ISOLATION AND SCREENING OF MICROORGANISMS FOR CITRIC ACID PRODUCTION

Citric acid is a weak acid and naturally occurring antioxidant. Due to its wide application in the food industry, cosmetics and manufacturing cleaning products, Citric acid is artificially synthesized by exploiting microorganisms, especially fungi. With an aim of isolating and screening the potential microorganisms for citric acid production, the present study was carried out by collecting samples of decaying fruits and vegetables from the local market of Kalaburagi, Karnataka, India viz., Citrus limon, Citrus sinensis, Vitis vinifera, Malus pumila, Ananas comosus, Solanum tuberosum, Punica granatum, Citrullus lanatus, Musa paradisiaca, Solanum lycopersicum, Phaseolus vulgaris, Mangifera indica, Manilkara zapota, Beta vulgaris, Saccharum officinarum, Ipomoea batatas, Daucus carota, Psidium guajava, Solanum melongena and Abelmoschus esculentus. A total of 20 isolates have been isolated from the samples, out of 20 fungal isolates, 7 isolates viz., KLV03, KLV04, KLV07, KLV10, KLV11, KLV13 and KLV14 showed significant acid unitage values of 3.00, 1.88, 3.36, 1.31, 2.35, 1 and 1 respectively. Among these, KLV07 isolate showed the highest acid unitage value of 3.36. Molecular identification of the isolate KLV07 was done by 18S rRNA gene sequencing and confirmed the isolate to be Aspergillus niger strain KRF9 small subunit ribosomal RNAgene with Sequence ID: MK611800.1. The next closest homologue was found to be Aspergillus awamori isolate RN 206 smallsubunit ribosomal RNA gene with Sequence ID: MK530131.1.


Introduction
Citric acid (2-hydroxy-propane-1,2,3-tricarboxylic acid) occurs in many plants, especially citrus plants, and it is an intermediate in the tricarboxylic acid cycle (TCA) of aerobic metabolism.Citric acid monohydrate has a molecular weight of 210.14 g mol -1 (Dhillon et al., 2011).Citric acid contains three carboxyl groups with three different pKa values (3.13, 4.76, and 6.40).Commercial citric acid is a colourless, transparent, or translucent crystal, or particle powder that is readily soluble in water and ethanol.It is widely used in food (75%), pharmacy (10%), and other industries (15%) (Ates et al., 2002;Tran et al., 2008).Citric acid can be produced through microbial fermentation, or it can be extracted from a natural source.Chemical syntheses of citric acid are possible but uneconomical because the starting chemical is usually more expensive than the desired product.The production of citric acid by microbial fermentation has many advantages compared to direct extraction from citrus fruits.
Citric acid manufacturing has grown significantly over the past century since the advent of biotechnology, which gives adequate comprehension of fermentation procedures and product recovery.Biochemistry, which gives understanding of several factors affecting citric acid synthesis and blockage, has also improved the production of citric acid and efficiency of recovery.In addition to biotechnology and biochemistry, molecular regulatory mechanisms and techniques had a significant role in increasing citric acid synthesis.Extensive literature studies and hundreds of publications on citric acid manufacturing have been published during the last 60 years (Prescott and Dunn 1959;Lockwood and Schweiger 1967;Miall 1978;Dawson 1986;Vandenberghe et al. 1999;Papagianni 2007;Max et al. 2010;Show et al. 2015;Tong et al. 2019).

MATERIAL AND METHODS
1. Collection of samples Decaying fruits and vegetables samples were collected from the local market of Kalaburagi, Karnataka, India.The samples were collected in the sterile sealed polythene bags and brought to the laboratory and stored at room temperature for further studies.

Isolation of fungi for citric acid production
1 gram of sample was taken in a 250 ml conical flask containing 100 ml of sterile distilled water and shaken well for 20 minutes.From the stock, Serial dilutions of the samples were prepared as per the standard method (Cochrane et al., 1961), up to 10-6 dilutions.0.5ml of 10 -5 and 10 -6 dilutions, 10 -4 and 10 -3 dilutions were inoculated on Nutrient Agar and Sabouraud Dextrose Agar respectively for the isolation of fungi.All the inoculated plates were kept for incubation in the incubator at 30 o C for 5 days.

Screening of fungi for citric acid production
Isolated cultures were subjected for screening of citric acid production by determining acid unitage value (AU).A loop full of fungal sporulation solution was inoculated on petri plates containing mineral salt agar acid indicator medium (Park et al.,1998) supplemented with bromoscerol green and incubated for five days for the formation of yellow zone around the mycelia growth.The acid unitage value for selection of potential fungal isolate was calculated using the formula given below.

AU = 4. Morphological and microscopic observation
Lactophenol cotton blue was used as a mounting medium and staining agent for preparation of slides and microscopic examination of fungi.Nagajyoti @el .Depending upon the morphology of the particles, there are two categories in Suspension. 5. Molecular identification a.DNA Extraction was carried out based on the standard protocol described by Rogers and Bendich, (1994).b.PCR Amplification of 18s Gene: 170 ng of Extracted DNA is used for amplification along with 10pM of each primer.c.Composition of TAQ Master MIX: • High-Fidelity DNA Polymerase • 2.5mM dNTPs • 3.2mM MgCl2 • PCR Enzyme Buffer d.The ~2kb, 18s-rDNA fragment was amplified using high-fidelity PCR polymerase (Table 1 and Table 2).The PCR product was sequenced Bi-directionally (Table 3).
The sequence data was aligned and analyzed to identify the organism and its closest neighbors.RESULT AND DISCUSSION Isolation of fungal strains from various sources A total of 20 samples of decaying fruits and vegetables (Table 4) were subjected to isolation of fungi for citric acid production on SDA medium and incubated under 25 o C for 120hrs.The prominent colonies of the fungi were selected for further screening.
The colour transition from purple to yellow on the fermentation medium indicates positive results for citric acid synthesis.Plate 2 illustrates the synthesis of organic acid on a modified mineral salt medium; a yellow halo forms around the citric acid-producing colonies.Out of 20 fungal isolates, 7 isolates viz., KLV03, KLV04, KLV07, KLV10, KLV11, KLV13 and KLV14 showed significant acid unitage values of 3.00, 1.88, 3.36, 1.31, 2.35, 1 and 1 respectively (Table 5).Among these, KLV07 isolate (Plate 3) showed the highest acid unitage value of 3.36 and was selected for morphological and molecular identification (Table 5).The culture of KLV07 isolate was maintained in slant (Plate 4) and was stored in 4 o C for further use.(Rahul jabnoor @ el) The contrasting in the image is due to the difference in spreading from variety surfaces area as an effect of geometrically difference and dispersed oxide sample.

Morphological identification
Basic morphological features of aerial mycelium, substrate mycelium and nature of colonies were observed under microscope as per the methods described in the Bergey's Manual of determinative Bacteriology (Goodfellow, 1989).Fungal elements are stained intensely blue.Under the microscope, several fruiting bodies and mycelia were observed (Plate 5).

Molecular Identification
Plate 3 depicts the genomic DNA of KLV07, PCR amplified gene and 100bp ladder for the isolate.
Plate 3. QC report for KLV07 isolate: A, genomic DNA of KLV07; B, PCR amplified gene; C, 100bp ladder for the isolate.
18S rRNA gene sequence confirmed the isolate to be Aspergillus niger strain KRF9 small subunit ribosomal RNA gene with Sequence ID: MK611800.1 ( Results shows that strain KLV07, together with the above-mentioned species do not form a distinct phyletic line with the neighbour-joining method.The summary of the accession number of the first ten closest strains to KLV07 is listed in table 6. In the present study, the potential isolate for citric acid production is identified as Aspergillus niger with acid unitage value 3.36 isolated from pineapple substrate.Citric acid has recently been commercially produced through fungal fermentation, primarily by Aspergillus niger.It is also produced commercially using mutant fungal strains of Saccharomycopsis lipolytica (Good et al., 1985), Penicillium simplicissimum and Aspergillus.foeitidus (Tran et al., 1998).A. niger are principally used to produce citric acid, however in culturing these fungi, the growth of Aspergillus in pellet form is desirable and this can be achieved by process optimization (Rohr 1998;Wayman and Mattey, 2003).Yarrowia lipolytica can produce citric acid from raw glycerol (Rymowicz, 2007), a byproduct of biodiesel production from rapeseed oil.Citric acid is commercially generated using various cost-effective and accessible raw materials such as molasses, carob pod extract, rape seed oil, corncobs, apple pomace, grape pomace, kiwi-fruit peel, mandarin orange, and brewery wastes as carbon sources (Roukas, 1998).
Due to increasing demand of citric acid, and there is a dire need for cost-effective and sustainable citric acid production methods.As therefore, from the standpoints of bioeconomy and environmental (white) biotechnology, it is crucial to develop a novel bioprocess for citric acid synthesis that has higher productivity and a lower environmental impact.
A. niger's ability to produce citric acid biochemical and physiological processes have attracted both biological and biotechnological interests, and studies have been performed by many research groups (Usami, 1978;Ruijter et al., 1996 andRoehr, 1998).Citric acid fermentation by A. niger is the most economical and exclusively used method to produce citric acid (Usami, 1978;Rohr et al., 1996;Ruijter et al., 1996;Roehr, 1998;Bentley and Bennett, 2008;Berovic, 2004 andKaraffa &Kubieck, 2003).There reports on A. niger's full genome sequences and comparative genomics studies by Pel et al., (2007) andDe Veries et al., (2027) making the organism open to targeted growth through genetic and metabolic engineering.Genetic selection of citric acid producing strains, or the development of strains with desirable traits, has been investigated by numerous research groups.Honda et al. (2011) established a high-frequency homologous transformation method for gene disruption and tailored gene substitution in citric acid generating A. niger.

Table 6 )
. The next closest homologue was found to be Aspergillus awamori isolate RN 206 small subunit ribosomal RNA gene with Sequence ID: MK530131.1 (Plate 6).