Putative glycogen-accumulating organisms belonging to the Alphaproteobacteria identified through rRNA-based stable isotope probing
Rikke Louise Meyer,3Aaron Marc Saunders3and Linda Louise Blackall
Correspondence
Linda Louise Blackall blackall@awmc.uq.edu.au Advanced Wastewater Management Centre,The University of Queensland,St Lucia,QLD4072, Australia
智能手机解锁Received14August2005 Revised25October2005 Accepted1November2005Deterioration of enhanced biological phosphorus removal(EBPR)has been linked to the proliferation of glycogen-accumulating organisms(GAOs),but few organisms possessing the GAO metabolic phenotype have been identified.An unidentified GAO was highly enriched in a laboratory-scale bioreactor and attempts to identify this organism using conventional16S rRNA gene cloning had failed.Therefore,rRNA-based stable isotope probing followed by full-cycle rRNA analysis was used to specifically identify the putative GAOs based on their characteristic metabolic phenotype.The study obtained sequences from a group of Alphaproteobacteria not previously shown to possess the GAO phenotype,but90%identical by16S rRNA gene analysis to a phylogenetic clade containing cloned sequences from putative GAOs and the
isolate
Defluvicoccus vanus.Fluorescence in situ hybridization(FISH)probes(DF988and DF1020) were designed to target the new group and post-FISH chemical staining demonstrated anaerobic–aerobic cycling of polyhydroxyalkanoates,as per the GAO phenotype.The successful use of probes DF988and DF1020required the use of unlabelled helper probes which increased probe signal intensity up to6?6-fold,thus highlighting the utility of helper probes in FISH. The new group constituted33%of all Bacteria in the lab-scale bioreactor from which they were identified and were also abundant(51and55%of Bacteria)in two other similar bioreactors in which phosphorus removal had deteriorated.Unlike the previously identified Defluvicoccus-related organisms,the group identified in this study were also found in two full-scale treatment plants performing EBPR,suggesting that this group may be industrially relevant.
INTRODUCTION
Enhanced biological phosphorus removal(EBPR)is a microbial process widely used for removing phosphorus (P)from wastewater(Mino et al.,1998)and presents an environmentally friendly alternative to P removal by chemi-cal precipitation.EBPR is facilitated by polyphosphate-accumulatin
g organisms(PAOs)that take up P in excess of that required for normal cellular growth.When these micro-organisms are exposed to alternating anaerobic and aerobic conditions where carbon is available only under anaerobic conditions,they take up volatile fatty acids(VFA)in the anae-robic period without the use of an external electron acceptor and use the VFA to synthesize polyhydroxyalkanoates (PHAs)(Mino et al.,1998;Seviour et al.,2003).The energy and electrons needed for the uptake and transforma-tion of VFA under anaerobic conditions come from intra-cellular glycogen and polyphosphate,and orthophosphate is released by the cells(Seviour et al.,2003).In the following aerobic period,part of the intracellular PHA is converted to glycogen and part is oxidized,producing CO2and providing energy for growth and uptake of orthophosphate to replen-ish intracellular polyphosphate(Seviour et al.,2003).The uptake of P in the aerobic period is greater than the release of P in the anaerobic period and a net removal of P from the bulk liquid therefore occurs by the end of the process cycle. At the end of the aerobic period,a fraction of the biomass is removed for a net removal of P from the system. Glycogen-accumulating organisms(GAOs)are considered competitors to PAOs,as they can perform similar carbon transformations but do not take up P in excess of the requirement for growth(Liu et al.,1996;Satoh et al.,1992). GAOs possess the ability to take up VFA under anaerobic conditions,convert them to PHA,which is stored until the following aerobic period and then oxidized to CO2or
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3Present address:Department of Microbial Ecology,Aarhus University,
Ny Munkegade,8000Aarhus C,Denmark.
Abbreviations:COD,chemical oxygen demand;EBPR,enhanced
biological phosphorus removal;FISH,fluorescence in situ hybridization;
GAO,glycogen-accumulating organism;OTU,operational taxonomic
units;PAO,polyphosphate-accumulating organism;PHA,polyhydroxy-
alkanoate;SBR,sequencing batch reactor;SIP,stable isotope probing;
TFO,tetrad-forming organism;VFA,volatile fatty acids.
0002-8445G2006SGM Printed in Great Britain419 Microbiology(2006),152,419–429DOI10.1099/mic.0.28445-0
transformed to glycogen.The glycogen produced during the aerobic period provides energy and reducing equivalents for the carbon uptake and transformations that occur during the anaerobic peri
od(Filipe et al.,2001;Zeng et al.,2003b). GAOs therefore compete with PAOs for VFA,and GAOs are known to be abundant in systems being operated for EBPR, but where P-removal is poor(Liu et al.,1996;Oehmen et al., 2004).
Only a few GAOs have been identified and as result of this, the role of different GAOs in deterioration events of the EBPR process has not been fully characterized.A deep-branching group of Gammaproteobacteria was identified as containing putative GAOs(Crocetti et al.,2002;Kong et al., 2002a;Nielsen et al.,1999)and this group is known as the GB lineage(Kong et al.,2002a)or by the name‘Candidatus Competibacter phosphatis’(Crocetti et al.,2002).Using fluorescence in situ hybridization(FISH)probes targeting this group,Saunders et al.(2003)were able to show a negative correlation between the abundance of‘Candidatus Competibacter phosphatis’and the ratio of anaerobic VFA consumption to anaerobic P release in a number of full-scale wastewater treatment plants performing EBPR.This sug-gests that members of‘Candidatus Competibacter phos-phatis’are probably GAOs playing an important role in the competition for VFA between PAOs and GAOs in EBPR in full-scale treatment plants.
Other bacteria,described as‘G-bacteria’(Cech&Hartman, 1993)or‘tetrad-forming organisms’(TFOs)(Levantesi et al., 2002;Tsai&Liu,2002),are putative GAOs in EBPR systems. Organisms of this morp
hotype have been linked to the deterioration of EBPR in lab-scale systems(Kong et al., 2002b;Oehmen et al.,2005c).Numerous isolates of TFOs have been obtained,but none of these organisms has been shown to display the GAO phenotype(Seviour et al.,2003). Using culture-independent methods,two TFOs in the Alpha-proteobacteria were recently identified from anaerobic–aerobic activated sludge biomass and shown to possess aspects of the GAO phenotype.One group is in the Sphingomonadales family(Beer et al.,2004)and the other is related to the isolate Defluvicoccus vanus(Wong et al.,2004). While present in lab-scale systems,these organisms have been found in only a few full-scale EBPR plants and more knowledge of the diversity of GAOs that have the TFO morphotype is required.
In the study presented here,a directed approach using rRNA-based stable isotope probing(SIP)to target cells capable of anaerobic propionate uptake identified a distinct group of Alphaproteobacteria related to D.vanus,which displayed the PHA transformation component of the GAO phenotype. This study thereby adds to the diversity of known putative GAOs that may play a role in deterioration of EBPR. METHODS
Reactor operation.Biomass from three lab-scale sequencing batch reactors(SBRs)and two full-scale wastewater treatment plants were used in this study.Lab-scale SBR performance was monitored by
weekly cycle studies,in which VFA,PHA and glycogen were mea-sured at regular intervals over the cycle.Analytical methods are described by Oehmen et al.(2005a).
Details about each system are provided below,but only biomass from the Reactor1was used for SIP.
Reactor1was an8-litre SBR configured for enrichment of GAOs.It was operated with sequential anaerobic and aerobic periods,where the
initial concentrations of carbon and P in the reactor were132mg propionate l21[200mg chemical oxygen demand(COD)l21]and 2mg PO4-P l21.Influent was added only in thefirst5min of the
anaerobic period and propionate,which was the only carbon source, was fully consumed before the aerobic period.The reactor was operated in a6h cycle;130min anaerobic period,160min aerobic period(O2set-point=3?0±0?2mg l21)and70min settle and decant. Further details of the SBR operation and influent composition are given in the description of reactor GAO-2in Oehmen et al.(2005a). Reactor2was an8-litre SBR configured for enrichment of PAOs.It was operated with a similar cycle configuration and O2setpoint as Reactor 1.The influent composition was identical,except that P was added at an initial concentration of13?3mg PO4-P l21.Influent was added only in thefirst7min of the anaerobic period and propionate was fully consumed before the aerobic pe
riod.The reactor was monitored as described for Reactor1.A further description of this reactor is given in Oehmen et al.(2005b).
Reactor3was a5-litre SBR configured for simultaneous nitrification,
denitrification and phosphorus removal(Zeng et al.,2003a).The SBR was operated in a6h cycle,including a1h anaerobic period,a4h aerobic period(O2set-point of0?35–0?50mg l21)followed by a1h settle and decant period.Influent containing propionate as the only carbon source was added only in thefirst7min of the anaerobic period,leading to a propionate concentration of approximately99mg propionate l21(150mg COD l21),which was fully consumed before the aerobic period.
Full-scale plant A was configured as afive-stage Bardenpho process (Tchobanoglous&Burton,1991)treating12Ml wastewater per day. The influent contained240–360mg biochemical oxygen demand l21and4–21mg PO4-P l21;the effluent contained less than0?5mg PO4-P l21during the sampling period.
Full-scale plant B was an oxidation ditch with an initial anaerobic reactor,giving a‘three-stage Phoredox’-type process(Tchobanoglous &Burton,1991),which treated7?5Ml wastewater per day.The
influent contained approximately550mg COD l21and11mg PO4-P l21;the effluent contained approximately3mg PO4-P l21(de Haas, 2005).
SIP.Biomass(50ml)was sampled from Reactor1at the end of the aerobic period and transferred to a100ml bottle for13C labelling. Sequential anaerobic–aerobic conditions were facilitated by sparging with O2-free N2gas for3h followed by air sparging for2h. Biomass(6ml)was removed at the end of the aerobic period,but no settle or decant periods were included in the cycle.HEPES (3?0g l21;BDH)was added to buffer the medium and the pH was adjusted to7?0at the end of each anaerobic and aerobic period. Nutrients were added at the beginning of the anaerobic period proportionally to the nutrient concentration in the influent to the main SBR.13C-labelled propionate(1,2-13C sodium propionate; ICON Isotopes)was the only carbon source.At the end of each anaerobic period,a2ml sample wasfiltered(0?22m m polyethersul-fone;Millipore)for VFA analysis to confirm that no propionate was detectable when aeration commenced,thus restricting13C-uptake to organisms capable of anaerobic propionate uptake.VFA analysis was
420Microbiology152 R.L.Meyer,A.M.Saunders and L.L.Blackall
carried out by HPLC(Shimadzu)with an HPX-87H30067?8mm Bio-Rad Aminex ion exclusion column.吸波
After eight cycles,the incu-bation was terminated and the remaining biomass was divided into 2ml aliquots for nucleic acid extraction.
Nucleic acid extraction and measurement of13C-labelling. RNA was extracted and pooled from two aliquots of2ml biomass using the FastRNA blue kit(Qbiogene),following the manufac-turer’s instructions.RNA was further purified with an RNeasy spin column(Qiagen).DNA was extracted using the FastDNA SPIN kit (Qbiogene),following the manufacturer’s instructions,and quanti-fied by spectrophotometric analysis at260nm.
The degree of13C-labelling of the DNA and RNA was then measured on extracted nucleic acids from biomass sampled before and after incubation with13C-propionate.Labelling was assessed by determining the13C/12C ratio through online combustion of a sample in a Fisons elemental analyser(NA-1500NC)coupled to a Micromass IsoPrime continuousflow stable isotope ratio mass spectrometer(CF-IRMS). The CF-IRMS measures the13C/12C ratio(%)of the sample against a global standard,expressed as the d13C value.To obtain sufficient sample material for the analysis(>10m g-C was required),0?36m g-C DNA or RNA was mixed with10m g-C glucose,corresponding to an approxi-mately28-fold dilution of the sample carbon with glucose-carbon. Isolation of13C-labelled RNA.RNA concentration and integrity were determined using a2100
Bioanalyser(Agilent Technologies), following the manufacturer’s instructions.RNA(500ng)was mixed with gradient buffer(0?1M Tris/HCl,pH8;0?1M KCl;1mM EDTA)to obtain a total volume of0?9ml.Deionized formamide (0?175ml)and4?1ml caesium tri-fluoroacetate(CsTFA;Amersham) were then added,resulting in a mean density of1?794g ml21.The mixture was placed into5?1ml heat-sealable centrifuge tubes (Beckman)and centrifuged (130000g)for60h in an Optima TFX ultracentrifuge(Beckman).Immediately after centri-fugation,each tube was carefully fractioned by injecting sterile water into the top of the tube at0?2ml min21and collecting10fractions (0?4ml)from a hole pierced in the bottom.The density of each fraction was measured with a temperature-controlled RFM340 refractometer(Bellingham&Stanley)using CsTFA and MilliQ water as standards.
cDNA synthesis,cloning and sequencing.RNA was isolated from each fraction by2-propanol precipitation(Sambrook& Russell,2001),then cDNA was synthesized using the Superscript III kit(Invitrogen)and the27f primer(59-AGAGTTTGATCMTGG-CTCAG-39;Lane,1992),following the manufacturer’s instructions. 16S rRNA genes were then PCR-amplified from cDNA from each fraction using the27f and1492r(59-TACGGYTACCTTGTTACG-ACTT-39;Lane,1992)primers.The size of each PCR product was confirmed by gel electrophoresis.
A clone library was prepared from the PCR product from the fraction with the highest density using th
e pGEM-T cloning vector (Promega)and OneShot competent Escherichia coli cells(Invitrogen). Cloned inserts were amplified using vector-specific primers(T7 and SP6)and screened using restriction fragment length polymor-phism(RFLP)analysis with Msp I(New England Biolabs).Clones from the most abundant RFLP patterns were selected for full-length 16S rRNA gene sequencing using BigDye Terminator3.1reaction chemistry and the following primers(Lane,1992):27f and1492r (detailed above),and530f(59-GTGCCAGCMGCCGCGG-39)and 907r(59-CCGTCAATTCMTTTRAGTTT-39).
FISH probe design,optimization and evaluation.Sequence alignment and phylogenetic analysis of16S rRNA gene sequences were performed using the software ARB(Ludwig et al.,2004).BLAST (Altschul et al.,1997)was used to identify closely related sequences and these were also added to the database.Clone sequences were checked for chimeras by analysing fragments of each sequence in BLAST.
Phylogenetic trees were made using the maximum-likelihood algo-rithm(AxML)and bootstrap analysis was performed using maximum-parsimony with1000replicates.Sequences shorter than1200nt were added by the parsimony insertion tool after construction of the maximum-likelihood tree.These sequences are indicated by dashed lines.Probes were designed using the Probe Design
function in ARB. Probe length was adjusted to obtain a T m higher than57u C and helper probes were designed to have a melting temperature exceeding that of the labelled probes.
FISH was carried out according to the protocol of Amann(1995)and results visualized on a Zeiss LSM510Meta confocal laser scanning microscope(CLSM).FISH probes used in this study were EUBMIX for Bacteria(Amann et al.,1990;Daims et al.,1999),DF1MIX (TFO_DF218plus TFO_DF618)for a group of D.vanus-related Alphaproteobacteria(Wong et al.,2004),SBR9-1a for Sphingomonas-related Alphaproteobacteria(Beer et al.,2004),GAOMIX[probes GAOQ989,GAOQ431(Crocetti et al.,2002)and GB_G2(Kong et al., 2002a)]for‘Candidatus Competibacter phosphatis’,PAOMIX for ‘Candidatus Accumulibacter phosphatis’(probes PAO462,PAO651 and PAO846;Crocetti et al.,2000)and ALF1b(Manz et al.,1992)for Alphaproteobacteria.纳米铂金
FISH quantification was done by digital image analysis using Laserpix software(Bio-Rad).Samples were hybridized with the Cy3-labelled specific probe and Cy5-labelled EUBMIX(Amann et al.,1990;Daims et al.,1999)and40images were captured using the same CLSM settings.The abundance of cells targeted by the specific probe was calculated as the area of each image containing a positive signal for both the specific probe and the EUBMIX probes relative to the area o
nly containing a positive signal for the EUBMIX probes.The value presented is a mean value for the40images.
FISH with the DF988and DF1020probes was optimized with respect to formamide concentration and the effect of helper probes by quantifying the FISH probe signal intensity.Signal intensity was determined for>30individual cells using image analysis with ImageJ software(rsb.v/ij/)on images captured using the same CLSM settings.The effect of helper probes was tested using 35%formamide in the hybridization buffer and the effect of forma-mide in the range of20–50%was then tested at5%intervals while including the chosen unlabelled helper probes in the incubation.No pure cultures with less than4nt mismatches to either probe were available to be used as positive or negative controls.The probes therefore had to be optimized using the biomass from Reactor1.This approach has been used Erhart et al.,1997).
Post-FISH chemical staining was carried out as detailed by Crocetti et al.(2000)and the staining of intracellular PHA was done according to the method of Ostle&Holt(1982).
RESULTS AND DISCUSSION
Conventional cloning
In our laboratory,TFOs belonging to the Alphaproteo-bacteria were abundant in the mixed microbial commu-nities from three different anaerobic–aerobic lab-scale bioreactors that received propionate as the sole carbon source.Reactor1received a low influent PO4-P concentra-tion to enrich for GAOs.Reactors2and3received high
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Putative glycogen-accumulating organisms relevant to EBPR
influent PO4-P concentrations to enrich for PAOs,but EBPR had deteriorated in both of the latter reactors.In Reactor2,the effluent PO4-P concentration had increased from0?2to27mg l21over the4-week period prior to sampling for the present study,and in Reactor3,EBPR had gradually deteriorated over a2-month period,leading to no net P removal and an effluent PO4-P concentration of 17?5mg l21.
The biomass in all three reactors displayed anaerobic pro-pionate uptake,PHA production and glycogen consump-tion followed by aerobic PHA degradation and glycogen production,as per the GAO phenotype.However,the cells were not targeted by GAOMIX or SBR9-1a probes.Some DF1MIX-hybridizing cells were present in Reactor1(see below),but most of the TFOs in the reactors
were not targeted by these probes.Two rRNA gene clone libraries previously constructed from Reactor3biomass failed to identify the TFOs even though they comprised>50%of all Bacteria(C.Yeates,personal communication).Thefirst clone library used Bacteria-specific primers(27f and LS1608r;
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设备博鼎包
达标Lane,1992)to amplify the16S rRNA gene,intergenic spacer region and part of the23S rRNA gene.The second library used27f(Lane,1992)and the Alphaproteobacteria-specific primer ALF969r,a modification of ALF968(Neef et al.,1999).ALF969bound the abundant TFOs when used in FISH.Although51clones from19different operational taxonomic units(OTUs)were sequenced and analysed from these libraries,not one sequence was closely related to Defluvicoccus,and none of the16Alphaproteobacteria-targeting FISH probes designed from the sequences bound abundant populations in the sludge(C.Yeates, personal communication).Therefore,SIP was used to specifically target the bacteria in the reactor capable of anaerobic propionate uptake to make the cloning more directed.
Characteristics of biomass used for SIP experiment
Fig.1shows the analysis from a cycle study carried out on Reactor1prior to sampling biomass for the
SIP experiment. It demonstrates the anaerobic uptake of propionate and the cycling of intracellular PHA and glycogen during the anaerobic and aerobic periods,which are characteristics of the GAO phenotype.
Neither‘Candidatus Competibacter phosphatis’nor the Sphingomonas-related putative GAOs(according to probe SBR9-1a)were detected in the biomass,which was domi-nated by Alphaproteobacteria(61±14%of all Bacteria by FISH quantification).Some of the Alphaproteobacteria bound the DF1MIX probes targeting a group of putative GAOs related to D.vanus(see below).However,many of the Alphaproteobacteria in the reactor did not bind these probes, suggesting that other putative GAOs belonging to the Alphaproteobacteria were present.Assessment of13C-labelling of nucleic acids and separation of labelled RNA from other nucleic acids
13C-labelled propionate was supplied to the biomass over
eight reactor cycles,but was only available under anaerobic conditions as no propionate was detected in any of the samples taken at the end of the anaerobic period.After the 13C incubation,the d13C value of both DNA and RNA
extracted from the biomass had increased(Table1),indicat-ing that bacteria capable of anaerobic pro
pionate consump-tion had incorporated13C-labelled propionate.However, the d13C value in the RNA extracts was considerably higher than the d13C value in the DNA and the RNA was selected as the preferred nucleic acid for further analysis.This result demonstrates the advantage of using RNA-SIP compared to DNA-SIP,when the target organism is slow-growing but has a high metabolic activity.
Density-gradient centrifugation of RNA and subsequent fractionation of the gradient yielded10fractions,the density of which decreased linearly(r2=0?9911)between 1?979g ml21(fraction1)and1?872g ml21(fraction
10).
Table1.d13C values determined by isotope ratio mass spectrometry
An increase in the d13C value(more positive or less negative) indicates enrichment with13C in the nucleic acid.
Sample d13C value(%) Glucose control226 Glucose+DNA before13C labelling229 Glucose+DNA after13C labelling21 Glucose+RNA before13C labelling228 Glucose+RNA after13C labelling+643
422Microbiology152 R.L.Meyer,A.M.Saunders and L.L.Blackall
RNA purified from each fraction was used to synthesize cDNA,which was then used as template in PCR amplifica-tion of near full-length16S rRNA genes.PCR products were obtained from fractions5,6and8,but not from fraction7, indicating that the separation of the13C-enriched RNA from the unlabelled RNA had been successful.As fraction5had the highest density,this fraction was the most13C-enriched and the PCR product from this fraction was used to construct a clone library.
Analysis of16S rRNA sequences obtained from 13C-labelled RNA
cnc真空吸盘怎么做Eleven of13sequences(from the four most abundant OTUs by RFLP)were related to D.vanus(Maszenan et al., 2005),within the Alphaproteobacteria.The remaining two sequences were related to Gammaproteobacteria and Betaproteobacteria,respectively.Phylogenetic analysis of these sequences and published sequences showed that the sequences related to D.vanus formed a monophyletic group with two distinct clusters(Fig.2).Cluster1comprised one sequence from the present study(Meyer Clone17),D. vanus,four sequences from Wong et al.(2004)and four partial16S rRNA gene sequences obtained from an EBPR system(T.Zhang et al.,unpublished;direct submission to GenBank).Cluster2was monophyletic and contained10of the11Alphaproteobacteria sequences obtained in this study, one sequence from a lab-scale anaerobic–aerobic operated bioreactor(Wong et al.,2004)and three sequences obtained from a full-scale EBPR plant(K.D.McMahon et al., unpublished;direct submission to GenBank). The phylogenetic tree(Fig.2)contains representatives from each of the orders within the Alphaproteobacteria
.
Fig.2.Phylogenetic tree(maximum-likelihood)of the sequences obtained and highly identical sequences obtained from GenBank.Clusters1and2indicate the two distinct phylogenetic D.vanus groups identified.Bootstrap values are indicated as a percentage of1000analyses.Clone sequences in Clusters1and2are presented as author and clone name followed by accession number.Sequences on dashed lines were added after phylogenetic analysis by the parsimony insertion tool of ARB and were between311and962nt in length.All other sequences were at least1257nt in length.
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Putative glycogen-accumulating organisms relevant to EBPR
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