IBG 202

INDUSTRIAL MICROBIOLOGY

LAB 5

PRODUCTION OF CITRIC ACID

Prepared by : MUHAMMED AZHAR BIN AZIZAN LOW PIK KUAN TOH SEE MIN Group : 4

Prepared for : PN. WAN NADIAH WAN ABDULLAH

Objective To prepare fermentation culture and medium for citric acid production To determine the pH, total acid, sugar and citric acid content after fermentation To compare the effects of different pH of fermentation medium towards the production of citric acid

Introduction Citric acid is a commodity chemical produced and consumed throughout the world. It is used mainly in the food and beverage industry, primarily as an acidulant. Approximately 75 % commercial use of citric acid is for food and 12 % for pharmaceutical industries. Although it is one of the oldest industrial fermentations, its world production is still in rapid increasing. Global production of citric acid in 2007 was over 1.6 million tonnes. Commercial production of citric acid is generally by submerged fermentation of sucrose or molasses using the filamentous fungus A. niger or synthetically from acetone or glycerol. In the recent times, solid state fermentation (SSF) as an alternative to submerged fermentation in the production of microbial metabolites. Solid-state fermentations refer to the cultivation of microorganisms in a low-water-activity environment on a non-soluble materials acting as both nutrient source and physical support. Many microorganisms have been evaluated for the production of citric acid including bacteria such as Bacillus licheniformis, B. subtilis, fungi such as A. niger ,Penicillium restrictum.Yeast such as Saccharomyces cerevisiae. However, A. niger is a filamentous fungus remained the organism of choice for citric acid production due to ease of handling, its ability to ferment a variety of cheap raw materials, and high yields. A cost reduction in citric acid production can be achieved by using cheap agricultural wastes such as apple and grape pomace, orange peel, kiwi fruit peel, cotton waste, okara soy-residue and cane molasses. Materials and Methods 2. 2.1 : Inoculum preparation: a. One loopful of conidia from Aspergillus niger is grown on PDA ( Potato Dextrose Agar ) agar slant. b. Incubation is carried out at 30c for 7 days. c. After incubation, add 7 ml sterile distilled water and shake using rotamixer. d. Collect all conidial suspension for the subsequent fermentation. 2.2 : Preparation of fermentation medium a. Composition of the fermentation medium for the production of citric acid is as

follows : i. ii. iii. iv. v. vi. vii. Sucrose (14%) (NH4)2 CO3 (0.25%) KH2PO4 (0.25%) MgSO4 7H2O (0.025%) CuSO4 5H2O (0.06 mg/L) ZnCl2 (0.25 mg/L) FeCl3 6 H2O (1.3 mg/L)

b. Dissolve all the above components (i vii) in 1000ml, distribute into 4 parts and adjust the pH to 3.5, 4.0, 4.5, and 5.0 respectively using 1.0 M HCl or 1.0 M NaOH. c. Then transfer 100 ml medium of each Ph prepared into respective conical flasks. Prepare duplicates. d. Sterilize the solution using 121c for 15 minutes. 2.3 : Experimental methods: a. Fermentation Each group will only carry out the fermentation for one pH only. Class results are required to discuss the effects of ph on the production of citric acid. i. Aseptically transfer 5ml of the conidial suspension of A. niger into 100 ml medium (sterilized). For the control, use 5 ml sterile distilled water instead of the conidial suspension. Label carefully. Next, aseptically transfer out (using a pipette with a wide tip) 10 ml sample representative of the contents of the flask. Analyze for the cell mass determination. Repeat until all the flasks have been analyzed. ii. iii. Incubate the flasks in an incubator shaker for 7 days at 30c and 200 rpm. After incubation, pipette out (using a pipette with its tip widened) 10 ml sample that is representative of the flask. Analyze for the cell mass. Then, filter the remaining fermentation medium using a Whatman No. 1 filter paper to obtain the supernatant for subsequent analysis. iv. Analysis is carried out to determine the ph, total acid, sugar content and citric acid content after fermentation.

b . Analytical methods i. Cell mass determination

Take 10 ml of the fermentation medium that has been inoculated at the beginning and at the end of the fermentation. (mix well before sampling to ensure representative sample) Filter using Whatman No. 1 filter paper that has been dried and preweighed (using analytical balance) Dry the residue on the filter paper at 55C until constant weight. ii. pH determination Determine the pH using a pH meter. iii. Determination of total acid Determine the total acid using titration method, ie 10 ml sample (duplicates) is titrated with 0.2 M NaOH with phenolphthalein as the indicator. iv. Determination of sugar (sucrose) content a. Sample treatment Mix 10 ml sample with 5ml 1 M HCl. Boil for 50 min. Add distilled water to make up total volume to 15 ml. Adjust pH to 7.0 (using 1 M NaOH). Determine the final volume (v). Take 1 ml from the final volume and dilute to 10 ml (use a volumetric flask). Determine the sugar content (x g/L) b. Sugar content is determined using the DNS method (Dinitrosalicyclic acid) as follows: Add 1 ml sample into a test-tube, and mix in 1 ml DNS reagent. Add 3 ml distilled water and boil for exactly 5 min. Immediately cool the sample. Read the absorbance at 570nm using the spectrophotometer. It is suggested that a standard is scanned first, and the absorbance is read at the wavelength of maximum absorbance. Also prepare a set of standards with concentrations ranging from 0 2.5 mg/ml, and determine their sugar content as above. Calculation for sucrose content

Glucose content = X g/L x 10 x 15/10 x V/15. Residual sucrose content = 342/180 x glucose content = Y g/L Sugar (sucrose) utilized = (140 Y) g/L v. a. b. c. d. e. f. g. Determination of Citric acid content Add about 10 ml 1% Ca(OH)2 solution to 15 ml sample and heat at 80 90 C for 10 min. Carry out in duplicates. After heating, filter using Whatman No. 1 filter paper. Add about 15 ml 1% Ca(OH)2 solution to the filtrate. Heat again at 95 C for 1 hr with continuous mixing. Filter again using Whatman No. 1 filter paper that has been pre-dried and weighed on an analytical balance. Dry the residues on the filter paper at 55C until constant weight, before placing the filter paper in a vacuum dessicator. Note the weight when it is constant. Citric acid content = weight(g) x 1000 = ---------g/l 15 ml Percentage yield = citric acid content x 100% sugar utilised Results and Discussion Results i. Cell mass determination Sample Initial weight (g) (Dried Whatman No.1 filter paper) 0.8992 0.9148 Final weight (g) (Dried filter paper with cell mass) 1.2541 1.2697 Dried cell mass (g)

First Second ii. pH determination

0.3549 0.3549

pH of the fermented medium = 2.80 iii. Determination of total acid Trials Intial reading of NaOH in burette (ml) 30.1 33.8 Final reading of NaOH in burette (ml) 33.8 37.6 Total acid used (ml) 3.7 3.8

First Second

iv.

Determination of sugar (sucrose) content

Absorbance reading
0.6 0.5 Absorbance at 570nm 0.4 0.3 Absorbance reading 0.2 0.1 0 0 -0.1 0.5 1 1.5 2 2.5 3 Concentration of glucose dilution (mg/mL) Linear (Absorbance reading) y = 0.1929x - 0.0238 R = 0.9752

Graph of absorbance versus concentration of glucose dilution a. Final volume, V= 20 ml b. Absorbance reading (10 x dilution) sample 1= 0.104 sample 2=0.095 sample 3= 0.126 Calculation of sucrose content Glucose content = X g/L x 10 x 15/10 x V/15 X = (0.126+0.095+0.104)/3 = 0.1083 V ( final volume ) = 20ml Glucose content = 0.1083g/L x 10 x 15/10 x 20/15 = 2.167g/L Residual sucrose content = 342/180 x glucose solution = Y g/L Residual sucrose content = 342/180 x 2.167g/L = 4.117g/L Sugar ( sucrose ) utilized = ( 140-Y ) g/L Sugar ( sucrose ) utilized = ( 140-4.117)g/L = 135.883g/L

v.

Determination of Citric acid content Intial weight (g) 0.9192 0.9124 Final weight (g) 1.0387 1.0211 Weight of sample (g) 0.1195 0.1087

Sample First Second

mean weight of sample = ( 0.1195 + 0.1087 )/ 2 = 0.1141g Citric acid content = weight of selected mean sample(g) x 1000 15 ml Citric acid content = (0.1141g/15ml) x 1000 = 7.6067g/L Percentage yield = citric acid content x 100 sugar utilised Percentage yield = (7.6067/135.883) x 100 = 5.59%

Conclusions References