Isolation and phylogenetic analysis of heterotrophic nitrobacteria

Journal of Harbin Institue of Technology (New Series), Vol. 15, No. 6, 2008Isolation and phylogenetic analysis of heterotrophic nitrobacteriaSU Jun-feng'2 , MA Fang' , GUO Jing-bo' , HOU Ning' , CAO Shan-shan' , WANG Hong-yu'苏俊峰,马放，郭静波，侯宁高珊珊,王弘宇(1. School of Muniripal and Environmental Engineering, Harbin Instiute of Technology, Harbin 150090, China;2. School of Environmental andMuniceipal Engineering, Xi' an Universily of Architeture and Technology. Xi' an 710055, China;3. Dept. of Municipal Engineering.Wuhan University, Wuhan 430072 ,China)Abstract: A method about the isolation of heterotrophic nitrosobactria and the characteristics of heterotrophicnitrosobactria were studied. It can be seen from the fluorescence in situ hybrdization results of the bio-mem.brane sample from the bio-ceramic reactor, the spots of green nitrosobacteria are obviously more than those oforange nitrobacteria. Two heterotrophic nitrobacteria were isolated from the bio-ceramic reactor. By sequencing16SrDNA and establishing the phylogenic tree, they were identified physiologically and biochemically asPseudomonas sp.. After 12 d, the COD removal efficiency of wgy55 and wgy68 were 45. 03% and 50. 85% ,the NH.-N removal fficiency of them were 80. 12% and 85. 93%。and the TN removal eficiency of them were69. 71% and 64. 7% . The final concentration of NO2-N of wgy55 and wg>68 was 0. 753 mg/L and 0.601 mg/L, and that of NO3-N was 3.21 mg/L and 3.38 mg/L. These results show that wgy55 and wgy68 have the ca-pability of nitrifcation and they are heterotrophic nitrobacteria.Key words: bio-ceramic reactor; luorescence in situ hybridization; heterotrophie nitrificationCLC number: X703Document code: AArticle ID: 1005-91 13(2008 )06 0790-04As nitrogen pollution has become a gteater cause forThe aims of this work were (1 )to isolating hetero-concermEutrgphication due to thetrophic bacteria from bio-ceramic reactor. (2) to obtainpresence of nutrients including nitrogen was a wel-rec- information about characterization of these bacteria inognized environmental problem worldwide. As a result,terms of the ability of heterotrophic bacteria. (3) to i-stringent standards had been imposed on TN( total nitro-dentify 2 bacteria through their circadian and biochem-gen) concentration levels in efluent from wastewater istry detecting and 16S rDNA gene sequence checkingtreatment systems in many parts of the woad. In China,to genus and category.for example, new built plants were required to achieveammonia and TN concentration in the efluent of 5 mg/L 1 Materials and Methodsand 15 mg/L,respectively ; existing plants would need tobe upgraded to ulimately meet the. same standard， 1.1 Experimental SetupGB18918 - 2002( China ) researchers'2.A bio-ceramic reactor made by organic glass col-Nitrification, which is performed by ammonia-oxi-umn was used in the pilot-scale experiment. It wasdizing bacteria ( AOB) converting NH4-N to NO2-N and50 mm in inner diameter and 1200 mm in height. Atthen by nitrie-oxidizing bacteria ( NOB ) converting the the bottom, there was a 100 mm gravel supporting lay-latter to NO3-N, is a crucial process of the global N cy-er, and then fixed the column with 800 mm ceramisitecle l4J.It is generally thought to be performed by a situwhich was 3 -5 mm in diameter. Sampling ports wereof autotrophic bacteia over a long time. Since the intalld for each 200 mm over the height of the reac-growth rate of heterotrophic is much higher than that oftor. The compressed air was introduced to the reactorautotrophs, it would be important to find some hetero-from the bottom by aeration diffuser and the aeration a-trophs that have nirifieation acivitys.6. The phenome- mount was adjusted by an aerometer.non of heterotrophic nitrification was described as early.2 Synthetic Wastewateras 1894 as a fungus. Since than , numerous reports dem-The experiment was performed by using syntheticonstrated unequivocally that nitrite/ nitrate production iswastewater，which contained NH4CI， Na,HP02,not restrictte to autotrophic ammonia bacteria or nitriteKH2PO2, MgSO4●7H20, MnSO, , NaHCO3, NaAC●oxidizers but is a widespread phenomenon among differ-3H2中国煤化工osition of the yn-ent genera of fungji and heterotrophic bacteria'7-10.theticTYHCNMHGRereived 2006 -08 -25.Sponsored by the National Naturl Science Foundation of China( Crunt No. 50521 140075)..790.Journal of Harbin Institute of Technology (New Series), Vol. 15, No. 6, 2008Tab. 1 Composition of synthetic wastewaterpended in 200 μL.2 x PBS and added to an equal vol-ComponentConcenration/(mg.L")ume of 100% ethanol. The cell mixture was left over-COD106 -316.night at4-6 C for fixation. For paraformaldehyde fix-ation，the washed cell pellet was resuspended inNH,*-N42.78 -73.62250 μ山I x PBS by vortexing. Then, 750 μL of freshI-NO,"-N0-1.92prepared paraformaldehyde fixative was added (4% f-0-0.22.nal concentration ) and thoroughly mixed. The cellmixture was incubaled at 4 -6 C overmight. The fixedP3-6cells were centrifuged and washed with 1 x PBS andH7.2-8.0subsequenly resuspended in 100 - 200 μL of 2 x PBSand an equal volume of 100% ethanol. The fixed cells1.3 The Formation of Bioceramicswere stored at 20 C for later use.The donesticated NH,-N of oxidizing bacteria liq-1.4.2 Hybridizationuid culture was pumped to circulate in the bio-ceramicA 10 μL cell suspension was loaded in each gelat-column for 2 h, and then stopped pumping at 2 h inter-in-coated well of a 6-well Teflon-printed slide andval. Sterile water was then added to wash bio-ceramicdried for 10 min at 46 C. The cells were dehydratedbed and supporting layer after 18 h. Continue this pro-by successive passage through 50, 80, and 96% etha-cedure for several times. Clear efluent indicated thesenol washes (3 min each) and air-dried. To each wellfree cells had been immobilized on the bioceranics. Af-on the slide, 10 μL of freshly prepared hybridizationter 15 d, the NH/N removal eficiency was about 60%buffer ( containing the appropriate NaCl and formamideand microbe cell had attached on the bioceramics.concentration for the used probe, Tab. 1 ) and I μLWastewater was fed continuously into the system fromprobe ( at a concentration of 50 ng/L) working solutionthe bottom of reactor with flow rale of 0.6 m'/(m2 ●was added to a well. The hybridization was conductedh). The temperature was kept constantly at 20 -25 C.for 90 min at 46 C in a humidifed chamber. Follow-1.4 Fluorescence in Situ Hybridization (FISH)ng hybridization, a stringent washing step was per-1.4. 1 Cell fixalionformed for 15 min at 48 C in a buffer with the appro-Cell suspension/floc was centrifuged and washedpriate NaCl concentration (Tab. 1), 20 mmolVL Tris-twice with1 x PBS ( 145 mmol/L NaCI, 8. 7 mmol/LHCl, pH 7, 5 mmol/L EDTA, and 0.01% SDS. TheNa2HPO,，1.5 mmol/L NaH,PO, pH 7.4) prior todata were shown in Tab. 2.fixation. For alcohol fixation, the cell pellet was resus-Tab.2 Probes used for fluorescence in situ hybridization and hybridization solutionBacteriaProbeSequence (5'-3')5'-market Fomamide/% SDS/% Tis-HCV(mol.L-1) NacCV(mol.L"')Nirobacteria NS0190 CCACCTCTTTCTCC FAM550.01200.9Nirobacteria NIT3 CCTGTCTCCATCCTCCC HEX40CNTT3 CCTGTGCTCCACCCTCCCAll oligonucleotide probes were obtained from Mi-composition was identical to the liquid medium with thecrnosynth. Microscopy was perfomed on an Olympus BX50addition of 10% agar. The composition of the LB liquidFluorescence microscope. Digital images were taken withmedium was as follows: peptone 10 g/L, beef extracta CCD camera. The positive was orange and green.3 g/L and sodium chloride 5 g/L. The resulting isolates.5 Separate Mediaof bacteria were tested for their ability to produce nitrite1.5.1 LB mediaor nitrate by inoculation into the liquid ammonia medi-Meat-extract3g, NaCl5 g, peptone 10g, H2 0um. and cultivate the bacterium in the temperature of1000 mL,pH 7.0-7.2.30 C. Spot tests for nitrite were made on approximately1.5.2 Ammonia media2 mL medium using the Griess - losvay method everyNH,CI 0. 382 g, NaC00CH,2 g, MgS04●7H2Oweek. When the test proved positive for nitrite. A2 mL0.05 g, K2 HPO.0.2 g, NaCI0. 12 g, MnSO。●4H2aliquot of the enrichment cultures was transfefred to ni-00.01 g, FeSO, 0.01 g, H20 1000 mL, pH 7.0 -trite中国煤化irobial srains were8.0.sprea: sterilized nichrome1.6 Isolation of Bacteria Strains Capable of Het-loops:YHC N M H Grial strains were ierotrophic Nitrificationsolaleu and punilea Dy repeated streak plate.Pure isolates were obtained from the system by1.7 The Examination of the Trackin of the Nitrogenplating onto a peptone- meat extract( LB) agar; theThe tracking studies for the aerobie condition were.791.Joumal of Harbin Instiute of Technology (Nen Series)，Vol. 15, No. 6, 2008conducted to investigate the ability of heterotrophic ni-trification by the pure cultures isolated. Samples of ap-proximately 5 mL mixed liquid was added to 500 mLconical flasks containing 300 mL of the sterilized am-monia and nitrite media;the pH was adjusted to 7. 0 -8. 0 by the addition of filter sterilized 1 mo/L H2SO4or NaOH. The flasks were incubated at 30 Con a rota-ry shaker at around 120 r/ min. The medium was filtra-ted and analyzed colorimetrically for ammonium, ni-trite ，nitrate and total nitrogen each day.1.816SrDNA Sequence Analysis and lsogenyFig.2 Fish micrograph of bio-ceramic sludge using probeComparisonNIT3 and CNIT3Using universal primer ( BSF/20, BSR1451/20 )of 16sDNA gene for amplifying the DNA of the bacteri-2.Heterotrophic Nitrifying Ability of the Isola-m，taking 3 μL product and then detecting it by 1%ted Strainsagarose gel electrophoresis. After recycling, clone theDuring determination of characteristics for hetero-product to T-carrier ( Bao Shengwu), measure the se-trophic nitrifying bacteria， NaCOOCH, and ammoniumquence by sense and anti-sense universal primer. Com-chloride were used as carbon and nitrogen source. Thepare the 16sDNA of the sequencing results with the al-COD concentration declined significantly , These phenom-ready registered sequences by BLAST, and by usingena indicate that at the beginning of incubation , isolatedBlastn as well as Cluastx, PhyloDraw and other soft-microorganisms utilized a growing portion of the carbonware provided by NCBI, we' ve found isogeneic se-content in the mixed liquid to gain energy for growth. Itquences and estabolished the dendrogram.was a course of assimilation for the bacteria. After5 d theCOD concentration remained stable. The COD removal2 Results and Discussionefficiencies by wgy55 and wgy68 were 45. 03% an50. 85%，respectively. The above results indicated that21 Analysis of Fluorescence in Situ Hybridizationwgy55 and wg>68 had special heterotrophic ability to uti-As we can see from the results of fluorescence inlize organic substrate as their energy source.itu hybridization of the activated sludge from the bio-As shown in Figs.3 -5, we can find that at theceramic reactor. as shown in Figs.1 and2, green ni-beginning period the TN descent quickly, the TN re-trosobacteria spots were obviously more than orange ni-moval efficiency of strain wgy55 and wgy68 wertrobacteria, the nitrosobacteria distributed in the strip69.71% and 64. 7% respectively, and the removal ef-shape on the activated sludge， while the nitrobacteriaficiencies of ammonium nitrogen were 80. 12% anwere in spot shape, thus the quantity of nitrosobacteria85.93%. On the Ist or 2nd day，nitrification occurredwas far more than that of nitrobacteria. This indicatedand assimilation of the bactenia ceased, the nitrite ni-that the growth and reproduction of nitrobacteria can betrogen( NO2-N) concentration and the nitrate nitrogeninhibited by contolling the concentration of DO, while( NO3-N) concentration don' t change obviously. NO2-nitrosobacteria could be the predominant population,N concentration increased slowly in the first few daysthus the accumulation of nitrite carbon happened andand reached the peak at the lIth and 12th for strainShortcut Nitrification Denitrification had achieved.wgy66，wgy62 respectively. The concentration of NO3-NpeakedattheI2thdayforstrainwgy55andwgy68000一一NH-N (wg>68) --TN (wgy68)NHcN(wgySS) - ---TN (wgy5)150曾10050中国煤化工二一YHCNMHG9102Fg. 1 Fish micrograph of bio-ceramic studge using probeFg.3 Change in concentration of NH,-N and TN in am-NSO190monia media●792●Jourmal of Harbin Institute of Technology (New Series). Vol, 15. No. 6.20083 Conclusions.0 r.5s1) As can be seen from the fuorescence in situ.0hybridization results of the biomembrane sample from62.5◆一NO-N(wgy68) - 昌- NO-N (wg68)the bio-ceramic reactor, green nitrosobacteria spotswere obviously more than orange nitrobacleria, the ni-曾2.0NO-N(wgy55) - *- NO-N (wgy5)trosobacteria distributed in the strip shape on the acti-a 1.5vated sludge， while the nitrobacteria were in spotshape , thus the quantity of nitrosobacteria was far moreo.s tthan that of nitrobacteria.2) After 12 d, the COD removal efficiency ofwgyS5 and wgy68 were 45. 03% and 50.85% , NH.Nof wgy55 and wgy68 were 80. 12% and 85. 93%，TNFig.4 Change in concentration of NO-N and NO3-N inof were 69. 71% and 64. 7% . The final concentrationammonia mediaof NO2-N of wgy55 and wgy68 were 0. 753 mg/L and0.601 mg/L, and NO3-N were 3.21 mg/L and2500十wg68.3. 38 mg/L. These results showed that wgyS5S and000wgy68 have nitrification capability and they were heter-otrophic nitrobacteria.; 15003) With the aid of sequencing 16S rDNA and etablishing the phylogenic tree, wgy55 and wgy68 were出出台1000identified physiologically and chemically asPseudomonas sp00 tReferences:[1] MulderA, Graf van de A A, Robertson L A. et al. Anae-robic anunonium oxidation discovered in a deritrilying fluid-ized bed reactor. FEMS Microbiology Ecology , 1995 ,16:177Fig.5 Change in concentration in COD- 184.[2] SerraltaJ, Mbes J, Seco A, et al. A supervisory controlrespectively. As the NO2-N and NO3-N concentrationsystem for optimizing nitrogen removal and aeration energyincreased, the COD did so at the same time. This indi-consumption in wastewater treatment plantsl. Wat Scicaled that the organie matters might be released via theTeeh,2002 , 45(5) :309 -316.[3] Zhiguo Y, Adrian 0, Inglldsen P. 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