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HOA CHE BIEN DAU KHi Nghien cCru tong hop chdt hogt dong be mat sinh hoc hogt tinh coo ung dung trong cong nghiep dau khi ThS Vu Xuan Hoan, TS Nguyin Tan Hoa ThS Trjnh Thanh Sen, KS Nguyen Khanh Toan[.]
HOA - CHE BIEN DAU KHi Nghien cCru tong hop chdt hogt dong be mat sinh hoc hogt tinh coo ung dung cong nghiep dau ThS Vu Xuan Hoan, TS Nguyin Tan Hoa ThS Trjnh Thanh Sen, KS Nguyen Khanh Toan ThS Vu An, KS Nguyen Thj Van Anh KS Cii Thj Viet Nga Vien Diu Viet Nam Tom tat Bai bao trinh bay ket qua nghien eiru tong hgp chat hoat dgng be mat sinh hge (CHDBMSH) hoat tinh eao tren ehung vi sinh Pseudomonas aeruginosa dwgc phan lap tir nguon nw&e thai nhiim dau - Tong kho xang dau Dire Giang Cae yeu to anh hir&ng den hieu suit tong hgp, eau true va dae tinh hoa ly eiia CHDBMSH da dwgc nghien eiru Ket qua tot nhat thu dirge nuoi eay Pseudomonas aeruginosa moi trw&ng khoang eo bo sung 4% DO, pH giir on dinh & 8, nhiet th & 340C, toe lie 200 vong/phut; sau nuoi eay, hieu suit tong hgp CHDBMSH tho dat gia trj ewe dai la 12,50g/l; sinh khoi dat 8,60g/l CHDBMSH tong hgp b&i Pseudomonsa aeruginosa thuge hg rhamnollpid (RhamCioCio), eo kha nang lam giam sire cang be mat eua nw&e tir 72,0mN/m xuong 29,5mN/m, kha nang nhu hoa xylen dat 65,6% Kit qua nghien eiru bw&e dau da m& trien vgng m&i viee ehe tao CHDBMSH hoat tinh eao tip nguon vi sinh vat nw&e irng dung cho eong nghiep dau Viet Nam DAU KHi - 8/2010 PETROVIETNAM I Giai thieu Chit hoat dpng b l mat sinh hoc vi sinh vat tao la c h i t cd p h i n d u true da dang vdi b l mat r i t hoat ddng Vi cd cae uu d i l m la dpc tinh thip, phan hiiy nhanh, dp tao nhu cao va nhiiu tinh chat quy bau khac nhu b i n nhiet, chju m u l i , chju pH va kha nang tong hgp tii' nhii'ng nguyen lieu re tien nen CHDBMSH cang dugc nghien eiru va irng dung rong rai cdng nghiep, ndng nghiep, nhlt la cdng nghiep d i u vdi eae muc dich nhu nang eao he s l thu h i i d i u , xir ly mdi trudng, lam chit phan tan, nhu hda can d i u (Liang-Ming Whang et al, 2008; Jane-Yii Wu et al, 2008; Kishore Das et al, 2007) Trong nhii'ng nam gan day, cdng nghiep d i u d Viet Nam da cd nhO'ng budc phat t h i n manh me, dd nhu d u sir dung CHDBMSH cang tang Tuy nhien nhOng nghien ciru ve CHDBMSH cdn r i t han c h i Trong cdng trinh nay, chung tdi trinh bay mdt s l kit qua nghien ciru budc d i u v l kha nang tong hgp CHDBMSH hoat tinh cao tir nguon vi sinh vat phan lap nudc irng dung eho cdng nghiep d i u Viet Nam II ThiPC nghiem Phan lap vi sinh vat eo kha nang tong hg'p CHDBMSH mdi trudng khoang ed b l sung parafin Chpn ehiing cd hoat tinh cao nhlt d l djnh ten dya tren hinh thai t h i bao quan sat bing kinh hiin vi dien tu" DM-750 ciia Leica - Microsystems (My) va thu' phan ung sinh hda bang Kit chuin API 20E cua Biomerieux (Phap) Nuoi cay va tong hg'p CHDBMSH Cay vi sinh vat phan lap duge vao mdi trudng khoang ed b l sung 4% d i u diesel (DO), nudi lie vdng 110 gid, t i e dp lie 200 vdng/phut, tri nhiet dp d 34°C, pH = Djch nudi d y sau dd duge ly tam vdi t i c dp 10.000 vdng/phut 15 phut tren may ly tam sieu toe LHSC - 30 cua LABNICS (My) d l tach t l bao, thu djch huyin phu Phin t l bao tach duge say khd qua dem d 45°C sau dd can d l xae djnh sinh khii Phin djch huyIn phu dugc tiia vdi axit HCI (1N) d i n pH = De qua dem d 4°C de kit tua hoan toan, sau dd dem ly tam vdi tdc dp 8.500 vdng/phut 20 phut Phin k i t tua thu duge se rira bing nude elt d i n pH = thu dugc CHDBMSH thd (san pham thd) Tiip tuc tinh c h i CHDBMSH thd bing he dung mdi: Chloroform/metanol = 65/15 d nhiet dp phdng thu lay pha hiru ca Chuyen pha hiru ea vao binh d t quay ehan khdng tren may elt quay ehuyen dung Buchir - 125 (Dire) de loai bd dung mdi se thu duge CHDBMSH m i u mat ong Chiit lai vdi he dung mdi tren l i n , cuoi eung eat quay d l thu duge san phim CHDBMSH tinh k h i l t a) Ngudn gde miu Miu nudc thai nhiem d i u duge lay tu" Tong kho xang d i u Du'c Giang - Ha Ndi b) Mdi trw&ng nudi ciy vi sinh vat - Mdi trudng giau dinh dudng (MPA): Cao nIm men 1%, pepton 0,5%, glucose 0,5%, aga 18g/l, dieu chfnh pH - 7; - Mdi trudng khoang Gost khong dudng: MgS04 0,3g/l, CaCl2 0,03g/l, NaH2P04 0,5g/l, Na2HP04 0,5g/l, FeClj 0,05g/l, KNO3 4g/l, d i i u ehinh pH = 7; - Dung djch sinh ly: NaCI 99,5%: 0,85g/100ml Xae dinh dae tinh hoa ly eiia CHDBMSH a) Pho hdng ngoai IR P h i hdng ngoai eiia CHDBMSH dugc ghi tren may Nicolet 6700 ciia Thermo Electron (My) sir dung vien nen vdi KBr Dai 500 - 4000 e m ' \ dp phan giai 0,4 b) sic ky Idng khdi phd LCMS Pho LC - MS ciia CHDBMSH dugc ghi tren may Agilent 1100 - LC/MSD Trap Pha dpng A: Aeetonitril; pha dpng B: Nudc (cd chira % axit axetic) Vung quet 100 - 1000 m/z (2 giay/mdt l i n quet) c) Phan lap va dinh ten chung vi sinh vat c) Do sire cang be mat (SCBM) Miu dugc d y tren mdi trudng khoang Gost khdng dudng, nudi lac d nhiet dp 34°C, t i c dp lie 200 vdng/phut 72 gid, tach khuan lac tren dTa thach Thir hoat tinh tao CHDBMSH b i n g kha nang tao nhO SCBM cua dieh nudi d y dugc tren may SCBM chuyen dung Radian series 300 eua Thermoscientific (My) theo phuang phap ban mong Wihelmy Nhung d i u ciia thilt bj vao dung DAU KHi-so 8/2010 27 HOA - CHE BIEN DAU Khi dich, de mdt lat cho on djnh rdi dpc ket qua Tien hanh l i n va l l y gia trj trung binh d) Xac dinh chi sd nhO hda E24 o l xae djnh chf so nhu hda E24, t i i n hanh trpn 2ml djch nudi d y sau tach te bao vdi 2ml xylen I n g nghiem, trpn phut vdi t i c dp 2000 vdng/phut Mau dugc giii' d 4°C vdng 24 gid trudc dem d l xac djnh E24 Do lai m i u sau 30 de danh gia dp b i n nhu hda trudng khoang c h i t cd b l sung 4% glucose, d i u nanh hay DO nhu la nguon cacbon va nang lugng nhlt, chung P.aeruginosa cho k i t qua t i t nhlt nguln cacbon la DO, hieu s u i t san pham thd 12,50g/l, sinh k h i i dat 8,60g/l dau vdi dat Chi s l nhu hda E24 dugc tinh theo edng thirc sau: I HS CHDBMSH Itho) E24 - [(chieu cao cot nhu hda)/(ehilu eao tong so)riOO% III Ket qua va thao luan Phan lap va dinh ten ehiing vi sinh vat K i t qua quan sat dudi kinh hiin vi cho t h i y ehiing vi sinh vat phan lap dugc thude nhdm vi khuin Gram (-), t l bao ed dang hinh que, khdng tiem mao va kha linh dpng Dya tren dae diem ban d i u Kit chuin API 20E (Biomeheux - Phap) da dugc lya ehpn de djnh ten chiing K i t qua cho thiy vi sinh vat phan lap dugc la chung Pseudomonas aeruginosa Nhu vay tu" nguln nudc thai nhiem d i u , sau lam giau va phan lap theo phuang phap Koch, da thu dugc chung P aeruginosa thuin khilt, cd kha nang tao CHDBMSH cao Day cung la mdt nhirng chung vi sinh vat dang dugc nghien eiru va sir dung biln tren t h i gidi de t i n g hgp CHDBMSH hien (Misun Cha, et al., 2008; Mahna Sanschez, et al., 2007; Henny Kell, et al., 2007) Nghien cii'u tong hg'p CHDBMSH a) Lwa chgn mdi trw&ng dinh dwang de nudi ciy va tong hgp CHDBMSH Mdi trudng dinh dudng ddng vai trd quan trpng qua trinh tong hgp CHDBMSH b i n g phuang phap vi sinh, dd viee sir dung nguln cacbon nhu la mdt nguln dinh dudng va nang lugng se anh hudng tryc tiep d i n kha nang sinh trudng va tao CHDBMSH cua cac vi sinh vat (Sen, 1997; Misun Cha et al., 2008; Palashphya Das et al., 2009) Trong cdng trinh nay, kha nang sinh trudng va tao CHDBMSH ciia P.aeruginosa da duge khao sat vdi ba nguln cacbon la glucose, d i u dau nanh va DO K i t qua (Hinh 1) cho thiy sau nudi c l y mdi 28 DAU KHi - s d 8/2010 Oou nanh Nguon cac bon Hinh Kha nang sinh truang va tao CHDBMSH tren cac ngudn cacbon khac cua Pseudomonas O i l u cd the dugc giai thich la P.aeruginosa dugc phan lap tir nguln nude nhiem dau nen kha nang tan dung DO la nguln cacbon de sinh trudng va tong hgp CHDBMSH cao han so vdi nguln cacbon cdn lai (N.K.Bordoloi et al., 2008, lloh et al, 2005; Nguyen QuIe Viet, 2004) O mdt khia canh khac, sir dung nguln cacbon la carbonhydrate hoae d i u dau thyc vat thi san pham trung gian la cae axit htru ca se hinh thanh, lam giam pH ciia mdi trudng O i l u khdng thuan Igi eho viee sinh trudng va phat trien cua P.aeruginosa (M Abouseoud et al., 2008) b) Cac yeu to anh hw&ng din hieu suit CHDBMSH tong hgp Cung gilng nhu t i n g hgp theo phuang phap hda hpe, hieu s u i t t i n g hgp CHDBMSH b i n g vi sinh chju anh hudng ciia nhiiu y l u t l nhu nguln cacbon, thdi gian nudi cly, nhiet dp, pH mdi trudng, t i e dp lie Do dd, viec nghien ciru cac y l u t l anh hudng d i n hieu suit tong hgp d l toi uu hda quy trinh thyc nghiem la viee lam d n thilt Trong eong trinh nay, cae y l u t l ca ban anh hudng d i n kha nang sinh trudng va t i n g hgp CHDBMSH la ty le cacbon, thdi gian nudi d y , pH mdi trudng va nhiet dp da duge khao sat tren doi tugng la P.aeruginosa vdi n g u l n cacbon la DO DO dugc lya ehpn la n g u l n ea c h i t ban dau de tong hgp CHDBMSH vi day la n g u l n nguyen lieu s i n cd PETROVIETNAM re tiin d Viet Nam va chiing P.aeruginosa sinh trudng va tong hgp CHDBMSH t i t n h l t tren nguon cacbon * Anh hw&ng eua ty le cacbon o l khao sat anh hudng eiia ham lugng cacbon mdi trudng nudi cay d i n hieu suit tong hgp CHDBMSH, tac gia da tiin hanh thay doi ty le DO bo sung vao mdi trudng muli khoang tu* 2% d i n 8% Sau nudi cly, phin djch duge ly tam d l tach t l bao va thu CHDBMSH K i t qua thi nghiem dugc the hien Bang Bang Anh huang cua ty ie diu DO din kha nang sinh truang va tdng hap CHDBMSH ciia P.aeruginosa T y l e DO 2% 4% 6% 8% Hi0u suat sinh khoi, g/l 5,86 8,60 8,65 8,61 Hieu suat SPT, g/l 9,25 12.50 12,62 12,36 48,20 36,68 36,40 36.40 SCBM mN/m Diiu kien nudi cay: Nhiet 34^0, pH = 8, tde lie 200 vdng/phiit: Khi tang ty le DO tir 2% len 4%, hieu s u i t sinh khii va hieu s u i t san pham thd d i u tang manh, dd hieu s u i t t i n g hgp CHDBMSH cua P.aeruginosa tang tu' 9,25 len 12,50g/l, tuang ung vdi 26%, lam SCBM ciia djch nudi c l y giam tir 48,20mN/m xuong cdn 36,68mN/m Tuy nhien, t i i p tuc tang ham lugng cacbon len 6% va 8% thi hieu suit sinh khii va hieu s u i t san pham thd lai thay doi khdng dang ke Ngoai ra, tLi" Bang ed the t h i y hieu s u i t sinh k h i i va hieu suit tong hgp CHDBMSH eao thi SCBM eiia djch nudi c l y giam O i l u hoan toan phii hgp bdi vi vi sinh vat sinh trudng va tong hgp CHDBMSH tot thi ehinh CHDBMSH da lam giam SCBM ciia djch nudi d y Nhu vay, ed t h i t h i y ty le sir dung nguln cacbon t i i uu d i i vdi ehiing P.aeruginosa la khoang 4% * Anh hw&ng cua th&i gian nudi ciy Nhiiu nghien cu'u da ehirng minh t i e dp sinh trudng va hieu suit tong hgp CHDBMSH thay doi kha nhiiu theo thdi gian nudi c l y (Hun Yin et al, 2008; N K Bordoloi et al, 2008; Palashphya Das et al, 2009) Do dd, viec xac djnh thdi d i l m thich hgp de thu CHDBMSH qua thnh nudi d y la rit quan trpng Trong edng trinh nay, d l xac djnh thdi gian nudi d y t i i uu, hieu suit sinh khii va sy giam SCBM eua djch nuoi c l y da dugc khao sat theo thdi gian nudi d y Ket qua dugc t h i hien Bang Bang Anh huang cua thai gian nudi ciy din kha nang sinh truang va tdng hap CHDBMSH ciia P.aeruginosa 48 72 96 110 134 Hi$u su^t sinti kh6i, g/l 4.78 7.21 8.54 8,60 8.56 SCBIVI, mN/m 58.21 47.42 40.82 36,54 36.44 Thai gian, gib- Diiu kien nudi ciy: Nhiet 340C pH = toe lie 200 vdng/phiit Tir Bang cd t h i thiy, hieu suit sinh khii cua Paeruginosa tang manh 96 gid d i u nudi c l y sau dd tang cham lai va dat eye dai d 110 gid Khi keo dai thdi gian nudi c l y den 134 gid thi hieu suit sinh khoi lai giam cac chung vi sinh bude vao chu ky chit (Orathai Pornsunthorntawee et al, 2008) Oilu ehung td khoang thdi gian tu- 96 gid eho d i n 110 gid la khoang thdi gian t i i uu d l P.aeruginosa sinh trudng va tong hgp CHDBMSH K i t qua SCBM djch nudi c l y cOng hoan toan phu hgp vdi s l lieu sinh khdi SCBM eiia djch nuoi d y giam manh 96 gid d i u nudi d y trude budc vao trang thai on djnh sau 110 gid Oilu ed the dugc giai thich la CHDBMSH tao bdi eae chung vi sinh da lam giam manh SCBM cua djch nudi c l y giai doan d i u Khi ham lugng CHDBMSH tang dat d i n gia th can bing thi SCBM cua djch nudi cay gan nhu khdng thay d i i (Huy Yin et al, 2008; Kishore Das et al, 2007) Nhu vay can cir vao hieu suit sinh khii va kha nang lam giam SCBM cua djch nudi d y thi khoang thdi gian nudi c l y t i i uu de thu CHDBMSH d i i vdi P.aeruginosa la 96-110 gid (khoang - ngay) * Anh hw&ng trwang nudi ciy cua pH mdi Kha nang sinh trudng va tong hgp CHDBMSH ciia cac ehung vi sinh phu thupc kha nhieu vao gia tri pH ciia mdi trudng nudi d y Nhiiu nghien cuu eho thiy, d mdi trudng axit (pH thip) hoae kiem (pH cao), viec sinh trudng va tong hgp ciia P.aeruginosa se khdng thuan Igi so vdi mdi trudng trung tinh Vi vay, khoang pH = - 10 da dugc lya chpn de khao sat, k i t qua dugc trinh bay Bang DAU K H i - s o 8/2010 29 HOA - CHE BIEN DAU KHI Bang Anh hwang cua pH mdi trwang din kha nang sinh trwang va tdng hap CHDBMSH ciia P.aeruginosa pH m o i triro'ng t i 6,12 H i e u s u a t s i n h k h o i , g/l 8,38 8,60 8,56 10 8,27 H i e u s u a t S P T , g/l 10,24 11,14 12,50 12,35 11,28 SCBM, mN/m 40,78 36,35 36,46 36.40 38,13 Diiu kien nudi ciy: Nhiet 34°C, pH = - 10, toe lie 200 vdng/phut Bang Anh huang ciia nhiet mdi truang nudi ciy din kha nang sinh truang va tdng hap CHDBMSH ciia P.aeruginosa Nhieta^ 30 Hieu suat sinh khoi, g/l 7,68 j 32 7,91 34 8,60 T 36 38 8,15 7,24 T 40 6,86 Hieu suat SPT, g/l 11,43 12,18 12,50 12,37 11.48 10,31 SCBM, mN/m 39.02 36,40 36,43 36,67 37,80 40,10 Dieu kien nuoi cay: pH = 8; nhiet = 30 - 40^C, toe lie 200 vdng/phut 2S00 2000 Wa^cnumbers (cm-l) Hinh Pho IR ciia CHDBMSH tdng hap bai P.aeruginosa Tir Bang cd t h i thay, kha nang sinh trudng va t i n g hgp CHDBMSH ciia P.aeruginosa kha nhay cam vdi gia trj pH eua mdi trudng nudi d y Hieu suit sinh khii tang tu' 6,12g/l len 8,60g/l, tuang duang vdi g i n 40% tang pH mdi trudng tir d i n Khi tiip tue tang gia th pH d i n 10 thi hieu suit sinh khii thay d i i khdng dang k l nhung xu the giam K i t qua khao sat sy phu thupc hieu suit ting hgp CHDBMSH vao gia th pH cung co xu hudng tuang ty, tang kha manh khoang pH = - va dat eye dai tai pH = vdi 12,50 g/l, sau dd giam nhe tang pH d i n 10 (y khia canh khac, SCBM ciia djch nudi c l y khoang pH tu" -10 dudng nhu it thay doi mac du quan sat thiy sy thay doi kha Idn tang pH tu' len Kit qua eOng phu hgp vdi dac dilm sinh ly cua P.aeruginosa la sinh trudng kem mdi trudng axit va sinh trudng t i t han d mdi trudng trung tinh hoae kilm y l u (Hua Yin et al, 2008; Misun Cha et al, 2008) Nhu vay, qua khao sat ed t h i thiy gia trj pH t i i uu d l nudi c l y va thu CHDBMSH d i i vdi P.aeruginosa nam khoang - 9, t i t nhlt la pH = * Anh hwang cua nhiet dd nudi ciy Theo nghien ciru ciia Cameotra va cpng sy, nhiet dp nudi c l y anh hudng manh d i n kha nang sinh trudng va tong hgp CHDBMSH eiia cae vi sinh vat 6' nhiet dp t h i p , eae vi sinh vat sinh trudng cham, hieu s u i t tong hgp thudng thap, song d nhiet dp eao, nhiiu vi sinh vat lai khdng T 8/2010 ed kha nang sinh trudng Vi vay viec xac djnh nhiet dp nudi c l y toi uu la viee lam c i n thilt Trong cdng trinh nay, kha nang sinh trudng va tong hgp CHDBMSH cua chiing P.aeruginosa da duge khao sat khoang nhiet dp tu' 30 d i n 40°C, I sau nudi d y tii on nhiet, p h i n djch dugc ly tam de tach t l bao va xac djnh hieu suit CHDBMSH K i t qua dugc dua Bang I K i t qua khao sat cho thiy, nhiet dp thich hgp cho sinh trudng va tong hgp CHDBMSH ciia ehung P.aeruginosa n I m khoang 32 36°C, t i i uu la 34°C Khi tang nhiet dp tren 40°C, ea hieu suit sinh khii va hieu suit ting hgp CHDBMSH d i u giam manh, cd t h i nhiet dp eao khdng thuan Igi cho sy sinh trudng ciia P.aeruginosa Khi giam nhiet dp t h i p han 34°C, t i c dp sinh trudng thudng cham, sau nudi cly, hieu suit sinh khii cua P.aeruginosa la 7,68g/l chi bang 80% so vdi sinh khii nudi cly d nhiet dp t i i uu la 34°C Nhw vay diiu kien tdi wu de tdng hgp CHDBMSH la nudi ciy P.aeruginosa mdi trw&ng muoi khoang cd bd sung 4% DQ, pH giir on dinh & 8, nhiet tri a 34"C, tdc lie 200 vdng/phut Sau nuoi ciy & diiu kien tdi wu, hieu suit tdng hgp CHDBMSH dat cwc dai ciia P.aeruginosa la 12,50g/l Nghien ciru eau true hoa hgc va dae tinh hoa ly eua CHDBMSH 3.1 ciu true va phin hoa hoc cua CHDBMSH Pho h i n g ngoai (Hinh 2) ciia CHDBMSH t i n g hgp bdi Paeruginosa ed eae dao dpng dac trung ciia rhamnolipid vdi cac dfnh hap thu tai 2359 - 2938 em"\ 1731 va 1100-1350 cm-"" Dfnh h i p thu tai I fc3 wBvir^f, ,: