Plant Soil (2011) 346:259273

DOI 10.1007/s11104-011-0816-9

REGULAR ARTICLE

What happens to soil chemical properties after mangrove

plants colonize?
Tomomi Inoue & Seiichi Nohara &
Katsumi Matsumoto & Yasuharu Anzai

Received: 17 September 2010 / Accepted: 2 May 2011 / Published online: 21 May 2011
# Springer Science+Business Media B.V. 2011

K. Matsumoto
Kawakami Farm,
4041 Owari,
Tsukubamirai, Ibaraki, Japan

We detected oxidative effects in all three species,

including deposition of iron oxide on root surfaces,
lowered methane concentrations and increased oxidized inorganic nitrogen concentrations in the soil
pore-water, suggesting that radial oxygen loss from
mangrove roots had affected these soil chemical
properties. Besides the oxidative effects, enhanced
Fe2+ concentrations in the soil pore-water were
present in A. marina, and enhanced phosphorus
concentrations in the soil pore-water were present in
all three species, suggesting that mangrove roots
provide Fe- and phosphate-solubilizing substrates.
The most remarkable change was in soil nitrogen
enrichment. During the experimental period, amounts
of nitrogen in the mangrove soils increased four times
more than in uncolonized soil. Six months from the
start of cultivation, bacterial nitrogen fixation (nitrogenase activity) was significantly higher in soil
colonized by mangrove plants than in uncolonized
soil, suggesting that mangrove roots stimulated
bacterial nitrogen fixation. Among these properties,
Phosphate mobilization and soil nitrogen enrichment
are likely to be particularly important for the growth
of mangrove plants, because phosphate and nitrogen
are generally limited in mangrove ecosystems. This
self-supporting ability of mangroves observed in this
study could be one key to the high productivity of
mangrove ecosystems.

Y. Anzai
1-4-2 Azuma,
Tsukuba, Ibaraki 305-0031, Japan

Keywords Mangrove . Root . Soil chemicals . Iron .

Phosphorus . Nitrogen . Methane

Abstract Understanding soil chemical properties is

necessary to characterize the basic properties of
ecosystems. In mangrove ecosystems, soil iron,
phosphorus, methane and nitrogen have been well
studied under field conditions. However, it is difficult
to understand fundamental relationships between
mangrove root functions and soil chemical properties,
because of the multiple factors present in field data.
The aim of this study was to clarify what will happen
to soil chemical properties after mangrove plant
colonize. To examine the effect of mangrove roots
on these soil properties, three representative mangrove species (Avicennia marina, Rhizophora stylosa
and Bruguiera gymnorrhiza) were cultivated in a
greenhouse and selected soil chemical properties were
monitored in comparison with those in unplanted soil.

Responsible Editor: Katja Klumpp.

T. Inoue (*) : S. Nohara
National Institute for Environmental Studies,
16-2 Onogawa,
Tsukuba, Ibaraki 305-8506, Japan
e-mail: tomomi.inoue@nies.go.jp

260

Introduction
This study focused on mangrove plants that form
highly productive ecosystems in coastal areas of
tropical and subtropical regions. For several decades,
areas of mangroves have continuously declined
around the world, despite their important ecosystem
role (Spalding et al. 2010). To characterize these
productive ecosystems and provide scientific guidelines for their conservation, knowledge of their soil
chemical properties is necessary, because these
properties are the basis of the ecosystems.
When plant seeds germinate and start to grow, soil
chemical properties are affected. It is known that
plants excrete a variety of substrates that facilitate the
availability of macro- and micronutrients in the
rhizosphere, by enhancing absorption of appropriate
nutrients even under nutrient deficient conditions. For
instance, organic acids exuded from plant roots, such
as citrate and malate, are known to mobilize P from
sparingly soluble Fe, Al and Ca phosphates (Gardner
et al. 1983). Therefore, greater amounts of nutrients
such as P and Fe are sometimes observed in the plant
root zone compared with the bulk soil (Inderjit and
Weston 2003). Besides root exudates, plant roots
continuously provide organic matter such as decaying
root parts. These organic matter-rich root zones are
different from the bulk soil and provide niches in
which bacteria thrive (Lynch and Whipps 1991),
because heterotrophic bacteria can use these plantderived carbon compounds as electron donors to
generate energy. Therefore, soil microbial metabolic
processes also change in association with plant
colonization. In addition to the above mentioned root
functions, radial oxygen loss may be an important
characteristic factor in mangrove root zones. Coastal
habitats for mangroves are always affected by tidal
fluctuation, and thus, the soil surface is repeatedly
flooded. To cope with the hypoxia in root cells, most
mangrove species develop oxygen transporting mechanisms and an aerial root system. This allows
atmospheric oxygen to diffuse towards root tips
through internal lacunae (a gasspace continuum).
Supplied oxygen is partially utilized for aerobic
respiration of the roots, and the rest diffuses towards
the rhizosphere via the root surface. This results in the
formation of a thin oxidative layer around the oxygenreleasing root surface and affects soil microbial
processes. Radial oxygen loss from mangrove plant

Plant Soil (2011) 346:259273

root has been detected in Avicennia marina (Forsk.)

Vierh., Kandelia obovata, Lumnitzera racemosa
Willd, Bruguiera gymnorrhiza (L.) Lam., and
Excoecaria agallocha L. (Pi et al. 2009, 2010). So
far, the following four soil chemical properties, iron,
phosphorus, nitrogen and methane have been comparatively well studied in mangroves growing in
rooted soil.
Iron is basically found as Fe(II)/Fe(III) oxide complexes in soil. Ferrous ion (Fe2+) is released by
chemical reduction under conditions of low redox
potential and/or chelation with organic acids. There are
some reports that concentration of ferrous ion (Fe2+) in
mangrove soil pore water is positively correlated with
live root density (Alongi et al. 1999, 2005; Otero et al.
2006). These observations indicate that mangrove roots
lead to enhanced Fe mobilization. On the other hand,
Pi et al. (2010) reported that they observed iron oxide
deposition on the root surface in two mangrove
species, B. gymnorrhiza and E. agallocha, probably
because of oxygenation by oxygen-releasing roots.
Mangrove roots thus may form Fe2+-Fe(III) oxide
contrasts in the rhizosphere.
Phosphorus has been well studied, probably
because it is one of the most limiting elements in
mangrove ecosystems (Feller et al. 2003a, b;
Lovelock et al. 2004; Reef et al. 2010). In mangrove
soils, phosphorus tends to be adsorbed on Fe/Aloxides, Ca compounds and polymerized organic
matter (Paludan and Morris 1999; Prasad and
Ramanathan 2010). The immobilized phosphorus
cannot be used by plants unless it is released by
phosphate solubilizing bacteria and/or chelation with
organic acids. So far, phosphate-solubilizing bacteria
have been detected in three mangroves, A. marina,
Avicennia germinans (L.) Stearn, and Laguncularia
racemosa (L.) Gaertn. f. (Vazquez et al. 2000;
Kothamasi et al. 2006; El-Tarabily and Youssef
2010), but resultant phosphate mobilization in the
rhizosphere has not yet been measured.
Nitrogen is another limiting element in mangrove
ecosystems (Feller et al. 2003a, b; Lovelock et al.
2004; Reef et al. 2010). Coastal habitats for mangrove
plants are always exposed to tidal fluctuation, and
thus nitrogen is continuously exported to the ocean
(Boto and Robertson 1990). Under such circumstances, bacterial nitrogen fixation is a major nitrogen
input process in mangrove ecosystems (Hicks and
Silvester 1985). High nitrogen fixing activity has been

Plant Soil (2011) 346:259273

found associated with mangrove sediments, dead

leaves, and cyanobacterial mats covering sediment
surfaces (Zuberer and Silver 1979; Hicks and
Silvester 1985; Holguin et al. 2001; Lugomela and
Bergman 2002). These observations suggest a relationship between nitrogen fixers and mangrove
growth. However nitrogen fixers are also found in
many other systems, including terrestrial and marine
ecosystems, and it has not yet been confirmed
whether the high nitrogen fixation is partly attributed
to mangrove plant growth or not. Besides nitrogen
fixation, oxygen-releasing roots may promote the
nitrification process. In mangrove ecosystems in
Thailand, higher nitrate concentrations in soil porewater in an area vegetated by the mangrove Rhizophora apiculata Bl. than in an unvegetated area have
been found (Kristensen et al. 1998).
Methane production in mangrove ecosystems has
been studied because it is one of the most significant
greenhouse gases. Although there are many studies
measuring the amount of methane produced in
mangrove soils (Sotomayor et al. 1994; Purvaja and
Ramesh 2001; Alongi et al. 2004; Biswas et al. 2007),
mangrove root effects on methane production have
not yet been studied. In herbaceous wetland plants,
oxidation of methane to carbon dioxide around
oxygen-releasing roots has been reported (Gilbert
and Frenzel 1998; Bodelier et al. 2000). Oxygenreleasing mangrove roots may also have such a
methane-consuming effect.
As discussed above, information on soil chemical
properties is available from previous field studies.
However, it is difficult to interpret inter-relationships
between mangrove root functions and soil chemical
properties from field data, because the data reflect
multiple factors. From field observations, we hypothesized that (a) oxygen-releasing mangrove roots
would have iron oxide deposition on their surfaces,
promote nitrification process in the soil pore water
and decrease methane concentrations in soil pore
water, (b) mangrove roots would enhance Fe and P
mobilization and (c) mangrove roots would stimulate
soil bacterial N-fixation. The aim of this study was to
confirm these hypotheses in three representative
mangrove species (A. marina, Rhizophora stylosa
Griff., and B. gymnorrhiza). To eliminate external
factors (e.g. tidal fluctuations, precipitation and
intrusion of chemical entities from the surrounding
area) and focus on mangrove root effects on soil

261

chemical properties, we cultivated the three mangroves in a pot system under greenhouse conditions
and monitored soil iron, phosphorus, nitrogen and
methane values for 180 days in comparison with
those values in unplanted soil.

Materials and methods

Plant materials and cultivation
Plant cultivation was conducted under full sunlight
conditions in a greenhouse (27C air temperature,
70% humidity). Propagules of each mangrove species
were randomly collected from more than 30 different
mature trees in mangrove forest located at Kabira Bay
in north-west Ishigaki, Japan (242652 N, 12408
03 E) during the relevant fruiting season; September
for A. marina, July for R. stylosa and January for B.
gymnorrhiza. The mean dry weights of the propagules
were 0.960.09 in A. marina, 7.371.77 in R .stylosa
and 4.181.32 g in B. gymnorrhiza (mean SD, N=
20). The lower part (5 cm for R. stylosa and B.
gymnorrhiza, 1 cm for A. marina) of each propagule
was buried in the sediment in plastic pots (159 mm
diameter, 190 mm depth) and flooded with 25%
instant sea water (Marin Merit, Matsuda, Ltd., Osaka,
Japan) to 10 cm above the soil surface. After being
cultured for 2 years, 40 healthy plants for each species
were selected and individually transplanted into
separate plastic pots (250 mm diameter, 450 mm
depth) filled with 15 L (27.5 kg dry weight) of soil.
For each species, the transplanting was performed
exactly 2 years after the collection of propagules. At
transplanting, the height and leaf number of each
species was, respectively, 48.45.3 cm and 748 in
A. marina, 55.35.6 cm and 220 in R. stylosa and
624.8 cm and 180 in B. gymnorrhiza, (mean SD,
N=40). Another 40 pots without plants were prepared
as control treatments. The soil used for the cultivation
was a mixture of commercial potting compost and
sand (1:2 by volume). The soil texture was a sandy
loam and the organic matter content of the soil was
1.2%. The total nitrogen and sulfur concentrations of
the soil were determined as 5.0 and 40.0 mg g dry
weight1, respectively by an elemental analyzer
(Flash EA 1112, Thermo Fisher Science, Waltham,
USA). Concentrations of the total forms of other
elements in the soil were P: 20.0, K: 6.0, Al: 60.0, Ca:

262

200.0: Fe: 10.0, Mg: 30.0 mg g dry weight1

determined by an inductively coupled plasma atomic
emission spectrometer (ICP-JA, Nippon Jarrell-Ash,
Kyoto, Japan), following digestion by a mixture of
sulfuric, nitric and perchloric acids. The cultivation
experiment was run for 180 days. All pots were
maintained in the greenhouse under similar growth
conditions of a 10 cm flooding depth with 25% sea
water. The water level of each pot was monitored
daily and readjusted for evaporation by addition of
water. The salinity of the flooded water was also
monitored daily by an electrode sensor (WM-22EP,
HORIBA, Kyoto, Japan) and readjusted within 1%.
Thirty days from the start of the experiment, the pH
and redox potential in the control pots were 6.80.2
and 65.33.5 mv (mean SD, N=40), respectively.
For each species, 10 pots were randomly selected at
30, 60, 120, and 180 days from the start of the
experiment and chemical properties of the soil porewater, soil and plant material were measured.
Soil pore-water, soil and plant material
To collect soil pore-water, a water sampling tube was
inserted into each pot at the start of the experiment.
The water sampling tube comprised three parts: (a) an
unglazed tube (2 mm diameter, 10 cm length); (b) a
Teflon-lined tube (2 mm diameter, 30 cm length); and
(c) a hollow needle. The unglazed tube was buried in
the sediment at 1020 cm depth. When collecting soil
pore-water, the needle was inserted into the silicon lid
of the vacuum-sealed glass vessels, and soil porewater was drawn into the vessel. The first 2 ml of
water was discarded with the next 35 ml immediately
filtered through a 0.2-m mesh filter (DISMIC-25cs,
cellulose Acetate, ADVANTEC, Tokyo, Japan) with a
plastic syringe (ss-30ESZ, TERUMO, Tokyo, Japan)
to remove bacteria. Twenty milliliters of the sampled
water was used to measure dissolved NH4+, NO2,
NO3 and phosphate, by an auto-analyzer (AACS-II,
Bran+Luebbe, Norderstedt, Germany). Another 10 ml
of the sampled water was used for CH4 measurement.
The sampled water was transferred to 30 ml
sterilized glass vials and sealed with Teflon-silicon
septa. The vials were kept at 27C in a water bath
(NTS-4000B, Tokyo Rikakikai, Tokyo, Japan).
Methane concentrations in the head space of the
glass vials were determined by a gas chromatograph
equipped with a Porapak Q column and FID

Plant Soil (2011) 346:259273

detector (GC-4000, GL Science, Tokyo, Japan).

The remaining 5 ml of the sampled water was used
for Fe2+ measurement. After filtering, 5 ml of
10 mM o-phenanthroline hydrochloride (Wako Pure
Chemical Industries, Ltd, Osaka, Japan) and 5 ml of
sodium acetate buffer solution (pH=5.5) were immediately added to the water samples. The peak
absorbance at 510 nm of the Fe2+-phenanthroline
complex was determined by a UV/visible spectrophotometer (U1000, Hitachi, Tokyo, Japan).
After completion of water sampling, 50 ml of soil
(92 g dry weight) was collected from around the
submerged unglazed tube using a core sampler (2.5 cm
diameter, 10 cm height). The soils were dried at room
temperature and their nitrogen concentrations determined by the elemental analyzer. Each sample was
measured three times and the mean value determined.
At 60, 120 and 180 days from the start of the
experiment, the dry weights of roots, aboveground
parts and leaf litter in each pot were measured after
oven-drying at 80C for 72 h. Plant material nitrogen
concentrations were measured by the elemental
analyzer after fine grinding. Each sample was
measured three times and the mean value determined.
The relative growth rate (RGR) and the nitrogen
use efficiency (NUE) of the plants were calculated as
follows:


ln Wf  ln Wi
RGR
;
1
t
NUE

Wf  Wi
;
Nf  Ni

where Wi (g) and Wf (g) are the total dry weights at

the beginning (30 days) and end (180 days) of the
experiment, respectively, and t (days) is the growth
period (150 days). Ni (g) and Nf (g) are the amount of
nitrogen contained in plant material at the beginning
and end of the experiment, respectively.
The amounts of nitrogen in each pot were
calculated as follows:
Nj nj  Wtj ;

Where Nj (g) is the amount of nitrogen contained in j

in each pot (j = soil, water, plant, litter), nj (Ng g1) is
the nitrogen content of j and Wtj (g) is the weight of j
in each pot at t days from the start of the experiment
(t=30, 60, 90, 150 days).

Plant Soil (2011) 346:259273

Root Fe plaques
At 60, 120 and 180 days from the start of the
experiment, the amount of iron oxide deposition on
plant root surfaces caused by oxygen release from the
roots was determined by the cold DCB (dithionite
citrate bicarbonate) technique (Taylor and Crowder
1983). Fresh fine roots (corresponding to approximately 0.5 g dry weight) collected from each plant
were agitated in 45 ml of DCB reagent at 20C for
3 h, and the wash collected. The roots and beaker
were rinsed into the wash with deionized water. The
resulting solution was made to 100 ml and the iron
concentration was determined by an inductively
coupled plasma atomic emission spectrometer (ICPJA, Nippon Jarrell-Ash, Kyoto, Japan). Roots used for
measurement were less than 2 mm in diameter.
Nitrogenase activity
At the end of the experiment (180 days), soil nitrogen
fixation was indirectly assessed by the acetylene reduction technique (Hardy et al. 1968). A 5-ml soil core
(1 cm diameter, 5 cm height) from 16 cm soil depth in
each pot was transferred to a 50-ml glass vial together
with 5 ml of the water contained in the pot. Two vial
sets were prepared for each pot. The vials were purged
with argon to provide an anoxic atmosphere and sealed
with Teflon-silicon septa. For one of the vial sets, 4 ml
of the gas phase was evacuated and replaced with 4 ml
of acetylene through the Teflon-silicon septa. The other
vial was used as a control and incubated without
addition of acetylene to test the endogenous production
of ethylene. The vials were shaken in the dark at 30C
in a water bath and 0.2 ml of the gas phase was sampled
at 0.3, 3, 6 and 9 h after the acetylene injection.
Ethylene concentrations in the gas samples were
determined by the gas chromatograph equipped with a
Porapak N column and FID detector (GC-4000, GL
Science Inc., Tokyo, Japan). Nitrogenase activity was
determined from the accumulation of ethylene in the
vials with time, using linear regression analysis. A
similar procedure was used for fresh root materials to
determine the activity of nitrogen fixers on the roots.
Data analysis
In this study, 10 randomly selected pots from each
mangrove species were used for measurements on each

263

sampling day and then discarded to avoid pseudoreplicates. For each variable (Fe2+, CH4, PO43, NO2,
NO3, NH4+, soil N content and plant N content),
mean values of the 10 pots were calculated at each
sampling day. Two-way analysis of variance (ANOVA)
was used to test the effects of mangrove species, days
elapsed from mangrove colonization and interactions
between both on soil chemical properties and plant N
content. Differences among treatments means were
compared and tested for significance at the p<0.05
level by Tukeys test. Tukeys test was also used to
compare mean values of RGR, NUE, soil and root
nitrogenase activity, and the C/N ratio of plant
materials at the end of the experiment.

Results
Results from two-way ANOVA show that the presence of mangrove plants and elapsed time from
colonization both significantly affected all measured
chemical properties in the pots (Table 1). Furthermore, except for nitrite (NO2) concentrations in soil
pore-water, the probability of a greater F value (P
level) of the interaction factor was less than 0.05 in all
measured response variables indicating that periodic
change of the chemical properties significantly differed among the treatments.
Iron, phosphorus and methane
Iron oxide deposition (iron oxide plaque) was
detected on the root surfaces of all three mangrove
species at 60, 120 and 180 days (Fig. 1a). As time
passed, A. marina had increased iron oxide plaque on
its roots, which, at the end of the experiment, was
significantly higher than those of the other two
species, being 431% higher than for R. stylosa and
339% higher than for B. gymnorrhiza, respectively.
For A. marina, the concentration of ferrous ion (Fe2+)
dissolved in soil pore-water rapidly increased between
60 and 120 days after colonization (Fig. 1b). Although Fe2+ concentrations in the other three treatments also gradually increased as time passed, the
value under A. marina was 1,351% higher than that of
the control 120 days from colonization. After 120 days,
Fe2+ concentrations in A. marina did not significantly
increase, and the difference between A. marina and the
control decreased to 249%. Among the three mangrove

264

Plant Soil (2011) 346:259273

Table 1 ANOVA results for soil chemical properties in the

cultivated pots, showing degrees of freedom (d.f.), F-value (F)
and probability under a null model (P)
d.f.

[Fe2+] in soil pore-water

Treatment

24.2

0.000

Days

21.6

0.000

Treatment Days

6.1

0.000

Fe plaque on root surface

Treatment

40.5

0.000

Days

41.7

0.000

Treatment Days

22.7

0.000

Treatment

82.8

0.000

Days

35.8

0.000

Treatment Days

9.8

0.000

Phosphate in soil pore-water

[CH4] in soil pore-water

Treatment

20.6

0.000

Days

16.7

0.000

Treatment Days

8.0

0.000

Soil N content
Treatment

42.9

0.000

Days

173.8

0.000

Treatment Days

33.3

0.000

Treatment

126.1

0.000

Days

71.0

0.000

Treatment Days

20.8

0.000

Inorganic N in soil pore-water

Plant N content
Treatment

66.1

0.000

Days

170.6

0.000

Treatment Days

4.7

0.002

Treatment

198.7

0.000

Days

369.7

0.000

Treatment Days

198.7

0.000

Litter N content

[NO2] in soil pore-water

Treatment

9.0

0.000

Days

15.3

0.000

Treatment Days

1.9

0.055

[NO3] in soil pore-water

[NH4+]

Treatment

45.7

0.000

Days

34.0

0.000

Treatment Days

5.1

0.000

128.1

0.000

in soil pore-water
Treatment

Table 1 (continued)
d.f.

Days

72.6

0.000

Treatment Days

21.0

0.000

The effects of treatment (planted by A. marina, R. stylosa, B.

gymnorrhiza or control), elapsed time since colonization (30,
60, 120, 180 days), and both interactions on soil chemical
properties in the cultivated pots were tested by two-way
ANOVA. Values with P<0.05 are shown in bold

species, B. gymnorrhiza showed the lowest Fe2+

concentration at the end of the experiment.
After 30 days from colonization, the three
mangrove species enhanced dissolved phosphate
ion concentrations in the soil pore-water (Fig. 1c).
The degree of enhancement was significantly higher
in A. marina than in the other two species. At the end
of the experiment, phosphate ion concentrations
were 89, 95 and 304% higher in R. stylosa, B.
gymnorrhiza and A. marina compared with the
control, respectively. Although the greatest enhancement was observed in A. marina, the concentration
curve reached saturation 120 days after plant
colonization and mean values did not significantly
differ between then and 180 days.
The three mangrove species lowered methane
concentrations in the soil pore-water (Fig. 1d). In
the control pots, the CH4 concentration gradually
increased with elapsed time. At the end of the
experiment, CH4 concentrations in the soil pore water
were 24, 30 and 35% lower in R. stylosa, A. marina
and B. gymnorrhiza than in the control, respectively.
Nitrogen
Changes in the amounts of nitrogen with time are
shown in Fig. 2. Although soil nitrogen in the control
pots gradually decreased with elapsed time, it rapidly
increased in the mangrove planted pots between 120
and 180 days after colonization (Fig. 2a). At the end
of the experiment, the amounts of nitrogen contained
in the soil were 294, 329 and 338% higher in A.
marina, R. stylosa and B. gymnorrhiza pots than in
the control, respectively. In all mangrove colonized
pots, inorganic nitrogen dissolved in the soil porewater started to decrease between 30 and 60 days after
colonization, while the control pots maintained their
initial value throughout the experiment (Fig. 2b). At

Plant Soil (2011) 346:259273

265

16

A. marina
R. stylosa
B. gymnorrhiza
Control

12
8
4

Ferrous ion
(mg L-1)

b
b

no data
0

NH4+ (Fig. 3c), and thus the change in NH4+

concentration with time was of a very similar form
to that of total N (NO2+NO3+NH4+) (Figs. 2b and
3c). At the end of the experiment, NH4+ concentration
was 84, 95 and 98% lower in R. stylosa, A. marina
and B. gymnorrhiza pots than in the control pots,

60

120

180

Soil nitrogen
(gN pot-1)

Root Fe plaque
(gFeg root dry weight-1)

200

120

180

50
40

30
20

b
b

10

0
60

120

180

6
a

b
b
b

no data

120

180

b
Inorganic nitrogen in water
(mgN pot-1)

60

60

20
15

10
5
b
c
c

60

120

180

c
1.0

Plant nitrogen
(gN pot-1)

Phosphate ion
(gPL-1)

a
a
a

Methane
(mol L-1)

300

A. marina
R. stylosa
B. gymnorrhiza
Control

bc

400

100

no data

500

a
b
c

0.5
no data
0

60

120

180

d
0

60

120

40

180

the end of the experiment, the amount of nitrogen

contained in the soil pore-water was 98, 99 and 99%
lower in R. stylosa, A. marina and B. gymnorrhiza
pots than in the control pots, respectively. During the
experimental period, most of the inorganic nitrogen in
the soil pore-water was present as the ammonium ion,

Litter nitrogen
(mgN pot-1)

Fig. 1 Periodic changes in a amount of iron oxide plaque on

root surfaces, b Fe2+ concentration in the soil pore-water, c
phosphate ion concentrations in the soil pore-water and d CH4
concentrations in the soil pore-water. Results are expressed as
mean standard error (n = 10). Different letters indicate
significant differences (p<0.05) among treatments at the end
of the experiment

30
20
10
b
b

0
60

120

180

days

Fig. 2 Periodic changes in the amounts of nitrogen present in

cultivated pots; a soil nitrogen, b inorganic nitrogen dissolved
in the soil pore-water, c nitrogen in plant material and d
nitrogen in leaf litter. Results are expressed as mean standard
error (n=10). Different letters indicate significant differences
(p<0.05) among treatments at the end of the experiment

266

Plant Soil (2011) 346:259273

a
Nitrite ion
(gNL-1)

2.0

A. marina
R. stylosa
B. gymnorrhiza
Control

1.5

a
a
a

1.0
b

0.5
0

60

120

180

b
2.5

Nitrate ion
(gNL-1)

2.0

a
a

1.5

b
c

1.0
0.5
0

Ammonium ion
(mgNL-1)

60

120

180

0.8
a

0.6
0.4

Nitrogenase activity

0.2
0

mangrove plant materials increased as the plants grew

(Fig. 2c). Among the three species, nitrogen concentrations in aboveground material were highest in A.
marina followed by R. stylosa and B. gymnorrhiza
(Fig. 4). For belowground material, the nitrogen
concentration was basically higher in R. stylosa than
in B. gymnorrhiza while values for A. marina
fluctuated. For all three species, nitrogen concentrations were higher in aboveground than belowground
parts, as were biomass dry weights (Table 1). Between 120 and 180 days after colonization, all three
species began to supply leaf litter on the soil surface
(Fig. 2d). Dry weights of litter were highest in A.
marina, followed by R. stylosa and B. gymnorrhiza;
1.320.07, 0.530.06 and 0.410.04 g, respectively
(mean SE, N=10). The carbon to nitrogen ratio C/N
(g g1) of the litter was highest in A. marina, followed
by R. stylosa and B. gymnorrhiza (Fig. 5). The C/N
ratio of litter was significantly lower than that in the
aboveground parts of A. marina, while the other two
species had higher C/N ratios in the litter.

b
cc

60

120

180

days

Fig. 3 Periodic changes in inorganic nitrogen concentrations; a

NO2, b NO3 and c NH4+ dissolved in the soil pore-water.
Results are expressed as mean standard error (n=10).
Different letters indicate significant differences (p < 0.05)
among treatments at the end of the experiment

respectively. In contrast, the three mangrove species

enhanced concentrations of the oxidized forms of
nitrogen, nitrite and nitrate, in the soil pore-water
compared with the control pots (Fig. 3a, b). Both
NO2 and NO3 concentrations increased between 30
and 60 days after plant colonization, but remained
largely unchanged thereafter. At the end of the
experiment, NO2 concentrations were 79, 99 and
123% higher in R. stylosa, A. marina and B.
gymnorrhiza than in the control pots, respectively,
while concentrations of NO3 were 20, 55 and 55%
higher in A. marina, R. stylosa and B. gymnorrhiza
than in the control pots, respectively.
During the experimental period, the amounts of
nitrogen contained in the biomass of all three

At the end of the experiment, soil nitrogenase activity

significantly differed among the treatments (Fig. 6).
Mangrove colonization enhanced soil nitrogenase
activity with values being 16, 33 and 176 times
higher in A. marina, R. stylosa and B. gymnorrhiza
pots than in control pots, respectively. Root materials
of the three mangrove plants also showed nitrogenase
activity (Fig. 6). Compared on a volume basis,
nitrogenase activity of mangrove root materials was
significantly higher than in the control soil. In A.
marina, nitrogenase activity was higher in the soil
than in root materials, while there were no significant
differences between soil and root materials in R.
stylosa and B. gymnorrhiza.
Plant growth and nitrogen use efficiency
Thirty days after the start of the experiment, the three
mangrove species had similar total biomass dry
weights (Table 1). During the following 150 days,
the biomass of A. marina and R. stylosa increased to
about twice that at 30 days, while that of B.
gymnorrhiza increased about three times (Table 2).
As a result, the relative growth rate was significantly
higher in B. gymnorrhiza than in A. marina and R.

Plant Soil (2011) 346:259273

267

Fig. 4 Periodic changes

in nitrogen concentration in
a aboveground and
b belowground plant
material. Results are
expressed as mean standard
error (n=10). Different
letters indicate significant
differences (p<0.05) among
treatments at each sampling
time

Nitrogen concentration
(%)

Aboveground

A. marina
R. stylosa
B. gymnorrhiza

Belowground

a
a
ab
b

a
b
b

b
c

a
a
b

a
b
b

120

180

b
c

0
60

120

180

60

days

stylosa (P<0.05). The figure for B. gymnorrhiza was

5.901.11 mg g dry weight1 day1 (mean SD, N=
10), compared to 4.700.29 for A. marina and 4.46
0.45 mg g dry weight1 day1 (mean SD, N=10) for
R. stylosa, respectively. The nitrogen use efficiency
was also significantly higher in B. gymnorrhiza than
in A. marina and R. stylosa (P<0.05). The figure for
B. gymnorrhiza was 322.231.2 g gN1 (mean SD,
N=10), compared to 170.753.2 for A. marina and
142.726.2 g gN1 (mean SD, N=10) for R.
stylosa, respectively.

precipitation provide complex soil chemical conditions that develop fully over long time periods. The
absolute values obtained from this study will
therefore not necessarily apply to practical field
conditions. Although further field studies including
long term monitoring are needed to fully understand
how mangrove plants contribute to soil chemical
conditions under field conditions, the trends and
comparison among treatments observed in this study
provide important basic information.
Oxidative effects

Discussion
This study showed the effect of colonization by 2year-old mangroves on some soil chemical properties under a 10-cm flooded condition. Under field
conditions, a variety of factors such as tidal
fluctuation, salinity, nutrient intrusion from external
ecosystems, plant growth condition, temperature and

In this study, we detected several oxidative effects on

soil chemical properties and processes in the three
mangrove species: iron oxide plaque on root surfaces,
decreased methane concentrations in the soil porewater and increased oxidized inorganic nitrogen
200
Soil
Root

P= 0.817

200

C/N

150

Aboveground
Belowground
Litter

g
b

100
50

df
ae

de

bf

120
80

P= 0.086

40
P= 0.019

0
A. marina

Nitrogenazeactivity
(mmolC2H2L-1day-1)

160

R. stylosa

A.m R.s

B.g

Cont.

B. gymnorrhiza

Fig. 5 C/N ratios (g g1) of plant materials at the end of the

experiment. Results are expressed as mean standard error (n=10).
Different letters indicate significant differences (p<0.05)

Fig. 6 Nitrogenase activity of soil and plant root materials at

the end of the experiment under the four treatments: A. marina
(A.m.), R. stylosa (R.s.), B. gymnorrhiza (B.g.) and control
(Cont.). Results are expressed as mean standard error (n=10)

268

Plant Soil (2011) 346:259273

Table 2 Initial* and final** biomass of three species of 2-year-old mangrove plants (A. marina, R. stylosa and B. gymnorrhiza)
grown under greenhouse conditions (27C, 70% humidity) for 150 days
Aboveground (g dry weight)

Belowground (g dry weight)

Total (g dry weight)

Initial

Initial

Initial

Final

Final

Final

A. marina

20.70.4a

53.20.6a

19.50.1a

39.21.0a

40.20.5a

93.81.5a

R. stylosa

26.80.9

90.42.4a

B. gymnorrhiza

30.21.2c

40.00.3a

117.08.5b

54.80.8

71.64.3b

13.50.3

9.80.3c

35.02.3

46.04.8a

40.31.1

Values are mean SE (N=10). Different letters indicate significant differences (p<0.05) among species by Tukeys test
30 days* and 180 days** from the start of the experiment

(nitrite and nitrate) in the soil pore-water. According

to model studies of rice plant roots, most of the
oxygen released from the roots will be consumed by
the chemical oxidation of Fe2+, while the rest will be
consumed by aerobic heterotrophs (Van Bodegom et
al. 2001). In flooded soil, the reduced ferrous iron,
Fe2+, tends to accumulate and this will be chemically
oxidized to Fe(III) (hydroxyl) oxide precipitates on
oxygen-releasing root surfaces. Reddish brown colored iron oxide plaques covering wetland herbaceous
plant root surfaces have been observed in many
studies (Snowden and Wheeler 1995; Emerson et al.
1999; King and Garey 1999). In mangrove plants,
iron oxide plaque has been detected in two species, B.
gymnorrhiza and E. agallocha (Pi et al. 2010). This
study found that A. marina and R. stylosa can also
form iron oxide plaque on their root surfaces
(Fig. 1a). Even in the same plant species, the amount
of plaque fluctuates due to several factors, such as
root oxidizing ability and availability of Fe2+ in the
soil. For example, Mendelssohn and Postek (1982)
reported that amounts of root iron oxide plaque of
Spartina alterniflora varied from 32 g g1 root dry
weight in inland areas to 1,648 g g1 root dry weight
in stream side areas. Therefore, plaque on mangrove
plant roots would also be expected to fluctuate
according to the habitat and plant conditions. In this
study, A. marina rapidly increased its root iron oxide
plaque after 120 days from the start of the experiment.
The three species all began to produce aerial roots at
this time (data not shown): pneumatophores for A.
marina, prop roots for R. stylosa and knee roots for B.
gymnorrhiza. The great enhancement of iron oxide
plaque in A. marina after 120 days might be due to
the development of the effective pneumatophore
aerial root system. Furthermore, Fe2+ concentration

in soil pore-water increased remarkably after A.

marina colonization (Fig. 1b). This high availability
of Fe2+ might be another reason for the high
accumulation of iron oxide plaque on roots. Although
we measured iron oxide plaque using young fine roots
(<2 mm diameter), plaques on thicker roots, such as
mature roots and cable roots, should be different.
Root anatomy, such as diameter, length, root porosity
(aerenchyma), and surface lignification (a barrier for
radial oxygen loss from roots), are critical factors
affecting oxygen diffusion through roots to the
rhizosphere. Therefore, studies on relationships between iron oxide plaque and root anatomy are needed
to improve our understanding of mangrove soil
chemistry.
The remaining molecular oxygen that is not
consumed by Fe2+ oxidation may be directly used as
an electron acceptor by heterotrophic, methaneoxidizing and nitrifying bacteria for energy generation. This study suggests the possibility that mangrove plant roots act to lower methane production
(Fig. 1d). This is consistent with field observations
that methane production in mangrove forests is
comparatively low (Harriss et al. 1988; Strangmann
et al. 2008). In anaerobic soils, methane is the
terminal compound of carbon degradation. When
oxygen-releasing roots exist, methane-oxidizing bacteria can oxidize this methane to carbon dioxide. This
process would contribute to lower methane concentrations in the soil and prevent the diffusion of soil
methane to the atmosphere. Besides the contribution
of methane-oxidizing bacteria, there is a possibility of
a lowered contribution by methane-producing bacteria
(methanogens). Methanogens are activated under low
redox potentials, below 150 mv, (Yang and Chang
1998), and thus the oxic rhizosphere of mangroves

Plant Soil (2011) 346:259273

may be unsuitable for methanogenesis. In this study,

we observed gradually increased methane concentrations with elapsed time in control pots (Fig. 1d). This
suggests that the redox potential gradually decreased
in soil uncolonized by mangroves. Furthermore, high
production of Fe(III)-oxides on root surfaces stimulates bacterial Fe(III) reduction, because Fe(III)reducing bacteria can use Fe(III)-oxides as electron
acceptors to oxidize organic compounds (Lovley
1995). This microbial Fe(III)-oxide reduction may
possibly suppress methanogenesis and sulfatereducing bacteria, because these processes compete
for the same carbon in the root zone (Roden and
Wetzel 1996; Biswas et al. 2007; Huang et al. 2009).
The low methane concentrations in mangrove pots
observed in this study might be attributed to the
resultant combination of enhanced bacterial methane
oxidization, lowered methanogenesis and competition
with Fe(III)-reducing bacteria in the oxidized rhizosphere. The contribution of each process would vary
depending on many factors, including the amount
of nutrient input and plant growth condition.
Recently, it has been reported that human impacts,
such as inputs of high nutrient loadings from
sewage, aquaculture and agriculture, could be
contributing to enhanced methane production in
mangrove ecosystems (Kreuzwieser et al. 2003;
Alongi et al. 2005; Strangmann et al. 2008; Chauhan
et al. 2008; Penha-Lopes et al. 2010).
For inorganic nitrogen, the reduced form of NH4+
tends to be abundant in anaerobic soils. The presence
of oxygen will lead to oxidation of NH4+ to NO2 by
ammonia oxidizing bacteria, and the NO2 is then
continuously oxidized to NO3 by nitrite oxidizing
bacteria. Results of this study suggest that the roots of
the three mangrove species also possess the ability to
change nitrogen metabolism in rooted soil to produce
NO2 and NO3 (Fig. 3a, b). In addition to the aerobic
autotrophic nitrification process, recent studies have
suggested the possibility of anaerobic autotrophic
nitrification at the expense of electron acceptors such
as Fe and/or Mn (Mortimer et al. 2004; Krishnan et al.
2007). Krishnan and Bharathi (2009) reported that the
nitrification rate in mangrove sediments showed a
positive relationship with Fe (contained in the
sediment) and with the abundance of autotrophic
nitrifiers. Accumulation of iron oxide plaque on the
root surface observed in this study might also
contribute to anaerobic autotrophic nitrification. The

269

resultant NO2 and NO3 will be converted to

gaseous N2 and/or N2O (denitrification) that will
diffuse to the atmosphere. Besides denitrification,
assimilation by bacteria and absorption by plant roots
may also contribute to decreased NO2 and NO3
concentrations in the soil pore-water. In this study,
concentration curves of NO2 and NO3 seemed to be
saturated 60 days from plant colonization. This
suggests that the combination of input (nitrification)
and output (denitrification, bacterial assimilation and
plant absorption) processes had reached steady state.
Although the concentrations of NO2 and NO3 seem
to have reached steady state 60 days from plant
colonization, NH4+ concentrations decreased remarkably for the first 120 days from plant colonization
(Fig. 2b). Such a low NH4+ concentration in soil porewater is consistent with field observations (within the
M range) in tropical mangrove forests (Alongi et al.
1992). In our previous field study, we also observed
lower inorganic nitrogen concentrations in soil porewater in the root zone of the three mangroves
compared with areas having no roots (Inoue et al.
2011). In the field study, production of NO2 and
NO3 in the root zone was also detected as found
here. In this study, relatively lower NO2 concentrations in A. marina compared with those in the
other two species was observed (Fig. 3b). This
suggests that A. marina had low nitrite inputs
(nitrification) and/or high nitrite outputs (nitrite
oxidization and/or denitrification).
Fe and P mobilization
Along with the iron oxide plaque formation on root
surfaces, we also observed increases in Fe2+ concentrations in the soil pore-water of pots growing A.
marina (Fig. 1a, b). These observations suggest that
A. marina roots have the greatest ability to mobilize
Fe to form clear Fe2+-Fe(III) oxide contrasts in the
rhizosphere among the three species. Fe mobilization
is promoted by reduction under conditions of low
redox potential and chelation with organic acids such
as exudates from plant roots. Otero et al. (2006)
reported that Fe2+ concentrations were higher in the
upper soil layer where mangrove plant (L. racemosa,
Rhizophora mangle L., Avicennia schaueriana Stapf.
et Leechm. and S. alterniflora) live roots were
abundant. They also found that the redox potential
in the upper soil layer was higher than in lower layers.

270

These observations suggest that enhanced Fe2+ was

not due to chemical reduction, but to chelation with
root exudates. In this study, enhanced Fe2+ concentrations in A. marina might have also attributed to
chelation with organic acids released from roots. We
did not measure root exudates in the experimental
pots, and thus further investigation into root exudates
in the three mangrove species is needed. So far,
exudates from four mangrove species (K. obovata, B.
gymnorrhiza, E. agallocha and H. fomes) have been
characterized (Haoliang et al. 2007; Kumar et al.
2009). After 120 days from plant colonization, the Fe2+
curve for A. marina seemed to reach saturation. This
suggests the existence of processes consuming Fe2+
such as Fe oxide deposition and plant absorption, and
that input (Fe mobilization) and output (Fe2+ consumption) processes reached steady state.
Although enhancement of Fe2+ was observed only
in A. marina, phosphate mobilization was observed in
all three species in this study (Fig. 1c). As with
ferrous ion concentrations, phosphate ion concentration curves for A. marina reached saturation 120 days
after the plant colonization, suggesting the steady
state of input (P mobilization) and output processes (P
immobilization and plant uptake). Phosphate displacement from Al/Fe-P is promoted by chelation with
organic acids (Gyaneshwar et al. 2002). Enhancement
of phosphate in this study may be attributed to
organic acids from roots and/or phosphate solubilizing bacteria. Among the three species, enhancement
of phosphate in soil pore-water was remarkable in A.
marina. El-Tarabily and Youssef (2010) isolated rock
phosphate solubilizing bacteria from the rhizosphere
of A. marina and one of them showed a high ability to
produce a variety of organic acids, as well as acid and
alkaline phosphatases. Considering the different
phosphate enhancement between A. marina and the
other two species, phosphate-solubilizing substrates
may also differ among the three species.
Nitrogen enrichment
The most remarkable change mangrove plants made
in this study was in soil nitrogen enrichment (Fig. 2a).
Oxygen-releasing roots also support free-living heterotrophic nitrogen fixers associated with their roots
and rhizomes (Stoltzfus et al. 1997; Bagwell et al.
1998; Lovell et al. 2000). The nitrogen fixing process
requires great amounts of energy (carbon). Further-

Plant Soil (2011) 346:259273

more, the enzyme that mediates this reaction, nitrogenase, is extremely oxygen-sensitive. Therefore,
oxygen-releasing root zones that have abundant
carbon in micro-aerobic conditions may create ideal
conditions for nitrogen fixers. Nitrogen fixation has
been observed in sediments and algae in many
mangrove forests (Zuberer and Silver 1978, 1979;
Hicks and Silvester 1985; Holguin et al. 1992, 2001;
Pelegr and Twilley 1998; Lugomela and Bergman
2002). Under field conditions, nitrogen fixation
fluctuates spatially and temporally (Lee and Joye
2006), and there are complex relationships among
many factors. Consequently it has been difficult to
assess plant growth effects on nitrogen fixation. In
this study, we detected higher nitrogenase activity in
mangrove-colonized soils than in the unplanted soils
(Fig. 6, data at 16 cm soil depth). This suggests that
the three mangrove species have the ability to
stimulate nitrogen fixation. It also suggests that the
reported observations of nitrogen fixation in many
mangrove forests may be a consequence not only of
the intertidal habitat, but also of mangrove plant
colonization per se. Furthermore, nitrogenase activity
in the experimental pots differed significantly among
the three species in this study. This suggests the
possibility that rooted plant species may be a factor
determining the capability for nitrogen fixation. In
this study, we compared soil nitrogen fixation among
the plant species using a 16 cm soil depth. Soil
chemical and biological properties vary significantly
with soil depth (Alongi et al. 1992), and thus the
spatial profile of nitrogen fixation requires further
study. We also detected nitrogenase activity in the
roots of the three species. Although the mean
values for roots were significantly higher than that
of the uncolonized soil, standard deviations for root
nitrogenase activity of R. stylosa and B. gymnorrhiza
were comparatively high. This suggests that the
distribution of nitrogen fixers on roots is not uniform,
but differs with position on the root (root tip or base,
lateral or main root) and/or with root condition (fresh
or old root).
Among the three species, nitrogenase activity,
relative growth rate and nitrogen use efficiency were
all greatest in B. gymnorrhiza. Furthermore, B.
gymnorrhiza and R. stylosa might re-absorb or retranslocate nitrogen prior to leaf fall, because litter C/
N ratios of the two species were higher than those of
fresh aboveground parts. These observations suggest

Plant Soil (2011) 346:259273

271

that B. gymnorrhiza is the most nitrogen-economical

species (plants which have a high ability to enrich the
nitrogen content of rooted soil, and a high ability to
increase their biomass with low nitrogen supply)
among the three species. In contrast, A. marina
showed lower nitrogen use efficiency than B. gymnorrhiza, and had comparatively high nitrogen concentrations in the plant material. This suggests that A.
marina may have high bacterial nitrogen fixation
around its roots to meet its high nitrogen demand.
However, nitrogenase activities in A. marina soil and
root materials were lowest among the three species.
The nitrogenase activity of the decaying leaf litter was
not measured, but A. marina showed remarkably
higher nitrogen concentrations (low C/N) in its leaf
litter compared with fresh aboveground material in
this study (Fig. 5). This suggests that high bacterial
nitrogen assimilation might accompany degradation
of the A. marina leaf litter. After litter fall, mangrove
litter is gradually enriched in nitrogen via bacterial
nitrogen immobilization, which occurs during decomposition. The rate of nitrogen fixation varies among
plant species, probably because of differences in
chemical composition of the leaves. Generally, leaves
with higher nitrogen content (lower C/N) decompose
more rapidly (Melillo et al. 1984; Twilley et al. 1986)
and leaves with higher phenolics contents decompose
more slowly (Cundell et al. 1979; Gonzlez-Farias
and Mee 1988). In this study, the aboveground
nitrogen content was highest in A. marina, followed
by R. stylosa and B. gymnorrhiza. Previous studies on
the leaf composition of other mangrove species, for
example, Avicennia germinans and Rhizophora mangle have reported that A. germinans leaf litter has
higher initial nitrogen content and lower phenolics
content (Robertson 1988), while R. mangle leaf litter
has lower initial nitrogen (Twilley et al. 1986; Pelegr
et al. 1997) and higher phenolics content (Cundell et
al. 1979; Benner et al. 1986; Robertson 1988). From
these observations, Avicennia species are likely to
have rapidly decomposable leaves (high nitrogen and
low phenolics), thereby enriching the nitrogen content
of the litter layer accumulated on the soil surface.

although the extent of change differed among the

species. Colonization by the three mangrove species
resulted in effects on soil chemical properties associated with oxidation: deposition of iron oxide on
mangrove root surfaces, enhanced NO2 and NO3,
and decreased CH4 concentrations in soil pore-water.
Also, mobilization of ferrous ion (in A. marina) and
phosphate ion (in all three species) occurred, probably
due to root exudates. For nitrogen, the three mangrove species significantly stimulated bacterial nitrogen fixation in rooted soils, leading to soil nitrogen
enrichment. While the insoluble soil nitrogen content
increased, dissolved inorganic nitrogen in soil porewater significantly decreased.
Among these properties, phosphate mobilization
and soil nitrogen enrichment are likely to be particularly important for the growth of mangrove plants,
because phosphate and nitrogen are generally limited
in mangrove ecosystems (Feller et al. 2003a, b;
Lovelock et al. 2004; Reef et al. 2010). Strongly
weathered tropical soils tend to contain low soluble
phosphate concentrations, with most of the phosphate
existing as immobile Al/Fe-P, Ca-P and organic
matter-P. For nitrogen, export by repeated tidal
fluctuation occurs in seaward regions (Boto and
Robertson 1990). Although nitrogen and phosphate
supply to mangrove plants seems to be insufficient for
their optimum growth in many practical field conditions (Feller 1995; Feller et al. 2003a, b; Lovelock
et al. 2004), the self-supporting nitrogen and phosphorus ability of mangroves observed in this study
could be one key to the high productivity of
mangrove ecosystems.

Conclusion

Alongi DM, Boto KG, Robertson AI (1992) Nitrogen and

phosphorus cycles. In: Robertson AI, Alongi DM (eds)
Tropical mangrove ecosystems. American Geophysical
Union, Washington DC, pp 253292
Alongi DM, Tirendi F, Dixon P, Trott LA, Brunskill GJ (1999)
Mineralization of organic matter in intertidal sediments of

All three mangrove species showed changes in the

four measured soil chemical properties, iron, phosphate, nitrogen and methane, in their rooted soils,

Acknowledgments We thank Ms. S. Matsumoto for her

assistance with laboratory measurements. We also thank Mr.
M. Hashizume and Mr. T. Takagi for their comments and
advice. This work has been partly supported by a Grant-in-Aid
for Young Scientist (B) (No. 20770020) from the Ministry of
Education, Culture, Sports, Science and Technology (MEXT)
of the Japanese Government.

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