Influence of furfural concentration on growth and ethanol yield of Saccharomyces kluyveri

Availableonlineatwww.sciencedirect.com. ScienceDirectcN 11.6742529XJoumal of Environmental Sciences 19(2007)1528-1532Influence of furfural concentration on growth and ethanol yield ofSaccharomyces kluyverLU Peng, CHEN Li-jun', Li Guo-xue, SHEN Shi-hua2, WANG Li-1i'JIANG Qi-yang, ZHANG Jin-fengl. College of Resources and Envimnment China Agricultural University Beiing 100094, China. E-mail: hupeng 531@16Received 15 March 2007; revised 30 May 2007; accepted 5 June 2007AbstractFurfural is an important inhibitor in ethanol fermentation process using lignocellulosic hydrolysates as raw materials. In order to findout the furfural concentration range in which furfural inhibits the fermentation process, we used one strain Saccharomyces kluyverselected from soil and cultured in several different furfural content media under low glucose concentration condition. Experimentresults showed that microorganism growth was stimulated and dry cell weight decreased when furfural concentration in the mediumwas 0.25 mg/ml Furfural had negative effect on cell growth when its concentration was above 1.00 mg ml. At the same time, the straingrowed better and had a higher glucose consumption rate in 5% original glucose concentration condition than in 3% original glucoseconcentration condition, The results showed that appropriate exaltation of original glucose concentration in stalk hydrolysates willKey words: furfural concentration; Saccharomyces kluyveri; fermentation; growth; ethanol yieldIntroductionerate during hydrolyzing pretreatment process( Carlos andLeif, 2003; Nurdan and Yesim, 2000). Hemicellulose canIt is nearly 7x10t of crop stalks produced each year be hydrolyzed to xylose and glucose at low temperaturein China( Dong et aL, 2006). Crop stalks were replaced (below 200oC)and diluted acid concentration (less thanby coal, natural gas or other energy materials for changes 3% v/v). Then xylose and glucose can be hydrolyzed toof energy consumption structure in rural areas. Farmers furfural at such temperature and acid conditions. duringspend a lot of money in grinding stalks and plowing them traditional alcoholic fermentation process, furfural comesinto soil after crop harvesting, and composting occupies from various raw materials and accessories, such as grain,more land and needs more labor force. Stalks incineration rice chaff, wheat bran and rice bran etc. (lu, 1994).Theis common, and the total amount reaches 1.30x10ya( Cao analysis of furfural content in various distilled spirit showet al, 2006). Such phenomenon increases air pollution that: furtural in it generally does not exceed 100 mg/L andand resource waste. Using crop stalks as raw materials almost all are below 30 mg/L(Zhang ef al., 2002; Xufor ethanol fermentation can produce renewable energy. and Zhao, 1997). China distilled spirit Mao Tai has theAnd it also can contribute to the reuse of solid waste highest furfural concentration, about 209 mg/L(Xu andand environmental protection. This technology has good Zhao, 1997). It has no prominent inhibiting influence toeconomic and social performances.ethanol fermentation when furfural concentration is belowWhen using crop stalks as raw materials for ethanol 0. 2 g/ml(209 mg/L).fermentation, the hydrolysates acquired in the pretreatment The previous research results also show that furfuralprocess (diluted acid treatment) are different from tra- inhibiting effects are different in ethanol fermentation byditional ethanol fermentation using starch-based original using different yeast (Nurdan Eken-Saracoglu et aL., 2000;materialsMohammad et aL., 1999). It has prominent inhibitingFirstly, the saccharide in the hydrolysates is complicated effects to ethanol fermentation when furfural concentrationand total sugar content is low, generally not exceeding increases The yeast growth would be restrained and obvi.5%(w/v). Secondly, fermentation inhibitors such as fatty ous中国煤化工r( Steve er al,2003acids, furfural and phenolic compounds etc. always gen- Evntation(25treatmC NMHGhanol fermentationProject supported by the Nationaley Technology Research and Devel&oent Program of China(No, 2006BAD07AOL, 2006BADIOB05-02). ( Mohammad et al., 1999), but it is difficult to apply thisesponding author. E-mailhnology to commercial ethanol production. ThereforeInfluence of furfural concentration on growtb and ethanol yield of Saccharomyces kluyverifurfural concentration in hydrolysates is one of the most fluidimportant factors during the fermentationA Chinese researcher(Fang et al, 2005)found that 1.3.2 Determination of strain densityhen furfural concentration in culture medium was abFermentation broth was diluted 10 times using (.9%1 mg/ml, it had prominent inhibiting effects during xylitol Nacl solution. The turbidity of solution was detectedfermentation. It restrained fermentation severely when at 600 nm with a 752N spectrophotometer(Shanghaifurfural concentration in culture medium was above 2.0 Precision& Scientific Instrument Corp, China)mg/mL. Tbey have less related reports about the furfural 13.3 Determination of residual dextroseinhibiting effect during ethanol fermentation. Saccha-omyces kluyveri is one of microorganisms that haveThe fermentation solution cultivated 24 h was cen-high ability to convert raw materials to ethanol. In thisuged, and the supernatant fluid was extracted afterexperiment, we explored the growth and fermentation appropriate diluting. The residual dextrose corharacteristics of S. kluyveri under different furfural con-on was analyzed using 3-5 dinitrosalicylic acid (DNSliteratus1.3, 4 Determination of ethanol yieldI Materials and methodsOne part of the supernatant fluid was filtrated by 0.22um microporous film, Then put quantitative n-propyl1.1 Microorganismsalcohol (0.64% w/v) into 0.5 ml colature as internalo The source of strain S. kluyveri was colected from standard material. Gas chromatography with MS detectorhard soil in Haidian District, Beijing, and screened by (GCMS-QP2010, Shimadzu Corp, Japan)was used toenrichment and restrictive culture. The pure culture was analyze ethanol concentration(intermal standard method)identified by Microorganism Research Institute of ChineChromatographic setting: peg capillary column and FIDex Hansen, was selected as CK strain(bought from China ture: 200'C, detector temperature: 219; carrier gas: NzAgricultural Microbial Culture Collection Center. serial (60 kPa): flow rate: 31 ml/min; spilt ratio: 1/20: voltage ofnumber is 20251)detector: 75 kV; velocity of H2 flow: 30 ml/min; samplequantity:Iμ.1.2 Culture medium preparation and culture conditionAll of the experiments repeated thrice, and the data used1.2.1 Substrate medium constitutein the article were the average of repeats.The original sugar(3%)treatment culture medium (100 2 Results and analysisml) was prepared with peptone 1 g, powder yeast extrac.tion 0.5 g, and dextrose 3 gThe original sugar(5%)treatment culture medium(100 2.1 Influence of furfural concentration gradient onml) was prepared with peptone 1 g, powder yeast extrac-strain cell dry weighttion 0.5 g, and dextrose 5 gWith the furfural concentration increasing in the cul1.2.2 Culture and treatment conditionshure medium during fermentation process, strain cell dryweight increased at ]ow concentation and decreased atFurfural stock solution was prepared by filtration high concentration(Fig. 1 ) The maximum ceil dry weightthrough a 0. 22-um microporous film, put the sterilized occurred at 70.3 mg/ml when furfural concentration wasstock solution into culture medium by sterile operation, 0.25 mg/ml. When furfural concentration was higher thanprepared culture medium which contain different furfural 0.25 mg/ml, cell dry weight decreased. Apparently, the0.25, 0.50, 0.75, 1.00. 1.25 and increase of furfural concentration inhibited the increase1.50 mg/mI. Experiments were carried out thriceof cell dry weight. As furfural concentration was lowerThe microbial strain was activated in slant medium, than 0.25 mg/ml, furfural promoted the increase of cell dthen inoculated active strain to 50 ml triangular flasks, put weight in all treatmentsthe masks in the shaker and cultivated them at rotationn promote the increase of cell dryspeed of 140 r/min, and set cultural temperature at 30C. apparently under the condition of 3% original sugaStrain suspension fluid cultivated for 24 h was usedcell dry weight began to decrease gradually while furexperimental strain and 2.5 ml was inoculated in 50 m concentration exceeded 0.25 mg/mlIn 5% original sugar culture medium, higher cell dry1.3 Fermentation parameter testinggrowth weight could be obtained comparing with 3%original sugar treatment. The minimum difference between1.3.1 Determination of cell dry weightthe two groups of treatment of the cell dry weight wasStrain suspension 10 ml was cultivated for 24 h, cen-nean中国煤化工 e analysis show thattrifuged for 10 min(4000 r/min), collected the cells, then (Tabin culture mediumput it into bake oven, and dried at 80C until constant hadCNMHG weight Furfuralweight. Finally, cells were weighed by ten thousandth scale concentration gradient also had different inhibiting effectsand calculated the cells dry weight in 1 ml fermentation on two groups of treatment.LU Peng et al.Table 1 varance annlysis of cell dry weight of S kluyveri culture odifferent original sugar content treatmentnumber of cells of CK strain was more easily inhibited bythe furfural compared with S. kluyveri undered the sameBetween groups 476.1 1 476.1 20.6 0.001892 5.3 2.3 Influence of furfural concentration on glucose cou-In group823.660659sumption during fermentation process>Foos, there is marked difference between two treatment SS: sunGlucose can be converted into other materials rapidlysquares; MS: mean square.in normal fermentation environment. The consumption of2.2 Influence of furfural concentration on strain tur. glucose will decrease when fermentation function of straitis inhibited. The degree of inhibiting can be determined bybidity in fermentation brothmeasuring the residual glucose content in the fermentationThe strain density of fermentation solution can be exsolution under different furfural concentration( Carlos andpressed by turbidity. At different furfural concentrations. Leif, 2003; Liu, 2001).By analyzing the residual glucose concentration afterthe changing tendency of turbidity is similar to that of cell fermenting 24 h(Fig 3), we found that furfural had no ob-increased to 0. 25 mg/ml in all sugar content treatments vious inhibiting effects on glucose consumption if furfural(Fig. 2). With the furfural concentration rising continuous- concentration was below 1 mg/ml. But when furfural con-ly, turbidity began to decrease. Strain turbidity decreased centration was above I mg/ml, the glucose consumptionat a uniform speed(the rates of slope of ever point are decreased quickly(Fig. ) Therefore, the point of furfuralsimilar)when furfural concentration changed from 0.25 concentration at 1 mg/ml is very important for the strainto 1.25 mg/ml under 5% original sugar treatment. In 3%original sugar treatment, the absolute value of the rate of Comparing the residual glucose concentration in fer-increased in the same concentration changing scope. Fur- sugar in the same changing scope of furfural concentrationof different sugar treatments was similar. The reducingthe low original sugar concentration circumstance as it sugar concentration in 5%b original sugar treatment was lessThe reaction of S. kluyveri and that of CK strain are the concentration of the original sugar, utilization ratiodifferent at the same furfural concentration. The turbidityof glucose increased. The difference of residual glucoseof CK treatment decreased when furfural concentration concentration between the two treatments became obviincreased(Fig. 2). And it decreased rapidly when furfural ously when furfural concentration was above 1 mg/ml fromconcentration increased to 0. 5 mg/ml. It is inferred that the the furfural concentration increasing direction(Fig 3).Thisphenomenon showed that the change of furfural concentra-tion had more effects on 3 original sugar treatment than子没四on 5%. Appropriate increase of original sugar can promotethe conversion of glucose. And it also contributed to theaccomplishment of fermentation as furfural concentrationis higher than 1 mgmThe residual glucose after 24 h fermentation of 3% sugartreatment accumulated obviously when furfural concentra-tion reached 1.0 mg/ml(Fig 3). However, in CK treatment(S% original sugar content), the residual glucose increased0250.500751.00125rapidly when furfural concentration reached 0.75 mg/ml(Fig 3). The furfural tolerance ability of S. kluyveri wasFig. I Infuence of furfural concentration on cell dry weight of yeast prominently higher than that of CK strains under the same5% Orginal suger中国煤化工01231500751001251.50CNMHGmDig 2 Influence of furfural concentration on turbidity of S. kluyveri.Fig. 3 nfluence of furfural concentration on glucose consumption of S.Influence of furfural concentration on growth and ethanol yield of Saccharomyces kluyveri1531original sugar content conditions, The substrate utilization concentration of the inhibitor is low(Carlos and Leif,rate of S. kluyveri was higher than that of CK strains2003). The results of the experiment show that the ethanolThe furfural concentration had no prominent influence productivity of the strain was not influenced obviously.on ethanol output of S. kluyveri train in the experiment Besides, the inhibiting effects of the mixture of variousscope, but original sugar content had prominent influence inhibitors were much greater than that of single inhibitoron ethanol output (Table 2). Ethanol yield of S. kluyveri ( Carlos and Feif, 2003; Nurdan and Yesim, 2000; Steveunder 5% original glucose treatment was similar to that of et al, 2003; Lohmeier- Vogel et aL, 1998). The researchresults of Steve ef al. (2003)show that the co-inhibitioneffects of two mixed inhibitors are greater than each single3 Discussioninhibitors. In addition, the furfural and hydroxymethylfurfural has different influence on microbe stains( Carlos3.1 Influence of sugar content in the hydrolysates on and Leif, 2003)and needs more research in outcomesethanol fermentationduring hydrolytic processThere are less related reports on the scope offurfuralDuring the pretreatment process, the stalk hydrolysates concentration that inhibits ethanol fermentation. Becauseare a mixture which contains glucose, giucan, xylose, stalk hydrolysates are a mixture which contains variousxylan and many other low molecular weight materials. The saccharide and inhibitors, the constituents will changetotal sugar concentration of this mixture is generally less when the conditions in the pretreatment process change. Itthan 5%(w/v). The original sugar concentration is above is very difficult to study all inhibitors in the hydrolysatesethanol fermentation process. Glucose concentration in interact with each other. And we use similar system toethanol fermentation is from 10% to 12%(w/v). In this imitate the reaction in stalk hydrolyzing solution. It is anscope, yeast can make use of glucose sufficiently to pro- innovative point that the strain inhibited concentration canduce the highest ethanol productivity and keep the lowest be studied by setting furfural concentration gradientsugar remains (Liu, 2001). Therefore, the hydrolysates The lower limit value for S kluyveri strain growth andusing yeast in ethanol fermentation process. The sugar that of CK treatment, about 0.75 mg/ml. The furfuralconcentration should be lower than the above mentioned endurancing ability of S. kluyveri is higher than thatwhile using bacteria as fermentation microbe at 35reference strain. In the same time low concentration ofIf glucose concentration is above 5%, the ethanol yields furfural(0.25 mg/ml) can promote the growth of the straindecrease as the sugar content rises. The ethanol conversion (both S. kluyveri and CK), and the mechanics needs toberate is lower than 50% when original sugar concentration further studiedapproaches 10%(Tian et al., 2005). Concentrating hydrolysates need to be added in the producing procedures, 3.3 Infiuence of furfural concentration on ethanol fer-ncrease the ethanol production cost by using cellulosicmaterials as raw materials. When using xylose as substrate, Massive ethanol begins to yield after the furfural in thethe ethanol comversion rate is less than using glucose substrate has already been metabolized Steve et aL, 2003(Steve et aL 2003).In the designed scope of furfural concentration, the furfuraluse sugar content is always less than 5%companies with a lot of non-fermentation materials in the contrary, furfural can promote ethanol production andstalk hydrolysates after pretreatment process, the sugar can not anastomose with the growth curve of microbesconsumption rate in fermentation of the experiment may This phenomenon may be caused by the low furfuralbe higher than that of practical production. In addition, concentration in the experiment; or the disturbance of5% original sugar content can be easily reached by ad- volatilization of ethanoi. Therefore, using fermentationjusting solid-to-liquid ratio appropriately. Therefore, hetank to further study ishydrolysates can be converted to ethanol at low sugarontent directly is the priority research area. S. Kluyver 4 Conclusions3.2 Furfural influence on strain growthThe growth of S. kluyveri can tolerate low furfuralSome research results show that the furfural inhibitingoncentration. Furfural concentration at 0.25 mg/ml caninfluence on strain growth is greater than that on ethanol promote the growth of the strain. The inhibiting effectsfermentation(Eva and Barbel, 2000), especially when the on the strain growth appear when furfural concentrationTable 2 Ethanol content after 24 h ferm中国煤化工urfural concentration(mg/ml)100S kluyveri (3% original sugar)CNMHGS kluyveri(5% original sugar)2472532.35259LU Peng et adis above 1.00 mg/mlLiu QL 2001. The research of active-dry yeast tolerance to highThe ethanol yields are not influenced apparently with thesugar concentration[]. Liquor Making, 28(3): 46increasing of furfural concentration in the experimental. Lohmeier - Vogel E M, Sopher C R, Lee H. 1998. IntracellularAnd it has no prominent differences in different furfuralacidification as a mechanism for the inhibition by acidconcentration treatment. The ethanol yields of S. kluyverhydrolysis-derived inhibitors of xylose fermentation byand CK strain, S. cerevisiae, are similar.easts[]. Joumal of Industrial Microbiology Biotechnology20:7581The strain can be more easily inhibited by furfural LaS P, 1994. White wine producing process[M]. Beijing:Chinacultured in 3% original sugar medium compared withLight Industry Presscultured in higher sugar condition (5%). During the pre- Miler G L, 1959. Use of dinitro salicylic acid reagent fortreatment process, setting a proper stalk-to-water ratio willdetermination of reducing sugars[]. Anal Chem, 31: 426-increase the original sugar content in hydrolysates, which428.can increase the strain tolerance to furfural and promote Mohammad J T, Claes N, Gunnar L et al, 1999. 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