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

用与环境生物学报2009,15(4)563~5672009-08-25Chin J Appl Environ Bio/=/SSN 1006-687XDOI:10.3724SPJ1145.200900563Improvement of Ethanol Concentration and Yield by InitialAeration and Agitation Culture in Very highGravity fermentationLIU Yan, QI Tiansheng, SHEN Naikun, GAN Mingzhe, JIN Yanling ZHAO Hai(Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, ChinaAbstract 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 non-aeration condition, and ethanol concentration increased by 68.7%(from 85. 2 to 143. 8 g 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. 8 g L in 54 h, in a double feeding fermentation, and the yield ofethanol on substrate(Y )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,震荡是提髙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η5.8gL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to143.8gL)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0.47lgg(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, ethanolfor 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 oxygenbecause of its particular advantages, such as less energy was absent. In particular, S cerevisiae required a certain supplyconsumption 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 cellconcerned the ethanol concentration in the fermentation liquid envelopes 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 givendilutey1Received:2008-11-25 Accepted:2008-12-15by increasing air supply while*Supported by the National Key Sci& Tech Project of the 1lh 5-year Plan of ethanol concentration decreased both under oxygen-limited andChina(no 2007 BAD78B04)and the Provincial Key Sci& Tech Project of(No.027.X028-037)oxygen-unlimited continuous cultures. The inhibition of growthponding author(E-mail: zhaohai a cib. ac cn)by ethanol decreased under microaerobic conditions, where64应用与环境生物学报 Chin J Appl Environ Bio15卷oxygen 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 4. 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 562. 5 g L: was fed twice when fermented for 8 h and 24glucose was only 0.43 g/g 6h, 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 medium.growth 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 chromatoinvestigated its impacts specially.using a FId detector The samples taken from flasksThe 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 (Y400 with the methylene blue technique1 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 oflittle different1. 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 75. 8 g 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), SO4 1.3, MgSO4 7H, O 0.1 titre by 12.3%, biomass from 13.05 g L-" to 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(NH), SO, 1.5, KH, PO, 1.5, MgSO,,7H,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 glucosed other fermentation pa[ Ethanol] final (p/g L-)43.8Y (w/ g)0.4200.2790.471Stoichiometric yield (w82,292.2Glucose utilization(w/ %a86.05813Average rate of the glucose consumption during 4-8 h(p/g L-h)verage rate of the ethanol formation during 4-8 h(p/g L-h)7.12Average ethanol productivity (p/g L-h)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 underagitating culture. Y, the yield of ethanol on substrate; Stoichiometric yield, the ratio of produced mass of ethanol to theoretical mass of ethanol calculatedass of glucose to teI, Non-aeration and static culture: 2, Non-aeration and agitating culture; 3, Aeration and static culture; 4, Aeration and agitating culture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 by8.7%(from 85.2 to 143 8g L-). Among the four fermentations,distinction of the stoichiometric yield was obvious, toorould strongly implTo understand whether agitation affected fermentation byenhancing dissolved oxygen of the system, we replaced theoxygen of surface atmosphere with nitrogen. The result indicated1020304050that no obvious difference appeared(Data not shown). Soagitation mainly affected the fermentation process by forming aIn order to prove the effect of agitation on fermentationprocess, different stirring rates(0, 50, 200, 300 r/min)wereAt 0 r/min, biomass was 7. 81g Lapproximately 13. 5-14.0 g L, and ethanol concentration 143-144g L-l. So, different stirring rates almost had no obvious differencehad no fatal effect on fermentation system, and agitation mainlyamong four fermentatioEthanol 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 factorFig. I Time courses of ethanol, glucose and biomass concentrations durinfermentations with S cerevisiae9: I, Non-aeration and static culture: a: 2, Non-aeration and agitatingculture: A: 3, Aeration and static culture; x: 4, Aeration and agitating culture0000000000 8e0@@ooeea, Non-aeration and static culture; b, Non-aeration and agitating culture; c,eration 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 to19 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): No 6,AcFig 2 Change in dissolved oxygen concentration in initial aeration culture 12, Naphthol-AS-BI-phosphohydrolase(+, blue); No. 13, a-galactosidase(+,at the firstntation phaseviolet): No. 16, a-glucosidase(+, violet)566应用与环境生物学报 Chin J Appl Environ biol15卷for 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. Fromour research, biomass was the most direct faodifferent fermentation capabilities of yeast in term of ethanolconcentrationIn the VHg technique of our research, appropriate oxygenand feeding time were taken to regulate biomass, As a result,he yield of ethanol on substrate(Y, )reached 0.471 g/g(Thestoichiometric yield was 92.2%)in the best fermentation(4)h▲Then, to identify the characteristics of the differentstates in the four fermentations, through analyzing 4 groupsof tests by enzymatic detection kit (Obtained from biomerieuin France) which was a semi-micromethod designed for theresearch of enzymatic activities, the results indicated that thekinds of active enzymes were discrepant, moreover, the activitof the same enzyme was different. In positive assay, enzymactivity 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 revealed6420different 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-Lfermentations under initial aerationI and agitating cultureThe fed-batch experiment was performed in a 10-L Glucose (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 146 g L: to 85g L-. The average speed of consuming glucose achieved 7.6g Lh, and the maximal speed was about 18 g L-h, slower than thatof 100-ml fermentation , and the biomass was lower than that of100-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 reproductionat the same level for all the rest time, The lower ph value wasspecially in the VHG fermentation, where oxygen becamepropitious 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, oxygenconcentration and yield in VHG ethanol fermentation were would destroy the process of anaerobic metabolism. In that case,investigated 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 ethanol concentration and stoichiometric yield. In our期LIU Yan, et al. Improvement of Ethanol Concentration and Yield by Initial Aeration and Agitation Culture567research, initial aeration was taken, and the initial aeration factors in VHG. Oxygen was mainly related with yeast growth,trategy achieved appropriate dissolved oxygen concentration quick accumulation of biomass and cell activities. On the othe(4.0-9.0 mg L ). Within the range of oxygen concentration, this hand, agitation led to fermentation liquid mixing well, CO,strategy not only provided suitable oxygen for yeast growing releasing and nutrition consuming, etc. To summarize, thequickly, but also avoided aerobic respiration to a certain extent. two 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 (0-2, and to accelerate the consumption of nutritionReferencesIkegamai T, Yanagishita H, Kitamoto D, Haraya K. Accelerated ethanolby yeast. Under this condition, yeast would grow well andby Saccharomyces cerevisiae with addition of activatedcells were vigorous, so that the cells had good performance oncarbon. Biotechnol Lett. 2000. 22: 1661-1665ethanol production and endurance to bad circumstance includingThomas KC, Hynes SH, Ingledew WM. Practicatheoreticalhigher concentration of residual sugarirs. ethalol poisoning, byproducts and nutrition lacking, especially in the last period ofconsiderations in production of high concentrations of alcohol byfermentation. Proc Biochem. 1996. 31: 321-331fermentation3 Torrico D. Acevedo F Effect of agitation and Tween 80 addition in thehereas, in static culture, the yeast cells would subside tocontinuous alcohol fermentation. Mircen. 1988. 4: 393-399the bottom of the fermentor, thus they could not absorb nutrient4 Hoppe GK, Hansford GS. The effect of micro-aerobic conditions onwell. As a result. the biomass was so low and the cell size was sosmall that they were weak in ethanol productionLe,1984,6:681~686The glucose concentration in the broth of VHG was much5 Grosz R, Stephanopoulos G 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 yeastcells were very excessive, they would consume more sugars forcerevisiae. I: Hysteresis, oscillations culture. Biotechnol Bioeng, 1990,36:1006-1019growth, which would lower the stoichiometric yield. Moreove, 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 fermentsugars to ethanol fast and efficiently.Saccharomyces cerevisiae by a vitamin feeding strategy during fedbatch process. App/ Microbio/ Biotechnol, 2002, 60: 67-72Considering the results of the 10-L fermentation, the7 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 ofvery high ethanol performance in Saccharomyces cerevisiae fed-batchglucose consumpion and ethanol production were slower. Theprocess. App/ Microbiol Biotechnol, 2004, 63: 537-542reason must be the lower amount of yeast cells, which was relatedBai Fw, Chen L3, Zhang Z, Anderson WA, Moo-Young M. Continuouswith weak transfer capability of material elements in the 10-Lethanol production and evaluation of yeast cell lysis and viability underfermentorvery high gravity medium conditions. Biotechnol, 2004, 110: 287-293culture increy(r(威mhmL-),and made the stoichiometric yield(up to 92%)higher thanChin App! Environ Bio/(应用与环境生物学报,2007,13(1):69-72other general VHG ethanol fermentation processes, that was toJones rP, Greenfield PF. Effect of carbon dioxide on yeast growth andy, the equal raw material would obtain more ethanol under thatermentation. Enzyme Microbiol Techno 1982. 4condition, The increased stoichiometric yield could decrease11 Takezaki m. matsuura K. Hirotsune m, Hamachi M. effect of solids onhe growth of yeast. J J Brew Soc Japan, 1993. 88: 319-325Termentation time was shortened for only 54 hours. So, it became12 Fukuda T, Hiramatsu M, Sanmoto H, Fukuzaki S, Discharge ofa highly competitive technique.issolved carbon dioxide by the addition of activbon particleIn conclusion, oxygen and agitation were two importantand its effect on yeast. J Brew Soc Japan, 1996, 91: 279-28