PHN I: M U 1.1. Tnh cp thit ca ti Trong c th thc vt, ln ng vai tr quyt nh s bin i vt cht v nng lng.

. N tham gia cu to nn cc acid nucleic, coenzyme, adenosin triphosphate (ATP) l nhng cht cn thit cho s sng [2]. Ngoi ra ln cn ng nhng vai tr khc nh to mi trng m, nh hng n qu trnh ht cc cht khong khc ca cy. t cha khi lng ln cht cha ln. Tuy nhin khng phi hp cht cha ln no trong t cng c cy s dng d dng. c bit l t bazan nu Ty Nguyn vn rt giu ln tng s nhng ln d tan li rt thp. S chuyn ha ln xy ra ch yu di tc dng ca qu trnh ha hc v sinh hc [1]. Qu trnh chuyn ha hp cht phophat kh tan trong t c phn ng gp quan trng ca cc loi vi sinh vt. Vi sinh vt c kh nng chuyn ha qung phosphate kh tan thnh d tan cy trng hp th c. Do vy bn vi sinh vt phn gii phosphate kh tan s cung cp mt lng ln d tan cho cy trng v gip cy hp th cc cht dinh dng trong t tt hn [3]. Ngoi ra nhng vi sinh vt chuyn ha hp cht ln va c kh nng to cc cht dinh dng cho cy, sinh tng hp cht kch thch sinh trng thc vt ng thi cng c kh nng c ch mt s vi sinh vt gy bnh vng r cy trng [7]. Theo Fridland (1973) t bazan nu l loi t hnh thnh trn t bazan, trong iu kin nhit i m, cc baz b ra tri mnh, t c phn ng chua, st nhm tch ly nhiu, y l nguyn nhn ch yu ca qu trnh gi cht ln trn t bazan [13]. Theo on Triu Nhn (1999): t bazan nu Ty Nguyn c hm lng ln tng s t 0,02% P2O5 v lng ln d tiu l 4,12mg P2O5/ 100g t [13]. ak Lak vi hn 360.000 ha t bazan nu l mt loi t giu ln tng s rt thch hp cho vic pht trin cc cy cng nghip di ngy [21]. Tuy nhin cc qu trnh c nh ln t d tiu thnh dng kh tiu thng xuyn xy ra nn lng ln d tiu lun mc ngho. ng thi, nng dn trong thi gian qua thng ch dng phn ha hc

bn cho cy trng thiu bn phn hu c cng nh phn vi sinh vt lm cho t trng b thoi ha, chai cng, vi sinh vt t b suy thoi, gy nhim mi trng. Xut pht t nhng l do trn chng ti tin hnh nghin cu ti: Nghin cu phn lp v tuyn chn vi sinh vt phn gii phosphate kh tan trn t bazan nu ak Lak. 1.2. Mc tiu ti - Tuyn chn mt s chng vi sinh vt c hot tnh phn gii photphate kh tan cao trn t Bazan nu ti ak Lak. 1.3. ngha khoa hc, thc tin 1.3.1 ngha khoa hc Kt qu nghin cu ca ti gp phn lm sng t vai tr ca vi sinh vt phn gii ln kh tan . Cc nghin cu ca Vit Nam v th gii v phn gii ln kh tan dng phosphate st, nhm cn rt t nghin cu. ng ch ti nghin cu, tuyn chn v phn lp cc chng vi khun c kh nng phn gii ln kh tan hiu qu trn t bazan nu trong iu kin c th ak Lak v gp phn bo tn ngun gen vi sinh vt bn a hu ch. 1.3.2 ngha thc tin Thnh cng ca ti s gp phn vo vic sn xut v ng dng ch phm vi sinh vt phn gii ln kh tan trong sn xut nng, lm nghip nhm cung cp ln d tan cho cy trng, gim chi ph u t, gim s thoi ha t, gp phn ci thin i sng ca nng dn v bo v mi trng nng thn ak Lak. 1.4. Gii hn ca ti Trong qu trnh thc hin do thi gian, trang thit b, ha cht c hn nn chng ti ch tin hnh phn lp trn mt s mu t ca tnh ak Lak v ch theo di mt s ch tiu c bn. Mc khc, l sinh vin ln u tham gia nghin cu nn khng th trnh khi thiu st. Knh mong qu thy c v cc bn ng gp kin.

PHN II: TNG QUAN TI LIU 2.1 Cc dng ln trong t Ln rt quan trng i vi cy trng. Tuy nhin hiu sut s dng ln bi cy trng khng qu 25%, trong khi mt lng ln b c nh trong t v chuyn thnh dng kh hp th. Lng d tr ln trong t xp x 0,025 0,3% P2O5 nhng chng tn ti trong t dng khng tan trong nc cy kh hp th. Thnh phn ln d tan v kh tan trong t c quyt nh bi tnh cht m, thnh phn c gii v hm lng cht hu c quyt nh [5]. Theo Sepfe-Satsaben (1960) th hm lng phosphate trung bnh nhiu loi t thng t 0.02 0.08%. Do qu trnh tch ly sinh hc, hm lng phosphate trong lp t mt cao hn lp di [13]. Trong cc loi t khong , t l ln hu c thng t 25 65%. Cc c ht thuc thnh phn st thng cha nhiu ln hn cc c thuc thnh phn ct. Do : cc chn t nh, t bc mu c t keo st, th t l phosphate thng thp hn cc loi t khc [13]. T l ln trong t khc nhau thy theo tnh cht ca m v nhng tng pht sinh t m nh: Nai, mica, quartzit thng t l ln thp hn l t pht sinh t mu thch khng chua nh: Bazan, vi,[13] Qu trnh phn gii xc b ng thc vt cung cp cho t mt ngun phosphate quan trng. Nh vy vic b sung cht hu c vo t gip lm tng cng hm lng ln cho t. Trong t nhin ni chung v trong t ni ring , phosphate tn ti hai dng ch yu sau: 2.1.1 Ln hu c Ty loi t, t l phosphate hu c thng chim t 20 80% phosphate tng s trong t. lp t mt, phosphate chim khong 50% [5]. Phosphate hu c trong t ch yu trong thnh phn mn. t cng giu mn th cng giu phosphate hu c. Theo Kletcpki v Petecbuaxki (1964) th: Trong phosphate hu c ca t, dng ph

bin nht l dng fytat, c th chim n 50% tng s phosphate hu c. Ty theo mi trng acid hay kim m tn ti cc dng fytat khc nhau. t chua, phosphate hu c ch yu l fytat Fe, Al; t trung tnh, kim tn ti dng fytat Ca, Mg [13]. Phosphate hu c trong c th ng vt, thc vt, vi sinh vt thng gp cc hp cht ch yu nh fitin, phospholipide, acid nucleic. iu ng ch l phosphate vi sinh vt khng tham gia trc tip vo dinh dng cy trng m phi i vi sinh vt cht i, t bo b khong ha th cy trng mi hp thu c [5]. Khi thc hin khong ha c s tham gia ca vi sinh vt phn gii phosphate, nhng hp cht hu c (trong c ln hu c) s c khong ha gii phng ra ln v c hay hu c, l ngun dinh dng cung cp thc n cho cy trng. C 70 80 tp on vi sinh vt tham gia vo qu trnh phn gii phosphate [13]. Trong qu trnh khong ha cht hu c ca t, phosphate hu c c gii phng ra di dng acid phosphoric v mui d tan ca n. Nhng cc dng ln ny li b t hp ph v vi sinh vt ht li, nn trong t r t phosphate dng ha tan. Nhiu tc gi nghin cu cho rng: Nu cht hu c vi trong t l cht hu c ngho phosphate th qua qu trnh phn gii khng nhng hm lng phosphate hu c trong t khng tng m cn gim xung (Ivanop 1955) [3]. Theo nhng cng trnh nghin cu ca Kaila (1954): Nu cht hu c vi xung t cha t hn 0.2 0.3% P2O5 th qu trnh phn gii khng tng thm v phosphate d tancho cy v vi sinh vt s ht ht. Cng ht phosphate hu c ca t thng qua s phn gii ca vi sinh vt ph thuc rt nhiu vo nhit . Theo Sepfe-Satsaben (1960) trong iu kin nhit bnh thng, cc nc n i , s khong ha phosphate hu c tin hnh rt chm v lng phosphate cung cp cho cy t nhng hp cht hu c khng ng k. Tri li, nhit t 35 50oC th qu trnh khong ha tng ln rt mnh v cung cp cho cy c nhiu phosphate t nhng hp cht hu c. V th, nc ta bn phn chung cng l gii php cung cp phosphate cho cy trng [13].

2.1.2. Ln v c

Phosphate v c tn ti dng mui ca nhng nguyn t Ca, Fe, Al. t trung tnh v t kim th phosphate Ca l ch yu, cn t chua th phosphate Fe, Al l ch yu. Phosphate Ca d c huy ng lm thc n cho cy hn l phosphate Fe, Al. S tn ti ca ion phosphate trong mi trng t b chi phi bi ion phosphate b chuyn i ha tr [13]. Mi trng chua: H2PO4HPO42PO43-

Trong thc t, H2PO4- l dng cy trng d hp thu nht. Cc dng phosphate cn li thng l nhng loi kh ha tan m cy trng khng th ng ha c, mun cy trng s dng c phi qua qu trnh bin i thnh dng d tan [13]. Cng nh cc yu t khc, phosphate trong t nhin lun lun tun hon chuyn ha. Nh vi sinh vt, ln hu c c v c ha bin thnh dng mui ca acid phosphoric. Cc dng ln ny mt phn c cy trng s dng bin thnh dng ln hu c, mt phn b c nh di dng kh tan nh Ca3PO4, AlPO4, FePO4. Nhng dng kh tan ny trong cc mi trng c pH thch hp s chuyn thnh dng d tan. Trong qu trnh ny, vi sinh vt gi vai tr quan trng. 2.1.3 Vng tun hon ca ln trong t nhin Vng tun hon ca ln khng ging nh vng tun hon ca nit. Trong khi nit lun khan him trong t th ln tn ti nhiu trong t dng kh phn gii [15, 18]. Nit c a vo t nh vi sinh vt c nh m t khng kh, cn i vi ln, chng c cc vi sinh vt phn gii t cc ngun ln v c v hu c khc nhau. Vng tun hon ca ln c biu din trong s sau:

Cy xanh PO43- trong dung dch t PO43- b hp th Qu trnh khong Ha tan Ln v c C nh tm thi Cht hu c ti v t bo sinh vt Cht hu c mn ha 2.2. S chuyn ha ln trong t 2.2.1. i vi ln hu c

ng vt

Qu trnh c nh

Trong t c nhiu loi vi sinh vt khong ha c ln hu c. Cc vi sinh vt ny tit ra cc enzyme kh phosphoryl ng thi gii phng ion phosphate. Phn ng enzyme nhanh khi hp cht ln hu c va mi bn vo t v sau xy ra chm khi ln b ci bin. Ln s to cc phc lin kt vi Fe, Al, cc cht hu c phn t lng cao v b gi cht trn cc phn t st. Tc gii phng ln ph thuc vo cc yu t sau: - Bn cht cc hp cht hu c c ln: Acid nucleic d khong ha hn phytin. Nguyn nhn dohu ht cc vi sinh vt khong ha ln hu c u tit ra cc enzyme tng ng phn gii acid nucleic [3]. - Nu lng C/P cao hn 300 th ln s b cc vi sinh vt trong t c nh. Cn mc C/P nh hn 200, ln s tha nn c khong ha [3].

- pH ti thch l 6 7. mi trng kim ln v c c phng thch nhanh hn ln ha c. - Nhit cao cng thun li cho vic khong ha ln hu c. Ti thch l 40 50oC. Do , trong ma h tc khong ha ln mnh hn cc ma khc. 2.2.2. i vi ln v c S tn ti cc loi ion phosphate trong t ph thuc vo pH t. Do vy, thc t trong t, ln tn ti ch yu hai dng: H2PO4- v HPO42-. H2PO4Dung dch acid HPO42PO43Dung dch kim

pH = 7 t l 2 loi ion ny gn bng nhau. H 2PO4- d ng ha hn HPO42-, nn v mt l thuyt pH = 5 6 dinh dng ln ca cy thun li nht. Song trong t do c mt ca nhiu ion khc m vn tr nn phc tp hn. 2.2.2.1. S chuyn ha ln t chua Trong t chua ngho cht hu c: Fe, Al v Mn thng nm di dng ha tan phn ng vi H2PO4- to thnh hp cht khng tan cy khng ng ha c [13]. Al3+ + H2PO4- + 2H2O 2H+ + Al(OH)3.H2PO4 Khng tan

cc loi t rt chua, Al3+ v Fe3+vt cc ion H2PO4- nhiu lm cho phn ng trn cng nghing theo chiu thun, to thnh ln khng tan khin cho ch cn mt lng rt nh H2PO4- trong t. t chua, ion H2PO4- khng nhng phn ng vi Fe3+, Al3+ ha tan m cn phn ng vi cc oxit ngm nc ca cc nguyn t nh gibbsit (Al2O3.3H2O) v goethit (Fe2O3.3H2O). t chua s lng ln b cc oxit st, oxit nhm ngm nc c nh cn vt qu c s lng ln b kt ta vi Fe, Al v Mn ha tan.

Al(OH)3 + H2PO4-

Al(OH)2.HPO4- + H2O

iu ng lu l hu ht cc loi t u cha oxit st, nhm ngm nc nn y cng l kiu c nh kh nhiu ln v din ra trn phm vi rng. Trong mi trng chua cn c hai qu trnh c nh ln lin quan ti st. l do s tn ti cc ion OH- l trn b mt khong st. S c nh ny i km vi vic gii phng kim theo phn ng sau: St OH + Ca(H2PO4)2 st H2PO4- +1/2Ca(OH)2

Kh nng c nh thay i theo bn cht khong vt ca keo st theo th t sau y: Illit > Kaolinit > Montmorillonit. [Al] + H2PO4- + 2H2O 2H+ + Al(OH)2.H2PO4

Vai tr ca st v nhm th hin qua thc t l vic c nh cc anion mnh ln khi t l SiO2/seoquioxit gim, vi cc loi t mt vi nu cc seoquioxit cng mt i th kh nng c nh ln cng gim. Cn v vi, ngi ta thy khi cho st hp th Ca2+, t l anion phosphate c hp thu tng ln, bt chp ngng kt ta canxi phosphate. iu chng t rng : y l qu tnh c nh ion ch khng phi bng con ng ha hc. Tnh n nh ca qu trnh gi cht ny ph thuc vo iu kin mi trng v vic gii phng anion c th xy ra khi iu kin mi trng thay i v c bit l nu st b gii keo. t chua, cc hydroxit st, nhm lng tnh c th mt 1 nhm OH- tr thnh keo dng tnh tham gia hp ph trao i anion: Al(OH)3 + H+ = Al(OH)2+ + H2O 2.2.2.2. S chuyn ha ln t kim Trong mi trng kim giu Ca, ion H2PO4- phn ng mnh vi Ca to thnh cc hp cht t tan hn theo cc phn ng ln lt nh sau [13]:

Ca(H2PO4)2 + CaCO3 + H2O 2CaHPO4.2H2O + CO2 6CaHPO4.2H2O + 2CaCO3 + H2O Ca8H2(PO4)6.5H2O + 2CO2 Ca8H2(PO4)6.5H2O + CaCO3 Ca3(PO4)2 + CO2 + 6H2O Ln tr nn km ha tan hn khi gp iu kin thun li v thi gian Ca3(PO4)2 c th chuyn thnh cc hp cht khng tan hn na nh hydroxy, carbon v ngay c fluoro apatit. 2.3 Tng quan v vi sinh vt phn gii ln Vi sinh vt (microorganisms) l tn chung dng ch tt c cc loi sinh vt nh b m mun thy chng, ngi ta phi s dng ti knh hin vi. Theo Hong Lng Vit (1978) trung bnh mt gam t kh c n gn 200 triu t bo vi sinh vt. Vi sinh vt c cc nhm chnh sau: vi khun, x khun, nm men, nm mc, vi to, virus [6]. Cc nhm c kh nng phn gii phosphate l vi khun, x khun, nm men, nm mc [5]. Nghin cu ca Puneet v cng s cho thy: Vic nhim mt s chng nm si c kh nng ha tan phosphate nh Aspergillus flavus v Asp. niger vi vi khun c nh nit Azotobacter sp tng nng sut ht 17.7%, trong khi ch nhim Azotobacter sp ch lm tng 9% [18]. Kopoor v cng s cng t kt qu tng t khi nghin cu s phi hp gia chng Azotobacter sp vi cc vi khun phn gii phosphate thuc cc chi Asgrobacterium sp, Bacillus sp v Pseudomonas sp[20] Vi sinh vt phn gii ln c chia thnh hai nhm: Vi sinh vt phn gii ln hu c v vi sinh vt phn gii ln v c. 2.3.1 Vi sinh vt phn gii ln hu c S chuyn ha cc hp cht ln hu c thnh mui ca H 3PO4(Stoklaza 1991, Menkina 1952) theo s sau [1, 5]: 1. Nucleoprotein 2. Lxitin Nuclein acid nucleic H3PO4 Nucleotic H3PO4

Glixerphosphate

S ca qu trnh phn gii nucleoprotein c th nh sau: Nucleoprotein protein

Acid nucleic

4C5H10 O5

C6H5O5

C5H5O5O

C5H5O5O2

C4H5O5O

4H3PO4

Acid amin

NH3

CO2

H 2S

H 2O

Cht khc

Vi sinh vt phn gii ln hu c ch yu gm cc chng Bacillus v Pseudomonas. Ngoi ra cn c mt s x khun v nm khc. ng ch l B. megaterium var phosphatsum c kh nng phn gii ln hu c cao [13]. ng thi B. megaterium cn c kh nng hnh thnh bo t nn sc sng rt mnh [4]. 2.3.2. Vi sinh vt phn gii ln v c Nhiu vi khun nh Bacillus megaterium, Bacillus mycoides, Bacillus butyricus, Pseudomonas fluorescens, vi khun nitrat ha, mt s vi khun h r, x khun c kh nng phn gii Ca3(PO4)2 v bt apatit. Kh nng phn gii ln v c lin quan mt thit ti s sn sinh acid ca vi sinh vt. Qu trnh ln men to ra acid carbonic, l acid ch yu thc y qu trnh ha tan ln v c [3]. Ca3(PO4)2 + H2CO3 + H2O Ca(PO4)2H2O + Ca(HCO3)2

Trong t, vi khun nitrat ha v vi khun chuyn ha lu hunh cng c tc dng quan trng trong vic phn gii Ca3(PO4)2. V trong qu trnh sng, cc vi khun ny tch ly trong t HNO3 v H2SO4. Qu trnh ha tan c th biu th theo phng trnh sau: Ca3(PO4)2 + 4 HNO3 = Ca(H2PO4)2 + 2 Ca(NO3)2 Ca3(PO4)2 + 2 H2SO4 = Ca(H2PO4)2 + 2CaSO4 i vi nm th Aspergillus niger cho kh nng phn gii ln mnh nht. Ngoi ra cn c mt s chng khc nh Penicillin, Rhizopus 2.3.3. Cc iu kin nh hng ti kh nng phn gii ln ca vi sinh vt - pH: nhn chung pH t nh hng n kh nng phn gii ln. Tuy nhin pH trong khong 7.8 8.0 nh hng tt ti s pht trin ca h vi sinh vt phn gii ln [5]. - Nhit : cc chng vi sinh vt c nhit thch hp cho qu trnh phn gii ln l khc nhau. Nhn chung khong nhit thch hp nm trong khong 20 40oC [9]. - Hp cht hu c: cht hu c lm tng qu trnh sinh trng ca vi sinh vt. Do kh nng phn gii ln ca chng s tng ln. - m: nhng ni c m cao, do hot ng ca vi sinh vt mnh nn to ra nhiu acid hu c lm tng phn gii ln. - H r: h r cy trng khch thch s sinh trng ca vi sinh vt. Do phn gii ln cng c tng cng. Tuy nhin mt s loi cy c th tit ra cc cht c ngn cng s sinh trng, pht trin ca vi sinh vt. - T l N v C trong mi trng: N, C l nhng thnh phn cn thit cho s sinh trng v pht trin ca vi sinh vt. T l N, C trong mi trng cao s thc y kh nng phn gii ln. 2.4. Tng quan v t bazan nu ak Lak Theo h thng phn loi t ca FAO UNESCO (1995), ak Lak c 11 nhm v 84 n v t ai. Trong , nhm t xm chim din tch ln nht 44%. Nhng nhm

t sm thng phn b nhng vng t dc, v bn cht c ph thp, chua, hm lng ln tng s v ln d tiu thp. Nh vy, nhm t xm khng thun li cho vic trng cc loi cy trng. Nhm t chim din tch nhiu th 2 ak Lak l t bazan (Ferrasols). Din tch t bazan khong 311.340ha, chim 23.7% din tch t t nhin. Phn b tp trung ti cc khi bazan Bun Ma Thut. Nhm t ny c cc n v phn loi: Nu trn Bazan (Fk), nu vng trn bazan (Fu), l nhm t c tng B tch t nhm r nht. t c phn b tp trung khi bazan Bun Ma Thut chy t bc xung Nam, t ng sang Ty. Pha Bc cao nguyn (Ea H'Leo) c cao 800m, pha Nam cao 400 m, pha Ty cao 300m (khu vc huyn C M'gar). B mt cao nguyn rt bng phng [21]. t bazan nu hnh thnh v pht trin trn cc cao nguyn Ba zan phn ln c dc thp, tng t mn dy, c thnh phn c gii nng (t l st >40%), ti xp khi m, xp trung bnh 62-65%, kh nng gi nc v hp thu nc tt... Rt thch hp vi cc loi cy cng nghip di ngy c gi tr hng ho cao: c ph, cao su, tiu v nhng cy n qu khc... [21]. Tuy nhin, im hn ch ca nhm t ny l hm lng ln d tiu thp, lng P2O5 d tan nh hn 1,0 mg/100 g t. Trong khi , ln tng s ca nhm t ny li cao, P2O5 tng s trn 0.2% [21]. 2.5. Tnh hnh nghin cu trn th gii Hm lng ln trong cc loi t thng rt thp v vy ngi ta tm cch tng lng ln d tan trong t bng cch bn phn. Nhng 2/3 lng ln bn vo t b chuyn ha tr thnh ln kh tiu khin cy trng khng hp th c hoc b ra tri i. Do hiu qu ca vic bn phn ln b gim i nhiu. Cc vi sinh vt phn gii ln kh tan gii quyt c vn ny. Chng va gim c lng phn ln bn cho cy, ng thi huy ng c c lng ln kh tiu trong t. Vi nhng li ch nh vy, cc vi sinh vt phn gii ln c nhiu nc trn th gii quan tm. Nhiu cng trnh nghin cu chu u, M v n cho thy hiu qu to ln ca cc vi sinh vt phn gii ln. Cc nghin cu ca Sen v Paul, 1957 , Katznelson v Bose,1967; Ostwal v Bhide, 1999 cho thy: cc chng vi khun c bit thuc loi Pseudomonas v Bacillus,

cc chng nm thuc loi Penicillium, Aspergillus c kh nng chuyn ha photphat khng tan thnh dng d ha tan trong t nh tit ra cc acid hu c nh formic, acetic, lactic, propionic, fumaric, glucolic v acid succinic. Nhng acid ny lm gim pH v ha tan cc dng photphate kh tan [20]. Lin x (c) sn phm phn bn vi sinh vt thng mi phosphobacterin vi s c mt ca B. megateirum var phosphaticum c s dng rng ri Lin x v cc nc ng u , lm tng nng sut cy trng 5 10% so vi i chng.Vin nghin cu nng nghip n s dng phosphobacterium trn la m, la v ng cng cho kt qu tng ng k so vi i chng. Ngi ta tnh nu s dng vi sinh vt phn gii ln c tc dng tng ng vi vic bn 50kg P2O5/ha [3]. Kt qu nghin cu mi nht Canada cho thy bn vi sinh vt phn gii ln c th thay th 50 75% lng ln cn bn bng qung ngho P 2O5 m nng sut, cht lng khng h thay i (Gaur, 1992). S dng chng Pseudomonas striata khi bn qung phosphate v superphosphate cng lm tng ng k nng sut khoai ty (Gaur v Negi, 1980) [16]. Perez (2007) nghin cu phn lp c 130 chng vi khun c kh nng phn gii phosphat kh tan Venezuela. Tuy nhin, khng c chng no c hot tnh phn gii phosphate Fe v Al. Tc gi cng chn c 10 chng c tim nng nghin cu tip thuc cc chi Raltonia, Pantoea, Serratia [15]. Alvaro (2009) phn lp c 2 chng vi khun t r c Ty Ban Nha c hot tnh phn gii phosphate v kch thch sinh trng i vi cy trng thuc chi mi l Acenitobacter [17]. Hin nay, hai nc Trung Quc v n ang y mnh nghin cu v ng dng cng ngh sinh hc trong sn xut phn ln vi sinh vi quy m cng nghip, ng dng trn hng chc triu ha [3].

2.6. Tnh hnh nghin cu trong nc Nhm mc tiu pht trin nng nghip sinh thi bn vng. Vit Nam c kh nhiu nghin cu v vi sinh vt phn gii ln. Thp k 90 th k XX cc vi sinh vt phn gii ln sau khi c nhn sinh khi c tm nhim vo cht mang to thnh ch phm vi sinh vt phn gii ln hoc phi trn vi cht hu c to thnh phn ln hu c vi sinh vt. Kt qu nghin cu nhiu ni cho thy phn vi sinh vt phn gii photphate kh tan c th nng cao hiu qu s dng phn ln khong ln 20 30% so vi i chng ng thi c tc dng nng cao nng sut cy trng 5 10% ty loi t trng v cy trng. Vic s dng vi sinh vt phn gii ln c th thay th 30 50% lng ln cn bn bng qung phosphorit vi hm lng ln tng s tng ng m nng sut cy trng khng b gim st. Ngoi tc dng phn gii ln ,vi sinh vt phn gii ln cn c kh nng sn sinh ra cc cht kch thch sinh trng thc vt hoc cc cht khng sinh gip cy trng pht trin tt hn, chng chu tt hn i vi iu kin bt li t bn ngoi [7]. Nguyn Th Phng Chi, Phm Thanh H, Nguyn th Qunh Mai nghin cu kh nng tit enzyme photphataza ca 10 chng vi sinh vt ha tan ln v nhn thy rng ngoi kh nng ha tan ln kh tan cc chng vi sinh vt ny c kh nng sn sinh enzyme photphatase (ch yu l nm si v vi khun) enzyme ny ng vai tr xc tc khng th thiu cho qu trnh phn hy sinh hc cc hp cht hu c cha ln. V Thy Nga ,Nguyn Ngc Quyn, Trn Thy T, Phm vn Ton (2003) nghin cu kh nng sinh tng hp IAA v phn gii phosphate v c kh tan ca vi khun Bradyrhizobium. Kt qu cho thy chng c kh nng tng hp 20 100 microgam/ml mi trng nui cy. Phm Thanh H, Nguyn Th Phng Chi (1999) nghin cu nh hng ca cc ngun nit ln kh nng phn gii photphat kh tan ca hai chng nm si Aspergillus awamori MN1 v Penicillum cyaneofulvum T1.Tc gi nghin cu 7 ngun cung cp nit khc nhau ln kh nng phn gii photphat ca Aspergillus awamori MN1 v Penicillum cyaneofulvum T1. Kt qu cho thy KNO3, NaNO3, (NH4)2SO4 l nhng ngun nit tt nht cho mi trng nui chng MN1 to kh nng phn gii photphat cao. Cn i vi chng T1 l NH4Cl [10].

Nghin cu gn y nht i vi vi sinh vt phn gii ln trn t bazan nu l s dng ch phm vi sinh phn gii ln (50g) cho 1 ha c ph c tc dng tng ng vi 34.3kg P2O5/ha [13]. Nghin cu cng cho thy, vic bn thm vi sinh vt phn gii ln lm tng s lng vi sinh vt phn gii ln trong t, dn n tng cng phn gii ln kh tan trong t 23 35%.

PHN III: NI DUNG V PHNG PHP NGHIN CU 3.1 Ni dung nghin cu - Nghin cu phn lp cc chng vi sinh vt phn gii photphat kh tan trn t bazan nu ak Lak. - Nghin cu tuyn chn cc chng vi sinh vt phn gii phosphate kh tan mnh. - Nghin cu nh hng ca mi trng nui cy n hot tnh phn gii phosphate kh tan trong iu kin phng th nghim. - Nghin cu nh hng ca cc iu kin nui cy (nhit , pH, tc lc) n kh nng phn gii phosphate kh tan trong iu kin phng th nghim. - Nghin cu kho st hot tnh phn gii phosphate nhm kh tan ca cc chng vi sinh vt c tuyn chn. 3.2. Phng php nghin cu 3.2.1. Phng php thu mu a im thu mu: Ry c ph ca cc a bn sau: thn 4 - x Eahu - huyn C Kuin, thn 3 x C Su huyn C Mgar, Thn Xun Ho X Ph Xun Huyn Krng Nng. Thi gian thu mu: t ngy 29/01/2010 n ngy 31/01/2010. t c ly tng mt, su t 0 n 30cm. Lng t ly mi ln t 40 50g. Mu t c ng trong ti nilon polypropylen c kh trng. Cc mu sau khi ly c ghi nhn ni ly, ngy ly v ngi thu mu. Mu t sau khi thu c em ngay ti phng th nghim tin hnh phn lp [15]. 3.2.2. Phng php phn lp cc chng vi sinh vt phn gii phosphate kh tan Mi trng phn lp nui cy vi sinh vt chuyn ha phosphate kh tan c s dng l NBRIP [15]. NBRIP l mi trng ch cha dng phosphate kh tan nn cc vi

sinh vt mun sinh trng v pht trin c phi c kh nng s dng phosphate kh tan. Hay ni cch khc, cc vi sinh vt phi c kh nng phn gii phosphate kh tan. Thnh phn c bn ca mi trng NBRIP gm: - 5 g Ca3(PO4)2 - 0.25g MgSO4. - 0.2g KCl. - 0.1g (NH4)2SO4 - 5 gMgCl2.6 H2O. - 10g glucose - Nc 1lt. -Phn lp cc vi khun trn mi trng c a petri [8]: + t c nghin nh v phi kh. + Cn 1g t pha trong 99ml nc mui sinh l 0.9% v trng. Lc 10 pht. + Pha long dung dch t 10-2 10-5 ln. +Dng micropipette c u cn v trng ht 0.1ml dch t cc pha long cy tri trn cc a petri cha sn mi trng NBRIP. - Tuyn chn cc vi khun phn gii phosphate kh tan: sau 5 7 ngy, quan st khun lc, nhng khun lc no c vng phn gii c la chn. - m s lng t bo (CFU/ml) theo Trn Linh Thc (2007). m tt c cc khun lc xut hin trn cc a sau khi . Chn cc a c s m t 25 250 khun lc tnh kt qu. Mt vi sinh vt phn gii phosphate tng s c tnh theo cng thc sau [12]:

N n1Vf1 + + niVfi

A (CFU/ml) =

Trong :

A: s t bo (n v hnh thnh khun lc) trong 1 ml mu. N: tng s khun lc m trn c a chn. ni: s lng a cy ti pha long i. V: th tch dch mu (ml) cy vo trong mi a. fi: pha long tng ng.

- Lm thun: cy ria cc khun lc c la chn ln cc a petri cha mi trng NBRIP khc thu khun lc n. Qu trnh lm thun c tin hnh 2 3 ln. Chn khun lc n c lm thun cy truyn vo ng thch nghin gi ging. 3.2.3. Phng php nh gi hot tnh phn gii photphat kh tan ca cc chng vi sinh vt nh gi hot tnh phn gii phosphate ca cc chng vi sinh vt da trn nng PO4 c trong dch nui cy. Nng PO4 cng cao chng t lng phosphate kh tan b phn gii cng nhiu. Nng PO4 c xc nh bng phng php xanh molipdate [9]. Nguyn tc ca phng php l ion PO4 s phn ng vi (NH4)2SO4 to ra phc cht (NH4)2PO4.12MoO3 mu vng. Trong iu kin acid v c ion Sn 2+ phc mu vng s chuyn thnh phc mu xanh (NH4)3(4MoO2.2MoO3): PO43- + 12(NH4)2SO4 + 24H+ = (NH4)2PO4.12MoO3 + 21NH4+ + 12H2O (NH4)2PO4.12MoO3 + Sn2+ + 16H+ = (NH4)3(4MoO2.2MoO3) + Sn4+ + 8H2O Phc mu xanh c bc sng hp th cc i 690nm. S dng my UV Vis o bc sng 690nm xc nh hp th mu ca phc cht. hp th mu cng ln chng t nng ion PO4 cng cao.

 tnh tng quan gia ch s mt quang (OD) v nng PO4 cn thit lp phng trnh tng quan gia hai ch s bng KH2PO4. S dng dung dch KH2PO4 5mg/l pha dy nng theo bng 3.1. Bng 3.1. Bng nng KH2PO4 (mg/l) Nng PO4 (mg/l) 0 0.1 0.2 0.3 0.4 0.5 Th tch KH2PO4 5mg/l (ml) 0 2 4 6 8 10 Th tch nc ct (ml) 100 98 96 94 92 90

Thc hin phn ng xanh molipdate cc nng KH2PO4 ri o OD bc sng 690nm. ng chun v phng trnh tng quan gia ch s OD v nng mg/l c xy dng trn phn mm Excel. Chun b bnh tam gic 250ml, mi bnh cha 50ml mi trng NBRIP c kh trng. Cy cc chng vi sinh vt phn lp c vo v 5 ngy. Sau , ly 1.5ml dch mi trng nui cy em ly tm 10000rpm trong 10 pht. Ht cn thn 1ml dich pha trn lm phn ng xanh molipdate ri o OD xc nh nng PO4. Chn ra 6 chng vi sinh vt c hot tnh phn gii phosphate kh tan mnh nht tip tc th nghim.

3.2.4. Phng php quan st hnh thi khun lc v t bo - To khun lc n trn a thch: s dng k thut ria ch T ( T streak) [12]. Sau 30oC trong 72h. - Khun lc s c xc nh hnh thi theo Nguyn Ln Dng (1979). M t cc c im sau: hnh dng, kch thc, b mt, c tnh quang hc, mu sc, mt ct ngang, cu trc [6]. - Lm tiu bn t bo bng thuc nhum methyl blue v quan st bng knh hin vi quang hc phng i 40x10. 3.2.5. Phng php nghin cu mt s iu kin nh hng n kh nng phn gii phosphate kh tan ca cc chng vi sinh vt Cc th nghim u s dng mi trng NBRIP vi ngun phosphate kh tan l Ca3PO4. 3.2.3.1. Phng php nghin cu nh hng ca nhit n kh nng phn gii phosphate kh tan ca cc chng vi sinh vt Thc hin 54 ng nghim cha 10ml mi trng NBRIP. Hp kh trng cc ng nghim bng phng php nhit m 1atm trong thi gian 20 pht. Cy ging mi chng vi sinh vt vo 9 ng nghim. Chia cc ng nghim thnh 3 l. Mi l c 3 ng nghim ca mi chng vi sinh vt. Mi l th nghim c phn b nh sau: - L 1: t trong iu kin bnh thng ca phng th nghim. - L 2: t trong t m, chnh nhit ti 30oC. - L 3: t trong t m, chnh nhit ti 35oC. Sau 3 ngy nui cy, tin hnh thu dch mi trng ca cc ng nghim lm phn ng xanh molipdate, xc nh nng PO4.

3.2.3.2. Phng php nghin cu nh hng ca pH n kh nng phn gii phosphate kh tan ca cc chng vi sinh vt S dng mi trng NBRIP cho th nghim. o pH ca mi trng bng my o pH meter. iu chnh pH mi trng bng KOH 1N v HCl 1N. Th nghim trn 3 l: - L 1: S dng mi trng NBRIP khng c iu chnh pH (pH = 6.8). - L 2: Mi trng c iu chnh pH v gi tr 5.8. - L 3: Mi trng c iu chnh pH v gi tr 7.8. Mi l th nghim mi trng c phn phi vo 18 ng nghim, mi ng nghim cha 10ml mi trng. Sau hp kh trng 1atm trong 20 pht. Cy ging vi sinh vt vo 3 l. Cy mi ging vi sinh vt vo 3 ng nghim ca mt l. Sau 3 ngy nui cy, tin hnh thu dch mi trng ca cc ng nghim lm phn ng xanh molipdate, xc nh nng PO43-. 3.2.3.3. Phng php nghin cu nh hng ca tc lc n kh nng phn gii phosphate kh tan ca cc chng vi sinh vt Chun b 3 l th nghim, mi l th nghim gm 12 bnh tam gic 250ml. Phn phi vo mi bnh tam gic 50ml mi trng NBRIP, em hp kh trng 121 oC, 1atm. Cy ging vi sinh vt vo cc bnh tam gic. Mi ging cy vo 2 bnh tam gic trn 1 l. Cc l c lc cc tc khc nhau: - L 1 : trng thi tnh. - L 2 : Lc tc 75rpm. - L 3 : Lc tc 150 rpm. Sau 3 ngy nui cy, tin hnh thu dch mi trng ca cc bnh tam gic lm phn ng xanh molipdate, xc nh nng PO43-.

3.2.6. Phng php nghin cu mt s thnh phn mi trng nui cy cc chng vi sinh vt phn gii phosphate kh tan 3.2.6.1. Phng php xc nh ngun carbon thch hp cho nui cy cc chng vi sinh vt phn gii phosphate kh tan Thnh phn mi trng da trn mi trng NBRIP nhng c thay i. Cc ngun carbon s dng l glucose v saccharose. Thc hin 2 l th nghim. - L 1 : s dng ngun carbon l glucose (10g/l). Mi trng c phn phi vo 18 ng nghim, mi ng cha 10ml mi trng, em hp kh trng 121 oC v 1atm. Cy ging vi sinh vt vo cc ng mi trng, mi ging lp li 3 ln. - L 2 : S dng ngun carbon l saccharose (10g/l). Mi trng c phn phi vo 18 ng nghim, mi ng cha 10ml mi trng, em hp kh trng 121oC v 1atm. Cy ging vi sinh vt vo cc ng mi trng, mi ging lp li 3 ln. Sau 3 ngy nui cy, tin hnh thu dch mi trng ca cc bnh tam gic lm phn ng xanh molipdate, xc nh nng PO4. 3.2.6.2. Phng php xc nh ngun nit thch hp cho nui cy cc chng vi sinh vt phn gii phosphate kh tan S dng mi trng NBRIP, tuy nhin thnh phn cha nit c thay i. Cc ngun nit s dng l ur v (NH4)2SO4. Thc hin 2 l th nghim. - L 1 : s dng ngun nit l ur ( 0.053g/l). Mi trng c phn phi vo 18 ng nghim, mi ng cha 10ml mi trng, em hp kh trng 121oC v 1atm. Cy ging vi sinh vt vo cc ng mi trng, mi ging lp li 3 ln.

- L 2 : S dng ngun nit l (NH4)2SO4 (0.1g/l). Mi trng c phn phi vo 18 ng nghim, mi ng cha 10ml mi trng, em hp kh trng 121 oC v 1atm. Cy ging vi sinh vt vo cc ng mi trng, mi ging lp li 3 ln. Sau 3 ngy nui cy, tin hnh thu dch mi trng ca cc bnh tam gic lm phn ng xanh molipdate, xc nh nng PO43-. 3.2.7. Phng php th nghim hot tnh phn gii phosphate kh tan ca cc chng vi sinh vt tuyn chn c trn ngun phosphate AlPO4 S dng mi trng NBRIP vi ngun phosphate kh tan c thay i l AlPO4(3.9g/l). Thc hin 2 l th nghim: - L 1 : s dng ngun phosphate kh tan l Ca3(PO4)2. Mi trng c phn phi vo 18 ng nghim, mi ng cha 10ml mi trng, em hp kh trng 121 oC v 1atm. Cy ging vi sinh vt vo cc ng mi trng, mi ging lp li 3 ln. - L 2 : S dng ngun phosphate kh tan l AlPO4. Mi trng c phn phi vo 18 ng nghim, mi ng cha 10ml mi trng, em hp kh trng 121oC v 1atm. Cy ging vi sinh vt vo cc ng mi trng, mi ging lp li 3 ln. Sau 3 ngy nui cy, tin hnh thu dch mi trng ca cc bnh tam gic lm phn ng xanh molipdate, xc nh nng PO4. So snh hot tnh phn gii phosphate kh tan khi s dng AlPO4 vi i chng l Ca3(PO4)2 l 1. 3.2.8. Phng php x l s liu thng k Cc s liu v kh nng phn gii phosphate kh tan ca cc chng vi sinh vt c x l trn phn mm MSTATC version 2.10 ca i hc bang Michigan. Cc s liu c phn tch ANOVA 1 v c trc nghim phn hng LSD vi mc ngha 0.01.

 th tng quan v phng trnh tng quan gia ch s mt quang OD v nng PO4 c x l trn phn mm EXCELL 2007 ca Microsoft. Cc biu khc cng c x l trn phn mm ny.

PHN IV: KT QU V THO LUN 4.1. Phn lp cc chng vi sinh vt phn gii phosphate kh tan Cc mu t bazan ca 3 huyn Krng Nng, C Kuin, C M gar c nghin nh ri pha long t 10-2 n 10-7. Mi pha long ly 100 l tri u trn a thch. Kt qu cho thy pha long 10-4 10-5 cho kt qu khun lc nm trong khong 25 250. Bng 4.1. Tng mt vi sinh vt phn gii phosphate kh tan 3 mu t: Krng Nng, C Kuin, C Mgar Mu Krng Nng C Kuin C Mgar a 1 a 2 a 1 a 2 a 1 a 2 S khun lc cc pha long 10-4 10-5 185 26 209 21 217 31 27 >250 25 248 37 Tng mt vi sinh vt (CFU/ml) 2.00 x 107 2.29 x 107 2.58 x 107

Kt qu bng 4.1 cho thy tng mt vi sinh vt phn gii phosphate trong t bazan C Mgar t cao nht, gn 26 triu CFU/ml. Sau l C Kuin, gn 23 triu CFU/ml. Cn tng mt vi sinh vt phn gii phosphate trong t bazan Krng Nng thp nht: 20 triu CFU/ml. S khc bit ny c th do t l ln kh tan v d tan trong t. t cha nhiu ln kh tan th s lng vi sinh vt phn gii ln nhiu hn t cha nhiu ln d tan. T l ln kh tan trong t li ph thuc vo nhiu yu t khc nhau nh: loi t, iu kin t nhin ca tng vng, qu trnh canh tc, cch bn phn. Tuy nhin, mt vi sinh vt phn gii phosphate kh tan mi vng t khc nhau cn do nhiu nguyn nhn khc nh: bn phn vi sinh c cha vi sinh vt phn gii phosphate kh tan, ch ncDo , a ra c kt lun chnh xc v t ca ba a phng trn cn thm nhng nghin cu khc. Cc khun lc s c phn loi s b da vo hnh thi ca khun lc v hnh thi t bo ca vi sinh vt. Nhng khun lc no c c im hnh thi ging nhau s c xp vo mt chng.

Bng 4.2. Cc chng vi sinh vt phn gii phosphate kh tan phn lp Nhm vi sinh vt Vi khun X khun K hiu chng M1, M4, M5, M6, M7, M8, M10, M11, M12, M13, M16, M17 X2, X3, X9, X14, X15

Theo kt qu bng 4.2, phn lp c 17 chng vi sinh vt phn gii phosphate kh tan thuc hai nhm vi sinh vt: vi khun v x khun. Trong gm 12 chng vi khun v 5 chng x khun. xc nh s lng chng vi sinh vt phn gii phosphate kh tan mi mu t, m s chng vi sinh vt trn cc a thch. Kt qu c ghi nhn trong bng 4.3 nh sau: Bng 4.3. S chng vi sinh vt phn lp c trn cc mu t t Krng Nng, C Mgar v C Kuin. STT 1 2 3 a im ly mu Krng Nng C Mgar C Kuin K hiu chng M8, M11. M1, X2, X3, M5, M6, X9, M10, M1, M12, X14, X15, M16, M17. M4, M5, M7, M8, M11, M12, M13, X15, M17. Mu t Krng Nng phn lp c 2 chng vi sinh vt, C Kuin c 9 chng cn C Mgar c 13 chng. Nh vy, t ry C Mgar c s lng cc chng vi sinh vt phn gii phosphate kh tan nhiu nht. Chng ti gii thch iu ny do iu kin t nhin ca tng vng lc thu mu, loi t, ch phn bn tm hiu c nguyn nhn s khc bit v s lng cc chng vi sinh vt phn lp cc a phng cn phi c nhng nghin cu v c im t ca tng vng, qu trnh canh cc, cch bn phn trong iu kin gii hn ca ti khng thc hin c. 4.2 nh gi v tuyn chn cc chng vi sinh vt c hot tnh phn gii phosphate kh tan cao

4.2.1. Xy dng ng chun v phng trnh tng quan gia ch s OD v nng mg/l ca dung dch PO4 Trong nghin cu ny, chng ti s dng phng php xanh molypdate nh gi kh nng phn gii phosphate kh tan ca cc chng vi sinh vt. Do , vic dng ng chun v phng trnh tng quan gia ch s OD v nng mg/l ca dung dch PO4 l cn thit cho cc nh gi sau ny cng nh c th so snh kt qu ca nghin cu ny vi cc nghin cu khc. KH2PO4 c pha cc nng khc nhau ri thc hin phn ng xanh molipdate. Kt qu th hin qua bng 4.4 nh sau: Bng 4.4. Ch s OD ca cc nng KH2PO4 khc nhau. STT 1 2 3 4 5 6 Nng KH2PO4 (mg/l) 0.5 0.4 0.3 0.2 0.1 0 Ch s OD 690nm 0.923 0.725 0.560 0.368 0.180 0

ng chun v phng trnh tng quan gia ch s OD v nng mg/l ca dung dch PO4 c th hin qua th 4.1.

Biu 4.1. ng chun v phng trnh tng quan gia ch s OD v nng mg/l ca dung dch PO4.

Theo th 4.1, phng trnh tng quan gia ch s OD v nng mg/l: y=1.84x Trong : x l nng PO4 (mg/l). y l ch s OD. 4.2.2. nh gi v tuyn chn cc chng vi sinh vt c hot tnh phn gii phosphate kh tan cao Cc chng vi sinh vt khc nhau c kh nng phn gii phosphate kh tan khng ging nhau. V vy, nh gi kh nng phn gii ca tng chng l rt cn thit. Chng ti tin hnh nui 17 chng vi sinh vt trong mi trng NBRIP trong 5 ngy, sau xc nh nng PO4 c phn gii. Kt qu v kh nng phn gii phosphate kh tan ca 17 c ghi nhn trong bng 4.5.

Bng 4.5. Hot tnh phn gii phosphate kh tan ca 17 chng vi sinh vt STT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 K hiu chng M1 X2 X3 M4 M5 M6 M7 M8 X9 M10 M11 M12 M13 X14 X15 M16 M17 Nng PO4 mg/l 17.93a 11.89bc 20.37a 0.09h 13.05b 5.67ef 18.09a 6.59def 8.97cd 1.23gh 2.16gh 7.83de 6.54def 1.13gh 14.26b 0.36h 3.87fg

(Cc tr s c cc ch ci ging nhau cng mt ct khng c s khc bit theo trc nghim phn hng LSD vi mc ngha 0.01)

Qua kt qu bng 4.5 chng ti xc nh kh nng phn gii phosphate kh tan ca 17 chng vi sinh vt nh sau: kh nng phn gii phosphate kh tan ca 17 chng vi sinh vt c chia thnh 8 nhm theo th t t cao n thp: a, b, c, d, e, f, g v h. Trong chng X3 c nng PO4 phn gii cao nht 20.37mg/l. Chng M4, M10, M11 v M16 cng cho kh nng phn gii phosphate thp nht h, nng PO4 m hai chng M16, M4 phn gii ln lt l 0.36mg/l, 0.04mg/l. So snh kt qu th nghim vi nghin cu ca Henri v cng s (2008) v kh nng phn gii phosphate kh tan ca Pseudomonas fluorescens sau 5 ngy nui cy pH bng 6.3 l 15.25mg/l [20], chng ti nhn thy c th tuyn chn mt s chng c hot tnh phn gii phosphate kh tan cao tip tc th nghim. Kh nng phn gii phosphate kh tan ca cc chng vi sinh vt c th hin qua biu sau:

a a bcc b ef h def cd gh de gh def fg gh h a b

Biu 4.2. Kh nng phn gii phosphate kh tan ca 17 chng vi sinh vt Nh vy, xc nh c 6 chng vi sinh vt c hot tnh phn gii phosphate kh tan cao nht l M1, X2, X3, M5, M7, X15. Trong X3, M7, M1 c kh nng phn gii cao nht a. Sau l hai chng M5, X15, X2 c mc phn gii phosphate thp hn b, bc. Cc chng ny s c chng ti s dng cho cc th nghim sau. 4.3. c im sinh hc ca cc chng vi sinh vt tuyn chn Su chng vi sinh vt tuyn chn s c cy ria ch T trn a petri. Sau 3 ngy, chng ti quan st cc c im khun lc ca cc chng v lm tiu bn t bo xem trn knh hin vi. Cc khun lc s c quan st trn vt knh 4x v 10x. Tiu bn t bo c quan st vt knh 40x, 100x. Hnh thi khun lc ca 6 chng vi sinh vt c m t trong bng 4.6. Bng 4.6. Hnh thi khun lc ca 6 chng vi sinh vt tuyn chn

K hiu chng M1 X2 X3 M5 HD Khng u Dng r Dng r Trn Trn M7 rng ca Dng r

Hnh thi khun lc sau 3 ngy nui cy B TQ Mu KT MKL MCN mt H sc Ln Vng 1cm Trn c Li sng nht 2 3 Gh Li c cnh Nu cm gh cong Gh Li 4 cm c Si Trng gh cong Gh Bng 2cm c Trng Phng gh phng 3cm Gh gh Gh gh c Ln sng Si Trng Li

CT ht nh Si Si Ht ln ng nht Si

X15

1cm

Trng

Phng

Hnh thi t bo ca cc chng vi sinh vt tuyn chn c m ta trong bng 4.7. Bng 4.7. Hnh thi t bo ca 6 chng vi sinh vt tuyn chn c. K hiu chng M1 X2 Hnh thi t bo sau 3 ngy nui cy Hnh dng Kch thc Hnh cu 1 - 2m Khun ti khng vch ngn, Khun ti kh sinh: 0.5 - 1m. cung sinh bo t xon, mc Cung sinh bo t:0.5 - 1m x 10 vng - 20m Khun ti khng vch ngn, Khun ti kh sinh: 1.5 - 2m. X3 M5 M7 X15 cung sinh bo t nhnh Cung sinh bo t: 1.5 - 2m x thng, mc n. Hnh cu Hnh que Khun ti khng sng. 15 - 20m. 3 - 4m. 5 - 6m x 2 - 3m. vch Khun ti kh sinh: 0.5 - 1m. - 40m.

ngn,cung sinh bo t ln Cung sinh bo t:0.5 - 1m x30

4.4. Nghin cu mt s iu kin nh hng n kh nng phn gii phosphate kh tan ca cc chng tuyn chn 4.4.1. nh hng ca nhit n kh nng phn gii phosphate kh tan ca cc chng tuyn chn Mi c th vi sinh vt u chu tc ng ca nhit ba gii hn khc nhau: nhit thp nht, nhit ti thch v nhit cao nht. Chnh v vy nghin cu nh hng ca nhit n kh nng phn gii phosphate ca cc chng vi sinh vt l cn thit. Chng ti tin hnh nui cy vi sinh vt trn mi trng NBRIP trong trng thi tnh 3 mc nhit khc nhau: nhit thng, 30oC, 35oC. Sau 72h nui cy, em ly tm dch mi trng ri thc hin phn ng xanh molypdate v o OD bc sng 690nm. Kt qu c trnh by trong bng 4.8.

Bng 4.8. nh hng ca nhit n kh nng phn gii phosphate kh tan ca 6 chng vi sinh vt c tuyn chn. Nhit 270C K hiu chng M1 X2 X3 M5 M7 X15 2.09f 1.38g 2.79e 3.12e 1.41g 1.09g 30oC 2.81e 2.17f 2.99e 4.2d 2.73e 1.34g 35oC 5.41c 6.29b 5.76c 5.75c 7.77a 6.52b

(Cc tr s c cc ch ci ging nhau cng mt ct khng c s khc bit theo trc nghim phn hng LSD vi mc ngha 0.01)

a b c e f g e e c d e g f e g g c b

Biu 4.2. nh hng ca nhit ln kh nng phn gii phosphate kh tan ca 6 chng vi sinh vt tuyn chn. Qua bng 4.8 cho thy c 6 chng vi sinh vt cc mc nhit khc nhau th u c s khc bit v kh nng phn gii phosphate kh tan. S khc bit th hin r trong biu 2. Theo biu 2, kh nng phn gii phosphate ca 6 chng vi sinh vt mnh nht nhit 35oC v thp nht nhit bnh thng. iu ny cho thy cc vi sinh vt mi vng khc nhau ph hp vi iu kin kh hu ca vng [9]. Cc chng

vi sinh vt c phn lp trn a bn tnh ak lak, ni c nhit hng ngy cao [22], nn c kh nng sinh trng v phn gii phosphate kh tan tt nhit cao. 4.4.2. nh hng ca pH n kh nng phn gii phosphate kh tan ca cc chng tuyn chn ngha nghin cu nh hng ca pH l tuyn chn chng ph hp vi chua ca tng loi t hoc xut bin php ci to chua ca t nng cao hot tnh phn gii phosphate kh tan ca vi sinh vt t. Trong nghin cu ny, chng ti kho xc nh hng ca pH n kh nng phn gii phosphate ca cc chng vi sinh vt 3 gi tr pH khc nhau: 5.8, 6.8 v 7.8. Cc th nghim c trng thi tnh. Sau 72h nui cy, chng ti thu dch mi trng, thc hin phn ng molypdate v o OD bc sng 690nm, kt qu thu c c trnh by trong bng 4.9. Bng 4.9. nh hng ca pH n kh nng phn gii phosphate kh tan ca 6 chng vi sinh vt c tuyn chn. pH pH = 5.8 K hiu chng M1 X2 X3 M5 M7 X15
phn hng LSD vi mc ngha 0.01)

pH = 6.8 3.07d 2.34f 3.18cd 3.15d 3.59c 1.77g

pH = 7.8 13.16a 2.55ef 2.56ef 3.16d 1.82g 2.88de

2.35f 2.25f 2.94de 3.09d 6.59b 1.11h

(Cc tr s c cc ch ci ging nhau cng mt ct khng c s khc bit theo trc nghim

Qua bng 4.9 chng ti xc nh: cc chng X2, M5, X3 cc pH khc nhau th kh nng phn gii phosphate kh tan l khng khc bit. Cc chng M1, M7, X15 c s khc bit r rt v kh nng phn gii phosphate cc pH khc nhau. Hai chng M1v X15 pH 7.8 c kh nng phn gii phosphate kh tan cao nht v thp nht pH 5.8. Chng M7 c kh nng phn gii phosphate kh tan tt nht pH 5.8 v thp nht 7.8 iu th hin biu 4.3:

b d f cd ef c g h g de

ef f f

de

d d d

Biu 4.3. nh hng ca pH n kh nng phn gii phosphate kh tan ca 6 chng vi sinh vt tuyn chn. Nh vy, nh hng ca pH ln kh nng phn gii phosphate kh tan ca 6 chng vi sinh vt tuyn chn l khc nhau. Chng M1, X15 c hot tnh phn gii phosphate kh tan tt pH kim. Trong khi chng M7 li c hot tnh phn gii tt mi trng acid. Ba chng X2, X3, M5 a ti suy ngh c th pH nh hng n kh nng phn gii phosphate kh tan ca chng nm gi tr khc ngoi khong pH 5.8 - 7.8. 4.4.3. nh hng ca tc lc n kh nng phn gii phosphate kh tan ca cc chng tuyn chn Cc loi vi sinh vt khc nhau c nhu cu vi O2 khng ging nhau. Nhng vi sinh vt hiu kh cn nhiu O2 hot ng. Ngc li, nhng vi sinh vt k kh khng cn hoc cn rt t O2. Do , trng thi nui cy s c nh hng n vi sinh vt v n kh nng phn gii phosphate ca vi sinh vt. Chng ti tin hnh th nghim nh hng ca tc lc n kh nng phn gii phosphate ca vi sinh vt 3 mc khc nhau: 0rpm, 75rpm, 150pm. Kt qu thu c trong bng sau. Bng 4.10. nh hng ca tc lc n kh nng phn gii phosphate kh tan ca 6 chng vi sinh vt c tuyn chn.

Tc lc 0 rpm K hiu chng M1 X2 X3 M5 M7 X15 1.03g 1.42fg 3.75b 1.72efg 2.1def 2.39cde 75 rpm 1.42fg 1.49fg 2.11def 2.4cde 1.74ef 3.14bc 150 rpm 7.25a 2.13def 1.76efg 1.08g 1.02g 2.78cd

(Cc tr s c cc ch ci ging nhau cng mt ct khng c s khc bit theo trc nghim phn hng LSD vi mc ngha 0.01)

T bng 10, xy dng biu 4.4 v nh hng ca tc lc n kh nng phn gii phosphate kh tan ca cc chng vi sinh vt. Theo kt qu trn, nh hng ca tc lc n kh nng phn gii ca cc chng vi sinh vt l khc nhau. iu th hin r trong biu 4.4. a

b def fg fg defefg cde efg g def ef g

bc cd cde

fg

Biu 4.4. nh hng ca tc lc n kh nng phn gii phosphate kh tan ca 6 chng vi sinh vt tuyn chn. Qua biu 4.4, chng ti nhn xt: su chng vi sinh vt u c kh nng phn gii phosphate kh tan iu kin tnh v lc. Nh vy c 6 chng vi sinh vt u cn oxy sinh trng. Do , chng l nhng vi sinh vt hiu kh hoc k kh khng bt

buc. Tuy nhin, nhu cu oxy ca mi chng l khc nhau. Chng M1 c kh nng phn gii phosphate kh tan cao tc lc 150rpm, tc cn s thng kh mnh. Trong khi , chng X3 li c kh nng phn gii phosphate cao khi trng thi tnh, tc khng cn s thng kh. C th chng X3 thuc nhm vi sinh vt k kh khng bt but. Chng M5 c kh nng phn gii phosphate kh tan cao khi lc tc 75rpm. iu ny c ngha khi nui cy chng M5 khng cn s thng kh mnh. Tng t, chng M7 c hot tnh phn gii phosphate kh tan cao khi iu kin lc 75rpm hoc trng thi tnh. Do nui cy chng ny cng khng cn thng kh mnh. Hai chng X2 v X15 khng c s khc bit v kh nng phn gii phosphate kh tan khi lc cc tc khc nhau. Ngha l hai chng ny kh nng phn gii phosphate kh tan khng ph thuc vo lng oxy. C th chng thuc nhm vi sinh vt k kh khng bt buc. Khi nui cy hai chng ny khng cn s thng kh.

4.5. Nghin cu nh hng ca thnh phn mi trng n kh nng phn gii phosphate ca chng vi sinh vt tuyn chn 4.5.1. nh hng ca ngun carbon thch hp cho nui cy cc chng vi sinh vt phn gii phosphate kh tan Thc hin hai l th nghim: l 1 s dng glucose, l 2 s dng saccharose. Kt qu thu c trong bng 4.11. Bng 4.11. nh hng ca ngun carbon n kh nng phn gii phosphate kh tan ca 6 chng vi sinh vt c tuyn chn Ngun Carbon Glucose K hiu chng M1 X2 X3 M5 M7 X15 1.1e 0.29g 6.23a 1.08e 1.6d 0.4fg Saccharose 0.78ef 4.64b 1.95d 3.95c 0.99e 0.03g

(Cc tr s c cc ch ci ging nhau cng mt ct khng c s khc bit theo trc nghim phn hng LSD vi mc ngha 0.01)

Theo bng 11, cc chng u c kh nng phn gii phosphate kh tan khi s dng ngun carbon l glucose. Trong , hai chng X3 v M7 c kh nng phn gii phosphate tt nht khi s dng ngun ng l glucose. Hai chng M1, X15 khng c s khc bit v nng PO4 phn gii khi s dng glucose v saccharose. Cn X2 v M5 li c kh nng phn gii phosphate tt nht khi s dng ngun ng l saccharose. Kt qu th hin r trong biu 4.5.

a b

c d

ef

e g

e fg g

Biu 4.5. nh hng ca ngun carbon n kh nng phn gii phosphate kh tan ca 6 chng vi sinh vt c tuyn chn. Tt c chng vi sinh vt u c kh nng phn gii phosphate kh tan khi s dng glucose c gii thch do glucose l ng n, vi sinh vt d dng s dng lm ngun dinh dng cho mnh. Tuy nhin, i vi hai chng X2 v M5 ngun carbon thch hp cho kh nng phn gii phosphate li l saccharose. iu ny c th do chng s dng ng fructose c trong saccharose hiu qu hn so vi glucose. Chng X15 gn nh khng c kh nng phn gii phosphate kh tan khi s dng ng saccharose, chng t ng ny khng phi l ngun carbon thch hp cho chng. 4.5.2. Xc nh ngun nit thch hp cho nui cy cc chng vi sinh vt phn gii phosphate kh tan. Vi sinh vt c th s dng nit t nhiu ngun khc nhau. Tuy nhin, khng phi ngun nit no cng thch hp cho vi sinh vt pht trin. V vy, xc nh ngun nit thch hp cho nui cy vi sinh vt l rt cn thit. Tin hnh 2 l th nghim khc nhau, s dng 2 ngun nit l ur v (NH4)2SO4. Kt qu th hin trong bng 4.12.

Bng 4.12. nh hng ca ngun nit n kh nng phn gii phosphate kh tan ca 6 chng vi sinh vt c tuyn chn. Ngun nit (NH4)2SO4 K hiu chng M1 X2 X3 M5 M7 X15
phn hng LSD vi mc ngha 0.01)

Ur 4.99b 0.51ef 0.01g 5.33b 0.78de 0.6ef

1.1d 0.29fg 6.23a 1.08d 1.6c 0.4efg

(Cc tr s c cc ch ci ging nhau cng mt ct khng c s khc bit theo trc nghim

Kt qu bng 4.12 cho thy cc chng vi sinh vt khc nhau th c ngun nit thch hp khc nhau. iu th hin trong biu sau: a b b

d fg ef
M1

d g
M5

c de efg ef

M7

Biu 4.6. nh hng ca ngun nit n kh nng phn gii phosphate kh tan ca 6 chng vi sinh vt c tuyn chn. Qua biu 4.6, chng ti nhn xt: su chng vi sinh vt phn lp u c kh nng phn gii phosphate kh tan khi s dng (NH4)2SO4. iu ny c gii thch nh sau: mui (NH4)2SO4 trong mi trng nui cy s phn ly ra ion NH4+, l dng c cht d

s dng cho cc vi sinh vt. i vi ur, cc vi sinh vt phi c enzyme urease phn gii ur th mi s dng c c cht ny. Cho nn khng phi vi sinh vt no cng c kh nng s dng ur nh ngun c cht. iu th hin biu 6, chng X3 gn nh khng c kh nng phn gii phosphate kh tan khi s dng ur l ngun cung cp nit duy nht, chng t chng X3 khng c enzyme urease. Nm chng cn li c kh nng phn gii phosphate kh tan khi s dng ur, chng t chng tit ra enzyme urease. Nhn xt i vi tng chng: chng M1 v M5 c kh nng phn gii phosphate kh tan khi s dng ur cao hn so vi s dng (NH4)2SO4. Nh vy, ur l ngun c cht tt cho kh nng phn gii phosphate kh tan ca hai chng ny. Chng X3 v M7 th ngc li, ngun c cht tt nht cho kh nng phn gii phosphate kh tan l (NH4)2SO4. Cn X2 v X15 khng c s khc nhau v kh nng phn gii phosphate kh tan khi s dng ngun c cht l (NH4)2SO4 v ur. 4.6. Th nghim hot tnh phn gii phosphate kh tan ca cc chng vi sinh vt tuyn chn trn ngun phosphate nhm t bazan nu c hm lng AlPO4 cao [21]. y l dng phosphate rt kh tan [13]. Do , tm ra chng vi sinh vt phn gii c dng phosphate kh tan ny c ngha quan trng. Chng ti tin hnh th nghim trn 2 l: l1 s dng Ca 3(PO4)2 lm i chng. L 2 s dng AlPO4. Kt qu th hin trong bng 4.13.

Bng 4.13. Kh nng phn gii phosphate kh tan ca 6 chng vi sinh vt c tuyn chn trn ngun phosphate l Ca3(PO4)2 v AlPO4 . Ngun phosphate Ca3(PO4)2 K hiu chng M1 X2 X3 M5 M7 X15 5.09a 0.61fgh 2.43c 1.53e 2.04d 3.44b
phn hng LSD vi mc ngha 0.01).

AlPO4 2.16cd 0.45gh 0.88f 0.77fg 0.32hi 0.10i

( Cc tr s c cc ch ci ging nhau cng mt ct khng c s khc bit theo trc nghim

Qua bng 4.13 cho thy kh nng phn gii phosphate kh tan ca cc chng u thp hn i chng. Kt qu ny ph hp vi Perez (2007): phosphate to thnh phc hp khng tan vi Al lm cho qu trnh phn gii AlPO4 kh hn so vi Ca3(PO4)2 [15].

b cd fgh c e gh f fg hi i d

Biu 4.7. Kh nng phn gii phosphate kh tan ca 6 chng vi sinh vt c tuyn chn trn ngun phosphate l Ca3(PO4)2 v AlPO4 . Qua biu 4.7, chng ti nhn xt: chng M1 c kh nng phn gii AlPO4 cao nht, sau l X3, M5, X2, M7 chng X15 c kh nng phn gii thp nht. Tuy nhin, kh nng phn gii phosphate ca cc chng u rt thp so vi kt qu nghin cu ca Henri (2008) trn Pseudomonas fluorescen [19]. Gii thch iu ny, c th do cc chng vi sinh vt tuyn chn tit rt t cc cht phn gii AlPO4. Nhng nghin cu ca Henri (2008) gi kh nng phn gii AlPO4 ph thuc rt nhiu vo pH: Pseudomonas fluorescens phn gii AlPO4 rt km pH bng 6 7, sau tng rt mnh pH bng 2.5 3.5 [19]. iu cho thy, mun tm hiu r hn v kh nng phn gii AlPO 4 ca cc chng vi sinh vt phn lp cn phi th nghim cc pH khc nhau.

PHN V: KT LUN V KIN NGH

5.1. Kt lun Qua qu trnh nghin cu chng ti rt ra cc kt lun sau: - phn lp c 17 chng vi sinh vt t cc mu t bazan nu ca 3 hun Krng Nng, C Mgar, C Kuin, trong c 12 chng vi khun v 5 chng x khun. - Tuyn chn 6 chng vi sinh vt (3 chng vi khun v 3 chng x khun) c kh nng phn gii phosphate kh tan mnh nht l M1, X2, X3, M5, X15. - Nhit c nh hng ln n kh nng phn gii phosphate kh tan ca 6 chng vi sinh vt, nhit thch hp cho 6 chng vi sinh vt tuyn chn l 35oC. - Kh nng phn gii phosphate kh tan ca cc chng vi sinh vt chu nh hng ca pH l khc nhau. Chng X2, M5, X3 t b nh hng khi pH thay i. i vi 4 chng cn li th M1, X15 c pH thch hp l 7.8 cn vi chng M7 l 5.8. - nh hng ca tc lc n kh nng phn gii ca cc chng vi sinh vt l khc nhau. X2 v X15 khng chu nh hng ca tc lc n kh nng phn gii phosphate kh tan . M1 c kh nng phn gii phosphate kh tan cao nht tc lc 150rpm, M5 l tc 75rpm, cn X3 v M7 th trng thi tnh. - Ngun carbon thch hp cho cc chng vi sinh vt l khc nhau. X3 v M7 c kh nng phn gii phosphate kh tan tt nht khi s dng ngun ng l glucose. Cn X2 v M5 li c kh nng phn gii phosphate tt nht khi s dng ngun ng l saccharose. Cc chng M1, X15 khng c s khc bit v kh nng phn gii phosphate kh tan khi s dng glucose v saccharose. - Ngun nit thch hp cho mi chng vi sinh vt l khc nhau. Hai chng X3, M7 c kh nng phn gii phosphate kh tan tt khi s dng ngun nit l (NH4)2SO4. Chng M1, M5, th kh nng phn gii phosphate kh tan tt hn khi s dng ngun nit l ur. Cn i vi X2 v X15 khng c s khc bit v kh nng phn gii phosphate khi s dng 2 ngun nit trn.

- Kh nng phn gii AlPO4 ca cc chng thp hn nhiu so vi kh nng phn gii Ca3(PO4)2. Chng M1 c kh nng phn gii AlPO4 cao nht. 5.2 Kin ngh. - Phn lp cc chng vi sinh vt phn gii phophate kh tan trn cc a bn c t bazan nu khc. - Tip tc nghin cu cc ch tiu c th i vi tng chng vi sinh vt c tuyn chn. - Xy dng quy trnh nhn nui mt s chng c kh nng phn gii phosphate cao. - Th nghim hot tnh phn gii phosphate kh tan trn ngun phosphate l FePO4. - Th nghim hiu qu ca cc chng vi sinh vt tuyn chn trn ng rung. - ng dng cc chng vi sinh vt phn gii phosphate kh tan trong sn xut phn vi sinh.

TI LIU THAM KHO

Ti liu ting Vit
1.

Bch Phng Lan. 2004. Gio trnh hot tnh vi sinh vt t. Trng i

hc Lt. 2.
3.

Bi Trang Vit. Sinh l thc vt 1. NXB HQG H Ch Minh. 2002. Minh.D, Anh.V.T. Vi sinh vt t. Gio trnh ging dy trc tuyn. Trng

i hc Cn Th.
4.

Nguyn Hu Hip. 2009. Bi ging vi sinh nng nghip. Vin nghin cu

v pht trin cng ngh sinh hc Cn Th. 5.

6.

Nguyn Kim Phng. Bi ging nng ha. Trng i hc Ty Nguyn. Nguyn Ln Dng. Phm Vn Ty. Dng c Tin. 1979. Vi sinh vt hc

tp 1. NXB i hc v Trung hc chuyn nghip.

7.

Nguyn Th Phng Chi, Phm Thanh H. 1999. Phi hp cc chng vi

khun c nh nit v vi khun ha tan photphate nng cao hiu qu phn vi sinh vt. Bo co khoa hc Hi ngh Cng ngh Sinh hcTon Quc. H Ni 1999. NXB KHKT. Trang 428 433
8.

Nguyn Xun Thnh, Gio trnh vi sinh vt hc cng nghip. NXB gio

dc. 2006.
9.

Phm Thanh H, Nguyn Th Qunh Mai, H Th Kim Anh, Nguyn Th

Phng Chi. 2003. nh hng ca nhit i vi vi sinh vt ha tan phosphate. Nhng vn nghin cu c bn trong khoa hc s sng. Bo co hi ngh CNSH ton quc. NXB KH& KT H Ni. 381 383.
10.

Phm Thanh H. Nguyn Th Phng Chi(1999) nh hng ca cc ngun

nit ln kh nng phn gii photphat kh tan ca hai chng nm si MN1 v T1. Nhng

vn nghin cu c bn trong sinh hc. Bo co hi ngh CNSH ton quc. NXB KH& KT H Ni. 434 440. 11. Phm Th Ngc Lan, Trng Vn Lung (2003). nh hng ca vic s

dng nm si phn gii ln kh tan n mt s ch tiu sinh l v sinh ha ca la Khang Dn v IR 17494. Nhng vn nghin cu c bn trong khoa hc s sng. Bo co hi ngh CNSH ton quc. NXB KH& KT H Ni. 308 312.
12.

Trn Linh Thc. 2007. Phng php phn tch vi sinh vt trong nc. thc

phm v m phm. NXB Gio Dc.

13.

V Th Li. 2006. Nghin cu nui cy v kh nng phn gii ln kh tan

ca vi khun Bacillus megaterrium. Lun vn tt nghip. Trng i hc Ty nguyn.

14.

V Thy Nga, Nguyn Ngc Quyn, Trn T Thy. Phm Vn Ton

(2003). Nghin cu kh nng sinh tng hp IAA v phn gii photphat v c kh tan ca vi khun Bradyrhizobium. Nhng vn nghin cu c bn trong khoa hc s sng. Bo co hi ngh CNSH ton quc. NXB KH& KT H Ni. tr.349 352. Ti liu nc ngoi 15. Elizabeth Perez. Miguel Sulbaran. Mara M. Ball. Luis Andres Yarza

bal. 2007. Isolation and characterization of mineral phosphate-solubilizing bacteria naturally colonizing a limonitic crust in the south-eastern Venezuelan region. Soil Biology & Biochemistry 39. 29052914. 16. F. Gil-Sotres, C. Trasar-Cepeda, M.C. Leiro s, S. Seoane, Different

approaches to evaluating soil quality using biochemical properties, Soil Biol. Biochem. 37 (2005). 877887.
17.

P. Alvaro, Elke Lang, Susanne Verbarg, Cathrin Sproer, Raul RivasJunli.

2009. Acinetobacter strains IH9 and OCI1, two rhizospheric phosphate solubilizing isolates able to promote plant growth, constitute a new genomovar of Acinetobacter calcoaceticus. Microbiol. (2009).

18.

Kapoor. K.K. 2006. Phosphate mobilization through soil microorganism.

Phant microbe. Interaction in sustainable agricultuare. Eds: R.K behl, A.L. Khurane, G.C. Dogra chau, Hisar & MMb, New Dehli, 46 61.
19.

FANKEM Henri, NGO NKOT Laurette, DEUBEL Annette, QUINN John,

MERBACH Wolfgang, ETOA Franois-Xavier and NWAGADieudonn. 2008. Solubilization of inorganic phosphates and plant growth promotion by strains of Pseudomonas fluorescens isolated from acidic soils of Cameroon. African Journal of Microbiology Research. Vol.(2) pp. 171-178.
20.

Reynaldo Fraga, Hilda Rodrguez , Reynaldo Fraga. 1999. Phosphate

solubilizing bacteria and their role in plant growth promotion. Biotechnology Advances 17 (1999), 319339. Ti liu tham kho Internet
21.

http://bk.dakdak.gov.vn/index.php?

option=com_content&task=view&id=86&Itemid=128
22.

http://www.laodong.com.vn/Home/Chat-luong-nuoc-suy-giam-do-su-dung-

phan-bon-bua-bai/20091/121260.laodong

PH LC 1. Cc ph bng Ph bng 1: Kh nng phn gii phosphate kh tan ca 17 chng vi sinh vt. K hiu Nng PO4 mg/l Ln 1 17.23 16.73 20.77 0.05 13.05 5.32 18.43 7.02 8.60 0.97 2.01 7.82 6.39 0.60 14.20 0.30 4.10 Ln 2 17.38 12.12 20.00 0.15 13.04 6.38 18.14 6.11 9.42 1.38 2.60 7.85 6.79 1.57 15.36 0.53 3.30 Ln 3 17.55 6.82 20.34 0.06 13.05 5.59 17.70 6.64 8.90 1.34 1.87 7.83 6.45 1.22 13.20 0.26 4.20

chng M1 X2 X3 M4 M5 M6 M7 M8 X9 M10 M11 M12 M13 X14 X15 M16 M17 Bng ANOVA 1
Function: ANOVA-1 Data case no. 1 to 51

One way ANOVA grouped over variable 1 (nt) with values from 1 to 17. Variable 2 (PO4) ANALYSIS OF VARIANCE TABLE Degrees of Freedom Sum of Squares Mean Square F-value Prob.

--------------------------------------------------------------------------Between Within 16 34 2091.403 55.022 130.713 1.618 80.771 0.0000

--------------------------------------------------------------------------Total 50 2146.425

Coefficient of Variation = 15.49% Var. 1 V A R I A B L E No. 2 Number Sum Average SD SE

-----------------------------------------------------------------1 2 3 4 5 6 7 8 9 10 11 12 13 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 52.160 35.670 61.110 0.260 39.140 17.290 54.270 19.770 26.920 3.690 6.480 23.500 19.630 17.387 11.890 20.370 0.087 13.047 5.763 18.090 6.590 8.973 1.230 2.160 7.833 6.543 0.16 4.96 0.39 0.06 0.01 0.55 0.37 0.46 0.41 0.23 0.39 0.02 0.22 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.73 0.73

14 15 16 17

3.00 3.00 3.00 3.00

3.390 42.760 1.090 11.600

1.130 14.253 0.363 3.867

0.49 1.08 0.15 0.49

0.73 0.73 0.73 0.73

-----------------------------------------------------------------Total Within 51.00 418.730 8.210 1.27 6.55 0.92

Trc nghim phn hng kh nng phn gii phosphate kh tan ca 17 chng vi sinh vt phn lp c.
Function : RANGE Error Mean Square = 1.618 Error Degrees of Freedom = 16 No. of observations to calculate a mean = 3 Least Significant Difference Test LSD value = 3.033 Original Order Mean Mean Mean Mean Mean Mean Mean Mean 1= 2= 3= 17.39 A 11.89 BC 20.37 A H at alpha = 0.010 Ranked Order Mean Mean Mean 3= 7= 1= 20.37 A 18.09 A 17.39 A 14.25 B 13.05 B 11.89 BC 8.970 7.830 CD DE

4 = 0.09000 5= 6= 7= 8= 13.05 B 5.760 18.09 A 6.590 EF

Mean 15 = Mean Mean Mean 5= 2= 9=

DEF

Mean 12 =

Mean

9=

8.970 1.230 2.160 7.830 6.540 1.130

CD GH GH DE DEF GH

Mean

8=

6.590 6.540 5.760 3.870 2.160 1.230 1.130

DEF DEF EF FG GH GH GH H H

Mean 10 = Mean 11 = Mean 12 = Mean 13 = Mean 14 = Mean 15 =

Mean 13 = Mean 6=

Mean 17 = Mean 11 = Mean 10 = Mean 14 = H

14.25 B

Mean 16 = 0.3600 Mean 17 = 3.870

Mean 16 = 0.3600 Mean 4 = 0.09000

FG

Ph bng 2: nh hng ca nhit n kh nng phn gii phosphate kh tan ca cc chng vi sinh vt phn lp. K hiu chng M1 X2 X3 M5 M7 X15 Ln 1 2.06 1.24 2.8 3.05 1.42 0.98 Ln 2 2.13 1.47 2.76 3.21 1.4 1.2 Ln 3
2.08 1.43 2.81 3.1 1.41 1.09

Nhit phng

30oC Ln 1
2.79 2.09 3.12 4.13 2.68 1.05

35oC Ln 2
2.95 2.19 2.84 4.37 2.51 1.73

Ln 3
2.69 2.23 3.01 4.1 3 1.24

Ln 1
5.39 6.3 5.73 5.7 7.06 6.49

Ln 2
5.5 6.35 5.79 5.83 7.82 6.71

Ln 3
5.34 6.22 5.76 5.72 8.43 6.36

Trc nghim phn hng nh hng ca nhit n kh nng phn gii phosphate kh tan ca 6 chng vi sinh vt tuyn chn.
Function : RANGE Error Mean Square = 0.04500 Error Degrees of Freedom = 17 No. of observations to calculate a mean = 3 Least Significant Difference Test

LSD value = 0.5020 Original Order Mean Mean Mean Mean Mean Mean Mean Mean Mean 1= 2= 3= 4= 5= 6= 7= 8= 9= 2.090 2.810 5.410 1.380 2.170

at alpha = 0.010 Ranked Order F E C G F Mean 15 = Mean 18 = Mean Mean 6= 9= 7.770 A 6.513 B 6.290 B 5.760 5.750 5.410 4.200 3.120 2.990 2.810 2.790 2.730 2.170 2.090 1.410 1.380 1.340 C C C D E E E E E F F G G G

Mean 12 = Mean 3=

6.290 B 2.790 2.990 5.760 3.120 4.200 5.750 1.410 2.730 7.770 A 1.090 1.340 G G E E E C E D C G

Mean 11 = Mean 10 = Mean Mean Mean 8= 2= 7=

Mean 10 = Mean 11 = Mean 12 = Mean 13 = Mean 14 = Mean 15 = Mean 16 = Mean 17 =

Mean 14 = Mean Mean 5= 1=

Mean 13 = Mean 4=

Mean 17 =

Ph bng 3: nh hng ca pH n kh nng phn gii phosphate kh tan ca 6 chng v sinh vt tuyn chn c. K hiu chng M1 X2 Ln 1 2.31 2.2 Ln 2 2.46 2.73 Ln 3 2.28 1.82 Ln 1 2.97 2.21 Ln 2 3.15 2.56 Ln 3 3.09 2.25 Ln 1 13.29 2.47 Ln 2 12.86 2.7 Ln 3 13.33 2.48 pH = 5.8 pH = 7.8 pH = 8.8

X3 M5 M7 X15

3.15 3.11 6.47 1.03

2.86 3.05 6.74 1.42

2.81 3.11 6.56 0.88

3.02 3.14 3.42 1.69

3.19 3.19 3.65 1.82

3.33 3.12 3.7 1.8

2.58 3.01 1.97 2.75

2.53 3.27 1.81 3.02

2.57 3.2 1.68 2.87

Trc nghim phn hng nh hng ca pH n kh nng phn gii phosphate kh tan ca 6 chng vi sinh vt tuyn chn.
Function : RANGE Error Mean Square = 0.03300 Error Degrees of Freedom = 17 No. of observations to calculate a mean = 3 Least Significant Difference Test LSD value = 0.4299 Original Order Mean Mean Mean Mean Mean Mean Mean Mean Mean 1= 2= 3= 4= 5= 6= 7= 8= 9= 2.350 3.070 13.16 A 2.250 2.340 2.550 2.940 3.180 2.560 3.090 3.150 3.160 F F EF DE CD EF D D D F D at alpha = 0.010 Ranked Order Mean 3= 13.16 A 6.590 B 3.590 3.180 3.160 3.150 3.090 3.070 2.940 2.880 2.560 2.550 C CD D D D D DE DE EF EF

Mean 13 = Mean 14 = Mean 8=

Mean 12 = Mean 11 = Mean 10 = Mean Mean 2= 7=

Mean 10 = Mean 11 = Mean 12 =

Mean 18 = Mean Mean 9= 6=

Mean 13 = Mean 14 = Mean 15 = Mean 16 = Mean 17 = Mean 18 =

6.590 B 3.590 1.820 1.110 1.770 2.880 C G H G DE

Mean Mean Mean

1= 5= 4=

2.350 2.340 2.250 1.820 1.770 1.110

F F F G G H

Mean 15 = Mean 17 = Mean 16 =

Ph bng 4: nh hng ca tc lc n kh nng phn gii phosphate kh tan ca 6 chng vi sinh vt tuyn chn. K hiu chng M1 X2 X3 M5 M7 X15 Ln 1 0.93 1.56 3.62 1.79 1.98 3.06 Ln 2 1.17 1.38 3.93 1.72 2.54 3.28 Ln 3 0.99 1.32 3.7 1.65 1.78 0.83 Ln 1 1.57 1.74 2.09 2.35 1.67 3.05 Ln 2 1.35 1.38 2.14 2.61 1.92 3.49 Ln 3 1.34 1.35 2.1 2.24 1.63 2.88 Ln 1 7.03 2.11 1.94 0.97 0.85 2.65 Ln 2 7.36 2.17 1.68 1.12 1.05 3.01 Ln 3 7.36 2.11 1.66 1.15 1.16 2.68 0rpm 75rpm 150rpm

Trc nghim phn hng nh hng ca tc lc n kh nng phn gii phosphate kh tan ca 6 chng vi sinh vt tuyn chn c.
Function : RANGE Error Mean Square = 0.1340 Error Degrees of Freedom = 17 No. of observations to calculate a mean = 3 Least Significant Difference Test LSD value = 0.8662 Original Order Mean 1= 1.030 G at alpha = 0.010 Ranked Order Mean 3= 7.250 A

Mean Mean Mean Mean Mean Mean Mean Mean

2= 3= 4= 5= 6= 7= 8= 9=

1.420 7.250 A 1.420 1.490 2.130

FG

Mean

7=

3.750 B 3.140 BC 2.780 2.400 2.390 2.130 2.110 2.100 1.760 1.740 1.720 1.490 1.420 1.420 1.070 1.030 1.020 CD CDE CDE DEF DEF DEF EFG EFG EFG FG FG FG G G G

Mean 17 = FG FG DEF Mean 18 = Mean 11 = Mean 16 = Mean Mean 6= 8=

3.750 B 2.110 1.760 1.720 2.400 1.070 2.100 1.740 1.020 2.390 DEF EFG EFG CDE G DEF EFG G CDE

Mean 13 = Mean 9=

Mean 10 = Mean 11 = Mean 12 = Mean 13 = Mean 14 = Mean 15 = Mean 16 = Mean 17 = Mean 18 =

Mean 14 = Mean 10 = Mean Mean Mean 5= 2= 4=

Mean 12 = Mean 1=

3.140 BC 2.780 CD

Mean 15 =

Ph bng 5: xc nh ngun carbon thch hp nui cy 6 chng vi sinh vt phn gii phosphate kh tan phn lp c. K hiu chng M1 X2 X3 M5 M7 Ln 1 0.92 0.27 6.22 0.96 1.95 Ln 2 1.13 0.31 6.37 1.17 1.48 Ln 3 1.25 0.29 6.1 1.11 1.37 Ln 1 0.69 4.37 2.06 4.01 0.53 Ln 2 0.85 4.81 1.87 3.94 1.06 Ln 3 0.8 4.74 1.92 3.9 1.38 Glucose saccharose

X15

0.38

0.42

0.4

0.02

0.05

0.02

Trc nghim phn hng xc nh ngun carbon thch hp nui cy 6 chng vi sinh vt phn gii phosphate kh tan.
Function : RANGE Error Mean Square = 0.03400 Error Degrees of Freedom = 11 No. of observations to calculate a mean = 3 Least Significant Difference Test LSD value = 0.4676 Original Order Mean Mean Mean Mean Mean Mean Mean Mean Mean 1= 1.100 E EF G at alpha = 0.010 Ranked Order Mean Mean Mean Mean Mean D E C D E FG G Mean Mean 5= 4= 8= 6= 9= 1= 7= 6.230 A 4.640 B 3.950 1.950 1.600 1.100 1.080 C D D E E E EF FG G G

2 = 0.7800 3 = 0.2900 4= 5= 6= 7= 8= 9= 4.640 B 6.230 A 1.950 1.080 3.950 1.600

Mean 10 = 0.9900 Mean 2 = 0.7800

Mean 10 = 0.9900 Mean 11 = 0.4000 Mean 12 = 0.03000

Mean 11 = 0.4000 Mean 3 = 0.2900

Mean 12 = 0.03000

Ph bng 6: xc nh ngun nit thch hp nui cy 6 chng vi sinh vt phn gii phosphate kh tan phn lp c.

K hiu

(NH4)2SO4

ur

chng Ln 1 Ln 2 Ln 3 Ln 1 Ln 2 Ln 3 M1 0.92 1.13 1.25 5.17 4.86 4.94 X2 0.27 0.31 0.29 0.43 0.74 0.36 X3 6.22 6.37 6.1 0.01 0.01 0.01 M5 0.96 1.17 1.11 5.21 5.64 5.14 M7 1.95 1.48 1.37 0.79 0.74 0.81 X15 0.38 0.42 0.4 0.63 0.58 0.59 Trc nghim phn hng xc nh ngun nit thch hp nui cy 6 chng vi sinh vt phn gii phosphate kh tan phn lp c.

Function : RANGE Error Mean Square = 0.02500 Error Degrees of Freedom = 11 No. of observations to calculate a mean = 3

Least Significant Difference Test LSD value = 0.4010 Original Order Mean Mean Mean Mean Mean Mean Mean Mean 1= 2= 1.100 D at alpha = 0.010 Ranked Order Mean Mean FG EF Mean Mean Mean G D Mean 5= 8= 2= 9= 1= 7= 6.230 A 5.330 B 4.990 B 1.600 1.100 1.080 C D D DE EF

4.990 B

3 = 0.2900 4 = 0.5100 5= 6.230 A

6 = 0.01000 7= 8= 1.080

Mean 10 = 0.7800 Mean 12 = 0.6000

5.330 B

Mean

9=

1.600

C DE

Mean

4 = 0.5100

EF EFG FG G

Mean 10 = 0.7800 Mean 11 = 0.4000 Mean 12 = 0.6000

Mean 11 = 0.4000 Mean Mean 3 = 0.2900 6 = 0.01000

EFG EF

Ph bng 7: Th nghim hot tnh phn gii phosphate kh tan ca cc chng vi sinh vt tuyn chn c trn ngun phosphate. K chng M1 X2 X3 M5 M7 X15 hiu Ca3(PO4)2 Ln 1
4.99 0.64 2.34 1.37 1.85 3.2

AlPO4 Ln 2
5.17 0.59 2.58 1.72 2.13 3.68

Ln 3
5.11 0.6 2.37 1.5 2.14 3.44

Ln 1
2.01 0.49 0.76 0.78 0.27 0.09

Ln 2
2.35 0.41 0.91 0.72 0.35 0.11

Ln 3
2.12 0.45 0.97 0.81 0.34 0.1

Trc nghim phn hng th nghim kh nng phn gii AlPO4 ca 6 chng vi sinh vt tuyn chn c.
Function : RANGE Error Mean Square = 0.01600 Error Degrees of Freedom = 11 No. of observations to calculate a mean = 3 Least Significant Difference Test LSD value = 0.3208 Original Order Mean Mean Mean 1= 2= 5.090 A 2.160 CD FGH at alpha = 0.010 Ranked Order Mean 1= 5.090 A 3.440 B 2.430 C

Mean 11 = Mean 5=

3 = 0.6000

Mean Mean Mean Mean Mean Mean

4 = 0.4500 5= 2.430 C

GH

Mean Mean

2= 9= 7=

2.160 2.040 1.530

CD D E F FG FGH GH HI I

6 = 0.8800 7= 1.530

F E FG D HI

Mean Mean Mean Mean Mean

6 = 0.8800 8 = 0.7700 3 = 0.6000 4 = 0.4500

8 = 0.7700 9= 2.040

Mean 10 = 0.3200 Mean 11 = 3.440 B

Mean 10 = 0.3200 I Mean 12 = 0.1000

Mean 12 = 0.1000

2. Cc hnh nh ca ti

Mu t ti Krng Nng Mu t ti C Kuin Mu t ti C Mgar Hnh 1. Cc khun lc mc trn mi trng NPRIP

Hnh 2. Hnh nh ng ging ca 17 chng vi sinh vt tuyn chn c

Hnh 3. Hnh nh gi ging 17 chng vi sinh vt trong eppendorf

Hnh 4. Hnh nh khun lc trn a thch ca 6 chng c la chn

Hnh thi t bo chng M1

Hnh thi t bo chng X2

Hnh thi t bo chng X3

Hnh thi t bo chng M5

Hnh thi t bo chng M7

Hnh thi t bo chng X15

Hnh 5. Hnh thi khun lc v t bo ca 6 chng vi sinh vt c tuyn chn.

Hnh 6. Hnh nh phn ng mu xanh molipdate ca 17 chng vi sinh vt.