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1 Bioeneficil Spectrum of Hlophyte Plnt Avicenni mrin s Secon Genertion of Bioethnol Prouction By Ael Ghny T. M., Neem I. Elhussieny n Ael Rhmn M.A. Shter ISSN (Print) ISSN (Online/Electronic) Inex Copernicus Interntionl Vlue IC Vlue of Journl 4.21 (Poln, Europe) (212) Glol Impct fctor of Journl:.587 (212) Scientific Journls Impct Fctor: J. Biol. Chem. Reserch Volume 31 (2) 214 Pges No Journl of Biologicl n Chemicl Reserch (An Interntionl Journl of Life Sciences n Chemistry) Pulishe y Society for Avncement of Sciences

2 J. Biol. Chem. Reserch. Vol. 31, No. 2: (214) An Interntionl Journl of Life Sciences n Chemistry Ms 31/2/44/214, All rights reserve ISSN Print ISSN Online/Electronic Dr. T. M. Ael Ghny jiolchemres@gmil.com info@jcr.in RESEARCH PAPER Receive: 26/5/214 Revise: 1/7/214 Accepte: 12/7/214 Bioeneficil Spectrum of Hlophyte Plnt Avicenni mrin s Secon Genertion of Bioethnol Prouction Ael Ghny T. M., *Neem I. Elhussieny n **Ael Rhmn M.A. Shter Botny n Microiology Deprtment, Fculty of Science, AL-Azhr University, Ciro, Egypt *Biology Deprtment, Fculty of Science, Jzn University, Jzn, KSA **Biology Deprtment, Fculty of Science n Technology, Thmr University, Thmr, Yemen ABSTRACT Bioethnol prouction from cellulosic mterils my offer solution to some of the recent environmentl, economic n energy prolems fcing in the worl, therefore the serch for renewle sources of fuel is very necessry for the progress of countries. The im of this stuy is to optimize the hyrolysis of lignocellulosic mterils of Avicenni mrin s the sl fermenttion meium for ioethnol prouction with using Scchromyces cerevisie. Hyrolysis of lignocellulosic of A. mrin with using sulphuric ci, nitric ci n their mixture giving reucing sugrs 3.9, 5.2 n 4.9 g/l respectively. Nitric ci ws the optimum ci for hyrolysis n their optimum concentrtion ws 5% which giving 9.9 g/l reucing sugr. Reucing sugrs yiels were 2.1 n 3.6 g/l t heting temperture 9 C n heting time 6 min. respectively. Ethnol prouction increse with incresing reucing sugr content, the optimum yiel otine ws g/l t 3 g/l sugr. Finlly, the mximum yiel of ioethnol otine ws 12.67, 12.52, n g/l uner optimum conitions of n inoculums size (2 ml), incution perio (72 h), ph (6) n temperture (3 C) respectively. Key wors: Avicenni mrin, Bioeneficil, Bioethnol Prouction n Secon Genertion. INTRUDUCTION Biofuels represent n lterntive to petroleum-se fuel; in prticulr, ioethnol is the most wiely use iofuel for trnsporttion (Blt, 21). Bio-ethnol is n importnt chemicl prouct with emerging potentil s io-fuel s sfe lternte to fossil fuels. Bio-ethnol cn e prouce y fermenttion of griculturl proucts (Rkin et l., 29). Pulishe y Society for Avncement of Science

3 Bioeneficil.Prouction Ghny et. l., 214 Ethnol cn e me of three min, unnt renewle feestock sources, scchrines, strch mterils n cellulosic mterils (Kumr et l., 25). First genertion iofuels re often criticise ue to their potentil verse impct on foo prices (Reuters, 28). To ese the conflict etween my tnk n your plte secon genertion iofuels like lingocellulosic ioethnol re promote (Bmelv, 28). The secongenertion iofuels (Biomss to Liqui) re me from orgnic mterils, such s strw, woo resiues, griculturl resiues, reclime woo, swust, n low-vlue timer. The secon ctegory is much more inexpensive n ecuse it is wste mteril, it is more ethicl to use for ioethnol prouction s compre to the first ctegory. Lignocellulose iomss, incluing woo wste, griculturl wste, househol wste, etc. represents renewle resource on erth which hs store solr energy in its chemicl ons (McKenry, 22). It hs gret potentil for ioethnol prouction, when compre to ethnol prouce from grin, tuers n sugr plnts, ecuse it is wiely ville chep feestock which oes not compete with humn foo proucts (Lyn et l., 1991). Zhng et l. (27) reporte tht the cellulosic mterils re renewle nturl iologicl resources which cn e use for the prouction of io-fuels. Mngroves re wooy trees n shrus tht grow in the intertil zones of tropicl n sutropicl regions (Duke, 1992). Mngrove forests hve een utilize for mny functions incluing woo prouction, firewoo n chrcol (Tomlinson, 1994). However, woo-relte ctivities or inustries se on lingo-cellulose s mnufcturing sustrte re very estructive n the rtes of mngrove renewl o not mtch this t ll (Kiro et l., 21). Plnt cell wlls re the most unnt renewle source of fermentle sugrs on erth (Sleem et l., 28). The mjor components of plnt cell wlls re cellulose, hemi cellulose n lignin, with cellulose eing the most unnt component (Hn et l., 23). Plnt iomss comprises on verge 23% lignin, 4% cellulose n 33% hemi cellulose y ry weight (S-Pereir et l., 23). In recent yers, much work hs een crrie out towrs efficient utiliztion of griculturl resiues n forest resiues to prouce io-fuels y microorgnism (Gnnsounou n Durit 21; Wng et l., 211). Accoring to stuies of Lee (1997) n Minhee et l. (211) the iologicl ctivities for converting plnt-erive lignocellulose to io-ethnol requires igestion to prouce hemicellulose n cellulose, epolymeriztion of crohyrte to lierte free monoscchries n fermenttion of mixe pentose n hexose to prouce ethnol. Biologicl n ci tretments of the lignocellulosic wste mterils improve the ioethnol prouction y Scchromyces sp. (Lei et l., 212). In mny scientific ppers, nitric ci n phosphoric ci t ifferent concentrtions n temperture were use to igest gsse of sugr cn (Sun n Cheng, 24). The yest S. cerevisie is the potentil yest for the ethnol prouction from lignocellulose ginst toxic chemicls present in the sustrte (Toms et l., 21). S. cerevisie is wiely use for the prouction of lcohol n ethnol fuels through fermenttion process tht converting sugr into ethnol. J. Biol. Chem. Reserch 87 Vol. 31, 2: (214)

4 Bioeneficil.Prouction Ghny et. l., 214 This microe cn prouce lot of ethnol n hs high tolernce to ethnol n other growth inhiitors compouns (Blt et l., 28; Jsmn et l., 213; Srvnkumr et l., 213; Zhenglin et l., 214). Also, Jeffries (26) stte tht, S. cerevisie ue to their cell structure, high growth rte uner stress conitions of environmentl n nutritionl fctors n greter resistnce to contmintion re vntgeous over other microorgnisms for commercil prouction of io-ethnol. The ims of this present stuy were to investigte the use of lignocellulosic mterils of A. mrin in prouction of ioethnol using S. cerevisie n to optimize the process conitions for enhnce ioethnol prouction. MATERIAL AND METHODS Sustrte Employe n ci hyrolysis Shoot system incluing stems n leves of Avicenni mrin ws collecte from the muy costl re of Jzn, KSA. Collecte shoots of A. mrin were wshe with istille wter n ir rie for 24 h. then oven rie t 6 C until constnt weight. Dry plnt prts were grin in n electricl griner. The rie sustrte ws then elignifie y treting with sulphuric n nitric cis t 3% n mixture of these cis, then the reucing sugr yiels were estimte. Effect of ifferent concentrtions, heting temperture n heting time of the optimum ci on reucing sugr yiels were estimte. NOH (.5 M) ws then use to neutrlize the ciic hemicelluloses hyrolyzte n lignin precipittes were remove y filtrtion through Whtmn filter pper.the pre-trete cellulosic resiue ws then wshe with istille wter to remove resiul ci. Microorgnism n their culturl conitions Scchromyces cerevisie (prouct of Frnce) ws colecte from mrket in KSA. One grm commercil lyophilize ker`s yest (S. cerevisie) ws issolve in1 ml 2% sterile sucrose solution uner septic conitions then su-culture on YEPD gr (1% Yest extrct, 2% Peptone, 2% Glucose, n 2% Agr (Atls, 24) pltes n incute t 25 C for 72 h. Single colony ws inoculte into 5 ml of YEPD meium in 25 ml Erlenmeyer flsk tht were incute in shking incutor t 25 C n 2 rpm for 24 h n then use in fermenttion processes. Growth ws mesure y Opticl Density (O.D.) t wvelength 6 nm. Optimiztion of fermenttion conitions (Osmn et l., 211) Different optimize conitions were pplie in the fermenttion processe. Ech of optimize conition in the experiment ws pplie in the nexet experiment s in the following: Reucing sugrs concentrtion: Different concentrtions of mngrove hyrolyste representing ifferent reucing sugrs concentrtions (5,1, 15, 2, 25, n 3g/l) were prepre s fermenttion meium in 25 ml Erlenmeyer conicl flsks ech contine 1 ml n inoculte with 1 ml yest culture, n incute in shking incutor t 25 C n 2 rpm for 72 h. J. Biol. Chem. Reserch 871 Vol. 31, 2: (214)

5 Bioeneficil.Prouction Ghny et. l., 214 Inoculum size: Fermenttion mei (ph 7) with 3 g/l reucing sugrs were prepre. Erlenmeyer flsks (25 ml) ech contine 1 ml mei were inoculte with ifferent volumes (.5, 1, 1.5, 2, n 2.5 ml) yest culture (initil concentrtion of yest culture is 7x1 7 cell ml -1 ), n incute in shking incutor t 3 C n 2 rpm for 48 h. Incution perio: Fermenttion mei contin 3 g/l reucing sugrs n ph 7 were prepre. Erlenmeyer flsks (25 ml) ech contine 1 ml mei were inoculte with 1.5 ml yest culture, n incute in shking incutor t 3 C n 2 rpm for ifferent perios (12, 24, 36, 48, 72, n 84 h.). Initil ph Vlue: Fermenttion mei contin 3 g/l reucing sugrs were prepre t ifferent ph vlues (3,4,5,6,7,8, n 9 ph). Erlenmeyer conicl flsks (25 ml) ech contine 1 ml mei were inoculte with 1.5 ml of yest culture, n incute in shking incutor t 3 C n 2 rpm for 72 h. Temperture: Fermenttion mei contin 3 g/l reucing sugrs n ph 6 were prepre. Erlenmeyer conicl flsks (25 ml) ech contine 1 ml were inoculte with 1.5 ml yest culture, n incute in shking incutor t 2 rpm n ifferent tempertures (2, 25, 3, 35, n 4 C) for 72 h. Estimtion of reucing sugrs: The Dinitro slicylic (DNS) metho of Miller (1959) ws use to estimte reucing sugrs. Quntittive estimtion of ethnol y potssium ichromte metho One ml of the fermente wsh ws tken in 5ml pyrex istilltion flsk contining 3 ml of istille wter. The istillte ws collecte in 5 ml flsk contining 25 ml of potssium ichromte solution ( g of K 2 Cr 2 O 7 issolve in 4 ml of istille wter with 325 ml of sulfuric ci n volume rise to 1 liter). Aout 2 ml of istillte ws collecte in ech smple n the flsks were kept in wter th mintine t 62.5 C for 2 minutes. The flsks were coole to room temperture n the volume rise to 5 ml. Five ml of this ws ilute with 5ml of istille wter for mesuring the opticl ensity t 6 nm using Uvikon Xs/6/ spectrophotometer. Astnr curve ws prepre uner similr set conitions y using stnr solution of ethnol contining 2 to 12% (v/v) ethnol in istille wter. Ethnol content of ech smple ws estimte n grph ws me. Sttisticl nlysis The t otine were sujecte to sttisticl nlysis ccoring to the proceure outline y Senecor n Cochrn (1981) n the mens were compre using Duncn`s multiple rnge test (Duncn, 1988). RESULTS AND DISCUSSION In the pst few yers, the environmentl issues, incresing of worl oil prices n shortge of fossil fuels re the min resons which re responsile for serch of lterntive energy sources, which re renewle cost effective n environmentlly sfe. J. Biol. Chem. Reserch 872 Vol. 31, 2: (214)

6 Bioeneficil.Prouction Ghny et. l., 214 The ethnol prouction from rw mterils of griculturl origin, with lower price, is the im of most of the stuies in the recent yers, performe in mny lortories. In our experiments, A. mrin ws use s rw mteril. The hyrolysis of lignocellulosic of A. mrin for mximum extrction of reucing sugrs using sulphuric ci, nitric ci n their mixture ws investigte (Fig.1), the otine reucing sugrs ws 3.9, 5.2 n 4.9 g/l respectively. In the current stuy, nitric ci ws significnt highly effective in elignifying of the iomss compre with sulphuric. Moiser et l. (25) reporte higher hyrolysis yiel from lignocellulose pretrete with ilute H 2 SO 4 compre to other cis. The ioconversion of lignocellulosic mterils to ioethnol prouces high yiel of glucose fter hyrolysis (McMilln, 1994, Ysuyuki et l., 211). An experiment ws crrie out y (Shfizeh, 1977) revele tht mximum mount of reucing sugrs (8-85%) ws otine with using H 2 SO 4 from hyrolysis of cellulose. A ifferent concentrtion of nitric ci ws lso crrie out for etection the optimum concentrtion of hyrolysis, where reucing sugr increse with incresing ci concentrtion (Fig. 2). Non significnt increse in reucing sugr content (9.9 n 9.7 g/l) t concentrtion 5 n 6% of nitric ci respectively. Accoring to Roriguez-Chong et l. (24), 6% HNO 3 ws the optiml concentrtion for hyrolysis of sugr cne gsse. Vrious fctors influence yiels of monomer sugrs from lignocellulose. Generlly, iologicl n ci tretments of the lignocellulosic wste mterils improve the ioethnol prouction y Scchromyces sp. (Srith et l., 211). Temperture, ph n mixing rte re the min fctors of enzymtic hyrolysis of lignocellulosic mteril (Therzeh n Krimi 27). Concerning the effect of heting t ifferent temperture, the otine results showe tht reucing sugrs yiels increse with incresing temperture up to 9 C (Fig. 3) with the pplie optimum concentrtion 5% of nitric ci compre to other nitrogen sources. This is ue to the fct tht high temperture increses hemicelluloses egrtion n lignin trnsformtion, thus incresing the potentil of cellulose hyrolysis (Li et l., 29). Accoring to Roriguez-Chong et l. (24), 122 C ws the optiml temperture for hyrolysis of sugr cne gsse with using 6% HNO 3. At ifferent heting time the reucing sugrs yiels re not significntly ifferent especilly t 6, 9 n 12 min where it ws 3.6, 3., n 29.8 g/l respectively (Fig. 4). On the other hn, reucing sugrs yiels (22.1 g/l) ecrese significntly when the iomss hyrolyze t heting for 3 min. with the pplie optimum conition of hyrolysis. It cn e oserve tht ioethnol yiels were significntly increse with incresing the content of reucing sugrs n they spen 3.97, 6.1, 8.84 n g/l with sugr content 15, 2, 25 n 3g/L. respectively (Fig. 5). Thus, the mount of reucing sugrs hs significnt role in the ioethnol prouction. Bioethnol prouction from the lignocellulosic wste mterils epens on their tretment for removl of the hemicelluloses n lignin from the iomss for the prouction of monosugrs such s glucose. This is chllenge to the current reserchers on the renewle fuel prouction (Meinit et l., 212). J. Biol. Chem. Reserch 873 Vol. 31, 2: (214)

7 Bioeneficil.Prouction Ghny et. l., 214 Reucing Sugrs (g/l) c 1 Control Sulphuric ci (3%) Nitric ci (3%) Sulphuric & Nitric cis Fig. 1 Effect of ifferent of cis n their mixture on reucing sugrs yeils. Mens followe y the sme letter re not significntly ifferent. 12 Reucing Sugrs (g/l) c 2 e Nitric ci concentrtion (%) Fig. 2 Effect of ifferent concentrtion of nitric ci on reucing sugrs yiels Mens followe y the sme letter re not significntly ifferent. J. Biol. Chem. Reserch 874 Vol. 31, 2: (214)

8 Bioeneficil.Prouction Ghny et. l., Reucing Sugrs (g/l) 15 1 c c c Temperture 6 ( C) Fig. 3 Effect of ifferent temperture on reucing sugrs yiels. Mens followe y the sme letter re not significntly ifferent Reucing Sugrs (g/l) Heting 6 time (Min.) 9 12 Fig. 4 Effect of ifferent heting time on reucing sugrs yiels. Mens followe y the sme letter re not significntly ifferent. J. Biol. Chem. Reserch 875 Vol. 31, 2: (214)

9 Bioeneficil.Prouction Ghny et. l., 214 On the other hn, the ethnol content increse grully s the mount of inoculums use increses to stilize eyon 2. ml. Thus, the ethnol content otine reche vlue of g/l using inoculums greter thn or equl to 2. ml. The wekest ethnol content i.e., 9.4 g/l ws otine with n inoculums level of.5 ml (Fig. 6). Our fining is n greement with those otine y Acourene n Ammouche (21) with using Bker s yest. The ethnol content evolve grully uring the fermenttion, reching to 4.75 g/l in 12 h n then 8.6 g/l in 24 h to stilize t g/l eyon 72 h (Fig. 7). The optimum fermenttion perio for mximum ethnol prouction is 72 h. Our fining is n greement with other results (Kongkittikjorn et l., 27; Acourene n Ammouche, 21). Recently, Zhenglin et l., (214) reporte tht uner ll process conitions, the ethnol concentrtion ecme constnt fter 72 h fermenttion. Mrine strin of S. cerevisie showe the mximum ioethnol prouction of 69.58% uner the optiml conitions of temperture (3 C) t 89 h of incution (Srvnkumr et l., 213). Bioethnol prouction y S. cerevisie is very sensitive to initil ph of fermenttion meium. The mintennce of fvourle ph is very essentil for the successful fermenttion of ioethnol. The otine result shows tht the mximum ioethnol yiel (13.68 g/l) ws chieve when the initil ph of fermenttion meium ws kept t 6 (Fig.8). Okeh et l. (214) foun tht initil ph 7 gve the mximum iogs yiel 357 ml/y. The present stuy ttine significnt mximum ioethnol prouction of g/l uner the optimize conitions of temperture t 3 C (Fig. 9), the ethnol yiel ecrese significntly with the increse of temperture.this might e cuse y the ecrese in the yest ctivity. Ethnol yiels with using S. cerevisie uring fermenttion perio showe higher vlues t 3 C compre to tht t 33 C (Suliemn et l., 213). Although, Zhenglin et l., (214) stte tht the fermenttion temperture h no significnt effect on the ethnol yiel y S. cerevisie n finl ethnol concentrtion, coul e set t 35 C to chieve the mximum fermenttion rte f 1 8 Ethnol (g/l) 6 e 4 c Reucing Sugrs 2 (g/l) 25 3 Fig.5 Effect of reucing sugrs content on oiethnol prouction. Mens followe y the sme letter re not significntly ifferent. J. Biol. Chem. Reserch 876 Vol. 31, 2: (214)

10 Bioeneficil.Prouction Ghny et. l., c 1 Ethnol (g/l) Inoculum size (ml) Fig.6 Effect of inoculums size on ioethnol prouction. Mens followe y the sme letter re not significntly ifferent c e e 8 Ethnol (g/l) Incution perio (h) Fig.7 Effect of incution perio on ioethnol prouction. Mens followe y the sme letter re not significntly ifferent. J. Biol. Chem. Reserch 877 Vol. 31, 2: (214)

11 Bioeneficil.Prouction Ghny et. l., e f e Ethnol (g/l) Intil 6 ph vlues c Fig.8 Effect of ph on ioethnol prouction. Mens followe y the sme letter re not significntly ifferent e 12 c Ethnol (g/l) Temperture ( C) Fig.9 Effect of temperture on ioethnol prouction. Mens followe y the sme letter re not significntly ifferent. J. Biol. Chem. Reserch 878 Vol. 31, 2: (214)

12 Bioeneficil.Prouction Ghny et. l., 214 CONCLUSION The prouction of io-ethnol from A. mrin seems very much possile n therefore it hs potentil to ecome future feestock for ioethnol prouction. Using the optiml conitions otine (uner hyrolysis n fermenttion), in continuous process, sustntilly increse the prouctivity of io-ethnol. ACKNOWLEDGEMENTS Thnks to Biology Deprtment, fculty of science, Jzn University, Jzn, KSA. REFERENCES Acourene S. n Ammouche A. 21. Optimiztion of Culture Meium for Bker s Yest, Ethnol, Citric Aci n Α-mylse Prouction from Dtes Syrup. Reserch Journl of Agriculture n Biologicl Sciences, 6(6): Atls, R.M. 24. Hn Book of Microiologicl Mei. 3r e. CRC Press, Boc Rton, Lonon, New York, Wshington, D.C. Blt M., Blt H. n Oz, C. 28. Progress in ioethnol processing, J. Prog. Ener. An Comust. Sci., 34, Bmelv, 28. Wrum ruchen wir Biokrftstoffe? [Why we nee iofuels?]. Bunesministerium für Ernährung,Lnwirtschft un Verrucherschutz, Cputi A., Ue M. n Brown T Spectrophotometric etermintion of ethnol in wine. Am J Enol Vitic 19: Duke, N.C Mngrove floristics n iogeogrphy. In: A.I. Roertson n D.M.Alongi (es) Tropicl Mngrove Ecosystems. Americn Geophysicl Union, Wshington DC., Duncn, D.B., Multiple rnge n multiple F tests. Biometrics, 11: Gnnsounou, E. n Durit A. 21. Techno-economic nlysis of lignocellulosic ethnol: review. Bioresour Technol, 11 (13): Hn, S.O., Yukw H., Inui M. n Doi R.H. 23. Regultion of expression of cellulosoml cellulse n hemicellulse genes in Clostriium cellulovorns. J. Bcteriol., 185: Jsmn, Irfn D. P., Chusnul H. n n Donny W Ethnol fermenttion on mixe sugrs using mixe culture of two yest strins Inonesin, Journl of Biotechnology, 18, (2): Jeffries, T.W. 26. Engineering yests for xylose metolism. Current Opinion in Biotechnology 17(3): Kiro, J.G., Dhouh-Gues F., Bosire J., Koem N. 21. Restortion n mngement of mngrove systems - lesson for n from the Est Africn region. South Africn J. Bot. 67: J. Biol. Chem. Reserch 879 Vol. 31, 2: (214)

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