The main objective of this review has been to provide some informative background on citric acid production by solid substrate fermentation. A brief introduction to the history of citric acid production, including a description of nutritional and environmental factors and the biochemistry is provided in order to understand the process.
The comparison among liquid surface, submerged and solid substrate fermentation technologies for citric acid production indicates potential advantages and our limited understanding of the solid substrate fermentation. Referring to current investigations and applications of solid substrate fermentation, our understanding of heat and mass transfer, and subsequent reactor designs, is also limited.
The ultimate aim of this thesis is to investigate solid substrate fermentation for citric acid production, including nutritional requirements and environmental factors, and to investigate the kinetic characteristics, so that a reactor may be selected. Based on the selected reactor, and optimized conditions, attempts will be made to understand the kinetic and mass transfer characteristics.
Chapter 3
Materials and Methods
3.1 Materials
3.1.1 Microbiological media
33
Two media were used for the sporulation of Aspergillus niger, sucrose-beef extract
medium for strain MH I5- 1 5, as shown in Table 3. 1 ; and malt extract agar medium, as supplied by Unipath Ltd. (Basingstoke, England), for strain Yang No.2.
Table 3 . 1 Sucrose-beef extract medium (Sanchez-MarroquIn et ai, 1970; Dawson, 1986, Dawson et ai, 1987, 1988) Component Sucrose Beef extract Sodium chloride Agar other 3.1.2 Chemicals Concentration (gil) 2.5 10.0 5.0 1 5.0 distilled water
Chemicals used for fermentation and analytical work are listed in Table 3.2.
3.1.3 Raw substrates
Potato, kumara and taro, which were purchased fresh at a local supermarket, were used in the experiments. The fresh substrates were packed in plastic bags to prevent serious moisture loss, and stored at 4°C for long term use (less than 2 months)
3.1.4 Gases
The following gases were used in the experiments, and were supplied and certified by New Zealand Gases Ltd., Palmerston North, New Zealand:
Name Formula
Ammonium sulphate (NH4)2S04
Amylase Termamyl- 120L
Amylose
Citric acid C6H07
Copper sulphate CuS04·5H2O
Ferrous sulphate FeS04·7H2O
Gluco-amylase San-240L
Hydrochloric acid HCI
Manganese sulphate MnS04·5H2O
Nitric acid HN03
Orthophosphoric acid H3P04
Potassium di-hydrogen
phosphate KH2P04
Sodium Chloride NaCI
Sodium Hydroxide NaOH
Soda Lime 'Corbosorb'
Soluble starch
Zinc sulphate ZnS04.3H2O
Table 3.2 Chemicals and suppliers
Grade Manufacturer
A.R. BDH Chemicals Ltd, England
NOVO, Nordisk,
Bagsaerd, Denmark
A.R. BDH Chemicals Ltd, England
A.R. Sigma Chemicals Co.
St. Louis, U.S.A.
A.R. BDH Chemicals Ltd, England A.R. BDH Chemicals Ltd, England
NOVO, Nordisk
Bagsaerd, Denmark
A.R. Ajax Chemicals Co., Australia A.R. BDH Chemicals Ltd, England
A.R. Ajax Chemicals Co., Australia A.R. Ajax Chemicals Co., Australia
A.R. BDH Chemicals Ltd, England
A.R. BDH Chemicals Ltd, England
A.R. Ajax Chemicals Co., Australia Ajax Chemicals Co., Australia
Ajax Chemicals Co., Australia
A.R. BDH Chemicals Ltd, England
Supplier
CHEMICOLOUR INDUSTRIES NZ. LTD.
CHEMICOLOUR INDUSTRIES NZ. LTD.
Materials and Methods 35
3.1.5 Organism
Two strains of Aspergillus niger were used in the experiments. Strain MH 15-15, was
isolated in this laboratory by Hossain et al (1983) as a mutant strain of A. niger
IMI4 1 874, and was used by Dawson ( 1 986) and Dawson et al ( 1 987, 1988) for citric
acid production in submerged fermentation. It was cultivated on slopes of sucrose-beef extract agar (Table 3. 1 ) at 30°C for 8-9 days. Another strain, Yang No.2, supplied by Professor H. Imai, Waseda University, Tokyo, Japan, has been used in liquid surface
fermentation for citric acid production (Sakurai and Imai, 199 1 ; Sakurai et aI, 1 99 1 ,
1992). It was cultivated on slopes of malt extract agar at 30°C for 5-6 days to obtain spores.
For long term preservation of the organisms, the techniques described by Hossain ( 1 983) and Dawson ( 1 986), were used:
a) The spores of the organism were harvested from agar slopes with sterilized distilled water. Then the spore suspension was shaken for 1 hour to break the spore chains and clumps. The concentration of the suspension was adjusted to
1 -2x 108 spores/rnl using sterilized distilled water.
b) Two rnl spore suspension was dispensed aseptically into 3 rnl of corresponding medium (no agar), containing 30% (v/v) glycerol, contained in a
1 0 rnl capacity screw cap bottle. The inoculated bottles were then stored at -20°C.
When spores were required for sub-culturing, a loopful from a thawed bottle was transferred to a slope of the corresponding medium, and incubated at 30°C for a different period dependent on different strains of the organism. A further subculture from this initial slope, onto a fresh slope, was carried out to obtain improved sporulation. This second slope was used for up to 2 months as the stock culture for inoculum preparation.
Materials and Methods 3.2 Media Sterilization
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Microbiological media were sterilized by autoclaving at 1 2 1 °C for 15 minutes. Fennentation media, unless otherwise stated, were sterilized at 12 1°C for 20 minutes.
3.3 Cleaning of Glassware
All glassware, including glass reactors and flasks, was washed in hot Pyroneg(R) solution, rinsed in tap water, then in distilled water, and hot air dried. Glassware used in experiments involving metal ions, was treated with 10% (v/v) nitric acid after the detergent wash, then rinsed thoroughly with deionized water, and hot air dried.