Nigerian Journal of Microbiology, Vol. 25: 2397 - 2402 2011www.nsmjlJllrnal.org

EFFECT OF SUGAR SUPPLEMENTATION ON CITRIC ACIDPRODUCTION FROM ORANGE PULP BY ASPERGILLUS NIGER

USING SUBMERGED FERMENTATION

DASHEN M. M.\ S. A. ADO 2, J. B. AMEH2, J. D. MAWAK1

l. Dept. of Microbiology, Faculty of Natural Sciences, University of Jos,P.M.B. 2084 Plateau state. Tel.08035165995. E-mail

macvrendashcn@1yahoo.co.uk2. Dept. of Microbiology, Faculty of Science, Ahmadu Bello

University (ABU) Zaria, Kaduna state.

* Corresponding author.

ABSTRACTThe effect of sugar supplementation on citric acid production by Aspergillus niger fromorange pulp using submerged fermentation was studied. Highest citric acid yields wereobtained from three and five percent glucose supplemented orange pulp, the citric acidyields obtained were 14.S8g/1 and 13.72g/1 respectively, while orange pulp withoutglucose supplementation yielded the least amount of citric acid (11.S3g/I).

KEYWORDS: Citric acid, sugar supplementation, orange pulp, Aspergillus niger,submerged fermentation.

INTRODUCTIONCitric acid is the most important

organic acid produced in tonnage by '-...fermentation. Citric acid is a versatileand innocuous alimentary additive. It isaccepted world wide as GRAS(generally recognized as safe), approvedby the Joint FAOIWHO ExpertCommittee on Food Additives (Schusteret aI., 2002). Many by-products andresidues of the agro-industry can be usedin the production of citric acid. A costreduction in citric acid production can beachieved by using less expensivesubstrates. The used of agro-industrialresidues as support in solid-statefermentation is economically importantand minimizes environmental problems(Soccol et al., 2006). The study wasaimed at improving citric acid yield fromorange pulp using sugar (glucose)supplementation.

MATERIALS AND METHODSOrganism and culture maintenance

A cellulase producing A. nigerisolate obtained from the Microbiologydepartment, Ahmadu Bello University,Zaria, Kaduna state, was used for thiswork.Preparation and Proximate Analysisof the Agricultural wastes.

The orange pulp was obtained byexpressing the juice between the palmsof the hands. The pulp was removed,washed, sun-dried and ground usingpestle and mortar and sieved to obtainfine particles. Proximate analysis of thewaste was carried-out at theBiochemistry Laboratory, IndustrialChemicals Research Unit of the NationalResearch Institute for ChemicalTechnology (NARICT), Zaria, Kadunastate. The parameters determine are

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percentage carbohydrate, protein, lipids,crude fibre, ash and moisture.Inoculum Preparation

The spores of the isolates wereharvested from slant bottles of 4-6 daysold cultures by washing with steriledistilled water containing 0.8% Tween80(Poolyoxyethylene-sorbitanmonooleate) and enumeratedusing a haemocytometer (Lasure et al.,2003).Fermentation Technique

All fermentations were carried-out in 500ml flasks. Twenty grams ofeach of the wastes were placed in threesets of two flasks. The first set of twoflasks was without sugarsupplementation while the second andthird sets were supplemented with threeand five percent glucose respectively.The fermentation medium consisted of20g of the waste, 0.93g ammoniumnitrate (NH4N03), 0.045g potassiumdihydrogen phosphate (KH2P04), 0.66gmagnessium sulphate heptahydrate(MgS04.7H20) and 3% methanol in300ml of distilled water ( Lasure etal.,2003). The spore inoculum used was107 spores per ml. Agitation rate andlength of fermentation used were 450rpm (on orbital shaker) and 168h (7days) respectively. Potassiumferrocyanide at a concentration of 0.66gwas added to the fermentation mediumto reduce the deleterious effect of tracemetals on citric acid yield while refinedvegetable oil was added as an antifoamagent. Citric acid yield, residual sugarand dry mycelia weight were determineddaily.Analytical method

Citric acid was determined dailyby filtering 10mI of the culture mediumthrough Whatman filter paper no. 41. 2-3drops of phenolphthalein as indicatorwas added and the filtrate was titrated

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against O.lM NaOH and calculated as %according to the following formula(British Pharmacopea, 1973; Al-Delaimy and El-Holi, 2003):% Citric acid = 192.13 X MNaOHX VNaOH

Weight of substrateWhere,

192.13 = molar mass of citric acid.MNaOH= molarity ofNaOHVNaOH= volume of NaOH consumedduring titration.Concentration of citric acid in g/ 1=% citric acid calculated above x 1000

100Dry mycelia weight was determined asdescribed by AI- Delaimy and El-Holi,(2003), Haq et al., (2003).Residual sugar was determined from thefiltrate obtained in 2.5.1 above using the3, 5- Dinitrosalycilic acid (DNS) method(Miller, 1959).RESULTSProximate Analysis

The proximate composrtionof and orange pulp is as follows,59.03% carbohydrate, 9.47% crude fibre,5.25% crude protein, 0.70% fat content,3.76% ash content and 21.79% moisturecontent. These analysis shows thatorange pulp contain 68.50% of theportions (carbohydrate and crude fibre)that can be fermented to citric acid.Citric Acid YieldsFigures 1, 2 and 3 show citric acidyields, residual sugar concentration andmycelia weight of the A. niger isolatewithout glucose supplementation, withthree percent glucose supplementationand with five percent glucosesupplementation during submergedfermentation of orange pulp. The highestcitric acid yield of l4.58g/1 was obtainedfrom orange pulp supplemented withthree percent glucose, followed by acitric acid yield of 13.72g/1 from orangepulp supplemented with five percent

Dashen et aI., 2011

glucose in both cases after 144h (sixdays) of fermentation. The highest citricacid yield obtained from fermentationfrom orange pulp without glucose wasl1.53g/1 after 120h (five days) offermentation.

With respect to orange pulpwithout glucose, residual sugarconcentration increased steadily fromO.35g/l at day one to 3.60g/l at day fivethen reduced to 3.20g/l at day seven.However, mycelia weight increasedprogressively from 3.05g/l at day one to12.84g/l at day seven.

Both three and five percentglucose supplemented orange pulp showa similar pattern of residual sugarconcentration, a decreased from 21.90g/l

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and 27AOg/l at day one to O.90g/l andOAOg/l at day four followed by anincrease to 1.iOg/l and O.75g/l at dayfive and a decreased to 1.35g/l and1.25g1l at day seven respectively.Mycelia weight also increasedprogressively in both cases from 2A8g/lat day one to 11.60g/l at day seven withrespect to three percent glucosesupplemented orange pulp and from3.11 g1l at day one to 11.49g/l at dayseven with respect to five percentglucose supplemented orange pulp.There was no significant difference inmean citric acid yields from orange pulpwithout glucose, with 3% glucose andwith 5% glucose (P>O.05).

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Figure 1:Cltrte acid yiekf. residual sugar concentration & mycelia weight of A. niger during submerged fermentation oforange pulp without glucose

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Dashcn et al., 2011 Nig.J. Microbial Vol. 25: 2397 - 2402

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Figure 2:Citric acid yfekl, r8sidualsugar concentration & mycelia weight of A. nlger during submerged fennentation oforange pulp with 3-/0 glucose

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Figure 2:Citric acid yield, residual lugar concentration & mycelia weight 01A. niget' during lubmerged fermentation oforange pulp with 5% glucose

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DISCUSSIONBoth 3% and 5% glucose

supplemented rice bran gave highercitric acid yields than unsupplementedrice bran, this agrees with the findings ofKiel et al., (1981) on the possible use ofcotton wastes as a carbon source forcitric acid production by Aspergillusniger in which no citric acid wasproduced when Aspergillus niger wasgrown on cotton waste as a sole carbonsource but when supplemented with 1%,2%, 3%, 5%, 7%, 10% and 14% w/vsucrose, citric acid yield of 35mM,59mM, 63mM, 81mM, 66mM, 84mMand 87mM respectively was obtained.However, there is a dearth ofinformation on citric acid fermentationfrom orange pulp by submergedfermentation.The yields obtained from this work ishigher than that reported for two agro-industrial wastes, a citric acid yield of0.050g/l from undersized semolina (awheat waste) was reported by Erkmanand Alben (2004) and a yield of 2.86g/1from waste bread was reported by Lodhiet al. (2001). Other studies gave highercitric acid yields than this study; Hang etal., 1987 reported maximum citric acidyield of 100g/kg from dry Kiwi fruitpeel and 56g/kg from dry grape pomacefor various A. niger strains in solid statefermentation, Buzzini et al. (1993)obtained maximum citric acid yield of67.60g/l from rape grape must in batchculture (Ikenebomeh and Oshoma,2002).CONCLUSIONSugar supplementation was found toimprove citric acid production byAspergillus niger from orange pulpunder condition of submergedfermentation. Increasing the level ofsugar supplementation may further

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Increase the yield from the wastestudied.REFERENCES

AI-Delamy, K. S. and EI-Holi, M.(2003). Citric acid production fromwhey sugars and additives byA.niger. African Journal ofBiotechnology, 2(10) ,356 - 359.

Buzzini, P., Gobbetti, M., Rossi, J. andRibaldi, M. (1993). Utilization ofgrape must and concentratedrectified grape must to producegluconic acid by Aspergillus nigerin batch fermentations.Biotechnology letter 15:151 - 156.

Erkmen, O. and Alben, E. (2004).Production of citric acid from anew substrate, undersizedsemolina, by Aspergillus niger.Food Technology Biotechnology,42 (1) 19- 22.

Haq, I. U., Ali, S., Qadeer, M. A. andIqbal, J. (2003). Stimulatory effectof alcohols (methanoland ethanol) on citric acidproduction by a 2 - deoxy D -glucose resistant culture of A. nigerBCB 47. BioresourceTechnology, 86 (3) 227-228.

Hang, Y. D., Luh, B. S. and Woodams,E. E. (1987). Microbial productionof citric acid by solid - statefermentation of kiwi fruit peel.Journal of Food Science, 52:226-227.

Ikenebomeh, M. 1. and Oshoma, C. E.(2002). Citric acid production fromrice bran by Aspergillus niger.Nigerian Journal of Microbiology,16:1-2:35-41.

Lasure, L.L., Ziyu, D., Xingxue, M., andMagnuson, J. K. (2004).Identification of genes associatedwith morphology in A. niger byusing Suppression SubtractiveHybridization. Applied and

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Environmental Microbiology,70:2474-2485.

Lodhi, A. K., Ashar, M., Zia, M.,Ambreen, S. and Javed, A.(2001). Production of citricacid from waste bread by A.

niger. Online Journal ofBiological Sciences, 1 (4) 182-183.

Kiel, H., Guvrin, R. and Henis, Y.(1981). Citric acid fermentationby A. niger on low sugarconcentrations and cotton waste.Applied and EnvironmentalMicrobiology, 42 (1) 1-4.

Schuster, E., Dunn-coleman, N., Frisvad,J. C. and Van-Dijck, P. W.(2002). On the safety ofA. niger-« review. AppliedMicrobiology Biotechnology, 59(4-5) 426-430.

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Soccol, C. R., Vandenberghe, L. P. S.,Rodrigues, C. and Pandey, A. (2006).New perspectives for citric acidproduction and application. FoodTechnology Biotechnology, 44 (2) 141-149.

ACKNOWLEDGEMENTThe authors wish to acknowledge theFaculty of Natural Sciences ResearchGrant Committee of the University ofJos, Nigeria for sponsoring this researchwork.

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