zcitrus-processing industry

Papagianni (2007) citric acid is regarded as a metabolite of energy metabolism, of which the concentration will rise to appreciable amounts only under conditions of substantive metabolic imbalances. Citric acid fermentation conditions were established during the 1930s and 1940s, when the effects of various medium components were evaluated. The biochemical mechanism by which Aspergillus niger accumulates citric acid has continued to attract interest even though its commercial production by fermentation has been established for decades. Although extensive basic biochemical research has been carried out with A. niger, the understanding of the events relevant for citric acid accumulation is not completely understood. This review is focused on citric acid fermentation by A. niger. Emphasis is given to aspects of fermentation biochemistry, membrane transport in A. niger and modeling of the production process.

 

Jongh and Nielsen (2008) involved in the reductive branch of the tricarboxylic acid (TCA) cycle on citrate production by Aspergillus niger was evaluated. Several different genes were inserted individually and in combination, i.e. malate dehydrogenase (mdh2) from Saccharomyces cerevisiae, two truncated, cytosolic targeted, fumarases (Fum1s and FumRs) from S. cerevisiae and Rhizopus oryzae, respectively, and the cytosolic soluble fumarate reductase (Frds1) from S. cerevisiae. Overexpression of these genes in their native strain backgrounds has been reported to lead to alterations in the intracellular cytosolic dicarboxylate concentrations. It was found that all the transformant strains had enhanced yield and productivities of citrate compared with the wild-type strain. The transformants also had the ability to produce citrate in trace-manganese-contaminated medium, where the wild type was unable to produce. Overexpression of FumRs and Frds1 resulted in the best citrate-producing strain in the presence of trace manganese concentrations. This strain gave a maximum yield of 0.9g citrate per g glucose and a maximum specific productivity of 0.025g citrate per g DW per h. Overexpression of mdh2 alone resulted in an increased citrate production rate only in the initial phase of the fermentations compared with the other transformants and the wild type.

 

Rivas et al. (2008) reported that the citrus-processing industry generates in the Mediterranean area huge amounts of orange peel as a byproduct from the industrial extraction of citrus juices. To reduce its environmental impact as well as to provide an extra profit, this residue was investigated in this study as an alternative substrate for the fermentative production of citric acid. Orange peel contained 16.9% soluble sugars, 9.21% cellulose, 10.5% hemicellulose, and 42.5% pectin as the most important components. To get solutions rich in soluble and starchy sugars to be used as a carbon source for citric acid fermentation, this raw material was submitted to autohydrolysis, a process that does not make use of any acidic catalyst. Liquors obtained by this process under optimum conditions (temperature of 130 degrees C and a liquid/solid ratio of 8.0 g/g) contained 38.2 g/L free sugars (8.3 g/L sucrose, 13.7 g/L glucose, and 16.2 g/L fructose) and significant amounts of metals, particularly Mg, Ca, Zn, and K. Without additional nutrients, these liquors were employed for citric acid production by Aspergillus niger CECT 2090 (ATCC 9142, NRRL 599). Addition of calcium carbonate enhanced citric acid production because it prevented progressive acidification of the medium. Moreover, the influence of methanol addition on citric acid formation was investigated. Under the best conditions (40 mL of methanol/kg of medium), an effective conversion of sugars into citric acid was ensured (maximum citric acid concentration of 9.2 g/L, volumetric productivity of 0.128 g/(L.h), and yield of product on consumed sugars of 0.53 g/g), hence demonstrating the potential of orange peel wastes as an alternative raw material for citric acid fermentation.

Kumar and Jain (2008) treated sugarcane bagasse supplemented with sucrose medium was found 1.7 fold (citric acid based on sugar consumption) better substrate than untreated bagasse carrier. The performance of packed bed reactor at aeration rate of 0.75 l/min and apparent packing density of 35.0 g/l was superior with citric acid yield of 55.90% (w/w), overall productivity of 0.087 g/100 g DS.h and specific growth rate of 0.055 h-1. However, in flask fermentation citric acid yield of 41.56% (w/w) with overall productivity of 0.064 g/100 gDS.h and specific growth rate of 0.043 h-1 was observed. The system confirmed that citric acid production was Type-II fermentation. Citric acid recovery of 90.39% (w/w) was achieved from fermented broth.

sequential optimization strategy

Susana (2005) worked that Solid state fermentation (SSF) has become a very attractive alternative to submerged fermentation (SmF) for specific applications due to the recent improvements in reactor designs. This paper reviews the application of SSF to the production of several metabolites relevant for the food processing industry, centred on flavors, enzymes (α-amylase, fructosyl transferase, lipase, pectinase), organic acids (lactic acid, citric acid) and xanthan gum. In addition, different types of biorreactor for SSF processes have been described.

Ali and Haq (2005) investigated deals with the promotry effect of different additives and metallic micro minerals on citric acid production by Aspergillus niger MNNG-115 using different carbohydrate materials. For this, sugar cane bagasse was fortified with sucrose salt medium. Ethanol and coconut oil at 3.0% (v/w) level increased citric acid productivity. Fluoroacetate at a concentration of 1.0 mg/ml bagasse enhanced the yield of citric acid significantly. However, the addition of ethanol and fluoroacetate after 6 h of growth gave the maximum conversion of available sugar to citric acid. In another study, influence of some metallic micro-minerals viz. copper sulphate, molybdenum sulphate, zinc sulphate and cobalt sulphate on microbial synthesis of citric acid using molasses medium was also carried out. It was found that copper sulphate and molybdenum sulphate remarkably enhanced the production of citric acid while zinc sulphate was not so effective. However, cobalt sulphate was the least effective for microbial biosynthesis of citric acid under the same experimental conditions. In case of CuSO4, the strain of Aspergillus niger MNNG-115 showed enhanced citric productivity with experimental (9.80%) over the control (7.54%). In addition, the specific productivity of the culture at 30 ppm CuSO4 (Q(p) = 0.012a g/g cells/h) was several folds higher than other all other concentrations. All kinetic parameters including yield coefficients and volumetric rates revealed the hyper productivity of citric acid by CuSO4 using blackstrap molasses as the basal carbon source.

Haq et al. (2005) investigated is concerned with the optimization of nitrogen for enhanced citric acid productivity by a 2-deoxy D-glucose resistant culture of Aspergillus niger NGd-280 in a 15 l stirred tank bioreactor. Nutrients, especially nitrogen source have a marked influence on citrate productivity because it is an essential constituent of basal cell proteins. Citric acid has been known to be produced when the nitrogen source was the limiting factor. Ammonium nitrate was employed as a nitrogen source in the present study and batch culture experiments were carried out under various concentrations of ammonium nitrate. Specific growth rate was decreased and the biosynthesis of citric acid was delayed at higher concentrations of ammonium nitrate. Specific citric acid production rate was the highest when intracellular ammonium ion concentration was between 2.0 and 3.0 mmol g(-1) cells.

Xie and West (2006) determined which citric acid-producing strain of Aspergillus niger utilized wet corn distillers grains most effectively to produce citric acid. Citric acid and biomass production by the fungal strains were analysed on the untreated grains or autoclaved grains using an enzyme assay and a gravimetric method respectively. Fungal citric acid production on the grains was found to occur on the untreated or autoclaved grains. The highest citric acid level on the grains was produced by A. niger ATCC 9142. The autoclaved grains supported less citric acid production by the majority of strains screened. Biomass production by the fungal strains on the untreated or autoclaved grains was quite similar. The highest citric acid yields for A. niger ATCC 9142, ATCC 10577, ATCC 11414, ATCC 12846 and ATCC 26550 were found on the untreated grains. Treatment of the grains had little effect on citric acid yields based on reducing sugars consumed by A. niger ATCC  9029 and ATCC 201122. It is feasible for citric acid-producing strains of A. niger to excrete citric acid on wet corn distillers grains whether the grains are treated or untreated. The most effective citric acid-producing strain of A. niger  was ATCC 9142. The study shows that the ethanol processing co-product wet corn distillers grains could be utilized as a substrate for the commercial production of citric acid by A. niger without treatment of the grains.

Kim et al (2006) investigated that citric acid is an effective remediation agent for soils contaminated by heavy metals. The large-scale field use of this organic acid requires the development of efficient production techniques using low cost substrates such as sugar rich wastes. To develop such techniques, the objective was to simultaneously optimize the initial glucose, nitrogen (N), phosphorus (P) and NaCl levels of a nutrient solution used to wet peat moss (PM) simulating a sugar rich waste on which Aspergillus niger NRRL 567 was grown to produce citric acid. Seventeen different combinations of nutrients were tested to grow A. Niger at 30 °C for 48 and 72 h, and to measure the resulting citric acid production. With the central composite design method (CCD), the results were used to produce a second order equation defining citric acid production as a function of initial glucose, N, P and NaCl levels. Initial glucose and N levels were found to have a significant positive and negative effect on citric acid production, while P and NaCl were found to have a limited positive and an insignificant effect, respectively. A peak citric acid production of 82 g kg−1 dry peat moss (DPM) was reached after 72 h with the following optimized nutrient solution, in terms of g kg−1 DPM: 967.9 glucose, 15.4 (NH4)2SO4, 43.9 KH2PO4 and 4.0 NaCl. This represented a 50% increase in production, over a shorter fermentation period, compared to a basal nutrient solution optimize solely for initial glucose level.

Lofty et al. (2007) sequential optimization strategy, based on statistical designs, was employed to enhance the production of citric acid in submerged culture. For screening of fermentation medium composition significantly influencing citric acid production, the two-level Plackett-Burman design was used. Under our experimental conditions, beet molasses and corn steep liquor were found to be the major factors of the acid production. A near optimum medium formulation was obtained using this method with increased citric acid yield by five-folds. Response surface methodology (RSM) was adopted to acquire the best process conditions. In this respect, the three-level Box-Behnken design was applied. A polynomial model was created to correlate the relationship between the three variables (beet molasses, corn steep liquor and inoculum concentration) and citric acid yield. Estimated optimum composition for the production of citric acid is as follows pretreated beet molasses, 240.1g/l; corn steep liquor, 10.5g/l; and spores concentration, 10(8) spores/ml. The optimum citric acid yield was 87.81% which is 14 times than the basal medium. The five level central composite design was used for outlining the optimum values of the fermentation factors initial pH, aeration rate and temperature on citric acid production. Estimated optimum values for the production of citric acid are as follows initial pH 4.0; aeration rate, 6500ml/min and fermentation temperature, 31.50C.

solid state fermentation

Kumar et al. (2003) used solid state fermentation (SSF) method to produce citric acid by Aspergillus niger DS 1 using sugarcane bagasse as a carrier and sucrose or molasses based medium as a moistening agent. Initially bagasse and wheat bran were compared as carrier. Bagasse was the most suitable carrier, as it did not show agglomeration after moistening with medium, resulting in better heat and mass transfer during fermentation and higher product yield. Different parameters such as moisture content, particle size, sugar level and methanol concentration of the medium were optimised and 75% moisture level, 31.8 g sugar/100 g dry solid, 4% (v/w) methanol and particles of the size between 1.2 and 1.6 mm were found to be optimal. Sucrose and clarified and non-clarified molasses medium were also tested as moistening agents for SSF and under optimised conditions, 20.2, 19.8 and 17.9 g citric acid /100 g of dry solid with yield of 69.6, 64.5 and 62.4% (based on sugar consumed) was obtained in sucrose, clarified and non-clarified molasses medium respectively, after 9 days of fermentation.

Rodriguez and Sanroman (2004) invested that Solid-sate fermentation (SSF) has received new interest not only from researchers but also from industry. This technique has become a very attractive and alternative to submerged (SmF) for specific application due to the recent improvements in reactor designs

Vandenberghe et al. (2004) studied were conducted to evaluate citric acid production by solid-state fermentation (SSF) using cassava bagasse as substrate employing a fungal culture of Aspergillus niger LPB 21 at laboratory and semipilot scale. Optimization of the process parameters temperature, pH, initial humidity, aeration, and nutritive composition was conducted in flasks and column fermentors. The results showed that thermal treatment of cassava bagasse enhanced fungal fermentation efficacy, resulting in 220 g of citric acid/kg of dry cassava bagasse with only treated cassava bagasse as substrate. The results obtained from the factorial experimental design in a column bioreactor showed that an aeration rate of 60 mL/min (3 mL/[g.min]) and 60% initial humidity were optimum, resulting in 265.7 g/kg of dry cassava bagasse citric acid production. This was almost 1.6 times higher than the quantities produced under unoptimized conditions (167.4 g of citric acid/kg of dry cassava bagasse). The defined parameters were transferred to semipilot scale, which showed high promise for large-scale citric acid production by SSF with cassava bagasse. Respirometry assays were carried out in order to follow indirectly the biomass evolution of the process. Citric acid production reached 220, 309, 263, and 269 g/kg of dry cassava bagasse in Erlenmeyer flasks, column fermentors, a tray bioreactor, and a horizontal drum bioreactor, respectively.

Ikram et al. (2004) investigated deals with citric acid production by some selected mutant strains of Aspergillus niger from cane molasses in 250 ml Erlenmeyer flasks. For this purpose, a conidial suspension of A. niger GCB-75, which produced 31.1 g/l citric acid from 15% (w/v) molasses sugar, was subjected to UV-induced mutagenesis. Among the 3 variants, GCM-45 was found to be a better producer of citric acid (50.0 +/- 2a) and it was further improved by chemical mutagenesis using N-methyl, N-nitro-N-nitroso-guanidine (MNNG). Out of 3,2-deoxy-D-glucose resistant variants, GCMC-7 was selected as the best mutant, which produced 96.1 +/- 1.5 g/l citric acid 168 h after fermentation of potassium ferrocyanide and H2SO4 pre-treated blackstrap molasses in Vogel's medium. On the basis of kinetic parameters such as volumetric substrate uptake rate (Qs), and specific substrate uptake rate (qs), the volumetric productivity, theoretical yield and specific product formation rate, it was observed that the mutants were faster growing organisms and produced more citric acid. The mutant GCMC-7 has greater commercial potential than the parental strain with regard to citrate synthase activity. The addition of 2.0 x 10-5 M MgSO4x5H2O into the fermentation medium reduced the Fe2+ ion concentration by counter-acting its deleterious effect on mycelial growth. The magnesium ions also induced a loose-pelleted form of growth (0.6 mm, diameter), reduced the biomass concentration (12.5 g/l) and increased the volumetric productivity of citric acid monohydrate (113.6±5 g/l).

Gokhan et al. (2005) The production of citric acid was achieved by using Aspergillus niger conidiaspores, entrapped in Ca-alginate beads, and the factors that affect this production were investigated. The effects of starting sucrose concentration (100-180 g/l), nitrogen concentration (0-0.3 g/l), methanol concentration (0-6 ml) and finally ethanol concentration (0-5 ml) in 100 ml feeding medium on citric acid production were studied and optimum experimental conditions were determined. The starting nitrogen concentration (0.05 g/l) and the starting sucrose concentration (140 g/1) were optimized and maximum citric acid production observed under these given conditions. Maximum citric acid production was observed upon addition of 4.0 ml methanol and 3.0 ml ethanol.

solid state fermentation method

Pazouki and Panda (2002) studies have been considered very important in fungal fermentation. Morphological parameters and the type of mycelia present (free mycelia without any branches, branched mycelia and branched mycelia with conidiophore) were measured to correlate citric acid production with the morphology of Aspergillus niger. We observed that morphological parameters and the type of mycelia present varied with substrate concentration. They also depended on the type of substrate (molasses and glucose) used. Maximum citric acid (6.8 kg/m3) was produced when branched mycelia with conidiophore were the most available mycelia present in the broth of molasses containing medium. Citric acid was produced in lesser quantity (1.82 kg/m3) when glucose was used. The addition of methanol doubled citric acid production, increased slightly the percentage of branched mycelia with conidiophore and conditioned the surface of the mycelia.

Hang and Woodams (2002) investigated Corn husks could serve as a potential substrate for the production of citric acid by Aspergillus niger NRRL 2001. Combined treatments of corn husks with dilute NaOH and Rapidase Pomaliq (a commercial apple juice processing enzyme preparation) significantly enhanced the yield of citric acid. Under favorable conditions (pretreated with 0.5 mol/L NaOH, followed by 120 h of fermentation at 30°C in the presence of Rapidase Pomaliq), the yield of citric acid was 259±10 g per kg of dry matter of corn husks.

Pazouk et al. (2002) morphological studies have been considered very important in fungal fermentation. Morphological parameters and the type of mycelia present (free mycelia without any branches, branched mycelia and branched mycelia with conidiophore) were measured to correlate citric acid production with the morphology of Aspergillus niger. We observed that morphological parameters and the type of mycelia present varied with substrate concentration. They also depended on the type of substrate (molasses and glucose) used. Maximum citric acid (6.8 kg/m3) was produced when branched mycelia with conidiophore were the most available mycelia present in the broth of molasses containing medium. Citric acid was produced in lesser quantity (1.82 kg/m3) when glucose was used. The addition of methanol doubled citric acid production, increased slightly the percentage of branched mycelia with conidiophore and conditioned the surface of the mycelia.

Haq et al. (2003) study describes citric acid fermentation by Aspergillus niger GCB-47 in a 15-1 stainless steel stirred fermentor. Among the alcohols tested as stimulating agents, 1.0% (v/v) methanol was found to give maximum amount of anhydrous citric acid (90.02±2.2 g/l), 24 h after inoculation. This yield of citric acid was 1.96 fold higher than the control. Methanol has a direct effect on mycelial morphology and it promotes pellet formation. It also increases the cell membrane permeability to provoke more citric acid excretion from the mycelial cells. The sugar consumed and % citric acid was 108±3.8 g/l and 80.39±4.5%, respectively. The desirable mycelial morphology was in the form of small round pellets having dry cell mass 14.5±0.8 g/l. Addition of ethanol, however, did not found to enhance citric acid production, significantly. The maximum value of Yp/x (i.e., 5.825±0.25 g/g) was observed when methanol was used as a stimulating agent. The best results of anhydrous citric acid were observed, 6 days after inoculation when the initial pH of fermentation medium was kept at 6.0.

Kumar et al (2003) studied a solid state fermentation method was used to utilize pineapple, mixed fruit and maosmi waste as substrates for citric acid production using Aspergillus niger DS 1. Experiments were carried out in the presence and absence of methanol at different moisture levels. In the absence of methanol the maximum citric acid was obtained at 60% moisture level whereas in the presence of methanol the maximum citric acid was obtained at 70% moisture level. The stimulating effect of methanol was less at lower moisture level. The inhibitory effect of metal ions was also not observed and maximum citric acid yield of 51.4, 46.5 and 50% (based on sugar consumed) was obtained from pineapple, mixed fruit and maosmi residues, respectively.

viability and citric acid production

Najam (1994) stated that when three different agro industrial wastes (bagasse, maize bran and wheat bran) were utilized in the fermentation process to obtain citric acid, baggase acted as best source due to presence of sucrose and showed highest concentration of citric acid with 4.5 g/ml in 48 hours followed by wheat bran with 4.0 g/ml and maize bran with 3.0 g/ml. Maize bran and wheat bran showed less ferment ability for citric acid production as compared to baggase. Maximum production of citric acid was obtained at pH 5 (optimum) in 48 hours (optimum) with all substrates at 30oC.

 

El-Samragy et al. (1996) investigated the effect of pH value, methanol and salt concentration on the production of citric acid from cheese whey by two strains of Aspergillus niger. Lactose concentration, utilized lactose, citric acid concentration, conversion coefficient of lactose to citric acid and mycelial dry weight were measured during the fermentation process. The maximum citric acid concentration (1.06 and 0.82 g/l), were obtained at pH 3.5 after 9 days of fermentation for Aspergillus niger 1 and 2, respectively. The presence of 4% (v/v) methanol in the fermentation medium increased the amount of citric acid produced by Aspergillus niger 1 and 2 by 23% and 18% respectively. Both strains showed a high ability to utilize lactose for the production of citric acid when grown in the presence of 10% (w/v) salt, the conversion coefficient of lactose to citric acid was 28.24% for Aspergillus niger 1 and 25.60% for Aspergillus niger when the fermentation medium had a 10% (w/v level of salt. The cumulative effect of fermentation medium pH (3.5), methanol concentration (4% v/v) and salt concentration (10%, w/v) during the fermentation process of whey did not enhance the production of citric acid by Aspergillus niger strain, 1 while it did increases the production of citric acid by Aspergillus niger strain 2 by about 4-fold.

Vergano et al. (1996) studied the type of sporulation medium and time of incubation. They found that these had an effect on spore viability and citric acid production by mycelia grown from Aspergillus niger spores. They found that viability increased with time of incubation, but higher production of citric acid was achieved with spores incubated for less than 7 days.

 

Anonymous (1998) stated that Corn cobs could serve as a substrate for citric acid production by Aspergillus niger of the four cultures examined, Aspergillus niger was found to produce the highest amount of citric acid (250 g/kg dry matter of corn cobs) after 72 h of growth at 30oC in the presence of 3% methanol. They yield of citric acid was over 50% based on the amount of sugar consumed.

Roukas (1998) investigated the production of citric acid from carob pod extraction by Aspergillus niger in surface fermentation. A maximum citric acid production (4.07 g/l) was achieved at pH of 6.5 and temperature of 30oC. Other kinetic parameters, namely, citric acid yield, biomass yield, specific biomass production rate, and fermentation efficiency were maximum at pH 6.5, temperature 30oC and an initial sugar concentration of 100 g/l. The external addition of methanol into the carob pod extract at a concentration upto 4% (v/v) improved the production of citric acid.

Hang and Woodams (1998) that Corncobs could serve as a substrate for citric acid production by Aspergillus niger. Methanol had a significant effect on fungal production of citric acid from corncobs. Of the four cultures examined, A. Niger NRRL 2001 was found to produce the highest amount of citric acid (250 g/kg dry matter of corncobs) after 72 h of growth at 30°C in the presence of 3% methanol. The yield of citric acid was over 50% based on the amount of sugar consumed.

 

Karaffa et al. (2001) fungi, in particular Aspergilli, are well known for their potential to overproduce a variety of organic acids. These microorganisms have an intrinsic ability to accumulate these substances and it is generally believed that this provides the fungi with an ecological advantage, since they grow rather well at pH 3 to 5, while some species even tolerate pH values as low as 1.5. Organic acid production can be stimulated and in a number of cases conditions have been found that result in almost quantitative conversion of carbon substrate into acid. This is exploited in large-scale production of a number of organic acids like citric-, gluconic- and itaconic acid. Both in production volume as well as in knowledge available, citrate is by far the major organic acid. Citric acid (2-hydroxy-propane-1,2,3-tricarboxylic acid) is a true bulk product with an estimated global production of over 900 thousand tons in the year 2000. Till the beginning of the 20th century, it was exclusively extracted from lemons. Since the global market was dominated by an Italian cartel, other means of production were sought. Chemical synthesis was possible, but not suitable due to expensive raw materials and a complicated process with low yield. The discovery of citrate accumulation by Aspergillus niger led to a rapid development of a fermentation process, which only a decade later accounted for a large part of the global production. The application of citric acid is based on three of its properties: (1) acidity and buffer capacity, (2) taste and flavour, and (3) chelation of metal ions. Because of its three acid groups with pKa values of 3.1, 4.7 and 6.4, citrate is able to produce a very low pH in solution, but is also useful as a buffer over a broad range of pH values (2 to 7). Citric acid has a pleasant acid taste which leaves little aftertaste. It sometimes enhances flavour, but is also able to mask sweetness, such as the aspartame taste in diet beverages. Chelation of metal ions is a very important property that has led to applications such as antioxidant and preservative. Moreover, it is a "natural" substance and fully biodegradable.

production of citric acid from cane molasses

Abou-Zeid et al. (1984) studied corn steep liquor, urea and ammonium salts as nitrogen sources. These sources have been used in citric acid production by yeast.

Nawaz (1986) reported that optimal production of citric acid through cane molasses fermentation by Aspergillus niger was 4.2% at pH 5 with 15% sugar concentration. The optimum temperature observed was 30oC. The minimal yield 0.8 percent was observed at pH 2 with 25 percent sugar concentration at 35oC.

Pervez (1986) reported that production of citric acid from cane molasses by Aspergillus niger. Optimum pH was and optimal concentration of MgSO4, 7H2O KCl and KH2PO4 were 0.025, 0.01 and 0.008% respectively. The optimal fermentation period was 9 days. The yield of citric acid was 4.33% in cleared molasses and 4.09% in crude molasses.

Roukas and Harvey (1988) described that the effect of pH on the production of citric and gluconic acid from beet molasses by Aspergillus niger was studied using continuous culture. At pH values >2.5 gluconic acid was the major product, citric acid being the predominant product at low pH values. The optimum specific activities of citrate synthase, aconitase, NAD-linked isocitrate dehydrogenase, and NADP-linked isocitrate dehydrogenase occurred at pH 4 and of glucose oxidase at pH 5.

Hang and Woodams (1989) reported that a multiple contact countercurrent process was developed for leaching citric acid from apple fermented with Aspergillus niger in solid state culture. Acetone proved the most efficient among the different solvents examined as it yielded that greatest among of citric acid in the leachate and gave an extraction efficiency of 90%.

Begum et al. (1990) stated that the wild type strain CA16 and the mutants 133/40 and 277/30 were grown for 9 days in molasses media containing 12, 14 or 16% sugar, initially at pH 4, the medium was supplemented with Prescott salts (NH4NO3, KH2PO4 and MgSO4.7H2O), either at full strength (respectively concentration 2.23, 1.0 and 0.23 g/litre), half or quarter strength. Citric acid yield was always highest with 16% sugar, being 34 mg/ml for strain CA16 regardless of Prescott salt strength. With mutant 136/40, the highest yield was 63 mg/ml at full strength; with mutant 277/30, it was 88 mg/ml (55% on sugar) at quarter strength.

Roukas (1991) reported that spores of Aspergillus niger were immobilized in alginate gel beads and grown for 4 days at 30oC on molasses medium at pH 6.5 containing 20 g sucrose’s and 0.6 g nitrogenous compounds/liter, in flasks shaken at 250 rpm. The beads washed and incubated at 30oC, in the medium containing 14% total sugars, in shake-flasks aerated at0.5, 1.5 and 2.5 litres/min. pH was adjusted to 3.0 with HCl. Maximum yield of citric acid was observed after 28 days, being 35 g/litre in shake flasks and <28 g/litre (lower with less aeration) in the bioreactor. When the beads were reused in shake flasks, the citric acid concentration in successive batches reached 40, 37.5 and 30 g/litre.

Roukas and Alichanidis (1991) investigated the production of citric acid from beet molasses at a varying pH profile using cell cycle of Aspergillus niger. Best results in terms of citric acid concentration yield, productivity were obtained with a substrate pH of 3.0.

Yigitoglu (1992) worked on the citric acid production through submerged fermentation processes and described wide variation in conditions recommended for successful fermentation. The importance of the nature and quantity of trace metals, carbon and nitrogen sources and correct environmental conditions were found to be very important for citric acid fermentation.

Casida (1993) reported that beet or sugarcane molasses medium containing sugars in the range of 10-20% often was employed in citric acid fermentation and ammonium nitrate, magnesium sulphate and KH2PO4 were usually added into the medium. Hydrochloric acid then, was used to adjust the medium to low pH value and the fermentation were conducted at approximately 28oC to 30oC with proper oration. He also suggested that citric acid fermentation medium should slightly be deficient in phosphate, in one or more of the metals like manganese, iron, zinc and probably copper. Of these, manganese appeared to be particularly important. The molasses, however, contained high quantities of trace metals. Excess of these metals were reduced during pretreatment of the molasses by complexing the metals with ferrocyanide or ferricyanide.

Isolated Ctric Acid from Lemon Juice

Scheele (1784) isolated citric acid from lemon juice. Italy was the main was the main producer of citric acid from unripe lemon juice and some 90 percent of the world supply of calcium citrate came from the country. Citric acid from natural sources is now produced in a number of locations, especially, California, Hawaii and West Indies. The importance of natural citric acid has, however, greatly diminished since the development of fermentation process from sugar solution.

Wehmer (1893) described the production of citric acid by mould fermentation. He designated the mould as Citromyces and later reported that penicllium and Mucor could produce similar reaction.

Currie (1917) pointed out that strains of Aspergillus niger were infact best for the fermentative production of citric acid.

Porges (1932) reported the effect of different concentrations of sugar, inorganic nutrients, temperature and incubation periods on the production of citric acid from cane sugar. He found that the highest yield of citric acid was obtained with NaNO3, 4.0, K2HPO4 1.0, KCl 0.5 and MgSO4.7H2O gm when incubated for 7 days with 16 percent sugar concentration at 280C to 30oC.

 

Galbraith and Smith (1969) studied the filamentous growth of Aspergillus niger in submerged shake culture process for citric acid production and reported that Aspergillus niger grow in filamentous or pellet from depending on medium pH in submerged culture and concluded that pellet form is more suitable for citric acid production.

Khan et al. (1970) reported the effect of different concentrations of sugar, inorganic nutrients and different pH values of the fermentation medium on the citric acid production from cane molasses. The use of molasses in final sugar concentration of 12.5 to 15 percent was found to be best. The initial pH ranging from 3.5 to 6.0 in the molasses solution was found suitable for citric acid production. The concentration of added inorganic salts did not exceed, 4.0 g NaNO3 1.0 g KH2PO4, 7H2O 0.2 g, FeCl3 and 0.001 g MnCl2. H2O in the fermentation medium of local cane molasses. At higher concentration of salts fungal growth was increased and the citric acid production was decreased. As a source of nitrogen, peptone was inferior to sodium nitrate and potassium nitrate whose effect appeared to be the same in the citric acid production.

Banik (1975) during mutation studies on Aspergillus niger strains observed that Aspergillus niger AB 180 produced the high amount of citric acid (60.0 mg/ml) from sucrose at a level of 15 percent. The optimum concentration of NH4NO3 was 2.2 mg/ml. The optimal conditions were pH 3.5; temperature 27oC, incubation period 9 days. The yield was 80.2 mg/ml. Addition of sodium monofluoracetate (50 g/l) to the fermentation medium increased the citric acid production to 120.4 mg/ml.

Kubicek et al. (1977) studied the influence of manganese on enzyme synthesis and citric acid accumulation by Aspergillus niger and investigated that citric acid production is possible under excessive nitrogen condition provided that phosphate ions are limiting.

Jerzy et al. (1980) found that optimum temperature for 8 days citric acid fermentation on a molasses media by Aspergillus niger in flasks was 30-32oC.

Glushchenko et al. (1981) reported that production of citric acid by Aspergillus niger depended upon concentration of molasses in the medium. The growth yield biomass and the concentration of citric acid in the medium showed periodic changes during fermentation when sugar concentration was maintained at 13.5 percent.

Counting of Spores

Spore counting was done in the Microbiology Department using haemocytometer. For spore counting 0.1ml of spore suspention was poured on to heamocytometer under the cover slip. Using low power of microscope various regions of heamocytometer were observed spores were counted in mdium squares (each have 16 small squares.


3.2 Sugarcane molasses

Molasses obtained from Crescent Sugar Mill Faisalabad was used as the nutrient source and the substrate for citric acid production.

3.2 Carriers/inert support materials for solid state fermentation

Substrates used ascarriers for SSF is corn cobes. The substrate was dried to costant weight in vaccum oven at 70oC and groud through waley mill in department of soil and environmental sciences, UAF to get uniform particle size of 40mm mesh. The powdered substrate was stored in airtight plastic jars to keep them free of moisture.. .

3.3 Solid state Fermentation

Solid state fermentation was carried out in 250 ml conical flask. 5g of corn cobs was taken in separate duplicate flasks. 10 % molasses solution was prepared in the distilled water adjusted to pH 5.5 using 1MNaOH /1MHCl. The substrate in each flask was moistened by using molasses solution to 60% moisture level and autoclaved (121oC) for 15 min under 15 psi pressure.

Composition of medium for solid state fermentation of molasses medium using carrier substrates for citric acid production by A. niger.



Substrate Corn cobs

S No. T1a T1b


Substrate (g) 5 5

Molasses solution(10%) 15 15

pH, 5.5; temperature, 37oC.


After sterilization 5ml inoculum were added aseptically in each flask under the laminar flow with the help of sterilized pipette. The flask was incubated at 30oC for 3 days under still culture medium.

The substrates for citric acid fermentation using submerged technique

Several industrial important chemicals are produced via biological processes, an example of which is citric acid (an organic acid). Citric acid is ubiquitous in nature found and found in all plant and animal tissues. Citrus fruits contain citric acid in large quantities, ranging from 5% in the fruit to about 9% in juice. The sour taste of lemon juice is mainly due to the presence of citric acid (5 to 8%) and partly due to the presence of vitamin C.

Citric acid is solid at room temperature, melts at 153ºC and decomposes at higher temperatures into other products (Rajoka et al. 1998). It is non-toxic and easily oxidized in the human body

Citric acid, a tricarboxylic acid, is one of the world’s largest products of fermentation process. It is the most verstile industrial acid and being used in the food and beverage industries as an acidifying and flavour-enhancing agent and also in other industries such as detergents and pharmaceuticals (Shojaosadati & Babaeipour, 2002).

Citric acid (2- hydroxy-1, 2,3,propane tricarboxylic acid) is used in food, beverages, pharmaceuticals, chemical cosmetics and other industries for application such as acidulation, antioxident, flavour enhancement, preservation plastizer and as synergistic agent (1993;shakaranand and Lonsane, 1994).

It is non-toxic and easily oxidized in the human body. It’s wide spread industrial application are due to its high solubility, palatability and low toxicity. These uses have placed greater stress on increased citric acid production and search for more efficient fermentation process. The worldwide demand of citric acid is about 6.0 x 105 tons per year and it is bound to increase day by day (Ali et al. 2001), due to its increasing new uses in industrial process.

Citric acid (2-hydroxy-propane-1,2,3-tricarboxylic acid) is a true bulk product with an estimated global production of over 900 thousand tons in the year 2000. Till the beginning of the 20th century, it was exclusively extracted from lemons. Since the global market was dominated by an Italian cartel, other means of production were sought. Chemical synthesis was possible, but not suitable due to expensive raw materials and a complicated process with low yield. The discovery of citrate accumulation by Aspergillus niger led to a rapid development of a fermentation process, which only a decade later accounted for a large part of the global production. The application of citric acid is based on its three properties: (1) acidity and buffer capacity, (2) taste and flavour, and (3) chelation of metal ions. Because of its three acid groups with pKa values of 3.1, 4.7 and 6.4, citrate is able to produce a very low pH in solution, but is also useful as a buffer over a broad range of pH values (2 to 7). Citric acid has a pleasant acid taste which leaves little after taste. It sometimes enhances flavour, but is also able to mask sweetness, such as the aspartame taste in diet beverages. Chelation of metal ions is a very important property that has led to applications such as antioxidant and preservative. Moreover, it is a "natural" substance and fully biodegradable. Karaffa et al. (2001)

Production of citric acid from sugar solutions by aerobic bioprocesses was first realized by using Penicillium. Due to low yields obtained from Penicillium, Aspergillus niger was utilized in subsequently developed processes (Shuler,2002) Many microorganisms such as fungi and bacteria can produce citric acid. The various fungi, which have been found to accumulate citric acid in their culture media, include strains of Aspergillus niger, A. awamori, Penicillium restrictum, Trichoderma viride, Mucor piriformis and Yarrowia lipolytica (Arzumanov et al. 2000). But Aspergillus niger remained the organism of choice for the production of citric acid. (Mattey and Allan, 1990; Ali et al. 2001).

Currie (1917) pointed out that strains of Aspergillus niger were infact best for the fermentative production of citric acid. Aspergillus species are highly aerobic and are found in almost all oxygen-rich environments, where they commonly grow as molds on the surface of a substrate, as a result of the high oxygen tension. "In recent studies, increased levels of Reactive Oxygen Species (ROS) were shown to be correlated with increased levels of aflatoxin biosynthesis in Aspergillus parasiticus." (Reverberi, et al 2008) Commonly, fungi grow on carbon-rich substrates such as monosaccharides (such as glucose) and polysaccharides (such as amylose). Aspergillus species are common contaminants of starchy foods (such as bread and potatoes), and grow in or on many plants and trees

The development of a microbial process for the formation of citric acid is aimed at maximizing three things; the yield of product per gram of substrate, the concentration of product and the rate of product formation.

The substrates for citric acid fermentation using submerged technique of fermentation are beet or cane-molasses (Pazouki et al. 2000). Blackstrap sugarcane molasses is an econimically easily and abundantly available by-product of sugar industries and is a desirable raw material for citric acid fermentation because of its availability and relatively low price. Owing to the steadily increasing demand of citric acid for industrial purposes, its manufacture from cane or beet molasses has proved to be of great importance to the sugar industry (Pazouki et al., 2000). Sugar cane molasses is a complex medium and has high content of sugars and metal ions that inhibit the growth of Aspergillus niger in liquid cultures.

In the past decade or so, there has been an increasing number of reports on the use of solid-state fermentation processes for the production of a number of microbial products (Roussos et al., 1994; Nampoothiri and Pandey, 1996 and Pandey et al., 1999). This is partly because solid-state processes have lower energy requirements and produce much less wastewater and environmental concerns because disposal is of solid wastes.

Solid substrate fermentation involves “the growth of microorganisms on moist solid substrates in the absence or near absence of free flowing water” (Robinson et al., 2001).

Solid State Fermentation offers numerous advantages for production of bulk chemicals and enzymes. This is partly because solid-state processes have lower energy requirements and produce much less wastewater and environmental concerns related to disposal are for solid wastes. In addition, immobilized microbial cell systems have been the subject of extensive research during the last 20 years. This technology offers many advantages such as high yield, low risk of contamination and easy control. In short, various chemical, physical and biochemical techniques have been investigated for industrial citric acid production (Papagianni et al., 1999).

Solid-state fermentation has long been applied to the food industry. SSF is a process carried out with microbes growing on nutrient impregnated solid substrate with little or no free water. Solid state fermentation (SSF) can be directly carried out with low-cost biomaterials like corn Stover, corncobs, banana stalk, wheat bran etc. abundant and available in Pakistan with minimal or no pretreatment, and thus is relatively simple, uses less energy than submerged fermentation (SmF), and can provide unique microenvironments conductive to microbial growth and metabolic activities. The present project designed to use corncobs as carrier substrate for SSF of molasses based medium by Aspergillus niger.

Incubation temperature plays an important role in the production of citric acid. Temperature between 25-30ºC is usually employed for culturing of Aspergillus niger but temperature above 35ºC is inhibitory to citric acid formation because of the increased the production of by-product acids and also inhibition of culture development. Sanjay and Sharma (1994) reported that citric acid production by Aspergillus niger is sensitive to the initial pH of the fermentation medium.

Traditionally, SSF are characterized by the development of microorganisms in a low water-environment on a non-soluble material that acts both as physical support and source of nutrients; however it is not necessary to combine the role of support and substrate but rather reproduce the conditions of low water activity and high oxygen transference by using a nutritionally-inert material soaked with a nutrient solution (Pandey and Soccol, 1998).

Corncobs could can serve as a substrate solid state for citric acid production by Aspergillus niger in molasses based SSF. Factors will be characterized that limit growth of Aspergillus niger in SSF cultures by studying the mechanism of inhibition of microbial growth and citric acid synthesis and accumulation in cane molasses fermentation medium. It is hypothesized that development of solid state medium using corn cobs powder as solid matrix is experted to absorb sugars and minerals resulting in their slow release for Aspergillus niger and minimize the inhibition objective.