Diverse key nitrogen cycling genes nifH, nirS and nosZ associated with mangrove rhizosphere soils of Pichavaram as revealed by culture dependent and independent analysis.

PCR-DGGE and culturable diversity analysis of nitrogenase gene nifH and denitrifying genes nirS and nosZ aliated with heterotrophic and unculturable bacterial communities associated with rhizosphere of A. marina, R. mucronata, S. maritima and S. brachiata revealed the dominance of gammaproteobacterial community across the rhizospheres. Sequence analysis of the PCR-DGGE proles of nifH genes clustered to unculturables, while majority of the nirS and nosZ genes clustered with unculturables with few culturable groups viz., Pseudomonas sp. and Halomonas sp. Culturable analysis reected the dominance of Gammaproteobacteria as both nitrogen xers and denitriers while other groups like Firmicutes and Alphaproteobacteria were very less represented among nitrogen xers, and denitriers respectively. A total of 16 different genera were identied as nitrogen xers and denitriers. BOX-PCR analysis of Mangovibacter, Vibrio, Bacillus and Catenococcus isolated in this study showed varied ngerprinting patterns compared to their respective positive controls reported earlier from this ecosystem, indicating they may be novel. suggesting that mangroves harbor denitrifying bacterial communities from both tidal and urban ecosystems. The results showed that majority of the nirS and nosZ gene obtained through DGGE analysis belonged to uncultured denitrifying bacterial group. Phylogenetic analysis of both nirS and nosZ genes formed two different clusters with ocean, marine and estuarine sediments in one cluster and agricultural isolates in another cluster which indicates the wide distribution and yet to explore unknown bacterial lineages in this ecosystem.


Introduction
Nitrogen is one of the most important nutrient in any ecosystem; the nitrogen cycle mediated by microbes is a very complex process which involves transformation of nitrogen to different forms through nitrogen xation, nitri cation, denitri cation, ammoni cation, anaerobic ammonium oxidizing, and dissimilatory nitrate reduction to ammonium (Purvaja et al. 2008). Genomics research and high-throughput Illumina sequencing methods provide a broader prespective on the diverse microbial communities and their functional genes involved in the nitrogen cycling process (Zhang et al. 2017).
The mangrove ecosystems located between terrestrial and marine interface environments along the tropical and subtropical coastline are frequently inundated by oods and high tides (Holguin et al., 200;Giri et al. 2011), but play a major role in protecting the coasts in the tropical and subtropical regions. This ecosystem is partially anaerobic coupled with high salinity and oxido-reductive potential; the microbiota associated with mangroves is represented by a combination of terrestrial, freshwater and marine microorganisms that are crucial to the biogeochemical productivity (Vazquez et al. 2000). The bacterial and archaeal groups inhabiting this ecosystems play a major role in nutrient transformation, ecological and biogeochemical functions (Cao et al. 2011), and is in uenced by salinity (Silveira, 2011), organic carbon (Dunaj et al. 2012), nitrogen content (Carriero et al. 2012), climate, and chemical substances (Bragazza, 2015), which in turn determine the diversity, distribution and function of the microbial communities in this ecosystem. The mangroves serve as a hot spot for the discovery of novel microbes with novel ecological functions (Rameshkumar et al. 2014; Raju et al. 2016).
Nitrogen xation and denitri cation have been reported in phylogenetically diverse group of bacteria and archaea; their diversity and distribution can be determined by targeting the functional marker genes such as nifH, nirS and nosZ (Jenkins and Zehr, 2008). Diverse group of bacteria and archaea harboring nifH/nirS involved in nitrogen xation and denitri cation have been reported from the estuarine ecosystems. Since most of these remain unculturable, advanced molecular tools have been employed to understand the diversity, function and distribution of these microbial groups (Ren et al. 2018). Nitrogen xation, a process where gaseous nitrogen (N 2 ) is converted to biologically available forms such as ammonia (NH 3 ) by diazotrophs is considered to be the major source of combined nitrogen input in mangrove forest habitats. Thus, the high productivity of mangrove ecosystems might be partially attributable to the high rate of biological nitrogen-fixing activity of free living diazotrophs in rhizosphere of mangroves as well as the sediments (Holguin et al. 2001).
Denitri cation, a functional trait distributed among taxonomically diverse group of microbes, ) is primarily a bacterial respiratory process regulated by four different enzymatic steps and cataslysed by four mettaloproteins such as nitrate reductases, nitrite reductases (nir), nitric oxide reductase and nitrous oxide reductases (nos) (Braker et al. 2000). Denitiri cation is reported in a wide range of heterotrophic (e.g., Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitri cans) and autotrophic bacterial communties (e.g., Thiobacillus denitri cans) belonging to the phylum proteobacteria (Green et al. 2010). However, the microbe mediated denitri cation also acts as a sink by removing the excess anthropogenic N input, thus preventing the transport of excess nitrogen to the estauarine and coastal systems which pose a serious threat to these ecosystems.
With the culture based analysis only a minimal proportion of the microbial population can be retrieved from any sample, leaving the rest undetected or uncultured. Hence, recent studies on microbial community assessment, using a wide range of techniques such as classical cultivation procedure, ngerprinting, clone libraries and next generation sequencing (Andreote, 2012;Zhang et al. 2017) have revealed the extensive microbial diversity and its function that were not detected earlier. DGGE is one of the ngerprinting approaches that have been designed to study microbial communities which cannot be achieved with the cultivable fraction represented by >1% of the total number of prokaryotic species present in a given sample. DGGE has been used to exploit communities from a wide range of ecosystems and also the microbes that are involved in biogeochemical cycling of an ecosystem (Rastogi et al. 2010).
This study mainly focused on accessing the diversity and distribution of bacterial communities associated with mangrove rhizospheres that contribute to nitrogen xation by targeting the nifH genes and the denitri ers harbouring nirS and nosZ gene by both culture dependent and independent approaches. Therefore exploring the diversity and distribution of rhizosphere associated diazotrophs and denitrifying microbial communities that drive the nitrogen cycle is essential to understand the biogeochemical cycling of nitrogen.

Materials And Methods
Site description and sampling: The study site Pichavaram mangroves is located on the southeast coast of India near Chidambaram, situated about 250 km away from Chennai. It is an estuary located in between Coleroon and Velar estuary (GPS data) which covers a total area of 1350 ha colonized by true mangrove species and some halophytic plants. The rhizosphere soil samples of A. marina, R. mucoronata, S. maritima and S. brachiata were collected randomly using a soil core, and transferred to sterile polyethylene bags and transported to the laboratory on ice within 6 hrs. The geographical location of each sampling sites were recorded using a global positioning system instrument (GPS) (GARMIN Etrex, Taiwan). A total of 10 samples were collected from the rhizosphere region with a minimum distance range of 2 kms of which 3 samples were from A. marina, 3 from R. mucronata, 2 from the intersecting region of A. marina and R. mucronata, 1 sample each from S. maritima and S. brachiata. The samples were stored at -80 °C for deoxyribonucleic acid (DNA) extraction.
Soil DNA extraction and PCR ampli cation of nitrogen cycling genes.
Total genomic DNA was extracted from each rhizosphere soil sample by CTAB-SDS method as described by Ghosh et al. (2010) and puri ed using MO-BIO DNA (MO BIO Laboratories, USA) clean up kit as per manufacturers guidelines and stored at -20 °C for further analysis. The targeted genes were ampli ed using gene speci c primers and annealing conditions are given in table 2. Ampli cation was veri ed by agarose gel electrophoresis and was subjected to DGGE analysis.

PCR-DGGE analysis
About 45 µl of the ampli ed products were loaded onto DGGE gel in Dcode TM mutation detection system (Bio-Rad, Laboratories, CA, USA). The electrophoresis was carried out at a constant temperature of 60 °C for 17 hrs, in 8% polyacrylamide gel (100% denaturant, 7 M urea, and 40% (vol/vol) formamide) with a 50% to 60% denaturant gradient and stained with SYBR gold nucleic acid and kept in dark for 20 mins.
The gel was then rinsed with double sterilized dist. water and the gel images were documented using UV illuminator (Gel-doc XR+, Bio-Rad laboratories, CA, USA). The digitized gel images were analysed using Bio-Rad ngerprinting II software and the position of the bands were recorded and the variable bands were then eluted from the gel by marking the band position with a sterile scalpel and transferred to sterile micro centrifuge tubes with TE buffer (pH 8.0) and incubated overnight at 4 °C. To recon rm the purity and position of the bands, 6 µl of the eluted bands was used as a template, with the same combination of DGGE primers but without GC clamps, the total reaction volume was made upto 50 µl, reampli ed and run in the same gradient DGGE and its position was con rmed and the products were puri ed.

Cloning and sequencing of DGGE bands
The puri ed PCR products were then ligated into pTZ57R/T cloning vector (InsTAclone PCR cloning kit, Fermentas, Thermo scienti c) and the ligation mixture was prepared as per the manufacturers instruction with slight modi cation, which contained 1µl of 10 X ligation buffer, 5 µl of template DNA, 1 µl of vector, 1 µl ligase enzyme and 2 µl of nuclease free water. The mixture was then incubated at 4 °C overnight for e cient ligation and transformed into competent cells of E. Coli strain XL1-Blue (Novagen, Madison, WI, USA). Plasmids were extracted using the Favorgen plasmid DNA extraction Miniprep kit (Favrgen, Taiwan) as per manufacturer instruction and the positive clones were sequenced. All the sequences were analysed through BLAST-N for determining the taxonomic identity and tBLAST-X for determining the protein identity. Phylogenetic trees were constructed by neighbor joining method using Mega 5.0.

Data analysis
Digitized images of DGGE ngerprint were used to quantify diversity by using quantity one software which detects bands and quanti es the relative concentrations of ampli ed bands from cumulative pixel intensities within a given lane. The Shannon diversity index was calculated from the number of bands and relative intensities of bands present in each lane. In order to evaluate the correlation between the diversity of nifH, nirS and nosZ genes among the different rhizosphere regions, multivariate Principle Component Analysis (PCA) was performed from the data obtained from the DGGE patterns, based on band intensity and position, and were analyzed by adopting PCA. All the values were log transformed before subjecting to analysis and PCA was performed using PAST 3.0. The similarities between the DGGE pro les were displayed graphically as a dendrogram based on UPGMA algorithms (unweighted pair group method with arithmetic averages).

Culturable analysis of nitrogen xers and denitri ers
About 5g of the individual rhizosphere soil samples were transferred into test tubes containing 50 ml of sterile dist. water and vortexed for about 30 min and serially diluted upto 10 8 , about 0.1 ml was spread plated onto LB agar, Nitrogen free medium and BTB agar plates. The plates were incubated for 3-4 days at room temperature and based on colony morphology, individual colonies were picked, and maintained as pure cultures in their respective media and also stored in 25% glycerol stocks at -80 °C.
Genomic DNA isolation of culturable bacteria Genomic DNA was extracted from overnight grown cultures in 10 ml LB broth incubated at 32º C for 24 hrs. The isolated DNA were subjected to PCR based screening for the presence of nitrogen xers and denitri ers ( Table 2).
Screening for nifH, nirS and nosZ harboring bacteria: The nifH gene fragments were ampli ed using primers as described by Poly et al. (2001) and the denitri ers, nirS and nosZ gene were ampli ed using primers described by Braker et al. (2000). All the PCR reactions were carried out in BioRad T100 thermal cycler. Primers and PCR conditions are given in table (2) Genetic diversity analysis using BOX PCR ngerprinting The genetic diversity among nitrogen xing and denitri ers was determined by BOX-PCR pro ling using BOX A1R primers (BenHaim et al. 2003). Primer and ampli cation conditions are given in the table (2). About 5 µl of the PCR products were run in EtBr stained 2% agarose gel in 0.5X TBE buffer at a constant voltage of 80V for 3-4 hours. The BOX-PCR pro les were visualized under UV illuminator, followed by digital image capturing using BioRad gel documentation system. Normalization, recognition and band assignment were made using Fingerprinting II software (BioRad, USA) by Dice coe cient with optimum range of 0.5%. The cluster analysis of similarity matrices was performed by Unweighted Pair Group with Mathematical Average (UPGMA) for dendrogram analysis.

Sequencing and Data Analysis of culturable bacteria
The universal bacterial primers 27f and 1492r were used to amplify culturable bacterial 16S rDNA genes and sequenced byEuro ns India Pvt. ltd. The sequence similarities were compared in EzTaxon databases (Chun, 2007). The phylogenetic trees were constructed, using MEGA 5.0 (Tamura, 2011) to determine the taxonomic a liation.

Soil characteristics
The physico-chemical properties of the rhizosphere soils of Pichavaram are shown in table 1. The soil pH was 7.2, the organic carbon (OC) was <0.78% and the organic matter 1.34%. The total nitrogen content was 916 mg/kg while available nitrogen was low with 173 mg/kg. Available Phosphorus as P, zinc as Z, copper as Cu, manganese as Mn, molybdenum as Mo and boron as B were found to be below detection limit (BDL). It was found that the available potassium as K was the most abundant macronutrient (980 mg/kg) in Pichavaram soil (Table 1).

Culturable bacteria from mangroves
A total of 579 culturable bacterial isolates with different colony morphology were selected and screened for nitrogen xers and denitri ers. All the isolates were maintained in LB agar for further analysis and stored as glycerol stock in -80 C.
Culture independent analysis of nifH gene diveristy (PCR-DGGE) The DGGE pro les of nifH genes of all the 5 rhizosphere samples showed varied banding pattern with a total of 10-15 bands per lane (Fig. 1). The pro les represented rich diversity in all the rhizosphere samples except S. maritima rhizosphere which had only 3-5 bands,indicating a low level of diversity of nifH gene associated with this rhizosphere. A total of 29 DGGE ribotypes for nifH ( Fig. 1) were eluted and assigned a unique number with a pre x MSSRF ie MSSRF 1H to MSSRF 29H.

Cluster analysis of nifH DGGE ribotypes
The DGGE ribotypes of nifH gene formed three major clusters (i) cluster A represented nifH ribotypes of A. marina, S. maritima and R. mucronata rhizosphere (ii) cluster B represented nifH ribotypes of A. marina and R. mucronata rhizospheres, their intersecting region and S. brachiata rhizosphere (iii) cluster C represented nifH ribotypes of R. mucronata and intersecting region of both A. marina and R. mucronata at 60% con dence level with considerable variation observed among different rhizosphere samples (Fig.  1b.) Phylogenies of nifH gene sequences The nifH genes have been used as marker genes for studying the nitrogen xing bacterial diversity and a number of bacterial groups harboring nifH genes have been reported in mangrove sediments, revealing high diazotrophic diversity in mangrove ecosystems (Zhang, 2008;2017). The taxonomic identi cation of nitrogen xing bacterium that represents the unique DGGE bands are summarized in the table (S1). BLAST-N analysis of 29 sequences revealed that 10 sequences fell in the range of 80-89% similarity values and 19 sequences fell within the range of 90-99% and were similar to nifH gene of uncultured organisms reported from various ecosystems. However, protein analysis revealed that majority of the sequences fell between 94-100% with similarity to the known nifH sequences of various environmental origins especially from saline soil and marine sediments. This indicates that the mangrove rhizosphere region harbors nitrogen xing bacterial communities similar to saline and marine environments. Further phylogenetic analysis of nifH gene formed two major clusters with 11 subclusters indicating the presence of diverse nifH gene in this ecosystem. All the sequences in cluster 1 represented the sequences from marine sediments, saltmarsh, high and low saline soils and sea sediments while cluster 2 showed similarity to sequences from rhizosphere soil of paddy and other terrestrial ecosystem (Fig. S1) .
Previous reports suggested that phylum Proteobacteria particularly Gammaproteobacteria and Deltaproteobacteria are the predominant nifH genes harbouring groups in the rhizosphere sediments from many mangrove species, (Wu et al. 2016;Zhang et al. 2017), similarly in the present study also Proteobacteria were found to harbor nifH genes-predominantly, which may be contributing to nitrogen xation in the mangrove ecosystem. In addition to proteobacterial groups, Firmicutes were also found to harbor nifH genes, Similarly, nifH gene sequences a liated with alpha, beta and gamma proteobacteria, have been reported previously by Bagwell et al. (2002), from the tropical seabed grass which is in concurrence with our study. The ndings of Bird et al. (2005) suggested that gamma proteobacteria are predominant and acts as an important component of the heterotrophic nitrogen xing microbial community of the tropical and subtropical oceans. The sequence analysis of nifH DGGE showed similarity to uncultured nitrogen xing bacterial groups reported from high and low saline soils (Yousuf, 2014), marine sediments (Dang, 2013), rhizosphere of smooth cordgrass and salt marsh (Lovell, 2012), and agricultural soils (Pereira, 2013). None of the 29 nifH sequences reported in this study were related to earlier known nifH genes of cultured nitrogen-xing bacteria reported neither from mangroves nor from other ecosystems, indicating the abundance of unreported uncultured nitrogen-xing bacteria in the mangrove rhizosphere soils. The phylogenetic placement of the nifH gene sequences from the mangroves exhibited unique nifH gene types a liated with the phyla belonging to unculturables. The sequences were partially matching with the nitrogen xers described from marine environments, and also those found in other ecosystems. Thus, the distribution of the nifH gene in the mangrove ecosystems represented both the marine and the terrestrial ecosystems.
Diversity of culturable nitrogen xing bacteria Nearly 52 strains formed pellicle in nitrogen free medium and showed positive ampli cation for nifH gene with amplicon size of 360 bp compared to Ciceribacter lividus MSSRFBL1 T used as positive control. The BOX-PCR ngerprinting of the 52 strains showed genetic variation and formed 23 clusters at 80% con dence level (Fig. 2a). BOX-PCR based analysis has been widely recognized as one of the most common tools for determining the microbial diversity particularly between closely related groups (Ikeda, 2013).
Our current knowledge on the microbial community pertaining to the South Indian mangrove ecosystems, is still largely based on cultivation-dependent studies ( (2007) showed that the nitrogen xers isolated from the rhizosphere of mangroves were distributed to various genera such as Azospirillum, Azotobacter, Rhizobium, Clostridium, Klebsiella, Vibrio, Phyllobacterium, Oceanimonas, Paracoccus, Corynebacterium, Arthrobacter, Aeromonas, and Pseudomonas, while this study also reported similar groups in addition Mangrovibacter and Rhodobacter sp. were reported.The BOX-PCR pro ling of vibrio consisiting of V. plantisponsor, V. alginolyticus and V. neocledonicus (Fig. 3a) was supported by the phylogenetic analysis of these strains as they formed an outward clade with the type strains. From Ez-Taxon analysis, it is understood that the isolates of species V. alginolyticus and V. neoclaedonicus cannot be distinguished based on 16S rDNA analysis and the difference in BOX pro le of these strains suggest that these may be novel species. On a similar note, so far only two Mangrovibacter species has been reported (Rameshkumar, 2010; Zhang, 2015) from the mangroves but isolation of additional ve Mangrovibacter species in this study displayed divergence from the reported strains in BOX-PCR pro ling as well as phylogenetic analysis indicating they could possibly be novel species. The genus Bacillus obtained in this study, showed similarity to B.
aerophilus 28K T which has been previously reported in stratosphere region of earth's atmosphere by Shivaji et al. (2006). It is known that the strains B. aerophilus, B. startosphericus and B. altitudinis, (Fig.  3a) cannot be differentiated by 16S rDNA analysis which is also well supported by BOX-PCR ngerprinting analysis. Our results con ded the same thus suggesting that further experiments has to be donet in order to prove that these might be novel species exhibiting diazotrophic activity.
Culture dependent and independent analysis of denitrifying bacteria DGGE analysis of nirS and nosZ genes Culture-independent approaches have been adopted to analyze the diversity of denitrifying genes like nirK, nirS and nosZ (Li et al. 2020;Gao, 2016) from forest and marine sediments. In this study nirS and nosZ genes were used as molecular marker to determine the distribution and diversity of culturable and unculturable denitrifying populations of mangrove rhizosphere. A total of 31 DGGE ribotypes for cdnirS coding nitrite reductase (Fig. 3a) and 21 DGGE ribotypes for nosZ gene coding nitrous oxide reductase gene (Fig.4a) were eluted and assigned a unique number from MSSRF CD1 to MSSRF CD31 for cdnirS gene and MSSRF Z1 to MSSRF Z21 for nosZ gene.
Cluster analysis of cdnirS and nosZ gene ribotypes At 60% con dence level, both nirS (Fig. 3b) and nosZ (Fig. 4b) genes formed ve and four major clusters respectively, with a high degree of variation among the rhizosphere regions. The cluster A represented nirS genes from A. marina, R. mucronata and S. maritime rhizosphere, while cluster B represents nirS genes from A. marina and R. mucronata, whereas cluster C, D and E comprised ribotypes of intersecting region and S. brachiata. But the cluster analysis of nosZ gene exhibited a unique pattern with the individual rhizosphere region forming single cluster, eg., cluster A comprised of nosZ ribotypes from A. marina and its intersecting region, cluster B represented nosZ ribotypes of R. mucronata, with outward cluster of samples from both S. maritima and S. brachiata.
The nosZ gene, encoding N 2 O reductase, an enzyme catalyzing the nal step of denitri cation, is largely unique to denitrifying bacteria. It represents the process leading to the loss of biologically available N from the sediments and has been used as a marker gene for determining the diversity of denitri ers (Hong, 2019). The DGGE ribotypes of the nitrous oxide reductase gene (nosZ) showed rich diversity associated with A. marina rhizosphere. Nearly 21 prominent bands with 10-12 bands in each lane was eluted and sequenced. BLAST-N analysis of the nosZ gene and the protein derived sequences showed 85-99% similarity and 83-98% similarities to unculturable nosZ gene respectively. Nearly 18 sequences showed similarity to uncultured nitrous oxide reductase gene reported from various environmental sources while sequences of two bands MSSRF Z10 and MSSRF Z18 were present in all the rhizosphere samples and showed 95-98% similarity to Pseudomonas balearica DSM 6083 T genome and band MSSRF Z17 from S. maritima rhizosphere showed 99% similarity to H. nitroreducens LMG 24185 T nitrous oxide reductase gene which was also con rmed by protein derived sequences. which shared 75-100% identities to the closest matched nosZ sequences detected from variety of marine environments including ocean sediments, sea sediments, salt marsh, fresh water, paddy soil, sewage water, solar saltern, laizhou bay soil, rhizosphere soil, Puccinia distans soil, agricultural and wheat soil. Phylogenetic analysis of protein derived sequences showed six clusters forming monophyletic clade with different known environmental sequences (Fig. S3 & Table S3).. As reported in other environmental studies of the functional genes in the denitri cation pathway, most of the dominant nirS and nosZ types in our study clustered with other environmental clones. Majority of the sequences belong to uncultured denitrifying bacterial group reported from various environmental sources such as land ll leachate, estuarine sediments, activated sludge, salt marsh, forest soil (Bárta et al. 2010; Zheng et al. 2015) as well as sludge and agricultural ecosystem (Yoshida, 2012;Zhang et al. 2013), suggesting that mangroves harbor denitrifying bacterial communities from both tidal and urban ecosystems. The results showed that majority of the nirS and nosZ gene obtained through DGGE analysis belonged to uncultured denitrifying bacterial group. Phylogenetic analysis of both nirS and nosZ genes formed two different clusters with ocean, marine and estuarine sediments in one cluster and agricultural isolates in another cluster which indicates the wide distribution and yet to explore unknown bacterial lineages in this ecosystem.

Diversity of culturable denitrifying bacteria
About 112 strains grew in nitrate broth, of which 83 strains were selected based on nitrate/nitrite reduction and identi ed as true denitri ers using Greiss reagent (data not shown). All these strains were screened for nitrite reductase and nitrous oxide reductase genes as described in materials and methods. Among the 83 cdnirS positive isolates only 74 isolates harbored nosZ gene with the amplicon size of 1100 bp compared to Marinobacter hydrocarbanoclasticus SP17 T and thus indicating the presence of both nirS and nosZ genes in 74 isolates while 9 isolates contained only nirS gene. The genetic diversity among these 83 strains analyzed by BOX-PCR ngerprinting showed the presence of 24 clusters at 80% con dence level (Fig. 5a).

Taxonomy of denitrifying bacterial isolates
Denitri cation is well recognized as a dominant pathway for the removal of reactive nitrogen in an ecosystem. A number of studies upto date have reported denitri er communities from marine habitats but only from distinct geographic locations (Arce, 2013;Alcantara, 2014).. Of these, 96% of cultured denitri ers belonged to the gammaproteobacteria (Braker, 2000), most of them were the well-known  (Fig. 5b). The exploration of the culturable diversity of these nirS and nosZ in culturable heterotrophic bacterial isolates indicated the prominent distribution of these genes in the Gammaproteobacteria group (Qaisrani et al. 2019). The results obtained were on par with the previous studies on marine sediments (Bowman, 2005; Zhou 2009), which revealed that Gammaproteobacteria was the most abundant denitrifying population in mangroves.
Studies by Fernandes et al. (2012) also showed the dominance of gammaproteobacteria in culturable and non-culturable denitri ers from Tuvem and Divar estuary. Our results also were concurrent to earlier reports with predominant denitrifying community belonging to Gammaproteobacteria consisting of Pseudomonas and Halomonas groups as predominant denitri ers.
In this study we were able to successfully screen and characterize some of the aerobic culturable heterotrophic denitrifying bacterial population from this ecosystem. In culturable analysis of denitri ers, it was observed that majority of the isolates were from Gammaproteobacterial group which belonged to the genus Pseudomonas sp. Different group of bacterial genera like Halomonas, Labrenzia, Paracoccus, Nitratireductor, Bacillus, Virgibacillus, Shewanella, Staphylococcus were also observed to contribute to denitrifying activity. Previous known reports have shown that these microbial groups have been described from different ecosystems ie., Halomonas from hydrothermal vent (Kaye et al. 2011), Labrenzia from marine ecocystem as well as from halophytic plant Sueada (Bibi et al. 2014:) which were similar to the ndings in this study. Other groups like Paracoccus (Flores mirles, 2007), Nitratireductor (Labbe, 2004), and Virgibacillus (Yoshida, 2012 ) were reported from marine as well as mangrove ecosystems except Bacillus which has been reported from the stratosphere (Shivaji, 2006). It was observed that the genus Pseudomonas, Labrenzia, Halomonas, Paracoccus, Virgibacillus and Shewanella were found to harbor both nirS and nosZ gene whereas other genera like Bacillus, Staphylococcus and Nitratireductor harbored only nirS gene. The denitrifying Pseudomonas comprised diversi ed species such as P. balearica, P. bauzanensis, P. xiamanensis, P. stutzeri and P. xanthomarina. This is the rst study to describe the presence of P. balearica, P. bauzanensis, Labrenzia sp. and Paracoccus kondratievae from mangrove ecosystem and were found to be vigorous denitri ers as they can convert nitrate into gaseous form of nitrogen within 24 hrs of incubation under aerobic conditions.
A strong correlation between the DGGE pro les of denitri ers and culturable denitri ers was observed.
Some of the sequences of nirS showed similarity to P. balearica and nosZ gene to Halomonas nitroreducens which has been observed in culture dependent studies as well. The study revealed that 80% of the denitri ers belonged to Pseudomonas sp. and Halomonas sp. represented 16% indicating the dominance of these two species in the rhizosphere contributing to denitri cation.
Overall, the results obtained in this study coincides with the previous studies of marine sediments which showed Gammaproteobacteria as the most abundant nitrogen xing and denitrifying population.

Principal component anaylsis of DGGE ngerprints
Principal component analysis of all the three genes nifH, NirS and NosZ showed the qualitative differences in the distribution of genes among the rhizosphere regions. The PCA analysis clearly separated the microbial communities into three different groups, well supported by UPGMA clustering analysis which showed that the distribution of these genes in halophytic plants is unique when compared to mangrove plants. All the mangrove rhizosphere formed unqiue clustering pattern as is revealed in PCA analysis (Fig. S4a), Both the mangrove rhizospheres A. marina and R. mucronata exhibited almost similar gene distribution pro les and formed a unique clustering pattern. Individual DGGE pro le cluster analysis of these genes well corroborated with the PCA analysis and UPGMA analysis (Fig. S4b).

Conclusion
This is a basic study in an attempt to explore the diversity of culturable and unculturable microbial group involved in nitrogen xation and denitri cation process. To our knowledge this is the rst paper attempting to explore the microbial communities involved in nitrogen xation and denitri cation process through culture depdendent and indepdenent analysis from Pichavaram mangroves. The results presented here provide baseline data about nitrogen xing and denitrifying bacterial groups present in mangrove rhizosphere regions at the genetic level. It is essential to mention that two strains from Rhodobacteraceae family namely Rhodobacter johrii and Labrenzia aggregata involved in nitrogen xation and denitri cation process; sulfur cycling bacteria Catenococcus thiocycli involved in nitrogen xation and nitrate reducing bacteria Nitratireductor are being reported for the rst time from the mangrove ecosystem. Few novel groups belonging to Vibrio, Mangrovibacter, Catenococcus and Bacillus were identi ed based on BOX-PCR ngerprinting analysis. DGGE analysis revelaed the presence of many uncultured bacterial groups harboring genes involvd in nitrogen xation and denitri cation process.
Overall, PCR-DGGE in combination with culture dependent studies revealed known as well as unknown microbial groups involved in nitrogen cycling process in this study. Further research has to be carried out to understand the expression of these genes under different conditions which will give a complete data on the microbial communities involved in this process.

Declarations
Funding The authors did not receive support from any organization for the submitted work.
Availability of data and material-The datasets generated during and/or analysed during the current study are available in the Code availability-Not applicabale Acknowldegement This work was supported by Department of Biotechnology, Govt of India.
Author contributions. BV: Experiment designing, data analysis and writing. VRP: Supervision and crtical correction of the manuscript.
Compliance with ethical standards Con ict of interest: The author's declare that they have no con ict of interest.
Research involving Human Participants and/or Animals: The study is not related to animals or humans.  Tables   Tables 1-2 and Tables S1-S3 were not provided with this version of the manuscript.