Patent GB-653808-A (GB)
Title: Improvements in or relating to the production of citric acid by fermentation
IPC: C12P7/48
Filed: 1947-12-08
Abstract: Citric acid is made by submerged fermentation of a solution of a carbohydrate containing material by a citric acid producing strain of A. niger in which the Fe conc. of said solution is reduced to less than 1 p.p.m. prior to fermentation.  The cellular morphology of the organisms during fermentation is controlled by the initial adjustment of the nutrient balance to include ammonium carbonate for providing sufficient nitrogen for cell synthesis and inducing cell structure characterized by (a) abnormally short, stubby, forked, bulbous mycelium, (b) numerous swollen, oval to spherical-shaped cells well distributed throughout the mycelial structure, (c) mycelial structures all showing granulation, and numerous vacuoles or refractile bodies, (d) absence of normal reproductive bodies (vesicles or sterigmatar) and (e) formation of compact aggregates or colonies having a gross granular appearance and of sizes under 0.5 mm. in cross-section and averaging about 0.1 mm.  By this means at least 60 per cent of the total initial sugar may be converted to citric acid in a final concentration of at least about 7 per cent without significant formation of other acids such as oxalic and gluconic.  Slime-forming tendencies are also eliminated so that aeration and separation are easier.  Organisms used are a mild strain 18B2 and U.V. ray induced mutants (numbers 101, 127 and 164), of a strain ATCC?1015 all of which give good yields in surface fermentation techniques.  Stock cultures are maintained, and cells propagated, as slant cultures on Sabouraud's agar with serial transfers by mass inoculation on a five to seven day cycle. A concentration of 5-25 million spores per litre of fermentation medium is used for best results.  Carbohydrate media specified are refined sugars such as beet, cane, glucose or dextrose and incompletely refined sugars such as invert molasses, hydrolysed corn starch, raw sugar juices, and beet and and citrus molasses. The carbohydrate material should be of low ash content, particularly with respect to iron and invert molasses may be purified by cation-exchange resins operating on the hydrogen cycle.  The composition of the medium is favourably carbohydrate 10-15 per cent, (NH4)2CO3 0.2-0.15 per cent, KH2PO4 0.01-0.02 per cent, preferably 0.014 per cent, MgSO4.7H2O 0.08-0.15 per cent and Zn++ (added as SO4 or Cl), 0.002-0.004 per cent, the pH being adjusted to 2.5.  Ammonium carbonate is superior to other nitrogen containing nutrient salts because (a) it suppresses oxalic acid formation, (b) it eliminates pigmentation, (c) it eliminates slime-forming tendencies, and (d) it increases the efficiency of citric acid production per unit weight of mycelium. Tables are provided showing the effect on citric acid production of variations in the concentration of the above salts, carbohydrate and Fe and of the use of ammonium nitrate and chloride in place of carbonate. Figures are given illustrating suitable apparatus and fermenters for effecting the process humid sterile air (or other free oxygen containing gas), being provided for aeration at flow rates of 1/4 -1\ba1/4 vols. per vol. of medium per minute and warmth is obtained sufficient to humidify the air to a 95-100 per cent saturation.  One fermenter requires mechanical agitation, the other being in the form of a tower requiring no agitation. Antifoaming agents, e.g. n-octadecyl alcohol as a micro-crystalline suspension in mineral oil in amounts of 0.25-0.5 ml. of a 3 per cent suspension per litre of medium may be added.  In examples, invert molasses of sugar cone 17-19 per cent is passed through a synthetic cation exchange resin working on the hydrogen cycle to produce an effluent containing 16-18 per cent sugar, pH 1.35-1.45 and 5-10 p.p.m. Fe, which is again passed through a similar cycle with regenerated resin to produce an effluent contuining 14-15 per cent sugar content, pH 1.4-1.55 and less than 1 p.p.m. of Fe when it is passed through an anion exchange resin bed to elevate the pH to 2.75-2.90, the above nutrients added in the amounts specified, pH adjusted with HCl, the medium added to fermenters in which it is sterilized before inoculation with a spore suspension and fermentation effected at 24-34 DEG  C. for usually about 10-14 days with the passing of sterile humid air and in the presence of antifoam agents and mechanical agitation, in (2) refined cane sugar is treated as in (1) and (3) is a repeat of (1) using the column fermenter.  Tables are given of citric acid production using the two types of fermenter.