TWI510616B - Heat-resistant Pichia kudriavzevii - Google Patents
Heat-resistant Pichia kudriavzevii Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Description
本發明係關於耐高溫(40-45℃)酒精生產酵母菌Pichia kudriavzevii SI，尤其是該P.kudriavzevii SI菌株係可耐受纖維料源抑制物而進行纖維酒精醱酵。The present invention relates to a high temperature resistant (40-45 ° C) alcohol producing yeast Pichia kudriavzevii SI, in particular, the P. kudriavzevii SI strain is resistant to fibrous material source inhibitors and is subjected to fiber alcohol fermentation.
於酒精醱酵工業中，稀酸蒸氣爆裂前處理是目前常用處理纖維料源的方法之一，然而，於前該稀酸蒸氣爆裂處理過程中，往往會釋放許多纖維抑制物，諸如：香草醛(vanillin)、羥甲基糠醛(5-HMF)及呋喃甲醛(furfural)，該等抑制物會抑制醱酵菌株之生長，甚至導致醱酵菌株於醱酵過中死亡，而影響所欲之酒精醱酵結果。因此，為了避免纖維抑制物影響醱酵菌株之醱酵能力，現今許多纖維酒精製程於進行菌株醱酵前，往往得進一步進行去毒化之步驟，如：超鹼處理(overliming)來去除纖維抑制物，此等步驟往往會增加纖維酒精製程中之生產成本。In the alcohol fermentation industry, the pre-treatment of dilute acid vapor is one of the methods commonly used to treat fiber sources. However, in the process of this dilute acid vapor burst, many fiber inhibitors are often released, such as vanillin. (vanillin), hydroxymethylfurfural (5-HMF) and furfural (furfural), these inhibitors inhibit the growth of the fermentation strain, and even cause the fermentation strain to die in the fermentation, affecting the desired alcohol Fermentation results. Therefore, in order to avoid the fiber inhibitors affecting the fermentation ability of the fermented strain, many fiber alcohol processes today often require further detoxification steps before the strain is fermented, such as: overliming to remove fiber inhibitors. These steps tend to increase the production costs in the fiber alcohol process.
此外，於纖維酒精製程中之酵素水解步驟中，纖維水解酵素最適作用溫度為50℃，然而，習知之酒精生產酵母菌Saccharomyces cerevisiae 之最適醱酵溫度為30℃。若採用兩階段步驟，即分步水解與醱酵程序(separate hydrolysis and fermentation；SHF)來進行纖維酒精之生產，其製程上需要等待酵素水解完畢後，再將醱酵槽溫度自50℃降溫至30℃，醱酵菌株才得以進行酒精醱酵；反之，若採用單一階段之同步水解與醱酵程序(simultaneous saccharification and fermentation；SSF)來進行纖維酒精之生產，其製程上則需要將醱酵槽溫度控制於醱酵菌秼S.cerevisiae 菌株可耐受溫度之範圍內(30-35℃)，然而，於此溫度範圍內水解酵素之作用活性會明顯下降。In addition, in the enzyme hydrolysis step in the fiber alcohol process, the optimum temperature for the fiber hydrolyzing enzyme is 50 ° C. However, the optimum fermentation temperature of the conventional alcohol producing yeast Saccharomyces cerevisiae is 30 ° C. If a two-stage step, namely, separate hydrolysis and fermentation (SHF), is used for the production of fibrous alcohol, the process needs to wait for the hydrolysis of the enzyme to complete, and then the temperature of the fermentation tank is lowered from 50 ° C to At 30 °C, the fermented strain was allowed to carry out alcohol fermentation; on the contrary, if a single stage of simultaneous saccharification and fermentation (SSF) was used for the production of fibrous alcohol, the fermentation tank was required in the process. The temperature is controlled within the temperature tolerance range of the strain of S. cerevisiae (30-35 ° C), however, the activity of the hydrolyzed enzyme is significantly decreased in this temperature range.
因此，不論採取那一種製程來生產纖維酒精，於控溫過程往往需以大量冷卻水來降低醱酵槽體溫度，醱酵菌株才得以存活並進行酒精醱酵，此舉不但增加製程上的生產成本，低溫醱酵同時也容易增加雜菌污染所導致之敗槽現象。Therefore, no matter which process is used to produce fiber alcohol, the temperature control process often requires a large amount of cooling water to lower the temperature of the fermentation tank, and the fermented strain can survive and carry out alcohol fermentation, which not only increases the production on the process. Cost, low temperature fermentation and easy to increase the damage caused by the contamination of bacteria.
有鑒於此，為解決上述現有技術的缺失，本發明之目的即為篩選出一株高效率酒精生產酵母菌Pichia kudriavzevii SI，其耐高溫(40-45℃)及耐高濃度纖維抑制物之特性以進行纖維酒精之生產，以期減少製程成本上的支出與增加操作上的便利性。In view of the above, in order to solve the above-mentioned deficiency of the prior art, the object of the present invention is to screen out a high-efficiency alcohol-producing yeast Pichia kudriavzevii SI, which is resistant to high temperature (40-45 ° C) and high-strength fiber inhibitors. To produce fiber alcohol, in order to reduce the cost of the process and increase the convenience of operation.
為了克服纖維料源在前處理後產生之抑制物而造成菌株醱酵被抑制，及醱酵槽體控溫於野生型S.cerevisiae 可耐受溫度之範圍(30-35℃)所導致多能耗增加生產成本之問題，現今技術多以突變菌株基因之方式，如：利用突變劑或照射高能量之光線、以纖維水解液馴化菌株，藉此得到突變菌株以提升菌株對於纖維抑制物或高溫之耐受度。然而，此等過程耗時長，且於後續菌株培養的過程中，易因為菌株之基因修復現象，導致原突變菌株之基因不穩定性，進而喪失纖維抑制物或高溫之耐受性。In order to overcome the inhibitory effect of the fiber material source after the pretreatment, the strain fermentation is inhibited, and the temperature of the fermentation tank is controlled by the wild type S. cerevisiae tolerate the temperature (30-35 ° C). The problem of increasing production cost is that the current technology mostly uses mutant gene genes, such as: using a mutagen or irradiating high-energy light, acclimating the strain with a fiber hydrolysate, thereby obtaining a mutant strain to enhance the strain for fiber inhibitors or high temperature. Tolerance. However, these processes are time consuming, and in the process of culturing the subsequent strains, it is easy to cause gene instability of the original mutant strain due to the gene repair phenomenon of the strain, thereby losing the tolerance of the fiber inhibitor or high temperature.
故以其他原生優勢菌種如P.kudriavzevii 具耐高濃度抑制物及耐高溫之性質，來進行纖維酒精之生產，才能改善此問題。Therefore, the production of fiber alcohol can be improved by using other dominant species such as P. kudriavzevii with high concentration inhibitor and high temperature resistance.
本發明之目的即針對上述問題，提供一種經篩選所得之具耐高濃度抑制物及耐高溫性質之酒精醱酵菌株，該菌株係耐高溫及抑制物而同時進行纖維酒精之生產，可有效減少製程的成本及增加操作上的便利性。The object of the present invention is to provide an alcohol-fermented strain which is resistant to high-concentration inhibitors and high-temperature-resistant properties, which is resistant to high temperature and inhibitors and simultaneously produces fibrous alcohol, which is effective for reducing the above problems. The cost of the process and increased operational convenience.
為達上述目的及其他目的，本發明係提供一種德里阿兹威氏畢赤酵母菌(Pichia kudriavzevii SI)，該德里阿兹威氏畢赤酵母菌寄存於中華民國食品工業發展研究所，寄存編號為BCRC 920094，該德里阿兹威氏畢赤酵母菌具耐高性及耐纖維抑制物性，其可耐受高溫40-45℃，亦可耐受纖維抑制物vanillin、5-HMF及furfural。To achieve the above and other objects, the present invention provides a Pichia kudriavzevii SI, which is deposited in the Republic of China Food Industry Development Institute, the registration number. For BCRC 920094, the P. azensis has high tolerance and fiber inhibiting properties, can withstand high temperatures of 40-45 ° C, and can also tolerate fiber inhibitors vanillin, 5-HMF and furfural.
且上述之德里阿兹威氏畢赤酵母菌(Pichia kudriavzevii SI)，其中該纖維抑制物vanillin濃度為3g L-1 、5-HMF濃度為5g L-1 及furfural濃度為3g L-1 。And the above-mentioned Pichia kudriavzevii SI, wherein the fiber inhibitor has a vanillin concentration of 3 g L -1 , a 5-HMF concentration of 5 g L -1 and a furfural concentration of 3 g L -1 .
為達上述目的及其他目的，本發明同時提供一種纖維酒精生產之方法，係以如上所述之德里阿兹威氏畢赤酵母菌(Pichia kudriavzevii SI)進行醱酵，其醱酵料源係為稀酸蒸氣爆裂前處理後且未去毒化之纖維稻稈、木片及蔗渣，並經由40℃醱酵可生產濃度為22.6-35.4gp L-1 及產率為86.6-95.5%之纖維酒精。To achieve the above and other objects, the present invention also provides a method for producing fibrous alcohol, which is fermented by Pichia kudriavzevii SI as described above, and the source of the mash is Fiber straw, wood chips and bagasse which have not been detoxified after pre-explosion of dilute acid vapor, and fermented at 40 ° C can produce fiber alcohol with a concentration of 22.6-35.4 g p L -1 and a yield of 86.6-95.5%.
如上所述方法，該方法中之醱酵溫度為40℃時，纖維酒精之生產濃度為22.6-35.4gp L-1 ，纖維酒精之產率為86.6-95.5%。As described above, in the method, when the fermentation temperature is 40 ° C, the production concentration of the fiber alcohol is 22.6-35.4 g p L -1 , and the yield of the fiber alcohol is 86.6-95.5%.
如上所述方法，其中該稀酸蒸氣爆裂前處理之纖維料源，係預先經0.7-1%(v/v)稀硫酸混酸前處理程序，再送入高溫反應槽通入介於180-200℃之間之蒸汽進行蒸煮1-10分鐘後，立即釋放壓力以破壞其結構。The method as described above, wherein the fiber source of the pre-treatment of the dilute acid vapor is pre-treated by a 0.7-1% (v/v) dilute sulfuric acid mixed acid pretreatment process, and then sent to the high temperature reaction tank for introduction at 180-200 ° C. After the steam was steamed for 1-10 minutes, the pressure was immediately released to destroy its structure.
藉由如上所述之德里阿兹威氏畢赤酵母菌(Pichia kudriavzevii SI)及纖維酒精生產之方法，係可有效降低纖維酒精製程的成本及增加操作便利性。By the method of producing Pichia kudriavzevii SI and the fiber alcohol as described above, the cost of the fiber alcohol process can be effectively reduced and the operation convenience can be increased.
第1圖係為本發明中之Pichia kudriavzevii SI菌株最大比生長速率比較圖。Fig. 1 is a graph comparing the maximum specific growth rates of the Pichia kudriavzevii SI strain in the present invention.
第2a圖係為本發明菌株P.kudriavzevii SI與對照組S.cerevisiae 之纖維抑制物香草醛耐受性比較圖。Fig. 2a is a graph comparing the tolerance of vanillin to the fiber inhibitor of the strain P. kudriavzevii SI of the present invention and the control group S. cerevisiae .
第2b圖係為本發明菌株P.kudriavzevii SI與對照組S.cerevisiae 之纖維抑制物羥甲基糠醛耐受性比較圖Figure 2b is a comparison of the tolerance of the fiber inhibitor hydroxymethylfurfural of the strain P. kudriavzevii SI of the present invention and the control group S. cerevisiae
第2c圖係為本發明菌株P.kudriavzevii SI與對照組S.cerevisiae 之纖維抑制物呋喃甲醛耐受性比較圖。Fig. 2c is a graph comparing the resistance of the fibrinogen to the fiber inhibitor of the strain P. kudriavzevii SI of the present invention and the control group S. cerevisiae .
第3a圖係為本發明菌株P.kudriavzevii SI與對照組S.cerevisiae 之纖維稻稈酒精生成比較圖。Figure 3a is a comparison of the ethanol production of the fiber stalk of the strain P. kudriavzevii SI of the present invention and the control group S. cerevisiae .
第3b圖係為本發明菌株P.kudriavzevii SI與對照組S.cerevisiae 之纖維木片酒精生成比較圖。Figure 3b is a comparison of the alcohol production of the fiber wood chips of the strain P. kudriavzevii SI of the present invention and the control group S. cerevisiae .
第3c圖係為本發明菌株P.kudriavzevii SI與對照組S.cerevisiae 之纖維蔗渣酒精生成比較圖。Figure 3c is a comparison of the alcohol production of the fiber bagasse of the strain P. kudriavzevii SI of the present invention and the control group S. cerevisiae .
第3d圖係為本發明菌株P.kudriavzevii SI與對照組S.cerevisiae 之纖維蔗渣中菌體生長情形的比較圖。Fig. 3d is a comparison of the growth of the cells in the fiber bagasse of the strain P. kudriavzevii SI of the present invention and the control group S. cerevisiae .
為充分瞭解本發明之目的、特徵及功效，兹藉由下述具體之實施例，並配合所附之圖式，對本發明做一詳細說明，說明如後：本發明之菌株來源係篩選自台北市信義區果菜市場之家庭廚餘，經高效液相層析儀(High-performance liquid chromatography)分析經該菌株醱酵所得之醱酵樣品數據，得知該菌株為一酒精生產菌株。而本發明經篩選後所得之菌株經菌種鑑定之結果如表1所示(中華民國食品工業發展研究所)，而根據食品 工業發展研究所利用酵母菌26s rDNA之D1/D2序列比對，鑑定本發明所篩選出菌株為德里阿兹威氏畢赤酵母菌Pichia kudriavzevii ，遂命名其為Pichia kudriavzevii SI，並將該德里阿兹威氏畢赤酵母菌寄存於中華民國食品工業發展研究所，寄存編號為BCRC 920094。In order to fully understand the object, features and effects of the present invention, the present invention will be described in detail by the following specific examples, and the accompanying drawings, The household food waste of the fruit and vegetable market in Xinyi District was analyzed by high-performance liquid chromatography, and the strain was obtained as an alcohol production strain. The results of strain identification of the strains obtained by the screening of the present invention are shown in Table 1 (Research Institute of Food Industry Development of the Republic of China), and according to the D1/D2 sequence alignment of yeast 26s rDNA according to the Food Industry Development Research Institute, The strain selected by the present invention was identified as Pichia kudriavzevii , which was named Pichia kudriavzevii SI, and the Pichia pastoris was deposited in the Republic of China Food Industry Development Institute. The deposit number is BCRC 920094.
而菌種鑑定報告指出，該Pichia kudriavzevii SI菌株與文獻(Kurtzman C.P.,J.W.Fell and T.Boekhout.2011.The Yeasts,a Taxonomic Study.Fifth edition.Elsevier Science publishers,B.V.Amsterdam.)試驗過Pichia kudriavzevii 的生理特性總和有所差異。文獻指出Pichia kudriavzevii 不可代謝Inulin，但可代謝glycerol；然而，本發明P.kudriavzevii SI菌株其生理特性為可代謝Inulin，但不可代謝glycerol，顯示該P.kudriavzevii SI菌株為一新穎性菌株。將該菌株經培養所得菌液與50%甘油等比例混合，製備成凍管並保存於-80℃冰箱中。The strain identification report indicated that the Pichia kudriavzevii SI strain and the literature (Kurtzman CP, JWFell and T. Boekhout. 2011. The Yeasts, a Taxonomic Study. Fifth edition. Elsevier Science publishers, BVAmsterdam.) tested the physiological characteristics of Pichia kudriavzevii . The sum is different. The literature indicates that Pichia kudriavzevii cannot metabolize Inulin, but can metabolize glycerol; however, the P. kudriavzevii SI strain of the present invention has physiological characteristics that can metabolize Inulin, but cannot metabolize glycerol, indicating that the P. kudriavzevii SI strain is a novel strain. The bacterial solution obtained by the culture was mixed with 50% glycerol in an equal ratio to prepare a frozen tube and stored in a -80 ° C refrigerator.
該菌株Pichia kudriavzevii SI之醱酵樣品數據分析，係利用高效液相層析儀之Coregel-87H3(Transgenomics,Co.)管柱，搭配流速0.8mL min-1 之4mM H2 SO4 沖提液於45℃進行樣品分離，以折射率偵測器(refractive index detector)偵測沖提流出液中待測化合物之訊號，紀錄分離出每種化合物的發生時間。The data of the fermentation sample of the strain Pichia kudriavzevii SI was analyzed by a Coregel-87H3 (Transgenomics, Co.) column of high performance liquid chromatography with a flow rate of 0.8 mL min -1 of 4 mM H 2 SO 4 extract. The sample was separated at 45 ° C, and the signal of the test compound in the effluent was detected by a refractive index detector, and the time of occurrence of each compound was recorded.
依據表2之培養基配方於250ml搖瓶中配製種菌培養基，在121℃下高壓滅菌20分鐘後，自凍管取出體積為培養基之千分之一P.kudriavzevii SI菌株，接種於培養基內並在溫度40℃、轉速150rpm下培養16小時，以得到醱酵實驗的前接菌液。According to the medium formula of Table 2, the inoculum culture medium was prepared in a 250 ml shake flask, and after autoclaving at 121 ° C for 20 minutes, a P. kudriavzevii SI strain of one thousandth of the medium was taken out from the frozen tube, and inoculated into the medium at a temperature. The cells were cultured for 16 hours at 40 ° C and a rotation speed of 150 rpm to obtain a pre-inoculation solution for the fermentation test.
參閱表3所示，本發明係以P.kudriavzevii SI培養於不同溫度及葡萄糖濃度之培養基，以250ml搖瓶測試菌株的溫度及葡萄糖濃度之耐受性。根據表2之培養基配方，將葡萄糖濃度調整至9、12或13%(w/v)，接種菌體濃度為0.2g L-1 之P.kudriavzevii SI前接菌液，並培養於溫度37、40、42或45℃、轉速150rpm，各醱酵時間點之醱酵液樣品以高效液相層析儀進行數據分析。醱酵24小時，該菌株於溫度40-42℃可代謝濃度為9-13%(w/v)之葡萄糖，其酒精產率為理論產率之九成以上。此外，該菌株可耐高溫至45℃，於葡萄糖濃度為9%及12%(w/v)之情況下，其酒精產率皆可達理論產率之九成。實驗結果顯示，Pichia kudriavzevii SI為一耐高溫且耐高葡萄糖濃度之菌株。Referring to Table 3, the present invention was cultured in a medium of different temperatures and glucose concentrations using P. kudriavzevii SI, and the temperature and glucose concentration tolerance of the strain were tested in a 250 ml shake flask. According to the medium formula of Table 2, the glucose concentration was adjusted to 9, 12 or 13% (w/v), and the P. kudriavzevii SI pre-bacterial solution having a cell concentration of 0.2 g L -1 was inoculated and cultured at a temperature of 37, 40, 42 or 45 ° C, 150 rpm, the fermentation time samples of each fermentation time point were analyzed by high performance liquid chromatography. After fermentation for 24 hours, the strain can metabolize glucose at a concentration of 9-13% (w/v) at a temperature of 40-42 ° C, and the alcohol yield is more than 90% of the theoretical yield. In addition, the strain can withstand high temperatures up to 45 ° C, and the glucose yield can reach 90% of the theoretical yield at a glucose concentration of 9% and 12% (w/v). The experimental results show that Pichia kudriavzevii SI is a strain that is resistant to high temperatures and high in glucose concentration.
菌株於不同培養溫度之最大比生長速率比較圖如第1圖所示，本發明係根據表2之培養基配方，以250ml搖瓶將葡萄糖濃度調整至9%(w/v)，接種菌體濃度為0.2g L-1 之P.kudriavzevii SI前接菌液，並培養於溫度37、40、42或45℃、轉速150rpm。利用分光光度計偵測不同培養時間點之菌濃度，依公式μ max (h-1 )=ln(X 2 /X 1 )/(t 2 -t 1 )計算得到菌株之最大比生長速率(maximum specific growth rate)。實驗結果顯示，相較培養於37、42及45℃，Pichia kudriavzevii SI於培養溫度40℃下有最快之最大比生長速率(0.405h-1 )，因此後續醱酵實驗皆以40℃作為菌株之最適培養溫度。The maximum specific growth rate of the strains at different culture temperatures is shown in Fig. 1. According to the medium formula of Table 2, the concentration of the glucose is adjusted to 9% (w/v) in a 250 ml shake flask, and the concentration of the cells is inoculated. The P. kudriavzevii SI pre-inoculation solution was 0.2 g L -1 and cultured at a temperature of 37, 40, 42 or 45 ° C and a rotation speed of 150 rpm. The concentration of the bacteria at different culture time points was detected by spectrophotometer, and the maximum specific growth rate of the strain was calculated according to the formula μ max (h -1 )=ln( X 2 / X 1 )/( t 2 - t 1 ) (maximum Specific growth rate). The experimental results showed that Pichia kudriavzevii SI had the fastest maximum specific growth rate (0.405h -1 ) at 40 °C compared with cultured at 37, 42 and 45 °C. Therefore, the subsequent fermentation experiments were carried out at 40 °C. The optimum culture temperature.
參閱第2圖所示，本發明係以P.kudriavzevii SI作實驗組，傳統酒精生產酵母菌株Saccharomyces cerevisiae 作為對照組，培養於不同纖維抑制物濃度之培養基，以250ml搖瓶、轉速150rpm測試菌株之纖維抑制物耐受性。根據表2之培養基配方，將葡萄糖濃度調整至6-7%(w/v)，並添加纖維抑制物vanillin濃度0.5-3g L-1 、5-HMF濃度1-5g L-1 或furfural濃度1-5g L-1 至培養基中，接種菌體濃度為0.5g L-1 之前接菌液，P.kudriavzevii SI培養於40℃，S.cerevisiae 培養於30℃。P.kudriavzevii SI可耐受抑制物vahillin濃度3g L-1 、5-HMF濃度5g L-1 及furfural濃度3g L-1 ，其酒精醱酵能力與產率可達1.7-3.3gp L-1 h-1 及81-88%；然而同此條件下，S.cerevisiae 卻無法醱酵。實驗結果顯示，相較於S.cerevisiae 該P.kudriavzevii SI擁有較高耐纖維抑制物之能力。Referring to Fig. 2, the present invention uses P. kudriavzevii SI as an experimental group, a traditional alcohol-producing yeast strain Saccharomyces cerevisiae as a control group, cultured in a medium of different fiber inhibitor concentration, and tested in a 250 ml shake flask at a rotation speed of 150 rpm. Fiber inhibitor tolerance. According to the medium formula of Table 2, the glucose concentration was adjusted to 6-7% (w/v), and the fiber inhibitor vanillin concentration was added to 0.5-3 g L -1 , 5-HMF concentration 1-5 g L -1 or furfural concentration 1 -5 g L -1 to the medium, the inoculum concentration was 0.5 g L -1 before the inoculum, P. kudriavzevii SI was cultured at 40 ° C, and S. cerevisiae was cultured at 30 ° C. P.kudriavzevii SI tolerant inhibitor vahillin concentration 3g L -1 , 5-HMF concentration 5g L -1 and furfural concentration 3g L -1 , its alcohol fermentation capacity and yield can reach 1.7-3.3g p L -1 h -1 and 81-88%; however, under the same conditions, S. cerevisiae could not be fermented. The experimental results show that the P. kudriavzevii SI has a higher ability to resist fiber inhibitors than S. cerevisiae .
為檢測菌株Pichia kudriavzevii SI之多重壓力耐受度及多源纖維料源之酒精醱酵可行性，本發明係以稀酸蒸氣爆裂法進行纖維料源的前處理，係經0.7-1%(v/v)稀硫酸處理過的料源，送入高溫反應槽以180-200℃的高溫蒸氣蒸煮1-10分鐘後，立即釋放壓力以破壞其結構，此泥狀物經壓濾過後所得之固體供後續纖維素水解酵素作用。酵素水解程序，係配製稀酸蒸氣爆裂前處理之纖維料源至固液比為15%，以NaOH調整pH值至5，接著添加每克纖維素15 FPU之纖 維水解酵素，在50℃、轉速200rpm下反應3天，可得酵素水解液，此水解液即可做菌株Pichia kudriavzevii SI之醱酵用培養基。In order to detect the multiple pressure tolerance of the strain Pichia kudriavzevii SI and the feasibility of alcohol fermentation of the multi-source fiber source, the present invention performs the pretreatment of the fiber source by the dilute acid vapor burst method, which is 0.7-1% (v). /v) Dilute sulfuric acid treated feed source, sent to a high temperature reaction tank for steaming at a high temperature of 180-200 ° C for 1-10 minutes, immediately release pressure to destroy its structure, the solid obtained by pressure filtration of the mud For subsequent cellulose hydrolyzing enzymes. The enzyme hydrolysis process is to prepare the fiber source of the pre-treatment of dilute acid vapor to a solid-liquid ratio of 15%, adjust the pH to 5 with NaOH, and then add 15 FPU of fiber hydrolyzate per gram of cellulose at 50 ° C. After reacting at 200 rpm for 3 days, an enzyme hydrolyzate can be obtained, and this hydrolyzate can be used as a fermentation medium for the strain Pichia kudriavzevii SI.
參閱第3圖所示，本發明係以稀酸蒸氣爆裂前處理後且未去毒化的纖維料源(稻稈、木片及蔗渣)，進行P.kudriavzevii SI之醱酵能力評估。P.kudriavzevii SI生產纖維稻稈酒精如第3a圖所示，前處理後暨未去毒化之纖維稻稈含抑制物furfural濃度0.17g L-1 、acetic acid濃度1.68g L-1 ，以5L醱酵槽轉速200rpm進行40℃醱酵，接種菌體濃度為0.5g L-1 之前接菌液，其醱酵72小時之纖維稻稈酒精濃度及產率分別為35.4gp L-1 和86.6%。Referring to Fig. 3, the present invention evaluates the fermentation capacity of P. kudriavzevii SI by a fiber source (rice stalk , wood chip and bagasse) which has been dehumidified after pre-explosion of dilute acid vapor. P.kudriavzevii SI produces fiber straw stalk alcohol as shown in Figure 3a. The pre-treatment and undetoxified fiber stalk contain the furfural concentration of 0.17g L -1 and acetic acid concentration of 1.68g L -1 , 5L酦The fermenter was rotated at 200 rpm for 40 °C fermentation. The inoculum was inoculated with 0.5 g L -1 before the inoculum, and the alcohol concentration and yield of the fiber stalks after fermentation for 72 hours were 35.4 g p L -1 and 86.6%, respectively. .
第3b圖係以P.kudriavzevii SI作為實驗組，S.cerevisiae 作對照組，比較菌株之多源纖維酒精醱酵的可行性。前處理後且未去毒化之纖維木片含抑制物furfural濃度1.21g L-1 、5-HMF濃度0.72g L-1 、acetic acid濃度4.8g L-1 ，以250ml搖瓶轉速150rpm進行P.kudriavzevii SI之40℃醱酵，接種菌體濃度為0.5g L-1 之前接菌液，其醱酵28小時之纖維木片酒精濃度及產率分別為26.3gp L-1 和95.5%；以同料源進行S.cerevidiae 之30℃醱酵，其纖維木片酒精生成速率於8小時內較P.kudriavzevii SI慢1.74倍。In Fig. 3b, P.kudriavzevii SI was used as the experimental group and S. cerevisiae was used as the control group to compare the feasibility of the multi-source fiber alcohol fermentation of the strain. The fiber wood chips after pretreatment and not detoxified contained inhibitors furfural concentration 1.21g L -1 , 5-HMF concentration 0.72g L -1 , acetic acid concentration 4.8g L -1 , P.kudriavzevii at 250ml shaker speed 150rpm The 40°C fermentation of SI was carried out, and the inoculum concentration was 0.5g L -1 before the inoculum, and the alcohol concentration and yield of the fiber wood chips after fermentation for 28 hours were 26.3g p L -1 and 95.5%, respectively; The source was subjected to S. cerevidiae 30 °C fermentation, and the rate of alcohol production of the fiber chips was 1.74 times slower than that of P. kudriavzevii SI within 8 hours.
第3c圖係以P.kudriavzevii SI作為實驗組，S.cerevisiae 作對照組，培養於前處理後且未去毒化之纖維蔗渣，內含抑制物furfural濃度為1.07g L-1 、5-HMF濃度0.2g L-1 、acetic acid濃度7.65g L-1 ，以250ml搖瓶轉速150rpm進行P.kudriavzevii SI之40℃醱酵，接種菌體濃度為0.5g L-1 之前接菌液，其醱酵56小時之纖維蔗渣酒精濃度及產率分別為22.6gp L-1 及91.2%；以同料源進行S.cerevisiae 之30℃醱酵，而如第3d圖所示，S.cerevisiae 細胞生長幾近停止且無法順利生產纖維蔗渣酒精，即顯示P.kudriavzevii SI比S.cerevisiae 擁有較佳之纖維抑制物耐受度及多源纖維酒精醱酵之可行性。In Fig. 3c, P.kudriavzevii SI was used as the experimental group, and S. cerevisiae was used as the control group. The fiber bagasse was cultured in the pretreated and not detoxified, and the concentration of the furfural inhibitor was 1.07 g L -1 and 5-HMF. 0.2g L -1 , acetic acid concentration 7.65g L -1 , P.kudriavzevii SI 40 ° C fermentation at 250ml shaker speed 150rpm, inoculum concentration before the inoculum concentration of 0.5g L -1 , its fermentation The 56-hour fiber bagasse alcohol concentration and yield were 22.6 g p L -1 and 91.2%, respectively; S. cerevisiae was fermented at 30 °C with the same source, and as shown in Figure 3d, S. cerevisiae cells grew several times. The near stop and the inability to produce fiber bagasse alcohol smoothly show that P.kudriavzevii SI has better fiber inhibitor tolerance and multi-source fiber alcohol fermentation than S. cerevisiae .
本發明在上文中已以較佳實施例揭露，然熟習本項技術者應理解的是，該實施例僅用於描繪本發明，而不應解讀為限制本發明之範圍。應注意的是，舉凡與該實施例等效之變化與置換，均應設為涵蓋於本發明之範疇內。因此，本發明之保護範圍當以申請專利範圍所界定者為準。The invention has been described above in terms of the preferred embodiments, and it should be understood by those skilled in the art that the present invention is not intended to limit the scope of the invention. It should be noted that variations and permutations equivalent to those of the embodiments are intended to be included within the scope of the present invention. Therefore, the scope of protection of the present invention is defined by the scope of the patent application.
國內寄存資訊Domestic deposit information
中華民國食品工業發展研究所生物資源保存及研究中心(Bioresource Collection and Research Center,BCRC)Bioresource Collection and Research Center (BCRC), Republic of China Food Industry Development Institute
2014年8月27日August 27, 2014
BCRC 920094BCRC 920094
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