<figure><img src="image_35.png" /><figcaption>**Fig. 24** Wild Thai mushrooms cultivated in compost media. **a** *A. subrufescens* (Thongklang et al. 2014a). **b** *Agaricus flocculosipes* (Thongklang et al. 2014a), **c** *Lepista sordida* (Thongbai et al. 2017), **d** *A. subtilipes* (Thongklang et al. 2016), **e** *Macrolepiota dolichaula* (Rizal et al. 2016)</figcaption></figure>

including decumbens grass, sugarcane straw, brizantha grass, wheat straw, and sugarcane bagasse, supplemented with and without wheat bran as the primary substrates, were studied and brizantha grass was found to be the optimal substrate for cultivating *P. ostreatus*. Supplementing the compost with wheat bran improved yield in all substrates tested. In addition, the conditioning of compost after 7 days for all substrates showed higher yield and biological efficiency for *P. ostreatus*. The composts supplemented with 20–40% composted or 20% raw two-phase olive mill waste were reported as potential substrates for *Pleurotus* cultivation (Zervakis et al. 2013). In addition, semi-compost made from wheat straw and sunflower husks (2:3) can be used to grow *Pleurotus* sp. The substrate was fermented by aerobic fermentation for 36 h at 65 °C followed by cooling to 25 °C over 24 h (Myronycheva et al. 2017).

*Other genera*

Thongbai et al. (2017) used a rice straw based compost to cultivate *Lepista sordida*. Rice straw mixed ammonium phosphate, calcium carbonate, calcium sulfate, rice bran and urea were used as main substrates. The mycelium of *L. sordida* was inoculated into the pasteurized compost, which was later covered with a casing layer mixture of 15% sand with humus. *Lepista sordida* produced fruiting bodies after 31 days at 25 °C with 95–97% humidity after casing (Fig. 24). *Macrolepiota dolichaula* is a wild edible mushroom that is widely consumed in China, India and northern Thailand. This mushroom also can grow well in rice straw based compost. Mushroom production occurred

at 41 days with four flushes at 25 °C with 92% relative humidity (Fig. 24, Rizal et al. 2016).

Mushrooms have long been used for consumption, medicinal purposes and functional foods; however, the recognition that they are nutritionally a very healthy food group that contains important sources of biologically active compounds of medicinal value is more recent. Mushrooms have become a hot issue, especially in Asia. Sustainable success of mushroom growing depends on research and development to establish new ideas and methods for improving cultivation. Agricultural waste can be used as substrate for growing mushrooms, resulting in lower cost as well as the productive utilization of waste.

At present, most domesticated mushroom species are international species or variants, and few local strains are grown. The price of those mushrooms is quite low and there is little potential for export. Therefore, developing novel species or strains for local cultivation has become important. New wild species or strains of mushrooms will provide high value products with nutritional and medicinal potential. However, the successful development of a newly cultivated mushroom depends on economical and biological factors, the ease of production and acceptance in the market. After establishing edibility and cultivability, the best conditions for growth must be established. Best cultivation techniques include finding the optimal growth media, pH, temperature, C source, N source, C:N ratio, as well as the best spawn, compost and casing.
## 24. Growing mushrooms in bags

Wood-inhabiting mushrooms can be cultivated in any type of lignocellulosic material, such as straw, sawdust or rice hulls (Victor and Olatomiwa 2013). Pathmashini et al. (2008) showed that the cultivation of the oyster mushroom (*Pleurotus ostreatus*) on various sawdust types produces different fruiting yields. Sawdust is the most commonly used material for the cultivation of oyster mushrooms and is the preferred medium for commercial production (Oei 2005). Maximum biological efficiency of oyster mushroom cultivation is gained from growth on the sawdust of rubber trees (Pathmashini et al. 2008). It has also been shown that softwood sawdust such as coconut, cashew, mango and rubber are more suitable than hardwood sawdust (Custodio and Cristopher 2004). The availability of raw materials such as sawdust, rice straw, sugarcane wastes are key factors in the choice of agricultural waste for growing mushrooms (Pathmashini et al. 2008). The most commonly and easily cultivated mushrooms in South East Asian countries are oyster mushrooms (*Pleurotus*), ear mushrooms (*Auricularia*), and straw mushrooms (*Volvariella*), *Lentinula edodes* (shiitake), as well as *Lentinus* (e.g. *Lentinus squarrosulus*), *Ganoderma*, *Macrolepiota* and “Agrocybe” (i.e., *Cyclocybe*) spp. (Kwon and Thatithatgoon 2004).

Different types of sawdust are used as the growing medium in tropical areas, depending on the characteristics of the area and the trees available. Rubber tree sawdust is the most popular (Kwon and Thatithatgoon 2004; Klomklung et al. 2012; Nguyen 2004), followed by *Acacia auriculiformis* (Tapingkae 2005), *Mangifera indica* (Tong and Rajendra 1992) and *Tamarindus indica*. For every 1 kg

sawdust bag, additions are made of 10 g of calcium carbonate, 50 g of ricebran, 10 g of pumice, 10 ml of molasses, 10 g of flour and 10 g of brewer's waste. These components are then mixed with water to obtain a water content of 65-70%. Each 800 g of substrate is then tightly packed in a 25.8 cm polypropylene bag and capped with a plastic ring or bottle neck, leaving space to later inoculate with mycelium (Kwon and Thatithatgoon 2004; Stamets 2000; Klomklung et al. 2012; Yamamaka 1997). Each sawdust bag is sealed with a cotton wool plug, covered with newspaper, and tied with a rubber band. The sawdust bags are sterilized at 121 °C for 15 min or at 90-100 °C for 3 h. After the temperature drops to 25 °C, the bags are inoculated with spawn (Fig. 25c) that comprises 10% of the weight of the sawdust bag. Sawdust bags are kept at room temperature (25 °C) at 70-80% humidity to produce fruiting bodies (Klomklung et al. 2012).

When new wild mushrooms are introduced to the market, it is important to conduct fruiting tests. Depending on the type of mushroom, a choice can be made between compost or sawdust media. As a rule of thumb, for wood-inhabiting mushrooms (e.g. *Lentinula*, *Auricularia*) it is better to use sawdust media in bags (Fig. 25a, b), while for soil-inhabiting mushrooms (e.g. *Agaricus*, *Macrolepiota*) it is better to use straw compost.

For wood-inhabiting mushrooms, protocols adapted from Klomklung et al. (2012) are followed. The surface of sawdust growing bags is inoculated with spawn. The bags are kept in a dark incubation room at the optimum temperature and relative humidity of the particular mushroom. Bags are opened when the mycelium has completely colonized the substrate. The surface of the substrate is scraped slightly with a sterile teaspoon to remove the thin whitish

<figure><img src="image_36.png" /><figcaption>**Fig. 25** a *Auricularia thailandica* growing on sawdust substrate bags; b *Auricularia cornea* white variety growing on sawdust substrate bags; c mushroom spawn bottles</figcaption></figure>

<figure><img src="image_37.png" /></figure>
<figure><img src="image_38.png" /><figcaption>**Fig. 26** Field preparation for cultivation of *Stropharia rugosoannulata* in Honghe County, China</figcaption></figure>

<table><caption>**Table 12** Examples of mushroom species suitable for field cultivation, including soil characteristics, substrates, and climatic requirements for the listed species</caption><thead><tr><th>Species</th><th>Soil type</th><th>Soil pH</th><th>Substrate</th><th>Temperature (°C)</th><th>Humidity (%)</th><th>References</th></tr></thead><tbody><tr><td>Coprinus comatus</td><td>Sandy</td><td>7</td><td>Cottonseed meal</td><td>16–22</td><td>85–95</td><td>Chen (2000)</td></tr><tr><td>Dictyophora indusiata</td><td>Loam</td><td>6.5–7</td><td>Bamboo litter, straw, sawdust</td><td>22–30</td><td>60–65</td><td>Chen (2000)<sup>a</sup></td></tr><tr><td>Ganoderma sp.</td><td>Sandy</td><td>4.2–5.3</td><td>Sawdust</td><td>23–34</td><td>80–90</td><td>Mayzumi et al. (1997), Cha and Yoo (1997)</td></tr><tr><td>Lentinula edodes</td><td>Clay loam</td><td>4.5–6</td><td>Sawdust, wheat/rice bran</td><td>5–20</td><td>80–90</td><td><sup>a</sup></td></tr><tr><td>Morchella spp.</td><td>Sandy loam</td><td>7–7.5</td><td>Sawdust, wheat bran, humus</td><td>&lt; 20</td><td>50–70</td><td>Liu et al. (2018a)<sup>a</sup></td></tr><tr><td>Oudemansiella radicata</td><td>Clay loam</td><td>6.5–7.2</td><td>Wood chips/corn cobs</td><td>20–30</td><td>60–80</td><td>Hao et al. (2016)</td></tr><tr><td>Phlebopus portentosus</td><td>Clay loam, humus</td><td>4–6</td><td>Sawdust</td><td>27–37</td><td>55–80</td><td>Ji et al. (2011), Kumla et al. (2011, 2015)<sup>a</sup></td></tr><tr><td>Polyporus umbellatus</td><td>Sandy loam, humus</td><td>5.5–7</td><td>Leaves, humus,</td><td>18–24</td><td>60–80</td><td>Bandara et al. (2015)<sup>a</sup></td></tr><tr><td>Stropharia rugosoannulata</td><td>Humus rich sandy</td><td>5–6</td><td>Wood chips/rice straw</td><td>22–28</td><td>70–75</td><td><sup>a</sup></td></tr><tr><td>Volvariella volvacea</td><td>Clay</td><td>7.5–8</td><td>Rice straw</td><td>22–40</td><td>90</td><td><sup>a</sup></td></tr></tbody></table>

mycelia. The substrate bags are then placed on a shelf and covered with black cloth to allow appropriate ventilation. They are maintained in a growing house at 80–85% relative humidity, and sprayed daily with water until pin heads appear and eventually develop into fruiting bodies. The fruiting bodies are manually harvested, counted and weighed (Klomklung et al. 2012).

## 25. Growing mushrooms in the field

With the increase in consumer awareness and subsequent demand for cultivated mushrooms, there now exists a need for alternative, cost-effective strategies of mushroom cultivation. The global mushroom industry is forecast to grow from a value of about $35 billion USD in 2015 to nearly $60 billion USD in 2021, thus clarifying the need for a diverse range of production styles in order to develop the

industry in a sustainable manner (Research and Markets 2018). Alternatives to more traditional high-volume production techniques include the inoculation of logs, cultivation in forest understories, and use of managed forests, as well as field-grown mushrooms.

Field-grown mushrooms offer an effective use of space, allowing for the production of mushrooms in agricultural fields, alongside crops, or between cropping cycles (Fig. 26). Offering additional income to farmers, this practice has become popular with rural development programs (Zhang et al. 2014a, b; Brum and Brum 2017). Additionally, field cultivation allows for improved soil systems in agricultural fields due the increased rates of nutrient cycling and provision of organic matter into the soils (Phan and Sabaratnam 2012; Zhang et al. 2012).

In this section, we outline some of the basic principles and considerations for field cultivation while listing some

<footnote><sup>a</sup>Rows include unpublished findings/data</footnote>
of the key species that can be successfully cultivated in a field environment (Table 12). We will limit our discussion to the cultivation of mushrooms in agricultural fields and soils, not the broader practices of outdoor cultivation using bags or inoculated logs.

There are a range of mushrooms that can be cultivated outdoors, and appropriate species can be selected according to local growing conditions (Table 12). For example, *Coprinus comatus* or *Stropharia rugosoannulata* can be grown during the autumn and winter months when temperatures are milder, and *Volvariella volvacea* should be grown during the warmer summer months. Consideration for soil pH, texture and health is also critical. The pH and texture will partially determine which species of mushroom will be grown, and also the degree of irrigation required to maintain desired soil water levels (Table 12). Soil health is crucial to the final product, as many species of fungi can hyperaccumulate heavy metals (Cocchi et al. 2006; Turkekul et al. 2004).

After the selection of an appropriate growing area, a few basic steps are required for the preparation of the field and subsequent cultivation. After light tilling of the field, compost is applied, followed by the application of the fungal material (spawn or colonized bags), this is then covered in compost and a final layer of soil over that (a mulch of straw or shade cloth could further protect against desiccation and drying out) (Fig. 26).

There are a wide range of applications for the field cultivation of mushrooms, but most relate to the intensification of land use systems, generation of additional sources of income, and appeal to low income groups that cannot afford the costs of establishing grow houses. This method of production has become popular in many parts of Asia where climates are suitable and smallholder farmers need alternative sources of income. For example, in Cambodia, farmers have adopted the practice of spreading spawn produced from *Lentinus* or *Volvariella* species over rice

straw waste in the paddies (with supplemented watering). This provides an additional harvest of mushrooms from the paddies during the off season. Another program run in Honghe County, Yunnan Province, China, assists local farmers with the cultivation of *Stropharia rugosoannulata* in their fields; this is a seasonal mushroom crop produced mostly in autumn and spring when the temperatures are suitable (Fig. 27, Table 12).

The nature of growing mushrooms outdoors means that the mushroom crop is subject to ambient environmental conditions, with no climate controls in place, for example, temperature and humidity fluctuate with no real mechanisms in place to control these parameters. In addition to climatic variability, outdoor cultivation exposes the mushrooms to non-sterile conditions, allowing easy access for secondary infection by slime molds, predatory fungi, and insects (Table 12).

Field cultivation of mushrooms is applicable to a wide range of habitats and conditions, depending on the desired species to be grown. By offering novel and exciting alternatives to plant-based agriculture, and providing additional income to farmers, field cultivation of mushrooms is becoming popular in programs aimed at improving the livelihoods of smallholder farmers and for incorporation into projects looking to diversify the agricultural output of a unit of land. However, growing mushrooms outdoors is not without challenges and requires careful species selection and training of practitioners before any such programs can commence.

## 26. Modern mushroom production: an automated factory process

Limited harvests during unproductive seasons and the risk of mushroom poisoning prevent wild mushrooms from entering the food market value chain in a major way. Domestication and industry production has therefore

<figure><img src="image_39.png" /><figcaption>**Fig. 27** *Stropharia rugosoannulata* emerging from soil substrate mix and mature crop shown. Shade cloth is pulled back to reveal harvest</figcaption></figure>
<figure><img src="image_40.png" /><figcaption>**Fig. 28** Different mushroom species cultivated worldwide. **a**, **b** *Hy-menopellis* sp., **c**, **e** *Pleurotus* sp. (Oyster mushrooms), **f** *Ganoderma* sp. (Lingzhi). Photo credit: T. Luangharn</figcaption></figure>

become essential for incorporating mushrooms into dietary diversity and nutritional security. The global production has increased from 30.2 million tons in 2010 to 48 million in 2017 (FAO). As a leading producer, China increased from 22.6 million tons in 2010 to 38.4 million in 2017, accounting for 75% of global production (CIRI 2017). Of the 16,000 known mushroom species (Hawksworth 2012), about 7000 species have varying degrees of edibility, more than 3000 species are major edible mushrooms, and 700 are regarded as safe medicinal mushrooms (Chang and Wasser 2017).

Global demand for mushrooms has rapidly increased and thus large-scale growers have been established, with year-round marketing dominating commercial mushroom production. In China, there are six groups of mushrooms each with a production capacity of over one million tons per year (Zhang et al. 2015): 8.2 million tons for *Auricula* species; 7.7 million tons for *Lentinula edodes* (Shiitake mushroom); 5.9 million tons for *Pleurotus* species; 3.4 million tons for *Agaricus bisporus*; 2.6 million tons for *Flammulina* spp.; and 1.4 million tons for *Pleurotus eryngii*. The growth of the commercial mushroom industry depends on productivity, efficiency, and competitiveness within the entire market value chain. Most of the more recently domesticated species can recycle waste substrates

<figure><img src="image_41.png" /><figcaption>**Fig. 29** Equipment for manufacturers for mushroom cultivation. **a** Substrate media mixer, **b** mushroom bag filling machine, **c** transporter and conveyors carry out substrate bags, **d** steam sterilization machine, **e** inoculation, **f** auto-transportation, **g** incubation. Photo credit: F. Huang</figcaption></figure>
<figure><img src="image_42.png" /><figcaption>**Fig. 30** Mushroom industry zone with controlled environment for rural employment and rural development</figcaption></figure>

and therefore suit smallholders in agroecosystems (Dai et al. 2010b) (Fig. 28).

In the last decade, there have been remarkable advances in mushroom growing technology. Not only have strains been improved, but the whole mushroom production process has changed from manual to automated systems. The mixing of substrates, filling of bags, growing of liquid spawn, inoculation of bags, moving of bags, temperature and humidity control, and packing are all automated processes (Fig. 29).

Reliable substrate sources and machinery are two of the factors that determine the scale of operation and productivity. Substrates can be harvested from nearby forests through sustainable forest management, from recycled agricultural residue, sugarcane factories, livestock feed and waste, and the mulberry industry (Fig. 29) (Zhou et al. 2012). Although mushrooms can be cultivated using different methods, such as sawdust bags, bottles, shelves, and logs (see preceding chapters) there is an increasing demand for the establishment of modern mushroom facilities, which provides a range of 1 ~ 10 million bags annually with well-equipped enclosed cooling and sterilized buildings. This enables proper growing conditions to be maintained. The most suitable conditions for mushroom

production must be establish and are temperature, humidity, uniform ventilation, substrate moisture levels and or light to promote the formation of fruit bodies. For example, blue light-emitting diodes impact the quality of *Lentinus sajor-caju* (Huang et al. 2017a). However, maintaining and running such growing houses requires high volumes of electricity and water, making the production process costly and having a large impact on the release of greenhouse gases. Future trends should focus on the use of renewable energy sources.

The development of new technologies, such as photovoltaics for heating and cooling, artificial intelligent and technology for controlling the environment to optimize temperatures and moisture and even light formula by LED, will maximize the production season, enhance the productivity and quality of mushrooms, and reduce energy costs (Fig. 30). Artificial intelligence or automation includes substrate bag filling, inoculation, cultivation, scanning and picking up contaminated bags, and robots for packing and transportation. Both small- and large-scale production lines should embrace integrated systems, for example incorporating the use of recycled materials, such as agricultural waste, or making use of a sustainable supply
of woody substrates from managed plantations, coupled
with the integration of renewable energies.

Edible mushrooms have been cultivated for many cen-
turies, and it is expected that their production will increase
further due to market demand. The improvement and
development of modern technologies, such as computer-
ized control systems to control environmental parameters,
automated harvesting, techniques for the production of
mushrooms in a non-composted substrate, and new meth-
ods for substrate sterilization and spawn preparation, will
increase the productivity of mushrooms (Sánchez 2004).
However, the modern automated factory processes for
mushroom production require a significant initial invest-
ment of fixed assets. Typically, this large capital outlay of
fixed assets requires a recovery period of at least 5 years
(Li and Hu 2014). It is unreasonable for farmers in low-
and middle-income countries to own automated equipment,
since the upfront capital investment required for these
items is economically unsustainable, even in the long term
(Higgins et al. 2017). A more viable, long-term solution is
the granting of funds to farmers via either public or pri-
vately funded organizations (Zied and Pardo-Giménez
2017).

27. New edible mushrooms

Due to their culinary, nutritional, and health benefits, the global market for mushrooms continues to grow, from US $34.1 billion in 2015 to US$69.3 billion by the end of 2024 (Valverde et al. 2015; Bal 2018). Mushrooms also show potential for use in waste management, as discussed elsewhere. However, mushrooms have life cycles very different from those of green plants. The choice of mushroom species for cultivation depends both on the growth media available and on market considerations (Beetz and Greer 1999; Rosmiza et al. 2016; Sánchez 2004). Oyster mushrooms, which grow on many substrates, are easiest (Patil et al. 1989). Shiitake mushrooms have already garnered considerable consumer demand (Teng 2008). To date, only two mycorrhizal mushrooms, morels (Morchella spp.) and truffles (Tuber spp.), have been commercially cultivated (Selosse et al. 2017).

Several new species of wild edible mushrooms have
been successfully domesticated over the last few years,
especially in tropical areas (Klomklung et al. 2012;
Thongklang et al. 2014a, b, 2016; Rizal et al. 2016; Ban-
dara et al. 2017) (Fig. 31A). Luangharn et al. (2017),
Thongbai et al. (2017) and Klomklung et al. (2012) have
shown that it is possible to domesticate local strains of
Pleurotus giganteus that can grow at temperatures

<figure><img src="image_43.png" /><figcaption>**Fig. 31** A *Auricularia thailandica*; B Hybrid from Thai and French strains of *Agaricus subrufescens*; C White *Auricularia cornea*; D *Lepista sordida*; E *Agaricus flocculosipes*; F *Agaricus subtilipes*; G Fruiting bodies of *Macrolepiota dolichaula* strain MFLUCC-13-</figcaption></figure>

0579. a different stages of fruiting body development, b pileus with annulus, c squamules on pileus, d and e bud stages of fruiting bodies; f mature fruiting bodies. a = 25 cm, b = 10 cm, c, d, e, f = 5 cm
consistent with Thai farm production. A new hybrid developed from Thai and French strains of *Agaricus subrufescens* was developed successfully between INRA, France and Mae Fah Luang University, which fructifies in tropical climates (Thongklang et al. 2014b, Fig. 31B). For the first time, a new white strain of *Auricularia cornea* was successfully domesticated at the Kunming Institute of Botany, Chinese Academy of Sciences (Fig. 31C). In Thailand, strains of *Auricularia thailandica* (Fig. 35), *Lepista sordida* (Fig. 31D), *Agaricus flocculosipes* (Fig. 31E), *A. subtilipes* (Fig. 31F) and *Macrolepiota dolichaula* (Fig. 31G) have successfully been cultivated. Mushrooms are not only used in traditional medicines, but are known to contain various bioactive components which can be used in cosmetics (Kwon and Thatithatgoon 2004; Hyde et al. 2010) and in medicine (De Silva 2013; Wisitrassameewong et al. 2012b). People in most parts of the world enjoy eating mushrooms, and therefore there is enormous potential for introducing new tropical mushrooms to the global market.

The number of fungi worldwide is estimated to be between 2.2 to 3.8 million, but only 120,000 species have been described, so it appears that about 92% of fungi have yet to be described (Hawksworth and Lücking 2017). Over the past 5 years, numerous new fungi have been described, especially within tropical areas (Ariyawansa et al. 2015; Liu et al. 2015; Li et al. 2016; Hyde et al. 2016; Tibpromma et al. 2017, 2018; Hyde et al. 2017; Wanasinghe et al. 2018). One recent paper published by Hyde et al. (2018a, b, c) showed that up to 96% of fungi in northern Thailand may be new to science. On the other hand, the State of the World's Fungi report 2018 (Willis 2018) states that about 350 species of edible fungi are collected and eaten worldwide annually, but this figure should be higher. To address this knowledge gap, the Center of Excellence in Fungal Research in Mae Fah Luang University and the Soil Biology group of the Kunming Institute of Botany are working on updating the global list of edible and medicinal fungi. There are many reasons for being optimistic about the future of wild edible fungi: they help maintain the health of forests and are a valuable source of nutrition and income.

## 28. Agaricus subrufescens

*Agaricus subrufescens* is an important medicinal mushroom (Wisitrassameewong et al. 2012a; De Silva et al. 2013), which belongs to section *Arvenses* (subgenus *Flavoagaricus*) of *Agaricus*. The main synonyms of *A. subrufescens* are *A. blazei sensu* Heinemann (misapplied), *A. brasiliensis* (illegitimate), and *A. rufotegulis*. This species has a broad climatic and geographical distribution range in Asia, Europe, Oceania and Africa (Thongklang et al. 2016). *Agaricus subrufescens* was discovered in

America in the late nineteenth century and later in Sao Paolo, Brazil; it was called Piedade mushroom. One mushroom (named *A. blazei* Murrill) was introduced in Asia in 1965 by T. Furomoto, who sent it to Japan for medicinal investigation but turned out to be conspecific with *A. subrufescens* (Kerrigan 2005). The common name for *A. subrufescens* is the almond mushroom. In addition, other common names are Himematsutake in Japan, Cogumelo do Sol in Brazil, and Royal Sun Agaricus in several other countries (Wisitrassameewong et al. 2012a, b). The main characters of *A. subrufescens* are a reddish-brown cap covered with silk-like fibres, a two-layered and floccose annulus, the odour of almond, yellow staining, and a positive Schäffer's reaction (Thongklang et al. 2014b).

*Agaricus subrufescens* has been commercially cultivated in Brazil, Japan, China, Korea and Taiwan (Kerrigan 2005; Gregori et al. 2008). In Brazil, the mushroom is a particularly important export mushroom with a higher price than other commercial mushrooms (Souza Dias et al. 2004). Button mushroom compost is normally used to cultivate *A. subrufescens* commercial strains. Other forms of agricultural waste are also used as alternative substrates to grow this mushroom. González Matute et al. (2011) produced *A. subrufescens* based on spent oyster mushroom substrate mixed with sunflower seed hulls, vermicompost and supplements. Wild strains of *A. subrufescens* have been studied for cultivation. Zied et al. (2011) reported the success of wheat straw with chicken manure and black peat+soil (4:1) as casing to cultivate a Brazilian/French *A. subrufescens* strain and their hybrids. Thongklang et al. (2014b) isolated a wild Thai *A. subrufescens* strain and successfully cultivated it in wheat straw/horse manure based compost at 25 °C and 95% humidity (Fig. 26a). Thongklang et al. (2014b) reported that samples from Brazil, France and Thailand are interfertile. Hybrid strains of Thai × Brazilian and Thai × French showed higher yields than Thai parental strains.

The mushroom is both nutritious and medicinal. Zied et al. (2017) evaluated the chemical composition of *A. subrufescens*, and reported that the stipe of the mushroom contained 69.56% of total carbohydrates, 63.89% of available carbohydrates, and an energy value of 363.97 kcal 100 g⁻¹ DM). The pilei comprised 33.96% protein, 7.75% nitrogen, 8.24 and 2.44% ash and crude fat. Medicinal benefits have also been reported for this mushroom, for example in cancer care, as antitumor agents, and in cholesterol reduction (Wisitrassameewong et al. 2012a, b; De Silva et al. 2013; Stadler and Hoffmeister 2015). *Agaricus subrufescens* produces several bioactive compounds, such as lectin, riboglucan, glucomannan, agaritine and blazien, all of which were shown to reduce tumor growth. In addition, spiro triterpenoids of the blazeispirol type are produced in mycelial cultures of this mushroom (Hirotani et al.
1999, 2000, 2001, 2002). Extracts were found to be highly selective antagonists of Liver X receptors (LXR) alpha (Grothe et al. 2011). Thongklang et al. (2017) reported that Thai-Brazilian and Thai-French hybrid strains produce higher yields of blazeispirol A in culture than their Thai parental strain. However, blazeispirols have never been found in the basidiomes of *A. subrufescens* (Thongklang et al. 2017). This is important, as if strains could be found with blazeispirols in the basidiomes, then eating the mushroom would have important medicinal benefits.

Mushrooms have been used as food and health products for millions years for their perceived medicinal properties. *Agaricus subrufescens* is widely cultivated in Brazil (Souza Dias et al. 2004; Mendonça et al. 2005), and the mushroom is exported from Brazil to various countries such as Australia, Bolivia, Germany, Korea, India, Japan, South Africa, Thailand, and the USA (Mendonça et al. 2005). The mushroom normally requires compost for cultivation (Thawthong et al. 2014). *Agaricus subrufescens* is consumed worldwide in fresh, dried and powder form from mycelium/fruiting bodies. Fresh mushrooms have been consumed as food because of their potential medicinal property and pleasant almond flavour. Moreover, dried or powdered mycelium/fruiting bodies are used as nutrient supplements. In Japan, 100,000–300,000 kg of dried almond mushrooms is produced every year (Takaku et al. 2001).

The mushrooms have been used as complementary and alternative medicine for cancer care. Private companies such as King Agaricus 100, Sen-Sei-Ro Gold, and ABMK sell products from *A. subrufescens*, and these products are currently being used by around 500,000 people for cancer treatment and prophylaxis (Hyodo et al. 2005).

## 29. Using fungi to enhance food value

For thousands of years, human societies have been utilizing fungi as food sources (Chang 1980; Moon and Lo 2014; Siddiq et al. 2018). In particular, edible mushrooms are ideal for vegetarians, because they are excellent sources of protein (typically 20–30% crude proteins as a percentage of dry matter), have a low-fat content, are free of cholesterol, and contain most of the amino acids essential to human and animal nutrition (Kaur et al. 2018). Fungal taxa are utilized in the food industry taking advantage of their metabolism and metabolites (Gilbert and Robinson 1957; Moore and Chiu 2001; Adrio and Demain 2003; Ghorai et al. 2009). In particular, fungal species are used in the production of fermented foods and beverages in many traditional and indigenous cultures around the world (Abe et al. 2008; Dupont et al. 2016). For instance, the yeast *Saccharomyces cerevisae* is the oldest and best known fungal species used for wine and bread making, in drug production, as bio-control agents, in enzyme biotechnology, as well as for

research and development (Ghorai et al. 2009; Sharma et al. 2018). A brief description of some of the most important fermented food industries is provided below.

### Mold-ripened cheese

Cheese is a solid or semi-solid protein food product manufactured from milk. Fungal species are important for the manufacture and ripening of two types of cheese, the Camembert and Blue-veined varieties (Fox et al. 2017). The name “blue-veined cheese” derives from the blue-green mold growing throughout the product (Martín and Coton 2016). There are various kinds of blue-veined cheese products, viz. Roquefort, Gorgonzola, Stilton, Danish Blue, and Blue Cheshire. Most Blue cheese is produced from unpasteurized milk, and *Penicillium roqueforti* is added to the cheese prior to storage at controlled temperature and humidity. *Penicillium roqueforti* grows throughout the cheese and produces proteolytic and lipolytic enzymes (viz. proteases, lipase and beta-ketoacid decarboxylase) and methyl ketones, particularly 2-heptanone, as the major flavour and odour compounds (Kinsella et al. 1976; Dupont et al. 2016; Ropars et al. 2017). *Penicillium roqueforti* grows at a low oxygen tension and low pH values, while having the ability to use both pentoses and hexoses as substrate. These characteristics make the fungus ideally suited for the industrial Roquefort-type cheese production (Babel 1953; Kinsella et al. 1976, García-Estrada and Martín 2016). Camembert cheese is rather different from blue-veined cheese, as it characterized by a white mold growth on the surface and a soft texture. *Penicillium canidum* and *P. camemberti* are used for the camembert cheese production (Bourdichon et al. 2012).

### Fermented products

#### Soy sauce (Shoyu) and Miso

Soy sauce is one of world's oldest flavor ingredients; made from fermented soybeans/wheat. It is a dark brown liquid that is stable at ambient temperature and does not require refrigeration during storage due to its low water activity and high salt content. Soy sauce has been used in cooking for over 1000 years, in particular in China, Japan, Korea and other Asian countries (Luh 1995; Hong et al. 2015; Liu 2017). The soy sauce production process comprises three major steps, which are Koji production, brine fermentation and refining. In the first step, soaked, cooked, mashed soy beans are mixed with an equal amount of roasted, lightly crushed wheat, and inoculated with 0.1–0.2% starter mold (*Aspergillus oryzae* or *A. sojae*) in wooden trays. The fermented mixture (Koji) then undergoes brine fermentation using the lactic acid bacterium, *Pediococcus halophilus*, and yeasts including *Zygosaccharomyces rouxii* and *Candida* species (Liu 2017). The final step of soy sauce fermentation (refining), includes pressing, filtration, pasteurization and packaging. The
fermented soybean paste is called miso, has a similar aroma and flavor to soy sauce.

## Indonesian tempeh

Tempeh is a mould-fermented product prepared from fermented soy beans and is consumed mostly in Indonesia (Babu et al. 2009). Tempeh possesses some unique characteristics, including its flavor, sliceable meat-like texture, and nutritional properties (Astuti et al. 2000; Nout and Kiers 2005). *Rhizopus oligosporus* is the dominant fungal species used in the soy bean fermentation process. Initially, husk-free, soaked and pressed soybeans are inoculated with *R. oligosporus* and fermented for 1–2 days. During the fermentation process, the white mycelia of *R. oligosporus* invade and cover the substrate mass to bind the soy bean (Shurtleff and Aoyagi 1979; Steinkraus 1995; Nout and Kiers 2005; Babu et al. 2009; Shah and Patel 2017). Tempeh provides many health benefits, in particular for heart diseases, strokes, osteoporosis, cancer and digestive disorders, loss of excess weight), as it contains essential fatty acids, numerous vitamins, fiber and minerals.

## Quorn

Quorn is made from myco-proteins, which are produced by a fermentation process using the filamentous fungus *Fusarium venenum* (Finnigan et al. 2017; Kozubal et al. 2019). Quorn mycoprotein strains were previously misidentified as *Fusarium graminearum*, but were later found to represent *F. venenum* (Wheelock 1993; Trinci 1994; Wiebe 2004; Finnigan et al. 2017). With a low fat content and being free of cholesterol, quorn provides an alternative protein option that is meat-free and nutritious (Garodia et al. 2017). In addition, it is high in dietary fibre, which has been found to lower blood cholesterol (Denny et al. 2008; Ruxton and McMillan 2010; Garodia et al. 2017; Kozubal et al. 2019). The production process of Quorn is similar to that of beer, requiring less wheat and water for production, as well as resulting in lower carbon emissions, compared with meat sourced proteins (Finnigan 2011). During the production process, mycoprotein is mixed with vegetable flavorings and a small amount of egg albumen. To obtain its characteristic texture, a series of steaming, chilling and freezing processes are used to obtain the meat-like texture of Quorn products (Wiebe 2004; Finnigan 2011). Initially, Quorn products originated in the UK, but currently are available worldwide (Apostolidis and McLeay 2016).

## Rennet

Rennet is used by food companies for the milk clotting process within cheese production (Ogel 2018). Calf rennet was traditionally used for cheese-making worldwide (Thakur et al. 1990; Mamo and Balasubramanian 2018). Subsequently, the global increase in cheese production,

along with a decrease in animal rennet production, raised the price of traditional rennet. Therefore, food researchers tried to find alternatives for the milk clotting process within cheese production (Moschopoulou 2017; Ogel 2018). As a consequence, microbial rennet was produced using *Rhi-zomucor miehei* and *R. pusilus* (Thakur et al. 1990; Silveira et al. 2005; De Lima et al. 2008). Microbial rennet is cheaper than the rennet produced by animals, and the best option for vegetarians.

Other than those fermented food products, there are many commercial food industries in the world. Some food products which use fungal species are listed in Table 13.

## 30. Food colouring from filamentous fungi

Synthetic colorants are widely used in food production to enhance the appearance of food colors. Although synthetic colorants are stable and inexpensive, the demand for these products has decreased due to their potential or perceived harmful effects on human health (Bateman et al. 2004). Today, many synthetic colorants have been replaced with safer natural colorants. Pigments from plants are often used as food colorants (Shamina et al. 2007; Ambati et al. 2014; Leong et al. 2018; Upadhyay 2018). Although these pigments can generate various colors, production processes are limited due to plant growth needs. Filamentous fungi can be grown in fermenters, and are also being investigated as important sources of pigments (Table 14, Fig. 32). Some examples are given below.

**Red mold rice**, also known as red yeast rice, is an Asian traditional fermentation product of steamed rice fermented with filamentous fungi belonging to the genus *Monascus* (Fig. 33). Red mold rice has been widely used in East Asian countries with a very long history as both food coloring and medicine. The red colour from red mold rice is used in a variety of Chinese, Korean and Japanese foods, such as fermented bean curd, preserved dry fish, pork stew, roast duck, and roast pork (Chen et al. 2015). Red mold rice is also used as substrate for the production of “Hong Qu glutinous rice wine,” which gives it a bright-red colour and fine sweet flavor (Liu et al. 2018b; Park et al. 2016). After the optimization of biotechnological production processes, some species can easily produce pigments at a large scale in bioreactors, relying on relatively short fermentation times, and resulting in acceptable costs-of-goods. For example, the optimization of red pigment production in a submerged culture by *Monascus ruber* in complex culture media resulted in the development of a medium containing 10 g/l glucose, 5 g/l corn steep liquor and 7.6 g/l monosodium glutamate, generating the highest amount of red pigment at 20.7 U (Hamano and Kilikian 2006).
<table><caption>**Table 13** Fungal species used in the food and beverage industries</caption><thead><tr><th>Application</th><th>Product</th><th>Fungal species</th></tr></thead><tbody><tr><td rowspan="2">Beverages</td><td>Beer, Rum, Wine</td><td>Saccharomyces cerevisiae</td></tr><tr><td>Sake</td><td>Aspergillus oryzae</td></tr><tr><td rowspan="2">Cheeses</td><td>Roquefort, Blue cheese</td><td>Penicillium roqueforti</td></tr><tr><td>Camembert, Brie, soft ripened</td><td>Penicillium camemberti</td></tr><tr><td rowspan="10">Oriental food fermentations</td><td>Ang-kak</td><td>Monascus purpureus</td></tr><tr><td>Doenjang</td><td>Aspergillus oryzae</td></tr><tr><td>Hamanatto</td><td>Aspergillus oryzae</td></tr><tr><td>Miso</td><td>Aspergillus oryzae, A. sojae</td></tr><tr><td>Ontjom</td><td>Neurospora intermedia</td></tr><tr><td>Rennet</td><td>Rhizomucor miehei<br>Rhizomucor pusilus</td></tr><tr><td>Shoyu (soy sauce)</td><td>Aspergillus oryzae, A. sojae</td></tr><tr><td>Tempeh (Indonesian)</td><td>Rhizopus oligosporus</td></tr><tr><td>Quorn</td><td>Fusarium venenum</td></tr></tbody></table>

<table><caption>**Table 14** Examples of pigments produced by filamentous fungi</caption><thead><tr><th>Color</th><th>Pigments</th><th>Fungal producers</th><th>References</th></tr></thead><tbody><tr><td>Blue</td><td>Sanguinone A</td><td>Mycena sanguinolenta</td><td>Peters and Spiteller (2007)</td></tr><tr><td>Green</td><td>Xylindein</td><td>Chlorociboria aeruginosa</td><td>Saikawa et al. (2000)</td></tr><tr><td>Orange</td><td>Monascorubrin<br>Rubropunctatin<br>β-Carotene</td><td>Monascus sp.</td><td>Vendruscolo et al. (2017)</td></tr><tr><td>Red</td><td>Rubropunctamine<br>Monascorubramine<br>Lycopene</td><td>Mucor circinelloides Neurospora intermedia<br>Monascus sp.</td><td>Torres et al. (2016)<br>Hamano and Kilikian (2006), Dikshit and Tallapragada (2013)</td></tr><tr><td>Yellow</td><td>Ankaflavin<br>Monascin</td><td>Blakeslea trispora<br>Monascus sp.</td><td>Feofilova et al. (2006)<br>Yongsmith et al. (2013), Klinsupa et al. (2016)</td></tr></tbody></table>

Red mold rice also generates orange pigment. Pigment production is partially associated with cell growth and a dissolved oxygen concentration of between 0.894 and 1.388 mg O₂/l at 30 °C. Limiting conditions of dissolved oxygen decreases the production of orange pigments (Vendruscolo et al. 2017). An induced mutation of *M. purpureus* led to production of yellow pigments, which can be added to Chinese fresh noodles, providing a yellow shade with no discoloration (Yongsmith et al. 2013). Klinsupa et al. (2016) investigated fusing intraspecific protoplasts of a yellow mutant of *Monascus* spp. with a white prototroph, and a strain with high yellow pigment production was produced. Another mutant strain of *M. ruber* 10910, generated through UV mutagenesis, improved the production of extracellular hydrophilic yellow pigment tenfold (Wu et al. 2015b).

Unfortunately, the mycotoxin citrinin is often found in red mold rice, and has a negative impact on health. This

mycotoxin has been investigated as nephrotoxic and hepatotoxic to human cells (Bilgrami et al. 1988). The regulation of citrinin concentration in commercial red mold rice occurs in many countries, such as Commission Regulation (EU), Taiwan, US FAD, and Japan (Le Bloc’h et al. 2015; European Commission 2014). Therefore, the production of red mold rice with low citrinin is important to increasing its value and safety. Pengnoi et al. (2017) evaluated the effects of various purple rice varieties on the production of citrinin and red pigments by *M. purpureus* CMU002U (UV-mutant strain). The lowest value of citrinin concentration (132 ppb) was found in the Na variety, which passed the standards of Japan, Taiwan, and the European Union. The highest red pigment yield was obtained from the fermented Doi Muser variety. These results demonstrate that fermented purple rice has a high potential to be developed as a safe food colorant.
<figure><img src="image_44.png" /><figcaption>**Fig. 32** Chemical structures of pigments from filamentous fungi</figcaption></figure>

β-Carotene (50; orange pigment) is a precursor to vitamin A, and of great importance for healthy skin, the immune system and vision, and is abundant in plants and fruits, such as carrots, pumpkin and sweet potatoes. This pigment is often added to various foods and drinks for its coloring effect and antioxidant activity (Hameed et al. 2017). The filamentous fungi *Blakeslea trispora*, *Fusarium sporotrichioides*, *Mucor circinelloides*, *Neurospora crassa* and *Phycomyces blakesleeanus* are all excellent producers of β-carotene (Torres et al. 2016). The pigment production by *Neurospora intermedia* was successfully manipulated by varying several factors. The formation of β-carotene was strongly influenced by light, carbon, pH, and the co-factor zine ion. The highest pigmentation (1.19±0.08 mg carotenoids/g dry weight biomass) was achieved in a bubble column reactor (Gmose et al. 2018). Goksungur et al. (2002) investigated the effect of the pretreatment of

molasses, nitrogen sources, natural oils, fatty acids, antioxidant, and precursors on β-carotene production by *B. trispora*. The maximum β-carotene concentration (790.0 mg/dm³) was obtained in culture grown in molasses solution supplemented with linoleic acid (30.74 g/dm³), kerosene (27.79 g/dm³) and antioxidant (10.22 g/dm³). Thakur and Azmi (2013) investigated the different strategies for the extraction of β-carotene from *Mucor azygosporus* (strain MTCC 414). Various combinations of cell disruption and extraction methods were employed. The disruption of cells by sonication, followed by extraction with hexane and ethyl acetate (1:1, v/v), gave a maximum yield of β-carotene (985 µg/ml). Zhang et al. (2016d) investigated a mutant strain of *M. circinelloides* for β-carotene production. The deletion in two mutants (MU206 and MU218) of the *crgA* gene, a well-known repressor of carotenoid biosynthesis, resulted in increased β-carotene
<figure><img src="image_45.png" /><figcaption>**Fig. 33** *a* Red mold rice produced by *Monascus purpureus*. *b* *Monascus purpureus* strain MU002</figcaption></figure>

production. One strain derived from MU218 was able to accumulate up to 4 mg/g of β-carotene.

**Lycopene** (51; red pigment) is the strong natural antioxidant among carotenoids often found in tomatoes. Unlike β-carotene, it is not converted to vitamin A and remains in the blood plasma (Feofilova et al. 2006). The filamentous fungus *Blakeslea trispora* is a potential producer of lycopene. Enhanced lycopene production in *B. trispora* was achieved when treated with nitrogen ion implantation and N-methyl-N'-nitro-N-nitrosoguanidine. Mutant I5 with nitrogen ion implantation had a maximum lycopene yield of 28.8 mg/g, which was 64% higher than the parent strain (Wang et al. 2013a). Shi et al. (2012) improved the lycopene production of *B. trispora* by adding isopentenyl alcohol, dimethyl allyl alcohol, geraniol, and mevalonic acid to the medium in optimal concentrations. The highest lycopene production in mated cultures was 578 mg/l, achieved by adding 42 mg geraniol/l to the medium after 48 h of growth. The optimization of lycopene production by *B. trispora* in bath culture was based on response surface methodology. The highest yield was observed at 50 mg/l, by the addition of 2-methyl imidazole in a substrate supplemented with crude soybean oil (Pegklidou et al. 2008).

Many other species of filamentous fungi have the potential to produce various types of natural pigments for use in food coloring. However, to make these compounds commercially viable, much work on bioprocess technology and toxicity studies remains necessary before these pigments can be launched as products globally. Interestingly, the molecular background of biosynthesis of some fungal pigments, including the azaphilones from *Monascus* spp. (54–59; Mapari et al. 2010; Balakrishnan et al. 2013), has been elucidated, which may allow for a more straightforward and concise optimization of the production processes.

Aside from the above examples of fungal metabolites that are actually amenable to biotechnological production, there are many other pigments that have been described over the past century. A comprehensive overview of this

topic is not possible, and therefore we refer to the reviews by Gill and Steglich (1987) and the updates by Gill (1994, 2003). Many fungal pigments have been shown to be heterogeneous mixtures of polymers, complexes of organic compounds with metal ions, or have been proven to be extremely unstable and can therefore not serve as a basis for the development of commercial colorants. The same holds true of stable compounds, which have so far only been encountered in ectomycorrhizal mushrooms that cannot be subjected to sustainable biotechnological production. In fact, some very interesting pigments have been discovered from saprotrophic fungi that have rather rare colors, such as the green pigment xylindein (52 in Fig. 32; Table 14, Saikawa et al. 2000) from *Chlorociboria aeruginosa*, or the blue pigment sanguinone A (53) from fruiting bodies of the mushroom *Mycena sanguinolenta* (Peters and Spiteller 2007). Since such green and blue pigments are especially in demand as natural food coloring agents, further research on these aspects may prove rewarding. Recently even over 1000 years old stromata of the ascomycete *Hypoxylon fragiforme* have yielded novel pigments such as rutilin C (60) (Surup et al. 2018), which can be isolated from freshly collected stromata in substantial amounts.

## 31. Food flavouring

Bio-flavors that are of natural origin are preferable and in higher demand when compared with chemically synthesized products, even though they often constitute the same compound, because of the phobias of consumers to synthetic food ingredients (Christen and López-Munguía 1994; Bluemke and Schrader 2001; Vandamme 2003; Ravasio et al. 2014). Plant extracts have previously been used as a main source of flavor and fragrance compounds (Carroll et al. 2016). Microbial fermentation can also be used to produce flavors and aromas (Kim 2005), such as aldehydes, esters, methyl ketones and terpenoids (Gupta et al. 2015; Carroll et al. 2016).
Microbial cultures and their enzymes produce flavors naturally, as secondary metabolites of fermentation. Enzymes also act as biocatalysts in wide range of food flavor productions (Christen and López-Munguía 1994). These flavors are mostly used as food additives. The first synthetic flavor and fragrance compounds in the food industry were vanillin in 1874 and coumarin in 1868 (Gupta et al. 2015). Vanillin synthesis from ferulic acid uses a bioconversion process with two steps using two filamentous fungi (Lesage-Meessen et al. 1996). Ferulic acid is converted to vanillin acid using *Aspergillus niger* and reduced to vanillin using *Pycnoporus cinnabarinus* (Lesage-Meessen et al. 1996; Poornima and Preetha 2017).

Both filamentous fungi and yeasts play a significant role in producing flavoring compounds (Bluemke and Schrader 2001). In the wine industry, mixed fermentation of *Lachancea thermotolerans* and *Saccharomyces cerevisiae* are used to enhance the level of 2-phenylethanol, which has a rose-like odor (Ravasio et al. 2014). *Saccharomyces cerevisiae* and the filamentous basidiomycete *Tyromyces chioneus* are used for the production of grape wine and wine off flavors (Poornima and Preetha 2017). Off flavors are defined as “a flavor that is not natural or up to standard owing to deterioration or contamination” (Nijssen 1991; Christoph et al. 1999).

During amino acid catabolism in yeasts, isoamyl alcohol (banana flavor) and 2-phenylethanol (floral-rose flavor) are produced as natural flavors (Ravasio et al. 2014). Through the esterification of the lipase enzyme, banana, green apple and pineapple flavors are synthesized. *Rhizomucor miehei* is mainly used to obtain immobilized lipase (commercially available enzyme) in solvent free systems. This lipase was initially produced to be used in the food industry (Güvenç et al. 2002; Radzi et al. 2011; Goulet et al. 2012; Poornima and Preetha 2017). Through the lyophilisation of the culture fluid of *Pleurotus sapidus* (edible mushroom), the sesquiterpene (+)-valencene is deoxygenated into (+)-nootkatone (citrus-type odor), which is an industrial flavor component (Krügener et al. 2010).

The main biotechnological techniques for food flavoring with microorganisms are de novo synthesis (fermentation) and biotransformation (bioconversion) (Krings and Berger 1998; Kim 2005). In de novo synthesis, simple, inexpensive substrates are used as starter media without adding precursors of the products. Many primary metabolites and mixtures of (small amounts of) many aroma compounds are normally produced in the media (Krings and Berger 1998). During biotransformation, precursors of the products are added to the cultivation media in large amounts, leading to production of a single major component (Kim 2005). Flavor active substances are considered to be volatile organic compounds, such as higher alcohols,

esters, fatty acids derivatives and terpenes (Bluemke and Schrader 2001; Ravasio et al. 2014).

There are many commercialized enzymes for the biotechnological production of food flavor compounds, and a high concentration of aroma compounds. Enzymes are mainly used in food flavor synthesis because of the great reaction speed of catalytic activity in enzymes (Kim 2005). The contributions of fungi to flavor and aroma compound production are provided in Table 15.

Fungi produce a wide range of metabolites that can be used in the food industry (Vandamme 2003; Carrau et al. 2015). Microbial flavor phenotypes should be designed with the integration of natural yeast diversity, sensory analysis practices, and evolutionary engineering in mind (Carrau et al. 2015). Research should be implemented to better understand the molecular modifications of salivary composition, and receptor proteins and fats, which are stimulating and sensing odorants and tastes (Hofmann et al. 2018).

## 32. What is mushroom stock? Products, processes and flavours

Mushroom stock is a seasoning or food additive comprised of various mushroom flavours. In China, mushroom stocks are probably used more than anywhere else in the world. Mushroom stock products can be made from various species, including *Agaricus bisporus* (Guo et al. 2011), *Termitomyces albuminosus* (Zhou and Liu 2011) *Tuber magnatum* (Fantozzi et al. 2015) and many others. Mushroom stock products can mainly be divided into five types: condensed mushroom extracts, mushroom powders, mushroom sauces, preserved fried mushrooms, and instant mushroom soups. Mushroom extracts and powder are often used as a seasoning or ingredients in hot pot, while mushroom sauce and preserved fried mushrooms are commonly used as additives to rice, noodles and bread.

Several studies were carried out on processing technology, raw material ratio, nutrient composition and flavour components of mushroom stocks. Mushroom stock powder or liquids are made from various odorous mushrooms species. Normally, gas chromatography coupled with mass spectrometry (Largent et al. 1990; Rapior et al. 2002; Du et al. 2014) and electronic nose technology (Fujioka et al. 2013; Zhou et al. 2015b) is usually used for the detection of volatile organic compounds (VOC) within mushrooms. Small hydrosoluble molecules, such as amino acids and nucleotides (Ribeiro et al. 2008; Wang et al. 2009), as well as volatile aroma substances, such as $C_8$ derivatives, sulfur-containing compounds, terpenes and other VOCs, are mainly associated with mushroom aroma (Rapior et al. 1996, 1997a, b, c, 2002; Liu et al. 2016; Palazzolo et al. 2017). These numerous potential combinations bring
<table><caption>**Table 15** Contribution of fungi or fungal enzymes to volatile organic compound production</caption><thead><tr><th>Fungi</th><th>Volatile organic compounds</th><th>References</th></tr></thead><tbody><tr><td>Aspergillus niger</td><td>Methyl ketones</td><td>Armstrong and Brown (1994)</td></tr><tr><td>Aureobasidium pullulans</td><td>(creamy type odor)</td><td>Hagedorn and Kaphammer (1994)</td></tr><tr><td>Penicillium sp.</td><td></td><td>Krings and Berger (1998)</td></tr><tr><td>Aspergillus niger</td><td>Vanillin</td><td>Ramachandra and Ravishankar (2000)</td></tr><tr><td>Phanerochaete chrysosporium</td><td>(vanilla odor)</td><td>Poornima and Preetha (2017)</td></tr><tr><td>Pycnoporus cinnabarinus</td><td></td><td></td></tr><tr><td>Beauveria bassiana</td><td>Raspberry ketone<br>(berry type odor)</td><td>Gupta et al. (2015)</td></tr><tr><td>Diutina rugosa (reported as "Candida")</td><td>(-)-Menthol<br>(mint flavor)</td><td>Gupta et al. (2015)</td></tr><tr><td>Ischnoderma benzoinum</td><td>Benzaldehyde<br>(bitter almond flavor)</td><td>Fabre et al. (1996)</td></tr><tr><td></td><td>4-Methoxybenzaldehyde<br>(coumarin-like odor)</td><td>Gupta et al. (2015)</td></tr><tr><td>Kluyveromyces sp.</td><td>Phenylethanol<br>(rosary-like odor)</td><td>Krings and Berger (1998)</td></tr><tr><td>Pithomyces sp.</td><td>Nor-patchoulenol (woody type odor)</td><td>Gupta et al. (2015)</td></tr><tr><td>Yarrowia lipolytica</td><td>γ-Decalactone<br>(peach flavor)</td><td>Gupta et al. (2015)</td></tr><tr><td>Zygosaccharomyces rouxii</td><td>Furaneol (caramelly type odor)</td><td>Hecquet et al. (1996)</td></tr><tr><td></td><td></td><td>Krings and Berger (1998)</td></tr></tbody></table>

intense and elegant flavour to the mushroom, e.g. an almond-like aroma from *Agaricus subrufescens* (Kerrigan 2005) and a maple syrup-like odour in *Lactarius fragilis* var. *rubidus* (Wood et al. 2012). These unique combinations can alleviate the potent smell of any meat, poultry, or seafood dish and are perfect choices for vegetarian and vegan dishes.

The rationale why mushroom stocks can be attractive ingredients for many consumers is based not only on their flavour profile but also on a broad spectrum of their bioactive compounds (De Silva et al. 2013; Greeshma et al. 2018). These bioactive compounds may include unsaturated fatty acids (Günç et al. 2013), free amino acids (Ribeiro et al. 2008), volatile components (Politowicz et al. 2018), sulfur-sensory active compounds (Kupcová et al. 2018), antioxidant components like phenolics (Sevindik et al. 2018), antiproliferative and antitumor molecules like polysaccharides (Pandya et al. 2018; Morel et al. 2018), anti-lung cancer peptides (Prateep et al. 2017), and various neuroactive compounds (Thongbai et al. 2015; Sabaratnam and Phan 2017), depending on the mushrooms used to make the stock.

## 33. Fungi used in making tea

Tea is one of the most popular beverages in the world. Tea contains relatively high amounts of chemical substances,

such as caffeine and vitamins, which can accelerate the oxidation of fat. Therefore, almost all teas have a slimming and weight loss effect. Fermentation is the key factor in the tea manufacturing process. The fermentation of tea leaves alters their chemistry, affecting the organoleptic qualities of the tea. Fermentation affects the smell of the tea and typically mellows its taste, reducing astringency and bitterness while improving mouthfeel and aftertaste. The microbes may also produce metabolites with health benefits. According to different degrees of fermentation, tea can be divided into green tea (non-fermented), oolong tea (semi-fermented), black tea (fully fermented by oxidizing enzymes) and dark tea (post-fermented by microbes). Unlike other teas, dark teas, such as Pu-erh tea and Ceylon tea, are microbially fermented teas.

Fungi play an important role in the post-fermentation process of dark teas. Fungi can change some sensory properties in the post-fermentative process, as well as influence the contents of polyphenols, catechins, gallic acid, and caffeine. Additionally, fungi can improve the ripping speed, success rate, and quality stability of the tea (Chen et al. 2008; Hou et al. 2010). *Aspergillus* and *Blastobotrys* spp. have been reported to be effective in enhancing the nutrition of Pu-erh tea through fermentation (Zhao and Zhou 2005; Zhao et al. 2006; Abe et al. 2008). Some *Aspergillus* species, e.g. *A. aureolatus*, *A. egyptiacus*, *A. japonicus*, *A. niger*, *A. penicillodes*, *A. restrictus*, *A.*
wentii var. fumeus and Penicillium spp., Rhizopus spp. and yeasts were isolated during Pu-erh tea processing (Chen et al. 1985, 2006). In addition to these fungal isolates, Zhang et al. (2016c) reported two new species, A. mar-vanovae and Candida mogii, from a solid-state fermenta-tion of Pu-erh tea. Most fungal isolates can convert the tea polyphenols to bioactive theabrownins via a 4-day shaking fermentation in flasks at 40 °C, 250 rpm (Zhang et al. 2016c).

Tea bushes grow only in tropical and subtropical cli-mates. The time to pick tea leaves is related to the local temperature and humidity. For example, the picking period of Pu-erh tea is from the end of February to the end of November, and the best time for picking fresh leaves is half an hour after sunrise. Enzymatic inactivation is performed for freshly recovered leaves. After rolling and sun-drying, pile fermentation, the most important step, is carried out. Finally, the process of Pu-erh compressed tea (cake tea) is completed after a series of drying and compression steps. Water availability, temperature, and their interactions are important for mycelial growth. Chen et al. (1985) simu-lated the pile fermentation process of Pu-erh tea in the laboratory. During this process, Aspergillus niger, Peni-cillium spp. and Rhizopus spp. began to multiply at the beginning of fermentation. The maximum fungal quantity occurred in the first and second turning periods and then gradually decreased (Chen et al. 1985). Aspergillus niger is the predominant species in the whole pile fermentation, comprising about 80% of the mass of microorganisms (Chen et al. 1985; Liu et al. 1986); later, A. glaucus, a low-temperature xerophilous species, begins to grow. Next, yeasts take a dominant position in the middle of pile fer-mentation (Zhao et al. 2015). The mass reproduction of molds and yeasts further suppresses the growth of bacteria (Liu 2009). The enzymatic activities of polyphenol oxi-dase, peroxidase and ascorbic acid oxidase are positively correlated with the growth status of fungi (Chen et al. 1985; Zhou et al. 2004; Zhao et al. 2015). High amounts of phenolic substrates, catechins and the oxidising enzymes can be released when the cellulolytic enzymes produced by fungi increase the maceration of plant tissues. Fungi are also involved in the transformation of components of Pu-erh tea within the pile fermentation process. Wang et al. (2008) proposed that fungi can convert theophylline into caffeine. In addition, fungal metabolites are an important part of Pu-erh tea, for example in dimethoxy-4-methyl benzene, the specific metabolite of A. niger (Gong et al. 1993). However, toxic microbial metabolites were also detected from Pu-erh tea samples, such as Andrastin A, Emodin and Malformin C (Zhang et al. 2016c).

The commercially available tea also contains many fungal populations. Abe et al. (2008) found that Aspergillus niger and Blastobotrys adeninivorans exists in the final Pu-

erh tea products. This is due to the influence of production environments and unstable fermentation processes, which are difficult to control and regulate. However, on the basis of traditional post-fermentation, through inoculating spe-cial preponderant fungal agents, not only can this suppress harmful microbes, but also can shorten the fermentation time and ensure the stability of product quality (Fig. 34).

## 34. Wine, beer and spirits

Brewing was invented about 8000 years ago, and became widely used as early as 5000 years ago (Corran 1975). At present, alcoholic beverages have become an integral part of numerous people's daily lives (Hornsey 2003, 2012). The three major well-known alcohol beverages are wine, beer and spirits, with beer being the most common and popular worldwide. These alcoholic beverages can be divided into many categories and subcategories based on their characters and fermentation processes. In the meta-bolic processes of brewing alcoholic beverages, the most important biological agents belong to the genus *Saccha-romyces*. Some traditional beers have used the genera *Candida*, *Dekkera*, *Saccharomycopsis* and *Schizosaccha-romyces* (Van Oevelen et al. 1977; Verachtert and Dawoud 1990) in production. Yeast has been used in the ethanol formation and the process to finish brewing wine, beer and spirits worldwide (Campbell 1996; Arora 2003; Kurtzman et al. 2011; Blomqvist 2011).

### Wine

Wine seems to be the most easy to produce among the three beverages due to the fact that wine can be naturally fer-mented by sugar from ripe fruits and ambient yeasts. In the fermentation process, yeasts (natural or commercially cultured yeasts) use sugar from fruits, and release alcohol and carbon dioxide. Winemakers filtrate the large particles in the wine and bottle the wine or allow for additional aging (Fig. 35). However, the winemaking process can be complicated in cases of premium, healthy and specialty wine, e.g. types of grape fruits, oak-aging or steel tank, pumpovers or punch downs (Mosedale et al. 1999; Fischer et al. 2000; Zoecklein 2002a, b; Sacchi et al. 2005; Downey et al. 2006; Hicks 2011; Casassa and Sari 2014; Guerrini et al. 2017).

### Beer

Beer is the most common alcoholic beverage, with the largest market share and consumption worldwide (Fig. 36). Goldammer (2008) reported that beer in the United States provides over $91.6 billion U.S dollars annually in retail sales. The global market share of beer consumption has grown rapidly over the past decade worldwide. However, some reports state that people will tend to drink less beer in the near future due to a combination of factors, such as
drinking-age population, GDP of countries and health concerns (IWSR, Economist.com 2017). Beer is mostly made from barley and other cereal grains, e.g. corn, rice, wheat. The brewing process is mainly about transforming carbohydrates from these grains into sugar. The primary product from grains is a sugary liquid called as “wort”. Yeasts and wort will be fermented to produce alcohol and fizzy beers. This beverage was classified into two major categories, i.e. the “top-” (*Saccharomyces cerevisiae*) and the “bottom-” (*S. pastorianus*) fermenting types (Arora 2003). The top-fermenting type (or “ale”) mostly produces darker beers, while the bottom-fermenting type (or “lager”) produces lighter beers. Some other different characteristics

of ale and lager are shown in Table 16. Recently, hybrid and other specialty beers that have new flavors added or generated from the hybrid yeasts have been established and accommodated into the third category. For example, lager yeasts are cold-tolerant strains, but they have higher ethanol sensitivity than ale yeasts (warm-tolerant strains). Hybrid strains of regular *S. cerevisiae* × a cold-tolerant *Saccharomyces* species have been used to brew ale at lager (cool) temperatures (Magalhães et al. 2017). Some hybrids of yeast strains useful in the fermentation environment were produced, e.g. by Le Jeune et al. (2007), Gibson and Liti (2015), Krogerus et al. (2017). Presently, *Saccharomyces* contains hundreds of varieties and strains, which need to be studied further to understand the different expression of numerous genes for improving production in the brewing industry. In addition, many flavours have been recommended to add in the beer brewing process, including dried mushrooms. Many kinds of fungi have been tested and will be used in brewing in the near future (Okamura et al. 2001; Okamura-Matsui et al. 2003; Leskošek-Čuka-lović et al. 2010; Moon and Lo 2014).

## Spirits

Hard alcohol or spirits have higher alcoholic content than beers and wines. Although yeasts can only survive at up to 15% alcohol by volume (Nguyen et al. 2009), a secondary process, “distilling,” has been used to provide much stronger beverages than beers and wines. Brewers take advantage of the different boiling points of alcohol and other materials in the mixture to obtain spirits. Heating mixtures separates the alcohol as distilled liquids, while leaving water, grain particles and others in the boiling

<figure><img src="image_46.png" /><figcaption>**Fig. 34** Pu-erh compressed tea (cake tea) molded into a Table ornament. The compressed tea shown at the front is broken for use</figcaption></figure>

<figure><img src="image_47.png" /><figcaption>**Fig. 35** Pictures of the winemaking process by Jamie Goode (reproduced with kind permission by the publisher Jamie Goode, wineanorak.com)</figcaption></figure>
<figure><img src="image_48.png" /><figcaption>**Fig. 36** Global market share of beer consumption in 2016 by regions (reproduced with kind permission from Kirin Holdings Company, Limited)</figcaption></figure>

vessel. The liquids distilled are cooled in a condenser and often be re-boiled to ensure their purity (Forbes 1970; Lembeck 1983; Birch and Lindley 1985; Thomas and Shipman 2016). The raw materials result in a variety of flavours and aromas in the spirits, but the aging processes and periods are also important (Christoph and Bauer-Christoph 2007; Rosso et al. 2009; Robert 2010). The

variety of spirits is showed in Table 17; however, there are many sub-categories within each type of spirit as well.

The brewed alcoholic beverage industry has grown rapidly over recent decades, and it is increasingly producing competitive products heightened by consumer requirements worldwide. Creating unique experiences for beer consumers is one of the most important goals of alcoholic beverage manufactories. Brewers have established numerous new techniques and processes for improving the flavours, colours, and nutrients in wines, beers, and spirits. Numerous new materials and processes in brewing that can affect flavours and aromas are being developed, and there will likely be hundreds of different kinds of alcoholic beverages in the future. The development of biotechnology and new discoveries will be the most important tools to reach the goals of future brewing.

## 35. Functional foods and nutraceuticals

The average age of the global population is increasing, and society is becoming more aware of the potential side effects of prescription medicines, and therefore are seeking alternative therapeutics (Ekor 2014; Rowe et al. 2016; WHO 2004a, b, 2015). Various new nutritional concepts, such as functional foods and nutraceuticals have been developed over recent decades. By definition, functional foods are conventional or everyday foods consumed as part of the normal daily diet, targeting the enhancement of the well-being and quality of life and thus reducing the risk of

<table><caption>**Table 16** The major differences between ales and lagers</caption><thead><tr><th>Beers<br>types</th><th>Brewing<br>process</th><th>Types of yeast<br>(Saccharomyces)</th><th>Fermentation<br>temperature</th><th>Brewing<br>times</th><th>Colour</th><th>Example</th></tr></thead><tbody><tr><td>Ale</td><td>Top-fermenting</td><td>S. cerevisiae</td><td>Warm (15.5 to<br>25.5 °C)</td><td>7 days</td><td>Brown to dark</td><td>Goose Island<br>Bourbon County Stout<br>Samuel Smith's Taddy<br>Porter<br>Sierra Nevada<br>Three Floyds Dark Lord</td></tr><tr><td>Lagers</td><td>Bottom-<br>fermenting</td><td>S. pastorianus</td><td>Cool (7.2 to 12.8 °C)</td><td>Several<br>months</td><td>Blight gold or<br>yellow</td><td>Carlsberg<br>Corona<br>Dunkel<br>Heineken<br>Kirin<br>Miller<br>Pilsner Urquell<br>Schwarzbier<br>Singha<br>Tsingtao</td></tr></tbody></table>

<figure><img src="image_49.png" /></figure>
disease (El Sohaimy 2012). The term “nutraceuticals” is defined as food constituents that provide medical or health benefits, including the prevention and treatment of disease (Kalra 2003). Nutraceuticals can be food in the form of extracts, single, pure natural compounds, or nutrients that have been turned into a pharmaceutical formulation (e.g. pills or tablets), and they can be applied as dietary supplements or as part of a specific diet (Giavasis 2014; Reis et al. 2017). Therefore, the role of functional foods is mainly related to reducing the risk of disease, while nutraceuticals are usually consumed to promote well-being through the prevention or treatment of diseases and disorders (Bagchi et al. 2014).

Fungi, and especially edible mushrooms, are well known for their nutritional value and health-promoting properties. Mushrooms are ideal dietary supplements because they are relatively rich in proteins, contain almost no fat, and possess a variety of fibers and vitamins (Valverde et al. 2015). Many studies have been conducted to prove the benefits of functional foods and nutraceuticals derived from fungi in various pathological complications, such as diabetes, atherosclerosis, cardiovascular diseases, cancer, infection, renal, and gastrointestinal and neurological disorders (Banik et al. 2015).

Polysaccharides such as beta-glucanes are one of the most widely studied and recognized groups of bioactive molecules derived from mushrooms. For instance, the glucanes from *Lentinula edodes* (shiitake, Fig. 37) were successfully used as a partial replacement of wheat flour in baked foods to produce a low calorie, fiber-rich, functional food (Guillamón et al. 2010). Among all studied mushrooms species, *L. edodes* is also known to have antibacterial activities against both Gram-positive and Gram-negative bacteria (Alves et al. 2012a, b). In

another study, the beta glucane lentinan (65, Fig. 38) of *L. edodes* was added to noodles as a partial wheat flour replacement, and it was claimed that their anti-oxidant and hypocholesterolemic effects in vitro would improve the quality of the noodles (Kalač 2013). Extracts from *Ganoderma glucans*, on the other hand, have been reported to act as free radical scavengers, thereby preventing lipid peroxidation, and to stimulate interferon synthesis in human blood cells after consumption (Kozarski et al. 2011).

In other studies, glucans from various edible mushrooms including *Agaricus bisporus*, *Auricularia auricula-judae*, and the Asian “*Flammulina velutipes*” (Enokitake: current name: *F. filiformis*; cf. Wang et al. 2018a), were shown to possess notable antioxidant capacities and free radical scavenging potential in vitro, and hence were proposed as natural antioxidants in food applications. Other snack foods enriched with a powder from extracts of the Chestnut Mushroom (*Cyclocybe aegerita*) as a partial starch replacement exhibited a low glycemic response after consumption, which was correlated to the dietary fiber content (Brennan et al. 2012). *Agaricus subrufescens* (synonyms: *A. blazei*; *A. brasiliensis*) is another mushroom which has been suggested as a safe immunostimulant and for ameliorating obesity or diabetes (Yamanaka et al. 2013). Lastly, Japanese researchers managed to produce a functional cheese-like food containing *Schizophyllum comune*. The final product contains a beta-glucan, which was claimed to have a significant antithrombotic function (Okamura-Matsui et al. 2001).

The term “mushroom nutraceuticals” was coined by Chang and Buswell (1996) to describe those compounds that have considerable potential as dietary supplements and are used for the enhancement of health and prevention of

<table><caption>**Table 17** Differences of major types of spirits</caption><thead><tr><th>Spirits</th><th>Raw materials</th><th>Mashing</th><th>Fermentation</th><th>Distilling</th><th>Aging</th><th>Most % ABV</th></tr></thead><tbody><tr><td>Brandy</td><td>Grapes and fruits etc.</td><td>✓</td><td>✓</td><td>✓ (distilled wine)</td><td>In oak</td><td>40% (80 proof)</td></tr><tr><td>Gin</td><td>Vodka+juniper berries</td><td>✓</td><td>✓</td><td>✓</td><td>×</td><td>40-47% (80-94 proof)</td></tr><tr><td>Liqueur</td><td>Spirits+sugar+flavor (fruit, cream, herbs, spices, flowers or nuts)</td><td>✓</td><td>✓</td><td>✓</td><td>×</td><td>15-55%</td></tr><tr><td>Rum</td><td>Sugarcane or Molasses</td><td>✓</td><td>✓</td><td>✓</td><td>×</td><td>40% (80 proof), up to 75% (150 proof)</td></tr><tr><td>Tequila</td><td>Juices from blue Agave plant</td><td>✓</td><td>✓</td><td>✓ (distilled Agave)</td><td>×</td><td>40-50% (80-100 proof)</td></tr><tr><td>Vodka</td><td>Grains, rye, corn, wheat, potatoes etc.</td><td>✓</td><td>✓</td><td>✓</td><td>×</td><td>40-50% (80-100 proof)</td></tr><tr><td>Whisky</td><td>Grains (corn, rye, wheat, barley, etc.)</td><td>✓</td><td>✓</td><td>✓ (distilled beer)</td><td>✓ in charred oak, or used whiskey or wine barrels</td><td>40-50% (80-100 proof)</td></tr></tbody></table>

Summarized from Graham (https://www.thespruceeats.com/quick-guide-to-distilled-spirits-760713)
<figure><img src="image_50.png" /><figcaption>**Fig. 38** Examples of biologically active compounds from fungi used in functional foods and nutraceuticals. **61:** Synthetic compound inspired from cordycepin (Alapi and Fischer 2006); **62:** Triterpenoid from *Ganoderma* sp. (Sato et al. 1986); **63:** Main active principle of "Phellinus" (i.e. *Tropicoporus*) *linteus* (Shao et al. 2015); **64:** Blazeispirils (triterpenoid from cultures of *Agaricus subrufescens*) (Thongklang et al. 2017); **65:** Lentinan, a beta-glucane from *Lentinula edodes* (Ina et al. 2013)</figcaption></figure>

<figure><img src="image_51.png" /><figcaption>Didanosine (61), Synthetic compound (based on the *Coryceps* spp compound) (Alapi EM and Fischer J 2006)</figcaption></figure>

<figure><img src="image_52.png" /><figcaption>Ganoderiol A (62), *Ganoderma* sp. (Sato et al. 1986)</figcaption></figure>

<figure><img src="image_53.png" /><figcaption>Hispidin (63), *Phellinus linteus* (Shao et al. 2015)</figcaption></figure>

<figure><img src="image_54.png" /><figcaption>Hericenone A (64), *Hericium erinaceus* (Thongbai et al. 2015)</figcaption></figure>

<figure><img src="image_55.png" /></figure>
various human diseases (Taofiq et al. 2016). The important edible mushrooms with remarkable nutraceuticals properties include the species of *Auricularia*, *Grifola*, *Hericium*, *Lactarius*, *Pisolithus*, *Pleurotus* and *Tremella*. Some studies suggest mushrooms as inhibitors of mycotoxin production (Reverberi et al. 2005). The bioactive glycoprotein (SX-fraction) extracted from *Grifola frondosa*, or maitake mushrooms, have been investigated in animal and clinical trials. It is particularly encouraging that the glycemic control of several diabetic patients under oral medications was significantly improved with a SX-fraction regimen that may primarily target or act on the insulin signal transduction pathway, overcoming insulin resistance (IR and IRS-1) (Konno et al. 2013). Other formulations exist on the market, such as a new product for dementia, particularly Alzheimer's disease, based on a proprietary standardized extract that contains hericenones (e.g. **64**) and amyloban (both from *H. erinaceus*) (Thongbai et al. 2015). The popular culinary oyster mushroom *Pleurotus ostreatus* and other species of this genus synthesize bioactive pleuran, which is another potential candidate for the development of nutraceuticals (Giavasis and Biliaderis 2006; Zhang et al. 2001). Some examples of biologically active compounds from fungi used in functional foods and nutraceuticals are given in Fig. 38.

Today, it is very easy to find functional foods and nutraceuticals derived from mushrooms in the market. Many of these products can even be purchased via the Internet. However, it must be kept in mind that these studies do often not rely on standardized material and that the “health claims” derived from them are often not substantially justified, e.g. because the studies were conducted with rodents or even only in vitro (cell-based or enzyme based assays), and were never validated by treatment on humans in a similar manner as the clinical trials that are mandatory in development of ethical drugs. Serious diseases should therefore not be treated be self-medication and patients should by all means seek advice from a medical doctor. Among the various side effects reported for patients that have used such preparations from TCM mushrooms as self-medications, pancytopenia (i.e. a decrease in the number of all blood cells) has recently been frequently recorded (Yoon et al. 2011; Jung et al. 2013). Even though the symptoms disappeared after the consumption of the mushroom product stopped, these cases should give a warning to everybody who uses mushroom nutraceuticals not to overdose them.

## 36. Harvesting the untapped probiotic potential of fungi

Gastrointestinal disorders and diseases constitute a major cause of morbidity and mortality worldwide (Kaplan 2015;

Kirk et al. 2015; Peery et al. 2015). Diseases of the gastrointestinal tract may or may not be contagious. In the case of non-communicable gastrointestinal tract diseases, there is no transmissible organism involved. These include inflammatory bowel disease, irritable bowel syndrome, colon cancer, Crohn's disease and colitis. In the case of communicable gastrointestinal tract diseases, the aetiological agent is an infectious organism. Sufferers may exhibit one or several of a broad array of gastrointestinal tract disturbances and perturbations, including flatulence, abdominal pain, vomiting, nausea, gastrointestinal tract bleeding, constipation, and diarrhoea (Wadsworth et al. 2011).

Several gastrointestinal tract disorders are becoming increasingly difficult to treat due to ambiguous overall symptomatology or increased resistance of pathogens to administered pharmaceutical drugs, mainly antibiotics (Prestinaci et al. 2015). Given these issues, there is a critical need for therapeutics that will complement/replace existing strategies. In that light, probiotics present an attractive option. The Food and Agriculture Organization of the United Nations and the World Health Organization along with the International Scientific Association for Probiotics and Prebiotics define probiotics as “live microorganisms, which when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO 2001; Hill et al. 2014).

Probiotics can be delivered as dietary supplements (e.g. capsules, tablets, and powder) or food ingredients (e.g. yogurts and kefirs) (Szajewska et al. 2016). The health benefits of probiotics include strengthening of the gut mucosal barrier followed by gut pathogen colonization resistance, replenishing of beneficial gut microbes after diarrheic episodes, and enhancing overall health of the digestive tract (Hill et al. 2014). Probiotics have been used to a limited extent for the treatment/prevention of gastroenteritis, inflammatory bowel disease, irritable bowel syndrome, colorectal cancer, and colitis (Brown and Valiere 2004; Hill et al. 2014).

Most probiotics currently in use are limited to bacterial strains with members of the genera *Bacillus*, *Bifidobacterium*, *Enterococcus*, *Lactobacillus*, *Propionibacterium* and *Streptococcus* being the most common (Ouwehand et al. 2002; Brown and Valiere 2004; Szajewska et al. 2016). A notable exception is the yeast *Saccharomyces boulardii*, which was initially isolated to combat cholera-associated diarrhoea, and is now being used to treat diarrhoea of varying aetiology (McFarland and Bernasconi 1993). The health benefits of *S. boulardii* have been well-documented in several randomized clinical trials (Table 18). Nonetheless, the probiotic potential of various other fungal species is also well known. For instance, members of the genera *Kluyveromyces* and *Yarrowia* are
<table><caption>**Table 18** Fungal probiotics and their health benefits</caption><thead><tr><th>Fungi</th><th>Health benefits</th><th>References</th></tr></thead><tbody><tr><td><i>Saccharomyces boulardii</i></td><td>Considerable reduction of duration of diarrhoea-RCT<br>Risk reduction of antibiotic-associated diarrhoea-RCT<br>Risk reduction of diarrhoea associated with <i>Clostridium difficile</i> in children-RCT<br>Reduction of diarrhoea associated with <i>Helicobacter pylori</i> infection-RCT<br>Amelioration of abdominal pain in IBS sufferers-RCT</td><td>Szajewska et al. (2016) and references therein</td></tr><tr><td><i>Saccharomyces cerevisiae</i></td><td>Absorption of mycotoxins</td><td>Moslehi-Jenabian et al. (2010)</td></tr></tbody></table>

RCT randomized clinical trials

promising candidates due to their strong activity against bacterial pathogens and their ability to tolerate the inhospitable environment of the gut (Kumura et al. 2004; Chen et al. 2010). However, this avenue of research remains largely unexplored (Huseyin et al. 2017).

A largely untapped source of potential fungal probiotics is the human gut mycobiome, the collection of fungi residing in the gut. Members of the genera *Candida* and *Saccharomyces* are consistently prevalent across studies, but *Cladosporium*, *Malassezia* and *Penicillium* seem to be also abundant depending on the population under study (Hoffmann et al. 2013; Rodriguez et al. 2015; Nash et al. 2017). Future research should focus on identifying and precisely characterizing appropriate fungal probiotic strains. Outcomes of each probiotic fungal strain should be determined explicitly and optimal doses defined according to age group and disease state. Research efforts should also expand to examine the use of fungal probiotics in the aquaculture and animal husbandry industries.

## Saving the planet

Waste is a common and major global problem. Anthropogenic activities result in the generation of millions of tons of plastic waste each year. Extensive agricultural practices have also contributed to the generation of enormous quantities of waste, which are being disposed of in non-cost-effective and environmentally unfriendly ways. There are various ways in which fungi can help decrease waste and reduce pollution. There is therefore an urgent need to tap into the power of fungal organisms and devise smart strategies regarding how they can reduce the negative effects that pollutants have on the environment. The section below outlines how fungi contribute to the health of our planet and have the ability to curb a number of environmental problems, most notably pollution. Examples are given as to how agricultural waste (which was previously disposed of or burnt, resulting in air pollution) can be

converted into useful substrates on which to grow edible mushrooms at an industrial scale. Mycologists have also conducted research on resilient fungal microorganisms and explored their ability to degrade plastics, textile effluents, and hydrocarbons, all of which are polluting our environment at an alarming rate.

## 37. Agricultural waste disposal

Agricultural waste, the residue from planting until harvest, generally contains a mixture of plant waste, animal/insect waste, and toxic residues from the chemicals used as pesticides, insecticides, and herbicides. Asia is one of the world's most agriculture-intensive areas: from 2010 to 2016, 46.9% of emissions in Asia came from crop residue, compared to 28.2% and 16.2% from America and Europe, respectively. According to the FAOSTAT<fnref n="2" /> emissions database, 90.6% of rice produced within Asia generated more than a hundred million tons of residual material. This agricultural waste accounted for over 50% of the nitrous oxide generated from the decomposition of crop residue on cultivated soils, one of the main contributors of the greenhouse gas effect and a cause of global warming. In the Sacramento Valley of California, rice and wheat straw are also major contributors of generated agricultural waste, with 1.5 tons of rice straw waste produced annually (Zhang et al. 2002). In Thailand, the main crops are sugarcane, cereal, rice, cassava and oil palm, and these contribute 80% of the total solid waste generated from crop cultivation.

A number of processes have been developed or proposed for agricultural waste management (such as composting, which is a sustainable process), while the common practice of burning the rice straw, husk, stalks, corn cobs, stubble and grasses is considered a cheap and easy means for the disposal of excess residues (Obi et al. 2016; Walia et al. 1999). Unfortunately, burning agricultural waste generates many poisonous and harmful oxides and hydrocarbonates into the atmosphere (Shaban and Omaima

<figure><img src="image_56.png" /></figure>

<footnote n="2">² http://www.fao.org/faostat/en/?#data/GA.</footnote>
2010), which in turn increases air pollution and causes respiratory problems, which then impact the economy through higher medical costs and employee absences. Therefore, there is an increasing demand for sustainable processes that utilize agricultural waste in more eco-friendly ways.

Biological processes are considered both natural and sustainable, and are also important in carbon recycling. Fungi are some of the most effective decomposers in an ecosystem and are extremely efficient in the degradation of plant-based agricultural waste such as rice straw, wheat straw, maize stoves and sugarcane residue (Dinis et al. 2009). Mushroom cultivation using agricultural waste, e.g. wheat, rice, paddy straw, rice bran, coffee straw, tea leaves, cotton straw sawdust, provide profitable agri-business opportunities for small farmers and offers an eco-friendly alternative to disposing of waste, which is normally burnt (Zhang et al. 2002). Various commercial mushrooms are cultivated worldwide, and agricultural residues are used as cheap and renewable substrates, and a selection is given in Table 19.

Agricultural waste consists of large quantities of lignocellulosic waste, and is composed of approximately 60–70% fermentable sugars and 20–30% aromatic compounds. Lignocellulosic waste is a renewable and important source for biomass/ethanol production (Kim and Dale 2004). Wheat straw, corn stover, sugarcane bagasse, and rice straw are the most common substrates for biofuel production, and filamentous fungi are key components of the fermentation process. *Pichia stipites* (strain NRRL

Y-7124) is used in the fermentation process to produce ethanol from wheat straw and rice straw (Moniruzzaman 1995), and *Fusarium oxysporum*, *Neurospora crassa*, and *Paecilomyces* sp. are typically used to ferment cellulose directly to ethanol (Szczodrak and Fiedurek 1996). Therefore, various fungi have the potential to produce lignocellulosic enzymes and play important roles in agricultural waste practice. White-rot fungi can also be used to pre-treat the agricultural waste to enhance the enzymatic saccharification process due to the selective removal of lignin (Miyauchi et al. 2018; Rouches et al. 2018; Zhou et al. 2015b, 2018). The screening of the secreted enzymes from fungi can also be used to enhance the saccharification of agricultural waste by commercial enzyme cocktails, and this approach has led to the discovery of new activities such as lytic polysaccharide oxidases which open up the lignocellulose structures to facilitate further access of cellulolytic enzymes (Berrin et al. 2012; Couturier et al. (2012, 2018). Alternatively, fungi can be used to transform compounds in agricultural waste into higher value commercial molecules such as vanillin (Taira et al. 2018) and cannolo (Odinot et al. 2017).

In most countries, some waste, such as cereal residue, is universal, whereas other forms of agricultural waste are more region-specific. In the French region of Provence, for example, lavender is grown on a large scale for the production of essential oils. Globally, these oils are among those most commonly used for industrial purposes, including in perfumes, pharmaceuticals and cosmetics.

<table><caption>**Table 19** Agricultural waste use as substrate for mushroom cultivation</caption><thead><tr><th>Fungal taxa</th><th>Agricultural waste</th><th>References</th></tr></thead><tbody><tr><td>Agaricus bisporus</td><td>Button mushroom</td><td>Wheat straw<br>Kamthan and Tiwari (2017)</td></tr><tr><td>Ganoderma sp.</td><td>Chinese mushroom</td><td>Sawdust<br>Shashitha et al. (2016)</td></tr><tr><td>Lentinula edodes</td><td>Shiitake mushroom</td><td>Bracts of pineapple crown, sugarcane bagasse, sugarcane leaves, wheat straw, corn-cobs, oak-wood sawdust<br>Salmones et al. (1999)<br>Philippoussis et al. (2007)</td></tr><tr><td>Pleurotus eryngii</td><td>King oyster mushroom</td><td>Supplementation of wheat bran with wood chips. Barley straw and sugar beet pulp with rice bran<br>Jeznabadi et al. (2016)</td></tr><tr><td>Pleurotus djamor</td><td>Pink oyster mushroom</td><td>Supplementation of wheat bran with sugarcane bagasse<br>Hasan et al. (2015)</td></tr><tr><td>Pleurotus ostreatus</td><td>Oyster mushroom</td><td>Paddy straw, rice straw, wheat straw, date-palm leaves empty fruit bunch, olive cake, tomato tuff, banana leaves, pine needles, sugarcane bagasse<br>Alananbeh et al. (2014)<br>Ananbeh and Almomany (2005, 2008)<br>Ali et al. (2010)<br>Marlina et al. (2015)<br>Rezania et al. (2017)<br>Yang et al. (2013a)</td></tr></tbody></table>
After steam distillation, the residual straw is considered to be agricultural waste, though it can also be used as a source of terpene derivatives (e.g. τ-cadinol, β-caryophyllene), lactones (e.g. coumarin, herniarin), and phenolic compounds of industrial interest, including rosmarinic acid (through fungal treatments) (Lesage-Meessen et al. 2018). Fungal-derived enzymes represent over 60% of the world market in industrial enzymes. Lavender residues, like most agricultural waste residues, are both suitable catalysts for the secretion of a wide panel of lignocellulose-acting enzymes, such as cellulases, laccases and xylanases, and therefore can provide a local and cheap source of degradative and transforming biocatalysts. Even plant pathogens like *Phyllosticta capitalensis* produce arabinase, cellulase, laccase, pectinase and xylanase, especially when cultivated on lavender residues. The initial growth of *P. capitalensis* on lavender waste was higher in comparison to its growth on other agricultural waste materials, such as miscanthus, rice straw, rice husk, sorghum, and wheat straw, suggesting further stimulation of growth by a compound remaining within the lavender residue (Wikee et al. 2017). This opens up further avenues to investigate the presence of microbial growth enhancers within agricultural waste for utilization in biotechnology. Therefore, the aims of this present review are to reveal the capacity of fungi for agricultural waste management and to explore the varieties of fungi and their lignocellulolytic production, taking full advantage of various agricultural waste products as substrate.

## 38. Mycoremediation: Fungi to the rescue

Man-made activities have resulted in extreme environmental pollution, and therefore environmentally friendly remediation techniques are more necessary than ever before (Rhodes 2013). Fungi, the most vigorous decomposers found in nature, are a potential solution to this problem (Singh 2006). Bioremediation with fungi, known as mycoremediation, is an innovative biotechnological approach which uses live fungi to clean up contamination through total mineralization using enzymes, or through the total removal of the contaminant via absorption (Stamets 2005). Pollutants released into the environment due to man-made activities pose a significant threat to the sustainability of the environment and to human health. The burning of wood and fossil fuels, as well as coal mining and oil drilling all release recalcitrant pollutants such as polycyclic aromatic hydrocarbons (PAH), which are not easy to remove once they have been released. In addition, pesticides that leak into natural waterways and industrial effluents containing heavy metals, that have been released into the environment without proper treatment can be effectively removed if fungi are utilized as a remediation tool.

The vegetative part of the fungi, called mycelium, has the ability to exude powerful extracellular enzymes and acids that are able to decompose many organic substrates, including lignin and cellulose (Pointing et al. 2001; Bucher et al. 2004). With a simple screening procedure, the most suitable fungal species for the target pollutant can be identified (Matsubara et al. 2006). Many species of fungi, such as *Aspergillus niger*, *A. terreus*, *Cladosporium oxysporum*, *Fusarium ventricosum*, *Rhizopus oryzae*, *Trichoderma harzianum* as well as the model white rot fungus, *Phanerochaete chrysosporium*, have been tested for their ability to degrade pesticides (Bhalerao and Puranik 2007; León-Santiesteban et al. 2016). Polychlorinated biphenyls, which are highly toxic chemicals previously used in many industries and one of the more persistent organopollutants, can be degraded by non-ligninolytic enzymes from fungi such as *Fusarium solani*, *Penicillium chrysogenum* and *Scedosporium apiospermum* (Tigini et al. 2009).

Different species of basidomycete white rot fungi are best suited for mycoremediation, as their extracellular lignin modifying enzymes have low substrate specificity (Lang et al. 1995). Not only do they target lignin, but they also are capable of targeting many organic pollutants that are similar in structure to lignin, like DDT, lindane, chlorodane and PCBs (Arisoy 1998; Pointing 2001; Mansur et al. 2017). Enzymes like lignin peroxidases (LiP), manganese peroxidases (MnP) and laccases (LAC) secreted by white rot fungi are excellent at degrading organopollutants held together by hydrocarbon bonds (Kües 2015). White rot fungi such as *Lentinus subnudus*, *Phlebia acanthocystis* and *Pleurotus ostreatus*, and many others have been successfully used separately as well as in mixtures to treat sites contaminated with pesticides and herbicides (Kamei et al. 2011; Nyakundi et al. 2011; Xiao et al. 2011). Since white rot fungi grow by hyphal extension, they can better reach the pollutants in contaminated sites, unlike other organisms with a low colonizing capacity (Reddy and Mathew 2001). Furthermore, the lignin degrading enzymes used by white rot fungi are extracellular (Novotný et al. 2004). These fungi can tolerate high concentrations of the pollutant, as the pollutant does not need to be internalized in order to start the degradation process.

Lamar and White (2001) recommend a four-step strategy for the practical implementation of fungi for the mycoremediation of contaminated sites. The steps include laboratory scale experiments to establish preparation methods; on-site pilot testing to understand sound technical and engineering techniques; and the production of inoculum fortified with nutrients to ensure growth and finally full-scale application (Fig. 39).

Because of their tolerance to extreme pH conditions, temperatures, and nutrient availability, fungal species such
as *Aspergillus* sp. and *Sterigmatomyces halophilus* have been tested for their ability to absorb heavy metals from contaminated sites (Bano et al. 2018). The cell walls of these fungi have excellent metal binding capabilities, and can absorb contaminants which then can be physically removed by harvesting the fungus (Baldrian 2003). The utilization of the biosurfactants of fungal species has been a success for the removal of heavy metals such as Fe, Zn and Pb (Igiri et al. 2018). For example, an anionic biosurfactant from *Candida sphaerica* was tested on cleaning soil collected from an automotive battery industry, and the heavy metal removal success rate was 95%, 90% and 79% for Fe, Zn and Pb, respectively (Luna et al. 2016).

The textile industry is one of the primary contributors to environmental pollution. Untreated textile effluents, inefficiency in the dyeing process, and the poor handling of spent dye stuff effluents are the main causes of surrounding natural water and land contamination, both of which adversely affect plant, human and animal life. Textile dyes, such as azo dyes (acid red) or anthraquinonic dyes (basic

blue) are highly toxic and mutagenic in nature (Delclos et al. 1984). Biological treatment of these harmful pollutants through microbial decolourization and degradation have attracted attention as the conventional treatment methods produce hazardous by-products and are not very cost effective. *Trichoderma harzianum* (Singh and Singh 2011) has been successfully bench tested for the decontamination of various textile dyes.

Apart from the absorbance process, enzymatic degradation of waste is another feasible approach that can be used for the removal of slow-degrading waste. Various types of cellulose-based urban solid waste are primarily disposed of via incineration. One study conducted by Espinosa-Valdemar et al. (2011) used *Pleurotus ostreatus* (Oyster mushroom) to reduce the mass and volume of used disposable diapers via cellulose degrading enzymes secreted by the fungi, which simultaneously produced a pathogen-free, high-protein food source in the form of a mushroom fruiting body that could be used for human consumption or as a food supplement for animals.

<figure><img src="image_57.png" /><figcaption>**Fig. 39** The process of mycoremediation</figcaption></figure>
Other conventional strategies for the decontamination of polluted sites, such as the use of chemicals or through incineration, are comparatively less effective than mycoremediation. The washing and extracting used in the chemical method only shifts the contaminant from one place to another (Valentín et al. 2013); likewise, incineration can be highly energy intensive, not cost effective, and has the potential to cause harm through the formation of dioxides (Shibamoto et al. 2007).

There are ongoing studies to test whether edible mush-
rooms could be used first for mycoremediation and then for
human consumption. In one study, for example, *Pleurotus
pulmonarius* was successfully tested for cleaning crude oil
(Olusola and Anslem 2010); in another study, *Agaricus
bisporus* and *Lactarius piperatus* were successfully used
for the biosorption of Cd²⁺ under laboratory conditions
(Nagy et al. 2013). However, these mycoremediation pro-
cesses remain impractical due to the fact that the absorp-
tion of pollutants, such as heavy metals might render the
edible part of the mushrooms highly contaminated (Kul-
shreshtha et al. 2014). However, if means of utilizing
mushrooms with anti-mutagenic and anti-genotoxic abili-
ties could be developed (Mendez-Espinoza et al. 2013),
then it would be possible to simultaneously reduce the
toxicity of the pollutants, clean the lands deemed unfit for
agriculture, and produce crops or animal feed.

Some fungi not being able to grow in the presence of the
natural microbiota of the contaminated site and the need to
have precise understanding the nutrient requirements of the
fungus for it to thrive in the contaminated site might be
some small setbacks for this technology. In comparison to
the alternative methods, these problems are negligible. And
they can be readily solved with improved *in-situ* and *ex-
situ* trails.

The lack of more comprehensive information about
degradation mechanisms and the enzymes involved in the
pollutant degradation by different fungal species hinders
the possible use of them in mycoremediation in a more
specific manner. More in depth studies should be done to
recognize the oxidative enzymes, organic acids and
chelators excreted by fungi during the mineralization and
degradation process. Another way to increase efficiency of
mycoremediation is using large scale studies to verify the
results of the bench scale studies that would provide
insights in to efficient use of the existing species of fungi
and identifying new species suitable for bioremediation.
More resilient fungal species adapted to harsh environ-
mental conditions should be tested for their adaptability as
bioremediators as they are more physiologically active. For
example in some areas of the world, the soil is high in salt
concentration due to inefficient drainage and this makes the
ecosystems in these areas very simple and fragile. To make
these areas more agriculturally feasible, halophilic or

halotolerant fungi can be used to clean up the soil of
the excessive salt. Up until now most of the studies done
regarding halophilic fungi only focus on the characteriza-
tion. Fungi such as *Aspergillus penicillioides*, *Gymnascella
marismortui*, *Cladosporium cladosporioides* and *Penicil-
lium westlingii* are well characterized as halophilic but their
potential use as soil mycoremediators should be done with
more focused studies specifically designed to check their
salt remediation ability (Zhang and Wei 2017).

By studying the genes related to enzyme degradation, mineralization, absorbance and adaptability to extreme environmental conditions, transgenic fungi can be produced with superior mycoremediation capabilities. However, since the remediation capacity of naturally occurring fungi is still very much understudied, isolating and screening suitable fungi from their natural habitats should carried out in order to reclaim polluted sites and to sustain a better environment for future generations.

39. Mycofumigation using the genus *Muscodor*

Mycofumigation is the use of antimicrobial volatiles pro-
duced by fungi for the control of other organisms (Stinson
et al. 2003). The prototypes of the mycofumigant agents
belong to strains of the genus *Muscodor*, on which most of
the activities in research and development in this area have
so far been focused. *Muscodor* species are endophytes that
belong to the order Xylariales (Ezra et al. 2004; González
et al. 2009; Kudalkar et al. 2012; Suwannarach et
2013a; Wijayawardene et al. 2018; Chen et al. 2019), but
the taxonomic and phylogenetic position of the genus
remains unsettled (Daranagama et al. 2018; Wendt et al.
2018). *Muscodor* cultures do not sporulate, their sexual
state is as yet unknown, and their most salient common
feature is their ability to produce volatile organic com-
pounds. All strains of this genus can inhibit and kill other
fungi, including human and plant pathogenic species, as
well as bacteria and insects, through the production of a
mixture of volatile organic compounds. These mixtures are
primarily composed of various small molecular weight
alcohols, organic acids, esters, ketones, and several aro-
matic hydrocarbons as determined by gas chromatography-
mass spectrometry. The mixture of VOCs produced from
*Muscodor* species has been reported to be antimicrobial
volatiles (for a selection see compounds 66–72 in Fig. 40;
for original literature see, Atmosukarto et al. 2005;
Kudalkar et al. 2012; Mercier and Jiménez 2004; Mitchell
et al. 2008; Ramin et al. 2005; Siri-Udom et al. 2017;
Suwannarach et al. 2013a, 2015a, 2017; Worapong et al.
2001; Zhang et al. 2010b). Studies by Alpha et al. (2015)
and Hutchings et al. (2017) suggested that VOC produced
by *Muscodor* may act in the bacterial DNA damage
through inducing DNA alkylation and DNA methylation
processes; however, the active ingredients yet remain to be identified. If such biological activities would also be observed in eukaryotes, including mammalian cells, this might constitute a future challenge for the registration of products based on the respective strains because of their mutagenic potential.

The most advanced project of this kind has focused on *Muscodor albus* (strain 620), originally isolated from *Cinnamomum zeylanicum*. This strain was shown to be an effective biofumigant capable of controlling lemon decay caused by *Penicillium digitatum*; lemon sour rot caused by *Geotrichum citri-aurantii*; apple decay caused by *Penicillium expansum* and *Botrytis cinerea*; as well as peach brown rot caused by *Monilinia fructicola* (Mercier and Jiménez 2004; Mercier and Smilanick 2005). The strain also can control grain smut fungi (Goates and Mercier 2009) and act as an insecticide against potato tuber moths (Lacey and Neven 2006). Another strain, *Muscodor suthepensis* (Fig. 41a), isolated from *Cinnamomum bejolghota* in Thailand, has potential application as a biofumigant for controlling blue mold (*Penicillium digitatum*) decay of the tangerine fruit (Fig. 42a) (Suwannarach et al. 2015a, b).

*Muscodor inoculum* also has the potential to replace methyl bromide (a toxic chemical fumigant and pesticide) fumigation in controlling soil-borne plant diseases. *Muscodor albus* (strain MFC2), isolated from *Myristica*

*fragrans*, has the potential to control kale root rot disease caused by *Pythium ultimum* (Worapong and Strobel 2009). *Muscodor albus* (strain 620) and *M. roseus* (isolated from *Grevillea pteridifolia*) showed the ability to control disease severity caused by a variety of fungal and oomycete plant pathogens, e.g. *Aphanomyces cochlioides*, *Rhizoctonia solani*, *Pythium ultimum* and *Verticillium dahliae* (Stinson et al. 2003; Mercier and Jiménez 2009). *Muscodor roseus* also has nematicidal and nematostatic application against four plant-parasitic nematode species (*Meloidogyne chitwoodi*, *M. hapla*, *Paratrichodorus allius*, and *P. penetrans*) found on economically important vegetable crops (Riga et al. 2008). *Muscodor cinnamomi* (Fig. 41b) completely controlled damping-off symptoms on plant seedlings and Rhizoctonia-root rot in tomato plants, (Fig. 42c) (Suwannarach et al. 2012, 2015b). *Muscodor heveae* isolated from rubber trees (*Hevea brasiliensis*) is effective at controlling white root rot disease in rubber trees caused by *Rigidoporus microporus* (Siri-Udom et al. 2017).

The application of *Muscodor* in the control of soil-borne disease might be of concern due to the phytotoxic activity which depends on the mixture of volatile organic compounds and type of plant (Suwannarach et al. 2012; Siri-Udom et al. 2017). Suwannarach et al. (2017) suggested that *M. cinnamomi* could be used in fumigation with an active culture of a biological control agent to reduce the number of microorganisms present on eggshell surfaces

<figure><img src="image_58.png" /><figcaption>**Fig. 41** Colonies of *Muscodor suthepensis* (a) and *M. cinnamomi* (b) on potato dextrose agar at 25° C after 12 days. Scale bar = 1 cm</figcaption></figure>

<figure><img src="image_59.png" /></figure>
<figure><img src="image_60.png" /><figcaption>**Fig. 42** Mycofumigation by *Muscodor* species. a Control of blue mold decay of tangerine fruit at 15 days of storage after 24 h fumigation by *Muscodor suthepensis*. b Eggs and microorganism on eggshell surfaces at 3 days of storage after 24 h fumigation with *M. cinnamomi*. c Control of tomato root rot disease by *M. cinnamomi* at 4 months after planting. T1 infected control experiment, T2 *Muscodor* experiment, T3 *M. cinnamomi*+*R. solani* experiment, T4 non-infected control experiment</figcaption></figure>

without producing adverse effects on egg quality (Fig. 42b). *Muscodor* mycofumigants, through the use of the cereal grain inoculum, are being developed to control fruit decay and soil-borne diseases (Mercier and Smilanick 2005; Suwannarach et al. 2015a, b). Currently, the WAG BAG® Waste Kit, produced by *M. albus*, is a commercially available product for human waste disposal for application in aeronautics and by the military, which is regarded as non-toxic, biodegradable, and therefore environmentally friendly (Phillips et al. 2010).

Several other endophytic fungi, e.g. *Gliocladium* spp. *Hypoxylon* spp., *Nodulisporium* spp., *Phomopsis* spp., and *Oxyporus latemarginatus*, have been reported to produce volatile compounds that control fruit decay (Lee et al. 2009a; Park et al. 2010; Tomscheck et al. 2010; Singh et al. 2011; Suwannarach et al. 2013b). However, the identification of volatiles is often tentative, because no matching factors for GC/MS analytics have been given, and further effort on the characterization of the active principles are needed (cf. Helaly et al. 2018b). One way to safely identify the active components and assess their risk would be

through total synthesis and subsequent biological testing in a manner similar to that in Wang et al. (2018b).

## 40. Biomass to biofuel: Unmasking the potential of lesser-known fungi

The conversion of lignocellulosic biomass (grasses, sea-weeds, woody plants) to biofuel involves three phases: pre-treatment, saccharification, and fermentation (Himmel et al. 2007). Lignocellulosic biomass is primarily made up of lignin, cellulose, and hemicellulose. Lignin, which imparts structural rigidity and integrity to plants, is a heterogeneous phenolic polymer with a variety of ether and carbon-carbon linkages. The delignification of biomass is necessary to expose the cellulose and hemicellulose polymers for their subsequent enzymatic hydrolysis into monomeric sugars (saccharification). Lignin's recalcitrance to depolymerization has prevented the cost-effective conversion of lignocellulosic biomass to sugars, and in some ways has undermined the viability of biorefinery operations. While the ability of a few white-rot fungi (basidiomycetes) to delignify lignocellulosic biomass has been explored in pre-treatment strategies (Tian et al. 2012; López-Abelairas et al. 2013), and cellulases and hemicellulases of *Trichoderma reesei* and *Aspergillus niger* (filamentous ascomycetes) have been extensively utilized for saccharification (de Souza et al. 2011; Keshavarz and Khalesi 2016), the emphasis on selected fungi has left the full potential of this vast kingdom largely untapped. By drawing attention to examples of less-studied fungi from unusual ecological niches, we highlight the payoffs from such initiatives to broaden the biocatalyst arsenal and to increase the prospects for new pre-treatment and saccharification enzyme cocktails and methods (Fig. 43).

The harsh physicochemical pre-treatment of lignocellulosic biomass often involves milling, as well as treatment with acid or alkali at high temperatures (Himmel et al. 2007; Wilson 2009; Wi et al. 2015). An alternative pre-treatment entails the use of ionic liquids (e.g., 1-ethyl-3-methylimidazolium acetate) to dissolve the lignocellulosic biomass and decrease lignin interference (Swatloski et al. 2002; Dadi et al. 2006; Wahlström and Suurnäkki 2015). Thus, ionic liquids afford a one-pot pre-treatment with saccharification (Shi et al. 2013), and are expected to increase the yield of sugars for fermentation. However, because ionic liquids inhibit the cellulolytic enzymes (Elgharbawy et al. 2016), ionic liquid-tolerant saccharification enzymes are necessary. Indeed, ionic liquid-tolerant endoglucanases, cellobiosidases, and β-glucosidases (the cellulolytic trio) from thermophilic/halophilic bacteria and archaea have been identified (Datta et al. 2010; Zhang et al. 2011b; Ilmberger et al. 2012; Park et al. 2012; Gladden
<figure><img src="image_61.png" /><figcaption>**Fig. 43** Novel roles for fungi in biofuel production. Adapted in part with permission from Ribeiro et al. (2016)</figcaption></figure>

et al. 2014). For ionic liquid-tolerant hemi-cellulolytic enzymes, we directed our bio-prospecting efforts to marine-derived endophytic fungi, which have gainfully leveraged millions of years of co-evolution with marine plants/algae to dominate the host microbiome and create a powerful catalytic repertoire that permits them to function as primary degraders of lignocellulosic biomass. We demonstrated that a mesophilic *Trichoderma harzianum*—isolated as an endosymbiont of the brown seaweed *Sargassum wightii*—produces ionic liquid-tolerant β-xylosidase, an enzyme needed for hemicellulose breakdown (Sengupta et al. 2017). Considering the structural difference in the xylans of brown seaweed and red/green algae growing in salt-rich habitats (Kloareg and Quatrano 1988), targeted bio-prospecting of associated endophytes should uncover ionic liquid-tolerant enzymes for lignocellulosic biomass deconstruction and saccharification.

The biological pretreatment of lignocellulosic biomass with white-rot fungi has been investigated owing to the fact that these organisms secrete lignin-degrading peroxidases and laccases (López-Abelairas et al. 2013; Yang et al. 2013b). While generic wood-rot basidiomycetes are useful in this regard, the idea that endophytic fungi (ascomycetes) isolated from a specific plant/tree might be evolutionarily fine-tuned for the deconstruction of its host biomass is supported by recent studies. For example, *Ulocladium* sp.

and *Hormonema* sp., which are laccase-producing endophytes isolated from eucalyptus trees, were superior delignification agents relative to *Trametes* sp., an established laccase producer (Martín-Sampedro et al. 2015). In another example, *Pestalotiopsis* sp. was isolated from a mangrove (Arfi et al. 2013), and wood chips from *Rhizophora stylosa* mangrove trees were used to support the growth of this fungus. A proteomic analysis of this *Pestalotiopsis* sp. secretome revealed that 40% and 15% corresponded to glycosyl hydrolases and lignolytic enzymes, respectively. Endophytes isolated from such targeted bioprospecting are excellent tools for the deconstruction of their plant hosts, especially when used in conjunction with typical pretreatment methods that can now be performed at lower alkali/acid levels or temperatures. Second-generation variants, in which the aforementioned endophytes are genetically manipulated to overexpress specific enzymes (e.g., laccases) relative to the parental strain, will be worth developing. Transcriptome analysis of endophytic fungi capable of surviving as saprotrophs in abscised plant organs (Reddy et al. 2016; Guerreiro et al. 2018) would help to identify candidate cell wall polysaccharide-degrading enzymes that merit up-regulation in fungi that will be customized as pre-treatment agents. It would be instructive to investigate whether different genera of endophytic fungi exhibiting a biphasic life
style have conserved a specific suite of enzymes for bio-
mass degradation, as was discovered in the case of basid-
iomycetous fungi (Peng et al. 2018; López et al. 2018).

Pre-treatment with dilute acid at high temperature
releases organic acids, phenolic derivatives and furalde-
hydes (furfural and 5-hydroxymethylfurfural) (Palmqvist
and Hahn-Hägerdal 2000). The furaldehydes cause DNA
damage, inactivate glycolytic enzymes, and inhibit down-
stream saccharification and fermentation, thus reducing the
efficiency of biomass utilization (Caspeta et al. 2015).
Although these inhibitors could be removed by washing
and alkali treatment, or by ion exchange (Almeida et al.
2009), such methods are expensive, inefficient, and, sig-
nificantly, wash away fermentable sugars. Under these
conditions, the use of microbes which can metabolize such
inhibitors offers a bioabatement strategy (Suryanarayanan
et al. 2017).

Because furaldehydes are the most abundant and com-
mon volatile organic compounds released during biomass
burning, we postulated that fungi in forests experiencing
episodic fires can tolerate furfural and 5-hydroxymethyl-
furfural. Indeed, these two furaldehydes could be utilized
by both endophytic and litter fungi from these forests,
including those belonging to different taxonomic orders
(Govinda Rajulu et al. 2014). These findings are consistent
with those demonstrating the ability of *Coniochaeta lig-
niaria* (López et al. 2004), *A. niger* and *T. reesei* (Rumbold
et al. 2009), and *Amorphotheca resinae* ZN1 (Zhang et al.
2010a) to use furfural or 5-hydroxymethylfurfural for
growth. Using any of these fungi as bioabatement agents,
however, will require construction of sugar-transport
mutants that will minimize utilization of sugars and max-
imize consumption of furaldehydes.

Some of the currently used saccharification enzymes
from *Trichoderma reesei* and *Aspergillus niger* exhibit low
activity under industrial conditions (Druzhinina and
Kubicek 2017), motivating the search for alternatives that
are better suited for a specific biomass. Upon identification
of an abundant biomass (in a given locale) for use as the
main feedstock for biofuel production, the resident endo-
phytes should be isolated and characterized for their
delignification and saccharification capabilities. For
example, *Talaromyces borbonicus*, a new species found in
the naturally degrading biomass of *Arundo donax* (a tall
cane), was sequenced and found to use 4% of its genome to
code for 396 enzymes, all of which were linked to the
breakdown, modification, or synthesis of glycosidic bonds
(Varriale et al. 2018). In addition to *A. donax* deconstruc-
tion, this new palette of catalysts (once validated) will add
to a growing inventory of saccharification enzymes. It is
also useful to explore newer classes of enzymes, as
exemplified by the fungal lytic polysaccharide mono-oxy-
genases, which revealed a novel oxidative (rather than

hydrolytic) route to polysaccharide degradation (Couturier
et al. 2018). These lytic polysaccharide mono-oxygenases
(together with expansins and swollenins, which help loosen
the cellulose microfibrils) are grouped under non-hy-
drolytic cellulose active proteins that collectively enhance
the activity of cellulases in biomass hydrolysis (Ekwe et al.
2013). In light of these early successes with fungal non-
hydrolytic cellulose active proteins (Moncalro and Filho
2017; Santos et al. 2017), it is essential to screen different
ecological groups of fungi for superior variants.

New lessons have emerged from studying fungi not
deemed model platforms for biofuel production. For
instance, the anaerobic gut fungi of herbivores (e.g., goats,
horses), which extract nutrients from seemingly
intractable foliage, have many biomass-degrading enzymes
that permit the utilization of a broad range of substrates
(Solomon et al. 2016; Haitjema et al. 2017). Importantly,
the synergy among carbohydrates in Neocallimastigota
members (e.g., *Piromyces*, *Neocallimastix*) is the basis for
the superior biomass-degradation capabilities of the her-
bivore gut. In another parallel, the fungus-cultivating ter-
mite symbiosis complex exemplifies a remarkable
cooperation among different microbes for lignocellulosic
biomass utilization (Li et al. 2017). Within a colony, young
termites use their gut microbiome to degrade lignocellu-
losic biomass, most notably the typically refractory lignin
side-chains, and use their lignocellulosic biomass remnant-
rich faeces to build a fungal comb. The fungal microbiome
in the comb cleaves lignocellulosic biomass polysaccha-
rides and utilizes only xylose. The oligosaccharides in the
comb sustain the older termites, which forage and transport
plant material to the colony. This step-wise anaerobic and
aerobic tandem deconstruction of lignocellulosic biomass
occurs first within the gut of young termites and then in the
fungal comb. This accounts for the comparatively faster
pace with which termites degrade woody biomass when
compared with herbivores.

While enzymes in model fungi (e.g., *Trichoderma*) have
fostered advances for biofuel production, the two instances
described above (Solomon et al. 2016; Li et al. 2017)
demonstrate that lignocellulosic biomass deconstruction
efficiency is due to synergy rather the catalytic arsenal per
se; therefore, mimicking such consortia for industrial
applications will be profitable. Thus, it would be worth-
while to screen the biomass-degrading ability of consortia
of specific plant litter fungi at defined intervals during
deconstruction, as different fungal species may contribute
to different stages in the sequential breakdown (Voříšková
and Baldrian 2013). Indeed, such a temporal orchestration
of biomass degrading enzymes has also been reported for a
bacterial consortium growing on sugarcane bagasse
(Jiménez et al. 2018).
Fungi are the primary degraders of plant biomass. Due to the complex structure of plant biomass and the long-standing interaction of fungi with plants (Lange et al. 2018), fungi have evolved a wide variety of biomass-de-construction enzymes. However, since only a few fungal species have thus far been harnessed for their lignocellulolytic potential, it is essential to mine aerobic and anaerobic fungi from less-explored habitats [e.g., biogas plants (Young et al. 2018)]. Also, while the production of biomass-degrading enzymes by fungi is tightly controlled, the regulatory mechanisms are not highly conserved, as might be expected based on the diversity of ecological niches and lifestyles exhibited by fungi (Benocci et al. 2017). Thus, it is important to conduct omics studies on fungi from different habitats with varying lifestyles (saprobic, symbiotic and parasitic) in order to develop superior enzyme cocktails, or tailor pre-treatment agents. The finding that a single base pair difference among *Trichoderma* species could affect the expression and catalytic performance of biomass-degrading enzymes (Horta et al. 2018) affirms the need for a firm understanding of the underlying mechanisms for controlling gene expression.

## 41. Packed-bed bioreactor for mycomaterial production

Solid-state bioreactor systems have generally been considered the lesser alternative to liquid culture bioreactors for scaled generation and extraction of target proteins from bacteria and yeasts. Liquid culture allows for more efficient dissipation of heat, homogenization of cultures, and incremental addition of feedstock. While liquid culture provides a high degree of functionality for product extraction, they are largely limited to the production of discrete hyphal pellets or tissue sheets, making solid-state-fermentation bioreactors optimal for applications leveraging the three-dimensional structure of mycelium or modifying a solid substrate. Numerous solid-state bioreactor designs have been implemented in industry, but the details of their development and application are seldom reported due to their proprietary nature (Mitchell et al. 2010).

For 10 years, Ecovative Design (Green Island, NY, USA) has been manufacturing mushroom composite materials which harness the structure of mushroom mycelium to produce products for protective packaging, furniture componentry, and other goods. These products utilize the structure of mycelium—a tenacious combination of cell wall chitin–glucan matrices and filamentous inter-cellular crosslinking—to bind discrete lignocellulosic particles into mycelium composites of defined geometry (Islam et al. 2018) with sufficient compressive and flexural strength to withstand use in a variety of high stress applications. Initially, these mycelium products were manufactured in a

Type I (Mitchell et al. 2010) passively aerated molded tray incubation system, wherein the maximum dimensionality was governed by the limitations of passive metabolic heat and gas diffusion. Since 2016, Ecovative has invested in the development of an actively aerated solid-state bioreactor system designed to enable gas-exchange and heat-dissipation within large masses of mycelium composite through forced aeration with conditioned air. The adoption of this large, solid-state bioreactor (coined the Bulk Bin Reactor; see Fig. 44) has enabled the production of large geometry structural products that were impossible to produce with tray-based passively aerated systems. In addition, the necessary asepsis required for the production of this material has been greatly reduced compared to the former tray-based system. The 0.7 m³ blocks produced by the Bulk Bin Reactor system can be cut using a horizontal band saw mill into billets as thin as 3 cm thick, yielding multiple units from a single bin, and opening a variety of product opportunities for affordable flat stock panels. This represents the first report of the development and application of a Type II solid-state bioreactor system for mycelium material production, including a summary account of engineering and biological considerations.

The physical system consists primarily of an air pretreatment system and a vessel including air distribution (Mueller 2018). Pre-treatment of the air is critical for controlling temperature, humidity, and gas concentrations. Air is introduced to the system through a coarse particulate filter for protection of the blower. Critically, the blower is capable of providing air at a range of pressures which enables not only passage through the loose substrate prior to growth, but passage through the myceliated material at the end of the process cycle when pressures are highest. From the blower, the air is cooled to a programmable temperature via an intercooler or fan ventilator. This allows the system to run in an environment with fluctuating external temperatures, and also controls for the variable amount of heat added by the fan, which may change depending on load. Temperature controlled air can then be split into a plurality of flows for the support of multiple vessels. Here, flow (vol/vol/min) is also measured within each vessel to ensure that the desired flow rate is achieved.

Air at temperature next enters a humidification chamber wherein it is bubbled through a column of water. The humidification chamber is designed with a depth and size such that it can provide sufficient moisture into the air to fully saturate it. Additionally, as the process of evaporating water into the air stream requires heat, a heater is implemented to add the energy required to continually humidify the air, even at very high flow rates. By varying this energy input, it is possible to control with precision the humidity level during steady state operation. The humidification chamber (and all parts of the airflow pretreatment system)
<figure><img src="image_62.png" /><figcaption>**Fig. 44** a The bulk bin reactor system showing the air pretreatment system for air distribution and conditioning, and a bin incubation unit with a growing mycelium-lignocellulose composite block; b example of a bulk bin reactor system mycelium-lignocellulose block, after extraction from the bin incubation unit and prior to post-processing; c light micrograph of epoxy thin section of mycelium-wood composite, showing a two-dimensional section of the inter-particle hyphal matrix; d panels cut from a bulk bin reactor system mycelium-lignocellulose block during post-processing, showing examples of a sealed surface (top), an applied laminate (middle), and an unfinished surface (bottom)</figcaption></figure>

must be designed to handle the pressures which will be sustained at the end of the process when inter-particle colonization has reduced porosity.

The pre-treated air can be connected to a variety of vessel designs, from custom-molded shapes to generic blocks for later processing into panels. Design of the vessel depends on the nature of the substrate being used, particularly on its porosity, and on other variables such as the metabolic heat generated with the given combination of process parameters. In large vessels, an array of nozzles is used, each providing equal flow for the generation of uniform materials. The nozzles used are specially designed in order to provide uniform back pressure against free flow, therefore maintaining even flow rates through each nozzle and minimizing effects of random porosity variations in the material. The cross-sectional area of the nozzle and expansion area is selected such that even with partial blockage, the remaining cross-sectional area is still greater than the cross-sectional area of the flow restriction zone. This minimizes flow variation between nozzles caused by the random orientation of the chips.

It is important to note here that if $P_{D_S}+P_{D_G}$ exceeds the force of gravity on the substrate, the growing material will lift, opening low resistance air-flow channels that will

bypass the material and increase gas-exchange heterogeneity. This is colloquially termed “burping”, and whether it occurs in operation is a combined function of the porosity of the substrate; the density of inter-particle colonization; the air flow rate required; and the density of the substrate—all of which combine to dictate the burping back-pressure.

One critical dimension of the vessel is its height. If aeration is introduced on a single side, for example on the bottom of a rectangular open top vessel, and if the vessel is sufficiently large in its length and width dimensions that heat loss through the walls cannot be considered for the central material, then its core is essentially a one dimensional thermodynamic and fluid dynamic system. In such a system, with heat being generated by each successive unit layer of material, the delta between the temperature of the material at the bottom of the vessel ($T_{bot}$) and at the top ($T_{top}$) will be directly correlated with the height of the vessel. By the same logic, it is true that there will always be a temperature difference between the bottom and top of the vessel, so long as the material is generating heat and being cooled by aeration. It is important that the air flow rate, metabolic conditions, energy availability of the substrate, organism selected for growth, and height of the vessel all be selected in concert in order to ensure that the

<figure><img src="image_63.png" /></figure>
temperature delta between top and bottom is small enough that final properties of the top and bottom of the material are within the desired specifications.

A primary consideration for substrate and nutrition selection is the minimization of systemic sensitivity to contaminant organisms. The choice of a suitable primary substrate, composing the bulk of the raw material, is key to achieving stable and productive operation. Attributes to consider include density, porosity, nutritional availability, phytochemical composition, and cleanliness. From a holistic perspective, substrate composition—including spawn type, spawn rate, substrate species, supplemental nutrition, and temporal staging of supplemental nutritional inputs (i.e. pre- or post-priority effect)—is critical to the exclusion of contaminant organisms and to achieving a mechanically relevant stage of myceliation without strict aseptic control (Winiski 2015).

Strain selection involves a number of considerations, including ecological and metabolic habit, growth rate, hyphal morphology, and temperature sensitivity. Critically, the selected strain should have metabolic access to the recalcitrant lignocellulose in the base substrate while demonstrating growth rates that allow for exclusion of competitor species (Winiski 2015). Additionally, the selected strain should result in an inter-particle hyphal morphology that produces the desired mechanical properties. The organism selected must also be able to grow at a range of temperatures, but with generally similar growth rates at the range between $T_{bot}$ and $T_{top}$, thus enabling a uniform product.

One particular consideration in the production of mycelium materials with actively aerated static bioreactor systems involves the inherent conflict between the selection for inter-particle hyphal growth for material properties, and the resultant effect on inter-particle porosity and backpressure. Within this context, the morphology of inter-particle growth governs air flow through the particle mass, ultimately affecting oxygen availability, heat dissipation, and thereby material heterogeneity, as a result of air channeling during inter-particle densification. For this reason, it is critical to understand the specific dynamics

among the inter-particle hyphal network, mechanical properties, and system dynamics, in order to negotiate and optimize system operation and product properties. In this effort, simple and accessible techniques are employed for direct observation of inter-particle hyphal morphology by epoxy embedment and thin-sectioning, utilizing adaptations of well-established methodologies (Richardson et al. 1960). Open source image analysis software (Barry et al. 2015; Puspoki et al. 2016) is leveraged to perform quantitative analysis of the inter-particle hyphal matrix via light micrographs of the epoxy thin section (Fig. 44c). These methods and software applications provide accessible tools for extracting large quantities of morphological data with high sampling frequency (thousands of data-points on a scale of minutes), where manual measurement would be prohibitively laborious, and from which inter-particle morphological features such as hyphal length and area density, node density, inter-particle porosity, cell or strand thickness, and network orientation coherency and anisotropy can be related to system operation, environmental conditions, and material properties.

The described bulk bin reactor system is being leveraged to efficiently produce solid blocks of mycelium composite material (Table 20, mCore Panels and Blocks 2018), which is post-processed to panels of varying geometries using a horizontal band saw mill for applications including acoustic panels and wetland rafts, with further applications potentially envisaged for packaging, temporary signage and constructions, and insulation cores for doors. This material is manufactured within a 10-day processing time in $0.7 m^3$ blocks using the current bulk bin reactor paradigm, with plans for scaling to >56 $m^3$ with modification of existing lane composting system infrastructure with a projected cost of <$142 USD/m$^3$ for operations above 42,000 $m^3$ per year. For comparison, production costs for the Type I passively aerated molded tray system ranges from $350–700 USD/m$^3$.

Previous research conducted by Pelletier et al. (2013) identified the potential application of mycelium composites produced with agricultural substrates as acoustic absorbers. In addition to the added benefits of a low-energy

<table><caption>**Table 20** Material blends and properties</caption><thead><tr><th>Property</th><th>Standards</th><th>Hemp blend</th><th>Aspen chip</th><th>Aspen shaving</th></tr></thead><tbody><tr><td>Density (kg/m<sup>3</sup>)</td><td></td><td>102–129</td><td>192</td><td>96</td></tr><tr><td>Modulus of elasticity (MPa)</td><td>ASTM C165-07, D1621-16</td><td>0.89–1.72</td><td>3.44–4.20</td><td>0.27–0.48</td></tr><tr><td>10% compressive strength (MPa)</td><td>ASTM C165-07, D1621-16</td><td>0.06–0.15</td><td>0.20–0.41</td><td>0.01–0.02</td></tr><tr><td>Flexural modulus (MPa)</td><td>C203-05a</td><td></td><td>10.34–15.85</td><td>5.51–9.65</td></tr><tr><td>Flexural strength (MPa)</td><td>C203-05a</td><td></td><td>0.12–0.20</td><td>0.07–0.11</td></tr><tr><td>Noise reduction coefficient (0–1)</td><td>ASTM C423</td><td>0.6</td><td>0.4</td><td>0.9</td></tr><tr><td>Fire rating</td><td>ASTM E84</td><td>Class A</td><td></td><td></td></tr></tbody></table>
manufacturing process and biodegradability at the pro-
duct’s end-of-life stage, it was found that optimum per-
formance occurred at the critical automotive road noise
frequency of 1000 Hz. Subsequently, testing was per-
formed with panels produced within the bulk bin reactor
paradigm with three substrate blends (Table 20), according
to ASTM C423 using an IBM HVAL with a Norsonics
NE840 real time analyser. The highest sound absorption
was found with the aspen shaving blend, which boasted an
NRC rating of 0.9, thus demonstrating a Class A absorption
coefficient and suggesting potential application for manu-
facture of low VOC, biodegradable, acoustic panels. State-
of-the-art acoustic panels are produced with polyurethane
foam, with mineral wool and fiberglass providing low-
VOC options, but there is a growing interest in develop-
ment of natural alternatives due to human health and pro-
duct end-of-life concerns (Kaamin 2017).

A critical property for acoustic panels for indoor
applications is fire rating. Jones et al. (2017) reported
meaningfully lower CO2 and CO release, along with
reduced smoke density, for mycelium-rice hull composites,
as compared to XPS by cone calorimeter. Additionally, fire
performance can be improved by amendment with glass
fines (Jones et al. 2018).

Constructed floating wetland panels represent another
application opportunity for the mechanical and end-of-life
properties of mycelium composites. Constructed floating
wetlands are primarily utilized to improve wetland water or
wastewater quality, though they are also used for a variety
of other objectives, including habitat creation, shoreline
protection, and landscape improvement. Floating wetlands
are typically constructed from a variety of materials,
including non-biodegradable materials such as extruded
polystyrene, PVC pipes, and high-density polyethylene
(Pavlineri et al. 2017). In this consideration, mycelium
composite panels may provide a biodegradable option with
the required mechanical and hydrophobic properties (Ha-
neef et al. 2017). This application is being tested by the
Green Futures Research and Design Lab at the University
of Washington (Seattle, WA, USA), with mycelium com-
posite panels currently in field deployment.

42. Fungal degradation of plastics: A hidden
treasure for a greener environment

In the modern plastic era, primary plastic production has
increased to 335 million tonnes per year, with Asia
accounting for 50% of the total worldwide production
(PlasticsEurope 2017). According to a recent estimate,
from 1950 to 2015 about 9150 million tonnes of primary
plastics were produced, resulting in the accumulation of
about 6945 million tonnes of plastic waste on the surface of

the earth. Of the total waste generated, only a small per-
centage was recycled (9%) and incinerated (12%), while
79% amassed in landfills and other terrestrial and marine
environments (Geyer et al. 2017).

The majority of petroleum-based plastics, such as polyethylene (PE), poly(ethylene terephthalate) (PET), polystyrenes (PS), polyurethanes (PUs), and polyvinyl chloride (PVC), are resistant to biodegradation, and therefore persist in the environment with various consequences (Wei and Zimmermann 2017). Biodegradable plastics are considered eco-friendly alternatives to petroleum-based non-biodegradable polymers. However, limited information is available regarding the exact mechanisms underlying the biodegradation process, the time scale of biodegradation, and the optimal environmental conditions required for their biodegradation (Yang et al. 2014a). Thus, the careless disposal and degradation of both biodegradable and synthetic plastics result in the accumulation of waste in terrestrial landfills and marine environments, and pose a serious threat to surrounding ecosystems (Andrady 2015). Nonetheless, numerous studies have reported that plastics are vulnerable to microbial attack (Barratt et al. 2003; Krasowska et al. 2012; Mathur and Prasad 2012; Zafar et al. 2013, 2014; Restrepo-Flórez et al. 2014; Khan et al. 2017).

The conservational and ecological roles of fungi are not
only limited to energy or elemental cycling, but can also be
used in the biodegradation of various types of plastics.
Numerous studies have mentioned fungi as the predomi-
nant microorganisms responsible for the biodegradation of
both bio- and synthetic- plastics (Barratt et al. 2003; Khan
et al. 2017). According to the findings of one survey, the
majority of plastic degrading fungi belong to the genera
Aspergillus, Fusarium, Paecilomyces and Penicillium (Kim
and Rhee 2003). The mechanical forces exerted by the
fungi during growth, hyphal penetration into the plastic
layer, as well as the simultaneous secretion of various
enzymes and radicals, are all characterized as the fungal
degradation of plastics (Moore et al. 2000). The mecha-
nism of fungal degradation begins with the attachment of
hyphal filaments to the surface of a plastic substrate. The
hyphal tips then extend, using mechanical force to secrete
enzymes and radicles that allow the penetration of the
fungus into the substrate (see Fig. 45).

The next step is the absorption of small molecules,
polymers, radicles, or atoms through the porous tips of the
hyphae, followed by their transportation through the
underlying plasma membrane (Moore et al. 2000; Khan
et al. 2017). The enzymes are known to hydrolyze the
polymeric substrate and in turn provide nutrients, which
facilitates the growth of fungi or other microorganisms
(Santerre et al. 1994; Lucas et al. 2008; Banerjee et al.
2014). The various fungal species and the associated enzymes reported to degrade various types of plastics are listed in Table 21.

Most of the enzymes known for the degradation of plant polymers are able to depolymerize synthetic polymers, such as PE and PU, by hydrolyzing the ester bonds present in the polymer backbone. The surface area of polymers exposed to fungal enzyme attack has an advancing effect on biodegradation. Therefore, plastic particles in the range of 0.5 to 0.25 mm are most suitable for enzyme biodegradation. Although purified enzymes are known to breakdown the C–C bond in polyvinyl chloride (PVC), the use of microorganisms is favored for the biodegradation of polymers. Moreover, the mixed cultivation of several strains is known to have a greater impact on biodegradation efficiencies than the use of single strains (Wei and Zimmermann 2017).

The overall process of plastic biodegradation is dependent on the presence of optimal environmental conditions to facilitate the growth of fungi and maximize the activities of the enzymes to depolymerize the polymeric materials. The biodegradation process is also affected by the physicochemical properties of the plastics, such as surface

topology, molecular weight, hydrophobicity, and the degree of crystallinity (Manzur et al. 2004; Brueckner et al. 2008; Jenkins and Harrison 2008; Ronkvist et al. 2009; Restrepo-Flórez et al. 2014; Wei and Zimmermann 2017). Higher degrees of crystallinity strongly reduced the biodegradation of specific plastics. Aliphatic carbon chains are more easily biodegradable than aromatic polymers. The hydrophobicity of plastics makes the surface water repellant, reducing the success of the attachment, growth, and propagation of fungal hyphae, and thereby reducing the degree of plastic biodegradation. Similarly, abiotic factors such as environmental temperature; availability of oxygen; exposure to light or UV; and availability of radicles in the environment all affect the mechanisms of plastic biodegradation (Wei and Zimmermann 2017).

The diversity, zonal distribution, and niche partitioning of fungal strains in the environment also affect the biodegradation process. Furthermore, the amount and type of enzymes secreted by the fungal strain to digest the polymer also strongly affect the biodegradation process (Fig. 10). The secretion of specific types of enzymes or various types of enzymes by fungi to degrade polymers

<figure><img src="image_64.png" /><figcaption>**Fig. 45** Fungal degradation of polyester polyurethane (PEU) films. **a** PEU film not exposed to fungal degradation (control); **b–d** PEU films exposed to different *Aspergillus* spp. on malt extract agar medium incubated for 28 days in dark at 30 °C. The extent of biodegradation differed for each strain. Fungal hyphal growth is observed on the PEU surface and some portions of the PEU films are degraded due to fungal activities</figcaption></figure>
<table><caption>**Table 21** List of fungal species with their enzymes responsible for biodegradation and types of plastics used</caption><thead><tr><th>Fungal strain</th><th>Enzyme</th><th>Plastic type</th><th>References</th></tr></thead><tbody><tr><td>Aspergillus flavus</td><td>Glucosidases</td><td>PCL</td><td>Tokiwa et al. (2009)</td></tr><tr><td>Aspergillus niger</td><td>Catalase, protease</td><td>PCL</td><td>Tokiwa et al. (2009)</td></tr><tr><td>Aspergillus terreus</td><td>Esterase, urethane hydrolase</td><td>PU</td><td>Boubendir (1993)</td></tr><tr><td>Aspergillus tubingensis</td><td>Esterase, lipase</td><td>PU</td><td>Khan et al. (2017)</td></tr><tr><td>Bipolaris (Cochliobolus) sp.</td><td>Laccase</td><td>PVC</td><td>Sumathi et al. (2016)</td></tr><tr><td>Chaetomium globosum</td><td>Esterase, urethane hydrolase</td><td>PU</td><td>Boubendir (1993)</td></tr><tr><td>Curvularia senegalensis</td><td>Polyurethanase</td><td>PU</td><td>Crabbe et al. (1994)</td></tr><tr><td>Fusarium sp.</td><td>Cutinase</td><td>PCL</td><td>Shimao (2001)</td></tr><tr><td>Pestalotiopsis microspora</td><td>Serine hydrolase</td><td>PU</td><td>Russell et al. (2011)</td></tr><tr><td>Phanerochaete chrysosporium</td><td>Manganese peroxidase</td><td>PE</td><td>Shimao (2001)</td></tr><tr><td>Talaromyces funiculosus (formerly Penicillium funiculosum)</td><td>Glucosidases</td><td>PHB</td><td>Tokiwa et al. (2009)</td></tr><tr><td>Talaromyces pinophilus (formerly Penicillium pinophilum)</td><td>PHB-depolymeras</td><td>PHB</td><td>Panagiotidou et al. (2014)</td></tr><tr><td>Trichoderma sp.</td><td>Urease, protease, esterase and laccase</td><td>PU</td><td>Loredo-Treviño et al. (2011)</td></tr></tbody></table>

Disclaimer: Some of the fungal species in the table are pathogenic and it is out of question to use them in biotechnological processes

PCL polycaprolactone, PE polyethylene, PHB polyhydroxybutyrate, PU polyurethane, PVC polyvinyl chloride

**Fig. 46** Polyester polyurethane (PEU) films with fungal growth and signs of biodegradation on the surface. **a** PEU film not exposed to *Aspergillus* species: **b** fungal growth on the surface of PEU; **c**, **d** PEU films after the washing of fungal hyphae from the surface of the films

<figure><img src="image_65.png" /></figure>

<figure><img src="image_66.png" /></figure>
vary from one species to another, or even within the same species (Doi 1990; Howard 2012).

Currently, more than 100 fungal species are known to degrade different types of plastics, and many more are still in the process of being isolated and identified (Howard 2012). Most of the fungal strains discovered were studied for their enzyme biodegradation abilities at the laboratory scale. No efforts have yet been made to develop large scale or industrial scale bioreactors for specific types of plastic degrading fungi. Therefore, this research is of great importance for industrial development, as well as for future research purposes. Collaborations between chemists, biologists, engineers and physicists could result in the development of a large-scale bioreactor for plastic biodegradation utilizing fungi. At present, such processes are too expensive to be applied at large scale, and collecting and burning plastics under high heat remains more economical. However, once such bioreactors are successfully developed, new industries will emerge for both the degradation of various plastics as well as for the utilization of the degraded compounds in the manufacturing of new products. There is a need to properly collect disposed plastics and develop sophisticated large-scale bioreactors in which fungi could be used to degrade various types of plastics separately in controlled conditions using modern biotechnological techniques. Attention should be given to research and development on immobilized enzyme technologies with regard to fungal-degradation of plastics through enzymes. More knowledge is needed concerning the connection between specific-plastic types and specific fungal species, to determine the actual food web of various fungi. Integration of low-cost and easily degradable bio-plastics in markets around the globe is important. The mechanisms of fungal degradation of plastics need to be clarified using isotopic marking of plastics. The structure and active sites of biodegrading enzymes of various fungi need to be determined, and methods to increase the catalytic degradation of polymers need to be developed.

## 43. Polycyclic aromatic hydrocarbon degradation by basidiomycetes

Polycyclic aromatic hydrocarbons are ubiquitous contaminants widely distributed in the environment, with suspected carcinogenic and mutagenic effects. The clean-up of polycyclic aromatic hydrocarbon contaminated sites is of great importance to protecting human health. Polycyclic aromatic hydrocarbons are organic compounds composed of two or more fused benzene rings which are formed during the combustion of organic molecules and their subsequent recombination (Haritash and Kaushik 2009).

Low molecular weight polycyclic aromatic hydrocarbons are composed of 2–3 aromatic rings. They are

commonly found in petrogenic sources (e.g. gasoline, kerosene) and can be introduced to the aquatic environment through oil spillage and discharge from ships, tankers, motorboats, etc. Contamination can also come from municipal and urban runoff (Vieira et al. 2018). Polycyclic aromatic hydrocarbons of high molecular weight, composed of 4–6 aromatic rings (e.g. pyrene and benzo[a] pyrene), are often released into the environment through pyrogenic sources (Souza et al. 2015). Such polycyclic aromatic hydrocarbons are considered priority pollutants because they are recalcitrant in the soil, due to their high affinity and low water solubility (Rothermich et al. 2002).

The biodegradation of polycyclic aromatic hydrocarbon (Fig. 46) contaminants is a complex process which is highly dependent on the number of contaminants, natural conditions (e.g. temperature, humidity and climate), and presence of organisms consuming the available hydrocarbons. Polycyclic aromatic hydrocarbons tend to bind to soil components, thus becoming difficult to remove and degrade (Pandey et al. 2016). Hydrocarbons differ in their susceptibility to microbial attack, in which some of the high molecular weight polycyclic aromatic hydrocarbons may only be partially degraded or not degraded at all (Atlas and Bragg 2009).

Fungi can turn hydrocarbon contaminants into an energy source. In the hydrocarbon contaminated environment, those fungi capable of degrading hydrocarbon have better chance of surviving by utilizing this carbon source for growth (Fernandez-Luqueno et al. 2010). Some fungi, and in particular basidiomycetes, are efficient degraders of high molecular weight polycyclic aromatic hydrocarbons. In this way, fungi are unlike bacteria, which are better equipped for degrading smaller molecules (Peng et al. 2008).

The biodegradation of polycyclic aromatic hydrocarbons can be enhanced through extracellular oxidation. White rot fungi are the most efficient producers of oxidative extracellular enzymes. White-rot fungi are wood degrading basidiomycetes which produce special oxidases or enzymes that help in the degradation of lignin and other plant polymers. These enzymes can also degrade a variety of chemicals, including environmental pollutants (Crawford 2006) such as polycyclic aromatic hydrocarbon.

Successful polycyclic aromatic hydrocarbon biodegradation is brought about by different catabolic activities, such as metabolic capabilities, the induction of specific enzymes, and the presence of favourable organisms capable of degradation (Al-Hawash et al. 2018) under favourable conditions.

The degradation of polycyclic aromatic hydrocarbons can be affected by conditions such as temperature, oxygen, pH, and nutrient availability. Temperatures can affect the physical and chemical composition of polycyclic aromatic hydrocarbons: at low temperatures, the degradation rate is
slow due to the decreased enzymatic activities (Bisht et al. 2015). Polycyclic aromatic hydrocarbon degradation can actually occur at a wide range of temperatures according to medium: the maximum degradation was documented to be in the range of 30 to 40 °C in soil environments, 20 to 30 ° C in marine environments, and 15 to 20 °C in fresh water environments (Al-Hawash et al. 2018). Oxygen concentration is also important in the biodegradation process. Biodegradation can be at its best in highly oxygenated environments. Though the degradation of polycyclic aromatic hydrocarbons can also occur at negligible rates under anaerobic environments (Haritash and Kaushik 2009), the process is not as rapid as under aerobic conditions (Grishchenkov et al. 2000). The pH value should also be taken into account in the successful degradation of polycyclic aromatic hydrocarbons, as it regulates the passage of small molecules and ions in the biological membrane and affects the catalytic reaction balance. In general, fungi would prefer nearly neutral pH to maximize their degradation activity, although they can also tolerate acidic conditions (Bonomo et al. 2001). Nutrients can also become a limiting factor in the process of biodegradation. The concentration as well as the ratio of certain nutrients such as carbon, nitrogen, iron and phosphorous can affect the growth and sporulation of hydrocarbon-degrading fungi (Zafra and Cortés-Espinosa 2015). The chemical composition of polycyclic aromatic hydrocarbons is yet another factor that can greatly affect the success of polycyclic aromatic hydrocarbon degradation. The higher the molecular weight, the more recalcitrant polycyclic aromatic hydrocarbons are in the environment, thus taking much longer to be degraded (Fedorak and Westlake 1981).

Basidiomycetes use a variety of mechanisms to com-
pletely degrade organic compounds such as hydrocarbons.
In general, both oxidative and reductive reactions are
required for the complete metabolism of hydrocarbon
compounds. The first step in degradation occurs at the
intra-cellular level in the form of oxidation and the inte-
gration of oxygen with the aid of the enzymatic catalysts
peroxidases and oxygenates (Al-Hawash et al. 2018).
Basidiomycetes secrete these enzymes to catalyse both the
direct and indirect oxidation of chemicals. The enzymes,
which are oxidoreductases, aid in the detoxification of toxic
organic compounds through biochemical reactions, which
in turn transform polycyclic aromatic hydrocarbons step by
step into intermediates of the central intermediary meta-
bolisms (such as the tricarboxylic acid cycle). The action of
oxidoreductases renders the contaminants and xenobiotics
into harmless compounds that can be easily degraded fur-
ther (ITRC 2002). For instance, the ligninolytic fungus,
*Pleurotus ostreatus*, can degrade phenanthrene into less
harmful compounds, including 2,2'-diphenic acid (Fig. 47).

White-rot basidiomycetes produced non-specific extra-
cellular ligninolytic enzymes consisting of three groups:
lignin peroxidase (LiP), manganese-dependent peroxidases
(MnP), and laccase (Hofrichter et al. 2001), all of which
are essential for the transformation and mineralization of
organic pollutants (Wang et al. 2009) such as polycyclic
aromatic hydrocarbons. The genera *Ganoderma* (Agrawal
et al. 2018), *Armillaria* (Hadibarata and Kristanti 2013),
*Coprinus* (Li et al. 2009), *Marasmiellus* (Vieira et al.
2018), *Pleurotus* (Hadibarata and Teh 2014; Li et al. 2009),
*Pycnoporus* (Munusamy et al. 2008) and *Phanerochaete*
(Wang et al. 2009) were able to degrade polycyclic aro-
matic hydrocarbon moieties such as anthracene, pyrene,
phenanthrene and benzo[a]pyrene (Table 22).

Many fungal species, and in particular white-rot basidi-
iomycetes, have demonstrated the ability to degrade poly-
cyclic aromatic hydrocarbons under laboratory conditions;
however, these processes require further investigation in
the field, and to date a limited number of studies have
tested the efficacy of these fungi in the degradation of
polycyclic aromatic hydrocarbons within typical environ-
mental conditions. The placement of fungi into the envi-
ronment requires further studies to evaluate their impact on
other organisms. Intensive studies should also be con-
ducted into analytical chemistry and genetic engineering
tools which could help in successful bioremediation to
increase fungal efficiency in degrading hydrocarbons and
other recalcitrant contaminants. Furthermore, through the
optimization of adsorption, bioavailability and mass
transfer of polycyclic aromatic hydrocarbons, scientists
could enhance the rate of bioremediation. This could pro-
vide one effective means of mitigating environmental
pollution.

44. Can fungi help modify the sustainable soil enhancer biochar?

Biochar has achieved popularity as a sustainable soil
enhancer. Biochar is derived from biomass such as leaves,
forestry and agricultural residue, and animal manure
(Spokas 2010; Shackley et al. 2012). This biomass is
converted into a highly porous, carbonaceous product
through a controlled pyrolysis process, namely thermo-
chemical decomposition in the absence of oxygen (pre-
venting combustion). In this way, it is different from
charcoal. While charcoal produces energy, biochar is used
in carbon sequestration, environmental management, and
soil amendments (Lehmann and Joseph 2009; Warnock
et al. 2007).

Biochar is added to poorly performing or contaminated
soil as a means of increasing the recalcitrance of organic
soil matter in a sustainable manner. Due to its polycon-
densed aromatic structures, the added biochar can remain
<figure><img src="image_67.png" /><figcaption>**Fig. 47** Proposed pathway for the degradation of phenanthrene by the ligninolytic fungus *Pleurotus ostreatus* (Bezalel et al. 1996; Aust et al. 2003; Gupte et al. 2016)</figcaption></figure>

in the soil for centuries to millennia, thus contributing higher levels of aromatic compounds to the soil over long periods of time. Important physicochemical properties for the evaluation of biochar utilized in soil have been defined: pH, volatile compounds content, ash content, bulk density, water-holding capacity, pore diameter and volume, and specific surface area (Sohi et al. 2010). The origin of the biomass or feedstock and the pyrolysis conditions, therefore, are the key factors governing the physicochemical properties of biochar. Within the pyrolysis process, temperatures between 400 and 500 °C produce higher amounts of biochar (Gaunt and Lehmann 2008; Qambrani et al. 2017). While pyrolysis occurs more rapidly at high temperatures, a slowed pyrolysis process can produce a substantially higher quantity of biochar product. The yields of ash-free biochar were positively correlated with the cellulose, hemicellulose and lignin contents of feedstock; the

biomass samples possessing higher lignin content produced higher amounts of biochar, and the samples of high ash content hampered biochar production (Sun et al. 2017).

When incorporated into soils, biochar exhibits a natural oxidation through the formation of functional groups. These groups are dependent on the temperature employed to produce the biochar. Thus, biochars are redox active and as such contribute to the reductive transformation of organic contaminants in soil by facilitating electron transfer from bulk chemical electron donors (e.g. phenolic moieties arising from the lignin in the original biomass) to the receiving organic compounds (e.g. quinone–hydroquinone moieties) and redox-active metals (e.g. iron, manganese, copper), reversibly donating and receiving up to 2 mmol electrons per gram of biochar (Klöpfel et al. 2014). Biochar-mediated microbial electron shuttling has also been considered as an important process in soil
<table><caption>**Table 22** Degradation of polycyclic aromatic hydrocarbons by enzymes from basidiomycetes</caption><thead><tr><th>Enzyme</th><th>Basidiomycete strain</th><th>Polycyclic aromatic hydrocarbon moieties</th><th>Percentage of degradation (%)</th><th>References</th></tr></thead><tbody><tr><td rowspan="10">Laccase</td><td rowspan="2">"Ganoderma lucidum"<sup>a</sup></td><td>Phenanthrene</td><td>99.65</td><td rowspan="2">Agrawal et al. (2018)</td></tr><tr><td>Pyrene</td><td>99.58</td></tr><tr><td rowspan="3">Pycnoporus sanguineus</td><td>Phenanthrene</td><td>90.00</td><td rowspan="3">Munusamy et al. (2008)</td></tr><tr><td>Pyrene</td><td>96.00</td></tr><tr><td>Anthracene</td><td>37.00</td></tr><tr><td>Armillaria sp.</td><td>Pyrene</td><td>63.00</td><td>Hadibarata and Kristanti (2013)</td></tr><tr><td rowspan="2">Pleurotus eryngii</td><td>Anthracene</td><td>99.90</td><td rowspan="2">Li et al. (2009)</td></tr><tr><td>Benzo[a]pyrene</td><td>87.50</td></tr><tr><td rowspan="2">Agaricus bisporus</td><td>Anthracene</td><td>89.80</td><td rowspan="2"></td></tr><tr><td>Benzo[a]pyrene</td><td>48.60</td></tr><tr><td rowspan="2">Pleurotus ostreatus</td><td>Anthracene</td><td>38.00</td><td rowspan="2"></td></tr><tr><td>Benzo[a]pyrene</td><td>31.00</td></tr><tr><td rowspan="2">Coprinus comatus</td><td>Anthracene</td><td>9.80</td><td rowspan="2"></td></tr><tr><td>Benzo[a]pyrene</td><td>9.30</td></tr><tr><td rowspan="5">Lignin peroxidase</td><td rowspan="2">"Ganoderma lucidum"</td><td>Phenanthrene</td><td>99.65</td><td rowspan="2">Agrawal et al. (2018)</td></tr><tr><td>Pyrene</td><td>99.58</td></tr><tr><td rowspan="3">Phanerochaete chrysosporium</td><td>Phenanthrene</td><td>72.77</td><td rowspan="3">Wang et al. (2009)</td></tr><tr><td>Pyrene</td><td>51.16</td></tr><tr><td>Benzo[a]pyrene</td><td>25.50</td></tr><tr><td rowspan="5">Manganese peroxidase</td><td rowspan="2">Marasmiellus sp.</td><td>Pyrene</td><td>100.00</td><td>Vieira et al. (2018)</td></tr><tr><td>Phenanthrene</td><td>99.65</td><td rowspan="2">Agrawal et al. (2018)</td></tr><tr><td rowspan="3">"Ganoderma lucidum"</td><td>Pyrene</td><td>99.58</td></tr><tr><td rowspan="3">Phanerochaete chrysosporium</td><td>Phenanthrene</td><td>72.77</td><td rowspan="3">Wang et al. (2009)</td></tr><tr><td>Pyrene</td><td>51.16</td></tr><tr><td>Benzo[a]pyrene</td><td>25.50</td></tr></tbody></table>

remediation and other biogeochemical reactions (Yu et al. 2016), leading to an increase in microbial growth while reducing Fe(III) minerals in the soil (Kappler et al. 2014).

The effects of biochar on soil fertility have been demonstrated through the pH increase of the acid found in soils (Van Zwieten et al. 2010), and the benefits in high nutrient levels and retention through the ions absorption (Liang et al. 2006). However, biochar has also been shown to both stabilize and change soil biological community composition and abundance, depending on the biomass source and the temperature used in the pyrolysis reaction (Kim et al. 2012; Lorenz and Lai 2014). Such changes may affect soil structure and nutrient cycles (Gaskin et al. 2010; Rillig and Mummey 2006; Steiner et al. 2008), thereby indirectly affecting plant growth (Warnock et al. 2007). Rhizosphere bacteria and fungi may also directly promote

the growth of plants (Compant et al. 2010). Soil fungi (e.g. saprotrophs, pathogens and mycorrhizae) respond differently to biochar applications. Saprotrophic fungi have the potential to modify biochar in the soil through the colonization of the pores present in the structure, and this can lead to decomposition (Atkinson et al. 2010; Lipczynska-Kochany 2018). Certain arbuscular mycorrhizae are able to increase their root colonization sites in the presence of biochar, enhancing the availability of phosphate to the plant, and thereby negating the need to add artificial fertilizers.

Fungal extracellular enzymes are the agents of both colonization and decomposition, and are becoming increasingly common tools for examining soil microbial response in climate change experiments (Weedon et al. 2011). Saprotrophic fungi are considered to be efficient

<figure><img src="image_68.png" /></figure>

<footnote><sup>a</sup>The taxonomy of "Ganoderma lucidum" given in the paper by Agrawal et al. (2018) is doubtful since this species has never been safely recorded from India</footnote>
degraders of lignocellulosic biomass due to the wide spectrum of extracellular enzymes they produce. Their production and activities are strongly affected by temperature, moisture and pH. Many studies have been performed on the influence of temperature and moisture on the microbial ecosystem of soils and the concomitant effect on global enzyme pool composition and size (Schimel et al. 2007; Sowerby et al. 2005). Enzyme pool size is controlled by the rate at which enzymes are produced by microbes relative to the rate at which they are degraded by the environment. The production of these extracellular enzymes incurs a cost to the microorganism in terms of energy and nutrients, and so microbes produce certain enzymes targeting specific compounds rich in carbon, nitrogen or phosphorous. Moisture levels influence the diffusion of substrates, the hydration state of the individual enzymes, and the rate of water reactivity.

Due to the chemical nature of biochar, it may be possible to use fungi directly attached to the biosourced material or to use fungi in solid-state fermentation conditions to pretreat the biochar prior to soil addition (a type of composting). Figure 48 provides a scanning electron microscopy (SEM) image of field-aged biochar buried in agricultural soil in which hyphal fragments of fungi were fixed on the biochar surface. It is also possible, through a more biotechnologically sophisticated approach, to harness the power of these secreted enzymes to modify the physicochemical properties of the exposed hydroxyl and carbonyl groups on the surface of the biochar. This will make them more functionally active as a soil amendment for the stimulation of the existing microbial communities.

The structure of biochar is similar to lignin, and so the redox-responsive enzymes, such as laccases and heme-peroxidases produced by Basidiomycetes, Ascomycetes and soft-rot fungi such as *Chaetomium globosum*, *Phialophora malorum*, and *P. mutabilis* are designed to modify these structures, and to generate hydroxyl radicals (Gao et al. 2018). These fungi also control the availability of metal ions in the system, either through their incorporation into the protein structure as co-factors, or chelated in organic acids. While this extracellular process is designed to aid the fungus in the lignin degradation process, these enzymes and chelators could also be a way to control the release or captivation of metals in soils. A modified biochar, with increased surface-active groups, would enhance the soil even further, most notably the activity of the natural soil microbiome, including mycorrhizal fungi. This in turn would further improve the water dynamics, nutrient cycling, and suppression of crop diseases, thereby enhancing the productivity of the soil and the crops that are cultivated in it, while at the same time contributing to carbon sequestration and reduced air contamination in rural areas of the world.

## Commodities

Fungi have been exploited both industrially and commercially in many ways especially when valuable commodities are involved. These organisms offer unique advantages in biotechnology as they can be easily cultured, reproduce quickly and have short life cycles. In this section we

<figure><img src="image_69.png" /><figcaption>**Fig. 48** The scanning electron micrographs of field-aged biochar buried in agricultural soil, showing the outer surface of biochar soil interface (Quilliam et al. 2013; with permission), with the arrow indicating an example of pore blockage (a); spatial heterogeneity and sparsity of internal microbial colonisation (b); internal colonisation by hyphal and single-celled microbes (arrows) (c, d)</figcaption></figure>
discuss some of the many non-food commodities derived
from fungi.

45. Fungi and cosmetics

Fungi are used as ingredients in numerous cosmetic prod-
ucts, including in some of the very expensive brands. The
beneficial claims are all-encompassing, but many have yet
to be proven. As the uses of fungi in cosmetics was
reviewed by Hyde et al. (2010), which includinges, skin
whiteners, moisturizers, anti-aging, shampoos and many
others were reviewed by Hyde et al. (2010), we briefly
summarize the topic here. The demand for cosmetic
products has rapidly increased, and; hence cosmetics has
have become a worldwide industry (Hyde et al. 2010).
Cosmetics are mainly classified as cosmeceuticals, applied
externally to the skin, such as creams, lotions or ointments,
and nutricosmetics, which are consumed as dietary sup-
plements (Hyde et al. 2010). Apart from makeup, cosme-
ceuticals are categorized as anti-aging, anti-wrinklinge,
skin revitalizingation, skin whitening, anti-oxidant and
moisturizing products. To avoid any carcinogenic effects,
there has been a recent trend towards natural cosmetics,
such as fungi-based products (Hyde et al. 2010; Imhoff
et al. 2011; Mohd-Nasir and Mohd-Setapar 2018). Fur-
thermore, the potential of fungi to be utilized as “bio fac-
tories” to produce nanoparticles in the cosmetic industry is
currently being explored (El Enshasy et al. 2018).

Ascomycota and Basidiomycota are extensively used in the cosmetic industry (Hyde et al. 2010). Secondary metabolites extracted from the mycelia or fruiting bodies and ingredients from fungal fermentation are used as cosmetic ingredients. *Aspergillus* species (Hyde et al. 2010) and *Rhizopus* species (Mucoromycota) are used in the production of lactic acid, which is a main ingredient in both anti-aging and skin whitening cosmetics. Lactic acid is mainly used to hydrate and make the skin smooth. Additionally, in peeling lotions, lactic acid is contained in higher concentrations and helps to remove the outer layer of the skin (Zhang et al. 2007). Fungi are utilized in the production of anti-oxidants, fatty acids and polysaccharides in anti-aging products, such as the chitin-glucan complexes of *Aspergillus niger* and some mushroom species (Synytsya et al. 2009; Vysotskaya et al. 2009).

Eicosapentaenoic acid is a rare omega fatty acid used in
anti-aging products. It is extracted from *Mortierella* species
(Wang et al. 2007). *Mortierella* and *Rhizopus* species
produce γ-linolenic acid. This compound is used as an anti-
inflammatory agent and facilitates healthy skin (Krištofí-
ková et al. 1991). *Sporotrichum pruinosum* is used to
produce melanocytic enzymes through a submerged aero-
bic fermentation process. Melanocytic enzyme is used in
some skin whitening cosmetics to activate the

<figure><img src="image_70.png" /><figcaption>**Fig. 49** The King's Cordy Serum contains *Cordyceps militaris* extract. The extract is claimed to facilitate anti-wrinkle effects and add moisture and antioxidants to the skin. It is produced in the Centre of Excellence in Fungal Research in Mae Fah Lung University, Chiang Rai, Thailand</figcaption></figure>

depigmentation of the skin (Mohorčič et al. 2007). To treat
neurodermatitis and sclerodermatitis, an extract obtained
from *Tremella* sp. is used. Extracts of *Ophiocordyceps
sinensis* and *Tremella fuciformis* are utilized to increase the
moisturising effect in certain cosmetic products (Hyde
et al. 2010) (Fig. 49).

46. Agarwood

Agarwood is an economically valuable resinous heartwood product derived from wounded trees of the family Thymalaceae (Novriyanti et al. 2010; Subasinghe et al. 2012; Peng et al. 2015a; Chowdhury et al. 2016; Chen et al. 2018b). Agarwood incenses are used for fragrance in soaps and shampoos and have a pleasant aroma and general perfume and are an element of important religious rituals in Ayurvedic, Tibetan and traditional East Asian medicine (Subasinghe et al. 2012; Rhind 2013; Chowdhury et al. 2016; Lee and Mohamed 2016; López-Sampson and Page 2018) and as aromatic food ingredients (Liu et al. 2013; Tan et al. 2019). India as well as the Southeast Asian countries are the main manufactures of agarwood products, while China, India, the Middle East and Japan are the primary consumer countries. Species of *Aquilaria* (Adams et al. 2014; Mohamed et al. 2014; Selvan et al. 2014: Azren et al. 2019), *Gyrinops*, *Aetoxylon* and *Gonystylus* are used for the production of agarwood (Subasinghe et al. 2012; Mohamed et al. 2014; Mohamed and Rasool 2016). Currently, agarwood producing *Aquilaria* species are cultivated from the home garden level to large scale plantations in Southeast Asia, India and southern China (Lee and Mohamed 2016; Azren et al. 2019).

Naturally, agarwood formation occurs through wounds.
The infected tissues produce oleoresin which is converted
to odoriferous aromatic agarwood resin (Peng et al. 2015a;
Chowdhury et al. 2016). When microbial pathogens enter
<figure><img src="image_71.png" /><figcaption>**Fig. 50** Barks of *Gyrinops walla* Gaertn. (Thymelaeaceae) in Sbaragmuwa University premises, Sri Lanka 2018
**a** Healthy bark, **b**, **c** damaged bark (Photo credit: H.A.T. Chinthaka)</figcaption></figure>

the wound, the defense mechanism of the trees is triggered (Mohamed et al. 2014; Chowdhury et al. 2016; Azren et al. 2019). Sesquiterpenes and 2-(2-phenylethyl) chromone derivatives are the key active compounds in agarwood (Chen et al. 2011; Naef 2011; Mohamed et al. 2014; Li et al. 2019; Tan et al. 2019).

Agarwood causal agents are divided into chemical, physical, and biological agents. Fungi are the biological agents (Novriyanti et al. 2010; Chhipa et al. 2017). Depending on the stress, the tree forms either physical or chemical defense mechanisms (Mohamed and Rasool 2016; Chhipa et al. 2017). The defense substances produced act as biochemical or biological defense agents (Mohamed and Rasool 2016). The wounded or infected tree stem turns dark brownish or black (Fig. 50, Adams et al. 2014). After infection agarwood resin is secreted by the tree and deposited around the wound for number of years. Resinous agarwood form perfumed compounds and is a rare natural mechanism which is poorly understood (Sen et al. 2015). Volatile compounds eventually result in agarwood (Tan et al. 2019). Other than natural agarwood formation, artificial agarwood inducing methods have been developed. Biological inocula such as microbes and fungi are key agents for non-conventional artificial agarwood formation (Azren et al. 2019).

Research has been carried out to determine which fungi are responsible for agarwood production and some of the isolated taxa are listed in Table 23. However, the role of individual fungi needs extensive research to establish which species are important in the process.

## 47. Fungal enzymes

Enzymes are biocatalysts that are involved in catalysis reactions without needing extreme conditions, such as very high temperatures, high pressures or corrosive environments, all of which are often required in chemical processes. Enzymes often offer a competitive advantage when

compared to chemical catalysts. The enzymatic approach is environmentally friendly, as it requires mild conditions and does not normally result in the production of toxic by-products (Chapla et al. 2012). Enzymes are used to catalyze reactions in production processes of several sectors including industrial bioconversion (biocatalyst), environmental bioremediation, agricultural sectors and also bio-transformations of numerous compounds such as flavonoids (Das and Rosazza 2006; Wohlgemuth 2010; Choi et al. 2015). There are several sources of enzymes including animals, plants and microorganisms (bacteria, fungi and protists). Microbial enzymes have generally been used because of their easier isolation in high amounts, low-cost production, stability at various extreme conditions, and their co-compounds, which are also more controllable and less harmful. Microbial enzymes secreted into the media are highly reliable for industrial processes and applications. Microbes isolated from different sources even among species and strains of the same genus may produce varying levels of enzymes of differing properties.

Fungal enzymes have attracted attention for several applications because fungi can grow on low cost materials and secrete large amounts of enzymes into the culture medium, which eases downstream processing (Anitha and Palanivelu 2013). Several fungal enzymes are available commercially including amylases, cellulases, lipases, phytases, proteases, and xylanases (Saxena et al. 2005; Srilakshmi et al. 2015). The positive environmental impact of the production processes is of general interest and the use of enzymatic reactions instead of organic solvents or chemical reactions is highly valued. Figure 51 and Table 24 show examples of important fungal enzymes and the enzyme sources that are used in many applications, but only a few fungal strains meet the criteria for commercial production.

Most applications of enzymes in the food industry have focused on hydrolytic reactions (Akoh et al. 2008; Choi et al. 2015). Glycoside hydrolases and β-galactosidase are
<table><caption>**Table 23** Fungal species potentially responsible for the formation of agarwood in *Aquilaaria* species</caption><thead><tr><th>Host species</th><th>Agarwood responsible fungi</th><th>References</th></tr></thead><tbody><tr><td rowspan="6">Aquilaaria sp.</td><td>Chaetomium globosum</td><td>Budi et al. (2010)</td></tr><tr><td>Fusarium oxysporum</td><td>Azren et al. (2019)</td></tr><tr><td>F. moniliforme</td><td rowspan="2">Tamuli et al. (2000)</td></tr><tr><td>F. sambucinum</td></tr><tr><td>F. solani</td><td></td></tr><tr><td>F. tricinctum</td><td></td></tr><tr><td rowspan="5">A. agallocha</td><td>Aspergillus tamarii</td><td>Bhattacharyya et al. (1952)</td></tr><tr><td>Fusarium solani</td><td>Gibson (1977)</td></tr><tr><td>Botryodiplodia theobromae</td><td>Jalaluddin (1977)</td></tr><tr><td>Phialophora parasitica</td><td>Azren et al. (2019)</td></tr><tr><td>Cytosphaera mangiferae</td><td></td></tr><tr><td>A. crassna</td><td>Acremonium sp.</td><td>Azren et al. (2019)</td></tr><tr><td rowspan="6">A. malaccensis</td><td>Fusarium sp.</td><td></td></tr><tr><td>Alternaria sp.</td><td>Ma et al. (2012)</td></tr><tr><td>Cladosporium sp.</td><td rowspan="2">Premalatha and Kalra (2013)</td></tr><tr><td>Curvularia sp.</td></tr><tr><td>Fusarium proliferatum</td><td></td></tr><tr><td>Phaeoacremonium sp.</td><td></td></tr><tr><td>Trichoderma sp.</td><td></td></tr><tr><td rowspan="7">A. sinensis</td><td>A. niger</td><td>Lin et al. (2010)</td></tr><tr><td>Botryosphaeria dothidea</td><td>Cui et al. (2013)</td></tr><tr><td>Fusarium solani</td><td>Tian et al. (2013)</td></tr><tr><td>Lasiodiplodia theobromae</td><td>Zhang et al. (2014a, b)</td></tr><tr><td>Melanotus flavolivens</td><td>Mohamed et al. (2014)</td></tr><tr><td>Paraconiothyrium variabile</td><td>Chen et al. (2017)</td></tr><tr><td>Arthrinium sp.</td><td>Azren et al. (2019)</td></tr><tr><td rowspan="2">A. subintegra (agarwood oil)</td><td>Colletotrichum sp.</td><td>Monggoot et al. (2017)</td></tr><tr><td></td><td>Azren et al. (2019)</td></tr></tbody></table>

involved in the production of prebiotics, a dietary substance composed of non-starch polysaccharides and oligosaccharides, such as inulin fructo-oligosaccharides, galacto-oligosaccharides, lactulose, and breast milk oligosaccharides, that selectively promote the growth of beneficial intestinal microorganisms in humans (Torres et al. 2010). Cellulases, pectinases, and xylanases are widely used for the clarification of juices and wines. Amylases hydrolyze starch in the starch liquefaction process and convert starch into glucose syrups (Souza and Magalhaes 2010). Tannases are used to reduce tannin levels in food products such as fruit juices, tea, beer, and wines (Yao et al. 2014). These enzymes are also used to hydrolyze gallic acid esters and produce gallic acid, which is used as a substrate for the synthesis of food preservatives (Belmares et al. 2004; Srivastava and Kar 2009; Yao et al. 2014; Dhiman et al. 2017).

Starch is used as the strengthening agent in the desizing process to prevent the breaking of the warp thread used

within the weaving process in the textile industry. Amylases are used for desizing starch in the textile industry because they yield products that do not degrade the fibers. The use of α-amylases in the pulp and paper industry is for the modification of starch of coated paper and textiles. The quality of textiles and paper coated with desizing starch is protected against mechanical damage during processing and finishing. The recycling of waste paper is an eco-friendly trend in the paper industry. The enzymatic reaction of cellulases, lipases, pectinases and xylanases aid in the removal of contaminated ink (Bhat 2000). Proteases and cellulases are used in the polishing step for clear dyeing to improve color and surface vividness, and resist textile wrinkling. Lignin peroxidases, manganese peroxidases, laccases and xylanases are all used to enhance the quality of the pulp by removing lignin and hemicelluloses, which are typical impurities in the pulp and paper industries (Choi et al. 2015). Protease is used in leather processing for the dehairing and debating of skins and hides instead of

<figure><img src="image_72.png" /></figure>
<figure><img src="image_73.png" /><figcaption>**Fig. 51** Fungal enzymes and applications</figcaption></figure>

chemical processes that have negative implications for the environment. Tannases are used for high-grade leather tannin preparation in the leather industry (Dhiman et al. 2017). This enzymatic process is eco-friendly and helps to manage waste resulting from production processes.

Proteases, lipases and amylases are used for dishwashing detergents (Souza et al. 2015). Amylases are the second type of enzymes used in the formulation of enzymatic detergent, and 90% of all liquid detergents contain amylases (Souza and Magalhães 2010).

Cellulases, xylanases and β-glucanases have been used in cereal-based feed for monogastric animals such as pigs and poultry, which are unable to fully degrade and utilize plant-based feeds containing high amounts of cellulose and hemicellulose (Bhat 2000; Kirk et al. 2002). Adding phytases to the feed improves utilization of natural phosphorus bound in phytic acid in cereal-based feed for monogastrics. Normally, 85–90% of the total plant phosphorus is bound to phytic acid, which makes it difficult to monogastrics to utilize. Therefore, available inorganic phosphates have to be supplemented to the feed in order to reach the required concentration. Phytases can minimize the need for supplementation of inorganic phosphorus in the feed (Kirk et al. 2002).

Basidiomycetes can enzymatically attack the polymers in the complex-structured lignocellulose. The appearance of white rot fungi involves the nutrient recycling of wood required for new plant growth with evolutionary impact on plant diversification (Kües 2015). Dioxygenases, laccases,

peroxidases, and phenoloxidases are enzymes used in wastewater treatment (Durán and Esposito 2000). Fungi are recognized as one of the best lipase sources among microorganisms (Facchini et al. 2015). The most important step to improving the biological degradation of fatty wastewater is pretreatment procedures in order to hydrolyze and dissolve fats, which can accelerate the process by decreasing the fat adsorption to the surface of the anaerobic sludge while not limiting the transport of the soluble substrate to the biomass (Valladao et al. 2009; Facchini et al. 2015). Effluents from slaughterhouses contain high concentrations of biodegradable organic matter, most of which consists of lipids and proteins with low degradability. Consequently, before effluents are released into the environment, it is necessary to reduce fat oil and protein from these wastewaters. A potential application of enzyme treatments is a reduction of the organic matter, which contributes to a cleaner effluent (Valladao et al. 2011). Moreover, white rot fungi produce lignin degrading enzymes, such as manganese peroxidases, lignin peroxidases and laccases that have been used in biotechnology for degradation of broad-spectrum refractory organic pollutants and bioremediation of polycyclic aromatic hydrocarbons (PAHs) and chlorinated hydrocarbons in the environment (Gao et al. 2010).

Biotechnological applications for practical use have limiting factors, as they require large amounts of enzymes. Hence, the production of low-cost and readily available enzymes possessing satisfactory operating characteristics is
<table><caption>**Table 24** Examples of enzyme sources and applications of fungi</caption><thead><tr><th>Enzymes</th><th>Applications</th><th>Fungal sources</th><th>References</th></tr></thead><tbody><tr><td rowspan="5">Amylases</td><td rowspan="5">Hydrolysis of starch in starch processing industry; food and dairy industry; textile industry; pulp and paper industry; detergent industry; pharmaceutical industry; animal feed industry</td><td>Aspergillus fumigatus</td><td>Goto et al. (1998)</td></tr><tr><td>Aspergillus niger</td><td>Ko et al. (2005)</td></tr><tr><td>Cylindrocephalum sp.</td><td>Kathiresan and Manivannan (2006)</td></tr><tr><td>Lentinula edodes</td><td>Sunitha et al. (2012)</td></tr><tr><td>Penicillium citrinum</td><td>Sahoo et al. (2014)</td></tr><tr><td rowspan="4">Cellulases</td><td rowspan="4">Animal feed industry; pulp and paper industry; detergent industry; food processing; juices and wines clarification; textile industry; biomass conversion into biofuels</td><td>Penicillium fellutanum</td><td>Saleem and Ebrahim (2014)</td></tr><tr><td>Rhizopus stolonifer</td><td>Saleem and Ebrahim (2014)</td></tr><tr><td>Aspergillus niger</td><td>Ko et al. (2005)</td></tr><tr><td>Lentinula edodes</td><td>Pachauri et al. (2017)</td></tr><tr><td rowspan="7">Keratinases</td><td rowspan="7">Biomass conversion into biofuels; hydrolysis of keratinous wastes such as feather, hair, and horn; eliminating horny epithelial cells adhering to textile fibers; reducing the environmental pollution</td><td>Trichoderma longibrachiatum</td><td>Puntambekar (1995)</td></tr><tr><td>Volvariella diplasia</td><td>Wang and Hsu (2006)</td></tr><tr><td>Aspergillus oryzae</td><td>Anitha and Palanivelu (2013)</td></tr><tr><td>Aspergillus parasiticus</td><td>Farag and Hassan (2004)</td></tr><tr><td>Doratomyces microspores</td><td>Friedrich et al. (2005)</td></tr><tr><td>Paecilomyces marquandii</td><td>Gradisar et al. (2000)</td></tr><tr><td>Pleurotus ostreatus</td><td>Kim (2007)</td></tr><tr><td rowspan="5">Laccase</td><td rowspan="5">Biopulping biobleaching deinking in pulp and paper industry; lignin degradation; pharmaceutical industry; removal of phenolic substances and stabilize the beverage; biomass conversion into biofuels</td><td>Aspergillus sclerotiorum</td><td>Santos et al. (1996)</td></tr><tr><td>Cladosporium cladosporioides</td><td>Veselá and Friedrich (2009)</td></tr><tr><td>Mucor racemosus</td><td>Arora et al. (2002)</td></tr><tr><td>Phanerochaete chrysosporium</td><td>Ergun and Urek (2017)</td></tr><tr><td>Phanerochaete latifolia</td><td>Lee et al. (2004)</td></tr><tr><td rowspan="7">Lignin peroxidase</td><td rowspan="7">Lignin degradation; biomass conversion into biofuels</td><td>Aspergillus sp.</td><td>Songulashvili et al. (2007)</td></tr><tr><td>Coniophora puteana</td><td>Songulashvili et al. (2007)</td></tr><tr><td>Ganoderma sp.</td><td>Ullrich et al. (2005)</td></tr><tr><td>Omphalotus olearius</td><td>Ullrich et al. (2005)</td></tr><tr><td>Phanerochaete floridensis</td><td>Ullrich et al. (2005)</td></tr><tr><td>Pleurotus ostreatus</td><td>Ullrich et al. (2005)</td></tr><tr><td>Aspergillus sclerotiorum</td><td>Bonugli-Santos et al. (2010)</td></tr><tr><td rowspan="5">Lipases</td><td rowspan="5">Degradation of fat in wastewater treatment; animal feed industry; pulp and paper industry; detergent industry; food processing; leather processing; textile industry; pharmaceutical industry</td><td>Cladosporium cladosporioides</td><td>Chandrasekaran et al. (2014)</td></tr><tr><td>Mucor racemosus</td><td>Johjima et al. (1999)</td></tr><tr><td>Phanerochaete chrysosporium</td><td>Wen et al. (2009)</td></tr><tr><td>Phanerochaete latifolia</td><td>Wen et al. (2009)</td></tr><tr><td>Aspergillus sp.</td><td>Costa and Peralta (1999)</td></tr><tr><td>Curvularia sp.</td><td>El-Ghonemy et al. (2017)</td></tr><tr><td>Fusarium solani</td><td>Facchini et al. (2015)</td></tr><tr><td>Fusarium verticillioides</td><td>Maia et al. (2001)</td></tr><tr><td>Penicillium sp.</td><td>Marques et al. (2014)</td></tr><tr><td>Penicillium restrictum</td><td>Nwuche and Ogbonna (2011)</td></tr><tr><td>Penicillium wortmanii</td><td>Ul-Haq et al. (2002)</td></tr><tr><td>Rhizopus oligosporus</td><td>Ülker et al. (2011)</td></tr><tr><td>Trichoderma sp.</td><td>Valladao et al. (2011)</td></tr><tr><td>Trichoderma atroviride</td><td>Valladao et al. (2011)</td></tr><tr><td>Trichoderma harzianum</td><td>Valladao et al. (2011)</td></tr><tr><td>Mucor sp.</td><td>Valladao et al. (2011)</td></tr></tbody></table>

<figure><img src="image_74.png" /></figure>
<table><caption>**Table 24** continued</caption><thead><tr><th>Enzymes</th><th>Applications</th><th>Fungal sources</th><th>References</th></tr></thead><tbody><tr><td rowspan="4">Manganese peroxidase</td><td rowspan="4">Lignin degradation; biomass conversion into biofuels</td><td>Lentinula edodes</td><td>Arora et al. (2002)</td></tr><tr><td>Phlebia radiata</td><td>Grabski et al. (1998)</td></tr><tr><td>Omphalotus olearius</td><td>Songulashvili et al. (2007)</td></tr><tr><td>Phellinus robustus</td><td></td></tr><tr><td rowspan="5">Pectinases</td><td rowspan="5">Juices and wines clarification; textile industry</td><td>Aspergillus japonicus</td><td>Banu et al. (2010)</td></tr><tr><td>Aspergillus oryzae</td><td>Biz et al. (2016)</td></tr><tr><td>Penicillium viridicatum</td><td>Martins et al. (2002)</td></tr><tr><td>Penicillium chrysogenum</td><td>Semenova et al. (2003)</td></tr><tr><td>Thermoascus aurantiacus</td><td>Silva et al. (2002)</td></tr><tr><td rowspan="7">Proteases</td><td rowspan="7">Detergent industry; food processing; pharmaceutical industry; leather processing; textile industry</td><td>Aspergillus sp.</td><td>Cui et al. (2007)</td></tr><tr><td>Fomitella fraxinea</td><td>Lee et al. (2006)</td></tr><tr><td>Humicola sp.</td><td>Souza et al. (2015)</td></tr><tr><td>Mucor sp.</td><td></td></tr><tr><td>Penicillium sp.</td><td></td></tr><tr><td>Pleurotus citrinopileatus</td><td></td></tr><tr><td>Rhizopus sp.</td><td></td></tr><tr><td>Thermoascus sp.</td><td></td></tr><tr><td>Thermomyces sp.</td><td></td></tr><tr><td rowspan="5">Phytases</td><td rowspan="5">Feed supplement in diets in animal feed industry; improvement of soil fertilization and nutrient uptake by plants; reduction the excretion of phosphorus in manure; reducing phosphate pollution in soil and water</td><td>Aspergillus sp.</td><td>Bei et al. (2009)</td></tr><tr><td>Aspergillus niger</td><td>Brugger et al. (2004)</td></tr><tr><td>Mucor sp.</td><td>Casey and Walsh (2004)</td></tr><tr><td>Rhizopus oligosporus</td><td>Chadha et al. (2004)</td></tr><tr><td>Rhizomucor pusillus</td><td>Saxena et al. (2005)</td></tr><tr><td rowspan="10">Tannases</td><td rowspan="10">Food processing; juices and wines clarification and removal of phenolic substances and stabilize the beverage; reduction of hydrolysable tannin in poultry feeds; production of gallic acid from gallotannins</td><td>Aspergillus aculeatus</td><td>Ahmed and Rahman (2014)</td></tr><tr><td>A. awamori</td><td>Bagga et al. (2015)</td></tr><tr><td>A. caespitosus</td><td>Batra and Saxena (2005)</td></tr><tr><td>A. niger</td><td>Srivastava and Kar (2009)</td></tr><tr><td>A. versicolor</td><td>Mahapatra et al. (2005)</td></tr><tr><td>Penicillium charlesii</td><td></td></tr><tr><td>P. crustosum</td><td></td></tr><tr><td>P. variable</td><td></td></tr><tr><td>P. restrictum</td><td></td></tr><tr><td>Aspergillus foetidus</td><td>Chapla et al. (2012)</td></tr><tr><td rowspan="3">Xylanases</td><td rowspan="3">Pulp and paper industry; animal feed industry; bread-making; juice and wine industries; xylitol production; prebiotics production; food processing; textile industry; juices and wines clarification; biomass conversion into biofuels</td><td>A. niger</td><td>de Alencar Guimaraes et al. (2013)</td></tr><tr><td>Talaromyces amestolkiae</td><td>Nieto-Domínguez et al. (2017)</td></tr><tr><td></td><td></td></tr></tbody></table>

challenging. After all, each industrial application may require specific properties of the biocatalysts. Moreover, researchers still face challenges in finding new enzymes that could result in novel applications through better isolation, study of enzyme stability at extreme conditions and purification procedures. The selection of enzymes for thermostable and stringent conditions is important for

industrial processes and applications. Therefore, the search for new microorganisms that contain the desired properties is a continuous process. Furthermore, the production and expression of recombinant enzymes through protein engineering technology should also be investigated in order to obtain enzymes of desired characteristics in a specific host cell.