Influence of alcohols on citric acid production by Aspergillus niger A-9 entrapped in polyacrylamide gels

Journal of Food Engineering 70 (2005) 518–522 www.elsevier.com/locate/jfoodeng

Influence of alcohols on citric acid production by Aspergillus niger A-9 entrapped in polyacrylamide gels Ku¨rsßat Og˘uz Yaykasßlı a, Go¨khan Demirel a

b,*

, Ahmet Yasßar

b

Department of Chemistry, Faculty of Arts and Science, Middle East Technical University, 06531 Ankara, Turkey b Department of Chemistry, Faculty of Arts and Science, Gazi University, 06500 Ankara, Turkey Received 9 February 2004; accepted 5 October 2004 Available online 19 March 2005

Abstract In this study, the production of citric acid has been achieved by using Aspergillus niger conidiaspores, entrapped in polyacrylamide gels, and the factors that affect this production have been investigated. On citric acid production the effect of starting sucrose concentration (100–180 g/l), the initial nitrogen concentration (0–0.3 g/l), the effect of the methanol concentration in 100 ml feeding medium (0–6 ml), and finally the effect of ethanol concentration (0–6 ml) in 100 ml feeding medium were studied and optimum experimental conditions were determined. As a result of the experiments, the starting nitrogen concentration (0.05 g/l) and the starting sucrose concentration (140 g/l) were optimized and maximum citric acid production was observed for the given conditions. On the other hand, the maximum citric acid production was observed by the addition of 4.0 ml methanol and 3.0 ml ethanol.
2004 Published by Elsevier Ltd. Keywords: Entrapment; Polyacrylamide; Citric acid; Methanol; Ethanol; Aspergillus niger

1. Introduction Citric acid, a tricarboxylic acid, is one of the world
s largest tonnages of fermentation products. It is used in the food and beverage industries as an acidifying and flavour-enhanching agent and also in other industries such as pharmaceuticals (Shojaosadati & Babaeipour, 2002). Citric acid present in citrus fruits was first crystallized from lemon juice in the form of calcium citrate. Production of citric acid from sugar solutions by aerobic bioprocesses was first realized by using Penicillum. Due to low yields obtained from Penicillum, Aspergillus niger was utilized in subsequently developed processes. Citric acid has been produced by a conventional submerged culture, in which the biomass is suspended in the medium (Ro¨hr, Fesle, Sinkha, & Panda, 2000). There*

Corresponding author. Tel.: +90 312 212 6030; fax: +90 312 212 2279. E-mail address: [email protected] (G. Demirel). 0260-8774/$ - see front matter
2004 Published by Elsevier Ltd. doi:10.1016/j.jfoodeng.2004.10.006

fore, its separation from the medium and the biomass, provides ease of separation for the product and thus, continuous production of citric acid can be readily achieved. By using immobilized cells, the process can be controlled more easily than with a batch system of free cells. In addition, immobilized cells are more stable than free biomass (Ates, Dingil, Bayraktar, & Mehmetog˘lu, 2002). The successful use of immobilized micro-organisms as living biocatalysts, often having higher production rates than free micro-organisms, has led to a rapid development of immobilization techniques in the last few years. Many studies have been carried out, mainly on the production of organic acid (Eikmeier & Rehm, 1984; Fujii, Yasuda, & Sakahibara, 1994; Sankpal, Joshi, & Kulkarni, 2001; Sankpal & Kulkarni, 2002), enzymes and oligosaccharides, using mycelia immobilized on various support materials by entrapment or adsorption techniques. With respect to citric acid production, significant studies have been carried out to achieve

K.O. Yaykasßlı et al. / Journal of Food Engineering 70 (2005) 518–522

2. Materials and methods 2.1. Microorganism Lyophilized cultures of A. niger A-9 were obtained from Ankara University Agriculture Faculty (Ankara/ Turkey). A loopful of A. niger A-9 was spread on potato–dextrose–agar (PDA) and incubated for seven days at 30 C. After seven days of incubation, 40 ml of sterilized cold water were added to each Petri dish and then the conidia suspension was prepared. 2.2. Fermentation medium The fermentation medium for citric acid production had the following composition (g/l): sucrose, 140; NH4NO3, 0.05; KH2PO4, 1.0; MgSO4 Æ 7H2O, 0.25; CuSO4 Æ 5H2O, 0.6 · 10
4; CaCl2, 3.0; ZnSO4 Æ 7H2O, 2.5 · 10
4; FeSO4 Æ 7H2O, 1.3 · 10
3. The experiments were carried out in a constant-temperature shaker at 30 C and 100 rpm. 2.3. Immobilization A chilled cell suspension was prepared by adding 2 ml spores to 10 ml chilled sterile water. To 10 ml sterile potassium phosphate buffer (pH 7.0, 0.2 M) the following chemicals were added: acrylamide, 2.85 g; bisacrylamide, 0.15 g; ammonium persulphate, 10 mg and 1 ml N,N,N 0 ,N 0 -tetramethylethylenediamine (TEMED). The chilled cell suspension and chilled potassium phosphate buffer were mixed well and poured into sterile flat bottom 10 cm diameter petriplates. After polymerization, the acrylamide gel was cut into equal size cubes (5 · 5 · 5 mm3). The acrylamide cubes were cured in sodium phosphate buffer (pH 7.0, 0.2 M) for 1 h in a

refrigerator (Ellaiah, Prabhar, Ramakrishna, Thaer, & Adinarayana, 2004). These cubes were washed thoroughly two to three times with sterile water and used for the production of citric acid. 2.4. Preactivation of immobilized cells Ten grams of immobilized cells were suspended in a 250 ml Erlenmeyer flask containing 100 ml of citric acid production medium. Immobilized cells were incubated in a constant-temperature shaker at 30 C and 100 rpm over two days. The pH of the medium was 2.0 and airflow rate was 0.5 l/min (Ates et al., 2002). 2.5. Citric acid production Ten grams of the preactivated cells were washed thoroughly (three times) with distilled sterile water and then placed in a 250 ml Erlenmeyer flask containing 100 ml of substrate solution. The pH of the medium was 2.0 and the O2 flow rate was 0.5 l/min. The experiments were carried out in a constant-temperature shaker at 30 C and 100 rpm. Citric acid in the aqueous phase was analyzed by a spectrophotometric method (Marier & Boulet, 1958).

3. Results and discussion 3.1. Effect of fermentation period on citric acid production The experimental results of citric acid production with immobilized A. niger cells at pH 2.0 is shown in Fig. 1. According to experimental results, maximum citric acid production is observed at 4 days. After 4 days, citric acid production decreased because of feed back inhibition. At the end of the 4 days, a great amount of citric acid is produced, and cannot penetrate to the outside CITRIC ACID CONCENTRATION (g/l)

higher volumetric productivities under conditions of submerged and surface modes of growth (Eikmeier & Rehm, 1987; Garg & Sharma, 1992). Several studies report the use of Aspergillus niger immobilized on various kinds of solid supports, e.g. glass carriers (Heinrich & Rehm, 1982), entrapment in calcium alginate (Cruz, Cruz, Belini, Belote, & Vieira, 1998; Vaija, Linko, & Linko, 1982), polyurethane foams (Lee, Lee, & Chang, 1989), agar (Borglum & Marshall, 1984), agarose (Khare, Jha, & Gandhi, 1994), cellulose carriers (Fujii et al., 1994; Sankpal et al., 2001), or attachment on cotton fibres, metal screens, polyester felts (Liu, Li, Ridgway, Gu, & Moo-Young, 1998; Papagianni, Joshi, & Moo-Young, 2002). In this study, the production of citric acid has been achieved by using Aspergillus niger A-9 conidiaspores, entrapped in polyacrylamide gels, and the factors that effect this production has been investigated.

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FERMENTATION PERIOD (DAY)

Fig. 1. Effect of fermentation period on the citric acid production. (T = 30 C, N = 100 rpm, pH = 2.0, Qv = 0.5 l/min. air, 0.1 g of pellet/ml of fermentation media, CSu = 140 g/l, CN = 0.05 g/l). T = Temperature (C); CSu = Sucrose concentration (g/l); CN = Nitrogen concentration (g/l); N = Stirring rate (rpm); Qv = Aeration rate (l/min.); t = Time (day).

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of the cell. Citric acid accumulated in the cell inhibits citrate synthesis (C ¸ evrimli, 2000; Jianlong, 2000).

3.3. Effect of initial sucrose concentration on citric acid production

3.2. Effect of initial nitrogen concentration on citric acid production

The effect of initial sucrose concentration on citric acid production with immobilized A. niger cell over the 4 days fermentation period was studied under optimum condition (pH 2.0, nitrogens concentration 0.05 g/ l) at a constant-temperature of 30 C. According to Fig. 3, maximum citric acid production was obtained at 140 g/l sucrose containing medium. Tsay and To (1987) reported that maximum citric acid production was obtained at 140 g/l sucrose containing medium. The present results are consistent with this finding. At higher levels of sucrose containing media than 140 g/l, citric acid production decreases because of the polyalcohol forming (Gutierrez-Rozas, Cordova, Auria, Revah, & Favela-Torres, 1995). At lower levels of sucrose containing medium than 140 g/l, citric acid production decreases because of oxalic acid forming (Honecker, Bisping, Yang, & Rehm, 1989). 3.4. Reuse number of immobilized A. niger on citric acid production One of the most important benefit immobilized cells is their repeated use. This can be possible with media replacement. Reuse numbers of immobilized A. niger was investigated in citric acid production. After 4 days, immobilized A. niger was taken from the fermentation media and put into a new sterile fermentation media. This process was repeated 6 times and experimental results obtained are shown in Fig. 4. It can be concluded that while reuse number increases, citric acid production decreases, because A. niger immobilized polyacrylamide gel increase their number inside the gel and clog the pore of the gel. Citric acid produced inside the gel accumulated and can not penetrate to the outside of the gel. So, that accumulation inhibits the citric acid production (Ates et al., 2002). The accumulation of the citric acid inside the cell inhibits the

CITRIC ACID CONCENTRATION (g/l)

CITRIC ACID CONCENTRATION (g/l)

The effect of initial nitrogen concentration on citric acid production with immobilized A. niger cell over the 4 days fermentation period was studied at a constant-temperature of 30 C. The experimental results are shown in Fig. 2. To investigate the effect of initial nitrogen concentration on citric acid production, a low nitrogen concentration interval is used due to prevent a high level micelle forming. It can be concluded from Fig. 2, that the maximum citric acid production is obtained at 0.05 g/l nitrogen concentration. At higher levels of nitrogen concentration than that citric acid production decreased. Finally, citric acid production significantly decreased at 0.3 g/l nitrogen containing media. The influence of nitrogen on the production of citric acid can be explained by the observations of Kristiansen and Sinclair (1979). Accordingly, the cytoplasm in the hyphae flows toward the tip where the new cells are formed. Meanwhile, aged cells suffer from nitrogen limitation, become carbon stores, and will produce citric acid. The number of cells produced will increase with the nitrogen concentration, and a similar increase will be observed in the flow of cytoplasm toward the new cells. If the nitrogen concentration were increased, the rate of formation of storage cells would increase, resulting in higher yields of citric acid. It is known that citric acid is produced in the mitochondria. If the flow is apparently significant, streaming of cytoplasm is transported to the nonproducing tip of the hyphae, which is not suffering nitrogen limitation and citric acid production. According to these facts, citric acid concentration would decrease at both lower and higher nitrogen levels. For this reason, the optimum nitrogen concentration must be used.

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0.05

0.1

0.15

0.2

0.25

NITROGEN CONCENTRATION (g/l)

Fig. 2. Effect of initial nitrogen concentration on the citric acid production (T = 30 C, N = 100 rpm, pH = 2.0, Qv = 0.5 l/min. air, 0.1 g of pellet/ml of fermentation media, CSu = 140 g/l, t = 4 days).

1.2 1 0.8 0.6 0.4 0.2 0 80

100

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140

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SUCROSE CONCENTRATION (g/l)

Fig. 3. Effect of initial sucrose concentration of the citric acid production (T = 30 C, N = 100 rpm, pH = 2.0, Qv = 0.5 l/min. air, 0.1 g of pellet/ml of fermentation media, CN = 0.05 g/l, t = 4 days).

CITRIC ACID CONCENTRATION (g/l)

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REUSE NUMBER

Fig. 4. Reuse number of immobilized A. niger on citric acid production (T = 30 C, N = 100 rpm, pH = 2.0, Qv = 0.5 l/min. air, 0.1 g of pellet/ml of fermentation media, CSu = 140 g/l, CN = 0.05 g/l, t = 4 days).

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methanol and 3 ml ethanol per 100 ml fermentation media. Maximum citric acid production is observed at these levels. The observed increases in citric acid concentration show that methanol and ethanol have a profound effect on the metabolism of sugars by A. niger. The mechanism by which methanol and ethanol stimulate citric acid production from sugars is not clear. Maddox, Hossain, and Brooks (1986), reported that the effect of methanol and ethanol are at the cell permeability level, allowing metabolites to be excreted from the cell (Demirel, Yaykasli, & Yasar, 2005; Haq, Ali, Qadeer, & Iqbal, 2003; Rouskas, 2000).

References citrate synthesis found in the citric acid cycle (Lehninger, Nelson, & Cox, 1993). 3.5. Effect of methanol and ethanol concentration on citric acid production

CITRIC ACID CONCENTRATION (g/l)

Several concentrations of methanol and ethanol were added to optimum citric acid production media to investigate their effect on citric acid production. According to Figs. 5 and 6, citric acid production was increased by adding methanol and ethanol up to 4 ml

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METHANOL CONCENTRATION (%) (v/v)

CITRIC ACID CONCENTRATION (g/l)

Fig. 5. Effect of methanol concentration on the citric acid production (T = 30 C, N = 100 rpm, pH = 2.0, Qv = 0.5 l/min. air, 0.1 g of pellet/ml of fermentation media, CSu = 140 g/l, CN = 0.05 g/l, t = 4 days).

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ETHANOL CONCENTRATION (%) (v/v)

Fig. 6. Effect of ethanol concentration on the citric acid production (T = 30 C, N = 100 rpm, pH = 2.0, Qv = 0.5 l/min. air, 0.1 g of pellet/ml of fermentation media, CSu = 140 g/l, CN = 0.05 g/l, t = 4 days).

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