Improving dietary citric acid production by the wild‐type Aspergillus niger ASP26 strain isolated from date by‐product

Abstract This research investigates citric acid (CA) synthesis using the indigenous strain Aspergillus niger ASP26, which was isolated from date by‐products. The study initially involved isolating fungi capable of CA production and identifying the most potent strain based on its characteristic enzymatic activity. A. niger ASP26 was acknowledged in a previous study for its remarkable ability to produce extracellular enzymes, such as cellulase and amylase, which enable it to degrade organic materials effectively. After the identification phase, these isolates were screened for CA production using a modified Czapek‐Dox medium. The research identified significant factors affecting CA production in submerged fermentation, including pH, carbon source, inoculum size, and fermentation time. Optimal conditions were determined for A. niger ASP26, resulting in a maximum CA yield of 16.89 g/L. These conditions included a 2.5% spore suspension at 2 × 107 spores/mL, an initial glucose concentration of 125 g/L, and incubation at 30°C for 144 h. Notably, A. niger ASP26 demonstrated the ability to produce CA under stress conditions as well. Citric acid is essential for various biological processes, such as cellular respiration, and is naturally present in citrus fruits. It also serves as a preservative and flavor enhancer in processed foods and beverages. The ability of A. niger ASP26 to produce CA from agricultural residues positions it as a viable candidate for sustainable CA production, harnessing the value from organic waste materials.


| INTRODUC TI ON
Citric acid (CA) holds a crucial position in the chemical industry, with an annual production capacity reaching approximately 2 million tons (Karaffa & Kubicek, 2019).The sustained growth in demand for CA by the food, cosmetics, and pharmaceutical sectors, with an annual growth rate of 3-4% (Soccol et al., 2006), underscores its significance in various industrial applications.
CA is a naturally occurring, weak organic acid commonly found in many fruits and vegetables, with citrus fruits being particularly rich sources.It boasts a molecular weight of 210.14 g/mol and possesses three carboxylic functional groups, each with distinct pKa values (3.1, 4.7, and 6.4), profoundly influencing its solubility and stability across diverse environmental conditions.Within biological processes, CA serves as a primary metabolic product generated through the tricarboxylic acid cycle.Its production, predominantly achieved via fermentation, caters to the needs of industries such as food and beverage, pharmaceuticals and chemicals, textiles, and electroplating.
Notably, CA plays a multifaceted role in food product formulation, contributing as an acidulant, antioxidant, emulsifier, or preservative (Radwan et al., 2010;Soccol et al., 2006).Its versatile applications in these sectors stem from its advantageous attributes, encompassing low toxicity, high solubility, biodegradability, and pleasing taste characteristics (Ali et al., 2002).Despite its widespread use, CA's production predominantly relies on fermentation as the most cost-effective and widely adopted method, representing over 90% of global CA production.However, this method falls short in meeting the surging demand for CA, necessitating the exploration of more cost-efficient and sustainable production methodologies (Kim, 2004).
While numerous microorganisms, including fungi, bacteria, and yeasts, exhibit CA-producing capabilities, only a select few can do so on an industrial scale (Soccol et al., 2006).Among these, A. niger stands out as the primary microbe chosen for large-scale CA production due to its remarkable productivity, capability to ferment economical raw materials, ease of handling, and the ability to produce CA at low pH levels without releasing toxic metabolites (Lotfy et al., 2007).As a result, the primary objective of this study is to advance CA production through submerged cultivation techniques, harnessing A. niger strains sourced from date by-products.This endeavor represents a pivotal step toward addressing the growing demand for CA while aligning with sustainability and cost-effectiveness imperatives.

| Isolation and culturing of fungi strains
Thirty-six A. niger (ASP) strains were isolated from date by-products in a previous study (Bellaouchi et al., 2017).Each strain's spore solution was plated on Potato Dextrose Agar (PDA) medium (Biokar, France) and incubated at 25°C for 3-5 days.Subsequently, these strains were preserved on a slant PDA medium at 4°C and reactivated on PDA before utilization.

| Inoculum preparation and standardization
The chosen A. niger strains, originally isolated from date by-products, were reactivated on PDA medium and incubated at 25°C for 7 days.
Following incubation, the spores were harvested using a solution of distilled water containing 0.1% Tween 80 (Sigma, St. Louis, MO, USA).The spore concentration was then adjusted to 2 × 10 7 spores/ mL using a hemocytometer (Thoma cell) for subsequent analysis.

| CA production using Czapek-Dox agar medium
A qualitative screening was conducted to assess the CA production capability of 36 A. niger strains, following the procedure outlined by Ali (2004).This involved using the Czapek-Dox Agar culture medium containing (g/L: Sucrose 30.0,NaNO 3 2.0, K 2 HPO 4 1.0, MgSO 4 .7H 2 O 0.5, KCl 0.5, FeSO 4 0.01, Bromocresol green 40 mL at a concentration of 1%, Agar 20, pH 6.0). 100 μL of the spore suspension of 2 × 10 7 spores/mL was inoculated at the center of Petri dishes and incubated them at 25°C for 3-5 days.The development of a yellow halo around the colony was considered a positive result.

| Assessment of acidification capacity of A. niger
The acidification activity of 14 pre-selected A. niger strains was assessed using the submerged fermentation method.We employed serves as a preservative and flavor enhancer in processed foods and beverages.The ability of A. niger ASP26 to produce CA from agricultural residues positions it as a viable candidate for sustainable CA production, harnessing the value from organic waste materials.

K E Y W O R D S
A. niger, by-product, citric acid, date, fermentation Czapek-dox broth containing 50 g/L glucose, 2 g/L NaNO 3 , 1 g/L K 2 HPO 4 , 0.5 g/L MgSO 4 .7H 2 O, 0.5 g/L KCl, and 0.01 g/L FeSO 4 .This broth was inoculated with 1% (v/v) of a spore suspension at a concentration of 2 × 10 7 spores/mL and adjusted to a pH of 5 using 4 M hydrochloric acid.The inoculated broth was then cultivated at 30°C under agitation at 200 rpm.The acidification activity of the selected strains was evaluated by measuring the pH at specific time points from 0 to 240 h of incubation.

| Fermentation conditions
A submerged fermentation method was employed to quantitatively assess the ability of three selected A. niger strains (ASP 23, ASP 26, and ASP 29) to produce CA.A 1% (v/v) spore suspension of 2 × 10 7 spores/mL was inoculated into Czapek-dox broth, with pH adjustment to 5 using hydrochloric acid (4 M), followed by incubation at 30°C.pH, residual sugars, and CA levels were measured at 24-h intervals over a 240-h fermentation period, with an agitation speed of 200 rpm.The biomass growth of the strains was determined after the fermentation process.

| Measurement of pH
To evaluate the acidification activity of the three selected strains, the pH of the fermentation medium was measured every 24 h during the culture period using a calibrated VWR Symphony SB70P pH meter.

| Determination of residual sugars
Changes in residual sugars during fermentation were determined using the colorimetric DNS method.The optical density was measured at 523 nm using a spectrophotometer (UV-1601).The intensity of the orange color generated was directly proportional to the content of reducing sugars (Miller, 1959).A calibration curve was prepared using standard glucose solutions ranging from 0 to 1.2 g/L.

| Measurement of CA concentration
CA concentration was determined using the colorimetric assay method developed by Marier and Boulet (1958).A 0.5 mL sample of the filtrate, obtained by centrifugation at 8000 g for 10 min, was mixed with 0.65 mL of pyridine and vigorously stirred before adding 2.85 mL of acetic anhydride.Tubes were incubated at 32°C for 30 min in a water bath.The optical density was measured at 420 nm, with the intensity of the color being directly proportional to the citrate concentration.

| Determination of biomass
The biomass of A. niger strains (ASP 23, ASP 26, and ASP 29) was determined by filtering the fermentation products through Whatman No. 1 filter paper and subsequently drying the wet biomass at 105°C until a constant weight was achieved.

| Incubation period
The production of CA by A. niger strain ASP26 was monitored over a 336-h incubation period.In a 250 mL Czapek-dox broth adjusted to a pH of 5 using 4 M hydrochloric acid, we inoculated with 1% (v/v) of 2 × 10 7 spores/mL of A. niger (ASP26) before incubating at 30°C.At the end of the incubation period, residual sugar, biomass growth, and CA concentration were determined.

| Initial pH
The impact of initial pH on CA production was assessed using 250 mL shake flasks containing 150 mL of Czapek-dox broth, and adjusted to initial pH levels of 3, 4, 5, 6, and 7 using 4 M hydrochloric acid and 4 M NaOH, then inoculated with 1% (v/v) of 2 × 10 7 spores/mL of A. niger (ASP26).A control culture was prepared with Czapek-dox broth adjusted to pH = 5.Following inoculation, cultures were incubated at 30°C for 144 h, with CA concentration determined using the colorimetric assay method as previously mentioned.

| Incubation temperature
The effect of temperature on CA production was evaluated in 250 mL shake flasks containing 150 mL of liquid medium.A. niger (ASP26) spores were inoculated at a density of 2 × 10 7 spores/mL (1%, v/v) into Czapek-Dox broth.The cultures were then incubated at temperatures of 20°C, 25°C, 30°C, 35°C, and 40°C for a period of 144 h.Post-incubation, the concentration of CA was quantified using the previously described colorimetric assay method.

| Glucose concentration
The effect of different glucose concentrations on CA production was evaluated in 250 mL shake flasks containing 150 mL of the Czapek-dox broth containing 25, 50, 75, 100, 125, 150, 175, and 200 g/L of glucose was inoculated with 1% (v/v) of 2 × 10 7 spores/ mL of A. niger (ASP26).The CA concentration was determined using the above colorimetric assay after incubating the culture at 30°C for 144 h.

| Inoculum size
To assess the influence of inoculum size on CA production, A. niger (ASP26) was cultured in 250 mL shake flasks each containing 150 mL of Czapek-Dox broth.The broth's pH was adjusted to 5.0 using 4 M HCl and 4 M NaOH.Inoculation was carried out with varying volumes of the spore suspension: 1%, 1.5%, 2%, 2.5%, and 3%, corresponding to a concentration of 2 × 10 7 spores/mL.The cultures were incubated at 30°C for a duration of 144 h.Post-incubation, CA concentration was quantified employing the earlier mentioned colorimetric assay method.

| Optimization of CA production
The parameters for producing CA by A. niger (ASP26) in a modified Czapek-dox broth were chosen based on previous optimal results.
The broth contained 125 g/L glucose, 2 g/L NaNO 3 , 1 g/L K 2 HPO 4 , 0.5 g/L MgSO 4 .7H 2 O, 0.5 g/L KCl, and 0.01 g/L FeSO 4 , and was adjusted to an initial pH of 5 using 4 M hydrochloric acid and 4 M NaOH.The culture was inoculated with 2.5% of a spore suspension (2 × 10 7 spores/mL) and incubated at 30°C for 192 h.The CA concentration was determined using a colorimetric assay method.A control experiment was also conducted using unmodified Czapek-dox broth under the same conditions.

| Statistical analysis
All experiments were conducted three times to ensure the validity of all the work carried out.The results were reported as means ± standard deviation.One-way ANOVA analysis was employed to compare the means, with significance defined at p < .05.To identify groups of means, the Student-Newman-Keuls (S-N-K) post hoc test was utilized.All analyses were carried out using SPSS 10.0 for Windows (SPSS Inc., Chicago, USA).

| Isolation of A. niger strains
In this study, a set of 36 strains of A. niger was successfully isolated from date by-products and previously cataloged in Bellaouchi et al. (2021) prior to research.The meticulous selection of these particular strains played a pivotal role in shaping the research outcomes.
This deliberate choice was underpinned by the strains' established capacity to produce enzymes crucial for decomposing organic matter, as highlighted in Bellaouchi et al. (2021) work.Specifically, these strains were handpicked due to their proficiency in generating amylase and cellulase enzymes, which are instrumental in breaking down complex carbohydrates, such as starch and cellulose.The scrupulous curation of these strains was imperative to ensure the optimal efficiency of the experimental procedures undertaken in our research.The yellow halo observed for some strains (ASP5, ASP8) is more intense than that obtained for others (ASP26) (Figure 1), indicating their higher production of organic acids.Among the 36 strains, 14 had a positive result (Figure 2), with a diameter of yellow halo ranging between 25 and 67 mm.

|
The results of the acidification activity of the selected A. niger strains obtained after 240 h of submerged fermentation using a Czapek-Dox medium are presented in Table 1.The investigation disclosed that following a 24-h incubation period, all strains manifested a substantial decrease in pH, ranging from an initial pH of 5 to a final range of 3.1-3.9.Furthermore, a significant decrease in pH (p < .001) was observed after 120 h of incubation for the three selected A. niger strains (ASP23, ASP26, and ASP29) with pH values of 1.9, 1.8, and 2.0, respectively (Table 1).This decrease in pH results from the acidification process caused by the breakdown of organic compounds in the Czapek-Dox medium by the A. niger strains, which leads to the release of organic acids (Aboyeji et al., 2020).The high acidification potential of these strains makes them suitable for the quantitative analysis of CA production.

| Quantitative screening and optimization of citric acid production by ASP26 strain in Czapek-Dox broth
The evaluation of CA production potential through submerged fermentation involved an examination of three strains of A. niger, namely, ASP23, ASP26, and ASP29.This comprehensive assessment encompassed the quantification of CA concentrations, pH dynamics, and residual sugar content, as graphically depicted in Figures 3   and 4. The outcomes revealed a noteworthy range in CA production among these strains, ranging from 4.5 to 8.8 g/L, accompanied by a mycelium dry weight varying between 8.9 and 12.4 g/L (Figure 4).Of particular significance was the ASP26 strain, which emerged as the most prolific CA producer, yielding an impressive 8.8 g/L.This represented a yield efficiency of 28.38% based on the consumed sugar.
Furthermore, the fermentation process with ASP26 demonstrated a gradual decline in pH, reaching its nadir after 96 h.The mycelium dry weight reached its zenith at 8.9 g/L on the final day of fermentation, while residual sugars measured 18.37 g/L.In comparison, antecedent studies by Ali et al. (2001) delved into CA production by A. niger strains, revealing a diverse spectrum of CA concentrations ranging from 2.63 to 47.50 g/L and 0.24 to 16.04 g/L, respectively.The robust CA production observed with the ASP26 strain in our investigation aligns with and contributes to the expanding scholarly discourse on the diverse capabilities of A. niger strains in CA biosynthesis.
The production of CA by A. niger is a complex process that depends on several factors, including the nutritional conditions of the growth medium.These conditions include the carbon source's concentration, dissolved oxygen level, hydrogen ions level, and optimal concentrations of phosphate and trace metals (Max et al., 2010;Soccol et al., 2006).The process of CA production is closely linked to the glycolytic pathway, and excessive production of citrate can occur under specific conditions.Research has shown that citrate can act as a glycolysis inhibitor; therefore, its inhibition has been a topic of interest.Under certain conditions, the inhibition of citrate can be reduced by the positive effects of the phosphofructokinase gene (PFK-1) (Käppeli et al., 1978;Kristiansen & Sinclair, 1979).A manganese deficiency can also lead to protein breakdown, increasing the intracellular concentration of NH 4 + , known as the "ammonium pool."This can inhibit the enzyme phosphofructokinase, essential for converting fructose and glucose to pyruvate, leading to an increased flux through glycolysis and CA formation (Figure 5).High glucose and NH 4 + can also inhibit the formation of 2-oxoglutarate dehydrogenase, ultimately limiting the catabolism of CA present in the tricarboxylic acid (TCA) cycle (Rohr & Kubicek, 1981).

F I G U R E 1
Qualitative screening of organic acids production, indicated by the yellow halo, by Aspergillus niger strains on Czapek-Dox Agar medium.

| Effect of initial pH and temperature on CA production
An experiment was undertaken to assess how initial pH values, ranging from 3 to 7, influenced CA production by the ASP26 strain in the Czapek-Dox medium.The choice of this strain for further studies was based on its superior performance in CA production compared to other strains.The ASP26 strain showcased the highest CA yield, reaching 8.8 g/L, with an efficiency of 28.38% based on the consumed sugar.Moreover, the fermentation process demonstrated a gradual decline in pH, reaching its lowest point after 96 h.These significant findings establish ASP26 as the preferred strain for subsequent studies, underscoring its outstanding performance and potential for further exploration in our ongoing research.The results in  (Baei et al., 2008).Thus, a maximum quantity of CA (8.22 g/L) was reached at an initial pH value of 5, with a lower value of the dry weight of the mycelium (4.34 g/L).The sugar consumed was 30.80 g/L, with a yield of 26.62%.When the initial pH value of the fermentation medium is greater than 5, the CA production gradually decreases, then drops sharply after the value of 6.
Roukas (2005) reported that when Aspergillus niger is used for CA production, the initial pH depends on the employed medium: in synthetic medium, the initial pH is adjusted to 2.5-3.5.Meanwhile, in a by-product medium (molasses, fruit extract, syrup, etc.), the initial pH of the medium should be neutral or slightly acidic to ensure the germination and growth of the microorganism.Franz et al. (1993) found that A. niger produces CA at low pH in a synthetic medium, while Penicillium simplicissimum excretes CA at a higher pH range of 4-7.Furthermore, Papagianni (2007) noted that pH is crucial at two different stages of fermentation.Spores require a pH of 5 to germinate, and CA production requires a low pH of 2. Therefore, it is important to carefully determine the initial pH level to achieve maximum CA production (Soccol et al., 2006).
Very highly significant differences (p < .001) in CA production were observed at various temperatures (Table 3).Temperature plays a critical role in CA production, and our study found that the highest CA production (7.63 g/L) by the ASP26 strain occurred at an incubation temperature of 30°C.At this temperature, the mycelium's dry weight was 4.67 g/L, and the sugar consumption was 28.7 g/L, resulting in a CA yield of 27.52%.Other researchers have also reported that 30°C is optimal for CA production (Amer et al., 1999;El-Hussein et al., 2009;Khosravi-Darani & Zoghi, 2008).However, deviating from this temperature may reduce CA yield due to the denaturation of the citrate synthase enzyme and the activation of the oxalic acid synthesis pathway.Chioma and Agwa (2019) reported that the most favorable temperature for oxalic acid production was 35°C, but it completely inhibited the accumulation of CA at this temperature.Therefore, maintaining an incubation temperature of 30°C is crucial for achieving optimal CA production.

| Effect of the incubation period on the CA production
To investigate the effects of the incubation period on CA production, a fermentation process was carried out using the selected strain ASP26 for 336 h, and the results are presented in Figure 6.
Highly significant differences (p < .001) in CA production were observed during the incubation period (Figure 6).The study noted that the biosynthesis rate of CA production increases gradually during fermentation, reaching its peak value of 15.43 g/L at 144 h postinoculation.These findings are consistent with previous studies by Ali et al. (2002) who also stated that the highest productivity of CA is attained following 144 h of fermentation.In contrast, researchers like Arzumanov et al. (2000), Alvarez et al. (2007), Lotfy et al. (2007), and Nadeem et al. (2010) reported that the maximum concentration of CA was obtained after 192 h of fermentation.At the optimal CA production with ASP26 strain, the corresponding pH, residual sugar, and mycelial dry weight values were 2.08, 56.66, and 15.25 g/L, respectively (Figure 6).Increasing the fermentation time did not increase the production of an additional amount of CA.
Consequently, a decrease in productivity may be due to the reduction of the nitrogen available in the fermentation medium, the age of the strain, the exhaustion of sugar contents, and the degradation of the enzymatic system responsible for CA biosynthesis.

| Effect of glucose concentration on CA production
The isolated A. niger ASP26 was cultivated on the Czapek-Dox medium with glucose concentrations ranging between 25 and 200 g/L.Highly significant differences (p < .001) in CA production were observed between glucose concentration values (Table 4).
The maximum concentration of CA (15.46 g/L) was obtained with an initial glucose concentration of 125 g/L.The dry weight of the mycelium at this concentration was 12.13 g/L, while the sugar consumed was 59.69 g/L, with a yield of 25.79%.An increase or decrease in the initial glucose concentration above 125 g/L would decrease the yield of CA produced.This can be explained by the formation of oxalic acid and polyalcohols (Haq et al., 2003).
The presence of elevated concentrations of carbon sources frequently leads to the inhibition of α-ketoglutarate dehydrogenase (α-KGDH) activity (Papagianni, 2007).This enzyme plays a crucial

| Effect of inoculum size on CA production
The A. niger ASP26 strain was inoculated on a Czapek-Dox medium with different inoculum volumes ranging from 1% to 3%.Table 5 indicates the impact of the inoculum volume on the production of CA, the sugar consumed, and the dry weight of the mycelium.Highly significant differences (p < .001) in CA production were observed between the inoculum size values.A maximum of CA (16.89 g/L) was obtained when 150 mL of the culture medium were inoculated with 3.75 mL (2.5%) of spore suspension with a 2 × 10 7 spores/mL concentration.Ali et al. (2002) obtained the maximum CA from molasses using an inoculum rate of 1%, while Bari et al. (2009) obtained a maximum concentration of CA from 10% palm fruit clusters.High inoculum density can result in overcrowding, heightened competition, and the rapid depletion of nutrients (Uyar & Baysal, 2004).
Typically, the production of metabolites tends to increase with inoculum density up to a certain threshold (Nampoothiri et al., 2004).
Conversely, at low inoculum density, metabolite production declines, and the risk of contamination rises due to an inadequate cell population (Adham, 2002;Ruijter et al., 2000).

| CON CLUS ION
The present study highlights the critical role of key factors, including initial sugar concentration, pH levels, and specific fermentation conditions, in citrate production.Among the 36 strains of A.
Selection of A. niger producing CA 3.2.1 | Qualitative screening of CA production using Czapek-Dox agar medium The qualitative screening of CA production by A. niger strains was based on their production of organic acids in the Czapek-dox Agar medium supplemented with 0.04% Bromocresol purple (BCP).The color change from green-blue to yellow, obtained for most of the A. niger strains, indicates their production capacity of organic acids.
tion substrate.El-Hussein et al. (2009) reported a contradictory result and wrote a lower pH (3.5) for maximum CA production from F I G U R E 2 Diameter (mm) of yellow halo formation in strains treated on Czapek-Dox Agar medium.TA B L E 1 pH changes during the culture of Aspergillus niger strains in Czapek-Dox liquid medium at pHi 5, 200 rpm, and 30°C.

F I G U R E 3
Citric acid production by Aspergillus niger strains (ASP23, ASP26, ASP29) in Czapek-Dox liquid medium at pHi 5, 200 rpm, and 30°C.F I G U R E 4 Evolution of pH, glucose concentration, and dry cell mass of Aspergillus niger strains (ASP23, ASP26, ASP29) during fermentation in liquid medium at pHi 5, 200 rpm, and 30°C.F I G U R E 5 Schematic representation of the main metabolic reactions involved in citric acid production by Aspergillus niger (PC: phosphoénolpyruvate carboxykinase, ACO: Aconitase, PFK: phosphofructokinase).
role in the CA (Krebs) cycle by facilitating the conversion of αketoglutarate into succinyl-CoA.With ample carbon sources, the cell often prioritizes other metabolic pathways, such as glycolysis, at the expense of the CA cycle.Consequently, α-KGDH activity tends to be downregulated when exposed to high carbon source concentrations(Papagianni, 2007).It is hypothesized that citric acid production (CA) by A. niger during fermentation can be influenced by the physiological stress experienced by the cells, particularly in response to the specific carbon source utilized.This stress-driven response may divert cellular resources toward CA production, potentially at the cost of mycelial growth (biomass).Notably, as the glucose concentration exceeds 125 g/L, the strain seems to prioritize biomass formation over CA production, gradually decreasing CA concentration.This observation underscores the significance of the carbon source's type and concentration in modulating the cellular response.Thus, it underscores the critical importance of judicious carbon source selection when enhancing the efficiency of CA production.
niger studied, the wild-type ASP26 isolate demonstrated remarkable performance in producing CA within the fermentation medium.Under controlled conditions, maintaining an initial pH of 5 and utilizing the ASP26 strain, we achieved a citrate concentration of 8.22 g/L.This achievement soared to an impressive 16.89 g/L when inoculating the culture medium with a 2.5% spore suspension at 2 × 10 7 spores/mL.These conditions involved an initial glucose concentration of 125 g/L, a temperature of 30°C, and a 144-h fermentation period.Despite these exciting results, there is ample room for improvement.Future research should explore alternative F I G U R E 6 Biochemical (pH, citric acid, sugars) and biomass (mycelium dry weight) changes during the submerged culture of Aspergillus niger ASP26 strain.

Table 2
Note: Results are means ± SD (n = 3).Values of the same column, followed by the same letter, are not statistically different (p < .05)as measured by Student-Newman-Keuls test.Effect of inoculum size on citric acid biosynthesis by Aspergillus niger ASP26 in submerged Czapek-Dox liquid culture at pHi 5, 200 rpm, initial glucose concentration of 125 g/L, incubated at 30°C.
Glucose, g/L Citric acid, g/L Residual sugar, g/L TA B L E 4 Effect of initial glucose concentration on citric acid biosynthesis by Aspergillus niger ASP26 in submerged Czapek-Dox liquid culture at pHi 5, 200 rpm, and 30°C.Note: Results are means ± SD (n = 3).Values of the same column, followed by the same letter, are not statistically different (p < .05)asmeasured by Student-Newman-Keuls test.TA B L E 5