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Anaerobic uptake of glutamate and aspartate by enhanced biological phosphorus removal activated sludge
l@)
Pergamon
Waf. Sci. T~ch. Vol. 37. No. 4-5. pp. 579-582.1998. @
1998 IAWQ. Published by Elsevier Science Ud
PH: S0273-1223(98)00163-2
Printed in Great Britain.
0273-1223/98 S19'00 + (}oo
ANAEROBIC UPTAKE OF GLUTAMATE AND ASPARTATE BY ENHANCED BIOLOGICAL PHOSPHORUS REMOVAL ACTIVATED SLUDGE Hiroyasu Satoh, Takashi Mino and Tomonori Matsuo Department of Urban and Environmental Engineering, University of Tokyo, 7-3-/ Hongo, Bunkyo-ku. Tokyo //3. Japan
ABSTRACT Mechanisms of the anaerobic uptake of glutamate and aspartate by enhanced biological phosphorus removal activated sludge were investigated. Sludge hydrolysate with hydrochloric acid was analyzed by reverse phase HPLC after pre column derivatization with dabsyl-chloride. The experimental results indicated that glutamate is accumulated as a polymer consisting of y-aminobutyric acid and an unknown amino acid. On the other hand, aspartate was found to be deaminated and then accumulated as PHA. (C) 1998 IAWQ. Published by Elsevier Science Ud
KEYWORDS Enhanced biological phosphorus removal; anaerobic substrate uptake; glutamate; aspartate; metabolism; polyhydroxyalkanoates; dabsyl-CI. INTRODUCTION Microorganisms in enhanced biological phosphorus removal (EBPR) activated sludge take up organic materials under anaerobic conditions while consuming polyphosphate as the energy source. When volatile fatty acids are taken up by EBPR activated sludge under anaerobic conditions, the sink of carbon is polyhydroxyalkanoate (PHA) (Satoh et ai., 1992). Although volatile fatty acids are believed to be the main carbon source for the growth of microorganisms that are responsible for EBPR in full-scale EBPR plants treating domestic sewage, other kinds of short chain organic compounds are taken up by EBPR activated sludge. Although several kinds of amino acids are taken up by EBPR activated sludge, the biochemical mechanisms of the anaerobic uptake and the storage products are not yet clear (Arun et ai., 1989). In the present study. we investigated the anaerobic uptake mechanisms of two kinds of amino acids, glutamate and aspartate. MATERIALS AND METHODS Activated sludge was acclimatized in a continuous EBPR activated sludge process fed with synthetic wastewater containing glutamate, aspartate, peptone, and yeast extract as the carbon sources. After an acclimatization period of several months, activated sludge was sampled from the end of the aerobic zone. 579
580
H SATOH elat.
Batch anaerobic expenments were conducted with the sampled sludge by adding glutamate or aspanate. The fate of the carbon sources taken up was identified by monitoring the behaviour of PHA and polypeptide compounds in sludge. free amino aCids stored in sludge. and ammonia released into the bulk. PHA was analyzed by gas chromatography after methanolytic decomposition of the whole sludge (Satoh et al.• 1996). Free ammo aCids m sludge were extracted With 0.5 N perchlonc aCid (PCA) at 5°C, and for polypeptide compounds the activated sludge was hydrolyzed wllh 6N hydrochlonc aCid. Free amino aCids m supernatant and m the PCA extract, and amino acids m the hydrolysate of sludge were analyzed by reverse phase high performance liquid chromatography (RP HPLC) followed by precolumn derivatization wllh dabsyl-CI after hydrolysis (Knecht and Chang. 1986). Ammona-nllrogen was analyzed by the phenate method. Pr('('ollllll11 tll'ri\'(/tl~(/ti01I with clahw/-Cl. Dabsyl-C1 (4-dlmethylammoazobenzene-4'-sulfonyl chlonde) was obtamed from Pierce Co. An aqueous sample of 150 ~I was put m a vial, to which 100 !tl 125 mM bicarbonate buffer (pH 9) and 500!t1 acetomtnle solution of dabsyl-CI (1.4 mg/ml) was added. The Vial was closed with a teflon hned screw cap. and heated at 70°C for 20 mm. After cooling to room temperature, acetonllrile was added for dilution. If necessary. The same procedure was used for the analySIS of amino aCld~ m ,Iudge hydrolysate. and m the PCA extract of sludgl' after neutralilation and removal of PCA with pOlas'iUm carbonate
HPLC. The HPLC analy,e, of dab'ylated ammo aCid, were conducted on a Waters Module I HPLC system with a Nova-Pal.. C I H column (I.d. 3.9 nllTl. length 150 mm) and a UV -Vis detector at 436 nm. Eluents of A:
25 mM ,odium acetate (pH 65) and B acetonitrile were used for the separation. Linear gradient wa, applied with 'fi B as follow,: I Wi,. at 0 mm. I<)'k at I mm, 23o/c, at 1.1 mm. 38% at 9 mm. 43% at 9.1 mm. 65% at 16 mm. and HO% at 22 nun.
:::....
250
1"
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200
(;
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~ 100
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• Catt>ohydr'OlOS
PtlA TOC WI PCA rrxtoon Re J~ A.mmOnIa R
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o
h~ure
-[}
200
-
~100
~
~ 6
~IUlal1lale
I
PtlA TOCWI PCAr,aetoon
R......., Amrno,,,o
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~ ~
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-"
.........~.....................J 4 5 6 7
O::.........,.:::.......L.~-_
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I Anae,,,oll upl,lke "I
50
Catt>ohydr'o'os
R"':P!lo'llhO'·o -;:--r •
I
150
~
2 3 4 T,me (hour)
250
t r <
IS.ct Phosphate
-:::::-
-1
r:--_.
---DOC
DOC
C-
0
2
3
T,me (hour)
IIdll amI a'parl,lIe (rogol)
RESllLTS AND DISCl'SSION Glutamate Figure I
Anaerobic uptake of glutamate and
581
a~panate
Figure 2 shows chromatograms of the hydrolysate of activated sludge right before the addition of glutamate and after 6 hr of incubation. As can be seen, two peaks, peak I and peak II were found to increase. The retention time of peak I wa~ the same as that of dabsyl y-aminobutyrate, strongly indicating the formation of y-aminobutyrate as the result of glutamate uptake.
6 hours after the I u~!!'l2~te
_~~-1!.9"_of
peak"
t
I ~
lJl~ 0.0
5.0
i
30.0
Figure 2. Ammo acid analysIs of the hydrolysate of activated sludge hefore and aller the uptake of glutamate.
The experimental result~ shown in Figure 2 strongly indicate that some part of the glutamate is accumulated as y-aminobutyrate. This is reasonable since conversIOn of glutamate into y-aminobutyrate is a single step decarboxylation. Another component appearing as peak II should have one or more amino groups, because the amino group marked with the dabsyl group is detected using the employed analytical method. The amino acid component for peak II needs to be identified. Alipartate Figure I (right) shows an example of the batch experiments with aspartate as the ~ubstrate. Withm the 6 hr batch experiment. 140 mgC/1 of DOC was utilized, and 30 mgC/1 of carbohydrates wa~ consumed, where 130 mgC/1 of PHA was accumulated and a 10 mgC/1 increase of TOC was found in the PCA fraction. Most of the DOC and the increa~ed TOC in the PCA fraction was aspartate. Although carbon balance was not followed perfectly, it is reasonable to assume that PHA is the major fate of the carbon in aspartate. The reduced amount of DOC within the 6 hr batch experiment was equivalent to about 40 mgNlI, which balanced well with the increase of ammoma. Thi~ observation indicates that aspartate is metabolized through deamination. The results indicated that aspartate is metabolized through deamination. The composition of the accumulated PHA was 14% 3HB(3-hydroxybutyrate), 13% 3H2MB (3-hydroxy-2methylbutyrate), 48% 3HV(3• hydroxyvalerate), and 25% 3H2MV(3-hydroxy-2-methylvaleratel. Since two m:>lecules of acetyl-CoA yields a 3HB unit, one molecule of acetyl-CoA and propionyl-CoA yields a 3HV unit or a 3H2MB unit, and two molecules of propionyl-CoA yields 3H2MV, 45% acetyl-CoA and 55% propionyl-CoA were converted into PHA in this case. Aspartate is converted to fumarate through deamination (Gottschalk, 1985). If the conver~ion of aspartate into PHA is by the metabolic pathway shown in Fig. 3, the ratio of acetyl-CoA to proptonyl-CoA ~hould be 1.0, which is in fairly good agreement with the observed values.
582
H. SATOH et al.
1 oxaloacetate - . 1 phosphoenolpyruvate-. 1 pyruvate
t
+2(H) 1 malate 2 aspartate
~
+
2 fumarate " -2(H)
++2(H) 1 acetyl-CoA
f4
PHA
1 succinate ~ 1 succlnyl-CoA - - . 1 propionyl-CoA Figure 3. Metabolic model to explain the conversion of aspartate into PHA.
CONCLUSIONS Anaerobic uptake mechanisms of glutamate and aspartate by EBPR activated sludge was investigated. Glutamate wa~ accumulated as a polymer containing y-aminobutyrate and an unknown amino acid. The unknown component still has to be identified. Aspartate was accumulated as PHA, and a metabolic model shown in Fig. 3 was postulated. REFERENCES Arun, V., Mino, T. and Matsuo. T. (1989). Metabolic pathway of anaerobic uptake of amino acids in the enhanced biological phosphorus removal process. In: Advances in Water Pollution Control. Water Pollution Control in Asia. T Panswad et al. (eds), IAWPRC, 313-319. Gottschalk, G. (1985). Bacterial Metabolism. 2nd edn., Springer-Verlag, New York. Knecht, R., Chang, J. Y. (1986). Liquid chromatographic determination of amino acids after gas-phase hydrolysis and derivatization with (dimethylamino)azobenzenesulfonyl chloride. Anal. Chern., 58, 2375-2379. Satoh, H., Mino, T. and Matsuo, T. (1992). Uptake of Organic Substrates and Accumulation of Polyhydroxyalkanoates Linked with Glycolysis of Intracellular Carbohydrates Under Anaerobic Conditions in the Biological Excess Phosphate Removal Processes. Wat. Sci. Tech., 26(5-6), 933-942. Satoh, H.. Ramey, W. D.. Koch, F. A., Oldham, W. K., Mino, T. and Matsuo, T. (1996). Anaerobic Substrate Uptake by the Enhanced Biological Phosphorus Removal Activated Sludge Treating Real Sewage. Wat. Sci. Tech., 34(1-2), 9-16.
Pergamon
Waf. Sci. T~ch. Vol. 37. No. 4-5. pp. 579-582.1998. @
1998 IAWQ. Published by Elsevier Science Ud
PH: S0273-1223(98)00163-2
Printed in Great Britain.
0273-1223/98 S19'00 + (}oo
ANAEROBIC UPTAKE OF GLUTAMATE AND ASPARTATE BY ENHANCED BIOLOGICAL PHOSPHORUS REMOVAL ACTIVATED SLUDGE Hiroyasu Satoh, Takashi Mino and Tomonori Matsuo Department of Urban and Environmental Engineering, University of Tokyo, 7-3-/ Hongo, Bunkyo-ku. Tokyo //3. Japan
ABSTRACT Mechanisms of the anaerobic uptake of glutamate and aspartate by enhanced biological phosphorus removal activated sludge were investigated. Sludge hydrolysate with hydrochloric acid was analyzed by reverse phase HPLC after pre column derivatization with dabsyl-chloride. The experimental results indicated that glutamate is accumulated as a polymer consisting of y-aminobutyric acid and an unknown amino acid. On the other hand, aspartate was found to be deaminated and then accumulated as PHA. (C) 1998 IAWQ. Published by Elsevier Science Ud
KEYWORDS Enhanced biological phosphorus removal; anaerobic substrate uptake; glutamate; aspartate; metabolism; polyhydroxyalkanoates; dabsyl-CI. INTRODUCTION Microorganisms in enhanced biological phosphorus removal (EBPR) activated sludge take up organic materials under anaerobic conditions while consuming polyphosphate as the energy source. When volatile fatty acids are taken up by EBPR activated sludge under anaerobic conditions, the sink of carbon is polyhydroxyalkanoate (PHA) (Satoh et ai., 1992). Although volatile fatty acids are believed to be the main carbon source for the growth of microorganisms that are responsible for EBPR in full-scale EBPR plants treating domestic sewage, other kinds of short chain organic compounds are taken up by EBPR activated sludge. Although several kinds of amino acids are taken up by EBPR activated sludge, the biochemical mechanisms of the anaerobic uptake and the storage products are not yet clear (Arun et ai., 1989). In the present study. we investigated the anaerobic uptake mechanisms of two kinds of amino acids, glutamate and aspartate. MATERIALS AND METHODS Activated sludge was acclimatized in a continuous EBPR activated sludge process fed with synthetic wastewater containing glutamate, aspartate, peptone, and yeast extract as the carbon sources. After an acclimatization period of several months, activated sludge was sampled from the end of the aerobic zone. 579
580
H SATOH elat.
Batch anaerobic expenments were conducted with the sampled sludge by adding glutamate or aspanate. The fate of the carbon sources taken up was identified by monitoring the behaviour of PHA and polypeptide compounds in sludge. free amino aCids stored in sludge. and ammonia released into the bulk. PHA was analyzed by gas chromatography after methanolytic decomposition of the whole sludge (Satoh et al.• 1996). Free ammo aCids m sludge were extracted With 0.5 N perchlonc aCid (PCA) at 5°C, and for polypeptide compounds the activated sludge was hydrolyzed wllh 6N hydrochlonc aCid. Free amino aCids m supernatant and m the PCA extract, and amino acids m the hydrolysate of sludge were analyzed by reverse phase high performance liquid chromatography (RP HPLC) followed by precolumn derivatization wllh dabsyl-CI after hydrolysis (Knecht and Chang. 1986). Ammona-nllrogen was analyzed by the phenate method. Pr('('ollllll11 tll'ri\'(/tl~(/ti01I with clahw/-Cl. Dabsyl-C1 (4-dlmethylammoazobenzene-4'-sulfonyl chlonde) was obtamed from Pierce Co. An aqueous sample of 150 ~I was put m a vial, to which 100 !tl 125 mM bicarbonate buffer (pH 9) and 500!t1 acetomtnle solution of dabsyl-CI (1.4 mg/ml) was added. The Vial was closed with a teflon hned screw cap. and heated at 70°C for 20 mm. After cooling to room temperature, acetonllrile was added for dilution. If necessary. The same procedure was used for the analySIS of amino aCld~ m ,Iudge hydrolysate. and m the PCA extract of sludgl' after neutralilation and removal of PCA with pOlas'iUm carbonate
HPLC. The HPLC analy,e, of dab'ylated ammo aCid, were conducted on a Waters Module I HPLC system with a Nova-Pal.. C I H column (I.d. 3.9 nllTl. length 150 mm) and a UV -Vis detector at 436 nm. Eluents of A:
25 mM ,odium acetate (pH 65) and B acetonitrile were used for the separation. Linear gradient wa, applied with 'fi B as follow,: I Wi,. at 0 mm. I<)'k at I mm, 23o/c, at 1.1 mm. 38% at 9 mm. 43% at 9.1 mm. 65% at 16 mm. and HO% at 22 nun.
:::....
250
1"
~
200
(;
150
<:: z
---0. -
~ 100
<::
'5, .§.
1 __ x
50 0
• Catt>ohydr'OlOS
PtlA TOC WI PCA rrxtoon Re J~ A.mmOnIa R
:::-
- -- -..
o
h~ure
-[}
200
-
~100
~
~ 6
~IUlal1lale
I
PtlA TOCWI PCAr,aetoon
R......., Amrno,,,o
r ~~.- --~. E
~ ~
1
-"
.........~.....................J 4 5 6 7
O::.........,.:::.......L.~-_
-1
7
I Anae,,,oll upl,lke "I
50
Catt>ohydr'o'os
R"':P!lo'llhO'·o -;:--r •
I
150
~
2 3 4 T,me (hour)
250
t r <
IS.ct Phosphate
-:::::-
-1
r:--_.
---DOC
DOC
C-
0
2
3
T,me (hour)
IIdll amI a'parl,lIe (rogol)
RESllLTS AND DISCl'SSION Glutamate Figure I
Anaerobic uptake of glutamate and
581
a~panate
Figure 2 shows chromatograms of the hydrolysate of activated sludge right before the addition of glutamate and after 6 hr of incubation. As can be seen, two peaks, peak I and peak II were found to increase. The retention time of peak I wa~ the same as that of dabsyl y-aminobutyrate, strongly indicating the formation of y-aminobutyrate as the result of glutamate uptake.
6 hours after the I u~!!'l2~te
_~~-1!.9"_of
peak"
t
I ~
lJl~ 0.0
5.0
i
30.0
Figure 2. Ammo acid analysIs of the hydrolysate of activated sludge hefore and aller the uptake of glutamate.
The experimental result~ shown in Figure 2 strongly indicate that some part of the glutamate is accumulated as y-aminobutyrate. This is reasonable since conversIOn of glutamate into y-aminobutyrate is a single step decarboxylation. Another component appearing as peak II should have one or more amino groups, because the amino group marked with the dabsyl group is detected using the employed analytical method. The amino acid component for peak II needs to be identified. Alipartate Figure I (right) shows an example of the batch experiments with aspartate as the ~ubstrate. Withm the 6 hr batch experiment. 140 mgC/1 of DOC was utilized, and 30 mgC/1 of carbohydrates wa~ consumed, where 130 mgC/1 of PHA was accumulated and a 10 mgC/1 increase of TOC was found in the PCA fraction. Most of the DOC and the increa~ed TOC in the PCA fraction was aspartate. Although carbon balance was not followed perfectly, it is reasonable to assume that PHA is the major fate of the carbon in aspartate. The reduced amount of DOC within the 6 hr batch experiment was equivalent to about 40 mgNlI, which balanced well with the increase of ammoma. Thi~ observation indicates that aspartate is metabolized through deamination. The results indicated that aspartate is metabolized through deamination. The composition of the accumulated PHA was 14% 3HB(3-hydroxybutyrate), 13% 3H2MB (3-hydroxy-2methylbutyrate), 48% 3HV(3• hydroxyvalerate), and 25% 3H2MV(3-hydroxy-2-methylvaleratel. Since two m:>lecules of acetyl-CoA yields a 3HB unit, one molecule of acetyl-CoA and propionyl-CoA yields a 3HV unit or a 3H2MB unit, and two molecules of propionyl-CoA yields 3H2MV, 45% acetyl-CoA and 55% propionyl-CoA were converted into PHA in this case. Aspartate is converted to fumarate through deamination (Gottschalk, 1985). If the conver~ion of aspartate into PHA is by the metabolic pathway shown in Fig. 3, the ratio of acetyl-CoA to proptonyl-CoA ~hould be 1.0, which is in fairly good agreement with the observed values.
582
H. SATOH et al.
1 oxaloacetate - . 1 phosphoenolpyruvate-. 1 pyruvate
t
+2(H) 1 malate 2 aspartate
~
+
2 fumarate " -2(H)
++2(H) 1 acetyl-CoA
f4
PHA
1 succinate ~ 1 succlnyl-CoA - - . 1 propionyl-CoA Figure 3. Metabolic model to explain the conversion of aspartate into PHA.
CONCLUSIONS Anaerobic uptake mechanisms of glutamate and aspartate by EBPR activated sludge was investigated. Glutamate wa~ accumulated as a polymer containing y-aminobutyrate and an unknown amino acid. The unknown component still has to be identified. Aspartate was accumulated as PHA, and a metabolic model shown in Fig. 3 was postulated. REFERENCES Arun, V., Mino, T. and Matsuo. T. (1989). Metabolic pathway of anaerobic uptake of amino acids in the enhanced biological phosphorus removal process. In: Advances in Water Pollution Control. Water Pollution Control in Asia. T Panswad et al. (eds), IAWPRC, 313-319. Gottschalk, G. (1985). Bacterial Metabolism. 2nd edn., Springer-Verlag, New York. Knecht, R., Chang, J. Y. (1986). Liquid chromatographic determination of amino acids after gas-phase hydrolysis and derivatization with (dimethylamino)azobenzenesulfonyl chloride. Anal. Chern., 58, 2375-2379. Satoh, H., Mino, T. and Matsuo, T. (1992). Uptake of Organic Substrates and Accumulation of Polyhydroxyalkanoates Linked with Glycolysis of Intracellular Carbohydrates Under Anaerobic Conditions in the Biological Excess Phosphate Removal Processes. Wat. Sci. Tech., 26(5-6), 933-942. Satoh, H.. Ramey, W. D.. Koch, F. A., Oldham, W. K., Mino, T. and Matsuo, T. (1996). Anaerobic Substrate Uptake by the Enhanced Biological Phosphorus Removal Activated Sludge Treating Real Sewage. Wat. Sci. Tech., 34(1-2), 9-16.