Comparative Bacterial Community Analysis in Relatively Pristine and Anthropogenically Influenced Mangrove Ecosystems On The Red Sea

Page 1 of 33
For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.
Comparative bacterial community analysis in relatively pristine and
anthropogenically influenced mangrove ecosystems on the Red Sea
Riaz Ullah†1,2, Muhammad Yasir1†*, Imran Khan1,2, Fehmida Bibi1, Sayed Sartaj Sohrab1,
Ahmed Al-Ansari3, Fahad Al-Abbasi2, Abdulmohsin A. Al-Sofyani4, Ihsanullah Daur5,
Seon-Woo Lee6, Esam I Azhar1,7

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by MacEwan University on 04/05/17
1
Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz
University, Jeddah, Saudi Arabia.
2
Biochemistry Department, Faculty of Science, King Abdulaziz University, Jeddah,
Saudi Arabia.
3
Department of Environmental Sciences, Faculty of Meteorology, Environment and Arid
Land Agriculture, King Abdulaziz University, Jeddah, Saudi Arabia.
4
Marine Biology Department, Faculty of Marine Science, King Abdulaziz University,
Jeddah, Saudi Arabia.
5
Department of Arid Land Agriculture, King Abdulaziz University, Jeddah, Saudi Arabia.
6
Department of Applied Biology, Dong-A University, Busan 49315, Republic of Korea.
7
Medical Laboratory Technology Department, Faculty of Applied Medical Sciences,
King Abdulaziz University, Jeddah, Saudi Arabia.
†Authors contributed equally to this study
*Corresponding author: Muhammad Yasir
E mail: yasirkhattak.mrl@gmail.com
1
For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 2 of 33
Abstract
Mangrove habitats are ecologically important ecosystems that are under severe pressure
worldwide because of environmental changes and human activities. In this study, 16S
rRNA gene amplicon deep-sequencing was used to compare bacterial communities in

Red Sea mangrove ecosystems at anthropogenically influenced coastal sites and a
relatively pristine island site. In total, 32 phyla were identified from the mangrove
rhizospheres, with Proteobacteria predominating at each of the studied sites; however, the
relative abundance was significantly decreased at the coastal sites (Mastorah, MG-MS
and Ar-Rayis, MG-AR) compared with the pristine island site near Dhahban (MG-DBI).
The phyla Actinobacteria, Firmicutes, Acidobacteria, Chloroflexi, Spirochetes, and
Planctomycetes were present at a relative abundance of >1% at the MG-MS and MG-AR
sites, but their concentration was <1% at the MG-DBI site. A total of 1659 operational
taxonomic units (OTUs) were identified at the species level, and approximately 945
OTUs were shared across the different sampling sites. Multivariate principal coordinate
data analysis separated the MG-DBI site from the MG-AR and MG-MS cluster. Specific
bacterial taxa were enriched at each location, and in particular, the genera
Pseudoalteromonas and Cobetia were predominantly identified in the MG-DBI site
compared with the anthropogenically influenced coastal sites.
Key words: mangroves, bacterial community, metagenomic, rhizosphere, Red Sea
2
Page 3 of 33
Introduction
Mangrove forests occur at the interface of marine and terrestrial ecosystems and represent
relatively unexplored areas of microbial diversity (Andreote et al. 2012; Nedwell 1994).
Mangroves can be found in more than 100 countries and cover an area of approximately
150,000 km2; the American continent harbors the greatest area of mangroves, followed
by Brazil, with 13,000 km2 and representing 8.5% of the global total (Colares and Melo
2013). Mangrove forests are highly productive environments and are known for their
high levels of organic matter (OM) and nutrients (Nedwell 1994). Mangrove sediments
provide breeding and growth habitats as well as food and shelter for fish (Getter et al.
1984). They act as a buffer zone between the terrestrial and marine environments, and
they play an important role in maintaining sea levels (Duke et al. 2007). In addition,
mangrove forests function as a terminal sink for local waste and nutrient-rich sediments.
These unique environments take up excess nutrients and restrict their entry into coastal
waters, but they do not undergo any major changes in the process (Bouchez et al. 2013).
Moreover, they protect the coast from erosion and reduce the effect of tsunamis.
Unfortunately, mangrove forests are at high risk and are diminishing at the high rate of 1–
2% per year (Kathiresan and Rajendran 2005).
Mangrove ecosystems are complex and provide a dynamic microbial habitat. The
microbial community is very diverse and adaptive because of the high humidity and
periodic tidal flooding. Environmental factors including nutrient availability, light,
salinity, and temperature are the main contributors to the formation of mangrove habitats
(Holguin et al. 2006b). Mangrove bacterial communities mainly consist of the phyla
Proteobacteria, Actinobacteria, Bacteroidetes, Planctomycetes, Acidobacteria,
3
Chloroflexi, Cyanobacteria, Nitrospira, and Firmicutes. Proteobacteria are the most
common, with Gammaproteobacteria, Deltaproteobacteria, and Alphaproteobacteria
being predominant (Basak et al. 2015a). A metagenomic analysis of Brazilian mangrove
forests has revealed that they are rich in Deltaproteobacteria and Gammaproteobacteria;
these Proteobacteria inhibit the growth of Planctomycetaceae, Burkholdreaceae, and

Rhodobacteraceae, which are implicated in sulfur, nitrogen, and methane metabolism
(Andreote et al. 2012).
Previous studies have demonstrated the dependence of plants on soil microbes and for the
restoration ecology, the interaction of plants with rhizosphere microbes has become a
topic of intense study (Harris 2008). To fully understand mangrove habitats, knowledge
of the microbial communities and their activities is vital, particularly in the context of
rehabilitation and restoration (Ghosh et al. 2010). The Red Sea is a unique ecosystem
with unusual and extreme environments, and it differs from other seas and oceans
because of its hypersalinity, low nutrient concentrations, high average temperatures, and
constant high levels of solar radiation (Antunes et al. 2011). Mangrove forests in Saudi
Arabia are situated fragmentally along the Red Sea coast, and are dense in the south
where the main mangrove species is Avicennia marina (El-Juhany 2009). These forests
are under pressure from increased population growth in the coastal regions due to
industrial development and urbanization during the last decade. In this study, we have
analyzed for the first time the bacterial communities in rhizospheres of mangroves
present at two anthropogenically influenced coastal sites and one relatively pristine site
on a Red Sea island, using deep 16S rRNA gene amplicon sequencing to define a
baseline bacterial community profile for healthy mangroves. Increased rhizosphere
4
Page 5 of 33
bacterial diversity was observed for the Red Sea mangroves. Ignavibacteriae,
Thermotogae, Acetothermia, Fusobacteria, and Saccharibacteria were detected in the Red
Sea mangroves; these phyla were not reported in earlier 16S metagenomic studies from
mangroves in other geographical regions (Alzubaidy et al. 2016; Andreote et al. 2012;
Fernandes et al. 2014). In addition, substantial enrichment of specific taxa was observed
in the Red Sea mangroves habitats.
Methodology
Study area and samples collection
Mangrove vegetation occurs in areas along the Red Sea coast of Saudi Arabia. In this
study, three different mangrove fields were selected for sampling based on the mangrove
vegetation and how pristine they were. Sampling site 1 (MG-MS) was located on the
Mastorah coastal area (23°05′09″N, 38°48′41″E) and was densely vegetated compared to
sampling site 2 at Ar-Rayis (MG-AR; 23°36′43″N, 38°31′26″E). The average height of
the mangroves was between 1 and 3 m at MG-MS and 0.5 and 1.5 m at MG-AR. These
two sites were close to an urban area and located between the two major coastal industrial
cities of Rabigh and Yanbu. The sites were polluted with sewage, construction material
from a newly built coastal park, plastic debris, and human litter. Sampling site 3 (MG-
DBI) was an island (22°02′24″N, 38°57′40″E) near Dhahban in the Red Sea (Fig. S1).
Mangrove vegetation was quite dense at MG-DBI compared with the coastal sites and the
average height was 1.5–4 m. Human access to this site is limited, and MG-DBI is
considered to be a relatively clean island without external contamination. The mangrove
fields at all three sites were commonly vegetated by only A. marina. Annual precipitation
in the studied locations is scarce, with an average rainfall of 50–100 mm/year (Hasanean
5
and Almazroui 2015). In general, tides along the central Red Sea coast are a mixed type
and mainly semidiurnal, fluctuating between 0.2 and 0.7 m and inundating the coastal
mangroves as a thin sheet of water. In June 2015 sediment cores (3 cm in diameter and 15
cm long) were collected from the rhizosphere of the selected mangrove fields during low
tide. For each site three sediment cores were collected within a distance of 5 m of each
other. Each replicate was thoroughly mixed in a sterile plastic bag and then stored at
−20°C.
Physicochemical analysis
The environmental parameters of pH, temperature, and conductivity were measured for a
1/10 (w/v) saturated colloidal solution of the sediment in water using the Martini portable
meter (Martini, Australia). The sediment samples were air-dried and then crushed for
uniformity and analytical processing. OM was determined through loss of ignition
according to the protocol of Dean (Dean 1974), while levels of nitrogen were determined
using the Kjeldahl method; for other elements, a 0.5-g sample was digested with a nitric
acid–perchloric acid (HNO3-HClO4) mixture (Alzubaidy et al. 2016). Following
digestion, the solution was diluted with 50 ml of distilled water, and the level of
phosphorus was determined colorimetrically and that of potassium by atomic absorption
spectroscopy.
DNA extraction and 16S rRNA gene amplicon sequencing
Total DNA was extracted from a 0.3-g sediment sample for each replicate using a Power
Soil DNA extraction kit (MoBio Laboratories, Carlsbad, CA) according to the
manufacturer’s instructions. The 16S rRNA genes present in the samples were sequenced
by targeting the V3–V4 region using the overhanging adapter primers 341F
6
Page 7 of 33
(TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG)
and 785R
(GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTA
ATCC) and following the procedure of Yasir et al. (Yasir et al. 2014). DNA
concentrations were measured using a Qubit Fluorometer (Thermo Fisher Scientific,

Massachusetts), and dual-index barcodes and Illumina (San Diego, CA) sequencing
adapters were subsequently joined to the products using a limited PCR cycle. Following
purification with Agencourt AMPure beads (Agencourt, Indianapolis), libraries were
normalized using the Nextera XT protocol. Samples were pooled into a single flow cell
for sequencing via the MiSeq sequencing platform (Illumina) following the
manufacturer’s protocol. Automated cluster generation and paired-end sequencing with
dual index reads were performed in a single run with a read length of 2×300 bp. Raw
FASTQ files were obtained from the Illumina MiSeq, and paired-end reads were
collected using PANDAseq (Masella et al. 2012). Sequences were cleaned of primers and
barcodes, all reads of “N” and those with sequences <250 bp were deleted, and high-
quality sequences were dereplicated (Yasir et al. 2014). The cleaned sequences were then
clustered at k=10 (97% similarity), followed by deletion of chimeras and singleton reads.
Finally, operational taxonomic units (OTUs) were classified using the BLASTn
algorithm against a curated database derived from GreenGenes, RDPII, and NCBI.
Sequence data of this study is available in the NCBI Sequence Read Archive under
accession no. SRP097132.
7
Statistical analysis
The OTU biodiversity and richness were calculated using R package phyloseq (1.7.24),
which was implemented using nonparametric Chao1 and rarefaction analysis (McMurdie
and Holmes 2012). The R VEGAN package (version 2.2-1) was implemented with
package “rich” (0.3) and used to check the richness of the OTUs. Multivariate principal
coordinate analysis (PCoA) was performed using R VEGAN (http://cran.r-project.org,
http://vegan.r-forge.r-project.org/), and the Kolmogorov-Smirnov D test was used to
ascertain the normality of the data. One-way analysis of variance (ANOVA) and Tukey
honest significant difference (HSD) tests were performed to identify significantly
different mangrove rhizosphere bacterial taxa at the three different sampling sites. The
Statistical Package for the Social Sciences (SPSS) version 20 was used for the statistical
analysis.
Results
Physicochemical analysis
The habitats at the studied mangrove sites were relatively similar for most environmental
factors, except for presence of OM and levels of phosphorus (Table 1). Significantly
(<0.05) higher percentages of OM and phosphorus were detected at MG-MS compared
with MG-DBI and MG-AR. The salinity was marginally higher at MG-MS (19.2 ± 1.6
PSU), but the difference was not statistically significant. Temperatures at the study sites
were in the range of 31.7–33.4°C.
8
Page 9 of 33
Bacterial community composition
In total, 1.016 million raw sequence reads were obtained using the MiSeq system
(Illumina). After filtration, approximately 1.015 × 106 high quality (>250 bp) sequence
reads were obtained and assigned to bacterial domains. In total, 13,189 distinct OTUs
were identified with 97% sequence identity, and overall, 32 phyla were identified in the
rhizosphere sediment of the mangrove fields located at the coastal sites and an island in
the Red Sea. Thirty-one phyla were found at all sites, but the phylum Fibrobacteres was
not detected in the MG-DBI. The relative abundance of dominant phyla varied
significantly between the coastal sites and the island (Fig. 1A). The majority of the
sequencing reads were representative of the phylum Proteobacteria, which dominated the
bacterial community at each of the studied sites. The relative abundance of Proteobacteria
was significantly (p<0.05) decreased in the coastal samples MG-MS (64.9±6.8%) and
MG-AR (58.2±2.9%) compared to the island site MG-DBI (89.6±6.7%). Among the
Proteobacteria, Gammaproteobacteria was the dominant class present at each site
(75.9±18.8% MG-DBI, 34±9.5% MG-MS, and 27±5.3% MG-AR), followed by
Deltaproteobacteria (11.3±11.5% MG-DBI, 23.4±6.9% MG-MS, and 25.1±3.4% MG-
AR) (Fig. 1B). The relative abundances of Actinobacteria, Firmicutes, Acidobacteria, and
Spirochaetes were increased at the MG-MS and MG-AR sites compared with the MG-
DBI site. Chloroflexi was present with a significantly (p<0.001) higher abundance at the
MG-MS site (7.1±1.9%) compared with the MG-AR (1.8±0.5%) and MG-DBI
(0.8±0.3%) sites, while Cyanobacteria was dominantly (p<0.005) present at the MG-AR
site (13±4.8%) compared with the MG-MS (0.7±0.5%) and MG-DBI (0.7±0.8%) sites.
Bacteroidetes were commonly present at >3% for all the sampled mangrove fields.
9
In total, 959 OTUs were identified at the genus level, with a greater number of genera
detected in the coastal site samples MG-AR (831) and MG-MS (806) compared with the
island sample MG-DBI (761). The 633 OTUs at the genus level were common among the
three sites, and 65 OTUs were unique to MG-AR, 54 OTUs to MG-MS, and 34 OTUs to
MG-DBI (Fig. S2). Seventy-nine OTUs were common specifically between the MG-MS
and MG-AR, 40 between MG-DBI and MG-MS, and 54 between MG-DBI and MG-AR.
In the island sample, MG-DBI, Pseudoalteromonas (58.9±15.6%) and Cobetia
(9.3±6.4%) genera were predominantly present, but these genera were detected at a
significantly (p<0.05) lower abundance (<1%) in the MG-MS and MG-AR samples.
Oceanospirillum predominated in the MG-MS sample (10.2±108%) compared with the
MG-AR (1.2±1.7%) and MG-DBI (0.1%) samples, while the genera Marinobacterium,
Dehalococcoides, and Syntrophobacter were present at significantly (p<0.05) higher
levels in the MG-MS sample compared with the MG-AR and MG-DBI samples (Fig. 2).
In the MG-AR sample, Pseudomonas, Microcoleus, and Dermocarpella were all present
with a significantly (p<0.05) higher relative abundance compared with the other sites.
The genera Thioprofundum, Spirochaeta, Acidobacterium, Thiohalophilus, Caldithrix,
and Cytophaga were present at relatively the same abundance in the MG-MS and MG-
AR, but they were significantly lower in the MG-DBI. The genus Desulfosarcina was
dominantly present in each of the studied sampling sites.
We observed 1659 different OTUs, with approximately 945 OTUs common among all
the different sampling sites (Table S1). In total, 1576 OTUs were found at the species
level in the anthropogenically influenced costal mangrove ecosystems (Table S2). The
highest number of OTUs was observed at the MG-AR site (1362) followed by the MG-
10
Page 11 of 33
MS site (1320). The lowest level of species diversity was observed for the island
mangrove rhizosphere MG-DBI (1213 OTUs), and species diversity was significantly
(p≤0.05) lower for the island site compared to the coastal sites (Fig. 3). At the MG-DBI
site, Pseudoalteromonas tetraodonis (41.5±6.4%), unclassified Pseudoalteromonas spp.
(15.1±8.9), and Cobetia marina (9.3±6.4%) were present at a relatively higher

abundance, but they were detected at significantly (p<0.05) lower levels of abundance
(<1%) at each of the coastal mangrove sampling sites (Fig. 4). At the MG-MS site
Oceanospirillum linum, Dehalococcoides spp., Sulfurovum spp., and Desulfobacterium
spp. were present at relatively higher levels of abundance compared with the MG-AR and
MG-DBI sites, while at the MG-AR site, Pseudomonas spp., Microcoleus spp., and
Alteromonas spp. were present at relatively higher abundance levels compared with the
MG-MS and MG-DBI sites. The Desulfosarcina spp. were dominantly present at levels
>5% for each of the studied sampling sites; 83 unique OTUs were present at MG-DBI,
135 at MG-MS, and 150 at MG-AR.
Alpha diversity analysis
Alpha diversity was estimated through rarefaction analysis, the Shannon index, and the
Chao1 index (Fig. 5). The lowest OTU richness was observed at the MG-DBI site, while
the highest Chao1 value was observed for the MG-AR site followed by the MG-MS and
MG-DBI sites. Rarefaction analysis indicated variation among the samples and showed
sufficient data coverage for diversity within the samples. The Shannon Wiener index,
calculated at 3% dissimilarity, showed the lowest value of evenness for the MG-DBI site
and the highest value of evenness for the MG-AR and MG-MS sites. The identified
OTUs from all sequence reads were compared using UPGMA distance matrix clustering
11
analysis. The OTUs from MG-DBI at the species level were found to form a separate
cluster from those from MG-MS and MG-AR, and PCoA of the data clustered the MG-
MS and MG-AR species distantly from those found at the MG-DBI site (Fig. 6).
Pseudoalteromonas tetraodonis, Pseudoalteromonas spp., Cobetia marina, and
Pseudoalteromonas mariniglutinosa at the MG-DBI site and Desulfosarcina spp.,

Oceanospirillum linum, Pseudomonas spp., and Microcoleus spp. at the MG-MS and
MG-AR sites were the species OTUs that mainly contributed to the separate clustering of
the coastal mangrove rhizospheres from the island rhizosphere.
Discussion
Mangrove forests are ecologically important ecosystems that are under severe pressure
worldwide because of environmental changes and human activities, such as urban runoff,
wastewater, and municipal sewage (Holguin et al. 2006a). Earlier studies revealed that
anthropogenic activities affect the microbial communities present in the mangrove
sediment that are crucial for the health and balance of this ecosystem (Maliao and
Polohan 2008). In recent decades industrial activities and urban development have
significantly increased the quantities of waste, plastic debris, and other human litter along
the Red Sea coast of Saudi Arabia (Al-Obaid et al. 2016). This study reports for the first
time on the difference in bacterial community structure between anthropogenically
influenced coastal mangrove ecosystems and a relatively more pristine mangrove
ecosystem on an island in the Red Sea.
The analysis of mangrove sediments from the Red Sea using 16S rRNA gene amplicon
deep-sequencing grouped the bacterial community into 32 different phyla. Overall,
Proteobacteria were the most commonly identified, followed by Bacteroidetes,
12
Page 13 of 33
Actinobacteria, Firmicutes, Acidobacteria, Chloroflexi, Spirochaetes, Planctomycetes,
and Cyanobacteria. Consistent with our findings, these phyla have been commonly
observed in culture and nonculture studies of mangrove rhizospheres in different parts of
the world (Alzubaidy et al. 2016; Andreote et al. 2012; Basak et al. 2015b). In
comparison to a well-studied Brazilian mangrove ecosystem, an increased number of taxa

and higher relative abundance of Proteobacteria were identified in the studied samples
from the Red Sea (dos Santos et al. 2011); however, the relative abundance of
Proteobacteria was significantly lower in the coastal mangrove sediments compared with
the relatively pristine island site MG-DBI. In agreement with earlier studies,
Gammaproteobacteria and Deltaproteobacteria were the dominant classes observed in the
studied samples (Alzubaidy et al. 2016; Andreote et al. 2012). Molecular analysis of
Chinese mangrove sediments showed that the largest part of their clone library was
composed of Gammaproteobacteria-affiliated sequences (Liang et al. 2007). The
identified predominant Gammaproteobacteria members are reported as active mediators
of the nitrogen, sulfur, and carbon cycles, indicating that these bacteria are probably
essential for the maintenance of mangrove ecosystems (Fernandes et al. 2014). At the
MG-DBI site, 59.9% of the Gammaproteobacteria were from the order Alteromonadales,
which were consisted mainly of the genus Pseudoalteromonas. Alteromonadales are
gram-negative bacteria mainly found in the marine environment (Bowman and
McMeekin 2005); Pseudoalteromonas tetraodonis was the dominant species detected
from this order. Further investigation is needed to elucidate the role of P. tetraodonis
within this particular mangrove ecosystem. We found relatively higher abundances of the
orders Oceanospirillales, Chromatiales, and Pseudomonadales at the anthropogenically
13
influenced MG-MS and MG-AR sites compared with the relatively pristine MG-DBI site,
similar to the results previously reported by Fernandes et al. (Fernandes et al. 2014) for
the anthropogenically influenced Divar mangrove ecosystems in Goa, India. Previously,
members of the genera Marinobacter, Alcanivorax, and Pseudomonas have been
described as hydrocarbonoclastic (Fernandes et al. 2014), and those taxa were present at
relatively higher abundances at the MG-MS and MG-AR sites than MG-DBI.
In this study, sequences belonging to Deltaproteobacteria and other sulfate-reducing
bacteria were commonly identified at all the sites, indicating importance of the sulfur
cycles in this ecosystem. However, the relative abundance of Deltaproteobacteria was
significantly increased in the coastal mangrove sediments. Consistent with our findings,
members from the orders Desulfobacterales and Desulfovibrionales were found to be
abundant in anthropogenically influenced and polluted sites in mangrove forests in São
Paulo and Goa, India (Fernandes et al. 2014; Varon-Lopez et al. 2014), where they were
associated with degradation of hydrocarbons and aromatic compounds (Muyzer and
Stams 2008). Members from the Desulfovibrionales order have been reported to be
strongly adapted to environmental stresses, such as oil and anthropogenic heavy metal
contamination (Muyzer and Stams 2008; Varon-Lopez et al. 2014). The unclassified
sulfate-reducing Desulfosarcina spp. from Deltaproteobacteria were commonly present in
each studied sample. Moreover, sequences affiliated with sulfate-reducing
microorganisms from other bacterial phyla such as Thermodesulfobacteria and
Nitrospirae (Thermodesulfovibrio spp.) were identified in this study but were not detected
in previous studies (Andreote et al. 2012; Fernandes et al. 2014). The genera
Marinobacterium and Marinobacter from Alteromonadales family were detected at
14
Page 15 of 33
relatively higher abundances in the Masthora site and have been previously reported from
oil-contaminated areas, including mangrove fields (dos Santos et al. 2011; Yakimov et al.
2005). Similarly, the abundance of Rhodobacterales in the Alphaproteobacteria was
increased at coastal sites that had previously experienced an oil spill on the Gulf coast
(Lamendella et al. 2014). The presence of such organisms and the shifts observed in these
orders, along with location of the MG-MS and MG-AR sites near one of the largest oil
refineries at Yunbu, suggest the effects of oil contamination, together with other
anthropogenic activities and environmental factors on the coastal mangrove bacterial
communities in the Red Sea. In a recent study, Alzubaidy et al. also reported degradation
of aromatic compounds at apparently clean-looking coastal sites by the mangrove
rhizosphere microbiome present in the Red Sea (Alzubaidy et al. 2016).
In various studies, bacterial community enrichment is observed in coastal samples
because human industry is an additional source of nutrients (Fernandes et al. 2014;
Rigonato et al. 2013). In agreement with previous studies, relatively more orders and the
increased abundance of Bacteroidetes, Actinobacteria, Firmicutes, and Acidobacteria
were found in the coastal mangrove sediments in this study. Bacteroidetes are very often
identified in near-shore sediments as well as in hydrocarbon-contaminated environments,
although only a few species have been confirmed as hydrocarbon degraders (Alzubaidy et
al. 2016). The increased abundance of Bacteroidetes in the rhizosphere of mangrove
fields and other plant species has been previously described (Gomes et al. 2010).
Actinobacteria are commonly reported in marine environments and have exceptional
metabolic and biodegradation capabilities, which may explain their enrichment at the
MG-MS and MG-AR sites (Andreote et al. 2012; Viggor et al. 2013). Viggor et al. found
15
more Actinobacteria in an oil-treated microcosm experiment in Baltic Sea coastal
seawater (Viggor et al. 2013).
Cyanobacteria act as primary producers of carbon and nitrogen in nutrient-poor
ecosystems, and they play a critical role in sustaining productivity of mangrove

ecosystems through photosynthesis (Rigonato et al. 2013). Cyanobacteria were detected
at significantly higher levels at the MG-AR coastal mangrove site compared with the
other sites, with the next highest levels observed at the other coastal site MG-MS. They
are autotrophic, with niche characteristics, and anthropogenic activities seem to perturb
the structure of a cyanobacterial community (Rigonato et al. 2013).
We also detected the phyla Ignavibacteriae, Thermotogae, Acetothermia, Fusobacteria,
and Saccharibacteria, which were not identified in previous 16S amplicon sequencing and
metagenomic studies conducted in mangrove fields in Brazil, the Red Sea, or any other
geographical region (Alzubaidy et al. 2016; Andreote et al. 2012; Fernandes et al. 2014).
Ignavibacteriae, Acetothermia, Fusobacteria, and Saccharibacteria members detected at
relatively higher abundance levels in the coastal samples have previously been reported
in high-temperature oil fields and in sewage samples or in association with cellulose
degradation (Hu et al. 2016; Kindaichi et al. 2016; Podosokorskaya et al. 2013). The
comparatively higher temperatures and constant solar irradiation at the studied sites in
Saudi Arabia may support the growth of thermophilic bacteria that have not been
reported in other studies.
In addition to anthropogenic activities, variation in environmental factors and the amount
of litter can influence the microbial community in mangrove rhizospheres (Pupin and
Nahas 2014). We physicochemically analyzed the sediment samples and found them to
16
Page 17 of 33
be slightly basic, with the observed pH values (8.08–8.2) being in the range of those
reported for mangrove rhizospheres on the Thuwal cost of the Red Sea in Saudi Arabia
(Alzubaidy et al. 2016). Similarly, the amount of OM and the salinity of the studied
samples were comparable to the values previously reported for the Red Sea (Alzubaidy et
al. 2016). However, variable concentrations of OM observed for different locations;

23.8–102.8 g kg−1 was reported in China and 27–36 g kg−1 in Sao Paulo, Brazil (Pupin
and Nahas 2014; Zhang et al. 2009). OM concentrations in the Red Sea are very low
compared with the generally observed medium and high OM content in this type of
ecosystem (Alzubaidy et al. 2016; Pupin and Nahas 2014). In this study, relatively high
levels of phosphorus were observed, particularly in the coastal MG-MS sediment. Pupin
and Nahas highlighted that physicochemical characteristics, together with the biological
activity of plant species, can regulate the composition of microbial communities (Pupin
and Nahas 2014). Gomes et al. evaluated bacterial community profiles for three sites
within Guanabara Bay in Brazil via denaturing gradient gel electrophoresis, and they
found that each location produced a different community profile with different relative
contributions of bacterial groups, which highlighted the importance of site location to
explain microbial community profiles (Marcial Gomes et al. 2008). The researchers
emphasized that anthropogenic effects did not completely alter the dominant bacterial
groups, but they may cause an effect in conjunction with other environmental and
biological factors (Marcial Gomes et al. 2008). Although our analysis was limited to
single point sampling from three different locations in this study, the deep 16S amplicon
sequencing revealed higher bacterial diversity in the mangrove ecosystems of the Red
17
Sea and an alteration in bacterial community composition as a result of recent
urbanization and industrialization of the Red Sea coast of Saudi Arabia.
Conclusions
We observed relatively increased and diverse bacterial community composition in the

studied mangrove rhizospheres of Red Sea compared with other geographical regions.
Overall, distribution of the dominant OTUs varied by sampling site, and a relatively high
abundance of sulfate reducers was observed in coastal mangrove sediments. This finding
may be attributable to environmental factors and increased anthropogenic activities at the
coastal sites compared with the island site of Red Sea. Moreover, identification of
specifically enriched taxa at the studied sites such as Pseudoalteromonas and
Oceanospirillum provides a good starting point for further research to understand their
functions in the unusual extreme environment of the Red Sea and its associated mangrove
rhizosphere.
Acknowledgement
This project was funded by the National Plan for Science, Technology and Innovation
(MAARIFAH) - King Abdulaziz City for Science and Technology - the Kingdom of
Saudi Arabia - award number (12-BIO3090-03). The authors also, acknowledge with
thanks Science and Technology Unit, King Abdulaziz University for technical support.
Declaration of competing interests
The authors declare no competing financial interests.
18
Page 19 of 33
References
Al-Obaid, S., Samraoui, B., Thomas, J., El-Serehy, H.A., Alfarhan, A.H., Schneider, W., and
O'Connell, M. 2016. An overview of wetlands of Saudi Arabia: Values, threats, and perspectives.
Ambio. doi: 10.1007/s13280-016-0807-4
10.1007/s13280-016-0807-4 [pii].
Alzubaidy, H., Essack, M., Malas, T.B., Bokhari, A., Motwalli, O., Kamanu, F.K., Jamhor, S.A.,
Mokhtar, N.A., Antunes, A., Simoes, M.F., Alam, I., Bougouffa, S., Lafi, F.F., Bajic, V.B., and
Archer, J.A. 2016. Rhizosphere microbiome metagenomics of gray mangroves (Avicennia
marina) in the Red Sea. Gene 576(2 Pt 1): 626-636. doi: S0378-1119(15)01240-8 [pii]
10.1016/j.gene.2015.10.032.
Andreote, F.D., Jiménez, D.J., Chaves, D., Dias, A.C.F., Luvizotto, D.M., Dini-Andreote, F.,
Fasanella, C.C., Lopez, M.V., Baena, S., and Taketani, R.G. 2012. The microbiome of Brazilian
mangrove sediments as revealed by metagenomics. PLoS One 7(6): e38600.
Antunes, A., Ngugi, D.K., and Stingl, U. 2011. Microbiology of the Red Sea (and other) deep-sea
anoxic brine lakes. Environ Microbiol Rep 3(4): 416-433. doi: 10.1111/j.1758-
2229.2011.00264.x.
Basak, P., Majumder, N.S., Nag, S., Bhattacharyya, A., Roy, D., Chakraborty, A., SenGupta, S.,
Roy, A., Mukherjee, A., and Pattanayak, R. 2015a. Spatiotemporal analysis of bacterial diversity
in sediments of Sundarbans using parallel 16S rRNA gene tag sequencing. Microbial ecology
69(3): 500-511.
Basak, P., Pramanik, A., Roy, R., Chattopadhyay, D., and Bhattacharyya, M. 2015b. Cataloguing
the bacterial diversity of the Sundarbans mangrove, India in the light of metagenomics. Genomics
Data 4: 90-92.
19
Bouchez, A., Pascault, N., Chardon, C., Bouvy, M., Cecchi, P., Lambs, L., Herteman, M.,
Fromard, F., Got, P., and Leboulanger, C. 2013. Mangrove microbial diversity and the impact of
trophic contamination. Marine pollution bulletin 66(1): 39-46.
Bowman, J.P., and McMeekin, T.A. 2005. Alteromonadales ord. nov. In Bergeyâ€™s Manual‫®آ‬
of Systematic Bacteriology: Volume Two The Proteobacteria Part B The Gammaproteobacteria.

Springer US, Boston, MA. pp. 443-491.
Colares, G.B., and Melo, V.M.M. 2013. Relating microbial community structure and
environmental variables in mangrove sediments inside Rhizophora mangle L. habitats. Applied
soil ecology 64: 171-177.
Dean, W.E. 1974. Determination of carbonate and organic matter in calcareous sediments and
sedimentary rocks by loss on ignition: comparison with other methods. Journal of Sedimentary
Research 44: 242–248.
dos Santos, H.F., Cury, J.C., do Carmo, F.L., dos Santos, A.L., Tiedje, J., van Elsas, J.D.,
Rosado, A.S., and Peixoto, R.S. 2011. Mangrove bacterial diversity and the impact of oil
contamination revealed by pyrosequencing: bacterial proxies for oil pollution. PLoS One 6(3):
e16943. doi: 10.1371/journal.pone.0016943.
Duke, N.C., Meynecke, J.-O., Dittmann, S., Ellison, A.M., Anger, K., Berger, U., Cannicci, S.,
Diele, K., Ewel, K.C., and Field, C.D. 2007. A world without mangroves? Science 317(5834):
41-42.
El-Juhany, L. 2009. Present status and degradation trends of mangrove forests on the southern
Red Sea coast of Saudi Arabia. American-Eurasian Journal of Agricultural and Environmental
Science 6(3): 328-340.
Fernandes, S.O., Kirchman, D.L., Michotey, V.D., Bonin, P.C., and LokaBharathi, P. 2014.
Bacterial diversity in relatively pristine and anthropogenically-influenced mangrove ecosystems
(Goa, India). Brazilian Journal of Microbiology 45(4): 1161-1171.
20
Page 21 of 33
Getter, C.D., Cintron, G., Dicks, B., Lewis, R.R., and Seneca, E. 1984. Recovery and Restoration
of Salt Marshes and Mangroves Following an Oil Spill. Restoration of Habitats Impacted by Oil
Spills, Butterworth Boston. 1984. Edited by John Cairns, Jr. and Arthur L. Buikema, Jr., p 65-
113, 7 Fig, 4 Tab, 121 Ref.
Ghosh, A., Dey, N., Bera, A., Tiwari, A., Sathyaniranjan, K., Chakrabarti, K., and
Chattopadhyay, D. 2010. Culture independent molecular analysis of bacterial communities in the
mangrove sediment of Sundarban, India. Saline systems 6(1): 1.
Gomes, N.C., Cleary, D.F., Pinto, F.N., Egas, C., Almeida, A., Cunha, A., Mendonca-Hagler,
L.C., and Smalla, K. 2010. Taking root: enduring effect of rhizosphere bacterial colonization in
mangroves. PLoS One 5(11): e14065. doi: 10.1371/journal.pone.0014065.
Harris, J. 2008. Soil microbial communities and restoration. Oikos 117: 1833.
Hasanean, H., and Almazroui, M. 2015. Rainfall: Features and Variations over Saudi Arabia, A
Review. Climate 3: 578-626.
Holguin, G., Gonzalez-Zamorano, P., de-Bashan, L.E., Mendoza, R., Amador, E., and Bashan, Y.
2006a. Mangrove health in an arid environment encroached by urban development--a case study.
Sci Total Environ 363(1-3): 260-274. doi: S0048-9697(05)00406-7 [pii]
10.1016/j.scitotenv.2005.05.026.
Holguin, G., Gonzalez-Zamorano, P., De-Bashan, L.E., Mendoza, R., Amador, E., and Bashan,
Y. 2006b. Mangrove health in an arid environment encroached by urban development—a case
study. Science of the Total Environment 363(1): 260-274.
Hu, P., Tom, L., Singh, A., Thomas, B.C., Baker, B.J., Piceno, Y.M., Andersen, G.L., and
Banfield, J.F. 2016. Genome-Resolved Metagenomic Analysis Reveals Roles for Candidate Phyla
and Other Microbial Community Members in Biogeochemical Transformations in Oil Reservoirs.
MBio 7(1): e01669-01615. doi: mBio.01669-15 [pii]
10.1128/mBio.01669-15.
21
Kathiresan, K., and Rajendran, N. 2005. Coastal mangrove forests mitigated tsunami. Estuarine,
Coastal and Shelf Science 65(3): 601-606.
Kindaichi, T., Yamaoka, S., Uehara, R., Ozaki, N., Ohashi, A., Albertsen, M., Nielsen, P.H., and
Nielsen, J.L. 2016. Phylogenetic diversity and ecophysiology of Candidate phylum
Saccharibacteria in activated sludge. FEMS Microbiol Ecol 92(6). doi: fiw078 [pii]
10.1093/femsec/fiw078.
Lamendella, R., Strutt, S., Borglin, S., Chakraborty, R., Tas, N., Mason, O.U., Hultman, J.,
Prestat, E., Hazen, T.C., and Jansson, J.K. 2014. Assessment of the Deepwater Horizon oil spill
impact on Gulf coast microbial communities. Front Microbiol 5: 130. doi:
10.3389/fmicb.2014.00130.
Liang, J.-B., Chen, Y.-Q., Lan, C.-Y., Tam, N.F.Y., Zan, Q.-J., and Huang, L.-N. 2007. Recovery
of novel bacterial diversity from mangrove sediment. Marine Biology 150(5): 739-747. doi:
10.1007/s00227-006-0377-2.
Maliao, R.J., and Polohan, B.B. 2008. Evaluating the impacts of mangrove rehabilitation in
Cogtong Bay, Philippines. Environ Manage 41(3): 414-424. doi: 10.1007/s00267-007-9021-2.
Marcial Gomes, N.C., Borges, L.R., Paranhos, R., Pinto, F.N., Mendonca-Hagler, L.C., and
Smalla, K. 2008. Exploring the diversity of bacterial communities in sediments of urban
mangrove forests. FEMS Microbiol Ecol 66(1): 96-109. doi: FEM519 [pii]
10.1111/j.1574-6941.2008.00519.x.
Masella, A.P., Bartram, A.K., Truszkowski, J.M., Brown, D.G., and Neufeld, J.D. 2012.
PANDAseq: paired-end assembler for illumina sequences. BMC Bioinformatics 13: 31. doi:
1471-2105-13-31 [pii]
10.1186/1471-2105-13-31.
McMurdie, P.J., and Holmes, S. 2012. Phyloseq: a bioconductor package for handling and
analysis of high-throughput phylogenetic sequence data. Pac Symp Biocomput: 235-246. doi:
9789814366496_0023 [pii].
22
Page 23 of 33
Muyzer, G., and Stams, A.J. 2008. The ecology and biotechnology of sulphate-reducing bacteria.
Nat Rev Microbiol 6(6): 441-454. doi: nrmicro1892 [pii]
10.1038/nrmicro1892.
Nedwell, D. 1994. Dynamic nature of the turnover of organic carbon, nitrogen and sulphur in the
sediments of a Jamaican mangrove forest. Marine Ecology Progress Series 110: 223-231.
Podosokorskaya, O.A., Kadnikov, V.V., Gavrilov, S.N., Mardanov, A.V., Merkel, A.Y.,
Karnachuk, O.V., Ravin, N.V., Bonch-Osmolovskaya, E.A., and Kublanov, I.V. 2013.
Characterization of Melioribacter roseus gen. nov., sp. nov., a novel facultatively anaerobic
thermophilic cellulolytic bacterium from the class Ignavibacteria, and a proposal of a novel
bacterial phylum Ignavibacteriae. Environmental Microbiology 15(6): 1759-1771. doi:
10.1111/1462-2920.12067.
Pupin, B., and Nahas, E. 2014. Microbial populations and activities of mangrove, restinga and
Atlantic forest soils from Cardoso Island, Brazil. J Appl Microbiol 116(4): 851-864. doi:
10.1111/jam.12413.
Rigonato, J., Kent, A.D., Alvarenga, D.O., Andreote, F.D., Beirigo, R.M., Vidal-Torrado, P., and
Fiore, M.F. 2013. Drivers of cyanobacterial diversity and community composition in mangrove
soils in south-east Brazil. Environ Microbiol 15(4): 1103-1114. doi: 10.1111/j.1462-
2920.2012.02830.x.
Varon-Lopez, M., Dias, A.C., Fasanella, C.C., Durrer, A., Melo, I.S., Kuramae, E.E., and
Andreote, F.D. 2014. Sulphur-oxidizing and sulphate-reducing communities in Brazilian
mangrove sediments. Environ Microbiol 16(3): 845-855. doi: 10.1111/1462-2920.12237.
Viggor, S., Juhanson, J., Joesaar, M., Mitt, M., Truu, J., Vedler, E., and Heinaru, A. 2013.
Dynamic changes in the structure of microbial communities in Baltic Sea coastal seawater
microcosms modified by crude oil, shale oil or diesel fuel. Microbiol Res 168(7): 415-427. doi:
S0944-5013(13)00021-9 [pii]
10.1016/j.micres.2013.02.006.
23
Yakimov, M.M., Denaro, R., Genovese, M., Cappello, S., D'Auria, G., Chernikova, T.N.,
Timmis, K.N., Golyshin, P.N., and Giluliano, L. 2005. Natural microbial diversity in superficial
sediments of Milazzo Harbor (Sicily) and community successions during microcosm enrichment
with various hydrocarbons. Environ Microbiol 7(9): 1426-1441. doi: EMI829 [pii]
10.1111/j.1462-5822.2005.00829.x.
Yasir, M., Angelakis, E., Bibi, F., Azhar, E.I., Bachar, D., Lagier, J.C., Gaborit, B., Hassan,
A.M., Jiman-Fatani, A.A., Alshali, K.Z., Robert, C., Dutour, A., and Raoult, D. 2014.
Comparison of the gut microbiota of people in France and Saudi Arabia. Nutr Diabetes 5: e153.
doi: nutd20153 [pii]
10.1038/nutd.2015.3.
Zhang, Y., Dong, J., Yang, B., Ling, J., Wang, Y., and Zhang, S. 2009. Bacterial community
structure of mangrove sediments in relation to environmental variables accessed by 16S rRNA
gene-denaturing gradient gel electrophoresis fingerprinting. Scientia Marina 73(3): 487–498.
24
Page 25 of 33
Figure Legends
Figure 1. Average relative abundance of the dominant bacterial phyla. (A) The x-
axis shows the mangrove sampling sites, and the y-axis the average percentages of
sequence reads. The cutoff point for selecting the dominant phyla was set to ≥1%;
‘others’ indicates minor phyla. (B) Distribution of different classes of Proteobacteria at
each site. MG-DBI, Dhahban; MG-MS, Mastorah; MG-AR, Ar-Rayis.
Figure 2. Comparative analysis of the dominant and significantly different genera
among the different mangrove sites. The cutoff point for selecting the dominant phyla
was set to ≥1%. MG-DBI, Dhahban; MG-MS, Mastorah; MG-AR, Ar-Rayis.
Figure 3. Comparative analysis of cumulative species richness in the mangrove
rhizosphere at different sampling sites from the Red Sea. MG-DBI, Dhahban; MG-
MS, Mastorah; MG-AR, Ar-Rayis.
Figure 4. Phylogenetic heat map of the dominant and significantly different species
among the different mangrove sites. The cutoff point for selecting the dominant species
was set to ≥1%. MG-DBI, Dhahban; MG-MS, Mastorah; MG-AR, Ar-Rayis.
Figure 5. Alpha diversity of the sequence reads. Rarefaction curves of the
experimentally observed OTUs versus diversity estimated by Chao1. Boxplots of the
Shannon index. MG-DBI, Dhahban; MG-MS, Mastorah; MG-AR, Ar-Rayis.
Figure 6. Multivariate principal coordinate data analysis of bacterial communities
at the different sampling sites. The projecting vectors represent a mangrove site and its
variability. MG-DBI, Dhahban; MG-MS, Mastorah; MG-AR, Ar-Rayis.
25
Fig. S1. Satellite map of the sampling location. MG-DBI, Dhahban; MG-MS,
Mastorah; and MG-AR, Ar-Rayis. Map and pictures were obtained from Google maps.
Fig. S2. Cytoscape based networks analysis of OTUs interaction at genus level. The
white circle nodes represent bacterial OTUs commonly found in respective samples and
connected with more than one edge. Unique bacterial OTUs to a specific sample were
linked with a respective sample node by single line.
26
Page 27 of 33
Table 1. Physicochemical parameters of sampling sites in this study.
Temp Salinity pH OM% TN% TP% K%
(ºC) (PSU)
MG- 31.7 17.8 ± 8.4 ± 0.1 5.8 ± 0.1 0.065 ± 0.057 ± 0.25 ±
DBI 1.9 0 0.01 0.01
MG-MS 33.4 19.2 ± 8.3 ± 0.1 6.3 ± 0.2 0.069 ± 0.13 ± 0.29 ±
1.6 0 0.01 0.02
Mg-AR 32.9 15.9 ± 8.5 ± 0.1 5.2 ± 0.2 0.064 ± 0.045 ± 0.27 ±
0.8 0 0.01 0.03
Abbreviations: PSU, practical salinity unit; OM, organic matter; TN, total nitrogen; TP,
total phosphorus; K, potassium; MG-DBI, Dhahban; MG-MS, Mastorah; MG-AR, Ar-
Rayis.
Table S1. A list of bacterial species recorded common at the three study sites.
Table S2. A list of bacterial species found in the anthropogenically influenced mangrove
rhizospheres at costal sites of the Red Sea.
27
Page 29 of 33
Figure 2. Comparative analysis of the dominant and significantly different genera among the different
mangrove sites. The cutoff point for selecting the dominant phyla was set to ≥1%. MG-DBI, Dhahban; MG-
MS, Mastorah; MG-AR, Ar-Rayis.
153x129mm (300 x 300 DPI)

177x152mm (96 x 96 DPI)

sampling sites from the Red Sea. MG-DBI, Dhahban; MG-MS, Mastorah; MG-AR, Ar-Rayis.
Figure 3. Comparative analysis of cumulative species richness in the mangrove rhizosphere at different
Page 30 of 33
Page 31 of 33
Figure 4. Phylogenetic heat map of the dominant and significantly different species among the different
mangrove sites. The cutoff point for selecting the dominant species was set to ≥1%. MG-DBI, Dhahban;
MG-MS, Mastorah; MG-AR, Ar-Rayis.
132x218mm (300 x 300 DPI)

MG-AR, Ar-Rayis.
127x279mm (300 x 300 DPI)

Figure 5. Alpha diversity of the sequence reads. Rarefaction curves of the experimentally observed OTUs
versus diversity estimated by Chao1. Boxplots of the Shannon index. MG-DBI, Dhahban; MG-MS, Mastorah;
Page 32 of 33
Page 33 of 33
Figure 6. Multivariate principal coordinate data analysis of bacterial communities at the different sampling
sites. The projecting vectors represent a mangrove site and its variability. MG-DBI, Dhahban; MG-MS,
Mastorah; MG-AR, Ar-Rayis.
254x190mm (96 x 96 DPI)

Comparative Bacterial Community Analysis in Relatively Pristine and Anthropogenically Influenced Mangrove Ecosystems On The Red Sea

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Comparative Bacterial Community Analysis in Relatively Pristine and Anthropogenically Influenced Mangrove Ecosystems On The Red Sea

Uploaded by

Copyright:

Available Formats

Page 1 of 33

Comparative bacterial community analysis in relatively pristine and

anthropogenically influenced mangrove ecosystems on the Red Sea

Ahmed Al-Ansari3, Fahad Al-Abbasi2, Abdulmohsin A. Al-Sofyani4, Ihsanullah Daur5,

Seon-Woo Lee6, Esam I Azhar1,7

University, Jeddah, Saudi Arabia.

Land Agriculture, King Abdulaziz University, Jeddah, Saudi Arabia.

Jeddah, Saudi Arabia.

King Abdulaziz University, Jeddah, Saudi Arabia.

†Authors contributed equally to this study

*Corresponding author: Muhammad Yasir

rRNA gene amplicon deep-sequencing was used to compare bacterial communities in

Red Sea mangrove ecosystems at anthropogenically influenced coastal sites and a

The phyla Actinobacteria, Firmicutes, Acidobacteria, Chloroflexi, Spirochetes, and

Pseudoalteromonas and Cobetia were predominantly identified in the MG-DBI site

compared with the anthropogenically influenced coastal sites.

Key words: mangroves, bacterial community, metagenomic, rhizosphere, Red Sea

2% per year (Kathiresan and Rajendran 2005).

periodic tidal flooding. Environmental factors including nutrient availability, light,

Proteobacteria, Actinobacteria, Bacteroidetes, Planctomycetes, Acidobacteria,

Chloroflexi, Cyanobacteria, Nitrospira, and Firmicutes. Proteobacteria are the most

common, with Gammaproteobacteria, Deltaproteobacteria, and Alphaproteobacteria

being predominant (Basak et al. 2015a). A metagenomic analysis of Brazilian mangrove

these Proteobacteria inhibit the growth of Planctomycetaceae, Burkholdreaceae, and

Rhodobacteraceae, which are implicated in sulfur, nitrogen, and methane metabolism

(Andreote et al. 2012).

baseline bacterial community profile for healthy mangroves. Increased rhizosphere

Thermotogae, Acetothermia, Fusobacteria, and Saccharibacteria were detected in the Red

in the Red Sea mangroves habitats.

Study area and samples collection

sampling site 2 at Ar-Rayis (MG-AR; 23°36′43″N, 38°31′26″E). The average height of

considered to be a relatively clean island without external contamination. The mangrove

uniformity and analytical processing. OM was determined through loss of ignition

acid–perchloric acid (HNO3-HClO4) mixture (Alzubaidy et al. 2016). Following

phosphorus was determined colorimetrically and that of potassium by atomic absorption

DNA extraction and 16S rRNA gene amplicon sequencing

concentrations were measured using a Qubit Fluorometer (Thermo Fisher Scientific,

purification with Agencourt AMPure beads (Agencourt, Indianapolis), libraries were

manufacturer’s protocol. Automated cluster generation and paired-end sequencing with

accession no. SRP097132.

coordinate analysis (PCoA) was performed using R VEGAN (http://cran.r-project.org,

http://vegan.r-forge.r-project.org/), and the Kolmogorov-Smirnov D test was used to

honest significant difference (HSD) tests were performed to identify significantly

(<0.05) higher percentages of OM and phosphorus were detected at MG-MS compared

were in the range of 31.7–33.4°C.

Bacterial community composition

Proteobacteria, Gammaproteobacteria was the dominant class present at each site

(75.9±18.8% MG-DBI, 34±9.5% MG-MS, and 27±5.3% MG-AR), followed by

Deltaproteobacteria (11.3±11.5% MG-DBI, 23.4±6.9% MG-MS, and 25.1±3.4% MG-

In the island sample, MG-DBI, Pseudoalteromonas (58.9±15.6%) and Cobetia

Oceanospirillum predominated in the MG-MS sample (10.2±108%) compared with the

Dehalococcoides, and Syntrophobacter were present at significantly (p<0.05) higher

The genera Thioprofundum, Spirochaeta, Acidobacterium, Thiohalophilus, Caldithrix,

dominantly present in each of the studied sampling sites.

site, Pseudoalteromonas tetraodonis (41.5±6.4%), unclassified Pseudoalteromonas spp.

(15.1±8.9), and Cobetia marina (9.3±6.4%) were present at a relatively higher

Oceanospirillum linum, Dehalococcoides spp., Sulfurovum spp., and Desulfobacterium

135 at MG-MS, and 150 at MG-AR.

Alpha diversity analysis

Pseudoalteromonas tetraodonis, Pseudoalteromonas spp., Cobetia marina, and

Pseudoalteromonas mariniglutinosa at the MG-DBI site and Desulfosarcina spp.,

the coastal mangrove rhizospheres from the island rhizosphere.

anthropogenic activities affect the microbial communities present in the mangrove