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Mushroom cultivation as a prominent biotechnological process for the valorization of agro-industrial residues generated as a result of agro-forestry and agro-industrial production. A huge amount of lignocellulosic agricultural crop residues and agro-industrial by-products are annually generated, rich in organiccompounds that are worthy of being recovered and transformed. A number of these residues have been employed as feedstocks in solid state fermentation (SSF) processes using higher basidiomycetus fungi for the production of mushroom food, animal feed, enzymes and medicinal compounds. Likewise, the above-mentioned microorganisms have been successfully employed in processes related with the bioremediation of hazardous compounds and waste detoxification. Mushroom cultivation presents a worldwide expanded and economically important biotechnological industry that uses efficient solid-state-fermentation process of food protein recovery from lignocellulosic materials. Several aspects of mushroom physiology along with impacts of different environmental and nutritional conditions on mycelium growth and fruiting bodies production are highlined. Moreover, cultivation technologies of Agaricus bisporus, Pleurotus spp and Lentinula edodes, comprising spawn (inoculum) production, substrate preparation and mushroom growing process i.e. inoculation, substrate colonization by the cultivated fungus, fruiting, harvesting and processing of the fruiting bodies, are outlined. Finally, the efficiency of residues conversion into fruiting bodies are outlined in two medicinal mushroom genera, Pleurotus and Lentinula, widely cultivated for their nutritional value and extensively researched for their biodegradation capabilities. Experimental data concerning residue-substrates used, as well as biological efficiencies obtained during their cultivation were considered and discussed.
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Production of Mushrooms
Using Agro-Industrial Residues as Substrates
Antonios N. Philippoussis
9.1 Introduction . . . . . . . . . . . . .................................................... 164
9.2 Residue-Based Substrates and their Solid-State Fermentation by Mushroom Fungi. . . . . 166
9.2.1 Types, Availability and Chemical Composition of Raw Materials. . . ........... 166
9.2.2 NutritionalandEnvironmentalAspectsofMushroomGrowing ............... 169
9.2.3 OutputandStagesofMushroomCultivation............................... 174
9.3 Bioconversion of Solid Residue-Substrates Through Mushroom Cultivation . . . . . . . . . . 177
9.3.1 Commercial Mushroom Production Processes.............................. 177
9.3.2 Efficiency of Residue Conversion to Pleurotus sp. and L. edodes
FruitingBodies....................................................... 182
9.4 ClosingRemarks............................................................ 185
References................................................................. 187
Abstract Mushroom cultivation as a prominent biotechnological process for the
valorization of agro-industrial residues generated as a result of agro-forestry and
agro-industrial production. A huge amount of lignocellulosic agricultural crop
residues and agro-industrial by-products are annually generated, rich in organic
compounds that are worthy of being recovered and transformed. A number of these
residues have been employed as feedstocks in solid state fermentation (SSF) pro-
cesses using higher basidiomycetus fungi for the production of mushroom food,
animal feed, enzymes and medicinal compounds. Likewise, the above-mentioned
microorganisms have been successfully employed in processes related with the
bioremediation of hazardous compounds and waste detoxification. Mushroom culti-
vation presents a worldwide expanded and economically important biotechnological
industry that uses efficient solid-state-fermentation process of food protein recovery
from lignocellulosic materials. Several aspects of mushroom physiology along with
A.N. Philippoussis (B)
National Agricultural Research Foundation, I.A.A.C., Laboratory of Edible and Medicinal Fungi,
13561 Ag, Anargyri, Athens, Greece
P. Singh nee’ Nigam, A. Pandey (eds.), Biotechnology for Agro-Industrial Residues
Utilisation, DOI 10.1007/978-1-4020-9942-7
Springer Science+Business Media B.V. 2009
164 A.N. Philippoussis
impacts of different environmental and nutritional conditions on mycelium growth
and fruiting bodies production are highlined. Moreover, cultivation technologies of
Agaricus bisporus, Pleurotus spp and Lentinula edodes, comprising spawn (inocu-
lum) production, substrate preparation and mushroom growing process i.e. inoc-
ulation, substrate colonization by the cultivated fungus, fruiting, harvesting and
processing of the fruiting bodies, are outlined. Finally, the efficiency of residues
conversion into fruiting bodies are outlined in two medicinal mushroom genera,
Pleurotus and Lentinula, widely cultivated for their nutritional value and exten-
sively researched for their biodegradation capabilities. Experimental data concern-
ing residue-substrates used, as well as biological efficiencies obtained during their
cultivation were considered and discussed.
Keywords Fungi · Mushroom cultivation · Biotechnology · Agricultural residues ·
By-products · Solid state fermentation · Fruiting bodies · Yield · Biological
efficiency · Agaricus spp. · Pleurotus spp. · Lentinula edodes
9.1 Introduction
On the surface of our planet, around 200 billion tons per year of organic matter are
produced through the photosynthetic process (Zhang 2008). However, the majority
of this organic matter is not directly edible by humans and animals and, in many
cases, becomes a source of environmental problem. Moreover, today’s society, in
which there is a great demand for appropriate nutritional standards, is character-
ized by rising costs and often decreasing availability of raw materials together
with much concern about environmental pollution (Laufenberg et al. 2003). Con-
sequently, there is a considerable emphasis on recovery, recycling and upgrading of
wastes. This is particularly valid for the agro-food industry, which furnishes large
volumes of solid wastes, residues and by-products, produced either in the primary
agro-forestry sector or by secondary processing industries, posing serious and con-
tinuously increasing environmental pollution problems (Boucqu
e and Fiems 1988,
Koopmans and Koppejan 1997, Lal 2005). It is worth mentioning that only crop
residues production is estimated to be about 4 billion tons per year, 75% originating
from cereals (Lal 2008).
Nevertheless, residues such us cereals straw, corn cobs, cotton stalks, various
grasses and reed stems, maize and sorghum stover, vine prunings, sugarcane and
tequila bagasse, coconut and banana residues, corn husks, coffee pulp and coffee
husk, cottonseed and sunflower seed hulls, peanut shells, rice husks, sunflower
seed hulls, waste paper, wood sawdust and chips, are some examples of residues
and by-products that can be recovered and upgraded to higher value and use-
ful products by chemical or biological processes (Wang 1999, Fan et al. 2000a,
Pandey et al. 2000b, c, Webb et al. 2004). In fact, the chemical properties of such
lignocellulosic agricultural residues make them a substrate of enormous biotech-
nological value. They can be converted by solid state fermentation (SSF) into
various different value-added products including mushrooms, animal feed enriched
with microbial biomass, compost to be used as biofertilizer or biopesticide, en-
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates 165
zymes, organic acids, ethanol, flavours, biologically active secondary metabolites
and also for bioremediation of hazardous compounds, biological detoxification of
agro-industrial residues, biopulping etc. (Pandey et al. 2000a, Bennet et al. 2002,
anchez et al. 2002, Tengerdy and Szakacs 2003, Howard et al. 2003, Kim and
Dale 2004, Nigam et al. 2004, Zervakis et al. 2005, Manpreet et al. 2005,
Krishna 2005).
Among applications of SSF, mushroom cultivation has proved its economic
strength and ecological importance for efficient utilization, value-addition and bio-
transformation of agro-industrial residues (Chang 1999, 2001, 2006, Chiu et al. 2000,
Zervakis and Philippoussis 2000). Current literature shows that lignocellulose de-
grading mushroom species are used in various SSF applications such as biore-
mediation and biodegradation of hazardous compounds (P
erez et al. 2007), bio-
logical detoxification of toxic agro-industrial residues (Pandey et al. 2000d, Fan
et al. 2000b, Soccol and Vandenberghe 2003), biotransformation of agro-industrial
residues to mushroom food and animal feed (Moore and Chiu 2001, Albor
et al. 2006, Okano et al. 2006), compost and product developments such as biologi-
cally active metabolites, enzymes, and food flavour compounds (Ooi and Liu 2000,
Cohen et al. 2002, Silva et al. 2007, Nikitina et al. 2007). Moreover, recent research
work indicates medicinal attributes in several species, such as antiviral, antibac-
terial, antiparasitic, antitumor, antihypertension, antiatherosclerosis, hepatoprotec-
tive, antidiabetic, anti-inflammatory, and immune modulating effects (Wasser and
Weis 1999, Wasser 2002, Daba and Ezeronye 2003, Paterson 2006).
Commercial mushroom production, carried out in a large or small scale, is an effi-
cient and relatively short biological process of food protein recovery from negative-
value lignocellulosic materials, utilizing the degrading capabilities of mushroom
fungi (Mart
ınez-Carrera et al. 2000, Chiu and Moore 2001). Among mushroom
fungi, L. edodes and Pleurotus species reveal high efficiency in degradation of a
wide range of lignocellulosic residues, such as wheat straw, cotton wastes, coffee
pulp, corn cobs, sunflower seed hulls wood chips and sawdust, peanut shells, vine
prunings and others into mushroom protein, (Ragunathan et al. 1996, Campbell
and Racjan 1999, Stamets 2000, Poppe, 2000, Philippoussis et al., 2000, 2001a, b),
the productivity of the conversion being expressed by biological efficiency (Chang
et al. 1981). Their mycelium can produce significant quantities of a plethora of
enzymes, which can degrade lignocellulosic residues and use them as nutrients
for their growth and fructification (Bushwell et al. 1996, Elisashvili et al. 2008).
However, the nature and the nutrient composition of the substrate affect mycelium
growth, mushroom quality and crop yield of this value-added biotransformation pro-
cess (K
ues and Liu 2000, Philippoussis et al. 2001c, 2003, Baldrian and Val
The focus of this work is to highlight significant aspects of utilization of low- or
negative-value agro-industrial residues in mushroom biotechnology, emphasizing
on their biotransformation to fruiting bodies that are nutritious human foodstuff
regarded also as functional food. Aspects to be reviewed in this article include:
an overview of availability, sources and types as well as chemical composition of
solid lignocellulosic agro-residues suitable for mushroom cultivation, some back-
166 A.N. Philippoussis
ground on mushroom degrading abilities and of their nutritional and environmental
demands, an outline of commercial production technologies of A. bisporus, Pleurotus
spp. and L. edodes mushrooms, and finally a consideration and discussion of experi-
mental data regarding productivity (biological efficiency) on various agro-industrial
residues during cultivation ofPleurotus spp. and L. edodes.
9.2 Residue-Based Substrates and their Solid-State
Fermentation by Mushroom Fungi
As a result of agro-forestry and agro-industrial production, a huge amount of live-
stock waste, agricultural crop residues and agro-industrial by-products are annually
generated, the major part being lignocellulosic biomass (Kuhad et al. 1997). Al-
though agro-industrial residues contain beneficial materials, their apparent value is
smaller than the cost of collection, transportation and processing for beneficial use.
However, if residues are utilized, such as to enhance food production, they are not
considered as wastes but new resources. A number of agro-industrial residues have
been employed as feedstocks in SSF processes, using high basidiomycetus fungi for
the production of valuable metabolites (Rajarathnam et al. 1998, Howard et al. 2003,
olker et al. 2004). However, mushroom production is one of the areas with great
potential for exploitation of forest and agricultural residues (Moore and Chiu 2001,
Chang 2006, Gregori et al. 2007, Silva et al. 2007).
9.2.1 Types, Availability and Chemical Composition
of Raw Materials
Reddy and Yang (2005) and very recently, Zhang (2008), reviewing the global world
information about lignocellulose availability, estimated the production of lignocel-
lulosic biomass to be more than 200 × 10
tons per year. Especially, the amount
of crop residues produced annually in the world from 27 food crops is estimated at
about 4 × 10
tons, from which 3 billion tons account per annum for lignocellu-
losic residues of cereals (Lal 2005, 2008). Cereals, accounting 75% of global world
production (FAO 2004), furnish these outstanding amounts of waste products as
wheat residues, rice straw and hulls, barley residue, maize stalks and leaves, millet
and sorghum stalks. Sugar cane provides the next sizeable residue with two major
crop wastes, leaves and stalk, and bagasse, which is the crop processing residue.
The cotton crop also provides significant residue in the form of stalks and husks,
while no negligible are the residues furnished by minor crops as sunflower, oil palm,
coconut, banana, vines, groundnut and coffee. In fact, this generation of residues is a
result of the limited portions of the crops that are actually used. To give the order of
magnitude, 95% of the total biomass produced in palm and coconut oil plantations
is discarded as a waste material; the respective values for sisal plant and sugar cane
biomass are 98% and 83% (Chang 1998). Moreover, in the flax industry only 2% of
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates 167
the produced biomass is effectively used, less than 9% in the palm oil industry and
only 8% in the brewing industry (L
opez et al. 2004).
Biomass availability is a primary factor for bio-based industrial production. In-
deed, the available resource potential (the amount of residues used for various pur-
poses) is smaller than the one generated. The quantities of crop residues that can
be available for bioprocesses are estimated using total grain production, residue to
product ratio (RPR), moisture content, and taking into consideration the amount of
residue left on the field to maintain soil quality (i.e. maintain organic matter and
prevent erosion), grazing and other agricultural activities (Koopmans and Koppe-
jan 1997, Giljum et al. 2005). Concerning cereal straw, the RPRs for rice, barley,
wheat and corn are 1.4, 1.2, 1.3 and 1 respectively (Mulkey et al. 2008). Assuming
that one quarter of the residues can be harvested and that roughly one third of the
harvested straw is used in animal husbandry, 0.22 tons straw per ton cereal grain
and 0.25 ton residues per ton maize are available biomass for other uses, as energy,
enzyme production or mushroom growing.
As agro-industrial residues accumulate in fields and factories, availability is-
sue tends to become a regional and local matter. Geographical distribution of crop
residues is skewed by large crop productions in India and China, where increased
quantities of crop residues and agro-industrial by-products are generated because
of expanding agricultural production. Furthermore, Asia along with Europe, North
America and Australia are world leader mushroom producing regions (Chang 2006)
and consequently the major residue demanding for this bio-based industrial activ-
ity. Among countries in the Asian and Pacific Region, China produces the largest
quantities of agricultural and forest residues, mainly by-products of rice, corn and
wheat (Zhang 2008). China’s quantities, estimated to reach about 1 billion tons/year
(Qu et al. 2006), are followed by India’s yielding at least 200 million tons/year
of agricultural residues according to Das and Singh (2004), while according to
Mande (2005) India’s total amount of agro-industrial residues reaches 600 mil-
lion tons. This quantity comprises 480 tons of crop residues (rice, wheat, millet,
sorghum, pulses, oilseed crops, maize stalks and cobs, cotton stalks, sugarcane trash
etc.) and 120 tons of processing–based residues (mainly groundnut shells, rice husk,
sugarcane bagasse, cotton waste, coconut shell and coir pith). Rice and sugar are
Asia’s rest southeast countries dominant crops.
Moghtaderi et al. (2006) report that Australian agro-industrial biomass reaches
100 million tons/year, including bagasse, cane trash, wood residues, energy crops
etc. As far as Africa is concerned, wheat and barley predominate in the north, millet
and sorghum are the main crops in sub-Saharan Africa, while farther south maize is
the dominant crop. Kim and Dale (2004), estimated Africa’s annual lignocellulosic
biomass from rice straw, wheat straw and sugar cane bagasse to be about 40 million
tons, indicating that the fraction of most crop residues collectable is less than 30%
because of low yields. In the same work, Central and South America’s lignocel-
lulosic residues were estimated to be about 140 million tons from rice and wheat
straw, corn stover and sugar cane bagasse, not taking into account coffee, banana
and other agricultural residues. Concerning North America, according to USDA-US
DOE report (2005), USA is able to produce 1.3 billion tons of dry residues per year,
168 A.N. Philippoussis
including agricultural (933 million tons) and forest resources (368 million tons).
Main lignocellulosic by-products in considerable quantity are corn stover, the most
abundant agricultural residue in USA, wheat, rice, barley straw, sorghum stalks,
coconut husks, sugarcane bagasse, pineapple and banana leaves. Canada, the second
largest supplier of wood lignocellulosic biomass, supplies more than 200 million m
of lignocellulose annually through commercial operations (Mabee et al. 2005). Fi-
nally, Europe is not only a great wheat straw producer, but also outstanding quanti-
ties of lignocellulosic residues from barley, maize, sunflower, rapeseed, cotton, olive
trees and vines, summarized as 120 million tons/ year (Nikolaou et al. 2003).
Regarding the types of wastes, according to Mande (2005), agricultural residues
can be divided into two groups: crop-based residues (generated in the field) and
processing-based residues (generated during wood and industrial processing). Crop-
based residues, which are plant materials left behind in the field or farm after re-
moval of the main crop produce, are consisted of different sizes, shapes, forms, and
densities like straw, stalks sticks, leaves, haulms, fibrous materials, roots, branches,
and twigs. Crop-based residues are produced from various sources such as field
and seed crops (including straw or stubble from barley, beans, oats, rice, rye, and
wheat, stalks or stovers from corn, cotton, sorghum, grasses and reeds, soybeans
and alfalfa), fruit, nut, vegetable or energy crops (brushes and orchard prunings, e.g.
vine shoots or leaves that remain on the ground after harvesting), and livestock ma-
nure. Processing-based agro-industrial residues are by-products of the post-harvest
processes of crops such as cleaning, threshing, linting, sieving, and crushing. They
are in the form of husk, dust, stalks etc. Food processing wastes that come from
plant materials are culls, rinds, seeds, pits, pulp, press cakes, marc, malts, hops
and a variety of other by-products from mass food production processes. Some
examples of these materials are coffee processing by-products, sugarcane bagasse,
hulls and husks, wheat middlings, corncobs, seed meals etc. Moreover, this category
comprises wood residues produced either from the primary processing or from sec-
ondary manufacturers (producing bark, chips, sawdust, coarse residues, and planer
shavings). During the sawing of a log at a typical sawmill, approximately 50% of
the initial log volume is converted into wood products and 50% is converted into
wood residues (Alderman 1998).
In general, solid agro-industrial residues are heterogeneous water insoluble ma-
terials having a common feature, their basic macromolecular structure being cel-
lulose, hemicellulose and lignin and to a lesser extend pectin, starch and other
polysaccharides (Thomsen 2005). Cellulose, the most abundant renewable organic
resource comprising about 45% of dry wood weight, is a linear homopolymer of
glucose units linked with 1,4-glucosidic bonds (Baldrian and Val
a 2008).
Hemicelluloses, heteropolysaccharides containing two to four different types of
sugars, are divided in three major groups: xylans, mannans and galactans. They
consist of short-branched chains of hexoses, e.g. mannose units in mannans and
pentoses such as xylose units in xylans (Kuhad et al. 1997). After cellulose, lignin
is the second most abundant renewable biopolymer in nature. Lignin, representing
between 26 to 29% of lignocellulose, is strongly bounded to cellulose and hemi-
cellulose, imparting rigidity and protecting the easily degradable cellulose from
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates 169
the hydrolase attack (Raimbault 1998). Lignin is an aromatic polyphenol macro-
molecule, 3-dimensional and amorphous (P
erez et al. 2002). As the proportions of
these three structural components characterize residue biomass, their percentages in
mushroom substrate ingredients, along with nitrogen content and carbon to nitrogen
ratios, are shown in Table 9.1. Additionally, crop residues contain, on a dry weight
basis, approximately 0.5–1.5%N, 0.15–0.2%P, 1%K, 1%Ca, 0.5%Mg, 0.2% S,
Mn, 100mgKg
Fe, 30mgKg
Zn, 5mgKg
Cu, 20mgKg
B and
about 1mgKg
Mo (Mills and Jones 1996). However, these values differ with crop,
plant part, season, soil moisture as well as other factors that affect plant growth.
Substrates used in mushroom cultivation include both field-based residues and
processing based-residues (Table 9.1). However, as the nutrient composition of the
substrate is one of the factors limiting colonization as well as quantitative and
qualitative yield of cultivated mushrooms (Philippoussis et al. 2000, 2002), sup-
plements containing sugars and starch (easily available carbohydrates) and fats
(slower degraded and time-lasting nutrient sources) are added to the basal ingre-
dient. Supplements are used to increase nutritional content, speed-up growth and
increase mushroom yield, especially in the cultivation of the white-rot mushroom
fungi L. edodes (Royse et al. 1990, Royse 1996) and Pleurotus spp. (Naraian
et al. 2008). The various organic supplements used in mushroom cultivation com-
prise molasses, brewer’s grain, grasses and waste paper, cotton and coffee wastes
etc. However, soybeans and cereal grains or their milling by-products are the most
commonly used supplements, as they are generated in considerable amounts and
contain increased levels of protein, fats and easily metabolized carbohydrates: soy-
beans (carbohydrates 21.5%, N 6.3%), wheat bran (carbohydrates 49.8%, N 2.4%),
rice bran (carbohydrates 37.0%, N 2.0%) and millet (carbohydrates 57.3%, N 1.9%),
(Przybylowicz and Donoghue 1990).
9.2.2 Nutritional and Environmental Aspects
of Mushroom Growing
From about 14000 mushroom-forming fungal species, at least 2000 are edible, of
which 80 species are grown experimentally and around 20 are cultivated commer-
cially (Chang 1999, Silva et al. 2007). The most cultivated worldwide species are
A. bisporus, P. ostreatus and L. edodes, followed by Auricularia auricula, Flam-
mulina velutipes and Volvariella volvacea. Other mushroom species produced suc-
cessfully on various substrates include Agrocybe aegerita, Ganoderma spp., Grifola
frondosa, Hericium erinaceus, Hypsizygus marmoreus, Lepista nuda, Coprinus co-
matus, Pholiota nameko and Stropharia spp. (Stamets 2000, Royse 2004). Although
the mentioned mushroom species have the ability to degrade lignocellulosic residues
in their original or composted form (Rajarathnam et al. 1998), they exhibit differ-
ences regarding production of enzymes necessary to degrade lignocellulosic sub-
strates and thus different abilities to grow and fruit on residue-substrates (Bushwell
et al. 1996, Chen et al. 2003, Baldrian and Val
a 2008).
170 A.N. Philippoussis
Table 9.1 Chemical properties (based on dry matter) of agro-industrial residues used as substrate ingredients in mushroom cultivation
Residue-substrates Cellulose Hemicellulose Lignin (%)
lignin Ash (%) N (%) C/N References
Field-based residues
Corn (maize)
36.4–40.0 25.0–29.0 13.0–21.0 2.1–2.3 3.6–7.0 0.6–0.9 55.8–77.3 1, 15, 17, 20
Grass residues 25.0–40.0 13.0–38.0 6.4–17.6 2.4–3.9 4.2–6.2 1.3–2.5 28.0–42.0 1, 8, 20, 22
Reed stems/ residues 34.4–42.6 28.4–30.6 17.1–19.7 1.7–2.0 4.3–4.9 0.3–0.5 150.0–170.0 12, 33
Rice straw 22.8–38.4 17.7–28.5 6.4–18.0 3.6–5.9 8.3–17.8 0.5–1.1 51.4–57.8 8, 14, 17, 29
Vine shoots 34.0–60.8 17.0–21.0 20–22.9 2.0–2.8 NA NA NA 7, 10, 31
Wheat straw 31.5–39.5 21.2–29.0 5.6–15.0 2.2–5.3 5.6–8.0 0.4–0.8 48.8–59.6 1, 20, 21, 22, 30, 31
Brewers grains 16.0–18.0 26.4–30.4 27.5–28.1 0.6–0.8 4.6–5.0 4.1–4.5 11.6–12.2 5, 9, 16
Coconut husk/coir 21.0–36.0 12.0–22.7 41.0–48.0 0.6–1.3 2.7–10.2 0.4–1.1 77.6–124.2 1, 24, 25, 29
Coffee pulp/husk 23.0–29.1 15.1–17.1 13.0–26.0 0.89–0.94 4.5–6.3/1.0–6.0 1.4–1.9/0.9–1.0 53.5–59.4 2, 26, 27
Corncob 28.0–45.0 35.0–43.0 11.0–17.0 2.5–2.7 4.4–4.8 0.4–1.1 64.2–71.6 6, 8, 11, 21, 22, 31
Cotton wastes (gin trash
52.0–90.0 5.0–20.0 4.0–12.0 5.0–11.2 2.6–8.4 0.3–1.4 40.0–59.0 8, 21, 22, 23
Hazelnut husk 24.5–37.5 20.6–24.9 29.6–35.1 0.7–1.2 8.2–8.7 0.8–0.9 50.6–58.6 3, 19, 28
Paper (waste) 54.3–70.0 12.4–25.0 11.3–29.7 3.0–6.0 NA
NA NA 8, 15, 34
Rice husk 28.0–43.0 17.5–20.6 21.5–22.5 1.3–1.9 16.7–21.4 0.3–0.4 100.0–136.0 9, 13, 31
Sugarcane bagasse 26.6–40.0 19.0–30.0 19.0–23.3 1.4–2.2 1.5–5.0 0.2–0.8 120.0–190.0 8, 18, 24, 26, 31
Sunflower seed hull 31.3–42.7 24.0–25.2 23.2–28.7 1.1–1.8 3.0–3.3 0.6–0.9 60.0–72.4 4, 28
Wood chips/ sawdust
37.7–49.5 10.7–25.0 26.1–29.5 1.4–1.7 0.4–0.5 0.1–0.1 310.0–520.0 20, 32, 34
Wood chips/ sawdust
42.9–45.1 22.0–33.0 24.0–26.0 1.7–2.0 0.2–0.3 0.1–0.2 150.0–450.0 6, 20, 21, 32
NA: Data not available
References: [1] USDA-US DOE 2005:, [2] Brand et al. 2000, [3] C¸
ur et al. 2007, [4] Curvetto et al. 2005, [5]
Demeke 2007, [6] Gabriel 2004, [7] Ga
nan et al. 2006, [8] Howard et al. 2003, [9] Anonymous 2006:,
[10] Jim
enez and Gonz
alez 1991, [11] Laufenberg et al. 2003, [12] Lin 2005, [13] Liou et al. 1997, [14] Mata and Savoie 2004, [15] Mosier et al. 2005, [16]
Mussato et al. 2008, [17] Obodai et al. 2003, [18] Ortega et al. 1992, [19]
Ozc¸elik and Peks¸en 2007, [20] Palonen 2004, [21] Philippoussis et al. 2001a,
[22] Poppe 2004, [23] Quian 2004, [24] Ragunathan et al. 1996, [25] Reddy and Yang 2005, [26] Salmones et al. 1999, [27] Salmones et al. 2005, [28]
Saura-Calixto et al. 1983, [29] Shashirekha and Rajarathnam 2007, [30] Singh 2000, [31] Thomsen 2005, [32] Tisdale et al. 2006, [33] Ververis et al. 2004,
[34] Ward et al. 2000.
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates 171
In plant residues, cellulose and hemicellulose are the main sources of carbohy-
drates, often incrusted with lignin, which forms a physical seal around these two
components. Lignocellulose is physically hard, dense and recalcitrant, the degra-
dation of which is a complex process requiring a battery of hydrolytic or oxidative
enzymes. Taking into consideration that the substrates are insoluble, degradation oc-
curs extracellularly, by two types of extracellular enzymatic systems: the hydrolytic
system, which produces hydrolases and is responsible for cellulose and hemicel-
lulose degradation; and a unique oxidative lignolytic system, which depolymerizes
lignin (P
erez et al. 2002, Baldrian 2005). The hydrolytic breakdown of cellulose
by fungi is catalyzed by extracellular cellobiohydrolases, endoglucanases and -
glucosidases, which hydrolyze the long chains of cellulose, liberating cellobiose
and finally glucose, while the major hemicellulose-degrading enzymes are endoxy-
lanases and endomannanases (Tengerdy and Szakacs, 2003). Most of these enzymes
have been detected in both wood-degrading mushroom fungi (WDF), like P. o s -
treatus and L. edodes (Elisashvili et al. 2008) and litter-decomposing mushroom
fungi (LDF), such as A. bisporus or V. volvacea (Steffen et al. 2007). Due to its
complicated structure, lignin is more difficult to break down than cellulose or hemi-
cellulose. The main extracellular enzymes participating in lignin degradation are
lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Hatakka 1994),
with MnP, prooving to be the most common lignin-modifying peroxidase produced
by almost all wood-degrading basidiomycetes (Steffen et al. 2007). In addition,
litter-decomposing basidiomycetes can degrade lignin e.g. A. bisporus produces at
least two lignolytic enzymes, laccase and MnP, however, the overall lignin degra-
dation rate by these fungi is lower compared to that of white-rot fungi (Lankinen
et al. 2005, Steffen et al. 2007). Besides the lignocellulosic enzyme complex, ligno-
cellulolytic fungi also produce other enzymes, such as pectinases, proteases, lipases
and phytases on lignocellulosic substrates (Tengerdy and Szakacs, 2003).
Basidiomycetous fungi comprise diverse ecological groups, i.e. WDF (white rots,
brown rots) and LDF, which may insure their nutrition in different ways. White-rot
fungi are able of a simultaneous degradation of all wood components (cellulose,
hemicellulose and lignin), while brown-rot fungi, a relatively small group of Ba-
sidiomycetes, degrade only cellulose and hemicellulose. Given that the majority of
cultivated higher basidiomycetes is WDF, while few of them are LDF, emphasis
is given here to the nutritional behaviour and degradation potentials of these two
groups, represented by the most cultivated species A. bisporus, Pleurotus spp. and
L. edodes.
White rot mushroom-forming fungi, comprising cultivated species like Pleuro-
tus spp., L. edodes, Ganoderma spp. etc., are the most efficient degraders, due to
their capability to synthesize relevant hydrolytic (cellulases and hemicellulases)
and unique oxidative (lignolytic) extracellular enzymes. Their general strategy is
to decompose the lignin in wood, so that they can gain access to the cellulose and
hemicelluloses embedded in the lignin matrix (Hatakka 1994). However, laccase
expression in fungi is influenced by culture conditions, such as nature and con-
centration of carbon and nitrogen sources, media composition, pH, temperature,
presence of inducers and lignocellulosic materials, etc. (Revankar et al. 2007).
172 A.N. Philippoussis
A wide variety of lignin degradation efficiency and selectivity abilities, enzyme
patterns and substrates enhancing lignin degradation are reported from white-rot
fungi (Hatakka 2001, Baldrian and Val
a 2008). An interesting category of
white-rot fungi are selective degraders that degrade lignin rather than cellulose, like
Pleurotus spp., which are used in a wide range of biotechnological applications
(Cohen et al. 2002). Lignin degradation by these fungi is thought to occur dur-
ing secondary metabolism and typically under nitrogen starvation (Hammel 1997).
Non-composted, chopped and water-soaked straw is sufficient for the cultivation
of Pleurotus spp., while L. edodes is cultivated on logs or in bags on moisturized
sawdust supplemented with cereal bran (Philippoussis et al. 2000, 2004). Although
not necessarily optimal, since they are low in readily accessible nutrients, these
commercially used substrates satisfy the needs of the fungi for growth and fruiting,
and most importantly, help to withstand microbial competitors (K
ues and Liu 2000).
In basidiomycetous LDF, comprising cultivated mushroom species like Agaricus
spp., Agrocybe spp., Coprinus spp., Stropharia spp. and V. volvacea, degradation
involves a succession of biodegradative activities that precede attack by lignocel-
lulose degraders. However, the ability to break down lignin and cellulose enables
some of the LDF to function as typical “white-rot fungi” in soil (Hofrichter 2002).
Well known mushroom forming LDF are A. bisporus and V. volvacea, both grown
commercially on composted lignocellulose. As A. bisporus contains lignolytic en-
zymes, degrades both cellulose and lignin, the former more rapidly (Cai et al. 1999,
Lankinen et al. 2005). Compost prepared from straw, horse or chicken manure, cal-
cium sulphate (gypsum), water and some nutritional supplements is a cheap cultural
substrate for A. bisporus and some other saprophytic basidiomycetes. Manure in the
compost serves as N source, straw as C source. It must be pointed out that after the
initial medium preparation stage, little control can be exerted over the composition
of the solid substrate medium. In composted substrates this is particularly crucial
since the nutrient composition of the initial medium ingredients has to allow both
a successful composting process and good fungal colonization and fruiting (Wood
and Smith 1987).
Mushrooms have a two-phase life cycle, the mycelium (vegetative or coloniza-
tion phase) and the fruiting body (reproductive phase that bears the spores). The
mycelium grows through the substrate, biodegrades its components and supports
the formation of fruiting bodies. Mushroom growers call the switch from mycelial
extension to the production of mushroom primordia “pinning”, thesuccessive devel-
opment of primordia into mushrooms “fruiting”. While growth of mycelium lasts for
several days, weeks or months, production of fruiting bodies is short lived, and the
phenomenon is called ‘fructification’. However, both vegetative and reproductive
phases are very much influenced by the physiological condition and nutritional state
of the mycelium (Wood and Smith 1987).
Since the carbon sources utilized by basidiomycetes are usually of a lignocellu-
losic character, fungi during vegetative growth produce a wide range of enzymes
to degrade the lignocellulosic substrates. Data obtained in various studies demon-
strate that the type and composition of lignocellulosic substrate appear to determine
the type and amount of enzyme produced by basidiomycetous fungi during vegeta-
tive growth (Baldrian 2005, Baldrian and Val
a 2008, Elisashvili et al. 2008).
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates 173
Moreover, cellulose/lignin ratios of wheat straw and cotton waste substrates were
positively correlated to mycelial growth rates and mushroom yields of P. ostreatus
and P. pulmonarius and with the yield of V. volvacea (Philippoussis et al. 2001a).
According to K
ues and Liu (2000), considerable changes in enzyme activities occur
during fruiting, indicating a connection to the regulation of fruiting body develop-
ment. For example, in A. bisporus and L. edodes, laccase activities are highest just
before fruiting body initiation and decline rapidly with primordia formation. Cellu-
lase activities are highest when fruiting body develops (Ohga et al. 2001). Regarding
the influence of nitrogen availability, recent studies revealed a positive correlation
between the C/N ratio and P. eryngii mushroom yield (Philippoussis et al. 2000).
They also demonstrated that mycelium growth rates of Pleurotus spp. and L. edodes
were positively correlated to C/N ratio (Philippoussis et al. 2001a, 2003). Similar
conclusion was drawn by Silva et al. (2005), indicating that L. edodes extension rate
is related to bioavailability of nitrogen and is enhanced by supplementation with
cereal bran. Moreover, both nature and concentration of nitrogen sources are factors
regulating enzyme production by wood rotting basidiomycetes, e.g. in L. edodes
cultivation on wheat straw, nitrogen supplementation represses MnP and enhances
laccase activity (Kachlishvili et al. 2005). According to K
ues and Liu (2000), for
fruiting body induction it is of importance to keep a balance between C and N
sources, e.g. in A. bisporus compost, the optimal C/N ratio for fruiting has been
determined to lie between 80:1 and 10:1. In addition, substrate supplementation with
protein-rich materials proved to enhance yield of Agaricus, Pleurotus and Lentinula
strains (Rodriguez-Estrada and Royse 2007, Naraian et al. 2008).
Apart from nutrition, mycelial growth and fruiting of basidiomycetous fungi are
also regulated by temperature, gaseous environment, water activity and in certain
cases by light. During substrate colonization, the effect of environmental parame-
ters plays an essential role on mycelium growth, and hence confers significantly to
the success of the entire cultivation process. In addition, the duration of the sub-
strate colonization phase is of direct economic importance, since media that are
non-thoroughly impregnated with the hyphae, are sensitive to fungal and bacterial
infections resulting in reduced yields (Philippoussis et al. 2001a, Diamantopoulou
et al. 2006). Production of the vegetative mycelium usually occurs over a wide
range of temperatures. Zervakis et al. (2001) examined the influence of temperature
on mycelium linear growth of P. ostreatus, P. eryngii, P. pulmonarius, A. aegerita,
L. edodes, V. volvacea and A. auricula-judae. Their temperature optima were found
to be 35
CforV. volvacea strains, while P. eryngii grew faster at 25
C, P. ostreatus
and P. pulmonarius at 30
C. Moreover, A. aegerita grew faster at 25
and A. auricula-judae at 20
C depending on the nutrient medium used, and
L. edodes at 20
C depending on the strain examined. It is generally believed
that basidiomycetes tolerate relatively high levels of salts for growth, but fruiting
body development can be more sensitive. Likewise, mycelial growth is less affected
by pH but fruiting body development of several species occurs best at neutral or
slightly acidic pH values around 6–7 (Wood and Smith 1987) or, in L. edodes,
at a pH 4.0 (Ohga 1999). On lignocellulosic substrates, Pleurotus and Lentinula
species are growing with a linear rate (Philippoussis et al. 2001a, Diamantopoulou
and Philippoussis 2001), which is influenced by substrate salinity and porosity
174 A.N. Philippoussis
(Philippoussis et al. 2002). Measurements of electric conductivity through the en-
tire colonization process of three residue-substrates by L. edodes strains revealed an
increase of salinity values until mycelium colonized 60 to 75% of the substrate, and
then it slightly declined or remained constant until the end of incubation, presenting
the highest and lowest values in the wheat straw and oak sawdust media respec-
tively. In addition, a negative correlation was established between final salt content
of the substrates and mycelium extension rates. Furthermore, monitoring of CO
concentrations in pilot-scale cultivation of L. edodes on synthetic blocks, revealed
higher respiration rates on oak sawdust and corncobs than on wheat straw, which
are further correlated with substrate colonization rates (Philippoussis et al. 2003).
Following colonization of the substrate, fruiting is induced by environmental
and/or cultural manipulation. The optimal environmental parameters for mycelial
growth and the subsequent fruiting are usually very distinct. Depending on the
species and the degree of investment in environmental control technology, tempera-
ture is normally manipulated by heating or cooling systems to maintain the optima
for vegetative growth or fruiting. However, fruiting body development is often in-
duced after drastically altering the environmental parameters, usually favoured by
reducing the temperature by at least five
C compared to mycelium growth. In fact,
fruiting is typically induced, after vegetative growth, e.g. in A. bisporus to 16–18
ues and Liu 2000), in P. ostreatus to 15
C (Zadrazil et al. 2004), and in L. edodes
to 10–16
C for the cold temperature strains and 16–21
C for the warm temperature
strains (Chen et al. 2000). Other parameters of fruiting body initiation and matura-
tion include CO
concentration, humidity, salinity and pH. High humidity (90–95%)
is favorable for pinning and fruiting but the moisture content of the substrate might
be even more critical. The optimal water content for wooden substrates is 35–60%
and for other substrates 60–80%. The lower values reflect the oxygen demand of
the fungi in the substratum, balanced against their requirement for water (K
ues and
Liu 2000). Carbon dioxide (CO
) level is also critical for efficient mycelial growth,
fruit body initiation and fruit body development. Higher CO
concentrations (e.g.
1% v/v in air) may stimulate mycelial growth and inhibit fruiting. Increased aeration
is used to reduce CO
levels, which otherwise produces increased elongation of stipe
growth and abnormality of cap development (Wood and Smith 1987). Light has been
implicated in the fruiting of several mushroom genera e.g. Lentinula and especially
Pleurotus species have an obligate requirement for light for fruiting induction. Brief
exposure of the culture to daylight or suitable artificial light is sufficient. Usually,
light positively influences hyphal aggregation and fruiting body maturation (K
and Liu 2000). However, light is not needed for the fruiting of A. bisporus (Wood
and Smith 1987).
9.2.3 Output and Stages of Mushroom Cultivation
Mushroom industry presents a worldwide expanded and economically important
biotechnological application, which can be divided into three main categories: culti-
vated edible mushrooms, medicinal mushroom products and wild mushrooms, with
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates 175
an annual global market value in excess of $45 billion (Chang 2006). The global
annual mushroom output (including production and wild mushroom collection)
surpass nowadays 10 million metric tons, with China being the top world pro-
ducer (about 8.000.000 tons), followed by Europe and USA (Desrumaux 2007,
Huang 2007). Commercial mushroom production is an efficient solid state fermen-
tation process of food protein recovery from lignocellulosic materials carried out
on a large or small scale (Mart
ınez-Carrera et al. 2000, Chiu et al. 2000). Taking
into account the value and volume of the product, the number of people involved
in the industry, or the geographical area over which the industry is practiced, mush-
room cultivation is the greatest application of exploitation of filamentous fungi us-
ing SSF and the biggest (non-yeast) biotechnology industry in the world (Moore
and Chiu 2001). The economic strength of mushroom cultivation derives from the
successful use as feedstocks of a variety of low- or negative-value residues from
agriculture, forestry or industry. These wastes are processed using relatively cheap
microbial technology to produce human foodstuff,which could also be regarded as a
functional food or as a source of drugs and pharmaceuticals (Wood and Smith 1987).
Moreover, the effective exploitation of resources from agricultural solid wastes and
by-products, rich in organic compounds that are worthy of being recovered and
transformed, is a sound environmental protection strategy (Zervakis and Philippous-
sis 2000).
There are three major stages involved in mushroom cultivation: (1) inoculum
(spawn) production, (2) substrate preparation, and (3) mushroom growing i.e. inocu-
lation of the substrate with propagules of the fungus, growth of the fungal mycelium
to colonise the substrate, followed by fruiting, harvesting and processing of the fruit-
ing bodies (Wang 1999, Mart
ınez-Carrera et al. 2000).
Inoculum (spawn) production. In order to achieve reliable and vigorous fungal
growth and fruiting bodies production of good quality, inoculum fungal cultures
are necessary. Inoculum is produced by inoculation of sterilized cereal grains (usu-
ally wheat, rye or millet) from high quality stock mycelial cultures (Stamets 2000,
Mata and Savoie 2005b). Essential prerequisite is the selection and breeding work
to acquire suitable biological material for commercial cultivation, which ensures
good yield and quality. The various mushroom inocula are often the only micro-
biologically pure part of the whole technology (Wood 1989). Spawn-making is a
rather complex task, not feasible for the common mushroom grower, and is pro-
duced by specialist companies (spawn-makers) using large scale bulk autoclaving,
clean air and other microbiological sterile techniques for vegetative mycelia cultures
onto cereal grains, wood chips and plugs or other materials. The colonized cereal
grain/mycelium mixture is called spawn and is grown under axenic conditions in
autoclavable polyethylene bags, ensuring gas exchange, or rarely in jars. Finally,
after quality control to assure biological purity and vigor, spawn is distributed from
the manufacturer to individual mushroom farms in the same aseptic containers used
for spawn production (Wood and Smith 1987, Royse 2002).
Substrate preparation. Fermentation process involves cultivation on specific
substrates imitating the natural way of life of mushroom fungi (Tengerdy and
Szakacs 2003). Regarding the litter-decomposer A. bisporus, this has come to mean
176 A.N. Philippoussis
cultivation of a mushroom crop on composted plant litter (Moore and Chiu 2001).
On the other hand, the white-rot mushroom fungi Pleurotus spp and L. edodes are
cultivated on non-composted lignocellulosic substrates, using methodologies that
exploit their ability to produce enzymes capable of degrading all wood components
(Chen et al. 2000, Zadrazil et al. 2004). The first stage of mushroom production has
to do with assembly and treatment of the substrate to prepare a growth medium. The
substrates used for mushroom production, varying according to cultivated species,
are prepared from waste agricultural or forest product materials using ingredients
such as manures, cereal straws or other crop residues, sawdusts etc. (Wood and
Smith 1987). In certain cases the substrate is be directly inoculated and require very
little pre-treatment, e.g. L. edodes production using logs. In other cases, the sub-
strate is microbiologically or physically pretreated. Microbiological pre-treatment
normally comprises some form of controlled bulkcomposting process (Wood 1989).
Physical pre-treatment couldinclude steam treatment or sterilization by autoclaving.
Substrates for fungal growth can be prepared as sterile materials, to produce an
axenic growth medium, e.g. bottle cultures of F. velutipes, or be non-sterile, e.g.
compost substrates to produce A. bisporus. One of the aims of substrate preparation
is to introduce sufficient water into the substrate to ensure that the water activity of
the final medium is optimal for fungal growth. The scale of substrate preparation
varies according to the type of species to be cultivated and the size of the production
unit. For A. bisporus production, many tons of straw are processed per day to pro-
duce compost. Thus, large-scale bulk handling machinery is used for this process.
After the initial preparation stage, little control can be exerted over the composition
of the solid substrate medium. In composted substrates, this is particularly crucial
since the nutrient composition of the initial medium ingredients has to allow both a
successful composting process and good fungal colonization and fruiting. Although
nutrient balances and status, e.g. for carbon, nitrogen, pH and other components,
can be measured on the initial ingredients, little can be done to regulate the quan-
tity or feed rate of these once the production processes are under way (Wood and
Smith 1987).
Mushroom growing. This stage deals with the two phases of mushrooms life
cycle i.e. the mycelium (vegetative phase) and the fruiting body formation (repro-
ductive phase). Following inoculation, the mycelium, grows through the substrate,
biodegrades its ingredients and supports the formation of fruiting bodies. Mycelial
growth and fruiting during this stage are regulated by temperature, gaseous envi-
ronment, nutrient status, water activity and in certain cases by light e.g. Pleurotus
spp. has an obligate requirement for light for fruiting induction, Agaricus spp. have
no light requirement (Wood 1989, Zadrazil et al. 2004). The level of environment
and cultural control used is determined by the type of production technology. In
controlled environment growing system, temperature is manipulated by heating or
cooling systems to maintain the optima for vegetative growth or fruiting. Carbon
dioxide (CO
) level and humidity are also controlled. Basidiomata production on
the culture medium surface occurs as a series of cycles (flushes). Depending on the
fate of the harvested product as fresh or preserved material, the fruit bodies are har-
vested either by hand or mechanically and processed accordingly. After harvesting,
mushrooms are normally cooled down to retard fruiting body metabolism, packed
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates 177
and sent to the fresh market, or processed further through freezing, canning, drying
etc., depending on marketing strategies (Mart
ınez-Carrera et al. 2000).
9.3 Bioconversion of Solid Residue-Substrates
Through Mushroom Cultivation
9.3.1 Commercial Mushroom Production Processes
In the suite, the principles of production of the litter decomposingAgaricus spp.
and. of two wood-degrading mushroom species (Pleurotus spp. and L. edodes)are
Agaricus bisporus cultivation. It is the most commonly cultivated mushroom
worldwide, mainly cultivated in Europe, North America, China and Australia
(Chang 1999). A. bisporus, belonging to the Agaricaceae family, is a litter-
decomposing basidiomycete that in nature usually grows on grasslands and forests
(Kirk et al. 2001). Western countries, focused on Agaricus for consumption, led to
progress in cultivation technology including farm design, quality control in compost
production, microprocessor control and records for growing, mechanical harvesting
and processing. The Netherlands, practising high-technology cultivation systems,
has the highest mushroom yield per unit area worldwide (Chiu et al. 2000). The
substrate used for A. bisporus cultivation is a complex culture medium made from
straw- and manure-based compost. Its preparation is a two-stage process in which
the first stage includes composting of the raw material consisting of straw, horse
or poultry manure and gypsum (S
anchez 2004). During composting that lasts about
3 weeks, the lignocellulose waste is modified by various bacteria and fungi to a
better-digested form suitable for A. bisporus. In the second week stage, the compost
is pasteurized before inoculation with A. bisporus spawn. The final mushroom com-
post is a selective growth medium for this organism. Natural drop in temperature
and lack of free ammonia are signs that the composting process has been completed
(Moore and Chiu 2001).
Cultivation begins with inoculation (spawning; the process that introduces the
mushroom mycelium into the compost) and growth of the mushroom mycelia into
the compost under high humidity and temperature 25
C. At complete colonization,
after 2–3 weeks, a casing layer containing peat moss and limestone is spread on
the top of the compost. ‘Casing’ is needed only by Agaricus, the procedure is not
necessary when cultivating other species such as Pleurotus spp. and L. edodes.After
allowing 7 to 9 days for the Agaricus mycelium to grow into the casing layer, a
machine with rotating tines is run across the mushroom bed to mix the casing layer
thoroughly. The above process is called ‘ruffling’ and it serves in breaking up the
mycelial strands and encourages the mushroom mycelia to grow and colonize the
surface of the casing layer. The mushroom mycelium grows into the casing layer in
similar conditions to those of compost colonization, and when it reaches the upper
surface of the casing layer the fruiting process starts comprising environmental ma-
nipulation. The growing room is ventilated to decrease the concentration of carbon
178 A.N. Philippoussis
dioxide (usually to < 0.1%) and to help reduce the temperature to 16–18
C. The
temperature, humidity and CO2 level are then adjusted to trigger fructification and
to favor the development of mushrooms. The first pin initials begin to appear about
2 weeks after casing. One layer of compost produces 2–4 crops called flushes. In
general, the production of A. bisporus is time-consuming due to the long composting
stage (S
anchez 2004).
In modern mushroom growing process, a specialist compost producer may com-
plete the outdoor stages of composting and the ready-to-use compost (spawn run or
not) can be delivered in bulk to a mushroom farm. Therefore, the mushroom produc-
tion industry comprises spawn makers, phase I, phase II and phase III compost sup-
pliers. Phase III compost is completely colonized by the mushroom mycelia, which
if placed in a suitable environment in a mushroom farm will produce the fruit body
crop readily. For a commercial mushroom farmer, the use of phase I compost gives
the most flexibility to optimize farm conditions for cultivation of any mushroom
strain. Purchase of phase II compost enables a farmer to choose which mushroom
strain to spawn. The use of phase III compost, though it is obviously more costly, it
guarantees the production of a crop in a short time and requires the least investment
in facilities (Moore and Chiu 2001). Moreover, there are different growing systems,
while the process can be separated into specialized stages. In the shelf-bed growing
system, shelving is usually made of metal and arranged to give four to six layers
of 1.4 m wide fixed shelves in a cropping room with centre and peripheral access
gangways. Special machinery for compost filling, emptying, spawning, casing and
other cultivation operations is necessary. In the bag growing system, growingbags of
about 25 kg are usually supplied to the farm already spawned and may be arranged
on the floor of the cropping house or on tiered shelving. Each arrangement makes
its own demands on techniques and equipment.
Pleurotus spp. cultivation. Pleurotus species (like P. ostreatus, P. sajor-caju,
P. pulmonarius, P. eryngii, P. cornucopiae, P. tuber-regium, P. citrinopileatus and
P. flabellatu ) are commercially very important edible mushrooms, found all over
the world. These mushrooms present several advantages related with rapid mycelial
growth, high ability for saprophytic colonization, simple, inexpensive cultivation
techniques and several kinds of species available for cultivation under different
climatic conditions. The production of Pleurotus mushrooms is a sharp contrast
with the technology used for Agaricus production. Both pasteurized and sterilized
substrate of a wide range of residues can be used (Fig. 9.1)and no casing is required.
The primary ingredients used for Pleurotus spp. production is chopped wheat straw
(Triticum aestivum L.) or cottonseed hulls (Gossypium hirsutum L.)ormixturesof
them. For production on wheat straw, the material is chopped from 2 to 6cm, water
is added and pH of the material is adjusted with limestone to about 7.5 or higher to
provide selectivity against Trichoderma green mold (Royse 2004). The substrate is
then pasteurized in tunnels with aerated steam at 60–70
C for 12hr by passing the
air-steam mixture through the substrate. After pasteurization is complete (a prox-
imate two-day process), filtered air is passed through the substrate for cooling to
C. Then grain spawn (inoculum) is added at about 3–5% of the fresh weight and
the substrate, packed into 15–20kg plastic bags or blocks, is placed in a dark room at
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates 179
Fig. 9.1 Biological efficiency (BE; % ratio of fruiting body fresh weight over the dry weight of substrate) of P. o s t r e a t u s and P. pulmonarius cultivated on
residue-based substrates. References: [1] Couti
no et al. 2004, [2] Curvetto et al. 2002, [3] Croan 2003, [4] Darjania et al. 1997, [5] Das and Mukherjee 2007,
[6] Gezer et al. 2007, [7] Hern
andez et al. 2003, [8] Kalmis¸ and Sargin 2004, [9] Lara et al. 2002, [10] Mandeel et al. 2005, [11] Marino et al. 2003, [12]
ınez-Carrera et al. 1985, [13] Mart
ınez-Carrera et al. 2000, [14] Moda et al. 2005, [15] Obodai et al. 2003, [16] Pant et al. 2006, [17] Philippoussis
et al. 2000, [18] Philippoussis et al. 2001a, [19] Ragunathan et al. 1996, [20] Royse and Schisler 1987, [21] Salmones et al. 2005, [22] S
anchez et al. 2002,
[23] Serra and Kirby 1999, [24] Shah et al. 2004, [25] Soto et al. 1987, [26] Upadhyay et al. 2002, [27] Vel
no et al. 2002, [28] Vetayasuporn 2006,
[29] Yildiz et al. 2002, [30] Zhang et al. 2002, [31] Zervakis and Balis 1992
180 A.N. Philippoussis
C with 80 per cent humidity. Depending on the strain, complete colonization
of the substrate is achieved in 2–3 weeks. In Asia, small containers are used, e.g. in
Japan, bottle production of Pleurotus mushrooms is common. Substrate is filled into
bottles, sterilized and inoculated with Pleurotus spawn. Upon completion of spawn
run, bottle lids are removed and mushroom emerge from the surface of the substrate
(Wood and Smith 1987, Royse 2003a, 2004).
Fructification of P. ostreatus is triggered by lowering the air temperature to
C (cold-shock treatment) although no such treatment is required for other
Pleurotus spp. The fruiting is light-dependent, requiring a 8–12 hour light cycle
(solar or fluorescent lamp light) and adequate ventilation is given to keep CO
levels lower than 500 ppm. Insufficient ventilation generally leads to mass primor-
dial development with little differentiation into fruit bodies (Zadrazil et al. 1996).
Three to four weeks after spawning depending on strain, amount of supplement used
and temperature of spawn run, mushrooms begin to form around the edges of bag
perforations and they are harvested from the substrate (Royse 2004). Throughout
cropping, mushroom houses are kept at 12–17
C, the substrate usually being a few
degrees higher. Under ideal growing conditions, 1 kg of well-colonized substrate
will yield about 1 kg of marketable mushrooms (after two flushes) the completely
growing cycle being completed in about 70 days. Due to the absence of a velum
covering the gills of Pleurotus fruitbodies, spore discharge begins at a very early
stage. Very large spore deposits within mushroom houses can cause allergy prob-
lems (Wood and Smith 1987).
Lentinula edodes cultivation. L. edodes (Berk.) Pegler is the second most popular
edible mushroom in the world because of its flavour, taste, nutritional and medicinal
properties. (Smith et al. 2002). This fungus can grow on synthetic logs as well as
natural logs. The most traditional but laborious cultivation is carried out in wood
logs, mainly oak. The wood logs are holed and the mycelia plugs are inserted in
these holes. After inoculation the logs are stored several months for mycelium col-
onization and finally for the formation of fruit bodies. This method is still used
because of its high quality mushroom product (Royse 2001, Silva et al. 2007),
although leads to a severe threat to natural forests (Chiu et al. 2000). The last
decades, new methods for L. edodes cultivation on residue-based substrates have
been developed using milled wood residues (e.g. oak, hornbean, sweetgum, poplar,
alder, ironwood, beech, willow, pine, maple and birch sawdust) supplemented with
nitrogen sources (e.g. rice bran). The main advantages of using synthetic medium
over natural logs are time and efficiency (Royse 2004). Some formulations used
consist of 80% sawdust and 20% bran; 80% sawdust, 10% bran and 10% wheat or
millet; and 84% sawdust, 5% rice bran, 5% wheat bran, 3% soybean and 3% lime
(Kalberer 1987).
Other agricultural wastes that can be used as substrates (alone or in combi-
nation with other supplements) in L. edodes cultivation are cereal straw, corn
cobs, sugarcane bagasse, tea waste, sunflower seed hulls, peanut shells, vine-
yard prunings, cotton straw and seed hulls etc. (Fig. 9.2; Curvetto et al. 2002;
Philippoussis et al. 2003; Rossi et al. 2003, Gait
andez and Mata 2004,
Mata and Savoie 2005a, b, Fan and Soccol 2005,
Ozc¸elik and Peks¸en 2007, Royse
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates 181
Fig. 9.2 Biological efficiency (BE; % ratio of fruiting body fresh weight over the dry weight of substrate) of L. edodes mushroom cultivated on residue-based
substrates. References: [1] Curvetto et al. 2005, [2] Diehle and Royse 1986, [3] Donoghue and Denison 1995, [4] Donoghue and Denison 1996, [5] Fan and
Soccol 2005, [6] Gait
andez and Mata 2004, [7] Gait
andez et al. 2006, [8] Hiromoto 1991, [9] Kalberer 2000, [10], Kawai et al. 1996, [11]
KirchhoffandLelley 1991, [12] Levanon et al. 1993, [13] L
opez et al. 2004, [14] Mart
ınez-Carrera et al. 2000, [15]
Ozc¸elik and Peks¸en 2007, [16] Philippoussis
et al. 2003, [17] Pire et al. 2001, [18] Rossi et al. 2003, [19], Royse and Bahler 1986, [20] Royse and Sanchez 2007, [21] Royse and Sanchez-Vazquez 2001,
[22] Royse and Sanchez-Vazquez 2001, [23] Royse et al. 1990, [24] Royse 2002, [25] Salmones et al. 1999, [26] Worrall and Yang 1992
182 A.N. Philippoussis
and Sanchez 2007). The principle of the cultivation method comprises mixing and
compacting ingredients into plastic bags, followed by sterilization, inoculation with
fungal mycelia, incubation in dark rooms with controlled temperature and humidity
for 30 to 80 days and finally fruiting induced by temperature reduction. Regardless
of the main ingredients used, starch-based supplements such as wheat bran, rice
bran, millet, rye, corn, etc. are added to the mixture in a 10 to 40% ratio to the main
ingredient. These supplements serve as nutrients to provide an optimum growing
medium (Royse et al. 1990, Royse 1996, 2003b). Substrate’s ingredients are mixed,
watered to gain a moisture content around 60% and filled into polypropylene bags
1–3kg/bag. The filled bags are stacked on racks, loaded into an industrial-sized
autoclave, sterilized for 2 hours at 121
C, cooled and inoculated with spawn. Af-
ter a 20 to 25 days spawn run, the bags are removed and the substrate blocks are
exposed to an environment conducive for browning of the exterior log surfaces.
As the browning process reaches completion (4 weeks), primordia begin to form
about 2mm under the surface of the bag-log indicating that it is ready to produce
mushrooms (Royse 2004). Primordia maturation is stimulated by soaking the sub-
strate in water (12
C) for 3 to 4 hours (or 3 to 4 min if vacuum soaking is used;
Royse 2002). Soaking allows water rapidly to displace carbon dioxide contained
in air spaces, providing enough moisture for one flush of mushrooms. Approxi-
mately 9 to 11 days after soaking, mushrooms are ready to harvest (Royse 2001).
This method decreases the production time and increases productivity. While in the
traditional cultivation the logs need 8 months to 1 year of cultivation to produce
10–15kg/100kg of substrate, the cultivation on agro-forestry residues can furnish
a yield of 60–80 kg/100 kg of substrate in 80 days harvest period (Israilides and
Philippoussis 2003, Royse 2004, Silva et al. 2007).
9.3.2 Efficiency of Residue Conversion to Pleurotus sp.
and L. edodes Fruiting Bodies
Two particular basidiomycetus mushroom genera that have received considerable
attention for their nutritional value, medicinal properties and biodegradation abil-
ities are Pleurotus and Lentinula (Elisashvili et al. 2008). These widely cultivated
edible mushrooms are efficient colonizers and bioconverters of lignocellulosic agro-
industrial residues into palatable human food with medicinal properties
(Zervakis and Philippoussis 2000, Philippoussis et al. 2004, Zadrazil et al. 2004,
Silva et al. 2007, Gregori et al. 2007). The efficacy of this value-added biocon-
version process and the productivity of the mushroom crop are assessed by the
biological efficiency (Chang et al. 1981). Biological efficiency (BE) expresses the
bioconversion of dry substrate to fresh fruiting bodies and indicates the fructification
ability of the fungus utilizing the substrate (Fan et al. 2000a). BE is calculated as
the percentage ratio of the fresh weight of harvested mushrooms over the weight
of dry substrate at inoculation (Chang and Chiu 1992, Philippoussis et al. 2001b,
Diamantopoulou et al. 2006). Yet, in a scarce number of reports, biological effi-
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates 183
ciency has been defined in terms of dry fruit bodies yield over the dry weight of
the substrate used (Bisaria et al. 1987, Wang et al. 2001). Nevertheless, it should
be made clear that apart from the type of substrate and stain used, yield response is
determined by the duration of the cropping period and cultivation practice applied
e.g. high spawn levels enhance mushroom yields (Obodai et al. 2003). Nevertheless,
for considering the Pleurotus cultivation profitable, BE value must be over 50%
(Patra and Pani 1995).
The genus Pleurotus comprises some the most popular edible mushrooms due
to their favourable organoleptic and medicinal properties, fast mycelial growth and
undemanding cultivation conditions. These mushrooms are commercially grown on
pasteurized straw-based substrates or hardwood sawdust, fermented or not, with
added supplements. However, as these fast-growing mushrooms display a complete
lignocellulolytic enzyme system (Bushwell et al. 1996, Elisashvili et al. 2007), they
can use a wide spectrum of agricultural and industrial wastes that contain lignin
and cellulose for growth and fruiting (Poppe 2000). A significant number of agro-
industrial lignocellulosic materials are used as substrates for the production of Pleu-
rotus spp., like corn cobs, various grasses and leaves, reed stems, maize and sorghum
stover, rice and wheat straw, vine shoots, cardboard and paper, wood sawdust and
chips, coffee pulp, cottonseed hulls, peanut shells, sunflower seed hulls, sugarcane
and tequila bagasse etc. Average experimental BE values varying from 14.5–126.0%
are presented in Fig. 9.1. Further evaluation of the overall BE values obtained on
these residue-substrates for P. ostreatus and P. pulmonarius strains indicated that
among all residues cardboard, coffee pulp, paper wastes and softwood residues, pre-
sented the highest ( 100%) biological efficiencies (Mart
ınez-Carrera et al. 2000,
Croan 2003, Mandeel et al. 2005). BEs between 75% and 100% were recorded on
cotton wastes and wheat straw (Upadhyay et al. 2002, Philippoussis et al. 2001a).
Regarding straw pre-treatment, data demonstrated an approximate 20% reduction
of overall BE when P. ostreatus is cultivated on non fermented wheat straw, as
compared to fermented substrate (mean values 70.5% and 85.5% respectively).
On pretreated wheat straw, supplementation with cotton seed cake and soybean
cake proved to enhance productivity of P. ostreatus (Upadhyay et al. 2002, Shah
et al. 2004). Finally, satisfactory productivity (BEs 50–75%) is demonstrated by
most of agro-industrial residues, namely corncobs, various grasses and reed stems,
vine shoots, cottonseed hulls and sugarcane bagasse. From the considered data it
can be assumed that P. pulmonarius furnished significantly better yields than P.
ostreatus on cardboard and soft-wood residues (respective BEs: 126.0 and 124.3%).
Moreover, yield of P. pulmonarius is favored on coffee and cotton wastes as well as
on wheat straw (Pant et al. 2006, Zervakis and Balis 1992, Philippoussis et al. 2001a,
no et al. 2002).
Our previous studies, concerning evaluation of a wide range of residues available
in the Mediterranean region as P. ostreatus, P. eryngii and P. pulmonarius cultivation
substrates, demonstrated significantly higher colonization rates of these mushrooms
on wheat straw and cotton waste (Philippoussis et al. 2000). Moreover, faster colo-
nization was achieved on non-composted than on composted wheat straw and cotton
waste substrates. Cellulose/lignin ratios in substrates were positively correlated to
184 A.N. Philippoussis
mycelial growth rates and mushroom yields of P. ostreatus and P. pulmonarius.
In addition, there was a positive correlation between the C/N ratio and P. eryn-
gii mushroom yield (Philippoussis et al. 2001a). Additional data furnished by the
‘race-tube’method experiments provided an estimate of the potential of wheat straw,
cotton gin-trash, peanut shells, poplar sawdust and corn cobs to serve as alternative
mushroom cultivation substrates (Zervakis et al. 2001). Furthermore, in recent stud-
ies conducted to evaluate different grass and reed stalks as cultivation substrates
of Pleurotus species (Philippoussis et al. 2007, Diamantopoulou et al. 2007), bean
plant residues (BRP) and a mixture of reed-grass maces (TCP) supported fast col-
onization rates for both genera strains, while BRP enhanced laccase and endoglu-
canase activities(Diamantopoulou et al. 2007). Moreover, fructification assay by the
‘tube fruiting technique’ provided a quick estimate of the potential of these wastes
to support basidiomata formation. Basidiomata produced by both fungi, on all tested
residues, with TCP supporting remarkably better fruiting results compared to wheat
straw (Philippoussis et al. 2007).
Lentinula edodes (Berk.) Pegler is one of the best-known species among culti-
vated mushrooms, grown on natural or artificial logs, composed of either sawdust
or of locally available agricultural wastes. L. edodes produces hydrolytic and ox-
idative enzymes responsible for lignocellulose degradation (Ohga and Royse 2001,
Mata and Savoie 2005c). The production of enzymes is specifically related to and
dependent on substrate composition and environmental factors such as tempera-
ture and moisture (Bushwell et al. 1996, Silva et al. 2005, Elisashvili et al. 2008).
Since L. edodes is an efficient wood degrader, it can be grown on a variety of
agro-industrial residues such as oak, ash, poplar, alder, eucalypt, beech, pine, maple
and birch sawdust, cereal straws (mainly barley and wheat), corn cobs, sugarcane
bagasse, sunflower seed hulls, peanut shells, cotton straw and seed hulls, vine
shoots, coffee husk and pulp etc. Figure 9.2 presents comparatively the biologi-
cal efficiencies obtained on these substrates during productivity evaluation experi-
ments. Data indicate that the nature of the substrate affects remarkably L. edodes
basidiomata yield. The highest average biological efficiencies were achieved with
sunflower seed hulls (BE: 107.5%; Curvetto et al. 2005), followed by sugarcane
bagasse (BE: 87.4%; Salmones et al. 1999). More or less similar interesting re-
sults (BE 80%) appeared to be obtained with hard-wood residues (beech) and
barley straw (Kirchhoff and Lelley 1991, Kawai et al. 1996, Gait
et al. 2006). Among other substrates, progressively lower BEs, in the range of
65–50% and in descending order, were furnished by cereal grains, wheat straw,
vine shoots, hard-wood residues of various trees and hazelnut husk (Hiromoto 1991,
Philippoussis et al. 2003, Gait
andez et al. 2006,
Ozc¸elik and Peks¸en 2007).
Oak sawdust (comprising all types of oak), pine sawdust and coffee residues (all
types) exhibited BEs in the range of 40–50% (Donoghue and Denison 1996, Royse
and Sanchez-Vazquez 2001, Royse and Sanchez 2007), while the overall lower
BE values (around 20%) were detected on apple pomace and corncobs (Worrall
and Yang 1992, Philippoussis et al. 2003). Nevertheless, reliable estimations of the
residues impact on L. edodes yield cannot be withdrawn from the presented aver-
age BE values, which only as indicative could be regarded. The main problem is
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates 185
that data from different substrate formulas, co-substrates and supplements, media
treatments, strains used, cultivation periods, experimental conditions etc. are com-
pared. It is well known that apart from substrate nature and composition, strain and
length of incubation (to mention only a few factors), are important parameters for L.
edodes production on artificial substrates (Royse and Bahler 1986, Zadrazil 1993,
Kalberer 1995, Sabota 1996, Chen et al. 2000, Philippoussis et al. 2002, 2003).
Comparing Figs. 9.1 and 9.2, BEs obtained on residue-substrates used for both
L. edodes and Pleurotus spp. cultivation, the overall assumption is that wood
chips and wheat straw, followed by sugarcane bagasse and vine prunings can sup-
port good basidiomata yield of L. edodes,aswellasofP. ostreatus and P. pul-
monarius strains. Coffee pulp and corncobs supported significantly higher yields
for Pleurotus strains, while sunflower seed hulls favoured higher productivity for
L. edodes.
Our previous studies, evaluating six commercial and wild L. edodes strains as
regards their efficacy of mycelium growth on wheat straw, cotton wastes, oak-wood
sawdust and corncobs, have demonstrated that oak-wood sawdust and wheat straw
supported faster growth than corncobs and cotton wastes (Philippoussis et al. 2001c).
In addition, a strain-dependent behaviour was detected since three strains per-
formed much better on oak-wood sawdust and wheat straw, while one commercial
strain performed satisfactorily on the other two substrates. In general, significantly
lower linear growth rates were recorded for corncobs and cotton wastes (Philip-
poussis et al. 2003). Results were verified by the fruiting technique conducted in
glass tubes, that furnishes a remarkable reduction in the time necessary for the
first fructification ( 2 months), conducing to a quick evaluation of the produc-
tion potential of tested substrates. This, along with the use of growth rate mea-
surement for valorization of the substrate incubation efficacy, renders the ‘glass-
tube’ method a dependable technique for screening-selecting purposes. Addition-
ally, measurements of mycelium respiration during the incubation phase in bag-log
cultivation demonstrated very low respiration rates on cotton wastes, irrespective of
the strain used (Philippoussis et al. 2002), while further experiments demonstrated
the suitability of wheat straw and to a lesser extend supplemented corncobs for
the cultivation of L. edodes (Philippoussis et al. 2003, 2004). Nevertheless, cotton
wastes generated in large quantities in many countries, have proved to be a very
good substrate for the cultivation of Pleurotus species (Philippoussis et al. 2001a,
Cohen et al. 2002).
9.4 Closing Remarks
Current mushroom industry is based on both application of techniques for the pro-
duction of mushroom fruiting bodies and the application of modern biotechnological
techniques to produce medicinally beneficial compounds and nutraceutical products
(Chang 2006, 2007). The medicinal properties of bioactive substances, like polysac-
charides with antitumor and immunostimulating properties occurring in higher
186 A.N. Philippoussis
basidiomycetes have become a subject of numerous recent reviews (Mizuno 1999,
Wasser and Weis 1999, Kidd 2000, Ooi and Liu 2000, Wasser 2002, Daba and
Ezeronye 2003, Paterson 2006). Among them, the commercial polysaccharide of
L. edodes, lentinan, has been researched extensively as it offers the most clinical
evidence for antitumor activity (Wasser 2002, Nikitina et al. 2007). Recently, some
mushroom polysaccharides have shown to exert a direct cytotoxic effect on cancer
cells in vitro (Jiang et al. 2004, Wong et al. 2007, Israilides et al. 2008).
Moreover, solid-state fermentations other than fruiting body production are sug-
gested for upgrading and valorizing lignocellulosic residues using basidiomyce-
tous cultures, either through protein enhancement and transformation of residues
into animal feed (Zadrazil et al. 1996, Zadrazil 2000), or for enzyme production
(Revankar et al. 2007, Elisashvili et al. 2008). In the first case, agro-industrial
residues such as rice straw, coffee pulp, sugarcane bagasse, banana leaves etc.
have been fermented by white-rot basidiomycetes to improve the digestibility of the
residues for use as ruminate feed supplement (Vega et al. 2005, Albor
es et al. 2006,
Okano et al. 2006). In the second case, lignocellulose degrading mushroom species
like Pleurotus sp, Lentinula edodes, Trametes versicolor, Flammulina velutipes are
used for the production of enzymes of industrial importance, such as cellulases,
xylanases and laccases, using as substrates agro-industrial residues, from which
wheat straw and bagasse are the most commonly used substrates (Krishna 2005,
Silva et al. 2007). Most results, however, come from laboratory, or semi-pilot-scale
experiments (Pandey et al. 2000a, Cohen et al. 2002). Additionally, lignocellulolytic
mushroom fungi likePleurotus ostreatus and Trametes versicolor have been inves-
tigated for bioremediation and biodegradation of toxic and hazardous compounds
like caffeinated residues (Fan et al. 2000a, b) as well as toxic chemicals such as
pesticides, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls
(PCBs), chlorinated ethenes (CIUs) etc., in polluted soils or contaminated ground
water (Pointing 2001, P
erez et al. 2007, Rigas et al. 2007).
In terms of food production process, the aim of mushroom growing should be
to follow the holistic concept of production, according to Laufenberg et al. (2003).
This approach tries to connect differing goals, such as highest product quality and
safety, highest production efficiency and integration of environmental aspects into
product development and food production. An outstanding example of integrated
crop management practice of mushroom cultivation is the use of spent substrate,
that is the residual growth medium after cropping (Rinker 2002) (1) as animal
feed, since the mushroom mycelium boosts its protein content (Zhang et al. 1995),
(2) as soil conditioner and fertilizer as it is still rich in nutrients and with poly-
meric components that enhance soil structure (Castro et al. 2008), (3) as a source
of enzymes (Ko et al. 2005), (4) for the biological control of plant pathogens
(Philippoussis et al. 2004, Davis et al. 2005) and even (5) used for bioremediation
purposes as to digest pollutants on land-fill waste sites because it contains popu-
lations of microorganisms able to digest the natural phenolic components of lignin
(Eggen 1999, Fermor et al. 2000). In this concept, solid state fermentation processes
are not only the methods of mushroom production for food and nutraceutical pur-
poses but also examples of an organic system integrated with waste treatment that
9 Production of Mushrooms Using Agro-Industrial Residues as Substrates 187
contributes to sustainability and benefits the human population, health and environ-
Acknowledgments This work was partially financial supported by the General Secretariat of Re-
search and Technology through the 05 PAV 105 project. The author wish to express his gratitude
to Diamantopoulou Panagiota MSc and to Kontogiorgi Ioanna, members of Laboratory of Edible
and Medicinal Fungi of NAGREF, for their great assistance in the preparation of the manuscript.
In addition, Dr. Serapheim Papanikolaou is thanked for his comments and improvements made to
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... The process of photosynthesis generates approximately 200 billion tons of organic compounds every year (Philippoussis 2009). This organic bulk can pollute the environment in many cases especially the lignocellulosic components as they are not readily degraded by organisms (Hatakka 1994;Philippoussis 2009). ...
... The process of photosynthesis generates approximately 200 billion tons of organic compounds every year (Philippoussis 2009). This organic bulk can pollute the environment in many cases especially the lignocellulosic components as they are not readily degraded by organisms (Hatakka 1994;Philippoussis 2009). Waste materials from agriculture and industry can be used to grow mushrooms (Abid et al. 2020;Iwuagwu et al. 2020). ...
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Mushrooms are a popular food source as they are highly nutritious and flavorful with a high content of proteins, vitamins, and minerals. Mushrooms could be an alternative solution to the world's food crisis as they are inexpensive to grow on different types of substrates including waste materials. Pleurotus ostreatus, frequently known as oyster mushrooms, are the second most cultivated mushroom in the world. This species is known for its high protein content and easy cultivation. Oyster mushrooms have the potential to produce protein-rich biomass when grown on various substrates. There is a need to identify substrates that are cost-effective for the commercial production of nutritious oyster mushrooms as the substrates used currently are either costly or inadequate to produce oyster mushrooms in the required quantity or quality. Thus, the effects of 6 different lignocellulosic substrates on the growth and nutritional composition of P. ostreatus were reviewed and analyzed in this article. The substrates included in this review were wheat straw, sugarcane bagasse, corncob, softwood sawdust, hardwood sawdust, and general sawdust. Based on the analyzed data, sugarcane bagasse was concluded as the most suitable substrate to grow P. ostreatus. These substrates contain a high amount of nutrients and are also likely to produce a significantly high yield of oyster mushrooms in addition to enhancing the nutritional quality of the mushroom. However, these findings must be evaluated and confirmed through further research in this field.
... Residues/wastes from major staple crops in South-Kivu are among those listed as promising candidates for recycling and valorization elsewhere. For instance, Philippoussis et al. (2000) and Philippoussis (2009) mentioned that residues of crops such as cereal straws, naked corn cobs, cotton stalks, various grasses, and reed stems, maize and sorghum stover, vine prunings, sugarcane bagasse, coconut and banana residues, corn husks, coffee pulp and coffee husk, cotton seed and sunflower seed hulls, peanut shells, rice husks, waste paper, wood sawdust, and chips, etc. are some examples of agricultural residues and by-products that can be recycled and valorized in useful products by chemical or biological processes. Their chemical properties (such as lignocellulosic agricultural residues composition) make them a substrate of enormous biotechnological value, and in our case, for oyster mushroom production. ...
... Mushroom cultivation has proved its economic potential and ecological importance for efficient utilization, valueaddition, and biotransformation of agro-industrial residues. Production and commercialization of mushrooms, at a large or small scale, is an efficient and relatively short biological process of food J o u r n a l P r e -p r o o f protein recovery from negative value lignocellulosic materials, exploiting the degrading capabilities of mushroom fungi (Philippoussis et al., 2000;Philippoussis, 2009). ...
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Food security is challenged by low agricultural productivity in eastern Democratic Republic of Congo (DRC). This study aimed at contributing to food security of rural households in South-Kivu by valorizing residues of four staple crops, including cassava, maize, banana, and common bean. The study was conducted in two steps: (1) monitoring of farmers' fields throughout the cropping season to record weight of crop residues and yields, and (2) assessment of the potential of staple crop residues for mushroom (Pleurotus ostreatus) production. Results showed that the four major staple crops had low yields and low biomass productivity in the study area. Residues of these staple crops were mainly used by farmers as fodder, compost, incinerated, or left on the farm for nutrient recycling. In addition to target plant parts (tubers or grains), cassava and common bean leaves were harvested for household consumption (as vegetables) or traded at local markets for income generation. Substrates based on maize residues, combined with manure as additive, gave highest yield of Pleurotus (2.4 kg kg⁻¹) compared to residues of other three staple crops. In contrast, substrates from banana leaves had consistently lowest yields, regardless of used additives (1.1 and 1.2 kg kg⁻¹ with soybean flour and cow manure, respectively). This study showed that valorizing residues of staple crops could help improve households’ food security and income in rural areas of eastern DRC. This practice should, therefore, be encouraged and scaled across the country and other parts of the world facing food shortages and poverty.
... Asia along with Europe, North America, and Australia are world leader mushroom producing regions because of the major residue demanding for this bio-based industrial activity. Among countries in the Asian and Pacific Region, China produces the largest quantities of agricultural and forest residues, mainly by-products of rice, corn, and wheat and estimated quantities of waste products was to reach about 1 billion tons/year in China, are followed by India's agricultural residues was at least 200 million tons/year while the total amount of agro-industrial residues reaches 600 million ton in India [12]. ...
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Soil is the fundamental and necessary natural resource for the agricultural production system. Due to the increasing global population and the impact of climate changes, natural resources are the major limiting factor to use widely for food production. The major factors responsible for the deterioration of natural resources are extreme events caused by man-made activities and unexpected and unpredictable adverse natural forces of nature. Among the different degradation processes, soil erosion is one of the serious threatens to the deterioration of soil for the agricultural sector and healthy ecosystem conservation. Intensive agricultural practices are particularly caused by the acceleration of the soil erosion process. Therefore, the good and systematic management of soil resources is indispensable not only for sustainable agriculture or conservation agriculture but also for the protection and reduction of the natural ecosystem. Covering crop residues on soil enhances organic matter, protects the soil surfaces, maintains water and nutrients, improves soil biological activity and chemical composition, and contributes to pest management. Therefore, crop residue management is one of the conservation practices and is designed to leave sufficient residue on the soil surface to reduce wind and water erosion. It includes all field operations that affect the amount of residue, its an orientation to the soil surface and prevailing wind and rainfall patterns and the residue distribution throughout the period requiring protection. This paper especially highlights the status of soil erosion, crop residues, and management in crop residues in sustainable agriculture.
The objective of this paper is to study potential application of spent mushroom substrate (SMS) in production of Pleurotus ostreatus mushroom. A various mass ratio mixture of rubber sawdust (RS) and SMS with wheat bran supplement were prepared for mushroom cultivation. The mushroom house was kept at an optimal condition with temperature between 28 and 33 ℃ and humidity in the range between 80 and 95%. The results showed that the 50% SMS + 50% RS had a significantly higher of average total yield at 156.12 g/bag, biological efficiency (B.E) of 55.76% and required only 26 ± 2 days for mycelium growth in comparison to 100% RS, a control substrate. It is also found that potassium (K) was the most concentrated macroelement and all heavy metal concentrations were below the maximum permitted by WHO/FAO in the first fresh mushroom collected. Therefore, it can be proven that SMS can be a great solution to the disposal issue, and it can be an excellent medium for the cultivation of Pleurotus spp. when it is combined with the RS.
Dealing with horticultural waste has become an urgent issue that coincides with the keen desire of the world today to achieve sustainable development, but the question arises: How and what is the appropriate tool? The answer will be provided by this review, through which it will focus on the possibility of benefiting from solid-state fermentation (SSF) as an eco-friendly technique on the one hand and as an effective tool in treating horticultural waste on the other hand. Also, SSF characterized over the other waste-managing techniques by low capital investment, reduced energy requirements, improved product recovery and reducing environmental problems. Despite the several advantages of SSF, it faces many challenges, including biomass estimation, heat transfer, scale-up, recovery and operational control. This work summarizes the various applications of SSF used for bioconversion of the horticultural wastes into value-added/innovative bioproducts such as enzymes, antibiotics, bioactive compounds, organic acids, bioethanol, etc. Furthermore, advantages, challenges and future directions associated with developing SSF technology in horticultural by-products management are presented and discussed. Additionally, SSF could be suggested as a strategic and promising approach for producing various added-value products from horticultural wastes that contribute effectively to the reduction of operational costs and decrease the environmental pollution.
The potential of selected Pleurotus ostreatus and P. eryngii wild-type and commercial strains to colonize and produce carposomes after solid-state fermentation of five substrates constituted of agro-residues namely wheat straw (WS), beech wood shavings (BWS), coffee residue (CR), barley and oats straw (BOS), rice bark (RB), supplemented with wheat bran was investigated. The effect of substrate composition on bioprocess feasibility was assessed for the different strains by quantitative (i.e., mushroom yield and Biological Efficiency-BE %) and carposome qualitative parameters (i.e., weight, size, colour, firmness). P. ostreatus strains produced carposomes earlier than P. eryngii ones. Early fruiting formation was promoted in WS for P. ostreatus strains, whereas for P. eryngii the lowest values of earliness were observed on BOS substrate. As for crop productivity, P. ostreatus strain AMRL 150 provided the highest BE (>70%) in all substrates except for RB, while P. ostreatus strain AMRL 144 achieved the highest yield and BE in BOS and BWS (75.30 and 64.26%, respectively). P. eryngii stains produced less number but heavier carposomes. The highest values of BE% for these strains were recorded on WS and BOS substrates. The BE was further correlated to growth parameters of fungal vegetative phase. As for mushroom colour, P. ostreatus were lighter than P. eryngii and BOS substrate promoted lightness in both strains. Firmer P. ostreatus mushrooms were produced at CR. No differences in the whiteness and firmness were detected among P. eryngii mushrooms and at any substrate used. The data included in this paper showed that Pleurotus mushrooms can be cultivated on low- or zero-value agro-industrial residues of great financial and environmental importance towards the production of value-added food products. Solid-state fermentation, oyster mushrooms, glucosamine, endoglucanase, laccase, carposomes, colour, texture
The influence of ten agro-residues, i.e., wheat straw (WS), poplar wood sawdust (PWS), grape pomace (GP), beech wood shavings (BWS), cotton cake (CK), corn cobs (CC), coffee residue (CR), olive pulp (OP), barley and oats straw (BOS) and rice bark (RB), used as alternative substrates for the cultivation of Pleurotus mushrooms, was examined during mycelial growth (colonization phase). Several native and commercial strains of P. ostreatus and P. eryngii were subjected to screening regarding their mycelial growth rates, biomass production and endoglucanase and laccase activities. The experimental results showed that the highest growth rates of P. ostreatus strains were noticed on BWS, BOS, CC and RB substrates, with the time of complete colonization varying between 16 and 36 days, whereas for P. eryngii a high linear growth rate was observed on CC, OP, CR and BOS with the colonization period ranging between 26 and 51 days. The maximum biomass production obtained on the various substrates for both Pleurotus species ranged from 115.32 to 454.42 mg/g d.w. for P. ostreatus and from 108.50 to 422.59 mg/g d.w. for P. eryngii. As for endoglucanase, the highest activities were observed in P. eryngii AMRL 166 cultures on BOS and RB, i.e., 0.18 and 0.14 U/g d.w., respectively. P. eryngii AMRL 173-6 and P. ostreatus AMRL 150 cultivated on RB and BOS, synthesized significant laccase amounts, i.e., 2172.28 and 1987.25 U/g d.w., respectively. The effect of the substrate components on the growth parameters was considered and discussed. This study showed the industrial potential to convert important low-value agro-residues to fungal biomass and enzymes and eventually to valuable food products.
The waste-to-wealth concept has attracted remarkable attention for generating values out of waste materials along with the effective management of notorious agri-food wastes. Globally, agri-food industries are generating daily mammoth pre- and postprocessing wastes, of which most of the untreated waste fractions are severely leading to environmental problems. This waste can be designed to be valorized in a sustainable way with cutting-edge technologies not only to generate value-added products but also to offer jobs. Valorization of agri-food wastes into alternative and renewable energy generation is a popular practice in several industries to meet the in-house energy requirement as well as for returns to offset the economic constrain of the ongoing process. Moreover, bio-energy from waste has been an efficient alternative resource for the depleting fossil fuel usage, which also improves the carbon footprint of the bioprocess. Today, food waste is a comparatively less explored resource mainly due to its high organic nature. The technological hurdles are associated with utilizing it as the main source for generating valuable bioproducts. The agro-food processing waste has been utilized for the production of bioactive molecules, platform chemicals, biofertilizers, enzymes, etc.
The present chapter shows an overview of the production of bioactive peptides (BAPs) obtained from food matrices, using fermentation processes. It shows that it is possible to obtain BAPs from milk, meat, and vegetable proteins and emphasizes scientific production and the proven benefits that milk protein-derived BAPs provide to health. It also emphasizes a promising outlook in BAP production by fully using meat and vegetable proteins using food industry by-products, which also helps to mitigate waste environmental issue. For viable and safe BAPs industrial production, advances about in vivo research and adaptations of biotechnological processes for this scale of production are required.
Fermented food and beverages constitute a significant part of the human diet (5%–40%) worldwide. Fermentation has been used for preservation and to augment the flavor, texture, and nutritional qualities of the food, since antiquity. During fermentation, the bioavailability of vitamins, minerals, and other constituents increases due to the microorganisms’ metabolic activities. Besides enhancing nutritional quality, fermented foods contain live organisms reported to prevent/treat many health disorders. Types of the fermentation process are also classified based on these microorganisms. In developing countries, fermented foods were usually prepared using traditional methods without any standardized techniques. Considering the beneficial effects of fermented foods, industrial-level production requires consistent specific microorganisms, fermentation methods, evaluation of nutritional compositions, and food safety testing. This chapter discusses the fermented foods and associated organisms, different sources available for the consumption of fermented foods, and food component’s effect on microorganism’s efficacy.
At first some general considerations about specificity and characteristics of SSF, their advantages and disadvantages as compared to LSF, are presented. Microorganisms involved in solid substrate fermentations are identified, considering the better performances of filamentous fungi. The solid substrates and their basic macromolecular compounds are detailed in relation to this complex and heterogeneous system. Biomass measurement is examined in detail, as well as environmental factors, both essential for studying and optimising solid substrate fermentations.
The increasing expansion of agro-industrial activity has led to the accumulation of a large quantity of lignocellulosic residues all over the world. In particular, large quantities of rice straw (300.000 t) and citric bagasse (50.000 t) are annually produced in Uruguay. In this work we present the study of the bioconversion of these substrates with the edible mushroom Pleurotus spp so as to increase nutritional values and digestibility for its use as animal feed. The SSF process was optimized and the products after different periods of mushroom growth were evaluated. The microbial counts (cfu/g) for the inoculated substrates 44 days after incubation were 15 x 10 4 , E. coli , respectively. After 14 days of SSF the percentage of dry matter, ADF and NDF decreased, and the content of protein increased. These results show that vegetal cell-wall components were degraded during the period of mushroom incubation. PCR - RFLP analysis of the ITS region was used to characterize the Pleurotus species produced in Uruguay and discriminate between DNAs of Pleurotus ostreatus 814 and other fungi from the different substrates.