Chelating Ability of the Chitosan-glucan Complex from Aspergillus niger NRRL595 Biomass Recycling in Citric Acid Production

Transcription

1 Reserch Journl of Agriculture nd Biologicl Sciences, 2(3): 13236, , INSInet Puliction Chelting Aility of the Chitosn-glucn Complex from Aspergillus niger NRRL595 Biomss Recycling in Citric Acid Production Motz, M. Sd Microil Chemistry Deprtment, Ntionl Reserch Center, Dokki, Ciro, Egypt Astrct: The production of citric cid frommethnol (3%, v/v) treted crude dte syrup (24%sugrs, ph, 6.5) y Aspergillus niger NRRL595 iomss recycle ws investigted. By reuse of the iomss for 5 cycles (75 dys), the citric cid concentrtion, the citric cid yield, the citric cid productivity nd consumed sugrs decresed initilly from the 2 nd cycle until reched the miniml vlues t 5 th cycle. The A. niger iomss dry weightproduced from5 recycles (75 dys) ws g L 1, the insolule chitosn-glucncomplex wsisolted in 25% yield of the iomss. The extrcted polymer ws frctionted to its constituents (chitosn, 3.65% yield nd β, 1-3 glucn, 80.85% Yield), whose infrred re reported. Copper ions re chelted y chitosn- glucn complex with higher inding % from 81.7 to 88%. Key words: Aspergillus niger, Citric cid, Recycling,Chitosn-glucn complex, Cu, Chitosn, Glucn INTRODUCTION Despite vrious dvnces in process development, the surfce nd sumerged tch culture techniques re still eing used for production of citric cid; however, in the tch techniques significnt mount of productive iomss is discrded ech time. Cell recycling techniques hve dvntges over tch techniques ecuse the iomss is conserved, tch downtime is mostly eliminted nd productivity is often incresed [14]. The wste fungl mycelium mts produced s y products of fermenttion industries represent promising potentil source for the isoltion of chitin nd /or chitosn [11]. Nowdys, Aspergillus niger is lmost exclusively used for industril scle production of citric cid. More thn 600,000 metric tons re produced nnully world wide [1]. No informtion is reported on the rtio of citric cid to mycelil wste ut it is resonle to ssume tht it is 5:1 [11]. Since most of the fungl iomss produced in industril fermenttions is currently disposed of either y lndfiling or incinertion, employment s iosorent would offer n ttrctive potentil use of this wste mteril [8,11,17]. Specificlly, out 1/3 of the myceliumof Aspergillus niger is n lkli- resistnt, ssocition of chitin with glucn contining 1-3 (85-90%) nd 1-4 linkges [16]. Chitosn is currently otined y the decetyltion of chitin (poly- β,4d-ncetylglucosmine).decetylted chitosn is produced y treting chitin in concentrted lkline solution (40-50%,wt/vol) nd oiling it for severl hrs [11,13]. Chitosn hs dvntges over chitin ecuse of its high soluility in cidic solutions nd its polyctionic nture. Inthe lst two decdes, pplictions of chitosnhve een developed in mny industries; chitosn hs emerged s new iomteril for food, phrmceuticl, textile nd other industries, s well s for wste wter tretment [5,7,11,15]. In the present study, the extrction of iopolymers from A. niger NRRL595 iomss recycle technique for surfce fermenttion of dte syrup to citric cid re involved in the inding of Cu. MATERIALS AND METHODS Orgnism nd culture conditions: Aspergillus niger NRRL 595 ws otined from the culture collection of Northern Regionl Reserch Lortory, Deprtment of Agriculture, Peori. Illinois, U.S.A. The fungus ws mintined on potto dextrose gr (PDA) slnts t 4 N C nd sucultured t intervls from dys. Inoculum: The culture ws incuted on potto dextrose gr Petri dishes t 30 N C for 5 dys. Circulr mycelil plug (4mm in dimeter) ws trnsferred to ech of sterile methnol (3%, v/v) treted dte syrup flsk. Preprtion of dte syrup: Semi-dried dtes were otined from the locl mrket. Dtes were chopped into smll prticles nd heted in distilled wter (solid/ liquid rtio, 1:3) t 80 N C for 2h. nd the mixture ws mintined t room temperture for 24h. fter tht the mixture ws homogenized in wrring Blender for 3 min. The extrct ws filtered in filter press nd it ws diluted with distilled wter to otin 24% sugrs, ph, 6.5. Citric cid production nd iomss recycle: The fermenttion ws performed in 250 ml conicl flsks ech of it contining 50 ml methnol (3%, v/v) treted dte syrup. The medium ws sterilized t 121 N C for 15 min. Corresponding Author: Motz, M. Sd, Microil Chemistry Deprtment, Ntionl Reserch Center, Dokki, Ciro, Egypt. 132

2 nd inoculted with one disk of the inoculum. The flsks were incuted sttic t 30±1 N C under sttionry conditions. Methnol ws dded on the thired dy of fermenttion. When the fermenttion ws complete (fter 15 dys), the originl medium ws withdrwn from ech flsk nd replced with 50 ml of fresh sterile medium (ph djusted to 6.5 with 1N NOH). The fermenttion ws stopped t 15 dys nd the fermented liquid sustrte ws removed from ech flsks, replced with fresh sustrte t the pproprite ph nd new cycle strted. Five cycles were crried out in totl. Hrvesting of fungl iomss: Mycelil mts were recovered from the 5 th cycle fter 75 dys y filtrtion, wshed severltimes with distilled wter, plotted etween filter pper. Preprtion of chitosn- glucn complex: The method of Muzzrelli et. l. [11] ws used. Wet iomss (325 g fresh wt=100g dry wt.) ws refluxed with 40% NOH solution for 6h. t 100 N C. Such tretment simultneously decetyltes the chitinous frction, dissolves the protein, removes the solule glucns nd hydrolyses the lipids. The mixture ws filtered nd the residue ws wshed severl times with distilled wter untilneutrl nd once with ethnol (84%), then twice with distilled wter. The product ws dried y lyophiliztion for 72 h. nd the dried powder ws weighed nd chrcterized y IR-spectroscopy. Chemicl frctiontion of chitosn-glucn complex: Chitosn-glucn complex ws roken into pieces nd refluxed with 1N cetic cid t 1:100 (w/v) for 30 min., the procedure ws replced for four times with fresh cid used for ech step, s the method descried y White et. l. [18] nd modified method y Arcidicono nd Kpln [2]. After centrifugtion of the mixture, the insolule frction s β,3 glucn ws discrded nd wshed severl times with distilled wter nd lyophilized. The cid solule superntnt ws djusted to ph8.5 with 3NNOH nd se- insolule precipitte (chitosn) ws collected y centrifugtion t 4,000g for 20 min. The precipited frction ws wshed severl times with distilled wter nd lyophilized. The lyophilized mterils were chrcterized y IR-spectroscopy. Copper cheltion y chitosn- glucn complex in continuous flow system: Biosorption of Cu from its solutions (1 μg nd 10 μg/ml) ws chieved in continuous flowsystem, 200 mg chitosn-glucncomplex ( mesh) were plced on lyer of glss wool in glss column (2Cm-ID nd 40 Cm height), rinsed with distilled wter. Aqueous solutions of Cu in pproprite concentrtions (1 μg or10 μg ml 1 ) were pssed through the columnt flow rtes of 10 ml /5 or 10 min.frctions (10 ml) were collected nd nlyzed for their Cu content. Anlyticl techniques: By the end of ech fermenttion period (15 dys), fermenttion flsks were removed nd the contents nlyzed. Citric cid, ph nd residul sugrs were determined. Citric cid ws determined y the method of Mrier nd Boulet [9] nd residul sugrs were determined s glucose y the method of Duois et. l. [4]. RESULTS AND DISCUSSIONS Citric cid production y Aspergillus niger NRRL 595 iomss recycle: In our previous work [10] the highest vlues of citric cid concentrtion, citric cid yield, productivity nd iomss dry weight (31.1 g L 1, 12.54%, nd 30.2 g L 1, respectively) were otined in culture grown in 3% (v/v) methnol treted crude dte syrup for 15 dys (360 h).as cn e seen from Tle 1, y reuse of the iomss, for 5 cycles (75 dys) thecitric cid concentrtion, the citric cid yield, the citric cid productivity nd the consumed sugrs decresed initilly from the 2 nd cycle until reched the miniml vlues t 5 th cycle, consequently, the ph of cultures ws incresed. This mens tht the orgnismlost their ctivity to produce citric cid. The low citric cid concentrtion oserved my e due to the high concentrtion of iomss in fermenttion roth. These resultswere gree with those otined y Rouks [14]. Preprtion of chitosn glucn complex from the iomss: The A. niger iomss dry weight produced from 5 recycles (75 dys)ws g L 1. The insolule polymer ws isolted in 25% yield of the iomss. The tretment of myceli with 40% NOH simultneously decetylted thechitinous frction, dissolved the proteins, removes the solule glucn nd hydrolyzed the lipids [11]. Chemicl frctiontion of chitosn-glucn complex:as shown in Tle 2, the totl mount of chitosn extrcted Tle 1: Citric cid production from methnol (3%, v /v) treted dte syrup y Aspergillus niger NRRL 595 iomss recycle. Citric Citric cid Finl cid Citric cid productivity Consumed Cycle ph (g L 1 ) yield (%)* (g/l/h) sugr (%) Initil sugr concentrtion = 240 (g L 1 ), Initil ph = 6.5, Process time (h) =360 *Citric cid yield (%), expresses % citric cid formed of initil sugr in medium., Ech vlue is n verge of results from two individul determintions nd four experiments. Tle 2: Isoltion of chitosn from 1.0g of chitosn- glucn complex. No of tril Chitosn yield (mg) Chitosn yield (%) Totl

3 Cu - ind ing % Time (min.) 1 mg Cu /L 10 mg Cu /L Fig. 1: Collection of Cu from Cu -solution 1 mg Cu /l nd 10 mg Cu /l t ph y200 mg ( Mesh) chitosn-glucn complex with 1.0N cetic cid ws 36.5 mg (3.65%). To determine thetime of extrction 1.0g of theisolted chitosn- glucn complex ws extrcted four times, with fresh cid used for ech step. The chitosn yield ws high during the first 30 min., fter tht the yield ws little. No dditionl chitosn ws otined fter totl 2.0h. The insolule frction (β, 3 glucn) ws discrded nd lyophilized. The mount of this frction ws 808.5mg (80.85%). Cu -cheltion y chitosn-glucn preprtion from Cu -solutions in continuous flow system: Since microil polyscchrides re known s potent metl dsorents [11], it ws of interest to investigte the reltive Cu -inding of the test polymer. Cu dsorption y the respective fungl polymer ws monitored over wide rnge of metl concentrtion(1nd 10 mg Cu L 1 solutions) (Fig. 1). It ws oserved tht the percentge of ction inding y the chitosn-glucn rpid during the first 5min.(55.77%) for 1.0 mg Cu L 1 nd 50.41% for 10 mg Cu L 1 ) nd then incresed with the pssge of time until reched the mximl t 30 min(88.46% for 1.0 mg Cu L 1 nd 81.7% for 10.0 mg Cu L 1 ). No dditionl Cu - inding ws occurred fter 1 h. These resultswere similr to tht for Cu collection y extrcted chitosn-glucn complex from A. niger industril wstes [11], it is seen tht copper is reltively collected (87-99%). Infrred nlysis of the isolted iopolymers nd metl ions contct chitosn-glucn complex preprtion: Figures 2-4 shows spectr of chitosnglucn complexefore nd fter Copper contct, chitosn s reference, chitosn preprtion, lminrin s reference nd β,1-3 glucnpreprtionetween 4000 nd c Wve no. (cm ) Fig. 2: Infrred nlysis of extrcted components () chitosn glucn complex, 1mg l Cu -inding, () chitosnglucn complex nd (c) 10mg l Cu inding chitosn glucn complex. 134

4 Wve no. (cm ) Fig. 3: Infrred nlysis of chitosn stndrd () nd chitosn preprtion () Wve no. (cm ) Fig. 4: Infrred nlysis of lminrin () nd Extrcted B1-3 glucn preprtion () 135

5 400 cm 1. Such oservtions re sed on the identicl nture of mjor sorption peks of polymer smples. An intense pek t round cm region indictes the sorption of γ(o-h) groups, while other eks t ~ 2900 cm 1 nd t the finger print region (i.e. elow 2000 cm 1 ) denote the chrcteristic sorption of γ(c-h) group due to the presence of croxyl ( cm 1 ) or cronyl (1640 nd cm 1 ) compounds (Fig. 2) indicting the present preprtion is contminted y chitin. The second mide peks t pproximtely 1660 cm 1 (C=O, mide 1) nd 1550 N-H mide II my e considered to represent the proteinfrction ofthe isoltedchitosn-glucn. The mide ndmine nds re more significntto theunderstnding metl ions sorption [3,11,12]. The shift in spectr etween chitosn-glucn preprtion nd metl ions contct chitosn-glucn is relted to n evolution of copper species ccumultion on the extrcted polymer: the hydroxylted form or orgnic complexed form (Fig. 2). Spectr of chitosn nd lminrin used s references shownin Fig. 3,4. The pek t 1380 cm 1 is proly the strong O-H group stretch pek. Between 1000 nd 1100 cm 1 is the C-O stretch pek, which my e considered to represent the crohydrte component (i.e., the oxygen in the glucose ring). The virtion t 1635 cm nd 1381 cm 1 cn e ttriuted to NH- C = N nd CH 2 indicting chitosn polymer (Fig. 3). However no sorption nds t 845 nd 820 cm were oserved chrcter of β,1-3 glucn (Fig. 4). The spectr reflected close chemicl similrity of the extrcted iopolymers (chitosn nd B,1-3 glucn) with the reference polymers. These nds re evident in the spectr of the isolted polymers y Knetsun nd Cronell [6,11]. Conclusions: Mycelil mts from citric cid production plnts re suitle for the production of chitosn derivtive.chitosn-glucn complex is esily otined y simply treting the prewshed myceli with 40-50% queous NOH, preferly t oiling tempertures for 4-6 h. It hs high ility to collect trnsition metl ions from solutions. REFERENCES 1. Anstssidi, S., A. Aivsidis nd C. Wndrey, Citric cid Production y Cndid strins under intrcellulr nitrogen limittion. Appl. Microiol. 60: Arcidicono, S. nd D.L. Kpln, Moleculr weight distriution of chitosn isolted from Mucor rouxii under different culture nd processing conditions. Biotech. Bioeng., 39: Brdy, D., A.D. L. Strke nd J.R. Ducncn, Chemicl nd enzymtic extrction of hevy metl inding polymers from isolted cell wlls of Scchromyces cervisie. Biotechnol. Bioeng., 44: Duois, M., K.A. Gilles, J.K. Hmilton, P.A. Reers nd F. Smith, Colorimetric method for determintion of sugrs nd relted sustnces. Anl. Chem., 28: Hudson, S.M., Applictions of chitin nd chitosn s fier nd textile chemicls, pp: In A. Domrd, G.A.F. Roerts nd K.M. Vrum(ed.) Advnces in chitin Science. Jcques Andre Pulisher, Lyon, Frnce. 6. Knetsun, F. nd L.M. Cronell, Cell wll glucns of the yest nd mycelil from of Prcoccidioes rsiliensis. J. Bcteriol., 101: Knorr, D., Recovery nd utiliztion of chitin nd chitosn in food processing wste mngement. Food Technol., 45: Luef, E., T. Prey nd C.P. Kuicek, Biosorption of Zinc y fungl mycelil wstes. Appl. Microiol., 34: Mrier, J.R. nd M.R. Boulet, Direct determintion of citric cid in milk with n improved pyridine cetic nhydride method. J. Diry Sci., 41: Motz, M.S., Citric cid production from pretreting crude dte syrup y Aspergillus niger NRRL595. J. Appl. Sci. Res.,(in press), 2(2): Muzzrelli, R.A.A., F. Tnfn nd S. Ginfrnco, Chelting, Film forming nd Cogulting ility of the chitosn glucose complex from Aspegillus niger industril wstes. Biotechnol. Bioeng., 22: Guil, E., C. Roulph nd P. Cloirec, Infrred spectroscopic study of urnyl iosorption y fungl iomss nd mterils of iologicl origin. Environ. Sci. Technol., 29: Roerts, G.A.F., Chitin chemistry. Mcmilln Press, Bsingstoke, United Kingdom. 14. Rouks, T., Citric cid production from cro pod extrct y cell recycle of Aspergillus niger ATCC9142. Food Biotechnol., 12: Shhidi, F., J.K.V. Archchi nd J.J. You, Food pplictions of chitin nd chitosn. Trends in Food Sci. Technol., 10: Stgg. M.C. nd S.M. Fether, The D-glucn chrcteriztion of α-chitin ssocited from the wlls of Aspergillus niger. Biochem. Et. Biophys. Act., 320: Tsezos, M. nd B. Volesky, Biosorption. (Eccles, H. nd S. Hunt, Eds.), Ellis Horwood, Chichester, pp: , 18. White, A.S., F.R. Peter nd F. Inge,1979. Production nd isoltion of chitosn from Mucor rouxii. Appl. Environ. Microiol., 38: