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Improved Citric Acid Production by Radiation Mutant Aspergillus niger Using Sugarcane Bagasse Extract



Abdullah-Al-Mahin , A.B.M. Sharifuzzaman, M.O. Faruk, M.A. Kader, J. Alam, R. Begum and Harun-Or-Rashid
 
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ABSTRACT

Due to huge demand of citric acid, attempts are taking to introduce its efficient production either by using low cost substrates or by improving the potency of the fermentation microorganisms. In this study, sugarcane bagasse extract was used for citric acid production using wild type Aspergillus niger CA16 and its radiation mutant 79/20 by submerged fermentation. Fermentation was carried out up to 15 days using 5, 10, 15 and 20% of sugarcane bagasse extract which contained 21.06, 32.60, 43.50 and 53.20 g L-1 sugar, respectively. The fermentation medium was supplemented with prescott salt. With the increasing concentration of baggase extract, total titratable acidity and citric acid production was increased. Moreover, radiation mutant A. niger 79/20 had higher citric acid production than A. niger CA16. Maximum amount of citric acid (12.81 g L-1) was produced in the 20% bagasse extract medium by A. niger 79/20, whereas, CA16 produced 10.25 g L-1 citric acid in the same fermentation medium. Maximum substrate uptake, growth yield co-efficient and productivity were also found higher in case of the strain 79/20. Thus, radiation mutation induced improved citric acid production in A. niger 79/20.

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Abdullah-Al-Mahin , A.B.M. Sharifuzzaman, M.O. Faruk, M.A. Kader, J. Alam, R. Begum and Harun-Or-Rashid , 2012. Improved Citric Acid Production by Radiation Mutant Aspergillus niger Using Sugarcane Bagasse Extract. Biotechnology, 11: 44-49.

DOI: 10.3923/biotech.2012.44.49

URL: https://scialert.net/abstract/?doi=biotech.2012.44.49
 
Received: October 22, 2011; Accepted: December 17, 2011; Published: February 15, 2012



INTRODUCTION

Citric acid is one of the most commonly used carboxylic acid intermediary of metabolism in most plants and animals. Due to its inoffensive nature, citric acid is extensively used in food preparations, pharmaceuticls and cosmetics. About 70% citric acid is used in food industry and remaining 30% in other industries (Dhillon et al., 2010). It also acts as an antioxidant synergist in fatty foods. Nowadays, citric acid is also increasingly utilized as a monomer for the manufacturing of eco-friendly polymers which are extensively used in various medical applications (Yang et al., 2004; Namazi and Adeli, 2005; Djordjevic et al., 2009; Naeini et al., 2010). As this organic acid has received the status of “Generally Recognized as Safe” (GRAS), utilization of citric acid is expanding in biomedicine, biopolymer production and various other applications. Considering high consumption rate and slight increase in price, the market value for this commodity chemical was expected to exceed $2 billion in 2009 (Partos, 2005).

A large number of microorganisms including bacteria, such as, Arthrobacter paraffinens, Bacillus licheniformis, Corynebacterium ssp., fungi, such as, Aspergillus niger, A. aculeatus, A. carbonarius, A. awamori, A. foetidus, A. fonsecaeus, A. phoenicis, Penicillium janthinellum and yeasts, such as, Candida tropicalis, C. oleophila, C. guilliermondii, C. citroformans, Hansenula anamola and Yarrowia lipolytica have been employed for citric acid production (Grewal and Karla, 1995; Kubicek and Rohr, 1986; Pandey et al., 2001; Vandenberghe et al., 1999; Maryam et al., 2011a, b; Xie and West, 2007a, b; Afifi, 2011; Lodhi et al., 2001; Ali et al., 2001; Mazhar et al., 2003). However, the fungus A. niger has remained the leading candidate for commercial production of citric acid because of its higher productivity. Recently, many (natural or mutant) Y. lipolytica strains have also been reported to produce citric acid using various substances. A major disadvantage of producing citric acid by yeasts is the simultaneous formation of isocitric acid. In contrary, application of A. niger has many advantages, i.e., easy to handle, can ferment a variety of cheap raw materials, give high yields (Soccol et al., 2006).

Citric acid is one of the most widely used organic acid which is produced by using fermentation technology on an industrial scale. Large-scale production of citric acid has been carried out solely by the A. niger in submerged fermentation on beet or cane molasses, sucrose or glucose syrup. In recent years, solid state fermentation process has gained global attention as an alternative to submerged fermentation (Soccol et al., 2006). Low-cost agro-industrial wastes can be utilized to produce different value added products by solid state fermentation. Thus, the process saves money as well as solves environmental problems. This method can also be adopted to produce citric acid using less expensive raw materials. Large amounts of sugarcane bagasse are produced world wide as a by-product of sugar industries. In our country, bagasse is mainly used as fire fuel. Previously, we reported a successful production of citric acid from sugarcane bagasse in presence of 14% sucrose by solid state fermentation with both wild type and radiation mutant A. niger strains (Abdullah-Al-Mahin et al., 2008). In this study, we aimed to find out the feasibility of using sugar cane bagasse for citric acid production by submerged fermentation.

MATERIALS AND METHODS

Analysis of the substrate used: In this study, Sugarcane baggase, collected from Rajshahi Sugar Mill, was used as substrates for citric acid production by submerged fermentation. Total Reducing Sugars (TRS) of the substrate were determined as glucose using the Nelson (1944). Fermentable Reducing Sugars (FRS) and Nonfermentable Reducing Sugars (NRS) were determined by the method of Saeman et al. (1945). Here, potential FRS and NRS are closely related to the contents of cellulose and hemicellulose, respectively. The lignin was gravimetrically estimated by Mooreand Johnson (1967). Moisture, protein, lipid, ash and fibre contents were determined according to the methods described in AOAC (1980).

Microorganisms used: Two citric acid-producing strains of A. niger were used in the present study. The strain CA16 was used as the original parent strain which was a natural isolate from local soil (Abdullah-Al-Mahin et al., 2008; Vandenberghe et al., 1999; Hannan, 1972; Hannan et al., 1973). Another strain of A. niger, designated as 79/20, was the second step radiation mutant of A. niger CA16 (Abdullah-Al-Mahin et al., 2008; Hannan et al., 1973). The strains were revived onto potato dextrose broth medium (PDB) for 7-8 days at 30±1°C and 200 rpm. The microorganisms were maintained on Potato Dextrose Agar (PDA) plates for 96 h at 30±1°C and stored at 4±1°C in a refrigerator for future use. The cultures were renewed every four weeks.

Citric acid production medium: Collected sugarcane bagasse was sundried, cut into small pieces, grounded and screened to collect a particle size of about 1.2-1.6 mm. Bagasse powder was then taken in 500 mL Erlenmeyer flasks and mixed with distilled water to make a concentration of 5, 10, 15 and 20% and allowed to soak for 2 h. The suspensions were then filtered through thin cloth and supplemented with Prescott salt (NH4NO3, 2.23 g L-1; K2HPO4, 1.00 g L-1 and MgSO4.7H2O, 0.23 g L-1). The pH of the medium was adjusted to 5.0, as was suggested by Begum and Chowdhury (2000), for better citric acid production by the studied A. niger strains.

Submerged fermentation: One hundred fifty milliliter of liquid substrate was dispensed in a 500 mL Erlenmeyer flask and thoroughly mixed and autoclaved at 121±1°C for 30 min (Fig. 1). The substrates were inoculated with spore suspension of 1x107 spores/25 mL. After thorough mixing, Erlenmeyer flasks and their contents were incubated in a shaker at 30±1°C at 200 rpm for 15 days (Papagianni, 2007).

Fig. 1: Flow sheet of fermentation process for the production of citric acid by A. niger CA16 and 79/20

Unless specified otherwise, these fermentation conditions were maintained throughout the study. All the experiments were conducted in duplicates.

Fermentation analysis: Sugar concentrations at different interval of fermentation were determined according to the method described by Morse (1947) using sucrose as standard. Total Titratable Acid (TTA) values in the media were determined against freshly prepared 0.1N NaOH. Citric acid concentrations were determined spectrophotometrically by the modified method of Marier and Boulet (1958). Appropriately diluted 1 mL sample of fermented filtrate was taken in a test tube and 1.3 mL pyridine was added. The contents were mixed manually by swirling and 5.7 mL acetic anhydride was added to the test tubes. The contents were again mixed by swirling and immediately placed in a constant-temperature (22°C) water bath and incubated for 30 min. The optimal density was recorded at 420 nm with the blank set on 100% transmission. Citric acid concentration was determined by referring to a standard curve for citric acid concentration. Citric acid concentrations were expressed as grams per liter (g L-1).

RESULTS

Analysis of sugarcane bagasse: Total reducing sugar, fermentable reducing sugar, non-fermentable reducing sugar and lignin of sugarcane baggase were 68, 43.5, 20.2 and 24%, respectively. The amounts of moisture, protein, lipid, ash and fibre of the agricultural waste were 45, 1.4, 0.37, 2.3 and 48.5%, respectively (Table 1).

Production of citric acid by A. niger CA16: Table 2 shows the production of citric acid by A. niger CA16. With the increase of incubation period sugar concentration in the medium was reduced and maximum reduction was found on day 15. Maximum utilization of sugar was detected in case of 20% sugarcane bagasse extract on 15th day. pH values were also decreased with the increase of fermentation period and maximum decrease was recorded in case of 20% bagasse extract medium.

However, TTA value and citric acid production were increased with the increase of time. These values were found just proportional to the utilization of sugar in the fermentation media. Most of the cases, the highest TTA value was obtained on the 12th day. In case of 5% bagasse extract, the highest TTA was obtained on the 7th day. Like TTA values, maximum citric acid production values for 10, 15 and 20% bagasse extract media were detected on the 12th day and for 5% bagasse extract on 7th day of fermentation. After that, these values tended to decrease again. Accumulations of citric acid by the strain CA16 in 5% bagasse extract medium on the 4th, 7th, 12th and 15th day of fermentation were 2.50, 4.65, 3.84 and 2.46 g L-1, respectively (Table 2). In 10% bagasse extract, sugar were estimated periodically. Here, also maximum utilization was detected in case of 20% sugarcane bagasse extract on 15th day (Table 3). However, if we compare the sugar utilization by CA16 and 79/20 (Table 2, 3), we find that 79/20 was more potential in utilizing sugar from all the studied bagasse extract media.

As expected, the decrease in pH during fermentation by the strain A. niger 79/20 (Table 3) was higher than that of CA16 (Table 2). Lowest pH was reached after 15 days of fermentation and the values were 4.0, 4.2, 3.25 and 3.3 for the media with 5, 10, 15 and 20% bagasse extract, respectively.

The TTA value during citric acid fermentation by A. niger 79/20 in case of 5% bagasse extract was highest on 7th day and the value was highest on 12th day in case each of 10, 15 and 20% bagasse extract (Table 3). Citric acid values were also highest in these days for the respective bagassa extract medium.

Increase of cell biomass during citric acid production: After inoculation of A. niger strains in the fermentation media, the amounts of cell biomass were found to increase with the increase of incubation period.

Table 1: Proximate analysis of sugarcane bagasse

Table 2: Citric acid production by A. niger CA16

Table 3: Citric acid production by A. niger 79/20

Table 4: Kinetic parameters and coefficients of citric acid fermentation by A. niger CA16 and 79/20
Qs: g substrate consumed/L/h, Yp/s: (gram citric acid produced/gram sugar consumed), Yx/s: (gram cell mass/gram sugar consumed), Qp: g citric acid produced/L/h

Fig. 2: Cell biomass of A. niger CA16 and 79/20 at the end of fermentation

Maximum cell dry weight was detected on 15th day of fermentation (Fig. 2). However, citric acid production was not necessarily increased with the increase of cell biomass for all concentrations of bagasse extracts. Highest cell weight was found in case of 20% bagasse extract in both of the strains. Expectedly, A. niger 79/20 had higher biomass than that of CA16 which also produced higher citric acid.

Fermentation kinetics: On the basis of kinetic parameters as shown in Table 4, in case of both of the A. niger strains, substrate uptake rate, product yield co-efficient, growth yield coefficient and productivity were found to increase with increase concentration of bagasse extract in fermentation media. A significant improvement in product yield coefficient and productivity were also detected in case of fermentation with A. niger 79/80 than that of the wild type strain CA16 in all the media used.

DISCUSSION

In this study, two A. niger strains were used for citric acid fermentation using an agricultural wastes which has almost no use in biotechnological purpose in Bangladesh even having a high sugar content (Table 1) for acid or biomass production. Reports of Li et al. (2002) also showed similar pattern of constituents in sugarcane bagasse. In the present study, there was a quantitative relationship between sugar utilization and citric acid production. The sugar concentration was decreased throughout the process indicating the utilization of sugar for citric acid production. However, the radiation mutant strain A. niger 79/20 utilized sugar to produce citric acid more rapidly than A. niger CA16. This finding was in accordance with the findings of Lotfy et al. (2007) where they reported improved citric acid production by a radiation induced mutant A. niger strain than that of mother strain. Begum and Chowdhury (2000) reported 1.6 fold improved production yield by radiation mutant A. niger compared to wild type strain. Our previous study also showed more efficient production of citric acid from sugarcane bagasse (Abdullah-Al-Mahin et al., 2008) than the wild type one by solid-state fermentation.

In this study, pH was adjusted to 5.0 in bagasse extract media for optimum production of citric acid as suggested by Begum and Chowdhury (2000). With the increase in incubation period the pH of the fermented medium was found to decrease gradually. This indicated the increase of acid production. Expectedly, the decrease of pH in the media inoculated with 79/20 was more than that with CA16. Again, this decrease was also higher with increased concentration of bagasse indicating higher citric acid production as a result of higher sugar utilization. The temperature of fermentation media is one of the critical factors for citric acid fermentation. A temperature of 30°C was reported to be optimum for citric acid fermentation by A. niger (Dalmau et al., 2000). When the temperature of medium is increased above 30°C, the biosynthesis of citric acid is decreased. It might be due to the accumulation of by-products such as oxalic acid (Kubicek and Rohr, 1986). In the present study, 30°C was maintained for maximum growth of A. niger strain CA16 and 79/20.

Total Titratable Acidity (TTA), a quantitative measure of citric acid production, was found just proportional to the utilization of sugar in the fermentation media. However, TTA values were decreased after 12th days of incubation. This might be due to product inhibition. The high level of citric acid in the medium caused an inhibition of product formation (Table 2, 3). For the first four days after incubation the growth rate was very slow (data not shown). From 4th to 12th days there was a sharp increase in citric acid level and from 13th to 15th the citric acid concentration was decreased in the fermentation media. This phenomenon can be explained by the inhibition of fungal enzymes by increased concentration of citric acid (Agrawal et al., 1983). Agrawal et al. (1983) reported that the activity of citric acid synthase, the terminal enzyme of the pathway responsible for citric acid synthesis, decreased after 8 days when citric acid accumulation was highest.

Table 4 showed that with higher concentration of bagasse extract citric acid production was increased by both of the strains indicating that the production of citric acid is dependent on substrate concentration. Higher values of maximum substrate uptake rate, maximum citric acid production, product yield co-efficient and productivity in case of the strain 79/20 than CA16 clearly indicates improved potential of the strain for citric acid production. In our study, sugar concentrations of different bagasse extracts were relatively lower than the optimum level, therefore some impurities of oxalic acid (Rohr et al., 1983) may present in the medium which was not detected in our experiments.

CONCLUSION

Finally, it can be concluded that radiation mutant A. niger 79/20 had improved sugar utilization and citric acid production ability. However, it is necessary to unveil the molecular mechanism of improved citric acid production by radiation mutation. The appropriate use of this radiation mutation can help us to generate more robust citric acid producing strains.

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