Development of Mycelium Materials Incubating Pleurotus Ostreatus Fungi With Different Substrates Composed of Poplar Sawdust and Cottonseed Hull

: The mycelium materials incubating Pleurotus ostreatus fungi based on different 13 substrate compositions were developed, the main components of which were poplar sawdust and 14 cottonseed hull in different proportions. The hyphae on the surface of the samples become dense 15 from appearance due to the addition of cottonseed hull. The Fourier Transforms Infrared analysis 16 revealed that the cellulose, hemicellulose and lignin in substrates of all samples were degraded in 17 different degrees owing to utilization by hyphae growth. The morphology and mechanical 18 properties of the mycelial materials changed as the substrate compositions varied. The difference 19 of properties among all mycelium materials was mainly attributed to the growth of mycelium and 20 different substrate compositions. And the mycelium material (the ratio of poplar sawdust to 21 cottonseed hull was 1) exhibited highest strength and lowest compression set, indicating that its 22 size recovery capability was best. In comparison, the substrate of this material was more favorable 23 to the growth of the mycelium and it showed optimal comprehensive performance among all 24 samples. The mycelium material showed good potentiality for packaging application.


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With the development of the global commodity economy, a large number of plastic cushion 29 packaging materials were widely used to protect commodities during transportation, storage and 30 distribution, such as expanded polystyrene (EPS) and polyurethane (PU), owing to their low 31 density, moisture resistance and excellent cushioning properties (Chen et al., 2015). However, 32 these packaging materials are basically petroleum-based materials of organic synthetic polymers, 33 which have caused serious environmental pollution due to their abuse and non-biodegradability, 34 which was one of the environmental problems that need to be solved urgently (Singh et al., 2016). 35 Therefore, many scholars had turned their attention to natural origin polymers, such as cellulose 36 foam (Li et  plants, grasses, vines and their processing residues and wastes as raw materials, biomass materials 39 were produced as new materials with excellent performance through physics, chemistry and 40 biology technologies (Narayan, 2006). In recent years, mycelium materials were initiated by 41 incubating saprophytic fungi with plant waste to fabricate bio-degradable porous materials. As a 42 new type of biomass material, the mycelium material was considered to be a new alternative to 43 petroleum-based materials (Melorose et al., 2015). It was a field with little attention but full of 44 research potential (Fratzl and Barth, 2009). The mycelium material was obtained by inoculating 45 saprophytic fungi on agricultural wastes rich in cellulose and lignin (López Nava et al., 2016), and 46 the fungal vegetative hyphae secrete cellulase, lignin peroxidase and laccase, which were used to 47 degrade cellulose, hemicellulose and lignin in agricultural wastes such as wood chips, straw and 48 cottonseed hull to obtain vegetative growth substances (Hoa and Wang, 2015;Jiang et al., 2016). 49 During this process, the mycelium penetrated into the matrix and continuously grew staggered to 50 form a three-dimensional network structure that wrapped the matrix (Bonfante and Genre, 2010). 51 After the growth of the mycelium was completed, it was dried to obtain the mycelium material. 52 Considering the capacity to consume and utilize agricultural waste, the fast growing rate of 53 mycelium, its biodegradability and reusability as fertilizer after being discarded, its unique 54 mechanical and aesthetic properties (Attias et al., 2020), which can fully meet the requirements of 55 sustainable development (Teixeira et al., 2018). 56 The performances of mycelium material were affected by many factors ( example, adding glucose to the standard medium was more easily absorbed by the mycelium than 66 cellulose And it promoted the biosynthesis of lipids and proteins in the mycelium that acted as a 67 "plasticizer", thereby improving the ductility of the mycelium (Haneef et al., 2017). It was also 68 reported that the incorporation of glucose would increase the porous structure of the mycelium and 69 the addition of lignin made the hyphae with slender characteristic (Antinori et al., 2020). Similar 70 to mycelia cultured on culture medium, the properties of mycelia cultured by different agricultural 71 by-products were also different. For example, the compressive strength of mycelium materials 72 cultured on sawdust and bagasse was higher than that of pure sawdust and bagasse (Joshi et al., precisely because of the many possibilities of mycelium materials that it had become a popular 78 choice for replacing some petroleum-based materials.

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The purpose of this study was to develop the mycelium materials using Pleurotus ostreatus 80 mycelium based on different substrates composition, which was mainly composed of poplar 81 sawdust and cottonseed hull. By comparing morphological characteristic, physical property, 82 chemical composition, thermal degradation and mechanical properties of the mycelium materials, 83 the effect of substrates composition on the properties of the materials were investigated.

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The microstructures of all samples were analyzed by SEM using SU-5000 (Hitachi Co. Ltd, 104 Matsuda, Japan). Before testing, the samples were cut into size of 5mm 5mm in liquid nitrogen. 105 After sputtering gold plating, the measurement was performed using SU-5000 at an accelerating 106 voltage of 5kv. 107

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The sample was placed in an environment of 25°C and 50% RH to complete the 109 measurement. The density of the materials was calculated by the following formula. 110 where d is the density in g/cm 3 , m is the mass of the sample material in g, and v is the sample 112 volume in cm 3 113

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A Konica Minolta CR-400 Chroma meter (Minolta Co. Ltd, Tokyo, Japan) was used to 115 measure the color of the material surface, where L* represents brightness, a* represents red and 116 green, and b* represents yellow and blue. The white standard plate (L*=94.28, a*=-1.39, b*=5.22) 117 was used as a comparison. The total color difference (△E) was calculated using formula as 118 follows: 119 (2) 120

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The Fourier infrared spectrophotometer Spotlight 400 (PerkinElmer, Waltham, Massachusetts, 122 USA) was used for identification of the materials in the range of 4250 cm -1 to 500 cm -1 with a 123 resolution of 4 cm -1 , and repeated scanning 32 times. 124

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The TGA was performed on a NETZSCH TG 209 F1 thermal analyzer (NETZSCH Scientific 126 Instruments Trading (Shanghai) Co., Ltd., Germany) at a heating rate of 10°C/min under a 127 nitrogen atmosphere from 30°C to 800°C. The weight of the samples was approximately 5-10 mg. 128

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An electronic universal testing machine DDL-100 (Changchun Institute of Mechanical 130 Science Co., Ltd., Changchun, China) was used for static compression test. The samples with 131 100 100 20mm (length width height) were placed on the platform of the machine for 132 compressing with a pressure plate at a speed of 12 mm/min. The stress-strain curves of the 133 materials were obtained from these measurements. 134

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The CS was tested on the electronic universal testing machine DCP-KY3000 (Sichuan 136 Changjiang Papermaking Instrument Co., Ltd., Sichuan, China). The samples were placed on the 137 platform of the machine for compressing with a pressure plate at a speed of 12 mm/min. The 138 compression was 20% of the sample thickness. After 15 minutes of compression, the pressure was 139 released and the thickness was measured. The CS of the materials were calculated by the 140 following formula. 141 where CS is the Compression set in %, is the initial thickness of the sample in mm and is 143 the final thickness of the sample in mm. 144

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The statistical analysis of the data was performed through ANOVA using SPSS software 146 (Version 20.0, Inc., Chicago, IL, USA). The differences among mean values were evaluated by 147 Duncan's multiple range tests. Significance level was defined at 5%. The data were represented as 148 means ± standard deviation. cottonseed hull. The hyphae on the surface of S10C0 was relative sparse, which can't wrap the 155 culture medium well and the internal hyphae had poor adhesion. The SEM result showed that the 156 mycelia of S10C0 were slender and it became thick as the content of cottonseed hull increased. It 157 suggested that the internal adhesion of the material would increase compared with S10C0. This was 158 attributed to that the mixture of cottonseed hull and sawdust was conducive to the growth of 159 hyphae and a certain amount of cottonseed hull would promote the colonization of strains. On the 160 one hand, cottonseed hull is a by-product of cotton and it contain more crude protein, crude fat 161 and soluble sugar (Yu et al., 2020). On the other hand, the addition of cottonseed hull would 162 reduce the C/N ratio of culture medium, which was propitious to promote hypha growth better. 163 However, the growth of S1C9 and S0C10 were weaker than S7C3, S5C5 and S3C7, which may be due 164 to the existence of free gossypol in cottonseed hull and the increase of gossypol content affects the 165 growth of hyphae using cottonseed hull. So, the growth of mycelium was closely correlated with 166 the substrate composition. Similarly, Sisti (Sisti et al., 2021) uses different proportions of bran to 167 verify the effect of the matrix on the surface morphology of the material. It was found that the 168 width of the mycelium increased by up to 38% in the material with 30% wheat bran. 169 The SEM image at 5um scale in Fig. 1 showed the growth of hyphae attached to matrix 170 particles. Mycelia decomposes and digests the matrix by its own enzyme, and the hypha was 171 macroscopically wrapped around the matrix, just like glue bonding the matrix. It can be seen from 172 the SEM image of 20um scale in Fig. 1 that the hyphae on the surface of mycelium material 173 gradually change from the intersecting and winding three-dimensional network structure to the 174 hyphal membrane structure, which were stacked layer by layer. This was similar to the SEM 175 results of freeze-dried mushroom slices reported by Liuqing (Liuqing et al., 2018). It reflected that 176 the surface layer of mycelium in the final material were similar to mushroom solid, which was one 177 of the reasons for the soft elasticity of the material texture. 178 179 Fig. 1 Visual and scanning electron micrograph of the sample. S10C0, S9C1, S7C3, S5C5, S3C7, S1C9, 180 S0C10 eye view and scanning electron micrograph (50um); Growth image of hyphae attached to 181 substrate particles at 5um; The phenomenon of "hyphae film" on the surface of the sample at the 182 20um. 183

Density and color
184 Fig. 2 showed the density and surface color difference of all mycelium material samples. 185 With the increase content of cottonseed hull, the density gradually increased, and the density of 186 pure cottonseed hull sample (S0C10) was the highest, which was attributed to the fact that the 187 density of cottonseed hull material was higher than that of sawdust. The density of all samples 188 ranged from 0.21 to 0.29 g/cm 3 , which were comparable to the previous reported results that were be noted that there were many factors that affected the material density of mycelium, such as 194 fungus species, substrate formula and process conditions. 195 The mycelium of Pleurotus ostreatus is white and the substrate is yellow-black. The surface 196 of a well-grown sample would be covered with white mycelium. Therefore, the degree of   substances such as cellulose, hemicellulose and lignin in sawdust and cottonseed hull to obtain 216 nutrition. Therefore, the growth of hyphae can be reflected by infrared analysis of substrate after 217 culture. Fig. 3 was the infrared spectrum of the substrate. Table3 showed the distribution of 218 infrared spectral bands of all sample substrates. 219 The infrared spectrum of S10C0 band was similar to that of poplar (Demcak et al., 2017), 220 suggesting that S10C0 matrix was close to uncultured state. Compared with S10C0, the C-O 221 deformation absorption peak (1030cm -1 ) (Mohan et al., 2006) and the C-H stretching vibration 222 absorption peak (1370cm -1 ) in the secondary alcohols and fatty ethers characterizing cellulose and 223 hemicellulose in other samples were weakened to some extent, and the weakest absorption peaks 224 appeared in S7C3 and S5C5. This indicated that cellulose and hemicellulose in the substrate were 225 degraded and utilized by hyphae growth. And the absorption peak of C=O stretching vibration 226 (1731 cm -1 ) (Saetun et al., 2017) in the non-conjugated ketones and ester groups of hemicellulose 227 was also obviously weakened. These results indicated that hyphae grew well with substrate in 228 S7C3 and S5C5, and hemicellulose degraded more than cellulose. In addition, the methylene (CH2) 229 bending vibration absorption peak (1454cm -1 ), lignin phenolic ether bond C-O stretching vibration 230 (1241cm -1 ) and carbonyl conjugated aryl ketone C=O (1638cm -1 ) (Fungi, 2005) which 231 characterized lignin weakened or even disappeared in other samples. The characteristic absorption 232 peak (1326cm -1 ) (Kubo and Kadla, 2005), which characterized the vibration of syringyl (S) and 233 the condensation of guaiacyl (G) and syringyl (S), also weakened to varying degrees. It indicated 234 that lignin in other samples were degraded and utilized by the growth of hyphae to varying 235 degrees. To sum up, the FTIR results revealed that the cellulose, hemicellulose and lignin of other 236 samples were degraded in different degrees comparing with S10C0. The colonization degree of 237 mycelium in other samples were better than S10C0. In comparison, the substrate of S5C5 was more 238 favorable to the growth of the mycelium. 239 240 Fig. 3 Infrared spectrum of all mycelium materials 241 Table3 Infrared spectrum characteristic peaks and their assignments of all mycelium materials 242

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The TGA curves of all samples were shown in Fig. 4. The mycelium material exhibited 245 similar thermal degradation behavior. It can be determined that all samples showed four stages of 246 quality loss. In the first stage, the mass loss temperature was between 30℃ and 134℃, which was 247 mainly the loss of free water, adsorbed water and crystal water (Joseph et al., 2003). In the second 248 stage, the temperature was between 230℃ and 310℃, which was mainly the decomposition of 249 organic components such as polysaccharide, chitin, amino acids and lipids in hyphae. The 250 temperature of the third stage occurs at 320℃-410℃, which was attributed to the pyrolysis of 251 cellulose and hemicelluloses (Yang et al., 2007). The second and third stages were the main stages 252 in the thermal degradation process. The temperature in the fourth stage was 440℃-800℃, owing 253 to the pyrolysis of lignin, thermal degradation of sample residues and oxidative decomposition 254 products. The thermal degradation curve of S10C0 was different from that of other samples in the 255 range of 370℃-440℃. It was speculated that the hyphae of S10C0 grew poorly, and the wood chip 256 substrate still contained relative many lignin that had not been decomposed by the hyphae (Borsoi 257 et al., 2013). into two stages. When the strain was less than 0.16 (S5C5 and S3C7 were 0.1), the stress increased 264 slightly. When the strain increased sequentially, the stress increased rapidly, the samples collapsed 265 and was compacted. The S5C5 showed highest stress and strain level, followed by S3C7, which 266 means that they can bear more stress under the same strain. The low stress and strain levels of 267 S10C0 and S9C1 indicated that under the same stress, the material had greater strain. Combined 268 with morphological analysis, the colonization level of S10C0 and S9C1 strains was poor, the ability 269 of mycelium to bind to the matrix was weak, and the material collapses rapidly under low stress.

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This difference of mechanical property among all samples were mainly attributed to the growth of 271 mycelium and different substrate. The mycelium material was viscoelastic material instead of 272 single elasticity or viscosity. From the microscopic performance of materials, when the material 273 was subjected to external force, on the one hand, the molecular chain deforms and after the 274 external force was removed, the deformation recovers and shows elasticity; On the other hand, the 275 molecular chain slips and after the external force was removed, the deformation can't be 276 completely restored, resulting in permanent deformation, which shows viscosity.

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The CS of all samples was presented in Fig. 6. It showed that the CS increased firstly and 278 then decreased with increasing of cottonseed hull. The CS of S10C0 was 19.41%, which was the 279 largest of all samples. The larger CS meat the anti-compression deformation capacity and 280 resilience of the material was worse. The CS value of S5C5 was significantly lower than that of the 281 other samples, indicating that its size recovery capability was best. It was ascribed to its high 282 degree of colonization. The mycelium grew more flourishing, the cohesiveness of material was 283 much stronger, leading to the enhanced resilience and size recovery capability of the material. It 284 also suggested that the S5C5 material possessed best cushioning property among these samples.

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The mycelium materials were successfully developed by incubating Pleurotus ostreatus fungi with 292 different substrates compositions, which were mainly composed of poplar sawdust and cottonseed 293 hull in different ratios. The difference of properties among all samples was mainly attributed to the 294 growth of mycelium and different substrate compositions. The hyphae on the surface of the 295 samples become dense from appearance for the addition of cottonseed hull. The FTIR results 296 revealed that the cellulose, hemicellulose, and lignin in substrates of all samples were degraded in 297 different degrees due to utilization by hyphae growth. The hyphae of S5C5 grew most abundant on 298 the surface of the material and the S5C5 sample exhibited the highest stress and strain level, 299 followed by S3C7, which meant that they can bear more stress under the same strain. The CS of 300 S5C5 was significantly lower than that of the other samples, indicating that its size recovery 301 capability was best. In comparison, the substrate of S5C5 was more favorable to the growth of the 302 mycelium and it showed optimal comprehensive performance among all samples. As a green 303 material, the mycelium material showed good potentiality through different processing processes 304 in the application of some areas, such as packaging materials, insulating materials and building 305 materials. It is also a good topic and many issues are worth studying in the future.