215
15 Biogeochemical Indicators of Nutrient Enrichments in Wetlands …
1. Dehydrogenase activity (global metabolic activity
of the microbial community; Enzyme Commision
Number (EC) 1.1.1.1): using the substrate 2-p-iodophenyl-3-p-nitrophenyl 5-phenyl tetrazolium cloride
(INT) as electron acceptor it occurs the reduction of
tetrazolium salts and the appearance of formazan
(Skujins 1976; Garcia et al. 1993): add 0.4 mL of distilled water and 0.2 mL of INT (0.4 % mass/volume)
to 1 g of soil sample and incubate for 20 h in darkness
at 20 °C. Then add 10 mL of methanol and stir vigorously during 1 min, then filter. Controls are prepared
adding 0.6 mL of distilled water. Read spectrophotometrically the absorbance of the extract at 490 nm
against a calibration curve prepared with INTF (iodonitrophenyl formazan) at different concentrations.
The results are expressed as mg INTF g
−1
.
2. β-glucosidase activity (C-acquiring enzyme –degradation of cellulose; EC 3.2.2.21): using the artificial substrate p-nitrophenyl-β-D-glucopyranoside
(PNG) and measuring spectrophometrically the
released p-nitrophenol (Tabatabai 1994): add 2 mL
of 0.1 M maleate buffer (pH = 6.5) and 0.5 mL of
PNG (0.05 M) to 0.5 g of soil sample. Incubate and
shake in a water bath at 30 °C for 1.5 h. Chill at 2 °C
for 15 min. Then add 0.5 mL of 0.5 M calcium chloride and 2 mL of 0.5 M sodium hydroxide. Centrifuge for 15 min at 2,000 g. Dilute if appropiate and
spectrophotometrically read at 398 nm. Controls
must be prepared by adding the substrate just after
incubation and prior to cooling tubes. The standard
curve is performed with p-nitrophenol (PNP). The
results are expressed in µmol PNP g −1 h
−1
.
3. Phenol oxidase (EC 1.10.3.2) and peroxidase (EC
1.11.1.7) (C-acquiring enzyme–oxidation of lignin
an other poliphenol) activities: using L-3,4-dihydroxyphenylalanine (DOPA) as the substrate and
measuring spectrophotometrically at 450 nm. For
phenol oxidase, add 50 µL of 25 mM of DOPA to
soil samples. For peroxidase assays add 50 µL of
25 mM DOPA plus 10 µL of 0.3 % H 2 O 2 . Negative control samples for phenol oxidase must be
prepared containing 200 µL of acetate buffer and
50 µL of DOPA solution; blank samples must
contain 200 µL of sample suspension and 50 µL
of acetate buffer. For peroxidase, negative control and blank samples also must contain 10 µL
of H 2 O 2 . Microplates are incubated in the dark at
20 °C, approximately 4 h for peroxidase and 18 h
for phenol oxidase. Finally, activity is quantified
by measuring absorbance at 450 nm using a microplate spectrophotometer. The results are expressed
in nmol h
−1
g
−1
.
4. Protease activity (N-acquiring enzyme): using
sodium caseinate as substrate and measuring spectrophotometrically the released tyrosine, using the
Folin-Ciocalteu reagent (Ladd and Butler 1972):
add 2.5 mL of 50 mM Tris buffer solution (pH 8.1)
and 2.5 mL of sodium caseinate (2 %) to 0.5 g of
soil sample. Incubate and shake in a water bath at
50 °C for 2 h. Cool at 2 °C for 20 min to stop the
reaction. Add 2.5 mL of trichloroacetic acid (15 %)
and later centrifuge at 8,000 g for 10 min. Subsequently 2.5 mL of supernatant are taken and added
to tubes with 3.75 mL of an alkaline reagent solution, which are incubated for 15 min at ambient
temperature. After this time add 2.5 mL of FolinCiocalteu reagent (33 %) and finally centrifuge at
2,000 g for 10 min. Dilute if appropriate, and read
in the spectrophotometer at 700 nm. The results are
expressed in mg tyrosine g
−1
h
−1
.
5. Leucine aminopeptidase activity (N-acquiring
enzyme; EC 3.4.11.1): using L-Leucine p-Nitroanilide (LPN) as substrate the hydrolysis of the peptide bond of leucinamide is measured spectrophotometrically at 410 nm (Mitz and Schlueter 1958):
add 2 mL of 50 mM Tris buffer solution (pH 8) and
2 mL of LPN (2 mM) to 0.5 g of soil sample in
a 5 mL polypropylene tube. Cap tubes, place them
in zip lock bags, put bags in the platelet mixer and
incubate for 2 h at 20 °C. Spin the reaction suspensions for 2–3 min in table top centrifuge and pipette
2 mL of supernatant into a 15 mL test tube. Add
0.2 mL of 1.0 N NaOH to each test tube to terminate
the reaction and develop the color. Add 8 mL of
distilled water to each test tube and vortex. Finally,
read absorbance of each tube at 410 nm. Zero the
spectrophotometer with distilled water. The results
are expressed in µmoles LPN g
−1
h
−1
.
6. Alkaline phosphatase activity (P-acquiring enzyme;
EC 3.1.3.1): using as substrate p-nitrophenyl phosphate (PNPP) and measuring spectrophometrically
at 398 nm the absorbance of released p-nitrophenol
gives a yellow coloration (Tabatabai 1994): Add
2 mL of 0.1 M maleate buffer (pH 6.5) and 0.5 mL
of PNPP (0.115 M) to 0.5 g of soil sample. Incubate and shake in a water bath at 30 °C for an hour
and a half. Cool at 2 °C for 20 min to stop the reaction. Add 0.5 mL of 0.5 M calcium chloride and
2 mL of 0.5 M sodium hydroxide and centrifuge at
2,000 g for 10 min. Read spectrophotometrically the
absorbance at 398 nm. Controls are prepared adding substrate just after the incubation and prior to
cooling tubes. The results are expressed in µmoles
PNPP g
−1
h
−1
.
15 Biogeochemical Indicators of Nutrient Enrichments in Wetlands …
1. Dehydrogenase activity (global metabolic activity
of the microbial community; Enzyme Commision
Number (EC) 1.1.1.1): using the substrate 2-p-iodophenyl-3-p-nitrophenyl 5-phenyl tetrazolium cloride
(INT) as electron acceptor it occurs the reduction of
tetrazolium salts and the appearance of formazan
(Skujins 1976; Garcia et al. 1993): add 0.4 mL of distilled water and 0.2 mL of INT (0.4 % mass/volume)
to 1 g of soil sample and incubate for 20 h in darkness
at 20 °C. Then add 10 mL of methanol and stir vigorously during 1 min, then filter. Controls are prepared
adding 0.6 mL of distilled water. Read spectrophotometrically the absorbance of the extract at 490 nm
against a calibration curve prepared with INTF (iodonitrophenyl formazan) at different concentrations.
The results are expressed as mg INTF g
−1
.
2. β-glucosidase activity (C-acquiring enzyme –degradation of cellulose; EC 3.2.2.21): using the artificial substrate p-nitrophenyl-β-D-glucopyranoside
(PNG) and measuring spectrophometrically the
released p-nitrophenol (Tabatabai 1994): add 2 mL
of 0.1 M maleate buffer (pH = 6.5) and 0.5 mL of
PNG (0.05 M) to 0.5 g of soil sample. Incubate and
shake in a water bath at 30 °C for 1.5 h. Chill at 2 °C
for 15 min. Then add 0.5 mL of 0.5 M calcium chloride and 2 mL of 0.5 M sodium hydroxide. Centrifuge for 15 min at 2,000 g. Dilute if appropiate and
spectrophotometrically read at 398 nm. Controls
must be prepared by adding the substrate just after
incubation and prior to cooling tubes. The standard
curve is performed with p-nitrophenol (PNP). The
results are expressed in µmol PNP g −1 h
−1
.
3. Phenol oxidase (EC 1.10.3.2) and peroxidase (EC
1.11.1.7) (C-acquiring enzyme–oxidation of lignin
an other poliphenol) activities: using L-3,4-dihydroxyphenylalanine (DOPA) as the substrate and
measuring spectrophotometrically at 450 nm. For
phenol oxidase, add 50 µL of 25 mM of DOPA to
soil samples. For peroxidase assays add 50 µL of
25 mM DOPA plus 10 µL of 0.3 % H 2 O 2 . Negative control samples for phenol oxidase must be
prepared containing 200 µL of acetate buffer and
50 µL of DOPA solution; blank samples must
contain 200 µL of sample suspension and 50 µL
of acetate buffer. For peroxidase, negative control and blank samples also must contain 10 µL
of H 2 O 2 . Microplates are incubated in the dark at
20 °C, approximately 4 h for peroxidase and 18 h
for phenol oxidase. Finally, activity is quantified
by measuring absorbance at 450 nm using a microplate spectrophotometer. The results are expressed
in nmol h
−1
g
−1
.
4. Protease activity (N-acquiring enzyme): using
sodium caseinate as substrate and measuring spectrophotometrically the released tyrosine, using the
Folin-Ciocalteu reagent (Ladd and Butler 1972):
add 2.5 mL of 50 mM Tris buffer solution (pH 8.1)
and 2.5 mL of sodium caseinate (2 %) to 0.5 g of
soil sample. Incubate and shake in a water bath at
50 °C for 2 h. Cool at 2 °C for 20 min to stop the
reaction. Add 2.5 mL of trichloroacetic acid (15 %)
and later centrifuge at 8,000 g for 10 min. Subsequently 2.5 mL of supernatant are taken and added
to tubes with 3.75 mL of an alkaline reagent solution, which are incubated for 15 min at ambient
temperature. After this time add 2.5 mL of FolinCiocalteu reagent (33 %) and finally centrifuge at
2,000 g for 10 min. Dilute if appropriate, and read
in the spectrophotometer at 700 nm. The results are
expressed in mg tyrosine g
−1
h
−1
.
5. Leucine aminopeptidase activity (N-acquiring
enzyme; EC 3.4.11.1): using L-Leucine p-Nitroanilide (LPN) as substrate the hydrolysis of the peptide bond of leucinamide is measured spectrophotometrically at 410 nm (Mitz and Schlueter 1958):
add 2 mL of 50 mM Tris buffer solution (pH 8) and
2 mL of LPN (2 mM) to 0.5 g of soil sample in
a 5 mL polypropylene tube. Cap tubes, place them
in zip lock bags, put bags in the platelet mixer and
incubate for 2 h at 20 °C. Spin the reaction suspensions for 2–3 min in table top centrifuge and pipette
2 mL of supernatant into a 15 mL test tube. Add
0.2 mL of 1.0 N NaOH to each test tube to terminate
the reaction and develop the color. Add 8 mL of
distilled water to each test tube and vortex. Finally,
read absorbance of each tube at 410 nm. Zero the
spectrophotometer with distilled water. The results
are expressed in µmoles LPN g
−1
h
−1
.
6. Alkaline phosphatase activity (P-acquiring enzyme;
EC 3.1.3.1): using as substrate p-nitrophenyl phosphate (PNPP) and measuring spectrophometrically
at 398 nm the absorbance of released p-nitrophenol
gives a yellow coloration (Tabatabai 1994): Add
2 mL of 0.1 M maleate buffer (pH 6.5) and 0.5 mL
of PNPP (0.115 M) to 0.5 g of soil sample. Incubate and shake in a water bath at 30 °C for an hour
and a half. Cool at 2 °C for 20 min to stop the reaction. Add 0.5 mL of 0.5 M calcium chloride and
2 mL of 0.5 M sodium hydroxide and centrifuge at
2,000 g for 10 min. Read spectrophotometrically the
absorbance at 398 nm. Controls are prepared adding substrate just after the incubation and prior to
cooling tubes. The results are expressed in µmoles
PNPP g
−1
h
−1
.