Current Developments in

Biotechnology and Bioengineering:

Filamentous Fungi Biorefinery
Mohammad Taherzadeh
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Current Developments
in Biotechnology
and Bioengineering
Series Editor:
Professor Ashok Pandey
Centre for Innovation and Translational Research
CSIR-Indian Institute of Toxicology Research
Lucknow, India
&
Sustainability Cluster
School of Engineering
University of Petroleum and Energy Studies
Dehradun, India
 Current Developments
in Biotechnology
and Bioengineering
Filamentous Fungi Biorefinery

Edited by
Mohammad J. Taherzadeh
Swedish Centre for Resource Recovery, University of Borås, Borås, Sweden

Jorge A. Ferreira
Swedish Centre for Resource Recovery, University of Borås, Borås, Sweden

Ashok Pandey
Centre for Innovation and Translational Research, CSIR-Indian Institute
of Toxicology Research, Lucknow, India
Sustainability Cluster, School of Engineering, University of Petroleum and
Energy Studies, Dehradun, India
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Contributors

Ruchi Agrawal The Energy and Resources Institute, TERI Gram, Gwal Pahari, Haryana,
India
Hamid Amiri Department of Biotechnology, Faculty of Biological Science and
Technology; Environmental Research Institute, University of Isfahan, Isfahan, Iran
K. Amulya Bioengineering and Environmental Sciences, Department of Energy and
Environmental Engineering, CSIR-Indian Institute of Chemical Technology, Hyderabad,
India
Elisabet Aranda Institute of Water Research; Department of Microbiology, University of
Granada, Granada, Spain
Mohammadtaghi Asadollahzadeh Swedish Centre for Resource Recovery, University of
Borås, Borås, Sweden
Aparna Banerjee Centro de investigación en Estudios Avanzados del Maule (CIEAM),
Vicerrectorı́a de Investigación Y Posgrado, Universidad Católica del Maule, Talca,
Chile
Parameswaran Binod Microbial Processes and Technology Division, CSIR-National
Institute for Interdisciplinary Science and Technology (CSIR-NIIST),
Thiruvananthapuram, Kerala, India
Kamalpreet Kaur Brar Department of Civil Engineering, Lassonde School of Engineering,
York University, Toronto, ON; Industrial Waste Technology Center, Abitibi Temiscamingue,
QC, Canada
€ lru Bulkan Swedish Centre for Resource Recovery, University of Borås, Borås,
Gu
Sweden
Gustavo Cabrera-Barjas Universidad de Concepción, Unidad de Desarrollo Tecnológico
(UDT), Coronel, Chile
Marta Cebrián AZTI, Food Research, Basque Research and Technology Alliance (BRTA),
Parque Tecnológico de Bizkaia, Derio, Bizkaia, Spain
Chiu-Wen Chen Department of Marine Environmental Engineering, National Kaohsiung
University of Science and Technology, Kaohsiung City, Taiwan

xiii
xiv Contributors

Eduardo Coelho CEB—Centre of Biological Engineering, University of Minho, Braga,

Portugal
Gabriela Ángeles de Paz Institute of Water Research, University of Granada, Granada,
Spain
dric Delattre Universite
Ce  Clermont Auvergne, Clermont Auvergne INP, CNRS, Institut
Pascal, Clermont-Ferrand; Institut Universitaire de France (IUF), Paris, France
Ratih Dewanti-Hariyadi Department of Food Science and Technology, IPB University,
Bogor, Indonesia
Lucı́lia Domingues CEB—Centre of Biological Engineering, University of Minho, Braga,
Portugal
Cheng-Di Dong Department of Marine Environmental Engineering, National Kaohsiung
University of Science and Technology, Kaohsiung City, Taiwan
Pascal Dubessay Universite Clermont Auvergne, Clermont Auvergne INP, CNRS, Institut
Pascal, Clermont-Ferrand, France
Laurent Dufosse  Chemistry and Biotechnology of Natural Products (CHEMBIOPRO),
University of Reunion Island, ESIROI Food Science, Saint-Denis Cedex 9, Reunion
Island, France
Jorge A. Ferreira Swedish Centre for Resource Recovery, University of Borås, Borås,
Sweden
Shakuntala Ghorai Department of Microbiology, Raidighi College, Raidighi, India
Daniel G. Gomes CEB—Centre of Biological Engineering, University of Minho, Braga,
Portugal
Sharareh Harirchi Swedish Centre for Resource Recovery, University of Borås, Borås,
Sweden; Department of Cell and Molecular Biology & Microbiology, Faculty of
Biological Science and Technology, University of Isfahan, Isfahan, Iran
Jone Ibarruri AZTI, Food Research, Basque Research and Technology Alliance (BRTA),
Parque Tecnológico de Bizkaia, Derio, Bizkaia, Spain
Sajjad Karimi Swedish Centre for Resource Recovery, University of Borås, Borås, Sweden
Vinod Kumar School of Water, Energy, and Environment, Cranfield University, Cranfield,
United Kingdom
Patrik Roland Lennartsson Swedish Centre for Resource Recovery, University of Borås,
Borås, Sweden
Hanifah Nuryani Lioe Department of Food Science and Technology, IPB University,
Bogor, Indonesia
 Contributors xv

Sara Magdouli Department of Civil Engineering, Lassonde School of Engineering, York

University, Toronto, ON; Industrial Waste Technology Center, Abitibi Temiscamingue,
QC, Canada
Manikharda Department of Food and Agricultural Product Technology, Universitas
Gadjah Mada, Yogyakarta, Indonesia
Philippe Michaud Universite Clermont Auvergne, Clermont Auvergne INP, CNRS, Institut
Pascal, Clermont-Ferrand, France
Marzieh Mohammadi Swedish Centre for Resource Recovery, University of Borås, Borås,
Sweden
Rafael León Morcillo Institute of Water Research, University of Granada, Granada,
Spain
Soumya Mukherjee University of Toledo, Toledo, OH, United States
Vivek Narisetty School of Water, Energy, and Environment, Cranfield University,
Cranfield, United Kingdom
Seyyed Vahid Niknezhad Burn and Wound Healing Research Center; Pharmaceutical
Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
Ashok Pandey Centre for Innovation and Translational Research, CSIR-Indian Institute
of Toxicology Research, Lucknow; Sustainability Cluster, School of Engineering,
University of Petroleum and Energy Studies, Dehradun, India
Mohsen Parchami Swedish Centre for Resource Recovery, University of Borås, Borås,
Sweden
Anil Kumar Patel Department of Marine Environmental Engineering; Institute of Aquatic
Science and Technology, National Kaohsiung University of Science and Technology,
Kaohsiung City, Taiwan
 Clermont Auvergne, Clermont Auvergne INP, CNRS, Institut
Guillaume Pierre Universite
Pascal, Clermont-Ferrand, France
Endang Sutriswati Rahayu Department of Food and Agricultural Product Technology,
Universitas Gadjah Mada, Yogyakarta, Indonesia
G. Renuka Department of Microbiology, Pingle Government Degree College for Women,
Warangal, India
Saddys Rodriguez-Llamazares Centro de Investigación de Polı́meros Avanzados (CIPA),
Edificio Laboratorio CIPA, Concepción, Chile
Neda Rousta Swedish Centre for Resource Recovery, University of Borås, Borås,
Sweden
xvi Contributors

Taner Sar Swedish Centre for Resource Recovery, University of Borås, Borås, Sweden
Behzad Satari Department of Food Technology, College of Aburaihan, University of
Tehran, Tehran, Iran
Ulises Conejo Saucedo Institute of Water Research, University of Granada, Granada,
Spain
Zohresadat Shahryari Swedish Centre for Resource Recovery, University of Borås, Borås,
Sweden; Avidzyme Company, Shiraz, Iran
Pooja Sharma Environmental Research Institute, National University of Singapore;
Energy and Environmental Sustainability for Megacities (E2S2) Phase II, Campus for
Research Excellence and Technological Enterprise (CREATE), Singapore, Singapore
Rui Silva CEB—Centre of Biological Engineering, University of Minho, Braga, Portugal
Raveendran Sindhu Microbial Processes and Technology Division, CSIR-National
Institute for Interdisciplinary Science and Technology (CSIR-NIIST),
Thiruvananthapuram, Kerala, India
Reeta Rani Singhania Department of Marine Environmental Engineering, National
Kaohsiung University of Science and Technology, Kaohsiung City, Taiwan
Mohammad J. Taherzadeh Swedish Centre for Resource Recovery, University of Borås,
Borås, Sweden
 A. Teixeira CEB—Centre of Biological Engineering, University of Minho, Braga,
Jose
Portugal
Sunita Varjani Gujarat Pollution Control Board, Gandhinagar, Gujarat, India
S. Venkata Mohan Bioengineering and Environmental Sciences, Department of Energy
and Environmental Engineering, CSIR-Indian Institute of Chemical Technology,
Hyderabad, India
Rachma Wikandari Department of Food and Agricultural Product Technology,
Universitas Gadjah Mada, Yogyakarta, Indonesia
Akram Zamani Swedish Centre for Resource Recovery, University of Borås, Borås, Sweden
Preface

Advances in Filamentous Fungi Biorefinery is a book in the Elsevier series on Current

Developments in Biotechnology and Bioengineering (Editor-in-Chief: Ashok Pandey). This
book explores various fundamental and industrial aspects of filamentous fungi for the
manufacture of different products used in our society.
Fungi are part of the ecosystem, and the world would look totally different without
them. Most people know only mushrooms and their fruiting bodies as fungi. However,
the main fungal biomass is their filaments, which can have many applications. Filamen-
tous fungi can grow on a large variety of materials that contain carbohydrates, proteins,
fats, etc., by degrading the macromolecules and then assimilating the monomers to grow
and produce various enzymes and metabolites. This means that there are a large number
of substrates on which to grow fungi, from agricultural and forest residuals to industrial
residuals and products, to household wastes and wastewaters. Depending on the ecosys-
tem and the environmental or cultivation conditions, fungi can grow in various morphol-
ogies, and many fungal strains are dimorphic, meaning that they can grow both like yeast
and filaments. In addition, they can grow in various environmental conditions, such as
aerobic or anaerobic. They adapt their enzyme machinery as required to these conditions,
producing a variety of metabolites that are necessary for the fungi to grow.
As fungi grow on a large number of substrates, they can produce various extracellular
enzymes such as hydrolytic enzymes to degrade biopolymers. Therefore, fungi are an
industrial source of enzyme production.
Ultimately, we should not forget that the only goal of fungi is to grow. However, in cer-
tain conditions they can produce, for example, enzymes and/or various metabolites,
which can be used as products. Bearing in mind the single goal of fungi, one of their major
products is always fungal biomass or mycelium. This biomass normally contains protein,
fat, and other biopolymers such as chitosan or beta-glucan in its cell wall, and a variety of
bioactive compounds. As a result, the biomass of many filamentous fungi can be a good
source for food and feed. Some of these fungi, particularly among the zygomycetes and
ascomycetes, are edible and can be used for different food preparations such as tempeh,
oncom, and koji. However, there is also an interest nowadays in developing new food and
feed such as fish feed from fungi as an environmentally friendly and healthy alternative to
meat, chicken, or even soy-based vegetarian products, for example. However, as some
fungi produce mycotoxins, the fungal strain and the process conditions should be chosen
carefully in order to avoid any risks to humans or animals.
The aim of this book is to explore comprehensively the advances in using filamentous
fungi as the core of industrial biorefinery. The book provides a thorough overview and

xvii
xviii Preface

understanding of fungal biology, biotechnology, and ecosystems, and covers a variety of

industrial products that can be developed from fungi.
The contents of this book are organized in 18 chapters to cover: (a) the fungal ecosys-
tem, biology, and biotechnology; (b) the fungal growth and process in solid state and sub-
merged fermentation, particular aspects of bioreactors, and sampling, preservation, and
process monitoring; (c) mycotoxins as an important aspect to choose the fungal strains for
processes; (d) products of biorefinery that the fungi including food, feed, organic acids,
alcohols, bioactive pigmented compounds, and antibiotics; and (e) fungi in novel
processes.
We are grateful to the authors for compiling the pertinent information in their chap-
ters, which we believe will be a valuable resource for both the scientific community and
readers in general. We are also grateful to the expert reviewers for their useful com-
ments and scientific insights, which helped shape the book’s organization and which
improved the scientific discussions and overall quality of the chapters. The Editors
(Mohammad J. Taherzadeh and Jorge A. Ferreira) acknowledge support from the
Swedish Agency for Economic and Regional Growth (Tillva €xtverket) through a European
Regional Development Fund “Ways2Tastes.” Finally, our sincere thanks go to the staff
at Elsevier, including Dr. Kostas Marinakis (former Senior Book Acquisition Editor),
Dr. Katie Hammon (Senior Book Acquisition Editor), and Bernadine A. Miralles (Editorial
Project Manager), and the entire Elsevier production team for their support in publishing
this book.
Editors
Mohammad J. Taherzadeh
Jorge A. Ferreira
Ashok Pandey
 1
World of fungi and fungal
ecosystems
Gabriela Ángeles de Paza, Ulises Conejo Saucedoa,
Rafael León Morcilloa,#, and Elisabet Arandaa,b
a
INSTITUTE OF WATER RESEARCH, UNIVERSITY O F GRANADA, GR ANADA, SPAIN
b
DEPARTMENT OF MICROBIOLOGY, UNIVERSITY O F GRANADA, GR ANADA, SPAIN

1. Introduction
Fungi, belonging to Eukarya, are highly diverse and less explored. They are cosmopolitan
and play important ecological roles as saprotrophs, mutualists, symbionts, parasites, or
hyperparasites. Advances in molecular phylogeny have allowed to clarify the complex
relationships of anamorphic fungi (fungi imperfecti) and to place some of them outside
the fungi. Exiting progress have been made in developing fungi for modern and postmod-
ern biotechnology, such as obtaining enzymes, alcohols, organic acids, pharmaceuticals,
or recombinant deoxyribonucleic acid (DNA). Filamentous fungi and yeasts are exten-
sively used as efficient cell factories in the production of bioactive substances and metab-
olites or for native or heterologous protein expression. This is due to their metabolic
diversity, secretion efficiency, high-production capacity, and capability of carrying out
post-translational protein modifications. The commercial exploitation of fungi has been
reported for multiple industrial sectors, such as those involved in the production of anti-
biotics, simple organic compounds (citric acids), fungicides or food and beverages.

2. Fungal morphology
Fungi can colonize new places by growing as a system of branching tubes, known as
hyphae, whose aggregates form the mycelium (filamentous fungi). Mycelium can be found
in the substrates where the fungi growth or belowground and play an important role in
obtaining nutrients for growth and development. The hyphae are characterized by the pres-
ence or absence of septa, cross-walls that are distinctive among different taxonomic groups.
They are absent in Oomycota and Zygomycota, known as coenocytic hyphae (koinos ¼
shared, kytos ¼ a hollow vessel). The presence of septa is a common feature of Basidiomy-
cota and Ascomycota, in which the exchange of cytoplasm or organelles is ensured by septal

#
Current affiliation: Institute for Mediterranean and Subtropical Horticulture “La Mayora” (IHSM), CSIC-
UMA, Campus de Teatinos, Málaga, Spain.

Current Developments in Biotechnology and Bioengineering. https://doi.org/10.1016/B978-0-323-91872-5.00010-7 1

Copyright © 2023 Elsevier Inc. All rights reserved.
2 Current Developments in Biotechnology and Bioengineering

FIG. 1 Septum types in fungal hyphae.

pores. These pores can be simple or dolipores, pores with a distinctive morphology that
have a barrel-shaped swelling that surrounds the central pore (Fig. 1). Under senescence
processes, differentiation or simply under mechanical breaking off, different organelles
act as septal pore plugs, preventing the detrimental effect of trauma senescence or permit-
ting differentiation processes. They include Woronin bodies, hexagonal crystals, elongated
crystalline bodies, nuclei, mitochondria, or de novo deposition of plugging material
(Markham, 1994). The septum represents a specialized structure for cell division.
Not all fungi grow as hyphae; some occur as yeasts (yeast-like fungi). They usually
grow on surfaces where penetration is not required (such as the digestive tract). Such fungi
have attracted the attention of biotechnologists because they grow rapidly and can easily
be manipulated. Other fungi can switch between yeast-like fungi and filamentous fungi;
they are known as dimorphic fungi and include some pathogens such as the plant path-
ogen Ustilago maydis or the human pathogen Candida albicans. This attribute is highly
important as a model of differentiation in eukaryotic organisms (Bossche et al., 1993).
Dimorphism is a common treat in pathogenic fungi (animals and plants pathogen) and
usually is regulated by different factors such as temperature, glucose, pH, nitrogen source,
carbon dioxide levels, chelating agents, transition metals and inoculum size or initial cell
density (Romano, 1966). However, a number of fungi with unknown pathogenic activity
have important industrial applications such as the production of chitosan, chitin from
Saccharomyces, bioremediation process by Yarrowia, ethanol or enzyme production
(Doiphode et al., 2009). This fact is of special interest in industry, since (i) it is possible
to overcome the operational problems generated during hyphal growth in bioreactors,
(ii) morphology could be an indicator of biotechnological process (enzymes secretion
or proliferation vs penetration), or (iii) can be an advantage for biocontrol formulations
(Doiphode et al., 2009).
From a point of view of biotechnology, the morphology of fungi has an important
implication since the adaptation of the cultivation system must be optimized.
 Chapter 1 • World of fungi and fungal ecosystems 3

Filamentous fungi can growth as disperse mycelial or pellets, depending on the mechan-
ical conditions of the cultivation. Industrial cultivation processes with fungi have been
optimized over decades to increase productivity (Walker and White, 2017).
The fungal cell wall is a dynamic and complex structure and usually based on glucans
and chitin (Ruiz-Herrera, 1991). However, the chemical composition varies among differ-
ent taxonomic groups, with implications for biotechnological processes since different
enzymatic activities occur in the cell wall. The balance between wall synthesis and lysis
could influence hyphal morphology and cell growth, with impacts on the retention of
chemical compounds through bio-adsorption processes, enabling the successful indus-
trial fermentation of filamentous fungi. In addition, some components of the cell wall,
such as chitosan and chitin are considered high-value products for their use in biomed-
icine, agriculture, paper making, food industry, and textile industry that can be easily
extracted using different technologies (Nwe et al., 2011; Table 1).

2.1 General aspects of reproduction

Fungi can reproduce sexually or asexually (vegetative reproduction). These two reproduc-
tion modes differ according to the fungal morphology and taxonomic group (yeast, fila-
mentous, or dimorphic fungi) (Hawker, 2016). In yeasts, the most frequent mode of
vegetative reproduction is budding, which has been studied in detail for Saccharomyces
cerevisiae. This mode can be multilateral, bipolar, unipolar, or monopolar budding. How-
ever, fission by forming a septum can also occur in yeasts such as Schizosaccharomyces
pombe (host for heterologous expression). This fission can be binary or a bud fission,
in which a cross-wall at the base of the bud separates both cells. Ballistoconidiogenesis
is a specific vegetative reproduction in species such as Bullera (β-galactosidase) or Spor-
obolomyces (carotenoids and fatty acids). Pseudomycelia are typical in dimorphic species
in which a single filament is produced when cells fail to separate after budding or fission
(Walker and White, 2017). In filamentous fungi, such as basidiomycetes, this vegetative
reproduction can occur by fragmentation of the hyphae; in some ascomycetes, the forma-
tion of mitotic spores has been observed. Sexual reproduction is a complex mechanism
and differs according to the taxonomic groups; it involves the formation of a meiotic spore
by planogametic copulation, gametangial contact, gametangial copulation, spermatiza-
tion, and somatogamia.

Table 1 Percentages of dry weight of the total cell wall fraction of the main
components (chitin, cellulose, glucans, protein, and lipids) in different groups of fungi.
Group Chitin Cellulose Glucans Protein Lipids

Oomycota 0 25 65 4 2
Chytridiomycota 58 0 16 10 n/a
Zygomycota 9 0 44 6 8
Ascomycota 1–39 0 29–60 7–13 6–8
Basidiomycota 5–33 0 50–81 2–10 n/a

n/a (Data not available).

Data adapted from Ruiz-Herrera, J., Ortiz-Castellanos, L., 2019. Cell wall glucans of fungi. A review. Cell Surf. 5, 100022.
4 Current Developments in Biotechnology and Bioengineering

2.2 Fungal nutrition

Filamentous fungi and yeasts are chemo-organotroph microorganisms with relatively sim-
ple nutritional needs. Fungi are heterotrophic organisms since they lack photosynthetic
pigments. Most fungi are aerobes, but we can find representatives of obligate anaerobes
(Neocallimastix) or facultative anaerobes (Blastocladia). In aerobic respiration, the termi-
nal electron acceptor is oxygen; however, based on oxygen availability, we can find obligate
fermentative or facultative fermentative fungi, including Crabtree-positive (Saccharomyces
cerevisiae), Crabtree-negative (Candida utilis), non-fermentative (Phycomyces, Rhodotor-
ula rubra) or obligate aerobes (most fungi) (Walker and White, 2017).

2.2.1 Nutrient uptake

Filamentous fungi and few yeast species obtain their nutrients via extracellular enzymes;
they absorb smaller molecules produced after extracellular digestion. These enzymes can
be wall-bound-enzymes or may diffuse externally into the environment, depending on the
lifestyle of the fungus (Section 3). Nutrient distribution through the hyphae might occur
by passive (diffusion-driven) or active translocation (metabolically driven) through the
protoplasm (Olsson and Gray, 1998; Persson et al., 2000). In both cases, nutrient translo-
cation allows filamentous fungi to growth in habitats where the spatial distribution of
nutrients and minerals is irregular and variable, including environments with low nutrient
concentrations or polluted areas, by exploiting the resources available in other parts of the
mycelium (Boswell et al., 2002).
The enzymatic system in fungi depends on the taxonomic group. Some ecophysiological
artificial groups have been established based on the capability to produce enzymes. These
groups include the former ligninolytic fungi because of their ability to secrete a set of enzymes
involved in the degradation of lignin. These enzymes play a significant role in biotechnology
since they can be used in biorefineries. They include lipases produced by some yeasts
(Candida, Yarrowia lipolytica), hydrolytic enzymes such as glycoside hydrolases (GHs), poly-
saccharide lyases (PLs), glycosyltransferases (GTFs), carbohydrate esterases (CEs), lytic poly-
saccharide monooxygenases (LPMOs), non-catalytic carbohydrate-binding modules
(CBMs) and enzymes with “auxiliary activities,” including all enzymes involved in lignocel-
lulosic conversion such as laccases, peroxidases, manganese peroxidase, lignin peroxidases,
versatile peroxidases, DyP-type peroxidases (Levasseur et al., 2013). These enzymes are clas-
sified in the CAZymes database, which describes the families of structurally related catalytic
and CBMs of enzymes that degrade, modify, or create glycosidic bonds (http://www.cazy.
org/). These enzymes are important in biotechnology, particularly in biorefineries, for the
deconstruction of plant biomass into simple sugars to obtain fermentation-based products
and for the recovery of value-added compounds (Contesini et al., 2021).

3. Lifestyles of fungi
Fungi, as heterotrophic eukaryotic microorganisms and efficient producers of enzymes,
can live in different habitats and on different organic substrates. In general terms, fungi
 Chapter 1 • World of fungi and fungal ecosystems 5

are either saprophytic—they feed on nutrients from organic, non-living matter in the sur-
rounding environment-, symbiotic—they share a mutually beneficial relationship with
another organism-, parasitic—they feed off a living host that may survive (biotrophs)
or die (necrotrophs)- or hyperparasitic—they live at the expense of another parasites.
Saprotrophic fungi are important for the recycling of nutrients, especially phosphate
minerals and carbon incorporated in wood and other plant tissues. Their role as decom-
posers of organic matter is fundamental, since together with bacteria, they prevent the
accumulation of organic matter, ensure the distribution of nutrients and play a crucial role
in the global carbon cycle in terrestrial and aquatic ecosystems (Kjøller and Struwe, 2002;
Cebrian, 2004; Moore et al., 2004). Besides, filamentous fungi play other significant roles in
natural ecosystems. For instance, in terrestrial systems, fungi maintain the soil structure
due to their filamentous branching growth and participate in the transformation of rocks
and minerals (Gadd, 2008), incorporating new elements into the ecosystem that may be
used by other organisms.
In addition to the important role in natural processes, and as stated before, the decom-
position of organic matter by fungi represents an important trait for biotechnological pur-
poses due to the potential use of individual microbial strains or enzymes for the use of
renewable resources, such as plant biomass. In general terms, saprotrophic basidiomy-
cetes can degrade plant litter and wood more rapidly than other fungi because of their
high capacity to decompose lignin and other plant polymers, allowing them to spread rap-
idly in the environment (Osono and Takeda, 2002; Martı́nez et al., 2005; Baldrian, 2008).
Nevertheless, litter and wood decomposition is a successive process, of which basidiomy-
cetes and ascomycetes govern different phases (Osono, 2007; Vo
rı́šková and Baldrian,
2013). This capacity has been widely used in at industrial scale in various applications
because of the oxidation of phenolic and non-phenolic lignin-derived compounds. Few
examples are fungal laccases, ligninolytic enzymes which degrade complex recalcitrant
lignin polymers and are widely used in the food industry (Minussi et al., 2002; Mayolo-
Deloisa et al., 2020), in cosmetic, pharmaceutical, and medical applications (Golz-Berner
et al., 2004; Niedermeyer et al., 2005; Hu et al., 2011; Ueda et al., 2012; Sun et al., 2014), in
the paper and textile industry (Bourbonnais et al., 1995; Ozyurt and Atacag, 2003;
Rodrı́guez Couto and Toca Herrera, 2006; Virk et al., 2012) or in the nano-biotechnology
(Li et al., 2017; Kumari et al., 2018). Apart from industrial applications, the potential capac-
ity of saprophytic fungal intra- and extracellular enzymes to degrade/transform complex
polymers, such as lignin, is used in the biodegradation of organic xenobiotic pollutants.
Among them, oxidoreductases represent the most important group of enzymes used in
xenobiotic bioremediation transformations, including peroxidases, laccases, and oxyge-
nases, and can catalyze oxidative coupling reactions using oxidizing agents to support
the reactions (Sharma et al., 2018; Baker et al., 2019). These enzymes are produced by a
wide diversity of fungi, which are some of the most extensively fungi used to detoxify
xenobiotic compounds; they belong to the basidiomycetes group called “white rot fungi”
and include the genera Trametes, Pleurotus, and Phanerochaete spp. (Aust, 1995; Pointing,
2001; Baldrian, 2003; Asif et al., 2017), but also ascomycetes genera such as Aspergillus or
Penicillium (Aranda, 2016; Aranda et al., 2017).
6 Current Developments in Biotechnology and Bioengineering

Pathogenic and parasitic fungi virtually attack all groups of organisms, including bac-
teria, other fungi, plants, and animals, including humans. According to their nutritional
relationship with the host, parasitic fungi can be divided into biotrophic parasites, which
obtain their sustenance directly from living cells, and necrotrophic parasites, which first
destroy the parasitized cell and then absorb its nutrients. Besides, fungi might be facul-
tative parasites, which are capable of growing and developing on dead organic matter
and artificial culture media, or obligate parasites, which can only obtain food from living
protoplasm and, therefore, cannot be cultured in non-living media (Brian, 1967; Lewis,
1973) Fungi possess the broadest host range spectrum of any group of pathogens. For
instance, the filamentous ascomycetous fungus Fusarium oxysporum causes vascular wilt
on many different plant species (Pietro et al., 2003), but it is also responsible for causing
life-threatening disseminated infections in immunocompromised humans (Boutati and
Anaissie, 1997). Nonetheless, there are many examples of fungal pathogens that infect
only one host (Shivas and Hyde, 1997; Zhou and Hyde, 2001), highlighting the high host
specificity of diseases produced by certain fungal infections. To explain this dual aspect of
fungal infection specificity, the pathogenic strains of parasitic fungi are divided into for-
mae speciales, defining the existence of different subgroups within species based on their
host specificity (Armstrong and Armstrong, 1981; Anikster, 1984).
In terms of agriculture, the estimated crop losses due to fungal diseases would be suf-
ficient to feed approximately 600 million people a year (Fisher et al., 2012). To colonize
plants, fungi secrete hydrolytic enzymes, including cutinases, cellulases, pectinases and
proteases that degrade these polymers and permit fungal entrance through the external
plant structural barriers. These enzymes are also required for the saprophytic lifestyle
of fungi. As mentioned above, some fungi are facultative parasites and may attack plant
roots from a saprophytic base in the soil through the mycelium, progressively causing the
death of the host and thereafter living as saprophytes (Zhou and Hyde, 2001). Some fungi
have developed other mechanisms to colonize plant hosts, such as via specialized pene-
tration organs, called appressoria, or via penetrating through wounds or natural openings,
such as stomata (Knogge, 1996).
Fungal pathogens are responsible for numerous diseases in humans and for the extinc-
tion of amphibian and mammal populations (Brown et al., 2012; Fisher et al., 2012). For
example, Batrachochytrium dendrobatidis, an aquatic chytrid fungus that attacks the skin
of over 500 species of amphibians, and Geomyces destructans, a ascomycete fungus that
attacks numerous bat species, seriously threaten the survival of these animals and might
lead to the decline in the populations of other species (Colón-Gaud et al., 2009; Fisher
et al., 2009; Lorch et al., 2011).
For humans, fungal infections are rarely life-threatening; however, superficial fungal
infections of the skin, hair and nails are common worldwide and affect approximately
one-quarter of the human population (Schwartz, 2004). Airborne pathogenic fungi can
also cause different respiratory diseases and also be lethal in immunocompromised
patients (Mendell et al., 2011). The infection process is, generally, similar to that in plants.
However, and contrary to plant infections, appressoria have not been described for
 Chapter 1 • World of fungi and fungal ecosystems 7

animal-pathogenic fungi, except for a few similarly shaped structures formed by Candida
albicans (Kriznik et al., 2005). Instead to appressoria, in animals, fungal pathogens use
other mechanisms, such as the binding of specific receptors that facilitate the endocytosis
of host cells and, therefore, their entrance into the living tissue (Woods, 2003; Nu€ rnberger
et al., 2004). Nonetheless, and similar to fungal infections in plants and other fungal life-
styles such as saprophytes, this process may also be mediated by lytic enzymes, such as
proteases, that degrade the surface of the host cells and permit fungal penetration into the
living host (Nu € rnberger et al., 2004; Schaller et al., 2005).
A special type of parasitic fungi is represented by hyperparasites, fungi that live at the
expense of another parasite, which is highly common among fungi ( Jeffries and Young,
1994). Hyperparasitism is often used in agriculture for plant protection as an alternative
to chemical treatments (Brožová, 2004). A classic example is Trichoderma harzianum, a
fungus extensively used as biological agent against a wide range of fungal parasites. None-
theless, it is estimated that 90% of all fungi used in plant protection products belong to the
genus Trichoderma (Benı́tez et al., 2004).
Fungi may also live as mutualistic symbionts, associating with other organisms with
benefits for both parties. Remarkable examples of these symbioses are mycorrhizae and
lichens. Mycorrhizae are the symbiotic association of soil fungi with the roots of vascular
plants. Generally, fungi colonize plant roots and provide nutrients and water, which are
captured from the soil through the external hyphal network, whereas plants supply
organic molecules derived from photosynthesis, such as sugars or fatty acids, to the obli-
gate biotrophic fungi (Harrison, 1999; Keymer et al., 2017). This represents a universal
symbiosis, not only because almost all plant species are susceptible to form the symbiosis,
but also because such symbioses can be established in the majority of terrestrial ecosys-
tems, even under highly adverse conditions (Mosse et al., 1981). Moreover, this symbiosis
contributes to global carbon cycles as plant hosts divert up to 20% of photosynthates to
the host fungi (Smith and Read, 2010). There are three different types of mycorrhizae:
endomycorrhizae, ectomycorrhizae, and ectendomycorrhizae.
Endomycorrhizae are characterized by the presence of hyphae inside the cells of the
root cortex. It is estimated that at least 90% of all known vascular plants, (about
300,000 species), form this type of mycorrhizae. On the other hand, in the ectomycorrhi-
zae symbiosis, the hyphae of the fungus do not penetrate the cells of the cortex of the roots
and form a dense hyphal sheath, known as the mantle, surrounding the root surface. It is
believed that at least 3% of vascular plants develop this type of mycorrhizae, including
almost all species of the most important forest tree genera. Finally, the ectendomycorrhi-
zae present characteristics of both endo- and ectomycorrhizae, namely a mantle sur-
rounding the plant roots and fungal hyphae that penetrate the root cells (Smith and
Read, 2010).
Mycorrhizal symbiosis plays an essential role in the establishment and functioning of
terrestrial ecosystems, being involved in natural processes such as nutrient cycling and, in
part, in the structure and dynamics of populations and plant communities (Newman,
1988; Klironomos et al., 2011). In terms of agriculture, mycorrhizae improve the
8 Current Developments in Biotechnology and Bioengineering

productive capacity of poor soils, such as those affected by desertification, salinization,

and wind erosion, because of the fungal capacity to obtain and translocate nutrients
and water to the host plants (George, 2000). In addition, the symbiosis enhances soil
aggregation and structure (Miller and Jastrow, 2000) and contributes to defence against
diverse plant pathogens (Elsen et al., 2001; Azcón-Aguilar et al., 2002; Garcı́a-Garrido
and Ocampo, 2002) and abiotic stresses, such as drought or salinity (Ruiz-Lozano et al.,
1996; Ruiz-Lozano, 2003).
Another form of symbiotic association of fungi is represented by lichens, which are
composite organisms composed of algae or cyanobacteria, called photobionts, living
among filaments of a fungus, the so-called mycobiont. The algae, as an autotrophic organ-
ism, provides the fungus with organic compounds and oxygen derived from its photosyn-
thetic activity, whereas the fungus, as a heterotroph, supplies the algae with carbon
dioxide, minerals and water, since contrary to plants, lichens lack vascular organs to
directly control their water homeostasis (Proctor and Tuba, 2002; Lutzoni and Miadli-
kowska, 2009). Most of the lichenized fungal species (98% approximately) belong to the
phylum Ascomycota, whereas only few orders are in the phylum Basidiomycota and
mitosporic fungi (Hawksworth et al., 1996). Although some lichens inhabit partially
shaded areas and forests (Neitlich and McCune, 1997), most lichens often live in highly
exposed places under intense light intensities, such as deserts or arctic and alpine ecosys-
tems. For this reason, the mycobiont normally produces secondary colored compounds,
called lichen compounds, that strongly absorb UV-B radiation and prevent damage to the
algae’s photosynthetic apparatus (Fahselt, 1994). These compounds are a source of struc-
turally diverse groups of natural products, with a wide range of biological activities includ-
ing antibiotic, analgesic, and antipyretic activities (Yousuf et al., 2014) and have
traditionally been used in the cosmetic and dye industry as well as in food and natural
remedies (Oksanen, 2006).
In nature, lichens are important as early-stage primary succession organisms. For
instance, they are the pioneers in the colonization of rocky habitats and, after dying, their
organic matter might be used by other organisms (Lutzoni and Miadlikowska, 2009; Muggia
et al., 2016). As poikilohydric organisms, their water status passively follows the atmo-
spheric humidity (Nash, 1996), and they can tolerate irregular and extended periods of
severe desiccation. This allows them to colonize habits that cannot be colonized by most
plants. Despite this, many lichens also grow as epiphytes on plants, mainly on the trunks
and branches of trees. However, they are not parasites or pathogens since they do not con-
sume or infect the holding plant (Ellis, 2012). In addition, lichens adsorb and are sensitive to
heavy metals and pollutants (Garty, 2001), making them perfect environmental indicators.

4. Taxonomy of fungi
Understanding how fungi have adapted to so many ecosystems, the way in which they
have evolved, but above all, taxonomic classification, has not been an easy task. Fungi,
after plants and animals, are one of the most diverse and dominant groups in almost
 Chapter 1 • World of fungi and fungal ecosystems 9

all ecosystems. It is estimated that there are between 1.5 and 5.1 million species. However,
a new estimation of the number of fungi ranges between 500,000 and almost 10 million
(Hawksworth and Lu € cking, 2017), although only almost 10% have been identified so far
(Blackwell, 2011; Hibbett et al., 2016)
In the middle of the 18th century, the scientist Carl von Linnaeus implemented a bino-
mial system to classify living beings (Systema naturae). This system is based on the clas-
sification of organisms according to their morphological characteristics and phenotypic
traits. Fungi were considered as part of the plant kingdom (Linnaeus, 1767). Some years
after, Whittaker classified the fungi as an independent group, which he called “true fungi”
(Eumycota) (Whittaker, 1969). Then, different taxonomic classifications continued until
the middle of the 19th century. Advances in the classification of fungi have always gone
hand in hand with the development of new technologies, such as electron microscopy,
new biochemical, and physiological analysis methods, the study of secondary metabo-
lites, cell wall composition and fatty acid composition, as well as molecular technologies,
among others (Guarro et al., 1999).
In the last two decades, the development of PCR techniques and, later, genome
sequencing, has significantly contributed to the advance in fungal taxonomy. This pro-
moted rapid changes, and therefore, different proposals for the reclassification of fungi
have been made, tripling the number of phyla from 4 to more than 12. However, less than
5% of the identified species have been taxonomically classified ( James et al., 2020). Next-
generation sequencing tools have allowed fungal genome sequencing, transcriptomes,
and mitochondrial genomes that provided relevant information for phylogenetic studies
in fungi. Additionally, specific regions of ribosomal RNA (rRNA), such as internal tran-
scribed spacers (ITSs), large subunit (LSU), small subunit (SSU) and intergenic spacer
of rDNA, as well as various markers including translation elongation factor 1 (TEF1),
glycerol-3-phosphate dehydrogenase (GAPDH), histones (H3, H4), calmodulin gene,
RNA polymerase II largest subunit (RPB1) genes and mitochondrial genes (cytochrome
c oxidase I and ATPase subunit 6), have played an important role in the development
of the fungal taxonomy (Zhang et al., 2017).
The organization of such information (genomes) by the scientific community has
required different efforts. On the one hand, the Y1000 + project aims to sequence the
genomes of 1000 yeast species (https://y1000plus.wei.wisc.edu), and on the other hand,
the 1000 Fungal Genomes project (http://1000.fungalgenomes.org/home) has the objec-
tive of sequencing 1000 fungal genomes. The database UNITE is a recent database that
concentrates the sequences of the ITS ribosomal region of fungi included in the Inter-
national Nucleotide Sequence Database (http://www.insdc.org/). This database resulted
from the collaboration of researchers and taxonomic specialists who collected and
deposited fungal sequences, specifically with the purpose of building a database that
registers, analyses, and shares this information with the scientific community (Kõljalg
et al., 2020).
In the last 14 years, the taxonomy of fungi has been under major changes. The kingdom
of fungi, proposed by Hibbett et al. (2007), includes one subkingdom (Dikaria) and seven
Another random document with
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 Seuraavassa silmänräpäyksessä olin oven ulkopuolella ihan
pyörällä päästäni, ja sydämessäni kiisteli kaksi tunnetta, kiitollisuus
ja tyytymättömyys. Mielelläni olisin tahtonut viipyä miettimään
asemaani, mutta siihen en saanut aikaa. Totellen oppaani viittausta
seurasin häntä usean käytävän läpi, ja kaikkialla tapasin saman
äänettömyyden, saman luostarimaisen hiljaisuuden. Nyreänä
aprikoidessani lähetettäisiinkö minut Bastiljiin vai Chåteletiin, hän
pysähtyi erään oven eteen, pisti kirjeen käteeni, nosti säppiä ja
viittasi minua käymään sisälle.

Astuin huoneeseen kummeksuen ja pysähdyin tyrmistyneenä.

Edessäni, vasta nousseena tuolilta ja kasvot milloin kalpeina, milloin
purppuranpunaisina, seisoi made moiselle de Cocheforêt. Huusin
häntä nimeltään.

»Herra de Berault», sanoi hän vapisten. »Te ette odottanut

näkevänne minua täällä?»

»En odottanut näkeväni ketään ja kaikkein vähimmin teitä,

mademoiselle», vastasin yrittäen koota malttiani.

»Teidän olisi toki pitänyt ymmärtää, ettemme voineet hylätä teitä

tykkänään», vastasi hän nöyrän nuhtelevalla, äänellä, joka liikutti
sydäntäni. »Olisimme todellakin käyttäytyneet alhaisesti, jollemme
olisi koettaneet pelastaa teitä. Kiitän taivasta, herra de Berault,
yrityksen onnistumisesta sikäli, että tuo kummallinen mies on
luvannut minulle säästää henkenne. Oletteko tavannut häntä?»
jatkoi hän innokkaasti ja sävyltään muuttuen, ja hänen silmänsä
laajenivat äkkiä levottomuudesta.

»Olen, mademoiselle», sanoin minä. »Olen tavannut hänet, ja se

on totta: hän on lahjottanut minulle henkeni.»
 »Ja…?»

»Ja lähettänyt minut vankilaan.»

»Kuinka pitkäksi aikaa?» kuiskasi hän.

»Sitä en tiedä», vastasin minä. »Niin pitkäksi kuin kuningasta

miellyttää, pelkään minä.»

Hän värähti.

»Olen kenties tehnyt enemmän vahinkoa kuin hyötyä», mutisi hän

itsekseen ja katseli minua surkuttelevasti. »Mutta tein sen hyvässä
tarkoituksessa. Kerroin hänelle kaikki, ja kuitenkin olen kenties
tuottanut vahinkoa.»

Mutta kuulla hänen soimaavan itseään noin, tehtyään tämän

pitkän ja yksinäisen matkan minun pelastuksekseni, voitettuaan
vastenmielisyytensä vihollistaan kohtaan ja — siitä olin varma —
nöyrryttyään hänen edessään, sitä minun oli mahdoton sietää.

»Vaietkaa, mademoiselle, vaietkaa!» huudahdin kiivaasti. »Te

haavoitatte sydäntäni. Te olette tehnyt minut onnelliseksi, ja kuitenkin
soisin, että te ette olisi täällä, vaan Cocheforêtissa. Te olette tehnyt
puolestani paljoa enemmän kuin olisin voinut toivoa ja sata kertaa
enemmän kuin olen ansainnut. Mutta tähän täytyy sen loppua. Olin
auttamattomasti mennyttä miestä, ennen kuin tämä tapahtui, ennen
kuin olin teitä koskaan nähnyt, ja niin olen vieläkin, en enempää
enkä vähempää, enkä minä tahdo, että teidän nimeänne mainitaan
minun nimeni yhteydessä täällä Pariisissa. Sentähden — hyvästi.
Jumala varjelkoon minua sanomasta enempää teille ja antamasta
teidän viipyä täällä, missä joutilaat kielet pian ahdistaisivat teitä.»
 Hän katseli minua hiukan ihmetellen; sitten hän sanoi hymyillen ja
lempeästi:

»Se on myöhäistä.»

»Myöhäistä?» huudahdin. »Kuinka niin?»

»Sentähden että… muistatteko, herra de Berault, mitä

tienristeyksessä Agenin ulkopuolella kerroitte minulle
rakkaustarinastanne? Ettei se voinut saada mitään onnellista
loppua? Samasta syystä en minäkään hävennyt kertoa kardinaalille
omaani. Nyt se on joka miehen suussa.»

Katselin häntä, hänen seistessään ihan vastapäätä minua. Hänen

silmänsä säteilivät kiharaisten hiusten alta, jotka melkein peittivät ne.
Hän painoi kasvonsa alas, ja kuitenkin väreili hymy hänen huulillaan.

»Mitä kerroitte hänelle, mademoiselle?» kuiskasin läähättäen.

»Että rakastan», vastasi hän uljaasti ja käänsi kirkkaat silmänsä

minuun. »Ja että sentähden en hävennyt kerjätä — vieläpä
polvillani.»

Lankesin hänen jalkoihinsa ja tartuin hänen käteensä, ennen kuin

viimeinen sana oli kirvonnut hänen huuliltaan. Silmänräpäykseksi
unohdin kuninkaan ja kardinaalin, vankilan ja tulevaisuuden, kaikki,
kaikki, paitsi että tämä nainen, niin puhdas ja kaunis, kaikessa niin
suuresti yläpuolelleni kohonnut, rakasti minua. Silmänräpäykseksi,
sanon. Sitten palasi muistini. Minä nousin pystyyn ja irtauduin
hänestä äkillisessä tunteitteni vaihdoksessa.

»Te ette tunne minua!» huudahdin. »Te ette tiedä, mitä kaikkea
olen tehnyt!»
 »Juuri sen tiedän», vastasi hän ja katseli minua ihmettelevästi
hymyillen.

»Ei, te ette tiedä!» vakuutin taas. »Ja sitäpaitsi… on tämä meidän

välillämme!» Otin kardinaalin kirjeen, joka oli pudonnut lattialle.

Hän kääntyi ja kalpeni hiukan. Sitten hän huusi nopeasti:

»Avatkaa se! Avatkaa! Se ei ole suljettu eikä sinetöity.»

Tottelin koneellisesti, kauheasti peloissani siitä, mitä saisin nähdä.

Vielä silloinkin kun pidin sitä avoinna edessäni, loin vain pelokkaan
silmäyksen kaunismuotoisiin kirjaimiin. Se kuului näin:

»Kuningas näkee hyväksi käskeä, että herra Gil de Berault, joka

on sekaantunut valtion asioihin, viipymättä vetäytyy takaisin
Cocheforêtin herrastilalle ja pysyy sen alueella, kunnes kuningas
armollisesti toisin määrää.

Kardinaali Richelieu.»

Me menimme naimisiin seuraavana päivänä, ja neljätoista päivää

myöhemmin olimme Cocheforêtin kellastuneissa metsissä korkeiden
vuorten suojassa, suuren kardinaalin, joka oli vielä kerran voittanut
vihollisensa, katsellessa kylmäkiskoisesti hymyillen koko maailman
tungeksimista vastaanottohuoneessaan. Hänen onnensa nousua
kesti kolmetoista vuotta siitä ajasta lukien, ja se päättyi vasta hänen
kuollessaan. Sillä maailma oli kokemuksesta viisastunut, ja vielä tällä
hetkelläkin on se päivä, jona minä olin yksinäni vastaanotossa,
nimeltään »narrien päivä.»
*** END OF THE PROJECT GUTENBERG EBOOK KARDINAALIN
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