Insights on aquatic microbiome of the Indian Sundarbans mangrove areas.

BACKGROUND: Anthropogenic perturbations have strong impact on water quality and ecological health of mangrove areas of Indian Sundarbans. Diversity in microbial community composition is important causes for maintaining the health of the mangrove ecosystem. However, microbial communities of estuarine water in Indian Sundarbans mangrove areas and environmental determinants that contribute to those communities were seldom studied.
METHODS: Nevertheless, this study attempted first to report bacterial and archaeal communities simultaneously in the water from Matla River and Thakuran River of Maipith coastal areas more accurately using 16S rRNA gene-based amplicon approaches. Attempt also been made to assess the capability of the environmental parameters for explaining the variation in microbial community composition.
RESULTS: Our investigation indicates the dominancy of halophilic marine bacteria from families Flavobacteriaceae and OM1 clade in the water with lower nutrient load collected from costal regions of a small Island of Sundarban Mangroves (ISM). At higher eutrophic conditions, changes in bacterial communities in Open Marine Water (OMW) were detected, where some of the marine hydrocarbons degrading bacteria under families Oceanospirillaceae and Spongiibacteraceae were dominated. While most abundant bacterial family Rhodobacteracea almost equally (18% of the total community) dominated in both sites. Minor variation in the composition of archaeal community was also observed between OMW and ISM. Redundancy analysis indicates a combination of total nitrogen and dissolved inorganic nutrients for OMW and for ISM, salinity and total nitrogen was responsible for explaining the changes in their respective microbial community composition.
CONCLUSIONS: Our study contributes the first conclusive overview on how do multiple environmental/anthropogenic stressors (salinity, pollution, eutrophication, land-use) affect the Sundarban estuary water and consequently the microbial communities in concert. However, systematic approaches with more samples for evaluating the effect of environmental pollutions on mangrove microbial communities are recommended.

1. Introduction

Sundarbans, the largest mangrove forest of the world, is situated in the joint delta of Ganges, Brahmaputra and Meghna rivers at Bay of Bengal [1,2]. This UNESCO World Heritage site comprises the Indian state of West Bengal and southwest Bangladesh [3]. Livelihood and well being of millions of people live in and around of Sundarbans, depends on its status and ecological services. Despite its high ecological and economical values, Sundarbans is seriously threatened by different anthropogenic activities. Since the early 19th century, landscapes of Sundarbans have also been changing due to saline and freshwater imbalances. Water quality of this ecosystem is largely affected by sewage pollutant originated from industries located upstream and urban areas of West Bengal. Sewage entering into coastal water contains diverse chemical and microbiological pollutants and a wide variety of organic and inorganic wastes [4,5], driving changes on its ecological and physiological health.

Microbial communities of mangroves are responsible for nutrient cycling and play a vital role in productivity, conservation and rehabilitation of mangrove ecosystems [6]. Therefore, understanding their responses to environmental changes is essential to predict changes in service-provisioning [7]. Several recent studies described the microbial community compositions of surface sediments and water of Indian Sundarban mangrove areas. Surface sediments this area dominanted with Deltaproteobacteria followed by Gammaproteobacteria, Alphaproteobacteria, Betaproteobacteria, and Epsilonproteobacteria under phylum Proteobacteria. Abundant bacterial orders are Desulfobacterales, Desulfuromonadales, Myxococcales, and Bdellovibrionales. [8-10]. While bacterioplankton communities in the water of this region were found to be abundant with Gammaproteobacteria and Alphaproteobacteria. At the family level dominancy of Hyphomicrobiaceae, Rhodobacteraceae, Pseudomonadaceae, Erythrobacteraceae, Kordiimonadaceae, Hyphomonadaceae, and Ruminococcaceae were observed [11-13]. However, sampling locations of those studies on microbial communities in the Indian Sundarban mangrove water mainly restricted near to an island (Sagar Island) and other estuary of Mooriganga, Thakuran, Matla and Harinbhanga, therefore the major conclusions of these studies were made based on a limited number of samples. Moreover, not much effort has been made to investigate the archaeal community of this region except single report by [3] on surface and subsurface sediments of Indian Sundarban mangrove forest. Moreover, the above studies have rarely analyzed the bacterial and archaeal community structures of the same samples at the same time. Therefore, our knowledge on those communities as well as information on how they are controlled by environmental parameters is limited. In order to assess the microbial communities of marine ecosystem via high-throughput sequencing of amplified 16S rRNA genes with high resolution and fidelity, it is extremely important to select the proper primer set that can’t underestimated or overestimated any common marine taxa [14]. However, this will be the first attempt to visualize the accurate and well-resolved picture of bacterial and archaeal communities simultaneously of marine water in Sundarbans mangrove using next-generation amplicon sequencing of the 16S rRNA gene using recently developed 515F-Y/926R primers that target V4-V5 region of 16S rRNA gene. We also tried to explore the environmental determinants that contribute to the variation of their microbial communities. This study will provide baseline knowledge on microbial ecology of the World Heritage site and serve as a baseline for monitoring programs and predicting changes at impacted sites.

2. Material and methods

2.1 Ethics statement

The water samples were brought to ZMT, Germany for its environmental parameters and nutrients measurements. The extracted mutagenic DNA were also carried to ZMT, Germany for sequencing and further analysis. In this reference, The National Biodiversity Authority (NBA), Government of India is well aware of this activity (NBA/Tech Appl/9/Form B/52/17/17-18/2985) that the above mentioned samples are being used only for research purpose. Further no permits were prerequisite for the defined field studies, which complied with all relevant regulation neither the studied locations are not privately owned.

2.2 Study sites and sample collection

In the present study sampling was conducted in the Sundarbans mangrove ecosystem that shared between India and Bangladesh and lies in the Ganga-Brahmaputra-Meghna (GBM) delta. This mangrove ecosystem contains over 102 islands with a network complex of many rivers, rivulets, and creeks [15]. Sampling was carried out at two different locations on Thakuran River—Matla River estuarine complex of Maipith coastal areas in the Indian Sundarbans mangroves during March 2017 during low tide (Fig 1). They are designated as Island of Sundarban Mangroves (ISM) and Open Marine Water (OMW). ISM is an uninhabited small island with lesser anthropogenic disturbance situated in Thakuran and Matla river complex in low-lying coastal plain. This river has no reports for perennial fresh water source [15]. Water from three different coastal regions of this island is selected for sampling. OMW is an open marine site around same regions, which is supposedly continually influenced by the wastewaters from upstream regions of Matla River. Three independent replicated water samples (1 L) from each of three different sampling sites of both ISM and OMW were collected in sterile containers and immediately stored at a chilled box until further laboratory analysis.

Fig 1

Map of the sampling area: Water samples were collected from three stations (KL 1, KP 1 and TH1) of a small Island are named as ISM and open marine water samples named as OWM (TH 2, KL 2 and BL 1).

Three biological replicates from each of the six stations; therefore, total eighteen (18) samples (nine from ISM and another nine from OMW) were collected for this investigation. The GPS data of sampling sites were collecting during sampling and compiled together in an ArcGIS10.3 software environments and finally map has been prepared using open source database of GADM (https://gadm.org/).

2.3 Environmental parameters and nutrients analysis

Physiochemical parameters (salinity and pH) of all collected samples were measured using Eureka 2 Manta multiprobe (Eureka Environmental Engineering, Texas, USA). Total 50 mL of each sample was filtered through a 0.7 μm syringe filter and poisoned with 200 μL of 3.5 g/100 mL HgCl2 solution for nutrient analysis. Now each of the treated samples was filtered through a 0.7 μm pore size GF/F filter (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) for DOC and nutrients measurements. The total nitrogen (TN) content of each the samples were measured using the EuroVector EA 3000 elemental analyzer. For measurements of DOC, the filtered samples were acidified with concentrated HCl (pH <2) and analyzed by high-temperature catalytic oxidation method method using a TOC-VCPN analyzer (Shimadzu, Mandel, Canada). Seawater standards (Hansell laboratory, RSMAS University Miami, USA) were used for calibration and quality control, and ultrapure water as a blank. The Dissolved inorganic nutrients that includes combined nitrate and nitrite (NOx), phosphate (PO43-P), and silicate (Si(OH)4-) were analyzed using a continuous flow analyzer (Flowsys by Unity Scientific, Brookfield, USA) and detected spectrophotometrically as a colored complex [16] (https://doi.pangaea.de/10.1594/PANGAEA.889699).

2.4 DNA extraction, PCR amplification, and Illumina MiSeq sequencing

From each site, water (1 L) was filtered (0.2 μm) and DNA was extracted using the PowerWater® DNA Isolation Kit according to the manufacturer’s instructions (MoBio Laboratories Inc., Carlsbad, CA, USA). DNA concentrations and purity were measured spectrophotometrically. Presence of bacterial and archebacterial 16S rRNA gene was in the extracted metagenome was verified following previous method [17]. In order to classify taxonomically both bacterial and archaeal community structure simultaneously, sequencing of V4–V5 hypervariable regions of 16S rRNA gene were generated using primers 515F-Y (5′-GTGYCAGCMGCCGCGGTAA-3′) and 926R (5′-CCGYCAATTYMTTTRAGTTT-3′) [14] on the Illumina MiSeq platform (CeBiTec Bielefeld, Germany), in a 2 × 300 bp paired-end run.

2.5 High throughput sequencing data processing

Primer sequences were removed using cutadapt from the raw paired-end reads [18]. The primer-trimmed sequences are available on Sequence Read Archive (SRA) (accession no. SRP144285). Sequences were quality trimmed with trimmomatic v0.32 [19] using a sliding window of 4 bases and a minimum average quality of 15, and merged with PEAR v0.9.5 [20]. Quality-filtered sequences were clustered into OTUs with swarm algorithm using default parameters [21]. One single representative sequence per OTU was taxonomically classified with SINA (SILVA Incremental Aligner; v1.2.11; Silva reference database release 132) at a minimum alignment similarity of 0.9, and a last common ancestor consensus of 0.7 [22]. OTUs that were unclassified on the domain level and those matching to chloroplast and mitochondrial sequences were excluded from the analysis using well-standardized r script [23]. The final OTU tables are accessible at (https://doi.pangaea.de/10.1594/PANGAEA.890757).

2.6 Statistical analysis

Principal component analysis (PCA) was performed to cluster the sampling sites based on their environmental parameters. Differences in environmental parameters among ISM and OMW were assessed using general linear mixed models (GLMM) with sampling station as a random factor [24].

Alpha-Diversity indices were calculated to assess richness and evenness of the microbial communities [25] in the studied samples, based on repeated random subsampling of the amplicon data sets after randomly rarefying the data set to the minimum library size (50517 sequences). Significant differences in alpha-diversity indices between the studied stations were determined by using the non-parametric Kruskal test followed by p-value adjusted Wilcoxon tests [26].

To assess the differences in community structure between two sampling sites (beta-diversity), Bray–Curtis dissimilarities were calculated using the relative OTU abundances and also non-metric multidimensional scaling (NMDS) plot was produced. Analysis of similarity (ANOSIM) was calculated to assess the separation of bacterial communities between the two sites. P-values of all multiple pairwise comparisons were adjusted using the false discovery rate (fdr) correction method by [27]. In order to evaluate the environmental parameters as drivers of the variations in community compositions, redundancy analysis (RDA) was used with centered log ratio (clr)-transformed sequence counts using the R function aldex.clr of the ALDEx2 package via median values of 128 Monte-Carlo instances [28]. To compare the explanatory power of all measured environmental parameters, additional RDA models were constructed with environmental parameters as predictors. Forward model selection was used after checking for variance inflation to determine which of parameters would be included in the RDA models. When more than one parameter was included, pure effects were also tested accounting for the variation explained by the other factors in the model. Collinearity among predictors was determined via Variance inflation factors (VIFs) of the individual parameters. All of the parameters in any of the RDA models displayed VIFs less than 10. The adjusted R2 is provided as goodness-fit-stat. All statistical analyses were conducted in R using the core distribution, version 3.3.2 and R-Studio, version 1.0.153,with following packages: vegan [29], lmerTest for the GLMM [24], ALDEx2 [29] and multcomp [28].

3. Result

3.1 Environmental characterization

Environmental parameters (pH, salinity) and nutrients (including DOC, TN, NOx, nitrate, DIN, phosphate: PO43-P, and silicate: Si(OH)4-) concentrations for all samples were measured (. Samples were slightly alkaline (pH 8.0 to 8.7) in nature. The GLMMs analysis indicated that the measured water nutrients that differed significantly among the two sampling station were mainly TN, DIN and PO43-P (Table 1; ). The PCA ordination ( showed that first two principal components (PC1 and PC2) represented 74.4% of data variation among sites. PC1 alone represents 60.1% of total variation and influenced by most of the measured parameters, while pH showed a strong correlation with PC2 (. Noteworthy to mention, the samples were separated into two clusters by PC1. One cluster is mainly composed with the samples from ISM (except TH2.3 of OMW) and other cluster accommodating samples collected from OMW. This ordination probably indicates elevated eutrophication in samples from the OMW compared to ISM.

Table 1

Environmental conditions in Indian Sundarban mangroves and details of the sampling sites.

Station id	Site Id	GPS (DD COORDINATES)	Sample ID	Salinity	pH	DOC (μM)	TN (μM)	NOx (μM)	NO3 (μM)	DIN (μM)	PO4 (μM)	Si (μM)
Open Marine Water (OMW)	TH 2	N 21.82389	TH2.5	26.6	8.2	112.6	12.5	1	1	1	0.4	11.7
		E 88.50585	TH2.2	27.0	8.2	111	11.4	1.4	1.4	1.5	0.2	13.1
			TH2.3	25.7	8.3	66.2	6.4	0	0	0	0.1	7.7
	KL 2	N 21.8172	KL2.3	26.6	8.3	129.5	13.6	2.6	2.5	2.6	0.2	14.3
		E 88.53658	KL2.6	26.9	8.1	162.4	10	1.2	1.2	1.3	0.1	26.3
			KL2.1	26.3	8.2	116.3	12.1	1.5	1.4	1.5	0.6	15.9
	BL 1	N 21.78962	BL1.6	26.8	8.5	121.1	14.8	1	1	1.1	0.2	16.3
			BL1.5	26.9	8.4	114.1	13.2	1.7	1.7	1.8	0.2	12.6
		E 88.50534
			BL1.2	26.5	8.7	111.4	12.7	2.3	2.2	2.3	0.2	16.1
Island of Sundarban Mangroves (ISM)	KL 1	N 21.85189	KL1.3	23.6	8.3	56.1	4.4	0.2	0.2	0.3	0.1	6.2
			KL1.6	22.9	8.0	58	6	0.9	0.8	0.9	0.1	6.1
		E 88.51168
			KL1.2	24.3	8.1	62	5.1	0.2	0.2	0.2	0.1	6.5
	KP 1	N 21.85604	KP1.6	25.8	8.1	78.5	5.7	0	0	0.1	0.1	12.2
		E 88.51191	KP1.1	25.3	8.1	92.5	9	0	0	0.1	0.2	15.4
			KP1.3	24.9	8.1	81.2	7	0.4	0.4	0.5	0.1	12.3
	TH1	N 21.85706	TH1.4	21.5	8.3	91.9	8.3	0	0	0.1	0.2	8.7
		E 88.51638	TH1.3	21.8	8.3	86.8	8.9	0.1	0	0.1	0.1	8.7
			TH1.2	21.2	8.3	84.8	7.4	0.6	0.6	0.6	0.1	9.5

Principal component analysis (PCA) to ordinate the eighteen collected water samples collected samples from ISM and OMW based on their environmental parameters.

The arrows show the direction of the environmental parameters. DIN, dissolved inorganic nitrogen; TN, total nitrogen; DOC, dissolved organic carbon.

3.2. Microbial communities

Total numbers of reads generated per sample ranged between 50517 to 90468 (after merged) corresponding to 3,644 to 6,470 swarmed, non-singleton 16S OTUs (. After rarefaction, numbers of bacterial and archaeal OTUs ranged between 3390 to 5415 and 37 to 91, respectively (). None of the measured diversity indices (Average Shannon diversity and inverse Simpson indices) were found significance differences in between OMW and ISM (; ) indicated by Kruskal—Wilcoxon test (p > 0.5), although values varied considerably.

Alpha diversity of the water microbial community at two different sites (ISM and OMW) of Sundarban mangrove forest areas.

Values are calculated based on repeated random subsampling to the lowest number of sequences per sample. The median per group presented in black line.

The microbial community of marine estuary water from Sundarbans was dominated with bacteria occupying more than 96% of total community and archaea represented only 4%. Bacterial assemblage of two different sites this area showed to have a distinct community. In class level, among the dominant bacterial groups, Flavobacteria (ISM: 15.9% vs OMW: 8.6%), Alphaproteobacteria (ISM: 29.5% vs OMW: 28%), and Acidimicrobiia (ISM: 6.6% vs OMW: 5.0%) were dominant in ISM while OMW was dominated with mainly with Gammaproteobacteria (ISM: 22.6% vs OMW: 35.3%) (. At higher taxonomic resolution levels (, bacterial communities were composed with a total of 474 and 915 different bacterial family and genus, respectively. The most dominant bacterial family was Rhodobacteraceae (18.6%), almost equally distributed between studied two sites. Other dominant bacterial families of ISM were Flavobacteriaceae (14.8%) and OM1 clade (5.2%) whereas in OMW, Oceanospirillaceae (16%) and Spongiibacteraceae (4%) were the most abundant.

Taxonomic composition of dominant bacterial taxa on family level across eighteen samples under sites ISM and OWM (nine samples each).

Ten (10) most abundant bacterial families for each of the samples were reported here and rests less dominant members are label as “other”.

We observed dominancy of Marine Group (MG I) (currently known as Thaumarchaeota) and Euryarchaea MG II in archaeal community assemblages with 78.5% and 16.9% of relative abundance, respectively while the presence of Woesearchaeota (2.1%) was also evident (. MG I was found in relatively higher abundant at ISM constituting on average 82.1% of sequences as opposed to 75% at OMW. Euryarchaeota MG II comprised about 20.3% at OMW compared to 13.7% at ISM. Among total twenty-nine (29) archaeal genera, Candidatus Nitrosopumilus and Candidatus Nitrosopelagicus accounted for the 40.7% and 21.4% of total relative abundance, respectively ().

Taxonomic compositions of dominate archaeal phyla across eighteen samples represents two sites ISM and OWM (nine samples each).

Ten (10) most abundant phyla for each of the samples were reported here and rests less dominant members are label as “other”.

3.3 Environmental drivers of bacterial communities

At OTU resolution level also, distinct microbial communities were observed between OMW and ISM based on changes in community structure (beta diversity) which is quantified by non-metric multidimensional scaling (NMDS) plot by calculating Bray–Curtis dissimilarly (). This pattern is confirmed by the ANOSIM test that indicated a significant difference in microbial community structure between ISM and OMW (ANOSIM, R = 0.24, p < 0.001). Redundancy analyses attempted to identify the water quality parameters that had strong explanatory power for microbial communities. We observed that total nitrogen (TN) and dissolved inorganic nutrients (DIN) accounted for almost 10% of the variability in microbial community of OMW where TN alone explain 6% variation of microbial community (RDA, R2 = 0.06, F(1,7) = 1.34, p < 0.05). In contrast, salinity and TN explained approximately 9% of the variability in community composition of ISM (RDA, R2 = 0.06, F(1,7) = 1.34, p < 0.05) and alone salinity responsible for explaining 7% microbial variation of this site ().

4. Discussion

The pH values (8.0–8.7) indicates the water of ISM and OMW slightly alkaline in nature which supports the previous findings in similar samples from Sundarbans Mangrove forest areas [30]. Such ranges of pH may be attributed by the buffering capacity of water that support high biological activity [31,30]. The water of Sundarbans is characterized by elevated salinity values in line with previous reports [31]. The long-term changes in water properties in the eastern part of Sundarbans, sampling regions of our study, indicating increased trends on salinity and pH [3]. Our result shows differences in measured environmental parameters between two sites represented by three sampling stations and nine samples each leading to their segregation into two clusters (in PCA analysis) along with their sampling sites. This ordination as a result of different nutrient loads and this is reflected by potential eutrophication in water from OWM. Influences from the Thakuran and Matla rivers reported to have a strong impact on the estuary water quality represented by OWM [4,5], that also reflected in our study. The perennial discharge in Thakuran and Matla Estuary from upstream regions brings in a high suspended matter load throughout the year [30]. Those estuaries severely contaminated with huge organic load and sediment flux originated from upstream domestic sewage, aquaculture, intensive trawling activities, agricultural runoff as well as soil erosion [32,33] may supports the prospective cause for eutrophication in the OWM sites.

Because of the relevance of microbial community of Indian Sundarbans, several investigators attempted on surface sediments samples [34,8,3,10,35] as well as recently on water column of this regions [12,13] using 16S rRNA gene metagenomic approaches. Unlike previous studies, this investigation attempted to asses both the bacterial and archaeal community at a same time of water from relative less anthropogenic disturbance sites using an efficient primer set to target V4-V5 variable region of 16S rRNA gene in order to avoid the problems of underestimated or overestimated common marine taxa [14], therefore our investigation gives more accurate and well-resolved picture of microbial communities of these sites.

Although insignificant differences, elevated trends of α-diversity of the marine estuary water samples (OWM) might be an indications of relatively rich bacterial community compared to ISM of Sundarbans might be attributed toward their elevated eutrophication. This observation was supported by previous reports that indicate a higher diversity and equitability in the human impacted estuary because of proliferation of several different microorganisms [36,37].

The bacterial assemblages of the studied samples (specially OMW) showed similarity with the previously reported bacterial community of marine sediments and water samples of Sundarban Mangrove areas [8, 10–12]. The most dominant bacterial family presents both the station with almost equal proportion is Rhodobacteraceae. Dominancy of members of this family in marine water microbial community previously reported and known to be associated with marine phytoplankton blooms where it plays important role in transforming phytoplankton-derived organic matter [34,38,39]. The abundant OTUs of Rhodobacteraceae are classified as anoxygenic phototrophs Nautella, reported to serve an indicator of marine eutrophication, predominantly found in higher eutrophic OMW samples. The other dominant one under the same family is marine heterotrophs Ruegerias (almost equally distributed among both ISM and OMW) serve as the model of marine sulfur and carbon cycle [38,40,41].

Interestingly enrichment of several OTUs from the Flavobacteria is observed in the oligotrophic ISM compared to the eutrophic OMW. They are specialized in utilization of biopolymers and organic substances in oligotrophic environment i.e., when organic substances present even at very low concentrations. Higher abundance of these polymers degrading bacteria responsible for releasing the nutrients heterotrophs/copiotrophic organisms thus helps in the growth of heterotrophic bacterial community at oligotrophic marine environments [42-44]. Therefore, they might plays central role for balancing the heterotrophs/copiotrophic microbial ecology of ISM. The dominant OTUs under family Flavobacteriaceae were mainly classified as Aureimarina and NS5 marine group genus. Roles of Aureimarina in marine biogeochemistry has not been investigated much although few studies reported on their presences in coastal seawater and saline estuarine [45,46]. However, this is the first report of their abundance in marine water of Sundarbans. The NS5 marine group which are equally dominated in both the studied sites are reported to be ubiquitous in the seawater-related samples and known for phytoplankton-derived macromolecules [47-49].

This investigation identified Actinobacteria constituted a predominant fraction both in OMW and ISM but elevated amount in the later samples. Bacteria under this group are consisted of both copiotrophic and oligotrophic members with higher abundance in oligotrophic marine environments [50,51]. Their presence in mangrove estuary regions is well documented [52-54]. As marine Actinobacteria are the richest sources of secondary metabolites thus, have been well reported as potential sources of bioactive compounds [55]. Therefore, their abundance in our studied sites (specially ISM) would be potential hotspot for isolating bioactive molecules from Indian Sundarban mangrove forest. The OM1 clade (dominating in ISM), an uncultured Actinobacterial clade, frequently recovered from various marine environments with higher abundance at near coastal sites than open marine areas however supports our reports [56-58]. The dominant OTUs of this family were classified as Candidatus Actinomarina. Those photoheterotrophs are one of smallest free-living prokaryotes are reported to be ubiquitous in marine systems. Not many reports are described their role in the marine biogeochemical cycle but Actinobacteria are reported to be associated in carbon cycling to decompose the plant biomass via degrading the cellulose and hemicellulose materials, a dominate resources materials in mangrove, however may supports their dominancy in ISM. They are also known as chitin, hydrocarbons and organic contaminants degrader [59,60].

The OTUs affiliated to families Oceanospirillaceae and Spongiibacteraceae of Gammaproteobacteria showed increase abundance in the impacted site OMW. Bacteria from these families are known to be present in eutrophic marine environments. They are known as polymer degraders and can utilize polyhydroxy alkanoate compounds and proteorhodopsin, for harvesting an additional energy, supports their living in eutrophic water samples [61-63]. The dominant OTUs of Oceanospirillaceae are affiliated to chemoheterotrophic genus Marinobacterium. Their presence in mangroves as well as surface seawater have already been described in previous studies and known to be associated with hydrocarbon biodegradation [64,65]. The other dominated bacterial family in the samples from OMW is Spongiibacteraceae. They comprise mainly marine bacteria known as Oligotrophic Marine Gammaproteobacteria (OMG) group [66,67]. We recorded the dominant OTUs of this family are affiliated with BD1-7 which is a cosmopolitan group of Gammaproteobacteria is mostly autochthonous, reported to inhabits at diverse marine habitats [68,65,69]. In line with previous reports this investigation, therefore, indicates proliferation of bacterial groups under Gammaproteobacteria with respond to increased nutrient concentrations in estuary [36].

However, in contrast to sediments reported in previous investigations, an archaeal community of marine waters in the Sundarban mangroves is dominated with Thaumarchaeota Marine Group (MG I) and Euryarchaea MG II. The chemolithoautotrophic MG I which are in higher in number on ISM are responsible for oxidation of ammonia and showed ability in inorganic carbon fixation [70] thus important players in global Carbon (C) and Nitrogen (N) biogeochemical cycles. While the heterotrophic MG II, dominated in OMW, previously reported to be abundant in the marine aquatic environment [71]. Their abilities to acquired energy in presences of light through organic carbon degradation in the photic zone is also documented [71]. Dominant OTUs of MGI group are affiliated with ammonia-oxidizing archaeal, Candidatus Nitrosopumilus and Candidatus Nitrosopelagicus, play important roles in nitrogen and carbon cycling of marine ecosystem [3]. However, this investigation reports first on their presence in this areas. Therefore, the biological and geochemical processes at estuary water habitats in the Indian Sundarban Mangrove areas have likely influenced the archaeal community structure.

Overall, our study indicates along with the elevated level of average pH and salinity, the open marine water (OMW) showed eutrophication probably leads to an observed bacterial shift toward more copiotrophic and photoheterotrophic bacterial (Oceanospirillaceae and Spongiibacteraceae) and archaeal community (Euryarchaea MG II) and compared to the more oligotrophic microbial community (Aureimarina, NS5 marine group, OM1 clade and Thaumarchaeota MG I) of costal water of a small Island of Sundarban Mangroves (ISM). These microbial assembles thus might represent key players in biogeochemical cycle of this mangrove and the studied areas represent a hotspot for bacterial having potential to produce the commercially important secondary metabolites. This investigation also reports that total nitrogen and dissolved inorganic nitrogen are the major environmental contributors on determining the microbial communities for OMW and for ISM it is combination of total nitrogen and salinity.

5. Conclusion

This investigation provides the first details description of bacterial and archeal communities concurrently of Thakuran and Matla river complex of Maipith coastal areas in the Indian Sundarbans mangroves areas. This study indicates the eutrophication in open marine water (OMW) dominated with more copiotrophic and photoheterotrophic bacterial and archaeal community (Euryarchaea MG II) and while oligotrophic microbial community abundant in costal water of the Island of Sundarban Mangroves (ISM).

However, given the rising burden on Indian mangrove coastal ecosystems, this study suggests that sewages from urban areas lacking proper treatment can alter microbial communities that may play vital role in biogeochemical cycle (nitrogen cycle) of mangrove ecosystem and consequently may impact on the climate in the tropical country.

6. Acknowledgments

Foremost PKD would like to thank NAM S&T Centre New Delhi, India and ZMT, Bremen for selection. PKD also acknowledge to Jadavpur University for granting the leave to avail this fellowship. We sincerely thank the faculty and technical staff of ZMT Bremen for their generous help to carry this research work. Many thanks to Dr Halina Tegetmeye of CeBiTec Bielefeld, Germany for sequences. The assistance of conducting analyses at the laboratories at the ZMT in Bremen by Matthias Birkicht, Sonja Peters and Achim Meyer also acknowledge. Dr. Debajit Datta, Assistant professor, Jadavpur University is also acknowledged for helping in designing the map of the sampling location.

Heatmap of pairwise correlations between the different environmental parameters and three principal components.

Levels of correlations are indicated with different color bar.

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The rarefaction curve of the eighteen (18) samples, indicated by the number of OTUs as a function of the number of reads.

The curve approaching plateau indicates that the number of reads are enough to describe the OTUs representing the community.

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Taxonomic composition of the ten (10) most abundant bacterial phyla in the studied two sites ISM and OWM represented by eighteen samples (nine samples each).

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Taxonomic composition of the ten (10) most abundant archaeal genus in the studied two sites ISM and OWM represented by eighteen samples (nine samples each).

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Kruskal-Wallis test for Environmental parameters at the six sampling sites.

(DOCX)

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Kruskal-Wallis test for alpha diversity of the six sampling sites.

(DOCX)

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4 Oct 2019

PONE-D-19-22253

Insights on aquatic microbiome of the Indian Sundarbans mangrove areas

PLOS ONE

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Reviewer #2: Partly

**********

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**********

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Reviewer #1: PLOS ONE

Manuscript: PONE-D-19-22253

In this manuscript, the authors examined environmental parameters, microbial community composition in the island of Sundarban mangroves (ISM) and open marine water (OMW) using 16S rRNA gene-based amplicon, and attempted to assess the capability of environmental parameters for explaining the variation in microbial community composition. The authors reported that a higher eutrophic condition changes in bacterial communities in OMW. The authors did not make a solid conclusion. The authors concluded that multiple environmental/anthropogenic stressors (salinity, pollution, eutrophication, land-use) affect the estuary water and consequently the microbial communities in concert. Suggest also sample open seawater (or seawater standard), conduct examinations of water parameter and microbial community in open seawater, ISM, and OMW, and make comparison among three areas, and make full discussion. Suggest report clearly on pollution and eutrophication (Line 54). Examination on the environmental determinants that contribute to the microbial communities is unclear (Line 103-104). Suggest examine total phosphorus, organic phosphorus, total alkalinity, hardness, dissolved solids, and major ions and substances like sodium, potassium, calcium, magnesium, sulfate, carbonate, and chloride. There are many grammatical errors and typo errors.

1. Line 53: Rewrite the sentence.

2. Line 112, Line 117: Reference “Das 2011” is not listed in the Reference section.

3. Lines 117-119: Suggest examine total phosphorus (TP). Suggest examine orthophosphate-phosphorus (Orthophosphate-P, PO43--P).

4. Lines 127-136: Suggest rewrite the sentences. Suggest write detail about each parameter assay. It said “continuous flow analyzer (Flowsys by Unity Scientific, Brookfiels, USA)” (Line 129-130). It said “spectro-photometrical analysis with a Flows continuous flow analyzer (Systea, Anagi, Italy)” (Lines 133-134).

5. Line 129: It is “phosphate (PO43-)” or “orthophosphate (PO43-).

6. Line 128: Report content of dissolved inorganic nutrients (DIN).

7. Line 128: Report clearly on nitrate and nitrite (NOx).

8. Line 129: The statement “silicate (Si)” is not correct. Report it is silicon (Si), Silica (SiO2), or silicate.

9. Line 132: Suggest report examination of major water compositions like sodium, potassium, calcium, magnesium, sulfate, bicarbonate, carbonate, chloride.

10. Line 156: Reference “Maher et al 2014” is not listed in the Reference section.

11. Line 265: Suggest report clearly on high suspended matter.

12. Line 272: Reference “Eloc-Fadrosh et al. 2015” is not listed in the Reference section.

13. Line 273: Change to “Ghosh and Bhadury 2018, 2019”.

14. Lines 288-289: Change to “Basak et al. 2015, 2016; Ghosh and Bhadury 2017, 2018”.

15. Line 203: Reference “Buchan et al. 2010” is not listed in the Reference section.

16. Lines 302-306: Rewrite the sentence.

17. Lines 351-356: Rewrite the sentences.

18. Lines 251-358: The discussion is disjointed. Suggest link water parameters and microbial community composition in Family, make full discussion, and make discussion in these areas and open seawater.

19. Lines 359-377: The conclusion is lengthy. Suggest make a solid conclusion.

20. Lines 394-540: Suggest follow the Journal format, and write reference. Suggest write all authors.

Reviewer #2: In this study the authors have investigated aquatic microbial community structure from two estuaries of Sundarbans, world's largest contiguous mangrove ecosystem. While the findings of this study are potentially very interesting, I have highlighted some points that the authors need to address along with further strengthening of the discussion section.

The reference cited in the first line of Introduction is not correct. For example, Spalding et al (1997) and Gopal and Chauhan (2006) references would be more appropriate.

Introduction: However, sampling locations of those studies on microbial communities.... a single site specific with limited number of samples. This sentence need to be correct. The paper by Ghosh and Bhadury (2019) has looked in bacterioplankton community composition from four estuaries- Mooriganga, Thakuran, Matla and Harinbhanga.

Was the sampling undertaken during high or low tide? This should be clearly mentioned. What factors were taken into consideration for selection of sites? These points should be clearly stated.

How was the calibration of Eureka 2 Manta multiprobe undertaken?

Why V4-V5 region of 16S rRNA was targeted? This needs to be written upfront in introduction section given the importance of scales of taxonomic resolution among variable regions of this molecule.

The authors have reported dominance of Actinobacteria-like OTUs/sequences in their studied samples. I believe it is important to compare this finding with previous study undertaken in the same estuary as well as from other mangroves (e.g. Gong et al 2019, Scientific Reports). It is also important to highlight the specific roles of this group in terms of biogeochemical cycling (e.g. breakdown of complex forms of carbon and resulting role in remineralization).

One of the important groups that was reported from this mangrove is the presence and dominance of Firmicutes. However, I do not see any discussion on this point given Firmicutes signal were found to be much higher in Sundarbans compared to other mangroves.

It is really interesting to see the relative dominance of Thaumarchaeota Marine Group (MG I) and Euryarchaea MG II in this ecosystem. I am just curious if the authors can link this finding with reported dissolved ammonium concentration in this sector of Sundarbans given the availability of ammonia seems to be episodic in nature and thus there are consequences for nitrification rates. Also, the selection of primers improved the resolution in terms of encountering these groups? It is also important to highlight given the sampling has been focused only one time point, the observed community structure may also exhibit temporal variability.

**********

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Reviewer #2: No

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20 Dec 2019

Journal Requirements

Q 1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf. and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf.

Answer: We have tried to make all necessary changes as needed.

Q 2. We note that Figure(s) 1 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

Answer: Thanks for rising this issue. You may notice the above said Figure (i.e Figure 1) is a map of the sampling sites. This is to bring your kind notice that this figure neither copied / collected from any other sources nor it is a previously copyrighted maps. The concern figure has been prepared manually in laboratory using open source database of GADM (https://gadm.org/). The GPS data of sampling sites were collecting during sampling and those were compiled together in an ArcGIS10.3 software environments. Therefore, I think this figure do not need any copywriter permission. The revised manuscript is updated with this information in legend section of the Figure 1.

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1:

P1. In this manuscript, the authors examined environmental parameters, microbial community composition in the island of Sundarban mangroves (ISM) and open marine water (OMW) using 16S rRNA gene-based amplicon, and attempted to assess the capability of environmental parameters for explaining the variation in microbial community composition. The authors reported that a higher eutrophic condition changes in bacterial communities in OMW. The authors did not make a solid conclusion. The authors concluded that multiple environmental/anthropogenic stressors (salinity, pollution, eutrophication, land-use) affect the estuary water and consequently the microbial communities in concert.

Answer: We agree with the reviewer comments that this manuscript may need more concrete conclusion. The microbial communities of world largest mangrove sites demands more extensive investigations. However, our investigation few first attempt to reports the changes of microbial community that majorly impacted by the multiple environmental/anthropogenic stressors of the mangrove estuary water. In line with the reviewer suggestions we have included few more important observations on microbial communities in the discussion part of current manuscript. (Line 262-262, Line 295-299, Line 310-325, Line 348-354, Line 357-367)

P2. Suggest also sample open seawater (or seawater standard), conduct examinations of water parameter and microbial community in open seawater, ISM, and OMW, and make comparison among three areas, and make full discussion.

Answer: Thanks for this rising this issue and we also agree with the reviewer suggestions to on include the samples from open seawater. In this connection, this is to bring your notice that, OMW represents the samples collected the open marine water were included in the current investigation. The water parameters and microbial community of OMW were measured and compared with ISM which is marine water collected from coastal regions of a small Island of Sundarban Mangroves.

P3. Suggest report clearly on pollution and eutrophication (Line 54).

Answer: Thanks for suggestions. Now in the current manuscript updated information on the pollution and eutrophication is clearly stated (Lines 262)

P4. Examination on the environmental determinants that contribute to the microbial communities is unclear (Line 103-104).

Answer: In microbial ecology, redundancy analysis (RDA), a multivariate statistical analysis, is considerably being used to determine which of the environmental factors are the most significant to explain variation in microbial community composition (Alban Ramette, 2007; Chen et al 2018; Lee et al 2019; Shankar et al 2019). However, in the present study we have also attempted to evaluate the role of environmental parameters as drivers of the variations in community compositions in the studied sites. Our result indicates, total nitrogen (TN) and dissolved inorganic nitrogen (DIN) accounted for almost 10% of the variability in microbial community of Open Marine Water where TN alone explain 6% variation of microbial community (RDA, R2 = 0.06, F(1,7) = 1.34, p < 0.05). In contrast, salinity and TN explained approximately 9% of the variability in community composition of the water collected from costal area of an small Island of Sundarban Mangroves (RDA, R2 = 0.06, F(1,7) = 1.34, p < 0.05) and alone salinity responsible for explaining 7% microbial variation of this site .

P5. Suggest examine total phosphorus, organic phosphorus, total alkalinity, hardness, dissolved solids, and major ions and substances like sodium, potassium, calcium, magnesium, sulfate, carbonate, and chloride.

Answer: We would also like to thanks the reviewers for rising this issue. Those are the water parameters that should be measured. But unfortunately in the current situation we can’t do so. All those works had been perform at The Leibniz-Zentrum für Marine Tropenforschung in Bremen, Germany (ZMT) under expert supervisions. After completion of all the experiments were over we were asked to discard all remaining water samples, as per The National Biodiversity Authority (NBA), Govt. of India rules. Therefore, we do not have any more samples to measure the suggested water parameters.

Q1. Line 53: Rewrite the sentence.

Answer: The mentioned sentence rewritten now.

Q2. Line 112, Line 117: Reference “Das 2011” is not listed in the Reference section

Answer: We agree with the reviewer views and sorry for this typo. It would be “Das 2016” and necessary changes has been made.

Q3. Lines 117-119: Suggest examine total phosphorus (TP). Suggest examine orthophosphate-phosphorus (Orthophosphate-P, PO43--P).

Answer: Thanks for your kind suggestions, but I afraid I can measure total phosphorus (TP) and orthophosphate-phosphorus (Orthophosphate-P, PO43—P) in the current perspective as mentioned previously (lacking of the samples).

Q4. Lines 127-136: Suggest rewrite the sentences. Suggest write detail about each parameter assay. It said “continuous flow analyzer (Flowsys by Unity Scientific, Brookfiels, USA)” (Line 129-130). It said “spectro-photometrical analysis with a Flows continuous flow analyzer (Systea, Anagi, Italy)” (Lines 133-134).

Answer: Sorry for this unintentional mistake. The lines are corrected now. I was a mistake.

Q5. Line 129: It is “phosphate (PO43-)” or “orthophosphate (PO43-).

Answer: It is phosphate PO43-. We were not dealing with orthophosphate.

Q6. Line 128: Report content of dissolved inorganic nutrients (DIN).

Answer: The dissolved inorganic nutrients (DIN) content already reported in the present manuscript. Their amount varies among the samples as indicated in Table 1 and Figure 2 (PCA plot).

Q7. Line 128: Report clearly on nitrate and nitrite (NOx).

Answer: This manuscript in line with the previous reports (Kegler et al 2018) that uses the NOx to represent the combined amount of nitrate and nitrite content. The nitrite (NO2-) content in the studied samples is very insignificant therefore nitrate (NO3-) content along with NOx were reported in this manuscript (Table 1 and Figure 2).

Q8. Line 129: The statement “silicate (Si)” is not correct. Report it is silicon (Si), Silica (SiO2), or silicate.

Answer: It’s a mistake. Necessary changes has been made as silicate (SiO44−).

Q9. Line 132: Suggest report examination of major water compositions like sodium, potassium, calcium, magnesium, sulfate, bicarbonate, carbonate, chloride.

Answer: I fully agreed with the reviewers suggestions. When the experiments were carried out, we had no plans to measure them as they were not included in the proposed project. Unfortunately in the current situation we cannot measure those parameters because of lacking of samples.

Q10. Line 156: Reference “Maher et al 2014” is not listed in the Reference section.

Answer: The above mentions citation is “Mahe et al. 2014” which is available in Reference section.

Q11. Line 265: Suggest report clearly on high suspended matter.

Answer: Yes. We fully agreed with the reviewers suggestions. I am again sorry to inform you we are unable to measure any other environmental parameters.

Q12. Line 272: Reference “Eloc-Fadrosh et al. 2015” is not listed in the Reference section.

Answer: It’s a mistake. The concern citation deleted from the updated manuscript.

Q13. Line 273: Change to “Ghosh and Bhadury 2018, 2019”.

Answer: thanks for this corrections. Necessary change has been made as per suggestion.

Q14. Lines 288-289: Change to “Basak et al. 2015, 2016; Ghosh and Bhadury 2017, 2018”.

Answer: Necessary correction made in manuscript as suggested. Thank you.

Q15. Line 203: Reference “Buchan et al. 2010” is not listed in the Reference section.

Answer: It’s a typos. It’s would be Buchan et al. 2005.

Q16. Lines 302-306: Rewrite the sentence.

Answer: The sentence are rewritten now.

Q17. Lines 351-356: Rewrite the sentences.

Answer: The sentences in these lines are rewritten now.

Q18. Lines 251-358: The discussion is disjointed. Suggest link water parameters and microbial community composition in Family, make full discussion, and make discussion in these areas and open seawater.

Answer: Thanks for the suggestion. The discussion part of the revised manuscript is updated with modified suggestions. I think, after incorporating the reviewer suggestions, the revised manuscript is now much improved one. (Line 262-262, Line 295-299, Line 310-325, Line 348-354, Line 357-367)

Q19. Lines 359-377: The conclusion is lengthy. Suggest make a solid conclusion.

Answer: Again many thanks for the suggestions. The conclusion part is modified now.

Q20. Lines 394-540: Suggest follow the Journal format, and write reference. Suggest write all authors.

Answer: We have tried to make all necessary changes as needed

Reviewer #2:

P1: In this study the authors have investigated aquatic microbial community structure from two estuaries of Sundarbans, world's largest contiguous mangrove ecosystem. While the findings of this study are potentially very interesting, I have highlighted some points that the authors need to address along with further strengthening of the discussion section.

Answer: We fully agreed with the reviewers’ comments and suggestions. We tried to address all the issues raised by the reviewer and this eventually improved the quality of the manuscript.

Q1: The reference cited in the first line of Introduction is not correct. For example, Spalding et al (1997) and Gopal and Chauhan (2006) references would be more appropriate.

Answer: We agreed with the reviewer’s comments and changes has been made in the modified manuscript with the more appropriate citation accordingly.

Q2: Introduction: However, sampling locations of those studies on microbial communities.... a single site specific with limited number of samples. This sentence need to be correct. The paper by Ghosh and Bhadury (2019) has looked in bacterioplankton community composition from four estuaries- Mooriganga, Thakuran, Matla and Harinbhanga.

Answer: Sorry for this mistake and necessary changes have been made in the revised manuscript. (Line 85-89)

Q3: Was the sampling undertaken during high or low tide? This should be clearly mentioned. What factors were taken into consideration for selection of sites? These points should be clearly stated.

Answer: The samples were collected during low tide condition and this information mentioned in the revised MS. The rational for selecting the sites as also stated.

Q4: How was the calibration of Eureka 2 Manta multiprobe undertaken?

Answer: This experiment was performed at ZMT under the expert supervision. This multiprobe kit has the respective calibration solution and calibration was done with those solution.

Q5: Why V4-V5 region of 16S rRNA was targeted? This needs to be written upfront in introduction section given the importance of scales of taxonomic resolution among variable regions of this molecule.

Answer: Thanks for the suggestions. We agree with reviewer’s view to incorporate justification behind aiming the V4-V5 region of 16S rRNA gene in the introduction section of this manuscript. In line with the reviewers suggestion, the importance of using the primer set (that targeting V4-V5 region of 16S rRNA gene) used in the current investigation has been discussed in the manuscript. (Line 97-101 and Line 267-273)

Q6: The authors have reported dominance of Actinobacteria-like OTUs/sequences in their studied samples. I believe it is important to compare this finding with previous study undertaken in the same estuary as well as from other mangroves (e.g. Gong et al 2019, Scientific Reports). It is also important to highlight the specific roles of this group in terms of biogeochemical cycling (e.g. breakdown of complex forms of carbon and resulting role in remineralization).

Answer: We agreed with the reviewers comments compare their presences in mangrove and to highlight the specific roles in carbon biogeochemical cycling. That would improve the quality of the MS. Therefore, we follows the suggestions and modified the discussion part (Line 307-318).

Q7: One of the important groups that was reported from this mangrove is the presence and dominance of Firmicutes. However, I do not see any discussion on this point given Firmicutes signal were found to be much higher in Sundarbans compared to other mangroves.

Answer: The members from Firmicutes not much dominated in these study sites. As mentioned the manuscript the top ten dominated bacterial phylum are Proteobacteria, Bacteroidetes, Actinobacteria, Planctomycetes, Verrucomicrobia, Cyanobacteria, Acidobacteria, Chloroflexi, Gemmatimonadetes, and Nitrospirae.

Q8: It is really interesting to see the relative dominance of Thaumarchaeota Marine Group (MG I) and Euryarchaea MG II in this ecosystem. I am just curious if the authors can link this finding with reported dissolved ammonium concentration in this sector of Sundarbans given the availability of ammonia seems to be episodic in nature and thus there are consequences for nitrification rates. Also, the selection of primers improved the resolution in terms of encountering these groups? It is also important to highlight given the sampling has been focused only one time point, the observed community structure may also exhibit temporal variability.

Answer: Thank you for asking this question. This issue would be an interesting perspective to investigate with reference to current data. In fact in the subsequent project we are proposing to investigate this objective. With the current data set, I am afraid we can really do that, as we have not measured the concentration of dissolved ammonium during the investigation on 2017. But we have framed one collaborative project to investigate the nitrification rate and responsible microbial community structure in Sundarban Mangrove.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

21 Jan 2020

PONE-D-19-22253R1

Insights on aquatic microbiome of the Indian Sundarbans mangrove areas

PLOS ONE

Dear Dr. Dhal,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Mar 06 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Jiang-Shiou Hwang, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: PLOS ONE

Manuscript: PONE-D-19-22253-R1

The authors have revised and improved the manuscript. Several parameters are concerned. Suggest conduct minor modification.

1. Lines 121-133: It said physicochemical parameters (salinity and pH), DOC, nutrient, nitrate, nitrite, combined nitrite and nitrate etc. However, they did not report measurements of TN and DIN (Table 1).

2. Line 131-132: It said nitrite, nitrate, phosphate, silicate were analyzed using continuous flow analyzer. Suggest report clearly the measurements.

3. Line 132: It said ‘phosphate (PO43-)”. In Table 1, it reported TN (total nitrogen), DIN (dissolved inorganic nitrogen), and PO4. Nitrogen and phosphorus are important nutrients. Suggest check the measurement is orthophosphate (PO43-), not including pyrophosphate and metaphosphate. It is fine to use phosphate (PO43-). The orthoposphate (PO43-) is commonly used and reported as phosphate (PO43-). However, suggest check the standard solution, and check the measurement is phosphorus or phosphate. Suggest change the term here to orthophosphate-P or phosphate-P (PO43--P) to match well with DIN. Suggest check the term PO4 in Table 1. It is not correct. Suggest use orthophosphate-P, or simply use PO43--P.

4. Line 132: It said silicate (Si). In Table 1, it said Si. They are not correct. The term of Si is silicon, not silicate. Readers know silicate is important, and the measurement of silica (SiO2) is used for evaluating content of silicate. Suggest check the standard solution and measurement of this parameter. I think it is silica (SiO2), not Si, not silicate. Please check it is silica (SiO2) or silicon (Si). Suggest report silica, not silicate, not silicon.

5. Line 125 and Table 1: It said DOC and said DOC (µM). Suggest report clearly the measurement. Suggest check the unit “µM”. Suggest check the standard and report which chemical is used as standard.

Reviewer #2: The authors have addressed the points that I raised earlier. The manuscript is now acceptable for publication.

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Reviewer #1: No

Reviewer #2: Yes: Punyasloke Bhadury

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30 Jan 2020

Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1:

P1. Lines 121-133: It said physicochemical parameters (salinity and pH), DOC, nutrient, nitrate, nitrite, combined nitrite and nitrate etc. However, they did not report measurements of TN and DIN (Table 1).

Answer: Yes. Its mistake. As I mentioned earlier, this work has been carried out in ZMT, Germany under expert supervision. The total nitrogen (TN) content of each the samples were measured using the EuroVector EA 3000 elemental analyzer. The DIN measurements were done based on the methods described by Grasshoff et al. 1999. (Page 6, line 127-128)

P2. Line 131-132: It said nitrite, nitrate, phosphate, silicate were analyzed using continuous flow analyzer. Suggest report clearly the measurements.

Answer: Thanks for this rising this issue and we also agree with the reviewer suggestions on including the details of this information. In this connection, this is to bring your notice that, the Dissolved inorganic nutrients that includes combined nitrate and nitrite (NOx), phosphate (PO43--P), and silicate Si(OH)4-) were analyzed using a continuous flow analyzer (Flowsys by Unity Scientific, Brookfield, USA) and detected spectrophotometrically as a colored complex (Grasshoff et al. 1999). To correct for the water color, absorption of the samples was measured without chemicals first, and the values were subtracted from those obtained with chemicals. Precision of the method was better and most of the nutrient concentrations were near the detection limit.

P3. Line 132: Line 132: It said ‘phosphate (PO43-)”. In Table 1, it reported TN (total nitrogen), DIN (dissolved inorganic nitrogen), and PO4. Nitrogen and phosphorus are important nutrients. Suggest check the measurement is orthophosphate (PO43-), not including pyrophosphate and metaphosphate. It is fine to use phosphate (PO43-). The orthoposphate (PO43-) is commonly used and reported as phosphate (PO43-). However, suggest check the standard solution, and check the measurement is phosphorus or phosphate. Suggest change the term here to orthophosphate-P or phosphate-P (PO43--P) to match well with DIN. Suggest check the term PO4 in Table 1. It is not correct. Suggest use orthophosphate-P, or simply use PO43--P

Answer: Thanks for suggestions but we are sorry for the confusion rises again. We checked with the methods again as I had little knowledge with such details. This part of comments is enriching. After getting detail information it is now clear that its actually orthophosphate (PO43-) that we measured and now in the current manuscript for both text and Table 1, this information is updated as PO43--P (Lines 262).

P4. Line 132: It said silicate (Si). In Table 1, it said Si. They are not correct. The term of Si is silicon, not silicate. Readers know silicate is important, and the measurement of silica (SiO2) is used for evaluating content of silicate. Suggest check the standard solution and measurement of this parameter. I think it is silica (SiO2), not Si, not silicate. Please check it is silica (SiO2) or silicon (Si). Suggest report silica, not silicate, not silicon.

Answer: Yes. Again thanks for raising this issue. Actually it’s a silicate (Si(OH)4-) that we measured. For the calibration we used Sodium hexafluorosilicate (Na2SiF6) during measurements.

P5. 5. Line 125 and Table 1: It said DOC and said DOC (µM). Suggest report clearly the measurement. Suggest check the unit “µM”. Suggest check the standard and report which chemical is used as standard.

Answer: Yes. The DOC is measured in µM. The DOC were measured by the high-temperature catalytic oxidation method on a Shimadzu TOC-VCPH total organic carbon analyser. The instrument was calibrated with a 10-point calibration curve of serial dilutions from a potassium hydrogen phthalate certified stock solution (1000 ppm Standard Fluka 76067-500ML-F). Consensus reference material provided by DA Hansell and W Chan of the University of Miami was used a positive control between every 10 samples. Each sample was measured with five replicate injections.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

5 Feb 2020

Insights on aquatic microbiome of the Indian Sundarbans mangrove areas

PONE-D-19-22253R2

Dear Dr. Dhal,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Jiang-Shiou Hwang, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

10 Feb 2020

PONE-D-19-22253R2

Insights on aquatic microbiome of the Indian Sundarbans mangrove areas

Dear Dr. Dhal:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Prof. Jiang-Shiou Hwang

Academic Editor

PLOS ONE

Table 2

Contribution of environmental parameters including nutrient content of six sampling sites to explaining the variation in microbial community composition based on redundancy analysis (RDA).

Sampling station	Explanatory variable	Adjusted R2	F	df	p-Value
OMW	TN + DIN	6%	1.24	2, 6	0.022*
	TN	4%	1.34	1, 7	0.029*
	DIN	-	1.40	1, 7	0.125
ISM	Salinity + TN	7%	1.31	2,6	0.043*
	Salinity	9%	1.77	1,7	0.004**
	TN	0.6%	1.05	1,7	0.326

p-Values defined as significant at a threshold of 0.05 are highlighted in asterisks mark

Adjusted R2 are provided as goodness-of-fit metrics

df degrees of freedom (numerator, denominator).