Page 1 of 13
European Journal of Applied Sciences – Vol. 12, No. 3
Publication Date: June 25, 2024
DOI:10.14738/aivp.123.11327.
Dasat, G. S. (2024). Carbon Sequestration and the Enzymic Latch Mechanism in Red, Black and White Mangrove Soils of Florida
USA. European Journal of Applied Sciences, Vol - 12(3). 411-423.
Services for Science and Education – United Kingdom
Carbon Sequestration and the Enzymic Latch Mechanism in Red,
Black and White Mangrove Soils of Florida USA
Dasat, G. S.
Department of Science, Plateau State Polytechnic Barkin Ladi,
Plateau State PMB 2023 Jos, Nigeria
ABSTRACT
Mangrove swamps are important habitat types providing vital ecological services,
such as protection of coastlines from erosion and extreme weather conditions
including hurricanes, recycling of nutrients and sequestration of carbon.
Mangrove swamps support a wide range of biodiversity, improve water quality,
provide fish for local communities. They are also used as a source of wood fuel,
medication as well as for harvesting honey for the local population. Soil samples
for laboratory analyses were collected from red (Rhizophora mangle), black
(Avicennia germinans) and white (Laguncularia racemose) mangroves in Florida,
USA to determine the biogeochemistry processes. Results of analyses indicated
that the red mangrove soil is the most efficient for carbon sequestration. It had the
lowest phenol oxidase activity (206.15 nmol dicq g-1 h-1), highest phenolic
concentration (262.33 μg g-1) and lowest hydrolase enzyme activity (β- glucosidase) (3.04 nmol g-1 min-1) and, as a result, the highest concentration of soil
organic matter (SOM) (57.9%). It is believed that the high soil water content
(84.2%).) of the red mangrove, due to its proximity to the sea, is a key driver of
these observations. The 'enzymic latch' mechanism appears to be prevalent in the
red mangrove soil, in particular, allowing these ecosystems to be effective at
carbon storage hence, could serve as an important natural tool in mitigating the
effect of climate change. Preservation and conservation of mangrove swamps is
vital in balancing the effect of global warming.
Keywords: Mangroves, Carbon, Sequestration, Enzymic latch, Enzymes, Decomposition
INTRODUCTION
Mangroves swamps are considered biologically active wetland ecosystems with high
potentials to sequester and store huge quantities of carbon and other greenhouse gases
(GHGs) as soil biomass [1, 2]. Mangroves are found between latitudes 30° North and South of
the equator in several countries [3, 4]. From a global standpoint, mangroves inhabit a total
coastal area of 157-160,000 km2 [4]. Consequently, the world's mangroves are predominantly
found along the coasts of Indonesia, Australia, Brazil, and Nigeria [5]. Mangroves forests
provide vital coastal ecosystem services including water filtration, support biodiversity,
protection for coastal communities against heavy storms including hurricanes, wood
production, among many others [6, 7].
Donato et al.& Alongi [4 & 5] opined that mangrove swamps play a central role in carbon
sequestration globally capturing about 18.4 Tg C yr-1 to 23.2 Tg C yr-1 [1]. Therefore,
Page 2 of 13
Services for Science and Education – United Kingdom 412
European Journal of Applied Sciences (EJAS) Vol. 12, Issue 3, June-2024
mangrove ecosystems are an efficient natural tool for mitigating the effect of climate change
[8, 9]. Saraswati et al., Bridgham et al., &Wiener et al. [10, 11, 12] posited that mangrove
swamps could hold about 45-98% of organic carbon and up to 5metres of peat as soil
biomass. Furthermore, Murdiyarso & Donato et al. [13, 4] revealed that mangrove swamps
have sequestered an average of 1023 Mg ha-1 of carbon which could be attributed to the high
rates of leaf litter input from mangrove trees. Furthermore, Donato et al. [4] noted that
mangroves soils have an average of three to four times the quantity of carbon in storage per
unit area when compared to upland tropical forests.
The anoxic conditions in wetland soils due to limited atmospheric oxygen supply and low
nutrient availability impairs microbial decomposition thereby encouraging the accumulation
of vast organic matter in such soil environments. Consequently, Marx et al. [14] stated that in
soils, extracellular hydrolytic enzymes activity is considered to be the key driver of organic
matter breakdown and nutrient cycling. Thus, such anoxic conditions place a constraint on
enzyme activities which boosts the build-up of inhibitory phenolics in wetland soils, as a
result, the rate of organic matter decomposition is slowed down resulting in the accumulation
of organic material as peat. Freeman et al. [15] opined that the entire process is controlled by
oxygen limitations on a single enzyme, phenoloxidase ensuing in the phenolic build up and as
a result placing a constraint on the activities of hydrolases which are the main enzymes
implicated with decomposition, a process known as the 'enzymic latch' mechanism. As a
result of the above scenario, mangrove wetlands are considered as significant global sinks of
carbon. Saraswati et al. [10] investigated the influence of the 'enzymic latch mechanism' in
red mangrove soil from Florida, USA, and found the mechanism to be active.
Anthropogenically induced activities such as harmful agricultural practices and land
reclamation, oil spills, overexploitation of forest resources by local communities among others
pose a serious threat to mangroves [16, 17]. Accordingly, the aforementioned alongside
natural causes account for the loss of almost one-third of the world's mangrove forest in the
last 50 years [18, 19, 10, & 20]. Similarly, Pendleton et al. [21] reported a 50% loss of
sediment carbon within 8 years after clearing mangrove swamps in Panama. Such losses of
vegetation alongside centuries of buried carbon could have the undesired impact of
converting the mangrove wetlands from net sinks into net sources of GHGs with dire
consequences on the global climate, which may include a rise in atmospheric temperatures
and an increase in sea levels [18].
Valiela et al. & FAO, [22, 23] posited that without a good and sustained conservation program
in place, mangrove forest could be lost at even a greater rate than tropical forest ecosystems.
So also, Duke et al. [24] stated that 100% of mangrove forests, as well as 30-40% of coastal
wetlands, is likely to be lost in the next 100 years if urgent steps are not taken to curtail the
current rate of losses. Therefore, Onyena and Sam [7] suggested the establishment of effective
or enabling conditions for the protection and conservation of mangrove swamps in Nigerias'
Niger delta which would involve local communities to participate in a robust conservation
framework aimed at preservation and conservation of the mangrove ecosystem such as is
practised in India, where the government engaged rural counties in implementing
preservation and conservation programs of mangrove forest reserves.
Page 3 of 13
413
Dasat, G. S. (2024). Carbon Sequestration and the Enzymic Latch Mechanism in Red, Black and White Mangrove Soils of Florida USA. European
Journal of Applied Sciences, Vol - 12(3). 411-423.
URL: http://dx.doi.org/10.14738/aivp.123.11327
There remains much to be understood about the carbon sequestration dynamics of different
mangrove forest species. This investigation is built-up on the work of Saraswati et al. [10] by
determining the rate of microbial decomposition of organic matter on one hand and
examining the impact of the 'enzymic latch' mechanism in the soils of red, black, and white
mangroves in Florida, USA. It is, therefore, hypotheses that white mangrove soil has the
highest rate of decomposition, whereas soils from the red mangrove have the lowest, due to
their closeness to the sea and resultant period of inundation.
METHODOLOGY
A sampling of soils was done in southern Florida, the USA, close to Barefoot Beach County,
Bonita Springs, in an area comprising all three mangrove tree species; red (Rhizophora
mangle), black (Avicennia germinans) and white (Laguncularia racemose). The red mangrove
is situated closest to the sea, frequently flooded by tidal action, and closely followed by the
black mangrove. The white mangrove is located inland at a higher gradient than the red and
black mangroves and as a result, less frequently flooded. The three different areas had dense
vegetation dominated by species peculiar to their area. The mean annual temperature range
of the site is 18-29°C, and rainfall 1318 mm [25]. Soils were randomly sampled from the
following locations in five replicates at a distance of about three meters apart: Red: Latitude:
26.29553, Longitude: -81.83155, Black: Latitude: 26.29600, Longitude: -81.83200, White:
Latitude: 26.29465, Longitude: -81.83157.
Sample Collection
About 500g of the soil sample from a depth of 10-12 cm using a trowel was collected in five
locations randomly and placed in labelled plastic bags and sealed. Samples were further
packaged in cooler boxes containing ice packs and sent back to the UK and maintained at 4°C
until analyses within 2 weeks of sampling.
Laboratory Analyses
Before commencement of analyses all soil samples, water and reagents were placed in an
incubator at field temperature for 24 hours.
Water Extraction
Unwanted debris from soil samples were removed and hand hamogenised, followed by
placing a 5gram sub-sample into accordingly labelled 50 ml falcon tube, followed by the
addition of 40 ml deionised water and placed on a shaker (KIKA®-Werke, Gmbh & CO.KG,
Germany) at 300 rpm for 24 hours. This was followed by the determination of pH and
conductivity on an aliquot of the samples using SevenEasy and FiveGo (Mettler-Toledo,
Leicester, UK) bench-top meters. After which, the remaining samples were centrifuged at
5000 rpm using a Sorall ST-16R centrifuge (Thermo Scientific, UK) and 20 ml of sample
filtered through 0.45 μm cellulose nitrate filters (Cole Palmer, St. Neots, UK).
Soil Water and Organic Contents
The determination of soil water and organic matter weights were carried as detailed by
Frogbrook et al. [26]. Briefly, 10g of soil were placed in pre-weighed crucibles, and placed in
an oven at 105°C for 24 hours to ensure thorough evaporation of water content. Thereafter,
they were placed in a muffle furnace for 120 minutes at 550°C weighing at each point. The
Page 4 of 13
Services for Science and Education – United Kingdom 414
European Journal of Applied Sciences (EJAS) Vol. 12, Issue 3, June-2024
weights were used to calculate the water and organic contents as a percentage of the original
sample.
Phenol Oxidase Enzyme Assay
This was done by preparing a 10 mM solution of phenolic amino acid L-3, 4-
dihydroxyphenylalanine (L-DOPA) (Sigma Aldrich Ltd, UK) substrate by dissolving 0.986g of
powder and transferring into a 500ml capacity Duran bottle and making up the volume with
deionised water as described by Dunn et al. [27].
Two 1g homogenised soil sub-samples were placed into two separate stomacher bags, one
labelled as blank (B) and the other as substrate (S) followed by the addition of 9 ml deionised
water to each bag and homogenised in stomacher equipment. This was followed by the
addition of 10 ml L-DOPA solution and 10ml deionized water into the S and B bags
respectively, homogenised and then placed in the incubator for 10 minutes. After the period
of incubation, three 1.5 ml microcentrifuge tubes were filled with solution from each bag and
centrifuged at 10000 rpm for 5 minutes. 300 μL of the supernatant was then pipetted from
each microcentrifuge tube into separate wells of a clear 96 well microplate and the
absorbance at 475nm read using a SpectrMax M2e (Molecular Devices, Wokingham, UK) plate
reader. The activity of the enzyme was calculated by subtracting the average blank
absorbance value from the average substrate absorbance value and correcting for the dry
weight of soil, to give an activity expressed as nmol of product formed (dopachrome or 2-
carboxy-2,3-dihydroindole-5,6-quinone,) per minute (min-1) per gram (g-1) of soil (dry
weight).
Hydrolase Enzyme Assay
Methylumbelliferone-based enzyme-substrate solutions were prepared for all 5 enzymes
(400μM for β-glucosidase, β-xylosidase, sulphatase and chitinase; 200μM for phosphatase)
were prepared according to Dunn et al. [27]. For each sample and each enzyme, 1 g of soil was
placed in labelled stomacher bags and 7 ml of the relevant substrate was added. The bag was
then homogenised and incubated for 1 hour (45 minutes for phosphatase) at field
temperature. Following incubation, soil slurries were dispensed into 1.5 ml microcentrifuge
tubes and centrifuged at 10,000 rpm for 5 minutes. 250 μl supernatant was extracted from
each and added to separate wells of a black 96 well microplate, to which 50μl of deionised
water had been previously added. A similar procedure was adopted for the standard
solutions, but instead using deionised water rather than enzyme substrate in the stomacher
bags and 50 μl of varying concentrations of MUF-free acid solution in the microplate wells.
The concentration of the samples and standards was then measured using the SpectraMax
M2e plate reader and converted into a value of activity following Dunn et al. [27].
Gas Fluxes
Soil respiration was performed by placing 10g of homogenized soil into a 50 ml falcon tube
fitted with rubber septa in the lids and incubated at field temperature for 60 minutes. After
the allotted 60-minute period of incubation, gases were collected from the tubes using a 10
cm3 syringe fitted with a short bevel hypodermic needle (Sigma Aldrich Ltd, Dorset, UK) and
transferred into labelled (Time 2; T2), pre-evacuated 10 ml Exetainers (Labco Ltd, Lampeter,
UK) fitted with screw caps with rubber septa. An air sample from above the centrifuge tubes
Page 5 of 13
415
Dasat, G. S. (2024). Carbon Sequestration and the Enzymic Latch Mechanism in Red, Black and White Mangrove Soils of Florida USA. European
Journal of Applied Sciences, Vol - 12(3). 411-423.
URL: http://dx.doi.org/10.14738/aivp.123.11327
containing the samples at the start of the experiment was collected into five exetainers
labelled as Time 1 (T1).
Gas samples were analyzed on a Varian model 450 gas chromatograph (GC) instrument, fitted
with a flame ionization detector (FID) and a catalytic converter (methaniser) to measure CO2
and CH4 concentrations, and an electron capture detector (ECD) for N2O. Oxygen-free
nitrogen is used as the carrier gas. CH4, CO2, and N2O (retention times 1.08, 1.87 and 2.25
minutes respectively) were quantified by comparison of peak area with that of the three
standards of known concentrations, prepared by Scientific and Technical Gases Ltd
(Newcastle under Lyme, Staffordshire, UK), used in the preparation of a standard curve
STATISTICAL ANALYSIS
Data were analysed by one-way ANOVA to test for the effect of one factor on the measured
parameters; site (three levels, white, and black, red). Relationships between the enzyme
activities and physico-chemical factors across the three mangrove zones were determined by
correlation analysis. SPSS v22 (IBM Corporation, New York, USA) was used for all analyses. A
p-value of <0.05 was used to denote significance for the ANOVA analysis, but <0.01 for the
correlation analysis.
RESULTS
The red mangrove soil had a greater water content (84.2%) than soils in the black mangrove
stands (73.1%), but this difference was not significant (p>0.05; figure 1). The soil beneath
white mangroves had a much lower soil water content (44.1%), which is significantly
different to the red and black (p<0.05). This trend was mirrored for the SOM (Figure 1), with
the red mangrove having the highest SOM (57.9%), followed by the black mangrove (36.5%)
and the white (9.9%), however, only the red and white mangroves sites were significantly
different (p<0.05).
Figure 1: Bar chart showing mean soil water and soil organic matter contents in each mangrove
zone (n=5, error bars + SD).
Page 6 of 13
Services for Science and Education – United Kingdom 416
European Journal of Applied Sciences (EJAS) Vol. 12, Issue 3, June-2024
The mean soil pH differed substantially between the three zones (Figure 2), with ANOVA
analysis demonstrating significant differences for all comparisons (p<0.05). The white was
the most alkaline (mean pH 8.09), followed by black (7.40) and red (6.32).
Figure 2: Bar chart showing mean soil pH in each mangrove zone (n=5, error bars + SD)
The white mangrove soil had a more than threefold greater activity of phenol oxidase (853.74
nmol dicq g-g h-h) in comparison to the red mangrove soil (206.15 nmol dicq g-g h-h), which
was a significant difference (p<0.05) while, black mangrove had more than twice the activity
(439.48 nmol dicq g-g h-h) compared to the red soil, but this was not statistically significant
(Figure 3).
Figure 3: Bar chart showing mean activity of phenol oxidase in each mangrove zone (n=5, error
bars + SD)
Page 7 of 13
417
Dasat, G. S. (2024). Carbon Sequestration and the Enzymic Latch Mechanism in Red, Black and White Mangrove Soils of Florida USA. European
Journal of Applied Sciences, Vol - 12(3). 411-423.
URL: http://dx.doi.org/10.14738/aivp.123.11327
The concentration of soil phenolics (Figure 4) was similar in the black (282.06 μg g-1) and red
(262.33 μg g-1) mangrove soils but for the white was significantly lower than both (45.03 μg g- 1) (p<0.05).
Figure 4: Bar chart showing mean concentration of soil phenolics in each mangrove zone (n=5,
error bars + SD)
Similarly, the activity of β-glucosidase a hydrolase enzyme (Figure 5) does not differ much
between the black (4.41 nmol g-1 min-1) and red (3.04 nmol g-1 min-1) mangrove soils but
there was a significantly higher rate of activity in the white soil (9.42 nmol g-1 min-1)
(p<0.05). The other four hydrolase enzymes showed similar trends, with the white mangrove
soil always having the highest activity and the lowest activities usually being in the red
mangrove.
Figure 5: Bar chart showing mean activity of the enzyme β-glucosidase in each mangrove zone
(n=5, error bars + SD)
Page 8 of 13
Services for Science and Education – United Kingdom 418
European Journal of Applied Sciences (EJAS) Vol. 12, Issue 3, June-2024
The flux of CO2 (Figure 6) does not show the same pattern as the previous parameters, with
the highest flux measured from the red mangrove soils (9.33 μg g-1s-1) and the lowest from the
black (4.39 μg g-1 s-1), but these differences are not statistically significant (p>0.05).
Figure 6: Bar chart showing mean CO2 flux in each mangrove zone (n=5, error bars + SD)
A nonlinear (r=0.953, p<0.001); strong positive relationship between water percentage and
SOM contents was observed (Figure7). While no measured parameter correlated significantly
with the CO2 flux.
Figure 7: Scatterplot showing the relationship between % water and % soil organic matter
across the 3 mangrove zones. The trend line shows an exponential relationship