characterization of the microbial community in an anaerobic imp fish

8/6/2019 Characterization of the Microbial Community in an Anaerobic Imp Fish

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JOURNAL OF BIOSCIENCE AND BIOENGINEERINGVol. 94, No. 5, 4124 18. 2002

C h a r a c t er iz a t io n o f t h e M i c r o b i a l C o m m u n i t y i n an A n a e r o b i cA m m o n i u m - O x i d i z i n g B i o f i l m C u l t u r e d

o n a N o n w o v e n B i o m a s s C a rrier

TA K A O F U JI I, 1 . H I R O Y U K I S U G I N O , 1 J O S E P H D . R O U S E , 2AND K E N JI F U R U K A WA 2

Department o f Applied Life Science, Faculty o f Engineering, Sojo University, 4-22-1 Ikeda, Kumam oto 860-0082,Japan I and Department of Civil Engineering and Architecture, Kum amoto University,

2-39-1 Kurokami, Kumam oto 86 0-8555, Japan 2

Received 27 May 2002/Accepted 6 August 2002

T h e e n r i c h m e n t a n d c h a r a c t e r i za t i on o f an a e r o b i c a m m o n i u m - o x i d i z i n g b i o f il m c u l t u r e s a reo n g o i n g i n o u r l a b o r a t o r i e s . B i o m a s s , w i t h a p r e d o m i n a t e l y r e d c o l o r, d e m o n s t r a t i n g s i m u l t a -n e o u s r e m o v a l o f a m m o n i u m a n d n i t r it e u n d e r a u t o t r o p h i c a n d a n o x i c c o n d i t io n s , w h i c h i s c h a r -a c te r is ti c o f a n a er o b i c a m m o n i u m - o x i d i z i n g p l a n c t o m y e e t e s , w a s e n r i c h e d a n d m a i n t a i n e d f o r ane x t e n d e d p e r i o d o n a p o l y e s t e r n o n w o v e n c a r ri er. To i n v e s t i g a te t h e b a c t e r i a l c o m p o s i t i o n o f t h em a t u r e b i o fi lm c o m m u n i t y, 1 6 S r D N A s e q u e n c e s w e r e a m p l i f e d b y P C R a n d c o m p a r a t iv e a n a -l y se s u s i n g D N A d a t a b a s e s w e r e c o n d u c t e d . O n l y o n e s e q u e n c e h a d a n o t a b l e s im i l a r it y ( 9 2 . 2 % )t o t h a t o f t h e fi r st d i s c o v e r e d a n a e r o b i c a m m o n i u m - o x i d i z i n g p l a n c t o m y c e t e a n d l es s er , y e t s ig -n i f i c a n t , s i m i l a r it i es t o t h e 1 6 S r D N A s e q u e n c e s o f o t h e r r e c e n t ly r e p o r t e d a n a e r o b i c a m m o n i u m -o x i d i z i n g s tr a i n s . T h e n e w l y d is c o v e r e d s t r a in ( d e s i g n a t e d K S U - I ) r e p o r t e d h e r e w a s d o m i n a n ta m o n g d e t ec t a b le m e m b e r s o f t h e b i o f l m c o m m u n i t y. B y f lu o r e s c e n c e i m a g i n g , K S U - 1 w a ss h o w n t o fo r m s p h e r i c a l c l u s t er s w r a p p e d i n a t h i n la y e rofZoogloeas p . P o s s ib l e i n t e r a c t i o n s a n di n t e r d e p e n d e n c i e s o f t h e s e t w o s p e c ie s a r e d i s c u s s e d w i t h r e g a r d t o th e p u t a t i v e u n c u i t u r a b i l i t y o ft h e a n a e r o b i c a m m o n i u m - o x i d i z i n g p l a n c t o m y e e t es .

[Key words : anaerobic ammon ium oxidation, anammox , 16S rDNA , phylogenetic analysis, planctomycetes,Zoogloeasp.]

The r ecen t ly d i s cove red anae rob i c ammonium ox ida t i on(anammox ) p roces s i s one o f t he mos t i nnova t ive t e chno-log i ca l advances fo r t he r emova l o f n i t rogenous con t ami -nants f rom wastewater (1 , 2) . In the anammox reac t ion ,ammonium and n i t r i t e con t r i bu t e i n nea r ly equa l mo la ramou nts to the format ion o f d in i trogen gas (N2) . The s to-i ch iome t ry o f t he r eac ti on was de t e rmined by S t rouset al .(3) to be

NH4++ 1 .32 NO 2-+0.066 HC O 3 +0.13 H +1.02 N2+0 .26 NO 3 +0 .06 6 CH200.sN0.15+2.03 H2 0

(1 )

Compared wi th a t rad i t ional t rea tment sys tem for the re-mova l o f amm onium tha t coup le s n i t r if i c a ti on w i th den i t r i-f ica t ion , the anammox process (coupled wi th par t ia l n i t r i f i -ca t ion) i s econom ical ly favorable in tha t i t has o nly ha l f theoxygen demand and r equ i r e s no o rgan i c ca rbon . The bac -t e r ia r e spons ib l e fo r anam mox a re ve ry s low g rowing o rga -n i sms , d iv id ing on ly on ce eve ry two weeks , wh ich canno tbe cu l t iva t ed u s ing conven t iona l m ic rob io log i ca l t e chn iques(4 ). Compara t i ve s equence ana ly s is o f 16S rDNA was u sedto i den t i fy t he anae rob i c ammonium-ox id i z ing bac t e r ium

* Corresponding author, e-mail: [email protected]: +81 -(0)96-326-3111 fax: +81-(0)96-323-1331

412

f i r s t d i s cove red i n The Ne the r l ands a s a nove l , deep lyb ranch ing p l anc tomyce t e (4 , 5 ) f o r wh ich t he n amePla nc to -myceta les Candida tusBrocad ia anammoxidans has beenproposed (6) . Subsequent ly, an increas ing number of 16SrDNA sequences f rom o the r anae rob i c ammonium-ox id i z -ing bac ter ia enr iched f rom habi ta ts in Europe have a lsobeen repor ted (5 , 7) , wi th a l l of the bac ter ia be ing phy loge-ne t i ca ll y g rouped wi thCand ida tu sB. anammoxidans .

In our labora tory, resul t s of ba tch s tudies un der ana erobicand oxygen-s t ressed auto t rophic condi t ions us ing a s ludgeseed o r ig ina ti ng f rom K umam oto , r evea l ed n i t rogen t r ans -format ion s tha t could not be ex pla ined by t radi t ional n i t r i fi -

ca t ion and deni t r i f ica t ion (8) . Most of the t ransformat ionsiden t if i ed in ou r ea r ly work were v e ry s low and appea red t obe due to auto t rophic de ni t r i f ica t ion by s t ressed n i t r i fy ingorganisms. In subsequent cont inuous-f low exper iments , apo lyes t e r nonw oven b iomass ca r r ie r w i th a p o lymer coa t i ngdes igned to enhance r e t en ti on o f mic roo rgan i sms was u sedas the f i xed med ium fo r b io f i lm a t t achmen t. The n onwo venmater ia l was se lec ted because i t has been shown to be effec-t ive in enhancing b iomass re tent ion in t rea tment sys temsdes igned for n i t r i fica t ion (9) and the deg radat ion o f p-n i t ro-pheno l (10 ) . Us ing t he s ame s ludge (Kum amoto sou rce ) asseed i n t he con t inuous - f l ow syst em, t he r emova l o f ammo-nium and n i t r i te, wi th re la t ive ly low produ ct ion of n i t ra te a t


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voc. 94, 2002 ANAEROBIC AMMONIUM-OXIDIZING BIOFILM COMMUNITY 413

ratios strongly resembling that of the anammox reaction,was achieve d (11). Furthermore, a distinct ly red biomasswas formed, which is characteristic of anaerobic ammo-nium-oxid izing planctomycetes (12).

Similar results in a continu ous-f low system in which thebacteria were attached to a nonwoven carrier were also ob-tained using a sludge seed from a distinctly different geo-

graphical region (13). In that work, the dominant anaerobicammoni um-oxi dizi ng strain (designated A8) was identifiedas having a 16S rDNA sequence simil arity of 99.1% withthe archetypal C a n d i d a t u s B. anammoxidans.

The objective of this research was to characterize themicrobial community in a well-established anaerobic am-monium-ox idizin g biofilm on a nonw oven biomass carrierenriched from a seed sludge originating in Kumamoto,Japan. This objective was met, in part, by using 16S rDNAcomparative sequence analysis to determine the phyloge-netic identities of the dominant members of the microbialcommunity. Real-time PCR amplification with fluorescentdyes was used for quantification of the community mem-bers. In addition, fluorescent i n s i t u hybridization (FISH) inconjuncti on with epifluorescence microscopy and confocallaser microscopy were used for imaging of i n s i t u coloniza-tion.

M A T E R I A L S A N D M E T H O D S

Acc l ima t ion o f b io f i im The source of the seed sludge usedin this research was a laboratory-scale fill-and-draw denitr ificat ionreactor that used methanol as the electron donor. The biofilm hadbeen enriched for over one year of continuous-flow treatment intwo 2-I reactors (11) on support media consist ing of nonwov enporous polyester material (2 34 cm strips, 0.7 cm material thick-ness) with a pyridin ium-type polymer (Japan Vilene, US patent

5,185,415, 1993) designe d to enhance the retention of biomass.For this phase of research, 2 5 strips were transferred and sus-pended from a wire frame in a new larger reactor for a total one-sided sheet area o f 1700 cm 2. The reactor was made of glass andhad an inner diameter of 9.0 cm and height (to effluent port) o f43 cm (Fig. 1 . The total liquid volume was 2 . 7 l and the reactionzone (excluding the upper 3.0-cm settling zone) was 2.5 l . The re-actor was operated at 30C in the continuousflow mode withoutrecycling and maintained in the dark using a black vinyl sheetenclosure. The influe nt was supplemented with NH4CI and NaNO=at various concentrations, 12 5 mg 1 ~ KHCO3, 54 mg I ~ KH2PO 4and 9 mg/-~ FeSO 4. 7H20 (with 5 mg l ~ EDTA. 2Na as a chelat ingagent) mixed in groundwater of low total hardness containingapproximately 3 mg N l ~ of nitrate (groundwate r further describedb5 Rouse e t a l . [[4]). Airtight integrity was maint ained in the

capped reactor using an effluent water trap. Oxygen intrusion viathe influent, however, was not complete ly prevented but the oxi-dation reduc tion potential (ORP) was regulated to 238_+55 mV(n=40, hydrogen reference) as a result of approximately 10 rain ofdaily purgi ng with N 2. Effluent ORP levels were slightly lower(225_+ 51 mV), which could indicate further biochemical reductionof dissolved oxygen in the reactor. Theoretically, the biologicalnitrification of only I mgN FJ of ammo nium (to nitrite) wouldconsume 3.4 nag / ~ oxygen, which would facilitate the generati onof anoxic conditions.

Phy logen e t i c ana lys i s o f 16S rDNA f rom b io f i lm bac te r i aBiofilm material was removed from the support matrix by gentlymixing in TE buffer (10ra m Tris-HCI [pH8.0], l mM EDTA).The support was then removed and the biofilm material was pre-

inflt

FIG. I. Glass reactor with 9.0-cm inner diameter. Height to efflu-ent port, 43 cm. Nonwoven biomass carrier strips suspended from awire frame (see text).

cipitated by centrifugation for l min at 5000g. Total genomicDNA was extracted using the Isoplant kit (Nippongene, Tokyo)tbllowing the manufacturer's instructions.

Partial 16S rDNA fragments were amplified from the genomicDNA by PCR using 16S-1 (5'-t~AGTGGCGAAAGGGTGAGTAAI2-3') and 16S-2 ( 5 ' - 3 2 9 T G T C T C A G T C C C A G T G T G G C 3 1 % 3 ' )(DDBJ accession no. E05133; superscript numbers indicate E s e h -er ich ia co l t sequence positions) oligonucleotide primers, which

were designed from conserved sequences of known anaerobicammonium-oxid izing bacteria and E. colt . It was confirmed by anucleoti de-nu cleo tide Blast search of a DNA database (NCBI,http://www.ncbi.nlm.nih.gov/) that the 16S-1 sequence had at mosttwo nucleotide mismatches and the 16S- 2 sequence was com-pletely conserved in the 16S rDNA sequences o f prokaryotes.Thermal cycling consisted of 40 cycles each o f 15 s at 94C, 2 s at55C and 30 s at 68C.

To ampl ify full-length 16S rDNA sequences, PCR was per-lbrmed using an oligonucleotide primer pair based on conserved16S rDNA sequences close to the 5' and 3' temlina l regions of the16S rDNA of some anaerobic ammonium-oxidizing bacteria: 16S-5' (5'-3TGGCGGCGTGGffTTAGGC4L3 ) and 16S-3' (5'-1526GGTTACCTTGTTACGACTIS-3') (DDBJ accession nos. AB015552,AJ13189 and AJ250882; superscript numbers indicate AB015552

sequence positions). Thermal cycling consisted of 40 cycles eachof 15 s at 94C, 2 s at 50C, and 60 s at 68C. Other PCR condi-tions were the same as those described above.

The PCR products were ligated into the H i n c l l site of pBlue-script II SK(+) (Toyobo, Osaka), and E. colt XL-I Blue cells(Stratagene, CA, USA) were transformed using the constructedplasmids. The nucleotide sequences were determined using theBigDye termina tor cycle sequencing kit (Applied Biosystems,Tokyo) with the M13 RV primer (Takara, Kyoto), the -21M13universa l primer (Applied Biosystems) and an additional primerdesigned following the determinat ion of internal sequences. Thesequences determined in this study were compared with se-quences in the DDBJ database and phylogenetic analyses wereperformed using Clustal W and Treeview programs (15).


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414 FU JII ET AL . J. BIOSCt.BIOENG.,

TABLE 1. Designed primers used for real-time PCR targeting of 16S rDN As

Primer Nucleo tide sequence (5'-3') Sequence position TargetAna-F GGGATAACAACGTTCCGCAA 90-109 a KSU- 1Ana-R CGATACCGAAGCACCATGAGT 164-144a KSU- 1Zoo-F CGAGGTCGTTGCCAATCC 34-54 b Zoogloea sp .Zoo-R CCTGGCGAAGTCAGGCTAATA 100-83 b Zoogloea sp .1 6 S - F 3 A G C C AT T G TA T G C A C G T G T G 1 24 5-1 22 6 c B ac te ria16 S-R2 CGTCA GCTCGT GTCGTG AG 1081 - 1063c Bacteria

Al l prim er sequences were designed us ing Primer Express (version 1 .0) software (A ppli ed Biosystems)."KSU-I sequence numbering (this study, accession no. AB057453).b Zoogloeasequence numbering (this study, accession no. AB077826).

E. colisequence numbering (accession no. E05133).

Quant i t a t ive PCR ana lys i s To determine the frequency dis-t r ibut ion o f 16S rDNA phy lo types in the b io f i lm communi ty, r ea l-t ime PC R am plif icat ion was performed in 50%tl react ion mixturesusing buffers containing the f luorescent dye SYBR Green sup pl iedin the SY BR Green PCR Core Reagen t k i t (Appl ied Biosys tems)in Mic roAmp Opt ica l 96-wel l r eac t ion p la tes wi th op t i ca l caps(Appl ied Biosys tems) . The t emp la te DNA in the reac t ion mix tu reswas am plif ied and monitored using the ABI Prism 7700 SequenceDetec t ion sys tem (Appl ied Biosys tems) fo l lowing the manufac -turer ' s instruct ions (with AmpliTaq LD). The pr imer pairs Ana-F /Ana-R and Z oo-F /Zoo-R ( shown in Tab le 1 ) were des igned f romthe spec i fi c r eg ions o f the 16S rDNAs of s t r a in KSU-1 andZoo-gloea sp. , respect ively, that had low sim ilar i t ies with those of theother s t ra ins l is ted in Table 2 . Another pr imer pair, 16S-F3 and16S-R2, was also designed from conserved sequences amongs t ra in KSU -1 ,Candidatus B. anammoxidans ,Zoogloea sp. , and E.coli for enumerat ion of a l l bacter ia . I t was confirmed by a nu-c leo t ide -nuc leo t ide Blas t sea rch o f a DN A da tabase (NCBI) tha tthe 16S-F3 sequence had at most three nucleot ide mism atches andthe 16S-R2 sequence was co mple tely conserved in the 16S rDNAsequences of prokaryotes . Genomic DNA (0.5 ng) extracted fromall microorganisms in the biof i lm was used as a template . Cyclethresholds determined were compared to s tandard curves con-structed using several concentrat ions of template D NA fragmentstha t had been p rev ious ly ampl i f i ed . Re la t ive copy numbers amongtarget organism s were evaluated.

F l u o r e s c e n c ein s i tu h y b r i d i z a t i o n T h e d e t a ch e d b i o f i lmwas im media te ly imm ersed in a 4% (w/v) pa ra fo rmaldehyde so lu -t ion to f ix bacter ia l cel ls . 16S rDNA sequences were hybridizedwi th a n ewly des igned 16S rDNA -spec i f i c o l igonuc leo tide p r imerpa i r (Amersham Pharmac ia B io tech , Tokyo) : Ana- I (5 ' -~68CTCCCGAT ACCGA AGCA CCAT148-3 ' ) and Zoo-1 (5 ' -49GCCTGA CTTTCGCCAGGT32-3 ' ) (DDBJ accessio n nos. AB0778 26 andAB0778 27; sup erscr ipt numb ers indicate respect ive sequence po-si t ions) to detect KSU -1 an dZoogloea sp. s imultaneously. For useas a F ISH probe , the 3 ' - end o f Ana-1 w as l abe led wi th Texas Red-

TABLE 2. Groupings of amplified DN A fragments obtainedfrom the biof ilm using a bacterial 16S rDNA-specific

oligonucleotide pr ime r pair (see text)

NumberGroup of clones Bacterium with highest homo logy

11 CandidatusBrocadia anammoxidans (AJ 131819)11 Uncultured bacterium role 1-33 (AF 280861 )

Zoogloea ramigera(X 74913)1 Unidentified bacterium DNA (AB 021340)

Aquaspirillum metamorphum(AF 078757)3 Uncultured bacterium clone TCC 5 (AF 392634)

Rubrivivax gelatinosus(D 16213)2 Uncultured bacterium PHO-HE36 (AF 314435)I Thiobacillus denitrificans(AJ 243144)

12-dUTP or t e t ramethy l rhodamine (TMR)-5-dUTP us ing the D NATail ing ki t (Roche Diagnost ic , Tokyo) fol lowing the manufac-turer ' s instruct ions. For the same purpose, the 3 ' -en d of Zoo-1 waslabeled with f luorescein isothiocyanate (FITC)-12-dUTP. Hybrid-i za tions o f f ixed b io fi lm samples were pe r fo rmed in 20 mM Tr i s -HC1 buffer (pH 7.2) containing 0.9 M N aC1, 0 .01% SDS and 20%formamide us ing the l abe led p robes as desc r ibed by Amannet al.(16) a t 46C, fo l lowed by wash ing wi th 20 mM Tr i s -HC l buffe r(pH 7.2) containing 0.225 M N aCI, and 0.01% SD S.

F l u o r e s c e n c e a n d c o n f o c a l la s e r m i c r o s c o p yFor imag ingby f luorescence mic roscopy ( inc lud ing FISH) , spec imens werev iewed us ing a DMR f luorescence mic roscope (Le ica Microsys -t e rns , Tokyo) equ ipped wi th a G /R f i l t e r cube . For im ag ing by con-focal laser microscopy, a Leica TCS SP with hel ium-ne on andargon laser sources was used. Fo r acquis i t ion of the confocal lasermic roscope images , the s t andard so f tware package de l ive red wi ththe instrument was used.

O t h e r a n a l y t i c a l m e t h o d s Nit r i t e was quan t i f i ed by ionchromatography ( IA-100 ; TOA Elec t ron ics , Tokyo) o r the con-vent ional color ime tr ic method (17) . Nitra te was qu ant i f ied by ionchromatography o r the U V spec t ropho tomet r i c sc reen ing method(17) . Nitr i te was determ ined to have an interfer ing response in then i tr a t e UV sc reen ing method o f 25% of the n i t r at e r e sponse on ani t rogen weight basis , thus resul ts were corrected by calculat ion.Amm onium was quan t i f i ed by the phena te me thod ( l 7 ).

R E S U LT S A N D D I S C U S S I O N

C u l t iv a t io n o f a n a e r o b i c a m m o n i u m - o x i d i z i n g b i o f il mT h e b i o f i l m c u l t u r e u s e d i n t h is s t u d y h a d b e e n e n r i c h e d o na n o n w o v e n b i o m a s s c a r r i e r i n a r e a c t o r ( F i g . 1 ) o v e r ao n e - y e a r p e r i o d . G r o w t h o f a d i s t i n c t ly r e d b i o m a s s c o u p l e dw i t h n i t r o g e n t r a n s f o r m a t i o n p a t t e r n s a p p r o x i m a t i n g t h es t o i c h i o m e t r y s h o w n i n E q . 1 w a s o b s e r v e d a n d , i n a l o a d -i n g r at e s t u dy, v o l u m e t r i c a m m o n i u m a n d t o t a l - n i tr o g e n r e-m o v a l r a t e s o f 2 7 a n d 5 2 m g N / - 1 h 1, r e s p e c t i v e l y, w e r ea c h i e v e d ( 1 8) . S u b s e q u e n t l y, s t e a d y - s t a t e o p e r a t i o n w a sd e m o n s t r a t e d o v e r a p e r i o d o f si x m o n t h s a t 3 0 C w i t h th ei n f lu e n t a m m o n i u m a n d n i t ri t e c o n c e n t r a t i o n s a t a p p r o x i -m a t e l y 2 5 0 m g N / - 1 e a c h a n d a h y d r a u l i c r e t e n t i o n t im e o f7 . 4 h . Av e r a g e v o l u m e t r i c a m m o n i u m a n d t o t a l -n i t r o g e n re -m o v a l r a te s d u r i n g t h i s p e r i o d w e r e 2 1 + 3 . 4 a n d 4 1 + 5 . 2m g N L 1 h 1 , r e s p e c t i v e l y ( n = 4 2 ) , w h i c h a r e c l e a r l y w i t h i nt h e r a n g e f o r i n d u s t r ia l a p p l i c a t i o n . Wi t h t h e t o t a l - n i tr o g e nr e m o v a l e f f i c i e n c y c o n s i s t e n t l y a t 6 0 _ + 6 . 3 % , t h e s e r e s u l t si n d i c a t e d th a t t he m a x i m u m e f f i c i e n c y u n d e r t h e t e s t c o n d i -t i o n s b e i n g e m p l o y e d h a d b e e n a c h i e v e d a n d t h a t a n a n a e r o -b i c a m m o n i u m - o x i d i z i n g c u l tu r e h a d r e a c h e d a s t ab l e le v e li n th e b i o f i l m c o m m u n i t y. T h e s t o i c h i o m e t r i c r a ti o s o f n i-


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CandidatusKuenenia Stuttgartiensis (anoxic biofilm clone Plal-47) (AF202655)

CandidatusKuenenia Stuttgartiensis (anoxic biofilm clone P la 1-1 ) (A F202660)

Candidatus Kuenenia Stuttgartiensis (anoxic biofilm clone Pla2-48) (AF202663)

CandidatusKuenenia Stuttgartiensis (anoxic biofilm clone Pla2-19) (AF202661)

Ca~did~it~,~u~tlerli~S fl l t ~ ia~:'isilil f o i~i ~ ~ i fi~mC ~ii~1~2a12_2141A~2FI20626625)9

C a n d i d a t u s B r o c a d ia a n a m m o x i d a n s ( A J 1 3 1 8 1 9 )

Uncultured anoxic sludge bacterium KU1 (AB0540 06), A8

U n c u l t u r e d p l a n e t o m y e e t e s K S U - l ( A B 0 5 7 4 5 3 , t h is s tu d y )

CandidatusKuenenia Stuttgartiensis (anoxic biofilm clone Pla1-48) (AF202656)

-

CandidatusKuenenia Stuttgartiensis (anoxic biofilm clone Plal-44) (AF202657)Anaerobic ammon ium-oxidizing planctomycete GR-W P33-41 (AJ296629)

-- CandidatusKuenenia Stuttgartiensis (anoxic biofilm clone Pla2-10) (AF202658)

Anaerobic amm onium-oxidizing planctomycete KOLL 2a (AJ250882)

Anaerobic amm onium-oxidizing planctomycete GR -WP33 -37 (A 301578)

Anaerobic amm onium-oxidizing planctomycete GR-WP54 -11 (AJ296620)

Uncultured anoxic sludge bacterium K U2 (AB 054007)

- Anaerobic ammo nium-oxidizing planctomycete GR-W P33-59 (AJ296618)

Anaerobic amm onium-oxidizing planctomycete GR-W P33-66 (AJ296619)

0 .01

FIG. 2. Phylogenetic tree based on the results of 16S rDNA sequen ce com parisons. Scale bar, 0.01 substitutions per nucleotide positiocession numbers of 16S rDNA sequences corresponding to each strain are shown.

TABLE 3. Enumerat ion of KSU-1 andZoogloeasp. in the biofilm

Species C value Mean C~ value SDa Relative copy num berbKSU -1 24.7, 24.42, 24.43 24.52 0.168 5.68 10 6 (72.8%)

Zoogloeasp. 29.41, 26.81 28.11 1.84 3.72 10 7 (6.05% )Bacteria 24.72, 24.58, 24.53 24.61 0.0964 7.80 x 10~ (100%)a Standard deviation.b DN A am ounts were determ ined using standard curves and relative cop y numbers w ere calculated by dividing the amounts b y their te

base lengths. Standard curves were C t=A .log (D NA amo unts)+B , where [A, B] were [-3.39, 13.1], [ -3.32, 13.2], and [-3.67, 14.2] for KZoogloeasp., and bacteria, respectively.

t r i t e r e m o v a l a n d n i t r a t e p r o d u c t i o n t o a m m o n i u m r e m o v a l( w i t h a s s u m e d N 2 p r o d u c t i o n ) d u r i n g t h e s t e a d y - s t a t e p e r i o dc a n b e d e s c r i b e d i n a n ( u n b a l a n c e d ) e q u a t i o n a s

N H 4 + + l . 1 4 N O 2 --+ ( X ) N 2 + 0 . 2 1 N O 3 ( 2)

w h e r e ( X ) d e s i g n a t e s t h e u n d e t e r m i n e d N 2 p r o d u c t i o n .

T h e s e t e r m s a r e c o r r o b o r a t iv e o f th a t w h i c h i s e x p e c te d f o rt h e a n a m m o x r e a c t i o n ( se e E q . 1 ). T h e b i o m a s s d e m o n -s t r a t e d e s s e n t i a l l y 1 0 0 % a d h e s i o n t o t h e p o l y m e r - c o a t e dn o n w o v e n c a r r ie r i n d i ca t in g t h e a p p a r e n t p r e f e r e n c e o f t h em i c r o o r g a n i s m s d o m i n a t i n g t h i s s y s t e m f o r t h e a tt a c h e dm o d e o f g r o w t h . S a m p l i n g o f b i o m a s s f o r p h y l o g e n e ti c


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c )

I ) )

FIG. 3. FISH im ages of the biofilm bacteria: (a) phas e contrast microsco py. Bar indicates 10 ~m. (b) Epifluorescence microscop y w ith cellsstained w ith the general DNA stain DAP I. (c) Epifluorescence after hybridization with Tex as Red-labeled probe A na-1 and FITC -labeled-probeZoo-l. Strain KSU-1 was labeled red and theZoogloeastrain was labeled green.

a n a l y s e s w a s p e r f o r m e d a p p r o x i m a t e l y o n e -t h i r d o f t h e w a ythrough the no ted s teady-s ta te per iod .

M e m b e r s o f b i o f il m c o m m u n i t y To a m p l i f y 1 6Sr D N A s e g m e n t s i n a n o n - c o n s e r v e d r e g i o n a m o n g a w i d er a n g e o f o rg a n i s m s , a b a c te r i a l 1 6 S r D N A - s p e c i f i c p r i m e rpa i r (16S-1 and 16S-2) was used to se lec t the 190 to 225 bps e q u e n c e , a n d w a s u s e d f o r P C R . Tw e n t y - n i n e c l o n e s w e r eobta ined tha t could be grouped in to a t l eas t s ix ca tegor iesb a s e d o n t h e i r 1 6 S r D N A s e q u e n c e i d e n t it i es a s s h o w n i nTa b l e 2 . G r o u p s 1 a n d 2 c o n t a i n e d t h e m o s t n u m b e r o fc lones wi th e leven each . G roup 1 had a no tab le s imi la r i tyw i t h Candidatus B . a n a m m o x i d a n s , w h i c h w a s t h e f i r s tp l a n c t o m y c e t e r e p o r t e d t o h a v e a n a m m o x c a p a b i l i t y ( 1 ) .G r o u p 2 w a s a f f il i a te d w i t h t he u n c u l t u r e d b a c t e r i u m m l e 1 -33 and Zoogloea ramigera .T h e o t h e r g r o u p s h a d h i g h 1 6S

r D N A s e q u e n c e s i m i l a r i t i e s w i t hRubr iv ivax ge la t inosus(3c l o n e s ), a n u n c u l tu r e d b a c t e r i u m P H O S - H E 3 6 ( 2 c l o n es ) ,an Aquaspiri l lum spec ies (1 c lone) , andThiobacil lus deni-trificans (1 clone).

To m o r e s p e c i f i c a l ly p r o b e f o r a n a e r o b ic a m m o n i u m - o x i -d i z i ng o rg a n i s m s , f u l l - le n g t h 1 6 S r D N A w a s a m p l i f i e d u s -ing a pr imer pa i r des igned for th i s purpose ( see Mater ia l sa n d M e t h o d s ) . T h e r e s u l t i n g c l o n e ' s 1 6 S r D N A s e q u e n c e( f r o m s t r a in K S U - 1 ; D D B J r e g i s t r a ti o n n o. A B 0 5 7 4 5 3 ) c o r-r e s p o n d i n g t o G r o u p 1 h a d a 9 2 . 2 % s i m i l a r it y to t h a t o f th ea r c h e t y p a l Candidatus B . a n a m m o x i d a n s a n d c o n s i d e r a b l yless sequence s imi la r i t i es to those o f a l l o ther repor teda n a e r o b i c a m m o n i u m - o x i d i z i n g p l a n c t o m y c e t e s a s s h o w n

~ , ~ , I ! ~ )

C)

FIG. 4. Orthogonal representation of a dense assemblage of KSU -1 and Zoogloeasp. by con focal laser-scanning microscopy : (A) x -ysectional image, (B) y- z sectional image, (C) y -z sectional image. Barindicates 10 ~tm.


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in F ig . 2 . Bas ed on these resu l t s , the new ly ident i f ied s t ra in ,K S U - 1 , c u l t u r e d i n K u m a m o t o , q u a l i f i e s a s a u n i q u e a n -a e r o b i c a m m o n i u m - o x i d i z i n g s t r a i n a m o n g p l a n c t o m y c e t e s .

Q u a n t i f i c a t io n o f b i o f i l m b a c t e r i a S p e c i f i c p r i m e rpa i r s (Table 1 we re used to ta rge t KSU - 1 andZoogloeasp. ,w h i c h w e r e t h e m o s t n u m e r o u s o f t h e c l o n e s o b t a in e d u s i n gpar t ia l sequences , and a l l bac te r ia for enumera t ion by rea l -

t i m e P C R . T h e c o n c e n t r a t io n s o f t he p r i m e r s w e r e e m -pi r ica l ly op t imized to min imize the C, va lues . The C, va luesw e r e d e t e r m i n e d b a s e d o n t h e m e a n b a s e l i n e s i g n a l d u r i n ge a r l y a m p l i f i c a t i o n c y c l e s . T h e a m p l i f i c a t i o n e f f i c i e n c i e sf o r K S U - 1 , Zoogloea sp . and bac te r ia l sequences as de-t e r m i n e d b y t h e s l o p e s o f t h e s t a n d a r d c u r v e s w e r e - 3 . 3 9 ,- 3 . 3 2 , a n d - 3 . 6 7 ( = C / l o g [ r at io o f t e m p l a t e D N A a m o u n t s] ) ,respec t ive ly. The s im i la r i t ies o f these e ff ic ienc ies con f i rmt h a t t h e p r i m e r s A r i a - F / A r i a - R , Z o o - F / Z o o - R a n d t h e b a c -te r ia l p r imers were su i tab le for the ampl i f ica t ion of 16SrDNA by rea l - t ime PCR. The C, va lues were 24 .5_+0.168 ,28.1 _+ 1.84, ~md 24. 6+ 0.0 964 for K SU -1,Zoogloeasp. andbac te r ia , respec t ive ly. Us ing the s tandard curves , the de te r-m i n e d C, v a l u e s c o u l d b e u s e d f o r q ua n t i f ic a t i o n o f D N Af r a g m e n t s . T h e a m o u n t s w e r e t h e n d i v i d e d b y t he l e n g t h o fe a c h a m p l i f ie d D N A f r a g m e n t ( 7 5 b p f o r K S U - 1 , 6 7 b p t b rZoogloeasp . , and 189 bp for bac te r ia ) to ca lcu la te re la t ivec o p y n u m b e r s . T h e r e l a t i v e c o p y n u m b e r s w e r e d e t e r m i n e dt o b e 5 . 6 8 1 0 ~' ( n = 3 ) f o r K S U - 1 , 4 . 7 2 x 1 0 7 ( n : 2 ) f o rZoogloeasp . and 7 .80x 10 6 (n= 3) for bac te r ia as shown inTable 3, ind ica t ing tha t the KSU -1 c om pr ised 72 .8% o f thec o m m u n i t y p o p u l a t i o n w h i l eZoogloeas p . c o m p r i s e d a p -p r o x i m a t e l y 6 % . T h e s e r e s u l ts a g r e e w e l l w i t h t h o s e o f t h eFISH ana lys i s shown in F ig . 3 (d i scussed be low) and sug-g e s t t h a t K S U - 1 w a s t h e p r e d o m i n a n t b a c t e r i u m a n d w a sr e s p o n s i b l e f o r th e a n a m m o x r e a c t i o n i n th e b i o f i lm .

F l u o r e s c e n t in sRu h y b r i d i z a t i o n o f t h e b i o f i lm F o rima ging o f the loca l iza t ion of the bac te r ia l s t ra ins in the b io-f i l m , F I S H a n a l y s i s u s i n g t h e K S U - I - a n dZoogloea-spe-c i f ic D N A p r o b e s l a b e l e d w i t h Te x a s R e d a n d F I T C , r e s p e c -t iv e l y, w a s p e r t b r m e d . I n it ia l ly, c o n t a m i n a t i o n b y u n k n o w nm a t e r i a l s i n t h e b i o f i l m i n t e r f e r e d w i t h m i c r o s c o p i c i m a g -ing , however, t a i l l abe l ing of each pro be as descr ibed inM a t e r i a l s a n d M e t h o d s g r e a t l y i n c r e a s e d f l u o r e s c e n c e i n -t e n s i ty a n d i m p r o v e d i m a g e c o n t r a st . I m a g i n g u s i n g e p i f lu -o r e s c e n c e m i c r o s c o p y r e v e a l e d a h i g h d e n s i t y o f c e ll s g r o w -ing in c lus te rs and emi t t ing red f luorescence , ind ica t ing thed o m i n a n c e o f s t ra i n K S U - 1 w i t h i n th e b i o f il m . A c o m p a r a -t ive ly lesser sur face a rea w as s ta ined green , ind ica t ing alesser dens i tyofZoogloeasp. in the biofi lm (Fig. 3) .

F o r m o r e d e t a il e d l o c a l i z a ti o n , s e c t io n a l i m a g i n g o f s a m -p l e s u s i n g a c o n f o c a l l a s e r s c a n n i n g m i c r o s c o p e w a s p e r-l b r m e d u s i n g A n a - I l a b e l e d w i t h T M R a n d Z o o - 1 l a b e l e dwi th FITC as hybr id iza t ion probes . For ty hor izonta l p laneswere scanned a t 0 .4-1am in te rva ls a t a b iof i lm depth of 16la in f rom wh ich sec t iona l ima ges were recons t i tu ted . T hesec t iona l images shown in F ig . 4 c lear ly demons t ra te tha tK S U - 1 c e l l s a g g l o m e r a t e d i n s p h e r e s h a v i n g d i a m e t e r s o f1 0 - 3 0 p m a n d t ha tZoogloea sp . formed as a th in layera r o u n d t h e s e s p h e r e s. T h e c l u s t e r f o r m a t i o n b y K S U - I c e l l sm i g h t b e r e q u i r e d f o r m a i n t a i n i n g o f t h e h ig h m e t a b o l i c a c -t iv i ty observed . Others have proposed tha t the dens i ty -de-p e n d e n t a c t i v i t y o fCandidatusB . a n a m m o x i d a n s m a y i n-

d ica te tha t ce l l - to -ce l l communica t ion (quorum sens ing) i sc r i t i ca l tb r i t s metabol ic ac t iv i ty (4) . In addi t ion , theZoo-gloea c e ll l a y e r m i g h t s e r v e t o e l i m i n a t e m o l e c u l a r o x y g e nf r o m t he K S U - I h a b it a t, a n d K S U - 1 c e l l s c o u l d p r o v i d e e s-sen t ia l o rganic compounds to the ad jo in ingZoogloeacells .I t i s a l so cons idered tha t theZoogloea ce l l s , which a reknown for the i r n i t ra te resp i ra t ion capabi l i ty (19) , would

benef i t f rom the n i t ra te formed dur ing CO~ f ixa t ion byK S U - 1 d u r i ng t h e a n a m m o x r e a c t i o n (2 0 ). F u r t he r r e s e a r c hwi l l be necessa ry to assess these p oss ib i l it i es .

A C K N O W L E D G M E N T

This work was financially supported in part by a Grant-in-Aidtbr Scientific R esearch (no. 12480165) from Japan Society for thePromotion of Science.

R E F E R E N C E S

I . S t rous , M. , van Gerve n , E . , Kuenen , J . G . , and Je t t en , M. :Effects of aerobic and microae robic conditions on anaerobic

amm onium-oxidizing (anammox) sludge. Appl. Environ. Mi-crobiol., 63, 2446 2448 (1997).2 . v a n D o n g e n , L . G . J . M . , J e tt e n, M . S . M . , a n d v a n

L o o s d r e c h t , M .C .M .: The combined Sharon/anammoxprocess, a sustainable method for N-removal from sludgewater, a Stowa report. IWA Publishing, London (2001).

3 . S t rous , M. , He i j i nen , J . J . , Knenen , J .G . , and Je t t en ,M. S. M.: The sequencing batch reactor as a powerful tool forthe study of slowly growing anaerobic amm onium-oxidizingmicroorganisms. Appl. Microbiol. Biotechnol., 50, 589 596(1998).

4 . S t r o u s, M . , F u e r s t , J . A . , K r a m e r, E . H . M . , L o g e m a n n ,S . M . , M u y z e r , G ., v a n d e P a s - S c h o o n e n , K . T. , W e b b , R . ,K u e n e n , J . G . , a n d J e t t e n , M . S . M . : Missing lithotrophidentified as new planctomycete. Nature,400, 446-449 (1999).

5 . S c h m i d , M . , Tw a c h t m a n n , U . , K l e i n , M . , S t r o u s , M . ,Ju re t sc hko , S . , J e t t en , M . S. M. , M e tzge r, J . W. , Sch le i f e r,K. H., a n d Wa n g e r, M.: Molecular evidence fbr genus leveldiversity' of bacteria capable o f catalyzing anaerobic a mm o-nium oxidation. System. Appl. Microbiol., 23, 93 106 (2000).

6 . Kue nen , J . G . and Je t t en , M. S .M . :Extraordinary anaero-bic ammonium-oxidizing bacteria. ASM News, 67, 456-463(2001 ).

7. Egli, K., Fanger, U., Siegrist, H .R .,and van t i e r Me e t ,J. R .: Enrichment and characterization of an anamm ox bac-terium from a rotating biological contactor treating ammo-nium rich leachate. Arch. Microbiol., 175, 198-207 (2001).

8. Rouse, J . D. , Oda , H. ,a n d F u r u k a w a , K . :Autotrophic bio-logical transformations of nitrogen under oxygen-stressed andanaerobic conditions. Jpn. J. W ar. Treat. 13iol., 34, 215 230(1998).

9 . F u r u k a w a , K . , I c h i m a t s u , Y. , H a r a d a , C . , S h i m o z o n o , S. ,a n d H a z a m a ,M.: N itrification o f polluted urban river watersusing zeolite-coated nonwovens. J. Environ. Sci. Health,A 3 5 ,1267 1278(2000).

10. Bh atti, Z. l. , Toda, H.,a n d F u r u k a w a , K .: p-Nitrophenoldegradation by activated sludge attached on nonwovens.Water R es., 36, 1135-1142 (2002).

1 I . Fu r nkaw a , K . , R ouse , J . D . , Bha t t i , Z . I . , and Im a jo , U . :Anaerobic ammonium oxidation (anammox) in continuousflow treatment with non-woven biomass carrier.In Proceed-ings of the ISEB F ifth International Sym posium on E nviron-mental Biotechnology, Kyoto, Japan. The International Soci-ety for Environmental Biotechnology, Waterloo, ON, Canada,CD-ROM (2000).

12. v a n d e G r a a f , A . A . , d e B r u i j n , P., R o b e r t s o n ,L.A. ,


7/7

418 FUJ II ET AL . J. BlOSC|. B1OENG.,

Je t t en , M . S . M . ,a n d K u e n e n ,J . G . : A uto t roph ic g rowth o fanaerob ic am monium -ox id iz ing mic ro -o rgan i sms in a f lu id -ized bed reactor. Microbiolog y, 142, 2187 -2196 (1996).

13 . Furu kaw a, K. , Rou se , J . D . , Ima jo , U . , Na kam ura , K . , andIshida, H.: Anaerob ic ox ida t ion o f amm onium conf i rmed incont inuous f low treatment using a non-woven biomass car-rier. Jpn. J. Wat. Treat, Biol. , 38, 87-94 (2002).

14 . Rou se , J .D . , Sum ida , K . , Kida , K . , and Furu kaw a, K. :Ma intainabi l i ty of deni t r i fying granular s ludge in sof t to mar-ginal ly hard waters in an upflow sludge-blan ket reactor. Envi-ron. Technol . , 20, 219-225 (1999).

15. Page , R .D. M ." Treev iew: an app li ca t ion to d i sp lay phy lo -genet ic trees on personal computers . Com put . Ap plic . Biosci . ,12, 357-358 (1996).

16 . Amann , R . , Sna idr, J . , Wagner, M. , Ludwig , W. , andSchleifer, K . H . : In situ visual izat ion of high genet ic diver-s i ty in a natural microbial community. J . Bacter iol . , 178,3496-3500 (1996) .

17 . Eaton , A . D. , C lesoer i , L . S . , and G reenberg , A .E . :Stan-dard methods fo r the examina t ion o f wa te r and was tewater,19th ed. Ame rican Publ ic Heal th Associat ion, Washington,D.C. (1995).

1 8. F u r u k a w a , K . , R o u s e , J . D . , l m a j o , U . , S u g i n o , H . ,an dFuj i i , T. : Es tab l i shment o f an anae rob ic amm onium-ox id iz -ing cul ture in cont inuous f low treatment with non-wo ven bio-mass carr ier.In Bai l lod , C . R . ( ed .) , P roceed ings o f W EFTEC2001 74th Annu al Conference and Expo si t ion, Atlanta , Geor-g ia . Wate r Env i ronmenta l Federa t ion , Alexandria , VA, USA,CD-ROM (2000) .

19. Kr ieg , N . R . and Hol t , J .G . : Berg y ' s manua l o f sys temat icbacter iology, I s t ed. Wil l iam s and W ilkins , New York (1984).

20 . Je t t en , M . S . M. , S t rons , M . , Pas -Seh oonen , K. T. , Seha lk ,J . , D o n g e n, U . G . J . M . , G r a M , A . A . , L o g e m a n n , S .,M u y z e r, G . , L o o s d r e e h t , M . C . M . ,a n d K n e n e n ,J . G." Theanaerob ic ox ida t ion o f ammonium. FEMS Microb io l . Rev. ,22, 421-437 (1999) .

characterization of the microbial community in an anaerobic imp fish

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