Production of Important Organic Acids by Fermentation
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Some details on the production of important organic acids by fermentation are given below:
1. Citric Acid:
Citric acid was first discovered as a constituent of lemon. Today, we know citric acid as an intermediate of ubiquitous Krebs cycle (citric acid cycle), and therefore, it is present in every living organism. In the early days, citric acid was isolated from lemons (that contain 7-9% citric acid), and today about 99% of the world’s citric acid comes from microbial fermentation.
Applications of Citric Acid:
1. Citric acid, due to its pleasant taste and palatability, is used as a flavoring agent in foods and beverages e.g., jams, jellies, candies, desserts, frozen fruits, soft drinks, wine. Besides brightening the colour, citric acid acts as an antioxidant and preserves the flavors of foods.
2. It is used in the chemical industry as an antifoam agent, and for the treatment of textiles. In metal industry, pure metals are complexed with citrate and produced as metal citrates.
3. In pharmaceutical industry, as trisodium citrate, it is used as a blood preservative. Citric acid is also used for preservation of ointments and cosmetic preparations. As iron citrate, it serve as a good source of iron.
4. Citric acid can be utilized as an agent for stabilization of fats, oils or ascorbic acid. It forms a complex with metal ions (iron, copper) and prevents metal catalysed reactions. Citric acid is also used as a stabilizer of emulsions in the preparation of cheese.
5. In detergent/cleaning industry, citric acid has slowly replaced polyphosphates.
Microbial Strains for Citric Acid Production:
Many microorganisms can produce citric acid. The fungus Aspergillus Niger is most commonly used for industrial production of citric acid. The other organisms (although less important) include A. clavatus, A. wentii, Penicillium luteum, Candida catenula, C. guilliermondii and Corynebacterium sp.
For improved industrial production of citric acid, mutant strains of A. Niger have been developed. The strains that can tolerate high sugar concentration and low pH with reduced synthesis of undesirable byproducts (oxalic acid, isocitric acid and gluconic acid) are industrially important.
Microbial Biosynthesis of Citric Acid:
Citric acid is a primary metabolic product (of primary metabolism) formed in the tricarboxylic acid (Krebs) cycle. Glucose is the predominant carbon source for citric acid production. The biosynthetic pathway for citric acid production involves glycolysis wherein glucose is converted to two molecules of pyruvate. Pyruvate in turn forms acetyl CoA and oxaloacetate which condense to finally give citrate. The major steps in the biosynthesis of citric acid are depicted in Fig. 24.1.
Metabolic Pathway for the Biosynthesis of Citric Acid
Enzymatic regulation of citric acid production:
During the synthesis of citric acid, there is a tenfold increase in the activity of the enzyme citrate synthase while the activities of other enzymes (aconitase, isocitrate dehydrogenase) that degrade citric acid are reduced. However, recent evidence does not support the theory that reduction in the operation of tricarboxylic acid (i.e. degradation of citric acid) contributes to accumulation of citric acid.
Increased citric acid is more likely due to enhanced biosynthesis rather than inhibited degradation. Further, there are anaplerotic reactions that replenish the TCA cycle intermediates to keep the cycle continuously in operation. Pyruvate carboxylase that converts pyruvate to oxaloacetate is also a key enzyme in citric acid production.
Yield of citric acid:
Theoretically, the yield of citric acid for the most commonly used substrate sucrose has been calculated. It is worked out that from 100 g sucrose, 112 g of anhydrous citric acid or 123 g of citric acid — 1 hydrate can be formed. However, due to oxidation of sugar to CO2 during trophophase, the yield of citric acid is lower than the calculated.
Factors in the Regulation of Citric Acid Production:
Strict maintenance of controlled nutrient conditions is very crucial for maximal production of citric acid. The optimal conditions that have been worked out for A. Niger for the production of citric acid are briefly described (Table 24.1).
Optimal Parameters/ Conditions for Citric Acid Production
Carbohydrate source:
A wide range of raw materials can be used for the supply of carbohydrates. These include molasses (sugar cane or sugar beet), starch (from potatoes), date syrup, cotton wastes, banana extract, sweet potato pulp, and brewery waste and pineapple waste water.
A high yield of citric acid production occurs if the sugars that are rapidly metabolised are used e.g. sucrose, glucose, maltose. At present, cane molasses and beet molasses are commonly used. The variations in the composition of molasses (seasonal and production level), have to be carefully considered for optimising citric acid production.
The concentration of carbohydrate significantly influences citric acid production. Ideally, the sugar concentration should be 12-25%. At a concentration less than 5% sucrose, citric acid formation is negligible, and increases as the concentration is raised to 10% and then stabilizes (Fig. 24.2). It is believed that a high sugar concentration induces increased glucose uptake and consequently enhanced citric acid production.
Effect of Sugar Concentration on Citric Acid Production
Trace metals:
Certain trace elements (Fe, Cu, Zn, Mn, Mg, Co) are essential for the growth of A. Niger. Some of the trace metals particularly Mn2+, Fe3+ and Zn2+ increase the yield of citric acid. The effect of manganese ions has been investigated to some extent. These ions promote glycolysis and reduce respiration; both these processes promote citric acid production.
As regards iron, it is a cofactor for the enzyme aconitase (of TCA cycle). It is estimated that an Fe concentration of 0.05-0.5 ppm is ideal for optimal citric acid production. At higher Fe concentration, the yield is lower which can be reversed to some extent by adding copper.
pH:
The pH of the medium influences the yield of citric acid, and it is maximal when pH is below 2.5. At this pH, the production of oxalic acid and gluconic acid is suppressed. Further, at low pH, transport of citric acid is much higher. If the pH is above 4, gluconic acid accumulates at the expense of citric acid. And when the pH goes beyond 6, oxalic acid accumulates. Another advantage with low pH is that the risk of contamination is very minimal, since many organisms cannot grow at this pH.
Dissolved O2:
The yield of citric acid production substantially increases when the dissolved O2 tension is higher. This can be achieved by strong aeration or by sparging with pure O2. It has been observed that sudden interruptions in O2 supply (as occurs during power breakdowns) cause drastic reduction in citric acid production without harming the growth of the organism.
Nitrogen source:
Ammonium salts, nitrates and urea are the nitrogen sources used in the media for citric acid production. All the three compounds are equally good sources, as long as they do not adversely affect the pH of the medium. If molasses are used for nutrient supply, addition of extra nitrogen source is not required. However, some workers have shown that exogenous addition of ammonium ions stimulates citric acid production.
Production Processes for Citric Acid:
There are two processes by which citric acid can be industrially produced — the surface process and submerged process (Fig. 24.3).
Industrial Processes for the Production of Citric Acid
The surface process:
This is characterized by growing the microorganisms as a layer or a film on a surface in contact with the nutrient medium, which may be solid or liquid in nature. Thus, the surface process has supported-growth systems.
The submerged process:
In this case, the organisms are immersed in or dispersed throughout the nutrient medium. There are two types of submerged fermenters (bioreactors) stirred bioreactors and airlift bioreactors.
Surface Processes:
Solid surface fermentation:
Surface processes using solid substrates are particularly carried out in less developed areas of some Asian countries. The solid substrates such as wheat bran or pulp from sweet potato starch are used, as culture media. The pH of the medium is adjusted to 4-5, and then sterilized. Now the inoculum in the form of spores of A. niger is spread as layers (3-6 cm thickness) and incubated at 28°C.
The growth of the organisms can be accelerated by the addition of α-amylase. Solid-state fermentation takes about 80 to 100 hours for maximal production of citric acid. At the end of the process, citric acid can be extracted into hot water and isolated.
Liquid surface fermentation:
Surface fermentation using liquid as nutrient medium is the oldest method for citric acid production. It is still in use due to a simple technology, low energy costs and higher reproducibility. Further, the interference of trace metals and dissolved O2 tension are minimal. The labour costs are however, higher since the manpower requirements are more for cleaning the systems. About 20% of the citric acid in the world is produced by surface processes.
The nutrient supply for surface fermentation normally comes from beet molasses. The fermentation is usually carried out in aluminium trays filled with sterile nutrient medium. The inoculum in the form of spores is sprayed over the medium. A sterile air is passed for supplying O2 as well as cooling. The temperature is maintained around 30°C during fermentation.
As the spores germinate (that occurs within 24 hours of inoculation), a layer of mycelium is formed over the medium. The pH of the nutrient medium falls to less than 2, as the mycelium grows in size and forms a thick layer on the surface of the nutrient solution. The fermentation is stopped a