Different Bacillus species have been investigated for the production of γ-PGA. Based on the required nutrients, γ-PGA producing organisms have been classified into two groups: glutamic acid dependent bacteria and glutamic acid independent bacteria (Cao et al., 2011).
γ-PGA producing organisms that are glutamic acid dependent include: B. anthracis, B.
licheniformis ATCC 9945A, B. subtilis IFO 3335 and B. subtilis F-2-01. γ-PGA producing
organisms that are glutamic acid independent include: B. subtilis C1, B. subtilis 5E, B.
subtilis TAM-4, B. amyloliquefaciens LL3 and B. licheniformis A35 (Shih et al., 2002; Cao et al., 2011; Cao et al., 2013).
B. subtilis 5E is able to produce γ-PGA using L- proline as the sole carbon source and
nitrogen source in medium (Shih and Van, 2001). B. licheniformis A35 is able to produce γ-
PGA from glucose and ammonium chloride under denitrifying conditions and B. subtilis
TAM-4 is able to produce γ-PGA in a culture medium that contains salt and sugar as nitrogen
and carbon sources (Shih and Van, 2001).
Apart from glutamic acid, other factors such as carbon and nitrogen sources, ionic strength, agitation, aeration and pH of the medium affect the quality and production yield of γ-PGA (Bajaj and Singhal, 2011).
γ-PGA production has been studied extensively in glutamic acid dependent organisms, table 2.2 summarizes the nutrient requirements, cultivation conditions, yield and molecular weight of γ-PGA produced from the different γ-PGA producing organisms.
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Table 2.2: Report of the different strains of γ-PGA producing bacteria, showing the nutrients involved, conditions of cultivation, productivity and molecular weight of the produced strains of γ-PGA (Adapted from Shih and Van, 2001).
Strain Nutrients Cultivation Conditions
Yield (g/l) Molecular Weight
B. licheniformis
ATCC 9945
Glutamic acid, glycerol, citric acid, NH4Cl
30°C, 4 days 17-23 1.4x105-9.8x105
B. subtilis IFO3335 Glutamic acid, citric acid
37°C, 2 days 10-20 1.0x105-2.0x106
B. subtilis TAM-4 Fructose, NH4Cl 30°C, 4 days 20 6.0x105-1.6x106
B. licheniformis A35 Glucose, NH4Cl 30°C, 3-5 days 8-12 3.0~5.0x105 B. licheniformis A35 Glutamic acid, glucose 30°C, 2-3 days 50 1.20x106
B. subtilis natto Maltose, soy sauce, sodium glutamate
40°C, 3-4 days 35 Not determined
2.5.1 γ-PGA Production by Glutamic Acid Dependent Organisms
2.5.1.1 Production of γ-PGA B. licheniformis ATCC 9945A
B. licheniformis ATCC 9945A (NCIM 2324) is a common strain that has been used for the
production of γ-PGA. Factors such as glutamic acid, citric acid, inorganic salts, glycerol and size of inoculum have been reported to affect the production of γ-PGA by B. licheniformis ATCC9945A in shake flasks and static cultures (Ogunleye et al., 2015; Shih and Van, 2001).
γ-PGA yield in excess of 15g/l was reported when the organism was grown in shake flasks in medium C (medium C contains: 20g/l L-glutamic acid, 12g/l citric acid, 80g/l glycerol, 7g/l
NH4Cl, 0.5g/l MgSO4.7H2O, 0.04g/l FeCl3.6H2O and 0.5g/l K2HPO4), tap water and pH adjusted to 7.4 with NaOH. Investigations revealed that maximum polymer yield and
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maximum growth were obtained when a specific lot of FeCl3 and tap water were used during medium preparation, further studies then revealed that a significant amount of Ca2+ was present in the tap water and that the FeCl3 was contaminated with Mn2+ (Shih and Van, 2001).
Further investigations were carried out on the role and optimal concentrations of Mn+2 and Ca2+ in medium C for γ-PGA synthesis by B. licheniformis ATCC 9945A. Results showed that only 1.5 x10-7M of Mn2+ was required for maximum cell growth, increasing the concentration had an effect on cell viability and thus increased the γ-PGA yield. Increasing the concentration of Mn2+ to 6.15 x10-4M resulted in maximum yields of γ-PGA. Addition of 1.02 x10-3M of Ca2+ in the presence of 1.5 x10-7M of Mn+2M resulted in maximum yields of γ-PGA. Based on these results, medium C was modified into medium E (see table 4.3 for the composition of medium E) and this has become the choice medium for γ-PGA production (Shih and Van, 2001).
In another report, production of γ-PGA was optimised with B. licheniformis NCIM 2324 via the ‘one factor at a time’ method. This method was used to examine the effect of carbon sources, nitrogen sources and pH on γ-PGA production. The response surface method was then used to devise the optimum nutrient concentrations. γ-PGA yield of 26.12g/l with a molecular weight of 2.1x105 Da was obtained with the devised medium compared with a yield of 5.27 with the basal medium (Bajaj et al., 2009).
2.5.2 γ-PGA Production by Glutamic Acid Independent Organisms
Bacteria that do not require glutamic acid to produce γ-PGA are of importance because of the reduced cost of production and simplified process in industrial fermentor production systems.
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2.5.2.1 Production of γ-PGA by Bacillus subtilis TAM-4
B. subtilis TAM-4 that was isolated from the soil was reported by Ito and co-workers (1996)
to produce a large amount of γ-PGA if grown aerobically in a culture medium that contains sugar and ammonium salt as sugar and nitrogen sources respectively. In contrast to other γ- PGA producing strains, B. subtilis TAM-4 does not require biotin for its growth and does not
have strain degeneration challenges. This strain has the long ability to produce γ-PGA; it is
maintained by semi-annual sub-culturing on trypticase soy broth (TSB) slants at room
temperature. B. subtilis TAM-4 can use several sugars and organic/inorganic nitrogen sources for production of γ-PGA. Ammonium chloride and fructose are the most favourable nitrogen and carbon sources respectively (Shih and Van, 2001).
A maximum γ-PGA yield of 22.1g/l was obtained when B. subtilis TAM-4 was grown in medium M (1.8% ammonium chloride, 7.5% glucose, 0.15% K2HPO4, 0.035% MgSO4.7H2O, 0.005% MnSO4 · 5H2O, and 3.0% CaCO3 (pH 7.2)), at 30°C for 96 hours and at 150 rpm with shaking. This high yield is distinct especially with the absence of glutamic acid in the medium. Upon testing of the produced γ-PGA for polysaccharide by-products, less than 1% (w/w) was detected which suggested B. subtilis TAM-4 to scarcely produce
polysaccharide by-products. B. subtilis IFO3335 in contrast produces γ-PGA as well as a
polysaccharide by-product during growth in a glucose containing medium (Ito et al., 1996).
In addition, it was discovered that the ratio of D- and L- isomer of glutamic acid that makes up γ-PGA in γ-PGA from B. subtilis TAM-4 remains constant (78:22, D-isomer: L-isomer) throughout the cultivation period, this suggested that γ-PGA from B. subtilis TAM-4 was elongated without a change in the diastereoisomer ratio in the molecule (Ito et al., 1996).
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