content to less than 60% and then dried to reduce the moisture content to below 10% (Santos et aI., 2003). Oven drying is the most suitable drying method, however in addition freeze-drying (Bartolome et aI., 2002) and superheated steam (Tang et aI., 2005) have also been tested. Ovendrying has to be carried out at temperatures below 60Â°C in order to avoid unpleasant flavor which may be produced by higher temperatures and the risk of toasting and burning of dried BSG (Robertson et aI., 2010). A preservation study conducted by Bartolome et aI. (2002) with oven drying, freeze drying and freezing BSG showed that oven drying and freeze drying reduced

22 the volume of the product without altering its composition. Freezing affected the composition of some sugars such as arabinose and was thereby considered not to be suitable process for preserving BSG. Tang et aI. (2005) suggested thin-layer drying using superheated stearn (SS) as an alternative method to dry BSG. The process used a close-loop system of circulating superheated stearn that reduced energy wastage during hot-air drying. The exhaust stearn produced from the evaporation of moisture from the BSG could also be used in other operations. The drying in SS had no significant effect on the nutrient content ofBSG. Overall, the superheated stearn method has several advantages including the reduction of the environmental impacts, an improvement in drying efficiency, the elimination of fire or explosion risk, and a recovery of valuable volatile organic compounds (Aliyu & Bala, 2011). A membrane filter press with hot water (65Â°C) has also been used to dry BSG to moisture levels of between 20 and 30% (El-Shafey et aI., 2004). Al-Hadithi et aI. (1985) showed effective chemical preservation of BSG by using lactic, formic, acetic and benzoic acids and potassium sorbate without loss of quality and nutritional value ofBSG. Applications of BSG. BSG has been considered a waste product for a long time. With an increase in disposal cost due to legislation and corresponding decline in traditional disposal routes for the solid material (such as animal feed), alternative commercial uses for BSG are being sought. This drive is also supportive by the revelation of nutrient rich properties ofBSG. Various alternate applications of the BSG investigated for many years require targeted and practical technological developments. Animal feed. The primary application of BSG is in animal feed. Both wet and dry BSG can be used in ruminant animal feed, due to its high content of protein and fiber. Low cost BSG

23 when combined with inexpensive nitrogen sources, such as urea, can provide rich source of energy, essential amino acids, and other micronutrients to ruminant animals. BSG incorporation in the diet of cows reported an increase in milk yield, milk total solid content, and milk fat yield (Belibasakis & Tsirgogianni, 1996). Although BSG is primarily utilized for feeding dairy cattle, the feeding has been done on other animals such as poultry, pigs, and fish. Egg production and hatchability were improved with breeder turkey rations including 40% BSG (Keilbach, 2009). Kaur and Saxena (2004) observed that 30% BSG supplementation on diet containing rice bran showed increased body weight in fish compared to diet containing rice bran only which is attributed to the presence of essential amino acids like lysine, arginine and methionine in BSG. Fiber from BSG when fed to monogastric animals may significantly alter their fat and lean muscle content (Keilbach, 2009). Human nutrition. Despite its high nutritive value, BSG has limited use as an ingredient in human food applications because of its granular structure and color which imparts undesirable flavor and physical properties of the final products. Therefore, BSG needs to be dried and converted to flour before addition to human foods. Also, only relatively small quantities (510%) can be added in the food products. It is especially useful in the food that needs to be incorporated with high fiber contents. The addition of BSG has been evaluated in flakes, wholewheat bread, biscuits and aperitif snacks (Mussatto et ai., 2006). A high protein flour prepared from BSG was successfully incorporated into a number of bakery products, including bread, muffins, cookies, mixed grain cereals, fruit and vegetable loaves, cakes, waffles, pancakes, tortillas, snacks, doughnuts and brownies. BSG fiber has been suggested to provide human health benefits through changes in gastric emptying which helps in slower and efficient absorption of nutrients in the body

24 (Mussatto et aI., 2006). These health benefits have achieved great interest that the production of food-grade fiber products from BSG can be the greatest opportunity to upgrade the value of BSG (Keilbach, 2009). BSG flour in food delivers functional properties including ease in blending, lower calorie content, higher water absorption capacity, supplementation of protein, fiber and essential minerals, uniform tan color, bland flavor, and mildly roasted aroma (Keilbach, 2009). The ingestion ofBSG, or derived products, provides health benefits, which are associated with increased fecal weight, accelerated transit time, increased cholesterol and fat excretion and a decrease in gallstones (Mussatto et aI., 2006). Ajanaku et ai. (2010) conducted a histopathological study of rats after feeding with a diet containing BSG at 0, 3, 6, 9, 12, and 15% weight/weight. There were changes in body weight, haemoglobin, lymphocytes, WBC, RBC, and liver condition of rats. Based on their findings, they suggested that a BSG blend range of 13 % will be appropriate for utilization in human food without adverse effect on the liver.

Substrate for microorganisms and enzyme production. The presence of high moisture content, protein and polysaccharides make BSG particularly suitable for microbial growth. The protein fraction of BSG was used as medium for enhanced growth and sporulation of soil actinobacteria, especially Streptomyces (Szponar et aI., 2003). BSG was reported to be used for the cultivation of Bifidobacterium adolescenfis, Lactobacillus sp. (Novik et aI., 2007), and

Penicilliumjancaewskii (Terrasan et aI., 2010). The cultivation of Pleurotus ostreatus (oyster mushroom) was successful using BSG as basal substrate or as a supplement (Gregori et aI., 2008; Wang et aI., 200; Lara, Arias, & Villasenor, 2002). In order to degrade plant cell wall-derived material, microorganisms are required to produce a battery of enzymes, such as endoxylanases, b-xylosidases, a-arabinofuranosidases and esterases (Mandalari et aI., 2008). BSG, with the presence of digestible and non-digestible

25 organic residues is a potential substrate for the production of ~-amylases and amyloglucosidase by different amylolytic organisms (Aden iran et aI., 2008). Various other enzymes have also been reported to be produced using BSG. Nascimento et aI. (2009) showed the production of cellulase using Streptomyces malaysiensis. Feruloyl esterase (F AE) and xylanase production by Humicola grisea var. thermo idea and Talaromyces

Stipitatus in BSG were repOlied (Mandalari et aI., 2008). Adeniran & Abiose (2009) observed alpha-am lase activity by Helminthosporium oxysporium, Aspergillus jlavus, and other microorgansims on BSG. BSG was also a good substrate for production of alpha-amylase by

Aspergillus Olyzae NRRL 6270 in solid-state fermentation (Francis et aI., 2002). These studies indicated that BSG as low-cost substrate can be efficiently used for different enzyme production substantially reducing enzyme production costs.

Production of value-added products. BSG's constituents' cellulose, non-cellulosic polysaccharides and proteins can be degraded by hydrolytic procedures into various con-esponding simple compounds that are of industrial significance as precursors of food grade chemicals or as energy sources in microbial fermentations (Aliyu & Bala, 2011). Lactic acid has shown many applications in connection with foods, fermentations, pharmaceuticals, and the chemical industries. It is also used as a precursor of poly-lactic acid (PLA) production. BSG hydrolysate has been used as fermentation medium without any supplementation for Lactobacillus delbrueckii to produce L-Iactic acid (5.4 giL) at 0.73 gig glucose consumed (Mussatto et aI., 2007a). Hydroxycinnamic acids (ferulic and p-coumaric) have a number of potential applications such as natural antioxidants, food preservatives/antimicrobial agents, chemoprotectants, and as a food flavor precursors (Mussatto et aI., 2007b). Bartoleme et aI., (2002) extracted 0.17-0.24%

26 dry weight ferulic acid, which was lower than other sources like wheat bran, however the advantage of using BSG was that it does not require preliminary treatment like other sources do and hence could significantly reduce production costs. Enzymatic hydrolysis of BSG protein concentrate with the use of proteases generated BSG protein hydrolysates with improved technological properties (Celus et aI., 2007). Xylitol, a rare sugar with health benefits that could be an alternative to current conventional sweeteners was produced by Candida guilliermondii from xylose in acid hydrolysates ofBSG (Mussotto & Roberto, 2008). Technology for producing biofuels (such as ethanol, butanol, or various hydrocarbons) and bio-based chemicals from lignocellulosic material is experiencing significant advances in an effort to meet global energy and chemical needs. Ethanol production from BSG is possible after enzymatic conversion of cellulose to fermentable sugars. Xiros and Christakopoulos (2009) showed that Fusarium oxysporum converted cellulose and hemicelluloses in BSG directly to ethanol through the consecutive steps of hydrolysis of the polysachharides and fermentation of the resulting oligosachharides by secreting all the necessary enzyme systems. Other uses. Studies have been done to determine the possibility of using BSG to produce

charcoal bricks and evaluating their physical and chemical propeliies (Mussato et aI., 2007a). Charcoal made from BSG had various minerals and high calorific values, however was inferior to sawdust charcoal in burning properties. The low amount of ash coupled with the high amount of fibrous material (lignin, hemicelluloses and cellulose) makes BSG suitable for use in brick manufacture (Russ et aI., 2005). Bricks produced by using BSG had improved strength, and porosity, and lower density without changes in color and brick quality. Thus, it is suggested that BSG could be used as substitute for sawdust which is commonly used in brick-making operation.

27 Studies have been done to use BSG in paper towels, business cards and coasters with superior texture results on the products (Mussatto et aI., 2007a). Oyster Mushroom

Edible mushrooms belonging to the genus Pleurotus are commonly called oyster mushrooms and are considered specialty mushrooms in the United States. The cultivation of oyster mushrooms is becoming popular throughout the world because of its ability to grow in a wide range of temperatures utilizing various easily available lignocellulosic materials (Baysal et aI., 2003). It is the third largest commercially cultivated mushroom in the world. The increase in consumption of the oyster mushroom is largely due to its superior taste, medicinal and desirable nutritional propeliies (Obodai et aI., 2003). The cultivation of Pleurotus species does not require composed materials or a casing layer to induce fruit body formation (Kerem & Hadar, 1998). Since Pleurofus sp. can decompose lignocellulose efficiently without chemical or biological pretreatments, a large variety oflignocellulosic wastes can be used or recycled in the cultivation of this variety of edible fungi. Pleurotus eryngii Pleurofus eryngii is a saprophytic mushroom in the phylum Basidiomycota, class Homobasidiomycetes, order Agaricus, family Pleurofaceae (Cha et aI., 2010). The name eryngii

indicates where the mushroom can be found in the wild: on the roots ofumbelliferous plants, patiicularly Elyngium and Heracleum. It grows as a parasite on these plants - a big difference with the other oyster mushrooms which grow well on dead hardwood. It is widespread in southern Europe and the areas of central Asia and North Africa. Pleurotus eryngii has the ability to produce various biologically active compounds and possesses a well-developed ligninolytic

28 enzyme system responsible of the degradation of lignin and different aromatic compounds (Stajic et aI., 2009). Pleurotus eryngii is known by several common names such as King Oyster (United

States), Cardoncello or Cardarello (Italy) and Xing bao gu (China) among others. Presently, Japan, China, South Korea and Italy are the major producers of this mushroom. In the U.S., commercial production of this species began in 2000. By 2004, 85 tons were produced in the country (Royse et aI., 2005). The king oyster mushroom fully justifies its name: it is considered to be the most flavorful among the mushrooms belonging to the genus Pleurotus. The thick, firm stipe of this mushroom has a pleasant texture and a mild sweet taste. Pleurotus eryngU is less prone to mechanical damage and bruising, has a significantly longer shelf- life in comparison to other Pleurotus mushrooms and is therefore better suited for the logistics of food distribution.

Additionally, studies have shown the medicinal, and nutraceutical properties of the Pleurotus mushrooms as discussed in following section. Composition. The cultivated king oyster mushrooms on a suitable substrate can be seen growing as large fruiting bodies (LFB), small fruiting bodies (SFB), and the base (Mau et aI., 1998). They showed that LFB comprised 79.90% of total weight and the base comprised 15.47% of total weight. Out of the three parts of king oyster mushrooms, LFB and SFB were found to be similar in their proximate compositions and their compositions of volatile compounds and nonvolatile taste components. However, the base was remarkably different in its flavor profile (Mau et aI., 1998).

LI-Lrf!C fruitio<! bodies

- (LFF3l

Base

Small fnlllmg h\JJtes

(mij

(Mau et aI., 1998)

Figure 2. Structure of Pleurotus eryngii mushroom Pleurotus eryngii basidiomata is rich in carbohydrates (9.6% of fresh weight), mostly

dietary fibers (4.64% of fresh weight; 4.11 % is insoluble and 0.53% is soluble dietary fibers), followed by chitin (0.50% of fresh weight), and polysaccharides (0041 % of fresh weight) (Stajic et aI., 2009). The protein content is between 1.88% and 2.65%. Aspartic acid, glutamic acid, and arginine are the most abundant amino acids. Pleurotus eryngii basidiomata is also a good source of vitamins (C, A, B2 , B j , D, and niacin) and minerals (especially K, Mg, Na, and Ca). Freshly harvested P. eryngii basidiomata are high in moisture (86.6-91.7%) and low in lipids (0.8% of fresh weight). The composition of P. eryngii could vary depending upon strain, development stage, the type of substrate used for its cultivation and cultural and harvesting practices. Medicinal and nutraceutical characteristics of P. eryngii. Pleurotus eryngii synthesize

various biologically active compounds including

13-1, 3-glucans, lovastatin, pleureryn, eryngin,

ribonuclease, eryngeolysin, ergothioneine, and protein xb68AB (Stajic et aI., 2009). Pleurotus eryngii is demonstrated to have anti-hypertensive, antioxidant, anti-hyperchoesterolic, anti-

hyperglycemic, immunomodulating, antitumor, antibacterial, antiviral, antifungal, antiinflammatory, and anti-osteoporotic effects. Pleurotus eryngii hemolysin, known as

30 eryngeolysin was shown to exhibit anti proliferative action against leukemia cells and antibacterial activity against Bacillus spp. (Ngai & Ng, 2006). Pleurotus eryngii has been reported to exhibit anti-aging (Guillen et ai., 2000), and blood glucose-lowering properties (Kang et ai., 2001). It also inhibits angiogenesis-related enzymes (Kang et ai., 2003) and the proliferation of human colon cancer cells (Hawang et ai., 2003). It also has antioxidant and free radical scavenging activities (Hui et ai., 2002), and activates immune cells (Kang et ai., 2004). Cha et ai. (2010) investigated the biochemical and enzymatic activities of fibrinolytic enzyme obtained from P. eryngii. The enzymes showed a high degree of specificity towards fibrin, and were speculated to have applications in thrombolytic therapy as it is a directly acting thrombolytic agent. Enzyme System. Pleurotus species are one of the most efficient decomposers of lignocellulosic materials (Baysal et ai., 2003). For this reason, it is not necessary to pretreat substrates for cultivation of P. eryngii. The extensive extracellular enzymatic system of P. eryngii is responsible for lignin degradation by producing H20 2 needed for peroxidase activity. Lignolytic enzymes catalyze the one-electron oxidation of lignin units, resulting in various nonenzymatic reactions that include bond cleavage (Munoz et ai., 1997a). Pleurotus eryngii produces laccase (Lac), manganese oxidizing peroxidase (MnP), and aryl-alcohol oxidase (AAO) under submerged liquid cultures and under solid-state fermentation conditions. (Munoz et ai., 1997b). These enzymes are responsible for degradation oflignin durign growth stage of mycelia before primorida formation. Laccases (Lac) are glycoproteins which catalyze the one-electron oxidation of numerous organic and inorganic phenolic and nonphenolic substrates, as well as Mn2+, in the presence of mediators (Stajic et ai., 2009). Aromatic amines and phenols are the best substrate for Lac activity. Therefore, in P. eryngii cultivation,

31 laccases are responsible for the depolymerization of lignin, lignin-N complex as well as metabolizing aromatic compounds released during the process. This facilitates mycelia access to other nutrients in the substrate and produces lower molecular weight aromatic compounds such as soluble quinines and aromatics .

. . . . . . ... ·.. ..... ...

•

H,02

QH-

°~

'OH

,",vp,/

. Q.-.

:HYdrOqL~nOne 7La~ ·

Q +02'-

Autoxidation. Superoxide anion 02"" 'Semiquinone QUInone radical H20 2 radical H. Mn 2+ +H-

•

superoXi~d dismutase

K Mn 3+

02 H20 2

· ................ -_ ... _- ........ _----_ .... . I •• •

••••••••••••• ,

p.anysilalcohol

H;P2

0 2

'¢A~, poanYSaldehYdeL p.anlslc acid Oxidation

(Stajic et aI., 2009) Figure 3. Mechanism of compound degradation by laccase (Lac), versatile peroxidase (VP) and aryl-alcohol Oxidase (AAO) of Pleurotus eryngii The MnP of P. eryngii catalyzes the H 20 2-dependent oxidation of lignin and lignin derivatives. The Mn-oxidizing peroxidase group is composed of two enzymes: 1) Mn~dependent peroxidase (MnP) that oxidizes Mn2+ to Mn3+, which then directly oxidizes lignin and other phenolic aromatic compounds, and 2) versatile peroxidase (VP) that oxidizes phenolic and nonphenolic aromatic compounds, as well as Mn2+ to Mn3+ (Stajic et aI., 2009). Aryl alcohol oxidase oxidizes various aromatic compounds, as well as aliphatic polyunsaturated alcohols. During this oxidation, AAO provides H202 needed for the activity of

32 the Mn-oxidizing peroxidases and the generation of the 'OH radical that is involved in the initial attack on lignocellulose. AAO also has synergistic action with Lac in the process of oxidation of hydroquinines. Cultivation of P. eryngii. The cultivation of P. elyngii mushroom involves two phases: vegetative growth phase, and reproductive growth phase (Chang, 2008). The vegetative growth phase includes formation of filamentous branched hyphae known as mycelium on the substrate. After the maturity of mycelia, their growth tips are retarded by regulating the environmental factors to induce formation of fruiting bodies which is the reproductive growth phase. The induction of fruiting can be triggered by environmental shocks such as light exposure, increase in oxygen content, and lowering temperature (Chang, 2008). During the mycelia growth and the development to mature fruiting bodies, various biochemical changes occur as a result of which enzymes are secreted extracellularly to degrade the insoluble materials in the substrates into simple and soluble molecules which are subsequently utilized by the growing mushroom (Rash ad et aI., 2009). The substrates used in the cultivation of P. eryngii include sawdust, wheat straw, wheat and rice bran, corn cobs, cottonseed hulls, chopped rice straw, and soybean meal. Bag cultivation is the most used system for P. eryngii. Polypropylene or polyethylene bags (0.5-3 kg substrate/bag) with filter patches are used as containers (Rodriguez and Royse, 2008). After the primordial formation, the bags are folded to the sides for complete mushrooms development. The bottle system is used by many growers in Japan, South Korea, China, and in some modern production facilities in the U.S. and Canada (Chang, 2008). The substrate is contained in polypropylene bottles of 850-1050 ml capacity. This system is highly mechanized, with exception of the harvesting process that is performed manually. After colonization of the

33 substrate the lid is opened and the top layer of colonized surface is scratched to induce uniform fructification. Fresh King Oyster mushrooms are sold commercially in over-wrapped packages or in bulk (Rodriguez and Royse, 2008). Spawn

Spawn is the starter inoculum for commercial mushroom cultivation. It is prepared by growing mycelia on cereal grains usually wheat, rye, or millet. The purpose of spawn is to rapidly colonize the specific bulk-growth substrate (Sanchez, 2004). The success of mushroom production depends in great part on the quality of the spawn, which must be prepared under sterile conditions to diminish contamination of the substrate. Several studies have been done to improve and develop new techniques for the production of spawn (Royse, 2003). Spawn is made from secondary mycelium (dikaryon) (Royse, 2003). The reason behind doing this is uncertainty of performance by a new strain that could form from spores. Commercial spawn producers make spawn under aseptic conditions and ship it to growers in aseptic containers which are used to inoculate mushroom substrates.

Chapter III: Methodology Materials Two isotypes of P. eryngii (WC888 and WC943) were obtained from the Penn State University Mushroom Culture Collection (courtesy of Dr. Alma Rodriguez, University of Wisconsin-Green Bay). The fungal cultures were maintained on potato dextrose agar (PDA) plates at 4°C with periodic transfers to fresh PDA. Brewers' spent grain (BSG) was obtained from The Jacob Leinenkugel Brewing CO, WI. The wet BSG was dried at 55°C for 24 hrs to moisture content of 7% in a hot air-oven. Rye grain and Wheat bran (WB) were purchased from the Menomonie co-op market in Menomonie, WI.

Fungal Growth Studies in Undefined Media in Petri Plates BSG and WB were milled using a 1-L dry blending stainless steel vessel adapted to a Waring blender base to fine particle size. BSG was used as basal substrate with WB being added as supplement. Three combination formulations of BSG and WB were made, A = 100% BSG, B

= 75% BSG + 25% WB, and C = 50% BSG + 50% WB. These combinations were taken in order to examine the differences of BSG stand alone substrate to the WB supplemented substrates. First, the substrate formulations were mixed with water at three dilution levels (g of substrate/ml of water), 1:20, 1: 10, and 1:5. After addition of 1.5% agar, the substrate media were autoclaved at 121 ° C for 45 minutes. Petri plates containing 25ml of substrate media were prepared by gently pouring the tempered media into the plates. Plates made with the varying substrate formulations and the media dilution levels were used to study the main effects on the growth of P. eryngii. A water agar plate was used as an experimental control. The water activity (aw) of the substrate media in plates were determined using water activity meter (Aqua lab).

35 Apical Extension Rate

The substrate media plates (treatments) and water agar plates were inoculated with a plug of mycelia (6mm diameter) taken from an actively growing mycelial mat on PDA media. During incubation at 23±2°C, the diameter of the widest part of colony was measured from the third to the sixth day of growth. The radius vs. day plot was analyzed with linear regression and the slope of the line was recorded in Jlm/h which gave the apical extension rate,

U r of the

P. efyngii

colony (Sanchez & Gonzalez, 1996). The experiment was repeated twice with three replicates in each experiment. Fungal Growth Studies under Solid State Conditions Preparation of spawn. 100g of rye grain was mixed with 2g calcium carbonate and

120ml warm tap water in a 500ml flask. This preparation was autoclaved at 121°C for 45 minutes, cooled to room temperature and then inoculated with two 6 mm plug of P. eryngii mycelia grown on PDA agar. The inoculated preparation was incubated at 25°C until the P. eryngii fungal mycelia covered the outer surface of the grain. The flask was shaken to distribute

the P. eryngii mycelia for uniform colonization of the rye grain and re-incubated at 25°C. Following complete colonization of the rye grain with P. eyrngii mycelia, the flask was with the rye grain spawn was stored at 4°C and used in the experimental studies. Substrate preparation and inoculation. Un-milled BSG and WB were mixed dry to get

three substrate combinations as described above. The moisture content was adjusted to 65% and 2% calcium carbonate was added to regulate optimum pH. 200g of each substrate combination was taken into a glass bottle (473 ml), covered with aluminum foil and then autoclaved at 121 ° for 45 minutes. After overnight cooling, the substrates were inoculated with 5% rye spawn on the surface of substrate. Each substrate combination was tested in triplicate.

Cultivation of P. eryngii mushrooms. Inoculated substrate was incubated at 23±2°C in a no humidity control incubator (National Appliance Company, Portland, Oregon) until the substrate was completely colonized with mycelium (30 days). The bottles were opened and the surface of the substrate was scratched with a needle to remove aerial mycelium. Scratching stimulates mycelia to form primordial uniformly over the surface of the substrate. Next 15ml sterile water was added to each bottle and then covered with plastic sheeting. In this plastic sheeting, five small holes (1 mm diameter) were punched in all bottles holding the substrate with the colonized mycelia. This was done to maintain high humidity and oxygen flow. To induce fruit body formation (fruiting), the bottles were transferred to an environmental chamber (Espec, Hudsonville, Michigan) maintained at 18°C and 95% relative humidity with 12h lightl12h darkness cycles. The plastic cover was removed after the immature basidiomata touched the plastic cover. Mushrooms were harvested after 35 days of scratching when the basidiomata completely matured and the cap was flat. Fresh mushrooms from each bottle were weighed separately.

Biological efficiency (BE). Biological efficiency was determined as the percentage of conversion of dry substrate to fresh matter in fruit bodies as follows (Wang et aI., 2001): · I . Iff"" (01.) B10 oglca e IClency 10

100°/ = (Weightoffreshfruitbodiesharvested) .. . X Imtlal weight of dry subtrate

Statistical Analysis The mushroom cultivation experiment was a 3 (substrate combination) level)

3 (Dilution

2 (strain) factorial designs with three replicates per treatment. Linear regression line of

radial growth and generalized linear model procedure for analysis of variance (ANOV A) for apical extension rate were carried out. Tukey's test (p < 0.05) was used to separate the treatment

means. Data analyses were performed using the Minitab 16 statistical software (State College, PA).

Chapter IV: Results and Discussion Fungal Growth Studies in Undefined Media in Petri Plates

Pleurotus eryngii mycelial colonization was visible on the third day in the Brewers' spent grain (BSG)-Wheat bran (WB) substrate media plates with the colony extending in radial direction upon growth. There was no growth of P. elyngii in water agar plates. Figure 4 shows the P. eryngii colonization on BSG-WB media plates at different substrate combination and dilution levels. The variation in dilution level of the media changed the nutrient concentration in the media with higher dilution resulting in lowered nutrient concentration in the media. There were also differences in water activity (aw ) of the media with the dilution and compositional differences. Visual observation showed that the largest diameter occurred in the media treatment containing 50% BSG + 50% WB combination at 1:20 dilution level while the highest colony density occurred in plates with the 1:5 dilution levels. There was an inverse relationship between colony diameter and colony density (Figure 4).

100% BSG

75% BSG+ 25%WB

50% BSG + 50%WB

1:20

1:10 WC888

1:5

1:20

1:10 WC943

Figure 4. Growth of P. eryngii colony on BSG-WB media plates at different substrate combination and dilution levels

1:5

1:20

35 6'30

~25 rI)

A1:20

B1:20

.= 20 ~ 15 10 . 5 .+- .._.-.-._-,.-..........• --....-......--,,-.... -------,--.-- ...----, 3

IIC1 :20

1:10

35 6'30

~25 rI)

A1:10

B1:10

.= 20 ~ 15

IIC1:10

10 5 3

1:5

35 6'30

~25 rI)

+Al:5

Bl:5

.= 20 ~ 15

IIC1 :5

10 5 3

Figure 5. Pleurotus eryngii WC888 mycelia radial growth on BSG/WB substrate combination and varying dilution levels (1 :20, 1: 10 and 1 :5) at 23±2°C. A

100% BSG; B

75% BSG +

25% WB; C = 50% BSG + 50% WB. Standard elTors are marked with vertical bars.

1:20

35 9'30 525 ell .:: 20 "0

IIIB 1:20

l,CI :20

t':S

.Al:20

Days

1:10

35 ,-,30

525 ell .:! 20 "0 t':S

.Al:IO IIIB 1:1 0

.\CI:lO

10 5 3

40 35

··1

1:5

,-, 30

525

.AI:5

ell

;;= 20

IIIB 1:5

t':S

CI:5

10 5 3

Figure 6. Pleurotus eryngii WC943 mycelia radial growth on BSG/WB substrate combination and varying dilution levels (1 :20, 1: 10 and 1 :5) at 23±2°C. A = 100% BSG; B = 75% BSG + 25% WB; C

50% BSG + 50% WB. Standard errors are marked with vertical bars.

41 Figure 5 and 6 show the radial growth of two strains of P. eryngii in different substrate media plates. Among substrate combinations, the radial growth of each strain was highest in 50% BSG + 50% WB media at all dilution levels. Among substrate dilution levels, the radial growth was highest in 1:20 dilution level in all three substrate formulations. Apical Extension Rate

WC888 -1:20 !;31:10 0 1:5

A .-

250

-=........

0.992

0.991

[200

0.995

'-'

... ~

t':I

'"' 150

....= ~

...='" 100 ~

....t':I

CJ Q;

0 100%BSG

75% BSG + 25% WB

50% BSG + 50% WB

Figure 7. Mean apical extension rate of P. eryngii WC888 at different substrate combinations

and dilution levels. The value above the bar represents water activity of the substrate media. Upper case A, Band C and lower case a, b, and c indicate significant difference between the treatments.

WC943 .1 :20 !3 1: 10 01:5

--ยง.

250 -

0.992

0.991

200

a 0.995

'-'

.....~ ~

'"' 150

....o

~ 100 ..... ~

's, ~

o 100% BSG

75% BSG + 25% WB

50% BSG + 50% WB

Figure 8. Mean apical extension rate of P. eryngii WC943 at different substrate combination and

dilution levels. The value above the bar represents water activity of the substrate media. Upper case A, Band C and lower case a, b, and c indicate significant difference between the treatments. Figure 7 and 8 show mean apical extension rate of two strains of P. eryngii colony at various substrate compositions and dilution levels. Both substrate combination and substrate dilution level showed significant differences in apical extension rate, /lr of P. eryngii colony (p < 0.05). The /lr increased by wheat bran supplementation level with 50%BSG + 50% WB

composition giving the maximum value. Similarly, /lr increased by the substrate dilution level with 1:20 dilution giving the maximum value. Within one substrate combination, the /lr decreased with decreasing water activity level. The apical extension rate was significantly different between genotype also (p < 0.05). Strain WC888 showed faster growth than WC943. The apical extension rate ranged from 82.44/lmlh to 230.36/lm/h for WC888 while it ranged from 87.9!lmlh to 222.03/lmlh for WC943. A previous study by Lara et al. (2002) on apical

extension rate of Pleurotus spp using BSG reported that by increasing BSG content from 10 to 50% on maguey tequila bagasse, the apical extension rate of P. ostreatus and P. pulmonarius increased gradually with highest apical extension rate of 162.51 J.lm/h in 50:50 combination of BSG and maquey tequila bagasse. In that sense, P. eryngii showed faster growth than those two species but difference in species and type of substrate used i.e. wheat bran to maquey tequila bagasse needs to be considered. Based upon the results, the different substrate media tested could be arranged according to extension rate in descending order as follows, for substrate composition; 50% BSG + 50% WB, 75% BSG + 25% WB, and 100% BSG and for substrate dilution level; 1:20, 1: 10, and 1:5 accordingly. Higher water activity level produced higher apical extension rate. Water availability in substrate and surrounding environment plays a major role in the fungal growth. It is equally important factor affecting fruiting body formation (Kues & Liu, 2000). Wang et al. (2001) showed an increase in biological efficiency and mycelia extension of P. ostreatus with the increase in moisture content of substrate with an optimum level at 70% moisture. Similar to the current study where P. eryngii apical extension rate decreased at lowered water activity level, a previous repOli by Inch and Trinchi (1987) noted reduction in yield and colony radial growth rate of Paecilomyces farinosus with decrease in water activity of the media substrate. The mechanisms involved in the control and regulation of mycelial growth are better studied on solid medium than in submerged cultures, as fungi are adapted to growth on solid substrates (Reeslev & Kjoller, 1995). In solid medium, colony diameter and radial growth rate can be used as indicator parameters of fungal growth (Trinci, 1969). The fungal growth involves germinated spores elongating at the tip and continuous branching to form tubular hyphae. These hyphae reach to the nutrients available in the media plates extending the colony'S diameter. The

44 filamentous growth nature of fungi and nutrient composition of substrate can cause variation in colony density. Our results on substrate media plates showed that the growth rate of P. eryngii colonies was significantly affected by the nutrient that increased in concentration. At lower substrate dilution level, the nutrient concentration was higher which gave lower apical extension rate but increased colony density.

The results corroborate with the literature that fungi spread

across substrate containing low nutrient concentration at higher radial growth rates (Trinci, 1969). The higher nutrient concentration results in denser colony which was evident by highest colony density on least substrate dilution level (highest nutrient concentration). A growth study of Aspergillus niduluans with different concentration of glucose showed that the radial growth rate and hyphal density of the colony increased with increasing concentration of the glucose up to an optimum level and then decreased on higher concentration (Trinci, 1969). In a similar study done on a noble agar with different apple juice concentrations, the color of epiphytic fungi darkened and formed sclerotium-like bodies with increased apple juice concentration (Batzer et aI., 2010). This suggested that nutrient type and concentration in substrate influence the growth rate and colony pattern of the fungal mycelia in the substrate. Formation of secondary metabolites and products of autolysis at the centre of colony can inhibit mycelia growth thus inhibit colony radial growth rate if they diffuse through the medium and accumulate below the peripheral growth zone (Robson, Bell, & Kuhn, 1987). Nutrient concentration may also have an effect on colony growth rate by influencing the rate of hyphal septa formation (Trinci, 1971). Substrate inhibition and/or the accumulation of inhibitory substances and different rate of septa formation may have resulted in variation in the growth rate of P. eryngii in different substrate combinations and dilution levels.

Cultivation of P. eryngii mushrooms Pleurotus eryngii mycelia growth rate in the cultivation study, as in the plates study was observed to be highest in 50% BSG + 50% WB combination for both genotypes WC888 and WC943 (Figure 9). Denser substrate colonization was also visible with WB supplementation. The primordial formation occurred after 2 weeks and 3 weeks of scratching in 50% BSG + 50% WB and 75% BSG + 25% WB combination respectively. There were no primordial and fruiting body formations in the 100 % BSG substrate. The WC943 strain of P. eryngii had no fruiting bodies in any of the substrate combinations. One flush of mushroom and one fruiting body/bottle was produced.

Figure 9. P. eryngii (WC888 and WC943) mycelial growth on BSG/WB combination substrate. A = 100% BSG; B = 75% BSG + 25% WB; C = 50% BSG + 50% WB. The mushroom cultivation pictures are shown in Figure 10. The fruiting body produced from 50% BSG + 50% WB combination was larger than one produced from 75% BSG + 25% WB combination. The average length and diameter of mushroom stipe from 50% BSG + 50% WB combination were 10 cm and 3.6 cm respectively with cap diameter averaging 6.5cm. Likewise, the average length and diameter of mushroom stipe from 75% BSG + 25% WB combination were 6 cm and 2.4 cm respectively. The average cap diameter was 2 cm.

Figure 10. P. eryngii WC888 cultivation on BSG and WB substrates a) Rye spawn b) Bottles inside environmental chamber c) Mushroom growing in the bottles d) & e) Fruiting bodies produced from 50% BSG + 50% WB f) Fruiting body produced from 75% BSG + 25% WB

47 The data on Table 1 shows the yield and biological efficiency (BE %) of P. eryngii WC888 grown on different substrate composition. 50% BSG + 50% WB recorded the highest yield and BE being 46.2 g/kg and 66.97% respectively, while 75% BSG + 25% WB substrate had yield and BE of 6.32 g/kg and 9.37% respectively. Table 1. Effect ofgrowth substrate composition on yield and biological efficiency of P. eryngii.

Substrate Composition

Yield (g)

Biological Efficiency (BE) (%)

75 %BSG+25 % WB

6.32±1.59

9.37±2.36

50 % BSG + 50 % WB

46.20±1.28

66.97±1.86

100 % BSG

These results concluded that BSG when supplemented with WB had significant effects on P. eryngii mycelia growth and mushroom yield. This is in agreement with other studies

suggesting enhanced growth of mushroom mycelia by supplementation of the substrate with wheat bran. Hasan et al. (2010) observed that 25% wheat bran supplementation in substrate made of sawdust, soybean straw, and rice straw media resulted in maximum mycelium linear growth. Similarly, the wheat bran supplementation to the substrate produced higher sclerotia biomass in the Penicillium sp. solid-state fermentation (Han & Yuan, 2003). Schnurer (1991) measured fungal growth in terms of colony forming unit and ergosterol content and showed that the highest growth was visible in wheat bran compared to other components of wheat grain. In a study conducted by Hassan and Bullerman (2009), wheat bran gave highest mycelial growth and macro conidia formation by Fusarium species. Likewise, 10% addition of wheat bran as organic nitrogen supplement gave 20% higher biological efficiency by P. ostreatus var florida grown on

48 wheat straw (Upadhyay et aI., 2002). A nuclear magnetic resonance (NMR) biodegradation study of wheat bran during P. ostreatus growth showed changes in molecular composition of wheat bran with preferential loss of hemic elluloses and possibly other amorphous carbohydrates (Locci et aI., 2008). This suggested wheat bran as a good substrate for the cultivation of P. ostreatus.

The fungal species ability to grow and form fruiting bodies is dependent on both fungal and substrate associated factors. The fungal associated factors include its ability to produce necessary hydrolytic and oxidative enzymes necessary to break down and utilize major components of the substrate. The substrate associated factors include the substrate physical and chemical structure, nutrient content, and presence of inhibitory substances. Various authors have reported the increase in mushroom yield by the supplementation of substrate with protein rich, carbohydrate rich or oil-rich substances like soybean meal, cotton seed hull, wheat bran, rice bran, alfalfa meal, bagasse, or corn gluten meal (Rodriguez & Royse, 2007; Lara et aI., 2002; Wang et aI., 2001). Even in P. eryngii cultivation study, 20% rice bran supplementation to wheat straw-cotton straw yielded fruiting bodies with BE of 77.2% compared to BE of 48.6% for rice bran unsupplemented substrate (Kirbag & Akvuz, 2008). The need for substrate supplementation is to complement the nutrient requirement of the fungi in terms of both organic (Carbon and Nitrogen) and inorganic components. Although, both carbon concentration and C:N ratio in the substrate have significant effect on fungal growth, C:N ratio was shown to be more influential than carbon concentration (Gao et aI., 2007). The optimum C:N ratio requirements for growth on substrates varies among fungal isolates even within the same species. The P. eryngii being oyster mushroom requires more carbon and less nitrogen in the substrate than button mushroom (Kang, 2004). But still, all the substrate used in

production needs to be supplemented with nitrogen source like wheat bran and rice bran to reach optimal C:N ratio. A previous study by Philippoussis et aI. (2001) indicated positive correlation between P. eryngii mycelia growth and mushroom yield with substrate C:N ratio (Philippoussis, et aI., 2001). Nitrogen is an essential element for cellular functions for growth and various metabolic activities particularly protein and enzymes synthesis (Buswell et aI., 1993). It is also essential especially during the fructification of the mushroom and depletion of it in substrate can substantially affect the mushroom yield. Nitrogen is also considered a rate-limited factor for extension of fungal growth and decay in wood material showing the effects of nitrogen concentration on susceptibility of the wood to decomposition (Boyle, 1998). Wheat bran supplementation could have shown better growth due to both physical structure and nutrient content of the wheat bran. Substrate particle affects both vegetative growth and yield of mushrooms (Royse, 2001). Wheat bran particles increase the porosity within the substrate which allows better oxygen supply and hence higher biomass. In the study, the compositional and nutrient variation of WB with BSG could have largely affected the growth of P. eryngii in different substrate composition. Table 2 compares the composition of BSG with

WB and other substrates (cotton waste and saw dust) used in mushroom cultivation. The noticeable important differences observed in the BSG and WB are the nitrogen content and carbon to nitrogen ratio (C:N). WB (2.6%) has higher nitrogen than BSG (1.02%), C:N ratio being 21: 1 in WB and 35: 1 in BSG respectively. Wheat bran also contains about 10-20% of starch. Starch can be easily broken down into glucose providing simple nutrient available for fungal growth. In summation, the presence of some extra nutrients in the form of nitrogen, amino acids, starch resulted in better growth and yield of P. eryngii in WB supplemented substrate.

50 Table 2.

Proximate composition of agricultural substrates commonly used in the cultivation of edible fungi Composition

BSG

Cotton Waste

Saw dust

Cellulose (%)

Lignin (%)

Total Carbon (%)

Total Nitrogen (%)

1.02

2.6

0.41

0.1

C:N ratio (%)

35: 1

21 :1

59:1

491 :1

(Jang et aI., 2000; Choi, 2004; Aliyu & Bala, 2011; Locci et aI., 2008)

Table 2 also shows that BSG provides better nitrogen source than cotton waste and sawdust which are commercially used substrate for mushroom cultivation. However, cellulose content is lower in BSG than those two substrates. It indicated that BSG can be used as nitrogen source which will require some carbohydrate source to complement the substrate for the mushroom cultivation. Different strains of king oyster mushroom response differently to different substrates, supplements, supplementation amount and environmental factors in the aspects of mycelia growth, biological efficiency and quality (Moonmoon et aI., 2010). Basically, individual strains' growth requirement variation with respect to N or protein, and carbon in the substrate is the attributing factor. Therefore, P. eryngii WC888 and WC943 when grown on BSG-WB combinations behave differently and resulted in variation in growth rate and yield in the single substrate combination. This was in conformity with Rodriguez and Royse (2007) who also observed significant differences in the yield, BE, number of mushroom and mushroom size

51 between WC888 and WC846 strains of P. eryngii. Similarly, type of P. eryngii strain and growth substrate composition were shown to have effect on mycelia run rate, number of fruiting bodies, biological yield and efficiency (Moonmoon et aI., 2010). Our result is comparable with the study done by Wang et al. (2001) using BSG for the cultivation of P. ostreatus. In the study, the replacement of saw dust with BSG increased the biological efficiency of mushroom by almost 3 times (5.8% dry wet basis in sawdust substrate to 16.9% in BSG substrate). As with our result wheat bran supplementation increased the biological efficiency, an optimum supplementation level of 45%. Similarly, BSG without any supplementation resulted in fewer fruiting bodies. Lack of low molecular carbohydrates and/or vitamins in the BSG was thought to be responsible for the lower yield of BSG alone substrate. Rodriguez and Royse (2007) studied growth of P. eryngii WC846 and WC888 strain using cottonseed hulls/sawdust as basal substrate at various Mn, Cu, and ground soybean supplementation level. The highest biological efficiencies obtained at optimum level of supplementation were 64.8% for Mn supplements, 59% for Cu supplements, and 63.6% for soybean supplements. Elsewhere the biological efficiency of Pleurofus ostreatus and Pleurotus pulmonarius grown with 50% BSG + 50% maquey tequila bagasse were 29.2% and 25.9% respectively (Lara et aI., 2002). Gregori et al. (2008) showed highest biological efficiency of 51 % for Pleurotus ostreafus mushroom at 10% BSG addition to sawdust and wheat bran substrate. 50% BSG + 50% WB combination without any other supplementation showed higher biological efficiency (66.97%) than those obtained from previous work as described above. One reason for the BSG alone showing slower growth rate and not able to produce fruiting bodies could be higher content oflignin (28%) compared to that in wheat bran (8%). Lignin moiety binds cellulose and hemicellulose preventing fungal hydrolyzing enzyme's access

52 to those polysaccharides. This could have markedly reduced the ability of P. eryngii to grow and form fruiting body in presence of high lignin content ofBSG. Also lignocellulosic components might produce some phenolic monomers during lignin degradation which are thought to be inhibitory to fungal growth and hydrolytic enzymes responsible for breakdown of plant cell wall polysaccharides (Buswell et aI., 1993). Production of such inhibitory substances might have also resulted in lowered growth rate and yield of P. eryngii in BSG substrate. Boyle (1998) investigated nutritional factors affecting the growth pattern of various white-rot fungi on wood. Carbohydrate supplementation, regardless of type or concentration had no effect on the growth indices. However nitrogen added in the form of glutamate caused an increase in all the growth indices. He also suggested that nitrogen supplementation in a complex form caused lesser inhibition of lignin degradation as oppose to nitrogen supplementation in simple form. In general, N sources inhibited lignin degradation by white rot fungi except for P.

ostreatus and L. edodes. In our study, P. eryngii might have behaved like those species which can be said by that nitrogen supplementation (WB) produced slightly higher growth rate and yield indicating that the N supplementation did not largely inhibit the lignin degradation which otherwise would have reduced the growth rate of the P. eryngii in the WB supplemented substrate. The results with different dilution levels or substrate composition are supportive that nitrogen concentration variations in the substrate may affect lignin degradation and the resulting differences in the apical growth rate of P. eryngii.

Pleurotus eryngii colonization in 100% BSG in both media plates and cultivation substrate indicated that BSG was self-supporting nutrient substrate for mycelia development (and associated enzyme viz. laccase, MnP, AAO production). For the study BSG was dried to prolong the storage life until it was used. Instead, wet BSG sourced directly from breweries or

53 water-rinsed BSG may also be used as they have shown to increase the biological efficiency of the mushroom produced (Wang et aI., 2001). Therefore, in commercial production, the direct use of wet BSG may be preferable as it may avoid additional drying and treatment costs.

54 Chapter V: Conclusion

Brewers' spent grain was successfully used as basal substrate material for the cultivation of P. eryngii. BSG when supplemented with wheat bran increased P. eryngii colony extension rate in plates and in larger cultivation-scale bottles. Out of three combinations, 50% BSG and 50% WB substrate combination showed the highest mycelium growth rate of P. eryngii. This combination resulted in the highest biological efficiency (66.97%) for P. eryngii WC888 genotype. The BE of P. eryngii fruiting bodies produced from BSG was higher than the BE of many substrates currently being used in commercially mushroom production. The study showed that BSG alone can support growth of P. eryngii mycelia. This could draw wide interest for the use of BSG in various biotechnological applications other than mushroom cultivation like in enzyme, organic acids and other hydrolysis preparation, and also in bioremediation of organopollutant-contaminated sites. BSG should not be considered as industrial waste, instead should be taken as a prominent resource for recycling into a value-added product for generating a sustainable profit and utilization. The study also concluded that P. eryngii with its fast mycelia growth rate and multilateral enzyme system is able to utilize BSG as nutrient source for its growth and mushroom production. The nutrient type and concentration in substrate plays significant role in the P. eryngii mycelium growth and biological efficiency ofthe cultivated mushroom. The ability of P. eryngii to grow on substrate is largely dependent on the strain type also. Genotype and nutrient requirement variation of strain influences the growth pattern, yield and size of fruiting body of mushroom produced from different strains. The large continuous supply and low-cost availability of BSG make it potential substrate in the commercial cultivation of mushroom. This efficient recycling of BSG can lead to benefits

55 from both economic and environmental standpoints. Brewers can largely save their disposal cost and also pollution issues associated with BSG after its proper direction. Mushroom growers on other hand will have access to voluminous substrate locally at low cost that will generate high productivity and potential profit to their business. It is also more likely that BSG use in mushroom industry could markedly reduce the production cost and market price of the P. eryngii mushroom which would be in interest of the consumers also. Recommendations for future:

Further extensive work needs to be carried out before the larger scale utilization of the BSG in mushroom production. Following studies can be done for future research work with the aim of the optimization ofBSG as a basal substrate for cultivation of mushroom and other various biotechnological applications. 1. The growth rate study of P. eryngii showed BSG as suitable nutrient reserve for the mycelia growth of the P. eryngii. In addition to that, the extent of growth can be quantified by various techniques like measuring fluorescein diacetate hydrolyzing activity, protein and chitin content, lignin and dry weight losses, and others. This would give understanding of mycelia biomass and bioactivity of the P. eryngii on BSG. 2. The type and amount of enzymes determine the ability of the fungi to grow in that particular substrate. Enzyme production largely depends upon the type of substrate used. Determination of the activity of various extracellular enzymes like laccase, manganese dependent peroxidase (MnP) and aryl-alcohol oxidase (AAO) will help better understand the suitability of the growth of fungus for mushroom and even for other enzyme production from utilizing BSG as a substrate.

56 3. The biological efficiency ofthe mushroom produced from 50:50 combination ofBSG and WB was very high compared to most of the previous work. The proximate composition of fruiting body produced from BSG would be helpful to judge the nutritive value of BSG produced mushroom compared to produce from other substrates. 4. The study had shown that the supplementation of wheat bran significantly increased the growth rate and yield of P. eryngii. Similarly, the supplementation ofBSG with other nutritious supplements like wheat straw, cottonseed hulls, and soybean meals should be compared to determine optimum substrate combination for P. eryngii growth in BSG. Since total nitrogen, carbon:nitrogen content of substrate proved to be very essential for the mycelia formation and yield, BSG should be evaluated with other substrates to optimize the carbon:nitrogen ratio. 5. The bulky nature and high moisture content ofBSG are major challenges to its feasible utilization. Economical means of drying BSG in brewery and/or limited cost transportation locally to recycle location should be mechanized efficiently for sustainable BSG application.

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67 Appendix A: Apical Extension Rate of P. eryngii mycelia

Pleurotus eryngii WC888 Dilution levels

Substrate combinations 1:20

1:10

1:05

100% BSG

175.5 ± 6.22

137.6 ± 5.26

82.44 ±7.87

75% BSG + 25% WB

213.89 ± 14.6

196.13 ± 8.57

140.08 ± 12.59

50% BSG + 50% WB

230.36 ± 11.05

213.79 ± 9.68

149.6 ± 13.59

II.

Pleurotus eryngii WC943 Dilution levels

Substrate combinations 1:20

1:10

1:05

100% BSG

157.04 ± 3.38

124.4 ± 9.3

87.9 ± 2.98

75% BSG + 25% WB

210.12 ± 10.3

171.43 ± 10.37

112.7 ± 5.67

50% BSG + 50% WB

222.03 ± 3.1

180.16 ± 9.34

138.69 ± 10.04