Occurrence of PAOI in a low temperature EBPR
system
Wen-De Tian a ,b ,C.M.Lopez-Vazquez c ,⇑,Wei-Guang Li b ,D.Brdjanovic a ,c ,M.C.M.van Loosdrecht a
Department of Biotechnology,Delft University of Technology,Delft,The Netherlands
b
School of Municipal and Environmental Engineering,Harbin Institute of Technology,Harbin,China c
Environmental Engineering and Water Technology Department,UNESCO-IHE Institute for Water Education,Delft,The Netherlands
h i g h l i g h t s
货物税At 10°C and 6d net aerobic SRT,an enriched GAO culture shifted to PAO I.
PAO I did not switch to a GAO-like metabolism under P-limiting conditions at 10°C. Low temperature can be ud to enrich PAO I cultures for their metabolic rearch.
a r t i c l e i n f o Article history:
Received 29September 2012
Received in revid form 7May 2013Accepted 8May 2013
Available online 31May 2013前台英语
Keywords:
Accumulibacter Type I Accumulibacter Type II PAO I PAO II
Low temperature
Enhanced biological phosphorus removal
a b s t r a c t
The occurrence of Accumulibacter Type I (a known phosphorus-accumulating organism,PAO)has received incread attention due to the potential operating benefits associated with their denitrifying activity in enhanced biological phosphorus removal (EBPR)wastewater treatment plants.In this study,after a shift from an enriched glycogen-accumulating organism (GAO)culture (competitors of PAO)to a PAO-enriched system,Accumulibacter Type I (PAO I)became dominant in an anaerobic–aerobic EBPR system fed with acetate and operated at 10°C with a net aerobic solids retention time (SRT)of 6d.Since Accu-mulibacter Type II (PAO II)were not detected,the low temperature in combination with the net aerobic SRT applied appeared to have suppresd their growth as well.The stoichiometry of PAO I was in agree-ment with previous metabolic models,suggesting that it was the
main PAO organisms prent in previ-ous studies operated under similar conditions.Moreover,under poly-P limiting conditions,PAO I were unable to switch to a GAO-like metabolism at low temperatures.The results contribute to increa the understanding of the physiology,microbial metabolism and microbial ecology of PAO I.
Ó2013Elvier Ltd.All rights rerved.
1.Introduction
The enhanced biological phosphorus removal (EBPR)process in activated sludge systems has been implemented as an economical and environmentally friendly technology in wastewater treatment plants worldwide.Denitrifying dephosphatation carried out by denitrifying phosphorus accumulating organisms (DPAO)is con-sidered to be a highly desirable process for wastewater treatment since DPAO are capable of using nitrate instead of oxygen as elec-tron acceptor to simultaneously achieve anoxic phosphorus uptake and denitrification (Kuba et al.,1993;Zeng et al.,2004;Kong et al.,2004).This process requires less carbon for P-uptake (Kuba et al.,1996),has lower aeration requirements (Kuba et al.,1996;Tian et al.,2011),and leads to lesr sludge production (Kuba et al.,1994;Murnleitner et al.,1997).
The existence of DPAOs had been first obrved in anaerobic-anoxic-aerobic batch tests when alternating or switching the final electron acceptor (nitrate or oxygen)(Kerrn-Jespern and Henze,1993).Kuba et al.(1993)showed that DPAOs can utili either ni-trate or oxygen as electron acceptor for biological phosphorus re-moval in HAc-fed anaerobic-anoxic (A 2)or anaerobic–aerobic (A/O)systems.Until a few years ago,it was assumed that DPAOs and PAOs were emingly the same microorganisms when com-paring their stoichiometry and kinetics in A/O and A 2systems (Ahn et al.,2002;Zeng et al.,2003a ).However,tho obrvations were still in conflict with the results of Kerrn-Jespern and Henze (1993)and Meinhold et al.(1999),who suggested the existence of two types of PAOs (DPAOs and non-DPAOs)according to the anal-ysis of chemical transformations.
A recent study concerning the lineage of the organisms re-vealed the existence of two main Types of Accumulibacter (I and II)each one comprising veral clades (He et al.,2007;Peterson et al.,2008)with different denitrification capabilities (He et al.,2007;Flowers et al.,2009).Accumulibacter Type I (hereafter re-ferred to as PAO I)are able to u nitrate and/or nitrite as final
0045-6535/$-e front matter Ó2013Elvier Ltd.All rights rerved.dx.doi/10.1016/j.chemosphere.2013.05.009
⇑Corresponding author.Address:Environmental Engineering and Water Tech-nology Department,UNESCO-IHE Institute for Water Education,Westvest 7,2611AX Delft,The Netherlands.Tel.:+31(0)152151781.
朦胧诗派E-mail address:c.lopezvazquez@unesco-ihe (C.M.Lopez-Vazquez).
electron acceptor whereas Accumulibacter Type II(hereafter re-ferred to as PAO II)are capable of using only nitrite for anoxic P re-moval(Flowers et al.,2009;Oehmen et al.,2010).The u of nitrate as electron acceptor ems to favour the growth of PAO I over PAO II,but in most of the cas they tend to co-exist in EBPR systems (Carvalho et al.,2007;He et al.,2010;Acevedo et al.,2012).
Though Kuba et al.(1993)and Zeng et al.(2003a)were able to cultivate a DPAO culture(presumably PAO I)on acetate using ni-trate as electron acceptor,Carvalho et al.(2007)found that HPr in-stead of HAc was more suitable to sustain the anoxic P-removal process after switching from an A/O to an A2system,apparently due to the prence of PAO I(Oehmen et al.,2010).Lanham et al. (2011),using nitrate as electron acceptor in a HPr-fed reactor oper-ated under anaerobic/anoxic/aerobic conditions,confirmed the obrvations of Carvalho et al.(2007)by achieving a relatively high enrichment of PAO I(approx90%),with practically no PAO II pres-ent.In a recent study(Acevedo et al.,
2012),PAO I was enriched in a HAc-fed A/O system but eventually switched over to PAO II when poly-P decread drastically in short-term experiments.This re-sulted in low intracellular poly-P levels,reduced anaerobic P-re-lea/HAc ratios and higher anaerobic glycogen requirements for HAc uptake associated to a GAO-like metabolism performed by PAO.However,it was unclear whether the GAO-like metabolism occurred due to a population shift from PAO I to PAO II,or becau PAO II were able to adjust their metabolism under low intracellular Poly-P levels.The aforementioned information emingly suggests that propionate,nitrate asfinal electron acceptor,and higher influ-ent COD/P ratios provide competitive advantages to PAO I over PAO II.However,to the best of our knowledge,the effects of other fac-tors influencing the occurrence of PAO I and PAO II have not been documented yet(such as pH,temperature,dissolved oxygen con-centration and solids retention time(SRT),among others).The main objectives of this study were therefore:(1)to explore the population distribution of Accumulibacter sub-clades(namely, PAO I and PAO II)in a quencing batch reactor(SBR)where the dominant microbial populations shifted from a highly enriched GAO to an enriched PAO culture after reducing the temperature from20to10°C under P-limiting conditions and adjusting the to-tal SRT to16d,26d and then back to16d,that led to net aerobic SRT of6d,13.5d and6d,respectively(Lopez-Vazquez et al., 2009);and,(2)to evaluate the biomass metabolism of the SBR en-riched with PAO with particular emphasis on the anaerobic and aerobic stoichiometry of the
dominant Accumulibacter clade(s). The results of this study will contribute to get a better understand-ing about the environmental factors affecting the occurrence of Accumulibacter sub-clades,potential metabolic and microbial pop-ulation shifts in EBPR systems and ultimately about the perfor-mance of EBPR process.
2.Materials and methods
2.1.Operation of the SBR
A double-jacketed lab-scale SBR was inoculated with sludge from a highly enriched GAO reactor operated at20°C(parent SBR)(Lopez-Vazquez et al.,2009).After the sludge transfer from the parent SBR to the cond SBR,the temperature in the cond SBR was reduced to10°C.The cond SBR had a working volume of2.5L,it was operated at10±0.5°C and pH was maintained at 7.0±0.1.In order to avoid temperaturefluctuations,prior tofilling up the reactor,the temperature of the synthetic influent was ad-justed to10°C in another double-jacketed batch reactor equipped with a water bath.The SBR cycle consisted of2.25h anaerobic, 2.25h aerobic and1.5h ttling.The SBR was operated with a SRT of16d in pha I and pha III,and36d in pha II.Table1shows the operating parameters of interest during the three differ-
万物皆有灵性
ent experimental phas.The net aerobic SRT was determined as a fraction of the total time that the biomass spends under aerobic conditions in relation to the total applied SRT.Further details regarding the operation of the SBR can be found elwhere(Lo-pez-Vazquez et al.,2009).
2.2.Synthetic media
The synthetic media fed to the SBR(both parent and the cond SBR)contained in mg LÀ1:850NaAcÁ3H2O(12.5C-mmol,approxi-mately400mg COD LÀ1)and107NH4Cl(2mmol).2allylthiourea were added to inhibit nitrification.In phas I and II of the opera-tion of the cond SBR,the phosphorus concentrations in the syn-thetic media were limited to2.2mg PO3À
4
-P LÀ1(0.07mM P)(Liu et al.,1997).However,in pha III the influent phosphorus concen-
tration was incread to15mg PO3À
4
-
P LÀ1(0.48mM P)in order to favour the growth of PAO(Smolders et al.,1994a,b).Other minerals and trace metals prent in the media were prepared as described by Smolders et al.(1994b).Prior to u,the media were autoclaved for1h at110°C.
2.3.Chemical analys
On a regular basis,the performance of the SBR was monitored through the determination of mixed liquor suspended solids (MLSS),mixed liquor volatile suspended solids(MLVSS)and ortho-phosphate.For MLSS and MLVSS determination,grab samples were collected by triplicate in the end of the aerobic stage,whereas for
PO3À
4
-P analys samples were taken in the end of the anaerobic and end of the aerobic phas.When the SBR exhibited steady-state pha III),cycle measurements were executed in concutive days and the average values were reported(Table2). During the execution of the cycle measurements the parameters of
interest were PO3À
4韩国明星照片
-P,acetate,poly-hydroxy-butyrate(PHB),poly-hydroxy-valerate(PHV),glycogen and ammonium.They were measured by duplicate in addition to MLSS and MLVSS that were measured by triplicate.All off-line analys were performed in accordance with Standard Methods(APHA,1995).The polyhydrox-yalkanoates(PHA,concluding PHB and PHV)and glycogen contents were determined as described elwhere(Lopez-Vazquez et al., 2007).
西游记有哪些人物2.4.Microbial characterisation
Fluorescence in situ Hybridization(FISH)was performed as de-scribed in Amann(1995).In order to asss the evolution of micro-bial population from a highly enriched GAO culture to a PAO culture at10°C,the rRNA oligonucleotide probes ud for FISH in this study were EUBMIX(including EUB338,EUB338-II and EUB338-III)to target the entire bacterial population(Daims et al., 1999),and Accumulibacter Type I and Type II probes to target PAOI and PAOII,respectively(Flowers et al.,2009).Other probes such as PAOMIX and GAOMIX to target the PAO and GAO populations, respectively,are described elwhere(Lopez-Vazquez et al., 2009).The quantification of the populatio
n distribution was car-ried out as described in Lopez-Vazquez et al.(2008b).
3.Results and discussion
3.1.Shift from a GAO-to a PAO I-enriched system
Fig.1a illustrates thefirst cycle of pha I(after reducing the temperature from20to10°C at an SRT of16d).It showed the typical phenotype of an enriched GAO culture:full anaerobic
W.-D.Tian et al./Chemosphere92(2013)1314–13201315
HAc uptake,anaerobic glycogen consumption,PHA production and low P-relea/HAc uptake ratio(0.05mol P molÀ1C),whereas in the aerobic stage glycogen replenishment and PHA oxidation were obrved.This indicated that GAO were barely inhibited after reducing the temperature from20to10°C.However,HAc started to leak into the aerobic stage within thefirst20cycles (between day3and4)after the long-term experiment started (data not shown).With the objective of maintaining the GAO cul-ture,the SRT was incread to36d(pha II).However,the leak-age of HAc to the aerobic stage continued within the next16d (day20of operation of the reactor)(Fig.1b).Interestingly,the anaerobic P/HAc ratio ro from0.05to0.13mol P molÀ1C(Fig.1a and b,resp
ectively)implying that PAO began to take up higher HAc amounts that led to the accumulation of glycogen and PHA (by comparing Fig.1a and b).This indicated that they were not consumed in the anaerobic and aerobic stages,respectively.FISH results confirmed that the fractions of Competibacter and Accumu-libacter changed from93±1and3±1%(pha I on day zero), respectively to43±2%Competibacter and8±1%Accumulibacter (pha II on day20of operation of the SBR)(Lopez-Vazquez et al.,2009).Herein,by using Accumulibacter Type I and Type II FISH probes(Flowers et al.,2009),it was confirmed that around 90%of the Accumulibacter belonged to PAO I but PAO II were not detected(Fig.2a and b).
Since it was suspected that the PAO I biomass activity was sup-presd by the low influent P concentration(Fig.1b),the influent P concentration was incread to15mg LÀ1(0.48mM P)(Smolders et al.,1994a,b)and the SRT re-adjusted to16d on day21st of oper-ation of the reactor(pha III).After16d(day37),the fraction of PAO I incread and accounted for around38±1%of total biomass, PAO II could not be traced,and Competibacter made up to12±1% (Lopez-Vazquez et al.,2009).This validated the previous assump-tion that the low influent P concentration was suppressing the growth of PAO(Fig.2c).After about one month(day67),the bio-mass activity reached steady-state conditions and displayed the typical phenotype of an enriched PAO culture(Fig.1c):complete anaerobic HAc upt
ake,an anaerobic P/HAc ratio of0.56mol P molÀ1C,glycogen consumption and PHA production;as well as complete aerobic P-uptake,PHA degradation and aerobic glycogen replenishment.PAO I were the dominant microorganisms(81±2%) but PAO II were hardly detected(<1%)(Fig.2d).Meanwhile,the fraction of Competibacter was around18±1%(Lopez-Vazquez et al.,2009).Hereto,the SBR microbial population shifted from a highly GAO-enriched culture to a highly PAO I-enriched EBPR sys-tem,induced by reducing the temperature from20to10°C and influent P increa,while keeping a SRT of16d with a net aerobic SRT of6d.To confirm the obrvations,a similar experiment was carried out on a GAO-enriched culture(reducing the temperature
Table1
Operating conditions of the quencing batch reactor throughout the different experimental phas.
Experimental pha Start of the pha
(d)
End of the pha
(d)
Length of the
pha(d)
SRT
(d)
Net aerobic SRT
(d)
Influent HAc concentration
(mg LÀ1)
Influent P concentration
(mg LÀ1)
青春的名言
I044166375 2.2 II520153613.5375 2.2 III21674616637515
Table2
Anaerobic stoichiometry parameters obrved in this study and reported in literature.
Temperature (°C)Cycle Organisms SRT
(d)
P/HAc
(mol
P molÀ1C)
Glycogen/
HAc
PHA/HAc PHB/HAc
(mol
C molÀ1C)
PHV/HAc PHV/PHB Refs.
10Pha I
(Day0)Competibacter(12±2%)
PAO I(38±1%)
160.050.96 1.95 1.490.460.31This study
10Pha II
(Day37)Competibacter(43±2%)
PAO I(8±1%)
360.130.62 1.42 1.100.320.29
10Pha III
(Day67)PAO I(81±2%)
Competibacter(18±1%)
PAO II(<1%)
160.56±0.020.55±0.05 1.44±0.07 1.31±0.050.13±0.020.10±0.02
10Short-term
test Competibacter100.010.46 1.40 1.100.300.27Lopez-Vazquez
et al.(2007)
20Long-term PAO80.500.50 1.33 1.330.000.00Smolders et al.
(1994a)
20Long-term GAO 6.60.00 1.12 1.86 1.400.470.34Zeng et al.(2003b) 20Long-term a PAO I and PAO II100.16–0.520.50–0.69 1.10–1.440.76–0.960.14–0.36N/A Carvalho et al.
(2007)Oehmen
et al.(2010)
20Short-term
test2nd
stage PAO I(66±7%)PAO II
(8±3%)b
80.730.35 1.36 1.300.060.05Acevedo et al.
(2012)
20Short-term
test5th
stage PAO I(23±5%)PAO II
(36±7%)c
80.08 1.08 2.02 1.740.280.16Acevedo et al.
(2012)
20Long-term DPAO80.440.48 1.20 1.20N/A N/A Kuba et al.(1996) 20Long-term DPAO140.420.80 1.52 1.52N/A N/A Kuba et al.(1996) 20Long-term DPAO/DGAO150.350.64 1.48 1.350.130.10Zeng et al.(2003a) 20Long-term DPAO/DGAO80.320.76 1.52 1.340.180.13Zeng et al.(2003a)
Note:N/A=not applicable.
a pH varied between7.0and8.2.
b Poly-P/VSS=0.30;pH ranged from7.0to8.9.
c Poly-P/VSS=0.01;pH range
d from7.0to8.9.
1316W.-D.Tian et al./Chemosphere92(2013)1314–1320
from15to10°C)that resulted in the same shift from a GAO-to a PAO-enriched culture(data not shown)
(Lopez-Vazquez et al., 2009),indicating that the previous obrvations were reproducible.
3.2.Low temperature effects on the anaerobic stoichiometry of PAO and GAO
During the shift from an enriched Competibacter culture to-wards an enriched PAO I EBPR system(Fig.2),the stoichiometric ratios from the cycles carried out at10°C were determined and compared to previous experiments executed at20°C(Table2).In thefirst cycle of pha I performed at10°C,most of the stoichiom-etric ratios were similar to tho of the Competibacter culture en-riched at20°C,with the exception of the anaerobic glycogen-to-acetate ratio that was lower(0.96compared to1.12mol C molÀ1 C)(Lopez-Vazquez et al.,2009).In pha II,the anaerobic stoichi-ometric parameters were in the range of previous reports for PAO-GAO mix cultures with a glycogen-to-acetate ratio of 0.62mol C molÀ1C when PAO I and Competibacter made up to 8±1and43±2%of the total biomass,respectively(Smolders et al.,1994a;Zeng et al.,2003b;Acevedo et al.,2012).The latter provided evidence that PAO I started to become the dominant organisms and GAO began to be outcompeted.Meanwhile,in pha III(Table3),the anaerobic stoichiometric parameters were similar to tho reported for enriched PAO cultures(Smolders et al.,1994a),suggesting that it was the main PAO organisms pres-ent in previous studies operated under similar conditions.
The results indicate that likely the low temperature inhibited the anaerobic glycogen degradation pathway(Table2),limiting the acetate uptake of GAO and giving the opportunity to PAO to proliferate.The obrvations are in agreement with Erdal et al. (2008)who obrved that a low temperature(5°C)inhibited the glycolysis pathway of the dominant organisms prent in their study resulting in a slower ATP generation that was unable to sat-isfy the energy requirements for HAc uptake and PHA synthesis. Furthermore,becau Competibacter were outcompeted due to the inhibition of the anaerobic glycogen degradation pathway,it appears that they have a different glycolysis pathway than PAO I (either the Entner-Doudoroff or the Embden–Meyerhof–Parnas pathway).Moreover,PAO II were unable to proliferate under P-limiting conditions(by performing a GAO-like metabolism as ob-rved by Acevedo et al.(2012)).Though,it must be noticed that PAO II were hardly detected(even in the beginning of the test at 20°C)(Fig.2).Certainly,there is a strong need to obtain highly en-riched cultures of PAO I,PAO II and GAO(to minimize and prefer-ably avoid potential interferences)and make u of advanced molecular and genetic techniques to clarify the long-standing de-bate concerning the actual microbial pathways of PAO and GAO cultures.In this regard,low temperature could be ud as one of the key strategies to achieve this purpo.Moreover,the current study also suggests that PAO I and PAO II may have different tem-perature dependences.Further rearch must focus on the asss-ment of the effects of temperatur
e on enriched cultures of PAO I and PAO II.The prent study can rve as a basis for such an asssment and could start with the operation of an EBPR system at10°C and a net aerobic SRT of6d to enhance the enrichment of PAO I followed by a progressive increa in temperature to as-ss the temperature dependency of PAO I and asss when PAO II(and Competibacter)may appear.Similarly,an enriched PAO II culture could be enriched at20°C and the culture could be expod to progressive temperature changes to cover a wide temperature from10to30°C).
Concerning the potential metabolic shift obrved in certain PAO cultures to a GAO-like metabolism(Zhou et al.,2008;Lopez-Vazquez et al.,2008a),Acevedo et al.(2012)obrved such a met-abolic shift after the enriched PAO culture integrated by66±7% PAO I and8±3%PAO II changed to23±5%PAO I and36±3% PAO II,with no GAO prent.They speculated that PAO II could switch to a GAO-like metabolism while PAO I could not.In this study,PAO I became the dominant organisms under P-limiting conditions at10°C,but incomplete HAc uptake was obrved and glycogen and PHA tended to accumulate in the system (Fig.1b).Once the influent P concentration was incread,PAO I were able to take up HAc and perform the typical PAO phenotype (anaerobic acetate uptake coupled to P-relea,glycogen consump-tion and PHA storage)(Fig.1c).The obrvations suggest that PAO I cannot switch their metabolism to a GAO-like metabolism under P-limiting conditions while PA
O II may be able to do so (Acevedo et al.,2012).Thefindings can contribute to elucidate previous discrepancies concerning the u of glycogen as sole en-ergy source for anaerobic HAc transport and PHA synthesis under poly-P limiting conditions(Lopez-Vazquez et al.,2008a).Previ-ously,Brdjanovic et al.(1998b)had concluded that PAO were un-able to do it,meanwhile Zhou et al.(2008)obrved that, converly,it was feasible.Likely,PAO I were the dominant organ-isms in the study of Brdjanovic et al.(1998b),and PAO II in that of Zhou et al.(2008),which may explain the different obrvations.
Fig.1.The evolution of acetate,intracellular carbon compounds,ammonium and
orthophosphate concentrations in the experimental phas(a)I,(b)II and(c)III.
Acetate(}),glycogen(s),PHB(j),PHV(h)orthophosphate(4)and ammonium
(;).
92(2013)1314–13201317
经典诗文3.3.Low temperature effects on the aerobic metabolism of PAO and GAO
As shown in Table 3,in the first cycle of pha I,the aerobic stoi-chiometric coefficients of Competibacter at 10°C were similar to tho reported at 20°C by Zeng et al.(2003b),which suggested that the aerobic metabolism of Competibacter was not affected by the short-term temperature decrea.Meanwhile,the aerobic stoi-chiometric parameters of PAO I,measured in pha III and per-formed at 10°C,were consistent with tho reported for PAO at 20°C (Smolders et al.,1995).Unfortunately,in other studies where PAO I and PAO II became dominant (Acevedo et al.,2012)the aer-obic yields of the organisms have not been determined as a func-tion of the so-called delta-factor (reprents the ATP produced per NADH 2produced as a measure of the efficienc
y of the oxidative phosphorylation)(Smolders et al.,1994b ).This would have al-lowed to carrying out a direct comparison of the aerobic stoichi-ometric parameters of interest among the different PAO I and PAO II cultures.
Despite that the low temperature played a crucial role in the enrichment of PAO I,the effects of other factors such as the net aer-obic SRT and a potential ability of PAO I to take up acetate under aerobic conditions (when acetate was not consumed by GAO and leaked into the aerobic stage)cannot be discarded as determinant operating factors that favoured the (aerobic)metabolism of PAO I.Particularly,Brdjanovic et al.(1998a)found a correlation between the effects of temperature and the required net aerobic SRT (instead of the total applied SRT)for the growth of PAO
since
distributions obrved in the long-term effect tests performed at 10°C by applying Fluorescence in perature to 10°C;(b and f)pha II (day 20of operation of the SBR)at an SRT of 36d and 10°C;(c and g)day 37)and the influent P concentration was incread;and,(d and h)the 48th day of pha III (day 67).(a–d)superposition of EUBmix and PAO I probes),and PAO II in green.(e–h)EUBacteria appear in blue,PAO I probes),and GAOmix in green (or light green due to superposition with EUBmix).In all figures,the bar indicates figure legend,the reader is referred to the web version of this article.)
