Waste apple wood: A safe and economical alternative substrate for the cultivation of Pleurotus ostreatus and Lentinula edodes

P. ostreatus (LD0003) and L. edodes (LD0040) strains were preserved in the Key Laboratory of Edible Fungus Technology of College of Agriculture, Ludong University (Yantai, China).

The materials for the cultivation of edible mushrooms, including apple sawdust, oak sawdust, corn cob, cottonseed husk and wheat bran, were all purchased from QixiaXurui Biological Technology Co., Ltd (Yantai, China).

A total of 21 sampling areas were established in the main apple producing areas (Qixia, Penglai, Haiyang and Muping) in Yantai, Shandong Province, including 13 sampling areas in Qixia, 3 in Penglai, 2 in Haiyang and 3 in Muping (Figure 1). A total of 73 apple wood samples were collected. The collected samples were left to dry in the sun and then cut into thin pieces with a profile cutting machine. A few pieces were randomly selected based on the size of the apple wood. The small branches were cut short by pruning. The sliced apple wood or short branches were placed in a Petri dish in a 60°C-blast dryer fan and baked until a constant weight, crushed and then sifted through a 0.5 mm sieve.

Figure 1

P. ostreatus and L. edodes were cultivated with apple wood sawdust as the main materials (Table 1). Formulae 2 and 4 were the best formulae for P. ostreatus and L. edodes cultivated with apple sawdust. P. ostreatus was also cultivated with cottonseed husk as the control (Formula 1), while L. edodes was cultivated with oak (Quercus mongolica) wood sawdust (Formula 3) as the control. Three replicates were established for each recipe (50 bags per group). The biological efficiency of each formula (fresh weight of fruiting body/dry weight of culture material) was calculated.

The microwave digestion method was used to pretreat samples to detect heavy metals and other mineral elements (Siwulski et al., 2017; Falandysz and Treu, 2019). The digestion tube was cleaned according to the manufacturer's instructions (Multiwave3000; Austria Antompa Co., Ltd, Shanghai, China). A total of 0.3 g sample (0.3 g apple wood and cultivated raw materials and 0.2 g P. ostreatus and L. edodes powder) was added to the digestion tube; 6 ml (P. ostreatus and L. edodes, 4 ml) of concentrated HNO₃ was added; the cover was opened and the sample was soaked in the fume cupboard overnight. Next, it was placed in the microwave digestion instrument for digestion, heated to 130 °C over a 10-min period, incubated at this temperature for 5 min and then heated to 180 °C over a 10-min period, at this temperature for 30 min, and then the temperature was lowered to 60 °C. After the digestion, the digestion tube was opened and placed into an acid discharge meter (140 °C). After it had drained completely, 1 ml of concentrated HNO₃ was added to dissolve it, and then the volume was fixed to 50 ml with pure water. A total of 8–9 ml of supernatant was placed in a centrifugal tube and measured using a Prodigy full-spectrum direct-read inductively coupled plasma (ICP) emission spectrometer (Teledyne Leeman Labs, Hudson, NH, USA). The content of Hg was measured using ‘GB5009.17-2014 National Food Safety Standard – Determination of Total Mercury and Organic Mercury in Food’, while that of As was measured using ‘GB5009.11-2014 National Food Safety Standard–Determination of Total Arsenic and Inorganic Arsenic in Food’. Se was determined as described by ‘GB5009.93-2017 National Food Safety Standard – Determination of Selenium in Food’.

A total of 179 pesticide residues were detected in 21 apple wood samples. The samples were pretreated as described by ‘GB23200.113-2018 National Food Safety Standard – Determination of 208 Pesticides and Metabolites Residues in Foods of Plant Origin – Gas chromatography – Tandem Mass Spectrometry Method’ and ‘GB/T 20770-2008 Determination of 486 Pesticides and Related Chemical Residues in Grains – Liquid chromatography-tandem mass spectrometry (LC-MS-MS) Method’. Gas chromatography-mass spectrometry (GCMS-TQ8040 NX, Shimadzu, Tokyo, Japan) and liquid chromatography-tandem mass spectrometry (6460 Triple Quad LC/MS; Agilent Technologies, Santa Clara, CA, USA) were used to measure the pesticide residues.

Microsoft Excel 2007 (Redmond, WA, USA) and SPSS 17.0 (SPSS, Inc., Chicago, IL, USA) were used for data processing and statistical analysis, respectively. A Pearson's correlation coefficient was used for the correlation analysis. The contents of pesticide residues and heavy metals were calculated by dry weight (mg · kg⁻¹ dw). The results represent the means of three replicates ± SD (n = 3). In the significance analysis, * and ** represent significant differences at p < 0.05 and p < 0.01, respectively.

The contents of 10 heavy metals (Pb, Cd, Hg, As, Cr, Ni, Zn, Fe, Mn and Cu) in the 73 apple wood samples collected were measured (Table 2).

Cd was not detected in 20 of the samples, and the detection rate was 72.60%. Six samples did not have detectable Hg, and its detection rate was 91.78%. Ten samples lacked detectable As, and the detection rate was 86.30%. Two samples lacked Cr, which was detected in 97.26% of the samples. All the samples contained Pb, Ni, Zn, Fe, Mn and Cu (Table 2).

The average contents of Pb, Cd, Hg, As, Cr, Ni, Zn, Fe, Mn and Cu were 2.37 mg · kg⁻¹, 0.20 mg · kg⁻¹, 0.057 mg · kg⁻¹, 0.60 mg · kg⁻¹, 3.49 mg · kg⁻¹, 4.73 mg · kg⁻¹, 18.55 mg · kg⁻¹, 486.61 mg · kg⁻¹, 84.73 mg · kg⁻¹ and 34.38 mg · kg⁻¹, respectively. The inter-sample standard deviation and variation coefficient of each element were large, which reflected the large differences between samples (Table 2). The contents of heavy metals in the top five apple wood samples are also shown in Table 3. Some of the samples had extremely high levels of contamination, indicating that contamination is an individual phenomenon.

As shown in Table 4, the maximum positive correlation between the contents of Pb and Fe occurred in the 73 apple wood samples, with a similarity coefficient as high as 0.988. The contents of Pb, Fe and Cu positively correlate with each other and are not correlated with the contents of As, Cd and Ni, indicating the joint accumulation of Pb, Fe and Cu in apple wood.

Among the 179 pesticide residues detected, five types, including thifensulfuron-methyl, rimsulfuron, nicosulfuron, iprodione and benfuracarb, were not reported because the quality control results did not meet the requirements. Among the remaining 174 pesticide residues, only 11 types of pesticide residues, including chlorpyrifos, cyhalothrin, cyper methrin, difenoconazole, tebuconazole, chlorbenzuron, carbendazim, imidacloprid, acetamiprid, cymoxanil and prochloraz were detected, while the remaining 163 pesticide residues were not detected (Table S1 in Supplementary Materials).

Chlorpyrifos was detected in all the apple wood samples, with the content ranging from 0.01 mg · kg⁻¹ to 0.75 mg · kg⁻¹. Chlorpyrifos is an insecticide that is widely used in apple cultivation (Ho et al., 2020), which explains why all the samples contained detectable amounts of chlorpyrifos.

Carbendazim was detected in 19 samples and ranged from 0.015 mg · kg⁻¹ to 4.86 mg · kg⁻¹, and the detection rate was 90.48%. Tebuconazole was detected in 16 samples ranging from 0.017 mg · kg⁻¹ to 0.44 mg · kg⁻¹, and the detection rate was 76.19%. Imidacloprid was detected in 14 samples ranging from 0.031 mg · kg⁻¹ to 0.32 mg · kg⁻¹, and the detection rate was 66.67%. Acetamiprid was detected in 13 samples ranging from 0.01 mg · kg⁻¹ to 0.11 mg ·kg⁻¹, and the detection rate was 61.90%. Chlorbenzuron was detected in eight samples and ranged from 0.037 mg · kg⁻¹ to 0.21 mg · kg⁻¹ and the detection rate was 38.10%. Only cyhalothrin was detected in two samples at 0.14 mg × kg⁻¹ and 0.19 mg ·kg⁻¹, respectively; cypermethrin was detected in two samples at 0.11 mg · kg⁻¹ and 0.19 mg · kg⁻¹, respectively; difenoconazole was detected in two samples at concentrations of 0.034 mg · kg⁻¹ and 0.083 mg · kg⁻¹, respectively, cymoxanil was detected in two samples at concentrations of 0.056 mg · kg⁻¹ and 0.083 mg · kg⁻¹, respectively. Their detection rate was 9.52%. Prochloraz was detected in one sample at a content of 0.04 mg · kg⁻¹, and the detection rate was 4.76% (Table 5, Table S2 in Supplementary Materials).

The main nutrient in apple sawdust is crude fibre, which has low contents of proteins and lipids and almost no carbohydrates. It can be used as a carbon source to cultivate wood rot edible mushrooms (unpublished). However, apple wood is agricultural waste and may have heavy metal pollution. Therefore, the raw materials were analysed for the presence of heavy metals and other mineral elements. The contents of As (0.43 ± 0.19 mg × kg⁻¹) and Hg (0.021 ± 0.0082 mg × kg⁻¹) in apple sawdust was higher than those in other cultivation materials, which could be caused by the accumulation of As and Hg in the tree owing to the long-term use of some pesticides in apple orchards (Ian et al., 1994). The Mn content in the sawdust of wild oak was as high as 198.80 ± 33.24 mg × kg⁻¹, and the content of Pb (0.90 ± 0.19 mg × kg⁻¹) was relatively high. The Ni content of corn cob (61.45 ± 10.58 mg × kg⁻¹) was significantly higher than that in other cultivation materials. In terms of other mineral elements, the content of P (11,736.02 ± 2,074.40 mg × kg⁻¹) in bran was much higher than that of sawdust, cottonseed husks and corn cobs, and the content of Ca in sawdust was relatively high (Table 6).

A total of 179 pesticide residues were tested for each cultivation material. Chlorpyrifos (0.56 ± 0.075 mg × kg⁻¹), acetamiprid (0.16 ± 0.020 mg × kg⁻¹) and phoxim (0.053 ± 0.0047 mg × kg⁻¹) were only detected in apple sawdust. Phoxim (0.058 ± 0.0047 mg × kg⁻¹) was detected in bran, and pesticide residues were not detected in the other culture materials (Table 7).

The contents of heavy metals and other mineral elements (Pb, Cd, Hg, As, Cr, Ni, Zn, Fe, Mn, Cu, P, K, S, Ca, Mg and Se) in the fruiting body samples of P. ostreatus and L. edodes were analysed. Based on the ‘GB 2762-2017 National Food Safety Standard – Limits of Contaminants in Food’ and the ‘NY/T 749-2018 Green food – Edible Mushroom’, Pb (fresh edible mushrooms 1.0 mg × kg⁻¹), Cd (fresh edible mushrooms 0.2 mg × kg⁻¹, L. edodes 0.5 mg × kg⁻¹), Hg (fresh edible mushrooms and its products 0.1 mg × kg⁻¹) and As (edible mushrooms and its products 0.5 mg × kg⁻¹) limited the contents as evaluation criteria. Pb was not detected in any sample. The contents of Cd, Hg and As were measured by their dry weight. Utilising the ‘NY/T 749-2018’, the contents of Cd, Hg and As in all the samples did not exceed the standard.

The contents of Zn and Fe in P. ostreatus cultivated in apple sawdust were higher than those of the control group, reaching 75.47 ± 5.03 mg × kg⁻¹ and 92.35 ± 6.26 mg × kg⁻¹ (Table 8). The World Health Organization (WHO) and the Food and Agricultural Organization (FAO) recommend a daily Reference Nutrient Intake (RNI) of zinc for men and women of 14 mg and 10 mg per capita per day, respectively. The requirements for teenagers are even higher. The reference intake of Fe for adult men is 13.7 mg × day⁻¹, and the reference intake for women is even higher, reaching 29.5 mg · day⁻¹ (WHO and FAO, 2004). In addition, a lack of these mineral elements is recognised as “hidden hunger” (Gregory et al., 2017). P. ostreatus and L. edodes cultivated with apple sawdust can be used as good dietary sources of Zn and Fe. Therefore, based on the contents of heavy metals and other mineral elements, discarded apple sawdust can completely replace wild oak wood and cotton seed husks (Table 8).

Fruiting body samples of P. ostreatus and L. edodes were analysed for 179 pesticide residues. Chlorpyrifos was only detected in L. edodes of formula 4, and the national standard does not limit the amount of residues in edible mushrooms. However, the European Union's limit of chlorpyrifos in mushrooms is 0.01 mg · kg⁻¹ fresh weight (EU, 2021). In this study, the chlorpyrifos content of L. edodes cultivated in the formula 4 was 0.12 ± 0.025 mg · kg⁻¹ dry weight, which merits our attention. No pesticide residues were detected in other samples. The detection of chlorpyrifos may be related to residues in the apple sawdust. Chlorpyrifos primarily controls Eriosoma lanigerum. Owing to serious infestations of E. lanigerum, all fruit farmers use chlorpyrifos. The amount of apple sawdust in formula 4 reached 81%. Therefore, the pesticide residues in the apple wood itself may have a greater impact on the pesticide residues in the fruit bodies of L. edodes.

Apple sawdust and oak sawdust were used as the main materials to cultivate L. edodes. The biological efficiency of the three flushes of mushroom cultivation was approximately 80%, and the difference in yield was not significant (Table 9). However, the use of apple sawdust and cottonseed husk as the main materials to cultivate P. ostreatus resulted in significantly different yields. Among them, the biological efficiency of P. ostreatus cultivated with cottonseed husk can reach 112%, while the biological efficiency of P. ostreatus with apple sawdust was only 90% (Table 9). Therefore, the choice of ingredients to cultivate P. ostreatus must balance the two factors of price and yield.