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 fixers and denitrifiers. All the isolates were maintained in LB agar for further analysis and stored as glycerol stock in -80 ⁰C.
Culture independent analysis of nifH gene diveristy (PCR-DGGE)
The DGGE profiles 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 profiles 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 prefix 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% confidence 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 fixing 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 identification of nitrogen fixing 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 fixing 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 fixation in the mangrove ecosystem. In addition to proteobacterial groups, Firmicutes were also found to harbor nifH genes, Similarly, nifH gene sequences affiliated 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 findings of Bird et al. (2005) suggested that gamma proteobacteria are predominant and acts as an important component of the heterotrophic nitrogen fixing microbial community of the tropical and subtropical oceans. The sequence analysis of nifH DGGE showed similarity to uncultured nitrogen fixing 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-fixing bacteria reported neither from mangroves nor from other ecosystems, indicating the abundance of unreported uncultured nitrogen-fixing bacteria in the mangrove rhizosphere soils. The phylogenetic placement of the nifH gene sequences from the mangroves exhibited unique nifH gene types affiliated with the phyla belonging to unculturables. The sequences were partially matching with the nitrogen fixers 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 fixing bacteria
Nearly 52 strains formed pellicle in nitrogen free medium and showed positive amplification for nifH gene with amplicon size of 360 bp compared to Ciceribacter lividus MSSRFBL1T used as positive control. The BOX-PCR fingerprinting of the 52 strains showed genetic variation and formed 23 clusters at 80% confidence 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 (Rameshkumar, 2014; Viswanath et al. 2015; Raju et al. 2016;). This needs to be further expanded using current molecular tools to completely understand the diversity of the nitrogen fixers associated with this ecosystem.
Taxonomic classification of bacterial isolates harboring nifH gene
PCR based 16S rRNA amplification and sequencing analysis of 1350-1410 bp of the amplified product of the culturable nitrogen fixers assigned the taxonomic identification upto generic and species level. The EzTaxon analysis of 16S rRNA gene sequences of the positive strains were compared with available sequences of the type strains in the database and were assigned the respective taxonomic position. The positive nitrogen fixers predominantly belonged to Gammaproteobacteria particularly the genus Vibrio (31%) comprising of seven species ie., V. plantisponsor MSSRF 40T, V. alginolyticus NBRC 15630T, V. azureus NBRC 104587T, V. diabolicus HE800T, V. natrigenes DSM 759T, V. parahemolyticus NBRC 12711T, and V. neocaledonicus NC470T; followed by Mangrovibacter (12%) belonging to M. plantisponsor MSSRF 40T, Klebsiella (12%) comprising of K. pneumoniae (DSM 30104T), Serratia (6%) belonging to S. marcescens (KREDT), and Catenococcus thiocycli DSM 9165T; the Alphaproteobacteria group was represented by Rhodobacter johrii JA192T (2%), Azospirillum lipoferum NCIMB118161T (2%) (8%) (Fig. 2b). The second largest phylum was Firmicutes represented by Bacillus (21%) comprising of four species such as B. aerophilus 28KT, B. oceanisediminis H2T, B. subterraneus DSM 13966T and B. boroniphilus JCM 21738T; Staphylococcus (8%) comprising of S. epidermis (ATCC14990T).
Previous studies by Liu et al. (2012) showed that 55.6% of the nitrogen fixing bacterial community belonged to gammaproteobacteria, which substantiates the present results depicting the dominance of this microbial group in mangrove ecosystems. The results obtained in our study showed that majority of the culturable nitrogen fixers from the Pichavaram mangroves belonged to Gammaproteobacterial group coinciding with earlier reports (Rameshkumar 2014, 2010; Viswanath et al. 2015). Flores-Mireles et al. (2007) showed that the nitrogen fixers 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 profiling 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 profile 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 five Mangrovibacter species in this study displayed divergence from the reported strains in BOX-PCR profiling as well as phylogenetic analysis indicating they could possibly be novel species. The genus Bacillus obtained in this study, showed similarity to B. aerophilus 28KT 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 fingerprinting analysis. Our results confided 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% confidence level, both nirS (Fig. 3b) and nosZ (Fig. 4b) genes formed five 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.
Phylogenies of nirS and nosZ gene sequences
The unique nirS sequences recovered from mangrove rhizosphere shared 94-100% identities with known GeneBank sequences. After translation, the corresponding protein sequences shared 75-100% identities to the closest matched nirS sequences detected from variety of marine environments including estuarine sediments of pearl river estuary (cd13, cd30), Hai river (cd9, cd17,cd21), Yangtze river (cd4, cd10), Baltic sea sediments (cd6, cd15), lake sediments (cd20), South and East china sea (cd27, cd29), Salt marsh sediment (cd19, cd23), Solar saltern (cd25), sludge (cd7, cd8), Agriculture soil (cd26), coastal sediments (cd2, cd5, cd11, cd22), sediments (cd14, cd18), estuary (cd12, cd16), soil (cd24, cd31) and Landfill bioreactor respectively (cd1, cd3, cd28), all which showed similarity to uncultured nitrite reductase coding genes. However, majority of the nirS (28 bands) sequences didn’t match with culturable denitrifiers and showed similarity to uncultured nirS gene sequences. Only 3 bands namely CD29 showed 90% similarity to nitrite reductase gene of Pseudomonas, and two other bands CD25 and CD31 showed 96% and 92% similarity to nitrite reductase gene of Halomonas nitroreducens LMG 24185T and Halomonas cernia LMG 24145T strains respectively (Fig. S2 and table S2).
The nosZ gene, encoding N2O reductase, an enzyme catalyzing the final step of denitrification, 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 denitrifiers (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 6083T genome and band MSSRF Z17 from S. maritima rhizosphere showed 99% similarity to H. nitroreducens LMG 24185T nitrous oxide reductase gene which was also confirmed 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)..
The nirS (cd3afGC and R3Cd) and nosZ (nosZfGC and nosZ1773R) primer pair showed efficient amplification of the nirS and nosZ genes from the denitrifying populations of the four different mangrove rhizospheres. Studies by Lee et al. (2017) and Xie et al. (2020) showed the abundance of denitryfing bacterial communities in pearl river estuary and sanfransisco bay were correlated with the present study. As reported in other environmental studies of the functional genes in the denitrification 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 fill 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 identified as true denitrifiers 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 SP17T 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 fingerprinting showed the presence of 24 clusters at 80% confidence level (Fig. 5a).
Taxonomy of denitrifying bacterial isolates
Denitrification is well recognized as a dominant pathway for the removal of reactive nitrogen in an ecosystem. A number of studies upto date have reported denitrifier communities from marine habitats but only from distinct geographic locations (Arce, 2013; Alcantara, 2014).. Of these, 96% of cultured denitrifiers belonged to the gammaproteobacteria (Braker, 2000), most of them were the well-known genus Pseudomonas. BLAST-N analysis of the 16S rDNA of nitrate reducing bacterial groups revealed the predominant presence of genus Nitratireductor aquimarinus VL-SC21T(2), Staphylococcus hominis DSM 20328T(1) and Bacillus aryabhattai B8W22T(2). BLAST-N analysis of denitrifiers were mostly represented by groups such as Pseudomonas sp. (48 isolates) comprising of P. bauzanensis DSM 22558T, P. xanthomarina KMM1447T, P. baleriaca DSM 6083T, P. stutzeri ATCC 17588T and P. xiamenensis C10-2T, Paracoccus kondratievae GBTsp. (4 isolates), Labrenzia aggregata IAM 12614Tsp. (5 isolates), Halomonas venusta DSM 4743T and H. hydrothermalis Slthf2T (13 isolates), Virgibacillus dokdonensis DSW-10T (4 isolates) and Shewanella marisflavi SW 117T(3 isolates) (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 denitrifiers 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 denitrifiers.
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 denitrifiers, 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 findings 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 diversified species such as P. balearica, P. bauzanensis, P. xiamanensis, P. stutzeri and P. xanthomarina. This is the first study to describe the presence of P. balearica, P. bauzanensis, Labrenzia sp. and Paracoccus kondratievae from mangrove ecosystem and were found to be vigorous denitrifiers as they can convert nitrate into gaseous form of nitrogen within 24 hrs of incubation under aerobic conditions.
A strong correlation between the DGGE profiles of denitrifiers and culturable denitrifiers 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 denitrifiers belonged to Pseudomonas sp. and Halomonas sp. represented 16% indicating the dominance of these two species in the rhizosphere contributing to denitrification.
Overall, the results obtained in this study coincides with the previous studies of marine sediments which showed Gammaproteobacteria as the most abundant nitrogen fixing and denitrifying population.
Principal component anaylsis of DGGE fingerprints
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 profiles and formed a unique clustering pattern. Individual DGGE profile cluster analysis of these genes well corroborated with the PCA analysis and UPGMA analysis (Fig. S4b).