HOI NGHI KHOA HOC C O N G N G H E SINH HOC TOAN QUOC 2013
"NGHi£N"Ci>in"0ru'"uTr6A"Oi£LrKiEN NUorcArmcmcussuBTius,— — BACILLUS MEGATERIUM, NITROSOMONAS MARINA VA NITROBACTER
WINOGRADSKY! Bt TAO C H ^ P H A M XCf L V NUOC AO NUOI CA TRA
L6 Van NhJlVBCil H 6 n g Quan*, N g u y e n Dipc L u ^ n g ' ' 'Trubng Bd itpc Bach khoa, Dal hgc Quoc gia Tp. H6 Chl Minh '\ri$n C6ng ngh$ slnh hgc vk Thgc phim, B^i hgc Cbng Nghl$p Tp. Ho Chl Minh
T 6 M T A T
Myc tiSu c i a nghien ciiu n h i m t8i im qua trinh nu6i cay 04 chiing B. subtilis, B megaterium^ K marina v i N. winogradskyi c 6 k h i nang x i IJ nude ao nuoi ca Tra {Pangasianodon hypophthabnus) Chiing tfli d a sii dung thiSt ke thi nghifm Piacketl - Bimnan d£
ki^m fra miic Snh hudng ciia cac ygu t& h6a 1^ ISn sinh lch6i cCia tCmg lo?i vi khuia vk i p dyng phuong p h i p d i p ling be m^t vcd phuong i n clu fruc c6 tarn (RSM-CCD) dh tSi u u h6a moi frndng v i d i ^ I d ^ mioi cay n h i m Ihu sinh khoi vi khiian. Trong 6.6, gia tri t6i va cho B. subtilis GBDTS3 l i mgt ri duftng (10 g/1), pepton (4.979 g/1) v i MgS04.7HjO (0,48 g/1); B. megaterium G B D ' r e i 2 3 li glucose (20 g/1), pepton (7,5 g/I) v i K i H P O , (1,649 g/1); N manna GBDTS135 l i (NH4)iS04 (1,122 g/1), CaCli.OTjO (0,02 gfl), NaCI (1,938 g/1) v i NaHCOs (0,938 g/1); N. winogradskyi GBDTS027 l i NaNOj (1,993), KjHPO, (0,499) v4 FeS04.7H20 (0.001 g/1). B6n chiing vi khuan duijic t r V vcri g i i di^ CaCOj di tao chi phdm chiia l O ' CFU/^ B subtilis, 10* CFU/g B. megaterium, 3,3x 10' CFU/g N. manna v i 6,5x 10* CFU/g N. winogradskyi Thii nghidm 0;i906 g chS p h i m cho 1.2 lit nirdc ao nu6i c i Tra. sau 4 ngiy, n^ng d$ NH4* d i giim bi 4,40 mg/l xuSng dta 0,51 mg/L
Tit khda: Bacillus subtilis, B . megaterium, N . maiina, N . winogradskyi, Piackett - Burman. RSM-CCD
M d r o A U
Ck Tra (Pangas/anodon hypophthalmus) ik lodi cd nube nggt, chOng c6 kha nang si> dgng nhifeu lo?i thi>c fin khfic nhau.
Tuy n h l S n . c i c nghifin ciru da chi ra rang cA da born chl h^p thy du'pc t > 27 - 30% nlbogen. 16 - 3 0 % photpho vk khoang 25% chSt hOu c o d u ^ c dua vko tir thirc kn (Boyd, 1985; Gross et al., 2000). PhSn thiic Sn 6u thira con lai se dugc hda tan vk tlch l u bong bOn ao nufii. C h ^ u Minh Khdi et al. (2012) da nghien ciru trSn nir6c nu6i cA Tra va chF ra rSng hSm lu^ng N-NH4* dao dflng tCr 0,02 - 8,2 mg/l. hkm iugng N hoa tan tong so tir 0,5 - 11,6 mg/l, ikn hfia tan (P- POO-tiJ' 0,05 - 7,7 mg/l.
Nghifin ci>ucOa H i Thi Tru-frng Thy e( a/. (2011) cht rarSng chiing fi. subW/s c6 khd nang su-dyng ISm probiotic cho ca Tra. S. subtilis co k h i nang ti^t enzymfe ngoai bko giOp klch thfch b6u hda (Vaseeharan et ai. 2003) vk phSn g i i i protein vk carbonhydrate (Verschuere et al., 2000). T i r o n g tg, chung S. megaterium cung c6 khS nfing sinh enzyme ngoai bfio nhu-cellulase v&l hogt tfnh cao (V6 VSn Phu-orc Q u § v S Cao Ngoc Diep, 2011). kha nfing tfch IQy poly-P Ifim giam hfim lup'ng P 0 4 ^ dtf thiia tnang nmjrc (Bui The Vinh e ( a / . , 2011). 6. subtilis vk B. megaferionr ciS vai Jr6 thiic dSy qufi blnh phan c4l protein, bOt du'dng du> thira tir thirc an mOt ckch cb dinh h u d n g . Qufi trinh c h u y i n hda NHa thfinh NO3' nh£r qufi trinh nitrite h6a vfi nibate h6a bbi Nitrosomonas vk Nitrobaderdk dirge cfic tfic gifi di birdrc nghifin ci>u (Guen-ero ef ai, 1996; Magopian et aL, 1998). Do d d , 2 chung N. marina vk N. winogradskyi se nitrate hda ammonia tCr d d cai thi#n chSl iugng nubc ao nudi.
Trong nghiSn ci>u nfiy, c h i i n g tdi lfin l u v t li^n hin'h t i i i n j hda qufi trinh len men ciia tirng 4 chiing b i n g ma b$n Pladtett - Burman vk RSM-CCD. T i r n g u i n sinh kh6i Ihu dirgrc. ^ i i n g tdi ph6i tr$n de tgo che phSm sinh h^c vfi thCr n g h i ^ xO"
ly nud'c ao nudi c f i Tra 6 quy m S p h d n g t h l nghidm.'" * " ' ' - - - -• - ~ - — - - - -
NGUYgN LI$U VA PHU'ONG P H A P Nidi trud-ng nudI cay v f i m d i t r u i n g giiv g i d n g
Vi khuSn B. sufaWis GBDTS3 vfi B. megaterium GBDTS123 d u p c giii' tren mdi trud'ng NA. ChCing N. marina GBDTS135 d u p c glir vfi nhfin gidng trSn mdi bird'ng Winogradsky I. Chung N. winogradskyi GBDTS027 dirpc giO- vfi nhfin gi6ng trfin mdi tru'd'ng Winogradsky II. Tat ca cfic chung d d GBD Co.. Ltd cung d i p .
Thi^t k4 ma t r i n s ^ n g I9C Piackett - B u r m a n vfi phipomg phfip RSM-CCD f^;
Bk t6i m i hda di4u kl$n vfi mdi trudrng nudi cay thu nh^n sinh kh6i cua tirng 4 chiing vi khuSn. chOng tdi da thiet ke t h i nghi$m sfing IQC theo ma bfin Piackett - Bunnan vfi t6i u u hda bang RSM-CCD cho B. subtilis ( B i n g 1, B i n g 5 vk Bang 7), 6 . megaterium ( B i n g 2, Bang 5 va Bang 7). N. manna (Bang 3. Bang 5 vfi Bang 7) vfi N. winogradskyHBang 4 . Bang 5 v i B i n g 7).
B i n g 1. C i c yAu to trong ma t r | n Piackett - Bunnan cho B. subdlis vi anh htrarng
, , Gifi In E)d Qn KiJhi^u TSnySutd Mucanhhufrng •
Thap(.i) cao(-i-i) - '^y
Xi Glucose (sn) - 5 20 Xi MStridirimg(g/I) 0 10 X] Pepton (g/l) • 2.0 5,0 X, Cao nim men (gfl) -•• 2.0 5 0 X. (NH,)S04(gfl) 0.5 SO
0,0178"
0.0241' 0,0225' -0,0097"
0,0170"
0.0030 0,0022 0,0024 0,0055 0,0031
IIII
H p i NGHI KHOA HOC CONG NGHE SINH HQC TOAN QU6C 2013 K2HPO, (gfl)
KHjPO, (gfl) Caa2.2H,0 (gfl) HgS04 7HiO(gfl}
0,25 0.25 0.1
0.0134"
-0,0142*
0,0101*
0,0247*
-0,0001°
-0.0128'
0,0040 0,0037 0.0053 0.0021
.*:Cdyn^avddodnc$ya = 0,05 ": Khdng c6 y ngh^ vd 0^ tin i4y o - 0,05 B i n g 2. C i c ySu to trong ma trfn Piackett - Burman cho B. megaterium va i n h h u ^ g
Teny4ut6 Mi>c finh hubng
Glucose {gli) M$trldu«ng(g/I) Peplon(9/l) Cao nam men (g/l) (NH,)SO. {gll) K2HP04 (gfl) KHjPO, (gfl) CaCli-2H20 (gfl) Mg504 7H:0 (gfl) pH Ty 1# giang (% v/v)
0.0516"
-0,0001' -0,0306*
-0,0131*
-0,0085*
-0,0302"
-0.0079"
0.0010 ' -0.0248' 0.005r 0.0251"
0,0002 0,0006 0,0074 0,0006 0,0084 0,0009 0,0163 0.0009 ':C6f nghia vddd tin c$y a = 0,05 ". Khong cd y ngf^ vbi di^ Vn c^y a B i n g 3. C i c yeu t6 trong ma I r ^ Piackett - Burman cho N. marina v i anh h v d ^ g
T#ny4ut6
T h i p ( - I ) (NH4)2S04 (gfl)
KiHPO. (gfl) MgSO. 7H2O (g/l) NaCI (gfl) CaCli,2HiO (gfl) NaHCOi (gfl) FBS04.7H20 (g/I) Ty 1$ gifing (% v/v) Thfiri gran (h) pH CaC03(gfl)
3.0 1,0 0,50 2,0 0,05 1.0 0.010 7 240
10 1,00
-0,1958"
-0,1158"
-0,0882"
0.2292"
.0.1393* ••
-0,1816*
-0,0566*
0.0014"
0.0734*
-0,0662*
-0,0952°
0,0047 0,0079 0,0103 0,0040 0,0066 0,0050 0,0161 0,0124 0.0138 0,0096
". C6 y nghia vdi d^ tin c$y a = 0,05 -: Khdng c6 j'nghia • B i n g 4. Cic y4u't6 trong ma trfn Piackett - Burman cho N. winogradskyi v i i n h h u ^ g
Bdijdnc^y 0 = 0,05
NaNOj (g/1) KzHPO. (gfl) MgSO,.7H20 (gfl) NaCI (gfl) NaiCOj (gfl) CaCli 2H2O (gfl) FeSO, 7HJ0 (gfl) Ty l# giong (% v/v) Th&i gian (h) .pH CaCOa (gfl)
-0,3787' -0,4310*"
0,0016"
-0,0187*
-0,0111' 0,2669*
0,8388*
0,3279*
-0,5624"
0,2031"
0,3086"
')tinckya = 0.05
< 0.0001
<: 0.0001
0.0002
< 0,0001 0,0001
< 0,0001 0.0005 0,0001 '.• Cd y nghia vddobn c$y a = 0,05 : Khdng cb y nghia vbi dl?
Hfim dap ling dirpc chgn Ifi sinh kh6i khd (d6i vb\ S subtilis vfi B. megaterium) vfi m$t 6b tk bko {66i vbi N. marina vfi N. winogradskyi). Md hinh hda d i r p c bieu d i l n b i n g phircng trinh t}|c 2;
Y=b^-^2^i^i+2^»'^'"^ Z ^'"^"^
i=i i=i i»i. i = i
Trong do. n = 3 (hofic n = 4 cho/tf. marina), t jS j , j > i; bi Ifi cac h? so b^c 1; bi, Ifi cfic h§ so bfic 2; b j Ifi ( d c h# s6 t i / o n g tfic cua b>ng cfip y4u to; x,, Xi vfi jq l i c i c b i l n d^c l | p . K^t q u i thyc nghi$m dirpc p h ^ tich bang phan m ^ Design Expert* 7.0.0. TO' k i t qua phfin lich, xac djnh gifi tri t i i wu ciia cac y l u tb Ifim cho sinh k h i i dgt ci/c dgi.
Hi;*! N ( J H | KHUA HQC C O N G NtJHE: tJINH H U t ; I U A N O U O C 2013
Bang 5. K i t q u i bii^c ngbi$ffl v i auy d o i n san 1^9119 sinh kh6i bong thl nghif m Piackett - Burman Thi nghidm
Ky hi$u y6u t&
( X , - X„)
B, B, N l
N2 X, X, X.
X*
X, Xr X.
x«
x„
X,n T M 1 M 1 M T M1 +
;
* *
*
0.0771
*
0,0771 0,1695 0.1701 8,161 8,162 8,246 8,261
2 t
+
•
+
*
0.0645 0,0645 0,1119 0.1125 7.954 7,955 9,589 9,584
: 3 +
•
I
+ 0.0544 0J)544 0.1349 0.1343 8.061 8,060 9,653 9.6564 +
:
I
0,0666
*
0,0666 0,1107 0,1103 8,623 8,623 8,758 8,774
5 + t
*
+
+ 0,1021 0,1021 0.0768 0.0762 8.230 8.230 8.641 8.836
6 + +
-
+
0.0159 0.0159 0.1189 0.1194 8.255 8,256 9.651 9.656
7 +
t
+
• * •
0.0324
;-—
0.0324 0.2312 0.2306 8.079 8,079 9,904 9,901
8
*
+ +
I
0,0918 1^0918 0.1868 0,1864 7,839 7,838 8,146 8,132
9
*
+
*
+ 0,0894 0,0894 0.1580 0,1586 7,903 7,904 8,286 8,271
10 +
+
*
0,0486 0,0486 0,1522 0,1518 8.2ie 8.217 9,006 9,009
11 +
+
*
-+
+ 0,0741 0,0741 0,1639 0.1644 8,415 8,416 7,954
• 7,968 12
0,0146 0^0146 0,1642 0,1647 8.352 8.353 , 8.615 8.601
+ ; ky hi^u mOc cao cda yiu t6 •: ky hl$u mbc thSp cOa ySu t6
T: Kit qui Ihu Oirgc (i> ttnK nglii$ni.
Bing 6 . Ph|m vi nghiin ci
U Kit qua suy ra lit ra6 hlnh 'g/SOml} I N,: N. marina 'g/IOOmO I N»- N. mnogradskyis c i c y i u t i dilng trong RSM-CCD cho ti>ng chdng vl khuin Chung
VSV
a subWis
S. megaterium Biin
X l . X i
Xi Xt Xj
YSutfi MgS0,.7H20 (gfl) Mfil rfduerng (g/l) Pepton (gfl) Glucose (gfl) Pepton (gfl) KiHPO. (gfl)
Pham vi nghifin ci>u 0,195-1.205
1.95-12,05 0,977-6.023 3,909-24.091 0,796-9.204 0,489-3.011
•a 0,195
1.95
•' 0,977 3,909 0,796 ' 0,489
-1 0.4 4 2.0
8 2,5 1,0
. Mire 0 0,7
7 3,5 14 5 1,75
+1 1.0 10 5,0 20 7.5 2,5
+a 1,205 12,05 6.023 24.091 9,204 3.011 ( N H 4 ) i S 0 4 (gfl)
C a C l i 2 H 2 O ( g f l ) NaCI (g/l) NaHCOj (gfl)
0,318-3.682 0,010-0.060 0.659-2.341 0,330-1.170
0,318 0,010 0,659 0,330
1,0 0,02 1,0 0,50
2,0 0.035
1,5 0,75
3.0 0.05 2,0 1,00
3,682 0,060 2,341 1,170 N.winogradskyi NaN02 (gfl)
K^HPO. (gfl) FeS04 7HiQ (mg/i)
0,659-2.341 0,098-0.602 0,195-1.205
0,659 1.0 0,098 0,20 0,195 0.4
2.341 0,602 1,205 Bing 7. K i t q u i t i i iru hda thi,Fc nghiem thso RSM-CCD cho B. sabdlls, B. megaterium, N. winogradskyi
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Matron
-1 + t
+1 -a +a 0 0 0 0 0 0 0 0 0
Jli
+1
+1 u 0 -a +a 0 0 It 0 0 0 0
m i hda J j
+1
• 1 +1 0 0 0 0 +a 0 0 0
.14
+1 +1 +1 0 0 0 0 0 0 -a +0 u
S. subtils
TN MH
DCW (g/50m() 0,0806 0.0764 0,0971 0,0931 0,0921 0,0729 0,1104 0,0993 0.0898 0,0633 0.0807 0.1116 0.0908 0,0997
0,0863 0,0821 0,0754 0,0982 0,0916 0.0910 0,0733 0.1128 0.0952 0.0872 0,0667 0,0806 0,1126 0,0904 0,1009
0,0861 0,0861 0,0861
S. megaterium
TN MH
DCW (g/50ml) 0,0807 0,0854 • 0,0912 0,0946 0,0874 0,0892 0,0923 0,0961 0,0887 0,0960 0,0855 0.0946 0.0804 1 0,0844
0.0877 o.aeeo 0.0686
0,0811 0,0853 0,0897 0,0943 0,0866 0,0896 0.0913 0,0946 0,0899 0,0963 0,0850 0.0966 0.0807 0,0856
0.0880 0,0880 0,0880
N.vdn TN
ogradsk^
MH Log(CFU/ml) 8.362 7,954 7,699 7,903 7,544 7,875 7,544 8.477 7,477 7,978 7,778 8.021 7,778 7,845
7.398 7,477 7.544
8.245 7.654 ..
7,613 7,828 7,M0 7.892 7.575 8.524 7.542 8,011 7.949 7.949 7.861 7.861
7,480 7,460 7.480
N. manna
TN MH
Log(CFU/ml) 6,699 7,477 7,125 8.525 8.703 7.875 7.544 8.352 7,669 6.699 8.505 7.903 7.778 8,146 7,740 7.813 7,740 7,845 7.699
6.766 . 7,544
• 7,192 8.592 8.587 7,759 7.428 8.236 7.704 6,734 8,540 7,938 7,444 8,249 7,846 7,846 7.846 7,846 7,846
HOl NGHj KHOA H ^ CONG N G H f SINH HOC TOAN QUOC 2013 20
21 22
0 0 0
0 0 0
0 0 0 0 !
0 '.
0.0659 i 0.0862 0,0861
: 0.0861 . 0,0861
; 0,0861 1 0,0877 1 0,0686 1 0,0875
0,0880 1 0,0830 i 0,0880 1
7.398 7,602 7.477
7.480 7.480 7.480
1 7.699 i 7,903 : 8.041
7,846 7.846 7.846 -
Chiilhich: TN~KitquathuiIuvctirUn/cn'ghl$m;li«H-KitquisuyratCrm6hInn X,, xi, ji>- cAo B sutiHla, B meg^snum v i N urinogradskyi X,. » i Xi Xi" cho N. manna
P h u ' o n g phap p h o i t r ^ n v i thi> n g h i d m x 5 ^ y n u ^ ao n u o i cfi Tra
Tir k i t q u i t6l iru hda. chOng tdi da t i i n hfinh len men thu n h | n sinh kh6l 4 chung. Djch nudi i ^ y '^^^^J^^TJcb 0 St 5000 rpm trong 15 phOt vfi trpn v o l gifi t h l CaCOa. C h i p h i m sau khl b^n 6uac mang fli sfiy chan Knong u d i thu d u p c c h i p h i m dgng bpt vd'i m | t dO t i b i o dat 10^ te bfio/g c h l p h i m .
K6T QUA VA THAO LUAH ^ . .
Sfing Ipc vfi t & i n i h6a d i i u ki$n v i miM t r u i n g n u d i c i y Badllus subtilis GBDTS3
Thi nghidm sfing Ipc b i n g ma b i n Piackett - Burman du-a ra tifin dofin san tirpng sinh > * * ' ^ ^ ^ ' i f ° V ° ' ' f ^ ~ A l ° ^ l DCW g/50ml djch l#n men (Bfing 5). Gifi b l finh h u ^ n g cOa tirng y i u t6 Ifin sfin l i ^ r n g smh khdi khd 9_^J^l.^^2-w.rL.-.
Bfing 1 . M u i i MgSO* 7H2O. m&t ri du&ng vfi pepton cd mi>c i n h hubng Ibn n h i t d i n san l u p n g sinh i<h6i ^ 1 mirc y nghTa a = 0.05. Do do. MgS04.7H20, m j t ri d i r i m g vfi p ^ l o n dirpc chiing tdi chgn cho thifit kfi thl ngni?m 101 u'u noa sfin lupng sinh k h i i B. subtilis GBDTS3 theo RSM-CCD.
Chting tdi t i i n h i n h thl n g h i i m t i l iru hda 3 y i u td dS chgn theo phu-ong phap RSM-CCD bang p h i n m i m Design Expert* 7 0.0. Tir k i l q u i phfin lich vdi mu-c y nghTa a = 0.1. Phiro'ng trinh hdi quy d i r p c x i c djnh n h y sau-
YB.«rf,fln4(DCWg/50ml) = 0 , 0 8 6 - 6 , 0 8 2 x 1 0 ' ' X i + 9 , 5 0 9 x 1 0 ' X 2 + 3 , 1 1 0 x 1 0 % - 2 . 7 6 2 x 1 0 ' ' X , X 3 + . M 3 7 x 1 0 - ^ X 2 X 3 - 3 , 2 2 9 x 1 0 W + 3,701x10"*X2 + 3,382x10''Xi?
Trong dd X,, Xz, X3 lfin iugt ik MgS04.7H20, m$l ri dud'ng vfi pepton. H# s i l i i i quy (R^) tlnh d u p c la 0.9793. D i i u n i y t h i hi#n rang cd 97.93% s i lifiu t h y c nghl#m Urcrng thich vd'i s i l l | u tiSn dofin theo m d hlnh. Md hlnh dfi d y dofin san l u p n g sinh k h i l t i i da (0,1118 g DCW g/50ml) khi cac y i u t6: MgS04.7H20 (0.48 g/I); M$t ri dud'ng (10 g/1) v i pepton (4,979 g/l).
Thir nghifim m i hinh bfin thpc nghidm b quy md 100ml, nudi c i y l i e 150 rpm. nhifit dfi phdng trong 30h. K i t qua thu d u p c Y = 0,0983 DCW g/50 ml (bfing 87,9% so vd'i md hinh). N h u v$y, md hinh hofin tofin tucmg thich vd'i thi,rc nghifim.
Sfing Ipc vfi t i l u>u hoa dieu kifin v i m d i t r u ^ g n u d i c i y Bacillus megaterium GBDTS123
Trang Bang 5, thi nghifim sfing Ipc d u a ra tifin d o i n s i n lirpng sinh k h i i khd tir 0,0762 - 0,2306 DCW g/IOOml. Y l u t i nfio cd gifi lr[ Snh hud'ng duo'ng vfi Id'n sfi i n h hud'ng td'i s i n l u p n g sinh k h i l cua S. megaterium GBDTS123 d u p c trinh bfiy 6 Bang 2. Tir k i t q u i phfin tich, glucose, peplon v i K2HP0« d u p c chpn d i t h i l l k l thi nghifim t i i u u hda.
K i t qua phfin tich ANOVA vbi mire •y nghTa o = 0,1 dfi x i c djnh d u p c phucrng blnh hdi quy n h u sau:
Ya™.B.(«f<™ (DCW g/50ml) = 0,088 + 1,902x,*p^Xi + 3,427x10''X2 + 1,452x10"'X3 + 8,750 x10"'XiX2 - 3,125x10"*XiXi - 9 , 8 7 5 x 1 0 ' ' X 2 X j + 1,813x10'^Xi^ + 9,997x10'*X2^-1,705x10"*X3'
Trong dd X i , XT. X3 l i n l u p t Ifi glucose, pepton vfi K2HPO4. Hfi s i h i i quy (R^) tinh d u p c l i 0,9612. M6 hinh da d y dofin s i n lupng slnh k h i l khd dat cgc dai (0.0960 DCW g/50ml) khi glucose (20 g/1), pepton (7.5 g/i) v i i ^ H P 0 4 (1,649 g/l).
T h u nghifim md hinh bfin t h y c nghifim v d i IW ml djch Ifin men, nudi c i y l i e 150 rpm. nhifit d i phdng, 36h. K i t qua, Y = 0,0986 DCW g/50ml ( b i n g 102,7% md hinh) N h u vfiy, md hinh mfi chung t i i xfiy d u n g Ifi hofin tofin phii hpp.
Sang Ipc va t i l u u hoa d i i u kien v i m d i trirdrng n u d i c i y Nitrosomonas marina GBDTS135
Vd'i ChOng N. marina, chiing tdi da t i i n hfinh t i i uu hoa 4 y i u t i : NaCI, (NH4)2S04, NalHCOj vfi CaCl2.2l-l20. K i t qua phfin tlch b i n g p h i n m i m Design Expert* 7.0.0 vd'i mi>c •ji nghTa a = 0,1 dk xkc djnh p h u o n g trtnh h i i quy nhu s a u ;
Y R ^ r i n . (Log{CFU/ml))= 7.85 - 0,29Xi - 0.18Xz + 0,24Xj + 0,28XiXi - 0,27X,X3 - 0,28X1X4 - 0,27X2X3 - 0,56XzX4 - 0,13X3X4-0,22X1^ + 0,14X2^
Trong dd X,, X2. X3, X4 lan i u p t Ifi (NK.)2S04, CaCb.ZHjO, NaCI vfi NaHCOj, He s i h i i guy (R^) b i n g 0,9435. B i mfit dfip Ong (hlnh 1) t h i hifin s y Urong t i c cOa tOfng c$p y i u t i . Md hlnh da d p dofin mfit dfi t i b i o e y e dai d?t (4,7x10^
CFU/ml) khi (NH4)2S04 bfing 1,122 g/l; CaCl2.2H20 b i n g 0,02 g/l; NaCI b i n g 1,938 g/l vfi NaHCOs b i n g 0,938 g / l . Ben canh d i , d i chpn d u p c 4 y i u t i cho thl nghifim tfii u u hda d- bfin chiing tdi 6k b i n h i n h thi nghifim s i n g Ipc b i n g ma trfin Piackett - Bumian. V o i mO"c y nghTa a = 0,05. NaCI. (NH4)2S04. NaHCOa vfi CaCiz.2HzO la 4 y i u t i cd m i r e finh h u d n g I6n n h i t d i n sinh k h i i vl k h u i n N. marina GBDTS135 nen da d u p c chpn cho thi nghifim tdi u u h i a . Vk gifi b|
i n h hu&ng cua tirng y l u t i l i n san l u p n g sinh k h i i vi k h u i n d u p c trinh b i y d' Bang 3.
HQI NGHI KHOA HQC C 6 N G NGHE SINH H 0 & T Q A N - Q ( J 6 & 2 Q 4 3 -
Hlnh 1. Bk n i | t d i p Ong cua c i c y i u t6 l€n slnh k h i l vl khuin Nitrosomonas marina GBDTS135 . S i n g ipc vfi t i l u u hda dieu kifin v i m d i t r u d n g n u o i c i y M/trobacter winogradskyi GBDTS027
Thi nghifim sfing Ipc b i n g ma bfin Piackett - Bunnan dua ra BSn d o i n mfil dp t i bfio bfio tir 7,968 - 9,901, dan V\
log(CFU/ml) d j d i nudi e l y ( B i n g 5). Vcrl a = 0,05. NaNOi, K2HP04, FeS04.7H20 v i th&i gian Ifi cfic y i u t i ed mOc i n h hudng Id'n n h i t d i n m | t dfi t i bfio. T u y n h i i n . d i i vd'i y i u t i thfi'i gian, chdng tfil d i t i i n h i n h x i y d y n g d u d n g cong slnh trudng d i x i c djnh Ih&i glan thu nhfin sinh k h i i , dong th&i cung g i i m d u p c mire fl? phirc igp cila thl nghifim t i i u u hda. Do d i , NaN02, I^HP04 v i FeS04.2H20 d u p c chdng t i i chpn cho t h i i t k i thi nghifim t i i u u hda.
K i t q u i phfin tlch b i n g p h l n m i m Design Expert* 7.0.0 v 6 i mOc y nghTa a = 0,1 da xfic (Hnh.phuo'ng trinh h i i quy:
~ YKwinoffrwiiiw (Log(CFU/mI))= 7,48 + 0,139Xi + 0,152XiX2 + 0,183XiX3 + 0,164X2X3 + 0,105Xi^ + 0,166X2^ + 0,135X3^
Trong dd X i , X2, X3 l i n l u p t l i NaN02. K2HPO4 va FeS04.7H20. Hfi s i h i l quy (R^) Ifi 0,9315 Md hinh da d y dofin mfit d i t i b i o eye dai (3.1x10^ CFU/ml) khi NaN02 b i n g 1.993 g/1; K2HP04 b i n g 0,499 g/1; FeS04.7H20 b i n g 0,001 g/1.
ThO nghifim c h i p h i m xi> 1^ nuOc ao n u d i cfi Tra
o i dfinh g i i k h i n i n g xir I;? 4 ehiing vi k h u i n trfin 1,21 m i u nud'c dyng trong blnh 21 tir c i c ao nudi c i Tra trung binh 26 tuin t u i l d u p c l i y n g i u nhifin b dfi sfiu tCr 1 - 2,5". Phfin tlch mau nud-c ao nudi eho k i t q u i : NH4* (4,40 mg/l), N02"
(0.27 mg/l); NGj" (0,15 mg/l). Sau 4 ngfiy XCP Ij. n i n g 6b NH4* dfi g i i m tO 4,40 mg/l x u i n g 0,51 mg/l (so v d i m i u d i i chung t u 4,40 mg/l x u i n g 3.01 mg/l); n i n g dO N02' da g l i m tir 0.27 mg/l x u i n g 0,12 mg/l ( m l u d i i chOng: 0.27 mg/l xuing 0,23 mg/l) v i n i n g d i NO3' lang tir 0,15 mg/l len 0,2 mgfl (mau ddi chirng 0,15 mg/l len 0,16 mg/I). K i t qufi nfiy chOng minh c h i p h i m sinh hpc tao ra c6 khfi n i n g ' l i m g i i m nhanh n i n g dO 2 ion NH4* v i NO2'.
KtT LUAN
Nghifin cOu dfi sfing Ipc b i n g ma tr§n Piackett - Burman cfic y l u to anh hud-ng v i t i i u u hda d i l u kifin - mdi trudrng hufli c l y b i n g RSM-CCD n h i m thu nhfin sinh khoi 4 chiing vi k h u i n B. subtilis, B. megatenum, N. marina v i N.
winogradskyi. C h i "phim sinh hpc da d u p c tao t h i n h b i n g c i c h p h i i trfin 4 chiing (mfit d i m i l chCing dat I O ' t i bfio/g).
C h i p h i m c6 k h i n i n g Ifim g l i m n i n g dfi 2 ion NH4* vfi N O j ' m f i t each nhanh chdng v i dtmg dinh hud'ng. . - - , - LM cdm <ra
Ch£ng loi xin gui ldi cdm an den B^ mon CNSH- DHBK TPHCM dd tQO diiu ki$n vi PTN: Cty TNHH Thuy Sdn Quang Mmh. Q. 6 Mdn, Tp. CSn Thir da tgo diiu kien khao sdt. thu mdu tai cdc trd cd cua cong ty; sinh vien Hoang Thi Nggc Anh, V3 Thi Kim Ngan vd Chu DQ! Hifp dd hd tn? do dqc 1 sd chl tiiu.
TAI U $ U THAM KHAO
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OPTIMIZING THE CONDITIONS FOR INDIVIDUAL CULTURE OF BACILLUS SUBTILIS, BACILLUS MEGATERIUM, NITROSOMONAS MARINA AND NITROBACTER WINOGRADSKYI TO APPLY IN WASTEWATER TREATMENT OF CATFISH P O N D S ^
Le Van Nhat^, Bul Hong Quan^, Nguyen Due Luong^
' University of Techndogy, Vietnam National University, Ho Chl Mmh City
^Inditute ofBhtedtndogy and Food Techndogy, Ho Chi Mmh University of Industry
SUMMARY _ . ,' ,
This study was executed t o prepare a microbial mixture consisting of 04 strams: Bacillus subtilis. Bacillus megaterium, Nitrosomonas marina, and Nitrobacter winogradskyi, which can be used for water treatment of Tra {Pangasianodon hypophthalmus) fishpond. Plackett-Bunnan desipi was used lo evaluate the effect of physical and chemical fectors on biomass production. After being screened, these factors were subsequently optimized using Response Suiftce Methodology (RSM) and Central Composite Designs (CCD) to obtam maxiimun biomass yields. It was found that the optimal levels of tbe'screened factors were (g/I) molasses (10). peptone (4.979) and M g S 0 , . 7 H , 0 (0.48) for Bacillus subtilis GBDTS3, glucose (20). peptone (7.5) and K1HPO4 (1.649) for B.
megaterium GBDTS123; (1^4)3804 (1.122). CaCl3 2 H , 0 (0.02). NaCI (1.938) and I^aHCOj (0.938) J b r Nitrosomonas marina GBDTSI35. and NaNOi (1.993), KiHPO, (0.499) and FeS04.7HiD (0.001) for Nitrobacter winoff-adslgri GBDTS027. These abains were mbted with CaCOj to make a preparation containing 1 0 ' CFU/g B. sublilis, 10* CFU/g B. megaterium, 3.3xlO' CFU/g Nitrosomonas manna and 6.5x10* CFU/g Niirobacler winogradskyi. 0.2906g of this microbial preparatioo was used for the treatment of 1.2 litre of Tra fishpond water. After four days of treatment, the concentration of NH4* was efTectiveiy reduced fi-om 4.40 mg/l to 0.51 mg/i.
Keywords: Bacillus sublilij. B . megaterium, N . marina, N. winogradskyi, Piackett - Burman, Response Surface Methodology (RSM) - CenBal Composiie Designs (CCD)
'Author for correspondence: Tel 0948505090: email [email protected]
