合成氣發(fā)酵生產(chǎn)乙醇菌株的篩選

網(wǎng)絡(luò )優(yōu)先數字出版時(shí)間:2014-4-01網(wǎng)絡(luò )優(yōu)先數字出版地址http://www.cnkinet/kems/doi/10.13656/j.cnkigxkx.20140401.002.html廣西科學(xué) Guangxi Sciences2014,21(2):124~128合成氣發(fā)酵生產(chǎn)乙醇菌株的篩選Screening of a Microbial Strain Capable of producingEthanol from Syngas Fermentation郭鈴,黃艷燕,潘麗霞,李檢秀,孫菲菲,孫靚,黃日波GUO Ling, HUANG Yan-yan, PAN Li-xia, LI Jian-xiu, SuN Fei-fei. SUN LiangHUanG Ri-bo(廣西科學(xué)院,非糧生物質(zhì)酧解國家重點(diǎn)實(shí)驗室,國家非糧生物質(zhì)能源工程技術(shù)研究中心,廣西生物質(zhì)產(chǎn)業(yè)化工程院,廣西生物煉制重點(diǎn)實(shí)驗室,廣西南寧530007)(Guangxi Academy of Sciences, State Key Laboratory of Non-Food Biomass and Enzyme Technology, National Engineering Research Center for Non-food Biorefinery, Guangxi Biomass Industrialization Engineering Institute, Guangxi Key Laboratory of Biorefinery. Nanning, Guangxi, 530007, China摘要:【目的】從動(dòng)物糞便樣品中分離能利用合成氣生產(chǎn)乙醇的菌株,并對其進(jìn)行鑒定及初步發(fā)酵實(shí)驗,為工業(yè)利用合成氣生產(chǎn)乙醇奠定理論基礎?！痉椒ā坷酶患Z化培養技術(shù),以合成氣為唯一碳源,分離篩選合成氣利用菌,通過(guò)形態(tài)觀(guān)察、生理生化實(shí)驗及16 SrrNA序列分析鑒定菌株,并利用菌株進(jìn)行初步發(fā)酵實(shí)驗?！窘Y果】分離到1株可以發(fā)酵合成氣生產(chǎn)乙醇的菌株,分子鑒定結果表明,該菌株為 Clostridium ljungdahlii。生理生化分析結果顯示:該菌是嚴格厭氧型革蘭氏陽(yáng)性菌,短杄狀,有運動(dòng)性,芽孢很少見(jiàn),生長(cháng)必須因子包括酵母粉和維生素B族;最適碳源為果糖,最適生長(cháng)溫度35~37℃,最適pH值為6.0~7.0。合成氣發(fā)酵實(shí)驗結果顯示:3℃,厭氧靜止發(fā)酵30d乙醇最高產(chǎn)量達到2.58g/L,添加BFSA、莫能菌素、丁基橡膠顆?？梢允挂掖籍a(chǎn)量分別提高49%、70%、4.3%,最大產(chǎn)量達到4.31g/L?！窘Y論】從動(dòng)物糞便中分離到1株( clostridium ljungdahlii,該菌能夠利用合成氣生產(chǎn)乙醇,為進(jìn)一步研究和開(kāi)發(fā)新型生物質(zhì)材料提供了基礎資料。關(guān)鍵詞:合成氣厭氧發(fā)酵梭狀芽孢桿菌乙醇中圖分類(lèi)號:Q939文獻標識碼:A文章編號:1005-9164(2014)02-0124-05Abstract: (Objective]To isolate and identify a microbial strain capable of producing ethanol fromsyngas fermentation from animal waste. [Methods] The enrichment culture using syngas as thesole carbon source was utilized to isolate the strain, which was identified according to the ob-servation of morphology, physiological and biochemical tests, and analysis of 16S rRNa genesequences. The isolated strain was utilized for preliminary fermentation. [Results] a strain capable of fermenting syngas into ethanol was isolated successfully. The strain was identified asClostridium ljungdahlii, which is a strictanaerobe and gram-positive. The cells are rod收稿日期:2013-11-29shaped and show motility; spores are formed in-修回日期:2014-01-24frequently; the strain requires a minimum level作者簡(jiǎn)介:郭鈴(1983-),男,碩士,主要從事微生物發(fā)酵與酶工of yeast extract (0.1%) and B-vitamins for程研究growth; substrate optimum is fructose; temper廣西科學(xué)院基本科研業(yè)務(wù)費項目(11y2sw12、 ature optimum range is35~37℃; pH optimum11y2sW1),廣西自然科學(xué)基金項目(2013 GXNSFBA019089) range Is6.0~7.0; batch fermentation revealsthat under anaerobic conditions the highest yield**通信作者:黃日波(1958-),男,教授,博士生導師,主要從 of ethanol in serum bottles at37℃was2.58g/Lafter 30d by addition of bESA. monensin and分子酶工程研究。Email:ruhuang@gxas.ac.cnbutyl rubber particles yields of ethanol can inrease 49 %.70% and 4.3%.respectively, with the highest yield of ethanol at 4.31g/LICon-clusion] Clostridium Ljungdahlii was capable of producing ethanol from syngas fermentationKey words: syngas, anaerobic fermentation, Clostridium ljungdahlii, ethanol【研究意義】隨著(zhù)化石能源日益枯竭,尋找新的可勻后取ImL培養物接種到含有40mL篩選培養基的再生替代能源就成為全世界最關(guān)注的熱點(diǎn)。乙醇是血清瓶中,以合成氣為唯一碳源,37℃厭氧靜止培養種優(yōu)質(zhì)的液體燃料,單位質(zhì)量的乙醇完全燃燒時(shí)可3d,取有生長(cháng)趨勢的培養物,稀釋1×10·倍后接種釋放約30KJ的熱量,是目前為止最有效的化石能源到新鮮的篩選培養基中,重復培養30d,重復以上步替代品。目前,燃料乙醇的生產(chǎn)方法主要有化學(xué)合成驟3次后,取培養物稀釋1×10°倍后涂布于固體與微生物發(fā)酵兩種方式,化學(xué)合成法主要是乙烯直接CLM培養基上,以果糖為唯一碳源,37℃厭氧靜止培水合法;微生物發(fā)酵法主要有以糖質(zhì)原料和淀粉質(zhì)原養3d后,挑取單克隆于液體CLM培養基中,以果糖料為底物直接發(fā)酵和以纖維素原料水解發(fā)酵兩種方為唯一碳源,37℃厭氧靜止培養24~36h,用甘油保式。利用微生物發(fā)酵合成氣生產(chǎn)乙醇是以生物質(zhì)為存菌種原料生產(chǎn)乙醇的新工藝1,即先將生物質(zhì)完全氣化生1.3菌株的鑒定成以CO、CO2、H2、N2等為主要成分的生物合成氣,提取菌株基因組DNA,PCR擴增16 SrrNA(引再利用一些特殊的微生物將合成氣轉化為乙醇,該新物27: AGAGTTTGATCCTGGCTCAG和1492r:工藝發(fā)酵條件簡(jiǎn)單,原料來(lái)源廣泛,且大大降低了廢 TACGGCTACCTTGTTACGACTI),送上海生工液廢渣的排放2,屬于環(huán)保型新技術(shù)?！厩叭搜芯窟M(jìn)測序公司測序。展】20世紀80年代以來(lái),國外研究人員相繼在雞糞、1.4菌株生理生化分析兔糞等動(dòng)物糞便以及農業(yè)泄湖、沼氣池中發(fā)現了一些菌株生理生化分析的具體方法參見(jiàn)文獻可利用合成氣生產(chǎn)乙醇的菌株,主要研究的微生1.5菌株生長(cháng)曲線(xiàn)的測定物有以下幾種: Clostridium ljungdahlii L分別挑取 Clostridium ljungdahlii單菌落于Clostri -dium autoethanogenum, Moorella thermoRCM培養基和CLM培養基中,37℃厭氧靜止培養ilic sp. HUC22-1. Clostridium scatologenes24h,次日按1%的接種量接種于10 ML RCM培養基rain sI1l,( lostridium carboridivorans p7,這和CLM培養基中,以果糖為唯一碳源,37℃厭氧靜些菌株都是嚴格厭氧菌,可以在高濃度合成氣條件下止培養至OD6≈0.2時(shí),按2%的接種量接種到生長(cháng)并產(chǎn)生乙醇?！颈狙芯壳腥朦c(diǎn)】目前,國內尚未見(jiàn)50mL的相應培養基中,37℃厭氧靜止培養50h,每有關(guān)利用合成氣發(fā)酵生產(chǎn)乙醇菌株的相關(guān)報道?！緮M2~4h取樣檢測ODo值解決的關(guān)鍵問(wèn)題】從廣西象州家禽養殖場(chǎng)、沼氣池等1.6菌株初步發(fā)酵實(shí)驗地采集到的雞、牛等動(dòng)物糞便中篩選到1株能夠利用挑取 Clostridium ljungdahlii單菌落于CLM合成氣生產(chǎn)乙醇的菌株,對其進(jìn)行生理生化分析及初培養基中,以果糖為唯一碳源,37℃厭氧靜止培養步發(fā)酵實(shí)驗,摸索菌株的生長(cháng)發(fā)酵條件,為工業(yè)化發(fā)24h,按1%的接種量接種于10 ML CLM培養基中酵合成氣生產(chǎn)乙醇提供理論依據。以果糖為唯一碳源,37℃厭氧靜止培養至OD6?！?材料與方法0.6時(shí),按5%的接種量接種到發(fā)酵培養基中,抽出空氣,充入合成氣,密封血清瓶后,37℃厭氧靜止培養1.1材料30d,每3~7d補氣1次。樣品: Clostridium ljungdahlii由本研究篩選1.7乙醇測定獲得用注射器抽取2.5mL發(fā)酵液,12000r/min離心培養基:本研究所用培養基參考文獻[5~12],略10min,取上清,以0.1%的乙腈為內標,氣相檢測乙有修改醇產(chǎn)量,根據乙醇和乙腈峰面積比計算乙醇含量。合成氣:購自南寧國信氣體有限公司,含73%乙醇含量(g/L)=(乙醇峰面積/乙腈峰面積)CO,10%CO2,15%H2,2%CH4,或者80%H2,20%1.020,式中1.020為換算常數,根據同一條件下測定乙醇和乙腈的標準曲線(xiàn)回歸方程求得1.2菌株的富集與篩選測定乙醇含量采用 Agilent technologies6890N在厭氧條件下稱(chēng)取1.0g新鮮的動(dòng)物糞便,溶于 GC systen氣相色譜,檢測器采用FID,柱型號:Agi0.3m,載氣為氮氣,載氣流率20mL/min,氫氣流率、婚40mL/min,分流比1:50,色譜進(jìn)樣口和柱子的溫度分別為250℃和100℃Eo2結果與分析2.1菌株鑒定結果提取菌株基因組DNA后,PCR擴增16SrRNA,測序結果經(jīng) Genebank比對,比對結果(表1)顯示與 Clostridium ljungdahlii同源性為99%,可圖2革蘭氏染色后鏡檢結果以初步判定該菌株為 Clostridium ljungdahlii。Fig 2 Electron microscopy of the strain after gram staining表1菌株16 S rrNa片段序列比對結果表2不同酵母粉含量對菌株生長(cháng)的影響Table 2 Effect of different yeast powder on strain growthTable 1 Comparison of 16s rRNA sequence of strain by sequencing and Blast analysis碳源酵母粉 Yeast extracto%)Carbon source編號基因文庫編號最相似菌株相似度00.020.040.060.080.10.20.5No. Genbank Accession Most similar straiidentity(%)Fructose不添加果糖1NR074161.1538growth: w: Weak growth.(2)維生素B族及氨基酸3Y18178,1從表3可以看出, Clostridium ljungdahlii菌株061在添加維生素B族的條件下生長(cháng)良好,在添加氨基2.2菌株生理生化分析酸的條件下生長(cháng)很微弱,因此可以初步判定維生素族是 Clostridium ljungdahlii生長(cháng)必須因子,氨基2.2.1菌落特征菌落為白色或灰白色,邊緣呈不規則狀,中間凸酸對 Clostridium ljungdahlii的生長(cháng)不起到?jīng)Q定性的作用。起有光澤。顯微鏡下呈棒狀或短桿狀,有運動(dòng)性,芽表3維生素B族及氨基醱對菌株生長(cháng)的影響孢很少見(jiàn),嚴格厭氧菌,革蘭氏染色后顯紫色(圖1和Tab3 Effect of B-Vitamins and amino acid on strain growth圖2),表明為革蘭氏陽(yáng)性菌,與文獻[5]的報道一致添加情況維生素B族氨基酸Adding conditionB-vitamin solution Amino acid solution添加 Adding不添加 Without adding不生長(cháng);w:微弱生長(cháng)。+: Positive growth;growth: w: Weak growth(3)可用碳源從表4可以看出,( lostridium ljungdahlii在CLM培養中可以利用合成氣、木糖、果糖、阿拉伯糖丙酮酸鹽及乙醇作為碳源,生長(cháng)良好,果糖生長(cháng)最快,圖1顯微鏡下菌體情況而葡萄糖為唯一碳源時(shí)生長(cháng)非常緩慢。Fig 1 Electron microscopy of the strain2.2.3菌株最適生長(cháng)條件2.2.2菌株生長(cháng)必須因子圖3和圖4表明,菌株的最適生長(cháng)溫度為35~(1)酵母粉37℃,最適pH值為6.0~7.0。從表2可以看出, Clostridium ljungdahlii菌株2.2.4菌株耐氧實(shí)驗的生長(cháng)需要酵母粉,當酵母粉含量低于0.06%時(shí),在菌株耐氧實(shí)驗發(fā)現.當空氣含量為0.1%或含有碳源的條件下,菌株也不生長(cháng);但當酵母粉含量0.2%時(shí),菌株可以生長(cháng);當空氣含量為0.5%時(shí),菌大于等于0.2%后,即使不另外添加碳源也能微弱生株微弱生長(cháng);當空氣含量為0.75%或1.0%時(shí),菌株常生長(cháng);當空氣含量超過(guò)0.75%時(shí),菌株生長(cháng)受到抑長(cháng)狀況有較大差異,在RCM培養基中生長(cháng)迅速,且制,不能正常生長(cháng)菌體密度大,在CLM培養基中生長(cháng)相對緩慢,且菌表4 Clostridium ljungdahlii在單一碳源下的生長(cháng)狀況體密度低,其原因是由于RCM培養基是完全培養Table↓ Growth of Clostridium Ijungdahlii on substratesεs基,營(yíng)養豐富更適合菌株的生長(cháng)復蘇the sole carbon生長(cháng)狀況‖碳源生長(cháng)狀況Growth‖ CarbonGrowthsituation‖ sourcecuatro合成氣 SyngasCO2/H2木糖 Xylose+果糖 Fructose阿拉伯糖 Arabinose葡萄糖 Glucose蔗糖 Sucrose甘油 Glycerol00=°°L-乳酸鹽L- lactate丙酮酸鹽 Pyruvate.檸檬酸鹽 Citrate麥芽糖 Maltose時(shí)間Iieh)甲醇 Methanol乙醇 Ethanol圖5 Clostridium ljungdahlii生長(cháng)曲線(xiàn)淀粉 Starch山梨醇 SorbiteFig 5 Growth curve of Clostridium ljungdahli乳糖 Lactose●:RCM,a:CLM,:生長(cháng):-:不生長(cháng);w:微弱生長(cháng)。+: Positive growth;-:No2.4合成氣發(fā)酵乙醇的初步實(shí)驗growth: w: Weak growth氣相色譜檢測結果(圖6)顯示,發(fā)酵液中有乙醇生成,乙醇產(chǎn)量在0.68~2.58g/L之間,其中20mL血清瓶中幾乎沒(méi)有乙醇生成,250mL血清瓶中普遍較高,推測血清瓶體積越大,所能容納的合成氣越多,0.45乙醇產(chǎn)量越高H值 ph valud最適pH值Fig 3 Optimum pH of Clostridium ljungdahlvo5清瓶體積 volume of serum bottle ml.)醇產(chǎn)量分析Fig 6 Yield analysis of ethanol2.5乙醇產(chǎn)量?jì)?yōu)化實(shí)驗從表5可以看出,添加BESA、莫能菌素及丁基濕度 Tcimpcraturc()橡膠顆粒后,乙醇平均產(chǎn)量均有所提高,分別提高了圖4( Lostridium ljungdahlii最適生長(cháng)溫度49%、70%、4.3%,其中莫能菌素提高最多,BESA次Fig.4 Optimum temperature of( Lostridium ljungdahlii之,丁基橡膠顆粒最低,這是由于莫能菌素和BESA2.3 Clostridium ljungdahlii生長(cháng)曲線(xiàn)都可以抑制甲烷的生成,促進(jìn)乙醇和乙酸的生成,而按照1.7方法測定( clostridium ljungdahlii在丁基橡膠顆粒的主要作用是吸附氣體,提高氣體在培不同培養基中的生長(cháng)曲線(xiàn),結果(圖5)表明,在RCM養基中的溶解度,從而提高乙醇的產(chǎn)量,但是由于發(fā)培養基中,菌株幾乎沒(méi)有延滯期,2h后菌株OD。達酵過(guò)程是靜止的,丁基橡膠顆粒大多沉到底部,導致到0.2,然后迅速進(jìn)入對數期,28h后生長(cháng)速度開(kāi)始減吸附氣體量減少,對乙醇產(chǎn)量提高所起的作用不大緩進(jìn)入穩定期,OD6最大值達到1.93左右;而Clostridium ljungdahlii在CLM培養基中,前23討論8h菌株幾乎不生長(cháng),20h左右,OD達到0.2,24本研究成功地從動(dòng)物糞便中篩選到1株可以發(fā)3$h內為菌株生長(cháng)的對數期,4h后菌株生長(cháng)趨于穩酵合成氣生產(chǎn)乙醇的菌株,經(jīng)菌株生理生化分析以及定,OD0最大值達到0.89。從結果中可以看出,16 S rrNA鑒定為 Clostridium ljungdahlii,我們利表5添加BESA、莫能菌素和丁基橡膠顆粒對乙醇產(chǎn)量的影forward for biofuels and biomaterials[]. Science, 2006響(g/311(27):484-489Table 5 Effect of adding BESA, monensin and butyl rubl[3 Vega J L, Prieto S, Elmore BB, et al. The biological pro-articles on yield of ethanol(g/L)duction of ethanol from synthesis gas[JJ. Appl BiochemBiotech,1989,20-21(1):781-797添加情況BESA能菌素橡膠顆粒[4] Hurst K M. Lewis R S Carbon monoxide partial pres-particlesure effects on the metabolic process of syngas fermen添加 Addingtation[J]. Biochem Eng J, 2010, 48(2): 159-165不添加 Without adding2.54[] Gaddy J L, Clausen E C. Clostridium ljungdahlii,annaerobic ethanol and acetate producing microorganism的初步實(shí)驗,實(shí)驗結果顯示該菌株能夠在以合成氣為US,5173429[P].1992-12-22唯一碳源的培養基中生長(cháng)并產(chǎn)生乙醇,經(jīng)30d血清瓶61skns, Nakashimada Y. Yoshimoto. Ethanol production from H, and CO by a newly isolated thermophilic發(fā)酵,乙醇產(chǎn)量達到2.58g/L,添加BESA、莫能菌bacterium,Moorella sp. HUC22-1 [J]. Biotechnology素、丁基橡膠顆?？梢苑謩e使乙醇產(chǎn)量提高49%Letters,2004,26:1607-16170%、4.3%,最高產(chǎn)量達到4.31g/I[7 Kusel K, Dorsch T, Acker G, et al. Clostridium scato在實(shí)驗過(guò)程中,研究發(fā)現不同組分含量的合成氣logenes strain SIl isolated as anacetogenic bacteriumfrom acidic sediments [J]. Int J Syst Evol Microbiol會(huì )導致乙醇產(chǎn)量的不同1,同時(shí)合成氣中除了CO、2000,50:537-546CO2、H2等主要成分外,還含有硫化氫、焦油、NO等[8] Gaddy J I,ArkF. Biological production of ethanol from不可利用的有毒有害氣體,也會(huì )在發(fā)酵過(guò)程中影響菌waste gases with Clostridium ljungdahlii: US,6136577株的生長(cháng)和代謝。另外,發(fā)酵產(chǎn)物檢測結果顯示,菌[P.200010-24.株發(fā)酵合成氣最主要的產(chǎn)物是乙酸和乙醇,當pH值91aour, Younesi H. Ethanol and acetate synthesisfrom waste gas using batch culture of Clostridium為4~5時(shí),發(fā)酵主產(chǎn)物是乙醇,當pH值為6~7時(shí)ljungdahlii [J]. Enzyme and Microbial Technology發(fā)酵主產(chǎn)物是乙酸,然而 Clostridium ljungdahlii2006,38:223-2最適pH值是6~7,pH值過(guò)低會(huì )影響菌株的生長(cháng),[0 Younes, Najafpour G. Mohamed r. Ethanol and實(shí)驗結果也表明在pH值為4~5范圍內,單位菌體acetate production from synthesis gas via fermentationprocesses using anaerobic bacterium, Clostridi密度的乙醇產(chǎn)量更高,但是由于菌株生長(cháng)受到抑制,ljungdahlii [J]. 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