Abstract
The aim of this study was to isolate and screen citric acid producing yeasts using low cost substrates. Thirty three yeast isolates were obtained from pineapple, plantain and sugar cane waste and identified as; Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida tropicalis, Pichia guilliiermondii, Debaromyces sp., Candida parapsilosis, Candida rugosa, and Candida krusei. Candida tropicalis had zone of clearance of 49±2.1 mm in diameter, Pichia guilliermondii had 40±1.2mm. Saccharomyces cerevisiae produced citric acid with glucose with 105.0 mg/l. C. tropicalis yielded 132.2 mg/l with sodium nitrate. S. cerevisiae and C. tropicalis produced citric acid at pH 6 with 23.70mg/l and 23.80mg/l. P. gulliermondii at pH 4 produced 23.00mg/l. The temperature of 30°C favoured S. cerevisiae and C. tropicalis yielding 40.80mg/l and 39.80 mg/l. After extraction, the yield of the citric acid was 4.231g, 3g of which was recrystallized to yield 2.16g of pure citric acid resulting into 72% recovery. The result indicated that pineapple wastes, plantain wastes and sugarcane cane are potential sources of yeasts that can be used for the production of citric acid.
Keywords:Citric, acid, Yeast, Yield, Parameters, Recrystallized.
Received: 3 October 2018 / Revised: 8 November 2018/ Accepted: 11 December 2018 / Published: 31 December 2018
Contribution/ Originality
This study contributes in the existing literature by providing basic information for other researchers regarding the isolation and the screening of yeasts for the production of citric acid using low cost substrates, as well as to study the effect of various fermentation parameters on citric acid production.
1. INTRODUCTION
Citric acid (2-hydroxy-1, 2, 3-propanetricarboxylic acid) is an intermediate and important commercial product of metabolism and its traces are found in all plants and animals tissues (Kamzolova et al., 2008).Citric acid is a commercially valuable organic acid, widely used in food, pharmaceutical and beverage industries. It has several applications in cosmetic industries as an acidulant flavour enhancer, preservative, antioxidant and emulsifier and chelation agent (Roehr, 1998).
Citric acid (C6H8O7) is a natural component and common metabolite of plants and animals. It is the most versatile and widely used organic acid in foods, beverages, detergents and pharmaceuticals. Citric acid is accepted as GRAS (Generally Recognized as Safe) and approved by the Joint FAO/WHO Expert Committee on Food Additives (Dhillon et al., 2011). In its pure form, this citric acid is colourless, soluble in water and solid at room temperature. Due to its functionality and environmental acceptability, it is used in numerous industrial and research applications for chelation, buffering, pH adjustment, and also as a source of energy for controlled bacterial metabolism (Yoo et al., 2004).
The annual production of citric acid is estimated to be about 1.4 to 1.5 million tons per year and its demand is estimated to be growing at a rate of about 3.5 to 4.0% annually (Soccol et al., 2006; Lazar et al., 2011; Show et al., 2015) . Of the total amount of citric acid produced annually, about 70% is utilized by the food industry; about 12% is utilized by pharmaceutical industries as flavouring, anticoagulant and preservative while the remaining 18% is utilized by other industries such as cosmetics, detergent, textile, oil recovery and paper (Soccol and Vandenberghe, 2003; Silva et al., 2011). Citric acid is also used in the detergent industry as a phosphate substitute, because of less eutrophic effect, and in the cement industry to slow down the process of hardening (Max et al., 2010). Citric acid is responsible for the tart taste of various fruits in which it occurs (i.e. lemons, limes, figs, oranges, pineapples, pears and goose-berries) (Francis, 2000). Hence, citric acid is used to impart a pleasant tart flavours to foods and beverages. It is also used in the industries to achieve acidulation, antioxidation, emulsification, preservation, flavour enhancement, and as plasticizer and synergistic agent (Soccol et al., 2006).
Citric acid production by fermentation is the most economical and widely used way of obtaining this product. More than 90 % of the citric acid produced in the world is obtained by fermentation. Citric acid can be produced using three types of microbial fermentation. These are surface fermentation, submerged fermentation and solid-state fermentation (Papagianni et al., 2005). The use of citric acid in industries is attributed to its low/non-toxicity, high solubility; biodegradability and palatability (Ali et al., 2002).
Citric acid is known as the most important organic acid produced in tonnage by fermentation and is the most exploited biochemical product (Soccol et al., 2006; Lotfy et al., 2007). All developed countries follow a conventional procedure involving the use of Aspergillus niger (producer) and molasses (substrate) for citric acid production. Use of yeasts such as Y. lipolytica however has several advantages over the use of Aspergillus spp, since yeast strains show lower sensitivity to low dissolved oxygen concentrations and heavy metals, and give higher product yields.
Citric acid can be produced by microorganisms and related yeast species. Presently most citric acid is being produced using Aspergillus niger because of its high citric productivity but there is dearth of information on the use of yeast for the production of citric acid. The constant increase in citric acid consumption, compared to its natural supply and some problems in traditional production process (molasses treatment and environmental issues) leads to the need of exploring the use of alternative microorganisms like yeasts species. Use of yeasts such as Y. lipolytica however has several advantages over the use of Aspergillus spp, since yeast strains show lower sensitivity to low dissolved oxygen concentrations and heavy metals, and give higher product yields.
The aim of this study was to isolate and screen citric acid producing yeasts using low cost substrates, as well as to study the effect of various fermentation parameters on citric acid production.
2. MATERIALS AND METHODS
2.1. Sample Collection
Samples of pineapple wastes, plantain wastes and sugarcane were collected in a sterile polythene bag from Agbowo and Bodija market in Ibadan, Oyo State, Nigeria. The samples were transported to the laboratory immediately for analysis.
2.2. Sample Preparation
The samples were rinsed with sterile distilled water and then kept in a sterile container from which samples were analysed daily. Old samples were discarded after some days and replaced
2.3. Isolation of Citric Acid Producing Yeasts
Isolation was done by using pour plate method. One gram of the plantain and pineapple wastes and sugarcane waste was dispensed in 9mls of sterile distilled water and 1ml of the sugarcane juice was dispensed in 9mls of sterile distilled water. One ml from each sample was plated out using Potato Dextrose Agar (PDA) and then incubated at room temperature for 48-72hrs at 25 0C. The culture was maintained on Potatoe Dextrose Agar (PDA) plate at 4oC for further studies.
2.4. Characterization and Identification of Yeast Isolates
Different identification system or techniques were used in the identification of the isolates using standard morphological and physiological test and identification keys described by Kurtzman et al. (2003) and Barnett (2007) which includes: macroscopic identification, microscopic evaluation, sugar fermentation etc.
2.5. Screening of Isolates for Citric Acid Production in a Solid Medium.
The basal medium for screening of citric acid was modified as described by Finogenova et al. (2008). The yeast isolates were grown on a medium of the following composition (g/L): (NH4)2SO4 1.0; KH2PO4 1.0; K2HPO4 . 3H2O 0.16; MgSO4 • 7H2O 0.70; NaCl 0.50; Ca(NO3) . 4H2O 0.40; bromocresol green 0.40; glucose 20.0; agar 20.0 and 1 L of distilled water. The yeast growth, which showed yellow colour zones around them, was selected as the citric acid producers.
2.6. Fermentation Medium and Conditions
Fermentation was carried out in the laboratory in 250 ml Erlenmeyer flasks as small scale laboratory fermentor containing 100 ml of fermentation medium in each flask. The production medium used contained (g/l): Glucose 30, (NH4)2SO4 0.5, yeast extract (Fluka, nitrogen content: 7% (w/w) 0.5, KH2PO4 7, Na2HPO4 2.5, MgSO4.7H2O 1.5, CaCl2 0.15, FeCl3.6H2O 0.15, ZnSO4.7H2O 0.02, MnSO4.H2O 0.06 (Papa Nikolaou et al., 2002) and were cultivated at 30ºC in a rotary shaker at 180 rpm speed for 7days.
2.7. Measurement of Citric Acid:
The concentration of citric acid in the fermentation medium was estimated titrimetrically (AOAC, 1995) as reported by different researchers (Imandi et al., 2008). The readings were taken at regular intervals.
2.8. Citric Acid Assay
After fermentation process, the fermentation medium was centrifuged at 4000rpm for 15minutes. Titration was used to determine the amount of citric acid in the filtrate. 0.1 M NaOH was titrated against 10 mL of the filtrate and 2 - 3 drops of phenolphthalein was used as an indicator. Once the endpoint has been reached, the volume of NaOH used (titre) on the burette was read and the titration was completed and the final calculations can be done using citric acid, 1ml 0.1M NaOH is equivalent to 0.0064 g citric acid (Emeka et al., 2012) the amount of citric acid can be expressed in terms of g/l by citric acid factor (0.0064) formula. The titration was performed in duplicates and the average titre was determined and used for the calculation using the equation below:
g/l acid = Titre x 0.0064 x 100 x 10 = Titre x 0.64
10ml of analyte (Sample)
2.9. Optimization of Fermentation Medium:
The next phase of the study conducted was optimization of the culture conditions taking 4 different parameters i.e. pH, Carbon sources and Nitrogen sources, for citric acid production as it showed the maximum levels of acid production so far. The citric acid production was estimated after 7 days of incubation of the inoculated medium.
2.9.1. pH of the Medium
The pH of the fermentative medium was another criteria that was worked upon. A range of pH starting from 4, 5, 6, 7 and 8.0 were selected for citric acid production by the yeast isolates.
2.9.2. Carbon Sources
Five Carbon sources were chosen and tested for their suitability as substrates for citric acid production, which were; Glucose, Sucrose, Glycerol, Maltose and Fructose.
2.9.3. Nitrogen Sources
Five different Nitrogen sources were used and their suitability was checked for citric acid production. The sources selected were: Ammonium phosphate, Yeast extract, Sodium nitrate, Peptone, and Urea.
2.9.4. Temperature
Different temperature ranges were selected i.e. 30°C, 35°C, 40° and the fermentative media were allowed to produce citric acid under these temperature ranges. The amount of citric acid produced at different temperatures by the yeasts was estimated gravimetrically, using pyridine-acetic anhydride method as reported by Marier and Boulet (1958) and Ikram-ul et al. (2004) One ml of the culture filtrate along with 1.3ml of pyridine was added in the test tube and shacked then 5.70ml of acetic anhydride was added in the test tube. The test tube was placed in a water bath for 30min. The optical density was measured on a spectrophotometric (450 nm) and citric acid contents of the sample were estimated with reference (run parallel, replacing 2.0 ml of the culture filtrate with distilled water) to the standard.
2.9.5. Agitation
The effect of agitation on the citric acid production by incubating a set up containing the fermentation medium and the yeasts in an incubator (static) while another set up was incubated in a rotary shaker at 180rpm for 7 days (agitated).
2.10. Recovery of Citric Acid Process
After the completion of the fermentation process, the incubated broth (300mls) was filtered for the separation of pellet form of yeast culture and the fermentation broth, which acts as the source of citric acid. Lime (Ca (OH) 2) was added to the fermentation broth to allow precipitation of citric acid in the form of calcium citrate. The precipitate was recovered (by centrifuging the suspension at 3000 rpm for 10 minutes) and dried at room temperature.
2 C6H8O7 + 3Ca (OH)2 = (C6H5O7)2Ca3.4H2O+2H2O
Again, the precipitate was treated with concentrated sulphuric acid (H2SO4) to precipitate insoluble calcium sulphate, and then filtered, leaving citric acid in solution.
(C6H5O7)2Ca34H2O+3H2SO4= 2C6H8O7 +3CaSO42H2O+2H2O
The resultant supernatant (presumed to be citric acid in solution) was decanted and was kept in the refrigerator for 2 hours. The crystals were formed and washed with small amount water twice, dried at room temperature and weighed prior to the estimation of melting temperature (Jinglan et al., 2009).
2.10.1. Assessment of the Purity of the Crystals
A melting point apparatus was used to assess the purity of the product obtained. The temperature at which the product crystals melted was compared with the melting temperature of pure, anhydrous citric acid crystals (153°C; melting point of monohydrate approx. 75°C).
2.10.2. The Recrystallization Procedure
The purpose of recrystallizing the recovered citric acid was to remove any by-product and impurities that might be attached to the crystals. Most impurities are believed that the impurities would be soluble in the solvent, hence filtered out of the mixture.
To 3g of citric acid, 5ml of hot distilled water to totally dissolve the recovered citric acid crystals and 45ml of hot acetone, place on ice cubes where it will not be disturbed for about 10 minutes to allow the crystals to collect at the bottom of the beaker. It was then filtered and dried at room temperature.
2.10.3. Melting Point Determination
A melting point capillary was used to pick small amount of the citric acid until the crystals fall to the bottom of the capillary. The capillary was placed in the melting point apparatus (also melting- temperature apparatus) and the crystals was watched from the lens to determine the approximate range. The melting temperature range was determined from the first appearance of liquid in the capillary tube to the sample’s complete liquefaction. The melting points and the appearances of the sample with a standard (Laboratory citric acid) (Downham and Collins, 2000).
2.11. Data Analysis
All the data were analyzed using statistical analyses.
3. RESULT
3.1. Isolates Obtained From the Samples
The total number of isolates obtained from pineapple waste (PP) was eight, that of plantain waste (PL) was six, while the isolates obtained from sugarcane juice (SC) was nineteen. In the course of this study, the total number of yeast isolates obtained were 33, belonging to six (6) Genera and eight (8) species.
3.2. Distribution of Isolates Obtained From the Samples
Table 4.3 shows the distribution of yeast isolates obtained from the samples, Saccharomyces cerevisiae was found to occur throughout the sampling and also has the highest total count of 13. Also, most of the yeast isolates obtained were isolated from sugarcane juice (Saccharomyces cerevisiae, Candida parapsilosis, Candida tropicalis, Candida rugosa, Pichia guilliermondii, Debaromyces sp., and Candida krusei), while the least were obtained from pineapple waste (Saccharomyces cerevisiae, Schizosaccharomyces pombe and Candida parapsilosis).
3.3. Diameter of Zones of Inhibition and Percentage of Occurrence of the Yeast Isolates
Table 4.4 shows the diameter of zone of clearance (in mm) from screening of the yeast isolates for citric acid production. All the yeast isolates showed significant zones of clearance from the screening. SC9 (identified to be Candida tropicalis) gave the highest zone of clearance with 49±2.1mm, while the lowest zone of clearance was obtained from PL5 (identified as Saccharomyces cerevisiae) with 09±2.3mm.
Fig. 4.1 represents the percentage of occurrence of yeast isolates obtained from the samples (Pineapple waste, plantain wastes and sugarcane juice). S. cerevisiae had the highest occurrence of 39.4% and C. rugosa had the lowest with 3.03%.
3.4. Optimization of the Fermentation Medium
The effect of different carbon sources on citric acid yield by the yeast isolates was represented in fig. 4.2, where S. cerevisiae, P. guilliermondii and C. tropicalis produced the highest citric acid with glucose and gave the lowest yield with fructose and glycerol respectively.
In fig. 4.3, the effect of different nitrogen sources on the yield of citric acid was represented. Sodium nitrate was observed to favour the highest yield of citric acid by the yeast isolates (S. cerevisiae, P.
guilliermondii, and C. tropicalis). Ammonium phosphate gave the lowest yield of citric acid for S. cerevisiae and P. guilliermondii while C. tropicalis gave the lowest yield with Urea.
The effect of different pH on the yield of citric acid was also done and this was represented in fig. 4.4. S. cerevisiae produced highest at pH 7 and lowest pH 6, P. guilliermondii gave the highest yield at pH 4 and lowest at pH 6, while C. tropicalis gave the highest yield of citric acid at pH 6 and lowest at pH 5.
Fig. 4.5 represents the effect of agitation on the yield of citric acid by the yeast isolates. All the yeast isolates produced more when agitated, compared to when static fermentation was done.
In fig. 4.6, the effect of different temperatures on citric acid yield was represented. S. cerevisiae and C. tropicalis produced highest at 30°C, while P. guilliermondii produced highest at 35°C. Meanwhile, it was observed that at 40°C, the yeast isolates had the lowest yield.
The effect of different incubation period on the yield of citric acid was represented in fig. 4.7. P. guilliermondii and C. tropicalis produced highest on day 6, while S. cerevisiae gave the highest yield on the 5th day after which the yield declined.
Table-1. Distribution of Yeast Isolates Obtained from the Samples
Most Probable Organism |
Total Number (N) |
Pineapple Wastes |
Plantain Wastes |
Sugarcane Juice |
Saccharomyces cerevisiae |
13 |
+ |
+ |
+ |
Schizosaccharomyces pombe |
2 |
+ |
+ |
- |
Candida parapsilosis |
5 |
+ |
+ |
+ |
Candida tropicalis |
5 |
- |
- |
+ |
Candida rugosa |
1 |
- |
- |
+ |
Pichia guilliermondii |
3 |
- |
+ |
+ |
Debaromyces sp. |
2 |
- |
- |
+ |
Candida krusei |
2 |
- |
- |
+ |
Total |
33 |
Table-2. Yeast Isolates and Their Zones of Clearance from Screening For Citric Acid Production
No. |
Yeast Isolates |
Diameter of their Zones of Clearance (mm) |
1 |
PP1 |
34± 0.5 |
2 |
PP2 |
27±0.3 |
3 |
PP3 |
45±1.7 |
4 |
PP4 |
18±1.3 |
5 |
PP5 |
25±0.5 |
6 |
PP6 |
34±0.5 |
7 |
PP7 |
37±0.8 |
8 |
PP8 |
38±0.9 |
9 |
PL1 |
36±0.7 |
10 |
PL2 |
40±1.1 |
11 |
PL3 |
29±0.06 |
12 |
PL4 |
30±0.05 |
13 |
PL5 |
09±2.3 |
14 |
PL6 |
15±1.6 |
15 |
SC1 |
21±0.9 |
16 |
SC2 |
40±1.2 |
17 |
SC3 |
12±2.0 |
18 |
SC4 |
35±0.6 |
19 |
SC5 |
29±0.06 |
20 |
SC6 |
33±0.4 |
21 |
SC7 |
22±0.8 |
22 |
SC8 |
22±0.8 |
23 |
SC9 |
49±2.1 |
24 |
SC10 |
33±0.8 |
25 |
SC11 |
37±1.6 |
26 |
SC12 |
21±0.9 |
27 |
SC13 |
39±1.0 |
28 |
SC14 |
30±0.05 |
29 |
SC15 |
29±0.06 |
30 |
SC16 |
20±1.1 |
31 |
SC17 |
29±0.06 |
32 |
SC18 |
30±0.05 |
33 |
SC19 |
27±0.3 |
Figure-1. Percentage of occurrence of yeast isolates obtained from Pineapple waste, Plantain waste and Sugarcane juice.
Figure-2. Effect of different carbon sources on citric acid yield by the yeast isolates
Figure-3. Effect of different Nitrogen sources on citric acid yield by the yeast isolates
Figure-4. Effect of different pH on citric acid yield by the yeast isolates
Figure-5. Effect of Agitation on citric acid yield by the yeast isolates
Figure-6. Effect of different temperature on citric acid yield by the yeast isolates
Figure-7. Effect of different incubation period on citric acid yield by the yeast isolates
Table-3. Melting point range of the Citric acid crystals.
Melting point range of crystals |
||
Before recrystallization |
140 - 141°C |
|
After recrystallization |
151 - 152°C |
4. DISCUSSION
In the course of the study, a total number of 33 yeasts were isolated and identified. They include; Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida tropicalis, Pichia guilliiermondii, Debaromyces sp., Candida parapsilosis, Candida rugosa, and Candida krusei as shown in Table 4.1.
4.3 shows the distribution and total number of each isolate with Saccharomyces cerevisiae having the highest number of 13, followed by C. parapsilosis and C. tropicalis representing 5 isolates each, Pichia guilliermondii, with 3,Debaromyces sp., C. krusei and Schizosaccharomyces pombe being 2 each, while the lowest number was recorded with C. rugosa which was 1.
The samples employed in the course of this study were pineapple waste, plantain waste and sugarcane juice. The yeast isolates obtained from pineapple wastes (Table 4.3) has been reported by several researchers such as Chanprasartsuk et al. (2013) that isolated yeasts from the spontaneously fermentation of pineapple, and Santoshkumar and Patil (2006) who isolated wine yeast from pineapple fruits. Also the yeast species isolated from plantain wastes (Table 4.3) has been reported by Rao et al. (2008) who isolated and characterized ethanol-producing yeasts from fruits and tree barks. While the isolates from sugar cane juice (Table 4.3) correlates with the findings of Qureshi et al. (2007) that isolated Strains of S. cerevisiae from sugar cane based on their maltose utilization capacity. Ahmed et al. (2010) also conducted a study on the mycoflora associated with sugar cane juice isolated and Shehata (1960) where he isolated yeast from sugarcane juice to produce aguardente de cana where similar yeast were isolated and identified.
As shown in figure 4.1, Saccharomyces cerevisiae had the highest percentage of occurrence of 39.4%, followed by C. tropicalis and C. rugosa having 15.15% each, P. guilliermondii had 9.09% while Schizosaccharomyces pombe, Debaromyces sp. and C. krusei had 6.06%, and lastly C. rugosa had the least with 3.03%.This results were similar to the findings of Matapathi et al. (2010) who observed that Saccharomyces species dominates during fermentation more than any other yeast.
All the yeast isolates showed zones of clearance during the screening for citric acid production but with varying diameters. It was concluded that pineapple wastes, pineapple wastes and sugar cane juice is potentially a suitable source to select fermentative yeasts (Silva et al., 2011). It was observed that even isolates that was identified as the same species gave different diameters of clearance which could be attributed to being different strains of the same species. The yeast isolates which was used for further studies were, SC9 (Candida tropicalis) which was isolated from sugarcane juice gave the highest zone of clearance with 49±2.1mm followed by PP3 (Saccharomyces cerevisiae) which gave 45±1.7mm Yeasts (Saccharomyces spp.) have been used as an alternative to filamentous fungi to produce citric acid (Burden and Eveleigh, 1990) and PL2 (Pichia guilliermondii) isolated from plantain waste gave 40±1.1mm, all in diameter. The result of the screening was as shown in Table 4.4. It was observed that SC2 also gave a clearance of 40±1.2 but was not considered for further studies because it was also identified as Candida tropicalis (Table 4.2). This was in line with the previous work of Rymowicz et al. (2006) and Crolla and Kennedy (2001).
The carbon sources employed for the study were maltose, sucrose, glycerol, glucose and fructose. The three yeast isolates was observed to give the highest yield of citric acid utilizing glucose with S. cerevisiae yielding 105.0 mg/l, P. guilliermondii with 93.3 mg/l and C. gulliermondii with 69.5mg/l. This is similar to a previous study performed with C. lipolytica, by Hamissa et al., 1981, where it was reported that high citric acid yields were obtained when glucose was used as substrate. The least yield of citric acid for S. cerevisiae and P. guilliermondii was with fructose, yielding 16.2mg/l and 15.40mg/l respectively, while C. tropicalis gave the least yield with glycerol with 3.49mg/l. This was represented in Fig. 4.2.
Fig. 4.3 shows the yield of citric acid with different nitrogen sources .The sources of nitrogen were sodium nitrate, yeast extract, peptone, urea, and Ammonium phosphate. It was observed that the yeast isolates employed in the course of this study (S. cerevisiae, P. guilliermondii and C. tropicalis.) yielded the highest with Sodium nitrate with 99.70mg/l, 115.0mg/l and 132.2 mg/l respectively. These results agree with the report of Soccol et al. (2006) that inorganic sources of nitrogen have the potential of increasing the yield of citric acid. However, S. cerevisiae and P. guilliermondii yielded the least citric acid with 10.20mg/l and 11.60mg/l respectively when they utilized Ammonium phosphate, but C. tropicalis produced the least citric acid utilizing Urea with 21.60mg/l.
In Fig. 4.4, It was observed that at pH 6, S. cerevisiae, produced the highest citric acid with 23.70mg/l and the least yield was at pH 7, while P. guilliermondii produced highest at pH 4 with 23.80mg/l. C. tropicalis also produced highest at pH 6, yielding 23.00mg/l. It has been reported When working with yeasts, initial pH values above 5 should be used since citric acid production is adversely affected below pH 5 (Mattey, 1992).
The effect of agitation on the production of citric acid by the yeast isolates was also done, S. cerevisiae produced 55.90mg/l when agitated compared to when it was static with 26mg/l. This was also observed with P. guilliermondii and C. tropicalis which produced highest when the fermentation medium was agitated with 19.0mg/l and 75.70mg/l respectively compared to when it was static with 16.30mg/l and 38.90mg/l. This was as shown in Fig. 4.5. It is known that agitation increases the area available for oxygen transfer by dispersing air and insoluble substrate in the culture fluid in the form of fine bubbles and an optimum agitation speed is required to maximize product production (Kamzolova et al., 2003).
Temperature is also one of the factors that could affect both the growth of yeasts and the production of citric acid. The temperature that favoured the highest yield of citric acid for S. cerevisiae and C. tropicalis was 30°C yielding, 40.80mg/l and 39.80 mg/l while P. gulliermondii gave the highest yield at 35°C with 50.90mg/l. This was presented in Fig. 4.6. Temperature reported in various researches on citric acid production by yeasts was between 22-35°C and that optimum temperature for citric acid production may change relative to the used strain and medium conditions (Crolla and Kennedy, 2001).
The day on which the yeast isolates produced highest after incubation for S. cerevisiae was observed to be on day 5 with 24.32g/l, while both P. guilliermondii and C. tropicalis produced the highest on day 6 with 20.48g/l and 23.02 g/l yield of citric acid. Similar findings were obtained and reported by previous researchers such as Karasu-Yalcin et al., 2010 where they worked on the effect of fermentation conditions on growth and citric acid production kinetics of two Yarrowia lipolytica strains.
Lye and Woodley (1999) observed that many fermentation processes have low productivity and yield which may be due to product inhibition or hydrolysis of product by further catalytic reactions, which can be said of the yield of the 4.231 g of citric acid from 300ml of the fermentation medium in the course of this research.
A recovery of 72% (2.159 g) was obtained after the recrystallization of 3g of the extracted citric acid. This is in agreement with Karklin et al. (1984) when he reported that the yield of citric acid was between 64.8 - 92.9% when the classical method, precipitation was used in the extraction of citric acid from the fermentation of n-alkanes.
The solubility of the compound in the solvent used for recrystallization is important. In the ideal case, the solvent would completely dissolve the compound to be purified at high temperature, usually the boiling point of the solvent, and the compound would be completely insoluble in that solvent at room temperature or at 0 oC. In addition the impurity either would be completely insoluble in the particular solvent at the high temperature, or would be very soluble in the solvent at low temperature. In the former case, the impurity could be filtered off at high temperature, while in the latter case the impurity would completely stay in solution upon cooling. A suitable recrystallization solvent should also be partially volatile in order to be easily removed from the purified crystals. The solvent should not react with the compound being purified and it should have the boiling point below the melting point of the compound being purified because solid melts before it dissolves (oiling out) because recrystallization is a technique that has to be practiced and perfected. Hence, the choice of acetone in the course of this work, which has been reported by Vandenberghe et al. (1999) that better results was also achieved using extraction technique at normal temperature when acetone was used, followed by water, ethanol and then methanol.
A pure substance will always have the same melting point, no matter how it was prepared. An impure substance will melt differently than a pure substance in two ways (a) it will melt at a different, usually lower temperature than the pure sample (b) it will usually melt over a broad temperature range rather than a range of 1-3 degrees that is expected for a pure substance. Which as shown in table 4.11, the melting temperature of the crystals before recrystallization was between 140°C and 141°C, whereas the melting temperature of the recrystallized citric acid ranged between 151°C to 152°C and this is comparable to the standard melting point of citric acid, which is 153°C.
5. CONCLUSION
From the present study, it can be concluded that pineapple wastes, plantain wastes and sugarcane cane are cheap agricultural wastes that could be used as substrates for citric acid production using yeasts. In addition to the isolation and screening of yeast isolates for their ability to produce citric acid, the optimization of fermentation conditions such as the carbon sources, nitrogen sources, temperature, pH, agitation and the incubation period are of primary importance in the development of any fermentation process owing to their impact on the yield of citric acid obtained.
Funding: This study received no specific financial support. |
Competing Interests: The authors declare that they have no competing interests. |
Contributors/Acknowledgement: All authors contributed equally to the conception and design of the study. |
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