Title Ammonia-oxidizing archaea and ammonia-oxidizing bacteria in six full-scale wastewater ...

12 Pages·2011·0.75 MB·English

ApplMicrobiolBiotechnol(2011)91:1215–1225
DOI10.1007/s00253-011-3408-y
ENVIRONMENTAL BIOTECHNOLOGY
Ammonia-oxidizing archaea and ammonia-oxidizing bacteria
in six full-scale wastewater treatment bioreactors
Tong Zhang&Lin Ye&Amy Hin Yan Tong&
Ming-Fei Shao&Si Lok
Received:14March2011/Revised:20May2011/Accepted:22May2011/Publishedonline:25June2011
#TheAuthor(s)2011.ThisarticleispublishedwithopenaccessatSpringerlink.com
Abstract In this study, dideoxy sequencing and 454 high- Keywords Ammonia-oxidizing archaea.Ammonia-
throughput sequencing were used to analyze diversities of oxidizing bacteria.Municipal wastewater treatment
the ammonia monooxygenase (amoA) genes and the 16S plants.454 Pyrosequencing
rRNA genes of ammonia-oxidizing archaea (AOA) and
ammonia-oxidizingbacteria(AOB) insixmunicipalwaste-
watertreatmentplants.TheresultsshowedthatAOBamoA Introduction
genes were quite diverse in different wastewater treatment
plantswhilethe16SrRNAgeneswererelativelyconserved. Nitrification, the biological oxidation of ammonium to
BasedontheobservedcomplexityofamoA and 16S rRNA nitriteandnitrate,isanessentialprocessinnitrogencycling
genes,mostoftheAOBcanbeassignedtotheNitrosomonas and wastewater treatment bioreactors. Several groups of
genus, with Nitrosomonas ureae, Nitrosomonas oligotropha, microorganisms are involved in the two-step process:
Nitrosomonasmarina,andNitrosomonasaestuariibeingthe ammonia-oxidizing archaea (AOA), ammonia-oxidizing
fourmostdominantspecies.FromthesequencesoftheAOA bacteria (AOB), and nitrite-oxidizing bacteria (NOB). In
−
amoAgenes,mostAOAobservedinthisstudybelongtothe the first step, AOA and/or AOB oxidize NH to NO , and
3 2
− −
CGI.1bgroup,i.e.,thesoillineage.TheAOBamoAand16S in the second step NOB oxidize NO to NO . The first
2 3
rRNA genes were quantified by quantitative PCR and 454 step is rate limiting and has been relatively well studied
high-throughput pyrosequencing, respectively. Although the (Limpiyakorn et al. 2006; Park and Noguera 2004;
resultsfromthetwoapproachesshowsomedisconcordance, Tokutomi et al. 2010). Although some heterotrophic
they both indicated that the abundance of AOB in activated bacteria (Robertson and Kuenen 1990) and anaerobic
sludge was very low. ammonia-oxidizing (anammox) bacteria (Strous et al.
1999) can also oxidize ammonia to nitrite, AOA and
AOB are thought to be the main contributors for environ-
mental ammonia oxidation and in bioreactors (Nicol and
Schleper 2006). According to the previous reports, Nitro-
Electronicsupplementarymaterial Theonlineversionofthisarticle somonasandNitrosospiraarethemostimportant generaof
(doi:10.1007/s00253-011-3408-y)containssupplementarymaterial, AOB in activated sludge (Park and Noguera 2004;
whichisavailabletoauthorizedusers.
Purkhold et al. 2000). Of the two genera, Nitrosomonas
: :
T.Zhang(*) L.Ye M.-F.Shao has been shown to be the dominant AOB in many
EnvironmentalBiotechnologyLaboratory,
bioreactors (Limpiyakorn et al. 2006; Park and Noguera
TheUniversityofHongKong,
2004;Wells etal.2009).Bycontrast, Nitrosospirasp.were
HongKongSAR,China
e-mail:[email protected] rarely found in activated sludge (Schramm et al. 1999),
: probablyduetotheirrelativelowgrowthrate(Siripongand
A.H.Y.Tong S.Lok
Rittmann 2007) resulting in an underrepresentation in
GenomeResearchCenter,LiKaShingFacultyofMedicine,
bioreactors. The limited studies of AOA species in
TheUniversityofHongKong,
HongKongSAR,China activatedsludgeshowedacompositiondifferentfromthose
1216 ApplMicrobiolBiotechnol(2011)91:1215–1225
existing in other environments such as soil, water column, volume of 30 μl containing 15 μl of iQTM SYBR® Green
andsediment(Parketal.2006). Moreover, their abundances Supermix (Bio-Rad), 5 μl of DNA template with the
seemed to be much lower (four orders or more) than AOB concentration of about 1 ng/μl, and 0.3 μM of each primer
based on analysis of amoA gene copy number (Jin et al. usingthesamecyclingconditions.ThearchaealamoAgene
2010; Limpiyakorn et al. 2010; Wells et al. 2009). was amplified by PCR using the primer set Arch-amoAF
In this study, the AOA and AOB diversity in six (5′-STAATGGTCTGGCTTAGACG-3′) and Arch-amoAR
bioreactorsfromthewastewater treatmentplants (WWTPs) (5′-GCGGCCATCCATCTGTATGT-3′) (Francis et al.
in four countries was investigated using amoA and 16S 2005).PCRamplificationwasperformedina50-μlvolume
rRNA genes as the biomarkers. High-throughput pyrose- comprising 25 μl 2× MightyAmp Buffer (TaKaRa), 1 μl
quencing of the 16S rRNA genes was used to tabulate the (1.25 U) MightyAmp DNA Polymerase (TaKaRa), 0.3 μM
AOB diversity and their relative abundance in the total of each primer, and 20–50 ng of genomic DNA. PCR was
bacterial community. Quantitative polymerasechain reaction first incubated at 98 °C for 1 min and was followed by 35
(qPCR) was also used to quantify AOB amoA genes in the cyclesat98°Cfor10s,60°Cfor15s,and68°Cfor60s.
same activated sludge samples. Several SYBR green-based The PCR products were visualized by agarose (1%) gel
qPCRsystemshavebeentriedtoquantifyAOAamoAgene. electrophoresis in the presence of suitable size markers.
However, non-specific amplification and/or the formation of For high-throughput 454 pyrosequencing, the bacterial
primer dimer hindered the accurate quantification of AOA DNA was amplified with a set of primers targeting the
amoA using this approach. hypervariable V4 region of the 16S rRNA gene. The
forward primer is 5′-AYTGGGYDTAAAGNG-3′ and the
reverse primers are the mixture of four equally mixed
Materials and methods primers: 5′-TACCRGGGTHTCTAATCC-3′, 5′-TACCA-
GAGTATCTAATTC-3′, 5′-CTACDSRGGTMTCTAATC-3′,
Activated sludge sampling and DNA extraction and5′-TACNVGGGTATCTAATCC-3′(RDP'sPyrosequenc-
ing Pipeline, http://pyro.cme.msu.edu/pyro/help.jsp). Barc-
In this study, activated sludge samples were taken from odes that allowed sample multiplexing during
aeration tanks of eight full-scale WWTPs treating municipal pyrosequencingwereincorporatedbetweenthe454adaptor
wastewater in China, Singapore, Canada, and the USA. A and the forward primer.
Relevant parameters about these WWTPs are shown in
Supplementary Table S1. Sludge samples from the aeration Dideoxy DNA sequencing
tank were fixed with 50% ethanol (v/v) on site before
transporting to the laboratory for DNA extraction. Ten PCR products were purified by using PCRquick-spinTM
milliliters offixed activated sludge samples was centrifuged PCR Product Purification Kit (iNtRON Biotechnology,
at 4,000 rpm for 10 min at 4 °C. Approximately 200 mg of Korea). The purified PCR products were ligated to
samplepelletwasrecoveredforDNAextractioninduplicate pMD®18-T Vector (TaKaRa). Recombinant plasmid was
with a FastDNA® SPIN Kit for Soil (Qiagen, CA, USA), transformedintoE.coliandwhitecoloniesthatgrewonLB
which was found to be the most suitable DNA extraction plates containing ampicillin (60 μg/ml), X-Gal, and IPTG
methodforthesamplesinthisstudy,asbeingcomparedwith were picked to conduct colony PCR amplification with the
other commercial reagents. primer set M13F and M13R. The PCR products were
purifiedandsequencedbyABI3730xlcapillarysequencers
PCR and quantitative PCR (Applied Biosystems, Foster City, CA, USA) with the
primer M13F.
Primer set amoA-1F (5′-GGGGTTTCTACTGGTGGT-3′)
and amoA-2R (5′-CCCCTCKGSAAAGCCTTCTTC-3′) OTU definition and phylogenetic analysis
(Rotthauwe et al. 1997) was used to amplify bacterial
amoAgeneina30-μlmixturecontaining0.2μlofTaKaRa AnAOBamoAgeneclonelibrarywasconstructedforeachof
Ex TaqTM, 3 μl of 10× Ex Taq Buffer (TaKaRa), 3 μl of the eight activated sludge samples (sample IDs—CN-NJ-SJ,
10 mM dNTP mixture (TaKaRa), 0.2 μM of each primer, CN-HR-UN, SG-SG-UP, CN-QD-TD, CN-SH-TS, CN-BJ-
and 20–50 ng of genomic DNA. Thermal cycling param- JX, US-CO-CO, and CA-GP-GP). Individual AOA amoA
eters followed the protocol of Rotthauwe and colleagues geneclonelibrariesweresuccessfullyconstructedforsamples
(1997).AOBamoAgenecopynumberswerequantifiedby CN-NJ-SJ, CN-HR-UN, and SG-SG-UP, while the other
usinganiCyclerIQSystem(Bio-Rad,Hercules,CA,USA) samples failed to generate library products. Approximately
in triplicate with primer set amoA-1F/amoA-2R. Quantita- 20 clones in each clone library were selected randomly and
tive real-time PCR amplification was performed in a total sequenced. The resulting AOB amoA gene sequences were
ApplMicrobiolBiotechnol(2011)91:1215–1225 1217
aligned and the Jukes–Cantor distances between subsequent JF271927–JF271985. The pyrosequencing results are de-
pairs of sequences were calculated with DNADIST from posited into the NCBI short reads archive database
the PHYLIP package (http://www.phylip.com/), and were (accession number SRA026842.2).
grouped into 43 OTUs with a distance cut-off of 3%. In
order to construct the phylogenetic tree, one sequence in
each OTU was selected, merged, and aligned with the Results
reference sequences from NCBI Entrez Database. The
neighbor-joining phylogenetic tree of AOB amoA gene Diversity of AOB amoA gene and 16S rRNA gene
sequences was created by MEGA software (Kumar et al. in different activated sludge samples
2008). The translated protein sequences were assigned to
different OTUs with a 3% distance cut-off. The phyloge- Table S2 suggested that the coverage of the AOB amoA
netic tree of AOB AmoA protein sequences was also geneclonelibrarieswasover70%exceptsampleCN-SH-TS
created by MEGA software. AOA amoA gene and AOA witharelativelylowcoverage(65%).AsshowninFig. 1, a
AmoA protein sequences were analyzed in the same total of 43 OTUs were generated based on 163 AOB amoA
manner as that of AOB. The AOB 16S rRNA gene gene sequences in eight clone libraries with a 3% distance
sequences were also classified into different OTUs with a cut-off. Figure 1 reveals an interesting phenomenon that
3% distance cut-off. In addition, Good’s estimator of relativelyfewamoAOTUsweresharedamongtheactivated
coverage (Good 1953) was calculated for each AOA and sludge samples, indicating that the amoA genes were quite
AOB amoA gene clone library under 3% distance cut-off diverse and not widely disseminated.
(Table S2). Figure 2 illustrated that most of these bacterial amoA
OTUs were affiliated to Nitrosomonas genus with Nitro-
High-throughput pyrosequencing somonas ureae, Nitrosomonas oligotropha, Nitrosomonas
marina, and Nitrosomonas aestuarii being the four most
PCR amplicon libraries were prepared using a minimal dominant species. However, only two OTUs (OTU-17 and
number of amplification cycles (25 cycles) to minimize the OTU-32) were in the Nitrosospira lineage and these two
accumulation of PCR artifacts. Amplicons were purified OTUs represented only eight sequences in the total 163
usingPCRquick-spinTMcolumns(iNtRONBiotechnology). sequences, indicating the low abundance of Nitrosospira in
Equal amounts of amplicon products bearing individual various types of activated sludge.
sequence barcode for each sludge samples were combined Figure S1 shows the relative abundance and distribution
for multiplex pyrosequencing on the Roche 454 FLX of AOB AmoA protein OTUs in different activated sludge
Titanium platform (Roche, Nutley, NJ, USA). samples. Compared with AOB amoA gene OTUs (shown
in Fig. 1), the diversity of AmoA protein (a total of 26
Sequence analysis and phylogenetic assignment OTUs) was less than that of amoA gene (43 OTUs) due to
codon wobble positions. Twelve OTUs of AOB AmoA
Following pyrosequencing, Python scripts were written to protein were shared by at least two activated sludge
(1)removesequencescontainingmorethanoneambiguous samples. Our findings indicate that a number of different
base(‘N’),(2)checkthesequenceintegrityofthebarcodes amoA genotypes encode essentially the identical or very
and partitioned the multiplex reads to the individual similar proteins possibly reflecting the functional con-
samples, and (3) remove sequence reads shorter than 150 straints in efficient ammonia oxidation. As shown in the
bases.Theresultingfilteredreadswerethencomparedwith phylogenetic tree of AOB AmoA protein in Fig. S2, most
the Greengenes 16S rRNA gene database (DeSantis et al. OTUs were affiliated with N. ureae, N. oligotropha, N.
2006) using NCBI’s BLASTN tool (Altschul et al. 1990) marina, and N. aestuarii lineages, exactly the same as the
with default parameters set to a maximum hit number of amoAgenephylogenetictree.TherewereonlyafewOTUs
100 (Claesson et al. 2009). Sequences were then assigned inNitrosospiralineageandN.europaealineage,whichwas
to NCBI taxonomies with MEGAN (Huson et al. 2007) also identical with the above amoA gene phylogenetic
using the Lowest Common Ancestor (LCA) algorithm and analysis.
default parameters (absolute cut-off—BLAST bitscore 35; In addition to the amoA gene, the 16S rRNA gene was
relative cut-off—10% of the top hits). alsousedtoinvestigatetheAOBcommunityinsixsamples
(i.e., CN-NJ-SJ, CN-QD-TD, CN-HR-UN, US-CO-CO,
Accession numbers SG-SG-UP, and CA-GP-GP) by high-throughput pyrose-
quencing. As shown in Table 1, more than 20,000 16S
The sequences obtained from clone library in this study rRNA gene fragment sequences were obtained for each
were deposited in GenBank under accession numbers sample site. These sequences were assigned to NCBI
1218 ApplMicrobiolBiotechnol(2011)91:1215–1225
90%
80%
70%
60% e
g
50% nta
40% ce
r
e
30% P
OTOU-T4OU-3T4OU2-T4OU1-T4OU0-T3OU9-T3OU8-T3OU7-T3OU6-T3OU5-T3OU4-T3UO3-T3OU2-T3UO1-T3OU0-TA2UO9-TO2OU8-T2BUO7-T2 OU6-aT2UO5m-T2U4O-oT2UO3A-T2U2O -Tg2U1O-eT2U0On-T1Ue9O-T1 U8OO-T17UO-TT16UOU-T15UOs-T1U4O-1T3UO-T12UO-1T1U-O1T0UO-9TUO-8TUO-7TU-O6TUO-5TU-O4TU-O3TU-O2TU-1 CNC-NNCJ-Q-NSC-DJNHC--TRNSUD--HUSBS-N-JTGCC-SJ-OA0SX1%--G20CG0%-OPU%-PGP
Fig.1 RelativeabundancesanddistributionofAOBamoAgeneOTUsindifferentactivatedsludgesamples
taxonomies using BLAST and MEGAN software. A totalsequencesobtainedfrompyrosequencingandtheAOB-
tabulation of AOB-like sequences is shown in Table 1. like sequences in each of the six samples. AOB sequences
The filtered reads were merged, aligned, and classified into weresuccessfullyobtainedinfiveoutofthesixsamples,and
18 OTUs with a Jukes–Cantor distance cut-off of 3%. the percentages range from 0.29% to 0.64%. For US-CO-
Figure 3 shows that most (about 61%) AOB 16S rRNA CO, a sample from Columbia, USA, no AOB sequences
OTUs wereshared bymore than twosamplesites, and two were obtained, probably due to the low abundance of AOB
majorOTUs,i.e.,OTU-14andOTU-15,accountedfor49% in this wastewater treatment plant, in agreement with amoA
of total sequences. These results implied the AOB 16S gene quantification results.
rRNA genes are much more conservative than amoA gene
and AmoA protein. Diversity of AOA amoA gene in different activated sludge
samples
Abundance of AOB in activated sludge
In the present study, multiple experiments were attempted
Along with direct tabulation of AOB 16S rRNA genes by to amplify amoA gene from the eight samples using four
pyrosequencing, we also quantify AOB abundances in the PCR systems (including TaKaRa Ex Taq, TaKaRa Mighty
activated sludge samples by qPCR, normalizing the AOB Amp DNA Polymerase, Sigma Taq DNA Polymerase, and
amoA gene copy number against per nanogram of the Bio-Rad SYBR® Green Supermix) at different thermal
genomicDNAextractedfromactivatedsludge.Table1shows conditions (annealing temperature from 50 to 60 °C).
the abundances of AOB amoA genes in different activated Amplification was successful from only five samples as
sludgesamplesquantifiedbyqPCR.SampleCN-SH-TS has shown in Fig. S3 and only three samples were eventually
the most abundant AOB amoA gene copy numbers among successfully cloned and sequenced. Additionally, the
the eight activated sludge samples tested. Table 1 lists the presence of primer dimers was very serious in all samples
ApplMicrobiolBiotechnol(2011)91:1215–1225 1219
Fig. 2 Neighbor-joining phylo- 99 Nitrosomonas oligotropha (AF272406)
genetictreebasedonbacterial 50 OTU-18
amoAgenesequences.The 83 Nitrosomonas sp. NL7 (AY958704)
evolutionarydistanceswere 77 Nitrosomonas sp. Nm47 (AY123830)
computedusingtheJukes– OTU-35
Cantormethodandareinthe 1938 OTU-16
unitsofthenumberofbase 97 OTU-21
substitutionspersite.Bootstrap OTU-2
valuesareindicatedonbranch 3178 OTU-12
nodes.Sequencesobtainedin 69 OTU-38
thisstudyareshownwith 58 OTU-36
“OTU-”inthenames.Other 40 OTU-40
sequenceswereobtainedfrom 15 OTU-24
GenBank.Thetreewas 55 OTU-39 N. oligotropha lineage
out-groupedwithNitrosococcus OTU-23
13
halophilus(gammaproteobacte- 96 OTU-8
rialAOB)amoAsequence 99 OTU-14
7
Nitrosomonas sp. Nm59 (AY123831)
Nitrosomonas sp. Nm84 (AY123818)
OTU-43
12
29 36 OTU-13
99 Nitrosomonas sp. Nm86 (AY123819)
98 OTU-9
Nitrosomonas sp. JL21 (AF327919)
36 OTU-5
99 OTU-4
50 53 OTU-34
86 OTU-15
OTU-32
52 53 OTU-28 N. ureae lineage
49 OTU-31
OTU-42
98 Nitrosomonas ureae (AF272403)
54 Nitrosomonas sp. AL212 (AF327918)
20
75 Nitrosomonas aestuarii (AF272400)
Nitrosomonas marina (AF272405)
82 OTU-37
20
20 77 OTU-41
OTU-6
OTU-
48 11 N. marina and N. aestuarii
47 OTU-7
32 OTU-25
69 OTU-10
33
OTU-29
98 OTU-27
65
55 OTU-33
OTU-32
Nitrosospira multiformis (AY177933)
99
100 Nitrosovibrio tenuis (AY123824)
71 Nitrosospira tenuis (AJ298720)
Nitrosospira lineage
64 Nitrosospira briensis (AY123821)
49 Nitrosospira sp. CT2F (AY189143)
39 OTU-17
Nitrosomonas cryotolerans (AF272402)
99 OTU-1
100 OTU-3
Nitrosomonas-like
OTU-22
78 OTU-26
57 Nitrosomonas communis (AF272399)
40
32 Nitrosomonas sp. Nm58 (AY123820)
Nitrosomonas nitrosa (AF272404)
55 Nitrosomonas halophila (AF272398)
OTU-19
55
OTU-30
99 N.europaea lineage
98 Nitrosococcus mobilis (AF037108)
100 Nitrosomonas europaea (AF037107)
Nitrosococcus halophilus (AF272521)
0.05
1220 ApplMicrobiolBiotechnol(2011)91:1215–1225
A) and it was not possible to quantify amoA using SYBR®
N
eD Green qPCR approach applied in this study (Supporting
ng
ges/n informationFig.S3).
amoA(copie 34.5 0.538 2.81 142 5.55 1.29 74.3 1.72 assHesoswmeevnetr,ofitthisesdtiilvlerosfityintoefreAstOtAo haamvoeAagperneelimininathrye
activated sludge samples by exploring the sequences
ge obtained from the clone libraries of the samples CN-NJ-
a
ent SJ, CN-HR-UN, and SG-SG-UP, all of which had Good’s
c
OB pers estimator of coverage over 80% (Table S2). The 56
AbundanceofA 16SrRNAgeneintotalsequence 0.42%(20,955) 0.64%(24,437) 0.46%(22,593) ND ND 0%(25,499) 0.34%(23,928) 0.29%(22,593) wrsOfereoeTsqumruUeletssAnschiuOneassBritnehfdgiarsomaasmotmuAddotihysnOe,tgaiTntthUmcwreseuaelsc(tFiufspoitagl-uemo.nf1pfds)lae,otmhsfsaopwt3mlet%ehser.eeA(AJFcOulOidagAsgA.siian4afmg)ime.odofDArAoiinfmOfgteoeTrnetU1hen5esst
arenotasdiverseasAOBamoAgeneindifferentactivated
ce sludge samples. While it should be mentioned that the
n
Aue sample amount in this study was kind of limited, thus this
Nq
Rse issue needs more studies to be confirmed in the future. As
16Srgene 87 156 105 ND ND 0 81 66 shown in the phylogenetic tree of AOA amoA gene
sequences in Fig. 6, most of the sequences obtained in this
ofUs study were distinctly different from the previously reported
No.OT 8 8 7 9 12 12 5 3 sequences, especially the sequences recovered from marine
andsediment.OnlytwoOTUs(OTU-10andOTU-11)were
grouped into the marine and sediment lineages.
e
c
AOB amoAsequen 21 21 20 21 20 20 20 21
Discussion
s
U
T Diversity of AOB amoA gene and 16S rRNA gene
s O
WTP of in different activated sludge samples
W No. 6 ND 5 ND ND ND 10 ND
ht A previous study reported that the amoA gene similarities
g
ei among different AOB species ranged from 65% to 100%
AOBin Diversity AOA amoAsequence 20 ND 16 ND ND ND 20 ND o(Pbutariknheodldinetthails. s2t0u0d0y).wTehree 6s2im%ilatroitie1s00o%f.thTeheOhTiUghs
d diversity of the amoA gene found in this study was likely
n
a due to the different geographic locations of these sludge
undanceofAOA Cityandcountry Nanjing,China Qingdao,China Harbin,China Beijing,China Shanghai,China Columbia,USA Singapore Guelph,Canada strheapemTsopehrtlesWesrtWehasanuTtdlPtsssht.hoinewetdhdiifsfNesrietturnodtsyoompweoernraeatisocnoin/nsssetiewsataedgnetofwcNoitnhitdrioptsrioeovnsipsoiruians
b being the dominant AOB in nitrification bioreactors in
a
d WWTP (Park and Noguera 2004; Wells et al. 2009).
n
a al
y n Notably,sevenOTU-17-associatedsequenceswereallfrom
ersit egio the sample CN-HR-UN taken from Harbin, a city in
aryofthediv WWTP Suojincun Tuandao Harbin Jiuxianqiao Tianshan ColumbiaR UluPandan Guelph nacpmoorenroftsehniresgtrlenontwhtCowtsheeiimnthasp.aetmhrTaepthuleperdreelsavitoiniotfuu4tdsh–ei1rse0opsfo°tuCrttdhy(itsA.havOctriautNhyraitmorisobissoteehtsrepvaialrh.taii2og0nsh0pe3psist;.
m
Table1Sum Sample CN-NJ-SJ CN-QD-TD CN-HR-UN CN-BJ-JX CN-HR-UN US-CO-CO SG-SG-UP CA-GP-GP ND:NoData SnlOiuniTrmeiUpabog-e2ner6ga)onagfdnrodOsueTpvReUeidrtstamlfabaOrenTalnowUn2asgy0in(0Ofg7roT).mtUoW-t1heN,eiOtakrloTsnsoUoow-mo3nbo,snAOearOTsvBUee-usa2pr2oes,pcmaaienaeslad.l
ApplMicrobiolBiotechnol(2011)91:1215–1225 1221
Fig.3 Relativeabundancesand
distributionofAOB16SrRNA
geneOTUsindifferent
activatedsludgesamples
60%
50%
40% ge
a
nt
30% ce
er
20% P
OTU-O18TU-O17TU-O16TU-1O5TU-1O4TU-A1O3OTU-B1O2T1U6-1SO1T Ur-R10ONTAU-9 OgTeU-n8OeT UO-7TOTUU-s6OTU-5OTU-4OTU-3 OTU-2 OTU-1 CNC-NNJC--QNSUD-JHS-STR-CGD-CUO-SA0N-1CG-%G0O-UP%-PGP
ItwasreportedthattheAOB16SrRNAgenesimilarities obtainedinthisstudyweregroupedinNitrosomonasgenus.
generally ranged from 90% to 100%, much higher than There were 12 OTUs in Nitrosomonas oligotropha group,
those of amoA gene (about 65–100%) and AmoA protein four OTUs in the group of Nitrosomonas ureae, and one
(about 72.5–100%) (Purkhold et al. 2000). The phyloge- OTU in the N. marinaand N. aestuarii lineages, indicating
netic tree in Fig. 5 shows that all of these sequences the dominance of these species in the activated sludge
Fig.4 Relativeabundancesand
distributionofAOAamoAgene
OTUsindifferentactivated
sludgesamples
70%
60%
50% e
g
a
40% nt
e
30% erc
P
20%
OTU-15OTU-14OTU-13OTU-12OTAU-O11OATU-a10mOToUA-9 OgTeU-n8eOT OU-7TOUTUs-6 OTU-5 OTU-4 OTU-3 OTU-2 OTU-1 CN-NCJN-S-HJSRG0-1%-US0%NG-UP
1222 ApplMicrobiolBiotechnol(2011)91:1215–1225
OTU-1
25
17 OTU-5
N. ureae lineage
12 OTU-8
Nitrosomonas ureae(AF272414.1)
1 67 OTU-6
Nitrosomonas cryotolerans(AF272423.1)
OTU-10
21
Nitrosomonas aestuarii(AF272420.1)
71 N. marina and N.aestuarii lineage
8 63 Nitrosomonas marina(AF272418.1)
Nitrosomonas oligotropha(EF016119.1)
51
87 OTU-3
78 OTU-4
OTU-2
9 8 OTU-13 N.oligotropha Lineage
OTU-14
51
OTU-12
35
OTU-15
15
35 OTU-16
16
OTU-9
OTU-7 Nitrosomonas-like lineage
64
97 OTU-11
Nitrosomonas communis(AF272417.1)
93
14
Nitrosomonas nitrosa(AF272425.1)
45 OTU-17
Nitrosomonas-like lineage
100 OTU-18
Nitrosomonas halophila(AF272413.1)
Nitrosomonas europaea(HM446362.1)
73
86 Nitrosomonas eutropha(HM446363.1)
Nitrosospira briensis(M96396.1)
Nitrosococcus mobilis(M96403.1)
30
Nitrosospira tenuis(AJ298746.1)
46
Nitrosovibrio tenuis(M96397.1)
51
74Nitrosolobus multiformis(M96401.1)
Nitrosococcus halophilus(AF287298.1)
0.02
Fig.5 Neighbor-joiningphylogenetictreebasedonAOB16SrRNA substitutionspersite.Bootstrapvaluesareindicatedonbranchnodes.
genesequences.Theevolutionarydistanceswerecomputedusingthe Sequences obtained in this study are shown with “OTU-” in the
Jukes–Cantor method and are in the units of the number of base names.OthersequenceswereobtainedfromGenBank
samples.Thisalmostperfectlymatchestheaboveresultsof the betaproteobacterial AOB, no reads can be assigned to
this study using amoA gene as the biomarker. A minor thegammaproteobacterialAOB16SrRNAgenes,indicating
exception is that Nitrospira 16S rRNA gene was not found thedominanceofbetaproteobacterialAOBinsamplesofthis
by pyrosequencing, probably due to the low abundance of study.
these AOB species in activated sludge and that the
sequencing depth (>20,000 reads per sample) of the Abundance of AOB in activated sludge
pyrosequencing applied in this study was not of sufficient
depth to represent the complexity of the rare AOB TheabundancesofAOBinactivatedsludgewerequantified
sequences. Although we identified 495 sequence reads to usingqPCRandpyrosequencing.However,thetwodatasets
ApplMicrobiolBiotechnol(2011)91:1215–1225 1223
showacertaindegreeofdiscordancethatmightbeduetothe oneofthesesamples;(3)theqPCRnormalizationstepmade
followingreasons:(1)primerbiasinqPCRamplificationmay use of total activated sludge DNA, which contains nucleic
introducemanyuncertainties,especiallywhenthetargetgene acids from eukaryotes, bacteria, archaea, and viruses to
copynumbersareatlowlevels(Herrmannetal.2008; Smith which any variations in the relative distribution of these
et al. 2006); (2) the 454 pyrosequencing conducted in this components between samples would distort the qPCR
study may not be at a sufficient read depth to reflect the normalization process; and (4) 16S rRNA gene copy
AOB complexity since AOB sequences was not detected in numbers in prokaryotic microorganisms (Klappenbach et al.
OTU-8
100
59 R60-70 242 (DQ534853) [Soil]
91 OTU-4
89 OTU-5
92 OTU-14
OTU-3
41 95 OTU-6
OKR-C-4 (DQ148868) [Soil]
OTU-12
100 89
OTU-1
99
100 DI-1 (DQ278513) [WWTP]
64
OTU-2
34 100 HM-1 (DQ278530) [WWTP]
OTU-9
66 CB-23(DQ278512) [WWTP]
OTU-7
100
OTU-15
21 OTU-13
47 Alpine2 (DQ534698) [Soil]
AOAB sC01(EU339396) [Soil]
HF770 18E07(EF106900) [Marine]
100 95 OTU-10
DI-21(DQ278528) [WWTP]
62
48 BS2-130mA8(EF414239) [Marine]
100 MB C130m 3(DQ148821) [Marine]
88 SL-57(EU860282) [WWTP]
96 SouthBay-C1-4 (DQ278571) [Sediment]
100 SF06E-BA10-C02(EU650806)[Sediment]
87
Nitrosopumilus maritimus SCM1(EU239959) [Sediment]
MX 6 14v (DQ148614) [Marine]
96
99 MM-15 (DQ278564) [WWTP]
50 SF04-SU002S-H03(EU651171) [Sediment]
SF NB1 10(DQ148642) [Sediment]
85
OTU-11
50
CBSamoA1-2-5(EF450782) [Soil]
87
95 CBSamoA1-1-4(EF450780) [Soil]
Nitrosocaldus yellowstonii strain HL72.
0.05
Fig.6 Neighbor-joiningphylogenetictreebasedonAOAamoAgene Sequences recovered in this study are shown with “OTU-” in the
sequences. The evolutionary distances were computed using the names.OthersequenceswereobtainedfromGenBank.Thewordsin
Jukes–Cantor method and are in the units of the number of base squarebracketsindicatethesourcethesequencesoriginatedfrom
substitutionspersite.Bootstrapvaluesareindicatedonbranchnodes.
1224 ApplMicrobiolBiotechnol(2011)91:1215–1225
2001) are different and the amoA gene copy numbers in Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which per-
different AOB are also different (Norton et al. 2002).
mits any noncommercial use, distribution, and reproduction in any
medium,providedtheoriginalauthor(s)andsourcearecredited.
Diversity of AOA amoA gene in different activated sludge
samples
Although many reports described the diversity and abun-
References
dance of AOAs in the natural environments, only limited
information is available describing AOA diversity in the
activatedsludge(Limpiyakornetal.2010;Parketal.2006; AltschulS,GishW,MillerW,MyersE,LipmanD(1990)Basiclocal
alignmentsearchtool.JMolBiol215(3):403–410
Wells et al. 2009; Zhang et al. 2009). One possible reason
AvrahamiS,LiesackW,ConradR(2003)Effectsoftemperatureand
for this situation could be due to the difficulty in PCR
fertilizer on activity and community structure of soil ammonia
amplificationofAOAamoAfromDNAofsludgesamples. oxidizers.EnvironMicrobiol5(8):691–705
In this study, only five out of eight samples were ClaessonM,O'SullivanO,WangQ,NikkilaJ,MarchesiJ,SmidtH,
De Vos W, Ross R, O'Toole P (2009) Comparative analysis of
successfully amplified, and only three samples were
pyrosequencing and a phylogenetic microarray for exploring
eventually successfully cloned and sequenced.
microbial community structures in the human distal intestine.
Previous studies revealed that most of AOA should be PloSOne4(8):e6669
assignedtotheclustersofCGI.1a(themarineandsediment DeSantis T, Hugenholtz P, Larsen N, Rojas M, Brodie E, Keller K,
Huber T, Dalevi D, Hu P, Andersen G (2006) Greengenes, a
lineage) and CGI.1b (the soil lineage) in the phylum
chimera-checked 16S rRNA gene database and workbench
Crenarchaeota (Hatzenpichler et al. 2008). In this study, it compatible with ARB. Appl Environ Microbiol 72(7):5069–
was found that most of the AOA in the activated sludge 5072
samples have a closer distance to those AOA found in soil FrancisCA,RobertsKJ,BemanJM,SantoroAE,OakleyBB(2005)
Ubiquity and diversity of ammonia-oxidizing archaea in water
and thus belonged to the soil lineage. This result was in
columns and sediments of the ocean. Proc Natl Acad Sci USA
agreement with the previous studies on AOA communities 102(41):14683–14688
in activated sludge (Limpiyakorn et al. 2010; Park et al. GoodI(1953)Thepopulationfrequenciesofspeciesandtheestimation
2006), except for a study on the activated sludge in ofpopulationparameters.Biometrika40(3–4):237–264
Hatzenpichler R, Lebedeva EV, Spieck E, Stoecker K, Richter A,
bioreactors treating saline sewage (Jin, et al. 2010) which
Daims H, Wagner M (2008) A moderately thermophilic
had dominant AOA communities belonging to the marine
ammonia-oxidizing crenarchaeote from a hot spring. Proc Natl
and sediment lineage. AOA AmoA protein sequences were AcadSciUSA105(6):2134–2139
classifiedintonineOTUsusing3%distancecut-off.FigureS4 Herrmann M, Saunders AM, Schramm A (2008) Archaea dominate
the ammonia-oxidizing community in the rhizosphere of the
shows the relative abundance and distribution of AOA
freshwatermacrophyteLittorellauniflora.ApplEnvironMicrobiol
AmoA protein OTUs in different activated sludge samples, 74(10):3279–3283
indicating lower diversity compared with AOA amoA gene Huson D, Auch A, Qi J, Schuster S (2007) MEGAN analysis of
andthatmostoftheAOAAmoAproteinOTUswereshared metagenomicdata.GenomeRes17(3):377–386
Jin T, Zhang T, Yan Q (2010) Characterization and quantification of
amongthethreesamples.Theneighbor-joiningphylogenetic
ammonia-oxidizing archaea (AOA) and bacteria (AOB) in a
tree based on AOA AmoA protein sequences in Fig. S5
nitrogen-removing reactor using T-RFLP and qPCR. Appl
demonstrates the samepattern as shown by the AOA amoA MicrobiolBiotechnol87(3):1167–1176
gene tree (Fig. 6), that is, most of the OTUs in this study Klappenbach J, Saxman P, Cole J, Schmidt T (2001) rrndb: the
ribosomal RNA operon copy number database. Nucleic Acids
were grouped together and had a far distance from those
Res29(1):181–184
sequences of marine and sediment. Interestingly, there are
Kumar S, Nei M, Dudley J, Tamura K (2008) MEGA: a biologist-
twoOTUs(OTU-6andOTU-7inFig.S5)closelyrelatedto centric software for evolutionary analysis of DNA and protein
themarineandsedimentAOAlineage.ThetwoOTUswere sequences.BriefBioinform9(4):299–306
Limpiyakorn T, Kurisu F, Yagi O (2006) Quantification of ammonia-
only detected in the sludge SG-SG-UP, a sample from
oxidizing bacteria populations in full-scale sewage activated
Singapore, implying that the occurrence of these OTUs
sludgesystemsandassessmentofsystemvariablesaffectingtheir
mightbeduetothegeographiclocationofSingapore,which performance.WaterSciTechnol54(1):91–99
is near the ocean. The other two samples were taken from Limpiyakorn T, Sonthiphand P, Rongsayamanont C, Polprasert C
(2010)AbundanceofamoAgenesofammonia-oxidizingarchaea
cities far from the ocean and thus their AOA populations
andbacteriainactivatedsludgeoffull-scalewastewatertreatment
mainly come from soil. plants.BioresourTechnol102(4):3694–3701
Nicol G, Schleper C (2006) Ammonia-oxidising Crenarchaeota:
Acknowledgments The authors wish to thank the Hong Kong important players in the nitrogen cycle? Trends Microbiol 14
General Research Fund (HKU7197/08E) for the financial support of (5):207–212
this study. Lin Ye wishes to thank HKU for the postgraduate NortonJ,AlzerrecaJ,SuwaY,KlotzM(2002)Diversityofammonia
studentship. We would also like to thank W. Chan and C. K. Wong monooxygenaseoperoninautotrophicammonia-oxidizingbacteria.
fortechnicalhelpinpyrosequencing. ArchMicrobiol177(2):139–149