2012年第4期廣東化工第39卷總第228期www.gdchem.con87冰糖橙皮渣發(fā)酵產(chǎn)燃料乙醇的工藝優(yōu)化牛友芽12,高玉妹2,倪俊2(1.懷化學(xué)院生命科學(xué)系,湖南懷化418008:2.湘潭大學(xué)化工學(xué)院,湖南湘潭41115摘要采用纖維素酶、果膠酶和葡萄糖苷酶對(duì)冰糖橙皮渣進(jìn)行水解,所得還原糖液接種異常畢赤酵母進(jìn)行發(fā)酵,考察了酵母接種量、發(fā)酵時(shí)間、pH和發(fā)酵溫度等單因素對(duì)乙醇得率的影響。單因素結(jié)果表明:接種量為12%、發(fā)酵時(shí)間72h、pH45、發(fā)酵溫度33℃時(shí)乙醇得率最高。在此基礎(chǔ)上設(shè)計(jì)L4(3正交實(shí)驗(yàn)。結(jié)果表明,最佳工藝條件為pH4.5,接種量12%,發(fā)酵時(shí)間72h,發(fā)酵溫度30℃。在此條件下乙醇產(chǎn)率為0.2451gg,顯著高于單因素實(shí)驗(yàn)(0.263gg)和正交實(shí)驗(yàn)結(jié)果(02329gg)[關(guān)鍵詞]酶水解:發(fā)酵:冰糖橙皮渣:乙醇;工藝[中圖分類號(hào)TQ[文獻(xiàn)標(biāo)識(shí)碼]A[文章編號(hào)]007-1865(2012)040087-02Optimization of the fermentation Technology of Bio-ethanolProduction from Bingtang Sweet Orange Peel WasteNiu Youya, Gao Yumei, Ni Jun(L. Department of Life Science, Huaihua College, Huaihua 418008:2. College of Chemical Engineering, Xiangtan University, Xiangtan 411105, China)Abstract: The Bingtang sweet orange peel waste was hydrolyzed by celluase, pectinase and B-glucosidase. The resulting reducing sugar solution was inoculatedwith Pichia anomala and fermented. The effects of single factors including yeast inoculums, fermentation time, pH and fermentation temperature on ethanol yieldwere illustrated. Results showed that the optimum condition for single factor experiments were as follows: inoculums 12 % fermentation time 72 h; pH 4.5fermentation temperature 33 C. Then a Lg(3")orthogonal test was designed based on the forgoing results. Results showed the optimal fermentation technology wasoH 4.5; inoculums 12 %; fermentation time 72 h; fermentation temperature 30 C. Under these conditions, the ethanol yield could reach 0. 2451 g/g, significanthigher than those in single factor experiment(0. 2263 g/g) and orthodox experiments(. 2329 g/g)Keywords: enzyme- hydrolyze: ferment; bingtang sweet orange peel waste: ethanol: technology萬(wàn),第一次超過(guò)巴西成為世界第一大柑橘生產(chǎn)國(guó)。柑橘果實(shí)榨了233114.1接種量對(duì)乙醇得率的影響其他條件同上,考察酵母接種量(6%、8%、10%、12%和汁后會(huì)產(chǎn)生40%50%的皮渣,這些皮渣的主要組成為果膠、纖14%,Vv)對(duì)乙醇產(chǎn)率的影響維素和半纖維素等,約占柑橘皮渣干重的20%30%。特定條1.4.2發(fā)酵時(shí)間對(duì)乙醇得率的影響件下,果膠、纖維素和半纖維素等能轉(zhuǎn)化為可發(fā)酵性糖,在微生物作用下轉(zhuǎn)化成乙醇,美國(guó)、希臘等國(guó)家已逐步開始利用柑橘皮12h測(cè)乙醇產(chǎn)率固定其他條件,酵母接種量為12%VV),發(fā)酵24h后,每隔菌種的性能對(duì)發(fā)酵效率有重要影響,已用于柑橘皮渣發(fā)酵的14.3初始pH對(duì)發(fā)酵的影響將發(fā)酵水解液調(diào)整至不同pH(3.9、42、45、48和5.1)后菌種主要有釀酒酵母( Saccharomyces cerevisiae)378101、嗜鞣管囊接種12%的酵母菌懸液在33℃下靜置發(fā)酵72h,測(cè)定乙醇產(chǎn)率酵母( Pachysolen tannophilus)y、馬克斯克魯維酵母( Klvveromvce144發(fā)酵溫度對(duì)發(fā)酵的影marxianus、大腸桿菌( Escherichia coil)4和歐文氏菌( Erwinia/等將接種量為12%的酵母菌懸液接入到pH48的發(fā)酵水解液前期研究中,已從酒曲中分離得到一株可耐14%酒精度的異常畢赤中,在不同溫度(27℃、30℃、33℃、36℃和39℃)下靜置發(fā)酵母( Pichia anomala)菌株1,具備生產(chǎn)燃料乙醇的應(yīng)用潛力72h,測(cè)定乙醇產(chǎn)率本研究擬以冰糖橙皮渣酶水解物為底物,探討接種量、發(fā)酵1.4.5正交試驗(yàn)的設(shè)計(jì)時(shí)間、pH和發(fā)酵溫度對(duì)異常畢赤酵母發(fā)酵產(chǎn)燃料乙醇的影響,在在單因素實(shí)驗(yàn)的基礎(chǔ)上,分別選取對(duì)發(fā)酵影響較大的接種量此基礎(chǔ)上設(shè)計(jì)正交試驗(yàn),獲得最佳工藝條件,為利用柑橘皮渣和(%)、發(fā)酵時(shí)間(h)、pH、發(fā)酵溫度(℃)的4個(gè)因素的3個(gè)較優(yōu)水平,燃料乙醇生產(chǎn)提供理論參考。用L3正交試驗(yàn)對(duì)發(fā)酵條件作進(jìn)一步優(yōu)化。正交試驗(yàn)表設(shè)計(jì)如表11實(shí)驗(yàn)1實(shí)驗(yàn)材料表1正交試驗(yàn)設(shè)計(jì)表請(qǐng)之。糖 Citrus sinensis Osbeck)果實(shí)購(gòu)于湘潭大學(xué)購(gòu)物中心Tab. 1 The experimental design of orthogonal test因1.2冰糖橙皮渣的酶水解水平接種量(A)%時(shí)間B)hpH溫度①y℃生250mL錐形瓶中加入2g原料,分別加入100mLpH48磷酸檸檬酸緩沖液和纖維素酶(5mgg底物)、果膠酶(3mgg底物)和124.5葡萄糖苷酶酶液(2mgg底物),120rmin、48℃震蕩培養(yǎng)48h,反應(yīng)結(jié)束后,將三角瓶取出放入沸水中煮沸15min以終止反應(yīng),所得還原糖得率為6145%。1.3酵母菌懸液的制備1.5分析方法將活化好的異常畢赤酵母接種到Y(jié)PD液體培養(yǎng)基中,在28乙醇得率采用氣相色譜法測(cè)定,色譜條件為:安捷倫全溫振蕩培養(yǎng)箱中發(fā)GC-7890A,極性毛細(xì)管柱(25m×0025mm),氫火焰離子檢測(cè)rmin下離心10min,用生理鹽水將菌體洗滌3次后用無(wú)菌水稀釋到(FD,氫氣流量30mmin,載氣氮?dú)饬髁?50 mL/min,空氣流量4001.0×103個(gè)/mL。mL/min;程序升溫:40℃保持1min,然后以15℃min的升溫速率升14發(fā)酵條件至150℃;進(jìn)樣器溫度180℃,檢測(cè)器溫度200℃;分流比50:1,進(jìn)酶水解后,將水解液過(guò)濾,放入500mL三角瓶中,加入營(yíng)養(yǎng)物樣量04:以甲醇質(zhì)(蛋白胨5%、酵母膏3%NHSO40.05%MgSO40.25%KHPO41.6數(shù)據(jù)處理和中國(guó)煤化工以g底物表示025%),滅菌后接入酵母菌懸液,33℃、pH4.8靜置發(fā)酵84h每個(gè)實(shí)驗(yàn)3次軟件進(jìn)行將YHCNMHG采用SPSs30攵稿日期]2011-12-09[基金項(xiàng)目]湘潭大學(xué)大學(xué)生創(chuàng)新基金(201027資助[作者簡(jiǎn)介牛友芽(1976-),男,碩士,講師,主要從事應(yīng)用生物化學(xué)研究工作廣東化工2012年第4期88www.gdchem.com第39卷總第228期2結(jié)果與分析隨著接種量由6%增加到12%,乙醇產(chǎn)率由01756gg增加到2.1接種量對(duì)乙醇產(chǎn)率的影響02246gg。進(jìn)一步增加接種量對(duì)乙醇產(chǎn)率無(wú)顯著影響(p<0.05)。如圖1所當(dāng)增加異常畢赤酵母接種量能增加乙醇得率。0.250.250.200.15禮0.100100.0512%14%接種量溫度/℃c圖1接種量對(duì)乙醇產(chǎn)率的影響圖2發(fā)酵溫度對(duì)乙醇產(chǎn)率的影響Fig 1 Effect of Inoculum size on ethanol yieldig 2 Effect of Fermentation temperature on the ethanol yield22發(fā)酵溫度對(duì)乙醇產(chǎn)率的影響(p<0.05)。發(fā)酵溫度對(duì)乙醇產(chǎn)率的影響如圖2所示。隨著發(fā)酵溫度從2723pH對(duì)乙醇產(chǎn)率的影響℃上升到33℃,乙醇產(chǎn)率從0.983gg增加到0.2263gg;進(jìn)一步如圖3所示,當(dāng)pH為3.9和42時(shí),乙醇產(chǎn)率偏低,分別為0030升高溫度則會(huì)顯著降低乙醇產(chǎn)自發(fā)酵溫度為39℃時(shí),乙醇產(chǎn)gg和0.0384gg,顯著低于pH4.5時(shí)乙醇得率(.1885gg)。pH升高率為0.1594gg,顯著低于其他發(fā)酵溫度條件下的乙醇產(chǎn)率到4.8和51對(duì)乙醇產(chǎn)量無(wú)明顯影響(p<005)0.250.2000.150.10.出0.100.054.8發(fā)酵時(shí)圖3pH對(duì)乙醇產(chǎn)率的影響圖4發(fā)酵時(shí)間對(duì)乙醇產(chǎn)率的影響Fig 3 Effect ofof pH on ethanol yieldFig 4 Effect of Fermentation time on ethanol yield24發(fā)酵時(shí)間對(duì)乙醇產(chǎn)率的影響正交試驗(yàn)直觀分析見表2。由表2可以看出,因素C的極差R最發(fā)酵24h時(shí)乙醇產(chǎn)率僅為00532gg,發(fā)酵72h后的乙醇產(chǎn)率為的大小順序?yàn)?C>A>B>D。不同影響因子的比較:接種量以k20.1825gg。發(fā)酵時(shí)間延長(zhǎng)到84h,乙醇產(chǎn)率無(wú)明顯提高(P<0.05)。值最大,為0.2103;發(fā)酵時(shí)間以k3值最大,為0.1852;pH以k值最2.5發(fā)酵最佳工藝條件的選擇大,為0.2036:溫度以k值最大,為0.1739。方差分析的結(jié)果如表3所示。與直觀分析的結(jié)果一致,pH對(duì)表2正交實(shí)驗(yàn)結(jié)果乙醇產(chǎn)率影響極顯著(p<0.01),其次是接種量和時(shí)間(p<0.05),溫Tab 2 Results of orthogonal experiment序號(hào)因素乙醇產(chǎn)率最佳水平依次為C2A2B3D1。綜合直觀分析和方法分析結(jié)果,最佳A(gg°10.0912±00001h,發(fā)酵溫度30℃。為了確認(rèn)正交試驗(yàn)較優(yōu)組合條件的再現(xiàn)性以C2A2B3D為發(fā)酵條件進(jìn)行試驗(yàn),結(jié)果乙醇產(chǎn)率為0.2451gg313330.176340001顯著高于正交試驗(yàn)中的最高乙醇得率0.2329gg(p005)30.2164±0.0260310.2329±0.0016表3正交試驗(yàn)結(jié)果的方差分析5678920.1817±0.0240Tab 3 Orthogonal experiment results of analysis of varianc20.1348±0.0018顯著性30.08960.0008017112.66210.1977±0.002201283.150K10.46420.44230.5217K20.63090.51920.5132中國(guó)煤化工K3042220.5557k0.15470.14740.12080.1739errorYHCNMHGk20.21030.17310.20360.1711k30.14070.18520.18130.1608極差R0.06960.03780.082800131(下轉(zhuǎn)第90頁(yè))廣東化工2012年第4期www.gdchem.com第39卷總第228期在一氯乙酸與BCD的物質(zhì)的量配比為10:1,氫氧化鈉與一在氫氧化鈉與一氯乙酸物質(zhì)的量配比為2:1、反應(yīng)溫度為60氯乙酸的物質(zhì)的量配比為2:1,反應(yīng)時(shí)間4h條件下,改變反應(yīng)℃、反應(yīng)時(shí)間4b條件下,改變一氯乙酸與B環(huán)糊精物質(zhì)的量配溫度,分別在40℃,50℃,60℃,70℃,80℃下完成反應(yīng),觀比分別為7:1,8:1,9:1,10:1,11:1。由圖3可知,隨著察溫度對(duì)取代度的影響。如圖2可知,隨反應(yīng)溫度的增加產(chǎn)物的氯乙酸用量的增加產(chǎn)物取代度呈現(xiàn)上升的趨勢(shì),當(dāng)一氯乙酸超取代度和產(chǎn)率呈先上升后下降的趨勢(shì)。在溫度為60℃時(shí)取代度和過(guò)一定量時(shí),取代度反而下降。這種現(xiàn)象的原因可能是由于一氯率達(dá)到最高值。較低溫度致使取代度不高的原因可能是反應(yīng)達(dá)不到醚化溫而不利于親核試劑的進(jìn)攻,使SN2反應(yīng)受到一定影響,故過(guò)量的度,在反應(yīng)中產(chǎn)生了副反應(yīng)使取代度降低,而溫度高后可能導(dǎo)致氯乙酸會(huì)使取代度下降。整體而言,在一氯乙酸和BCD的物了一部分環(huán)糊精糊化,并發(fā)生交聯(lián),影響反應(yīng),使取代度降低質(zhì)的量配比為9:1時(shí),取代度和產(chǎn)率最高由這一系列可得知60℃是最佳的反應(yīng)溫度3結(jié)論23一氯乙酸與B-環(huán)糊精物質(zhì)的量配比對(duì)CMCD取代度的影響鑒于本實(shí)驗(yàn)氫氧化鈉與一氯乙酸的的物質(zhì)的量配比對(duì)反應(yīng)影響較小,在此不再對(duì)其進(jìn)行深入研究討論,對(duì)氫氧化鈉與一氯酸物質(zhì)的量配比參考文獻(xiàn)記載定為1:2,總結(jié)上述實(shí)驗(yàn)結(jié)果可知:在一氯乙酸物質(zhì)與B環(huán)糊精的量配比為9:1、反應(yīng)溫度為60m℃、反應(yīng)時(shí)間4h的條件下,是合成羧甲基B環(huán)糊精的最佳反應(yīng)條件參考文獻(xiàn)備3李文德,周俊俠,張力田,環(huán)糊精的發(fā)展與改性研究門,山西食品工業(yè)1996,1:10-152]王瑾,陳均志.羧甲基B環(huán)糊精的制備工藝研究[食品科學(xué),200912:98-1003]張毅民,張國(guó)棟,甘永江,等.系列單取代烷氧基2羥丙基B環(huán)糊精的合成與表征門,有機(jī)化學(xué),2006:60-65氯乙酸與環(huán)糊精的摩爾比[4王超.B環(huán)糊精作用下甾體化合物的生物轉(zhuǎn)化特性研究D].天津科技大學(xué)學(xué)報(bào),2005,13:33-39圖3一氯乙酸與B-CD的物質(zhì)的量配比[S]高士樣,王連生,黃慶國(guó).羧甲基環(huán)糊精的合成及對(duì)鹵代芳烴的增對(duì) CM-P-CD的取代度和產(chǎn)率的影響溶研究[,環(huán)境化學(xué),1999131-135Fig 3 Effects of molar ratio of monochloroacetic acid to B-CD(本文文獻(xiàn)格式:陳龍然,劉雪粉,王江,羧甲基環(huán)糊精的合ondegree of substitution and yield of CM-B-CD成研究[J].廣東化工,2012,39(4):89-90)(上接第88頁(yè))[7]Wilkins M R, Widmer WW, Grohmann K. Simultaneous saccharificationFo0s(2,2)=19F0n(2,2)=99and fermentation of citrus peel waste by Saccharomyces cerevisiae to produce3結(jié)論在單因素試驗(yàn)基礎(chǔ)上,通過(guò)正交試驗(yàn)優(yōu)化冰糖橙皮渣發(fā)酵產(chǎn)8]Wilkins M R, Suryawati L, Chrz D, et al. Ethanol production乙醇的工藝,以乙醇產(chǎn)率作為主要評(píng)價(jià)指標(biāo),得出各因素對(duì)提取Saccharomyces cerevisiae and Kluyveromyces marxianus in the presence效果影響的主次順序依次為:pH>接種量>發(fā)酵時(shí)間>發(fā)酵溫go. World Joumal Microbiology Biotechnology.20023(8)度。通過(guò)直觀分析和方差分析,得出冰糖橙皮渣接種異常畢赤酵母發(fā)酵產(chǎn)乙醇的最佳工藝條件為:pH4.5,接種量12%,發(fā)酵]sharma N, Kalra K L, Oberoi H S, et al. Optimization of fermentation parameters間72h,發(fā)酵溫度30℃,在此條件下乙醇產(chǎn)率為0.2451g/g,顯著for production of ethanol from kinnow waste and banana peels by simultaneous高于單因素實(shí)驗(yàn)和正交實(shí)驗(yàn)結(jié)果。本研究為開發(fā)和利用柑橘皮渣saccharification and fermentation[J]. Indian Joumal of Microbiology, 2007, 47(4提供了一定參考。310-316[10JBoluda-Aguilar M, Garcia-Vidal L, Gonzalez-Castaneda F del P,et參考文獻(xiàn)al. 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