Laboratory Experiment t Determine Batch Ethanol ：实验室实验确定批乙醇

1、laboratory experiment to determine batch ethanol production by s. cerevisiaebenjamin revesmay 11, 2005outlinelbackgroundltheorylprocedurelresultslconclusionslrecommendationslimpactlquestionsbackgroundneed for ethanollethanol used as raw material and solvent in the chemical, food, and pharmaceutical

2、industrieslfour million tons of ethanol are produced each yearleighty percent by fermentationlenergy information administration has predicted annual consumption growth in u.s. of 3.2% each yearsaccharomyces cerevisiaelcommon type of yeastleucaryoticlreproduces by buddinglcan grow aerobically or anae

3、robicallycurrent methodslbatch reactorlfed-batch reactorlcontinuous reactorlpacked bed reactortheoryglycolysislbreakdown of 6-c glucose into two molecules of 3-c pyruvatelfate of pyruvateaerobic conditions- tca cycleanaerobic conditions- converted to ethanol via acetaldehydecell growthlsubstrates +

4、cells extracellular products + more cellsbatch growthllag phaselexponential growth phase ldeceleration phaselstationary phaseldeath phaseyield coefficientslhelp to quantify growth kineticslyx/s=-x/slyp/s= -p/slyp/x= p/xproduct formationlgrowth-associated productslnon-growth-associated productslmixed

5、-growth-associated productsgxppydtdpxq/1pqgpqtemperature effectsloptimal temperatureslproduct formation affectedldiffusion limitationsobjectivesobjectiveslstudy ethanol production and glucose utilization by saccharomyces cerevisiaelstudy effect of temperature on fermentationlconstruct growth curves

6、methodsstages of experimentationlautoclavinglinoculationlgrowth of culturelanalyzing samplesautoclavinglsterility is a must!lsaturated steam at 121oc used to kill all sporesautoclavepreparing inoculumlinoculum is typically 5-10% of total working volumelyeast obtained from microbiology department on

7、plateslinoculating needle used to take yeast from plate into 800 ml of yeblplaced on shaker at 30oclimportance of inoculum conditioninoculum preparationyeast extract brothlundefined vs. defined medial1 l yeb contains:20 grams of glucose10 grams of yeast extract brothlglucose is carbon/energy sourcel

8、yeast extract provides cofactors and ionsfermentorl7.5 l bioflo 110 modular benchtop fermentorlcontroller and pcultemperature controlfermentorpcuheadplatetaking sampleslfermentor equipped with sample portlsample tubes had been autoclavedloptical density of sample measuredlcentrifuged for 5 minutes a

9、t 2000 rpmlliquid decanted and stored at 4occentrifugecentrifuged samplesestimating cell concentrationlspectrophotometer used to measured absorbance at 650 nmlsterile yeb used as blanklbeers law: a=bcllinear correlation between absorbance and concentration of cellslcalibration curve constructedspect

10、rophotometerconstruction of calibration curveloptical density measuredlwashed with 10 mm phosphate bufferldried in oven at 35 celsiusod calibration curveod calibrationx = 1.1435*abs. + 0.0358r2 = 0.996600.050.10.150.20.250.30.350.40.450.500.050.10.150.20.250.30.350.4absorbancex (mg/ml)glucose determ

11、inationlglucose assay kit ordered from sigmalbased on uv absorbance of nadh at 340 nmlglucose + atp glucose-6-phosphate + adplg6p + nad+ 6-phosphogluconate + nadh lsamples dilutedhexokinaseg6pdhethanol determinationlethanol assay kit ordered from r-biopharmlbased on absorbance of nadh at 340 nmlsamp

12、le dilutedlethanol + nad+ acetaldehyde + nadh + h+lacetaldehyde + nad+ + h2o acetic acid + nadh + h+adhal-dhresultsgrowth curve for 30 celsiusgrowth curve for 30 celsius0.0000.2000.4000.6000.8001.0001.2001.4001.6001.80005101520253035time (h)x (mg cells/ ml)xgrowth curve for all runsgrowth curve 00.2

13、0.40.60.811.21.41.61.801020304050time (h)x (mg cells/ ml)30 celsius37 celsius25 celsiusconcentration plot for 30 celsiusglucose and ethanol concentrations vs. time for 30 celsius 05101520253035404505101520253035time (h)cocentration (mg/ml)glucoseethanolglucose concentrationglucose concentration vs.

14、time010203040506001020304050time (h)glucose concentration (mg/ml)30 celsius37 celsius25 celsiusethanol productionethanol production02468101201020304050time (h)ethanol concentration (mg/ml)30 celsius37 celsius25 celsiusyield coefficients25oc30oc37ocyp/s (mg p/mg s)0.2290.2820.247yx/s (mg cells / mg s

15、)0.0270.0378 0.0293yp/x (mg p/ mg cells)8.447.458.44conclusionslyeast grew the fastest at 30 celsiusllag times were longer at 25 and 37 celsiuslglucose was fully used in each runlamount of ethanol produced was almost the same for each runlruns should be allowed to proceed longerrecommendationsldetermine growth and productivity effects due to other factors such as phldetermine optimal inoculum size and agelinvestigate better methods of analyzing samples loperat