21st Century Guidebook to Fungi, SECOND EDITION, by David Moore, Geoffrey D. Robson and Anthony P. J. Trinci

Table of Contents

1. Chapter 16: Fungi as pathogens of animals, including man
2. Chapter 17: Whole organism biotechnology
   1. Fungal fermentations in submerged liquid cultures
   2. Culturing fungi
   3. Oxygen demand and supply
   4. Fermenter engineering
   5. Fungal growth in liquid cultures
   6. Fermenter growth kinetics
   7. Growth yield
   8. Stationary phase
   9. Growth as pellets
   10. Beyond the batch culture
   11. Chemostats and turbidostats
   12. Uses of submerged fermentations
   13. Alcoholic fermentations
   14. Citric acid biotechnology
   15. Penicillin and other pharmaceuticals
   16. Enzymes for fabric conditioning and processing, amd food processing
   17. Steroids and use of fungi to make chemical transformations
   18. The Quorn fermentation and evolution in fermenters
   19. Production of spores and other inocula
   20. Natural digestive fermentations in herbivores
   21. Solid state fermentations
   22. Digestion of lignocellulosic residues
   23. Bread; the other side of the alcoholic fermentation equation
   24. Cheese and salami manufacture
   25. Soy sauce, tempeh and other food products
   26. Chapter 17 References and further reading
   27. Buy a PDF of Chapter 17
3. Chapter 18: Molecular biotechnology

17.22 Digestion of lignocellulosic residues

Lignocelluloses as agricultural, industrial and forest residues account for the majority of the total biomass present in the world. This is a renewable resource with a potential for bioconversion into many useful biological and chemical products. Accumulation of large quantities of this biomass every year results in both environmental deterioration and loss of potentially valuable materials that might otherwise be processed to yield energy, food, animal feed or fine chemicals.

Agricultural residues and forest materials containing high levels of lignocellulose are particularly abundant because the crop represents such a small proportion of what is grown (see our brief discussion of this in the section Contributions of fungi to ecosystems in Chapter 10; CLICK HERE to view the page). The major component of lignocellulose materials is cellulose, followed by hemicellulose and lignin, intermeshed and chemically bonded by non-covalent forces and by covalent cross-linkages. As fungi, particularly white-rot fungi (Basidiomycota), have particularly efficient lignocellulose degradation enzyme machinery they might be attractive components of low cost bioremediation projects (Kumar et al., 2008; Sánchez, 2009; Huberman et al., 2016; Rouches et al., 2016; Grace et al., 2020); and compare the use of laccase for remediation of petroleum-contaminated soil (Section 13.6) (Liu et al., 2017) .

White-rot basidiomycete fungi, such as Phanerochaete chrysosporium (= Sporotrichum pulverulentum) are able to mineralise lignin completely to carbon dioxide and water. Many microorganisms are capable of degrading and utilising cellulose and hemicellulose as carbon and energy sources, but only the white-rot fungi have the ability to breakdown lignin, the most recalcitrant component of plant cell walls. In nature these wood-and litter-degrading fungi play an important role in the carbon cycle and in addition to lignin, white-rot fungi are able to degrade a variety of persistent environmental pollutants, such as chlorinated aromatic compounds, heterocyclic aromatic and synthetic high polymers (See Using fungi to remediate toxic and recalcitrant wastes in Chapter 13; CLICK HERE to view the page). These additional capabilities of white-rot fungi are probably due to the strong oxidative activity and the low substrate specificity of their ligninolytic enzymes.

Fungi have two types of extracellular enzymatic systems: the hydrolytic system, which consists of hydrolases responsible for polysaccharide degradation; and a unique oxidative and extracellular ligninolytic system, which degrades lignin and opens phenyl rings. An important thought in understanding the mechanism(s) of degradation is that the enzymes concerned are too large to penetrate sound, intact wood; this task is performed by hydrogen peroxide and the active oxygen radicals derived from it (CLICK HERE to view the page). The most widely studied white-rot organism is the basidiomycete Phanerochaete chrysosporium. Trichoderma reesei and its mutants are the most studied ascomycete species, and the species is used for the commercial production of hemicellulases and cellulases. We have already dealt with the enzymes involved in biodegradation of the components of lignocellulose:

• cellulose (CLICK HERE to view the page)
• hemicellulose (CLICK HERE to view the page)
• lignin (CLICK HERE to view the page).

Here we wish to emphasise a few important points relating to the exploitation of these natural enzyme systems and illustrate a few of the biotechnologies to which they make a contribution.

In nature the various enzymes act synergistically to catalyse the release and hydrolysis of cellulose. Several physical factors, like pH, temperature, adsorption onto minerals, and chemical factors such as availability of oxygen, nitrogen and phosphorus, and presence of phenolic compounds and other potential inhibitors can influence the bioconversion of lignocellulose in both natural and artificial systems.

P. chrysosporium simultaneously degrades cellulose, hemicellulose and lignin, whereas other species, such as Ceriporiopsis subvermispora, tend to remove lignin in advance of cellulose and hemicellulose (and, of course, brown rot fungi rapidly digest cellulose, but only slowly modify lignin; this particularly affects softwoods and Piptoporus betulinus, Serpula lacrymans, and Coniophora puteana are examples). Consult Ecosystem mycology in Chapter 3; CLICK HERE to view the page, and Decay of structural timber in dwellings in Chapter 13; CLICK HERE to view the page (Peralta et al., 2017).

The steady growth of agro-industrial activity has led to the accumulation of a large quantity of lignocellulosic residues from agriculture, forestry, municipal solid wastes and various industrial wastes around the world (Table 14).

Bioconversion of lignocellulose into useful, higher value, products normally requires multi-step processes, the steps including:

• collection and mechanical, chemical or biological pretreatment;
• hydrolysis of polymers to produce readily metabolised (usually sugar) molecules (Bhattacharya et al., 2015);
• fermentation of the sugars to produce a microbial or chemical end-product;
• separation, purification, packaging and marketing.

Note that Table 14 shows official estimates of the annual production of these agricultural wastes. The amounts are staggering; the total of the entries shown in Table 14 is 1.2 billion metric tons, and does not include municipal solid wastes like waste paper or garden refuse collected for recycling. Several uses have been suggested for bioprocessed lignocellulosic wastes (Fig. 39), for example:

• use as raw material for the production of ethanol, in the hope that an alternative fuel manufactured using biological methods will have environmental benefits. Ethanol is either used as a chemical feedstock or as an additive enhancer for petrol. Softwood, the dominant source of lignocellulose in the Northern hemisphere, has been the subject of interest as a raw material for fuel ethanol production in Sweden, Canada and Western USA (Sanchez, 2009). Brazil and the USA produce ethanol from the fermentation of cane juice and maize, respectively. It is estimated that 25% of the maize and other cereals grown in America (107 million tonnes in 2009) is used to produce fuel ethanol). In the US, fuel ethanol has been used in gasohol or oxygenated fuels, containing up to 10% ethanol by volume, since the 1980s. It’s a very big business; 103 billion litres of fuel ethanol worldwide in 2017, of which 60 billion litres was produced in the USA, 27 billion in Brazil and 5.3 billion in the EU (source: www.statista.com/). Use of ethanol produced from cereals as an oxygenating fuel additive has been criticised on the grounds that using maize for this purpose has pushed the prices of the crop up to record levels in the commodities markets. This has led to an increase in some food prices because livestock feed produced from maize has increased in price correspondingly. Of course this criticism would not apply to processes that used crop wastes for the fermentation.

Fig. 39. Generalised process stages in lignocellulose waste bioconversion and the range of potential products. Adapted from Sanchez, 2009.

• High-value bioproducts such as organic acids, amino acids, vitamins and a number of bacterial and fungal polysaccharides, for example xanthans, are produced by fermentation using glucose as the base substrate but theoretically these same products could be manufactured from sugars derived from lignocellulose residues. Based on the known metabolism of Phanerochaete  chrysosporium, several potential high value products could be derived from lignin. Rumen microorganisms convert cellulose and other plant carbohydrates in large amounts to acetic, propionic and butyric acids, which ruminant animals then use as energy and carbon sources; these fungi also have promise for commercial bioprocessing of lignocellulose wastes anaerobically in liquid digesters.
• Compost making for cultivation of edible mushrooms. Good compost is the essential prerequisite for successful mushroom farming (see Industrial cultivation methods in Chapter 11; CLICK HERE to view the page). The basic raw material for mushroom compost in Europe is wheat straw, although straws of other cereals are sometimes used. Ideally, the straw is obtained already mixed with horse manure after being used as stable bedding. On commercial scale this is not possible and other animal wastes, like chicken manure, are mixed with the straw, together with gypsum (calcium sulfate) and large quantities of water. The excess calcium of gypsum precipitates the mucous and slimy components of manure and so prevents water logging of the compost and generally improves aeration and its mechanical properties that aid thorough mixing. All of this enables the compost to ferment uniformly, which itself results in large crops being grown reliably. Cultivation of edible mushrooms using lignocellulosic residues is a value addition process to convert these materials into human food. It is one of the most efficient biological ways by which these residues can be recycled. Mushrooms can be grown successfully on a wide variety of lignocellulose residues such as cereal straws, banana leaves, sawdust, peanuts hulls, coffee pulp, soybean and cotton stalk, indeed any lignocellulosic substrate that has a substantial cellulose component. Another advantage of this strategy is that the production unit can vary from the small-holder local farmer through to a multi-million pound mushroom farm.
• Lignocellulose bioconversion can be used to produce animal feeds. For example, fungi can be used to improve the nutritional quality of cereals like barley to compensate for the latter’s deficiency in the amino acid lysine. Inoculating soaked barley with Aspergillus oryzae or Rhizopus arrhizus increases the protein content as the fungus grows. The product is used as pig food. With a view to future product developments there is a good deal of research under way on growing fungi such as Trichoderma sp. on cheap lignocellulose residues. Growth of the mycelium of this fungus on, say agricultural wastes, releases components of lignocellulose residues and converts cellulosic and otherwise non-digestible materials into sugars and glycogen that are readily available to animals, and increases the protein content to the point where the product becomes a nutritious animal feed.
• Although not a ‘waste bioconversion’ process we finally want to mention the use of fungi to remodel timber to increase penetration by wood preservatives, providing environmentally friendly methods for wood protection. Construction timber tends to have high structural strength but low natural durability. Increasing durability by treatment with preservatives is lessened in efficiency by the timber’s low permeability because of closure of the pits between cells in the wood. These pits enable lateral permeability when the tree is alive, but they close when the timber is harvested and seasoned. Permeability is improved conventionally by mechanically incising the wood, but selective degradation of pits by white-rot fungus (Physisporinus vitreus) is a biotechnological alternative ‘bioincising’ process (Schwarze, 2007).

Further development of the bioprocessing potential of lignocellulose biodegradation is likely to depend on understanding the molecular mechanisms the organisms use. For instance, cloning and sequencing of the various cellulolytic genes could make cellulase production more economical. For instance, cloning and sequencing of the various cellulolytic genes could make cellulase production more economical (Chapter 18), and heterologous enzyme production of the enzyme cocktails required for efficient in vitro biomass digestion could promote adoption of lignocellulosic biomass as a feedstock to produce biofuels and other value-added products (Comyn & Magnuson, 2020).

Updated October, 2020