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Influence of pH on citric acid production by Aspergillus niger under submerged fermentation in carob pod extract

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Kattimani Lingappa at Gulbarga University
Kattimani Lingappa
Pramod T. at Jain University
Pramod T.
Syed Imtiaz Ali
Syed Imtiaz Ali
Syed Imtiaz Ali
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Abstract

Attempts have been made to optimize initial pH level for the production of citric acid using carob pod extract as substrate through submerged fermentation. Two strains of Aspergillus niger, MTCC 281 and KLP20, have been employed as the fermenting organisms. A. niger KLP20 produced maximum citric acid at pH 5.5. Introduction Citric acid, produced commercially by submerged fermentation using Aspergillus niger, is used in food, beverage, pharmaceutical, chemical, cosmetic and other miscellaneous industries. It has an estimated annual production of about 1000,000 tons 1-4 with good annual growth (3.5-4.0%) of demand/consumption rate 5-6 . For this purpose, different agro-industrial residues (apple pomace, orange processing waste, coffee husk, pineapple waste, cassava baggase, coconut cake, sunflower seed cake, palmyra jaggery, undersized semolina) have been used as substrates 7-13 . Carob bean (Ceratonia siliqua L) is an evergreen scleropyllous perennial and long-producing tree 14 . Carob pod is mainly cultivated in the Mediterranean and in many areas of North America with annual production of about 340,000-400,000 metric tons 15 . Carob bean contains 15 : moisture, 10-15; total sugars (glucose, fructose, sucrose and maltose), 40-50; protein, 3-4; pectin 1-2; cellulose, 7; hemicelluloses, 5; phenolic compounds, 20; fat, 0.5-1.0; and ash, 2-3%. For maximum production of citric acid, initial pH of the substrate 16 plays a very important role. This study presents the effect of initial pH of substrate, on the yield of citric acid by using carob pod extract with two strains of A. niger under submerged fermentation. Materials and Methods Two strains used were A. niger MTCC 281 procured from IMTECH, Chandigarh and A. niger KLP20 isolated from spoilt lemon and identified by Agharkar Research Institute, Pune. Strains were maintained on Potato Dextrose Agar at 4°C and sub-cultured every month. Both strains were grown on potato dextrose agar and incubated at 30°C for 7 days. The 7 day old culture slants were flooded with 5 ml of 0.01% Tween 80 (prepared in sterilized distilled water) and shaken vigorously to give inoculum, which was diluted (conc., 1x10 8 spores/ml) 17 . Carob pods (25 g) were finely grounded, powdered and added to 100 ml distilled water. The mixture was placed in a water-bath for sugar extraction from carob kibble. The extract thus obtained, contained a sugar concentration of 14-16° brix. This extract was adjusted to various pH levels using 0.1N NaOH and 0.1N HCl, and prepared extract was sterilized at 121°C for 15 min. This extract was then cooled and used for the production of citric acid.
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618 J SCI IND RES VOL 66 AUGUST 2007
Journal of Scientific & Industrial Research
Vol. 66, August 2007, pp. 618-620
*Author for correspondence
E-mail: lingappak1@rediffmail.com
Influence of pH on citric acid production by Aspergillus niger under submerged
fermentation in carob pod extract
K Lingappa*, T Pramod and Syed Imtiaz Ali
Department of Studies in Microbiology, Gulbarga University, Gulbarga 585 106
Received 31 January 2007; revised 22 March 2007; accepted 01 May 2007
Attempts have been made to optimize initial pH level for the production of citric acid using carob pod extract as substrate
through submerged fermentation. Two strains of Aspergillus niger, MTCC 281 and KLP20, have been employed as the
fermenting organisms. A. niger KLP20 produced maximum citric acid at pH 5.5.
Keywords: Aspergillus niger, Carob pod extract, Citric acid
Introduction
Citric acid, produced commercially by submerged
fermentation using Aspergillus niger, is used in food,
beverage, pharmaceutical, chemical, cosmetic and other
miscellaneous industries. It has an estimated annual
production of about 1000,000 tons1-4 with good annual
growth (3.5-4.0%) of demand/consumption rate5-6. For
this purpose, different agro-industrial residues (apple
pomace, orange processing waste, coffee husk, pineapple
waste, cassava baggase, coconut cake, sunflower seed
cake, palmyra jaggery, undersized semolina) have been
used as substrates7-13.
Carob bean (Ceratonia siliqua L) is an evergreen
scleropyllous perennial and long-producing tree14. Carob
pod is mainly cultivated in the Mediterranean and in many
areas of North America with annual production of about
340,000-400,000 metric tons15. Carob bean contains15:
moisture, 10-15; total sugars (glucose, fructose, sucrose
and maltose), 40-50; protein, 3-4; pectin 1-2; cellulose,
7; hemicelluloses, 5; phenolic compounds, 20; fat,
0.5-1.0; and ash, 2-3%. For maximum production of citric
acid, initial pH of the substrate16 plays a very important
role.
This study presents the effect of initial pH of substrate,
on the yield of citric acid by using carob pod extract with
two strains of A. niger under submerged fermentation.
Materials and Methods
Two strains used were A. niger MTCC 281 procured
from IMTECH, Chandigarh and A. niger KLP20 isolated
from spoilt lemon and identified by Agharkar Research
Institute, Pune. Strains were maintained on Potato
Dextrose Agar at 4°C and sub-cultured every month.
Both strains were grown on potato dextrose agar and
incubated at 30°C for 7 days. The 7 day old culture slants
were flooded with 5 ml of 0.01% Tween 80 (prepared in
sterilized distilled water) and shaken vigorously to give
inoculum, which was diluted (conc., 1x10 8 spores/ml)17.
Carob pods (25 g) were finely grounded, powdered
and added to 100 ml distilled water. The mixture was
placed in a water-bath for sugar extraction from carob
kibble. The extract thus obtained, contained a sugar
concentration of 14-16° brix. This extract was adjusted
to various pH levels using 0.1N NaOH and 0.1N HCl,
and prepared extract was sterilized at 121°C for 15 min.
This extract was then cooled and used for the production
of citric acid.
Production medium (extract), 100 ml, was taken in
Erlenmeyer flasks (250 ml) and inoculated with 1 ml of
spore suspension (1x108 spores/ ml). Flasks were
incubated at 30°C on a rotary shaker at 150 rpm for
5 days. Extracts were withdrawn periodically at every
24 h intervals aseptically. Extracts were then analyzed
for citric acid production18 and residual sugars19.
LINGAPPA et al: pH EFFECT ON CITRIC ACID PRODUCTION BY A. NIGER ON CAROB POD EXTRACT 619
Table 1—Effect of initial pH on the yield of citric acid by A. niger MTCC 281 on carob pod extract
pH Time, h
24 48 72 96 120
CA RS CA RS CA RS CA RS CA RS
050 050 050 050 050
2.0 535 726 920 613 510
2.5 5.2 36 728 8.8 21 5.8 14 511
3.0 636 826 10 20 716 510
3.5 635 7.8 26 11 22 714 612
4.0 6.2 34 827 10 21 613 5.5 10
4.5 734 927 13 20 914 610
5.0 733 10 25 14 22 815 611
5.5 7.9 13 11 26 17 21 11 16 711
6.0 6.8 32 826 12 23 915 610
CA: citric acid, g/l; RS: residual sugars, g/l
Table 2—Effect of initial pH on the yield of citric acid by A. niger KLP20 on carob pod extract
pH Time, h
24 48 72 96 120
CA RS CA RS CA RS CA RS CA RS
050 050 050 050 050
2.0 634 825 10.2 18 912 710
2.5 5.8 36 827 11 19 9.2 13 810
3.0 634 7.8 29 10 19 814 711
3.5 6.5 36 7.6 27 918 712 6.2 10
4.0 6.2 34 827 10 21 613 5.5 10
4.5 734 927 13 20 914 610
5.0 734 10 25 14 22 815 611
5.5 831 11 26 21 20 11 16 711
6.0 732 826 12 21 915 610
CA: citric acid, g/l; RS: residual sugars, g/l
Results and Discussion
In case of A. niger strain MTCC 281, yield of citric
acid increased with increase in pH (5.5) and thereafter
decreased at higher pH on all days (Table 1). Optimum
pH (5.5) gave maximum yield of citric acid (17 g/l) at
72 h of fermentation. In case of A. niger KLP20,
maximum citric acid production (21 g/l) was also highest
at pH 5.5 on all days at 72 h of fermentation (Table 2).
Both strains showed increased citric acid production
with a corresponding decrease in residual sugar content.
Therefore, sugar utilization and citric acid yield seems
to be a closely related phenomenon. A. niger MTCC
281 yielded maximum citric acid using residual sugars
(62%) at 72 h of fermentation. Strain KLP20 yielded
maximum citric acid utilizing residual sugars (68%) at
72 h of fermentation. Isolate KLP20 seems to be a
potential producer of citric acid as compared to A. niger
strain MTCC 281. Thus, both strains A. niger MTCC
281 and A. niger KLP20 seem to have similar optimum
pH levels for the maximum citric acid production.
Conclusions
Carob pod extract enhanced citric acid production. It
is highly rich in sugars, which favours the growth of fungi
and for the production of commercially important organic
acids/enzymes/ secondary metabolites, which otherwise
would have been left unused causing pollution problems.
Strain KLP20 has emerged as a potential producer of
620 J SCI IND RES VOL 66 AUGUST 2007
citric acid compared to MTCC strain 281. Initial pH of
5.5 has been observed optimum for the maximum
production of citric acid using strains KLP20 and MTCC
281.
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Article
  • Sep 1996
Carob bean is the fruit of Ceratonia siliqua L, an evergreen sclerophyllous perennial and long-producing tree, which naturally grows on barren, rocky and dry regions of the Mediterranean basin and other parts of world, with similar climate. Deseeded carob pod (husk), although rich in water-soluble sugars (mainly sucrose), has a very low protein content and high levels of tannins (mainly condensed), which minimize the nutritional value of carob beans. The present review attempts to appraise the current status and future potentials of the carob bean in food as well as feed industry and environment upgrading.
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Short Communication: Influence of inoculum preparation on citric acid production by Aspergillus niger
Article
  • Nov 1996
The type of sporulation medium and time of incubation had an effect on spore viability and citric acid production by mycelia grown from Aspergillus niger spores. Shu & Johnson agar (SJA) and potato dextrose agar gave higher citric acid titres than malt-extract agar. SJA also gave better germinability than the other media. Viability increased with time of incubation, but higher production of citric acid was achieved with spores incubated for less than 7 days.
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Ethanol production from carob pods by Saccharomyces cerevisiae
Article
  • Jul 1993
The production of ethanol from carob pods extract by Saccharomyces cerevisiae in static and shake flask fermentation was investigated. Shake flask fermentation proved to be a better fermentation system for the production of ethanol than static fermentation. The external addition of nutrients into the carob pods extract did not improve the production of ethanol. The maximum concentration of ethanol (75 g/l) was obtained at an inoculum amount of 0.3%, a pH of 4.5, 30°C and an initial sugar concentration of 200 g/1. Under the same fermentation conditions both sterilized and non‐sterilized carob pods extract gave the same final ethanol concentration.
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Coffee husk: An inexpensive substrate for production of citric acid by Aspergillus niger in a solid-state fermentation system
Article
  • Mar 1994
Aspergillus niger CFTRI 30 produced 1.3 g citric acid/10 g dry coffee husk in 72 h solid-state fermentation when the substrate was moistened with 0.075 M NaOH solution. Production was increased by 17% by adding a mixture of iron, copper and zinc to the medium but enrichment of the moist solid medium with (NH4)2SO4, sucrose or any of four enzymes did not improve production. The production of about 1.5 g citric acid/10 g dry coffee husk at a conversion of 82% (based on sugar consumed) under standardized conditions demonstrates the commercial potential of using the husk in this way.
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Direct Determination of Citric Acid in Milk with an Improved Pyridine-Acetic Anhydride Method
Article
  • Dec 1958
The determination of citric acid with pyridine and acetic anhydride has been investigated at reaction temperatures from 17 to 60° C. The optimum proportions of pyridine, acetic anhydride, water, and acetic acid for maximum color intensity and stability are given for each temperature. The procedure has been modified to eliminate the violent nature of the reaction, even when the analysis is done at a reaction temperature of 60° C. Details of a method for the determination of 25–200 μg. of citric acid, at a reaction temperature of 32° C., are presented. In comparison with previously published methods based on the reaction, the recommended technique results in improved sensitivity, stability, and reproducibility without requiring careful timing.
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Fermentation of orange processing wastes for citric acid production
Article
Press liquor from the dewatering of orange peel was used as a substrate for citric acid production. Of the three strains of Aspergillus niger examined, strain NRRL 599 produced the greatest amount of citric acid. The conditions favouring citric acid production were pH 5.0—6.2 in the presence of 40 ml kg−1 methanol. Under these conditions 30 g of citric acid per kg of press liquor were produced, representing a yield of 630 g kg−1 based on sugars consumed. The yield was further increased to 730 g kg−1 by thermal pretreatment of the press liquor.
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Pineapple waste - A novel substrate for citric acid production by solid-state fermentation
Article
  • Oct 1995
Citric acid production in solid-state fermentation by Aspergillus foetidus ACM3996 was better on pineapple waste than on apple pomace, wheat bran or rice bran. The highest citric acid content achieved on pineapple waste was 16.1 g per 100 g dried pineapple waste, with a moisture content of 70% and in the presence of 3% methanol. This represents a yield of 62.4% based on the sugar consumed.
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