Waste and Biomass Valorization

Citric Acid characterization on HPLC produced from Indigenous Fungal Strain through
Single and Co-Culture Fermentation
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Full Title: Citric Acid characterization on HPLC produced from Indigenous Fungal Strain through
Single and Co-Culture Fermentation

Article Type: Original research article

Keywords: Citric acid; Aspergillus niger; Rhizopus stolonifer; fermentation; HPLC

Corresponding Author: Muddassar Zafar, Ph.D

University of Gujrat Faculty of Sciences
Gujrat, Pujab PAKISTAN

Corresponding Author Secondary

Information:

Corresponding Author's Institution: University of Gujrat Faculty of Sciences

Corresponding Author's Secondary

Institution:

First Author: Fatima Arshad, M. Phil

First Author Secondary Information:

Order of Authors: Fatima Arshad, M. Phil

Seemab Faizi, Ph. D

Muhammad Imran, Ph. D

Raja Tahir Mehmood, Ph. D

Zahid Anwar, Ph. D

Muddassar Zafar, Ph.D

Order of Authors Secondary Information:

Funding Information:

Abstract: Agro-based waste materials are primarily composed of complex polysaccharides that
strengthen microbial growth to produce industrially relevant value-added products.
Therefore, in the present study, solid state fermentation (SSF) was carried out using
peanut shell, orange peel and mixture of both with 50:50 ratio as support for SSF to
enhance citric acid production from single and co-culture consortia of Rhizopus
stolonifera and Aspergillus niger. During initial trial, it was observed that growth media
supplemented with orange peel under solid state fermentation (SSF) process of co-
culture consortia revealed high yield (1593µg/mL) of citric acid. On partial optimization
of co- culture showed the maximum citric acid yields (7651µg/mL) in the presence of
orange peel base medium at 50% moisture content, 30 0C temperature, 5.5 pH and
25g substrate concentration after 48th hours of solid state fermentation. This amount of
citric acid produced in the culture was estimated through reversed-phase High
Performance Liquid Chromatography (HPLC) technique. Recent research activity
revealed that a suitable addition of fermentative substrate to the solid-state
fermentation media increased fungal growth, sugar utilization and citric acid production
when used in lower concentration.

Suggested Reviewers: Joachim Venus, Ph. D

Senior Scientist, Leibniz-Institut fur Agrartechnik und Biookonomie eV
jvenus@atb-potsdam.de
He is expert in agriculture engineering

D. A. Lightfoot, Ph.D
Professor, Room # 176, Ag. Building Southern Illinois University (SIU) Carbondale, IL

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 62901, USA
ga4082@siu.edu
Professor of Microbial Biotechnology

Olli H. Tuoviner, Ph. D

Professor, Ohio State University, 376 Biological Science Building, 484 West 12th AVE
Coloumbous USA, 43210-1292
tuoviner.1@osu.edu
He is expert in Microbiology

William B. Whitman, Ph. D

Professor, The University of Georgia 541 Biological Sciences Building Athens, GA
30602-2605, USA
Whitman@uga.edu
He is expert in microbiology

Wyatt Byrd, Ph. D

Assistant professor, University of New Mexico, MSC 10-5550, Albuquerque, NM
87131-000, USA
WyByrd@salud.unm.edu
He is expert in Microbiology

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4 Citric Acid characterization on HPLC produced from Indigenous Fungal Strain through Single and Co-
5
6 Culture Fermentation
7 Fatima Arshad1, Seemab Faizi2, Muhammad Imran3, Raja Tahir Mehmood4, Zahid Anwar1 and Muddassar
8
9 Zafar1*
10 1
Department of Biochemistry and Biotechnology, University of Gujrat, Gujrat, Pakistan
11
2
12 Department of Pharmacy, University of Lahore, Islamabad Campus, Pakistan
13 3
Institute of Biochemistry & Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan
14
4
15 Department of Biotechnology, Mirpur University of Science and Technology, Mirpur, Azad Jammu and Kashmir,
16 Pakistan
17
18
19 Corresponding author: muddassar.zafar@uog.edu.pk Ph # 0923346510834
20
21 Abstract
22 Agro-based waste materials are primarily composed of complex polysaccharides that strengthen microbial growth to
23
24 produce industrially relevant value-added products. Therefore, in the present study, solid state fermentation (SSF) was
25 carried out using peanut shell, orange peel and mixture of both with 50:50 ratio as support for SSF to enhance citric
26
27 acid production from single and co-culture consortia of Rhizopus stolonifera and Aspergillus niger. During initial trial,
28 it was observed that growth media supplemented with orange peel under solid state fermentation (SSF) process of co-
29
30 culture consortia revealed high yield (1593µg/mL) of citric acid. On partial optimization of co- culture showed the
31 maximum citric acid yields (7651µg/mL) in the presence of orange peel base medium at 50 % moisture content, 30
32
33 ⁰ C temperature, 5.5 pH and 25g substrate concentration after 48 th hours of solid state fermentation. This amount of
34 citric acid produced in the culture was estimated through reversed-phase High Performance Liquid Chromatography
35
36 (HPLC) technique. Recent research activity revealed that a suitable addition of fermentative substrate to the solid-
37 state fermentation media increased fungal growth, sugar utilization and citric acid production when used in lower
38
39 concentration.
40
41
Key words: Citric acid; Aspergillus niger; Rhizopus stolonifera; fermentation; HPLC
42
43
44 INTRODUCTION
45
46
47 Citric acid having chemical formula “1, 2, 3-tricarboxylic acid” is a very important organic acid, whose name comes
48 from the Latin word Citrus [1]. Worldwide citric acid requirements are divided between the main areas including food,
49
50 75% of confection and beverages, 10% of pharmaceuticals and 15% of other different industries. Nevertheless, it has
51 pleasant taste and its characteristic for improving the existing flavors strengthen central place in the market [2]. A lot
52
53 more investigation is required to examine the factors which are responsible for yielding low quantity of citric acid and
54 to advance the strategies for increment in yield reducing production costs. The process development can be solved by
55
56 improving the properties of the production from naturally occurring microbes or by generating hyper-producing
57 fermentation conditions [3]. Significant attention has been shown in consuming agricultural waste for production of
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59 citric acid [4]. For this purpose, various agro-industrial scums, including cassava bagasse, apple fill, wheat hay, kiwi
60 fruit bark, bananas and sugar beet cosset were studied as fermentation substrates [5]. In developing countries, these
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4 wastes are not eradicated and become an important pollutant for environmental [6]. According to a survey, yield of
5
6 citric acid is 500,000 tons annually using fermenting expensive raw resources such as sucrose, glucose. In developed
7 countries, 65% of citric acid is consumed in food and brews and 20% in domestic detergents with an estimated
8
9 increment in demand percentage is between 4 and 5% annually [7;8]. Different substrates like date fruit syrup, cane
10 syrup, carob, kurum and inulin are being used to produce citric acid from A. niger [7;8]. According to Business
11
12 Communication Co. (BBC) citric acid is the favorite most biological acid which is produced by the fermentation at
13 the world production rate round 1.4 million tons per year. Strong competition and comparatively low prices have
14
15 broken down many small citric acid businesses over the last ten years. Large manufacturers then profited from the
16 economy of larger scale manufacturing [9]. All manufacturers, including ADM and Tate & Lyle, have lowered citric
17
18 acid yield and the assets were closed in the Czech Republic due to hostile market situations [10]. Citric acid is produced
19
by submerged fermentation of starch or sucrose-based media using Aspergillus niger [11]. The last decade has been
20
21 of interest in the production of citric acid in solid state fermentation (SSF), since SSF offers numerous compensations
22
in the production of chemicals and enzymes [12]. This is partly because solid-state processes require lower energy
23
24 and produce much less wastewater and environmental wastes related to disposal for solid waste. Pakistan is the large
25
producer of lingo cellulosic wastes such as orange peel, peanut shells and mixture. Industrial residues based on
26
27 agricultural products from sugar cane, orange, coffee and rice processing are a suitable raw material for bioconversion
28
29 into chemicals, including citric acid through a fermentation process, which adds the value normally associated with
30 waste products [13] .
31
32 Materials and methods
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34 Substrate and fungal collection
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37 Orange peel and peanut shell were selected as substrates to produce citric acid. Different fungal species were isolated
38 from different spoil foods like bread, cake and from rotten fruits from local area of Sialkot, Pakistan to produce citric
39
40 acid.
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42
Media preparation and inoculums preparation
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45 Isolated fungus species were cultivated on PDA (potato starch-dextrose-agar) media on petri plates at 37 ⁰ C
46
temperature for 4-5 days and then store at 4 ⁰ C for future use. PDA slants were prepared by the method proposed by
47
48 Noomrio & Dahot [14] for inoculation of the substrate to make spore suspension. PDA broth was prepared, and pH
49
50 was adjusted to 5.5 and then autoclaved for 15 min at 121ᵒC temperature and leave for cooling. A loop full of fungal
51 culture was take from the sporulation medium of fungus that was raised on PDA slants aseptically into the 250ml flask
52
53 that contain 150ml sterilized inoculums medium. The flasks were placed in shaking incubator for 4 to 5 days at 37
54 ⁰ C [14].
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57 Substrate for citric acid production
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4 Orange peel, peanut shell and their different combinations were tried to produce citric acid. This experiment was
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6 performed in 250ml Erlenmeyer flask containing 5 g of the substrate.
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8 Fermentation protocol
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11 In initial substrate screening trial, 5 g of each above-mentioned substrate was added in a 250ml Erlenmeyer flask,
12 moistened with distilled water and inoculated with fungal spore suspension of Rhizopus stolonifer and Aspergillus
13
14 niger and co-culture consortia. All these experimental flasks were placed into an incubator at 37 ⁰ C± 1 for 3to 4 days.
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After 3 days, 50ml of distilled water was added in each flask and then placed into a shaking incubator at 37 ⁰ C for
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17 30 min at 180 rpm. The homogenized media were centrifuged at 10,000 rpm for 10 min at 4ᵒC. The clear supernatant
18
was used for further analytical studies.
19
20
21 Determination of biomass
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24 All samples tested in triplicates and their results presented as mean± standard error of mean. During extraction process,
25 the pellets obtained were re-suspended in 50mM phosphate buffer at 7.0 pH and re-centrifuged at 10,000 rpm for 10
26
27 min in falcon tubes and dried at 80 ⁰ C and final biomass weights were recorded in gram.
28
29 Optimization of fermentation parameters
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32 A classical method was used to optimize the product and to study the effect of incubation period (2 to 6 days) on the
33 production of citric acid from co-culture consortia in the presence of solid substrates. Citric acid production by co-
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35 culture consortia was also carried at different pH values (3 to 7) in triplet flasks. After pH adjustment, citric acid was
36
produced from co- culture consortia at different temperature (25 to 45 ᵒC). Effect of different inoculums concentration
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38 (2 to 8 ml) were also observed for the citric acid production. Effect of various amino acid (glutamine, Arginine,
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Tyrosine, Glycine and Aspartic acid) on the production of citric acid from co –culture consortia were also observed.
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42 Screening of fungal isolate for citric acid production
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45 Primary screening
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47 Screening of fungal species was done to identify the citric acid producing strains. Four strains of fungus (Rhizopus
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49 stolonifer, Aspergillus niger, Trichoderma cupreum and Trichoderma oryzae) were selected for screening and
50 evaluated on the base of maximum citric acid production. The screening experiment was performed on petri plates
51
52 containing czapek –Dox agar medium with bromocresol green as an indicator. The media was prepared by dissolving
53 all the ingredient and autoclaved. After being cooled, poured into sterile petri plates and allowed to cool at room
54
55 temperature. After solidifying, it contained 0.5 ml of conidial suspension was transferred to these plates. The plates
56 were rotated clockwise and anticlockwise to spread suspension uniformly and incubated at 37ᵒC for 3-5 days.
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59 Secondary screening
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4 Two fungal strains; Rhizopus stolonifer and Aspergillus niger were evaluated for single and co-culture consortia after
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6 primary screening. This experiment was done in 250ml Erlenmeyer flask containing 5 g and 10ml distilled water to
7 give moisture content and plugged with cotton for autoclave. Each flask contained 3ml inoculums of Rhizopus
8
9 stolonifer and Aspergillus niger and some flasks contained co-culture of both fungal strains.
10
11 Screening of substrate
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14 Orange peel (OP), peanut shell (PS) and their mixture were used as a substrate in citric acid production. Experiments
15
were performed in 250 ml Erlenmeyer flask that contained 5 g of substrate OP (1:0), PS (1:0) and MM (1:1) and added
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17 5 ml of water in each flask to give moisture content then plugged with cotton and autoclaved them at 121ᵒC for 15
18
min. The substrates were inoculated with 3ml of spore suspension which could yield maximum citric acid by co-
19
20 culturing of Rhizopus stolonifera & Aspergillus niger.
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22
23 Optimum incubation period
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25 Triplicate flasks were prepared containing 5g orange peel and 5ml distilled water in each flask to give moisture and
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27 plugged with cotton and autoclaved at 121ᵒC for 15 min to estimate the effect of fermentation period on citric acid
28 production. After being cooled, substrate was inoculated with 3ml of spore suspension containing 1.5ml each of both
29
30 inoculums and incubated for (2 to 6) days to select the optimum fermentation period for maximum production of citric
31 acid [15].
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34 Optimum pH
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Triplicate flasks were incubated at various pH solution (pH 1, 3, 4, 5, 5.5 and 7) to evaluate the effect of pH change
37
38 on citric acid production. Five gram of orange peel was added in 250 ml flask and moisturized it with distilled water
39
whose pH was maintained and then plugged with cotton and autoclaved at 121 ᵒC for 15 min. pH change was controlled
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41 using NaOH and HCl.
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44 Optimum Temperature
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46 For the evaluation of temperature effect on production of citric acid triplicate flasks were prepared at different ranges
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48 of temperature (25, 30, 35, 40 and 45ᵒC) having 5g of orange peel and 5ml of previously adjusted pH buffer was added
49 to moisturize it, plugged with cotton and autoclaved for 15min at 121ᵒC. After being cooled, 3ml of inoculums was
50
51 added to 1.5ml of each microorganism [15].
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53 Optimum moisture content
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56 Moisture is very important factor to produce citric acid from micro-organism. To find out the effect of optimum
57
moisture content, substrates were moisturizing at varying concentrations (50%, 60%,70% and 80%).
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60 Effect of different substrate quantities on Citric acid production
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4 Different quantity of orange peel (5g, 10g, 15g, 25g and 35g) was evaluated to estimate the effect of substrate
5
6 concentration on citric acid production. For each parameter, triplicate flasks were prepared to optimize substrate to
7 produce citric acid.
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10 Sample Harvesting
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12 After the incubation time, the flask in each experiment were harvested. In each flask 50ml of distilled water was added
13
14 and then culture was homogenized. The flasks were kept in shaking incubator at 120 rpm at 37 ⁰ C for 30min. The
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fermented biomass samples were filtered by filtration assembly apparatus. Then centrifuged at 10,000 rpm for10 min
16
17 to remove the spores of the organism. The supernatants were collected carefully and stored in refrigerator. Clear
18
supernatants were used for citric acid analysis [15-16].
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21 Determination of citric acid
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24 The amount of citric acid present in the fermented samples was determined by the HPLC technique [16].
25
26 Preparation of Citric acid standard (HPLC grade)
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29 HPLC grade citric acid standard was purchased from Sigma-aldrich (USA). A stock solution (500 ppm) of standard
30 was prepared. Different dilutions were prepared having concentrations of 100, 125, 200, 225, 300 and 325 ppm (Figure
31
32 1).
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34
HPLC analysis
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37 An HPLC (Hitachi, Japan) system was used for the determination of citric acid from samples. The detail of the HPLC
38
39 system is following; A pump (L-2130) was used to maintain and perform the analysis at a specific flow rate. Auto
40 sampler(L-2200) was used for the determination of citric acid. Column oven (L-2350Hitachi) was used to perform the
41
42 analysis at a specific temperature. Column was placed inside this oven. The range of this oven was 25-60 ᵒC. It is the
43 part of HPLC system in which actual separation of the analytes of sample mixture take place. Non –polar or reversed
44
45 phase(C-8) column was used for the detection of citric acid. A uv/visible detector (L-2420) Hitachi was used and the
46 wavelength was 212 nm. A software (D-200 Elite) was used to observe the presence of different analyte being
47
48 separated from the sample mixtures. This software displayed the integrated signals of detector in the form of peaks on
49 the chromatograph. Distilled deionized water (DD H2O) was used as mobile phase. The flow rate was set to 1.5ml/min
50
51 and absorbance was noted at 212nm using UV/visible detector. The flow rate was set to 1.5ml/min and absorbance
52 was noted at 212nm using uv/visible detector. The analysis of citric acid was performed at 25ᵒC using reversed phase
53
54 (C-8) column having 15cm length,4.6mm diameter and 5µ particle size. The results of different samples were
55 compared with standard result of citric acid for both quantitative and qualitative analysis. Retention time and peak
56
57 areas of standards were noted and calculated respectively. These calculations were employed for estimation of
58 amounts of citric acid present in fermented sample [16].
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4 RESULTS AND DISCUSSIONS
5
6
7 The present research work was performed at Industrial Biochemistry Lab, Department of Biochemistry and
8 Biotechnology, University of Gujrat, HH campus, Gujrat, Pakistan. The major goal of this study was to produce citric
9
10 acid by utilizing cheap, renewable agricultural wastes through the process of Solid-State Fermentation (SSF). Orange
11 peel and peanut shell were used as carbon sources. The Aspergillus niger and Rhizopus stolonifer was used to produce
12
13 citric acid. The conditions were optimizing under Response Surface Methodology (RSM) [17].
14
15
Screening of fungal isolate for citric acid production
16
17 Micro-organisms were screened on Czape –dox agar media with bromocresol green as an indicator. After 3-5 days
18
petri plates were observed yellow zones were appeared due to formation of citric acid. Two fungal species (Rhizopus
19
20 stolonifer, Aspergillus niger) were selected for further experiment.
21
22 Screening of microbial consortia
23
24
25 After the stipulated fermentation period, it was observed that citric acid continued to synthesize in the flask that were
26 supplemented with peanut shell and orange peel and mixture of both as a growth supported substrate at a significant
27
28 rate. It was observed that co-culture of Rhizopus stolonifer and Aspergillus niger incubated with orange peel gave the
29 maximal citric acid yield (Figure 2). Orange and orange peel revealed maximum yield of citric acid in solid state
30
31 fermentation process as compared with other substances (Table 1). On the base of optimal citric acid production, a co-
32
culture consortium was selected as a best yielded culture for further optimization of the various physiochemical and
33
34 nutritional factors [17].
35
36
37 Optimization of incubation temperature
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39 Maximum citric acid production was achieved (1012µg/mL) at 30ᵒC while lowest yield was observed at 45 ⁰ C.
40
41 Further increase in temperature suppressed the citric acid productivity. A similar kind of decreasing trend in the citric
42 acid synthesis was recorded at lower and higher temperature range than optimal level (Table 2). One possible reason
43
44 of this behavior was that at higher temperature the enzyme was denatured and hence caused the reduction in citric acid
45 production
46
47
48 Response surface methodology (RSM)
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50 RSM is an integrated technique in which combined effect of pH, incubated time, inoculums concentration, substrate
51
52 concentration and incubation moisture were observed at a time (Table 3). Citric acid production from co-culture of
53
Rhizopus stolonifer, Aspergillus niger was conducted at different time periods from 2 to 6 days to determine the effect
54
55 of different time period. Results revealed that maximum citric acid yield was obtained after 2 days of incubation
56
(7651µg/mL) with orange peel while peanut shell-based media produced second highest yield of citric acid. The
57
58 culture that was inoculated with mixed substrate showed least amount of citric acid production as compared to single
59
substrate. The possible reason of further reduction with increasing time period could be age of fungus, production of
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4 inhibitory metabolites, depletion of sugar contents and reduction of nitrogen in the fermentation medium [18]. The
5
6 optimum time period for maximum production of citric acid varied with co-culture organisms and growth supported
7 substrate [19]. The effect of pH was checked pH value range from 1 to 7 in triplicate flask to produce citric acid by
8
9 co-culture and maximum yield (7651µg/mL) was recorded at pH 5.5. However, results showed that citric acid
10 production decreased when pH level was above 5.5. pH is very critical parameter for fungal growth and for the working
11
12 of its enzymes. Quantities effect of pH medium on the citric acid production was reported by Bhattacharjee and Baruah
13 [20]. Moisture level was investigated to determine the effect of various incubation moisture (50 to 75%) on citric acid
14
15 production using orange peel. The results showed that 50% moisture level was found optimal to produce citric acid
16 because it gave maximum yield (Table 3). Further increase in moisture content changed its type form solid state
17
18 fermentation to semi-solid fermentation and then further increase its moisture up to 75% changed into semi-liquid and
19
then liquid state. It had already been reported in literature that SSF was better media for citric acid production than
20
21 liquid or semi-liquid type because in solid state fermentation the chances of contamination was lowered [21]. Present
22
data also supported the earlier phenomenon’s that citric acid production was increased under SSF at 50% moisture.
23
24 Citric acid production was decreased when the moisture content increased above 50 %. The maximum yield of citric
25
acid was achieved by co-culture of Rhizopus stolonifer & Aspergillus niger under substrate concentration of 25g
26
27 (Table 3). It was observed that high carbon source increased citric acid yield. It was observed that Rhizopus stolonifer
28
29 and Aspergillus niger were used reducing sugars present in the filtrate for its functional activity to produce citric acid
30 at higher titers by hydrolyzing polysaccharides [8; 21]. Present study revealed that the supreme production of citric
31
32 acid was attained using 25 g orange peel with 50 % moisture content inoculated with 6ml of inoculums, incubated for
33 48 hours at pH 5.5 and temperature 30 ⁰ C. Citric acid is very important in industries so it must be produced at low
34
35 cast or inexpensive method (Figure 3; Table 3). There are several environmental parameters that have great influence
36 on the production of citric acid for example concentration of substrate microorganism, moisture level, time period,
37
38 temperature and pH [22-23].
39
40 Conclusion:
41
42 The present study indicated that production of citric acid by co-culturing of Rhizopus stolonifer and Aspergillus niger
43 using orange peel as a substrate which had enough polysaccharides that were efficient, safe, renewable inexpensive
44
45 and abundantly available carbon and energy source for micro-organism. Analysis profile showed that the maximum
46 production of citric acid was obtained using 25 g orange peel with 50 % moisture content inoculated with 6ml of
47
48 inoculums, incubated for 48 hours at pH 5.5 and temperature 30 ⁰ C. Citric acid production through solid state
49 fermentation provided a key opportunity for achieving tremendous benefits of biomass utilization. Currently, two
50
51 significant points of organic acid production technologies were reaction conditions and the production cost of the
52
related system. In conclusion, the present research findings will be supportive in the improvement of low-cost system
53
54 for hyper-production of citric acid for industrial application.
55
56
57 References
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Figure Click here to access/download;Figure;figures.docx

4000000

3500000

3000000

2500000
Peak Area

2000000

1500000

1000000

500000

0
0 100 200 300 400

Concentration (µg/ml)

Fig. 1: Standard curve of Citric acid (HPLC grade)

Fig 2: HPLC chromatogram of Citric acid production in fermented sample (orange peel was used as a
substrate and inoculated with both strains
Fig 3; HPLC chromatogram of citric acid production in fermented sample (optimum conditions of RSM)
Table Click here to access/download;Table;tables.docx

Table 1: Amount of citric acid produce by single and co-culturing of substrate and fungal strain

Sr. No. Substrate Strains Amount (µg/mL) of

citric acid production
determined by HPLC
1 Orange +peanut Mixed strain 0.97
2 Orange + peanut Aspergillus niger 4.2
3 Orange +peanut Rhizopus stolonifer 1003.07
4 Orange + peel Mixed strain 1593.58
5 Orange peel Aspergillus niger 396
6 Orange peel Rhizopus stolonifer 10.7
7 Peanut shell Mixed strain 571
8 Peanut shell Aspergillus niger 451
9 Peanut shell Rhizopus stolonifer 580

Table: 2. Amount of citric acid production at various temperature

Sr No. Temperature Amount (µg/mL) of citric acid production
determined by HPLC
1 20ᵒC 34.09

2 25ᵒC 44.56

3 30ᵒC 1012

4 35ᵒC 34.3

5 40ᵒC 32.01

6 45ᵒC 31

Table 3: RSM optimization of pH, incubation period, moisture content, substrate concentration and
inoculums concentration
Amount (µg/mL) of citric
Sr no. pH Time (hours) Substrate Inoculums Moisture content acid production
concentration concentration determined by HPLC
1 3 48 5g 6ml 50% 2810

2 7 48 25 g 2ml 60% 4223

3 5.5 48 25 g 6ml 50% 7651

4 3 48 5g 2ml 55% 3883

5 3 48 25 g 2ml 50% 5533

6 7 48 5g 2ml 70% 363

7 5.5 48 25 g 6ml 65% 2034

8 3 48 25 g 6ml 55% 6033

9 5 96 10g 4ml 70% 19.7

10 5 96 35g 4ml 65% 30

11 5.5 96 15g 8ml 80% 10.3

12 1 96 15g 4ml 75% 47

13 4 96 15g 4ml 50% 3396

14 5.5 144 5g 2ml 60% 571.7

15 4 144 15g 2ml 75% 40.99

16 5.5 144 25g 6ml 70% 580

17 3 144 5g 2ml 75% 30

18 4 144 10g 6ml 65% 758

19 7 144 25g 6ml 50% 1876

20 3 144 25g 6ml 55% 40.30

21 5 144 15g 2ml 60% 175

Attachment to manuscript Click here to access/download;Attachment to manuscript;Title
Page.docx

Citric Acid characterization on HPLC produced from Indigenous Fungal Strain through Single and Co-
Culture Fermentation
Fatima Arshad1, Seemab Faizi2, Muhammad Imran3, Raja Tahir Mehmood4, Zahid Anwar1 and
Muddassar Zafar1*
1
Department of Biochemistry and Biotechnology, University of Gujrat, Gujrat, Pakistan
2
Department of Pharmacy, University of Lahore, Islamabad Campus, Pakistan
3
Institute of Biochemistry & Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan
4
Department of Biotechnology, Mirpur University of Science and Technology, Mirpur, Azad Jammu and
Kashmir, Pakistan

Corresponding author: muddassar.zafar@uog.edu.pk Ph # 0923346510834

Abstract
Agro-based waste materials are primarily composed of complex polysaccharides that strengthen microbial growth
to produce industrially relevant value-added products. Therefore, in the present study, solid state fermentation
(SSF) was carried out using peanut shell, orange peel and mixture of both with 50:50 ratio as support for SSF to
enhance citric acid production from single and co-culture consortia of Rhizopus stolonifera and Aspergillus niger.
During initial trial, it was observed that growth media supplemented with orange peel under solid state
fermentation (SSF) process of co-culture consortia revealed high yield (1593µg/mL) of citric acid. On partial
optimization of co- culture showed the maximum citric acid yields (7651µg/mL) in the presence of orange peel
base medium at 50 % moisture content, 30 ⁰ C temperature, 5.5 pH and 25g substrate concentration after 48 th
hours of solid state fermentation. This amount of citric acid produced in the culture was estimated through
reversed-phase High Performance Liquid Chromatography (HPLC) technique. Recent research activity revealed
that a suitable addition of fermentative substrate to the solid-state fermentation media increased fungal growth,
sugar utilization and citric acid production when used in lower concentration.

Key words: Citric acid; Aspergillus niger; Rhizopus stolonifera; fermentation; HPLC