初期通气和震荡培养提高高浓度乙醇发酵的乙醇浓度和产率

应用与环境生物学报2009,15(4563~5672009-08-25Chin J Appl Environ Bio/=ISSN 1006-687XDOI:10.3724SPJ1145.200900563Improvement of ethanol Concentration and yield by initialAeration and Agitation Culture in Very highGravity fermentation%LIU Yan, QI Tiansheng, SHEN Naikun, GAN Mingzhe, JIN Yanling ZhAo HaiChengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China)Abstract Effect of oxygen and agitation on ethanol production by Saccharomyces cerevisiae in a very high gravity(VHG)fermentation was studied. The results indicated that agitation was the most significant factor to improve ethanol concentrationand yield. Compared to static culture, agitation led to a 69.0 increase in ethanol titre(from 75. 8 to 128 1 g L- )under nonaeration condition, and ethanol concentration increased by 68.7 %(from 85.2 to 143 8g L- )under initial aeration condition.Aninitial aeration strategy and agitation culture were identified as an optimal condition for VHg in the fed-batch fermentationthat allowed a final ethanol concentration to achieve 143 8g L. in 54 h, in a double feeding fermentation, and the yield ofethanol on substrate (Yn )reached 0. 471 g/g(The stoichiometric yield was 92.2 %)which very few studies achieved. Theseoutstanding performances resulted from higher biomass and enhanced cell activities which were essential for VHG because ofaeration and agitation. Fig 5, Tab 1, Ref 12Keywords very high gravity fermentation(VHG); ethanol fermentation; initial aeration; agitation culture; oxygenCLC TQ920. 1: TK6初期通气和震荡培养提高髙浓度乙醇发酵的乙醇浓度和产率刘艳戚天胜申乃坤甘明哲靳艳玲赵海(中国科学院成都生物研究所成都610041)摘要研究了氧气和震荡条件对酿酒酵母高浓度♂醇发酵的影响.结果表明,震荡是提高发酵液乙醇浓度和产率的最重要因素,与静止培养相比,在不通气情况下震荡培养使乙醇浓度提高了69%(从75.8gL提高到128.gL),在通气条件下乙醇浓度提高了68.7%(从85.2gL提高到tol43.8gL)在最优条件下,两次补料,经54h发酵,发酵液中乙醇浓度达到143.8gL,乙醇产率与理论产率的比值为0.471gg(即92.2%).经分析,通气和震荡条件提高了发酵液中酿酒酵母的生物量和细胞活力.图5表1参12关键词高浓度发酵;乙醇发酵;初期通气;震荡培养;氧气CLC TQ920. 1: TK6With the rapid consumption of oil, people have to cast aboutAmong the factors affecting fermentation time, ethandfor substitutes for it. The production of bio-ethanol as one of the concentration and Y, oxygen was crucial, which affected cellalternative fossil fuel energy resources has been a subject of great growth, reproduction and activity, and also formation of by-interest The technology of very high gravity fermentation products including glycerol and ace acetate. The growth of(VHG) for fuel alcohol production is intensively concerned Saccharomyces cerevisiae would be inhibited when oxybecause of its particular advantages, such as less energy was absent. In particular, S cerevisiae required a certain supplyonsumption in distilling, more efficient utilization of equipments of elemental oxygen in order to synthesize unsaturated fattyand less wastewater to be processed 2. However, researchers only acids and sterols, which were important constituents of its celconcerned the ethanol concentration in the fermentation liquid envelopes 31. However, if oxygen was provided too much, Yin VHG, but usually ignored other parameters such as reducing would decrease sharply because of aerobic respiration Under fullglucose, fermentation time and the yield of ethanol production on aeration, yeast would consume more glucose to produce CO, andsubstrate(Y ) which was another determining factor for ethanol H,Oproduction costs. Improvement of Y by optimization was quiteThe impacts of oxygen supply on VHG have beenprospective to reduce production costsreported in some articles. In continuous cultures, at a givendilution rate, biomass concentration, biomass/glucose yieldReceived: 2008-11-25and viability were enhanced by increasing air supply while*Supported by the NatKey Sci& Tech Project of the IIth 5-year Plan of ethanol concentration decreased both under oxygen-limited andChina(no 2007 BAD78B04)and the Provincial Key Sci Tech Project ofSichuan, China(No 027. X028-037)oxygen-unlimited continuous cultures. The inhibition of growthk* Corresponding author(E-mail: zhaohai @cib ac cn)by ethanol decre中国煤化工 nditions, whereCNMHG564应用与环境生物学报 Chin J App/ Environ Bioloxygen limitation occurred even though aeration was carried 80 mg, pantothenic acid 60 mg, biotin 250 ug, thiamine 10 mg,out, compared to anaerobic conditions where oxygen was totally pyridoxine 30 mg, nicotinic acid 30 mg and para-aminobenzoicabsent 14. Specific ethanol productivity was stimulated when 6 mg. Before inoculation, aeration was done until dissolvedthe percentage of oxygen in the feed increased 5. In fed-batch oxygen concentration reached 4.0-9.0 mg L-in initial aerationprocess, higher ethanol concentration (147 g L. in 45 h) and fermentation process, Fermentations were provided in 250-mLaverage productivity were obtained in cultures without oxygen flasks with 60 mL media at the start, where glucose was aboutlimitation. Compared to micro-aerobic culture, full aeration led 150 g L- after inoculation, then glucose with concentrationto 23% increase in viable cell mass, but the yield of ethanol on of 5625g L. was fed twice when fermented for 8 h and 24glucose was only 0.43 g/g (6. 7.h, respectively. Finally, the total additive glucose was 305 gOn the other hand, in VHG fermentation, agitation affecting L-. Then the fed-batch fermentation was performed in a 10-Lfermentation system state was another important factor for cell fermentor with 8-L mediumgrowth and ethanol production by yeast Under VHG, the strategy 1.3 Sampling and analysisof agitation or supplying air to form a roll fermentation systemSamples from the fermentation media were taken regularlyhas been brought into effect in some articles (o-s. But few articles Ethanol concentration was determined by gas chromatographyinvestigated its impacts speciallyusing a fid detector 9. The samples taken from flasks wereThe aim of this work was to explore the effects of oxygen centrifuged at 5 000 r/min, and then dried at 105C to constantsupply and agitation on dynamic yeast behaviour in batch weight. Glucose concentration was determined using the DNSVHG alcohol fermentations, especially from the aspects of the method (3, 5-dinitro salicylic acid). Viable-cell counts wereparameters of ethanol concentration, reducing glucose, and the determined by a direct microscopic method at a magnification ofyield of ethanol on substrate(Y)400 with the methylene blue technique.Material methods2 Results1.1 Microorganism2.1 Impact of aeration on fermentation parametersS cerevisiae strain CCTCC M 206111, preserved in ChinaFour tests were designed for the factors of aeration andCenter for Type Collection, was separated from wine lees in our agitation(Table 1, Fig. 1-A-C). From Table 1, the effects oflaboratoryoxygen were a little different, compared agitating culture1.2 Medium and batch fermentationwith static culture. Under static culture, aeration slightlyYeast strain CCTCC M 206111 was maintained on malt improved both the final ethanol titre(85.2 vS 758g L )andwort agar medium at 4C. Before fermentation, the yeast was cell concentration(7.71 vs 7.58g L-). On the contrast, aerationincubated at 30C at rotary shaker (200 r/min)in 250-mL influenced fermentation process more strongly (143. 8 vS 128 1gErlenmeyer flasks with 50 mL medium containing in g L: L-)under agitating culture. Aeration improved the final ethanolglucose 100, yeast extract 8.5, (NH), SO 1.3, MgSO,7H, O 0. 1 titre by 12.3%, biomass from 13.05 g Lto 13. 82 g L, and theand CaCl, 0.06. After 16 h of growth, the yeast was used as an stoichiometric yield from 82. 2% to 92.2%Inoculum for fermentation so that the biomass concentrationCell viability was also quantified In fermentations 2 andat the start was 2.0-3.5 g dry weight L: Fermentation medium 4, the yeast cells were shapely and strong. Finally, over 98% ofexcept glucose contained in g L-: Yeast extract 8.5, peptone 5, viable cells were measured even until the ethanol concentration(NHD, SO, 1.5, KH, PO, 1.5, MgSO7H,O 0.65, CaCl, 2.8, inositol reached up to 143.8 g LTable 1 Effects of the initial aeration strategy and agitation on ethanol production, residual glucoseand other fermentation parameters[ Ethanol] final (p/g L-)143.8Residual glucose (p/g L-42.6124.3Ys(/ g)0.2490.2790.471Stoichiometric yield (w/%)92Glucose utilization(w/%)55.498.4Biomass (p/g L)Average rate of the glucose consumption during 4-8h(p/g L. h)5.13Average rate of the ethanol formation during 4-8 h(p/g L-h)3.48Average ethanol productivity (p/gLh)Total glucose concentration was 305 g L-. The initial aeration strategy made the saturation of oxygen reaching 4.0-9.0 mg L-l Stirring rate was 200 r/min undergitating culture. Y, the yield of ethanol on substrate; Stoichiometric yield, the ratio of produced mass of ethanol to theoretical mass of ethanol calculatedfrom total mass of glucose; Glucose utilization, the ratio of the consumed mass of glucose to total massI, Non-aeration and static culture; 2, Non-aeration and agitating culture; 3, Aeration and static cultureH中国煤化工CNMHG期LIU Yan, et al. Improvement of Ethanol Concentration and Yield by Initial Aeration and Agitation Culture565The initial aeration strategy, which led to dissolved oxygen sharply below 0.5 mg L-(Fig. 2)concentration reaching 4.0-9.0 mg L, was used in VHG in our 2.2 Impact of agitation on fermentation parametersresearch. The consuming rate of oxygen by yeast was rapidFrom Table 1, agitation played an important role in ethanolAfter inoculation for 3-4 hours, oxygen concentration decreased production, It strongly improved the final ethanol titre andbiomass concentration. Under non-aeration condition, agitationled to a 69.0% increase in ethanol titre(from 75.8 to 128.1 g L-)Under aeration condition, the final ethanol titre increased by85.2distinction of the stoichiometric yield was obvious, tooo doubt that agitation would stronglyethanol concentration from the results(Table 1, Fig. 1-A-CTo understand whether agitation affected fermentation byenhancing dissolved oxygen of the system, we replaced theoxygen of surface atmosphere with nitrogen. The result indicatedthat no obvious difference appeared(Data not shown). Sogitation mainly affected the fermentation process by forming arocess. different stites(0,50,200,300presented. At 0 r/min, biomass was 7.81 g L-, and ethanolconcentration 849g L-l, and at 50, 200, 300 r/min, biomass wasapproximately 13)gL, and ethanol concentration 143-144g L-.So, different stirring rates almost had no obvious differencein biomass and ethanol concentration. Therefore, stirring ratehad no fatal effect on fermentation system, and agitation mainlyformed a homogeneous system2. 3 Difference among four fermentationsEthanol is one of the products from yeast metabolismFermentation capability of yeast was correlated with biomassand cell activities. Different biomasses brought into differentfermentation capabilities (Table 1, Fig. 1-A-C). Generallyspeaking, enough biomass in the broth was an essential factor0102030405060Fig. I Time courses of ethanol, glucose and biomass concentrations duringfermentations with s cerevisiae0000000000 000000000oo: I, Non-aeration and static culture; u: 2, Non-aeration and agitatingture:A: 3, Aeration and static culture; x: 4, Aeration and agitating cultureta, Non ig. 3 Active enzymes under different fermentation conditionsa, Non-aeration and static culture; b, Non-aeration and agitating culture; cAeration and static culture; d, Aeration and agitating culture. In the picture( from left to right), No. I cell is the control, No. 2-20 are corresponding to9 kinds of enzymes. Colored wells was positive, and deeper colour onesmean higher activity of enzymes. No. 2, Alkaline phosphatase (+ violetNo. 3, Esterase(C4)(, violet); No, 4, Esterase lipase(C8)(+, violet): NoLeucine arylamidase (+ orange): No. 11, Acid phosphatase(+, violet); NoFig 2 Change in dissolved oxygen concentration in initial aeration cultureat the first fermentation phaseviolet): No. 16, a-gl中国煤化工17 a-galactosidase (CNMHG566应用与环境生物学报 Chin J App/ Environ Biolfor fast conversion to ethanol. More cells, more ethanol acquiredin definite time and volume. In fermentation 4. the amount ofcells was found the most and the ethanol titre the highest fromk…▲1140our research, biomass was the most direct factor leading todifferent fermentation capabilities of yeast in term of ethanolconcentrationIn the Vhg technique of our research80and feeding time were taken to regulate biomass, As a result,the yield of ethanol on substrate(Y )reached 0.471 g/g(Thestoichiometric yield was 92.2%)in the best fermentation(4)Then, to identify the characteristics of the differentstates in the four fermentations, through analyzing 4 greof tests by enzymatic detection kit(Obtained from biomerieuin France) which was a semi-micromethod designed for theresearch of enzymatic activities, the results indicated that the12kinds of active enzymes were discrepant, moreover, the activityof the same enzyme was different. In positive assay, enzymeactivity was higher in deeper color(Fig 3). For example, thea-glucosidase appeared in fermentations 2 and 4 in, in whichethanol concentration was higher, and there was also differencein enzyme activity. The results showed that S cerevisiae revealeddifferent fermentation performances in different states2.4 Kinetic parameters under aeration and agitationcondition in 10-L fermentationFig 4 Comparison of the parameters between 100-mL and 10-LThe fed-batch experiment was performed in a 10-Lfermentations under initial aeration and agitating cultureGlucose (real line), ethanol (broken line)and biomass in 100-mLfermentor with 8-L medium(Fig. 4-A-B). There was a short lag fermentation(), and 10-L fermentation(A)phase for yeast after inoculation, while they grew slowly andconsumed glucose slowly, too. Then after about four hours, theyeast came into log phase apparently, while the yeast consumedglucose quickly. From Fig 4, the speed of consuming glucosewas slow during the period from 0 to 4 h, and then it became fastAfter 8 h, glucose concentration descended from 146g L- to 85g L-. The average speed of consuming glucose achieved 7.6g Lh, and the maximal speed was about 18g L- h-l, slower than thatof 100-mL fermentation and the biomass was lower than that of0-mL fermentation, too. After the second feeding because theethanol concentration reached 13%. the circumstance was bad foryeast. The fermentation speed slowed down gradually, especiallyFig 5 pH change in the course of 10-L fermentationafter 48 h. At the end, the biomass decreased slightlyFrom Fig. 5. pH value descended from 6.5 to 3.5 when the cells quickly. Oxygen was one of crucial factors for the growth offermentation was conducted for 10 hours, and it was then kept S. cerevisiae, and it could accelerate its growth and reproduction,at the same level for all the rest time The lower ph value was especially in the VHG fermentation, where oxygen became apropitious to restrain other bacteria's growthlimiting factor Appropriate oxygen could promote biosynthesisof plasma membrane, and poly-unsaturated fatty acid and lipoid3 Discussionin chondriosome to protect the integrity of cell membrane fromThe effects of initial aeration and agitation on ethanol the poisoning by high concentration ethanol. However, oxygeconcentration and yield in VHG ethanol fermentation were would destroy the process of anaerobic metabolism. In that case,ivestigated in this study, Four kinds of tests were designed for the yeast produced less ethanol, which was not expected. Sothe investigation(Table 1). The results showed the initial aeration apropos oxygen was an important factor in ethanol productionstrategy shortened the lag phase and increased the amount of for final ethanolTH中国煤化工 c yield. In ourCNMHG期LIU Yan, et al. Improvement of Ethanol Concentration and Yield by Initial Aeration and Agitation Cultureresearch, initial aeration was taken, and the initial aeration factors in VHG. Oxygen was mainly related with yeast growth,strategy achieved appropriate dissolved oxygen concentration quick accumulation of biomass and cell activities. On the other(4.0-9.0 mg L"). Within the range of oxygen concentration, this hand, agitation led to fermentation liquid mixing well, CO2strategy not only provided suitable oxygen for yeast growing releasing and nutrition consuming, etc. To summarize, thequickly, but also avoided aerobic respiration to a certain extenttwo factors affected ethanol fermentation by biomass and cellFrom the research, agitating culture would make ethanol activities. Therefore, it was necessary to accelerate accumulationproduction well performed. It could be related with the status of of biomass, acquire optimal cell amount, and keep higher cellthe broth, which affected yeast growth and ethanol production. activities for fermentation by some means, so as to achieve highAgitation could create a symmetrical fermentation system. concentration and yield of ethanolMoreover, it did avail to release co, which was an inhibitorto yeast ( 0-12, and to accelerate the consumption of nutritionReferencesI Ikegamai T, Yanagishita H, Kitamoto D, Haraya K. Accelerated ethanolby yeast. Under this condition, yeast would grow well andfermentation by Saccharomyces cerevisiae with addition of activatedmance onethanol production and endurance to bad circumstance includingcarbon Biotechnol! 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Physiology, biochemical, and mathematicalhigher than that in common fermentation. Correspondingly, thestudies of micro-aerobic continuous ethanol fermentation Saccharomvcesbiomass in VHG should be, therefore, higher. But if the yeastcerevisiae. 1: Hysteresis, oscillations culture. Biotechnol Bioeng, 1990,cells were very excessive, they would consume more sugars for36:1006~1019growth, which would lower the stoichiometric yield. Moreover, 6 Alfenore S, Molina-Jouve C, Guillouet SE, Uribelarrea JL, Gomathe yeast cells would act on each others. Therefore, it was aG, Benbadis L. Improving ethanol production and viability ofrequired way to control optimized biomass in order to fermentSaccharomyces cerevisiae by a vitamin feeding strategy during fedsugars to ethanol fast and efficientlybatch process. App/ Microbiol BiotechnoL, 2002, 60: 67-72Considering the results of the 10-L fermentation, the 7 Alfenore S, Cameleyre X, Benbadis L, Bideaux C, Uribelarrea JL.biomass was lower than that in 100-mL fermentation at the initialGoma G, Molina-Jouve C, Guillouet SE. Aeration strategy: A need forphase, and the time was prolonged. From Fig. 4-A, the speeds ofery high ethanol performance in Saccharomyces cerevisiae fed-batchglucose consumpion and ethanol production were slower. Theprocess. Appl Microbiol Biotechnol, 2004, 63: 537-542reason must be the lower amount of yeast cells, which was related8 Bai Fw, Chen LJ, Zhang Z, Anderson WA, Moo-Young M. Continuouswith weak transfer capability of material elements in the 10-Lthanol production and evaluation of yeast cell lysis and viability underfermentvery high gravity medium conditions. BiotechnoL, 2004, 110: 287-29In this study, the initial aeration strategy and agitating9LuY(刘艳), Zhao h(赵海),QiTS(戚天胜) Tang QL(唐秋琳), Huangculture increased the final ethanol concentration (up to 143. 8 gL-), and made the stoichiometric yield (up to 92%)higher thanYF(黃宇峰) Fast production of ethanol by Zymomonas mobilis(zy1)Chin J Appl Environ Bio/(应用与环境生物学报,2007,13(1):69-72other general VHG ethanol fermentation processes, that was to10 Jones RP, Greenfield PF. Effect of carbon dioxide on yeast growth andsay, the equal raw material would obtain more ethanol under thatfermentation Enzyme Microbiol Technol, 1982, 4: 210-223condition. The increased stoichiometric yield could decrease1 Takezaki M, Matsuura K, Hirotsune M. Hamachi M, Effect of solids onthe bio-ethanol production cost effectively. Furthermore, thethe growth of yeast. J Brew Soc Japan, 1993, 88: 319-325fermentation time was shortened for only 54 hours. So, it became12 Fukuda T, Hiramatsu M, Sanmoto H, Fukuzaki S Discharge ofa highly competitive techniqudissolved carbon dioxide by the addition of activated carbon particlession,oxygen and agitation were two inand its effect on yeast. J Brew Soc Japan, 1996, 91: 279-283中国煤化工CNMHG