1
Influence of Macrofaunal Burrows on Extracellular Enzyme Activity and Microbial
2
Abundance in Subtropical Mangrove Sediment
3
Ling Luo1,2, and Ji-Dong Gu2*
4
1
5
Chengdu, Sichuan Province, People of Republic of China
6
2
7
The University of Hong Kong, Pokfulam Road, Hong Kong SAR, People’s Republic of
8
China
9
* Corresponding author
College of Environmental Sciences, Sichuan Agricultural University, Huimin Road,
Laboratory of Environmental Microbiology and Toxicology, School of Biological Sciences,
10
Mailing address: School of Biological Sciences, The University of Hong Kong, Pokfulam
11
Road, Hong Kong SAR, People’s Republic of China
12
Tel.: (+852) 2299-0605; fax: (+852) 2559-9114; e-mail address: [email protected]
13
14
Abstract
15
Bioturbation and bioirrigation induced by burrowing macrofauna are recongnized as
16
important processes in acquatic sediment since macrofaunal activities lead to the alteration of
17
sediment characterisitics. However, there is a lack of information on how macrofauna
18
influences microbial abundance and extracellular enzyme activity in mangrove sediment. In
19
this study, the environmental parameters, extracellular enzyme activities and microbial
20
abundance were determined and their relationships were explored. Sediment samples were
21
taken from the surface (S) and lower layer (L) without burrow, and crab burrow wall (W) and
22
bottom of crab burrow (B) locating at the Mai Po Nature Reserve, Hong Kong. The results
23
showed that the burrowing crabs could enhance the activities of oxidase and hydrolases. The
24
highest activities of phenol oxidase and acid phosphatase were generally observed in B
25
sediment, while the highest activity of N-acetyl-glucosaminidase was found in W sediment.
26
The enzymatic stoichiometry indicated that the crab-affected sediment had similar microbial
27
N and P availability relative to C, lower than S but higher than L sediment. Furthermore, it
28
was found that the highest abundance of both bacteria and fungi was showed in S sediment,
29
and B sediment presented the lowest abundance. Moreover, the concentrations of phosphous
30
and soluble phenolics in crab-affected sediment were almost higher than the non-affected
31
sediment. The alterations of
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envrionmental factors by the activities of crabs might be the main reasons for the changes of
33
enzyme activity and microbial abundance. Finally, due to the important role of phenol
phenolics, C:P, N:P ratio as well as undetermined
34
oxidase and hydrolases in SOM decomposition, it is necessary to take macrofaunal activities
35
into consideration when estimating the C budget in mangrove ecosystem in the future.
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Keywords Extracellular enzyme activity · Microbial abundance · Crab burrow · Mangrove
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sediment
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Introduction
39
In aquatic ecosystems, benthic macrofauna in sediment has attracted more and more attention
40
of scientists. It is recongnized that the burrows, consisting of macrofauna, including crabs,
41
shrimps, polychaetes, larvae and so on, have great implications for the microbiological and
42
biogeochemical processes in sediment [1-5]. Recently, numerous macrofauna has been
43
proposed as ecosystem engineers since they can reshape the structure of aquatic sediment by
44
digging burrows [3,6,7]. Moreover, they also play a key role in alterations of chemical and
45
biological properties of the sediment through a wide range of activities such as secreting
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mucus, excreting feces, feeding, and so on [1,3,8,9].
47
To date, there is an increasing evidence that the reworking of macrofauna in aquatic
48
sdiments can stimulate the nutrient cycling and the decomposition of sediment organic matter
49
(SOM), thereby enhance microbial activity [10-13]. However, a negative relationship
50
between bacteria and macrofauna/meiofauna has also been reported due to the competition
51
for food between them [14]. It is well known that extracellular enzyme activities are linkages
52
between microbial community dynamics and the ecosystem persperctives because they can
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catalyze SOM decomposition and the recycling of nutrients such as nitrogen (N) and
54
phosphrous (P) [3,15,16].
55
Extracellular enzyme activity is not only affected by environmental conditions but also
56
reflects the available resources for microbial community [16]. For example, phenol oxidase
57
(PHO), an “enzymic latch” of carbon (C) storage, is able to degrade recalcitrant phenolics
58
like lignin [17-19]. β-glucosidase (GLU), hydrolyzing cellulose and polymeric saccharides to
59
glucose,
60
N-acetyl-glucosaminidase (NAG) and acid phosphatase (ACP) often serve as indicators for N
61
and P acquisition, respectively [21,22]. Furthermore, the ratios of selective enzyme activities
62
are applied to estimate the recalcitrance of SOM and microbial nutrient acquisition [22,23].
is
the
most
commonly
measured
indicator
for
C
dynamics
[20].
63
Mangrove ecosystems, along most tropical and subtropical coastlines, are highly
64
productive due to the litter fall, debris production, trapping water suspension, and so on
65
[6,24-26]. Mangroves grow in seawater between land and sea, and support a number of
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microbial, meiofaunal and macrofaunal communities [6,10,25]. In recent years, the
67
importance of mangrove ecosystems in food source and global carbon budget has been
68
recognized increasingly [6,24,25]. Compared to freshwater and marine sediment, the
69
macrobethons of mangroves are relatively poorly known [4,8,27]. The existing research
70
mainly focuses on the distribution and abundance of macrofauna [27] across mangroves, their
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physical disturbance on sediment processes through burrowing [6], and the influence of
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macrofauna on the properties and availability of organic carbon by the foraging and feeding
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activities [24]. However, the effect of macrofauna on microbial diversity and activity in
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mangrove sediment is scarcely studied relative to freshwater and marine sediment. In
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mangrove forests, the benthic macrofauna is usually domintaed by burrowing crabs which
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can be largely seen. Generally, the crabs are herbivores, and act as workers who can retain,
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bury, macerate and ingest litter [6,24,25,28]. Based on the important role of crabs in
78
participating in litter decomposition, therefore, it is plausible to assume that the crabs could
79
affect the extracellular enzyme activity and microbial abundance in burrow sediment.
80
In this study, it aimed to explore how fiddler crabs influence the biogeochemical and
81
microbiological parameters of sediment in mangove. The physicochemical properties of
82
burrow sediment, including pH, water content, soluble phenolics, total carbon (C), total
83
nitrogen (N) and total phosphorus (P), were measured. Subsequently, the microbial
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abundance (both bacterial and fungal) was determined by quantitative polymerase chain
85
reaction (qPCR) analyses, and the measurement of enzymatic activities (including PHO,
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GLU, NAG and ACP involved in C, N and P cycling) was used to analyze the microbial
87
activities. Finally, we also explored which environmental variables correlated best with the
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changes of enzymatic and microbial parameters.
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Materials and Methods
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Study site and sampling procedure
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In January 2014, samples were collected from the intertidal mangrove at Mai Po Nature
92
Reverse of Hong Kong, China. The Mai Po Nature Reserve (lying between 22°29'N and
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22°31'N and between 113°59'E and 114°04'E) locates on the edge of Deep Bay at the North
94
West New Territories of Hong Kong. It is the largest stand of mangrove in Hong Kong,
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covering an area of 130 ha [25,26,29]. The most prominent crab is fiddler crab, Uca sp. and
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their burrows can be easily seen under the mangrove [25]. The burrows of Uca sp. extend
97
from 10- 20 cm into sediment and their shapes look like a “J”, similar with our determination
98
and observation [6].
99
Sediment from the burrows were assigned to one of the four categories: Surface (S)
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sediment was collected from the top 0-1 cm of the sediment 5 cm away from any burrows;
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Lower layer (L) sediment, usually defined as anoxic sediment, was taken at a depth of 5-6 cm
102
under Surface; Wall (W) sediment was sampled from the wall of burrow at a thickness of
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0-0.5 cm; and Bottom (B) sediment was collected from the bottom of burrows at a depth of
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15-20 cm [1,3,4]. The burrows (approximately 15 burrows) were randomly selected to take
105
sediment across the mangrove forest until the amounts of each sample were enough to
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conduct the following tests. Sediment subsamples were pooled together to form a composite
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sample (each type of sample consisting of 15 subsamples due to the small amounts of
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sediment sampling from each burrow) [3]. After collection, samples were immediately
109
transported on ice to the laboratory and stored at -20 °C for further analyses.
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Sediment properties
111
The pH was measured by using an IQ180G Bluetooth Multi-Parameter System (Hach
112
Company, Loveland, CO, USA). The water content was determined with the oven drying
113
method to a constant weight (approximately 105 ºC for 48 h). The procedures of determing
114
the concentration of soluble phenolics were according to the method of Toberman et al. [17]
115
(details of determining soluble phenolics showed in supplementary information).
116
Furthermore, elemental analyzer (Eurovector EA3028, UK) was employed to determine the
117
content of TC and TN, while TP was measured according to an analytical protocol developed
118
by the Standards Measurements and Testing Program of the European Commission (SMT
119
protocol) [26].
120
Enzyme assays
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In this study, four enzymes involved in C, N and P cycling were investigated, including
122
phenol oxidase (PHO), β-glucosidase (GLU), N-acetyl-glucosaminidase (NAG) and acid
123
phosphatase (ACP). The substrate and buffer used for enzyme assays are listed in Table 1,
124
and all enzymes were assayed spectrophotometrically, following the protocals described
125
elsewhere [30] (details of protocals showed in supplementary information). All enzyme
126
activities values were calculated on the basis of oven-dry (105 ºC) weight of sediment, and
127
expressed as μmol product released g-1 dry sediment h-1. The GLU:PHO ratio was applied as
128
the indicator for the recalcitrance of SOM [22]. In addition, the ratios of GLU:NAG and
129
GLU:ACP were calculated to represent N and P acquisition relative to C, repectively [23].
130
Quantitative PCR analyses
131
Whole community DNA was extracted from 0.25 g sediment using MoBio PowerSoil DNA
132
Isolation Kit (Carlsbad, CA, USA) and stored at -20 ºC. Quantitative polymerase chain
133
reaction (qPCR) assays were used to assess the gene abundance of the microbial communities.
134
To estimate the bacterial abundance, the PCR primers Eub338 and Eub518 were applied to
135
target the 16S rRNA gene [31]. For the fungal abundance, the PCR primers ITS1-F and 5.8S
136
were used [31]. Ten-fold serial dilutions of the plasmid DNA ranged from 109 to 103 were
137
subjected to qPCR assay in triplicate to generate external standard curves, and negative
138
control was also applied. Each sample was also determined in triplicate (details of qPCR
139
procedures
showed
in
supplementary
information).
In
addition,
the
ratio
of
140
fungal-to-bacterial abundance (F:B) was estimated to reflect the robust microbial community
141
composition [32].
142
Statistic analyses
143
Origin 8.0 was applied to analyze significance of enzyme activity and microbial abundance
144
among Surface, Wall, Lower layer and Bottom sediment by one-way analysis of variance
145
(ANOVA). In order to test the interrelationships among microbial abundance, enzyme
146
activity as well as environmental variables, Canoco 4.5 and SPSS 21.0 were employed to
147
perform redundancy analysis (RDA) and pearson correlation analysis, respectively. Moreover,
148
Tukey test was used to determine significant differences among samples. Statistical
149
significance was accepted at p < 0.05.
150
Results
151
Sediment properties
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Characteristics of these samples are summarised in Table 2. Highest water content and pH
153
value were recorded in Bottom sediment, while the lowest were found in Wall and Lower
154
Layer sediment. The concentration of soluble phenolics ranged from 12.45 (L) to 35.51 (B)
155
mg/kg dry sediment. Surface sediment showed highest TC (2.88%) and TN (0.27%), but
156
lowest TP (0.19%). Meanwhile, the lowest TC (2.01%) and TN (0.19%) were recorded in L
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sediment, and B sediment showed the highest value of TP (0.246%) which was close to W
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(0.243%).
159
Enzymatic activity
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Enzymatic responses to bioturbation of crabs were highly variable among different samples
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(Fig. 1a). Among them, mean activity of PHO ranged from 2.25 to 9.29 μmol product g-1 dry
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sediment h-1, and no significant differences of PHO activity were found (p > 0.05), with the
163
exception of Bottom sediment which showed extremely higher activity of PHO (p < 0.05).
164
For GLU activity, maximal value was recorded in Surface sediment (3.48 μmol product g-1
165
dry sediment h-1), while the lowest was observed in Lower Layer sediment (0.52 μmol
166
product g-1 dry sediment h-1). Moreover, notable differences were detected among all samples
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(p < 0.05) except that between Wall and Bottom sediment (p > 0.05). Interestingly, NAG
168
activtiy, ranging from 0.62 to 1.38 μmol product g-1 dry sediment h-1, showed a similar trend
169
with GLU activity, and the only exception is that there was no significant difference between
170
Surface and Bottom sediment (p > 0.05). And surprisingly, the trend of ACP activity was
171
inconsistent with GLU and NAG activity. The value of ACP activity, with a range between
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9.86 and 12.45 μmol product g-1 dry sediment h-1, ranked in a descending order of Bottom >
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Wall > Lower Layer > Surface sediment. Meanwhile, significantly higher activity of ACP (p
174
< 0.05) was presented in crab-affected sediment (i.e., Wall and Bottom) than non-affected
175
sediment (i.e., Surface and Lower Layer).
176
The enzymatic ratios were calculated and are showed in Fig. 1b. It is obvious that
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enzymatic ratios varied widely among these samples. The ratio of GLU:PHO ranged from
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0.23 (Lower Layer) to 1.25 (Surface), and significant differences (p < 0.05) were found
179
among samples, with the exception of that between Lower Layer and Bottom (p > 0.05). By
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contrast, the changing pattern of GLU:NAG (ranging from 0.84 to 2.50) and GLU:ACP (from
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0.05 to 0.35) were similar with the highest value in Surface sediment and the lowest value in
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Lower layer sediment of the no burrow sediment. Similarly, apparent differences were
183
evident among Surface, Wall and Lower Layer sediment (p < 0.05), but no significant
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difference between Wall and Bottom of the burrowing area (p > 0.05).
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Microbial abundance
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The changes of microbial abundance were analyzed and are showed in Fig. 2a. The bacterial
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abundance ranged from 1.95×1010 to 8.85×1010 copies per gram dry sediment, while the
188
range of fungal abundance was from 8.58×108 to 5.73×109 copies per gram dry sediment. It is
189
evident that bacterial abundance in Surface sediment was considerably higher (p < 0.05) than
190
the other three samples, and no differences were found among Wall, Lower Layer and
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Bottom sediment (p > 0.05). The trend of fungal abundance was quite different from bacterial
192
abundance and decreased in the order of Surface > Wall > Lower layer > Bottom sediment.
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Concomitantly, fungal abundance differed strongly from each other (p < 0.05). In Fig. 2b,
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F:B ratio varied with each other, and statistially significance was observed. The maximal
195
value (0.116) was recorded in Wall, while the minimal value (0.044) was in Lower layer
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which was close to the Bottom sediment. In addition, the ratio in Lower layer and Bottom
197
sediment were statistically different from that in Surface and Wall sediment ( p < 0.05). Also,
198
Surface and Wall sediment were evidently different (p < 0.05).
199
Interrelationships among sediment properties, enzymatic activity and microbial abundance
200
The redundancy analysis (RDA) and pearson correlation analysis were adopted to explore the
201
correlations relating environmental variables to enzyme activity and microbial abundance
202
(Fig. 3, Table 3 and Table 4). In Fig. 3a, NAG and GLU activity were closely related to TC,
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indicating a positive relationship, and the pearson correlation coefficients (Table 3) supported
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this positive relationship was statistically significant (p < 0.05). By contrast, PHO and ACP
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activity appeared to be more closely related to water content, phenolics and C:N, and
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observably postive relationships (p < 0.05) between these two enzyme activity and the
207
above-mentioned environmental factors were observed in Table 2. Furthermore, fungal and
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bacterial abundance were approached to N:P and C:P, but only bacterial abundance showed
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markedly positive relationship with C:P and N:P (p < 0.05).
210
The analyses relating environmental variables to enzymatic ratios and F:B ratios (Fig. 3b
211
and Table 3) indicated that GLU:NAG was notably related to pH (p < 0.05), while GLU:ACP
212
comparatively correlated with TC and TN (p < 0.05). Nevertheless, there was statistically
213
non-significant relationship between GLU:PHO and environmetnal conditions (p > 0.05), so
214
was the correlation between F:B and environmental variables (p > 0.05). The relationships
215
between enzymatic and microbial parameters were also investigated with RDA and Pearson
216
analysis (Fig. 3c and Table 4). A positive relationship was only observed between fungal
217
abundance and GLU:PHO (p < 0.05), which emphasized the role of fungi in the
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decomposition of recalcitrant organic matter.
219
Discussion
220
By far the most attention has focused on the microbial activities in burrow walls compared
221
with surrounding sediment, such as the rate of nitrification and denitrification [11], the
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distribution and diversity of ammonia-oxidizing microorganisms [33], the alteration of
223
bacterial community structure [3,4], and etc. However, there are no reports exploring the
224
influence of macrofauna on the activities of extracellular enzymes which are responsible for
225
the decomposition of organic matter and the cycling of nutrients. It is widely acknowleged
226
that the measures of extracelluler enzyme activities and ratios of commonly measured
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enzyme potentials can be used as indicators of microbial nutrient demand [15,23,34].
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Likewise, enzyme ratios can correlate the functional organization of microdecomposer
229
communities to environmental conditions since extracellular enzyme activity is not only
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affected by environmental variables but also feeds back on microbial community composition
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[16]. Therefore, the information provided by extracellular enzyme activities could help
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understaning the influence of crabs on the growth of microorganisms and the turnover of
233
SOM.
234
In the current study, we have explored how fiddler crabs affect the microbiological and
235
biogeochemical parameters of sediment in a subtropical mangrove ecosystem. Due to 5 cm
236
far away from any openings of the burrows, Surface and Lower layer sediment were assumed
237
as non-affected sediment, while Wall and Bottom sediment of burrows were considered as
238
crab-affected ones. Our results showed that the characteristics of sediment were indeed
239
altered by burrowing of fiddler crabs since the different characteristics of non-affected and
240
crab-affected sediment were observed (Table 2). Interestingly, soluble phenolics and TP
241
content, rather than TC and TN, were more affected by the burrows of fiddler crabs, which
242
were largely neglected in previous studies.
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The variations of C and N contents in burrow walls in relation to surface or lower layer
244
sediment have attracted much research interest [1,10,11,28,33]. For instance, macrofauna
245
(e.g., crab and earthworm) have been found to enhance TC and TN contents of burrow walls
246
in comparison to surrounding sediment, and also lower content of TC and TN in burrow walls
247
relative to surface sediment were reported [5,12,35]. Nevertheless, there is little information
248
referred to P content of burrow walls in comparison to surface or surrounding sediment.
249
Commonly, P has been accepted as the limiting factor in aquatic environments, and is not
250
readily replenished as P is derived primarily from weathering of rock and ecosystems have a
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relative constant supply and utilization of P [12,36,37]. As observed in Table 2, TP contents
252
in Wall and Bottom sediment, equal to each other, were higher than that of Surface and
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Lower Layer sediment, which are in agreement with previous findings [12,13]. In general, the
254
main reason for the increase of P in burrow wall sediment mighy be the oxidation of Fe2+ into
255
Fe3+ and the precipitation of P into Fe(OOH)-PO4 after the formation of Fe(OOH) [7,12,13].
256
On the other hand, the increase of P might be partially explained by the organic-rich
257
secretions of crabs, such as faecal pellets, mucus-linings, and so on [12,38]. The feces and
258
linings are usually rich in sulphate and phosphate [1,24]. Therefore, it is plausible that the
259
activities of burrowing carbs could directly impact the nutrient cycling in mangrove sediment,
260
thereby accordingly changing the microbial activity including enzymatic activity and
261
microbial abundance.
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This study showed the considerably higher concentration of soluble phenolics in Bottom
263
sediment than the other three samples (Table 2). However, until now, no related studies have
264
estimated or compared the concentration of soluble phenolics in Surface, Wall, Lower Layer
265
and Bottom sediment. By comparing these four samples together, only Bottom sediment had
266
higher water content, which was consisent with the findings that crab burrowing increased
267
soil water content [10]. Also, a markedly positive relationship between water content and
268
soluble phenolics was observed in the current study (p < 0.05) (Table S1), which was in
269
agreement with our previous finding that lower water content mighg reduce the leaching of
270
phenolics from litter and plant materials [26]. Hence, the retention of tide water in Bottom
271
sediment might explain the higher soluble phenolics. Moreover, it is well-known that
272
mangrove crabs play an essential role in the removal of leaf litter due to the foraging and
273
feeding activity of crabs, thereby affecting the availability of leaf litter on the forest floor and
274
its subsequent export. Beyond that, many crabs typically take the leaves down in their burrow
275
for storage, where they continue to decompose [10,24,27,39]. Considering both the higher
276
concentrations of phenolics in leaf litter and the leaching of phenolics through the
277
decomposition of leaf litter inside the burrows of crabs, it is reasonable that Bottom sediment
278
showed higher concentration of soluble phenolics [40].
279
Soluble phenolics have been proposed as inhibitors of hydrolase activities, and thus
280
contributing to the low rates of organic matter decomposition in several ecosystems (e.g.,
281
peatland soils) [41]. Unfornately, it seems that there was non-significant relationship between
282
soluble phenolics and hydrolase actvities in the current study. Instead, markedly positive
283
relationships between soluble phenolics and PHO as well as ACP activity were revealed (p <
284
0.05), and also between water content and these two enzyme activities (Table 3). At the same
285
time, PHO activity was found to be positively associated with ACP activity (p < 0.05) (Table
286
S2).
287
Based on the above-mentioned mechanisms of the highest TP and soluble phenolics in
288
Bottom sediment, two possible reasons might be proposed to explain these evidently positive
289
correlations among PHO, ACP, water content and soluble phenolics. One is because crab
290
activities could alter the oxidation reaction (such as oxidation of Fe2+) through transporting
291
oxygen, solutes or other oxidant from surface to burrow sediment [12,13]. Due to the
292
precipitation of P as Fe(OOH)PO4, microorganisms in Wall and Bottom sediment might
293
acquire soluble reactive P, thus increasing the activity of ACP. On the other hand, the
294
secretion of crabs probably contains high recalcitrant organic matter (e.g., phenolics) or is
295
rich in P substrates, thereby accordingly inducing higher activity of PHO or ACP in Bottom
296
sediment [12,38,40,42,43]. Moreover, PHO has been considered as an independent reagent
297
that catalyzes the oxidation of Fe2+, which presumably deciphered the remarkable
298
interrelationship between PHO and ACP activity. Finally, the variations of sediment C:N
299
were closely related with water content and soluble phenolics (Table S1), therefore, to some
300
extent this might account for the higher PHO and ACP activity in crab-affected sediment
301
(especially Bottom sediment).
302
GLU and NAG activity responded similarly to environmental variables and significantly
303
correlated with TC (Table 2). Several studies have showed the correlations relating soil C to
304
GLU as well as NAG, indicating the role of these enzymes in the conversion of total organic
305
matter stock [42,43]. Furthermore, we found that GLU was also remarkably related to NAG
306
(Table S2), which is in accrodance with the findings of Šnajdr et al. [44] and our previous
307
study [26]. This result implies that the production of GLU often accompanies with NAG due
308
to the maintennace of C:N ratio by microorganisms. Hence, the fluctuations of GLU and
309
NAG occurred in concert and showed the same or similar trends along with the changes of
310
environmental conditions. The same varaitions of ACP with GLU and NAG activity were
311
observed according to both the nutrients needed by microorganisms and the nutritional
312
condition of ecosystems [22,26]. However, this is not the case in the current study due to the
313
enhanced TP in burrow sediment through the activities of crabs.
314
In addition to investigating enzyme activity, the ratios of enzyme activity were
315
calculated to estimate the relative recalcitrance of sediment (i.e., GLU:PHO) and the
316
acquisition of N and P (GLU:NAG and GLU:ACP, respectively) in our study. With
317
comparison to global mean GLU:PHO ratio of sediment (0.202), all of four samples were
318
greater (Fig. 1a), indicating that these sediment are more labile, especially Surface sediment
319
with a value of 1.25 [22]. In contrast, it is evident that the GLU:PHO ratio of Wall sediment
320
was higher than its surrounding sediment (Lower Layer). This implies that the lability of
321
SOM was altered due to the mucoid secretions by crabs, which might be easily degradable
322
[22,45]. Furthermore, the analyses relating the ratio of GLU:PHO to environmental variables
323
and microbial parameters showed no remarkable correlations in this research, except with
324
fungi (Fig. 3b, Fig. 3c, Table 2 and Table 3). This being said, fungi appear to play an
325
important role in the labiality of mangrove SOM. And, coincidently, a strongly positive
326
relationship between GLU:PHO ratio and fungal abundance was also found when the
327
influence of mangrove roots on enzyme activity as well as microbial abundance in the same
328
ecosystems (unpublished data) was studied. Collectively, therefore, the role of fungi in the
329
labiality of SOM is substantially important, and is manifested as the greater labiality along
330
with higher fungal abundance increases rates of decomposition and microbial growth [22].
331
The ratios of GLU:NAG in Surface, Wall and Bottom sediment are approaching the
332
global average of 2.08, excpet for Lower Layer sediment with a lower value of 0.84. Thus, it
333
implies that microorganisms in Lower Layer sediment might acquire more N for growth and
334
cell maintenance in comparison to other three compartments, and also suggests that the
335
activities of crabs might modify the N acquisition of sediment. Meanwhile, the alteration of
336
GLU:ACP ratio indicates the similar trend with that of GLU:NAG, but strikingly lower
337
values than the global mean of 1.64 [22], suggesting that all sediment are P-limited. However,
338
relatively speaking, the P availability of Wall and Bottom are observably higher than Lower
339
Layer seiment, despite drasticlly lower than Surface sediment. Therefore, the enhanced P
340
availability by crabs has been manifested, and might stimulate the growth of microorganisms.
341
Collectively, the enhanced N and P availability in Wall and Bottom sediment could result in a
342
significantly increase of microbial growth [10].
343
Unexpectedly, in our study, a decreased bacterial abundance in burrow-affected
344
sediment (Wall and Bottom sediment) relative to non-affected sediment (Surface and Lower
345
Layer sediment) was observed .It is inconsistent with previous findings that the abundance of
346
microorganisms or at least certain fuctional groups in burrow-affected sediment were
347
elevated relative to that of non-affected sediment. Since burrow walls or linings, which have
348
characteristics including alteration of oxic-anoxic conditions, organic/nutrient content as well
349
as sediment structures, could provide an attractive and beneficial environment for
350
microorganisms living [1,3,8,12]. Nevertheless, there are still several literuatures reporting no
351
enhancement or reduction of bacterial abundance in burrow walls. And according to several
352
published literatures, it can be assumed that the possible reason for the lack of strong
353
bacterial enrichment in the burrow wall might result from the extensive meiofaunal grazing
354
along the burrow walls as well as the direct ingestion by macrofauna [1,8,9,46].
355
Simultaneously, the results of analyses relating bacterial abundance to environmental
356
conditions suggested that bacterial abundance closely correlated with C:P and N:P, but not the
357
content of TC, TN and TP. This implies that what the bacteria require is not only the
358
concentration of nutrient but also the equilibrium of sediment C:N:P ratio due to the different
359
C:N:P ratios of specific microbial taxa [47].
360
By contrast to bacterial abundance, our results showed fungal abundance was not
361
strongly related to any environmental factors, suggesting that the variations in fungal
362
community structure are probably caused by yet unknown environmental drivers and or by
363
stochastic events in sediment habitats [48]. In reality, the robust alteration of microbial
364
community composition (i.e. F:B ratio) was made up by the activities of crabs (Fig. 2b). The
365
ongoing studies have established that macrofauna could alter the structure and diversity of
366
microbial communities in coastal marine sediment or freshwater sediment [4,8], which was in
367
agreement with our findings, since the activities of macrofauna lead to the alterations in the
368
microbial transformation of important nutrients at the sediment-water interface. Besides the
369
above mentioned observations, no dominant environment factors showed any prominently
370
relationships with the F:B ratio. After considering both the changes of microbial abundance
371
as well as the possible reason for their variations, the grazing of bacteria by larger organisms
372
might be attributed to the weak relationships of F:B ratio with environmental parameters [9].
373
Alternatively, the reason is similar to fungal abundance that the variations of F:B ratio are
374
induced by yet unknown environmental drivers [48].
375
Results show that the burrowing crabs can change the nutrient cycling and quality of
376
SOM, as revealed by the shifts of sediment elemental composition and extracellular enzyme
377
activity in crab-affected sediment (Wall and Bottom) in comparison to non-affected sediment
378
(i.e. Surface and Lower Layer). Furthermore, this study shows that different extracellular
379
enzyme activity was strongly related to different measured environmental factors, indicating
380
that the changes of extracellular enzyme activity might be corresponded to the shifts of
381
certain relevant environmental parameters. Although the meiofaunal grazing and macrofaunal
382
ingestion presumably caused the underestimated microbial abundance, the alteration of
383
microbial abundance and robust microbial community composition indeed emphasized the
384
role of crabs in microbial growth, and thus the biogeochemical cycling of mangrove
385
sediment.
386
Until now, very little data exist with which to explore the extracellular enzyme activity
387
and microbial abundance in crabs’ burrow sediment in mangrove ecosystems. The findings
388
present here are novel but preliminary, and thus it requires further investigation over longer
389
time periods to get more information. Additionally, it is worthy to conduct relative research
390
that integrates the shift of functional grops (both diversity and abundance) with the nutrient
391
cycling and degradation of burrow SOM. Hopefully, it is expected to provide valuable
392
infromation for understanding C stock and estimating the contribution of macrofauna on
393
climate changes in mangrove ecosystems.
394
Acknowledgments
395
This research project was supported by a Ph.D. studentship from Graduate School, The
396
University of Hong Kong (LL) and financial support of Environmental Toxicology Education
397
and Research Fund of this laboratory.
398
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399
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Tables
535
Table 1 Substrate and buffer used for enzyme assay in sediment of this study
Enzyme
EC
Abbreviation
Phenol oxidase
1.14.18.1
PHO
β-glucosidase
3.2.1.21
GLU
3.2.1.14
NAG
3.1.3.2
ACP
N-acetyl-βglucosaminidase
Acid phosphatase
Substrate
Buffer
L-3,4-dihydroxy phenylalanine
Acetate buffer
(10 mM)
(50 mM, pH 5.0)
p-nitrophenyl-β-ᴅ-
MUB, pH 6.0
glucoside (50mM)
p-nitrophenyl-N-acetyl-
Acetate buffer
β-ᴅ-glucopyanoside (10mM)
(100mM, pH 5.5)
p-nitrophenyl phosphate
MUB, pH 6.5
(50 mM)
536
EC, enzyme commission classification; MUB, modified universal buffer prepared by
537
dissolving 12.1 g of Tris (hydroxymethyl)aminomethane (THAM), 11.6 g of maleic acid,
538
14.0 g of citric acid, and 6.3 g of boric acid (H3BO3) in about 800 mL of 0.5M sodium
539
hydroxide (NaOH), adjust to 1 L with 0.5 M NaOH, and store at under 4 ºC.
540
541
542
543
544
Table 2 Physicochemical properties of sediment in Mai Po Nature Reserve, Hong Kong
Water content (%)
pH
Phenolics (mg/kg)
TC(%)
TN(%)
TP(%)
Surface
53.71
6.21
15.82
2.88
0.27
0.190
Lower
53.92
4.88
12.45
2.01
0.19
0.197
Wall
53.50
6.05
15.18
2.27
0.21
0.243
Bottom
59.33
6.37
35.51
2.48
0.21
0.246
545
Table 3 Pearson’s correlation coefficients (r) relating environmental variables to microbial
546
abundance as well as enzyme activity.
Water content
pH
Phenolics
TC
TN
TP
C:N
C:P
N:P
PHO
0.994**
0.529
0.997**
0.190
-0.130
0.600
0.988*
-0.228
-0.380
GLU
0.150
0.801
0.310
0.963*
0.880
0.047
0.249
0.671
0.584
NAG
0.133
0.816
0.297
0.952*
0.872
0.072
0.241
0.650
0.565
ACP
0.958*
0.561
0.967*
0.027
-0.294
0.781
0.990**
-0.443
-0.582
Bacteria
-0.410
0.117
-0.339
0.786
0.926
-0.733
-0.441
0.988*
0.997**
Fungi
-0.643
0.244
-0.515
0.683
0.864
-0.469
-0.565
0.779
0.822
GLU:PHO
-0.550
0.463
-0.400
0.732
0.874
-0.321
-0.443
0.731
0.756
GLU:NAG
0.243
0.960*
0.411
0.894
0.771
0.246
0.371
0.508
0.411
GLU:ACP
-0.052
0.786
0.107
0.966*
0.950*
-0.120
0.041
0.772
0.713
F:B
-0.518
0.296
-0.386
0.027
0.129
0.350
-0.313
-0.149
-0.087
547
*
p < 0.05, ** p < 0.01, Pearson’s correlation coefficient (r) is given by 𝑟 =
∑𝑖( 𝑥𝑖 −𝑥̅ )(𝑦𝑖 −𝑦̅)
√∑𝑖(𝑥𝑖 −𝑥̅ )√(𝑦𝑖 −𝑦̅)2
548
549
550
Table 4 Pearson’s correlation coefficient (r) relating microbial parameters to enzymatic
551
parameters
552
*
PHO
GLU
NAG
ACP
GLU:PHO GLU:NAG GLU:ACP
Bacteria
-0.366
0.638
0.622
-0.559
0.800
0.475
0.763
Fungi
-0.569
0.656
0.664
-0.643
0.986*
0.554
0.797
F:B
-0.447
0.227
0.270
-0.267
0.565
0.309
0.283
p < 0.05, ** p < 0.01, Pearson correlation coefficient (r) is given by 𝑟 =
∑𝑖( 𝑥𝑖 −𝑥̅ )(𝑦𝑖 −𝑦̅)
√∑𝑖(𝑥𝑖 −𝑥̅ )√(𝑦𝑖 −𝑦̅)2
553
554
Figures captions
555
Fig. 1 Mean (± SD) of (a) extracellular enzyme activity and (b) enzymatic ratio in Surface,
556
Wall, Ambient and Bottom sediments. Statistical significance at p < 0.05 was showed by
557
different alphabets.
558
Fig. 2 Mean (± SD) of (a) microbial abundance and (b) fungi-to-bacteria (F:B) ratio in
559
Surface, Wall, Ambient and Bottom sediments. Statistical significance at p < 0.05 was
560
showed by different alphabets.
561
Fig. 3 Redundancy analyses (a) relating environmental factors to microbial abundance and
562
enzyme activity, (b) relating environmental parameters to the ratios of enzyme activity and
563
fungi-to-bacteria (F:B), and (c) correlating microbial parameters with enzymatic parameters.
564