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Abstract Phanerochaete chrysosporium was selected as the production strain of laccase， and the effects of stirring speed， ventilation volume， culture temperature， inoculation amount and initial pH of medium on laccase production by liquid fermentation in cylinder were studied. On the basis of single factor test， an orthogonal test was carried out to find optimal conditions for laccase production P. chrysosporium through liquid fermentation. These results showed that the stirring speed of fermentation cylinder had the highest effect on laccase production， and the optimal conditions were shown as follows： the temperature at 28 ℃， the rotating speed at 300 r/min， the ventilation volume of 5 L/min （ventilation ratio of 1.0 vvm）， the initial pH of medium of 5， and the inoculation amount of 15%， which gave the highest laccase level of 14.86 U/ml.
Key words White-rot fungi； Phanerochaete chrysosporium； Fermentation cylinder； Laccase； Industrial production； Optimization condition
Crop straw is a kind of feedstuff with wide source and low cost， but its lignin which is difficult to be degraded reduces its feeding value as fodder. Therefore， during the treatment of crop straw for preparing fodder， the degradation of lignin becomes a very important link. White-rot fungi are a type of fungi capable of releasing lignin and other wooden components[1]. Currently， three kinds of lignin-degrading enzymes produced by white-rot fungi， i.e.， manganedeperoxidase （MnP）， ligninperoxidse （LiP） and laccase are studied the most[2-3]. Laccase is the kind of polyphenol oxidase， which is produced at the secondary metabolism stage of white-rot fungi， and it is difficult to achieve a higher yield， which is also the main reason why the wide application of laccase is limited. In this study， Phanerochaete chrysosporium with better degradation effect on straw was selected for the experiment of laccase production by white-rot fungus in a mechanically-stirring type fermentation cylinder， to investigate the main factors including stirring speed， ventilation amount， temperature and pH value on the production of laccase by 2 kinds of P. chrysosporium. This study will provide a basis for the industrial production and the effective utilization of crop straw.
Materials and Methods
Experimental materials
Experimental strain P. chrysosporium was preserved in the microbiology lab of Jiangsu Agri-Animal Husbandry Vocational College.
Experimental medium PDA medium was prepared from potato 200 g， glucose 20 g， agar 20 g and distilled water 1 L， with natural pH value. Liquid fermentation medium was prepared from peeled potato 200 g by the steps of boiling for 30 min， filtering with gauze， adding 20 g of sucrose， and water to 1 000 ml after dissolution as well as 10 g of glucose， filling to 250 ml triangular flasks according to 100 ml per flask and performing intermittent sterilization at 121 ℃. Main instruments and equipment
Autoclave sterilizer （Shanghai Boxun Industrial Limited Company， YXQ-LS-50SII）； biochemical incubator （Shanghai Sanfa Scientific Instruments Co.， Ltd.， SHP-160）； digital thermostat oscillator （Shanghai Meixiang Instruments Co.， Ltd.， THZ-82A）； fermentation cylinder （Zhenjiang Dongfang Bioengineering Equipment Technology Co.， Ltd.， GBJT-7C）； air oil-less compressor （ZW-0.05/7-X）； air storage tank （Taizhou Fufang Compressor Co.， Ltd.）.
Experimental method
Activation of strain
Four slant seed strain tubes were inoculated on PDA medium. The strain was cultured at 28 ℃ for 5 d under inverted state.
Preparation of seed liquid
Plants with mycelia growing vigorously were selected， and the front end of lawn was inoculated on liquid seed medium， and cultured in a constant temperature shaker （160 r/min） at 28 ℃ for 3-5 d. When white mycelium pellet grew out， the culture could be used as seed liquid.
Inoculation and culture in fermentation cylinder for production of enzyme
When the liquid medium was cooled， the inoculation mouth was opened under the protection of fire， the cultured seed liquid was inoculated into a fermentation cylinder according to specific ratio. According to the experimental design， parameters including stirring speed， ventilation amount， temperature and pH value were set on the touchpad of the fermentation cylinder.
Determination of laccase activity
The culture was sampled every 24 h， filtered with gauze， and centrifuged at 4 000 r/min， obtaining crude enzyme[4]， which was determined with an ultraviolet-visible spectrophotometer for enzyme activity[5]， so as to observe the effects of various factors on the secretion of laccase.
Multi-factor orthogonal test
On the basis of single factor tests， factors having greater effects on laccase activity in the fermentation cylinder were selected for orthogonal test. The activity of laccase under different combinations of factor levels was determined， so as to find the optimal combination.
Results and Analysis
Single facotr experiments
Effect of rotating speed of fermentation cylinder on secretion of laccase
With other conditions unchanged， the activity of laccase was determined under the rotating speeds of fermentation cylinder at 200， 250， 300， 350 and 400 r/min， respectively， as shown in Table 1. It could be seen from Table 1 that during liquid fermentation in the fermentation cylinder， the stirring speed at 300 r/min was more suitable for the secretion of laccase by P. chrysosporium. From the 1st day of fermentation， differences between various factors appeared， and from the 4th day， the activity of laccase increased significantly compared with other levels， and exhibited a maximum value， averagely of 9.783 U/ml on the 7th day， which was significantly higher than the levels of laccase activity at other rotating speeds. It is indicated that when the rotating speed of the fermentation cylinder is too low， the shear force of stirring during the fermentation is weaker， resulting in overgrowth of mycelia， the fermentation liquid is viscous， which affects the microenvironment in the fermentation liquid， and oxygen and nutrient substance are distributed non-uniformly， thereby affecting normal enzyme secretion of P. chrysosporium； and when the rotating speed of the fermentation cylinder is too high， the shear force during the fermentation process is too strong， which affects the growth of mycelia， mycelium cells are even broken and could not secrete the enzyme normally， and the enzyme production level decreases. Furthermore， a too-high stirring speed has higher requirements for equipment and leads to consumption of too much energy， and the cost of production process is thus too high， which is not beneficial to the production of large-scale fermentation industry. Effect of ventilation volume on secretion of laccase in fermentation cylinder
With other conditions unchanged， experiments were carried out under the ventilation volumes of 3， 4， 5， 6 and 7 L/min， respectively， and the effect of ventilation volume on laccase secretion was observed. These results are shown in Table 2. It could be seen from Table 2 that the effect of ventilation volume on the enzyme production level was slightly lower than that of rotating speed. Specifically， from the 4th day， significant differences occurred between different ventilation volumes， and there were no significant differences in enzyme production level between the ventilation volumes of 6 and 7 L/min on the 5th day and the 6th day. The activity of laccase was the highest under the ventilation volume of 5 L/min， and the maximum value appeared on the 7th day， and was 9.180 U/ml， which was far higher than the laccase activity under other conditions. There were no remarkable differences in the enzyme production level between different ventilation volumes in the first 4 d， indicating that the effect of ventilation volume on the enzyme production level was lower than that of rotating speed on the enzyme production level， and the enzyme production trend of lower at first and higher in the end also accorded with the time sequence of laccase production by P. chrysosporium. The fermentation process of P. chrysosporium is an aerobic fermentation process， and therefore， a too-low ventilation volume would affect the growth of mycelia at the early stage， thereby affecting the enzyme production level at the later stage； and when the ventilation volume is too high， foam is produced in a large quantity， which affects the quiet production of laccase by mycelia at the later stage. Furthermore， the continuous high-strength running of the ventilation device increases the energy consumption of the whole fermentation process， the requirements for equipment are higher. Therefore， the optimal ventilation volume was 5 L/min， which could be converted to the ventilation ratio of 1.0 vvm.
Effect of temperature on secretion of laccase in fermentation cylinder
With other conditions unchanged， experiments were carried out under temperatures of 26， 28， 30， 32 and 34 ℃， respectively， and the effect of temperature on laccase secretion was observed. These results are shown in Table 3. It could be seen from Table 3 that the effect of temperature on laccase production was relatively lower than those of rotating speed and ventilation volume， and there were always non-significant differences in the enzyme production level under different temperatures. At the temperature of 28 ℃， the activity of laccase reached the maximum value of 8.880 U/ml； under 30 ℃ second to 28 ℃， the maximum value reached 8.770 U/ml； and at 34 ℃ with the lowest enzyme production level， the maximum value was 7.640 U/ml. It was indicated that 28 ℃ was the optimal fermentation temperature of P. chrysosporium. Research shows that southern areas are hot in summer， and when the fermentation temperature is set at 28 ℃， the fermentation equipment needs cool water for cooling the fermentation liquid， thereby increasing production cost. Meanwhile， the requirements for equipment are also higher. Therefore， in actual production process， it is necessary to consider from the point to adapt the efficiency of large-scale industrial production level. Effect of pH value on secretion of laccase in fermentation cylinder
With other conditions unchanged， experiments were carried out under pH of 4.0， 4.5， 5.0， 5.5 and 6.0， respectively， and the effect of pH value on laccase secretion was observed. These results are shown in Table 4. It could be seen from Table 4 that the initial pH value of medium had no big effect on the secretion of laccase， and there were remarkable differences between different levels. When the initial pH value of medium was 5.0， the activity of laccase reached the highest level of 9.253 U/ml on the 7th day， which was significantly different from the enzyme production levels under other conditions. Too-high or too-low pH values both would reduce enzyme production level. When the pH value is too small， the acidity is too high， mycelia grow difficultly， and the enzyme production level is thus affected； and when the medium is weakly acidic， the mycelia grow freely.
Effect of inoculation amounts on secretion of laccase in fermentation cylinder
With other conditions unchanged， experiments were carried out under inoculation amounts of 5%， 10%， 15%， 20% and 25%， respectively， and the effect of inoculation amount on laccase secretion was observed. The results are shown in Table 5. It could be seen from Table 5 that under the inoculation amount of 15%， the enzyme production level reached the highest value of 9.657 U/ml on the 7th day， which was significantly different from the levels under other conditions. On the 5th day of fermentation， significant differences appeared between various levels. It is indicated that when the inoculation amount is too small， nutrients in medium could not be utilized sufficiently， and it is impossible to achieve the highest enzyme production level； and if the inoculation amount is too large， the lack of space and nutrients affects the growth of mycelia on the contrary， and the enzyme production level is reduced.
Results of multi-factor orthogonal test
Comprehensively from the experimental results of the effects of single factors， stirring speed， ventilation volume， pH value and inoculation amount were selected as the main factors affecting the activity of laccase in the fermentation cylinder， so as to carry out the L9（34） orthogonal tests. The levels of the factors are shown in Table 6， and the experimental results are shown in Table 7. It could be seen from Table 7 that the optimal combination obtained from the multi-factor orthogonal test was A2B2C2D2， i.e.， rotating speed at 300 r/min， ventilation amount of 5 L/min （ventilation ratio of 1.0 vvm）， pH value of 5 and inoculation amount of 15%. This result accorded with results of single factor tests. It could be seen from range analysis that the most-important factor was rotating speed， the second factor was ventilation amount， and the effect of pH value on the enzyme production level was relatively the lowest. This result accorded with the results of single factor tests. Discussion and Conclusions
In this study， the conditions for laccase production by liquid fermentation of P. chrysosporium in fermentation cylinder were optimized through single factor tests and multi-factor orthogonal test， and comprehensively， the optimal conditions for laccase production by liquid fermentation of P. chrysosporium were temperature at 30 ℃， rotating speed at 320 r/min， ventilation amount of 5 L/min （ventilation ratio of 1.0 vvm）， pH value of 5 and inoculation amount of 15%. It was found from the experimental results that the stirring and ventilation of fermentation cylinder improved the disadvantage of fermentation in shaking flask such as little oxygen and non-uniform fermentation liquid， and released sufficient nutrients from the medium. At the early stage of liquid fermentation of P. chrysosporium， the mycelia grew mainly， the enzyme production level was lower， and at the later stage， the enzyme production level increased， resulting in significant differences under different conditions. Such results accord with reports in recent years. The fermentation culture in fermentation cylinder under the optimal conditions gave the highest laccase production level of P. chrysosporium， 14.86 U/ml， which is 32.0% higher than the result obtained by Wei et al.[6] through the optimized experiment using shaking flask.
Laccase production and the production cost in practical production are still not optimistic， production is mostly carried out in shaking flasks or reactors of small scale using microorganisms， and it is difficult to realize large-scale production. Therefore， the experimental conditions in fermentation cylinder decides the success of large-scale production.
References
[1]ZADRZIL F， GRINSBERG J， GONZALES A. "Palo-podrido"-decomposed wood， which was used as feed[J]. European Journal of Applied Microbiology and Biotechnology， 1982， 15（3）： 167-171.
[2] HUANG DL， ZENG GM， HUANG GH， et al. Recent research on white-rot fungi and its expected application in composting[J]. Microbiology China， 2004， 31（2）： 112-116.
[3] FAN WX， LIU XM. Advances in the research of the production and application of laccase[J]. Academy Periodical of Farm Products Processing， 2007（5）： 15-17， 84.
[4] FU K， FU SY， LI XY， et al. Mutational screening of white rot fungi for laccase production in liquid cultivation[J]. Paper Science & Technology， 2009， 28（5）： 21-24， 71.
[5] TANG Y， XIE B， LYU BY， et al. The culturing conditions and application of white rot fungi degrading the refractory organic pollutants[J]. World Sci-Tech R & D， 2008， 30（1）： 60-65.
[6] WEI LM， QIAO L， ZHANG L， et al. Progress of utilization of white-rot fungi in treatment of straw stalk[J]. China Feed， 2013（1）： 43-45.
Key words White-rot fungi； Phanerochaete chrysosporium； Fermentation cylinder； Laccase； Industrial production； Optimization condition
Crop straw is a kind of feedstuff with wide source and low cost， but its lignin which is difficult to be degraded reduces its feeding value as fodder. Therefore， during the treatment of crop straw for preparing fodder， the degradation of lignin becomes a very important link. White-rot fungi are a type of fungi capable of releasing lignin and other wooden components[1]. Currently， three kinds of lignin-degrading enzymes produced by white-rot fungi， i.e.， manganedeperoxidase （MnP）， ligninperoxidse （LiP） and laccase are studied the most[2-3]. Laccase is the kind of polyphenol oxidase， which is produced at the secondary metabolism stage of white-rot fungi， and it is difficult to achieve a higher yield， which is also the main reason why the wide application of laccase is limited. In this study， Phanerochaete chrysosporium with better degradation effect on straw was selected for the experiment of laccase production by white-rot fungus in a mechanically-stirring type fermentation cylinder， to investigate the main factors including stirring speed， ventilation amount， temperature and pH value on the production of laccase by 2 kinds of P. chrysosporium. This study will provide a basis for the industrial production and the effective utilization of crop straw.
Materials and Methods
Experimental materials
Experimental strain P. chrysosporium was preserved in the microbiology lab of Jiangsu Agri-Animal Husbandry Vocational College.
Experimental medium PDA medium was prepared from potato 200 g， glucose 20 g， agar 20 g and distilled water 1 L， with natural pH value. Liquid fermentation medium was prepared from peeled potato 200 g by the steps of boiling for 30 min， filtering with gauze， adding 20 g of sucrose， and water to 1 000 ml after dissolution as well as 10 g of glucose， filling to 250 ml triangular flasks according to 100 ml per flask and performing intermittent sterilization at 121 ℃. Main instruments and equipment
Autoclave sterilizer （Shanghai Boxun Industrial Limited Company， YXQ-LS-50SII）； biochemical incubator （Shanghai Sanfa Scientific Instruments Co.， Ltd.， SHP-160）； digital thermostat oscillator （Shanghai Meixiang Instruments Co.， Ltd.， THZ-82A）； fermentation cylinder （Zhenjiang Dongfang Bioengineering Equipment Technology Co.， Ltd.， GBJT-7C）； air oil-less compressor （ZW-0.05/7-X）； air storage tank （Taizhou Fufang Compressor Co.， Ltd.）.
Experimental method
Activation of strain
Four slant seed strain tubes were inoculated on PDA medium. The strain was cultured at 28 ℃ for 5 d under inverted state.
Preparation of seed liquid
Plants with mycelia growing vigorously were selected， and the front end of lawn was inoculated on liquid seed medium， and cultured in a constant temperature shaker （160 r/min） at 28 ℃ for 3-5 d. When white mycelium pellet grew out， the culture could be used as seed liquid.
Inoculation and culture in fermentation cylinder for production of enzyme
When the liquid medium was cooled， the inoculation mouth was opened under the protection of fire， the cultured seed liquid was inoculated into a fermentation cylinder according to specific ratio. According to the experimental design， parameters including stirring speed， ventilation amount， temperature and pH value were set on the touchpad of the fermentation cylinder.
Determination of laccase activity
The culture was sampled every 24 h， filtered with gauze， and centrifuged at 4 000 r/min， obtaining crude enzyme[4]， which was determined with an ultraviolet-visible spectrophotometer for enzyme activity[5]， so as to observe the effects of various factors on the secretion of laccase.
Multi-factor orthogonal test
On the basis of single factor tests， factors having greater effects on laccase activity in the fermentation cylinder were selected for orthogonal test. The activity of laccase under different combinations of factor levels was determined， so as to find the optimal combination.
Results and Analysis
Single facotr experiments
Effect of rotating speed of fermentation cylinder on secretion of laccase
With other conditions unchanged， the activity of laccase was determined under the rotating speeds of fermentation cylinder at 200， 250， 300， 350 and 400 r/min， respectively， as shown in Table 1. It could be seen from Table 1 that during liquid fermentation in the fermentation cylinder， the stirring speed at 300 r/min was more suitable for the secretion of laccase by P. chrysosporium. From the 1st day of fermentation， differences between various factors appeared， and from the 4th day， the activity of laccase increased significantly compared with other levels， and exhibited a maximum value， averagely of 9.783 U/ml on the 7th day， which was significantly higher than the levels of laccase activity at other rotating speeds. It is indicated that when the rotating speed of the fermentation cylinder is too low， the shear force of stirring during the fermentation is weaker， resulting in overgrowth of mycelia， the fermentation liquid is viscous， which affects the microenvironment in the fermentation liquid， and oxygen and nutrient substance are distributed non-uniformly， thereby affecting normal enzyme secretion of P. chrysosporium； and when the rotating speed of the fermentation cylinder is too high， the shear force during the fermentation process is too strong， which affects the growth of mycelia， mycelium cells are even broken and could not secrete the enzyme normally， and the enzyme production level decreases. Furthermore， a too-high stirring speed has higher requirements for equipment and leads to consumption of too much energy， and the cost of production process is thus too high， which is not beneficial to the production of large-scale fermentation industry. Effect of ventilation volume on secretion of laccase in fermentation cylinder
With other conditions unchanged， experiments were carried out under the ventilation volumes of 3， 4， 5， 6 and 7 L/min， respectively， and the effect of ventilation volume on laccase secretion was observed. These results are shown in Table 2. It could be seen from Table 2 that the effect of ventilation volume on the enzyme production level was slightly lower than that of rotating speed. Specifically， from the 4th day， significant differences occurred between different ventilation volumes， and there were no significant differences in enzyme production level between the ventilation volumes of 6 and 7 L/min on the 5th day and the 6th day. The activity of laccase was the highest under the ventilation volume of 5 L/min， and the maximum value appeared on the 7th day， and was 9.180 U/ml， which was far higher than the laccase activity under other conditions. There were no remarkable differences in the enzyme production level between different ventilation volumes in the first 4 d， indicating that the effect of ventilation volume on the enzyme production level was lower than that of rotating speed on the enzyme production level， and the enzyme production trend of lower at first and higher in the end also accorded with the time sequence of laccase production by P. chrysosporium. The fermentation process of P. chrysosporium is an aerobic fermentation process， and therefore， a too-low ventilation volume would affect the growth of mycelia at the early stage， thereby affecting the enzyme production level at the later stage； and when the ventilation volume is too high， foam is produced in a large quantity， which affects the quiet production of laccase by mycelia at the later stage. Furthermore， the continuous high-strength running of the ventilation device increases the energy consumption of the whole fermentation process， the requirements for equipment are higher. Therefore， the optimal ventilation volume was 5 L/min， which could be converted to the ventilation ratio of 1.0 vvm.
Effect of temperature on secretion of laccase in fermentation cylinder
With other conditions unchanged， experiments were carried out under temperatures of 26， 28， 30， 32 and 34 ℃， respectively， and the effect of temperature on laccase secretion was observed. These results are shown in Table 3. It could be seen from Table 3 that the effect of temperature on laccase production was relatively lower than those of rotating speed and ventilation volume， and there were always non-significant differences in the enzyme production level under different temperatures. At the temperature of 28 ℃， the activity of laccase reached the maximum value of 8.880 U/ml； under 30 ℃ second to 28 ℃， the maximum value reached 8.770 U/ml； and at 34 ℃ with the lowest enzyme production level， the maximum value was 7.640 U/ml. It was indicated that 28 ℃ was the optimal fermentation temperature of P. chrysosporium. Research shows that southern areas are hot in summer， and when the fermentation temperature is set at 28 ℃， the fermentation equipment needs cool water for cooling the fermentation liquid， thereby increasing production cost. Meanwhile， the requirements for equipment are also higher. Therefore， in actual production process， it is necessary to consider from the point to adapt the efficiency of large-scale industrial production level. Effect of pH value on secretion of laccase in fermentation cylinder
With other conditions unchanged， experiments were carried out under pH of 4.0， 4.5， 5.0， 5.5 and 6.0， respectively， and the effect of pH value on laccase secretion was observed. These results are shown in Table 4. It could be seen from Table 4 that the initial pH value of medium had no big effect on the secretion of laccase， and there were remarkable differences between different levels. When the initial pH value of medium was 5.0， the activity of laccase reached the highest level of 9.253 U/ml on the 7th day， which was significantly different from the enzyme production levels under other conditions. Too-high or too-low pH values both would reduce enzyme production level. When the pH value is too small， the acidity is too high， mycelia grow difficultly， and the enzyme production level is thus affected； and when the medium is weakly acidic， the mycelia grow freely.
Effect of inoculation amounts on secretion of laccase in fermentation cylinder
With other conditions unchanged， experiments were carried out under inoculation amounts of 5%， 10%， 15%， 20% and 25%， respectively， and the effect of inoculation amount on laccase secretion was observed. The results are shown in Table 5. It could be seen from Table 5 that under the inoculation amount of 15%， the enzyme production level reached the highest value of 9.657 U/ml on the 7th day， which was significantly different from the levels under other conditions. On the 5th day of fermentation， significant differences appeared between various levels. It is indicated that when the inoculation amount is too small， nutrients in medium could not be utilized sufficiently， and it is impossible to achieve the highest enzyme production level； and if the inoculation amount is too large， the lack of space and nutrients affects the growth of mycelia on the contrary， and the enzyme production level is reduced.
Results of multi-factor orthogonal test
Comprehensively from the experimental results of the effects of single factors， stirring speed， ventilation volume， pH value and inoculation amount were selected as the main factors affecting the activity of laccase in the fermentation cylinder， so as to carry out the L9（34） orthogonal tests. The levels of the factors are shown in Table 6， and the experimental results are shown in Table 7. It could be seen from Table 7 that the optimal combination obtained from the multi-factor orthogonal test was A2B2C2D2， i.e.， rotating speed at 300 r/min， ventilation amount of 5 L/min （ventilation ratio of 1.0 vvm）， pH value of 5 and inoculation amount of 15%. This result accorded with results of single factor tests. It could be seen from range analysis that the most-important factor was rotating speed， the second factor was ventilation amount， and the effect of pH value on the enzyme production level was relatively the lowest. This result accorded with the results of single factor tests. Discussion and Conclusions
In this study， the conditions for laccase production by liquid fermentation of P. chrysosporium in fermentation cylinder were optimized through single factor tests and multi-factor orthogonal test， and comprehensively， the optimal conditions for laccase production by liquid fermentation of P. chrysosporium were temperature at 30 ℃， rotating speed at 320 r/min， ventilation amount of 5 L/min （ventilation ratio of 1.0 vvm）， pH value of 5 and inoculation amount of 15%. It was found from the experimental results that the stirring and ventilation of fermentation cylinder improved the disadvantage of fermentation in shaking flask such as little oxygen and non-uniform fermentation liquid， and released sufficient nutrients from the medium. At the early stage of liquid fermentation of P. chrysosporium， the mycelia grew mainly， the enzyme production level was lower， and at the later stage， the enzyme production level increased， resulting in significant differences under different conditions. Such results accord with reports in recent years. The fermentation culture in fermentation cylinder under the optimal conditions gave the highest laccase production level of P. chrysosporium， 14.86 U/ml， which is 32.0% higher than the result obtained by Wei et al.[6] through the optimized experiment using shaking flask.
Laccase production and the production cost in practical production are still not optimistic， production is mostly carried out in shaking flasks or reactors of small scale using microorganisms， and it is difficult to realize large-scale production. Therefore， the experimental conditions in fermentation cylinder decides the success of large-scale production.
References
[1]ZADRZIL F， GRINSBERG J， GONZALES A. "Palo-podrido"-decomposed wood， which was used as feed[J]. European Journal of Applied Microbiology and Biotechnology， 1982， 15（3）： 167-171.
[2] HUANG DL， ZENG GM， HUANG GH， et al. Recent research on white-rot fungi and its expected application in composting[J]. Microbiology China， 2004， 31（2）： 112-116.
[3] FAN WX， LIU XM. Advances in the research of the production and application of laccase[J]. Academy Periodical of Farm Products Processing， 2007（5）： 15-17， 84.
[4] FU K， FU SY， LI XY， et al. Mutational screening of white rot fungi for laccase production in liquid cultivation[J]. Paper Science & Technology， 2009， 28（5）： 21-24， 71.
[5] TANG Y， XIE B， LYU BY， et al. The culturing conditions and application of white rot fungi degrading the refractory organic pollutants[J]. World Sci-Tech R & D， 2008， 30（1）： 60-65.
[6] WEI LM， QIAO L， ZHANG L， et al. Progress of utilization of white-rot fungi in treatment of straw stalk[J]. China Feed， 2013（1）： 43-45.