The substrates for citric acid fermentation using submerged technique

Several industrial important chemicals are produced via biological processes, an example of which is citric acid (an organic acid). Citric acid is ubiquitous in nature found and found in all plant and animal tissues. Citrus fruits contain citric acid in large quantities, ranging from 5% in the fruit to about 9% in juice. The sour taste of lemon juice is mainly due to the presence of citric acid (5 to 8%) and partly due to the presence of vitamin C.

Citric acid is solid at room temperature, melts at 153ºC and decomposes at higher temperatures into other products (Rajoka et al. 1998). It is non-toxic and easily oxidized in the human body

Citric acid, a tricarboxylic acid, is one of the world’s largest products of fermentation process. It is the most verstile industrial acid and being used in the food and beverage industries as an acidifying and flavour-enhancing agent and also in other industries such as detergents and pharmaceuticals (Shojaosadati & Babaeipour, 2002).

Citric acid (2- hydroxy-1, 2,3,propane tricarboxylic acid) is used in food, beverages, pharmaceuticals, chemical cosmetics and other industries for application such as acidulation, antioxident, flavour enhancement, preservation plastizer and as synergistic agent (1993;shakaranand and Lonsane, 1994).

It is non-toxic and easily oxidized in the human body. It’s wide spread industrial application are due to its high solubility, palatability and low toxicity. These uses have placed greater stress on increased citric acid production and search for more efficient fermentation process. The worldwide demand of citric acid is about 6.0 x 105 tons per year and it is bound to increase day by day (Ali et al. 2001), due to its increasing new uses in industrial process.

Citric acid (2-hydroxy-propane-1,2,3-tricarboxylic acid) is a true bulk product with an estimated global production of over 900 thousand tons in the year 2000. Till the beginning of the 20th century, it was exclusively extracted from lemons. Since the global market was dominated by an Italian cartel, other means of production were sought. Chemical synthesis was possible, but not suitable due to expensive raw materials and a complicated process with low yield. The discovery of citrate accumulation by Aspergillus niger led to a rapid development of a fermentation process, which only a decade later accounted for a large part of the global production. The application of citric acid is based on its three properties: (1) acidity and buffer capacity, (2) taste and flavour, and (3) chelation of metal ions. Because of its three acid groups with pKa values of 3.1, 4.7 and 6.4, citrate is able to produce a very low pH in solution, but is also useful as a buffer over a broad range of pH values (2 to 7). Citric acid has a pleasant acid taste which leaves little after taste. It sometimes enhances flavour, but is also able to mask sweetness, such as the aspartame taste in diet beverages. Chelation of metal ions is a very important property that has led to applications such as antioxidant and preservative. Moreover, it is a "natural" substance and fully biodegradable. Karaffa et al. (2001)

Production of citric acid from sugar solutions by aerobic bioprocesses was first realized by using Penicillium. Due to low yields obtained from Penicillium, Aspergillus niger was utilized in subsequently developed processes (Shuler,2002) Many microorganisms such as fungi and bacteria can produce citric acid. The various fungi, which have been found to accumulate citric acid in their culture media, include strains of Aspergillus niger, A. awamori, Penicillium restrictum, Trichoderma viride, Mucor piriformis and Yarrowia lipolytica (Arzumanov et al. 2000). But Aspergillus niger remained the organism of choice for the production of citric acid. (Mattey and Allan, 1990; Ali et al. 2001).

Currie (1917) pointed out that strains of Aspergillus niger were infact best for the fermentative production of citric acid. Aspergillus species are highly aerobic and are found in almost all oxygen-rich environments, where they commonly grow as molds on the surface of a substrate, as a result of the high oxygen tension. "In recent studies, increased levels of Reactive Oxygen Species (ROS) were shown to be correlated with increased levels of aflatoxin biosynthesis in Aspergillus parasiticus." (Reverberi, et al 2008) Commonly, fungi grow on carbon-rich substrates such as monosaccharides (such as glucose) and polysaccharides (such as amylose). Aspergillus species are common contaminants of starchy foods (such as bread and potatoes), and grow in or on many plants and trees

The development of a microbial process for the formation of citric acid is aimed at maximizing three things; the yield of product per gram of substrate, the concentration of product and the rate of product formation.

The substrates for citric acid fermentation using submerged technique of fermentation are beet or cane-molasses (Pazouki et al. 2000). Blackstrap sugarcane molasses is an econimically easily and abundantly available by-product of sugar industries and is a desirable raw material for citric acid fermentation because of its availability and relatively low price. Owing to the steadily increasing demand of citric acid for industrial purposes, its manufacture from cane or beet molasses has proved to be of great importance to the sugar industry (Pazouki et al., 2000). Sugar cane molasses is a complex medium and has high content of sugars and metal ions that inhibit the growth of Aspergillus niger in liquid cultures.

In the past decade or so, there has been an increasing number of reports on the use of solid-state fermentation processes for the production of a number of microbial products (Roussos et al., 1994; Nampoothiri and Pandey, 1996 and Pandey et al., 1999). This is partly because solid-state processes have lower energy requirements and produce much less wastewater and environmental concerns because disposal is of solid wastes.

Solid substrate fermentation involves “the growth of microorganisms on moist solid substrates in the absence or near absence of free flowing water” (Robinson et al., 2001).

Solid State Fermentation offers numerous advantages for production of bulk chemicals and enzymes. This is partly because solid-state processes have lower energy requirements and produce much less wastewater and environmental concerns related to disposal are for solid wastes. In addition, immobilized microbial cell systems have been the subject of extensive research during the last 20 years. This technology offers many advantages such as high yield, low risk of contamination and easy control. In short, various chemical, physical and biochemical techniques have been investigated for industrial citric acid production (Papagianni et al., 1999).

Solid-state fermentation has long been applied to the food industry. SSF is a process carried out with microbes growing on nutrient impregnated solid substrate with little or no free water. Solid state fermentation (SSF) can be directly carried out with low-cost biomaterials like corn Stover, corncobs, banana stalk, wheat bran etc. abundant and available in Pakistan with minimal or no pretreatment, and thus is relatively simple, uses less energy than submerged fermentation (SmF), and can provide unique microenvironments conductive to microbial growth and metabolic activities. The present project designed to use corncobs as carrier substrate for SSF of molasses based medium by Aspergillus niger.

Incubation temperature plays an important role in the production of citric acid. Temperature between 25-30ºC is usually employed for culturing of Aspergillus niger but temperature above 35ºC is inhibitory to citric acid formation because of the increased the production of by-product acids and also inhibition of culture development. Sanjay and Sharma (1994) reported that citric acid production by Aspergillus niger is sensitive to the initial pH of the fermentation medium.

Traditionally, SSF are characterized by the development of microorganisms in a low water-environment on a non-soluble material that acts both as physical support and source of nutrients; however it is not necessary to combine the role of support and substrate but rather reproduce the conditions of low water activity and high oxygen transference by using a nutritionally-inert material soaked with a nutrient solution (Pandey and Soccol, 1998).

Corncobs could can serve as a substrate solid state for citric acid production by Aspergillus niger in molasses based SSF. Factors will be characterized that limit growth of Aspergillus niger in SSF cultures by studying the mechanism of inhibition of microbial growth and citric acid synthesis and accumulation in cane molasses fermentation medium. It is hypothesized that development of solid state medium using corn cobs powder as solid matrix is experted to absorb sugars and minerals resulting in their slow release for Aspergillus niger and minimize the inhibition objective.