KHOA HOC CONG NGHE
PHAIV LAP VA OIMH DAIMH VI KHUAIM XV U AlVIIViaiMIUIVI TLf BLJIXl T H A I C H A M IVUOI H E O , BIJIM A O
CA T R A V A BiJiM A O IXIUOI TOIVI
Doan Thi TimS Dd Ngpc Anh HuyS Nguydn Thj DungS Dd Thanh D^it^, Nguyin Thi Mdn^ Chau T ^ Phat?
T6MTAT
Nghien ciiu tap trung sang loc vi khuan xif li ammonium tir cac mSu bim fliai tiong chan nuoi. Mau duoc thu t^ cac khu vuc tiiuoc TP. Ho Chi Mmh, Ti^n Giang va B^n Tre. Nhom vi khuan co kha nang xir ly ammonium phan lap tir nguon biin thai chan nuoi heo, bim ao ca tra, biin ao torn dao dong lan lupt trong khoang (1,5 - 2,4) x 10' MPN/g; (0,15 - 1,5) x 10' MPN/g va (0,93 - 2,4) x iC MPN/g. Dua vao dac diem hinh thai va sinh hoa, da tuyen chon duot 6 chung vi khuan co dgc diem gidng Paracoccus va 3 chung giong NiUosomonas. Bdn chiing co hoat tinh xir ly ammonium cao nhat (120,83 -123,66 mg/L) sau khi nuoi cay trong 168 h dupe chon de thuc hien giai trinh tu 16S rRNA Ket qua giai tiinh tu va so sanh ven du li^u tren ngan hang gen (GenBank) cho thay. 02 chiing S4 va S6 hiong dong voi Paracoccus communis (99%), chung S9 tirong ddng voi Paracoccus versutus (100%), chung S3 tuong ddng voi Nitrosomonas europaea (100%). Hai chiing S4 va S6 thuoc loai Paracoccus communis da cho thay kha nang xu li ammonium len d^n 74,9% va 76,65%. Day la lan dau Uen Paracoccus communis dirpc phan lap tir bim thai va duoc bao cao la vl khuan co kha nang xii li ammonium.
Tir kh6a: Paracoccus communis, Nitrosomonas eurojjaea, Paracoccus versutus.
1 . BAT VAN OE
Su gia tang dan sd, cac hoat ddng san xuat va do thi hda nhimg thieu quy hoach va cac bi6n phap xu li lam gia tang luong chat thai true tidp ra ngoai moi trudng gay nen tinh trang 6 nhidm ngay cang nghiem trpng. Ham lupng nito trong nude thai thudng hien dien dudi dang cac hpp chat hihi ca hay ion nhu NH4", NOa' va NO3'. Su xa thai khong qua xa li din den tich liiy su khoang boa nita cua eac hop chat him ea du thira lam tang ndng dp nita (NH3) gay suy giam ham lupng oxi hda tan eua ngudn nude. Ke tiep san pham cua qua trinh oxi hda ammonium la nitrite (NOg) gay dpc cho he tuan hoan he midn dich cua dong vat [ 15]. Ngoai ra, nitiite ed tiie chuyen hda thanh niti-osamin. mot hop chat gay ung thu [1].
Nhieu phuong phap da dupe ung dung de xu li ammonia cd ti-ong nude nhung phuong phap sinh hpc dupe chii y hon ca [19]. Nhidu nghien cim da chi
' Trung tdm Cong nghe Sinh hoc TP H6 Chi Minh - Tnrcrng E)ai hpc Cong nghiep Thirc phdm TP. H6 Chi Minh
rrucmg Dai hpc Van Lang Email: do:inlhitamSS@gmail,coni
ra da dang va tam quan hong cua cac quan xa vi sinh vat trong qua trinh chuyen hda nita [13], [9].
Gan day, mpt sd loai vi khuan di duong nhu Paracoccus versutus, Alcaligenes faecahs, Acinetobacter calcoaceticus, Thiosphaera pantotiopha. Bacillus sp., Microvirgula aerodenitrihcans, Pseudomonas stutzeri, Comamonas sp., Diaphorobacter sp. dupe md ta cd kha nang xir li ammonium bang each chuyen hda ammonium thanh khi nita [10], [18]. Trong khi dd.
cac vi sinh vat di duong cd kha nang chiu dupe ndng dp eao ammonium va chat hiru co [11]. Vi khuan hda tu duong Paracoccus sp. va Nitrosomonas sp. ed kha nang xu li ammonium phan lap duoc tir bim thai chan nuoi heo, bim ao nuoi ca tra va bim ao nuoi tom. Vi vay, da tidn hanh "Phan lap va dinh danh vi khuan xu li ammonium tii biin thai chan nudi beo, bun ao ca tia va bun ao nudi tdn/'.
Z. VAT UEU VA PHUONG PHAP niGHIBU CUU 2.1. Nguyfin vat U§u
Mau dupe thu bang he thdng dng PVC da tiet triing vdi dung dich edn 70%. Tai mdi ao, mau bim duoc tiiu d 3 vi tri: dau ao, giiia ao va cudi ao theo mpt dudng cheo. O mdi vi tri thu khoang IQQ a biin 1(X) N O N G NGHIEP V A PHAT TRIEN N O N G THON - KY 1 - THANG 6/2020
KHOA HOC CONG NGHE
Tai mdi ao ehua nude thai chan nuoi heo (HS), ao nudi ca tra (CS) va ao nudi tdm CTS) mSu duoc thu tuong tu nhu mSu biin. Miu dupe giir lanh bang nude da va chuyen vd phdng thi nghidm trong vdng 3 - 5 gid, bao quan d 4°C va xu ly h o n g vdng 2 gid.
Tr6n mdi chai thu mau ghi day du thong tin ve ngay, thang, dia diem thu man. Phuong phap thu mlu nen day [20].
2.2. Phuong phap nghifen cuu
2.2.1. Xac dinh mat sd vi khuan nitrite hda bdng phuong phap MPN-Most Probable Number [21
Mdi trudng ammonium-calcium-carbonate (SMI) (g/1): (NH4),S04 (0.5 g); K,HP04 (1 g);
FeS04.7H20 (0,03 g); NaCL (0,3 g); MgS04.7HaO (0,3 g); CaCOg (7,5 g) dupe sir dung de phan lap vi khuin Nitiosomonas dua theo phuong phap sd kha him (MPN) [2].
Thudc thu Griess - Ilosway: Dung dich A Hoa tan 0,6 g sunfanihc acid trong 10 ml nude cat ndng (90"C), de ngupi cho tii^m 20 ml HCl dam dac. Pha loang hdn hpp nay thanh 100 ml vdi nude cat rdi tron ddu; dung dich B. Hoa tan 0,6 g, a-naptiiylamine tiong 10-20 ml HCl dam dac, pha loang thanh 100 ml vdi nude eat va hdn deu; dung dich C: Hoa tan 16,4 g sodium acetate trong nude eat, pha loang thanh 100 ml vdi nude cat sau dd hpn deu. Bao quan rieng biet timg dung dich nay trong 3 chai tdi, giii trong tu lanh, thdi gian su dung khong qua 1 thang.
Hdn hop kem-ddng-manganese dioxide: trpn deu 1 g bpt kim loai Zn, Ig MnOj va 1 g bdt kim loai Cu.
Dem mat sd Nitiosomonas va Nitiobacter bing phuong phap MPN. Dem vi khuan hong 1 gam mlu bim hong cac dng nghiem chua mdi trudng tren (mau biin dupe pha loang d cac ndng do tir 10' den 10"^ 3 lan lap laQ. U cac dng nghiem d 28°C trong 21 ngay. Tra cuu bang 1-MPN tieu chuan de xac dinh sd lupng cd the cd cua nhdm AOB trong 1 gam mlu bim.
2.2.2. Phuong phap dinh luong ammonium Xac dinh ham lupng ammonium (NH/) bang may do cac chi tieu nude cam tay HACH DR 900 theo phuong phap SaUcylate.
2.2.3. Kiem tia sinh hoa vi khuan phan lap Vi khuan dupe nuoi cay tr6n moi trudng:
Tryptone (10 g/1), yeast extract (5 g/1) va NaCl (5 g/1), pH 7, nhiet dp 25"'C. Cac kiem tra eatalase, urease, khu nitrate, phan giai tinh bpt, phan giai gelatin, chuyen hda cacbonhydrate bing phan img 0X1 hda duac thuc hien theo phuong phap chuan [8].
2.2.4. Ky tiiuat PCR
Sau khi tach chiet DNA tong sd cac chimg vi khuan theo kit DNeasy® Blood & Tissue Handbook, tidn hanh phan ung khuech dai doan gen 16S rRNA vdi cap mdi chuyen biet dupe trinh bay trong bang 1.
Bang MJu Nitrosdmonas
. C§p primer Moi 357F 534R
5ii dung trong khuy&h d^ doan gen 16S rRNA Trinh tir
5'-CCTACGGGAGGCTGCAG-3' S'-ATTACCGCGGCTGCTGG-S'
Tham khao [141
Thanh phan phan img PCR bao gdm: 12,5 pi Mbc PCR, 2,5 pi Mbf primer, 2 pi DNA khuan va bd sung nude c^t vo triing de dat the tieh phan ung la 25 pi.
Phan ung PCR dupe thuc hien trong may luan nhiet tiieo quy trinh sau: Bien tinh genome d 94''C hong 5 phut, tidp den la 40 chu k^: 94°C/30 giay, 56°C/30 giay, 68°C/30 giay, cudi cimg la 72"C/10 phiit.
San pham PCR dupe kiem tra bang di&n di hen agarose gel 1% bd sung thudc nhupm Ethydium Bromide (EtBr) va phan tich hinh anh dien di bang h^ thdng Gel Documentation (Bio-Rad).
San ph^m PCR dupe gui giai trinh tir viing 16S rRNA tai Cdng ty Nam Khoa, Viet Nam.
Sau dd diing cong cu BLAST (Basic Local Alignment Search Tool) de so sanh va tim kiem nhanh nhung trinh tu tuong ddng vdi trinh tu eua timg mlu khao sat trong ngan hang dii lieu gen NCBI (GenBank). Sir dung phan mem ClustalX de so sanh miic tijong ddng trinh tu giiia cac loai khao sat vdi nhau va voi trinh tir cae loai tir GenBank. Dimg phin mdm Mega 5.2 de xay dung cay phat sinh loai.
Cudi cung dua vao cay phat sinh loai dd xac dmh cac mlu phan lap ddn muc loai.
S.KETQUiiVATHAOLUMI
3.1. Phan tich bidn dpng melt sd vi khuin nitrate hda bang phuong phap MPN-Most Probable Number Ket qua bien ddng mat dd vi khuan niti-ate hda bang phuong phap sd kha hiru (MPN) dupe the hien
KHOA HOC CONG NGHI qua bang 2 cho thay nhom vi khuan AOB phan lap tir
nguon nuoc thai chan nuoi heo (HS). bim ao ca tra (CS). bun ao t6m CIS) dao dong lan luot trong khoang (1.5 - 2,4) x Iff MPN/g; (0.15 - 1,5) x Vf MPN/g; (0.93 - 2.4) x 102 MPN/g. Ket qua nghien ciiu nay tuong tu voi mot s6 nghien cuu truoc do ve xac dinh mat do vi khuan nitrate hoa bang phuong phap MPN nhu Herbert va ctv (1999) ghi nhan mat
Bang 2. Kft qui tra bang MPN cac miu AOB phan lap a cac miic nong dp pha loang khac nhau do dao dong trong dat khoang 102 - 104 MPN/g 16];
Hesselsoe va ctv (2001) [7] khao sat thay trong khoang 17,9 x 102 MPN/g. Ngoai ra theo nghien cuu khao sat mat do va su da dang cua vi khuan nitrate hoa trong ao nuoi tom cua Pham Thi Tuyet Ngan va ctv (2011) [16] cho thay nhdm vi khuan AOB dao dong trong khoang (1,1- 460) x 102 MPN/g.
\ ^ N d n g dp Mlu ^ \
HSOl HS02 HS03 CSOl CS02 CS03 TSOi TS02 TS03
10' + + + + + + + + +
+ + +
+ + + + +
+ + + + +
+ + +
la^
+ + + + + + +
+ + +
+
+ +
+ +
+ +
+
10-^
+
+
+ +
+
+ MPN (MPN/g)
150 240 150 15 150 28 93 120 240 3.2. Khao sdt kha nang xu If ammonium tu cdc
chiing vi khu&i sau khi phan lip b ^ phuong phlp MPN
Bang 3. Kha nSng xu I^ cua cic chung vi khudn phSn Ijtp dupe sau 168 gid nudi c^y Chung
SI 32 S3 S4 S5 S6 S7 S8 S9
Ham lirang NH4 (mg/1) 104.33±3.61 96.83+2.29 121.45±0,76 120.83±1.32 91,33+2.65 123.66±1.53 89,16+1.26 99.50±2,75 125,78+1,74
CIc chiing sau khi phan lap bang phuong phap MPN dupe sang Ipe va thu nghiem kha nang chuyen hda ammonium hong moi tiirdng ammonium- calcium<-arbonate (SMI) long hong 7 ngay, Ket qua thu duoc (Bang 3) bdn chimg S4, S3. S6 va S9 cd hieu suat chuydn boa ammonium trong moi ti-udng ammonium-calcium-carbonate (SMI) Idng cao nhat lln lupt la 120,83; 121,45; 123,66 va 125,78 mg/1 sau 168h nuoi cly. didu nay phu tiiuoc vao nong dp vi sinh cic chung phan lap ban dau. Bdn chung nay sau
dd dupe thuc hien cac kiem tra sinh hda va dmh danh hep theo.
3.3. xac dinh d^c di^m hinh thai va smh hda ciia cic chiing vi khudn nitrite hda sau khi dupe phan l^p
Sau khi nhupm eac chung vi khuan phan lap dupe tii phuong phap MPN, kdt qua cac mlu nhupm the hien mau dd tia dieu nay cho thay ring cac chimg vi khuan nitrate hda phan lap dupe deu la vi khuan Gram-am (vi khuan Gram duong se cho mlu nhupm Gram mau xanh) (Hinh 1), cac ddng vi khuan nay cd hinh dang te bao hmh cau, kich thudc te bao rat nhd va khong hinh thanh bao tir. Khuan lac hinh trdn be mat bdng nhan, cd mau hoac khong mau.
Khao sat cac dac diem sinh hda (Bang 4) dupe so sanh vol eac bao eao mo ta ehi Paracoccus sp. deu trimg khdp [5], [17]. Cac kiem tira eatalase, urease, khir nitrate, phan giai tinh bpt. phan giai gelatin, chuyen hda cacbonhydrate bang phan ung oxi hda duoc thuc hien theo phuong phap ehuan [8]. Thong qua viec khao sat dac diem sinh hda, chung S3 khong phat tiien dupe ti-ong moi trubng thi nghiem.
Kiem tra eatalase cac chung S4, S6. 89 ed su phat trien Uiong qua moi tnrdng nuoi cay, khong cd chung vi khuan nao phat trien thong qua kiem ha urease, khir nitrate (S4, S6. S9), phan giai Unh bdt (S9), phan giai gelatin (S9).
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 6/2020
KHOA HOC CONG NGHE
rtx. • S3
*• " V " . ' f L ^ - ' - • > • . • • .
" ''
- r ^ . ^.^ .
'"A _
. ' •
54
•
k >
a 59
Hinh 1. Mlu nhuOm Gram cac ddng vi khudn sau khi dupe phan lap Chuyen hda cacbohydrate bang phan img oxi
hda duoe thirc hien theo phuong phap chuan [8]:
glucose (S4, S6); Glycerol (S4, S6, S9); Lactose (S4, S6,S9); Sucrose (S4,S6,S9).
Chuyen hda cacbonhydrate bang phan ling len men duoc thuc hien theo phuong phap ehuan [8]:
glucose (S9); Xylose (S4, S6, S9).
Bang 4. Dac di^m hinh thai va sinh hda ciia 4 chung S3. S4,S6vaS9 nitrite hda Dac didm
Catalase Urease Phan giai tinh bdt Phan siai eelatin Khir nihate Chuyen hda carbonhydrate bang each oxi hda hav I6n men Glucose Glycerol Lactose Sucrose Xylose
S3* S4 +
+
0x1 hda Oxi hda Oxi hda Oxi hda Len men
S6 +
+
0x1 hda Oxi hda Oxi hda Oxi hda Len men
S9 +
+ + +
Len men Oxi hda Oxi hda 0x1 hda Len men
*• Chung S3 khdng phat triin ti-ong mdi tiirdng dupe thi nghiem
3.4. Dinh danh cac chOng phan lap thdng qua phuong phap dung cay phan nhdm va so sdnh chudi trinh tu tuong ddng
Cac chimg vi khuan sau khi dupe phan lap thong qua phuong phap MPN ciing vdi cac thu nghiem kha nang xu ly ammonium, xac dmh dac diem binh thai va sinh hda sau khi phan lap, cac chung S3, S4, S6 va S9 se duoe ly hich DNA va thuc hien qua trinh nhan gen thong qua PCR. San pham PCR tir cac ddng nay se dupe giai trinh tu gen.
S.4.1. So sanh va tim kidm nhanh nhung trinh tu tuong dong vdi trinh tu cua tung miu khao sat trong ngan hang du lieu gen NCBI thdng qua cdng cu BLAST (Basic LocalAhgnment Search Tool)
Sau khi giai tiinh tu gen eua cae chiing vi khuan dupe phan lap. diing cong cu BLAST de so sanh va tim kiem nhanh nhimg trinh tu tuong ddng voi trinh tu cua timg mau khao sat trong ngan hang du heu gen NCBI. Cae chung phan lap dupe so sanh doan giai trinh tu 16S rRNA tren eo sd dir heu GenBank (Bang 5). Trong dd, tdng diem ciia eac chung S3, S4, S6 va S9 lan luat la 957, 821, 826 va 824.
Sir dung phan mdm ClustalX de so sanh muc tuong ddng trinh ttr giua cac loai khao sat voi nhau va vdi trinh tu cac loai tir GenBank. Ket qua cho thay:
02 chung S4 va S6 tuong dong vdi Paracoccus communis (99%), ehung S9 tuong ddng vdi Paracoccus versutus (100%), chung S3 tuong ddng vdi Nitrosomonas europaea (100%). Kdt qua dinh
danh loai Nitrosomonas europaea tren chiing S3 rang co sir hien dien cua gen amoA (mot gen chiu trong nghi6n ciiu nay cung duoc ciing co boi cong bd trach nhiem chinh ma hoa enzyme ammonia cua Ducey va Hunt (2013). Ducey va ctv (2011) cho monoxygenase).
Bangs. So sanh doan giai trinh iJJ gen 168 rRNA tren eg s6 d a li^u GenBank
STT MSu Ten chung gan Trinh tu tham chieu Maxscore Totalscore Tuong dong Nitiosomonas
europaea ATCC 19718 strain ATCC 19718 16S ribosomal RNA
100%
S4
Paracoccus communis strain S3 16S ribosomal RNA S6
Paracoccus communis strain S3 16S nbosomal RNA
99%
S9
Paracoccus versutus strain NJUST32 16S ribosomal RNA gene
100%
Th6m vao do. nghien ciiu phan lap dupe loai Paracoccus communis tren cac chiing S4, S6 la mot loai chua co nhieu thong tm ve kha nang xii li ammonium. Cac nghien cuu can tim hieu them v6 tiem nang ling dung trong xii li nuoc thai tir loai nay.
3.4.2. Dung cay phat sinh loai bdng phan mini MEGA 5.2 0inh 2&3)
Ket qua duoc the hien h-ong hinh 2 cho tiiay chung S4. S6 ciing nhom voi chung Paracoccus commums^m gia tri Bootstiap rat cao (99%) 'Cat hien dp tin cay cao ve thong tin nay. Chung S9 ciing nhom phan loai voi chiing Paracoccus versutus vdi gia tri Bootsti-ap rSt cao (100%) die hien dp tin cay cao v6 thong tin nay.
Hinh 2. C4y pha h^ dua ti^ ti-inh hi gen 16S rRNA giiia cic chiing S4,86,89 v i cic loii U6n quan. Gia tii Bootstiap CTh^ hifn phin ti«m cua 500 vong d(ii)
dugrc th^ hi&i a m6i nhinh
Hinh 3. Cay pha h^ dua frta trinh hj glen 16S rRNA giiia chung 83 va cac loai li6n quan. Gii tri Bootstrap CTh^ hita phan tram ciia 500 vdng dM) dupe th^ hi$n
a mdi nhanh
Kft qua dupe the hien ti-ong hinh 3 cho thay chung S3 ciing nhom voi chiing Nitiosomonas europaeavin gia tii Bootsti-ap rat cao (100%) the hien dp tin cay cao ve thong tm nay.
4. KET LUAN
Sau khi sang loc kha nang chuyen hoa ammonium tii cac m i u bim thu dupe tai cac khu vuc thupc TP. Ho Chi Mmh. Tien Giang va Ben Tre.
Chung S3 phan lap tir mau bim ao tdm thu a B&n Tre;
chiing S4 va 86 tir mau mrdc diai chan nuoi heo thu b TP. HCM, chiing S9 tir mSu biin ao ca tra d Tien Giang. Bdn chung nay co hoat tinh xu ly ammonium cao nhat (120,83 - 123.66 mg/I) sau khi nudi cay NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 6/2020
KHOA HOC C d N G NGHE
trong 168h duoc chpn de thuc hidn giai trinh tu 16S rRNA. Ket qua giai trinh tu, dung cay phan nhdm va so sanh miie tuong ddng hinh tu giiia cac loai khao sat vdi nhau va vdi trinh tu cae loai tir GenBank cho thay: 02 chiing S4 va S6 tuong dong vdi Paracoccus communis (99%), chung S9 tuong ddng vdi Paracoccus versutus (100%), ehung S3 tuong ddng vdi Nitrosomonas europaea (100%).
Cac vi khuan nay edn dupe nghi6n cuu sau hon ve kha nang ling dung trong xu li nude thai hoac tao cac chd pham xir li ammonium trong nudi hdng thuy san.
LOI M M ON
Nghien cuu nay duoc tai tru bdi Trung tam Cdng nghd sinh hoc TP. Hd Chi Minh.
TAI UEU THAM KHAO
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2. Ehrlieh G. G. (1975). Water quaUty: Analytical Methods Nihifying bacteria (most probable number, MPN, method), Qual water branch Tech Memo, 75, pp. 1-8.
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(2009). Microbial processes influencing performance of ti-eatment wetlands: a review. Ecological engineering, 55(6), 987-1004.
4. Gerardi M. (2002). Nitiification and Denitrification in the Activated Sludge Process.
Isted. Enviromental Protection, New York.
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Paracoccus mareusii sp. nov.. an orange gram- negative coccus. Intemational Joumal of Systematic and Evolutionary Microbiology, 4^2), 543-548.
6. Herbert R. A. (1999). Nihogen eyclmg in coastal marine ecosystems. FEMS Microbiology Reviews, 23(5), pp. 563 - 590.
7. Hesselsoe M., Pederson A., Bundgaad K. K.,&
Sorensen J. (2001). Nitiification hot-spots around degrading red clover (Tnfolium patense) leave in soil. Biology and Fertility of Soils. 33, 238-245.
8. Holding, A. J. & Collee, 1. G. (1971). Routine biochemical tests. Methods Microbio 6A, 1-32.
9. hibar. A , Sanchez-Perez, J. M., Lyautey, E., &
Garabetian, F. (2008). Differentiated free-livmg and sediment-attached bacterial community structure inside and outside denitrification hotspots in the nver-groimdwater interface. Hydrobiologia, 598{X), 109-121.
10. John, R. C , & OkpokwasiU, G. C. (2012).
Crude oJ-degradation and plasmid profile of nitrifying bacteria isolated from oil-impacted mangrove sediment in the Niger Delta of Nigeria. Bulletin of environmental contamination and toxicology, SS(6), 1020-1026.
11. Joo, H.S., Hirai, M.,& Shoda, M. (2005).
Characteristics of ammonium removal by heterohophie nitrifieationeaerobie denitrification by Alcaligenes faecalis no. 4. Joumal of Biosciences and Bioengineering 100,184-191.
12. Kim, D. J.. Lee. D. I., & KeUer. J. (2006).
Effect of temperature and free ammoma on nitrification and nitrite accumulation in landfill leaehate and analysis of its nitrifying bacterial community by FISH. Bioresource technology, 97^^, 459468.
13. KjeUin. J., Hailin, S., & Worman, A (2007).
Spatial variations in denitrification activity in wetland sediments explained by hydrology and denitrifying community structure. Water Research, 41(20), 4710- 4720.
14. Ueu, P. K., Homan, H., Kurogl, A., Kawagoshi, Y., Fujii, T., & Furukawa, K. (2006).
Characterization of sludge from single-stage nihogen removal usmg anammox and partial nitiitation (SNAP). Japanese Joumal of Water Treatment Biology, 42, 53-64.
15. Miller, D. N., & Smith, R. L. (2009).
Microbial characterization of nitrification in a shallow, nihogen contammated aquifer. Cape Cod, Massachusetts and detection of a novel cluster associated with nitrifying Betaproteobacteria.
The Journal of Contaminant Hydrology, 103,182-93.
16. Pham Thi Tuyet Ngan, Trdn Nhan Dung, Duong Mmh Vi^n (2011). Khao sat mat dp va su da dang cua vi khuan nitrate hda trong ao nuoi tdm. Tap chilOioahoc-Daihoc Can Tho, 20b, 69-78.
17. Poroshina, M. N., Doronina. N. V., Kaparullina, E. N., Kovalevskaya, N. P., &Trotsenko.
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ISOLATION AND IDENnnCATION OF AMMONIUM REMOVAL BACTERIA FROM PIGGEEY- CULTUKE, CATFISH-CULTUSE AND SHRIMP-CULTURE SLUDGE
Doan Thi Tam', Do Ngoc Anh Huy', Nguyen Thi Dung', Do Thanh Dat?, Nguyen Thi Men^ Chau Tan Phat=
'Biotechnology center of Ho Chi Mmh city 'Ho Chi Minh city University of Food Industry
^Van Lang University Summary
The study focused on screening for ammonium nittifying bactena from piggery-culture sludge, catflsh- culture sludge, and shrimp<:ultture sludge. Samples were collected in die surrounding areas of Ho Chi Minh city. Tien Giang and Ben Tre province. The isolates witii ammonium niuifying capability from those sources above were in a range of (1.5 - 2.4) X 10' MPN/g. (0.15 - 1.5) X W MPN/g and (0.93 - 2.4) x Iff MPN/g. respectively. Based on morphological and biochemical analysis, 6 strains which were similar to /•aracocci/sgenus and 3 sfrains which were sunilar to NiUosomonasgeaas were selected. Four strains had die highest ammonium removal acUvity (120.83 -123.66 mg/L) after culturing for 168 hours were chosen to perform 16S rRNA sequence The results of sequencing and comparing to data on GenBank showed that:
two strains, designated as S4 and S6. are homologous with Paracoccus communis (99%). the S9 strain is homologous with Paracoccus versutus (100%). and the S3 strain is homologous with Nitrosomonas europaea (10056). In our study, two strains S4 and S6. belongmg to Paracoccus communis genus, have shown their ammonium utlizing capability up to 74.9% and 76.65%. respectively. To our knowledge, this is the first time Paracoccus communis have been isolated from sludge and reported as ammonium nitrifying bactena.
Keywords; Paracoccus commums, Nitrosomonas europaea, Paracoccus versutus.
Ngu« phan bi^: PGS.TS. KhuSt HOu Thanh Ngiy nhjn bai: 21/02/2020
Ngiy thang qua phin bian: 23/3/2020 Ngiy duyet dang: 30/3/2020
106 N O N G NGHIEP VA PHAT TRIEN N O N G THON - KY 1 - THANG 6 / 2 0 2 0
