Folia Microbiol.36 (2), 136-140 (1991)
Determination of Nitrate by Conversion to Nitrite Using Paracoccus denitrificans I. MATCHOVA,I. C~RNA and I. KU~ERA Department of Biochemistry,Facultyof Science, Masaryk University,611 37 Bmo, Czechoslovakia ReceivedJanuary31, 1990 ABSTRACT. A new methodof determinationof nitrate was developed,utilizingthe nitrate reductase activityof Paracoccus denitrificans in whicha furtherreductionof nitrate is blockedeitherby a mutationaffectingformationof cytochromesc or by inhibitionof the electronflowto nitrite reductaseby mucidin.Afterdeproteinizationof the samplewithzincacetate the nitrite produced is determinedcolorimetrically.
With the aid of enzymes it is possible to determine some of inorganic compounds, eg. nitrate which is selectively reduced by membrane-bound nitrate reductase (EC 1.9.6.1) of Escherichia coil The nitrite produced can be determined colorimetrically in a diazo-coupling reaction yielding an azo-dye (Egami and Taniguchi 1970; Me Namara et al. 1971; Schild and Klemme 1985). In the present work we aimed at replacing nitrate reductase ofE. coli, which is difficult to isolate and unstable, with intact cells of the denitrifying bacterium Paracoccus denitrificans. Anaerobically grown cells of P. denitrificans are characterized by a high activity of nitrate reductase yet contain the dissimilatory nitrite reductase (EC 1.7.2.1) catalyzing a further reduction of nitrite. As c type cytochromes are involved in the interfering nitrite reductase reaction, as compared with the nitrate reduction reaction (Lain and Nicholas 1969), the former reaction can be inhibited by mucidin which blocks the electron flow through the bcl segment of the respiratory chain of P. denitrificans (Ku~era et al. 1984). It is also possible to use a cytochrome-c-deficient mutant strain.
MATERIAL AND METHODS
Microorganism. Wild strain of Paracoccus denitrificans NCIB 8944 was obtained from the Czechoslovak Collection of Microorganisms (Brno) as CCM 982. Cells were cultivated anaerobically and statically for 22 h at 30 *C in 1-L flasks containing a medium (Ku~era et al. 1983) in which glucose was replaced with sodium succinate (50 mmol/L). Cytochrome-c-deficient P. denitdficans HUUG25 (S1659) was obtained from Dr. H.W. van Verseveld of the Vrije Universiteit, Amsterdam. The mutant strain was cultivated aerobically in a nitrate-free medium for 12 h to the late exponential phase of growth. The grown cells were harvested by centrifugation (5 600 g, 20 min). Nitrate reductase in mutant HUUG25 cells was induced by a further 3-h incubation in the presence of nitrate (medium for anaerobic cultivation 200 mL, 250-mL Erleumeyer flasks, 30 ~ initial cell concentration was 1 mg dry mass per mL. Reduction of nitrate to nitrite proceeded at 30 ~ in closed test tubes containing 2 mL of the reaction mixture consisting of sodium phosphate buffer (0.1 mol/L), sodium succinate (40 mmol/L), bacterial cells and a nitrate sample. (When measuring nitrate reductase activity of cells the initial concentration of NO3- was 10 mmol/L and incubation time was 20 rain.) The reaction was stopped with 1 mL saturated zinc acetate, the precipitate was removed by centrifugation (14 000 g, 4 min) and the content of nitrite was determined photometrically in the supernatant. The nitrite concentration was determined by a modified diazo-coupling photometric method (Snell and Snell 1949) in a mixture containing 0.5 mL of 1 % sulfanilic acid and 0.5 mL of 0.02 % 1-naphthylamine (both solutions in HCI 0.1 tool/L) made up with water to a total volume of 5 mL. After'30 min at room temperature absorbance was measured at 524 nm using water as blank.
1991
DE'I~RMINATION OF NITRATE
137
The dry mass of cells in reserve suspension (0.1 tool/L, sodium phosphate, pH 7.3) was determined in 1-mL samples after a 3-h heating at 105 ~ The cell suspension was freezedried in a lyophiliTation apparatus (Virtis USA).
RESULTS A N D DISCUSSION Nitrate reductase and nitrite reductase activities of the mutant HUUG25
Aerobically grown cells of the mutant exhibit a low nitrate reductase activity. Under conditions of decreased aeration in a growth medium with nitrate (10 retool/L) the activity increased by two orders of magnitude in 3 h. At the same time, nitrate was reduced to nitrite (Fig. 1). In mutant ceils no nitrite reductase activity could be detected, either before or during adaptation. In this way, these cells significantly differ from wild-strain cells grown aerobically in which a sequential formation of nitrate reductase and nitrite reductase was observed after their adaptation to anaerobic conditions with only a temporarily increased nitrite concentration in the adaptation medium (Ku~era et al. 1986). The inability of mutant HUUG25 to perform dissimilatory nitrite reduction is probably associated with an impaired production of soluble periplasmic cytochromes c550 and cdl (Page and Ferguson 1989). This fact makes it possible to use adapted mutant cells for a two-electron reduction of NO3- to NO2-.
I
I
I
"
1.0
10
mm01/L
nkot~mg
--18
0.8
0.6
.
J'l
0.4
0.2
0
1
w
|
2
3
h
O 4
Fig. 1. Specific activity (nkat/mg) of nitrate rcductase (A1), nitrite reductase (A2) and nitrite concentration in the medium (NO2-, mmol/L) during anaerobic adaptation of aerobically grown cells of the mutant HUUG25.
1 341
I. MA~CHOV.~ et al.
Vol. 36
I
I
I
10
Conversion o f N O 3 - to NO2- and its dependence on p H
lamol 9
0
Quantitative conversion of NO3- to NO2- is essential for the use of nitrate reductase activity for the analytical determination. The time course of production of NO2from NO3- with an initial concentration of 5 m m o l / L is shown in Fig 2. The maximum degree of conversion is close to 100 %. A similar time relationship of NO3- reduction corresponding to a comparable specific activity was also found in anaerobically grown cells of the wild strain of P. denitn'ficans on the condition that a further reduction of NO2- was prevented by addition of mucidin (1 mg per g dry mass; data not shown). It follows from data shown in Fig. 3 that the rate of bacterial conversion of NO3- to
5
0
i 20
i ~0
rain
i 60
Fig. 2. Reductionof nitrate to nitrite Otmol)in the presence of 1.6 and 3.4 mg (numbers at curves) of cells of the mutant HUUG25 with induced nitrate reductase; initialamount of nitrate was 10/zmol(dashedl/m0. NO2- depends on pH, with a maximum at pH 7.3. Therefore, sodium phosphate buffer (pH 7.3) was used throughout.
Determination of nitrate
Fig. 4 is the calibration curve of determination of nitrate by the proposed method. As the absorbance values obtained were slightly lower than those when NO3- was replaced with an equivalent m o u n t of NO2added to denatured cells it can be assumed that on a long-term incubation of NO3- with an excess of cells certain losses of the produced nitrite may arise. However, they do not exceed several percent of the initial amount of NO3-. (In this way it can be explained why the prolonged part of the calibration curve does not pass through the origin.)
1.0
!
I
I
I 6
I ?
| 8
I
"
nkotlmg
0.9
0.8
0.7
0.6
pH
I 9
Fig. 3. Dependenceof specificactivityof nitrate reductase (nkat/mg) on pit (see Materialand Methods).
1991
DETERMINATION OF NITRATE
I
I
1.0-
A
0.5
0
9
9
-
I
i
5
lO
~tmot
Fig. 4. Calibration line for the determination of nitrate by enzyme reduction to nitrite (open symbols). The indicated amount of NO3- Ozmol) was incubated for 1 h with 6 mg of adapted cells of the mutant HUUG25 in 2 mL of reaction mixture. For determination of NO2- 50pL of the supernatant after deproteinization was taken; the determined absorbance values (A) are on the ordinate. In control experiments (closed symbols) zinc acetate was first added to the cells followed by nitrite.
139
To evaluate the sensitivity of the tested method a 1-mL sample containing nitrate at a maximum concentration allowable according to the valid Czechoslovak norm for drinking water (50 mg NO3per L) is considered. It follows from Fig. 4 that the resulting absorbance of 0.04 corresponds to the appropriate amount of NO3(0.8p, mol). Sensitivity can be further increased to about 20-30fold by taking volumes of the supernatant larger than 50/zL for the photometric determination. Sensitivity of NO3- determination reached in the model arrangement fulfils the requirements for analysis of real samples (e~g.water or extracts of vegetable). To express the accuracy of the method quantitatively we analyzed 10 identical samples containing 5 p,mol NO3-; the measured values served to calculate the variation coefficient - it was found to be 8 %.
Effect of lyophilization of the cells on their enzyme activity We also investigated the possible replacement of native bacterial cells with a lyophilized preparation. It could be shown that lyophili7ation of the cell suspension in sodium phosphate buffer (0.1 tool/L) is associated with a decreased specific activity of nitrate reductase by up to 80 %. Smaller losses of enzyme activity (by 50-60 %) were observed when phosphate was replaced with ammonium carbonate with an identical concentration. However, the lower specific activity of lyophilized cells can be compensated by using a higher amount of the preparation. Therefore, lyophilized cells of P. denitnficans could become a part of an economically undemanding set for the analytical determination of nitrate.
REFERENCES E~AMi F., TANIGUCHIS.: Nitrat, pp. 2179- 2184 in Methoden der Enzymatischen Analyse (H.U. Bergmeyer, Ed.), Vol. IlL Verlag Chemic, Weinheim 1970. KU(:ERA I., BOUBLfKOVAP., DADAKV.: The interaction of mucidin with anaerobically grown cells of Paracoccus denitrificans. Biochira.Biophys.Acta 767, 383-388 (1984). Kug:m~A I., DADAKV., DOnR'? R.: The distribution of redox equivalents in the anaerobic respiratory chain of Paracoccus denitrificans. EurJ.Biochem. 130, 359 - 364 (1983). KU~ERA I., MATYAgEKR., DvotLhKovA J., DADAKV.: Anaerobic adaptation of Paracoccus denitriftcans: Sequential formation of denitrification pathway and changes in activity of 5-aminolevulinate synthase and catalase. Curr.Microbiol. 13, 107-110 (1986).
140
I. MA~CHOV,A eta/.
Vol. 36
LAM Y., NICHOLAS DJ.D.: Aerobic and anaerobic respiration in Micrococcus denitriflcans. Biochim.Biophys.Acta 172, 450-461 (1969). Mc NAMARA A.L, MEEKER G.B., SHAW P.D., IIAGEMAN R.tt.: Use of a dissimilatory nitrate reductase from Escherichia coli and formate as a reductive system for nitrate assays. J.Agr.Food Chem. 19, 229-231 (1971). PAGE M.D., FERGUSO• SJ.: A bacterial c-type cytochrome can be translocated to the periplasm as an apt) form; the biosynthesis of cytochrome cdl (nitrite reductase) from Paracoccus denimficans. Mol.Microbiol. 3, 653-661 (1989). SCXILD J., KLEMME J.-H.: Enzymatic nitrate assay by a kinetic method employing Escher/ch/a coii nitrate rcductasr Z.Naturforsch. 40r 134-137 (1985). S~Et~ F.D., SNI~.J.C.T.: CoiorimetricMethods of Analysis, p.804. Van Nostrand, New York 1949.
Translated by J. Sp~ek
Determination of Nitrate by Conversion to Nitrite Using Paracoccus denitrificans I. MATCHOVA,I. C~RNA and I. KU~ERA Department of Biochemistry,Facultyof Science, Masaryk University,611 37 Bmo, Czechoslovakia ReceivedJanuary31, 1990 ABSTRACT. A new methodof determinationof nitrate was developed,utilizingthe nitrate reductase activityof Paracoccus denitrificans in whicha furtherreductionof nitrate is blockedeitherby a mutationaffectingformationof cytochromesc or by inhibitionof the electronflowto nitrite reductaseby mucidin.Afterdeproteinizationof the samplewithzincacetate the nitrite produced is determinedcolorimetrically.
With the aid of enzymes it is possible to determine some of inorganic compounds, eg. nitrate which is selectively reduced by membrane-bound nitrate reductase (EC 1.9.6.1) of Escherichia coil The nitrite produced can be determined colorimetrically in a diazo-coupling reaction yielding an azo-dye (Egami and Taniguchi 1970; Me Namara et al. 1971; Schild and Klemme 1985). In the present work we aimed at replacing nitrate reductase ofE. coli, which is difficult to isolate and unstable, with intact cells of the denitrifying bacterium Paracoccus denitrificans. Anaerobically grown cells of P. denitrificans are characterized by a high activity of nitrate reductase yet contain the dissimilatory nitrite reductase (EC 1.7.2.1) catalyzing a further reduction of nitrite. As c type cytochromes are involved in the interfering nitrite reductase reaction, as compared with the nitrate reduction reaction (Lain and Nicholas 1969), the former reaction can be inhibited by mucidin which blocks the electron flow through the bcl segment of the respiratory chain of P. denitrificans (Ku~era et al. 1984). It is also possible to use a cytochrome-c-deficient mutant strain.
MATERIAL AND METHODS
Microorganism. Wild strain of Paracoccus denitrificans NCIB 8944 was obtained from the Czechoslovak Collection of Microorganisms (Brno) as CCM 982. Cells were cultivated anaerobically and statically for 22 h at 30 *C in 1-L flasks containing a medium (Ku~era et al. 1983) in which glucose was replaced with sodium succinate (50 mmol/L). Cytochrome-c-deficient P. denitdficans HUUG25 (S1659) was obtained from Dr. H.W. van Verseveld of the Vrije Universiteit, Amsterdam. The mutant strain was cultivated aerobically in a nitrate-free medium for 12 h to the late exponential phase of growth. The grown cells were harvested by centrifugation (5 600 g, 20 min). Nitrate reductase in mutant HUUG25 cells was induced by a further 3-h incubation in the presence of nitrate (medium for anaerobic cultivation 200 mL, 250-mL Erleumeyer flasks, 30 ~ initial cell concentration was 1 mg dry mass per mL. Reduction of nitrate to nitrite proceeded at 30 ~ in closed test tubes containing 2 mL of the reaction mixture consisting of sodium phosphate buffer (0.1 mol/L), sodium succinate (40 mmol/L), bacterial cells and a nitrate sample. (When measuring nitrate reductase activity of cells the initial concentration of NO3- was 10 mmol/L and incubation time was 20 rain.) The reaction was stopped with 1 mL saturated zinc acetate, the precipitate was removed by centrifugation (14 000 g, 4 min) and the content of nitrite was determined photometrically in the supernatant. The nitrite concentration was determined by a modified diazo-coupling photometric method (Snell and Snell 1949) in a mixture containing 0.5 mL of 1 % sulfanilic acid and 0.5 mL of 0.02 % 1-naphthylamine (both solutions in HCI 0.1 tool/L) made up with water to a total volume of 5 mL. After'30 min at room temperature absorbance was measured at 524 nm using water as blank.
1991
DE'I~RMINATION OF NITRATE
137
The dry mass of cells in reserve suspension (0.1 tool/L, sodium phosphate, pH 7.3) was determined in 1-mL samples after a 3-h heating at 105 ~ The cell suspension was freezedried in a lyophiliTation apparatus (Virtis USA).
RESULTS A N D DISCUSSION Nitrate reductase and nitrite reductase activities of the mutant HUUG25
Aerobically grown cells of the mutant exhibit a low nitrate reductase activity. Under conditions of decreased aeration in a growth medium with nitrate (10 retool/L) the activity increased by two orders of magnitude in 3 h. At the same time, nitrate was reduced to nitrite (Fig. 1). In mutant ceils no nitrite reductase activity could be detected, either before or during adaptation. In this way, these cells significantly differ from wild-strain cells grown aerobically in which a sequential formation of nitrate reductase and nitrite reductase was observed after their adaptation to anaerobic conditions with only a temporarily increased nitrite concentration in the adaptation medium (Ku~era et al. 1986). The inability of mutant HUUG25 to perform dissimilatory nitrite reduction is probably associated with an impaired production of soluble periplasmic cytochromes c550 and cdl (Page and Ferguson 1989). This fact makes it possible to use adapted mutant cells for a two-electron reduction of NO3- to NO2-.
I
I
I
"
1.0
10
mm01/L
nkot~mg
--18
0.8
0.6
.
J'l
0.4
0.2
0
1
w
|
2
3
h
O 4
Fig. 1. Specific activity (nkat/mg) of nitrate rcductase (A1), nitrite reductase (A2) and nitrite concentration in the medium (NO2-, mmol/L) during anaerobic adaptation of aerobically grown cells of the mutant HUUG25.
1 341
I. MA~CHOV.~ et al.
Vol. 36
I
I
I
10
Conversion o f N O 3 - to NO2- and its dependence on p H
lamol 9
0
Quantitative conversion of NO3- to NO2- is essential for the use of nitrate reductase activity for the analytical determination. The time course of production of NO2from NO3- with an initial concentration of 5 m m o l / L is shown in Fig 2. The maximum degree of conversion is close to 100 %. A similar time relationship of NO3- reduction corresponding to a comparable specific activity was also found in anaerobically grown cells of the wild strain of P. denitn'ficans on the condition that a further reduction of NO2- was prevented by addition of mucidin (1 mg per g dry mass; data not shown). It follows from data shown in Fig. 3 that the rate of bacterial conversion of NO3- to
5
0
i 20
i ~0
rain
i 60
Fig. 2. Reductionof nitrate to nitrite Otmol)in the presence of 1.6 and 3.4 mg (numbers at curves) of cells of the mutant HUUG25 with induced nitrate reductase; initialamount of nitrate was 10/zmol(dashedl/m0. NO2- depends on pH, with a maximum at pH 7.3. Therefore, sodium phosphate buffer (pH 7.3) was used throughout.
Determination of nitrate
Fig. 4 is the calibration curve of determination of nitrate by the proposed method. As the absorbance values obtained were slightly lower than those when NO3- was replaced with an equivalent m o u n t of NO2added to denatured cells it can be assumed that on a long-term incubation of NO3- with an excess of cells certain losses of the produced nitrite may arise. However, they do not exceed several percent of the initial amount of NO3-. (In this way it can be explained why the prolonged part of the calibration curve does not pass through the origin.)
1.0
!
I
I
I 6
I ?
| 8
I
"
nkotlmg
0.9
0.8
0.7
0.6
pH
I 9
Fig. 3. Dependenceof specificactivityof nitrate reductase (nkat/mg) on pit (see Materialand Methods).
1991
DETERMINATION OF NITRATE
I
I
1.0-
A
0.5
0
9
9
-
I
i
5
lO
~tmot
Fig. 4. Calibration line for the determination of nitrate by enzyme reduction to nitrite (open symbols). The indicated amount of NO3- Ozmol) was incubated for 1 h with 6 mg of adapted cells of the mutant HUUG25 in 2 mL of reaction mixture. For determination of NO2- 50pL of the supernatant after deproteinization was taken; the determined absorbance values (A) are on the ordinate. In control experiments (closed symbols) zinc acetate was first added to the cells followed by nitrite.
139
To evaluate the sensitivity of the tested method a 1-mL sample containing nitrate at a maximum concentration allowable according to the valid Czechoslovak norm for drinking water (50 mg NO3per L) is considered. It follows from Fig. 4 that the resulting absorbance of 0.04 corresponds to the appropriate amount of NO3(0.8p, mol). Sensitivity can be further increased to about 20-30fold by taking volumes of the supernatant larger than 50/zL for the photometric determination. Sensitivity of NO3- determination reached in the model arrangement fulfils the requirements for analysis of real samples (e~g.water or extracts of vegetable). To express the accuracy of the method quantitatively we analyzed 10 identical samples containing 5 p,mol NO3-; the measured values served to calculate the variation coefficient - it was found to be 8 %.
Effect of lyophilization of the cells on their enzyme activity We also investigated the possible replacement of native bacterial cells with a lyophilized preparation. It could be shown that lyophili7ation of the cell suspension in sodium phosphate buffer (0.1 tool/L) is associated with a decreased specific activity of nitrate reductase by up to 80 %. Smaller losses of enzyme activity (by 50-60 %) were observed when phosphate was replaced with ammonium carbonate with an identical concentration. However, the lower specific activity of lyophilized cells can be compensated by using a higher amount of the preparation. Therefore, lyophilized cells of P. denitnficans could become a part of an economically undemanding set for the analytical determination of nitrate.
REFERENCES E~AMi F., TANIGUCHIS.: Nitrat, pp. 2179- 2184 in Methoden der Enzymatischen Analyse (H.U. Bergmeyer, Ed.), Vol. IlL Verlag Chemic, Weinheim 1970. KU(:ERA I., BOUBLfKOVAP., DADAKV.: The interaction of mucidin with anaerobically grown cells of Paracoccus denitrificans. Biochira.Biophys.Acta 767, 383-388 (1984). Kug:m~A I., DADAKV., DOnR'? R.: The distribution of redox equivalents in the anaerobic respiratory chain of Paracoccus denitrificans. EurJ.Biochem. 130, 359 - 364 (1983). KU~ERA I., MATYAgEKR., DvotLhKovA J., DADAKV.: Anaerobic adaptation of Paracoccus denitriftcans: Sequential formation of denitrification pathway and changes in activity of 5-aminolevulinate synthase and catalase. Curr.Microbiol. 13, 107-110 (1986).
140
I. MA~CHOV,A eta/.
Vol. 36
LAM Y., NICHOLAS DJ.D.: Aerobic and anaerobic respiration in Micrococcus denitriflcans. Biochim.Biophys.Acta 172, 450-461 (1969). Mc NAMARA A.L, MEEKER G.B., SHAW P.D., IIAGEMAN R.tt.: Use of a dissimilatory nitrate reductase from Escherichia coli and formate as a reductive system for nitrate assays. J.Agr.Food Chem. 19, 229-231 (1971). PAGE M.D., FERGUSO• SJ.: A bacterial c-type cytochrome can be translocated to the periplasm as an apt) form; the biosynthesis of cytochrome cdl (nitrite reductase) from Paracoccus denimficans. Mol.Microbiol. 3, 653-661 (1989). SCXILD J., KLEMME J.-H.: Enzymatic nitrate assay by a kinetic method employing Escher/ch/a coii nitrate rcductasr Z.Naturforsch. 40r 134-137 (1985). S~Et~ F.D., SNI~.J.C.T.: CoiorimetricMethods of Analysis, p.804. Van Nostrand, New York 1949.
Translated by J. Sp~ek
