lentinula edodes ( shiitake ) is the second most popular edible mushroom in the world market [ 1 , 2 ] and contains polysaccharides and substances for immune enhancement and antitumor activity [ 3 , 4 ] . l. edodes mycelium does not grow well where oxygen is limited , and when it grows actively the o2 demand becomes much higher than that of other mushrooms . o2 and co2 are important factors in the cultivation of mushrooms . in general , mushrooms consume o2 to secure the chemical energy needed for mycelial growth , and fruiting bodies then generate co2 . higher concentrations of co2 lead to elongation of the stipes of mushrooms , inhibited pileus development [ 6 , 7 ] and hindered basidiospore development [ 8 , 9 ] . growing shiitake in sawdust medium takes about 100 days , including about 60 days for mycelia growth and about 40 days for browning of the medium surface . this cultivation time is relatively long when compared to the 20 to 30 days required for flammulina velutipes and pleurotus ostreatus . because oxygen is in high demand during the mushroom fruiting period , the air permeability of the filters or plugs attached to the culturing bags greatly affects development of the mushroom . insufficient ventilation due to small or clogged holes ( fig . conversely , improving ventilation in shiitake cultural bags can shorten the mycelial growth period and thus reduce cultivation cost . this study was conducted to investigate the effects of ventilation of cultivation bags on carbon dioxide concentration , mycelial growth and medium weight change in the bags . the l. edodes sawdust cultivation bags used in this study were a round pillar type , 10 cm in diameter and about 20 cm height bags were filled with 1.4 kg medium composed of 8 : 2 mixtures of oak sawdust and rice bran , including 63% water content . the top of the bag had a traditional 27 mm diameter cotton plug , a 23 mm round ball , or a 8 cm diameter plastic lid with a 15 to 45 mm hole on or under each lid and was plugged with a sponge . 2 ) with three replicated bags each ventilation lid type and the bags were incubated at 25 to 30 for four months . the co2 concentrations were measured at wk 15 . specifically , the concentration of co2 in the middle part of the bags ( fig . 3 ) was measured with a gas sampler ( model gv-110s ; gasteko , ayase , japan ) and co2 core sword ( 2h , 2hh ; gasteko ) . in addition , the mycelial growth and change in medium weight were measured under a dissecting microscope every one or two weeks for 15 wk . the l. edodes sawdust cultivation bags used in this study were a round pillar type , 10 cm in diameter and about 20 cm height bags were filled with 1.4 kg medium composed of 8 : 2 mixtures of oak sawdust and rice bran , including 63% water content . the top of the bag had a traditional 27 mm diameter cotton plug , a 23 mm round ball , or a 8 cm diameter plastic lid with a 15 to 45 mm hole on or under each lid and was plugged with a sponge . 2 ) with three replicated bags each ventilation lid type and the bags were incubated at 25 to 30 for four months . the co2 concentrations were measured at wk 15 . specifically , the concentration of co2 in the middle part of the bags ( fig . 3 ) was measured with a gas sampler ( model gv-110s ; gasteko , ayase , japan ) and co2 core sword ( 2h , 2hh ; gasteko ) . in addition , the mycelial growth and change in medium weight were measured under a dissecting microscope every one or two weeks for 15 wk . the co2 concentration was 3.83  1.33% in the bags with a traditional cotton plug and 3.76  1.18% in the bags with a cotton ball plug ( fig . the average co2 concentration was 6.05  3.57% in the bags with a 15 mm hole and 3.64  1.23% in those with a 5 mm hole . the co2 concentrations were generally 0.06 to 1.17% lower in bags with holes on both the upside and underside on the lid than in bags with holes only on the underside . the carbon dioxide concentrations in the medium changed by 1.73~7.00% over 15 wk in the bags with the traditional cotton plugs and 1.50~7.28% in those with 5 mm or 10 mm ventilation holes . the co2 concentration in bags with only one ventilation hole underneath the lid was about 13.13% , while that of bags with holes on both sides of the lid was 11.35% . these findings indicate that as mycelia digest the substrate large amounts of co2 are produced . as shown in fig . 5 , mycelial growth was greatest in bags with a 36 mm hole in only the underside of the lid ( 3.05  0.17 mm / day ) , and slowest in those with a 5 mm hole ( 2.06  0.53 mm / day ) . except in the bag with 36 mm hole , the mycelial growth was higher in bags with holes on both sides of the lid than in bags with holes underside only . mycelial growth in bags with a traditional cotton plug did not differ from that of bags with cotton balls , as indicated by values of 2.35  0.64 mm / day vs. 2.44  0.56 mm / day ( data for fig . mycelial growth in bags with 5 to 10 mm small holes , traditional cotton plugs or cotton balls for ventilation was greatest at wk 2 , after which it decreased . conversely , mycelial growth in bags with 15~45 mm holes was maintained at 2.15 to 3.22 mm / day from week 1 to 5 . in bags with 5~20 mm holes , mycelial growth was highly positively correlated with carbon dioxide concentration ( r = 0.85~0.99 ) . massive amounts of co2 can stimulate mycelial growth , but the relationship between high co2 contents by active hyphal growth and relatively less aeration in the bags was complex . dikaryotic mycelium of l. edodes can be repressed under highly dissolved oxygen conditions . at various co2 levels , 0.03% , 12.5% and 25.0% , rhizopus oligosporus grow best at the lowest 0.03% co2 . after about five weeks , the medium gradually formed protrusions on its side owing to the growth of pure hyphal mass around the edges inside the medium . at about 10 weeks , inside the brown layer , there was a white hyphal mass 1.0 to 1.5 cm thick that was dense and compact at the edge with mushroom primordia ( fig . generally mushroom primordia formation responds to a reduction in co2 , while oxygen concentration affects the quality of stored mushrooms . for example , flammulina velutipes mushrooms stored in a package with 80% oxygen showed a delayed senescence process and decreased postharvest quality loss when compared to those with lower than 50% oxygen . the medium weight loss was 0.84  0.26 g / day in bags with traditional cotton plugs and 0.75  0.22 g / day in those with cotton balls . media with ventilation holes of 45 mm lost 1.76  0.54 g / day , while that with 5 mm holes lost only 0.55  0.20 g / day . generally , media with holes on both sides of the lid lost more weight than that with holes on only the underside of the lid . the rate of medium weight loss differed depending on hole size and cultivation period . bags with smaller holes such as cotton plugs , cotton balls or holes 5 to 10 mm size lost the greatest amount of weight at wk 15 , whereas those with holes larger than 20 mm lost the most weight at week 10 . at wk 10 , weight loss rates in bags with holes 5 to 10 mm and with 45 mm holes were about 0.7 and 2.7 g / day and 1.5 and 2.0 g / day , respectively ( data not shown ) . in conclusion the effects of aeration through lid hole filters on the hyphal growth of lentinula edodes in sawdust cultivation bags were investigated . an appropriate ventilation hole size and position for conventional cotton plugs can be effective for mycelial growth , ventilation of carbon dioxide and controlling water loss from the bags . the greater the hole size on the lid of the cultivation bags , the greater the water loss from the medium . however , mycelial growth was not proportional to the ventilation hole size , but rather to carbon dioxide production . thus , the appropriate ventilation hole size for the greatest mycelial growth in the bags should be determined by considering gas diffusion and water evaporation from the bags . the co2 concentration was 3.83  1.33% in the bags with a traditional cotton plug and 3.76  1.18% in the bags with a cotton ball plug ( fig . the average co2 concentration was 6.05  3.57% in the bags with a 15 mm hole and 3.64  1.23% in those with a 5 mm hole . the co2 concentrations were generally 0.06 to 1.17% lower in bags with holes on both the upside and underside on the lid than in bags with holes only on the underside . the carbon dioxide concentrations in the medium changed by 1.73~7.00% over 15 wk in the bags with the traditional cotton plugs and 1.50~7.28% in those with 5 mm or 10 mm ventilation holes . the co2 concentration in bags with only one ventilation hole underneath the lid was about 13.13% , while that of bags with holes on both sides of the lid was 11.35% . these findings indicate that as mycelia digest the substrate large amounts of co2 are produced . as shown in fig . 5 , mycelial growth was greatest in bags with a 36 mm hole in only the underside of the lid ( 3.05  0.17 mm / day ) , and slowest in those with a 5 mm hole ( 2.06  0.53 mm / day ) . except in the bag with 36 mm hole , the mycelial growth was higher in bags with holes on both sides of the lid than in bags with holes underside only . mycelial growth in bags with a traditional cotton plug did not differ from that of bags with cotton balls , as indicated by values of 2.35  0.64 mm / day vs. 2.44  0.56 mm / day ( data for fig . mycelial growth in bags with 5 to 10 mm small holes , traditional cotton plugs or cotton balls for ventilation was greatest at wk 2 , after which it decreased . conversely , mycelial growth in bags with 15~45 mm holes was maintained at 2.15 to 3.22 mm / day from week 1 to 5 . in bags with 5~20 mm holes , mycelial growth was highly positively correlated with carbon dioxide concentration ( r = 0.85~0.99 ) . massive amounts of co2 can stimulate mycelial growth , but the relationship between high co2 contents by active hyphal growth and relatively less aeration in the bags was complex . dikaryotic mycelium of l. edodes can be repressed under highly dissolved oxygen conditions . at various co2 levels , 0.03% , 12.5% and 25.0% , rhizopus oligosporus grow best at the lowest 0.03% co2 . after about five weeks , the medium gradually formed protrusions on its side owing to the growth of pure hyphal mass around the edges inside the medium . at about 10 weeks , inside the brown layer , there was a white hyphal mass 1.0 to 1.5 cm thick that was dense and compact at the edge with mushroom primordia ( fig . generally mushroom primordia formation responds to a reduction in co2 , while oxygen concentration affects the quality of stored mushrooms . for example , flammulina velutipes mushrooms stored in a package with 80% oxygen showed a delayed senescence process and decreased postharvest quality loss when compared to those with lower than 50% oxygen . larger hole sizes are associated with greater medium weight loss ( fig . 7 ) . the medium weight loss was 0.84  0.26 g / day in bags with traditional cotton plugs and 0.75  0.22 g / day in those with cotton balls . media with ventilation holes of 45 mm lost 1.76  0.54 g / day , while that with 5 mm holes lost only 0.55  0.20 g / day . generally , media with holes on both sides of the lid lost more weight than that with holes on only the underside of the lid . bags with smaller holes such as cotton plugs , cotton balls or holes 5 to 10 mm size lost the greatest amount of weight at wk 15 , whereas those with holes larger than 20 mm lost the most weight at week 10 . at wk 10 , weight loss rates in bags with holes 5 to 10 mm and with 45 mm holes were about 0.7 and 2.7 g / day and 1.5 and 2.0 g / day , respectively ( data not shown ) . in conclusion the effects of aeration through lid hole filters on the hyphal growth of lentinula edodes in sawdust cultivation bags were investigated . an appropriate ventilation hole size and position for the mushroom hyphal growth was 15 mm on underside of the lid . conventional cotton plugs can be effective for mycelial growth , ventilation of carbon dioxide and controlling water loss from the bags . the greater the hole size on the lid of the cultivation bags , the greater the water loss from the medium . however , mycelial growth was not proportional to the ventilation hole size , but rather to carbon dioxide production . thus , the appropriate ventilation hole size for the greatest mycelial growth in the bags should be determined by considering gas diffusion and water evaporation from the bags .
the effects of aeration through lid filters on the hyphal growth of lentinula edodes ( oak mushroom ) in sawdust cultivation bags were investigated . 
 the aeration treatment levels were traditional 27 mm hole cotton plugs , cotton balls and combinations of seven hole sizes  two hole positions ( up and under ) in the lids covering plastic bags containing 1.4 kg sawdust medium at 63% moisture that had been autoclaved for one hour and inoculated with sawdust spawn of l. edodes strain 921 . 
 aeration treatment effects were measured based on the co2 concentration at the 15th wk , as well as the hyphal growth rate and degree of weight loss of bags every 14 days for 15 wk . in bags with traditional cotton plugs , the co2 concentration was 3.8  1.3% , daily mean hyphal growth was 2.3  0.6 mm and daily mean weight loss was 0.84  0.26 g. in the bags with 15 mm diameter holes , the co2 concentration was 6.0  1.6% , daily hyphal growth was 2.8  0.2 mm and daily weight loss was 0.86  0.4 g. the bags with 15 mm holes had a higher co2 concentration and lower water loss than bags with other hole sizes , but the hyphal growth was not significantly different from that of other bags . the weight loss of bags increased proportionally relative to the lid hole sizes . taken together , these results indicate that traditional cotton plugs are economically efficient , but 15 mm hole lids are the most efficient at maintaining hyphal growth and controlling water loss while allowing co2 emissions .