Yang LlU，Dongmei XlONG，Xingyao XlONG，3，Xiaojun SU，Liu CAl，Lu ZENG*.College of Food Science and Technology，Hunan Agricultural University，Changsha 408，China；.Key Laboratory of Crop Germplasm Innovation and Utilization of Hunan Province，Hunan Agricultural University，Changsha 408，China；3.Institute of Vegetables and Flowers，Chinese Academy of Agricultural Sciences，Beijing 008，China

Effects of Mutagenesis by UV lrradiation and60Co-γ lrradiation on Fermentation of Xylose to Ethanol by Pichia stipitis

Yang LlU1，Dongmei XlONG2，Xingyao XlONG2，3，Xiaojun SU2，Liu CAl2，Lu ZENG2*
1.College of Food Science and Technology，Hunan Agricultural University，Changsha 410128，China；
2.Key Laboratory of Crop Germplasm Innovation and Utilization of Hunan Province，Hunan Agricultural University，Changsha 410128，China；
3.Institute of Vegetables and Flowers，Chinese Academy of Agricultural Sciences，Beijing 10081，China

In this study，effects of UV irradiation and60Co-γ irradiation on fermentation of xylose to ethanol by Pichia stipitis were analyzed to investigate the optimal mutagenic conditions.According to the growth curve and fermentation curve of P.stipitis，the optimal incubation duration and fermentation duration of P.stipitis mutant strain were 18 and 48 h，respectively.The cell concentration of original yeast liquid was 107cells/ml.After mutagenesis by UV irradiation and60Co-γ irradiation，yeast liquid was incubated in 20 g/L xylose media for 48 h.According to the results，after UV irradiation for 45-75 s，transformation efficiency reached 0.3794 g/g，which was 74.39%of the theoretical value；after irradiation with 800-1 000 Gy60Co-γ，transformation efficiency reached 0.3165 g/g，which was 62.06% of the theoretical value. Therefore，both UV irradiation and60Co-γ irradiation could improve the efficiency of xylose fermentation to ethanol by P.stipitis under appropriate conditions.

Pichia stipitis；Mutagenesis；Xylose；Fermentation；Ethanol

Materials and Methods

Strain

P.stipitis was provided by Key Laboratory of Crop Germplasm Innovation and Utilization of Hunan Province，Hunan Agricultural University.

Medium

Fermentation liquid media:xylose 20 g，peptone 15 g，yeast powder 10 g，（NH4）2SO41 g，KH2PO41 g，Mg-SO4·7H2O 0.5 g，CaCl2·2H2O 1 g，H2O 1 L.Xylose solid media:xylose 20 g，peptone 15 g，yeast powder 10 g，（NH4）2SO41 g，KH2PO41 g，MgSO4· 7H2O 0.5 g，CaCl2·2H2O 1 g，agar powder 20 g，H2O 1 L.TTC （2，3，5-triphenyltetrazolium chloride）upper media:TTC 0.05 g，xylose 0.5 g，agar 1.5 g，H2O 100 ml.TTC lower media: xylose 10.1 g，peptone 2 g，yeast extract 1.5 g，KH2PO41 g，MgSO4·7H2O 0.4 g，citric acid 0.27 g，agar 20 g，H2O 1 L.

Instruments

Thermostatic oscillator ZHWY-2102C，Shanghai Zhicheng Analytical Instrument Manufacturing Co.，Ltd.；UV-18000 spectrophotometer，Shanghai Mapda instrument Co.，Ltd.；biosensor analyzer SBA-40E，Biology Institute of Shandong Academy of Sciences；double-sided clean bench SW-CJ-2FD， Suzhou Purification Equipment Co.，Ltd.

Experimental methods

Determination of xylose residue content with DNS method The standard curve of xylose was drawn. After centrifugation of fermentation broth，an appropriate amount of supernatant was collected，diluted by 20 times and mixed evenly；1 ml of diluent was transferred to a 25 ml colorimetric tube with three replications，added with 3 ml of DNS liquid，incubated in a boiling water bath for 5 min，cooled to room temperature，added to a final volume and mixed evenly.The absorbance value was measured at 540 nm［13］.

Determination of ethanol content

Standard solution and buffer were prepared in accordance with the operational requirements of biosensor analyzer.After centrifugation of fermentation broth，1 ml of supernatant was collected，diluted by 10 times and mixed evenly，with three replications. The instrument was adjusted with standard solution；25 μl of samples were determined using a 50 μl injector.

Drawing of the growth curve Firstly，1 ml of 109cells/ml seed liquid was added to 100 ml of 2%xylose culture media and incubated at 28℃for 18-24 h with shaking at 120 r/min.The activated yeast liquid was inoculated by 1%to a tube containing 10 ml of 2% xylose media with three replications. Subsequently，the yeast liquid was incubated at 28℃with shaking at 120 r/min.Samples were collected every 2 h，mixed evenly and preserved in a refrigerator at-4℃.The absorbance value was measured at 600 nm to draw the growth curve.

Drawing of the fermentation curve

The yeast liquid was activated according to the above method，inoculated by 1%to an erlenmeyer flask containing 100 ml of 2%xylose liquid，and incubated at 28℃with shaking at 120 r/min.Samples were collected every 4-8 h，with three replications.All the collected samples were centrifuged；1 ml of supernatant was collected，diluted by 10 times and preserved in a refrigerator at-4℃ with three replications.Ethanol content was determined using biosensor analyzer to draw the fermentation curve.

Preparation of working liquid before mutagenesis In the logarithmic growth phase，yeast liquid was transferred to sterile saline and diluted to 107cells/ml before use.

Mutagenesis by UV irradiation

Firstly，3 ml of working liquid was transferred to a sterile petri dish to separate the cells，and placed under 15 W UV light with the vertical distance of 25 cm for different durations.UV irradiation was preformed in the dark. After UV irradiation，yeast liquid was placed in the dark for 2-3 h.Subsequently，mutagenic liquid was inoculated by 1%to 100 ml of 2%xylose media with three replications and incubated at 28℃for 48 h with shaking at 120 r/min.Contents of xylose residues and ethanol in fermentation broth were determined；after continuous subculture，stable value was recorded.Moreover，mortality rate was calculated with plate count method.

Mutagenesis by60Co-γ irradiation

Firstly，10 ml of working liquid was transferred to an 18×18 tube and irradiated with 24 kGy60Co-γ for different durations according to the experimental design.After irradiation with different doses of60Co-γ，yeast liquid was placed at room temperature for 2-3 h，inoculated by 1%to 100 ml of 2%xylose media with three replications，and incubated at 28℃for 48 h with shaking at 120 r/min.Contents of xylose residues and ethanol in fermentation broth were determined；after continuous subculture，stable value was recorded.The mortality rate was calculated with plate count method.

Verification of fermentation results

Sixteen grids were drawn on the prepared TTC lower media plate and inoculated by experimental strain with an inoculation loop，one cell per grid. After inoculation，TTC lower media was placed upside down in an incuba-tor at 28℃for 1-2 d.An appropriate amount of prepared TTC upper media was heated to about 50℃and poured on the inoculated TTC lower media. After solidification，the media was placed upside down in an incubator at 28℃for 1 d.The results of TTC color demonstration were observed and recorded.

Results and Analysis

Growth curve of Pichia stipitis

After inoculation，the number of P.stipitis cells in media，yeast liquid concentration and OD value measured using a UV spectrophotometer increased with the extension of incubation duration.Therefore，the absorbance value of yeast liquid measured after different incubation durations could reflect the growth trend of P.stipitis.

As shown in Fig.1，P.stipitis cells entered the logarithmic growth phase at 14 h post-inoculation，with a rapidly increasing cell concentration and maximum growth rate；P.stipitis cells entered the stable growth phase at 22 h post-inoculation，with a stable cell concentration；P.stipitis cells autolyzed and entered the declining growth phase at 40 h post-inoculation.In the logarithmic growth phase，microorganisms grow fastest with vigorous metabolism，high enzyme activity，consistent cell growth and good repeatability，which are sensitive to external stimuli and prone to mutation.Therefore，in this study，the strain was incubated for 18 h before mutagenesis.

Fermentation curve of Pichia stipitis

During the fermentation process，the yield of ethanol produced by xylose-fermenting strain varied with the extension of fermentation duration.In order to investigate the maximum yield of ethanol produced in fermentation process，ethanol-time curve of the fermentation broth was measured，thus determining the optimal fermentation duration of mutant strain.According to the fermentation curve of P.stipitis，experimental strain was incubated in 100 ml of 2% xylose media and changes in the ethanol content in fermentation broth with the extension of fermentation duration was determined（Fig.2）.

As shown in Fig.2，P.stipitis hardly produced ethanol within 8 h post-inoculation；subsequently，ethanol yield increased linearly with the extension of fermentation duration；at 48 h post-inoculation，ethanol yield reached the maximum；with continuous extension of fermentation duration，ethanol yield declined gradually. In order to reveal changes in the yield of ethanol produced by xylose-fermenting strain，the optimal fermentation duration should be selected.In this study，P.stipitis cells were fermented for 48 h.

Mutagenesis by UV irradiation

Mortality rate after UV irradiation for different durations According to the mortality rate after UV irradiation for different durations（30，45，60，75，90，105，120 and 135 s），the mortality curve was drawn（Fig.3）.As shown in Fig.3，with the extension of mutagenesis duration，the mortality rate of P. stipitis increased gradually.After UV irradiation for 45 s，the mortality rate of P.stipitis varied significantly（about 80%）；after UV irradiation for more than 90 s，the mortality rate of P.stipitis was higher than 95%；after UV irradiation for more than 120 s，the mortality rate reached nearly 100%.

Effects of different UV irradiation duration on xylose fermentation by P.stipitis According to related literatures［14-17］，UV irradiation duration is related with experimental strain and experimental conditions.Therefore，in accordance with experimental design，yeast liquid was treated by UV irradiation for 30，45，60，75，90，105，120 and 135 s respectively，and incubated in the dark for 48 h.Contents of xylose residues and ethanol in fermentation broth were determined（Table 1）.

As shown in Table 1，different UV irradiation durations exhibited significant effects on xylose fermentation by P.stipitis.Xylose consumption was reduced with the extension of UV irradiation duration，which might be due to that long-term UV irradiation exhibited a lethal effect on P.stipitis cells；fewer P.stipitis cells actually consumed less xylose within a certain period of time and produced less ethanol.Therefore，ethanol yield was almost positively correlated with xylose consumption in media.However，with the extension of UV irradiation duration，transformation efficiency of P.stipitis increased first and then declined.According to experimental data，transformation efficiency of P.stipitis reached the highest after UV irradiation for 45 s，indicating a good ability of P.stipitis to utilize xylose and produce ethanol；after UV irradiation for 90 s，P.stipitis produced an extremely small amount of ethanol that was not detected，indicating that UV irradiation for more than 90 s was not conducive to xylose fermentation to ethanol by P.stipitis.Overall，transformation efficiency of P.stipitis reached the maximum at 45 s post-irradiation，but UV irradiation for more than 90 s would hinder the production of positive mutant strain.

Table 1 Ethanol yield after UV irradiation

Table 2 TTC color demonstration results of P.stipitis after UV irradiation

Table 3 Ethanol yield after60Co-γ irradiation

Verification of UV irradiation result by TTC method

TTC is a chromogenic reagent. The amount or presence of dehydrogenase directly affects the color of colonies:yeast strains with higher fermentation rate exhibit more dehydrogenase and TTC accepts more hydrogen，resulting in deeper red color，otherwise the color is lighter［18-20］.Therefore，ethanol production capacity of yeast strains can be evaluated based on TTC color demonstration result.As shown in Table 1，ethanol yield varied among different UV irradiation durations，which indicated that ethanol production capacity of P.stipitis varied after UV irradiation.According to TTC verification method，randomly 20 single colonies were selected in each plate after UV irradiation for different durations.TTC color demonstration results of 200 colonies were shown in Fig.4 and Table 2.

As shown in Fig.4，dark red colonies were regarded as highly chromogenic；reddish colonies or those with light edges and slightly red middle parts were regarded as slightly chromogenic；colonies without red color were regarded as non-chromogenic. Finally，the number of highly chromogenic，slightly chromogenic and non-chromogenic colonies in each plate was recorded（Table 2）.

As shown in Table 2，the number of highly chromogenic colonies reached the maximum after UV irradiation for 45 s and declined significantly after UV irradiation for more than 90 s. According to the principle of TTC color demonstration，most P.stipitis strains with high ethanol production capacity were obtained after UV irradiation for 45 s，which was consistent with the efficiency of xylose fermentation to ethanol（Table 1）.Thus，TTC color demonstration showed that positive mutation rate of P.stipitis reached the maximum after UV irradiation for 45 s；positive mutation rate was higher after UV irradiation for 60 s than after UV irradiation for 30 and 75 s.Related literatures also confirmed that［21-22］most target strains were obtained after UV irradiation for 45 s.However，after UV irradiation for more than 90 s，the number of yeast strains with high ethanol production capacity was reduced，which was not conducive to screening positive mutant strains.

Mutagenesis by60Co-γ Co-irradiation

Mortality rate after irradiation with different doses of60Co-γ According to the mortality rate after irradiationwith different doses （100，200，400，600，800，1 000，1 200，1 400，1 600 Gy）of60Co-γ，the mortality curve was drawn（Fig.5）.As shown in Fig.5，with the increase of60Co-γ irradiation dose，the mortality rate of P.stipitis increased gradually.Compare with the mortality curve of P.stipitis after UV irradiation，60Co-γ irradiation exhibited greater stimuli on P.stipitis.The mortality rate of P.stipitis irradiated with 100 Gy60Co-γ was higher than 60%；the mortality rate of P.stipitis irradiated with 400 Gy60Co-γ exceeded 90%；the mortality rate of P.stipitis irradiated with more than 1 000 Gy60Co-γ was higher than 99%.

Effects of different60Co-γ irradiation doses on xylose fermentation by P.stipitis According to experimental design and related literatures［23-24］，yeast liquid was irradiated by 200，400，600，800，1 000，1 200，1 400 and 1 600 Gy60Co-γ，inoculated to 100 ml of 2% new xylose media and incubated for 48 h.Contents of xylose residues and ethanol in fermentation broth were determined（Table 3）.

As shown in Table 3，actual consumption of xylose was reduced gradually with the increase of60Co-γ irradiation dose and the transformation efficiency increased first and then declined.To be specific，transformation efficiency reached the maximum after irradiation with 1 000 Gy60Co-γ and declined continuously when60Co-γ irradiation dose was higher than 1 000 Gy，which indicated that P.stipitis irradiated with 1 000 Gy60Co-γ exhibited good ethanol production capacity；compared with UV irradiation，P.stipitis irradiated with Gy60Co-γ exhibited relatively low transformation efficiency，suggesting that UV irradiation was more suitable for screening positive mutant strains than60Co-γ irradiation. According to experimental data in Table 3，ethanol yield of P.stipitis irradiated with 800-1 000 Gy60Co-γ was higher than that irradiated with other doses；irradiation with 800-1 000 Gy60Co-γ was conducive to screening single mutant strain.In this study，three P.stipitis strains with high ethanol production capacity were obtained after irradiation with 800-1 000 Gy60Co-γ；transformation efficiency of P.stipitis reached about 0.30 g/g after fermentation in 5%xylose fermentation media for 36 h，which was improved by 30%compared with the original strain.

Discussions

In this study，P.stipitis mutagenesis was performed with UV irradiation and60Co-γ irradiation.Contents of xylose residues and ethanol in fermentation broth were adopted as analytical indicators to calculate transformation efficiency.According to the results，UV irradiation and60Co-γ irradiation had different effects on ethanol production performance of P.stipitis；different irradiation doses also exhibited varying effects on the mutation of P.stipitis flora.

Compared with60Co-γ irradiation mutagenesis，UV irradiation mutagenesis was more favorable for forward mutation of P.stipitis.According to the measured data，after UV irradiation，the maximum transformation efficiency reached 0.379 4 g/g，which was 74.39%of the theoretical value. TTC verification indicated that most highly chromogenic colonies were obtained after UV irradiation for 45-75 s，suggesting a good positive mutation effect，which could be used for single colony screening.In addition，experimental results showed that P.stipitis flora exhibited high negative mutation rate and low ethanol production capacity after60Co-γ irradiation mutagenesis； specifically， the maximum transformation efficiency reached 0.316 5 g/g，which was 62.06%of the theoretical value.However，experimental data indicated that substrate consumption and ethanol yield after60Co-γ irradiation was improved significantly compared with UV irradiation. Single strains with significant mutations in fermentation broth could be obtained by single colony screening. Ethanol yield of P.stipitis flora after60Co-γ irradiation mutagenesis was analyzed；results showed that irradiation with 800-1 000 Gy60Co-γ was conducive to obtaining positive mutant strains and screening single colonies，which provided a scientific reference for subsequent experiments.

References

［1］WANG DR（汪多仁）.Market perspective of bioethanol industry（生物乙醇汽油市场展望）［J］.Chemical Industry（化学工业），2009，27（10）:30-32；36.

［2］TIAN S（田沈），XU X（徐鑫），MENG FY（孟繁燕），et al.Research progress on ethanol fermentation from lignocellulose（木质纤维素乙醇发酵研究进展）［J］. Transactions of the Chinese Society of Agricultural Engineering（农业工程学报），2006，22（1）:221-224.

［3］CAO XH（曹秀华），RUAN QC（阮奇城），LIN HH（林海红），et al.Progress of xylosic fermentation of lignocellulosic materials for bioethanol production（纤维燃料乙醇生产中木糖发酵的研究进展）［J］. Plant Fiber Sciences in China（中国麻业科学），2010，32（3）:166-169

［4］WANG PY，SHOPSIS C，SCHNEIDER H.Fermentation of a pentose by yeasts［J］.Biochem B Biophys，1980，94:248-254.

［5］DELEGENES P，MOLETTA R.，NAVARRO JM.Effects on lingo cellulose degradation products on ethanol fermentation of glucose and xylose by Saccharomyces cerevisiae， Zymomonas mobilis，Pichia stipitis，and Candida shehatea［J］.Enzyme Microb. Technol.，1996，11:220-225.

［6］ZHANG YY（张亚云），DING CH（丁长河），LI LT（李里特），et al.Fermentation of D-xyiose for ethanol production by Pichia stipitis（树干毕赤酵母发酵木糖生产燃料乙醇）［J］.Liquor-making（酿酒），2009（1）:23-26.

［7］ZHONG GF（钟桂芳），FU XH（傅秀辉），SUN JS（孙军社），et al.Advance in producing ethanol by xylose-fermenting and its prospect（发酵木糖生产酒精的研究进展及其应用前景）［J］.Journal of Microbiology（微生物学杂志），2004，24（1）: 42-44.

［8］LI RJ（李荣杰）.Research progress of microbial mutation breeding techniques（微生物诱变育种方法研究进展）［J］. Journal of Hebei Agricultural Sciences（河北农业科学），2009，13（10）:73-76，78.

［9］WANG XL（汪杏莉），LI ZW（李宗伟），CHEN LH（陈林海）.The development of physical mutation techniques in industrial microbe breeding（工业微生物物理诱变育种技术的新进展）［J］.Biotechnology Bulletin（生物技术通报），2007，（2）: 114-115.

［10］LI ZQ（李志强）.Study on composite mutation breeding and fermentation conditions of Saccharomyces cerevisiae（酿酒酵母的复合诱变选育及其发酵条件研究）［D］.Hefei:Hefei University of Technology（合肥:合肥工业大学），2010.

［11］WANG YN（王秩男）.Study on mutation breeding of Candida shehatae capable of fermenting xylose to produce ethanol（发酵木糖产乙醇的休哈塔假丝酵母诱变育种研究）［D］.Harbin:Hei-

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紫外和60Co-γ诱变对树干毕赤酵母发酵木糖产乙醇的影响

刘阳1，熊冬梅2，熊兴耀2，3，苏小军2，蔡柳2，曾璐2*（1.湖南农业大学食品科技学院，湖南长沙 410128；2.湖南农业大学湖南省作物种质创新与资源利用重点实验室，湖南长沙410128；3.中国农业科学院蔬菜花卉所，北京10081）

以树干毕赤酵母为出发菌株，研究紫外短波照射和60Co-γ辐照2种诱变方法对该菌种利用木糖产乙醇性能的影响，探讨较佳诱变条件。通过绘制菌体生长曲线及发酵曲线，确定了诱变菌最适宜的培养时长为18 h，诱变菌最适宜的发酵时长为48 h。原菌液浓度为107个/ml时，根据诱变后菌液在木糖浓度20 g/L中培养48h的结果可知，紫外处理45～75 s乙醇产率可达0.379 4 g/g，为理论值的74.39%，辐照处理800～1 000 Gy，乙醇产率可达0.316 5 g/g，为理论值的62.06%。因此，紫外及辐照这2种诱变方式在合适的参数条件下均能提高树干毕赤酵母利用木糖产乙醇的转化率。

树干毕赤酵母；诱变；木糖；发酵；乙醇

W ith the rapid development of global economy，continuous reduction of petroleum storage and emergence of environmental issues，bioethanol has become a major source of clean and renewable energy［1］，which is included in the energy development plan in many countries. Cheap raw materials and efficient transformation technology are necessary conditions for commercial production of bio-fuel ethanol.In recent years，a large number of studies have been conducted on production of fuel ethanol with lignocellulose-rich agricultural waste （such as straws）.The content of xylose and other pentoses in plant hydrolysates reaches 30%［2-3］. Making full use of xylose in lignocellulose could improve ethanol yield by 25%.Therefore，producing ethanol by xylose fermentation is a key factor determining the economic feasibility of ethanol production with plant fiber resources.In 1959，Karczewska reported that xylose could be fermented by microbes.In 1980，Wang et al.［4］found that xylose could be fermented by some microbes to produce ethanol.So far，more than one hundred kinds of microbial species have been found to ferment xylose，including bacteria，filamentous fungi and yeasts［5］.Specifically，Pichia stipitis has been most frequently studied as one of the excellent strains with industrial application prospects，which exhibits high transformation efficiency and low content of xylitol byproduct in the final product of fermentation［6］.

At present，researches of xylosefermenting strains mainly involve screening strains from natural source，improving existing strains，and optimizing fermentation conditions［7］.Processing strains with mutagenesis tech-nology is the most direct and effective way to improve the transformation efficiency and screen dominant strains［8］. Many studies have been carried out on physical mutagenesis of ethanol-producing strains［9］.Li［10］processed Saccharomyces cerevisiae strain S31206 with ultraviolet-microwave compound mutagenesis and screened a mutant strain Y-9-3；the yield of ethanol produced by Y-9-3 was improved by 68.2% compared with the original strain.Wang［11］investigated the relationship between various physical mutagen dosages and mortality rate，positive mutation rate of Candida shehatae strain HDYXHT-01 and found that the yield of ethanol produced by mutant strain HN-3 was improved by 17.20%compared with the original strain.Mou［12］analyzed the relationship between mortality rate of P.stipitis protoplast and ultraviolet radiation dose and obtained a P.stipitis strain M-1 that produced high concentration of ethanol；the yield of ethanol produced by M-1 reached 7.52 g/L.However，little information is available on mutagenesis of P.stipitis by60Co-γ irradiation.In this study，effects of UV irradiation and60Co-γ irradiation on D-xylose fermentation to ethanol by P.stipitis were investigated，which laid a scientific foundation for enriching xylose-fermenting microbial genetic resources and optimizing the efficiency of xylose fermentation to ethanol.

国家973计划课题（2012CB723004）；湖南农业大学青年科学基金（12QN11）。

刘阳（1989-），女，湖南邵阳人，硕士研究生，研究方向：微生物发酵，E-mail：xiaoyangzi1012@163.com。*通讯作者，E-mail：happy7201@163.com。

2014-11-25

2015-01-10

Supported by National Key Basic Research Program of China （973 Program）（2012CB723004）；Youth Fund of Hunan Agricultural University（12QN11）.

.E-mail:happy7201@163.com

November 25，2014Accepted:January 10，2015