Screening of Citric Acid Producing Fungi from the Leaf Litter Soil of Sathuragiri Hills

Soil samples were collected from ten different locations of Sathuragiri Hills (Western Ghats, Tamilnadu). Percentage frequency of occurrence of fungal isolates was calculated. Cultural characteristics of fungal isolates on Sabouraud’s dextrose agar were observed. Fungal isolates were identified based on morphology, cultural characteristics and 18S rRNA sequencing. Citric acid production ability of fungal isolates was screened. Among the isolates Aspergillus niger found to produce the highest quantity of citric acid (2.5 ± 0.01 g/L) and it is selected for further work.


Introduction
Citric acid (C 6 H 8 O 7 , 2-hydroxy-1,2,3-propane tricarboxylic acid), a natural constituent and common metabolite of plants and animals, is the most versatile and widely used organic acid in the field of food (70%) and pharmaceuticals (12%). It has got several other applications in various other fields. Currently, the global production of citric acid is estimated to be around 736000 tones/year [1] and the entire production is carried out by fermentation. There is constant increase (3.5-4%) each year in its consumption, showing the need of finding new alternatives for its manufacture [2].
Citric acid is an intermediate of tricarboxylic acid (TCA) cycle which is obtained when carbohydrates are oxidized to carbon dioxide. It has three carboxylic acid functional groups with three pKa values at pH 3.1, 4.7 and 6.4. It is a ubiquitous intermediate product of metabolism and its traces are found virtually in all plants and animals [3].
Citric acid is known as the most important organic acid produced in tonnage by fermentation and is the most exploited biochemical/biotechnological product [4]. It has an annual production of 1.6 million tons [5] with annual growth demand /consumption rate of 3.5-4.0% [6].
Citric acid is responsible for the tart taste of various fruits in which it occurs (i.e. lemons, limes, figs, oranges, pears and goose-berries). Hence, citric acid is used to impart a pleasant tart flavours to foods and beverages. It is used in the industries to achieve acidulation, antioxidation, emulsification, preservation, flavour enhancement, and as plasticizer and synergistic agent [7]. The wide applicability of citric acid in industries is attributed to its low/non-toxicity, high solubility; biodegradability and palatability [8].
It is a product adjudged to be GRAS (Generally Recognized as Safe). The food industry consumes about 70% of total citric acid produced and pharmaceutical industries consume about 12% and the remaining 18% are consumed by other industries [6]. The demand for citric acid is increasing faster than its production and hence, more economical processes are required [9].
The supply of natural citric acid is very limited and the demand can only be satisfied by biotechnological processes [10]. Many microorganisms such as fungi, bacteria and yeast can produce citric acid. A large number of these microorganisms have been employed for citric acid production, but only a few of them can produce citric acid in industrial scale [7]. It is reported that Aspergillus niger is almost exclusively used for industrial scale production of citric acid. This is due to its high citric acid productivity at low pH, without secretion of toxic metabolites, ease of handling and ability to ferment a variety of cheap raw materials. Citric acid is commercially produced by large scale fermentation mostly using selected fungal or yeast strains in aerobe bioreactors [10].
A large number of microorganisms including fungi and bacteria such as Arthrobacter paraffinens, Bacillus licheniformis and Corynebacterium sp., Aspergillus niger, A. aculeatus, A. carbonarius, A. awamori, A. foetidus, A. fonsecaeus, A. phoenicis and Penicillium janthinellum; and yeasts such as Candida tropicalis, C. oleophila, C. guilliermondii, C. citroformans, Hansenula anamola and Yarrowia lipolytica have been employed for citric acid production [11]. Most of them, however, are not able to produce commercially acceptable yields due to the fact that citric acid is a metabolite of energy metabolism and its accumulation rises in appreciable amounts only under conditions of drastic imbalances. Among the mentioned strains, the fungus A. niger has remained the organism of choice for commercial production because it produces more citric acid per time unit. The problem in the production of citric acid for yeasts is the simultaneous formation of isocitric acid. The main advantages of using A. niger are its ease of handling, its ability to ferment a variety of cheap raw materials, and high yields. Industrial strains which produce commercial citric acid are not freely available and only a few can be obtained from international culture collections.
In this context, the isolation and identification of a potential citric acid producing fungal strain from the Western Ghats of Tamilnadu will be attempted in the present investigation considering the fact that novel and potential citric acid producing strain can be isolated from biodiversity niche. This species may be found to be very effective comparatively with the existing microbial strains. Microorganisms in Western Ghats of Tamil Nadu form a potential source for exploring novel microbial products, due to their unique natural habitat, distinct physiological characteristics, metabolic patterns and nutrient utilization.

Sampling Area
The leaf litter soil samples were collected out in Sathuragiri Hills (Western Ghats, Tamilnadu) is about 15 Kms from Srivilliputhur. The town is situated on latitude 9.727288300000001000" North and longitude 77.618638299999930000" East. The town is about 4500 feet above the sea level.

Sample collection
Soil samples were collected from ten different locations in leaf litter degrading soil environment of Sathuragiri Hills (Western Ghats, Tamilnadu). Using a sterilized spatula, 250g of soil sample was collected from each location into 300g-capacity plastic container which was previously washed and rinsed with 70% alcohol. The samples from the ten different sites were conveyed to the Microbiology Laboratory within same day, where they were analyzed.

Isolation and Identification of Fungal Isolates
Serial dilutions of soil samples from each site were prepared and inoculated in duplicates into Sabouraud's Dextrose Agar (SDA) plates by spread plate methods. The plates were incubated at 30 0 C for 3days and observations were made daily to determine the presence of filamentous fungi. Pure cultures of isolates were obtained by repeated sub-culture on SDA [12].
The fungal isolates were identified based on the basis of their cultural and morphological characteristics. The cultural characteristics were determined by their appearance on culture plates while the morphological features were determined microscopically. The isolates were identified with reference to the work of Domsch et al. [13].

Determination of Percentage Frequency of Occurrence of Isolates
The percentage frequency of occurrence for each species of fungus was determined by the method of using the formula: A/B X 100; where, A=Number of plates in which species appear and B=Total number of plates incubated for each sites [14].

Citric Acid Production Medium
Strains were screened for citric acid production in liquid culture which contained sucrose (g/l) 120g; NaNO 3

Selection of Strain for Citric Acid Production
Based on the colony morphology the fungal isolates were designated as AS1, AS2, AS3, AS4, AS5, AS6 and AS7. These strains were inoculated individually in the production broth and incubated at 37°C for 3 days and then they were analyzed for the citric acid production.

Isolation and Identification of Fungal Strains
The isolated colonies AS1, AS2, AS3, AS4, AS5, AS6 and AS7 were examined by lactophenol cotton blue technique and identified according to their morphological characteristics [16]

Screening of the Fungal Cultures
All the seven cultures were screened qualitatively for the production of citric acid as described by Ali, [17].
Czapek-Dox agar medium (20 mL) was poured into individual sterile Petri plates and allowed to cool at room temperature. The plates were incubated at 30°C for 3-5 days. The plates were observed after incubation for yellow zones due to citric acid formation. Strains of Aspergillus niger with the widest yellow zone were used for further studies.

Determination of Citric Acid Production by Fungal Isolates
Czapek-Dox broth (50 mL) was prepared in seven conical flasks and sterilized. They were allowed to cool at room temperature. The conical flasks were inoculated with seven different fungal isolates and incubated at 30°C for 3-5 days. The broth was centrifuged at 5000rpm for 10 minutes and the supernatant was taken for citric acid estimation. Citric acid was determined titrimetrically [18] by using 0.1 NaOH and phenolphthalein as indicator and calculated as % according to the formula: %CA = Normality×volume of NaOH×Equiv. wt. of CA/ Weight of sample×10

Results
The results of the soil samples of the different locations in Sathuragiri Hills (Western Ghats, Tamilnadu) are presented here. While Table 1 shows the results of the Total Fungal Counts in the soil samples of the different locations. The percentage frequencies of occurrence of fungal isolates are presented in Table 2, while the results of the cultural characteristics of isolates are presented in Table 3. The results of the citric acid levels produced by the fungal isolates are presented in Table 4  Penicillium sp + + - Cladosporium sp + -- Helminthosporium sp Aspergillus niger found to produce the highest quantity of citric acid (2.2 ± 0.001 g/L), followed by Aspergillus flavus (1.32 ± 0.01 g/L), Penicillium sp (1.28 ± 0.06 g/L), Helminthosporium sp. (1.22 ± 0.02 g/L). Least amount of citric acid was produced by Cladosporium sp. (0.92 ± 0.04 g/L). These findings have further confirmed the superior of Aspergillus niger as the industrial production species for the citric acid production (Table 4).
Equally, Aspergillus niger and Penicillium sp had the highest percentage frequency of occurrence of 60%, followed by Rhizopus stolonifer and Trichoderma viride which had 50%. Aspergillus flavus and Alternaria alternata had 40% frequency of occurrence respectively. Aspergillus fumigatus had 20%, while Absida corymbifera and Curvularia lunata had the lowest frequency of occurrence of 10% [16].
Whereas in a study, Aspergillus fumigatus, Cladosporium herbarum, Rhizopus stolonifer, Alternaria alternata and Curvularia lunata did not produce citric acid, while Absida corymbifera, Penicillium sp, Trichoderma viride, Aspergillus flavus and Aspergillus niger produced citric found to produce the highest quantity of citric acid (8.4μg ml -1 ), followed by Trichoderma viride (5.1 μg ml -1 ), acid at varying concentrations. Aspergillus niger was A. flavus (3.2 μg ml -1 ), Penicillium sp (2.0 μg ml -1 ) and Absida corymbifera (1.4 μg ml -1 ), respectively. These findings have further confirmed the superior of Aspergillus niger as the industrial species for the production of citric acid as reported by several workers [19,20]. The results also agree with Torres, [21] where it was reported that some species of fungi other than Aspergillus niger also do produce citric acid.

Conclusions
Aspergillus niger found to produce the highest quantity of citric acid (2.20 ± 0.01 g/L), followed by Aspergillus flavus (1.32 ± 0.01 g/L), Penicillium sp. (1.28 ± 0.06 g/L), Helminthosporium sp. (1.22 ± 0.02 g/L). Least amount of citric acid was produced by Cladosporium sp. (0.92 ± 0.04g/L). These findings have further confirmed the superior of Aspergillus niger as the industrial production species for the citric acid production.
Even though there are many microbial sources available for citric acid production only a few are recognized for commercial production. Their vast diversity and specific range of action have attracted the attention of biotechnologists' worldwide. Presently, in India, little effort is being made to prepare citric acid using microorganisms. The best possible solution considering cost effectiveness may be the utilization of the indigenous and cheaper substances as like agricultural wastes and by products as substrate for the production of this valuable organic acid and then its further use in application like food and pharmaceutical industry. Therefore, the plan for future research investigations has been chalked out to produce citric acid using Aspergillus niger.

Conflict of Interest
There is no conflict in this study.