Mushroom Biotechnology: Developments and Applications

By Marian Petre

Mushroom Biotechnology: Developments and Applications is a comprehensive book to provide a better understanding of the main interactions between biological, chemical and physical factors directly involved in biotechnological procedures of using mushrooms as bioremediation tools, high nutritive food sources, and as biological helpers in healing serious diseases of the human body.

The book points out the latest research results and original approaches to the use of edible and medicinal mushrooms as efficient bio-instruments to reduce the environment and food crises. This is a valuable scientific resource to any researcher, professional, and student interested in the fields of mushroom biotechnology, bioengineering, bioremediation, biochemistry, eco-toxicology, environmental engineering, food engineering, mycology, pharmacists, and more.

• Includes both theoretical and practical tools to apply mushroom biotechnology to further research and improve value added products
• Presents innovative biotechnological procedures applied for growing and developing many species of edible and medicinal mushrooms by using high-tech devices
• Reveals the newest applications of mushroom biotechnology to produce organic food and therapeutic products, to biologically control the pathogens of agricultural crops, and to remove or mitigate the harmful consequences of quantitative expansion and qualitative diversification of hazardous contaminants in natural environment

• Language: English
• Publisher: Academic Press
• Release date: Oct 14, 2015
• ISBN: 9780128028049

Marian Petre

Marian Petre is professor of biotechnology for environmental protection, microbial biotechnology, bioremediation, microbial ecology, and bioengineering at University of Pitesti, Faculty of Sciences. He has published more than 150 scientific articles, 73 of them in international journals and proceeding volumes all over the world; he is also the first author of 10 Romanian patents in mushroom biotechnology. So far, he has written and edited 25 books on applied biotechnology, environmental biotechnology, microbiology, bioremediation and microbial ecology.

Book preview

2015

Preface

Marian Petre, Editor, University of Pitesti, Pitesti, Romania

Mushrooms are considered one of the most diversified groups of biological species adapted for living in extreme environmental conditions all over the Earth. For centuries, many mushroom species have been used as outstanding sources of food and medicine, but in the recent past humans have discovered some of their powerful features to clean the environment through the bioconversion of organic residues from the habitats where they live and continuous recycling of chemical elements.

Nevertheless, for humankind, there is as an urgent need to sustain the efforts to change the current status of serious crises in food, human health, and environmental pollution through the beneficial applications of mushroom biotechnology!

In this respect, a better understanding of the main interactions between biological, biophysical, and biochemical phenomena and processes involved in biotechnological applications of using mushrooms as one of the most important biologic tools for maintaining environmental health will be a key solution for the future progress of humanity.

Mushroom biotechnology is defined as a component discipline of mushroom biology applications including mushroom cultivation, mushrooms for biocontrol of phytopathogens, and mushrooms as bioremediation agents. In this respect, a new field of using mushrooms in cleansing organic and inorganic wastes from the environment has been developed as mycoremediation.

The book Mushroom Biotechnology—Developments and Applications has been conceived as a synthetic mirror of recent scientific achievements in the fields of controlled cultivation of culinary and medicinal mushrooms as organic sources of food and medicines, automatic cultivation and processing of mushrooms, biocontrol of pathogens and pests, improvement of mushroom breeding by genetic methods, as well as biodegradation of recalcitrant contaminants through the application of advanced mycological biotechnology.

The content is divided into 12 chapters, each of which provides detailed information regarding scientific experiments carried out in various countries of the world to test novel applications designed to shed light on the beneficial effects of mushroom biotechnology.

The first three chapters are focused on biotechnology for conversion of organic agricultural wastes, both through submerged and solid-state cultivation of culinary and medicinal mushroom species. Chapter 4 has as its main subject the automatic cultivation and processing of mushrooms through a modular robotic prototype designed to produce both fruit bodies and sterilized and inoculated bags filled with mycelium of culinary and medicinal mushrooms. The next two chapters describe the biotechnology of Agaricus bisporus cultivation as well as specific methods for pathogen control in this button mushroom species. The seventh chapter presents current perspectives on sclerotia-forming mushrooms as an emerging source of medicines. Then, the next two chapters characterize medicinal mushrooms regarding their specific antiphytopathogenic and insecticidal properties as well as their cultivation in different types of bioreactors.

Chapter 10 relates to the use of Aspergillus niger extracts obtained by solid-state fermentation for enzyme production, and the next chapter highlights the identification and application of Volvariella volvacea mating type genes in mushroom breeding. The last chapter focuses on the biotechnological use of fungi for degradation of recalcitrant agro-pesticides.

This book is especially addressed to researchers, students, and specialists in mushroom biotechnology, mycological research, food biotechnology, environmental biotechnology, bioengineering, and bioremediation, but also all readers who want to improve their knowledge of biotechnological applications of mushrooms for the well-being of human society.

In conclusion, after a whole year of tremendous editorial activity, I would like to thank each of the contributors for their considerable efforts to present the most valuable achievements in their fields, and I really hope that readers will be interested in the scientific content of these chapters.

In addition, I take real pleasure in expressing my sincere gratitude toward Patricia Osborn, the Senior Acquisitions Editor of Elsevier Books Division, for her remarkable professionalism and kindness in support of this book project from the beginning of our cooperation in order to achieve such outstanding work!

Last but not least, my warm and sincere thanks are forwarded to Editorial Project Managers Jaclyn Truesdell, Lisa Jones and Carrie Bolger for their careful assistance and great patience during our joint work, as well as to whole staff of Elsevier Inc. for their professional involvement in publishing this book!

May, 2015

Chapter 1

Biotechnology of Mushroom Growth Through Submerged Cultivation

Marian Petre¹ and Violeta Petre²,    ¹Faculty of Sciences, University of Pitesti, Pitesti, Romania,    ²Department of Biology, Sfântul Sava College, Bucharest, Romania

Abstract

The submerged cultivation of mushrooms (SCM) requires full control of the bioprocess regarding the automatic tracking of all chemical and physical parameters, keeping them at optimal values. The main problem that needs to be solved by the intensive biotechnological process for submerged cultivation of edible and medicinal mushrooms on substrates made of agricultural wastes resulting from cereal grain processing is to convert these natural wastes of organic agriculture into nutritive biomass to be used as food supplements that are made through biological means only. The main stages of biotechnology to achieve highly nutritive mycelial biomass by controlled submerged fermentation are as follows: (i) preparation of culture substrates, (ii) steam sterilization of the bioreactor culture vessel, (iii) aseptic inoculation of sterilized culture media with the pure cultures of selected mushroom strains, (iv) running the submerged cultivation cycles under controlled conditions, and (v) collecting, washing, and filtering the fungal pellets which were obtained.

Keywords

Biotechnology; biomass; mycelium; mushrooms; submerged cultivation

1.1 Introduction

From the beginning of this century, the submerged cultivation of culinary and medicinal mushrooms has received a great deal of attention as a promising and reproducible alternative for the efficient production of mycelia biomass and fungal metabolites. Due to economic reasons, the submerged cultivation of mushrooms (SCM) has gained an ascending attention due to its significant potential for industrial applications, but its prospective success on a commercial scale depends on increasing product yields and development of novel production systems that address the problems associated with this biotechnology of mushroom cultivation.

In the recent literature, there are described several methods of growing strains of Basidiomycetes in submerged cultures, which provide an opportunity to get a huge production of biomass containing high concentrations of bioactive compounds with healthful effects on humans, such as proteins, essential amino acids, vitamins, and polysaccharides (Verstraete and Top, 1992; Smith, 1998; Stamets, 2000; Sanchez, 2004; Wasser, 2010).

Any technology for bioprocessing raw materials or their constituents into bioproducts requires the following three steps: process design, system optimization, and model development. To achieve all these steps, a biotechnological proceeding involves the use of biocatalysts, as whole microorganisms or their enzymes, to synthesize or bioconvert raw materials into new and useful products. At the same time, optimization of any submerged cultivation bioprocess is essential for biotechnology development in an industrial-scale application. In this respect, it should be taken into consideration that physical and chemical factors interact and affect the efficacy of the bioprocess regarding mycelia growth within the liquid medium. However, for the time being, in spite of research into optimizing the production of bioactive metabolites by synthesis by mushrooms, the physiological and engineering aspects of all submerged cultures are still far from being thoroughly studied (Wood, 1992; Wedde et al., 1999; Elisashvili, 2012).

1.2 The Concept of SCM

First of all, it is necessary to point out that the SCM has an exclusive and specific character concerning fungal cell growth and development in totally different conditions compared with the natural environment where all native mushrooms exist.

This means that the concept of SCM refers to a biotechnological process of mushroom growth inside an artificial environment represented by the volume of a liquid medium in which all physical and chemical factors needed for optimal development of mycelium are provided without any risk of chemical or biological contamination.

The specific status of all mushroom species as native or indigenous fungi is to grow and develop in natural habitats in terrestrial ecosystems; in other words, they are species adapted to colonize only solid substrates containing a certain amount of water and involving living organisms or organic structures accumulated outside or inside the soil (Vournakis and Runstadler, 1989; Wedde et al., 1999; Uphoff, 2002).

More precisely, no known mushroom species has any capability of growing and developing in natural aquatic habitats; more than that, none of them is adapted to form fruiting bodies inside a liquid medium. This is a restrictive living condition for all native mushroom species of planet Earth, by which they are compelled to live only inside terrestrial ecosystems from the natural environment due to their strictly specific adaptation to aerobic respiration.

The cellular metabolic processes of any mushroom species require permanent oxygen intake in appropriate concentrations, supplied from the outer environment of the mycelia, and this cannot be achieved inside a liquid volume of any natural environment where there does not exist the proper concentration of dissolved oxygen (DO) in order to maintain the mushroom’s life!

Mushroom species have great potential for adapting to any habitat which provides a solid support and containing only a small amount of water to sustain their natural life cycle. If this support is entirely formed by water, there is no chance for a mushroom strain to survive due to the lack of DO intake to the membrane surface of fungal cells. In such circumstances, the only way to artificially grow mushroom species inside a liquid medium is to keep the dissolved oxygen concentrations (DOC) at required levels to maintain the mushroom’s metabolic activities by using special devices to force oxygen penetration inside the liquid volume.

Thus, despite both the shear forces and turbulence generated by oxygen intake inside the liquid cultivation medium from the culture vessel of a bioreactor, the mycelium is forced to move circularly according to the specific rheology of such a medium. During the cultivation process, the fungal cells are able to grow in submerged conditions and, due to centrifugal force, these cells metabolize the nutritive particles from the cultivation medium and develop as a biomass containing many mycelium pellets of different sizes and almost rounded shapes.

1.3 Methods and Techniques used for SCM

As a general matter, SCM requires full control of the cultivation bioprocess regarding the automatic tracking of all chemical and physical parameters and keeping them at optimal values.

This biotechnological method permits fully standardized production of the fungal biomass with high nutritional value or the biosynthesis of mushroom metabolites with a predictable composition. At the same time, the downstream processing after submerged cultivation is very feasible and easier to carry out as compared with the classical procedure of solid-state cultivation. Inside the cultivation vessel of a bioreactor, it is possible to control the culture conditions, such as temperature, agitation, DOC, temperature, substrate and metabolite concentration, as well as the pH inside the liquid culture substrate (Kim et al., 2007; Elisashvili, 2012; Turlo, 2014; Homolka, 2014).

It is well known that the morphology of mycelia in submerged cultures has a significant influence on the rheology of the culture broth. At the same time, the initial viscosity of the liquid medium, as well as the stirring speed and air intake pressure, have important effects on fungal pellet formation during the cultivation cycle of mushroom spawn. Thus, the agitation rate and dispersion effect induced by shear forces upon the fragile structure of the mycelium, especially in the first period of time during a cultivation process, have determinant influence upon the fragile structure of the mycelium which is to develop inside the liquid culture medium as fungal pellets with different shapes and sizes. After many experiments to study the effects of stirring rate and share forces, it was noticed that an inverse relationship exists between agitation speed and pellet features. In fact, increased agitation determines the formation of small and very compact pellets; on the other hand, a vigorous agitation seems to prevent pellet formation (Park et al., 2001; Papagianni, 2004; Turlo, 2014).

Along the evolution of submerged cultures, the mushroom mycelia generate globular shaped aggregates called pellets. The morphological forms of pellets are characteristic of each mushroom species. In any submerged culture, the pellet size determines the oxygen and nutrient transport into its center. In the core region of a large pellet, the fungal cells stop their growth because of low DOC and nutrients, and for this reason the smaller pellet diameter could be advantageous in terms of increased mycelia biomass (Lee et al., 2004; Kim et al., 2007; Elisashvili et al., 2009; Xu et al., 2011; Turlo, 2014).

However, pellet size is influenced by various variables, such as agitation regime, density of the inoculums, and sugar concentration in the culture medium (Petre et al., 2010).

During the cultivation process, the culture viscosity increases significantly, and sometimes, mushroom mycelia start to wrap around impellers, spreading into the sampling devices and feed tubing with nutrients, causing functional blockages. All these problems limit the operation time of bioreactors, and they must be avoided by constant control and correction of the culture density (Shih et al., 2008; Elisashvili, 2012; Turlo, 2014).

While the SCM mycelia induce relatively high energy costs required for agitation, oxygen supply, and constant control of the temperature of the liquid medium during the whole cultivation process, this biotechnological method has significant industrial potential due to the possibility of process upscaling and operation of large-scale bioreactors.

There are many biotechnological methods for cultivating the mycelia of edible mushrooms in liquid media by applying various strategies. In this respect, batch culture is one of the most frequently used biotechnological methods for the SCM. In this cultivation method, no fresh nutritive elements are added to the culture composition and no end products of fungal metabolism are discharged during the process.

The simplest technique used for this kind of cultivation is based on shake flask cultures in order to get relatively small quantities of mycelia that can be used as inocula for the larger production of mycelia biomass by growing in the culture vessels of laboratory-scale bioreactors designed for batch cultures (Porras-Arboleda et al., 2009; Lin, 2010; Xu et al., 2011; Elisashvili, 2012; Petre and Petre, 2013; Homolka, 2014).

1.4 Biotechnology for Submerged Cultivation of Pleurotus ostreatus and Lentinula edodes

The main problem that needs to be solved for the intensive biotechnological process of submerged cultivation of edible and medicinal mushrooms on nutrient substrates made of agricultural wastes resulting from cereal grain processing is to convert these natural waste products of organic agriculture into nutritive biomass to be used as food supplements that are made only through biological means (Petre et al., 2014a; Petre and Petre, 2013).

In our recent studies on the application of laboratory-scale biotechnology for submerged cultivation of culinary mushrooms, we tested two Basidiomycetes species, described in the following lines.

Lentinula edodes (Berkeley) Pegler is a heterothallic mushroom species belonging to Basidiomycetes group. The optimum temperature for spore germination is 22–25°C, but for mycelial growth temperature can range from 5°C to 35°C. The species of the genus Lentinula can grow on various culture media, both natural and synthetic, depending on the cultivation procedure, and they have certain morphological and physiological characteristics that distinguish them from other types of mushrooms (Carlile and Watkinson, 1994; Hawksworth et al., 1995; Jones, 1995; Hobbs, 1995).

Pleurotus ostreatus (Jacquin ex Fries) Kummer, also known by its popular name as the oyster mushroom, is a Basidiomycetes species belonging to the family Pleurotaceae (Agaricales, Agaricomycetes). The species have carpophores with eccentric pileus and decurente blades showing white or hyaline enhanced with cylindrical or oval forms (Chahal and Hachey, 1990; Carlile and Watkinson, 1994; Hawksworth et al.,