Phin I: CONG NGHg BigN PQl SINH HOC 117
:^ NGHIEN ClTU LAM GIAU VA DINH DANH NHOM VI v^KHUAN OXY HOA AMONIUM Ky KHI (anammox) TlT
BUN CUA HE THONG XU* LY NU^OC THAI NUOI HEO
Le Cong Nhat Phuong, Tran Trung Kien, Ngo Ke Su-ong, Nguyin Tien Thing, Vi^n Sinh hoe Nhiet ddi
Kenji Furukawa, Pham Khic Lieu Kumamoto Universty, Japan
Takao FuJii
Sojo Universty. Japan.
MdDAU
Ngoai chu trinh bien ddi nito thdng thudng (1) cdn cd sy chuyen ddi ky khi trong qua trinh oxy hoa ammonium vdi sy cd mat ciia mdt chimg vi sinh vat ty dudng (anammox), ddng thdi nitrit ddng vai trd chat nhan tir. Theo phuong trinh ma Van de Graaf et al., 1995 va 1996; Strous et al., 1997 da dua ra vdi co che se trinh bay dudi day:
N02
^
^ . r
*•
NO3
Nitrification
~ • ~ *• Denitrification
• Fixation+AssimiiatioNitr
^ ification Anammox - — —>
Nitritation
^ Anammox
Hinh 1: Chu trinh bidn doi nitc
Cac he thong xir ly nito truyin thdng dya tren sy ket hgp 2 giai doan nitrat hda (nitrification) va denitrat'hda (denitrification).
Nam 1995, mdt phan img chuyin hda nito mdi chua timg dugc biet den trudc dd ve ca ly thuylt va thyc nghiem da dugc phat hien. Dd la phan ung oxy hda ky khi ammonium (Anaerobic Ammonium Oxidation, vilt tit la anammox) - frong dd ammonium dugc oxi hda bdi nitrit frong dilu kien ky khi, khdng cin cung cap chit hihi co, dk tao thanh nito phan tir (Sfrouss vacs., 1995).
Nhu da ndi d tren, phan img anammox da dugc xac nhan la sy oxy hda ammonium bdi nitrit, phan img hda hgc don gian vdi ti le moi NH4+ : NO2- =1:1,32 nhu d phuong trinh dudi day:
118 m nghi KHOA HQC VA C Q N G NGHg 2007
NH4* - 1.32 NO,' + 0.066 HCO3" + 0.13 IT -*
1.02 N: + 0.26 NOj" + 0.066 CHiOo.sNo.,,- + 2.03 H.O (1)
Trong do su tao thanh luong nhd nifrat tir nitrit dugc gia thiet la de sinh ra cac duong lugng khir khi ddng hda CO2. Phuong trinh nay da dugc chap nhan rdng rai nhu la dai dien cho phan ung anammox khi tinh toan, giai thich,. . .
NH2OH
-.—jr^z^i^** ^ti-'-i
Cytoplasm
i- i » A:>A!.:js^»,i.
Anammoxosome N2H4
''• aBriSSi.:Sjfe'.-.Sif:i.'X' frA.. 'i«
Hinh 2: Ca che sinh hoa qua trinh anammox
NR: enzyme khir nitrit (san pham gia thiet la NH2OH); HH: hydrazine hydrolase, enzyme xuc tac tao hydrazine tir ammonium va hydroxylamine; HZO: enzyme oxy hda hydrazine (tuong ty enzyme hydroxylamine oxidoreductase tuc HAO d cac Nitrosotnonas).
Den nay da cd 3 chi ciia vi khuan anammox dugc phat hien, gdm Brocadia, Kuenenia va Scalindua. Ve mat phan loai, cac vi khuan anammox la nhiing thanh vien mdi tao thanh phan nhanh sau cua nganh Planctomycetes, bd Planctoycetales (Schmid et al., 2005).
Mac dii ve nguyen tac, vi khuan anammox tdn tai frong ty nhien, frong mdi trudng va cac he thdng xir ly nude thai cd ndng do ammonium cao, nhung viec lam giau, nudi cay rat khd khan do chung sinh trudng cham.
Trong qua trinh tham gia de tai "Nghien cuu xu ly ammonium cd nong do cao bino cdng nghe sinh hgc trong nude thai nudi heo" va tham khao mdt sd tai lieu [1,2 4 va 5], Chimg tdi da quyet tam va tien hanh nghien cuu ve anammox.
Trong nghien cuu nay chiing tdi dya vao phuong frinh phan img (6)... dk khao sat trong cac loai bim ky khi va hieu khi d cac he thdng xu ly nude thai cd ndng dd ammonium cao nhu bim khi khi d be UASB cua he thdng xu ly nude thai chan nudi heo bim ky khi d be UASB ciia he thdng xu ly nude thai nude ri rac, bim hilu khi 0 bl hilu khi cua he thdng xir ly nude thai che bien thuy hai san, cd tdn tai nhdm vi sinh Anammox k±idng?. Bai bao nay chung tdi chi nghien cim lam giau bim ky khi cua he thdno xu ly nude thai nudi heo de oxy hoa ky khi ammonium (anammox) cd ndncr dd cao
Ph4n I: CdNG NGHg BigN Ddi SINH HQC
119 VAT LIEU VA PHU'ONG PHAP
I. Mo hinh thi nghifm
Thilt bi dimg thi nghiem lam giau vi khuin anammox d 2 giai doan vdi muc dich va thdi gian khac nhau nhu sau:
Giai doan h Giai doan thich nghi cua bim dugc thyc hien frong 120 ngay, vdi liru lugnc^
10 lit/ngay, frong mdt cdt hinh try D 200mm, H 800mm, thi tich chira 20 lif dung dich.
Giai doan Ih Giai doan tich luy lam giiu nhdm vi khuin anammox cd thdi gian van hanh frong thdi gian 270 ngay, vdi luu lugng 10 lit/ngay, frong cdt hinh fru co thi tich la 10 lit (D 100mm va H 1000mm)
• • ! £ - • • •
Kh( sinh hpc
Nifdc ra
c o t UASB
BOn ho^t tfnh
-"•"TPP**!
BO thu khi Bdm dinh lUcJng
Hinh 3: So* dd md hinh thi nghiem
2. Bdn hoat tinh ky khi
Bim dugc liy d bl UASB tir fram xu ly nude thai nudi heo Gd Sao - Quan 12, Tp. HCM.
dac diem cua bim va nude thai nudi heo cd thanh phin dugc trinh bay dudi bang sau day:
Bang 1: Th^nh phSn nu'dc thai nuoi heo cong nghifp s6tt
1 2 3 4 5 6 7 8 9 10
C i c chf tieu COD BOD5 DO SS NH4-N
T6ngP
NO2-N NO5-N Fe pH
D e n vl mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l
Ning d$
1.800-3.200 1000-1800
0-0.2 1.500-4.200
200-800 30-80
0-5 3-15 1.5-3.5 6.8-8.5
Ghi chu
[20 HQi nghj KHQA HQC VA C O N G NGHg 2007
3. Moi trudno lam 2iau
Bang 2: Moi tru-dng thich nghi va tfch luy lam giau nhom vi khuin anammox
Thanh pli^n moi tru'crng . (NH4)2S04 hoac NH4CI
NaN02 KHCO3 KH2PO4 CaCl2.2H20 MgS04-.7H20
Trace element solution 1* (g/L): EDTA:5, FeS04:5
Trace element solution 11* (g/L): EDTA :5, ZnS04.7H20: 0.43, C0CI2.6H2O: 0.24, IV1nCl2.4H20: 0.99, CUSO4.5H2O: 0.25, NaMo04.2H20: 0.22, NiCl2.6H20: 0.19.
NaSeO4.10H2O: 0.21, H3BO4:0.014
Ndng 6g 10-60 mg/L 10-60 mg/L 500 mg/L 27m g/L 0.180 g/L 0.120 g/L 1 mL/L 1 mL/L
Theo Van de Graaf er ai, 1996 4. Phuong phap phan tich
Phan tich thanh phan hda ly cac chi tieu frong nude trudc va sau md hinh nhu: N- NH4, N-NO3, N-NO2, COD, DO, pH, TDS, ...). Phan tich cac chi tieu frin hanh theo cac phuong phap trong "Standard methods fr)r the examination of water and wastewater". Tren may so mau Thermo Spectronic Moder 400I/4,USA, may TOA, Model 22, Japan va may do pH (Model 2000 VWR Scienfrfic, USA). Tai Phdng phan
tich mdi trudng, Vien Sinh hgc Nhiet ddi ;.
5. PhiroTig phap phan tich xac djnh vi khuan * Mau biin sau 9 thang tich luy lam giau dugc liy, bao quan lanh va mang ngay sang
Nhat de phan tich dinh tinh vi khuan anammox bang ky thuat sinh hgc phan tu.
Nguyen tac phan tich la mau dugc chilt xuit DNA, thyc hien phan ung PCR dac hieu khuech dai gen 16s rDNA, giai ma trinh tu va so sach vdi ngan hang gen thi gidi BLAST NCBI.
KET QUA VA THAO LUAN
1. Qua trinh thich nghi anammox ciia biin d giai doan I
Sd lieu theo ddi 3 thang van hanh khi nap hen tyc mdi trudng thich nghi cho anammox vdi N-NH4 va N-NO2 diu vao tang din (10-30) mg/l, d md hmh I dugc trmh bay d dd thi I va 2. Ket qua frong giai doan nay, da cd xuit hien sy tieu thu ddng thdi ca N-NH4 va N-NO2 va tao thanh mgt lugng nhd N-NO3. Hieu suit loai N-NTL tang din frr 0 - 8%, loai N-NO2 tang din tu 13,0-22,3% va lugng N-NO3 tao ra tu 1,0-I,5mg/1. Dilu do chinig minh ring kha nang xuat hien ciia phan img Anammox nhu 0 phuong trinh (6).
Ph^n I: CdNG NGHg BigN Ddi SINH HQC 121
^ Trong 3 thang va lugng bun (SS) giam tir 5.500mg/lit, cdn 3.200mg/lit, day la kit p qua ciia sy phan huy ky khi sinh khdi cua cac vi sinh vat di dudng trong dilu kien t khdng cung cip ngudn dinh dudng cacbon him co. Kit qua dd phii hgp vdi ndng do
COD dau ra. Biin chuyen tir mau den sang mau nau.
'¥
2. Qua trinh lam giau anammox ciia bun d giai doan II
^"' '" Bim tu md hinh I chuyin qua md hinh II cd thi tich 10 lit, vdi N-NH4 va N-NO2 I diu vao tang dan (30-60)mg/l, dugc trinh bay d bang 4. Tuong ty kit qua frong giai i doan nay, cd hieu suit cao hon va tang dan tir 9,3-29,3% vdi N-NH4 va 23,3-44,7% vdi
!•' N-NO2 va tao ra vdi lugng nhd N-NO3.
Mat khac lugng biin (SS) giam tir 3.200mg/lit xudng 1.120mg/lit va kem theo tilp tuc giam COD dau ra gan 50%. Dac biet, bim da chuyin tir mau nau sang mau nau L. hong. Day la dau hieu dac trung ciia vi khuan anammox
^.
3. Phan tich xac dinh vi khuan anammox bang PCR va 168 rDNA
Kit qua phan tich trinh ty gen I6S rDNA, phan 300bp diu 5' va phin 850bp diu 3' cho ket qua nhu bang 3.
Bang 3: K§t qua phan tich trinh tie gen 16S rDNA
TT Vi Ithu^n tu-cng tyf nhit
(Sd trong ngo^c 1^ accession number tren GenBank)
% tu-ang ty
Phan 300bp dau 5'
1 2 3 4
Anaerobic ammoium-oxidizing planctomycete K0LL2a (AJ250882) Uncultured anoxic sludge bacterium KU 1 (AB054007)
Candidatus Kuenenia Stuttgartiensis Candidatus Brocadia anammoxidans
100 100 100 95
Phan 850bp dau 3'
1 2 3 4
Anaerobic ammoium-oxidizing planctomycete K0LL2a (AJ250882) Uncultured anoxic sludge bacterium KU 1 (AB054007)
Candidatus Kuenenia Stuttgartiensis Candidatus Brocadia anammoxidans
100 1D0 99 94
Nhu vay, cd thi kit luan ring biin boat tinh da tich luy trong 270 ngay ciia chiing tdi cd sy hien dien ciia vi khuin anammox, tuong ty Candidatus Kuenenia SUittgartiensis da dugc xac dinh d Chau Au va cac ddng K0LL2a (phat hien cdng bd bdi phdng thi nghiem d Thuy ST), ddng KU2 (phat hien cdng bd bdi phdng thi nghiem d Nhat). Muc do tuong ty thip hon thu dugc vdi vi khuin anammox dugc phat hien dau tien d Ha Lan Candidatus Brocadia anammoxidans va ddng KUl (d Nhat).
122 H^i nghj KHOA HQC VA CONG NGHg 2007
KET LUAN
• Kit qua theo ddi sau 270 ngay d hai giai doan thi nghiem lam giau tir bim ky khi cua he thdng UASB xu ly nude thai nudi heo cho thiy cd sir hien dien cua phan ung anammox, vdi N-NH4 giam khoang 30% va N-NO2 giam khoang 40% va dong thdi trong mdi trudng, nude dau ra cd sinh ra N-NO3.
• Kit qua phan tich vi khuin bing ky friuat sinh hgc phan tir da xac dinh sir hien dien cua vi khuin anammox, tuong ty vdi cac vi khuan da biet fren the gidi. Day la nhdm vi khuin dugc tim thiy gin day, cd kha nang oxy hoa N-NH4 frong dilu kien ky khi ty dudng.
• Day la nghien cuu diu tien d Viet Nam vl Anammox, d budc phat hien, tich luy vi sinh vat. Trong thdi gian tdi chiing tdi se nghien cuu frep ve qua trinh thich nghi, lam giau cac loai bim khac nhau va kha nang ung dung nhdm vi khuan anammox de xu ly nude thai cd ndng do N-NH4 cao d Viet Nam.
TAI LIEU THAM KHAO
1. Egli, K., Franger, U., Alvarez, P. J. J., Siegrist, H., Vandermeer, J. R. and Zehnder.
A, J. B. (2001). Enrichment and characterization of an anammox bacterium from a rotating biological contactor freating ammonium-rich leachate. Arch.
Microbiol. 175, 198-207.
2. Fujji, T., H, Rouse, D. J, and Furukawa, K. (2002). Characterization of the microbial community in an anaerobic ammonium-oxidizing biofilm cultured on a nonwoven biomass carrier. J. Biosci.Bioeng., 94, 412-418
3. Furukawa, K., Rouse, J. D., Bhatti, Z. I., and Imajo, U. (2002). Anaerobic ammonium oxidation (anammox) in continuos flow freatment with non-woven biomass carrier. In Proceedings of the ISEB Fifth International Symposium on Environmental Biotechnology, Kyoto, Japan. The Intemafronal Society for Environmental Biotechnology Waterloo, ON, Canada, CD-ROM.
4. Hellinga C, Schellen AAJC, Mulder TW, van Loosdrecht MCM, Heijnen JJ (1998).
The SHARON process: an innovative method for nifrogen removal from ammoniumrich wastewater. Wat Sci Tech. 37:135-142.
5. Jetten, M.S.M., Wagner, M., Fuerst, J., Van Loosdrecht, M.C.M., Kuenen, G. and Sfrous, M. (2001). Microbiology and applicafron of the anaerobic ammonium oxidation ('anammox') process. Curr.Opin.Biotechnol. 12,283-288.
6. Mulder A. (2003). The quest for sustainable nifrogen removal technologies. Waste Science and Technology 48 (1), 67-75.
7. Schmid M. C , Maas B., Dapena A., and others (2005). Biomarkers for in situ detection of amaerobic ammonium-oxidizuig (anammox) bacteria Appl Envfron Microbiol 71(4) 1677-1684.
8. Strous M, Kuenen JG& Jetten MSM (1999). Key physiology of anaerobic ,
ammonium oxidation. Appl. Envfron. Microbiol.65,3248-3250. \
PhJIn I: CdNG NGHg BigN Ddi SINH HQC 123
9. Sfrous, M., Heijnen J. J., Kuenen J. G, and Jetten M. S. M. (1998). The sequencing batch reactor as a powerful tool for the sfridy of slowly growing anaerobic ammonium-oxidizing microorganisms. Appl. Microbiol. Biotechnol. 50, 589-596.
10. Van de Graaf AA, Mulder A, de Bruijin P, Jetten MSM, Roberston LA, Kuenen JG. (1995). Anaerobic oxidation of ammonium is a biologically mediated process.
Appl Environ Microbiol 61, 1246-51.
11. Van de Graaf AA, de Bruijin P, Robertson LA, Jetten MSM, Kuenen JG. (1996).
Autofrophic growth of anaerobic ammonium oxidizing microorganisms in a fluidized bed reactor. Microbiology 142, 2187-96.
12. Van Niftrik L. A., Fuerst J. A., Damstes J. S. S, Kuenen J. G., Jetten M. S. M. and Strous M. (2004). The anammoxosome: an infracytoplasmic compartment in anammox bacteria. FEMS Microbiology Letters 233, 7-13.
SUMMARY
The preliminary enrichment of anammox
(anaerobic ammonium oxidation) bacteria from sludge of UASB system
treating swine wastewater in Ho Chi Minh city
Le cong Nhat Phuong^'^, Tran Trung Kien^'^ Ngo Ke Suong^'^ Nguyen Tien Thang^'\
Kenji Furukawa^^', Pham Khac Lieu^^^ v^ Takao FuJii^^'
(1) Institute of Tropical Biology, (2)Kumaraoto Universty, Japan, (3)Sojo Universty, Japan
Data from 9 months of continuous enrichment showed the simultaneous consumption of ammonium and nitrite (0-30% and 13-45% of influent concentration,respectively) and the production of'small amount of nifrate (I,0-l,5mg/l), which meant anammox reaction had occurred.Analysis of enriched biomass by PCR and 16S rDNA sequencing techniques confirmed the existence of anammox bacteria with the high similarity with K0LL2a and KU2 strains and Candidatus Brocadia anammoxidans bacteria, all of which are well known as microorganisms having anammox activity.
