Acetic Acid Production by Fermentation

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Departemen Teknologi Industri Pertanian FATETA - IPB m.k TEKNOLOGI BIOINDUSTRI TIN 330 (2-3)ORGANIC ACID Production Technology and Its Prospect to be Developed in Indonesia


ORGANIC ACID Mayor Organic Acid : Citric acid, lactic acid, L-ascorbic acid, gluconic acid  Table In anaerobic bacteria  their formation parallels growth (growth associated product) In aerobic bacteria and fungi  accumulation of organic acids is the result of incomplete substrate oxidation and is usually initiated by an imbalance in some essential nutrients, e.g mineral ions Different physiological prerequisites for formation of organic acids




CITRIC ACID 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 However synthetic methods proved to be unsuitable because of expensive or hazardous raw materials or an excessive number of reaction steps leading to low yields. C6H8O72-hydroxypropane-1,2,3-tricarboxylic acid


APPLICATION Citric acid is produced either in the anhydrous form (crystallization from hot aqueous solutions ) or as the monohydrate (crystallization at temperatures below 36.6 ° C). Food : a. The dominant use of citric acid is as a flavoring and preservative in food and beverages (soft drinks). b. Citrate salts used to deliver those minerals in a biologically available form in many dietary supplements. Pharmaceutical : Used with sodium bicarbonate in effervescent formulae,for example in antacid and soluble aspirin preparation.


Cleaning and Chelating Agent excellent chelating agent, binding metals. It is used to remove scale from boilers and evaporators. soften water, which makes it useful in soaps and laundry detergents. By chelating the metals in hard water, it lets these cleaners produce foam and work better without need for water softening. active ingredient in some bathroom and kitchen cleaning solutions. Plastic Citric acid esters, particular triethyl, tributyl and acetyltributyl esters are employed as non-toxic plasticizers in plastic films used to protect food stuffs.



Microorganisms Many microorganisms have been evaluated for the production of citric acid including : Bacteria such as Bacillus licheniformis, B. subtilis, Corynebacterium spp. Fungi such as A. niger, A. awamori, A. foetidus, Penicillium restrictum Yeast such as Candida lipolytica, C. intermedia and Saccharomyces cerevisiae However, A. niger 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 of citric acid.


Medium : CHO source could be starch, starch hydrolysate, cane juice, glucose, sucrose or molasses  sucrose was the most favourable carbon source followed by glucose, fructose and galactose. Nitrogen source : ammonium salts are preferred, e.g. urea, ammonium sulfate, ammonium chlorite, peptone, malt extract, etc. Mineral : zinc, manganese, iron, copper and magnesium affect citric acid production. Potassium dihydrogen phosphate has been reported to be the most suitable phosphorous source.  Growth of A. niger on high concentrations of sugars and low concentrations of Fe3+ and Mn2+ gives high yield of citric acid


pH A low initial pH has the advantage of checking contamination and inhibiting oxalic acid formation. pH of 2.2 optimum for the growth of the mould as well as for the production of citric acid. Higher pH i.e. 5.4 and 6.0-6.5 has been found optimum for citric acid production in molasses medium. Aeration : Increased aeration rates led to enhanced yields and reduced fermentation time. It is important to maintain the oxygen concentration above 25% saturation and interruptions in oxygen supply may be quite harmful. Aeration is performed during the whole fermentation with the same intensity through the medium at a rate of 0.5 to 1.5 vvm.


Liquid fermentation Submerged fermentation (SmF) :  the commonly employed technique for citric acid production. It is estimated that about 80% of world production is obtained by SmF. Advantages : higher yields and productivity and lower labour costs are the main reasons for this. Two types of fermenters, stirred tank fermenters and tower fermenters are employed, although the latter is preferred due to the advantages it offers on price, size and operation



Inoculum (seed culture) Inoculation by adding : a suspension of spores, or of pre-cultivated mycelia. Spores are produced in glass bottle on solid substrate at 250C with incubation time of 10-14 days. When spores are used, a surfactant is added in order to disperse them in the medium. For pre-cultivated mycelia (germinate), an inoculum size of 10% of fresh medium is generally required. Normally, submerged fermentation is concluded in 5 to 10 days depending on the process conditions. It can be carried out in batch, continuous or fed batch systems, although the batch mode more frequently used.


Morphology of the mycelium : is crucial not only in relation to the shape of the hyphae , but also in the aggregation of the growth into small spherical pellets. The mycelial pellets should be small (0.2 to 0.5 mm) with a hard surface. This state of affairs is brought about by a deficiency of manganese in the medium or the obviously related additions of ferrocyanide ion.


Where a separate inoculum stage is employed, a suspension of spores of A. niger, usuaIly grown on a solid medium, is introduced into sterilized medium in the inoculum fermenter. The medium is aerated and, in some processes, agitated and the mould allowed to grow at a temperature of about 30°C for a period of from 18 to 30 hours as judged by pH level reached .Downstream Process


Surface Fermentation with A. niger  liquid surface culture (LSC) - Medium - beet molasses as raw material is still extensively employed by major manufacturers although somewhat labour intensive, the power requirements are less than in the submerged fermentation. - Additional nutrients and alkali ferrocyanide After sterilized and cooling, the prepared medium is run down into a series of trays supported on racks in a ventilated chamber. The trays are filled to a depth of between 0.05 and 0.20 m.


Surface Fermentation with A. niger Inoculum Spores of A. niger are obtained by growing a selected strain on a sporulation medium. The spores are collected and distributed over the surface of the medium in the trays. Physical Conditions Sterile air is supplied to the fermentation chamber. The air performs the dual function of supplying oxygen and carrying away fermentation heat and the rate of flow of the air is regulated accordingly. A temperature in the region of 30 °C is often employed.


The mycelium forms a coherent film on the surface of the liquid becoming progressively more convoluted. The removal of the heavy metals by the ferrocyanide severely restricts sporulation. · After a period of 7 to 15 days the trays are emptied and the mycelium separated from the fermented liquor. · The liquors are pumped forward to the recovery section. · Unwanted by-products of the process are gluconic and oxalic acids. In many processes oxalic, acid production is minimized by careful strain selection


Citric Acid Production using Surface Fermentation


Surface fermentation of citric acid Citric acid manufactures using microbial conversion of sugar beet molasses fermentation based on potassium hexacyanoferrate use for control of trace metal effect on fermentation ..


PRODUCT RECOVERY The recovery of citric acid from liquid fermentation is generally accomplished by three basic procedures : 1. Precipitation 2. Extraction Adsorption and Absorption (mainly using ion exchange resins). Separation of citric acid from the liquid : calcium hydroxide is added to obtain calcium citrate tetrahydrate → wash the precipitate→ dissolve it with dilute sulfuric acid, yield citric acid and calcium sulfate precipitate → bleach and crystallization → anhydrous or monohydrate citric acid.


Downstream Process of Citric AcidCa(OH)2calcium citrate tetrahydrate


Solid-state Fermentation (SSF) Citric acid production by SSF (the Koji process) was first developed in Japan and is as the simplest method for its production. SSF can be carried out using several raw materials. Generally, the substrate is moistened to about 70% moisture depending on the substrate absorption capacity. The initial pH is normally adjusted to 4.5-6.0 and the temperature of incubation can vary from 28 to 30°C. The most commonly organism is A. niger in simple tray type fermenters. One of the important advantages of SSF process is that the presence of trace elements may not affect citric acid production so harmfully as it does in SmF. Consequently, substrate pre-treatment is not required.


CONCLUSIONS AND PERSPECTIVES The use of alternative raw materials to produce citric acid by SmF, LSC, and SSF seems to be a suitable possibility. However, it is necessary to adapt the right type of raw material to the right technique e.g. cassava bagasse employed as substrate in SSF, or cellulose hydrolysate used in SmF. The need of some pre-treatment of raw materials may enhance the fermentation efficiency. Another area is the strain improvement with improved substrate utilization efficiency.

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Last Updated: 8th March 2018

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