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AbstractCitric acid (CA) is a weak organic acidfound naturally in the citrus fruits with a wide range of applications,including, preservation, flavor enhancement, bacterial inhibition, pHregulation and as an antioxidant. The main goal of this work was to producecitric acid from Ethiopian sugar cane molasses using an isolated fungal speciesvia submerged fermentation process.

The organism was identified as Aspergillus niger by 16S rDNAsequencing. In this study, the sugar cane molasses after dilution, is pretreatedwith  35 mL of 1N H2SO4 per liter, boiled, cooled, neutralizedwith CaO and clarified prior to fermentation. The effect of pH (5, 7 and 9) andthe fermentation time is recorded during the process and the yield of citricacid by the fungal species is determined. Keywords:Aspergillus niger,16S rDNA, Cane Molasses, Citric Acid, Submerged Fermentation 1.IntroductionSugarcane molasses is a black, viscous liquid by-product of refining sugarcane intosugar. It is  used as a substrate for theproduction of ethanol and citric acid due to its high reducing sugar contentwhich is about 62%.

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This content may vary based on the amount of sugar, methodof extraction and age of plant (cane). Citric acid (CA) has more health andeconomic benefits 1. They exist in two forms,monohydrous and anhydrous, which differs in their degree of hydration 2.Conventional citric acid production is carried outin three ways, from the citrus fruit extracts, chemical synthesis and by usingcarbohydrate source material such as sugar cane molasses, and other fruit peels.

  Ofall the methods, fermentation is the most economical method for producingcitric acid and is a predominant way of producing of citric acid as it accountsfor about 90% of world production. Though all the three types of fermentationprocesses have been widely employed, including the submerged, surfacefermentation and solid state fermentation 3,7,in the present study submerged fermentation was used to produce citric acid dueto the liquid nature of the substrate, the sugar cane molasses. Submerged fermentationtechnology has been reported by several workers as anattractive process to produce citric acid due to its advantage of low cost,short period and high yield. There are many sugar manufacturing industries inthe country, which provide cheap and ample cane molasses making the productionof citric acid reliable and consistent. The most important aspect  during the citric acid production is theclarification of the raw molasses that comes from the sugar milling factoriesand its pretreatment with sulfuric acid to form complex compounds of traceelements  in the molasses and thereby removingthem4,8.Aspergillusniger is one of the most common species used to utilize starchy and sugarsubstrates like molasses and converted into different products . It is the mostpromising microorganism used in production of citric acid compared to othermicroorganisms due to its ability of utilizing starchy and sugar substrates 5.

The production of citric acid using submergedfermentation depends strongly on an appropriate strain and on operationalconditions such as aeration, type and concentration of carbon source, nitrogenand phosphate limitation, pH, concentration of trace elements and morphology ofthe fungal species.2. Experimental section2.1.Microorganism-Inoculation and culture conditionsThefungus used in this study was obtained from the Ethiopian BiodiversityInstitute, Addis Ababa, Ethiopia.  The isolates were initially grown onPotato Dextrose Agar at 328 K for 24 h.

The fungus was further sub-cultured onthe Potato Dextrose Agar at regular intervals and incubated at 313 K.  For further propagation, the minimal medium composition employed in mg/L was,  0.223 NH4NO3,0.1 K2HPO4, and 0.023 MgSO4.7H2O. Canemolasses was employed as the chief carbon source.All  the submerged fermentations werecarried out using the isolated fungus.

 The strain was maintained on PDA slants at 30°C for 6 d.The strain was prepared as conidial suspensions by washing slant cultures with5 mL sterilized water. Spore suspension was counted at 25×106spores/mL by Haemacytometer.

  All the trials were carried out in 250 mLErlenmeyer flasks containing 100mL of molasses, at pH between 5 and 96. The flasks weresterilized prior to the inoculation with three milliliters of prepared sporesand incubated at a temperature of 28°C on a shaker incubator at100 rpm for 11days in succession.2.2.

Feedstock-MolassespretreatmentCane molasses contains the following contents: water,20%, sugar, 62%, and non-sugar, 10%, and inorganic salts (ash contents), 8%. Itis a blackish homogenous liquid with high viscosity20. Ash contents includeions such as Mg, Mn, Al, Fe and Zn in variable ratios. Sugar content wasdiluted to 24% with  deionized water.

Thiswas followed by the addition of 35 mL of 1NH2SO4 per liter, boiling for half an hour, coolingand neutralizing using the lime-water (CaO). The contents were then left tostand overnight for clarification 7. Theclear supernatant liquid was further diluted to 12-24 % sugar using deionizedwater.2.3. Processvariables for citric acid production             Citric acidproduction by fermentation can be divided into three phases, which includespreparation and inoculation of the raw material, fermentation, and recovery ofthe final product. After the pretreatment of the molasses,nutritive salts (like ammonium nitrate) are added and it is diluted withdistilled water to make a solution 8. This solutionis sterilized and after cooling down to 30°C, it is transferred to a sterilizedsubmerged fermenter and inoculated with the spore suspension of Aspergillus niger.

  pH of the substrate (solution) was adjustedto 5.5-5.9, and the temperature 28°C, the most suitable for the germina­tion ofthe conidial aggregation. This was followed by the addition of nutrients andmicroorganism to the bioreactor. Then the parameters such as pH and temperaturewere adjusted to an initial value of 2.5-3.0 and 28°C respectively.

The fermenteris then aerated, when a thin film of mycelium is created in 24 h (germinatingperiod). This was followed by the production period, when the mycelium getsstronger, and the temperature of the fermented solution rises9. During this phase, the exothermic citric acidproduction is initiated by the environment. 3. Results and discussions3.1. Microscopic observation andmolecular testingThe isolate was identified based on the colonymorphology, microscopic observation and molecular identification 12.

The fungus was identified as Aspergillus niger based on theproduction of clear carbon black /brown spores from the biseriate phialides. Thecolonies were fast-growing, whitish to blackish or brownish, and usually thick13. The morphology of the isolate wasexamined on potato dextrose agar (PDA in the dark. Colonies on PDA were fastgrowing with sporangiophores measuring 2.3 to 3.6 mm long and 1.2 to 2.

9 mmwide after 3 days. DNA was extracted from the hyphae of a 36 h culture on PDA slantsand suspended in UltraPURE distilled water in 2 ml Eppendorf tubes, eachcontaining one sterile 4.5-mm steel shot pellet.3.2. Fungal strain identification For the nucleotide sequence analysis, fungal genomicDNA was purified using the Fungi Genomic DNA Isolation Kit (MTK 08) (ModernScience Co., Nasik).

The fungal primer pairs annealing at the 50 and 30 end ofthe 18S rRNA, 50-GTAACCCGTT-GAACCCCATT-30 and 50-CCATCCAATCGGTAGTAGCG-30,respective-ly, were used for amplification. The PCR was run for 35 cycles in aDNA thermal cycler (Thermal Cycler Applied Biosystems 2720, USA). Amplified PCRproducts were then analyzed in a 1% (w/v) agarose gel and purified. Purifiedproducts were cloned and subsequently sequenced using an automated DNAsequencer (ABI 3130 Genetic Analyzer, USA). The 16S rDNA sequence obtained wascompared with the sequence obtained from the nucleotide database of the NationalCenter for Biotechnology Information (NCBI)12.The phylogenetic analysis of the strain, using its nucleotide sequence datashowed that this strain had the highest homology of 98% and 99% with the Aspergillus niger strains,  Aspergillus niger SH-2 and Aspergillusniger ATCC 10864, respectively.

Based on the evolution distance andpartial sequencing, this strain isolated was identified as Aspergillus niger.3.3. Citricacid production            After the citric acid production by the submergedfermentation, the fungal mycelium is separated from the solution, washed toprevent loss of considerable quantity of acid contained by the mycelium.

Afterthe hydrolysis, the liquid fraction of the hydrolysate samples were analyzedfor their reducing sugar content was determined using the phenol-sulfuric acidmethod. The absorption values of the samples were recorded at 490 nm on aspectrophotometer 3. The acid solution isthen separated from the mycelium and is reconditioned through filtering toremove the by-products such as oxalic acid. The calcium citrate is precipitatedusing lime and then the resulting solution is filtered. The resulting solutionis decolorized batch-wise by the addition of activated carbon. The percentageof citric acid produced by Aspergillusniger is then determined by the titrimetric method4.After the decolorization, thesolution is concentratedby evaporation and crystallized at a temperature of 22-26°C to form a whitemonohydrate citric acid powder as the final product.

3.4. Analysis of total reducingsugar contentIn this study, the amount ofreducing sugar in the molasses was investigated.

The total sugar content of molassessamples was determined using phenol sulfuric acid method. During the phenolsulfuric acid method, the reducing sugar content in the samples is dehydrateddue to the reaction with sulfuric acid and produced furfural derivatives3. Further reaction between furfural derivatives andphenol develops a detectible Yellow-Orange color.

The concentrations of unknownsugar content of samples were determined from the standard curve of glucose.The resulting molasses substrate was subjected to fermatation by the Aspergillus niger.3.

5. Analysis of citric acidcontent The fermented samples weresubjected to the assay for citric acid by the titrimetric method usingphenolphthalein as an indicator. Thefiltrate obtained is titrated against an alkali of known strength usingphenolphthalein as indicator.

The end point is the formation of pale pinkcolor. The volume of alkali used for neutralization is used to find thenormality and the percentage of acid in the sample. In this study, a solutionof 2.

1g citric acid per 100ml distilled water is prepared. And from theprepared solution, 10 mL of solution was pipetted in to a conical flask, withan addition of 2-3 drops of indicator, titration was carried out against 0.1NNaOH in the burette till a pale pink color was formed4.

The titration was repeated till expected value was obtained. During thesampling, a solution was prepared using 5 mL of the sample from each test tubesof citric acid per 20 mL distilled water. From the prepared solution, 10 mL of solution was pipetted into aconical flask and 2-3 drops of indicator were added and titrated following theprocedure previously mentioned. The percentage of citric acid produced is thencalculated.    3.6. Effect of pH on the citricacid productionIn this study, a considerable amount ofcitric acid was produced using molassesas a carbon substrate by the submerged fermentation from the molassescontaining about 62% of sugar.

  Stoichiometrically,by the conversion of 100 g sucrose with oxygen, 123g monohydrate or 112ganhydrous citric acid can be obtained. Biochemical conversion of sucrose intocitric acid by the microorganism is shown below, C12H22O11+4.95O2+0.133NH4NO3          1.56CH1.72O0.55N0.

17+3.54CO2+5.32H2O+1.

15C6H8O7      During the fermentation process, the pHof the medium was found to decrease initially at non regular intervals, fromthe initial pH of 7.0 to 4.32 on the eleventh day, indicating the acidity ofthe media and more evidently the citric acid stable pH (Table1). Figure. 1 shows the effect offermentation pH on the citric acid production.Table 1. Data collected throughout the fermentation process Day Biomass weight (g) Citric acid(%) Initial pH Final pH 3 3.42 24.

00 7 6.40 5 9.60 28.

88 7 5.90 7 1.98 38.40 7 4.60 9 5.29 67.20 7 4.52 9 1.

64 26.88 5 4.78 9 4.33 39.55 9 5.09 11 4.42 30.

72 7 4.32 Theproduction of citric acid increased exponentially until the day 9, at whichmaximum yield was observed. But as the fermentation time exceeded the day 9,the yield decreased due to the possible conversion of Citric acid into itsbyproducts. The production of citric acid was maximum at pH 7, implying thestability of the product at the favorable pH value of around 710. Fig.

1.  Effect of pH on the citric acid productionInthe present batch-wise fermentation of citric acid, the production startedafter  the lag phase of 1 day andreached maximum at the onset of stationary phase or late-exponential phase.Further, it was observed that there was no enhancement in the citric acidproduction during the increase in the incubation period.

It may be due to theage of fungus used and depletion of sugar contents in the culture broth. Theacid production was found to start at the initial stage of the idiophase(between 80-120 h) of fungal growth. At this stage, the pH was five.

Theyield gradually increased and reached to the maximum at the late idiophase (180-220 h).In this stage, the pH must be below 3 in order to suppress oxalic acid andgluconic acid formation. The acceleration of fermentation can be explained bythe higher starting biomass concentration in fermenter and by the adaptation ofbiomass to very high osmotic pressures11. 3.7. Effect of Temperature onCitric acid productionTemperatureplays an important role in the citric acid production.

Temperature between 25°Cand 30°C is usually employed for culturing the Aspergillus niger. Theoptimum temperature for citric acid production is 28°C, but during the citricacid production, the temperature of the medium increases above 30°C and thebiosynthesis of citric acid decreases. This may be due to high temperature,which causes the denaturation of enzyme-citrate synthase and accumulation ofother by-products such as oxalic acid. In addition to this, enzyme cataboliterepression, could be a possible inhibitor8. Fig.2.Effect of fermentation time on the production of citric acidNitrogenis another limiting factor in the citric acid production. Nitrogen is usuallysupplied in the form of ammonium nitrate, which is completely metabolizedduring fermentation periods.

Citric acid starts to appear when the nitrogenconcentration falls below a low limiting value6,11.Figure. 2 shows the effect of fermentation timeon the citric acid production at a pH of 7.

Earlier studies have reported thatthe factors affecting citric acid production by fermentation includes, thenutrient composition of the media, environmental conditions, deficiency ofmanganese, types and concentration of sugars, chelating effect of metal ions,ammonium nitrate and aeration10. The optimumtime of incubation for maximum citric acid production varies with the organismand fermentation conditions used.4.

ConclusionsThestudy shows that the concentration and type of molasses, influences the yieldof citric acid produced by Aspergillusniger. In the controlled production medium, the initial pH of 7 wasfound to decrease to 4.32 during fermentation confirming the production ofcitric acid. Sucrose in the molasses is the substrate responsible for the citricacid production in this medium. Aspergillusniger utilizes sucrose and produces 67.

2% citric acid. Based on ourstudy, the maximum amount of this citric acid was produced in 9 days at the pH7. References1              Young, M.

M., 1985. Comprehensivebiotechnology. Vol. 3.

Pergamon Press, Oxford, UK.2              Arzumanov, T.E., Shishkanova, N.V.,Finogenova, T.V.

, Biosynthesis of citric acid by Yarrowia lipolytica repeat-batchculture on ethanol, Applied Microbiologyand Biotechnology, 2000, 53(5) 525-529.3              Duboise, K. Sugar determination by phenolsulphuric acid method, Biotechnologyand Bioengineering, 1956, 10, 721-724.4              Kristiansen, B.

, Mattey, M., Linden, J.1999. Citric Acid Biotechnology,Taylor & Frances Ltd., London, UK, pp. 7-9.5              Pazouki, M., Felse, P.

A., Sinha, J., Panda,T. Comparative studies on citric acid production by Aspergillus niger andcandida lipolytica using molasses andglucose, Bioprocess Engineering, 2000,22(4) 353-361. 6              Prescott, S., Dunn’s, A.

Industrial Microbiology,4th edition, CBS Publishers and Distributors, New Dehli, India, 1987, pp.710-715.7              Doelger, W.P., Prescott, S.C.

, 1934. Citricacid concentration of Sucrose. Analytical Chemistry, 19: 1012-1013.8              Haq, I., Sikander, A.

, Qadeer, M.A., Javed, I., 2004. Citricacid production by selected mutants of Aspergillus niger from canemolasses. Bioresource Technology, 2004, 93, 125-30. 9              Watanabe, T.

A., Nakagawa, S.H., Kirimura, K., Usami S.,Citric acid production from cellulose hydrolysate by a 2-deoxy glucose-resistantmutant strain of Aspergillus niger, Applied  Microbiology and Biotechnology, 1998, 66, 271-274.

10          Panda, T., Kundu, S., Majumdar, S.K.

,Studies on citric acid production by Aspergillus niger using treatedIndian cane-molasses, Journal of Microbiology,1984, 5, 6-66.11          Pazouki, M., P.

A. Felse, J. Sinha and T. Panda, Comparativestudies on citric acid production by Aspergillus niger and Candidalipolytica using molasses and glucose, Bioprocess Engineering, 2000, 22,353-361.

12          AnuradhaJabasingh, S., Lalith, D., Pavithra, G.

, Sorption of chromium(VI) fromelectroplating effluent onto chitin immobilized Mucor racemosus sorbent (CIMRS) impregnated in rotating diskcontactor blades, Journal of Industrial and Engineering Chemistry, 2015, 23,79–92.13          Raper,K.B., Fennell, D.I., 1965. The genus Aspergillus.Williams and Wilkins.

Baltimore, Maryland. pp.686. 

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