Experiment 3: Microbial Growth

Mini Project 1Production of Citric acid

Objective To produce citric acid from Aspergillus niger

ApparatusMini Project 1CBD 20003 Introduction to Bioprocess Technology

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Incubator shakerSpectrophotometer500 ml Shake flaskUniversal BottleMicrocentifuge tubesHeating mantleMicro centrifuges CuvettCotton and gauzeWhatman No 1 Filter paperTest TubeWater Bath

Chemicals

An agar slant containing Aspergillus nigerPotato Dextrose agar (PDA)Sucrose(NH4)2CO3KH2PO4MgSO4.7H2OCuSO4.5H2OZnCl2FeCl36H2ODistilled waterNaOHHCLDNS Reagent

Preparation of inoculum1. Add 7 ml of sterile distilled water tp the Aspergillus niger conidia on Potato Dextrose agar slant.2. Shake using a vortex mixer.3. Collect the conidial suspension for the fermentation process.

Fermentation mediumMediumAmount (g/L)

Sucrose140

(NH4)2CO32.5

KH2PO42.5

MgSO4.7H2O2.5

CuSO4.5H2O0.06

ZnCl20.25

FeCl36H2O1.3

Procedures1. Prepare the medium accordingly. Adjust the pH to 5.0 respectively using 1.0 M HCl or 1.0 M NaOH. Bring the volume to 300 ml.2. Sterilize the medium at 121oC for 15 minutes.3. Transfer aseptically 15 ml of A. niger conidial suspension into the sterile medium. Sample out 35 ml for analysis.4. Incubate in the incubator shaker for 7 days, 30oC, at 200 rpm. Take 35 ml of samples every 24 hours.5. Perform final analysis on samples and fermented broth after the incubation period is done.

Analytical methods

1) Determination of cell mass (biomass concentration)1. Weigh the filter paper.2. Take 5 ml of unfiltered obtained earlier and filter separately using Whatman No 1 filter paper that has been dried and accurately weighed.3. Dry the cells on the filter paper at 55oC until constant weight.

2) pH determination Determine the pH using the pH meter3) Sugar (Sucrose) content determination

DNS Method1. Prepare the DNS reagent by dissolving 5 g of DNS and 8g of NaOH in 100ml of distilled water and top the volume to 300 ml. Add 150 g of potassium sodium tartarate slowly until all the DNS dissolve and top up the volume to 500ml.2. The sample was centrifuged at 3000 rpm for 10 minute to sediment the cells and other solutes.3. The supernatant was taken out and placed into a new universal bottle.4. Add 1.5 ml of sample (sugar) in a test tube and add 1.5 ml of the DNS reagent.5. Boil the reaction mixture for 5 min and allow to cool down. Add 10 ml of distilled water.6. Read the absorbance at 540nm. (Use the sample containing DNS reagent with distilled water as the blank).7. Prepare a standard curve for glucose using the concentration of pure glucose in the range 0-400mg/L by repeating steps 2-4.8. Use the standard curve to determine of unknown glucose content in the fermentation broth.

Calculation for sucrose content.Leftover sucrose content = 342/180 x glucose content x = Y g/lSugar (sucrose) utilized = (140-Y) g/l

4) Citric acid content1. Add approximately 10 ml 1% Ca (OH)2 to 15 ml sample, and heat to 80-90oC for 10 min. Filter using Whatman No1 filter paper.2. Add approximately 15 ml of 1% Ca (OH)2 to filtrate. Heat again at 95oC for 1 hr with continuous agitation. Filter using Whatman No1 filter paper that has been reweighed. Dry the filter paper and residue at 55oC until constant weight.3. Citrate content = weight (g) /15 ml x 1000 ml = weight in g/l4. % yield = citric acid content /sugar utilized x 100

Representation of report: (It must be followed consequently)1. Summary (Abstract)2. Methodology.3. Result consists of data and graph.a. Calculate and plot the cell biomass concentration, glucose concentration and citric acid concentration as a function of time.b. Plot graph to show trend of pH and temperature over time of fermentation.4. Discussion5. Conclusion6. Appendix (Consist of details calculation involve in the experiment)7. References