PRODUCTION OF CITRIC ACID FROM THERMOPHILLUS ASPERGILLUS FUMIGATUS THROUGH SINGLE AND CO-CULTURE FERMENTATION BY HANIA SHAH BANO ROLL#

PRODUCTION OF CITRIC ACID FROM THERMOPHILLUS ASPERGILLUS FUMIGATUS THROUGH SINGLE AND CO-CULTURE FERMENTATION
BY
HANIA SHAH BANO
ROLL#: 17071779-007
REGISTRATION#: 17021279001
DEGREE PROGRAM: M.PHIL BIOTECHNOLOGY
DEPARTMENT: BIOCHEMISTRY AND BIOTECHNOLOGY
FACULTY: FACULTY OF SCIENCE
CAMPUS NAME: HAFIZ HAYAT
SUPERVISOR NAME: DR. Muddassar Zafar
DEGREE ENROLMENT SEMESTER: OCTOBER 26, 2017
FIRST TIME THESIS ENROLMENT SEMSTER: 3 Sep, 2018
SEMESTER IN WHICH SUPERVISOR WAS ALLOTED: M.PHIL 3rd Semester
EXPECTED THESIS COMPLETITION SEMESTER: AUGUST, 2019
DATE OF SYNPSIS SUBMISSION TO DEPARTMENT: 2 Sep, 2018
DATE OF APPROVAL FROM DRRRC:
Scholar’s signature Supervisor’s signature Signature of convener DRRC
Date of submission to the Directorate of ASRB…………………….……
Date of Approval by ASRB………………………………………………..

Introduction
Citric acid (2-hydroxy propane 1,2,3-tricarboxylic acid) is very important organic acids, its name derived from the Latin word means Citrus. In 1784 Scheele Carl Wilhelm a Swedish chemist first isolate citric acid from lemon juice , and also produced citric acid by tri carboxylic acid metabolic pathways that take place in all living cell naturally ADDIN EN.CITE <EndNote><Cite><Author>Swain</Author><Year>2011</Year><RecNum>8</RecNum><DisplayText>(Swain, Ray, &amp; Patra, 2011)</DisplayText><record><rec-number>8</rec-number><foreign-keys><key app=”EN” db-id=”092esev9oz9zvherdepvw9fmwa0p5xs2ee2r” timestamp=”1536644410″>8</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Swain, Manas R</author><author>Ray, RC</author><author>Patra, JK</author></authors></contributors><titles><title>Citric acid: microbial production and applications in food and pharmaceutical industries</title><secondary-title>Citric Acid: Synthesis, Properties and Applications, Edition</secondary-title></titles><periodical><full-title>Citric Acid: Synthesis, Properties and Applications, Edition</full-title></periodical><pages>97-118</pages><volume>1</volume><dates><year>2011</year></dates><urls></urls></record></Cite></EndNote>(Swain, Ray, & Patra, 2011). Citric acid is an ubiquitous intermediate product of metabolism and traces of citric acid are present in all plants and animals ADDIN EN.CITE <EndNote><Cite><Author>Bhattacharjee</Author><Year>2015</Year><RecNum>2</RecNum><DisplayText>(Bhattacharjee &amp; Baruah, 2015)</DisplayText><record><rec-number>2</rec-number><foreign-keys><key app=”EN” db-id=”092esev9oz9zvherdepvw9fmwa0p5xs2ee2r” timestamp=”1535731427″>2</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Bhattacharjee, Ipsita</author><author>Baruah, PK</author></authors></contributors><titles><title>Isolation and Screening of Citric Acid Producing Aspergillus Spp and Optimisation of Citric Acid Production by Aspergillus Niger S-6</title><secondary-title>Journal of Environmental Science, Toxicology and Food Technology</secondary-title></titles><pages>19-23</pages><volume>9</volume><number>3</number><dates><year>2015</year></dates><urls></urls></record></Cite></EndNote>(Bhattacharjee & Baruah, 2015). This acid is biodegradable in nature, highly soluble and low toxic. Hence, it is reffered as GRAS (Generally Regarded As Safe). Citric acid, organic acid, is produced by fermentation ADDIN EN.CITE <EndNote><Cite><Author>Artmaktad?r</Author><Year>2010</Year><RecNum>3</RecNum><DisplayText>(Artmaktad?r, 2010; Yalcin, Bozdemir, &amp; Ozbas, 2009)</DisplayText><record><rec-number>3</rec-number><foreign-keys><key app=”EN” db-id=”092esev9oz9zvherdepvw9fmwa0p5xs2ee2r” timestamp=”1535734508″>3</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Artmaktad?r, A??rl?kl? Alkoller Kullan?ld???nda</author></authors></contributors><titles><title>Enhanced production of citric acid by Aspergillus niger M-101 using lower alcohols</title><secondary-title>Türk Biyokimya Dergisi Turkish Journal of Biochemistry–Turk J Biochem</secondary-title></titles><periodical><full-title>Türk Biyokimya Dergisi Turkish Journal of Biochemistry–Turk J Biochem</full-title></periodical><pages>7-13</pages><volume>35</volume><number>1</number><dates><year>2010</year></dates><urls></urls></record></Cite><Cite><Author>Yalcin</Author><Year>2009</Year><RecNum>4</RecNum><record><rec-number>4</rec-number><foreign-keys><key app=”EN” db-id=”092esev9oz9zvherdepvw9fmwa0p5xs2ee2r” timestamp=”1535734783″>4</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Yalcin, S Karasu</author><author>Bozdemir, MT</author><author>Ozbas, ZY</author></authors></contributors><titles><title>A comparative study on citric acid production kinetics of two Yarrowia lipolytica strains in two different media</title></titles><dates><year>2009</year></dates><isbn>0975-0967</isbn><urls></urls></record></Cite></EndNote>(Artmaktad?r, 2010; Yalcin, Bozdemir, & Ozbas, 2009). Citric acid can be produced by using many by-products and residues of the agro-industry ADDIN EN.CITE <EndNote><Cite><Author>Soccol</Author><Year>2006</Year><RecNum>6</RecNum><DisplayText>(Soccol, Vandenberghe, Rodrigues, &amp; Pandey, 2006)</DisplayText><record><rec-number>6</rec-number><foreign-keys><key app=”EN” db-id=”092esev9oz9zvherdepvw9fmwa0p5xs2ee2r” timestamp=”1536057795″>6</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Soccol, Carlos R</author><author>Vandenberghe, Luciana PS</author><author>Rodrigues, Cristine</author><author>Pandey, Ashok</author></authors></contributors><titles><title>New perspectives for citric acid production and application</title><secondary-title>Food Technology &amp; Biotechnology</secondary-title></titles><periodical><full-title>Food Technology &amp; Biotechnology</full-title></periodical><volume>44</volume><number>2</number><dates><year>2006</year></dates><isbn>1330-9862</isbn><urls></urls></record></Cite></EndNote>(Soccol, Vandenberghe, Rodrigues, & Pandey, 2006). For citric acid production many microorganisms have been used for example bacteria fungi and yeast many bacteria accumulate citric acid in their culture medium ADDIN EN.CITE <EndNote><Cite><Author>Iqbal</Author><Year>2016</Year><RecNum>5</RecNum><DisplayText>(Iqbal &amp; Utara, 2016)</DisplayText><record><rec-number>5</rec-number><foreign-keys><key app=”EN” db-id=”p92xr9eznts0a9evat4ptvr35pextxeapapw” timestamp=”1536900160″>5</key></foreign-keys><ref-type name=”Generic”>13</ref-type><contributors><authors><author>Iqbal, J</author><author>Utara, U</author></authors></contributors><titles><title>Isolation of Aspergillus niger Strains from Soil and their Screening and Optimization for Enhanced Citric Acid Production using Cane Molasses as Carbon Source. n</title></titles><dates><year>2016</year></dates><publisher>August</publisher><urls></urls></record></Cite></EndNote>(Iqbal & Utara, 2016).
Many bacterial strains including Arthrobacter paraffinens, Bacillus licheniformis and Corynebacterium ssp., fungi include strains of Aspergillus niger, Aspergillus fumigatus, Aspergillus awamori, Penicillium restrictum, Trichoderma viride, Mucorpiri formis, Candida tropicalis, C. oleophila, C. guilliermondii, C. citroformans, Hansenula anamola and Yarrowia lipolytic accumulate citric acid in their cultural medium ADDIN EN.CITE <EndNote><Cite><Author>Soccol</Author><Year>2008</Year><RecNum>7</RecNum><DisplayText>(Arzumanov, Shishkanova, &amp; Finogenova, 2000; Soccol et al., 2008)</DisplayText><record><rec-number>7</rec-number><foreign-keys><key app=”EN” db-id=”092esev9oz9zvherdepvw9fmwa0p5xs2ee2r” timestamp=”1536058525″>7</key></foreign-keys><ref-type name=”Book Section”>5</ref-type><contributors><authors><author>Soccol, Carlos R</author><author>Vandenberghe, Luciana PS</author><author>Rodrigues, Cristine</author><author>Medeiros, Adriane Bianchi Pedroni</author><author>Larroche, Christian</author><author>Pandey, Ashok</author></authors></contributors><titles><title>Production of organic acids by solid-state fermentation</title><secondary-title>Current Developments in Solid-state Fermentation</secondary-title></titles><pages>205-229</pages><dates><year>2008</year></dates><publisher>Springer</publisher><urls></urls></record></Cite><Cite><Author>Arzumanov</Author><Year>2000</Year><RecNum>1</RecNum><record><rec-number>1</rec-number><foreign-keys><key app=”EN” db-id=”50td0z0t1vzs21e29265s02v90xwravfedrz” timestamp=”1536898322″>1</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Arzumanov, TE</author><author>Shishkanova, NV</author><author>Finogenova, TV</author></authors></contributors><titles><title>Biosynthesis of citric acid by Yarrowia lipolytica repeat-batch culture on ethanol</title><secondary-title>Applied Microbiology and Biotechnology</secondary-title></titles><periodical><full-title>Applied Microbiology and Biotechnology</full-title></periodical><pages>525-529</pages><volume>53</volume><number>5</number><dates><year>2000</year></dates><isbn>0175-7598</isbn><urls></urls></record></Cite></EndNote>(Arzumanov, Shishkanova, & Finogenova, 2000; Soccol et al., 2008). Mostly candidates choose the species of Aspergillus and Candida for the production of citric acid ADDIN EN.CITE <EndNote><Cite><Author>Angumeenal</Author><Year>2013</Year><RecNum>5</RecNum><DisplayText>(Angumeenal &amp; Venkappayya, 2013)</DisplayText><record><rec-number>5</rec-number><foreign-keys><key app=”EN” db-id=”092esev9oz9zvherdepvw9fmwa0p5xs2ee2r” timestamp=”1536057529″>5</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Angumeenal, AR</author><author>Venkappayya, D</author></authors></contributors><titles><title>An overview of citric acid production</title><secondary-title>LWT-Food Science and Technology</secondary-title></titles><periodical><full-title>LWT-Food Science and Technology</full-title></periodical><pages>367-370</pages><volume>50</volume><number>2</number><dates><year>2013</year></dates><isbn>0023-6438</isbn><urls></urls></record></Cite></EndNote>(Angumeenal & Venkappayya, 2013). Citric acid is widely used in food preparations, biochemical, cosmetic and pharmaceuticals industries due to its inoffensive nature. Almost 70% citric acid is used in food industry, 12% in pharmaceutical industry and 18% in further applications ADDIN EN.CITE <EndNote><Cite><Author>Rodrigues</Author><Year>2013</Year><RecNum>2</RecNum><DisplayText>(Rodrigues et al., 2013)</DisplayText><record><rec-number>2</rec-number><foreign-keys><key app=”EN” db-id=”50td0z0t1vzs21e29265s02v90xwravfedrz” timestamp=”1536898524″>2</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Rodrigues, Cristine</author><author>Vandenberghe, Luciana PS</author><author>Sturm, Wilerson</author><author>Dergint, Dario EA</author><author>Spier, Michele Rigon</author><author>de Carvalho, Júlio Cesar</author><author>Soccol, Carlos R</author></authors></contributors><titles><title>Effect of forced aeration on citric acid production by Aspergillus sp. mutants in SSF</title><secondary-title>World Journal of Microbiology and Biotechnology</secondary-title></titles><periodical><full-title>World Journal of Microbiology and Biotechnology</full-title></periodical><pages>2317-2324</pages><volume>29</volume><number>12</number><dates><year>2013</year></dates><isbn>0959-3993</isbn><urls></urls></record></Cite></EndNote>(Rodrigues et al., 2013).
Scientists are trying to find the suitability of various waste food materials for bioconversions which may be cost effective for the production of different fermented and value added products. Thus, agro-based industrial waste from the processing of coffee, rice, sugarcane and orange use as a raw material for citric acid fermentation due to its availability at low price and also it is a basic substrate for citric acid fermentation ADDIN EN.CITE <EndNote><Cite><Author>Pazouki</Author><Year>2000</Year><RecNum>3</RecNum><DisplayText>(Pazouki, Felse, Sinha, &amp; Panda, 2000)</DisplayText><record><rec-number>3</rec-number><foreign-keys><key app=”EN” db-id=”50td0z0t1vzs21e29265s02v90xwravfedrz” timestamp=”1536898619″>3</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Pazouki, M</author><author>Felse, PA</author><author>Sinha, J</author><author>Panda, T</author></authors></contributors><titles><title>Comparative studies on citric acid production by Aspergillus niger and Candida lipolytica using molasses and glucose</title><secondary-title>Bioprocess Engineering</secondary-title></titles><periodical><full-title>Bioprocess Engineering</full-title></periodical><pages>353-361</pages><volume>22</volume><number>4</number><dates><year>2000</year></dates><isbn>0178-515X</isbn><urls></urls></record></Cite></EndNote>(Pazouki, Felse, Sinha, & Panda, 2000). Many nutritional and physiochemical factor such as moisture content, substrate particle sizes, temperature, nutrie0nt sources, pH and inoculum size are considered very critical which influence the growth of microbial strains and the end product PEVuZE5vdGU+PENpdGU+PEF1dGhvcj5CYXJpPC9BdXRob3I+PFllYXI+MjAwOTwvWWVhcj48UmVj
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ADDIN EN.CITE.DATA (Alam et al., 2010; Bari, Alam, Muyibi, & Jamal, 2009; Ellaiah, Srinivasulu, & Adinarayana, 2004; Lotfy, Ghanem, & El-Helow, 2007).

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ADDIN EN.CITE.DATA (Al-Mahin, Hasan, Khan, & Begum, 2008; Bayraktar & Mehmetoglu, 2000; Xu, Madrid, Röhr, & Kubicek, 1989). The efficiency of various fermented products is greatly influenced by the type of substrate and fermentation conditions like temperature, fermentation time and the type of culture/strain ADDIN EN.CITE <EndNote><Cite><Author>Ali</Author><Year>2002</Year><RecNum>1</RecNum><DisplayText>(S. Ali, Haq, Qadeer, &amp; Iqbal, 2002)</DisplayText><record><rec-number>1</rec-number><foreign-keys><key app=”EN” db-id=”p92xr9eznts0a9evat4ptvr35pextxeapapw” timestamp=”1536899549″>1</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Ali, Sikander</author><author>Haq, Ikram-ul</author><author>Qadeer, MA</author><author>Iqbal, Javed</author></authors></contributors><titles><title>Production of citric acid by Aspergillus niger using cane molasses in a stirred fermentor</title><secondary-title>Electronic Journal of Biotechnology</secondary-title></titles><periodical><full-title>Electronic Journal of Biotechnology</full-title></periodical><pages>19-20</pages><volume>5</volume><number>3</number><dates><year>2002</year></dates><isbn>0717-3458</isbn><urls></urls></record></Cite></EndNote>(S. Ali, Haq, Qadeer, & Iqbal, 2002). There is a vast need to investigate the factors that are responsible for low citric acid production and to develop strategies that increase the yield and reduce production cost ADDIN EN.CITE <EndNote><Cite><Author>Adham</Author><Year>2002</Year><RecNum>2</RecNum><DisplayText>(Adham, 2002)</DisplayText><record><rec-number>2</rec-number><foreign-keys><key app=”EN” db-id=”p92xr9eznts0a9evat4ptvr35pextxeapapw” timestamp=”1536899682″>2</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Adham, Nehad Z</author></authors></contributors><titles><title>Attempts at improving citric acid fermentation by Aspergillus niger in beet-molasses medium</title><secondary-title>Bioresource Technology</secondary-title></titles><periodical><full-title>Bioresource Technology</full-title></periodical><pages>97-100</pages><volume>84</volume><number>1</number><dates><year>2002</year></dates><isbn>0960-8524</isbn><urls></urls></record></Cite></EndNote>(Adham, 2002).

Review of Literature
A group of researchers carried solid state fermentation (SSF) using agro based waste materials (apple pomace and peanut shell and a mixture of both with 50:50 ratio) as support for SSF in order to increase the citric acid production by using single strain and co-cultures of Aspergillus species and Alternaria specie and it showed the substantial potential. By adding the fermentative substrate to the SSF medium increased fungal growth, citric acid production and sugar utilization. During initial screening trial it was observed growth media that was supplemented with apple pomace in SSF process of co-culture consortia gave the finest growth (0.46 ± 0.2 mg/mL) of citric acid. In the presence of arginine as a nutritional ingredient co-culture showed the maximum citric acid production (2.644 ± 0.99 mg/mL) ADDIN EN.CITE <EndNote><Cite><Author>Ali</Author><Year>2016</Year><RecNum>3</RecNum><DisplayText>(S. R. Ali, Anwar, Irshad, Mukhtar, &amp; Warraich, 2016)</DisplayText><record><rec-number>3</rec-number><foreign-keys><key app=”EN” db-id=”p92xr9eznts0a9evat4ptvr35pextxeapapw” timestamp=”1536899918″>3</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Ali, Sohaib Rafaqat</author><author>Anwar, Zahid</author><author>Irshad, Muhammad</author><author>Mukhtar, Saima</author><author>Warraich, Nabeela Tariq</author></authors></contributors><titles><title>Bio-synthesis of citric acid from single and co-culture-based fermentation technology using agro-wastes</title><secondary-title>Journal of Radiation Research and Applied Sciences</secondary-title></titles><periodical><full-title>Journal of Radiation Research and Applied Sciences</full-title></periodical><pages>57-62</pages><volume>9</volume><number>1</number><dates><year>2016</year></dates><isbn>1687-8507</isbn><urls></urls></record></Cite></EndNote>(S. R. Ali, Anwar, Irshad, Mukhtar, & Warraich, 2016).

Scientists have isolated and identified some local isolates of the fungus Aspergillus specie from the soil and leaving out the isolates that had ability to produce aflatoxins and then screened to select the more efficient isolate in producing citric acid and biomass. After growing the isolates on medium that contained sucrose supplemented with NH4NO3, KH2PO4 and MgSO4.7H2O and determined some of the optimal conditions for the production of citric acid from the selected isolation after growing it on the rice husks filtrate medium supplemented with some nutrients referred to in the synthetic medium. They obtained the highest production of citric acid which amounted to 19.447 g/l by using the rice husks filtrate medium containing 15% reducing sugars, 0.25% ammonium sulfate, temperature 30?C and primary pH 4 ADDIN EN.CITE <EndNote><Cite><Author>Alsudani</Author><Year>2015</Year><RecNum>4</RecNum><DisplayText>(Alsudani &amp; Al-Shibli, 2015)</DisplayText><record><rec-number>4</rec-number><foreign-keys><key app=”EN” db-id=”p92xr9eznts0a9evat4ptvr35pextxeapapw” timestamp=”1536899979″>4</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Alsudani, Ali A</author><author>Al-Shibli, Majid K</author></authors></contributors><titles><title>CITRIC ACID PRODUCTION FROM SOME LOCAL ISOLATES OF THE FUNGUS ASPERGILLUS NIGER BY RICE HUSKS FILTRATE MEDIUM</title></titles><dates><year>2015</year></dates><urls></urls></record></Cite></EndNote>(Alsudani & Al-Shibli, 2015).

Study had been done to describe the isolation, screening and strain improvement of Aspergillus specie for increase citric acid production in shake flask and using ferrocyanide treated cane molasses as a carbon source. By serial dilution method using malt extract one hundred and ninety-seven cultures were obtained from different soil samples. By qualitative analysis one hundred and twenty nine cultures were isolated out of one hundred and ninety-seven .The best citric acid producing strain was selected using cane molasses by shake flask method. Among all the cultures that were examined, the strain Aspergillus niger GCB-117 gave the maximum yield of citric acid i.e; 14.17 g/l. This culture was further optimized for cultural conditions i.e; sugar concentration, pH and incubation period. The maximum citric acid was obtained at sugar concentration 150 g/l (14.17 g/l), pH-5.5 (20.3 g/l) and incubation period 168h (20.3 g/l) ADDIN EN.CITE <EndNote><Cite><Author>Iqbal</Author><Year>2016</Year><RecNum>5</RecNum><DisplayText>(Iqbal &amp; Utara, 2016)</DisplayText><record><rec-number>5</rec-number><foreign-keys><key app=”EN” db-id=”p92xr9eznts0a9evat4ptvr35pextxeapapw” timestamp=”1536900160″>5</key></foreign-keys><ref-type name=”Generic”>13</ref-type><contributors><authors><author>Iqbal, J</author><author>Utara, U</author></authors></contributors><titles><title>Isolation of Aspergillus niger Strains from Soil and their Screening and Optimization for Enhanced Citric Acid Production using Cane Molasses as Carbon Source. n</title></titles><dates><year>2016</year></dates><publisher>August</publisher><urls></urls></record></Cite></EndNote>(Iqbal & Utara, 2016).

Aspergillus species are the most important fungi used in industrial microbiology for the commercial production of citric acid. Recovery of the citric acid greatly depends on the type of substrate as well as the fermentation conditions like temperature, pH, fermentation time and the type of strain. In this study, scientists investigated the prospective of Aspergillus niger to convert sugarcane molasses into citric acid through fermentation that was carried out for 8 days at three different temperatures 20°C, 24°C and 28°C and five substrate levels 0%, 6%, 12%, 18% and 24%. The optimum citric acid production was achieved after 6 days of fermentation the substrate level was 24% and temperature was 28°C. The maximum citric acid production was 6.87±0.12 g 100 mL-1 (68.7 g L-1) with a mean recovery of 51.62% with respect to initial total sugar contents of the media. However, the optimum recovery of citric acid (59.64%) with respect to initial total sugar contents was achieved from 18% substrate level after the completion of fermentation period of 6 days ADDIN EN.CITE <EndNote><Cite><Author>Ashraf</Author><Year>2013</Year><RecNum>6</RecNum><DisplayText>(Ashraf, 2013)</DisplayText><record><rec-number>6</rec-number><foreign-keys><key app=”EN” db-id=”p92xr9eznts0a9evat4ptvr35pextxeapapw” timestamp=”1536900621″>6</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Ashraf, Ejaz</author></authors></contributors><titles><title>CITRIC ACID PRODUCTION FROM SUGARCANE MOLASSES BY ASPERGILLUS NIGER UNDER DIFFERENT FERMENTATION CONDITIONS AND SUBSTRATE LEVELS</title><secondary-title>Int. J. Agric. Appl. Sci. Vol</secondary-title></titles><periodical><full-title>Int. J. Agric. Appl. Sci. Vol</full-title></periodical><volume>5</volume><number>1</number><dates><year>2013</year></dates><urls></urls></record></Cite></EndNote>(Ashraf, 2013).

A fungal strain was isolated from soil contaminated with fruit wastes and screened for citric acid production and it was identified as Aspergillus niger. The waste substrates like fruit peels (grapes and mosambi peel) and bagasse with varying particle sizes of M1­ (0. 250mm), M2 (0.150mm) and M3 (0.63mm) 70% moisture level and 4% methanol was maintained throughout the solid state fermentation process for the citric acid production from the fungal culture. The Aspergillus niger produces the higher yields of citric acid where grape peel with particle size 0.63mm as a substrate in the medium. Higher protein contents and fungal biomass estimated in grapes, mosambi and bagasse wastes substrates respectively in the solid state fermentation ADDIN EN.CITE <EndNote><Cite><Author>Goud</Author><Year>2012</Year><RecNum>7</RecNum><DisplayText>(Goud, Srilakshmi, Kumar, &amp; Narasimha, 2012)</DisplayText><record><rec-number>7</rec-number><foreign-keys><key app=”EN” db-id=”p92xr9eznts0a9evat4ptvr35pextxeapapw” timestamp=”1536900694″>7</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Goud, K Hariveeran</author><author>Srilakshmi, A</author><author>Kumar, PA</author><author>Narasimha, G</author></authors></contributors><titles><title>Citric acid production by Aspergillus niger through solid state fermentation using fruit wastes</title><secondary-title>Biotechnol. Indian J</secondary-title></titles><periodical><full-title>Biotechnol. Indian J</full-title></periodical><pages>93-96</pages><volume>6</volume><dates><year>2012</year></dates><urls></urls></record></Cite></EndNote>(Goud, Srilakshmi, Kumar, & Narasimha, 2012).
Aims and Objectives
The general objective of the research is the production of the citric acid in the laboratory by co-culturing from fungal strains
Selection of raw material such as orange peel and peanut shell to produce citric acid.

Production of citric acid from Aspergillus fumigatus by using solid state fermentation.

Optimization of different factors for citric acid by fungal strains.

Flow Chart of Materials and Method
123888578740Fungal culture and media preparation
00Fungal culture and media preparation

28508283559600010096504960620 Optimization of fermentation parameters
00 Optimization of fermentation parameters
16192504017645Determination of biomass
00Determination of biomass
838200725805Screening of fungal isolate for citric acid production
00Screening of fungal isolate for citric acid production
28371801200150009525001503045Spore suspension & inoculums preparation
00Spore suspension & inoculums preparation
28371801993900009525002331720Pretreatment of substrate for citric acid production
00Pretreatment of substrate for citric acid production
281813027317700018192753160395Solid state fermentation
00Solid state fermentation
28086053652520002808605454088500279908054686200018192755827395HPLC analysis of Citric acid
00HPLC analysis of Citric acid
Materials and Methods
Substrate Collection
In this study of citric acid production, orange peel and peanut shells will be selected as substrate. The selected substrates will contain high content of polysaccharides, carbohydrate and metal ions. These substrate will be collected from different juice shops at Gujrat, Pakistan. The collected substrates will be dried for 5 to 6 days and ground into powdered form. Then, this powder will be stored into air tight jar for further study.
Microorganism
For the production of citric acid, different fungal species will be collected from different spoiled foods like bread, cake and some fruits from local area of Gujrat, Pakistan.

Media Preparation
Then these isolated fungus species will be cultivated on PDA (potato starch-dextrose-agar) media on petri plate at 37°C temperature for 4-5 days and stored at 4°C for use.

Screening of Fungal Isolate for Citric Acid Production
Primary Screening
Screening of microorganism will be done to identify the maximum citric acid producing strain. The screening experiment will be performed in petri plates containing czapek-Dox agar medium with bromocresol green as an indicator. The media will be prepared by dissolving all the ingredient and will be autoclaved. After cooling, it will be poured into sterile petri plates and will be allowed to cool at room temperature. After solidify, it will be contained 0.5 ml of conidial suspension and will be transferred to these plates. Plate will be rotated clockwise and anticlockwise to spread suspension uniformly and incubated at 37°C for 3-5 days.

Secondary Screening
Strains will be evaluated for single and co-culture consortia. This experiment will be done in 250ml Erlenmeyer flask containing 5g of different substrates. 10ml distilled water will be added to give moisture content and will be plugged with cotton for autoclave. Each flask will contain 3ml inoculum in a way that some of these contained single organism and some of these will contain 3ml inoculum of both microorganisms which means each flask will contain 1.5ml of inoculum.

Screening of Substrate
Orange peel (OP), peanut shell (PS) and their mixture will be used as a substrate in citric acid production. Experiment will be performed in 250ml Erlenmeyer flask that will contain 5g of substrate OP (1:0), PS (1:0) and MM (1:I) and 5ml of water in each flask to give moisture content then will be plugged with cotton and will be autoclaved at 121°C for 15 min.

After cooling, 3ml of inoculums containing 1.5 ml each of both inoculums will be added. This experiment will be performed to assess that which substrate either orange peel, peanut shell or mixed substrate could yield maximum citric acid by co-culturing (Rhizopus stolonifer Aspergillus niger).

Spore Suspension & Inoculum Preparation
For Spore suspension, PDA slants will be prepared by following Noomrio & Dahot (1992) for inoculation of the substrate. As inoculums media PDA broth will be prepared and pH will be adjusted to 5.5 and then it will be autoclaved aerated for 15 min at 121°C temperature and will be left for cooling. A loop full of fungal culture will take from the sporulation medium of fungus that will be raised on PDA slants aseptically into the 250ml flask that container 150ml sterilized inoculums medium. To avoid contamination, the experiment will be performed in laminar air flow. Then the flask will be placed in shaking incubator for 4 to 5 days at 37°C. For better results, fresh inoculums will be prepared for each experiment. The ingredient of inoculums are shown in table 3.1Sr No.

Pretreatment Of Substrate For Citric Acid Production
Orange peel, peanut shell and their different combinations will be dried for the production of citric acid. This experiment will be performed in 250ml Erlenmeyer flask containing 5g of the substrate.

Citric Acid Production Method
For the production of citric acid by solid state fermentation (SSF) is a conventional method and this method will be used in this research work for the production of citric acid.

Fermentation Protocol
In initial substrate screening trial, 5g of each above mentioned will be added in a moisten 250ml Erlenmeyer flask and will be inoculated with fungal spore suspension of Rhizopus stolonifer and Aspergillus niger and co-culture consortia. All of these experimental Erlenmeyer flasks will be placed into an incubator at 37°C ±1 for 3 to 4 days. After 3 days, 50ml of distilled water will be added in each flask and then will be placed into a shaking incubator at 37°C for 30 min at 180 rpm. The homogenized media will be centrifuged again at 10,000 rpm for 10 min at 4°C .The clear supernatant will be used for further analytical studies.

Determination of Biomass
All samples will be tested in triplicates and their results will be presented as mean± standard error of mean. During extraction process, the obtained pellets will be re-suspended in 50mM phosphate buffer at pH 7.0, will be centrifuged at 10,000 rpm for 10 mining in falcon tubes and will be dried at 80°C and final biomass weights will be recorded in gram.

Optimization of Fermentation Parameters
A classical method will be used to optimize the end product and to study the effect of incubation period (2 to 6 days) for the production of citric acid from co-culture consortia in the presence of solid substrates. Citric acid production by co-culture consortia will be carried out at different pH values (3 to 7) in triplet flask. After pH adjustment, citric acid will be produced from co-culture consortia at different temperature (25 to 45 °C). Effect of different inoculums concentration (2 to 8 ml) will be also observed for the citric acid production. Effect of various amino acids (glutamine, Arginine, Tyrosine, Glycine and Aspartic acid) on the production of citric acid from co-culture consortia will also be observed.

Optimum Incubation Period
To estimate the effect of fermentation period on citric acid production, triplicate flask will be prepared containing 5g orange peel and 5ml distilled water in each flask to give its moisture content, will be plugged with cotton and will be autoclaved at 121°C for 15 min. After cooling, substrate will be inoculated with 3ml of spore suspension containing 1.5ml each of both inoculums and will be incubated for (2 to 6) days to select the optimum fermentation period for maximum production of citric acid.

Optimum pH
To evaluate the effect of pH change on citric acid production, flask will be incubated at various pH solution (1, 3, 4, 5, 5.5 and 7). Triplicate flask will be prepared for each pH. 5g of orange peel will be added in 250 ml flask and moisturized it with distilled water whose pH will be maintained, will be plugged with cotton and will be autoclaved at 121°C for 15 min. pH change will be controlled by using NaOH and HCI. To increase the pH, 1N NaOH will added and to decrease the pH, 1N HCI will be added drop by drop by the help of dropper. pH change will be measured by pH meter.

Optimum Temperature
For the evaluation of temperature effect on production of citric acid triplicate flask will be prepared at different ranges of temperature (25, 30, 35, 40 and 45°C) that contain 5g of orange peel and 5ml of this will be autoclaved for 15 min at 121°C. After cooling 3ml of inoculums will be added to 1.5ml of each distilled water with previously adjusted pH in order to moisturize it. Then these flasks will be plugged with cotton.

Optimum Moisture Content
Moisture is very important factor for the production of citric acid from micro-organism. To find out the effect of optimum moisture content, substrates will be moisturized at varying concentrations 50%, 60%, 70% and 80%.

Effect of Different Substrate Quantities on Citric Acid Production
To estimate the effect of substrate concentration on citric acid production, different quantity of orange peel (5g, 10g, 15g, 25g and 35g) will be evaluated. For each parameter, triplicate flask will be prepared to check on which quantity of substrate yield the maximum production of citric acid.

Sample Harvesting
After the incubation time, the flask in each experiment will be harvested. In each flask 50ml of distilled water will be added and then culture will be homogenized .The flasks will be kept in shaking incubator at 120 rpm at 37°C for 30min. The fermented biomass samples will be filtered by filtration assembly apparatus. Then will be centrifuged at 10, 0000 rpm for 10 min to remove the spores of the organism. Supernatants will be collected carefully and will be stored in refrigerator. Clear supernatants will be used for detection of citric acid using HPLC technique.

Determination of Citric Acid
The amount of citric acid present in the fermented samples will be determined by the HPLC technique.

HPLC Analysis
For the detection of citric acid in samples, HPLC analysis conditions will be optimized. Distilled deionized water (DD H2O) will be used as mobile phase. The flow rate will be set to 1.5ml/min and absorbance will be noted at 212nm using UV/visible detector. The analysis of citric acid will be performed at 25?C by using reversed phase (C-8) column having 15cm length, 4.6mm diameter and 5µ particle size. The results of different samples will be compared with standard result of citric acid for both quantitative and qualitative analysis. Retention time and peak areas of standards will be noted and calculated respectively. These calculations will be employed for estimation of amounts of citric acid present in fermented sample.

The results of different samples will be compared with standard result of citric acid for both quantitative and qualitative analysis. Retention time and peak areas of standards will be noted and calculated respectively. These calculations will be employed for estimation of amounts of citric acid present in fermented sample.

Expected Outcomes of The Work:
Increased amount of citric acid will be produced from Aspergillus fumigatus using orange peels, peanut shells and their different combinations through solid state fermentation (SSF). Such production of citric acid by co-culturing technique will be more economical as compared to simple fermentation technique.

End benefit to the users:
Final thesis submission (Date):
August 2017References:
ADDIN EN.REFLIST Adham, N. Z. (2002). Attempts at improving citric acid fermentation by Aspergillus niger in beet-molasses medium. Bioresource Technology, 84(1), 97-100.

Al-Mahin, A., Hasan, S. M., Khan, M. H., & Begum, R. (2008). Citric acid production by Aspergillus niger through solid-state fermentation on sugarcane bagasse. Bangladesh Journal of Microbiology, 25(1), 9-12.

Alam, M., Karim, A., Ismail, M., Jamal, P., Al-Mamun, A., & Bari, M. (2010). Production of Citric Acid from Oil Palm Empty Fruit Bunches by Solid State Bioconversion using Local Aspergillus niger.

Ali, S., Haq, I.-u., Qadeer, M., & Iqbal, J. (2002). Production of citric acid by Aspergillus niger using cane molasses in a stirred fermentor. Electronic Journal of Biotechnology, 5(3), 19-20.

Ali, S. R., Anwar, Z., Irshad, M., Mukhtar, S., & Warraich, N. T. (2016). Bio-synthesis of citric acid from single and co-culture-based fermentation technology using agro-wastes. Journal of Radiation Research and Applied Sciences, 9(1), 57-62.

Alsudani, A. A., & Al-Shibli, M. K. (2015). CITRIC ACID PRODUCTION FROM SOME LOCAL ISOLATES OF THE FUNGUS ASPERGILLUS NIGER BY RICE HUSKS FILTRATE MEDIUM.

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