Effect of different carbon sources on the enhanced biological phosphorus removal

E?ect of di?erent carbon sources on the enhanced biological phosphorus removal in a sequencing batch reactor

Juliane Hollender*,Doris van der Krol,Liane Kornberger,Edith Gierden and Wolfgang Dott Institut fu ¨r Hygiene und Umweltmedizin,RWTH Aachen,Pauwelsstr.30,52074Aachen,Germany *Author for correspondence:Tel.:+49-241-8088282,Fax:+49-241-8082477,E-mail:Juliane.Hollender@post.rwth-aachen.de

Received 9August 2001;accepted 25January 2002

Keywords:Enhanced biological phosphorus removal,glycogen,polyhydroxyalkanoate,polyphosphate

Summary

The e?ect of the di?erent carbon sources acetate,acetate/glucose or glucose on the enhanced biological phosphorus removal (EBPR)process was studied by experiments under alternating anaerobic–aerobic conditions in one sequencing batch reactor for each carbon source.The glucose was consumed completely within the ?rst 30min of the anaerobic phase whereas acetate degradation was slow and incomplete.Phosphate was released independently of the carbon source during the whole anaerobic phase.The highest phosphate release (27mg P l )1)and polyhydroxyalkanoate (PHA)storage (20mg C g )1dry matter (DM))during the anaerobic phase as well as the highest polyphosphate (poly-P)(8mg P g )1DM)and glycogen storage (17mg C g )1DM)during the aerobic phase were observed with acetate.In contrast to other investigations,glycogen storage did not increase with glucose as substrate but was signi?cantly smaller than with acetate.The PHA composition was also in?uenced strongly by the carbon source.The polyhydroxyvalerate (PHV)portion of the PHA was maximal 17%for acetate and 82%for glucose.Due to the strong in?uence of the carbon source on the PHA concentration and composition,PHA storage seems to regulate mainly the phosphate release and uptake.

Abbreviations:EBPR –Enhanced biological phosphorus removal;GAO –glycogen-accumulating organisms;PAO –polyphosphate-accumulating organisms;PHA –polyhydroxyalkanoates;PHV –polyhydroxyvalerate;poly-P –polyphosphate;SCVFA –short chain volatile fatty acids

Introduction

Since its discovery in 1960’s enhanced biological phosphorus removal (EBPR)has been investigated by many research groups and the process is now well established in practice.Nevertheless,fundamental un-derstanding of the process from microbiological and biochemical points of view is still incomplete.In general,in the anaerobic phase,polyphosphate-accumulating organisms (PAOs)take up the carbon sources and store them in the form of PHA accompanied by degradation of glycogen as well as poly-P and consequent release of phosphate.In the following aerobic phase,PAOs grow aerobically and take up phosphate to store poly-P using the stored PHA as the carbon and energy source.In addition,the glycogen level is replenished using PHA as the carbon source (Mino et al .1998).PHAs in PAO-enriched sludges have been veri?ed to be co-polymers composed of mainly 3-hydroxybutyrate (3-HB)and 3-hydroxyvalerate (3-HV)and with a lower quantity of 3-hydroxy-2-methylbutyrate and 3-hydroxy-2-methyl-valerate (Satoh et al .1992).The composition of PHA is dependent on the carbon source.When acetate is the only carbon source available in the anaerobic phase,the 3-hydroxybutyrate unit is the major unit in the PHA formed (Satoh et al .1992).3-Hydroxyvalerate is formed by conversion of glycogen to propionyl-CoA via the succinate–propionate pathway and subsequent reaction with acetyl-CoA (Pereira et al .1996).Glycogen func-tions as regulator of the redox balance in the cell.Con-version of glycogen to acetyl-CoA and CO 2generates reducing power,whereas conversion to propionyl-CoA via the succinate–propionyl-CoA pathway consumes reducing power (Mino et al .1998).

Sometimes deterioration of the EBPR in laboratory scale systems has been observed (Cech &Hartmann 1990,1993;Tasli et al .1997)and this has been attrib-uted to the carbon source supplied during the anaerobic phase of the EBPR process.In general,PAOs are known to consume short chain volatile fatty acids (SCVFAs)and are unable to utilize glucose,if glucose is not converted to SCVFAs by other organisms (Randall

World Journal of Microbiology &Biotechnology 18:355–360,2002.355

ó2002Kluwer Academic Publishers.Printed in the

Netherlands.

et al.1994).Glucose promotes the growth of glycogen-accumulating organisms(GAOs)although GAOs were reported to be able to grow even in acetate-fed sludge (Cech et al.1993;Satoh et al.1994,1996).The metab-olism of GAOs seems to be similar to that of PAOs, except that glycogen serves as the intracellular energy pool for anaerobic substrate uptake instead of poly-P (Mino et al.1994;Satoh et al.1994;Liu et al.1996).As a result PHA is synthesized without energy de?cit but with a net production of one mole of ATP in these GAOs.In contrast,several studies reported that a good phosphorus removal e?ciency can be achieved if glucose or a mixture of glucose and peptone are supplied as carbon source(Fukase et al.1984;Nakamura et al. 1991;Carucci et al.1995,1999).However,glucose decrease was not associated with the release of ortho-phosphate(Carucci et al.1995;Jeon Park2000).Re-cently,Jeon&Park(2000)proposed a metabolic model which postulates the accumulation of glucose as glyco-gen,conversion of glucose to lactate and the formation of a lactate polymer by one population.The conversion

of lactate to PHAs with a consumption of poly-P is performed by another population,the PAOs.

Due to the heterogeneous organic matter in waste-water which often reaches the treatment plant without complete acid fermentation,more attention must be paid to the type of carbon source in the wastewater and its e?ect on EBPR.The aim of this study was to compare the carbon sources acetate and glucose with respect to phosphorus removal e?ciency by a sludge derived from a wastewater treatment plant with ongoing EBPR.The storage of poly-P,glycogen and PHA in particular was investigated in more detail.The experi-ments with the di?erent carbon sources were performed simultaneously in one sequencing batch reactor for each carbon source.

Materials and methods

Laboratory system

The experiments were performed in2-l laboratory scale reactors(Figure1)with 1.5l medium containing 43.9mg l)1KH2PO4(10mg P l)1),101.4mg l)1 MgSO4?7H2O,14.0mg l)1CaCl2?2H2O,36.0mg l)1 KCl,38.18mg l)1NH4Cl,15ml vitamin solution(Eic-kelboom1975),1.5ml trace element solution(Pfennig& Lippert1966)and750ml activated sludge from the end of the aerobic step of the wastewater treatment plant Ju lich Hambach.Anaerobic and aerobic conditions were achieved by a continuous air or nitrogen?ow through two capillaries(8–15l h)1).The treatment cycle for all experiments consisted of a19.5-h aerobic phase, followed by a4-h anaerobic phase,and a0.5-h period for settling.Afterwards500ml of supernatant was drained through a glass outlet and threefold concen-trated fresh medium was added.The temperature was kept at20–23°C and the pH was adjusted30min before sampling to6.5–7.0.10Min after the beginning of the anaerobic phase,the organic substrate was added through a capillary.As organic substrates acetate (107mg C l)1),acetate+glucose(each53mg C l)1) or glucose(107mg C l)1)were used.Sampling was performed at the end of the aerobic and anaerobic phase,respectively.

Chemical analysis

Orthophosphate,nitrate and nitrite concentrations of the cell-free medium were measured with an ion chro-matograph DX-100(Dionex,Sunnyvale,USA)accord-ing to DIN38405part20.The total phosphate concentration was determined colorimetrically accord-ing to Herbert et al.(1971).The dry mass was deter-mined gravimetrically.Poly-P was extracted according to Clark et al.(1986)and determined colorimetrically by using Toluidine Blue.

Acetate analysis of the cell-free medium was per-formed enzymatically with a test-kit(Boehringer,Mann-heim,Germany).Glucose concentration of the cell-free medium was determined enzymatically by using glucose-6-phosphate dehydrogenase and hexokinase and mea-suring the NADPH formed.Glycogen analysis of the cell pellet was performed in the same way after digestion with amyloglucosidase(Keppler&Decker1974).Prior to the glycogen analysis cells were broken by a5-min ultrasonic treatment(100W,1700Hz). Polyhydroxyalkanoates(PHA)of lyophilized sludge samples were digested,ethylated,and extracted with chloroform according to Comeau et al.(1988).The ethyl esters formed were analysed by GC with?ame ionization detector(FID).Calibration of the whole method was performed using a3-hydroxybutyrate-3-hydroxyvalerate copolymer with a known composition as standard(Aldrich,Steinheim,Germany).The

ethyl Figure1.Experimental set-up of the experiments.

356J.Hollender et al.

ester of 2-methyl-3-hydroxybutyrate was synthesized as standard compound according to Fu chtenbusch et al .(1996).

Results and discussion Phosphate elimination

The experiments were inoculated with sludge from the end of the aerobic step of the wastewater treatment plant Ju lich Hambach which has an e?cient EBPR process.Three experiments were performed,di?ering only in the organic carbon source added at the begin-ning of the anaerobic phase.Besides acetate,the typical substrate used in studying the EBPR process,glucose which is known to support the growth of ‘G bacteria’(Cech &Hartman 1990,1993),and a mixture of acetate and glucose were used.Due to the high sampling volume,the dry matter (DM)content decreased during the experiment from 7.5to 4.4g l )1.In Figure 2the time course of phosphate concentration during 17cycles with alternating aerobic–anaerobic conditions is com-pared for the three di?erent carbon sources.During the aerobic phase phosphate has been taken up completely in all experiments whereas the phosphate release di?ered after three cycles.After 11cycles the release remained nearly constant for all substrates.The highest release was observed with acetate as substrate (23–27mg P l )1)and the smallest with glucose (13–19mg P l )1).The polyphosphate dynamics also showed with increasing time small but signi?cant di?erences (Figure 3).The highest poly-P-uptake (8mg P g )1DM)during the aerobic phase was found with acetate,just as for phosphate.After cycle 13,the total P-concentration of the pellet was also determined (Figure 3).The total P-content with glucose as carbon source was 8.5%at the end of the aerobic and 7.3%at the end of the anaerobic phase,similar to the results reported by Carucci et al .(1995,1997).The total P-content with acetate was not higher,in contrast to other results (Wentzel et al .1991).

In accordance with the poly-P degradation,the poly-P portion of the total P-content decreased during the anaerobic phase.The highest poly-P percentage of the total P-content was observed with acetate as carbon source (90%)followed by the substrate mixture.Poly-P contributed only 40%to the total P-content for the experiment with glucose.

Kinetics of the carbon source uptake and phosphate release

During the anaerobic phase of cycle 17sampling was performed every 10min after substrate addition and the glucose,acetate,phosphate and nitrate concentrations of the supernatant were determined (Figure 4).Acetate degradation was slow and incomplete after 190min whereas glucose was consumed completely within 30min.Phosphate concentration increased indepen-dently of the carbon source during the whole anaerobic phase.The release rate was higher during the ?rst 40min than during the following 160min.The phos-phate release is not correlated directly to the consump-tion of glucose,indicating that glucose is also used for other physiological processes like fermentation.Nitrate,which is known to interfere with the phosphate release (Kuba et al .1994),was formed from ammonium of the medium by nitri?cation.During the aerobic phase of cycle 17 4.1mg N-nitrate l )1was formed in the experiment with acetate and 2.6mg N-nitrate l )1in the experiments with the other carbon sources.The nitrate was consumed nearly completely during the ?rst 40min (experiment with acetate)and 30min (experi-ments with the other carbon sources)of the anaerobic phase,indicating denitri?cation.The results show that phosphate release was not signi?cantly inhibited by nitrate but obviously the e?ciency was reduced.Carbon storage

The highest PHA storage during the anaerobic phase was formed with acetate as carbon source (20mg C g )1DM)and the smallest with glucose (6mg C g )1DM)(Figure 5).This is in accordance with the

phosphate

Figure 2.Phosphate concentration in the medium under alternating anaerobic and aerobic conditions using di?erent carbon sources (19.5h anaerobic phase,4h aerobic phase and 0.5h settling period,

respectively).

Figure 3.Poly-P storage under alternating anaerobic and aerobic conditions using di?erent carbon sources.

Carbon sources and biological phosphorus removal 357

release because the energy for PHA formation is produced by poly-P hydrolysis(Figure2).According to the metabolic model of the EBPR,in the aerobic phase PHA is used as energy source for the phosphate uptake.Peterson et al.(1998)found that the increase of PHA in the biomass increases the phosphate uptake and subsequent poly-P formation.This explains the increas-ing phosphate uptake and poly-P storage observed in the sequence glucose,acetate/glucose,acetate corre-sponding to the PHA storage level.

The polyhydroxyvalerate(PHV)concentration as well as its percentage in the PHA increased with time in all experiments.As expected,the highest percentage was observed with glucose as carbon source.The PHV contributed up to88%to the PHA with glucose.This indicates that a major part of the glucose is converted by the succinate–propionate pathway to propionyl-CoA that together with acetyl-CoA produce3-hydroxyvale-ryl-CoA,the monomer of PHV.A maximum of17% PHV was found with acetate(Table1).This corre-sponds to the report from Satoh et al.(1994,1996)that after an anaerobic phase longer than2h with acetate as sole carbon source the3-HV portion of the PHA can increase up to40%.According to the metabolic model, glycogen supplies the carbon for3-HV formation(Mino et al.1998).The formation of PHAs containing the monomer3-hydroxy-2-methylbutyrate from acetyl-CoA and propionyl-CoA during the anaerobic phase and their degradation during the aerobic phase was estab-lished qualitatively for all carbon sources using the synthesized ethyl ester as standard for the GC-FID-analysis.3-Hydroxy-2-methylbutyrate formation was obviously also caused by the length of the anaerobic incubation time(Satoh et al.1994,1996).

Glycogen was degraded under anaerobic conditions and accumulated under aerobic conditions as shown in Figure5.The highest glycogen concentration (17mg C l)1)and the highest di?erence between the anaerobic and aerobic phase(12mg C l)1)was reached with acetate followed by the substrate mixture.In contrast,the experiment with glucose showed only a low level of glycogen storage,which decreased in the course of time.This contradicts the results of other investigations,which postulated the storage of

glycogen Figure4.Kinetic of the carbon source decrease and phosphate increase in the cell-free medium during the anaerobic phase of the cycle17using di?erent carbon

sources.

Figure5.PHA and glycogen storage under alternating anaerobic and aerobic conditions using di?erent carbon sources.

Table1.Ratios of carbon uptake to phosphorus release in the anaerobic phase of the cycle17.

Substrate uptake

(mol C)

Phosphate release

(mol P)

PHA accumulated

(mol C)

Ratio of3-HB

to3-HV

Glycogen

consumed(mol C)

Carbon recovery ratio

(PHA accumulated/glyco-

gen consumed+substrate

taken up)

1(acetate)0.15 1.5683:170.780.74

0.59(acetate)t0.41(glucose)0.12 1.3759:410.380.75

1(glucose)0.090.4112:880.060.22

358J.Hollender et al.

instead of poly-P with glucose as substrate combined with the deterioration of the EBPR(Cech et al.1993, Satoh et al.1992,1996).

Stoichiometry of C-and P-sources

The phosphorus release per mol substrate C-uptake was determined for cycle17(Table1).The values were signi?cantly smaller than reported by Mino et al.(1998), which can be explained by the denitrifying process taking place at the beginning of the anaerobic phase.A part of the carbon source was used for nitrate reduction.In addition, the carbon recovery ratio was calculated.This ratio determines which part of the carbon of the substrate taken up and the glycogen consumed during the anaerobic phase is transformed to PHA.Thereby the recovery ratio indicates whether the process corresponds to the meta-bolic model of EBPR.The recovery ratios of carbon from acetate and the acetate/glucose mixture(74–75%)are similar to the results of Satoh et al.(1996)determined for an EBPR.In contrast,the recovery ratio for glucose is only22%indicating a dominance of other processes. Again a part of the substrate is used for the denitrifying process and not for the formation of PHA.Additionally, glucose is obviously fermented during the anaerobic phase to metabolites which we did not analyse.Recently some studies detected acetate,propionate and lactate as fer-mentation product(Carucci et al.1999;Sudiana et al. 1999;Jeon&Park2000).Lactate fermentation is a quite general metabolism for facultative fermentative hetero-trophs under anaerobic conditions and probably occurred also in the experiment with glucose.

In conclusion,the e?ciency of the biological phos-phorus elimination is best with acetate as carbon source because the highest phosphate release per mol carbon, the highest PHA,poly-P and glycogen storage during the anaerobic and aerobic phases as well as a high carbon recovery ratio were observed with acetate.Due to the strong in?uence of the carbon source on PHA concentration and composition,the PHA storage main-ly seems to regulate phosphate release and uptake. Acknowledgements

This work was supported by grant DO237/11-1/2from the Deutsche Forschungsgemeinschaft.

We would like to thank U.Kaufmann,E.Ko rner,J. Meirick,M.Mo ller,and R.Zimmermann for their technical assistance and U.Dreyer for helpful discussion. References

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