Industrial Mycology

UNESCO – EOLSS

SAMPLE CHAPTERS

BIOTECHNOLOGY –Vol. VI - Industrial Mycology - J.S. Rokem

©Encyclopedia of Life Support Systems (EOLSS)

INDUSTRIAL MYCOLOGY

J.S. Rokem

Department of Molecular Genetics and Biotechnology, The Hebrew University of

Jerusalem, Jerusalem, Israel

Keywords: fungus, solid substrate, liquid substrate, metabolites, enzymes, flavors,

therapeutic compounds, food, cheese, heterologous protein

Contents

1. Introduction

2. Product Range

2.1. Metabolites

2.2 Enzymes

2.3. Biomass

2.4. More recent and potential products

3. Solid State Fermentation

3.1 Products from Solid State Fermentation

3.1.1 Gibberellic acid – GA

3.1.2 Glucoamylase

4. Submerged Fermentation

4.1. Selected metabolites produced by Submerged Fermentation

4.1.1 Lovastatin

4.1.2 Red Monascus Pigments

4.1.3 Rennet (Chymosin) from Mucor

4.1.4 Quorn

5. Other Developments of Industrial Mycology

5.1. Heterologous Proteins by Filamentous Fungi.

5.2 Flavoring Agents

5.3. Cheese Made with Fungi

5.4 Higher Fungi for Food Flavor and Medicine

6. Conclusions

Glossary

Bibliography

Biographical Sketch

Summary

Filamentous fungi are used by industry for manufacture of a large variety of useful

products, all for the benefit of humankind. Examples of how some of these products are

formed by an assortment of fungi and produced on a large scale are presented. The

products include metabolites, enzymes and food. Fungal cells can grow at different

environmental conditions. The chemical and physical conditions used for fungal

propagation will have a great impact on the capability of these cells to accumulate the

desired product(s).

Processes using solid state and submerged fermentations are described illustrated by a

UNESCO – EOLSS

SAMPLE CHAPTERS

BIOTECHNOLOGY –Vol. VI - Industrial Mycology - J.S. Rokem

©Encyclopedia of Life Support Systems (EOLSS)

few of the metabolites produced by industry. There are great economic benefits in the

use of filamentous fungi and there is a great potential for these organisms to produce

novel items.

Industrial processes using fungi are of great economical importance. The products are

unique and there is usually no other economic way to manufacture these products. In

this article only a few compounds will be described in greater detail, but it should be

remembered that there are many more that are manufactured using filamentous fungi.

1. Introduction

Fungi have several distinct characteristics that make them different from other life

forms. They grow in a filamentous branching mode, with apical growth, lateral

branching and by heterotrophic nutrition [see also – Mushroom Production]. Their life

cycle begins by germination of their resting structure or spore. Vegetative growth

follows; where the biomass increase as the substrate is utilized and novel cells (hyphae)

are formed. The fungal hyphae form a porous three-dimensional net that is known as

mycelium. At a certain stage in the life cycle, usually when there is lack of nutrients,

sporulation may occur, where morphologically distinct structures are produced that can

detach from the mycelium. Spore formation is not found for all filamentous fungi, in

those cases were spores are not found, it is usually due to incorrect nutritional

conditions and when those are found, spores will be produced. Most filamentous fungi

are found growing in their imperfect stage, where no sexual cycle is known, as for

others a perfect stage (a sexual cycle) is found, also dependent on the right nutritional

conditions The filamentous fungi play an important role in the biosphere, where their

decomposing action of organic material, leads to the restoration of substrates like

carbon, nitrogen, phosphorus and minerals to the biosphere. The filamentous fungi are

utilized for many different purposes, in the food industry, for the manufacture of useful

metabolites and in a variety of other processes (Table 1).

Food Applications Useful Products Other Processes

Baking Alkaloids Biobleaching/biopulping

Brewing Antibiotics Biological control agents

Cheese-making Ethanol Bioremediation of soils

Mushroom cultivation Enzymes Coal solubilisation

Oriental food fermentations Gibberellins Dyes/dye intermediates

Quorn myco-protein Immunomodulators Microencapsulation

Organic acids Mycorrhizal inoculants

Polysaccharides Steroid bioconversions

Vitamins Waste treatment

With permission. Fungal Biotechnology by P.F.Hamlyn, North West

Fungus Group (NWFG) Newsletter, April 1997.

Table 1: Uses of filamentous fungi

Yeast, that in specific cases also can grow in a filamentous mode, are dealt with in other

articles [see also – Production of Alcoholic Beverages]. The industrial production of

both primary and secondary metabolites from filamentous fungi is well established [see

UNESCO – EOLSS

SAMPLE CHAPTERS

BIOTECHNOLOGY –Vol. VI - Industrial Mycology - J.S. Rokem

©Encyclopedia of Life Support Systems (EOLSS)

also – Production of Organic Acids; – Production of Antibiotics]. There are also articles

describing different uses of filamentous fungi like Nutriceuticals from Mushrooms [see

Nutriceuticlas From Mushrooms] and Mushroom Production [see Mushrooms

Production]. In this article so called “micro fungi” are the main agents for which

production are described.

As can be seen in Table 1, there is a large assortment of metabolites produced by

filamentous fungi, including vitamins, polysaccharides, enzymes, immunosuppressive

agents, hypercholesterolemic agents, pigments, antibiotics and organic acids. For each

manufactured goods, a specific fungus and its growth conditions are characterized

thoroughly. The determination of optimal product conditions start with the initial

research finding, of a potentially commercial metabolite, followed by further research

and development to a full scale industrial process (see also - Process Optimization

Strategies for Biotechnology Products). For many cases, information of the actual

industrial production process of a specific compound by filamentous fungi is

confidential, and therefore it is not possible to acquire specific information for the

process. In this article knowledge is presented that is available in the public domain.

Information will be given for selected metabolites where more detailed information is

available on their industrial production. There is a plethora of fungal products and in

this article is described a few examples, considered to characterize some of the methods

and products of industrial mycology.

Some of the products of industrial mycology are made on a routine bases, however, the

detailed technical details are not publicly available. The products described are a biased

choice, where public information is available, containing more or less traditional

compounds. Other trends will also be discussed, specifically the attempts to use

filamentous fungi for the formation of heterlogous proteins for various applications and

higher fungi for food and therapeutical purposes.

2. Product range

The variety of metabolites produced by filamentous fungi by industry is vast. In this

article different categories of compounds are described with the intention to give a

representative presentation.

One group are called “metabolites”, by which is meant small molecular weight (> 1000

Dalton) compounds that can be either primary or secondary metabolites. The structural

complexity, including different stereochemistry and also chirality, is usually the main

reason why these “metabolites” are produced by biological methods and not by

chemistry. This is of course related to the economy of production, since the chemical

route would result in a much more expensive product.

The following products will be described in some more detail in this article.

2.1. Metabolites

The metabolite named gibberellic acid, a representative of the gibberellins, is used as

plant growth stimulators, produced by growing the fungus Giberella fujikuroi on solid

UNESCO – EOLSS

SAMPLE CHAPTERS

BIOTECHNOLOGY –Vol. VI - Industrial Mycology - J.S. Rokem

©Encyclopedia of Life Support Systems (EOLSS)

substrates. Gibberelic acid stimulates both cell division and cell elongation, and hastens

plant maturation and seed germination. It is applied to growing crops (field crops, small

fruits, vines and tree fruits), ornamental and shade tress, and ornamental plants, shrubs

and vines.

Another group of metabolites are the red pigments produced by Monascus species,

traditionally achieved by cultivation of the fungus on rice grains or bread. It is a

common process in South East Asia. Submerged fermentation methods have partially

replaced the solid state fermentation process [see also – Microbial Cell Culture] for

production of the Monascus red pigments (monascorubramine and rubropunctamine)

[see also – Natural Food Colorants].

Many of the most economically interesting metabolites are antibiotics [see also –

Production of Antibiotics]. There are other metabolites with different therapeutic

effects, like lovastatin (mevinolin), that belong to the group of statin drugs used for

hypercholesterolemia. Statins have a large market, estimated at $15 billion/year and was

first approved for clinical use by the FDA in 1987. The statins are manufactured by

submerged fermentation of filamentous fungi.

2.2 Enzymes

Enzymes have many beneficial characteristics which make them ideal as catalysts in a

large variety of reactions [see also – Enzyme Production]. Their main advantage is their

activity at ambient conditions, compared to the high temperature and pressure required

for many of the chemical reactions performed in industry. Their catalytic capability is

substantial and the amounts required are many times orders of magnitude less material,

than for chemical catalysis. Enzymes are versatile and are able to perform a multiplicity

of reactions. Novel activities are looked for and they are also compatible with

sustainable development. Enzymes are produced by all different cells and among them

also filamentous fungi. Enzymes from filamentous fungi are produced by industry and

also used by industry for many different purposes (Table 2).

Enzyme

Main Source

Asparaginase Aspergillus spp. and Penicillium spp.

Amylase

Aspergillus niger, Aspergillus. oryzae

Catalase A. niger, Penicillium spp.

Cellulase

A. niger, Trichoderma reesei, T. viride, Penicillium

finiculosum

Dextranase Penicillium spp.

ß-Glucanase

A. niger, Penicillium emersonii, T. reesei, T. viride

Glucoamylase

A. niger, A. oryzae

Glucose oxidase A. niger, Penicillium spp.

Hemicellulase

A. niger, A. oryzae, T. reesei, T. viride, P. emersonii

Laccase

Pyricularia oryzae

Lipase Several species including A.niger, A. oryzae

Pectinase Several species including A. niger, Rhizopus oryzae

Protease Several species including A. niger, A. oryzae

UNESCO – EOLSS

SAMPLE CHAPTERS

BIOTECHNOLOGY –Vol. VI - Industrial Mycology - J.S. Rokem

©Encyclopedia of Life Support Systems (EOLSS)

Rennet

Mucor miehei, Endothia parasitica

Tannase

A.niger, A. oryzae

Xylanase

A. niger, T. reesei

Table 2: Fungal enzymes of commercial importance

More and more enzymes are being introduced for novel purposes, and they are made by

other types of cells too. It is not possible to give a detailed description of the

manufacture of all the enzymes mentioned in Table 2. Therefore, in this article detailed

information will be presented for the production of glucoamylase. The glucoamylase

enzyme is a large volume product that is used by the sugar and starch industry to

generate glucose. The glucose is used for different purposes, for example the

conversion, by another enzyme, i.e. glucose isomerase, to high fructose syrup.

Glucoamylase is considered one of the main industrial enzyme products and the solid

state fermentation is described for the production of this enzyme (submerged

fermentations are also used for the production of this enzyme in industry).

Another enzyme of interest is the milk clotting enzyme, rennin (chymosin, rennet), used

in cheese production. The original source is the lining membrane of the fourth stomach

of the calf. There are many alternative sources for rennin that have been developed and

are also in use, since the supply of the fourth stomach of the calf is limited. One of the

ways to produce rennin is by submerged fermentation of the filamentous fungi of the

species Mucor.

2.3. Biomass

There are many foods produced with the aid of fungi, changing the texture and taste of

the material used as substrate (see also – Fermented Foods and Their Processing]. In

one case the growth of a filamentous fungus biomass under submerged conditions is the

source for a novel food. The fungal biomass has been named Quorn

to be described in

this article.

2.4. More recent and potential products

Other fungal products already in production and with a great promise for the future are

recombinant proteins [see also – Industrial Recombinant Protein Production], produced

by filamentous fungi, as well as the potential for growth of higher fungi in liquid

culture.

3. Solid State Fermentation

Solid State Fermentation (SSF) is defined as the growth of microorganisms on moist

solid substrate [see also – Microbial Cell Culture]. The growth can be on natural

substrates (termed Solid Substrate Fermentation) or on inert substrate used as solid

support. In both cases enough water is present to maintain microbial growth and

metabolism. There is no free-moving water and air is the continuous phase. SSF

technology provides many new opportunities, as it allows for the use of agricultural

waste products as fermentation substrates, without the need for extensive pretreatment

UNESCO – EOLSS

SAMPLE CHAPTERS

BIOTECHNOLOGY –Vol. VI - Industrial Mycology - J.S. Rokem

©Encyclopedia of Life Support Systems (EOLSS)

of the substrate.

SSF involves the heterogeneous interactions of microbial (fungal) biomass with

moistened solid support. In SSF, the microorganisms can grow between the solid

fragments, i.e., inside the matrix, or on the surface.

The microbial biomass inside the matrix and on the surface consumes substrates and

secretes metabolites and enzymes. As there is no active transport in the solid mass,

concentration gradients are the driving forces to supply the substrates and to remove the

products. Gradients in the concentrations of substrates and products may also cause

local differences in metabolic activity.

Similarly, gradients in the concentrations of inducers or repressors may affect

expression of different genes. These gradients are the most typical differences between

SSF and submerged fermentation (SmF) and it is assumed that the gradients contribute

to the observed differences in gene expression, metabolism, product spectrum, and

process efficiency between SSF and equivalent SmF processes.

The main disadvantage of SSF processes are that they are hard to reproduce compared

to SmF processes. However, SSF compared to SmF is claimed to be simpler, requires

lower capital, has superior productivity, reduced energy requirement, simpler

fermentation media, no need for rigorous control of fermentation parameters, uses less

water, produces lower volumes of wastewater and requires low cost for downstream

processing. In the SSF process, the solid substrate not only supplies the nutrients to the

culture, but also serves as an anchorage for the microbial cells.

Filamentous fungi are the most commonly used microorganisms in SSF. This is mainly

due to their high potential to excrete hydrolytic enzymes, their relatively high tolerance

to low water activities, and their morphology.

The filamentous fungi colonize the surface of the substrate and also penetrate into the

substrate matrix in search for nutrients, in those cases where the solid support is not

inert. SSF is ideal in cases where the purity of the product, for example in enzyme

production, is of less importance.

In many cases the enzyme formulation is the fungal biomass, where the enzyme activity

resides together with the substrate, after growth and production are terminated and used

as is as the “enzyme preparation”.

3.1 Products from Solid State Fermentation

3.1.1 Gibberellic acid – GA

Gibberellins are diterpenes synthesized from acetyl CoA via the mevalonic acid

pathway. The first gibberellin to be structurally characterized is referred to as GA

(Figure 1).

UNESCO – EOLSS

SAMPLE CHAPTERS

BIOTECHNOLOGY –Vol. VI - Industrial Mycology - J.S. Rokem

©Encyclopedia of Life Support Systems (EOLSS)

Figure 1: Chemical structure of gibberellic acid (GA

)

The elongation of rice plants induced by cell free culture filtrates of a fungus (Fusarium

moniliforme) was already demonstrated in 1926. Gibberelins function as plant growth

regulators, influencing a range of developmental processes in higher plants including

stem elongation, germination, dormancy, flowering, sex expression, enzyme induction

and leaf and fruit senescence. GA

is used in agriculture to eliminate dormancy of seeds,

in nurseries, for viticulture, in tea gardens, for induction of flowering and acceleration

of germination in the brewing industry. The annual world production of GA

estimated to exceed about 25 tons, with a market value of US $100 million. Improved

strains are available that produce GA

and also the precursors GA

and GA

concentrations of several grams per liter by specialized fermentation conditions.

The mechanism of action of the gibberellin GA

(the most studied gibberellin) is

thought to be due to molecular rearrangements in the cell wall matrix of the plants that

could promote wall extension.

The main producers are strains of Gibberella fujikuroi (the perfect stage of Fusarium

moniliforme) from which the genes have been cloned [see also– Genetic Engineering of

Fungal Cells] and well characterized. Certain agricultural and horticultural applications

specifically require GA

instead of the more traditional use of GA

. The final two

enzymes in the GA biosynthetic pathway leading to GA

in G. fujikuroi is of special

biotechnological interest. The inactivity of these enzymes allow for construction of

strains producing GA

and

as the main products of the G.fujikuroi, with the absence

or low concentrations of GA

. Such strains are of more interest for industry (see below).

Production of gibberellins by Gibberella fujikuroi is dependent on the quality and

quantity of the carbon and nitrogen source, and is stimulated by a high carbon to

nitrogen ratio. The initiation of gibberellins production is concomitant with the

exhaustion of the nitrogen source. The SSF fermentation is recommended by many

sources due to its advantages in use of agricultural waste and also with the claims for

higher concentrations of gibberellins. With substrates such as corn grains, wheat bran

and rice, the accumulation of GA

was reported to be 1.6 times higher than in

submerged culture, based on equivalent carbohydrate content of the medium. A

concentration of 3 g GA

per kg dry weight of the solid substrate was produced under

sterile conditions in a 50 liter fermentor with solids as substrate.

The use of submerged fermentation is well described in the literature and there are

several patents describing production conditions. The industrial process used for the

UNESCO – EOLSS

SAMPLE CHAPTERS

BIOTECHNOLOGY –Vol. VI - Industrial Mycology - J.S. Rokem

production of GA

is mainly based on submerged fermentation (SmF) techniques. The

fermentation conditions are growth of the fungus at a temperature of 28-32

C, at a pH of

between 5 and 7 and time of 4 to 6 days. A typical culture medium contains about 0.4 g

-1

of nitrogen source and about 75 g L

-1

of carbon source (usually a carbohydrate).

Despite the use of the advanced process technology, the yield of GA

reported for SmF

is lower then SSF. The presence of the product in dilute form in SmF was also

recognized as a major obstacle in economic manufacture of the product. This is due

mainly to the consequent higher costs of downstream processing and also disposal of

wastewater. The SSF technique is also at its advantage in countries with large

agricultural residues.

Recently mutants have been developed producing a dissimilar mixture of gibberellins,

with less GA

and more of GA

andGA

. GA

primarily stimulates the growth of stem

and leaves while GA

andGA

have their main effect on flowering and also cause fruit

cells to elongate, with the result of enhancement of fruit development. Concentrations of

over 1 g L

-1

are reported to be produced; where at least 50 % is GA

and GA

TO ACCESS ALL THE 23 PAGES OF THIS CHAPTER,

Visit: http://www.eolss.net/Eolss-sampleAllChapter.aspx

Bibliography

Bennet J.W. (1998). Mycotechnology: the role of fungi in biotechnology. Journal of Biotechnology,

66(2/3), pp. 101-107. [This is a description of the potential for use of recombinant DNA technology with

fungi]

Demain A.L., J. Velasco and J.L. Adrio (2004). Industrial mycology: past, present and future. In:

Handbook of Industrial Mycology. (ed Z. An), pp. 1-25, Marcel Dekker Publishers, New York, N.Y.,

USA. [This is an all-inclusive review of the potential of fungi in the biotechnological industry].

Gripon J.C. (1987). Mould-ripened cheeses. In: Cheese: Chemistry, Physics and Microbiology, vol. 2.

Major Cheese Groups. (Ed. P.F. Fox), Elsevier Applied Science, London, UK pp. 121–149. [This is a

detailed description of use of filamentous fungi for manufacture of cheese].

Hamlyn P.F. (1997) Fungal biotechnology. North West Fungus Group Newsletter (ISSN 1465-8054).

http://fungus.org.uk/nwfg/fungbiot.htm [This is a popular and comprehensive review of the potential of

fungi for industrial purposes]

Jůzlová P., L Martínková and Křen V. (1996). Secondary metabolites of the fungus Monascus : a review.

Journal of Industrial Microbiology, 16(3), pp. 163-170. [This describes the potential and risks with

Monascus pigments].

Manzoni M., S. Bergomi, M.Rollini and V. Cavazzoni. (1999). Production of statins by filamentous

fungi. Biotechnology Letters, 21(3), pp. 253-257. [This is a review of different fungi producing different

statins, used to lower cholesterol levels in humans].

Nevalainen H.K.M., V.S.J Te’o and P.L.Bergquist. (2005). Heterlogous protein expression in filamentous

fungi. Trends in Biotechnology, 23(9), pp. 468-474. [This describes the potential and the problems in the

UNESCO – EOLSS

SAMPLE CHAPTERS

BIOTECHNOLOGY –Vol. VI - Industrial Mycology - J.S. Rokem

use of filamentous fungi for heterologous protein production].

Robinson T., D. Singh and P. Nigam (2001). Solid state fermentation: a promising microbial technology

for secondary metabolite production. Applied Microbiology and Biotechnology, 55(3), pp. 284-289. [This

ia a review of the pros and cons of solid state fermentation]

Smith J.E., N.J. Rowan and R. Sullivan (2002). Medicinal mushrooms: a rapidly developing area of

biotechnology for cancer therapy and other bioactivities. Biotechnology Letters 24(22), pp. 1839-1845.

[This is a summary of achievements using higher fungi for medical purposes].

Wiebe M.G. (2002). Mycoprotein from Fusarium venenatum: a well established product for human

consumption. Applied Microbiology and Biotechnology, 58(4), pp. 421-427. [This is a summary of the

successful development of fungal cells for human consumption].

Tudzynski B. (2005) Gibberellin biosynthesis in fungi: genes, enzymes, evolution, and impact on

biotechnology. Applied and Microbiology and Biotechnology, 66(6), pp. 597-611. [This is a very

comprehensive review of the knowledge accumulated on all aspects of gibberellin biosynthesis in fungi].

Biographical Sketch

J. Stefan Rokem is a Senior Lecturer at the Department of Molecular Genetics and Biotechnology at the

Hebrew University of Jerusalem, Israel. He earned his M.Sc. in Microbial Biochemistry and Fermentation

Technology from the Imperial College of Science and Technology at the University of London, United

Kingdom. He completed his Ph.D. studies in Applied Microbiology at the Hebrew University of

Jerusalem, Israel with a thesis on “Production of Single Cell Protein”. In addition to his teaching duties at

the Hebrew University, Dr. Rokem serves as a scientific consultant to local biotechnology companies and

is a reviewer of several journals and granting agencies. He has been on the executive committee of the

International Organization of Biotechnology and Bioengineering for several years, serving as one of the

representatives of the Middle East. Dr. Rokem has given several guest lectures and courses in

Bioengineering and the use of Biotechnology for the improvement of the Environment in South and

Central America. Recently Dr. Rokem developed a novel curriculum in Biotechnology for the Open

University of Israel. During his academic career he has co-authored over 60 peer reviewed papers, written

several book chapters, co edited one book and written a monograph on the use of Biotechnology to

Improve the Environment. The main research interests of Dr. Rokem are the regulation of antibiotic

production in Actinobacteria, production of organic acids with filamentous fungi and the use of

Biotechnology to reach Sustainable Development, mainly novel methods for sewage treatment. He has

spent sabbaticals at Yale University, New Haven, CT, USA; Royal Institute of Technology, Stockholm,

Sweden; MIT, Cambridge, MA, USA and The Danish Technical University, Copenhagen, Denmark.