Nội dung

T(fp chi Cdng nghe Stnh hpc 10(1): 169-178, 2012 DAC D I E M C U A C H U N G V I K H U A N N D 1 5 3 S I N H F O L Y H V D R O X Y B U T Y R A T E ( P H B ) P H A N L ^ P TU" D A T RU"NG N G A P M A N H U V f N G I A O T H U Y , T I N H N A M D I N H Doin Van Thucyc, Nguyen Thi Binh Trudng Dgi hpc Suphgm Ha Npi T6M TAT Polyhydroxyalkanoate (PHA) la mgl dang polymer sinh hgc tich luy trong te bao ciia nhiSu loai vi smh vat voi vai tro Id nguon dy trir carbon va nSng lugng, thu6ng xiiy la khi moi Inrdng s6ng du thira carbon vd thicu mgt vai chat dinh dudng thiet yeu. vi dy nhu nitrogen, oxygen, phosphorus, sulfiir, hoac magnesium, Trong nghien ciiu ndy. 12 chung \ i khuan co kha ndng lich lily PHA da dugc phan lap tir dit rung ngap man huyen Giao Thiiy, tinh Nam Dinh, Voi khii nang sinh Inrong plidt tricn manh vd tich luy nhifiu hai PHA trong le bdo, chung vi khuan NDI53 da dugc Iga chgn de lien hdnh cdc nghien ciiu liep theo. Chung NDI53 co dgngtrgc khuan. Gram duong, di dgng, co khd ndng hinh Ihanh ngi bdo tii, thugc nhom vi sinh vat ua muoi nh?, ira pH trung tinh, va ua im trung binh Chiing vi khudn ND 153 co kha nang dong hoa nhieu nguon nitrogen va carbon khac nhau. Mau PHA tu chiing vi khuan NDI S3 da dugc tdch chiet va tinh sach Kel qud phan lich cau triic mlu polymer tinh s^ch bang pho cgng huong tir h^i nhan cho thdy chimg ND153 tich luy pol><3hydroxybulyrate) (PHB) - dang polymer dien hinh dugc tich luy bdi rat nhieu cac vi sinh vdi trong tu nhi^n. Chiing vi khuan ND153 co khd nang tich liJyhdm lugng PHB dgt 82% khoi lugng te bao kho sau 30h nuoi cay trong dieu kien han che MgSOj. Tirkhda: Baci"-s. PHA. phan huy sinh hpc, PHB. polymer sinh hgc DAT VAN DE Nhu cau sii dung polymer tren the gioi noi chung va b Viet Nam noi rieng ngay cang gia tang, tuy nhien nguon diu mo - nguyen lieu chinh de san xuat cac loai polymer nay dang co nguy co can kiet va gia thanh ngay cang tang cao. Them vao do, nhimg plastic co nguon goc tu diu mo khong c6 kha nang tu phan hiiy nen chiing la mpt trong nhiing nguyen nhan gay 6 nhiem moi truong. Giai phap dang dupe quan tam hien nay la phat irien va sii dung cac plastic co kha nang tu phan hiiy (thuong dugrc ggi la polymer sinh hpc) (Salehizadeh, Van Loosdrecht, 2004). Trong so cac polymer sinh hpc dang dupe nghien cuu sii dung flii polyhydroxyalkanoate (PHA) duoc quan tam nghien ciiii nhieu nhat. PHA Ik nhom polymer sinh hoc co kha nang phan hiiy tri?t de va CO nhieu tinh chit tuong tu cac plastic tong hpp. Chiing la polymer cua cac don phan hydroxyalkanoate, duac tich luy trong xk bao nhu la nguon du trii carbon va nang lupng boi rat nhieu vi sinh v£it, thuong diln ra trong diSu ki§n thigu mpt vai chat dinh duong nhu oxygen, nitrogen, phosphorus, sulfiir, hoac magnesium, va du thiia carbon (Lee 1996; Sudesh et ai. 2000). Nhi6u chung vi sinh vat c6 kha nang tich luy ham lupng PHA len toi 90% khoi lupng kho cua te bao (Thuoc, 2009). K I tu khi poly(3-hydroxybutyrate) (PHB)^ dupe tim ra vao nam 1926 (Lemoigne 1926), cho dSn nay da CO hon 150 loai PHA dupe phan lap tir hon 300 vi sinh vat (Valappil et ai. 2007). PHA c6 thl la polymer cua mpt loai monomer hoac ciia hai ho^c nhieu loai monomer. PHA dugc chia thanh hai nhom phu thuoc vao s6 lupng cac nguyen tii carbon co trong cac don phan monomer: nhiing PHA d^ng mach ngin (scl-PHAs) c6 khoang 3-5 nguyen tir carbon trong mgt monomer, nhiing PHA d?mg m^ch tnmg binh (mcl-PHAs) co khoang 6-16 nguyen tu carbon trong mpt monomer PHA m?ch ngan co tinh chit gi6ng voi cae loai plastic thong thudng nhu gion va ciing, trong khi do cac PHA mach trung binh CO tinh mim deo, nhiet dp soi va nhiet dp ket tinh thap (Philip et ai, 2007). Voi lich sir nghien cihi va phat triSn gan 100 nam, rit nhieu cac san pham tir PHA da dupe thuong mat hoa, hien nay co hang tram cong ty thuong mai Ion nho tren khap the gioi tham gia vao luih vi;c nay (Chen, 2010). iTng dung chinh ciia PHA la thay thl cac polymer c6 nguOn gdc tii diu mo dang dugc sir dyng de san xuit bao bi. Ngoai ra PHA la nguyen lieu dk san xuit cac loai d6 dung mpt lan nhu ta lot cho tre em, chai Ip dung my phim, cac san pham co dp ben cao nhu vo 169 Dodn Van Thupc & Nguyen Thj Binh may vi tinh, vo d^mg lop 6 to dy trir.. I);ic bigl hon, do khong gay phan img th:ii lo(ii khi dirpc cay gh6p vao ca the nen polyincr smh hpc c6 nhi>ng irng dyng quan trpng trong nginh y lc. C;lc sdn pham nhu chi khau phau ihuiit, dia xuong, ong gliip m,nch, van tim...Id nhihig vi tii,i dicii hinh (Philip el ai, 2007), Nhiing nghien ciWi vc PHA cung dii dupe tien hdnh (V Viv'l Nam irong ihoi gian gan day, hutVng nghien cim chii yen l^p trung vio PHB, Lc Ly Thiiy Tram \ a dong tic gia (2006) d;i phan l^ip dupe chimg Melhylobacteiium sp, en kha nfing lich luy PUB vtVi him lupng PHB tich luy c^c dgi trong tc bdo li 31,4% sau 4 ngay nuoi cay. Biii Thj Thanh Mai vd dong tdc gui (2010) da phan Igp dupe chimg vi khuan V23Xl.l lir xoii cdl c6 khi nang sinh tong hpp PHB, sa bp djnh logi da xic d)nh dupe chimg vi khuan niy thupc loai Bacillus cereus. vi him lugng PHB c\rc dgi do chung nay tich luy li 58% khoi lupng lc bio kho, Trong nghien cim nay chiing loi da tien hinh phan lap, tuyen chpn, v i nghien ciiu chiing vi khuan CO kha nang tich luy PHA lir dai rimg nggp m$n 6 Vict Nam. Day li nhuTig nghien ciiu tien de lam nen tang cho vi^c phat tnen san xuat \ a img dyng PHA 6 Viet Nam. VAT LIEU VA PHUONG PHAP Doi tuwng 20 mau dat dupe thu thap tir rimg ngap man huyen Giao Thuy, tinh Nam Dinh Cac mau dat d\mg trong cac hii kin va duac giii trong da lanh sau do chuyen ve phong thi nghi?m de tien hanh phan Igp Cac loai moi irucmg s u dung Moi truong phan Igp vi khuan (g/1): NaCl, 30; MgS04.7H20, 2,5; KBr, 0,06; KH2PO4, 2; Peptone, 2; Cao nam men, 2; Glucose, 20. Moi truong thu sinh kh6i (ki hieu la HM) (g/l): NaCl. 30; MgS04.7H20, 0,25; CaCl2.2H20, 0,09; KCl, 0,5; KBr, 0,06; Peptone, 5; Cao nam men, 10; Glucose, 1 M61 truong san xuat PHA (ky hi?u li MA) (g/l): NaCl, 30; MgS04.7H20, 1; CaCl2.2H20, 0,36; KCl, 2: KBr, 0,24; KHjPO^, I; Cao nam men, 2; NH4CI, 2; Glucose, 20. Cac thinh phan ciia moi Uuang dupe hoa tan trong 1000ml nude cat, pH ciia moi truong dupe dieu chinh den 7,0 bing dung djch 5M NaOH v i 5M HCl, moi truimg dac dupe b6 sung 2% agar. Phan lap, giir giong vi khuan Phan lap vi khuan theo cae buoc ca ban tham 170 khdo tir tii li^u ciia NguySn Thanh Dgt va Mai Thj I ling (2000). Giii- giong vi khuan bing cac phuang phdp: giO' trong 4"C vd cay huyen djnh ky 1,5 Ihing/ian, dong kho sinh khoi ic bio va giii 6 -SOT; bio quin sinh khoi te bio hong 15% glycerol is -80°C. I'uycn chpn chung vi khuSn cd tich luy PHA Cay chuyen vi khuan tir hpp petri giii giong sang hOp petri chira moi t r u ^ g HM d$c co b6 sung 0.5pg/ml dung djch thuoc nhupm Nile blue A lan trong DMSO (Dimcthylsulfoxide). Sau 24-48h khi vi khuan da mpc 161, soi c i c hpp petri niy duoi tia UV bu6c s6ng 254 nm, giii lai cdc chung vi khuan phat huynh quang mau vdng cam s i n ^ (Spiekermann el ai. 1999), De khang djnh thSm ket qua tuyen chgn, tien hinh lim tieu ban cit lirp v i quan sat cic hat PHA tich luy trong te bio chiing tuyen chpn duoi kinh hien vi di?n tit (HVDT) truyen qua (TEM), Mo ta hlnh thdi khu^n lac, hinh thai te hao chung \i khuan tuyen ch9n c i y vi khuan theo hinh dich dac tren moi truong HM dgc, ix a 35''C trong 24h de vi khuan mpc thinh khuan lac. Quan sat, lua chpn cac khuan Igc mgc neng re de mo t i hinh dang, m i u sac khuan Igc. Ldm tieu ban gipt ep, tieu bin nhMom don, tieu ban nhupm Gram, quan sal va mo ta dac diem hinh thai te bao ciia chimg vi khuan tuyen chpn tren kinh hien vi quang hpc. Hinh dang v i kich thuoc te bao cua chimg VI khuan tuyen c h ^ dupe \ a c djnh nhcr inh chyp tir kinh hien vi d i ^ quel (SEM). Tdch chiet, tinh sach, xac djnh cdng thirc phan tur ciia loai PHA tich luy trong ti- bao chung vi khuan tuySn chpn Nuoi cay chiing vi khuan tuyen chpn tren moi trucmg MA long a 35*^, toe dp lac 180 vong/phut. Sau 24-30h thu toin bg djch nuoi ciy, li tam 8000 vong/phiit Uong 10 phiit, giii Igi sinh Uioi te bao, rua sinh khoi bang nuac cat roi dong kho sinh khoi bdng mdy dong kho Plexy Dry (My). Tich chiet va tinh s^ch PHA bang dung dich chloroform trong h? thong Soxhiel (Verlinden et ai. 2007). Mau PHA tinh sach dupe giti tai Phong Phan tich ciu tnic, Vien Hoa hpc, Vi?n Khoa hpc va Cong nghe Viet Nam de xac dinh cong thiic phan tii bang may cpng huong tir hat nhan (Bruker ARX500, Bruker, Sikerstrifen, Germany). Nghien cihi anh hirung cua cac y4u th dinh dircmg v i dieu kif n moi t r u v n g den su- sinh t r u o n g cua chung vi khuan tuyen chgn Cay chimg vi khuan myen chpn tren moi truong Tgp chi Cong nghe Sinh hpc 10( 1): 169-178, 2012 HM dac 6 35''C trong 24h, ciy chuySn sang moi trucmg HM long, nuoi lie vol toe dp 180 v6ng/phut Cf 35°C trong 13h. Giong vi khuan dupe b6 sung v6i ty If 5% (v/v) vio cac moi truing thi nghi?m de thir inh huong cua nhi?t dp, pH, n6ng dp NaCl, ngufin carbon va nguon nitrogen. Moi thi nghigm dupe l$p lai 3 lan va lay ket qua trung binh, xu 1;^ s6 li?u bdng phan mem Microsoft Office Excel. D$ng thii sinh trurong ciia chiing vi khuSn tuy^n ch9n khi nuoi cay tren moi trudng c6 nguon carbon khic nhau khuan, ky hipu tir NDI den ND425. Tuyen chpn dupe 12 chiing vi khuin cd tich IGy PHA, gdm cdc chimg: ND97, ND153, ND156, ND199, ND201, ND203, ND 210, ND218, ND240, ND325, ND355, ND389. Trong 12 chiing vi khuan tren, chiing NDI53 cd khi n&ig sinh trudng, phat trien rat tot, anh TEM cho thay chiing ND153 cd nhieu hat PHA tich luy trong te bio (Hinh 1) do dd chiing niy dupe l\ra chpn dS tien hinh cic nghien cihi tiep theo. Nuoi cay chiing vi khuan tuyen chpn trong binh lam giac chiia moi trudng HM long c6 nong dp 4% NaCl, pH 7,0 vol 2 nguon carbon kliic nhau li glucose vi ri ducmg. D?it cdc binh tam giic trong miy lie vol t6c dp 180 v6ng/phiit a 3 5 ^ , thu mau o cac thoi diam Oh, I2h, 18h, 24h, 30h. 36h. Xac djnh kh6i lupng xk bao kho (CDW) va ham lupng PHB (wt%) tich luy trong te bio theo phucmg phip ciia (Law and Slepecky, 1961). PHB tinh sach dupe diing de xay dung do thi chuan. Nudi cay hai pha, xic djnh sir anh hirdng cua m$t s^ nguon dinh dir&ng den ham lirung PHB tich liiy trong te bao chung vi khuan tuyen chpn Pha sinh truong (pha 1): nuoi ciy chiing vi khuin mySn chpn trong binh tam giic 250 ml co chiia 50 ml moi trudng HM cd nguon carbon la ri dudng d 35°C, tdc dp lie 180 vdng/phiit. Sau 20h nuoi cay, ly tam va chuyen sinh khdi te bio sang pha 2 (pha tich luy). Pha tich liiy PHB: chiing vi khuin dupe tiSp tuc nudi trong binh tam giac 250 ml cd chiia 50 ml moi trudng MA nhimg him lugng ciia mgt sd chit dinh dudng trong mdi trudng lan lupt dupe giam di (thi nghi?m 1 - ndng dp NH4CI giam xudng 0,2 g/1; thi nghiem 2 - ndng dp KH2PO4 giam xudng 0,1 g^; thi nghiem 3 - ndng dp MgS04.7H20 giam xudng 0,1 g/l), ham lupng cac chit dinh duong khac dugc giir nguyfin. Sau lOh nuoi cay, bd sung 2ml dung dich glucose dam dac (50%) vio mdi binh rSi tiep tuc nudi d 35°C, tdc do lie 180 vdng/phiit. Thu djch nudi ciy va xac dinh gia trj CDW, ham lupng PHB tich luy trong tk bao d cac thcri diem 15h va 30 h nudi cay. KET QUA, THAO LUAN PhSn lap, tuyIn chpn vi khuan sinh PHA Tir 20 miu dit thu d rimg ngip man huy?n Giao Thuy, tinh Nam Djnh, phan lap dugc 425 chiing vi Hinh 1. Cdc h?t PHA tich luy trong \k b^o chiing NDI53 dudi kinh HVDT truyen qua ( x 20000 lan) Hinh thii khudn lac, hinh thii te bao chung, dinh loai chung NDI 53 Khuin lac ciia chung ND153 cd dang khdng trdn d^u, mep gpn sdng, hai ldi d tam, khuan lac cd mau kem, bi m$t tron bdng, hm nhiy udt (Hinh 2A). Anh SEM cho thi^y chung ND153 la true khuin ngin, kich thudc te bio dao dpng 0,9 - l,2jim x 2,03,5nm, cd tiem mao, dang don tr\rc khuin va song true khuin li phd bidn (Hinh 2B, 2C), cd kha nang hinh thanh bio tii, giir miu tim khi nhugm Gram chimg td chiing thugc nhom vi khuin Gram duong (Hinh 2D). Cong thirc phSn tu cua loai PHA tich lOy trong t^ bio chiing ND153 Mlu PHA tach chiet tir chiing ND153 cd mau tringfrong,mSm vi dai (Huih 3A). Kfit qui jihan tich miu polymer tinh sach cua chung ND153 bing may cpng hudng tir hat nhan dugc trinh biy d hinh 3.B. So sanh kit qua niy vdi nghien ciiu cua Thirumala vi ddng tic gia (2010) dua din kit luin: dinh d vj tri 5,27 ppm tuong img vdi nhdm methyne (-CH-), hai dinh kep d vi tri 2,5 vi 2,6 ppm tuong iing vdi nhdm methylene (-CH2-), mot dmh khic d vj tti 1,3 ppm Doin Vin Thugc & Nguyen Thj Binh tuong img vdi nhdm methyl (-CHj-). Nhu v$y cic monomer cau igo nen logi polymer tich luy trong 1^ bio ciia chung NDI 53 li 3-hydroxybutyric acid, va lo^i polymer niy cd ten li poly 3-hydroxybutyratc (PHB). Diy li d?mg PHA diin hinh dugc tich luy hong le bdo cua rit nhieu chimg vi khuan, vi li mgt trong 2 dgng polymer dang dugc sin xuat vi iing dyng tren quy md cdng nghifp. Hinh 2. (A) Khuin Igc vl khuin ND153 trfin mfli Irudng HM d^c. (B) vd (C) T6 bdo chung ND 153 du*i kinh HVDT qu^ (30000 \hn) (D) T6 bdo chung ND153 sau nhu$m Gram dudi kinh HVQH ( x 1000 lin) Hinh 3. (A) Mlu PHA tSch chi^ tir chung NDi 53 (B) PhA 'H-NRM cua miu PHA tmh s^ch t&ch chiAt tu chung NDI53. Hinh 4. Sinh trudng ciia ctiiing ND153 dr cic nhi^t 0^ khdc nhau. Anh hudng cua cic yiu th dinh dirS^g vi diiu ki^n mdi trudng din stf sinh trudng, phit triin cua Chung ND153 Anh hie&ng cda nhift dp Nhiet dp li tic nhin vit ly cd anh hudng sau sic den tdc dO ciia cic phan iing hda sinh trcaig ti bao vi sinh vgt, vi thi chiing tdi ti&i hanh nghien ciru inh hudng ciia nhi$t dg den khi ning sinh tnidng, [diit frien cua chung NDI S3 nhim tim ra khoang nhiet d$ toi uu de ip dyng cho nhung nghidn ciiu tiip flieo. Kit qua cho thiy chung ND153 cd khi ning sinh Tgp chi Congnghe Sinh hpc 10(1): 169-178, 2012 trudng, phit trien fren khoang nhiet dp rpng (Hinh NaCI cao c6 the thay doi phu thupc vao nhipt dd vi 4), d 4"C sau 3 ngay nudi ciy da thiy vi khuin mpc cic chat dinh dudng frong moi frudng do vay ndng len, tuy_ rang mpc kem. Trong khoing nhi^l dp td dp 4% NaCl dugc lya chpn di pha chi moi frudng 25"C din AS°C chiing NBI53 diu mpc kha tdt va nudi ciy chiing NDI53frongnhimg nghian ciru tiip mpc tdt nhat d 33-37''C. Can cir vao khoing nhi^l dg tlieo vdi 2 iy do: ndng dg muoi niy cd thi lic chi su toi uu nay c6 the ket luin chung ND153 thupc nhdm sinh trudng cua mpt so vi smh vat lap nhilm nhung vi sinh vat ua am trung binh (Nguyen Thanh Dgt, khdng inh hudng nhiiu den sy sinh trudng ciia Mai Thi Hing 2000). Nhipt dp 35''C dugc l\ra chpn chimg ND153, nudi cay vi khuan d nong dp mudi de tien hanh Cac nghien cuu tiep theo. cao se tao dieu ki?n thu^n Igi cho qui trinh tach chiit sau nay (khi hda sinh khdi te bio frong nude, nude Anh hirdng cua ndng do NaCl se khuech tan vio te bao do chenh l^ch ip suit thim Chung ND153 dugc phan lip td dat rimg nggp thau, khi do cic ti bio se dl bi phi vd de giii phdng man-mdi trudng song cd ndng dp NaCl cao. Him cic hgt PHB) lugng NaClfrongmdi trudng nhieu khi nang se anh hudng Idn den su sinli frudng va phdt friin ciia vi Anh hudng cua pH khuin, do vgy chiing tdi da tiin hinh thi nghiem pH cua moi frudng anh hudng den su phan li nghien ciiu su inh hudng cua ndng dp muoi den ciia cic ion, den cau fruc vi hoat tinh cua protein chiing ND153, kel qui dupe the hipn d hinh 5. nen cd j' nghia quyet dinh din sy sinh frudng vi Chiing NDI53 cd kha nang sinh frudng va phit frien sinh ting hgp cic chit ciia vi khuin. Xuit phit tir ly trong khoang ndng dp NaCl kha rpng (0% den 12%). do dd, chiing tdi tien hanh thi nghiem nghien cim Chiing sinh trudng tot d ndng dp 0% - 7,5% va tdt su anh hudng cua pH den chiing ND153. Hinh 6 nhit d ndng do 1,5% - 3% NaCl. Vdi nong dp NaCl cho thiy chiing ND153 sinh frudng, phitfrienfren toi uu nhu fren, theo sir phin chia cac nhdm vi sinh tit ci cic pH ma nghien ciiu tien hinh (4,0 den vat ua man ciia Gilmour (1990) thi cd the xep chimg 10,0) tuy nhien su sinh trudng dat tdt nhat d pH nay vao nhdm vi sinh vat ua man nhe (Slight 6,5-7,0 chiing td chung niy thugc nhom vi sinh vat halophilic microorganisms), day li nhdm vi sinh vat trung tinh. Can cii vio kit qua thuc nghiem, chiing dien hinh cua cic dai duang. Tren thuc te, nhu cau tdi lua chpn pH 7,0 de tien hanh cac nghien ciiu sir dung NaCl va khi nang chju dung ham lupng tiep theo 3 4.5 6 7.5 Nong do NaCI (%, w/v) Hinh 5. Anh hudng cua nong dp NaCI den sy sinh trudng ciia chung ND153 10.5 12 Doin van Thir(?c & Nguyen Thj Binh 3 4 5 6 7 pH Hinh 6. Anh hudng cud pH dfin sy sinh tru'dng ciia chung NDI 53 ,<^- *-%«>J.^^^-°\»*°' 0»^ Ngudn nitrogen (2%, w/v) Hinh 7. Anh hudng ciia cdc ngudn nitrogen khdc nhau d6n si/stnh irudng cuachCing NDI53. Anh hudng cua nguon nitrogen Trong mdi trudng phan Igp vi khuan vi mdi trudng thu sinh khdi, cao nam men dugc sii dyng li ngudn nitrogen. Tuy nhien theo Quillaguamin vi ddng tic gii (2008) thi sy cd mat ciia mpt ngudn ca chat tdng hgp nhu cao nam men giup vi sinh vgt sinh trudng tdt nhung lai inh hudng den khi ning tich liiy PHA vi da sd vi sinh vgt chi tich luy dugc him lugng PHA cao Idiifrongmdi trudng du thira carbon 174 vi thiiu mgt vii nguyen td dinh dudng thiet yeu, frong do cd nifrogen. Hon nua, cao nam men ti mfit ngudn CO chat dat tien nen khi su dyng se lim tang gii thinh ciia sin phim, do vgy nghien ciiu niy dugc tien hinh nham tim kiem mpt ngudn nitrogen khic, re tien cd the thay thi cao nam men. Tim nguwi nifrogen khic nhau da dugc thu nghidm, ket qui (Hlnh 7) cho thiy chung ND153 co thi sd dyng tit ci cic nguon nifrogen niy, tuy nhien cao nim men van li ngu(m nifrogen thich hgp nhit. Cic nguon Tgp chi Cdng nghe Sinh hpc 10(1): 169-178, 2012 nitrogen cd gdc amonium nhu NH4CI, NH4NO3,, NH4HCO3 anh hudng tdt hon din sy sinh trudng, phat frien cua chimg ND153 so vdi cac ngudn nifrogen cp gdc nifrate nhu KNO3 hay NaNOs, nhu vay CO the so bd ket luan chung ND153 ddng hoa g6c amonium tdt hen ddng hoa gdc nitrate. NH4C! dugc chon li ngudn nifrogen cho nghien ciru tiip theo (nghien ciiu inh hudng cua ngudn carbon) chiing niy. Nhicu tighien ciru cho thay gii thinh ciia ngudn carbon quyit dmh ding ki tdi gii thinh cua sin phim VUA (Thuoc, 2009) do dd vi?c tim ra ngudn carbon vira cd anh hudng tdt tdi su sinh frudng, phit trien cua chung vi khuan tuyin chgn, vira r^ tien la vi?c lim cin thiit. Ket qui nudi cay chiing ND153_tren cic nguon carbon khic nhau (hinh 8) cho thay chiing niy sinhfrudngvi phat friin tot nhat fren 3 nguon carbon la glucose, saccharose, ri dudng. Trong 3 ngudn carbon fren, ri dudng li Anh hirdng cda nguon carbon ngudn carbon rat dang quan tam, vi no vira inh Da sd vi khuan chi tich luy PHA khi trong mdi hudng tdt tdi sy sinh trudng, phit triin ciia chung, frudng nudi ciy du thira carbon, dieu dd chi ra vai vira cd gii thinh thap nen cd tiem nang iing dung frd rat quan frgng cua ngudn carbon ddi vdi cic lam nguyen li^u cho sin suit d quy md Idn. Nguon carbon (2%, w/v) Hinh 8. Anh hudng ciia mpl so ngufin carbon khac nhau den si/ smh trudng ciia chiing NDI 53 lam cin thiit. Kit qui nghien cuu dong thii smh trudng va khi nang tich luy PHB ciia chiing ND153 fren 2 ngudn carbon khac nhau duoc md ta frong Tren bi mat ciia cic h^t PHA ben canh hinh 10. Khdi lugng ti bio kho va him_ lugng PHB Polyester synthase cdn cd Depolymerase (Hinh 9) tich IQyfrongti bio dat gii fri cao nhat sau 24 va (Rehm, 2003). Polyester synthase thucmg hoat ddng 30h nudi cay. Ri duimg li ngudn co chit thuan lpi manh frong pha sinh trudng vi diu pha can bang, chg su sinh frudng va phit frien ciia chiing nhung frong khi do Depolymerase bat diu hoat ddng d glucose li ngudn carbon inh hudng tdt hon den sy CUOI pha cSn bang va hoat dpng manh d diu pha tich luy PHB. Khdi lugng ti bio khd cue dai la 3,9 suy vong. Nhu vay viec nghien cuu ddng thii smh g/l sau 24h nudi ciy vi khuin fren mdi frudng cd tnrdng visinh polymer cua cic chiing vi khuan de ngudn carbon la ri dudng, frong khi do ham lugng tim ra thdi diim tich liiy polymer cyc dai li viec PHB cue dai la 44% khdi lupng ti bio khd sau 30h Bong thai sinh trudng va ham lugng PHB tich luy trong ti bao chung Nfil53 Doin Vin Thugc & Nguyen Thj Binh nudi ciy tren mdi trudng cd nguon carbon li glucose. Nhu vgy cung giong nhu cao nim men, ri dudng li nguon co chat long hgp, nhiHig nguon co chat niy thudng thich hgp cho sy sinh trudng cua vi sinh v(it nhung khdng thich hgp cho sy tich luy polymer. PotyMtsr lynthM* |PhsC| Hinh 9. So ity of Education SUMMARY Polyhydroxyalkanoate (PHA) is a group of biopolymer thai accumulates inuacellularly as carbon and energy storage matenals in numerous microorganisms, usually when grown under the limitation of a nutrient such as nitrogen, oxygen, phosphorus, sulfur, magnesium and in the presence of excess carbon. In this study, PHA accumulating bactena were isolated from soil samples collected from mangroves in Giao Thuy district. Nam Dinh province. Twelve batenal strains were screened for PHA production with glucose as carbon source. The strain ND153 was chosen as a potential PHA producer. Strain ND153 is a slight halophilic. pH neuual, mcsophilic, molile, Gram-posilive, spore formation, and rod-shaped batcrium. The bactenum is a helerouoph, able to utilize various nifrogen and carbon sources. PHA from stmin NDI53 was punfied and used for magnetic resonance spectroscopic analysis. Based on 'H-nuclear magnetic resonance ( ' H - N M R ) spectroscqiic analysis, il was found that strain ND153 produced a homopolymcr of 3-hydroxybutyric acid (poly 3hydroxybulyrate - PHB) - a typical polymer accumulated by many microorganisms in nature. In a medium with glucose as carbon source, the maximum PHB conlenl of 82% of cell dry weight was oblained after 30h of cultivation in shake flask under MgSOj limitation. Keywords: Bacillus, biodegradable, biopolymer, PHA. PHB ' Author for correspondence: E-mail: doanvanthuoc&,vahoo. 178 com