El-Holi and Al-Delaimy. Citric acid production from whey with sugars and additives by Aspergillus niger. Afr. J. Biotechnol. 2003

Citric acid (CA) production by Aspergillus niger ATCC9642 from whey with different concentrations of sucrose, glucose, fructose, galactose riboflavin, tricalcium phosphate and methanol in surface culture process was studied. It was found that whey with 15% (w/v) sucrose with or without 1% methanol was the most favourable medium producing the highest amount (106.5 g/l) of citric acid. Lower CA was produced from whey with other concentrations of sugars and other additives used. Highest biomass of A. niger was produced with the addition of riboflavins. In general, extension of the fermentation (up to 20 days) resulted in an increase in CA and biomass, and decrease in both residual sucrose and pH values.

Key words: Citric acid, Aspergillus niger, whey fermentation, surface culture.

Introduction

Citric acid (CA), a carboxylic organic acid, soluble in water with a pleasant taste, is the most important acid used in the food industries. Until about 1920, all commercial CA was produced from lemon and lime juices (King and Cheetham, 1987). Rohr et al (1983) reported that CA can be produced by fermentation process using species of microorganisms namely Aspergillus niger, a fungus which was used commercially for the first time in 1923. They also indicated that factors affecting the production of CA by fermentation include the nutritional composition of the media, environmental conditions, deficiency of manganese and other metals, pH, and dissolved oxygen tension. The influence of types and concentrations of sugars (Hossain et al., 1984; Xu et al., 1989), chelating effect on metal ions (Roukas and Kotzekiodou, 1997), ammonium nitrate and aeration (Bayraktar and Mehmetoglu, 2000) on CA production by A. niger have also been studied. At present time CA is produced commercially using mutant strains of A. niger, and with a significant amount by Saccharomycopsis lipolytica (Good et al., 1985), Pencillium simplicissimum (Franz et al., 1993) and A. foeitidus (Tran et al., 1998). Other carbohydrates and wastes that have been considered, experimentally, to produce CA by A. niger includes inulin (Drysdale and McKay, 1995), date fruit syrup (Roukas and Kotsekiodou, 1997), sugar cane molasses (Gupta, 1994), soya whey (Khare, 1994), kumara (Lu, 1995), Carob pod (Roukas, 1998) and cheese whey (El-Samragy, 1996; Hossain et al., 1984).

Large amounts of whey are produced world wide as a by-product of cheese and other dairy products manufacturing. Whey in the Middle Eastern region is generally considered a waste and disposed in the sewage system leaving a small amount for drinking for domestic animals. The aim of this study was to produce citric acid by A. niger from cheese whey fortified with different concentrations of sucrose, glucose, fructose, galactose, tricalcium phosphates, methanol and riboflavin in a liquid surface culture process.

A. niger

A. niger ATCC 9642 stock culture was reactivated and cultivated by streaking a loopfull of the culture on Petri dishes containing solidified acidified(with 10% tartaric acid) potato dextrose agar (PDA) and incubated at 25⁰C for 5 days. Spores formed were washed out twice with 10 ml distilled sterilized water each time. Spore suspensions containing about log 8/ml were prepared and used as inoculums for the fermentation process.

Fermentation Media

Whey from the dairy plant of the University of Jordan was used as the basal fermentation media. Its proximate composition was determined. Sucrose, glucose, fructose and galactose sugar solutions of 5, 10, and 15 % (w/v) each were added to the whey in the fermentation process. Different concentrations of tricalcium phosphate (TCP), methanol (1, 2, 3, 4 and 5%), and riboflavin (10, 20, 30, 40 and 50 mg/L) were also used to fortify the fermentation media. Surface liquid culture fermentation process was carried out in a 500 ml Erlemyer flask containing 100 ml media. Each flask was inoculated with the given spore suspension and incubated at 30⁰C for up to 20 days.

Citric Acid Determination

Citric acid (CA) was determined titrimetrically (AOAC, 1995) by using 0.1 N NaOH and phenolphthalin as indicator and calculated as % according to the following formula:

Normality X volume of NaOH X Equiv. wt. of CA

%CA =

Weight of sample (g) X 10

Biomass, residual sugars and pH determination

Biomass, residual sugars and pH values were determined according to AOAC, (1995). To determine biomass, the whole fungal culture growth was filtered with Whatman filter paper #4, washed with distilled water (250 ml) and dried at 105⁰C to constant weight. Results was expressed as g/l. Residual sugars were determined using SL50 UV-Visible Spectrophotometer and expressed as g/l glucose. Culture pH was measured by Analoge WPA pH meter. The initial pH of the fermentation culture was adjusted to 3 using 1 N of HCl and/or NaOH.

Results and Discussion