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) 344-6783 ChE 124 L: Chemical Engineering Laboratory To be Performed by: Juvyneil E. Cartel Supervising Faculty: Engr. May V. Tampus Adviser: Engr. Ramelito Agapay Course&Yr. : MS ChE-1 Target Date to Start: May 14, 2008 Date of Completion: May 19, 2008
Experiment # 6: Describing Growth Patterns of Baker’s Yeast in a Batch Culture (Part 1: Using Batch Shake-Flask) I. Objectives: 1.) Identify the growth patterns of Baker’s Yeast during a batch culture. 2.) Mathematically describe these growth patterns in terms of biomass concentration in time. 3.) Determine the occurrence of by-product (ethanol and/or acetate) formation, if there is any, and to relate this phenomenon to oxygen consumption during the experiment. II. Materials and Methods 2.1 Micro-organism Active dry yeast (Saccharomyces cerevisiae) will be bought in the supermarket. This will be acclimatized and incubated first before culturing. Parameters will be monitored from time to time or in every sampling to determine its development. 2.2 Experimental Procedure [1-4] Mix nutrients (trace elements and vitamins) and Struktol J650 with distilled water (to 800ml mark) as shown in Table 1 and Table 2 in a 1-L Erlenmeyer flask. Cover it with cotton. Transfer 100 ml of the mixture into a 250-ml Erlenmeyer flask. Cover it also with cotton. Aside from that, prepare 200-ml glucose solution in 250-ml Erlenmeyer flask by mixing 20 grams of glucose with distilled water (to 200-ml mark). Mix it vigorously and cover it with cotton. Wrap each flask with tin foil including the glass funnel and sterilize them in AllAmerican Electric Pressure steam sterilizer model 25X at 15 psig for 20 minutes. After pressure and sterilization equalization, cool the flasks rapidly (e.g. to the temperature used for pouring). The flasks should be cooled under cool, running water. Pour 10 grams dry weight of Active dry Baker’s yeast ( Saccharomyces Cerevisiae) in the 100-ml nutrient mixture sterilized previously and mix well. Then, add it to the sterilized 2-L Erlenmeyer flask containing the 700-ml nutrient solution using a glass funnel aseptically. Shake at 250 rpm and acclimatize for 30 minutes in the New Brunswick Scientific rotary shaker model G25. Take sample after. After that, pour the sterilized glucose solution aseptically into the 1-L Erlenmeyer flask. Shake and incubate for 30 minutes. Take another sample. Carry out the reaction after incubating for 24 hours. Take 5-ml sample with a duplicate for every 30 minutes and every three hours onwards. Orion Model 210 A pH meter and ATI Orion Model 862 DO meter will be used in measuring pH and dissolved oxygen (DO), respectively in every after sampling.
2H2O FeSO4.2.HCl (Total) 15.1000 g/L = 1.00 0.50 0.3 Culture Media Table 1.0120 0.7500 g/L = 0.5000 g/L 0.0100 % (v/v) Amount in grams Table 2.7H2O CaCl2.5H2O CoCl2.0401 1.2500 g/L = 3.00 15.0000 g/L 0.0804 g/L 0.30 0.6H2O H3BO3 Na2MoO4.00 4.0008 0. Trace Elements and Vitamins Formulation [ 5 ] Component Concentration (g/L) of 2.0027 0.7H2O Trace Elements Vitamins Struktol J650 20 g/200 ml 5 g/800 ml 3 g/800 ml 0.2H2O KI (Total) Vitamins: Biotin Ca-pentothenate Nicotinic Acid Myo-Inositol Pyridoxine.0013 0.50 1.0011 0.5 g/800 ml 0.0401 0.0401 1.0401 0.67 ml solution Trace Elements: EDTA ZnSO4.0008 0.1715 2 .0804 0. Composition of the Culture Media  Component Initial Concentration Resulting Concentration after mixing Glucose (NH4)2SO4 KH2PO4 MgSO4.0020 0.0000 g/L 3.0080 0.00 375.4H2O CuSO4.75 15.6300 g/L = 0.1715 g/800 ml 0.30 1.0020 0.1715 g/L 0.0401 0.0027 0.00 0.0000 g/L 5.HCl Para-Amino Benzoic Acid Thiamine.0125 % 20.00 0.0003 0.10 0.7H2O MnCl2.40 3.00 15.00 3.00 4.1 ml/800 ml = 100.00 15.4600 g/L = 0.0804 g/800 ml 1.0120 0.0000 g/L = 6.
pipette 20 µl of distilled water (blank).3.1 and 3.4. This will be used for ethanol and acetate analysis using Shimadzu GC-8A. pathlength is 1. weigh them again in the analytical balance.2 Baker’s Yeast and Biomass Moisture Analysis [ 1.02.3. 0. temperature is 37 oC.3 PROCEDURE Into separate test tubes.5 ml.1 Reagent Preparation Prepare 50 ml working glucose enzyme reagent in a vial that contains ≥ 20.45 µm Whatman filter (or equivalent).2.1 Sample Preparation [ 6 ] Sample the fermentation so as to collect a representative slurry sample. ≥ 5000 U/L peroxidase. After which. Record the weights and compute the moisture content. Please refer to Section 3.3. 0.4 g/L.000 U/L glucose oxidase. Use deionized distilled water as solvent. Replace the rubber stopper and allow 5 minutes for reconstitution. 0.4. 2.4 g/L). Heat them for 20 minutes in the All American Microwave Oven Model AMW-210 LM at a power setting II. Put the biomass (retentate) in a dry crucible for biomass analysis.05. 2. Then.3. 0.2 for the calculation.4. Put the supernatant (filtrate) in the freezer. 0. Thus.1.2 mM 4-aminophenazone. 3 .4 g/L glucose. 2. 6 ] Weigh three 10 grams of dry yeast ( Saccharomyces cerevisiae) in the analytical balance and put them in the dry crucibles. HACH DR/2010 Spectrophotometer will be used in measuring the absorbance of the glucose standards and samples. 2. 0. Let it cool in the desiccator to initial temperature afterwards. Samples are to be centrifuged in Programmable IEC Centra Centrifuge (E0803) by spinning at 6000 RPM and 4 oC for five minutes. mode is endpoint.5 ml of working reagent to each test tube and mix. Filter the sample through a 0. Add 2. dilution will be made to samples which have higher glucose concentration than 0.2 TEST PROCEDURE CONDITIONS The test procedure conditions is as follows: wavelength is 505 nm.0 cm. reagent volume 2. or sample (serum.01. Following incubation for 3 minutes at 37°C in GFL water bath (#064700073) determine the absorbance of each standard sample at 505 nm using the distilled water sample as the reagent blank. reaction time is 10 min.52 ml. The procedure is linear to 0. sample volume is 20 µl. ≥ 0. may be diluted if necessary) to be assayed. glucose standards (0.2. weigh them again in the analytical balance. total volume 2.3 Glucose (Trinder) Assay [ 7 ] 2. and sample to reagent ratio is 1/100. Use the moisture content for yeast that you get in calculating for the actual amount of Baker’s yeast that is to be added in the Erlenmeyer flask. Collect 5-ml sample with a duplicate and put it in a sterilized vial with a cover.4. For biomass analysis.4. Record the weights and compute the moisture content of each and get the average. ≥ 10 mM p-hydroxybenzoate.4 Analytical Methods 2. Let it cool in the desiccator to initial temperature.4. Swirl gently until the contents of the vial are completely dissolved. put the crucible containing the biomass in the All American Microwave Oven Model AMW-210 LM at a power setting II and heat it for 20 minutes.
4 Ethanol and Acetate Analysis Using GC (Shimadzu GC-8A) [8-10] 2.1 minutes for isopropanol.15 (v/v) % (e. 0. 0. 0. Calculations  3. Use the equation of the line of the calibration curve in calculating the ethanol and acetate concentration in each sample. the internal standard concentration is 5 %v/v universally throughout this procedure. or blank. 0.g. run time is 5. 0.02. the internal standard concentration is 0. 0. Therefore. using reagent grade isopropanol ((≥95 %). 0.1 Calibration and Test Procedure Prepare 0.4.08.15%) and a blank all containing 0.6% eluent (≥88% formic acid).04.4. 2.08. Use the equation of the line of the calibration curve in determining the actual glucose concentration of each sample.1 %) glacial acetic acid. Add 100 µL of sample.Plot the absorbance of each glucose standard against its glucose concentration.02 to 0. and 60 minutes for formic acid.2 Operating Conditions Operating conditions for ethanol and acetate analysis are as follows: Oven temperature is 155oC (isothermal). Therefore. inlet and detector (FID) temperature is 250oC. 2. and injection volume is 1 µL.06.10. 55 minutes for acetic acid.9%v/v universally throughout this procedure.1%v/v internal standard spiking solution should be added to 100 µL part sample.02. This procedure specifies 900 µL of a 0.04.g. 4. Refrigerate analytical and calibration verification standards in sealed sampler vials for storage. .4. using 99. carrier (He) gas flow rate is 30 ml/min. 0. standard. standard.4. Prepare another standard for acetate analysis using the following method: Calibrate with six working standards over the range of 0. ranging from 0. or blank to 900 µL of 5.5 minutes. Using the blank and standards observe the peaks and make a calibration curve by plotting the peaks against the concentration of the standards. or sample into the GC.1%v/v concentration internal standard spiking solution.3 minutes for ethanol. Prepare six ethanol analytical standards.5%v/v ethanol. 0. Refrigerate analytical and calibration verification standards in sealed sample vials for storage Inject 1 µL of blank.005. standard.4.06. and 0. and 0.1 Baker’s Yeast Moisture Content The following formula is directly applied in calculating for the Baker’s yeast moisture content: %Moisture content of Baker’s Yeast = Initial Weight (g) – Final Weight (g) x 100 Initial Weight (g) 4 .9%v/v isopropanol. The internal standard spiking solution must be added in the same proportion to every standard or sample analyzed by this method.005 to 0. retention time is 2.1 %v/v (e. III. 0.
∆S in grams can be determined using the following formula: ∆S = [(CSo . and V is the final volume in liter. The following formula is used in determining the amount of biomass produced.3 Glucose Consumption The actual glucose or substrate consumption. and V is the final volume in liter. Vo – Cx . ∆X in grams: ∆X = [(Cxo . V] where Cxo is the initial concentration of biomass in grams per liter and Cx is the final concentration of biomass in grams per liter in the bulk liquid while Vo is the initial volume in liter .4 Amount Biomass Produced The actual amount of biomass produced determines the growth of yeast after some time or in every sampling.5 Yield Determination Biomass yield is the amount of dry weight yeast produced in grams per grams glucose consumed.3. biomass yield is calculated using the following formula: Ybiomass/glucose = ∆X/∆S 5 .2 Actual Amount of Baker’s Yeast to Add in the Shake Flask The following formula is directly applied in calculating for the actual amount of Baker’s yeast that is to be added in the Erlenmeyer flask: Weight Baker’s Yeast to add (in grams) = dry weight (g) Baker’s yeast Required [(100-%moisture content Baker’s yeast)/100] 3. After the experimental run. 3. Vo – CS . 3. V] where CSo is the initial concentration of substrate in grams per liter and C S is the final concentration of substrate in grams per liter in the bulk liquid while Vo is the initial volume in liter .
Chemical Analysis and Testing Task. 2 nd ed. RAMELITO AGAPAY Date: __________ Adviser. Fermentation Microbiology and Biotechnology. References:  Fabian et. NJ Date Submitted: May 13. TAMPUS Supervising Faculty Noted: ENGR.Berlin USC-ChE Faculty. 4th ed. University of San Carlos..IV. Cebu City  Dissolved Oxygen Test. Virgini. 17th ed. W.edu/~bradwood/eagles/dot. Analysis in Clinical Biochemistry.  Herwitz. 2004... Chemical Engineering Department. Laboratory Analytical Procedure. USA  Trinder. and Gagnon Y. Determination of Ethanol Concentration in Biomass to Ethanol Fermentation Supernatants by Gas Chromatography  El-Mansi and Bryce (1999).Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. LAP-011. University of San Carlos. City  MERCK Microbiology Manual. ChE 124 L 6 . Von Stockar U 2003 Quantitative comparison of transient growth of Saccharomyces cerevisiae. D. 81: 837-847. p. NIOSH Manual of Analytical Methods (NMAM). 2008  Herwig C. I and II. PDF. Biotechnol. 1994. D. The Effect of Nitrogen Source and Concentration on Biomass Yield by Baker’s Yeast (Saccharomyces Cerevisiae) in a Batch Shake Flask. Viewed April 18. 2008 Checked and Approved by: ENGR. USA  Foley. 1994. Vol. 2002. 2002. v. Bioprocess Engineering: Basic Concepts..htm. 2000. Prentice – Hall Inc.. Bioeng. and Kluyveromyces lactis. Springfield.24-26. MAY V. P. Saccharomyces kluyveri. al. Official Methods of Analysis of the AOAC International. http://www. Cebu. AOAC International. and Michael S. Chapter 5: Physical Processes in Bioreactors. 1973. ACETIC ACID: Method 1603.indiana.  Templeton. VA  Kargi F. 1969.6.
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