Citric Acid Fermentation by Aspergillus niger

Amal A. Almousa¹, Mohamed N. Abd El-Ghany², Eman H. Ashour¹

Introduction

Citric acid, or 2-hydroxy-propane-1, 2, 3-tricarboxylic acid (C₆H₈O₇.H O) is a naturally occurring weak organic 2 acid, with pH 0.2 found in all citrus fruits [1]. Citric acid in its pure form is readily soluble in water and colorless [2]. It is solid at room temperature. Citric acid has a melting point of 153°C and it decomposes at higher temperatures. Citric acid has a molecular weight of 210.14 g/mol and possesses three different pKa values, at pH 3.1, 4.7 and 6.4, owing to the presence of three functional groups of carboxylic acid in its structure [3].Citric acid fermentation is one of the primitive fermentations but still its production is increasing with passage of time.

In 1916, a study conducted by James Currie made a breakthrough for successful economic industrial production of citric acid from Aspergillus niger. He discovered that significant amounts of citric acid could be obtained from various strains of A. niger. The most important findings were the ability of A. niger to grow at a pH of around 2.5–3.5, which curbed the formation of gluconic and oxalic acid, and the increase in citric acid production with increasing sugar concentration. This single piece of research laid the foundation for presentestablished in the USA by the pharmaceutical company Pfizer in 1923 [4].

Materials and methods

Sample collection and isolation

Soils were collected into a clean polythene bag from King Saud University. Ten gram of each soil sample was suspended in 90 ml of sterilized distilled water. Subsequently, fungal cultures were isolated by serial dilutions in sterilized saline and plated onto Petri dishes with potato dextrose agar medium. The plates were incubated at 28-30°C for 4-7 days. Individual colonies were picked by repetitively streaked through a series of plates to isolate the individual member. For purification, the growing single colonies were picked up and streaked on slants of Potato Dextrose Agar, PDA (plus 4 ml per liter of chlorampenicol, 25 mg/ml, after autoclaving) and kept at 4°C until using. The isolated colonies were microscopically examined and identified according to their observed morphological characteristics [5].

Fungal cultures maintenance

Aspergillus niger strains used in the study include the local isolates which were obtained from various locations in Riyadh region in addition to fungal strain, Aspergillus niger EMCC132 that used throughout the experiments as a reference strain for citric acid production and it was obtained officially from Cairo Microbiological Resources Centre (Cairo MIRCEN), Ain Shams University, Cairo, Egypt. The cultures of Aspergillus niger were maintained on sterilized potato dextrose agar medium (PDA) and stored at 5ºC in the refrigerator. In general, all the used media, unless otherwise stated, were sterilized in autoclave at 15-lbs/inch2 pressure (121°C) for 15 min.

Preliminary screening

The A. niger culture was screened qualitatively for citric acid production by plate method on czapeck dox agar (contained (g/l): Sucrose: 20; NaNO³: 2; K²HPO⁴: 1; KCl: 0.5; MgSO.7H²O: 0.5; FeSO⁴.7H²O: 0.01 and agar: 15. Streptomycin (30μg/ml) was added to the above medium after sterilization and cooling to suppress bacterial growth, containing Bromocresol green as indicator for citric acid detection. The medium was prepared by dissolving all of the ingredients except agar; the pH was maintained at 6.0. Then, the medium was sterilized in the autoclave after the agar addition [6-7].

Citric acid production in submerged culture

The medium used was Czapek-Dox broth medium. The experiments were performed in shaking incubator using 50 ml of Czapek-Dox broth medium contained in 250 ml Erlenmeyer flask. All media were sterilized at 121°C for 15 min after cooling at room temperature and seeded with 1ml of inoculums (conidia suspensions). The flasks were incubated in a rotary shaker operating 100 rpm at 28°C. After incubation periods, the culture broth from each flask was filtered through Whatman filter paper for mycelia separation, dried and the yield of dry fungal biomass was recorded. The clear culture filtrate was employed for determination of citric acid and recorded final pH values.

Effect of some cultural conditions on extracellular citric acid production by Aspergillus niger

Eight factors were investigated to study their effects on citric acid production by A. niger. Those factors were, incubation periods at 24, 48, 72, 96, 120, 144 hrs; incubation temperature at 12, 20, 28, 36 and 44°C; initial pHs 3, 5, 8 and 10; Inoculum Size at (1.0, 2.0 and 3.0) 6 x10 spores/ml carbon source: sucrose, maltose, glucose, fructose, lactose and arabinose with initial concentration of carbon source (3% w/v); selected carbon source concentrations at 20, 25, 30, 35, 40 g/l; nitrogen source: NaNO3 (2g/l), NH⁴NO³ (1.9 g/l), KNO³ (2.4 g/l), NH⁴Cl (2 g/l), (NH⁴)²SO⁴ (2.1 g/l), glycine (1.8 g/l), or peptone (2.64 g/l); selected nitrogen source at 1, 2, 3, 4 or 5 g/l; Phosphorus Source (1.0 g/l) :NaH²PO⁴, Na²HPO⁴, Na3PO4, KH²PO⁴ and alcohol source (1%) Methanol, Propanol and Ethanol; selected alcohol concentrations at 1.0, 2.0, 0.0, 4.0, 0.0% added after sterilization. Triplicate flasks containing the optimized Czapek-Dox,s medium were prepared. The medium was inoculated with fungal discs and incubated under the previous successive conditions.

Usage of raw materials for citric acid production Raw material sources

Five different agricultural waste products (pineapple, sugar cane bagasse, sugar cane molasses, dates molasses, potato peels) used in this study was kindly obtained from various sources, sugarcane bagasse from sugarcane juice shops; potato peel from Saudi factory for the manufacture of potato and food – Riyadh - third industrial region; pineapple peel from vegetables markets – alraboa – Riyadh; dates molasses from Tuwaijri farm – Qassim and sugar cane molasses from the United Company for sugar – Jeddah.

Preparation of raw materials

Samples of sugar cane bagasse, potatoes peels and pineapple peels were dried in outside air (at the roof) for a week with a daily continuous stirring. After the samples completely dried, were milled using blinder, then sieved using sieve with narrowness holes. All samples were kept in clean bottles at a temperature of 25-28 until use.

Chemical analysis of the raw materials

All wastes samples were dried in oven at 50°C. For CHNS (carbon, hydrogen, nitrogen and sulfur) analysis, the samples were passed through 75 Micron Sieve and CHNS were analyzed by high combustion method using PERKIN ELMER Series II, CHNS/O Analyzer. Phosphorus (soluble) was analyzed by Ascorbic Acid method using HACH DR/U 4000 Spectrophotometer. Soluble components of Potassium, Calcium and Magnesium were analyzed by Ion Chromatography using Shimadzu Ion Chromatograph.

Raw materials and saccharification

The saccharification of waste materials used for the production of citric acid consisted of 5% in distilled water (w/v or v/v) of selected raw materials (i.e. sugarcane bagasse, pineapple peel, dates molasses and sugarcane molasses) except only 3% (w/v) of potato peel because of high viscosity of the suspended materials. Autoclaving (sterilization) of all prepared solutions were conducted for mild hydrolysis the raw materials. After cooling, the suspended insoluble materials were removed by filtration and the obtained filtrates were used in preparation of fermentation media for citric acid production.

Raw materials and production medium

Fermentation experiments were performed in 250 ml Erlenmeyer flasks containing Czapek-Dox broth medium components dissolved in 50 ml of the previous prepared filtrate (5 or 3% of the waste), then they were cotton plugged and sterilized at 15 lbs for 15 minutes at 121°C. Flasks were inoculated with isolated fungal, 1 ml of spores suspension used as inoculums. The flasks were incubated in rotary shaker operating at 100 rpm at the different temperatures for 5 days. After that the mycelia separation by filtration to assay the biomass dry weight and the filtrate was analyzed for pH and citric acid amounts.

Molecular identification of fungal isolates

Identification of Aspergillus niger species was identified according to Gilman, Joseph 1998, then identification was further confirmed using nuclear ribosomal DNA internal transcribed spacer (ITS) sequencing by CEQ 8000. The genomic DNA was obtained using the protocol of GeneJet Plant genomic DNA purification Kit (Thermo) # K0791 (http://www.thermoscientificbio.com/). Internal transcribed spacer (ITS) region of 5.8S rRNA was amplified using the primers ITS1 (5´TCC GTA GGT

GAA CCT TGC GG 3´) and ITS4 (5´TCC TCC

GCT TAT TGA TAT GC 3´). PCR sequencing of the amplified product was carried out at GATC Company (Germany). The resulting sequence was entered into the BLAST algorithm of National Centre of Biological Information (NCBI) database to obtain closely related phylogenetic sequences. A phylogenetic tree was constructed using MEGA 6 software. The obtained sequence was then submitted to the GenBank of National center for biotechnological information (NCBI) database. A strain identifier and an accession number were obtained for each isolate.

Results and discussion

Microfloral picture

The aim of this part is to isolate and get pure cultures of local fungus, A. niger strains. From twenty soil samples collected from different sites in Riyadh region, several Aspergilli strains were isolated and purified.

Among the colonies characteristics observed, the identification process was performed based on their morphological characteristics. Aspergillus niger, it was confirmed by Gillman fungi Manual Based on morphological characterization, ninety six isolates of Aspergillus niger from different sources were selected as mentioned in Table (1), the twenty sites and number of A. nigerisolates from each site with an abbreviation cods.

Preliminary screening of A. niger isolates producing citric acid

The experiment was conducted to choose citric acid producing-fungal isolates using plates of Czapek-Dox agar containing Bromocresol green as an indicator and yellow zones indicated citric acid production. To check from efficacy of the obtained isolates for citric acid production, the experiment was conducted using plates of Czapek-Dox agar containing Bromocresol green as an indicator and yellow zones indicated citric acid production. Ninety six isolates of Aspergillus niger were tested for their capability to produce citric acid using an indicator plates medium. All A. niger isolates understudying were varied in their capability to produce citric acid and in productivity amount at different time intervals i.e. 24, 48, 72, 96 hours incubation on indicator plats medium through the diameter of the formed yellow zone (mm). Most of A. niger isolates (70 isolates) reached to the maximum acid production, where yellow color filled the plates medium (90 mm), after 72 hour incubation, represented 66 72.9 % of the total isolates (Figure 1). However, some isolates (14 isolates) were slowly released acid in the indicator medium and acid production gave a maximum yellow zone diameter (90 mm) after 96 h incubation, represented 14.6 % of the total isolates. In the same time, twelve A. niger isolates proved to be the best citric acid producers, where gave a widest yellow zone diameter (90 mm) after only 48 hour incubation, represented 12.5 % of the total isolates.

On the basis of obtained results of the preliminary screening through used an easily and quick technique, It could be able to select six A. niger isolates, that obtained from different isolation sources, gave highest productivity of citric acid, ranged from 49 to 60 mm zone diameter, after 24 hours incubation. The favorable isolates of A. niger for citric acid production were SWH4, KSUD4, FTH1, FQWA1, FDRA3 and FAR4 selected for further experiments. In view of other workers, the citric acid is being produced by microorganisms during fermentation through their metabolic citric acid cycle [6, 8]. When microbes concern, there are many organisms which are producing citric acids. However, fungus is considered to be suitable producer of citric acid, especially, Aspergillus niger is utilized as much as it produces.

Selection and time course

One of the most important fungi used in industrial microbiology, Aspergillus niger, has been employed for many years for the commercial production of citric acid. However, the worldwide demand for citric acid is increasing faster than its production and more economical processes are required [9]. Citric acid production synthesis by fermentation is the most economical and widely used way of obtaining this product. More than 90 % of the citric acid produced in the world is obtained by fermentation, which has its own advantages: operations are simple and stable, the plant is generally less complicated and needs less sophisticated control systems, technical skills required are lower, energy consumption is lower and frequent power failures do not critically affect the functioning of the plant [10]. Especially, the submerged technique is widely used for citric acid production. It is estimated that about 80 % of world production is obtained by submerged fermentation. This fermentation process employed in large scale requires more sophisticated installations and rigorous control. On the other hand, it presents several advantages such as higher productivity, lower labour costs and lower out in batch, fed batch or continuous systems, although the batch mode is more frequently used. Normally, citric fermentation is concluded in 5 to 12 days, depending on the process conditions [11]. In addition, the submerged process has become the method of choice in the industrialized countries, because it is less labor intensive, yields higher production rate, and uses less space [4]. As well, the choice of a suitable strain is of great importance. Among the different fungal isolates of A. nigerhas the highest ability to produce citric acid [12].

Selection off citric acid producers

Therefore in this part, the best six citric acid producingfungal isolates (which selected after primary qualitatively screening for their potential as citric acid producers on plates of Czapek-Dox agar with Bromocresol green as an indicator) were selected from a group of indigenous A. niger population to study their capability for citric acid production in submerged culture, shake flask cultures, during three days incubation. The selected six A. niger isolates are FDR, SWH, FAR, FQW, KSUD and FTH. In addition to A. niger EMCC132 was used as a reference fungal strain for citric acid production. Data recorded in Table (2) shows that the production of citric acid by six locally isolated and selected reference strain A. niger EMCC132 were significantly varied. Citric acid production was in the range from 4.52 to 16.34 mg/10 ml culture filtrate. A highest significant citric acid concentration (16.34 mg/10ml) was produced by A. niger EMCC132 followed by KSUD, FQW and FDR that produced not significantly different amounts of citric acid, 9.98, 9.07 and 8.84 mg/10ml respectively. Dry fungal mass measurements were ranged from 0.24 to 0.41 g/50 ml. It noticed that no correlation between fungal biomass of all tested isolates and produced citric acid amounts (Table 2). The pH measurements of the tested fungal cultures after 3 days incubation reduced and reached to acid range (3.19 – 3.63) as shown in Table (2). The submerged fermentation process is desirable and fermentative production of citric acid arises from a primary energy metabolism although it is non-growth associated [13]. Based on the productivity of citric acid under these conditions, higher two local A. niger isolates, KSUD and FQW, were selected for further experiments comparing to the reference strain, A. niger EMCC132.

Time course of citric acid production under submerged fermentation condition

Although citric acid can be produced by submerged and solid state fermentation, industry still prefers submerged fermentation because of its continuous operation [14] [15]. For that, submerged fermentation in shaking flask cultures was used in all over current experiments for citric acid production.

Means in the same column followed by the same letter are not significantly To determine the effect of fermentation period on citric acid productivity and to define the optimum incubation time, fermentation was carried out for various time periods (1 – 6 days). As shown in figure 3. Shaking flask cultures were incubated at 28˚C. Samples were withdrawn every 24 hours. Citric acid production, change in pH and dry fungal biomass was also determined. The obtained results of citric acid production, pH values and fungal biomass that determined in shake flask cultures of the three molds under investigation (A. niger KSUD, FQW and EMCC132) are expressed in Table (3). The obtained data show that citric acid amounts produced by all A. niger tested isolates were gradually and significantly increased by increment of the incubation periods.

A. niger FQW was grown on Czapek-Dox broth medium and gave maximum biomass, 0.397 mg DW/50 ml, at the 6th day of incubation. In the same time, citric acid was detected and quantified starting from the 1st day and reached to a statistically maximum value, 13.19 mg/10ml, at 5th day shaking incubation (Table 3). Also the fungal biomass was grown progressively and recoded highest growth after 6 day incubation that was 0.40 g DW/50 ml.

In contrast, pH value was gradually reduced that recorded th lowest value, 2.29, at the 6 day. The recorded results in Table (3) indicate that the time course analysis of submerged culture of A. niger KSUD was slowly propagated in the first day, then gradually increased and gave a maximum fungal biomass (0.40 g DW/50 ml) in the 4th day of incubation. Although the fungal growth started reduces at 5th day incubation, citric acid amount increased and assayed statistically high value, 16.91 mg/10ml among 5 days incubation. The estimated pH values appeared to obviously decrease after first 24 hours and gradually reduced in parallel with acid production, that recorded lowest value, 2.43, at the 6th day incubation (as mentioned in Table 3). Comparing the previous data different based on LSD at p = 0.05 according to Duncan’s of local fungal isolates with the reference one, it was found that A. niger EMCC132 was produced highest amount of citric acid and reached to 25.51 mg/10ml at the 6th day incubation (Table 3), moreover, gave a maximum growth, 0.38 mg DW/50 ml, where pH values gradually reduced and recorded 2.73 after 6 days. Although, an international strain A. niger EMCC132, used as a reference fungal strain for citric acid production, had a pronounced capability to produce highest amount of citric in flask culture after 6 days incubation. However in case of A. niger KSUD and FQW, the maximum amounts of citric acid were at the 5th day and the higher amounts of citric acid at sixth day were insignificantly increased. But the variation was not significantly. Moreover, the quantity of citric acid produced varies with both microorganism and fermentation period. According to the pervious results, it is indicated that among all the tested fungal strains, citric acid production was varied. Citric acid was ranged between 14.09 – 25.51 mg/10ml. The tested fungal strains were produced citric acid in the following order, A. niger EMCC132 >A. niger KSUD >A. niger FQW. On comparing the time course of the citric acid production in the fermentation medium to that of incubation period where citric acid amounts reached statistically to maximum values on 5th day of fermentation. The optimal time of incubation for maximum citric acid production varies with both the organism. The obtained results of the time course analysis of tested fungal cultures indicated that optimal time incubation for the maximum citric acid production varies with both the tested fungal strains under the same condition and ranged between 5-6 days incubation period. Similarly, it was found by [16] who reported that viability increases with time of incubation, but higher production of citric acid was achieved and reached maximum at the stationery phase in less than 7 days incubation. While study of [17] revealed that the optimum time course is 192 hr (8 days) for citric acid production. In addition, our

result in agreement with the most recent study [18] where the quantity of citric acid produced varies with the type of tested microorganism. The rate of citric acid biosynthesis was studied and the maximum yield of citric acid (0.61 g/L) was after 5 days of fermentation. Extension of the fermentation period brought about depletion in the yield of citric acid produced. In batch fermentation of citric acid, the production started after a lag phase of one day and reached maximum at the onset of stationary phase or later. It might be due to the decreased available nitrogen in fermentation medium, the age of fungi, and depletion of sugar contents. These explanations were also in

acid production

In developing a fermentation process for obtaining a valuable product, the optimization of cultivation conditions and selection of appropriate substrates and the most favorable of their concentrations have primary importance due to their impact on the economy and feasibility of the process. Many studies have been intended to obtain high-yielded production using optimization processes with low cost as possible. The improvement strategies used in citric acid production by corroboration with earlier reports by [19] stated that much time causes the decrease of nitrogen and sugars in the substrate, thereby a reduction in citric acid production. This implies that the fermentation period used was suitable for the production of citric acid as higher yield is harvested at early stage thereby cutting down the cost needed to maintain the fermentation for longer time. The local fungal strains, A. niger KSUD and FQW which gave the maximum amounts of citric acid at the 5th day, therefore, this incubation period was applied in the further experiments.

classical optimization methods for citric acid production based on varying one-factor-at-a time [11, 17, 20].

Effect of incubation temperature

Temperature is known to influence the metabolic rate of the organism involved in the process, which in turn determines the amount of the end product. Therefore, the optimum temperature for citric acid production was defined by assaying the citric acid amounts at different incubation temperatures ranging from 12 to 44˚C, after 5

rpm for 5 days. Means in the same column followed by the same letter are not significantly different based on LSD at p = 0.05 experiment results. Data represented in Table (4) show a significant increment in citric acid production with the increase of incubation temperature up to 28˚C for the three tested fungal strains (A. niger FQW, KSUD and EMCC132) and thereafter, higher temperature resulted in a gradually lowering of citric acid formation. Maximum citric acid production by tested fungal strains, A. niger FQW, KSUD and EMCC132 were reached to 11.26, 15.25 and 22.02 mg/10ml, respectively at 28˚C. Obtained results indicated also that changing in pH values was accompanied by a noticeable of the yield of citric acid. Moreover, growth of fungal strains were significantly lowered at both 12 and 44˚C, but changing in fungal growth were insignificantly different in the temperature range 20-36˚C (Table 4). In general, temperature obviously influences the metabolic activity of cells. Where, the obtained results for growth of fungi at various incubation temperatures reveal that all tested A. niger according to Duncan’s multiple range test.

Moreover, Aspergillus niger has grown well at 30°C and produced maximum amount of citric acid (80.68 g l-1) followed by 20°C with 74.21 g l-1 of citric acid [17]. Also, [23] have reported maximum amount of citric acid production (99.56 ± 3.59 g l-1) achieved by Aspergillus niger GCBT7 at 30°C. [24] has also reported that maximum citric acid production was recorded at 30°C using Aspergillus niger NCIM 705. The effect of temperature plays an important role in the production of citric acid by Aspergillus niger [25]. According to obtained data in our experiments, incubation temperature strains could not tolerate higher temperature, that was appeared in the dry weight of the fungal biomass. Incubation temperature at 28˚C was considered as an optimum cultivation temperature for the growth. Temperatures between 25-30˚C are usually considered optimal for fungi. However, a temperature above 35˚C inhibits mycelial growth and favours oxalic accumulation. There is an exception for the thermo tolerant A. niger cultures which can produce citric acid even at 29-40˚C [21]. In addition, [22] reported that the temperature of fermentation medium is one of the critical factors that have a profound effect on the production of citric acid. Temperatures lower than 27°C slowed down growth and production substantially. When the temperature of medium was low, the enzyme activity was also low, giving no impact on the citric acid production. However when the temperature of medium was increased above 30°C, the biosynthesis of citric acid was decreased.

of 28°C was selected for further experiments for improvement of citric acid production.

Effect of initial pH

The metabolic activity of fungus is very sensitive to pH level of media. The initial pH of the medium was found to have an impact on citric acid production by A. niger [26]. Most filamentous fungi are observed to grow well under slightly acidic conditions, ranging from 3 to 6, but some fungi are able to growth at a pH value below 2 to better compete against bacteria [27].

The favorable initial pH is one of the most important steps for the successful progression of citric acid

days. Means in the same column followed by the same letter are not significantly different based on LSD at p = 0.05 according to evaluate the effect of the initial pH of the nutrient solution on citric acid production by A. niger local strains and the reference strain. The influence of initial pH value of the culture medium on growth and citric acid production by Aspergillus niger strains (FQW, KSUD and A. niger EMCC132) was investigated at initial pH values 3, 5, 6.5, 8 and 10. The initial pH values were adjusted by diluted acid or alkali. The flask cultures were incubated on rotary shaker for five days at optimal o temperature 28 C attained in the foregoing experiments.

The results on the effects of different initial pH values on citric acid production, mycelial dry weight and changing in cultures’ pH by A. niger strains are recorded in Table (5). The obtained results indicated that maximum citric acid concentrations by A. niger FQW, KSUD and EMCC132 (11.26, 15.25 and 22.27 mg/10 ml, respectively), was obtained at pH 6.5. Moreover, a very sharp decrease in citric acid yield was observed when the initial pH values were increased to 8.0 and to 10.0. This reduction was also significantly noticed and accompanied by a similar reduction in mycelia dry weight. The effect of the initial pH of nutrient solution on citric acid production was evaluated and concluded that the initial pH had a considerable effect on the growth of A. niger strains and production of citric acid. From the results of our work, it could be concluded that citric acid production and growth yield were strongly affected by initial pH level and apparently the maintenance of a favorable initial pH 6.5 is essential for successful production of citric acid 6 In Czapek-Dox broth medium.Inoculum density was ~1.0X10 spore/ml. Cultures were grown with shaking at 100 rpm at 28oC for 5 Duncan’s multiple range test.

in further experiments. In general, initial pH value of the culture medium mainly controls the fungal growth and citric acid production as well. Different pH optima have been report by several researchers. The obtained results are in line with [28, 29] studied the influence of low and high initial pH values and reported that decrease in initial pH value lower than 3.5 results in reduction in citric acid production. It might be due to that at low pH, the ferrocyanide ions were more toxic for the growth of mycelium. A higher initial pH leads to the accumulation of oxalic acid. Also Oxalic acid is an undesirable byproduct of citric acid fermentation and is considered to be a main impurity in the commercial citric acid process from A. niger at higher initial pH [30]. In addition, [23] insured that the maintenance of a favorable pH is very essential for the successful production of citric acid. Effect of different pH (4.5-7.0) on the citric acid production was studied and the highest value of citric acid yield (96.12 ± 3.5 g/l) when initial pH of the fermentation medium was kept at 6.0 by culture of Aspergillus niger GCBT7. A lower initial pH was reported to inhibit the growth of A. niger and is thus expected to negatively affect citric acid production. Similar results were explained in previous studies. Where the effect of different initial pH (4.5 - 7.0) of the fermentation media, it was found that maximum citric acid (47.63 g/l) were obtained when the initial pH of the fermentation medium was adjusted to 5.5. Decrease in pH caused reduction in both protein and citric acid yield [31].

Effect of inoculums density

Spore inoculum level is another parameter that influences citric acid fermentation and improvement. The objective of the present experiment was to evaluate the effect of the inoculums density on citric acid production by A. niger local strains and the reference strain. The influence of inoculums density on fungal growth and citric acid production by Aspergillus niger strains (FQW, KSUD and A. niger EMCC132) was investigated. The conidial density (from 5 days old cultures) of the used inoculums 6 were (1.0, 2.0 and 3.0) x10 spores/ml. The flask cultures were incubated on rotary shaker. The initial pH, incubation temperature and incubation period were maintained at optimal levels that attained in the foregoing experiments. Data present in Table (6) obviously indicate that citric acid yields by the three strains of A. niger were significantly increased by increment of the conidial density in the inoculums after 5 days of flask o fermentation at 28 C. Among different density of used inoculums, citric acid productivity was proportionally increased by conidial density increasing. Where citric acid yields were reached to the highest values with 6 3.0X10 inoculums size of all tested A. niger strains. Changing in pH values and fungal growth of A. niger FQW, KSUD and EMCC132 after 5 days of shaking flask cultures were not significantly appeared by increasing the inoculums density (as shown in Table 6). According to 6 obtained data, inoculums size 3.0X10 spores/ml was selected for further experiments for improvement of citric acid production. In agreement with our findings of 4 9 inoculums density between 1x10 to 1x10 spores/ml was found to be suitable for citric acid production by Aspergillus niger in submerged fermentation [32, 33]. According to the previous literatures, citric acid production is known to be affected by inoculums density. About 1.0% vegetative inoculums are adequate for optimal production of citric acid. The citric acid yield was lower at 5% of the vegetative inoculum [34]. This may be due to the clumping of Aspergillus niger at higher inoculum concentration. The increase in the number of inoculated spores primarily increases the level of acid production; however, on the long run this increase tends to force the process toward cellular crowding and to favor the consumption of sugar for biomass reproduction, thus resulting in a reduction in production of citric acid [31]. The excessive reduction in the level of inoculation also causes prolongation in the adaptation phase, therefore decreasing an ideal outcome. This result suggests that there appears to be an optimum amount of inoculum required for citric acid production. This disagrees with other findings who reported that the 5% conc. Of inoculum was optimum for maximum citric acid production (96.55 g l-1) using Aspergillus niger under submerged fermentation condition. The 5% conc. of the inoculums is seems to be optimum for citric acid population over-crowding, higher nutrient competition and rapid exhaustion of nutrients. Up to a specific limit, metabolite production generally increases with inoculum density [35]. At the lower inoculum density, metabolite production drops and contamination risks increase due to an insufficient cell population. In addition, the highest level of citric acid is produced when the age of spores is less than seven days, because older spores tend to consume the initially produced acid before the fermentation process is completed [36]. Otherwise, the increase in the number of inoculated spores primarily increases the level of acid production, however, in the long run this increase tends to force the process toward cellular crowding and to favor the consumption of sugar for biomass reproduction, thus resulting in a reduction in production of citric acid. The excessive reduction in the level of inoculation also causes prolongation in the adaptation phase, therefore decreasing an ideal outcome [37, 38]. By studying the effect of vegetative inoculum size (1–5%) on citric acid production by Aspergillus niger. They mentioned that the maximum citric acid production of 0.53 g/L was obtained with 3% inoculum size. As the inoculum size increased, citric acid production decreased [18].

Effect of carbon sources

Citric acid accumulation is strongly influenced by the type of carbon source. The carbon source for the citric acid fermentation has been the focus of much study, frequently with a view to the utilizing polysaccharide sources. Citric acid accumulation is strongly affected by the nature of the carbon source. The presence of easily metabolized carbohydrates has been found essential for good production of citric acid [10]. In general, only sugars that are rapidly taken up by the fungus allow a high final yield of citric acid. Polysaccharides, unless hydrolyzed, are generally not a useful raw material for citric acid fermentation because they are broken down too slowly to match the high rate of sugar catabolism required for citric acid production. The slow hydrolysis of polysaccharides is due to the low activity of the hydrolytic enzymes at the low pH that is necessary for producing citric acid [44]. There is several factors affect citric acid production and among these, carbon source has been found to play an important role in citric acid biosynthesis. So that for enhancing the production of citric acid in the present study, A. niger strains (A. niger FQW, KSUD and EMCC132) were cultivated on the Czapek-Dox broth medium amended with different carbon sources (sucrose, maltose, glucose, fructose, lactose and arabinose) in flask shack culture at 28˚C for 5 days to search for inducers of the citric acid overproduction.

In Czapek-Dox broth medium.Initial pH was 6.5. Cultures were grown with shaking at 100 rpm at 28 column followed by the same letter are not significantly different based on LSD at p = 0.05 according to Duncan’s multiple range test.

Data represented in Table (7) indicated that maltose followed by sucrose have been employed as the preferred carbon sources for citric acid production by A. niger FQW (23.02 and 17.97 mg/10 ml, respectively) and A.

niger KSUD (20.99 and 16.25 mg/10 ml, respectively) however in the case of reference strain A. niger EMCC132, maltose followed by glucose as the preferred carbon sources for citric acid production, 41.74 and 24.61 mg/10 ml, respectively. All of the other used sugars showed lower citric acid yield. Moreover, arabinose and lactose had shown significantly an inhibitory effect on citric acid production. In general, maltose served as the best carbon source for citric acid production by all tested A. niger strains (A. niger FQW, KSUD and EMCC132).

It is clear that citric acid production by fungal tested strains is dependent on nutritional conditions and especially on carbon source. The fungal biomass was varied widely among the carbon sources utilized. Where, the highest biomass values were obtained in the presence of maltose and sucrose, while the fungal growth were suppressed in the presence of lactose and arabinose that is reflect on the acid production. Among the used carbon substrates (which are mostly sugars of hexoses, mannoses, mono- or disaccharide nature), the previous studies were varied. For instance, [45] explained that the nature of the sugar source has marked effect on citric acid production by A. niger. He also showed that sucrose was the most favorable carbon source followed by glucose, fructose and galactose. Galactose contributed to a very low growth of fungi and did not favor citric acid accumulation. Other sources of carbon such as sorbose, ethanol, cellulose, manitol, lactic, malic and aacetoglutaric acid, allow a limited fungal growth and low citric acid production. Starch, pentoses (xyloses and arabinoses), sorbitol and pyruvic acid slow down growth, though the production is minimal [46]. Sucrose is the C for 5 days. Means in the same although glucose, fructose and maltose have also been used as substrates for citric acid production [46]. Generally, the presence of carbohydrates which are rapidly taken up by microorganisms has been found essential for a good production of citric acid [47]. The superiority of sucrose over glucose and fructose has been documented by [48]. Moreover, among the easily metabolized carbohydrates, sucrose is the most favourable carbon source followed by glucose, fructose and galactose for citric acid production [47, 49, 9]. However, it is also found that glucose is not suitable for higher production of citric acid [50]. In contrary of the obtained results [51] found that maximum citric acid production was recorded in the glucose incorporated medium and the low amount of citric acid was recorded in the maltose supplemented medium.

Maltose sugar which gave the highest citric acid production was used in further experiments for optimization of other medium components.

Effect of nitrogen sources

Nitrogen constituent has a profound effect on citric acid production because nitrogen is not only important for metabolic rates in the cells but it is also basic part of cell proteins. This report agreed with [25] that fermentation media for citric acid biosynthesis should consist of substrates necessary for the growth of microorganism primarily the carbon, nitrogen and phosphorus sources. This experiment was performed to evaluate the influence of different organic (urea, peptone, beef extract) and inorganic (sodium nitrate as a control, ammonium nitrate, ammonium chloride, ammonium sulfate) nitrogen sources on citric acid production by tested strains of A. niger (FQW, KSUD and EMCC132) at the previous favorable parameters.

Aspergillus niger strains on various carbon sources in shake flask culture. Data

In modified Czapek-Dox broth medium containing sucrose as control.Initial pH was 6.5. Inoculum density was ~2.0X10 A. niger Carbon pH c FQW Control 2.95 ±0.05 d Maltose 2.59 ±0.02 cd Glucose 2.78 ±0.08 c Fructose 2.93 ±0.07 b Lactose 3.60 ±0.12 a Arabinose 4.24 ±0.02 0.05 c KSUD Control 2.78 ±0.01 c Maltose 2.95 ±0.11 c Glucose 2.77 ±0.12 c Fructose 2.84 ±0.07 a Lactose 4.51 ±0.09 b Arabinose 3.64 ±0.14 c EMCC132 Control 2.94 ±0.01 c Maltose 3.16 ±0.19 c Glucose 3.43 ±0.10 c Fructose 3.40 ±0.06 b Lactose 5.15 ±0.64 a Arabinose 5.10 ±0.00 Cultures were grown with shaking at 100 rpm at 28C for 5 days.

The results were given in Table (8) demonstrated that peptone as an organic nitrogen significantly enhanced citric acid production and showed superiority over all organic and inorganic nitrogen sources. Where maximum citric acid yield were reached to 49.78, 56.07 and 52.23 mg/10 ml by A. niger FQW, KSUD and EMCC132, respectively. Mostly presences of organic nitrogen sources acted as enhancer for both citric acid production and fungal growth. However, inorganic nitrogen supplements had varied impact on production of the citric acid by all tested strains. Moreover, ammonium chloride significantly suppressed citric acid production that gave only 5.53, 5.32 and 3.65 mg/10 ml by A. niger FQW, KSUD and EMCC132, respectively (as shown in Table 8). Mostly complex nitrogen sources were better than inorganic nitrogen sources. This may be due to other nutrients and growth enhancers present in such organic b b a a c c e f d e d d cd b a a c c b c d d d d a c b a c b d d d e e f 6 spore/ml.

ncan’s high and reached to a maximum values in the presence of organic nitrogen source especially peptone and lowest fungal growth in the presence of inorganic nitrogen source, ammonium chloride. Citric acid production is directly influenced by the nitrogen source. Physiologically, ammonium salts are preferred, e.g. urea, ammonium sulfate, peptone, malt extract, etc. Acid ammonium compounds are preferred because their consumption leads to pH decrease, which is essential for the citric fermentation. However, it is necessary to maintain pH values in the first day of fermentation prior to a certain quantity biomass production. Urea has a tampon effect, which assures pH control [43]. The effect of nitrogen source on citric acid production has been intensively studied in solid substrate and submerged fermentation. Ammonium chloride, ammonium sulphate, ammonium nitrate, peptone and yeast extract were the

Aspergillus niger strains on various nitrogen sources in shake flask culture. Data

In modified Czapek-Dox broth medium.Initial pH was 6.5. Inoculum density was ~2.0X10 by fungus [56] Other sources of nitrogen that have been used include urea and yeast/malt extract [46]. Complex media such as molasses are rich in nitrogen-containing compounds and rarely need to be supplemented with a nitrogen source. The high purity media that are used mainly in research laboratories are generally A. niger Nitrogen Sources pH

Strains

FQW Control (Sodium nitrate) 2.96 Ammonium nitrate 2.69 Ammonium chloride 1.99 Ammonium sulfate 2.10 Beef extract 2.88 Peptone 2.77 Urea 4.25 0.05 KSUD Control (Sodium nitrate) 2.51 Ammonium nitrate 2.77 Ammonium chloride 2.09 Ammonium sulfate 2.86 Beef extract 2.59 Peptone 2.62 Urea 3.38 0.05 EMCC132 Control (Sodium nitrate) 3.84 Ammonium nitrate 3.10 Ammonium chloride 2.06 Ammonium sulfate 2.84 Beef extract 3.17 Peptone 3.33 Urea 4.07 0.05

Effect of phosphorus sources

Presence of phosphate in the medium has a great effect on the yield of citric acid. Low levels of phosphate have positive effect on citric acid production. This effect acts at the level of enzyme activity and not at the level of gene expression. On the other hand, the presence of excess of phosphate leads to a decrease in the fixation of carbon dioxide, which in turn increases the formation of certain sugar acids, and the stimulation of growth [9, 25, 43]. Several factors affect citric acid production and among of these, phosphorus source has been found to play an important role in citric acid biosynthesis. So for enhancing the production of citric acid in the present study, A. niger strains (A. niger FQW, KSUD and EMCC132) were cultivated on the Czapek-Dox broth medium amended with different phosphorus sources (NaH²PO⁴, Na²HPO⁴, Na³PO⁴ and KH²PO⁴) in the same supplemented with ammonium salts, particularly ammonium nitrate and ammonium sulfate, to provide the necessary nitrogen. An advantage of using ammonium salts is that the pH declines as the salts are consumed and a low pH is a requirement of citric acid fermentation [44].

b b b c c d d g f b e de bc f e bc a a a d c b b d cd c e e e f c de f bd b b bd a a a d c b d b d d d f f e e b b d e d c a a a c c 6 ncan’s amount of the initial phosphorus source, K²HPO⁴ (1.0 g/l) in flask shack culture at 28˚C for 5 days to search for inducers of the citric acid overproduction. Data represented in Table (9) indicated that Na³PO⁴ significantly employed as the preferred phosphorus source for citric acid production by A. niger FQW (96.60 mg/10 ml) and A. niger KSUD (77.81 mg/10 ml) however in the case of reference strain A. niger EMCC132, KH²PO⁴ was significantly as the preferred carbon sources for citric acid production, 91.33 mg/10 ml. All of the other forms of phosphate showed lower citric acid yields. Moreover, phosphate in the form of Na²HPO⁴ had shown significantly an inhibitory effect on citric acid production. In general, Na³PO⁴ served as the best phosphate form for citric acid production by local tested A. niger strains (A. niger FQW and KSUD). It is clear that citric acid production by fungal tested strains is dependent on nutritional conditions including phosphorus

Aspergillus niger strains on various phosphorus sources in shake flask culture. Data

In modified Czapek-Dox broth medium. Initial pH was 6.5. Inoculum density was ~2.0X10 salt source. The fungal biomass was varied widely among the various phosphorus sources utilized (Table 9). From results of the present experiment, phosphorus salt in the form Na PO has been recommended for the maximal 3 4 citric acid production from A. niger FQW, KSUD and EMCC132 strains and was selected as a control for the subsequent experiments. Phosphate is known to be essential for the growth and metabolism of A. niger [59].

The concentration of exogenous phosphorus in medium had significant effect on cell multiplication and metabolite production [43]. Presence of phosphate in the medium has a great effect on the yield of citric acid. In A. niger Phosphorus Sources pH

Strains

a FQW Control (K²HPO⁴) 2.54 ±0.04 b Na HPO 2.43 ±0.03 2 4 b Na3PO4 2.44 ±0.02 b NaH PO 2.46 ±0.01 2 4 b KH²PO⁴ 2.45 ±0.01 0.05 a KSUD Control (K HPO ) 2.33 ±0.01 2 4 c Na²HPO⁴ 2.24 ±0.00 bc Na3PO4 2.27 bc NaH²PO⁴ 2.25 ab KH PO 2.29 2 4 bc Control (K HPO ) 2.86 EMCC132 ^{2 4} b Na²HPO⁴ 2.90 ±0.02 bd Na3PO4 2.86 cd NaH PO 2.82 2 4 a KH²PO⁴ 2.96 ±0.00 0.05

Effect of enhancer (Alcohol addition)

Among all of the previous studied parameters were utilized as optimum conditions which led to enhance of citric acid production by A.niger local strains FQW and A. niger KSUD (Table 10). Besides the optimization of basal environmental parameters and nutrients as well as their concentrations, higher citric acid production levels can be obtained by applying stimulators, such as alcohols and chelating agents according to [61]. Several reports have shown the stimulatory effects of additives on fungal citric acid accumulation and secretion [26]. To improve citric acid production, stimulators have been used, such as organic solvents, phytate and lipids [26, 62, 63]. The objective of the present research was to determine the potential increase in citric acid production by A. niger selected strains (A. niger FQW, KSUD and EMCC132) in the present study, phosphorus salt in the form Na PO 3 4 was the favorite phosphorus source by the tested fungal stains however certain previous research mentioned that Potassium dihydrogen phosphate has been reported to be the most suitable phosphorous source. For instance KH PO and K HPO proved to be the best phosphorus 2 4 2 4 source [56]. It was found that phosphorous at concentration of 0.5 to 5.0 g/L was required by the fungus in a chemically defined medium for maximum production of citric acid [60]. In addition, phosphorus limitation induced higher citric acid production and yield [58].

a d a e a a a c a b c c b e a a b b b d c c bc d a b b b d a 6 ncan’s ethanol and methanol. Data recorded in Table (10) indicate that after 5 days of fermentation, impact of alcohols addition on citric acid production by all tested A. niger strains was significantly varied. Where methanol had obviously pronounced stimulatory effect, on contrary propanol was suppressed citric acid production while the effect of ethanol addition varied among the tested strains. Without methanol, the control produced 82.24 and 80.58 mg/10 ml while the addition of methanol significantly stimulated citric acid production to 128.2 and 103.58 mg/10 ml by A. niger FQW and KSUD, respectively. By comparing the effect of both ethanol and methanol on biomass growth of tested local fungal strains it was observed that methanol has fruitful effect on biomass growth but there is no stimulatory effect of ethanol on biomass, which means that ethanol was solely used by A. niger FQW and KSUD

Aspergillus niger strains in the presence of various alcohol sources (1%) in shake

In modified Czapek-Dox broth medium. Initial pH was 6.5. Inoculum density was ~2.0X10 findings were also reported that Alcohols have been shown to act principally on membrane permeability in microorganisms by affecting phospholipid composition [64]. Other studies showed that alcohols stimulate citric acid production by affecting growth and sporulation on space organization of the membrane or changes in lipid composition of the cell wall [65] The obtained results are in harmony with pervious data that showed the inductive effect of methanol in the fermentation media for citric acid by A. niger [66, 50] the stimulation of citric acid

flask culture. Data represented as mean ± standard error.

A. niger Alcohol pH b FQW Control 2.61 ±0.04 a Propanol 3.01 ±0.07 b Ethanol 2.72 ±0.03 b Methanol 2.68 ±0.01 d KSUD Control 2.43 ±0.02 a Propanol 3.21 ±0.02 c Ethanol 2.54 ±0.00 b Methanol 2.67 ±0.01 c Control 2.90 ±0.01

EMCC132

a Propanol 3.42 ±0.02 a Ethanol 3.42 ±0.03 b Methanol 3.02 ±0.00

Utilization off agricultural wastes for citric acid production by A. niger strains

A. niger a filamentous fungus is most commonly used for citric acid production due to ease of handling, its ability to utilize varieties of substrates because of its welldeveloped enzymatic system in addition to its ability to ferment a variety of cheep raw materials and high yield of citric acid [51, 68, 69]. Utilization of refined sugars such as glucose, maltose and sucrose are the most commonly used substrates for commercial production of citric acid by fermentation process; while they are expensive and can be replaced by various cheap and abundant substrates like agro-industrial wastes or by-products [4]. Several raw materials can be employed successfully for citric acid production. However, there are some critical factors that should be taken into account such as costs or need of pretreatment for choosing the type of substrate [10]. Thus, the objective of this part was to adopt the use of certain local raw materials as a cheap medium for the production of citric acid by tested A. niger. In this production by methanol in synthetic media is affected by cultural conditions, and especially by the mold strain used. The age and the amount of mycelia inoculum which is probably a reflection of its surface area may be critical. They reported that these factors must be investigated in applying the effect of the alcohol in any individual case [63]. From results of this experiment, methanol had significant favorable effect for the maximal citric acid production by local fungal strains (A. niger FQW and KSUD) and selected for the subsequent experiments.

b c c d b b a a b b d d c c a a b b d c c c a a 6 ncan’s production technique, which is still the major industrial rout to commercial citric acid production nowadays, tested strains of A. niger are cultivated on a raw materialcontaining modified Czapek-Dox broth medium to produce citric acid. Citric acid productivity by A. niger tested strains (A. niger FQW, KSUD and EMCC132) was determined after 5 days growing cultures in which 5% (w/v or v/v) of selected raw materials (pineapple peel, sugarcane bagasse, sugarcane molasses and dates molasses) or 3% (w/v) of potato peel was supplemented to the fermentative medium either carbon free or carbon and nitrogen free. The estimated results present in Table (11) indicate citric acid production by tested fungal strains in the presence of selected raw materials as a sole carbon source. The results showed that sugarcane molasses was significantly superior for citric acid production (256.94 and 188.45 mg/10 ml) by A. niger FQW and A. niger EMCC132, respectively.

In modified Czapek-Dox broth medium.Initial pH was 6.5. Inoculum density was ~2.0X10

Molecular identification of fungal isolates

The identification of the isolates was further confirmed using nuclear ribosomal DNA internal transcribed spacer (ITS) sequencing. The traditional Sanger technology and the new 454 technology were combined for sequencing

6 ncan’s the PCR products. The obtained nucleotide sequence was deposited at the NCBI GenBank and a strain identifier was given to each isolate. Thus, they were identified as Aspergillus niger MH368137 as shown in figure 2.

Conclusion

Fermentation parameter for citric acid production by A. niger depend on the type of process. The fermentation conditions are key to high and consistent production of citric acid. In the present study, Aspergillus niger MH368137 isolated from Farm way Qassim supported maximum production of citric acid. The optimal conditions for the maximum citric acid production reached statistically 5 days at 28˚C; initial pH 6.5; inoculums size 3.0×106 spores/ml; shaking culture (100 rpm); maltose as a carbon source 40 g/L; peptone as a nitrogen source 3 g/L; Sodium phosphate as phosphorus source; 2% of methanol as enhancer. It was also observed in this study that Potato peels supported the maximum production of citric acid.

Acknowledgment

This research project was supported by a grant from the “Research Center of the Female Scientific and Medical Colleges”, Deanship of Scientific Research, King Saud University.

References

References

1. Makut M. and Ekeleme I. Citric Acid Production by Aspergillus niger and Trichodermaviride Using Hydrolysed Potato Peels Substrate. Asian Journal of Advances in Agricultural Research 2018; 5(1): 1-7.
2. Angumeenal A., Kamalakannan P., Prabhu H., and Venkappayya D. Effect of transition metal cations on the production of citric acid using mixed cultures of Aspergillus niger &Candida guilliermondii. Journal of the Indian Chemical Society 2003; 80:903-906.
3. Papagianni M. Advances in citric acid fermentation by Aspergillus niger: Biochemical aspects, membrane transport and modeling. Biotechnology Advances 2007; 25:244–263.
4. Prasad M., Surendra Babu N., Sridevi V., Reddy O. and Prakasam R. Response surface methodology for optimization of sorghum malt media for citric acid production by improved strains of Aspergillus niger. International Journal of Advanced Research 2014; 2:498-507.
5. Watanabe, T. Pictorial Atlas of Soil and Seed Fungi. "Morphologies of Cultured and Key to Species", 2nd ed, CRC Press, Boca Raton London New York Washington D.C. 2002.
6. Ali S. Studies on the submerged fermentation of citric acid by Aspergillus niger in a stirred fermentor. Ph.D. Thesis. University of Punjab. Lahore. Pakistan. 2004; 59-60.
7. Kareem S., Akpan I. and Alebiowu O. Production of citric acid by Aspergillus niger using pineapple waste. Malaysian Journal of Microbiology 2010; 6(2):161–165.
8. Haq I., Ali S., Qadeer M.A. and Iqbal J.Citric acid fermentation by mutant strain of Aspergillus niger GCMC-7 using molasses based medium. Electronic Journal of Biotechnology 2002; 5(2):125-132.
9. VandenbergheL., Soccol C., Pandey A. and Lebeault J. Review: Microbial production of citric acid, Braz. Arch. Biol. Technol. 1999; 42:263–276.
10. Soccol C., Vandenberghe L., Rodrigues C. and Pandey A. New Perspectives for Citric Acid Production and Application. Food Technol. Biotechnol. 2006; 44(2):141–149.
11. Ambati P. and Ayyanna C. Optimizing medium constituents and fermentation conditions for citric acid production from Palmyra jiggery using response surface method. Journal of Microbiology and Biotechnology 2001; 17:331-335.
12. Ujcova E., Fencl Z., Musilcova M. and Scichert L. Morphology and citric acid production of Aspergillus niger. Biotechnology and Bioengineering 1980; 22:237-241.
13. Rane K. and Sims K. Citric acid production by Yarrowialipolytica: effect of nitrogen and biomass concentration on yield and productivity. Biotechnology Letters. 1996; 18(10):1139-1144.
14. Leangon S., Maddox I. and Brooks J. A proposed biochemical mechanism for citric acid accumulation by Aspergillus niger Yang No.2 growing in solid state fermentation. W. J. Microbiol. Biotechnol. 2022; 16:271-275.
15. Kumar D., Jain V.K., Shanker G., and Srivastava A. Utilisation of fruits waste for citric acid production by solid state fermentation. Proc. Biochem. 2003; 38:1725-1729
16. Vasanthabharathi V., Sajitha N. and Jayalakshmi S. Citric Acid Production from U-V Mutated Estuarine Aspergillus niger. Advances in Biological Research 2003; 7 (3):89-94.
17. Maharani V., Reeta D., Sundaramanickam A., Vijayalakshmi S. and Balasubramanian T. Isolation and characterization of citric acidproducing Aspergillus nigerfrom spoiled coconut. Int. J. Curr. Microbiol. App. Sci. 2014; 3(3):700-705.
18. Auta H., Abidoye K., Tahir H., Ibrahim A. and Aransiola S. Citric Acid Production by Aspergillus niger Cultivated on Parkiabiglobosa Fruit Pulp. International Scholarly Research Notices 2014; 762021:1-8.
19. Yadegary M., Hamidi A., Alavi S.A., Khodaverdi E., Yahaghi H., Sattari S., Bagherpour G., Yahaghi E. Citric Acid Production From Sugarcane Bagasse through Solid State Fermentation Method Using Aspergillus niger Mold and Optimization of Citric Acid Production by Taguchi Method. Jundishapur J. Microbiol. 2013; 6(9):7625.
20. Kishore K.A. and Reddy G.V. Optimization of incubation time, fermentation temperature & O flow rate in citric acid fermentation using 2 response surface methodology (RSM). International Journal of Advanced Engineering Technology 2012; III(1):64-67.
21. Sanjay K. and Sharma P. A high performance process for production of citric acid from sugarcane molasses. J. Microbiol. 1994; 23:211-217.
22. Vergano M.G.F., Soria M.A. and Kerber N.L. Influence of inoculums preparation on citric acid production by Aspergillus niger. World J. Microbiol. Biotechnol. 1996; 12:655–656.
23. Ali S., Haq I., Qadeer M.A. and Iqbal J. Production of citric acid by Aspergillus niger using cane molasses in a stirred fermentor. Electronic Journal of Biotechnology 2002; 5(3):258-271.
24. Kishore K.A., Kumar M.P., Krishna V.R., Ravi V. and Reddy G.V. Optimization of process variables of citric acid production using Aspergillus niger in a batch fermentor. Engineering Letters. 2008; 16 (4):572.
25. Grewal H.S. and Kalra K.L. Fungal production of citric acid. Biotechnology Advances 1995; 13 (2):209-234.
26. Kim J.W., Barrington S. and Lee B.H. Cheese whey for citric acid production by Aspergillus niger NRRL 567 under submerged fermentation. Part I. Optimizing nutrient supplementation. Appl. Microbiol. Biotechnol. 2004; 92(1):97-101.
27. Fawole O.B. and Odunfa S.A. Some factors affecting production of pectic enzymes by Aspergillus niger. International Biodeterioration and Biodegradation 2003; 52(4), 223–227.
28. Pessoa D.F.; Diasde C. and Angela C. Production of citric acid by
29. Pessoa F.F., Castro A.C. and Leite S.G. Citric acid fermentation with Aspergillus niger. Reviews in Microbiology 1984; 15:89-93.
30. Paul G.C., Priede M.A. and Thomas C.R. Relationship between morphology and citric acid production in submerged Aspergillus niger fermentations. Biochemical Engineering Journal 1999; 3(2):121-129.
31. El-Aasar S.A. Submerged Fermentation of Cheese Whey and Molasses for Citric acid Production by Aspergillus niger. International Journal of Agriculture & Biology 2006; 4:463–467.
32. Adham N.Z. Attempts at improving citric acid fermentation by Aspergillus nigerin beet-molasses medium. Bio resource Technology 2002; 84:97–100.
33. Papagianni M. and Mattey M. Morphological development of Aspergillus niger in submerged citric acid fermentation as a function of the spore inoculum level. Application of neural network and cluster analysis for characterization of fungal morphology. Microbial Cell Factories 2006; 5:3.
34. Van-Suijdam J.C., Kossen N.W.F. and Paul P.G. An inoculums technique for the production of fungal pellets. European Journal of Applied Microbiology and Biotechnology 1980; 10(3):211–221.
35. Nampoothiri M., Baiju T.V., Sandhya C., Sabu A., George S., Ashok P. Process optimization for antifungal chitinase production byTrichodermaharzianum. Process Biochem. 2004; 39:1583–1590.
36. Kirimura K, Honda Y, and Hattori T. Citric Acid. Comp. Biotechnol. 2011; 3:135–142.
37. Alam M., Bari M., Muyibi S., Jamal P. and Abdullah M. Development of Culture Inoculum for Scale-Up Production of CitricAcid from Oil Palm Empty Fruit Bunches by Aspergillus niger. ProcediaEnviron. Sci. 2011; 8:396–402.
38. Dhillon G.S., Brar S.K., Kaur S. and Verma M. Bioproduction and extraction optimization of citric acid from Aspergillus niger by rotating drum type solid-state bioreactor. Ind. Crops Prod. 2013; 41:78–84.
39. Papagianni M., Mattey M., and Kristiansen B. Morphology and citric acid production of Aspergillus niger PM 1. Biotechnol. Lett. 1994; 16(9):929-
41. Mitard A. and Riba A. Morphology and growth of Aspergillus niger ATCC 26036 cultivated at several shear rates. Biotechnol. Bioeng. 1988; 32:835-
43. Sakurai A., Gant H. and Hurochi I. Effect of oxygen tension on citric acid production by surface culture of Aspergillus niger. J. Chem. Technol. And Biotechnol. 1996; 65:432-437.
44. Papagianni M., Mattey M. and Kristiansen B. Citric acid production and morphology of Aspergillus niger as functions of the mixing intensity in a stirred tank and a tubular loop bioreactor. Biochemical Engineering Journal 1998; 2:197-205.
45. Kubicek C.P. and Rohr M. Citric acid fermentation, CRC Crit. Rev. Biotechnol. 1989; 3:331-373.
46. Mattey M. The production of organic acids. Crit. Rev. Biotechnol. 1992; 12:87–132.
47. Hossain M., Brooks J.D. and Maddox I.S. The effect of the sugar source on citric acid production by Aspergillus niger. Appl. Microb. Biotechnol. 1984; 19:393-397.
48. Xu D.-B., Madrid C. P., Rohr M. and Kubicek C. P. The influence of type and concentration of the carbon source on production of citric acid by Aspergillus niger. Appl. Microbiol. Biotechnol. 1989; 30:553-558.
49. Yokoya F. Citric Acid Production. In: Industrial Fermentation Series, Campinas, SP, Brazil 1992; 1–82.
50. Gupta J.K., Heding L.G. and Jorgensen O.B. Effect of sugars, hydrogen ion concentration and ammonium nitrate on the formation of citric acid by Aspergillus niger. Acta Microbiol. Acad. Sci. Hung. 1976; 23:63– 67.
51. Dasgupta J., Nasim S., Khan A.W. and Vora V.C. Production of citric acid in molasses medium: effect of addition of lower alcohols during fermentation, J. Microbiol. Biotechnol. 1994; 9:123–125.
52. Sukesh K, Jayasuni J.S, Gokul C.N and Anu V. Citric acid production from agronomic waste using Aspergillus nigerisolated from decayed fruit. Journal of Chemical, Biological and Physical Sciences 2013; 3(2):1572- 1576.
53. Boominathan S., Dhanaraj T.S. and Murugaiah K. Production and optimization of citric acid by Aspergillus niger using pineapple and tapioca waste. Herbal Tech. Industry 2012; 1(5):8-11.
54. Pazouki, M., Felse, P.A., Sinha, J., Panda, T. Comparative studies on citric acid production by Aspergillus nigerand Candida lipolytica using molasses and glucose. Bioproc. Eng. 2000; 22:353-361.
55. Demirel G., Yaykali K.O. and Yaar A. The production of citric acid by using immobilized Aspergillus niger A-9 and investigation of its various effects. Food Chemistry 2005; 89(3):393-396.
56. El-Hussein A.A., Tawfig S.A.M., Mohammed S.G., El-Siddig M.A. and Siddig M.A.M. Citric acid production from kenana canemolasses by Aspergillus niger in submerged fermentation. Journal of Genetic
57. Anwar S., Ali S. and Sardar A.A. Citric acid fermentation of hydrolyzed raw starch by Aspergillus niger IIB-A6 in stationary culture. Sindh University Research Journal 2009; 41(1):01-08.
58. Abou-Zeid A.A. and Ashy M.A. Production of citric acid: A review. Agr. wasters 1984; 9:51-76
59. Hang Y.D., Splittstoesser D.F., Woodams E.E. and Sherman R.M. Citric acid fermentation of brewery waste. J. Food Sci. 1977; 42:383-384.
60. Mirminachi F., Zhang A. and Roehr M. Citric acid fermentation and heavy metal ions: Effect of iron, manganese and copper. Acta Biotechnol. 2002; 22:363-373.
61. Shankaranand, V.S. and Lonsane, B.K. Ability of Aspergillus niger to tolerate metal ions and minerals in a solid-sate fermentation system for the production of citric acid. Process Biochem. 1994; 29:29-37.
62. Shu P. and Johnson M.J. The interdependence of medium constituents in citric acid production by submerged fermentation. J. Bacteriol. 1948; 54:161–167.
63. Navaratnam P., Arasaratnam, V. and Balasubramaniam, K. Channeling of glucose by methanol for citric acid production from Aspergillus niger. W. J. Microbiol. Biotechnol. 1998; 14:559-563
64. Jianlong W. and Ping L. Phytate as a stimulator of citric acid production by Aspergillus niger. Proc. Biochem. 1998; 33:313-316.
65. Nadeem A., Syed Q., Baig S., Irfan M. and Nadeem M. Enhanced Production of Citric Acid by Aspergillus niger M-101 Using Lower Alcohols. Turkish Journal of Biochemistry 2010; 35(1):7-13.
66. Barrington S. and Kim J. W. Response surface optimization of medium components for citric acid production by Aspergillus niger NRRL 567 grown in peat moss. Bio resource Technology 2008; 99:368–377.
67. Ingram L.O. and Buttke T.M. Effects of alcohols on microorganisms. Adv. Microb. Physiol. 1984; 25:253.
68. Hang Y.D. and Woodams E.E. Production of citric acid from corncobs by Aspergillus niger. Biores. Technol. 1998; 65:251-253.
69. Haq I., Ali S., Qadeer M.A. and Javed I. Stimulatory effect of alcohols (methanol and ethanol) on citric acid productivity by a 2-deoxy D-glucose resistant culture of Aspergillus niger GCB-47. Bioresource Technol. 2003; 86:227-233.
70. Schuster E., Dunn-Colema, N., Frisva, J C. and Van Dije, P W. On the safety of Aspergillus niger–A review. Applied Microbiology Biotechnology 2002; 59:426-435.
71. Femi-Ola T.O. and Atere V.A. Citric acid production from brewers spent grain by Aspergillus niger and Saccharomyces cerevisiae. International Journal of Research in BioSciences 2013; 2:30–36.
72. Chaudary K., Ethiraj S., Lakshminarayana K. and Tauro P. Citric acid production from Indian cane molasses by Aspergillus niger under solid state fermentation condition. Journal of Research Haryana Agricultural University 1978; 1:48–52.
73. Haq I., Ali S., Qadeer M.A. and Iqbal J. Citric acid production by selected mutants of Aspergillus niger from cane molasses. Bioresourse Technology 2004; 93(2):125-130.
74. Faruk M.O., Mumtaz T. and Rashid H. Supplementary effect of potato peel hydrolysate on the citric acid production by Aspergillus niger CA16. Biotechnology 2014; 9(8):308-310.