UNIVERSITY OF SAN CARLOS Department of Chemical Engineering Technological Center, Talamban, Cebu City, Philippines 6000 Telefax: (032)

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 26, 2008 Date of Completion: May 31, 2008

Experiment # 6: Describing Growth Patterns of Bakers Yeast in a Batch Culture (Part 2: Using Lab-Scale Fermentor) I. Objectives: 1.) Identify the growth patterns of Bakers 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. Fleischmann Active Dry Yeast will be used in the experiment since this exhibited high activity in the study of Castro, et al. (2003). This will be acclimatized and incubated first before cultivating. The parameters will be monitored from time to time or in every sampling to determine its development. The culture will be done in duplicate. 2.2 Experimental Procedure [1-5] Mix nutrients (trace elements and vitamins) and Struktol J650 with distilled water 700 ml mark as shown in Table 1 and Table 2 in a 1-L Erlenmeyer flask. Transfer about 100 ml of the nutrient broth to a 200-ml Erlenmeyer flask. 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 wick. One hundred-milliliter distilled water in 125-ml Erlenmeyer flask will also be prepared for rinsing trace amounts of substances or yeast on the funnel and container during transfer. Wrap the flasks with tin foil including the glass funnel, sample bottles and the lab-scale fermentor. Then, sterilize them in the LABSTAR 3x4x5 Autoclave at 15 psig for 20 minutes. After pressure and sterilization equalization, cool them rapidly to the temperature used for pouring. The flasks will be cooled under cool, running water. Transfer the sterilized nutrient broth in the 1-L Erlenmeyer flask to the fermentor using the sterilized funnel, aseptically. Rinse the flask with about 30 ml of sterilized distilled water using the same funnel. Pour 10 grams dry weight of Active dry Bakers yeast (Saccharomyces Cerevisiae) in the 100-ml nutrient broth sterilized previously and mix well. Then, add it to the sterilized 1-L Erlenmeyer flask containing about 600-ml nutrient solution using a glass a funnel aseptically. Again, rinse the flask with about 30 ml of sterilized distilled water using the same funnel. Stir the culture broth at 800 rpm, 6.0 pH, temperature

of 30 oC, and dissolved oxygen of 50%. Acclimatize for 30 minutes. After that, add sterilized glucose solution into the fermentor using the funnel aseptically. Rinse the flask with about 40 ml of the sterilized distilled water using the same funnel. Incubate for 30 minutes. Take 10-ml sample. Cultivate the yeast after incubating for 24 hours. Take 10-ml sample for every 30 minutes for the first hour, every hour for the next to the fourth hour, and every four hours onwards. Monitor and record parameters every sampling which is displayed on the LCD of the controller and the BIOxpert program on the computer. 2.3 Culture Media
Table 1. Composition of the Culture Media [1] Component Initial Concentration Resulting Concentration after mixing

Glucose (NH4)2SO4 KH2PO4 MgSO4.7H2O Trace Elements Vitamins Struktol J650

20 g/200 ml 5 g/800 ml 3 g/800 ml 0.5 g/800 ml 0.0804 g/800 ml 1.1715 g/800 ml 0.1 ml/800 ml

= 100.0000 g/L = 6.2500 g/L = 3.7500 g/L = 0.6300 g/L = 0.1000 g/L = 1.4600 g/L = 0.0125 %

20.0000 g/L 5.0000 g/L 3.0000 g/L 0.5000 g/L 0.0804 g/L 0.1715 g/L 0.0100 % (v/v)
Amount in grams

Table 2. Trace Elements and Vitamins Formulation [ 6 ] Component Concentration (g/L) of 2.67 ml solution

Trace Elements: EDTA ZnSO4.7H2O MnCl2.4H2O CuSO4.5H2O CoCl2.6H2O H3BO3 Na2MoO4.2H2O FeSO4.7H2O CaCl2.2H2O KI (Total) Vitamins: Biotin Ca-pentothenate Nicotinic Acid Myo-Inositol Pyridoxine.HCl Para-Amino Benzoic Acid Thiamine.HCl (Total)

15.00 4.50 1.00 0.30 0.30 1.00 0.40 3.00 4.50 0.10 0.75 15.00 15.00 375.00 15.00 3.00 15.00

0.0401 0.0120 0.0027 0.0008 0.0008 0.0027 0.0011 0.0080 0.0120 0.0003 0.0804 0.0020 0.0401 0.0401 1.0013 0.0401 0.0020 0.0401 1.1715

2.4 Analytical Methods 2.4.1 Sample Preparation [ 7 ] Sample the fermentation so as to collect a representative slurry sample. Collect 10ml sample and put it in the sterilized sample bottle. Samples are to be centrifuged in Programmable IEC Centra Centrifuge (E0803) by spinning at 6000 RPM and 4 oC for five minutes. Filter the solid portion and liquid portion of the sample through a 0.2 m Whatman filter (or its equivalent). In filtering the solid portion of the sample, rinse it with distilled water to eliminate soluble compounds mixed to it. Put the supernatant (filtrate) in the freezer. This will be used for ethanol and acetate analysis using Shimadzu GC-8A. Put the biomass (retentate) in a dry crucible for biomass analysis. 2.4.2 Bakers Yeast and Biomass Moisture Analysis [ 1, 7 ] Weigh three 10 grams of dry yeast ( Saccharomyces cerevisiae) in the analytical balance and put them in the dry crucibles. Heat them for 20 minutes in the All American Microwave Oven Model AMW-210 LM at a power setting II. Let it cool in the desiccator to initial temperature. After which, weigh them again in the analytical balance. Record the weights and compute the moisture content of each and get the average. Use the moisture content for yeast that you get in calculating for the actual amount of Bakers yeast that is to be added in the Erlenmeyer flask. For biomass analysis, 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. Let it cool in the desiccator to initial temperature afterwards. Then, weigh them again in the analytical balance. Record the weights and compute the moisture content. Please refer to Section 3.1 and 3.2 for the calculation. 2.4.3 Glucose (Trinder) Assay [ 8 ] 2.4.3.1 Reagent Preparation Prepare 50 ml working glucose enzyme reagent in a vial that contains 20,000 U/L glucose oxidase, 5000 U/L peroxidase, 0.2 mM 4-aminophenazone, 10 mM p-hydroxybenzoate. Use deionized distilled water as solvent. Replace the rubber stopper and allow 5 minutes for reconstitution. Swirl gently until the contents of the vial are completely dissolved. 2.4.3.2 TEST PROCEDURE CONDITIONS The test procedure conditions is as follows: wavelength is 505 nm, temperature is 37 oC, pathlength is 1.0 cm, mode is endpoint, reaction time is 3 min, sample volume is 20 l, reagent volume 2.0 ml, total volume 2.02 ml, and sample to reagent ratio is 1/100. TECHCOMP VIS 7200 Spectrophotometer will be used in measuring the absorbance of the glucose standards and samples. The procedure is linear to 0.4 g/L glucose. Thus, dilution will be made to samples which have higher glucose concentration than 0.4 g/L. 2.4.3.3 PROCEDURE Into separate test tubes, pipette 20 l of distilled water (blank), glucose standards (0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.4 g/L), or sample (serum, may be diluted if necessary) to be assayed. Add 2.0 ml of working reagent to each test tube and mix. Following incubation for 3 minutes at 37C in GFL water bath (#064700073) determine the absorbance of each standard sample at 505 nm using the distilled water sample as the reagent blank. 3

Plot the absorbance of each glucose standard against its glucose concentration. Use the equation of the line of the calibration curve in determining the actual glucose concentration of each sample. 2.4.4 Ethanol and Acetate Analysis Using GC (Shimadzu GC-8A) [9-11] 2.4.4.1 Calibration and Test Procedure Prepare 0.1%v/v concentration internal standard spiking solution, using reagent grade isopropanol ((95 %). The internal standard spiking solution must be added in the same proportion to every standard or sample analyzed by this method. This procedure specifies 900 L of a 0.1%v/v internal standard spiking solution should be added to 100 L part sample, standard, or blank. Therefore, the internal standard concentration is 0.9%v/v universally throughout this procedure. Prepare six ethanol analytical standards, using 99.5%v/v ethanol, ranging from 0.05 to 2.5 g/L (e.g. 0.05, 0.1, 0.5, 1.0, 1.5 and 2.5 g/L) and a blank all containing 0.9%v/v isopropanol. Refrigerate analytical and calibration verification standards in sealed sampler vials for storage. Prepare another standard for acetate analysis using the following method: Calibrate with six working standards over the range of 0.05 to 2.5 g/L (e.g. 0.05, 0.1, 0.5, 1.0, 1.5 and 2.5 g/L) glacial acetic acid. Add 100 L of sample, standard, or blank to 900 L of 5.6% eluent (88% formic acid). Therefore, the internal standard concentration is 5 %v/v universally throughout this procedure. Refrigerate analytical and calibration verification standards in sealed sample vials for storage Inject 1 L of blank, standard, or sample into the GC. Using the blank and standards observe the peaks and make a calibration curve by plotting the peaks against the concentration of the standards. Use the equation of the line of the calibration curve in calculating the ethanol and acetate concentration in each sample. . 2.4.4.2 Operating Conditions Operating conditions for ethanol and acetate analysis are as follows: Oven temperature is 155oC (isothermal), inlet and detector (FID) temperature is 250oC, run time is 5.5 minutes, retention time is 2.3 minutes for ethanol, 4.1 minutes for isopropanol, 55 minutes for acetic acid, and 60 minutes for formic acid, carrier (He) gas flow rate is 30 ml/min, and injection volume is 1 L. III. Calculations [12] 3.1 Bakers Yeast Moisture Content The following formula is directly applied in calculating for the Bakers yeast moisture content:
%Moisture content of Bakers Yeast = Initial Weight (g) Final Weight (g) x 100 Initial Weight (g)

3.2 Actual Amount of Bakers Yeast to Add in the Shake Flask The following formula is directly applied in calculating for the actual amount of Bakers yeast that is to be added in the Erlenmeyer flask:
Weight Bakers Yeast to add (in grams) = dry weight (g) Bakers yeast Required [(100-%moisture content Bakers yeast)/100]

3.3 Glucose Consumption The actual glucose or substrate consumption, S in grams can be determined using the following formula:
S = [(CSo . Vo CS . 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 , and V is the final volume in liter. 3.4 Amount Biomass Produced The actual amount of biomass produced determines the growth of yeast after some time or in every sampling. The following formula is used in determining the amount of biomass produced, X in grams: X = [(Cxo . Vo Cx . 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 , and V is the final volume in liter. 3.5 Yield Determination Biomass yield is the amount of dry weight yeast produced in grams per grams glucose consumed. After the experimental run, biomass yield is calculated using the following formula: Ybiomass/glucose = X/S

IV. References:
[1] Fabian et. al, 2004. The Effect of Nitrogen Source and Concentration on Biomass Yield by Bakers Yeast (Saccharomyces Cerevisiae) in a Batch Shake Flask, University of San Carlos, Cebu, City [2] Castro et. al, Screening Bakers Yeast for its Capacity to Catalyze the Reduction of 4Oxoisophorone, University of San Carlos, Cebu City, 2003 [3] MERCK Microbiology Manual, 2000.Berlin [4]USC-ChE Faculty, Chapter 5: Physical Processes in Bioreactors, PDF, Chemical Engineering Department, University of San Carlos, Cebu City

[5] Dissolved Oxygen Test, http://www.indiana.edu/~bradwood/eagles/dot.htm. Viewed April 18, 2008

[6] Herwig C, Von Stockar U 2003 Quantitative comparison of transient growth of Saccharomyces cerevisiae, Saccharomyces kluyveri, and Kluyveromyces lactis. Biotechnol. Bioeng. 81: 837-847. [7] Herwitz, W. 2002. Official Methods of Analysis of the AOAC International, 17th ed., Vol. I and II, AOAC International, Virgini, USA [8] Trinder, P. 1973.Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor., Analysis in Clinical Biochemistry, v.6, p.24-26, 1969. [9] Templeton, D. 1994. Chemical Analysis and Testing Task, Laboratory Analytical Procedure, LAP-011, Determination of Ethanol Concentration in Biomass to Ethanol Fermentation Supernatants by Gas Chromatography [10] El-Mansi and Bryce (1999), Fermentation Microbiology and Biotechnology, USA [11] Foley, D. and Gagnon Y. 1994. NIOSH Manual of Analytical Methods (NMAM), ACETIC ACID: Method 1603, 4th ed., Springfield, VA [12] Kargi F. and Michael S. 2002. Bioprocess Engineering: Basic Concepts, 2 nd ed., Prentice Hall Inc., NJ

Date Submitted: May 23, 2008

Checked and Approved by:

ENGR. MAY V. TAMPUS Supervising Faculty Noted:

ENGR. RAMELITO AGAPAY Adviser, ChE 124 L

Date: __________