Agronomy 2020 , 10 , x doi: FOR PEER REVIEW www.mdpi.comjournalagronomy [628093]

Agronomy 2020 , 10 , x; doi: FOR PEER REVIEW www.mdpi.com/journal/agronomy
Review 1
Use of spent mushroom substrate in new mushroom 2
crops to promote the transition towards a circular 3
economy 4
Diego Cunha Zied¹ ,* , Jose E. Sánchez ², Ralph Noble³ and Arturo Pardo-Giménez 4 5
1 Universidade Estadual Paulista (UNESP), Faculdade de Ciências Agrárias e Tecnoló gicas (FCAT), Câmpus 6
de Dracena, Dracena, SP, Brazil; [anonimizat] (D.C.Z.) 7
2 El Colegio de la Frontera Sur, Tapachula, Chiapas, Mexico; jesv2007@gm ail.com (J.E.S) 8
3 Pershore College, Warwickshire College Group, Pershore WR10 3JP, UK; rn [anonimizat] 9
(R.N.) 10
4 Centro de Investigación, Experimentación y Servicios del Champiñón (CIES), Quint anar del Rey, Cuenca, 11
Spain; [anonimizat] (A.P.-G.) 12
* Correspondence: [anonimizat] 13
† These authors are equal contribution to this review. 14
Received: date; Accepted: date; Published: date 15
Abstract: The use of spent mushroom substrate (SMS) in new cultivation cycles has already bee n 16
reported due to its economic and environmental viability. When consid ering the application of the 17
circular economy concept in the production of edible mushrooms, the re-use of the SMS within the 18
same process is highly attractive, because it allows a better use of the bioma ss and the energy 19
involved in the process and therefore, tends to improve energy efficiency and re source conservation. 20
However, this alternative generates important challenges, which derive from maint aining the 21
quality standards of the mushrooms produced and at the same time not incur ring excessive costs 22
that are detrimental to the process itself. In our opinion, the main di fficulty of the process in 23
achieving success is regarding the biological and agronomic parameters that invo lve the production 24
of the mushroom. It is useless to apply SMS in new cycles if the mushroom harv est is impaired and 25
farms become non-viable. However, numerous examples are reported here wh ere SMS was recycled 26
into new substrates for either the same or different mushroom species without negatively a ffecting 27
yield compared with using substrates prepared from 100% fresh raw material s. Thus, we suggest 28
that each farm has its own specific technological study, since a small va riation in the raw material 29
of the compost, and mushroom cultivation practices and casing layer used, ca n influence the entire 30
viability of the mushroom circular economy. 31
Keywords: eficiency process; waste reduction; compost production; casing layer; yield 32
33
1. Introduction 34
Mushroom cultivation has a relationship with the conversion of agricultural and agro-industrial 35
waste into food of high nutritional value; it stands out as an enviro nmentally sustainable option [1]. 36
This metabolic capacity of fungi takes place through degradative microbiological p rocesses, which to 37
achieve their highest economic viability, and optimal chemical, physical, en vironmental and 38
technological process/conditions must be controlled (Figure 1). 39

Agronomy 2020 , 10 , x FOR PEER REVIEW 2 of 20
40
Figure 1. Scheme of mushroom cultivation and the main chemical, physical, env ironmental and 41
technological process/conditions, which can influence the most important stages of the production. 42
Currently mushroom cultivation is carried out worldwide, adopting the most diverse 43
technologies possible, from the simplest to the most technologically advance d [2]. It should be noted 44
that the degree of economic investment and the local social condition s of each country are factors that 45
affect the technological degree of cultivation [3]. Despite this, the entire production process starts 46
from the selection of the raw materials that will be used for the production of the compost/substrate. 47
There is a specific criterion in the choice of raw materials that is related to the nutrition of 48
mushrooms, which are classified as primary and secondary decomposers. Primary decomposers 49
require materials with higher C/N ratio and lignin content and lower nitrogen content (sawdust, 50
sugarcane bagasse and straw), whereas secondary decomposers require material s with lower C/N 51
ratio and higher cellulose, hemicellulose and nitrogen contents (manure an d compost “mixture of 52
various agricultural wastes”) [4 -6]. Another specific aspect is that the secondary decomposers 53
develop in substrate degraded by bacteria or other fungi [7]. Even so, the versatility of mushrooms 54
can allow them to be primary and secondary decomposers at the same time, as is the case of Pleurotus 55
spp., which can be grown in sawdust/ wheat straw or in substrate (sugar can e bagasse, Brachiaria 56
straw, wheat and rice bran and limestone). This factor allows species to be disseminated and 57
cultivated in different regions with greater ease [8]. Therefore, in mushroom cul tivation, special 58
attention should be paid to which agricultural or agro-industrial wastes will be used to attain 59
successful production and economic viability in the activity [9]. 60
2. Circular economy 61
The circular economy's concept is not only to correctly choose the raw materials that will be the 62
basis of the activity, but also to define correctly all the actions invo lved during each production phase, 63
in order to apply again the wastes generated in the activity on the same compan y [10]. Using this 64
concept in mushrooms production, the main concern would be the spent mushroom substrate (SMS), 65
the principal waste generated in the cultivation [11]. However, some research has already been 66
developed aiming at the use of SMS as a complementary material to raw materials for n ew substrate 67
formulations, and as a casing layer for the cultivation of Agaricaceous mushrooms [12-15]. Figure 2 68
illustrates the dynamics of the process in the mushroom circular economy, wher e the only waste 69
during the crop cycle would be the plastic bags/bottle, which are used to grow certai n species 70
(Lentinula edodes , Pleurotus spp., Volvariella volvacea , Flammulina velutipes , and others). 71

Agronomy 2020 , 10 , x FOR PEER REVIEW 3 of 20
72
Figure 2. Mushroom circular economy representing the beginning of the process wit h the 73
introduction of raw materials, going through compost production , inoculation and production phase 74
(3 harvest flushes) until the end of cultivation, where the only waste of th e cycle is the plastic 75
bags/bottle. Red letters and arrows indicate the reuse of SMS in the formula tion of new substrates and 76
in the casing layer. 77
Finally, another waste in mushroom production, certainly generated in a small er quantity than 78
SMS, is the mushroom stipe base (bottom of the stipe which is in contact with the substrate/casing 79
layer), which will be described briefly in item 6, since it is not the general aim of thi s review  80
3. Spent mushroom substrate 81
After mushroom production, the substrate plus the cultivated mycelium generate the SMS 82
byproduct or waste [16], which is widely utilized in the agronomic sector [6]. In the cultivation of 83
Agaricaceous mushrooms, a casing layer based on peat or mineral soil + li mestone is incorporated in 84
the SMS. 85
Considering a commercial mushroom cultivation, i.e. Agaricus bisporus that can attain a yield of 86
40% of substrate weight (ratio of fresh mushroom weight harvested divid ed by the fresh weight of 87
the substrate, multiplied by 100), every 1 kg of fresh mushroom generates 2.5 kg of fresh SMS. 88
Emphasizing that in the cultivation of A. bisporus a casing layer is positioned over the compost, every 89
1 kg of fresh mushrooms generates 3.24 kg of fresh SMS, including the spent peat + limestone casing 90
[17]. Mushrooms that have a lower conversion efficiency than A. bisporus can result in 5 kg of fresh 91
SMS for every 1 kg of fresh mushrooms produced [18]. Significant amount s of wastes are therefore 92
available to be used again in mushroom production. 93
Among the possible uses for the substrates remaining after mushrooms cultivatio n, there are 94
references to its use in bioremediation (purification of air, water, soils an d degradation of pesticides), 95
for cultivating other crops (greenhouse flowers and vegetables, fruit and vegeta bles in the field, 96
among others), as a general amendment/fertilizer for soils, in nurseries and landscaping, as animal 97
feed or in aquaculture, in pest and disease management, as energy feedsto ck (alternative fuel, 98
production of biogas), in vermiculture, as source of degradative enzymes, and other diverse uses. Its 99
reuse in the cultivation of mushrooms, as component of casing material or ingredient of new growth 100

Agronomy 2020 , 10 , x FOR PEER REVIEW 4 of 20
substrates for further mushroom growing cycles, is also considered [6,19-23]. In this review, we will 101
discuss the use of SMS in new mushroom crops. 102
3.1. Spent mushroom substrate as bases of new substrates 103
To achieve maximum yield in commercial production of edible mushrooms, it is necessary to 104
know the natural ecological habitats of mushrooms in wood and straw (prim ary decomposers) or 105
compost (secondary decomposers), the nature of substrate materials and their prep aration, the 106
appropriate control of physical, chemical and biological parameters and the prope r management of 107
mushroom cultivation beds [24]. 108
As for the ecological habitats, cultivated mushrooms have two kinds of sa prophytic nutrition. 109
Most of them are primary decomposers that can be cultivated on pasteurized o r sterilized 110
lignocellulosic substrates. The others are leaf-litter secondary decomposers cultivated on composts 111
prepared from various agricultural wastes including manures [25]. 112
Mushrooms can produce a wide range of hydrolytic and oxidative enzymes. The proposal for 113
the reuse of SMS in new growing cycles is based on the different enzymatic activ ity of various species, 114
highlighting the importance of their sequence of introduction. For the use of these m aterials in the 115
preparation of substrates for the cultivation of different species of mushrooms , aspects such as their 116
enrichment and combination with other materials, the treatments to be applie d and the results 117
obtained must be taken into account. These applications allow the integration of th ese types of 118
materials, through new formulations and methodologies, with the double a dvantage of lowering 119
production costs and reducing environmental impact. 120
3.1.1. SMS of Agaricus spp. 121
A. bisporus and Agaricus subrufescens (syn A. blazei and A. brasiliensis ) are classic examples of 122
secondary decomposers. These mushrooms typically grow on composted material, and they rely on 123
the previous activity of other microorganisms to partially break down a sub strate to a state wherein 124
they can thrive. The actions of other fungi, actinomycetes, bacteria, and yeast s all operate within 125
compost. Once these microorganisms (especially actinomycetes) have completed their li fe cycles, the 126
compost is susceptible to colonization by a select secondary decomposer such as an Agaricus species 127
[26]. However, only a proportion of the nutrient content of the substrate is utili zed by an Agaricus 128
mushroom crop, leaving the remainder available for use in subsequent crops. Table 1 contains 129
references on reuse of SMS of Agaricus spp. in new substrate formulations. 130
Table 1. Reuse of SMS of Agaricus spp. in new substrate formulations. 131
Spent substrate Growing media for cultivation of References
Agaricus bisporus Agaricus bisporus [27-46]
Agaricus bisporus Pleurotus spp. [47]
Agaricus bisporus Pleurotus ostreatus [48,49]
Agaricus bisporus Pleurotus sajor-caju [50]
Agaricus bisporus Volvariella volvacea [51-57]
Agaricus bisporus Auricularia polytricha [47]
Agaricus bisporus Lentinula edodes [58]
Agaricus bisporus Agaricus blazei [59]
Agaricus bisporus 11 species [60]
Agaricus brasiliensis Pleurotus ostreatus [61]
Agaricus brasiliensis Agrocybe cylindracea [61]
Agaricus brasiliensis Stropharia rugosoannulata [61]
Agaricus brasiliensis Hericium erinaceus [61]
According to Till [27], A. bisporus SMS can be reused as a new substrate for Agaricus if it is 132
autoclaved and enriched with cottonseed meal and soybean meal. Murphy [ 29] developed a 133
succesfull SMS-based compost formula by supplementing SMS with corncobs and cottonseed meal 134
prior to pasteurization. Schisler [30] studied the behavior in new A. bisporus growing cycles of SMS 135
to which the commercial supplement Spawnmate II and Bonaparte peat were added. Diff erent works 136

Agronomy 2020 , 10 , x FOR PEER REVIEW 5 of 20
refer to the use of mixtures of non-composted substrate (NCS), based on oak s awdust, millet, and 137
SMS of A. bisporus . These substrate mixtures of NCS and SMS produced mushroom yields 138
comparable to standard non-supplemented phase I compost [34,62]. A mushroom yield of 27.2 kg m – 139
2 was obtained with a 50/50 mixture of NCS and SMS supplemented with 10% (d ry wt) of a 140
commercial delayed release nutrient at casing [33,34]. Moisture content of subs trates significantly 141
influenced mushroom yield [63]. Other works abound in the concept of do uble-cropping. This 142
consists of adding, after the 1st, 2nd, or 3rd flush, materials such as hydrolyzed protein, commercial 143
supplements, crystalline amino acids or phase II compost [12,35,36,38,40,4 1]. Double-cropping, and 144
even triple-cropping, offers growers an opportunity to increase bio-efficiency, reduce production 145
costs, and increase profitability [36]. Besides that, Royse [37] explo red the effect of using various 146
levels of SMS as an ingredient in compost formulations, incorporated at the beginin g of phase I or at 147
the time of filling the tunnel for phase II composting (at fill). 148
Loehr [42] evaluated the use of SMS added as an ingredient to milled co rn stover at fill in the 149
development of a phase II-only composting protocol. Warnstrom [43] and Bi shop et al. [45] 150
investigated the reuse of A. bisporus SMS as an ingredient in the preparation of fresh mushroom 151
composts based on straw-bedded horse manure and other comparatively high er lignocellulose-rich 152
materials. Zisopoulos et al. [17] compared the exergetic performance of a conv entional industrial 153
mushroom production chain with a mushroom production chain where part of the compost waste 154
was recycled and reused as raw material. Recently, Ahlawat and Kaur [46] studi ed the use of SMS in 155
partial replacement of wheat straw in compost formulation for A. bisporus cultivation. 156
In the UK, Noble et al. [64] removed the peat casing layer (which ha s no nutrient value) from the 157
cooked-out SMS during emptying of a mushroom crop. The separated SMS was then used in a phase 158
I composting process. Substrate prepared with 25%w/w of the wheat straw and poultry manure 159
substituted with SMS produced the same mushroom yield as the wheat straw + poultry manure 160
substrate. The separated casing layer was partially recycled with fresh casing for new mushroom 161
crops (Section 3.2). Machinery for separating the casing and compost layers o n shelf mushrooms 162
farms has been developed by the Dutch company MushComb [65]. 163
The use of Agaricus SMS in the production of different Pleurotus species has also been described 164
[47-50]. In Pleurotus ostreatus cultivation, substrates based on combinations of Pleurotus spent 165
substrate and Agaricus SMS mixed in proportions of 9:1 and 8:2 (w/w) provided a BE of 36.0% and 166
39.7% respectively, which was not significantly different from the values obtai ned with the 167
commercial substrate used as a control [48]. Picornell et al. [49] evaluated the quality parameters of 168
sporphores of P. ostreatus obtained from A. bisporus and P. ostreatus spent substrates mixed in different 169
amounts. Mueller et al. [50] used P. ostreatus spent substrate in the production of Pleurotus sajor-caju . 170
Oei [51,52] refers to the use in Taiwan of Agaricus SMS mixed with cotton waste, fermented 171
between 2 and 4 days and pasteurized, for the production of Volvariella “straw mushroom” . Poppe 172
[53,54] also suggests that spent substrate can be used to grow successive crops of mushro oms, like 173
Agaricus SMS amended with cotton waste for satisfactory cultivation of Volvariella . Oei et al. [22], refer 174
to Fan and Lu [55], who indicated that in some provinces of China, gro wers commonly cultivated 175
straw mushroom Volvariella with A. bisporus SMS. There are also references in the use of SMS of A. 176
bisporus in the production of other fungi, such as Auricularia polytricha [47], L. edodes [58] and A. blazei 177
[59]. 178
In Poland, the research of Jasinska and Smolna [61] proposed the use of sawdust or wheat straw 179
substrate with addition of SMS, after A. brasiliensis cultivation, for P. ostreatus, Agrocybe cylindracea , 180
Stropharia rugosoannulata and Hericium erinaceus . 181
3.1.2. SMS of Pleurotus spp. 182
Oyster mushrooms ( Pleurotus spp.), shiitake, and Jew's ear are examples of primary 183
decomposers. These mushrooms are typically fast-growing, sending out ropy strands o f mycelium 184
that quickly attach to and decompose plant tissue. Each species has developed spec ific sets of 185
enzymes to break down lignin-cellulose, the structural components of most plant ce lls, so that once 186
the enzymes of one mushroom species have broken down the lignin-cellulose to its fullest potential, 187

Agronomy 2020 , 10 , x FOR PEER REVIEW 6 of 20
other saprophytes utilizing their own repertoire of enzymes can reduce this materi al even further 188
[26]. References on reuse of SMS of Pleurotus spp. in new substrate formulations are compiled in Table 189
2. 190
Table 2. Reuse of SMS of Pleurotus spp. in new substrate formulations. 191
Spent substrate Growing media for cultivation of References
Pleurotus spp. Pleurotus spp. [21,66,67]
Pleurotus spp. Pleurotus sajor-caju [68]
Pleurotus spp. Auricularia polytricha [68,69]
Pleurotus spp.
P. ostreatus Stropharia rugosoannulata
A. bisporus [19,21,53,70]
[71]
Pleurotus ostreatus Pleurotus ostreatus [48,49,72-81]
Pleurotus ostreatus Pleurotus florida [74]
Pleurotus ostreatus Agaricus blazei [82]
Pleurotus pulmonarius Pleurotus abalonus [83]
Pleurotus pulmonarius Auricularia polytricha [83]
Pleurotus pulmonarius Agaricus blazei [13]
Pleurotus pulmonarius Agrocybe cylindracea [84]
Pleurotus sajor-caju Pleurotus sajor-caju [85]
Pleurotus sajor-caju Agaricus blazei [82]
Pleurotus sajor-caju Pleurotus citrinopileatus [86]
Pleurotus eryngii Pleurotus ostreatus [87]
Pleurotus eryngii Volvariella volvácea [56,57,88]
Pleurotus eryngii Pleurotus eryngii [89]
Pleurotus eryngii Agrocybe chaxingu [90]
Pleurotus eryngii Pleurotus geesteranus [91,92]
Pleurotus cornucopiae Pleurotus ostreatus [93]
Pleurotus cornucopiae Pleurotus cornucopiae [93]
Pleurotus eous Pleurotus sajor-caju [94]
Pleurotus eous Pleurotus florida [94]
Pleurotus eous Pleurotus flabellatus [94]
SMS of Pleurotus spp has been used in the cultivation of Pleurotus sajor-caju [68], A. polytricha 192
[68,69] and S. rugosoannulata [19,21,53,70]. Specifically, SMS of P. ostreatus has been used for P. 193
ostreatus [48,49,72,81,95,96], P. florida [74] and A. blazei [82]. 194
In Spain, the Mushroom Research, Experimentation and Services Center (CIE S) has carried out 195
different investigations on the reuse of a wheat straw-based substrate, prev iously used in growing 196
cycles of P. ostreatus , in new production cycles of the same fungus (double-cropping) [48,76-81] . For 197
this, the effect of the addition of different enrichment materials, such as comm ercial supplements, 198
wheat straw and wheat bran, as well as combinations with SMS of A. bisporus has been studied. In 199
the case of A. bisporus SMS, the material used was heat treated in the growing room before emptying 200
(cooking-out). P. ostreatus SMS was heat treated and neutralized with calcium carbonate (15-20 g kg- 201
1) prior to use. Gypsum (50 g kg -1) was added to the formulations as a structural agent. After mixing 202
and moistening, the new substrate was pasteurized at 60-65°C for 8 h before cooling over a period of 203
15 h to the spawning temperature (25°C). Table 3 lists some successful formul ations with indications 204
of BE. 205
Table 3. Some formulations of new substrates, based on SMSs, for cultivation of Pleurotus ostreatus. 206
Base material Supplements Biological efficiency (kg dt -1) Reference
SSP+SSA (9:1) 36.0 [48]
SSP+SSA (4:1) 39.7 [48]
SSP+WS (1:1) Promycel 600 (20 g kg -1) 42.8 [73]
SSP+WS (1:1) Champfood (20 g kg -1) 45.8 [73]
SSP+WS (1:1) Calprozime (20 g kg -1) 48.9 [73]
SSP+WS (1:1) Calprozime (20 g kg -1) 62.5 [76]
SSP Calprozime (20 g kg -1) 58.5 [76]
SSP Calprozime (10 g kg -1) 35.0 [77]
SSP Calprozime (20 g kg -1) 41.8 [77]

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SSP+WS (1:1) 65.5 [80]
SSP+WS+WB (9:9:2) 40.6 [80]
SSP 48.4 [80]
SSP+WB (9:1) 63.1 [80]
SSP: spent substrate of P. ostreatus ; SSA: spent substrate of Agaricus bisporus ; WS: wheat straw; WB: wheat bran. 207
SMS of P. pulmonarius has been used for Pleurotus abalonus and A. polytricha (alone or mixed with 208
rubber sawdust) [83], A. blazei (mixed with sunflower seed hulls) [13] and A. cylindracea (alone or 209
mixed with rubber sawdust) [84]. 210
SMS of P. sajor-caju for P. sajor-caju (double cropping with extra organic nitrogen in the form of 211
oil seed cakes) [85] , A. blazei (supplemented with urea, rice bran or ammonium sulfate) [82] and 212
Pleurotus citrinopileatus (with sawdust of Mangifera indica and rice bran) [86]. 213
SMS of Pleurotus eryngii for P. ostreatus (with poplar sawdust, beet pulp and cotton seeds) [87], 214
Volvariella volvacea (composted substrate and non-composted substrate supplemented with wheat 215
straw) [88]; mixed with cotton waste [56,57], P. eryngii (double cropping combined with cottonseed 216
hulls, ramie byproduct or ramie root mediums) [89], Agrocybe chaxingu (with wheat bran and Tenebrio 217
molitor feces) [90], and Pleurotus geesteranus (with sawdust, bran, sugar, gypsum and lime at different 218
rates) [91]; mixed with cottonseed shell and corncob [92]. SMS of Pleurotus cornucopiae for P. 219
cornucopiae (double cropping) and P. ostreatus [93]. SMS of Pleurotus eous for P. sajor-caju , P. florida and 220
P. flabellatus (mixed with fresh wheat straw) [94]. 221
3.1.3. SMS of Lentinula edodes 222
Shiitake ( L. edodes ) is another example of a primary decomposer. Traditionally, shiitake ha s been 223
cultivated on freshly cut logs, usually from the oak family. Heat treated substra tes consist of a mixture 224
of sawdust and/or other celulose-containig materials supplemented with grain, bran or other sources 225
of carbohydrates and nitrogen [97]. References on reuse of SMS of L. edodes in new substrate 226
formulations are presented in Table 4. 227
Table 4. Reuse of SMS of Lentinula edodes in new substrate formulations. 228
Spent substrate Growing media for cultivation of References
Lentinula edodes Pleurotus spp. [98]
Lentinula edodes Pleurotus ostreatus [26,99,100]
Lentinula edodes Pleurotus eryngii [26]
Lentinula edodes Pleurotus sajor-caju [101]
Lentinula edodes Pleurotus cornucopiae [100-102]
Lentinula edodes Pleurotus citrinopileatus [86]
Lentinula edodes Agaricus [19,103]
Lentinula edodes Flammulina velutipes [99,100]
Lentinula edodes Lentinula edodes [104,105]
Lentinula edodes Coprinus comatus [106]
Lentinula edodes Ganoderma lucidum [98]
Lentinula edodes Grifola frondosa [26]
Lentinula edodes Stropharia rugosoannulata [26]
Lentinula edodes Coprinus comatus [26]
Stamets [26] proposed the recycling of substrates by sequencing mushroom species on th e same 229
substrate. After shiitake mushrooms stopped producing, sterilized substrate was used in P. ostreatus , 230
P. eryngii and Grifola frondosa cultivation. After the second species in sequence had run its course, 231
sterilized substrate could be used for S. rugosoannulata or Coprinus comatus cultivation. 232
The use of the sawdust from waste shiitake bed logs with rice bran or wheat bran a s an additive 233
was investigated as a resource in the cultivation of P. cornucopiae , P. ostreatus and Flammulina velutipes 234
[99,100,102]. Rodríguez and Jaramillo [98] proposed to combin e the residual substrate of the shiitake 235
crop with coffee stalk sawdust for the cultivation of Pleurotus spp. and Ganoderma lucidum. 236
L. edodes spent substrate has been recycled in the production of other mushroom species : 237
supplemented with wheat bran, white millet and ground soybean for P. sajor-caju cultivation [101], 238
mixed into Agaricus substrate [19,103], partially replacing sawdust as substrate component for the 239

Agronomy 2020 , 10 , x FOR PEER REVIEW 8 of 20
cultivation of P. citrinopileatus [86], or partially replacing corn cob component in C. comatus substrate 240
[106]. 241
3.1.4. SMS of Volvariella volvacea and other mushroom species 242
References on reuse of SMS of other edible mushrooms in new substrate formulatio ns are 243
compiled in Table 5. Paddy straw mushroom ( V. volvacea ) is considered a primary decomposer. It 244
prefers high cellulose, low lignin containing substrate and produces a range o f cellulolytic enzymes. 245
However, common methods for cultivation use composted substrates based on cotton waste and/or 246
paddy straw [4,107]. According to Oei et al. [22], in Fujian pr ovince, China, many farmers use spent 247
mushroom compost from straw mushroom cultivation to grow Agaricus mushrooms, so they can save 248
on the cost of straw and cow manure as these are becoming too expensive. Oei et al. [22] cites Zeng 249
[108] who states that straw mushrooms use little nutrition from straw, which c an then be refermented 250
with cow manure to produce a good yield of C. comatus . After growing straw mushrooms, the spent 251
compost can also be mixed with rice bran to grow Pleurotus spp. [67,109] or dried and saved for 252
autumn to grow P. sajor-caju [4]. 253
Table 5. Reuse of SMS of other mushroom species in new substrate formulations. 254
Spent substrate Growing media for cultivation of References
Volvariella volvacea Agaricus bisporus [22]
Volvariella volvacea Pleurotus spp. [67,109]
Volvariella volvacea Pleurotus sajor-caju [4]
Volvariella volvacea Coprinus comatus [22,108]
Flammulina velutipes Agaricus bisporus [44,110,111]
Flammulina velutipes Pleurotus ostreatus [87,112]
Flammulina velutipes Lentinula edodes [113,114]
Flammulina velutipes Coprinus comatus [19,110,111]
Flammulina velutipes Flammulina velutipes [114]
Ganoderma lucidum Agaricus bisporus [110,111]
Ganoderma lucidum Coprinus comatus [19,110,111]
Hypsizygus marmoreus Hypsizygus marmoreus [115]
Hypsizygus marmoreus Pleurotus ostreatus [116]
Hypsizygus marmoreus Volvariella volvacea [56,57]
Armillaria spp. Pleurotus ostreatus [117]
Pholiota nameko Lyophyllum decastes [118]
Auricularia auricula Coprinus comatus [106]
Winter mushroom or enokitake ( Flammulina velutipes ), a primary decomposer, was initially 255
cultivated on wood logs, but now sawdust, corncob meal and cottonseed hulls a re used as the base 256
ingredients and mixed with nutritional supplements [4,119]. Ohga et al. [11 3] evaluated the cultural 257
waste from enokitake mushroom cultivation as a substitute for hardwood in shii take production. 258
Also Chai et al. [114] investigated the recycling and reutilization of enokitake cultural waste as 259
cultivating substrates, supplemented with fresh rice bran and oak sawdust for shiitake pr oduction, 260
and suppplemented with rice bran to enhance a second flush of enokitake. Utilization of spent straw 261
generated after the cultivation of F. velutipes has been recommended for the production of other 262
mushrooms, such as A. bisporus or C. comatus [19,110,111]. SMS of F. velutipes , supplemented with 263
unfermented cow dung, gypsum, and calcium superphosphate was tested for the cultivatio n of A. 264
bisporus [44]. Combinations of SMS of F. velutipes with corncob or with poplar sawdust, beet pulp and 265
cotton seeds were evaluated in P. ostreatus production [87,112]. 266
Reishi mushroom ( Ganoderma lucidum ), a species with numerous pharmacological effects, is 267
produced using wood-log cultivation technology or on substitutes using cottonseed husks, conrcobs, 268
sawdust and bagasse as main ingredients [120]. Utilization of SMS generated after the cultivation of 269
G. lucidum has been recommended for the production of A. bisporus and C. comatus [19,110,111]. 270
Buna-shimeji (Hypsizygus marmoreus ), another primary decomposer, is commonly cultivated on 271
a sterilized substrate of sawdust and/or corncobs [119]. SMS of H. marmoreus supplemented with 272
cottonseed hulls and wheat bran has been used in a second growing cycle [115] . It has also been 273

Agronomy 2020 , 10 , x FOR PEER REVIEW 9 of 20
utilized to substitute for cottonseed hulls as the substrate for P. ostreatus cultivation [116], and mixed 274
with cotton waste to cultivate V. volvacea [56,57]. 275
3.2. Spent mushroom substrate as a base of the casing layer 276
In commercial cultivation of Agaricaceous mushrooms and others species ( P. eryngii , C. comatus 277
and G. lucidum ), fructification occurs in the casing layer, material used as a top coverin g of the 278
compost usually after the substrate is colonized by mushroom mycelium, to ind uce the transition 279
from vegetative to reproductive growth [121,122]. A compost that is complete ly colonized by 280
mycelium will not on its own produce mushrooms. 281
Although the role of the casing layer has been imprecisely defined, it must have particular 282
physical, chemical and microbiological properties for it to function [123]. An ideal casing layer would 283
be high in porosity and water-holding capacity, and low in density an d salt concentration [124,125]. 284
Moreover, casing layers, made of materials of a very diverse nature, are an important source of 285
variation in terms of the yield, quality and uniformity of commercial croppi ng. 286
The success of using SMS as a casing layer has been reported by several authors [1 4,126,127]. 287
The environmental gain in replacing peat with SMS in its entirety or in percentage brings numerous 288
environmental and economic benefits. However, SMS should not be used dire ctly as a casing layer, 289
it must be submitted to some processes (thermal treatment, maturation, washing) , as shown in Figure 290
3. 291

Agronomy 2020 , 10 , x FOR PEER REVIEW 10 of 20
292
Figure 3. Steps that must be followed carefully before applying SMS as a casing layer. 293
Heat treatment (cooking-out) is done with the substrate still inside the cultiva tion rooms, at the 294
end of the harvest phase. The air temperature must be maintained for 70°C at 6-8 h ours [128,129]. 295
This process guarantees a significant reduction of pathogens and pests in the co mpost and cultivation 296
chambers; however, it represents a high-energy cost and can be considered an expensive practice. 297
Therefore, we have one more reason to use this SMS in new cultivation cycles of A. bisporus . 298
Then the SMS must be taken to undergo a maturation and washing process, whi ch takes 4 to 20 299
weeks, and requires specific facilities [130,131]. The area used for this ma turation must be close to the 300
mushroom industry, facilitating the transport of materials. Finally, some factors influence the time 301
for reusing SMS as casing, such as the origin material (raw materials used i n the composting, 302
presence/absence of the casing layer and type of casing), the conditions of recomposting (natural 303
maturation/accelerated controlled process, conditions of aerobiosis o r anaerobiosis, frequency of 304
turning and the weather), and performing an artificial washing, grinding or screening [132]. 305
Some quality indicators represent the ideal point of physical and chemical chara cteristics of 306
casing, such as electrical conductivity (EC) below 1600 µS cm -1, pH between 6.8 and 9 (ideal 7.8), 307
water retention capacity above 45%, porosity above 50% and absence of mites, nematodes and 308

Agronomy 2020 , 10 , x FOR PEER REVIEW 11 of 20
competitor molds [15]. If the SMS does not reach these quality parameters, some materials can be 309
added to obtain an optimal casing layer, for example peat, coconut fiber , limestone, and marl. 310
Pardo-Giménez et al. [14] studying the effect of sphagnum peat (SP) and SMS verified that 311
mixtures of SP + SMS in the proportions of 4:1, 3:2 and 2:3 had si milar A. bisporus yield to the control 312
casing layer (SP + limestone). The mixture of SP + SMS (1:4) produced a significantly lower yield due 313
to the high EC value (2426 µS cm -1). 314
The importance of the washing process was confirmed using two preparation method s of SMS 315
as the sole material in the casing layer. The first method (washed once based on its volume – W1) 316
produced an EC of 1616 µS cm -1 and the second method (washed twice based on its volume – W2) 317
produced an EC of 1306 µS cm -1. The Dutch commercial casing (Topterra) was used as a control and 318
it was found that mushroom yield was not significantly affected: Topterra ( 17.61 kg m -2), W1 (17.13 319
kg m -2), and W2 (16.78 kg m -2). With SMS casing, there was an increase in the dry matter of 320
mushrooms harvested: W1 (79.2 g kg -1) and W2 (78.3 g kg -1), compared to Topterra (67.0 g kg -1) [133]. 321
This is a particular benefit for mushroom destined for the canning industry, wh ere there is reduced 322
weight loss during processing. 323
To obviate the need for maturation and washing of the SMS, casing h as been separated from the 324
underlying compost at the end of mushroom crops. Nair and Bradley [134] a nd Jablonsky and Srb 325
[135] obtained similar mushroom yields from fresh casing and recycled casing. Noble and Dobrovin- 326
Pennington [65] separated the spent peat + lime casing from the underlying compost af ter cook-out 327
of a mushroom crop. After fully rewetting, the separated casing was mixed at 25% v/v with fresh peat 328
+ lime casing and produced the same mushroom yield as 100% fresh casing. Su bsequent tests (R. 329
Noble, unpublished) using 33% recycled casing showed a 4 to 5% (but not statistically significant) 330
reduction in mushroom yield compared with using 100% fresh casing. 331
Another option with regard to a circular economy is the the use of SMS for produccing worm 332
compost (vermicompost) and then using it as a substrate supplement in the culti vation of further 333
mushrooms. García et al. [71] used a non-conventional, non compost ed substrate to evaluate the 334
influence of adding vermicompost produced with SSP in the cultivation of A. bisporus . They applied 335
the vermicompost in both the substrate (0-12%) and the casing layer (0 -100%) to cultivate brown A. 336
bisporus and reported BE values as a high as 96% in three flushes. The vermicompost was produced 337
in three months, after finishing the cultivation of P. ostreatus , by adding worms to the SPP at 2000 m – 338
2 [136]. 339
4. Facilities that promote the use of spent mushroom substrate 340
Modern mushroom facilities and processes are already in place to allow the use of SMS (plus 341
casing) in new growing cycles of A. bisporus . Examples of this refer to the construction of shelves 342
(Dutch type) with mechanized nylon nets to move the compost with casing into the beds, without the 343
use of plastic films/bags (Fig. 4). 344

Agronomy 2020 , 10 , x FOR PEER REVIEW 12 of 20
345
Figure 4. A: Modern equipment for mechanicanized filling of phase III compost plus supplemen t and 346
casing together into growing rooms. The truck with compost and t he truck with casing soil from 347
different companies arrive together and one hour later the room is completely filled [137]; B: Nylon 348
nets that are below the compost (red arrow). 349
In the case of other species such as A. subrufescens , L. edodes , G. frondosa , Pleurotus spp., Agrocybe 350
aegerita , A. polytricha where production is based on plastic bags, it is recommended that they are 351
biodegradable. For F. velutipes, H. marmoreus, P. eryngii and P. nameko where production is with plastic 352
bottles, it is recommended that they be reusable (after washing and disin festation). Nevertheless, 353
there is a great challenge in the reuse and recycling of plastics for the mushroom circul ar economy. 354
5. Spent mushroom substrate impact assessment 355
Resources are getting scarce, ecosystems are threatened, and the consequences of clima te change 356
have a large impact on the living environment [9]. Thus, strategies aimed at sustainable production 357
are currently being proposed. As with all food products, mushrooms require energy, material, and 358
water inputs for their production [138]. 359
Zisopoulos et al. [17] evaluating the efficiency assessment of the industrial mush room 360
production chain found that the critical exergy loss points (CEPs) identif ied were the cooking-out 361
process of the SMS, and the phase I composting process which were related to chemical and physical 362
energy losses, respectively. In this sense, the use of SMS as a complementary materia l in the 363
formulation of new cultivation substrates can be efficient because it reduces the composting time in 364
phase I, since it is already degraded and has a high microbial community , which helps in the start of 365
the fermentation process during the phase I compost process. 366
Despite this, another high production cost in mushroom cultivation refers to transport. Several 367
authors mention that the ideal raw material used as a base for compost productio n must be close to 368
mushroom farms and available throughout the year. However, the cost of transport of raw materials 369
is 36% of the total transportation expense of mushroom farms [138]. Pea t casing accounts for 69% of 370
the raw materials transport costs [138], which further justifies the reuse of SMS as a casing layer, 371
especially after the high cost incurred in the cooking-out stage. 372
Finally another advantage of using SMS in new mushroom crops is to a void long distance 373
transport for disposal as a soil conditioner, as in the case of Netherlan ds, which generates about 374
800,000 tons of SMS per year [9,139]. Banasik et al. [9] verifi ed that adopting closing loop technologies 375
in industrial mushroom production by reusing SMS has the potential to increa se total profitability of 376
the chain by almost 11% while the environmental performance improves by alm ost 28%. 377

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6. Other waste of mushroom cultivation 378
In Brazil, the Mushroom Study Center (CECOG) has carried out different investi gations with 379
waste mushroom stipes: bottom 1 to 2 cm of the stipe which is in contact with th e substrate/casing 380
layer and is removed with peat remnants during harvesting. Due to being part of the mushroom, 381
nutrients absorbed from the compost and necessary for mushroom growth are pr esent in this biomass, 382
which can be used as a supplement in new mushroom growing cycles, if dehydrated and crush ed 383
(Fig. 5). As the quantity is insufficient for use with A. bisporus substrate supplement, we are 384
conducting studies for the supplementation of Pleurotus spawn, with A. bisporus stipe base + peat. 385
However, adaptations of technologies still need to be improved, such as t he amount of mushroom 386
stipe base to be added in the spawn, the possibility of using the mush room stipe base + soil as the 387
base of the casing layer, and the addition of mixtures of stipe base f rom different mushrooms species. 388
389
Figure 5. Use of A. bisporus stipe base as a supplement to spawn production in the cultivation of P. 390
ostreatus , a second example of the application of mushroom waste in a circular econo my . 391
7. Conclusions 392
The use of SMS in new mushroom cultivation cycles has already been reported due to its cost 393
saving and environmental viability, but in our opinion, the main d ifficultly of the process is to 394
identify the biological and agronomic parameters that affect mushroom yield. It is useless to apply 395
SMS in new cycles if the mushroom harvest is impaired and farms become n on-viable. However, 396
numerous examples are reported here where SMS was recycled into new substrates for eith er the 397
same or different mushroom species without negatively affecting yield compared wi th using 398
substrates prepared from 100% fresh raw materials. Thus, we suggest that each farm h as its own 399
specific technological study, since a small variation in the raw materi al of the compost, and 400
mushroom cultivation practices and casing layer used, can influence the entire viability of the 401
mushroom circular economy. 402
Acknowledgments: This research was supported by the Fundação de Amparo à pesquisa do Est ado de São 403
Paulo in Brazil (FAPESP 2018/21492-2, 2015/15306-3) and the Diputación Provincial de Cuenca in Spain. 404