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\setlength{\parindent}{0pt}\raggedright \textcolor{GJMediumGrey}{\rule{\textwidth}{2pt}} \vskip16pt \textcolor{GJBlue}{\large\bfseries\abstractname\space} }{% \vskip8pt \textcolor{GJMediumGrey}{\rule{\textwidth}{2pt}} \vskip16pt } \usepackage[absolute,overlay]{textpos} \makeatother \usepackage{lineno} \linenumbers \begin{document} \author[1]{Shamima Nasrin} \renewcommand\Authands{ and } \date{\small \em Received: 7 December 2016 Accepted: 1 January 2017 Published: 15 January 2017} \maketitle \begin{abstract} The worldwide demand for citric acid is increasing with the rising population and industrialization. The growing demand for citric acid and the need for alternative materials as substrates in the recent years have led to the choice of a novel and economically viable substrate, namely jackfruit (outer portion) and molasses for citric acid biosynthesis. Hydrolysis these substrates with 0.05N HCl followed by fermentation using two isolates of Aspergillus niger were investigated for citric acid production under submerged culture condition in a period of 15 days. The products of the microbial metabolism namely residual sugar, total titratable acidity (TTA), citric acid, and biomass contents were determined periodically. Maximum citric acid production was found after 12 days of fermentation for both isolates, namely Aspergillus niger CA16, the parent strain and gamma ray induced mutant Aspergillus niger 79/20. Citric acid production was found highest in the absence of Prescott salt by Aspergillus niger CA16 in mixed fermentation medium which was about 16.35 mg/ml and lowest in jackfruit medium, 12.88 mg/ml at day 12. Whereas in the presence of Prescott salt, lowest citric acid production was also found in jackfruit medium, 7.21 mg/ml and highest in mixed medium, 11.54 mg/ml. In case of the previously isolated gamma-ray induced mutant Aspergillus niger 79/20, the yield seems to be higher under similar experimental condition. In absence of Prescott salt highest production of citric acid was found by mutant Aspergillus niger 79/20 in mixed fermentation medium which was about 25.87 mg/ml and lowest in jackfruit medium, 22.12 mg/ml at the day 12 which was even higher than that found in case of the parent strain Aspergillus niger CA16. In the presence of Prescott salt highest production of citric acid was found in mixed media, 16.35 mg/ml and lowest in jackfruit medium, 13.94 mg/ml which was again higher than that was obtained in case of the parent strain. \end{abstract} \keywords{aspergillus niger 79/20, citric acid, titratable acidity.} \begin{textblock*}{18cm}(1cm,1cm) % {block width} (coords) \textcolor{GJBlue}{\LARGE Global Journals \LaTeX\ JournalKaleidoscope\texttrademark} \end{textblock*} \begin{textblock*}{18cm}(1.4cm,1.5cm) % {block width} (coords) \textcolor{GJBlue}{\footnotesize \\ Artificial Intelligence formulated this projection for compatibility purposes from the original article published at Global Journals. However, this technology is currently in beta. \emph{Therefore, kindly ignore odd layouts, missed formulae, text, tables, or figures.}} \end{textblock*} \let\tabcellsep& \section[{Introduction}]{Introduction}\par itric acid is one of the world's largest tonnages of fermentation products. It is widely used in the food beverage industries as an acidifying and flavor-enhancing agent, pharmaceutical, chemical, cosmetic and other industries for applications such as acidulation, antioxidation, flavor enhancement, preservation, plasticizer and as a synergistic agent. The worldwide demand for citric acid is met by fermentation mainly by the process involving the filamentous fungus A. niger. A number of carbon sources may be used for citric acid fermentation. For commercial reasons, the uses of molasses, sucrose or glucose syrups are favored. The use of molasses in particular is desirable because of its low cost availability.\par A. niger is capable of producing very high levels of citric acid, about 90\% of the theoretical yield from a carbohydrate source. For an efficient citric acid production, the growth of Aspergillus in pellet form is desirable and this can be achieved by process optimization. There is a great worldwide demand for citric acid consumption due to its low toxicity compared with other acidulants used mainly in the pharmaceutical and food industries. Global production of citric acid has now reached 1.4 million tones and there is annual growth of 3.5-4.0 \% in demand/consumption. A high rate of acidogenesis in A. niger is observed only under conditions of high glycolytic metabolism and can be induced by the addition of an excess amount of sucrose or other carbohydrates which induce a high rate of glycolytic catabolism. In this production technique, which is still the major industrial route to citric acid used today, cultures of Aspergillus niger are fed on a sucrose or glucose-containing medium to produce citric acid. The source of sugar is corn steep liquor, molasses, hydrolyzed corn starch or other inexpensive sugary solutions. Bangladesh, at present, imported cent percent citric acid from foreign countries. High production depends to a great extent on the strain used and its response to the composition of the medium can show a great deal of variability. Industrial production of this chemical by fermentation using cheap raw materials is helpful in economic development of our country. Keeping in view the future requirements and also the availability of cheap raw material, efforts were made to develop the process for citric acid fermentation, based on our local resources such as molasses from sugar mills and outer portion of jackfruit. So the purpose of present study describes the feasibility of using raw and cheap materials such as molasses and outer portion of jackfruit for citric acid fermentation and to use parent strain CA16 \& gamma-ray induced mutants for high citric acid yielding strain 79/20 of Aspergillus niger.\par Aspergillus niger is a haploid filamentous fungi and is a very essential microorganism in the field of biology. A. niger is cultured for the industrial production of many substances. Various strains of A. niger are used in the industrial preparation of citric acid (E330) and gluconic acid (E574) and have been assessed as acceptable for daily intake by the World Health Organization. A. niger is important because of its involvement in producing citric acid as well as industrial enzymes, such as amylases, proteases, and lipases. The uses of these enzymes are essential because of its importance for transformation to food enzymes. For example, A. niger glucoamylase is used in the production of high fructose corn syrup, and pectinases are used in cider and wine clarification. Glucose oxidase is used in the design of glucose biosensors, due to its high affinity for ?-D-glucose. A variety of carbohydrate sources such as beet molasses, cane molasses, sucrose, commercial glucose, starch hydrolysates etc., have been used for citric acid production. Among these, sucrose, cane and beet molasses have been found to be the best choice \hyperref[b32]{(Kapoor et al., 1982)}. \section[{II.}]{II.} \section[{Materials and Methods}]{Materials and Methods}\par This study was done in the research laboratory of the Department of Biochemistry and Molecular Miology at Jahangirnagar University and at Institute of Food and Radiation Biology, Atomic Energy Research Establishment, Bangladesh during July 2009 and June 2010.\par Parent strain Aspergillus niger CA16 and mutant strain Aspergillus niger 79/20 were first grown on agar slant medium. Each of the properly processed substrates [Molasses, jackfruit (outer potion) and mixed substrates] was hydrolyzed by 0.05 N HCl and filtered which were then used as medium for submerged fermentation. Each substrate were divided into two groups and were fermented separately, one in the presence of Prescott salt and the other in the absence. Each of the groups of the three types of media were further divided into another two subgroups and one of them was inoculated with the parent strain Aspergillus niger CA16 and the remaining one was inoculated with the mutant strain Aspergillus niger 79/20. All the flasks were then incubated for 15 days in an incubator under same conditions. Fermented media were collected on day 3, 6, 9, 12, 15 and were subjected to estimation of residual sugar, TTA value, citric acid concentration and pH determination. \section[{III.}]{III.} \section[{Chemical Reagents and Solutions}]{Chemical Reagents and Solutions}\par All chemicals and reagents used in this study were of analytical grade. All aqueous solutions were prepared with distilled water. Stock solution of Prescott salt (NH4NO3: 2.23g/L, K2HPO4:\par 1.00g/L, MgSO4.7H2O: 0.23g/L) used in the media were prepared 10 times the media concentration and diluted as its normal strength in the experiment. \section[{IV.}]{IV.} \section[{Analytical Determination}]{Analytical Determination}\par At the different stages of fermentation the culture flasks were taken out of the incubator and the medium was collected onto the screw cap test tubes by pipetting and preserved at 0oC. The appropriate amount of sample was used for the estimation of total titratable acidity, citric acid and amount of residual sugar present in the medium after fermentation. \section[{V. Determination of total Titratable}]{V. Determination of total Titratable}\par Acidity (TTA)\par Fermented medium (0.25ml) was diluted with 20ml of distilled water and was titrated against 0.1N NaOH solution using 2 to 3 drops of phenolphthalein as indicator. The value obtained was multiplied by 4 and total titratable acidity was expressed as ml of 0.1N NaOH required to neutralize 1ml fermented medium. The titrametric analysis of fermentation of each strain gave an indication of total acidity of the medium. The medium containing high TTA value i.e. higher acid content were then analyzed spectrophotometrically. \section[{VI.}]{VI.} \section[{Estimation of citric Acid from Fermentation Medium}]{Estimation of citric Acid from Fermentation Medium}\par Citric acid was estimated spectrophotometrically by the reference method of Marier and Boulet {\ref (1958)}. Citric acid forms a color complex of polyvinyl keto-anhydridepolymer when it reacts with acetic anhydride and pyridine which can be estimated spectrophptometrically \hyperref[b3]{(Auterhoff and Schwingel, 1975)}. Following the growth of the organism aliquots of the medium were diluted so as to have concentration in the range of \hyperref[b25]{25} \section[{Estimation of Residual Sugar}]{Estimation of Residual Sugar}\par Before inoculation and after completion of fermentation, samples were collected for initial and residual sugar estimation, respectively.\par Following the fermentation, amount of residual sugar in the medium was determined by diluting the aliquots of the medium so as containing sugar concentration range of 25-200?g per ml.\par Initial and residual sugar of the medium was determined spectrophotometrically by anthrone method \hyperref[b47]{(Morse, 1947)} using anthrone as the coloring agent with sucrose as standard. \section[{VIII.}]{VIII.} \section[{Results}]{Results} \section[{a) Estimation of residual sugar at different periods of citric acid fermentation}]{a) Estimation of residual sugar at different periods of citric acid fermentation}\par The residual sugar concentration was different in various media during citric acid fermentation by These results showed TTA value was comparatively higher in the absence of Prescott salt for all the three types of media and for each of the stain. Throughout the incubation period the TTA value was highest in case mixed fermentation medium followed by molasses and jackfruit fermentation medium. Again, fermentation by Aspergillus niger 79/20 resulted in a comparatively higher TTA value than by Aspergillus niger CA16 both in the presence and absence of Prescott salt (Figure \hyperref[fig_7]{19}). \section[{c) Estimation of citric acid accumulation at different period of citric acid fermentation}]{c) Estimation of citric acid accumulation at different period of citric acid fermentation}\par Accumulation of citric acid at different incubation periods on different media followed a very similar pattern as was seen in case of TTA value. Citric acid concentration was also different on different incubation periods with various fermentation media by the parent strain Aspergillus niger CA16 strain and the mutant strain 79/20. Citric acid concentration was found to increase gradually with the increase of incubation period and maximum citric acid concentration was found on day 12 in case of each of the three media. These results showed citric acid concentration was comparatively higher in the absence of Prescott salt for all the three types of media and for each of the strain. Throughout the incubation period the citric acid concentration was highest in case mixed fermentation medium followed by molasses and jackfruit fermentation medium. Again, fermentation by Aspergillus niger 79/20 resulted in a comparatively higher citric acid accumulation than by Aspergillus niger CA16 both in the presence and absence of Prescott salt (Figure -22).\par Fermentation of citric acid for commercial production is dependent on many factors like quality of strains, nutritional composition of the media, environmental conditions, deficiency of manganese and other metals, pH, temperature and dissolved oxygen tension . Usually, Aspergillus niger gives the best yield at around 25-28ºC. Increase in incubation period resulted in the increased citric acid production. A lower concentration of sugar leads to lower yield of citric acid as well as accumulation of oxalic acid \hyperref[b34]{(Kovats, 1960)}. But the use of wild type strain of Aspergillus niger is not cost effective. So, high yielding strains were searched which will give the best yield at around the room temperature. The superior strains Aspergillus niger CA16 and gamma ray induced mutants Aspergillus niger 79/20 seem to have fulfilled the requirement. Thus these strains can be conveniently exploited for the production of citric acid from cane molasses, jackfruit (outer portion) and a mixture of the two substrates.\par From the findings of this study it is clearly suggested that both fermentation medium and Prescott salt have a considerable effect on the production of citric acid. Among the media used in this study, the mixed fermentation medium was found to be most suitable for citric acid production followed by molasses and jackfruit (outer portion) media. Another important finding of the present study was that Prescott salt was found to have a negative effect on the citric acid production by the either strains of Aspergillus niger. Again the gamma-ray induced mutant strain, Aspergillus niger 79/20 had a yield efficiency more than that of the parent strain Aspergillus niger CA16 and thus considered superior to the parent strain Aspergillus niger CA16. Thus as far as citric acid production is \begin{figure}[htbp] \noindent\textbf{}\includegraphics[]{image-2.png} \caption{\label{fig_0}C}\end{figure} \begin{figure}[htbp] \noindent\textbf{}\includegraphics[]{image-3.png} \caption{\label{fig_1}}\end{figure} \begin{figure}[htbp] \noindent\textbf{14}\includegraphics[]{image-4.png} \caption{\label{fig_2}Figure 14 :}\end{figure} \begin{figure}[htbp] \noindent\textbf{15}\includegraphics[]{image-5.png} \caption{\label{fig_3}Figure 15 :}\end{figure} \begin{figure}[htbp] \noindent\textbf{16}\includegraphics[]{image-6.png} \caption{\label{fig_4}Figure 16 :C}\end{figure} \begin{figure}[htbp] \noindent\textbf{17}\includegraphics[]{image-7.png} \caption{\label{fig_5}Figure 17 :}\end{figure} \begin{figure}[htbp] \noindent\textbf{18}\includegraphics[]{image-8.png} \caption{\label{fig_6}Figure 18 :C}\end{figure} \begin{figure}[htbp] \noindent\textbf{19}\includegraphics[]{image-9.png} \caption{\label{fig_7}Figure 19 :}\end{figure} \begin{figure}[htbp] \noindent\textbf{}\includegraphics[]{image-10.png} \caption{\label{fig_8}}\end{figure} \begin{figure}[htbp] \noindent\textbf{} \par \begin{longtable}{P{0.85\textwidth}} Volume XVII Issue II Version I\\ D D D D )\\ (\\ Medical Research\\ Global Journal of\end{longtable} \par \caption{\label{tab_0}C}\end{figure} \footnote{© 2017 Global Journals Inc. (US)} \footnote{© 2017 Global Journals Inc. (US) Year 2017} \footnote{(Figure-20 \& 21). © 2017 Global Journals Inc. (US)} \backmatter \begin{bibitemlist}{1} \bibitem[ Aspergillus Niger. J. Gen. Microbiol]{b19}\label{b19} \textit{}, \textit{Aspergillus Niger. J. Gen. Microbiol} 14 p. . \bibitem[ J. Biotechnol. Bioeng]{b38}\label{b38} \textit{}, \textit{J. Biotechnol. Bioeng} 27 p. . \bibitem[Pp]{b40}\label{b40} \textit{}, J Pp . p. . \bibitem[Fermentae Junyi]{b45}\label{b45} \textit{}, Rony Fermentae Junyi . 12 p. . \bibitem[ Appl. Microbiol]{b49}\label{b49} \textit{}, \textit{Appl. Microbiol} 1 p. . \bibitem[ Enzyme. Micro. Technol]{b56}\label{b56} \textit{}, \textit{Enzyme. Micro. Technol} 6 p. . \bibitem[London et al. (1987)]{b54}\label{b54} \textit{}, London , S C Prescott , C G Dunn . \textit{Industrial Microbiology} 1987. August. CBS Publishers and Distributors. p. . Mcraw Hill book company. Inc. New York Toronto (4th edition) \bibitem[Kristiansen et al. ()]{b35}\label{b35} \textit{}, B Kristiansen , M Mattey , J Linden . \textit{Citric Acid Biotechnology} 1999. Taylor \& Frances Ltd. p. . \bibitem[Lakshminarayana et al. ()]{b37}\label{b37} \textit{A solid state fermentation method for citric acid production using sugarcane bagasse}, K Lakshminarayana , K Chaudhary , S Ethiraj , P Tauro . 1975. \bibitem[Hang and Woodams ()]{b20}\label{b20} ‘A solid state fermentation of apple pomace for citric acid production using Aspergillus niger’. Y D Hang , E E Woodams . \textit{J. Appl. Microbial Biolechnol} 1984. 2 (2) p. . \bibitem[Hannan et al. ()]{b24}\label{b24} ‘Analysis of some mutants of Aspergillus niger for citric acid production’. M A Hannan , F Rabbi , A T M Rahman , N Choudhury . \textit{J. Ferment.Technol} 1973. 51 (8) p. . \bibitem[Morse ()]{b47}\label{b47} ‘Anthrone in estimating low concentration of sucrose’. E E Morse . \textit{Anal. Chem} 1947. 19 p. . \bibitem[Wehmer (ed.) ()]{b66}\label{b66} \textit{As quoted in: Industrial microbiology}, C Wehmer . S. C. Prescott C. G. and Dunn (ed.) 1893. 1959. \bibitem[Aspergillus niger 506 J. Acta. Microbiol. Sin]{b71}\label{b71} ‘Aspergillus niger 506’. \textit{J. Acta. Microbiol. Sin} 21 (3) p. . \bibitem[Ali et al. ()]{b1}\label{b1} ‘Biosynthesis of citric acid by locally isolated Aspergillus niger using sucrose salt media’. S Ali , Ikram-Ul-Haq , M A Qadeer , J Iqbal . \textit{J, Biol. Sci} 2001. 1 (4) p. . \bibitem[Atkinson and Mavitane ()]{b2}\label{b2} \textit{Citri acid. Biochemical Engineering and Biotechnology hand book}, B Atkinson , F Mavitane . 1983. New York: The nature press. p. . \bibitem[Lockwood and Schweiger ()]{b39}\label{b39} ‘Citric acid and itaconic acid fermentation’. L B Lockwood , L B. ; H Schweiger . \textit{Microbiol. Technology} 1967. \bibitem[Snell and Schweiger ()]{b61}\label{b61} ‘Citric acid by fermentation’. R L Snell , L B Schweiger . \textit{British Patent} 1951. 653 p. 8719. (Chem Abstr.) \bibitem[Doelger and Prescolt ()]{b14}\label{b14} ‘Citric acid fermentation’. W P Doelger , S C Prescolt . \textit{Ind. Eng, Chem} 1934. 26 p. . \bibitem[Begum and Choudhury ()]{b6}\label{b6} ‘Citric acid fermentation by gamma-ray induced mutants of Aspergillus niger in sugarcane and jack fruit juice’. R Begum , N Choudhury . \textit{Bangladesh J. Life Sci} 1998. 10 (1\&2) p. . \bibitem[Ikram-Ul-Haq et al. ()]{b29}\label{b29} ‘Citric acid fermentation by mutant strain of Aspergillus niger GCMC-7 using molasses based medium’. Ali Ikram-Ul-Haq , S Qadeer , M Iqbal , J . \textit{EJB Electronic Journal of Biotechnology} 0717-3458. 2002. \bibitem[Hossain ()]{b28}\label{b28} \textit{Citric acid fermentation by some gamma ray induced mutants of Aspergillus niger use of Agro industrial residues and cassava as substrates}, D Hossain . 1970. p. . Dept. of Microbiology, University of Dhaka (M.Sc. Thesis) \bibitem[Begum and Choudhury ()]{b7}\label{b7} ‘Citric acid fermentation in different starchy substrates by radiation induced mutants of Aspergillus niger’. R Begum , N Choudhury . \textit{J. Asiat. Soc. Bangladesh. Sci} 2000. 26 (1) p. . \bibitem[Hang et al. ()]{b21}\label{b21} ‘Citric acid fermentation of brewery waste’. Y D Hang , D F Splittstoesser , E E Woodams , R M Sherman . \textit{J. Food Sci} 1977. 42 (2) p. . \bibitem[Masior et al. ()]{b44}\label{b44} \textit{Citric acid from potatoes by fermentation}, S Masior , J Surminski , J Abranik . 1968. Prezemysl. \bibitem[ Johnson ()]{b31}\label{b31} ‘Citric acid is produced industrially by Aspergillus niger’. Johnson . \textit{Medical hypothesis} 2003. 60 p. . \bibitem[Lu et al. ()]{b42}\label{b42} ‘Citric acid production by Aspergillus niger in solidsubstrale fermentation’. M Y Lu , I S Maddox , J Brooks , D . \textit{Bioresource Technol} 1995. 54 (3) p. . \bibitem[Xie and West ()]{b68}\label{b68} ‘Citric acid production by Aspergillus niger on condensed corn distillers solubles’. G Xie , T P West . \textit{Research J. Microbiol} 2007. 2 (5) p. . \bibitem[Xie and West ()]{b67}\label{b67} ‘Citric acid production by Aspergillus niger on corn Distillers' grains with solubles’. G Xie , T P West . \textit{Research J. Microbiol} 2006. McGraw Hill Book Co. 1 (3) p. . \bibitem[Drysdale and Mckay ()]{b15}\label{b15} ‘Citric acid production by Aspergillus niger on surface culture on inulin’. Cr \& Drysdale , M H Mckay . \textit{Lett Appl Microbiol} 1995. 20 p. . \bibitem[Mckay and Drysdale ()]{b46}\label{b46} ‘Citric acid production by Aspergillus niger on surface culture on inulin’. M H Mckay , C R Drysdale . \textit{Lett Appl Microbiol} 1995. 20 p. . \bibitem[Usami and Fukutomi ()]{b64}\label{b64} ‘Citric acid production by solid state fermentation method using sugar cane bagasse and concentrated liquor of pineapple waste’. S Usami , N Fukutomi . \textit{Hakkokogaku} 1977. 55 p. . \bibitem[Chaudhary et al. ()]{b8}\label{b8} ‘Citric acid production from Indian cane molasses by Aspergillus niger under solid state fermentation condition’. K Chaudhary , S Ethiraj , K Lakshminarayana , P Tauro . \textit{J. Ferment. Technol} 1978. 56 (5) p. . \bibitem[El-Holi and Al-Delaimy ()]{b16}\label{b16} ‘Citric acid production from whey with sugars and additives by Aspergillus niger’. Ma \& El-Holi , K S Al-Delaimy . \textit{Afr J Biotechnol} 2003. 2 (10) p. . \bibitem[Kapoor et al. (ed.) ()]{b32}\label{b32} \textit{Citric acid. In prescott and Dunn's. Industrial Microbiology, 4 th Ed}, K Kapoor , K Chandhary , K Tauro , P . G. Reed (ed.) 1982. 3 p. . \bibitem[Shadafza et al. ()]{b59}\label{b59} ‘Comparison of citric acid from beet molasses and date syrup with Aspergillus niger. Hakko Hogaka’. D Shadafza , T Ogawa , A Fazeli . \textit{Zasshi} 1976. 54 p. . \bibitem[Zhu et al. ()]{b70}\label{b70} \textit{Direct fermentation of citric acid from highly concentrated sweet potato mash: The strain selection of}, Zhu , Heng , Qinfany Hou . 1981. \bibitem[Haq and Ali ()]{b26}\label{b26} ‘Direct production of citric acid from corn starch by Aspergillus niger’. I Haq , S Ali , J . \textit{Process Biochem} 2003. 38 p. . \bibitem[Begum et al. ()]{b5}\label{b5} ‘Effect of addition of methanol in molasses medium on the production of citric acid by Aspergillus niger’. A A Begum , N Choudhury , M S Islam . \textit{Bangladesh J. Microbiol} 1988. 5 (1) p. . \bibitem[Moyer ()]{b48}\label{b48} ‘Effect of alcohols on the mycological production of citric acid in surface and submerged culture’. A J Moyer . \textit{Nature of the alcohol effect} 1953. \bibitem[Moyer ()]{b50}\label{b50} ‘Effect of alcohols on the mycological production of citric acid in surface and submerged culture. II. Fermentation of crude carbohydrates’. A J Moyer . \textit{Appl. Microbiol} 1953. 1 p. . \bibitem[Saha et al. ()]{b57}\label{b57} ‘Effect of cultural conditions on citric acid production by Aspergillus niger AJ 117173 in surface culture fermentation. Dhaka Univ’. M L Saha , Y Sakai , F Takahashi . \textit{J. Biol. Sci} 2006. 15 (2) p. . \bibitem[Shu and Johnson ()]{b60}\label{b60} ‘Effect of the composition of the sporulation medium on citric acid production by A. niger in submerged culture’. P Shu , M Johnson . \textit{J. Bacteriol} 1947. 54 p. . \bibitem[Dawson et al. ()]{b13}\label{b13} ‘Effects of interruptions to the air supply on citric acid production by Aspergillus niger’. M Dawson , W Maddox , I S Brooks , J . \textit{J. Enzyme Microb. Technol} 1986. 8 (1) p. . \bibitem[Noguchi and Banido ()]{b51}\label{b51} \textit{Effects of menthol and ethanol on the production of citric acid from cane molasses}, Y Noguchi , Y Banido . 1960. 38 p. . (Hakko Kogaku zusshi) \bibitem[Banik ()]{b4}\label{b4} ‘Fermentive production of citric acid by Aspergillus niger. Selection optimum cultural conditions for improved citric acid production’. A K Banik . \textit{J. Food. Sci. Technol} 1975. 12 p. . \bibitem[Hopwood et al. ()]{b27}\label{b27} ‘Genetic recombination through protoplast fusion in Streptomyces’. D A Hopwood , H M Wright , M J Bibb , S M Cohen . \textit{Narture} 1977. 268 p. 171. \bibitem[Wang ()]{b65}\label{b65} ‘Improvement of citric acid production by Aspergillus niger with addition of phytate to beet molasses’. J Wang . \textit{Bioresource Technol} 1998. 65 (3) p. . \bibitem[Rowalands ()]{b55}\label{b55} \textit{Industrial strain improvement : Mutagenesis and random screening procedures}, R T Rowalands . 1984. \bibitem[Sarwar ()]{b58}\label{b58} \textit{Isolation and characterization of Aspergillus niger mutants for high yield of citric acid from cane molasses}, M G Sarwar . 1973. Bangladesh. Dept. of Microbiology, University of Dhaka (M. Sc. Thesis) \bibitem[Chmiel ()]{b10}\label{b10} ‘Kinetic of citric acid production by preculturated mycelium of Aspergillus niger’. A Chmiel . \textit{Trans. Br. Mycol. SOC} 1977. 68 (3) p. . \bibitem[Chmiel ()]{b9}\label{b9} ‘Kinetic studies on citric acid production by Aspergillus niger II. The two stage process’. A Chmiel . \textit{Microbiologia potonica. Ser Br} 1975. 7 (24) p. . \bibitem[Fukuda et al. ()]{b17}\label{b17} ‘Mierobial preparation of citric acid’. H Fukuda , T Suzuki , Y Sumino , S C Akiyama . \textit{Ger. Pat} 1970. 2003. p. 331. \bibitem[Torres ()]{b62}\label{b62} ‘Modeling approach to control carbohydrate metabolism during citric acid accumulation by Aspergillus niger: I. Model definition and stability of the steady state’. N V Torres . \textit{Biotechnol and Bioeng} 1994. 44 (1) p. . \bibitem[Abarca et al. ()]{b0}\label{b0} ‘Ochratoxin: A production by strains of Aspergillus niger var, niger’. M Abarca , M Bragulat , G Castella , F Cabanes . \textit{Appl Environ Microbiol} 1994. 60 (7) p. . \bibitem[Gardner et al. ()]{b18}\label{b18} \textit{Production of citric acid by mutants of}, J F Gardner , L Valeric-James , S D Rubbo . 1956. \bibitem[Usami ()]{b63}\label{b63} ‘Production of citric acid by submerged culture’. S Usami . \textit{Mem School Sci. Eng. Waseda Univ} 1978. 42 p. . \bibitem[Hang and Woodams ()]{b22}\label{b22} ‘Production of citric acid from corncobs by Aspergillus niger’. Y D Hang , E E Woodams . \textit{Biores. Technol} 1998. 65 p. . \bibitem[Maddox et al. ()]{b43}\label{b43} ‘Production of citric acid from sugars present in wood hemicellulose using Aspergillus niger and Saccharomycopsis lipolytica’. I S Maddox , K Spencer , J M Greenwood , M Dawson , \textbackslash v Brooks , JD . \textit{J. Biotechnol. Lett} 1985. 7 p. 851. \bibitem[Lodhi et al. ()]{b41}\label{b41} ‘Production of citric acid from waste bread by Aspergillus niger’. A K Lodhi , M Asghar , M Zia , A Arnbreen , S Asad , MJ . \textit{J. Biol. Sci} 2001. 1 (4) p. . \bibitem[Auterhoff and Schwingel ()]{b3}\label{b3} ‘Reaction of citric acid with acetic anhydride and pyridine’. H Auterhoff , I Schwingel . \textit{Archiv. Der Phannazic} 1975. 308 (8) p. 583. \bibitem[Islam et al. ()]{b30}\label{b30} ‘Semipilot scale production of citric acid in cane molasses by gamma-ray induced mutants of Aspergillus niger’. M S Islam , R Begum , N Choudhury . \textit{Enzyme Microbial Technol} 1986. 8 p. . \bibitem[Das and Nandi ()]{b11}\label{b11} ‘Specific effects of mutagens on Aspergillus niger in producing citric acid’. A Das , P Nandi . \textit{Folia Microbiol} 1972. 17 p. . \bibitem[Hannan et al. ()]{b25}\label{b25} ‘Stepwise mutational improvement of Aspergillus niger for citric acid productivity in case of molasses’. M A Hannan , M G Sarwar , A Baten , N Choudhury . \textit{J. Folia Microbiol} 1976. 51 p. . \bibitem[Panda et al. ()]{b52}\label{b52} ‘Studies on citric acid production by Aspergillus niger using treated cane molasses’. T Panda , S Kundu , S K Majumdar . \textit{J. Process Biochem} 1984. p. 87. \bibitem[Khan et al. (19750)]{b33}\label{b33} ‘Studies on method of citric acid fermentation from molasses by Aspergillus niger’. M A A Khan , M M Hussain , S M A Khalique , M A Rahman . \textit{Pak. J. Sci. Ind. Res} 19750. 13 (4) p. . \bibitem[Kovats ()]{b34}\label{b34} ‘Studies on submerged citric acid fermentation’. J Kovats . \textit{Acta. Microbiol} 1960. 9 p. . \bibitem[Prescott and Dunn ()]{b53}\label{b53} ‘The citric acid fermentation’. S C Prescott , C G Dunn . \textit{Industrial microbiology}, 1959. p. . (3rd edition) \bibitem[Das-Gupta et al. ()]{b12}\label{b12} ‘The fermentative production of citric acid and oxalic acids from gur and molasses’. G C Das-Gupta , K C Shaha , B C Guha . \textit{J. Science and Culture India} 1938. 3 (7) p. . \bibitem[Xu et al. ()]{b69}\label{b69} ‘The influence of type and concentration of carbon source on production of citric acid by Aspergillus niger’. D P Xu , C P Madrid , M Röhr , C P Kubcek . \textit{Appl Microbiol Biotechnol} 1989. 30 p. . \bibitem[Kumar et al. ()]{b36}\label{b36} ‘Utilization of fruits waste for citric acid production by solid state fermentation’. D Kumar , V K Jain , G Shanker , A Srivastava . \textit{J. Process Biochem} 2002. 38 (12) p. 1731. \bibitem[Hannan ()]{b23}\label{b23} ‘Varients of Aspergillus niger induced by gamma rays’. M A Hannan . \textit{Ind. J. Expt. Biol} 1972. 10 p. . \end{bibitemlist} \end{document}