Shiitake (Lentinula edodes) cultivation in sawdust media consisting of kunugi (Quercus acutissima) mixed with sugi (Cryptomeria japonica): optimization of gaseous phase rate in media by three-phase-structure analysis
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The effects of the gaseous phase rate of kunugi (Quercus acutissima) sawdust media mixed with sugi (Cryptomeria japonica) determined by a three-phase-structure analysis of the fruiting body yields of shiitake were investigated. The fruiting body yield on kunugi media was significantly lower than that on commercially available hardwood-sawdust-mixture (HSM) media with 64 % water content. Three-phase-structure analysis showed that the gaseous phase rate in kunugi media was lower than that in HSM media. When the gaseous phase rate in kunugi media was increased to the level in HSM media by decreasing the water content to 56 %, the fruiting body yield on kunugi media also increased. These results suggested that kunugi sawdust could be used for shiitake cultivation if the gaseous phase rate in the media was optimized. Because sugi has a lower specific gravity and higher porosity than kunugi, mixing sugi sawdust up to 30 % with kunugi media caused an increase in the gaseous phase rate, and the fruiting body yield reached the same level as that in HSM media. These results suggested that kunugi media could be used for shiitake cultivation by mixing with sugi sawdust.
KeywordsLentinula edodes Mycelial block cultivation Three-phase-structure Quercus acutissima Cryptomeria japonica
Kunugi (Quercus acutissima) wood has been mainly used for the production of dried shiitake [Lentinula edodes (Berk.) Pegler] by bed-log cultivation in Kyushu, Shikoku, and west central Honshu, and has also been most commonly used in Miyazaki Prefecture. In the past, the nurturing of kunugi seedlings had been promoted by Miyazaki Prefecture to compensate for the increased demand for kunugi logs. However, dried shiitake production has recently decreased due to the decline in price and the aging of the growers. If the demand for kunugi-logs continues to drop, the current volume of growing kunugi forests would be too aged and they would eventually be left unused. The sprout growth function of kunugi is weakened in trees that are 30 or more years old as reported by Tanaka . Therefore, acceleration of the effective use of overgrown kunugi logs is required to keep kunugi forest habitats sound in the future. Shiitake production by means of sawdust-based cultivation has recently increased to about 90 % of the total fresh shiitake production in Miyazaki. The increase of prices due to the increase of the cost of transportation of raw materials is becoming a problem, since most of the sawdust for shiitake production is imported from other prefectures in hardwood-sawdust-mixture (HSM) form. The possible use of kunugi wood instead of HSM as the substrate for sawdust media would benefit the growers in Miyazaki, but kunugi sawdust is not popular with the growers because shiitake cultivation takes longer on this medium than on HSM media.
On the other hand, Miyazaki Prefecture is one of the most prosperous areas in Japan for the production of sugi wood (Cryptomeria japonica D. Don). The demand for sugi sawdust has recently increased for supply of the woody biomass generation [2, 3]. However, the use of a large quantity of forest residue and lower sugi wood is still issue, and a large quantity of sugi sawdust is produced in sawmills as factory waste every year. Thus, the price of sugi sawdust for mushroom cultivation is estimated to be around one-third of that of HSM . The cost of production of fresh shiitake would be reduced if the plentiful and inexpensive sugi sawdust could be used for the cultivation. However, Nakajima et al.  reported that ferruginol in methanol extracts of sugi inner bark inhibited shiitake mycelial growth. Matsui et al.  also showed that mycelial growth inhibition of shiitake is probably due to a synergistic effect of ferruginol and sandaracopimarinol, which are the major terpenoids in sugi wood. These results suggest that the poor shiitake mycelial growth on sugi wood meal compared with that on hardwood is caused by the inhibitory effects of extractives contained in sugi wood . In addition, the mycelial growth of shiitake on sugi media was found to be only about 60 % of that on konara (Q. serrata Murray) media, even if extract-free sugi sawdust was used . Therefore, shiitake cultivation on media using only sugi sawdust seems to be difficult.
In our previous study , three-phase-structure analysis was used to investigate the changes of the physical properties of the sawdust media that result from mixing them with volcanic ash. The results suggested that the change in the three-phase-structure of sawdust media by mixing it with ash or the decrease in volume of the gaseous phase caused the reduction in shiitake yields. Thus, the fruiting body yields of shiitake should be significantly affected by the physical properties of sawdust media illustrated as by their three-phase-structure. If the disadvantages of kunugi sawdust, which requires a longer incubation period than HSM, can be conquered by optimizing the three-phase-structure of the media, the growers could use kunugi sawdust instead of HSM as the substrate of media for shiitake cultivation.
In this study, the effects of the gaseous phase rate in kunugi sawdust media mixed with sugi, as determined by three-phase-structure analysis, on the fruiting body yields of shiitake were investigated.
Materials and methods
Strain and materials
A commercial dikaryotic strain of shiitake (L. edodes) No. 1791 (the variety registration number of Ministry of Agriculture, Forestry and Fisheries, Japan) was used in this study. This strain has been extensively used in the sawdust-based cultivation of shiitake in Miyazaki. The sawdust spawn was obtained from a commercial source and was used for the experiment involving the flushing of the fruiting body. A strain subcultured on potato dextrose agar media (Nissui Co., Ltd.) was used for the experiment in which mycelial growth was measured.
Commercially available sawdust, namely hardwood-sawdust-mixtures (abbreviated as HSM, Castanopsis spp.: Quercus spp. with the exception of Q. acutissima and Q. serrata: others = 5:3:2, wt/wt, dry weight), was used for the substrate of the media. HSM has been commonly used for the sawdust-based cultivation of shiitake in Miyazaki. Kunugi (Q. acutissima) and sugi (C. japonica) that were supplied from mushroom cultivator in Miyazaki and had been seasoned for over 3 months were also used. The particle size distribution of sawdust used for flushing the fruiting body was as follows: under 1.0 mm: 1.0–2.0 mm: over 2.0 mm = 2:7:1. The sawdust used for the measurement on mycelial growth and the analysis of the three-phase-structure was sieved to a particle size of 1.0–2.0 mm constituted with 70 % of sawdust used for flushing the fruiting body. Moreover, the particle size of the sawdust used in the experiment involving the measurement of physical properties was 0.25–1.00 mm, similar to that described by Hu et al. .
Wheat bran (Toku-Fusuma40, Nisshin Seifun Co., Ltd.) and rice bran were used as the nutrients in the media.
Preparation of the media
The control media were composed of 27 % (wt/wt, dry weight) HSM sawdust, 4.5 % (wt/wt, dry weight) wheat bran, 4.5 % (wt/wt, dry weight) rice bran and 64 % (wt/wt) tap water. In the experimental media, the kunugi sawdust was increased from 27 to 31 or 35 % (wt/wt, dry weight) and the water contents decreased from 64 to 60 or 56 % according to the mixing rate of the sawdust. In some of the experimental media, kunugi sawdust was replaced with sugi sawdust at rates of 10–50 % at a 64 % water content. The mixing rate of nutrients was the same as those in the control media.
Mycelial growth on sawdust media
Test tubes (internal diameter, 28 mm; length, 200 mm) were filled with 50 g (wet weight) media and were then autoclaved at 121 °C for 20 min. The strain mycelium was grown on PDA (potato dextrose agar, Nissui Co., Ltd.) agar media at 20 ± 1 °C with a relative humidity of 60 ± 3 % for 14 days. A disk of mycelia was cut out of the PDA agar media using a cork borer (internal diameter, 4 mm) and was then placed in the top center of the media for inoculation. The inoculated test tubes were incubated at 20 ± 1 °C with a relative humidity of 60 ± 3 %. The mycelial growth length was measured at predetermined periods on two points around the test tube using an electronic caliper. The results are given as the means of five replicates.
Flushing conditions of fruiting body
The prepared media (2.7 kg wet weight) were filled in a polypropylene bag with one filter (Sun-bag SS37, 37 mm diameter, Santomi Sangyo Co., Ltd.) and autoclaved at 121 °C for 50 min. The spawn (approximately 12 g) was inoculated onto the media in the bag. The inoculated media were incubated at 21 °C with a relative humidity of 70 % for 100 days under dark conditions. After incubation, only the upper side of the bag was removed. The water was poured between the mycelial block and the bag, and then a rubber band (O-band#370, Kyowa Co., Ltd.) was set at the side of the bag for fixing the mycelial block. The fruiting bodies were flushed 7–8 times for 180–200 days at temperatures 13 °C for 16 h and 22 °C for 8 h of a day with a relative humidity above 80 %. The illuminance was 500–900 lx at 22 °C, and was dark conditions at 13 °C. The harvest and recuperation period in each flush were approximately 25 days under the conditions described above. All fruiting bodies were harvested when 70–80 % of their gills were exposed, and the fruiting bodies were sorted for each the diameter (2L: 8 cm or more, L: 8–6 cm, M: 6–4 cm, S: 4–3 cm, 2S: less than 3 cm) of the pileus, and then the number and the weight of fresh fruiting bodies were recorded. Just after each flush, the mycelial blocks were inverted in a plastic container that was 6 cm in depth. Water was supplied for 4 h by soaking the flushing surface of the inverted block.
Physical properties of wood chips and sawdust
Specific gravity and porosity of wood chips
The specific gravity was determined using wood chips (approximately 1 cm3) according to the methods mentioned by Hu et al. . Wood chips were prepared using kunugi or Castanopsis spp., which was contained primarily in HSM instead of HSM.
Water retention of sawdust
Three-phase-structure analysis of sawdust media
The results were given as the means of three replicates.
All data collected were represented as mean ± standard deviation (SD). Statistical analyses were performed using the statistical analysis tool R (version 3.0.2). Differences in the data between the two groups were analyzed using a two-sided Student’s t-test. In addition, differences in the data among three or more groups were analyzed using Tukey’s HSD (honestly significant difference) test.
Results and discussion
Mycelial growth and fruiting body yield in HSM and kunugi media at 64 % water content
The physical properties of wood chips and sawdust, and the three-phase-structure analysis of sawdust media
Physical properties of HSM, kunugi and sugi
0.52 ± 0.02
0.64 ± 0.02***
0.33 ± 0.03
65.3 ± 1.26
57.2 ± 1.27***
77.8 ± 2.12
314 ± 12.7
246 ± 41.0**
389 ± 31.6
Three-phase-structure in HSM, kunugi and sugi sawdust media at 64 % water content
Indexes of three-phase-structure
Solid phase rate (%)
19.4 ± 0.8a
22.8 ± 0.3b
10.4 ± 0.5c
Liquid phase rate (%)
42.4 ± 1.3a
51.6 ± 1.6b
30.4 ± 1.0c
Gaseous phase rate (%)
38.1 ± 1.9a
25.6 ± 1.6b
59.2 ± 1.5c
Total porosity (%)
80.6 ± 0.8a
77.2 ± 0.3b
89.6 ± 0.5c
Degree of saturation (%)
52.6 ± 2.0a
66.9 ± 2.0b
33.9 ± 1.3c
The indexes of three-phase-structure were also significantly different between the two wood species. The solid phase of 64-kunugi media was 1.33 times (P < 0.001) and the liquid phase was 1.25 times (P < 0.001) those of 64-HSM media, but the gaseous phase was 0.63 times (P < 0.001) that of 64-HSM media. The total porosity of 64-kunugi media was 0.94 times that of 64-HSM media (P < 0.001). Therefore, despite the media having the same water content, the degree of saturation of 64-kunugi media was 1.34 times higher than that of 64-HSM media (P < 0.001).
These results suggested that the causes of the decrease of the fruiting body yields in kunugi media at 64 % water content could be attributed to the higher liquid phase and lower gaseous phase in kunugi media than in HSM media.
Three-phase-structure of the media with lower water content than the control media
The above results suggested that the gaseous phase rate in kunugi media could be increased to the same level as that in 64-HSM media (control media) by decreasing the water content to 60 or 56 %.
Effects of decreasing the water content on fruiting body yields in kunugi media
Three-phase-structure of kunugi media mixed with sugi sawdust
Effects of mixing sugi sawdust on fruiting body yields in kunugi media
Because the three-phase-structure in the media was markedly influenced by the physical properties of the wood, the specific gravity and water retention of the wood are important factors in selecting a substrate for the media for the sawdust-based cultivation of mushrooms. HSM would have a higher porosity compared with rates of kunugi sawdust since HSM was contained primarily Castanopsis spp., and so the water retention of kunugi sawdust must have been lower than that in HSM. In addition, kunugi media with a 64 % water content had a higher liquid phase rate and a lower gaseous phase rate due to the lower water retention of kunugi sawdust compared with the rates of HSM sawdust, and so ventilation through the media should be prevented. Shiitake mycelia seemed to be difficult to grow in the center part of the media due to a lack of oxygen. As shown in Fig. 4, the fruiting body yields on HSM media were highest in the second flush and gradually decreased as the number of flushes increased, but the yields on kunugi media with 60 or 56 % water content increased in the later flushes due to the improvement of ventilation in the media resulting from decay and evaporation. To increase the fruiting body yields in the earlier flush stages on kunugi media as occurs with HSM media, decreasing the initial water content to 60 or 56 % from the standard 64 % is important, as it promotes mycelial growth in the center part of the media.
The rate of the gaseous phase in kunugi media could also increase as a result of mixing with sugi sawdust because sugi wood has lower specific gravity and higher porosity as shown in Tables 1 and 2. Therefore, on the media in which 30 % of kunugi sawdust was replaced with sugi, the fruiting body yields were almost the same as on HSM media because the ventilation in kunugi media was improved through the mixing with sugi sawdust. Since sugi sawdust used to this experiment had been seasoned for over 3 months, it seemed that the inhibitory effects of extractives contained in sugi sawdust for the mycelial growth of shiitake is not almost. Physical properties of kunugi media would had been improved by the mixing of sugi sawdust which does not contained the inhibitory components.
As described above, the cost of sugi sawdust is relatively low, approximately one-third the cost of hardwood sawdust. Thus, the use of sugi sawdust for the sawdust-based cultivation of shiitake has been repeatedly attempted, but those attempts were not successful. If the method prepared by the mixing of sugi sawdust with kunugi media is used, it seems to be expected enough use of them in the sawdust-based cultivation of shiitake. Our results showed that the fruiting body yields in earlier flush stages in kunugi media partially mixed with sugi sawdust were better rather than those in the media only composed of kunugi sawdust. The effective utilization of unused overgrown kunugi wood and the cost reduction in the sawdust-based cultivation of shiitake could be achieved through its combination with inexpensive sugi sawdust, an industrial waste product.
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