RESEARCH ARTICLE

Ecological Role of Bacteria Involved in the Biogeochemical

Cycles of Mangroves Based on Functional Genes Detected
through GeoChip 5.0
Shanshan Meng,a Tao Peng,a Xiaobo Liu,b Hui Wang,a Tongwang Huang,a Ji-Dong Gu,a,b Zhong Hua,c

a Department of Biology, Shantou University, Shantou, Guangdong, People’s Republic of China

b Environmental Science and Engineering Research Group, Guangdong Technion-Israel Institute of Technology, Shantou, Guangdong, People’s Republic of China
c Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou, Guangdong, People’s Republic of China

Shanshan Meng and Tao Peng contributed equally to this study; author order is alphabetical.

ABSTRACT Mangroves provide a variety of ecosystem services and contribute greatly to

the global biogeochemical cycle. Microorganisms play important roles in biogeochemical
cycles and maintain the dynamic balance of mangroves. However, the roles of bacteria in
the biogeochemical cycles of mangroves and their ecological distribution and functions
remain largely uncharacterized. This study thus sought to analyze and compare the eco-
logical distributions and potential roles of bacteria in typical mangroves using 16S rRNA
gene amplicon sequencing and GeoChip. Interestingly, the bacterial community composi-
tions were largely similar in the studied mangroves, including Shenzhen, Yunxiao,
Zhanjiang, Hainan, Hongkong, Fangchenggang, and Beihai mangroves. Moreover, gamma-
proteobacterium_uncultured and Woeseia were the most abundant microorganisms in the
mangroves. Furthermore, most of the bacterial communities were significantly correlated
with phosphorus levels (P , 0.05; 20.93 , R , 0.93), suggesting that this nutrient is a

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vital driver of bacterial community composition. Additionally, GeoChip analysis indicated
that the functional genes amyA, narG, dsrA, and ppx were highly abundant in the studied
mangroves, suggesting that carbon degradation, denitrification, sulfite reduction, and pol-
yphosphate degradation are crucial processes in typical mangroves. Moreover, several
genera were found to synergistically participate in biogeochemical cycles in mangroves.
For instance, Neisseria, Ruegeria, Rhodococcus, Desulfotomaculum, and Gordonia were syn-
ergistically involved in the carbon, nitrogen, and sulfur cycles, whereas Neisseria and
Treponema were synergistically involved in the nitrogen cycle and the sulfur cycle. Taken
together, our findings provide novel insights into the ecological roles of bacteria in the
biogeochemical cycles of mangroves.
IMPORTANCE Bacteria have important functions in biogeochemical cycles, but studies on
their function in an important ecosystem, mangroves, are still limited. Here, we investi-
gated the ecological role of bacteria involved in biogeochemical cycles in seven repre-
sentative mangroves of southern China. Furthermore, various functional genes from bac- Editor Garret Suen, University of Wisconsin—
Madison
teria involved in biogeochemical cycles were identified by GeoChip 5.0. The functional
Copyright © 2022 Meng et al. This is an open-
genes associated with the carbon cycle (particularly carbon degradation) were the most access article distributed under the terms of
abundant, suggesting that carbon degradation is the most active process in mangroves. the Creative Commons Attribution 4.0
International license.
Additionally, some high-abundance bacterial populations were found to synergistically
Address correspondence to Zhong Hu,
mediate key biogeochemical cycles in the mangroves, including Neisseria, Pseudomonas, [email protected].
Treponema, Desulfotomaculum, and Nitrosospira. In a word, our study gives novel insights The authors declare no conflict of interest.
into the function of bacteria in biogeochemical cycles in mangroves. Received 30 November 2021
Accepted 15 December 2021
KEYWORDS GeoChip, bacterial community, biogeochemical cycle, functional Published 12 January 2022
characterization, mangrove

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Meng et al.

B iogeochemical cycles are critical components of ecosystem dynamics and contrib-

ute to the degradation of refractory organic materials as well as the recycling of
nutrients, toxic elements, carbon, nitrogen, sulfur, and phosphorus. Biogeochemical
cycles can be either directly or indirectly altered by human activities. Direct effects
include changes in the biological, chemical, and physical properties and processes of
the environment (1). However, global warming and climate change may threaten the
balance of biogeochemical cycles (2). For example, global warming might lead to the
loss of large organic carbon stocks in soils (3). Mangroves store larger amounts of car-
bon (up to 1,023 Mg/ha) than other major global forests (4). However, global man-
grove carbon storage decreased by 158.4 metric tons from 1996 to 2016 (5).
Furthermore, nitrogen (N) and phosphorus (P) delivery along the land-water contin-
uum are increasing due to climate change. Climate change is likely to increase terres-
trial biomass delivery into water bodies and accelerate aquatic biomass production
and turnover, thereby potentially increasing the magnitude and frequency of nutrient
(P and N) release events (6, 7). Additionally, the sulfur cycle promotes iron deposition
and further phosphorus release in freshwater ecosystems. The recent rise in the global
sea level threatens to disrupt coastal wetlands, thus altering biogeochemical cycling in
mangrove ecosystems (8). Moreover, several studies have indicated that bacteria play
an important role in biogeochemical cycles. For example, particle-associated bacteria
seem to play a much more important role in biogeochemical cycles than free-living
bacteria (9).
Bacteria are present in almost all ecosystems, both terrestrial and aquatic, and play
crucial ecological roles (10). For example, bacteria mediate the mineralization of labile
carbon (C) (11). Moreover, anammox bacteria play a vital role in the nitrogen cycle (12),
and rhizobia are involved in nitrogen fixation (13). Bacteria affect local and global bio-
geochemical cycles by absorbing organic carbon and nutrients (14, 15), and therefore,
the study of these microorganisms is key to understanding ecosystem dynamics.
Mangroves are intertidal wetlands that play crucial ecological roles in tropical and
subtropical coastlines worldwide (16). These ecosystems have immense ecological im-
portance due to not only their biodiversity but also their increasing association with
human activities (17). Furthermore, mangroves also provide several ecosystem services,

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including the amelioration of wind effects and coast protection (18, 19). Recently, 16S
rRNA gene amplicon sequencing studies have substantially contributed to our knowl-
edge of the phylogeny and community structure of mangrove bacteria (10, 20).
Additionally, previous studies have demonstrated that mangroves are uniquely rich in
microbial diversity, which in turn contributes greatly to their ecosystem dynamics (21,
22). Another study reported that biogeochemical processes were impacted by many
factors such as human activities, microbes, and viruses (23). Therefore, disruption of
natural biogeochemical cycles would result in insurmountable damage. Recent studies
have reported that mangroves are important carbon sinks (24, 25). Mangroves contrib-
ute 10% to 15% (24 Tg C year21) and 10% to 11% of carbon to oceans and terrestrial
ecosystems, respectively (26). Furthermore, large quantities of organic matter may
accumulate in mangroves, which promotes microbial growth and increases biodiver-
sity. Other studies have demonstrated that biogeochemical cycles are largely driven by
microbial activity (27). For example, the dominance of communities involved in the
nitrogen cycle and CO2 fixation has been found to vary between drought and rewet-
ting cycles (28). Moreover, Pseudomonas, Stenotrophomonas, and Serratia effectively
removed nitrogen in volcanic scoria (29). Bacterial communities can also affect biogeo-
chemical cycles (30). For example, Syntrophobacter, Sulfurovum, Nitrospira, and
Anaerolinea could potentially drive the coupling of the carbon, nitrogen, and sulfur
cycles in the Yunxiao (YX) mangrove (10). However, there is a lack of research on other
mangroves. Furthermore, 2% of the world’s mangroves are located in China. The
southeast coast is the main distribution area of mangroves in China. Therefore, we
selected seven research areas along the southern coastline of China that covered the
main distribution of mangroves (31, 32).

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Bacteria Involved in Biogeochemical Cycles

Here, we sought to investigate the ecological distribution and potential roles of

bacteria in the biogeochemical cycles of representative mangroves of southern China
based on the expression of functional genes detected by GeoChip analysis to (i) char-
acterize the distribution of functional genes in south China mangroves; (ii) explore the
key ecological functions involved in the carbon, nitrogen, sulfur, and phosphorus
cycles of typical mangroves of China; and (iii) identify bacteria that synergistically par-
ticipate in biogeochemical cycles in typical mangroves of China. Therefore, the findings
of this study provide novel insights into the ecological roles of bacteria in biogeochem-
ical cycles in mangroves, thus establishing a theoretical basis for the development of
strategies to safeguard the ecological stability of mangroves.

RESULTS
Correlation between bacterial communities and physicochemical parameters.
The bacterial community distribution in the studied mangroves was described in our
previous study (33). Concretely, our previous findings demonstrated that the bacterial
community structures of the different mangroves were largely similar. Many bacteria in
these mangroves were unclassified and uncultured; however, members of gamma pro-
teobacterium_uncultured and the Woeseia genus were abundant in most of the studied
mangroves (see Fig. S1 in the supplemental material). To explore the interactions between
bacterial communities and physicochemical parameters, a network was constructed and an-
alyzed. There were more interactions between P and bacterial communities. For example,
Hoppeia, gamma proteobacterium_uncultured, ADur.Binl 20, Marinifilaceae_uncultured, and
Draconibacterium were negatively correlated with P (P , 0.05; 20.82, R, 0.86). Marini-
filaceae_uncultured also exhibited a negative correlation with salinity. Particularly, Hoppeia
had a significant negative correlation with P. Moreover, subgroup 23, Desulfobact-
eraceae_uncultured, Aestuariivivens, the Sva0081 sediment group, Bacteroidetes_uncultured,
Steroidobacteraceae_unclassified, and Desulfatiglans were positively correlated with P.
Steroidobacteraceae_unclassified and subgroup 23 had a significant positive correlation with
P, whereas Steroidobacteraceae_unclassified had a negative correlation with NO32 (nitrate).
Furthermore, Bacteroidetes BD2-2, Marinilabiliaceae_uncultured, and Lentimicrobiaceae_
unclassified had a positive correlation with total nitrogen (TN) and total carbon (TC). The cor-

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relation between Lentimicrobiaceae_unclassified and Bacteroidetes BD2-2 with TN was
extremely significant. S exhibited a positive correlation only with Woeseia, which was most
abundant in the Fangchenggang mangrove (FCG-S) (Fig. S1). NO22 (nitrite) had a significant
positive correlation with Flavobacteriaceae_unclassified. Moreover, there was a significant
negative correlation between salinity and Robiginitalea. Interestingly, Marinilabiliaceae_
uncultured was not only significantly positively correlated with TN but also significantly neg-
atively correlated with salinity (Fig. 1).
Ecological functions of genes detected in south China mangroves. A total of 17
ecological functions were detected in the studied mangroves, most of which (over
20%) were associated with metal homeostasis. Particularly, these ecological functions
were mostly associated with metal homeostasis, stress, the carbon cycle, antibiotic re-
sistance, organic contaminant degradation, and the nitrogen cycle, whereas electron
transfer, plant growth promotion, and protists were limited (Fig. 2). Biogeochemical
cycles are very important in maintaining the stability of mangroves, particularly the
carbon, nitrogen, sulfur, and phosphorus cycles. Furthermore, the abundance of car-
bon cycle-associated genes exceeded 15%, whereas the abundance of those associ-
ated with the nitrogen cycle reached 5%. In contrast, the abundance of sulfur cycle
and phosphorus cycle genes was ,5% (Fig. 2).
(i) Carbon cycle genes and ecological functions. Carbon cycle processes included
carbon degradation, carbon fixation, and methane metabolism. Furthermore, the rela-
tive abundance of functional genes associated with carbon degradation was generally
high (69%) in the mangroves, whereas that of genes associated with methane metabo-
lism was the lowest (3%) (Fig. S2A). A total of 13 processes were linked to carbon deg-
radation (Fig. S2B), among which functional genes associated with starch degradation
were the most abundant (26%). These starch-degrading functional genes included the

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FIG 1 Correlation network between bacterial communities and environmental factors. Pink dots, bacterial
communities; green squares, environmental factors; red lines, negative correlation; black lines, positive correlation.
Thicker lines indicate P values of ,0.001.

amyA, amyX, apu, cda, glucoamylase, isopullulanase, nplT, and pulA genes. Among
these, amyA was the most abundant in starch degradation and even carbon degrada-
tion, whereas apu was the least abundant in starch degradation (Fig. 3a). Moreover,
the chitinase gene, which is involved in chitin degradation, was also abundant in the
studied mangroves (Fig. 3a). In carbon fixation, the genes with the highest relative
abundances were those associated with the Calvin cycle (Fig. S2B), whereas those asso-

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ciated with the 3-hydroxypropionate/4-hydroxybutyrate cycle had the lowest relative
abundances. Moreover, tktA was most abundant in carbon fixation (Fig. 3b). In the
methane cycle, the genes with the highest relative abundances were those associated
with methanogenesis (Fig. S2B). harB was the most abundant gene associated with the

FIG 2 Relative abundances of genes linked to 17 ecological functions in typical mangroves of China.

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FIG 3 Relative abundances of functional genes in biogeochemical cycling. (a) Carbon degradation; (b) carbon fixation (red)
and methane (yellow); (c) nitrogen cycling; (d) sulfur cycling; (e) phosphorus cycling. FBP, fructose 1,6 bisphosphate aldolase;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PRK, phosphoribulokinase; cnorB, genes encoding the nitric oxide
reductase catalytic subunit; 5f1_htxA, genes determining phosphorus oxidation; 5f1_ptxD, genes determining phosphorus
oxidation.

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methane cycle (Fig. 3b). Interestingly, the numbers of genes involved in the carbon
cycle were similar among various mangroves.
(ii) Nitrogen cycle genes and ecological functions. Functional genes involved in
the processes of denitrification, nitrogen fixation, ammonification, assimilatory N
reduction, dissimilatory N reduction, nitrification, assimilation, N assimilation, and
anammox were detected in this study. Genes associated with denitrification were the
most abundant (45%) (Fig. S3). Moreover, nitrogen fixation accounted for 16.33%,
whereas anammox accounted for only 0.36% (Fig. S3). Additionally, the functional
genes involved in denitrification, including cnorB, narG, nirK, nirS, norB, nosZ, and
p450nor, were found to be highly diverse. Among these, narG was the most abundant
denitrification gene (Fig. 3c). However, the abundances of the above-mentioned func-
tional genes were largely similar among the mangroves. The nifH gene, which is
involved in nitrogen fixation, was also abundant in the studied mangroves. Regarding
ammonification, the ureC gene was the most abundant, whereas the most abundant
gene associated with assimilatory N reduction was nraB. The most abundant nitrifica-
tion gene was amoa_quasI. The nitrate reductase gene was the most abundant in the
process of N assimilation, whereas the hzo gene was the least abundant in the nitrogen
cycle (Fig. 3c).
(iii) Sulfur cycle genes and ecological functions. Several genes associated with
sulfur cycle-associated processes were detected in the mangroves, including dimethyl-
sulfoniopropionate (DMSP) degradation, sulfite reduction, reduction, sulfur assimila-
tion, other sulfur cycles, adenylylsulfate reductase, and sulfite oxidation (Fig. S4). The
genes involved in sulfite reduction were the most abundant (48.5%), whereas those
involved in DMSP degradation exhibited the lowest abundance (2.8%) (Fig. S4).
Interestingly, functional genes related to sulfite reduction, including dsrA, dsrB, and sir,
were highly abundant in these mangroves, indicating strong sulfite reduction activity
in the mangroves. Particularly, dsrB was the most abundant in the Beihai (BH) man-
grove (Fig. 3d). Moreover, the functional genes soxV (sulfur oxidation), cysJ (reduction),
and cysI (other sulfur cycles) also exhibited high abundances in the mangroves
(Fig. 3d), suggesting high sulfur oxidation and reduction activities in the mangroves.
(iv) Phosphorus cycle genes and ecological functions. Regarding phosphorus

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cycle genes, our study detected functional genes associated with polyphosphate deg-
radation (76.3%), polyphosphate synthesis (15.3%), phytic acid hydrolysis (7.9%), and
phosphorus oxidation (0.46%). Those associated with polyphosphate degradation
were the most abundant (Fig. S5), of which the ppx gene was the most abundant. In
contrast, 5f1_htxA and 5f1_ptxD (phosphorus oxidation) were the least abundant
(Fig. 3e), suggesting that the mangroves had low phosphorus oxidation activity.
Bacteria synergistically participate in biogeochemical cycles in mangroves.
More than one bacterial species is commonly involved in any given biogeochemical
process. In the studied mangroves, a total of 12 highly abundant bacteria were known
to synergistically participate in biogeochemical cycles, including Neisseria, Treponema,
Nitrosospira, “Candidatus Kuenenia,” Agrobacterium, Desulfotomaculum, Rhodococcus,
Gordonia, Ruegeria, Nitrosomonas, “Candidatus Brocadia,” and Pseudomonas (Fig. 4).
Neisseria and Treponema are both involved in sulfur reduction and anammox, whereas
Treponema also participates in ammonification and nitrification. Neisseria is also
involved in denitrification. Furthermore, Neisseria and Treponema were both linked to
the sulfur and nitrogen cycles. “Candidatus Kuenenia,” Desulfotomaculum, Rhodo-
coccus, Gordonia, and Ruegeria potentially drive the carbon, nitrogen, and sulfur cycles.
Moreover, some processes were carried out by unique bacteria. For instance,
Agrobacterium was involved in methane oxidation in the studied mangroves. In con-
trast, Nitrosomonas, “Candidatus Brocadia,” and Pseudomonas, which are involved in
denitrification, were more highly abundant. Additionally, certain highly abundant func-
tional genes could be linked to specific processes. For example, the highly abundant
hdrB, mmoX, amyA, and tktK genes are involved in the carbon cycle, whereas nifH, ureC,
nifA, and amoA participate in the nitrogen cycle (Fig. 4).

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FIG 4 Major functional genes and functional communities involved in biogeochemical cycles in typical mangroves. Dots, bacterial community
(only functional genes and communities with high abundances are shown).

DISCUSSION
In this study, the bacterial community of the studied mangroves was primarily rep-
resented by members of gamma proteobacterium_uncultured and the Woeseia genus.
However, unclassified and uncultured bacteria accounted for a sizeable proportion of
the bacterial community, suggesting that mangroves harbor a wide variety of novel
bacteria. Some studies have demonstrated that gamma proteobacterium_uncultured
participates in biogeochemical cycles in various environments such as factories and
soil (34, 35). Furthermore, recent studies have indicated that gamma proteobacteriu-
m_uncultured has a negative correlation with P. In fact, most bacteria also have a corre-
lation with P (Fig. 1), suggesting that bacteria are primarily driven by P. The genus
Woeseia belongs to the Gammaproteobacteria subgroup, as described previously by
Zhang et al. (36), and was highly abundant in the Fangchenggang mangrove. Woeseia
possesses a repertoire of genes for hydrocarbon degradation, as described previously
by Bacosa et al., suggesting that this genus is linked to the carbon cycle (37). However,
this genus was more significantly correlated with S in this study (P , 0.01) (Fig. 1), sug-

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gesting that it could be involved in the sulfur cycle. Additionally, Liu et al. proposed
Steroidobacteraceae as a new family (38), and many unclassified members of this family
were found in this study. These bacteria exhibited a positive correlation with P and a
negative correlation with NO32 in the studied mangroves (Fig. 1). This indicated that
the mangroves were rich in novel microbial resources, which was consistent with the
observations of Li et al. (39). Some bacterial communities that have also been corre-
lated with other physiochemical parameters (TN, TC, and NO22) are thought to be
closely related to key biogeochemical cycles. For example, Marinifilaceae_uncultured,
which reportedly utilizes a wide range of carbon sources (40), was correlated with TN
and TC in the studied mangroves (Fig. 1). Flavobacteriaceae have been reported to uti-
lize diverse carbon sources in marine environments, suggesting a correlation with the
carbon cycle (41). However, Flavobacteriaceae_unclassified was significantly correlated
with NO22 in our study, suggesting that it may be involved in the nitrogen cycle.
Furthermore, salinity is an important driver of global bacterial community structures
(42). Mangroves often exhibit extremely high salinities, and these conditions shape the
local bacterial communities (43, 44). In our study, Marinifilaceae_uncultured and
Robiginitalea were negatively correlated with salinity (Fig. 1), indicating that salinity
mainly affects their distribution.
Many functional genes associated with metal homeostasis were identified in a pre-
vious study (33). However, our study determined that mangroves are uniquely rich in
functional genes associated with biogeochemical cycles (e.g., the carbon, nitrogen, sul-
fur, and phosphorus cycles) via GeoChip 5.0 analysis. GeoChip is a functional gene
array that enables the analysis of the functional diversity, composition, structure, and
metabolic potential/activity of bacteria, which can also be used to establish links

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between functional genes and microbial communities (45, 46). However, the efficiency
of this approach largely depends on the number of probes. GeoChip has been used to
detect functional genes in various environments, such as high-arsenic groundwater
(47), pastures (48), and freshwater ecosystems (49). Bai et al. studied the functional
gene diversity and metabolic potential in the Zhanjiang (ZJ) mangrove using GeoChip
4.0 (50). In our study, the functional genes were detected in the mangroves using
GeoChip 5.0, which contains more than three times more probes than GeoChip 4.0
and can thus provide novel insights into the biogeochemical cycles of mangroves. Our
results demonstrated that carbon cycle-associated genes were the most active in the
studied mangroves. Mangroves sequester large amounts of carbon and can therefore
become a significant source of greenhouse gases when disturbed by changes in land
use. These ecosystems thus play an important role in climate mitigation (51, 52).
Therefore, maintaining the carbon cycle balance in mangroves is of crucial importance.
Starch is among the main organic compounds in mangrove sediments and can be
degraded into glucose and maltose (53), thus providing a carbon source for microbes
to grow. The relative abundance of carbon degradation-associated genes was high in
the studied mangroves, particularly those linked to starch degradation (see Fig. S2A in
the supplemental material). The high abundance of amyA, which participates in the
degradation of starch, could help maintain a stable carbon cycle in mangroves while
also providing a rich carbon source to support the growth of mangrove microbes. Few
studies have characterized the importance of carbon degradation in mangroves, and
therefore, our findings provide important insights into mangrove dynamics. Our study
demonstrated that carbon degradation was the most active process in the mangroves.
Methane is the second most important greenhouse gas after CO2. Rising global tem-
peratures and human activities may increase the CH4 efflux rate in coastal ecosystems
(54). According to Zhang et al., hydrogenotrophic and methylotrophic methanogenesis
are the dominant pathways in mangrove sediments, as demonstrated by functional
gene analysis (55, 56). In our study, the abundance of functional genes associated with
methane metabolism was generally low (Fig. 3d), indicating that the methane cycle
processes were less active in the studied mangroves. Among the genes involved in the
methane cycle, the relative abundance of hdrB was higher than that of mmoX, suggest-

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ing that methane synthesis was more active than methane oxidation.
Mangroves are also important nitrogen sinks that can remove approximately 6% of
anthropogenic nitrogen inputs into the environment (57). Functional genes involved
in denitrification, nitrogen fixation, ammonification, assimilatory N reduction, dissimila-
tory N reduction, nitrification, assimilation, N assimilation, and anammox were
detected in the studied mangroves. The denitrification process oxidizes nitrates to
nitrogen and it is very important to maintain a dynamic nitrogen balance in mangroves
(58). Functional genes associated with denitrification were also highly abundant
(Fig. S3), suggesting that denitrification was a highly active process in the mangroves.
The narG gene, which is involved in denitrification, was highly abundant in the man-
groves, indicating that this gene contributed greatly to the nitrogen cycle. Moreover,
the nifH gene (involved in nitrogen fixation) was also highly abundant in the man-
groves (Fig. 3c), indicating that nitrogen fixation was also a vital process in the man-
groves. In contrast, hzo had the lowest relative abundance. This gene is involved in the
anammox process, suggesting that anammox occurs slowly in the mangroves. These
results also indicated that the occurrence of denitrification-associated genes might be
explained by the availability of nitrite, as reported previously by Luvizotto et al. (59).
Moreover, nitrogen fixation contributed substantially to the nitrogen input in man-
grove sediments (60). Taken together, our findings indicate that denitrification and
nitrogen fixation are dominant in the mangroves, both of which maintain the dynamic
balance of nitrogen and further preserve the stability of the biogeochemical cycles of
mangroves.
Additionally, functional genes associated with the sulfur cycle were also detected in
the mangroves. The ecological functions linked to the sulfur cycle include sulfite

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reduction, sulfide oxidation, other sulfur cycles, reduction, adenylylsulfate reductase,

sulfur assimilation, and DMSP degradation. Compared with other processes of the sul-
fur cycle, sulfite reduction-associated genes had the highest abundances in the man-
groves, whereas those linked to DMSP degradation were the least abundant, suggest-
ing that sulfite reduction was dominant in the studied mangroves, whereas DMSP
degradation was slow in the mangroves. Some studies have indicated that DMSP is a
major driver of the sulfur cycle and may have a strong impact on climate (61, 62).
However, although DMSP degradation is very important in the sulfur cycle, this process
was not dominant in the studied mangroves, indicating that the accumulation of
DMSP does not drive the sulfur cycle in these ecosystems.
Regarding the phosphorus cycle, the relative abundance of genes associated with
polyphosphate degradation was the highest, suggesting that polyphosphate degrada-
tion is the main process in the mangroves. The ppx gene, which is involved in poly-
phosphate degradation, was also found to be highly abundant, and therefore, we con-
cluded that the ppx gene dominated the polyphosphate degradation process in the
mangroves. Interestingly, functions and genes were not significantly correlated within
different mangroves.
A wide variety of bacteria have been linked to several biogeochemical cycles (10).
In this study, the bacterial community compositions across different mangroves were
very similar; however, their relative abundances varied. Furthermore, this study dem-
onstrated that a large number of bacterial species were involved in the biogeochemi-
cal cycles of mangroves. Some bacteria have been found to be synergistically involved
in biogeochemical processes. For example, Gordonia, Desulfotomaculum, Neisseria,
Rhodococcus, and Ruegeria are mainly involved in carbon fixation, denitrification, and
adenylylsulfate reduction in mangroves (Fig. 4). Ruegeria is a denitrifying marine bacte-
rium with the potential ability for cyanophycin synthesis (63). We speculate that
Ruegeria utilizes carbon for growth and oxidizes sulfur into sulfites, after which it oxi-
dizes sulfites to sulfate and hydrogen sulfide in mangroves. Our findings also sug-
gested that Ruegeria played crucial roles in the mangrove ecosystem dynamics as it
synergistically participated in the carbon and sulfur cycles in the mangrove.
Rhodococcus is known to reduce hydrogen sulfide but has also been reported to de-

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grade a wide variety of natural and synthetic organic compounds (64). Our results
demonstrated that Rhodococcus could be involved in the nitrogen cycle in mangroves,
suggesting that this genus is likely involved in various functional metabolic pathways
in mangrove ecosystems. Furthermore, Treponema was also found to be involved in
the nitrogen cycle, as reported in a previous study (65). In our study, Treponema was
found to be involved in the nitrogen and sulfur cycles in the studied mangroves.
Moreover, both Treponema and Neisseria participated in the nitrogen and the sulfur
cycles. Treponema and Nitrosospira also participated in the nitrogen cycle (Fig. 4), sug-
gesting that Treponema plays an important role in the nitrogen and sulfur cycles in the
mangroves. Furthermore, a previous study reported that Nitrosospira could be involved
in the nitrogen cycle (66). Our findings indicated that Nitrosospira was also involved in
the sulfur, carbon, and nitrogen cycles in the mangroves, indicating that Nitrosospira
played a key role in the biogeochemical cycles of the studied mangroves. Additionally,
our findings indicated that Pseudomonas participated only in denitrification (nitrogen
cycle), as demonstrated by the high abundance of denitrification-associated functional
genes. Agrobacterium and Streptomyces also exhibited high abundances of genes
involved in the carbon cycle.
Conclusion. This study investigated the community structures and ecological func-
tions of bacteria in seven representative mangroves of southern China. No significant
differences in bacterial community composition were observed among the mangroves,
suggesting that there were no geographic differences in the functional genes of the
studied mangroves. Moreover, the functional genes associated with the carbon cycle
(particularly carbon degradation) were the most abundant, suggesting that carbon
degradation is the most active process in mangroves. Additionally, some high-

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Meng et al.

abundance bacterial populations were found to synergistically mediate key biogeo-

chemical cycles in the mangroves, including Neisseria, Pseudomonas, Treponema,
Desulfotomaculum, and Nitrosospira. These findings thus provide novel insights into
the ecological functions of bacteria in mangrove biogeochemical cycles.

MATERIALS AND METHODS

Sample collection. Superficial (0- to 5-cm) sediment samples were collected in February 2018 from
seven different southern China mangroves, Beihai (BH), Fangchenggang (FCG), Zhanjiang (ZJ), Hainan
(HN), Shenzhen (SZ), Yunxiao (YX), and Hongkong (HK), using sterile plastic tubing. Three samples were
collected from each mangrove (the rhizosphere of Kandelia candel). All sediment samples were stored at
4°C and taken back to the laboratory within 48 h after collection. The physiochemical parameters of the
mangroves were investigated in our previous study (33). Detailed information is provided in the supple-
mental material.
Functional gene analysis. Functional genes putatively associated with biogeochemical cycles were
detected via GeoChip 5.0 analysis as described previously by Meng et al. (33). Total DNA was extracted
from mangrove sediment samples as described previously by Zhou et al. (67). Briefly, the samples were
freeze-dried in a vacuum freeze-drier. Next, 0.25 g of sediments was weighed from each sample to con-
duct phenol extractions. The DNA was then evaluated and labeled using random primers specific for
functional genes associated with biogeochemical processes. Afterward, the labeled DNA was purified
and transferred to a Labconco Centrivap concentrator (Labconco Corp., Kansas City, MO) to dry at 50°C
for 45 min. Prior to hybridization, the DNA was incubated at 95°C for 5 min and maintained at 42°C. The
labeled DNA was then placed into a hybridization station (Maui; BioMicro Systems, Salt Lake City, UT,
USA) and preheated at 42°C for at least 5 min. Finally, optical signals were obtained with a NimbleGen
MS200 scanner (Roche, Madison, WI, USA) and then converted to digital signals using ImaGene 6.0 soft-
ware (Biodiscovery Inc., El Segundo, CA, USA) to obtain the probe signal intensity. Samples were consid-
ered positive if spots with a signal-to-noise ratio of .2.0 were detected in at least 2/3 of the replicate
sets. The data were then normalized using logarithm transformation, and the mean signal intensity of
each sample was determined. Next, each probe’s intensity was normalized to the average intensity of
the corresponding sample. Functional gene data (GeoChip 5.0) are provided in the supplemental
material.
16S rRNA gene sequences. The primer pair 515F (59-GTG YCA GCM GCC GCG GTA A-39) and 806R
(59-GGA CTA CNV GGG TWT CTA AT-39) was used to amplify hypervariable region 4 (V4) of bacterial 16S
rRNA genes. Afterward, clean data were analyzed using QIIME 2 version 2-2020.2 (68). Representative
sequences of different operational taxonomic units (OTUs) were aligned with the SILVA SSU database
138 for taxonomy.
Data analysis. Correlation was calculated using the Spearman method. Furthermore, an interaction
network of bacterial communities and physicochemical parameters in mangrove sediments was con-

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structed using Cytoscape 3.7.2 software (69). Functional gene expression heat maps were created with
the TBtools tool kit (70).
Data availability. All 16S rRNA gene sequences were submitted to the Sequence Read Archive
(SRA) database under accession number PRJNA556990.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
FIG S1, TIF file, 0.3 MB.
FIG S2, TIF file, 0.3 MB.
FIG S3, TIF file, 0.1 MB.
FIG S4, TIF file, 0.1 MB.
FIG S5, TIF file, 0.1 MB.
FIG S6, TIF file, 0.9 MB.
DATA SET S1, XLSX file, 3.3 MB.

ACKNOWLEDGMENTS
We thank the sequencing company Magigene for technical support and the
National Natural Science Foundation for support.

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