苏云金杆菌ZLL13晶体蛋白分析及其菌糠液态培养基的优化
|
摘要
食用菌栽培废料,简称菌糠(spent mushroom substrate, SMS)是食用菌栽培和生产的残留物,其含有丰富的甲壳素、木质纤维和蛋白质等,可为苏云金芽孢杆菌(Bacillus thuringiensis, Bt)的生长提供所需的营养物质。本研究以优化后的前处理条件制备的菌糠浸提液(2%硫酸, 121℃, 1 h)作为主要碳源,通过单因素试验、Plackett-Burman设计、最陡爬坡和响应面分析等方法来优化最佳培养基组分,结果表明,54%SMS浸提液,31.9 g/L大豆饼粉、0.88 g/L CaCO_3、0.4 g/L MnSO_4、0.5 g/L K_2HPO_4和0.4 g/L吐温100为最佳培养基配方,且优化后培养基(1.8×10~8/mL)产生的孢子数是原始SMS培养基(0.065×10~8/mL)的27倍,这不仅为菌糠的二次利用提供一种新的有效方法,而且也可以大大降低生产Bt所需要的发酵成本,具有良好的应用前景。
Spent mushroom substrate(SMS) has been demonstrated that the entomotoxicity and chitinase activity of Bacillus thuringiensis based biopesticides may be increased by fortified with chitin during production. To develop a cost-effective biopesticide, spent mushroom substrate extract was studied as a potential chitin and carbon source for cultivating Bacillus thuringiensis. Subsequently, Spent mushroom substrate extract(2% H2 SO4, 121℃, 1 h)was selected for BtiZLL13 fermentation, Plackett-Burman design(PB) and response surface methodology(RSM)were employed to optimize the medium components for production of Bti-based biopesticide. The optimal composition was as followes: 54% SMS extract, 31.9 g/L soybean meal, 0.88 g/L CaCO3, 0.4 g/L MnSO4, 0.5 g/L K2 HPO4, and 0.4 g/L Tween 100. Under the optimized conditions, more than 27 times improvement in production of spores was achieved compared with the original SMS medium. Results from this study suggest that SMS is a highly cost-effective raw material for mass production of Bti.
Spent mushroom substrate(SMS) has been demonstrated that the entomotoxicity and chitinase activity of Bacillus thuringiensis based biopesticides may be increased by fortified with chitin during production. To develop a cost-effective biopesticide, spent mushroom substrate extract was studied as a potential chitin and carbon source for cultivating Bacillus thuringiensis. Subsequently, Spent mushroom substrate extract(2% H2 SO4, 121℃, 1 h)was selected for BtiZLL13 fermentation, Plackett-Burman design(PB) and response surface methodology(RSM)were employed to optimize the medium components for production of Bti-based biopesticide. The optimal composition was as followes: 54% SMS extract, 31.9 g/L soybean meal, 0.88 g/L CaCO3, 0.4 g/L MnSO4, 0.5 g/L K2 HPO4, and 0.4 g/L Tween 100. Under the optimized conditions, more than 27 times improvement in production of spores was achieved compared with the original SMS medium. Results from this study suggest that SMS is a highly cost-effective raw material for mass production of Bti.
引文
Adjalle K.D.,Brar S.K.,Tyagi R.D.,Valero J.R.,and Surampalli R.Y.,2009,Photostabilization of Bacillus thuringiensis fermented wastewater and wastewater sludge based biopesticides using additives,Acta Tropica,11(16):7-14
Berry C.,O'Neil S.,Ben-Dov E.,Jones A.F.,Murphy L.,Quail M.A.,Holden M.T.G.,Harris D.,Zaritsky A.,and Parkhill J.,2002,Complete sequence and orga niza tion of pBtoxis,the toxin-coding plasmid of Bacillus thuringiensis subsp israelensis,Appl.Environ.Microb.,68(10):5082-5095
Bialas W.,Szymanowska D.,and Grajek W.,2010,Fuel ethano production from granular corn starch using Saccharomyces cerevisiae in a long term repeated SSF process with full stillage recycling,Bioresour.Technol.,101(9):3126-3131
Box G.E.P.,and Behnken D.W.,1960,Some new three level designs for the study of quantitative variables,Technometrics2(4):455-475
Brar S.K.,Verma M.,Tyagi R.D.,Valero J.R.,and Surampalli R.Y.2006,Efficient centrifugal recovery of Bacillus thuringiensis biopesticides from fermented wastewater and wastewater sludge,Water Research,40(6):1310-1320
Hathout Y.,Demirev P.A.,Ho Y.P.,Bundy J.L.,Ryzhov V.,Sapp L.,Stutler J.,Jackman J.,and Fenselau C.,1999,Identification of Bacillus spores by matrix-assisted laser desorption ionization-mass spectrometry,Applied and Environmenta Microbiology,65(10):4313-4319
Lee J.W.,Koo B.W.,Choi J.W.,Choi D.H.,and Choi I.G.,2008Evaluation of waste mushroom logs as a potential biomass resource for the production of bioethanol,Bioresour.Technol.,99(8):2736-2741
?zkan M.,Dilek F.B.,Yetis.,ü and ?zcengiz G.,2003,Nutritional and cultural parameters influencing antidipteran deltaendotoxin production,Res.Microbiol.,154(1):49-53
Qiao J.J.,Zhang Y.F.,Sun L.F.,Liu W.W.,Zhu H.J.,and Zhang Z.,2011,Production of spent mushroom substrate hydrolysates useful for cultivation of Lactococcus lactis by dilute sulfuric acid,cellulase and xylanase treatment,Bioresource Technol.,102(17):8046-8051
Vu K.D.,Yan S.,Tyagi R.D.,Valero J.R.,and Surampalli R.Y.,2009,Induced production of chitinase to enhance entomotoxicity of Bacillus thuringiensis employing starch industry wastewater as a substrate,Bioresource Technology,100(21):5260-5269
Wu S.,Lan Y.,Wu Z.,Peng Y.,Chen S.,Huang Z.,Xu L.,Gelbic I.,Guan X.,Zhang L.,and Zou S.,2013,Pretreatment of spent mushroom substrate for enhancing the conversion of fermentable sugar,Bioresource Technology,148:596-600
Zhang C.K.,Gong F.,and Li D.S.,1995,A note on the utilisation of spent mushroom composts in animal feeds,Bioresource Technology,52(1):89-91
Zhang L.,Huang E.,Lin J.,Gelbic I.,Zhang Q.,Guan Y.,Huang T.,and Guan X.,2010,A novel mosquitocidal Bacillus thuringiensis strain ZLL13 isolated from the phylloplane of Magnolia denudata,Microbiological Research,165(2):133-141
Berry C.,O'Neil S.,Ben-Dov E.,Jones A.F.,Murphy L.,Quail M.A.,Holden M.T.G.,Harris D.,Zaritsky A.,and Parkhill J.,2002,Complete sequence and orga niza tion of pBtoxis,the toxin-coding plasmid of Bacillus thuringiensis subsp israelensis,Appl.Environ.Microb.,68(10):5082-5095
Bialas W.,Szymanowska D.,and Grajek W.,2010,Fuel ethano production from granular corn starch using Saccharomyces cerevisiae in a long term repeated SSF process with full stillage recycling,Bioresour.Technol.,101(9):3126-3131
Box G.E.P.,and Behnken D.W.,1960,Some new three level designs for the study of quantitative variables,Technometrics2(4):455-475
Brar S.K.,Verma M.,Tyagi R.D.,Valero J.R.,and Surampalli R.Y.2006,Efficient centrifugal recovery of Bacillus thuringiensis biopesticides from fermented wastewater and wastewater sludge,Water Research,40(6):1310-1320
Hathout Y.,Demirev P.A.,Ho Y.P.,Bundy J.L.,Ryzhov V.,Sapp L.,Stutler J.,Jackman J.,and Fenselau C.,1999,Identification of Bacillus spores by matrix-assisted laser desorption ionization-mass spectrometry,Applied and Environmenta Microbiology,65(10):4313-4319
Lee J.W.,Koo B.W.,Choi J.W.,Choi D.H.,and Choi I.G.,2008Evaluation of waste mushroom logs as a potential biomass resource for the production of bioethanol,Bioresour.Technol.,99(8):2736-2741
?zkan M.,Dilek F.B.,Yetis.,ü and ?zcengiz G.,2003,Nutritional and cultural parameters influencing antidipteran deltaendotoxin production,Res.Microbiol.,154(1):49-53
Qiao J.J.,Zhang Y.F.,Sun L.F.,Liu W.W.,Zhu H.J.,and Zhang Z.,2011,Production of spent mushroom substrate hydrolysates useful for cultivation of Lactococcus lactis by dilute sulfuric acid,cellulase and xylanase treatment,Bioresource Technol.,102(17):8046-8051
Vu K.D.,Yan S.,Tyagi R.D.,Valero J.R.,and Surampalli R.Y.,2009,Induced production of chitinase to enhance entomotoxicity of Bacillus thuringiensis employing starch industry wastewater as a substrate,Bioresource Technology,100(21):5260-5269
Wu S.,Lan Y.,Wu Z.,Peng Y.,Chen S.,Huang Z.,Xu L.,Gelbic I.,Guan X.,Zhang L.,and Zou S.,2013,Pretreatment of spent mushroom substrate for enhancing the conversion of fermentable sugar,Bioresource Technology,148:596-600
Zhang C.K.,Gong F.,and Li D.S.,1995,A note on the utilisation of spent mushroom composts in animal feeds,Bioresource Technology,52(1):89-91
Zhang L.,Huang E.,Lin J.,Gelbic I.,Zhang Q.,Guan Y.,Huang T.,and Guan X.,2010,A novel mosquitocidal Bacillus thuringiensis strain ZLL13 isolated from the phylloplane of Magnolia denudata,Microbiological Research,165(2):133-141