206
S. Sánchez-Carrillo et al.
emissions, which have a global warming potential 310 times
that of CO 2 (IPCC 1996), can be enhanced in the future as
nitrate availability in wetlands continues to be high owing
to increased agriculture pollution. When nitrate availability
is high, reduction of nitrate instead of N 2 O is energetically
favorable for denitrifyers (Barnard et al. 2005). This potential negative consequence of nitrate-rich wetlands is often
ignored or downplayed. Thus, there is a great need for additional information about the risk of nitrogen emissions from
nitrogen-loaded wetlands (Verhoeven et al. 2006).
15.3 Changes on Carbon, Nitrogen, and
Phosphorus Microbial Biomass during
Wetland Eutrophication
Most of the biogeochemical transformations are mediated by
microbes and are actively involved in regulating transformation and storage of nutrients. Microbial communities respond
to vertical gradients caused by the successive depletion of
electron acceptors and electron donors conditioned by the
oxygen availability. Because of its dynamic nature, microbial biomass (see Box 1 for methods of measurements) has
the potential to be a sensitive indicator to detect changes resulting from either anthropogenic impacts or ecosystem disturbance (Reddy and DeLaune 2008). In fact, MBC, MBN,
and MBP found in wetlands have been cited to be very sensitive to changes in the substrate nutrient content, according to
the nutrient limitation of the microbial communities (Reddy
and DeLaune 2008). In nutrient-poor wetlands, P has been
reported as the limiting of microbial activity (Amador and
Jones 1993; Reddy et al. 1993; Corstanje et al. 2007).
Unlike, MBP, MBC, and MBN may not be sensitive indicators of eutrophication. In a study by Wright et al. (2009)
it was reported that MBC and MBN was not responsive to
P loading in the Everglades wetland system. Increased response of MBP to P loading in wetlands of oligotrophic Everglades has been reported by Qualls and Richardson (2000).
MBC:MBP ratios have been found to range from 25
(Singh and Singh 1993) to 79–279 in humus-rich soils (He
et al. 1997). The MBC:MBP ratio in soils has been suggested
to be in the range from 10 to 35 (He et al. 1997). In general
terms, all soils reflect higher C:P ratios in P-limited soils (Joergensen et al. 1995a, b; He et al. 1997). Several studies carried out in the Everglades revealed that microbial biomass at
oligotrophic wetland soils exhibits significantly higher C: P
ratios than that at intermediate and impacted sites (White and
Reddy 2000; Corstanje et al. 2007). Significant correlations
were observed for MBC and MBN with soil total P ( r = 0.50;
0.70, p < 0.05) and with total P for the detrital layer ( r = 0.65;
0.41, p < 0.05), providing evidence that P was likely the limiting nutrient to the microbial biomass in natural Everglades
peat soils (Corstanje et al. 2007).
Box 1: Carbon, Nitrogen, and Phosphorus Microbial
Biomass by the Chloroform Fumigation Extraction
Procedure
Carbon, nitrogen, and phosphorus content of the microbial biomass can be determined by the 24-h chloroform fumigation-extraction (CFE) technique (Brookes
et al. 1985; Witt et al. 2000; Truu et al. 2009). Soils
are fumigated with chloroform vapor inducing a lysis
of microbial cell membranes and, thus, the subsequent
release of microbial constituents can be extracted and
quantified. While efficient lysis of microbial cells in
aerobic soils can easily be achieved with chloroform
vapour, Inubushi et al. (1991) proposed the direct addition of chloroform to anaerobic soil in order to overcome limitations in the fumigation efficiency when
exposing water-saturated soil to chloroform vapour.
First, ethanol-free chloroform needs to be prepared,
removing the ethanol from the chloroform by mixing
500 mL over 25 g of basic grade 1 alumina in a flask
and stir for 10 min. Triplicate, 15–20 g (wet weight)
soil subsamples are taken to carry out the analysis.
Fumigations must be done in the fume hood because
chloroform is toxic. Samples to be fumigated are
placed in 50 mL glass beakers which must be marked
with pencil. The beakers with sample soils are put into
a vacuum dessicator (if the sample number is high, beakers can be stacked in the dessicator by layering with
shelf). A 50-mL beaker or scintillation vial containing
~1 spatula boiling chips and 20 mL of chloroform is
placed in the dessicator. Using a vacuum the air inside
the dessicator must be evacuated until chloroform
boils, This procedure is repeated three more times,
venting at the end of each step except at the last time.
Then the dessicator (covered with a black garbage bag
because darkness prevents the chloroform from breaking down) is left in the dark for 5 days. After this time,
the vacuum is released, drawing a vacuum for 1 min
and releasing to remove the chloroform (it must be
repeated three times); chloroform is then evacuated to
the outside.
Sample extraction is done with 30 mL of 0.5 M
K 2 SO 4 for 30 min on a longitudinal shaker and vacuum filtered through #42 Whatman filter paper. The
extract is analyzed for total organic C on a TOC analyzer and subjected to Kjeldahl-N digestion for total
nitrogen analysis. MBP is extracted using 25 mL
0.5 M NaHCO 3 and is thereafter analyzed by the
ascorbic acid colorimetric procedure. MBC is determined by subtracting the extractable total organic C
(TOC) in the triplicate controls (nonfumigated) from
the triplicate chloroform-treated samples, using an
S. Sánchez-Carrillo et al.
emissions, which have a global warming potential 310 times
that of CO 2 (IPCC 1996), can be enhanced in the future as
nitrate availability in wetlands continues to be high owing
to increased agriculture pollution. When nitrate availability
is high, reduction of nitrate instead of N 2 O is energetically
favorable for denitrifyers (Barnard et al. 2005). This potential negative consequence of nitrate-rich wetlands is often
ignored or downplayed. Thus, there is a great need for additional information about the risk of nitrogen emissions from
nitrogen-loaded wetlands (Verhoeven et al. 2006).
15.3 Changes on Carbon, Nitrogen, and
Phosphorus Microbial Biomass during
Wetland Eutrophication
Most of the biogeochemical transformations are mediated by
microbes and are actively involved in regulating transformation and storage of nutrients. Microbial communities respond
to vertical gradients caused by the successive depletion of
electron acceptors and electron donors conditioned by the
oxygen availability. Because of its dynamic nature, microbial biomass (see Box 1 for methods of measurements) has
the potential to be a sensitive indicator to detect changes resulting from either anthropogenic impacts or ecosystem disturbance (Reddy and DeLaune 2008). In fact, MBC, MBN,
and MBP found in wetlands have been cited to be very sensitive to changes in the substrate nutrient content, according to
the nutrient limitation of the microbial communities (Reddy
and DeLaune 2008). In nutrient-poor wetlands, P has been
reported as the limiting of microbial activity (Amador and
Jones 1993; Reddy et al. 1993; Corstanje et al. 2007).
Unlike, MBP, MBC, and MBN may not be sensitive indicators of eutrophication. In a study by Wright et al. (2009)
it was reported that MBC and MBN was not responsive to
P loading in the Everglades wetland system. Increased response of MBP to P loading in wetlands of oligotrophic Everglades has been reported by Qualls and Richardson (2000).
MBC:MBP ratios have been found to range from 25
(Singh and Singh 1993) to 79–279 in humus-rich soils (He
et al. 1997). The MBC:MBP ratio in soils has been suggested
to be in the range from 10 to 35 (He et al. 1997). In general
terms, all soils reflect higher C:P ratios in P-limited soils (Joergensen et al. 1995a, b; He et al. 1997). Several studies carried out in the Everglades revealed that microbial biomass at
oligotrophic wetland soils exhibits significantly higher C: P
ratios than that at intermediate and impacted sites (White and
Reddy 2000; Corstanje et al. 2007). Significant correlations
were observed for MBC and MBN with soil total P ( r = 0.50;
0.70, p < 0.05) and with total P for the detrital layer ( r = 0.65;
0.41, p < 0.05), providing evidence that P was likely the limiting nutrient to the microbial biomass in natural Everglades
peat soils (Corstanje et al. 2007).
Box 1: Carbon, Nitrogen, and Phosphorus Microbial
Biomass by the Chloroform Fumigation Extraction
Procedure
Carbon, nitrogen, and phosphorus content of the microbial biomass can be determined by the 24-h chloroform fumigation-extraction (CFE) technique (Brookes
et al. 1985; Witt et al. 2000; Truu et al. 2009). Soils
are fumigated with chloroform vapor inducing a lysis
of microbial cell membranes and, thus, the subsequent
release of microbial constituents can be extracted and
quantified. While efficient lysis of microbial cells in
aerobic soils can easily be achieved with chloroform
vapour, Inubushi et al. (1991) proposed the direct addition of chloroform to anaerobic soil in order to overcome limitations in the fumigation efficiency when
exposing water-saturated soil to chloroform vapour.
First, ethanol-free chloroform needs to be prepared,
removing the ethanol from the chloroform by mixing
500 mL over 25 g of basic grade 1 alumina in a flask
and stir for 10 min. Triplicate, 15–20 g (wet weight)
soil subsamples are taken to carry out the analysis.
Fumigations must be done in the fume hood because
chloroform is toxic. Samples to be fumigated are
placed in 50 mL glass beakers which must be marked
with pencil. The beakers with sample soils are put into
a vacuum dessicator (if the sample number is high, beakers can be stacked in the dessicator by layering with
shelf). A 50-mL beaker or scintillation vial containing
~1 spatula boiling chips and 20 mL of chloroform is
placed in the dessicator. Using a vacuum the air inside
the dessicator must be evacuated until chloroform
boils, This procedure is repeated three more times,
venting at the end of each step except at the last time.
Then the dessicator (covered with a black garbage bag
because darkness prevents the chloroform from breaking down) is left in the dark for 5 days. After this time,
the vacuum is released, drawing a vacuum for 1 min
and releasing to remove the chloroform (it must be
repeated three times); chloroform is then evacuated to
the outside.
Sample extraction is done with 30 mL of 0.5 M
K 2 SO 4 for 30 min on a longitudinal shaker and vacuum filtered through #42 Whatman filter paper. The
extract is analyzed for total organic C on a TOC analyzer and subjected to Kjeldahl-N digestion for total
nitrogen analysis. MBP is extracted using 25 mL
0.5 M NaHCO 3 and is thereafter analyzed by the
ascorbic acid colorimetric procedure. MBC is determined by subtracting the extractable total organic C
(TOC) in the triplicate controls (nonfumigated) from
the triplicate chloroform-treated samples, using an