Diversity and activity of culturable nitrogen fixing heterotrophic bacteria from estuarine and coastal environments of Southeastern Arabian Sea (SEAS)

Diversity and activity of culturable nitrogen fixing heterotrophic bacteria from estuarine and coastal environments of Southeastern Arabian Sea (SEAS)

Journal Pre-proof Diversity and activity of culturable nitrogen fixing heterotrophic bacteria from estuarine and coastal environments of Southeastern ...

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Journal Pre-proof Diversity and activity of culturable nitrogen fixing heterotrophic bacteria from estuarine and coastal environments of Southeastern Arabian Sea (SEAS) Jesmi Yousuf, Jabir Thajudeen, Aneesa P.A, Ajith Joseph, Divya P.S, Abin Varghese, Mohamed Hatha A.A

PII: DOI: Reference:

S2352-4855(18)30699-6 https://doi.org/10.1016/j.rsma.2019.100973 RSMA 100973

To appear in:

Regional Studies in Marine Science

Received date : 22 December 2018 Revised date : 4 November 2019 Accepted date : 25 November 2019 Please cite this article as: J. Yousuf, J. Thajudeen, Aneesa P.A et al., Diversity and activity of culturable nitrogen fixing heterotrophic bacteria from estuarine and coastal environments of Southeastern Arabian Sea (SEAS). Regional Studies in Marine Science (2019), doi: https://doi.org/10.1016/j.rsma.2019.100973. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier B.V.

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Diversity and activity of culturable nitrogen fixing heterotrophic bacteria from estuarine and

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coastal environments of Southeastern Arabian Sea (SEAS)

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Jesmi Yousuf1*, Jabir Thajudeen2#, Aneesa P.A2, Ajith Joseph2, Divya P.S2, Abin Varghese1, and

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Mohamed Hatha A.A2*

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University of Science and Technology, Cochin, 682016, Kerala, India.

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School of Environmental Sciences, Mahatma Gandhi University, Kottayam, 686560, Kerala, India. Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin

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#Present Address: National Centre for Polar and Ocean Research, Headland Sada, Vasco-da-Gama, Goa.403 804, India

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Jesmi Yousuf

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School of Environmental Sciences

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Mahatma Gandhi University

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Kottayam, Kerala, India

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Pin Code -686560

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Email:[email protected],

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Ph:-+919249963078

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Dr. A. A. Mohamed Hatha

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Professor

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Dept. of Marine Biology, Microbiology and Biochemistry

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School of Marine Sciences

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Cochin University of Science and Technology

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Kochi, Kerala, India

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Pincode-682016

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Email:[email protected]

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Ph:-04842368120 Fax:-04842368120

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Compliance with ethical standards

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Conflicts of interest

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Authors declare that they have no conflicts of interest.

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*Corresponding Authors

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Abstract Nitrogen fixation by diazotrophic bacteria serve as an important source of fixed nitrogen in an

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aquatic ecosystem and thereby directly influence the carbon flux and primary production. Currently there

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is little information about the cultivable heterotrophic diazotrophs and the eco-physiological roles of

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nitrogen fixing bacteria in aquatic environments. The focus of the present study was to understand the

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diversity of cultivable heterotrophic diazotrophs and to evaluate their nitrogen fixation capability in the

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estuarine and coastal environments of the Southeastern Arabian Sea. The heterotrophic diazotrophic

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bacteria were isolated on nitrogen-free media and the potential activity of nitrogen fixation was estimated

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by acetylene reduction assay. The molecular basis of the nitrogen fixation capability among the above

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isolates was analyzed testing for the presence of dinitrogenase reductase (nifH) gene. The 16S rRNA gene

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based identity revealed that the cultivable heterotrophic diazotrophs belonged to α, β, γ-Proteobacteria

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and Firmicutes. The results also revealed that α-Proteobacterium Nitratireducter kimnyeongensis was

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found to be a potential diazotroph (190.3±4.55 nmol C2H4/mg protein/day), which was isolated from the

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coastal ecosystem. Various strains of γ-Proteobacteria such as Klebsiella pneumonia, Klebsiella

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quasipneumonia and Klebsiella variicola also exhibited relatively high nitrogen-fixing activity compared

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to that of Pseudomonas flavescens and Halomonas meridiana. Other cultivable diazotrophs encountered

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in the study area were Oceanobacillus iheyencis, Bacillus aerius, Exiguobacterium profundum,

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Staphylococcus warneri, Bacillus amyloliquefaciens, Staphylococcus arlettae and Staphylococcus caprae.

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The results revealed that Gram-negative bacterial strains possessed relatively superior nitrogen fixation

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activity over Gram-positive isolates (p<0.01). In the light of our observations, we hypothesize that

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heterotrophic diazotrophs play key role in nitrogen fixation process and contribute to primary production

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in estuarine and coastal environments of Southeastern Arabian Sea.

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Keywords

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Acetylene reduction assay; Biological nitrogen fixation; Cultivable bacteria; Heterotrophic diazotrophs;

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nifH gene.

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1. Introduction Biological nitrogen fixation is the process by which a specialized group of microorganisms

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(called diazotrophs) enzymatically transform atmospheric nitrogen into biologically available ammonium

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(Zehr et al., 2003) with the help of the enzyme nitrogenase. The diazotrophs include a wide range of

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archaeal and bacterial lineages, which possess the enzyme nitrogenase (Cobo-Diaz, 2015). This enzyme

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consists of multiple subunits that are encoded by various genes such as nifH, nifD and nifK (Rubio and

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Ludden, 2002) in which the nifH gene (encoding the dinitrogenase reductase subunit) is highly-conserved.

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Hence, this gene is widely used as a molecular marker for tracing the active diazotrophic organisms in the

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natural environment (Raymond et al., 2004; Zehr and Paerl, 2008; Gaby and Buckley, 2012).

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While the role of cyanobacteria as the most important group carrying out nitrogen fixation in the

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marine ecosystem is well documented, knowledge about non-cyanobacterial diazotrophs remains sparse.

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However, very recently reports have emerged highlighting the potential nitrogen fixing activity of

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heterotrophic bacteria and archaea (Bombar et al., 2016; Delmont et al., 2018). It is reported that nitrogen

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fixing capability of several heterotrophic diazotrophic bacteria such as Azotobacter, Bacillus, Clostridium

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and Klebsiella is significant because they provide considerable amount of fixed nitrogen into the

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biosphere (Emmyrafedziawati and Stella, 2018).

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Although the presence of potential heterotrophic diazotrophs was previously reported in the

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pelagic water column and sediments (Farnelid et al., 2013; Thajudeen et al., 2018), the activity and

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importance these heterotrophic nitrogen fixers needs to be determined (Riemann et al., 2010; Mirza and

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Rodrigues, 2012; Moisander et al., 2017). The majority of recent studies on heterotrophic nitrogen

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fixation mainly targeted the diversity of nifH gene and composition of the bacterial communities by

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molecular cloning and next-generation sequencing (Tai et al., 2013; Thajudeen et al., 2017). However, the

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expression of nif gene cannot be linked to nitrogen fixation rates without evidence of cell-specific activity

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(Moisander et al., 2017). A culture-dependent analysis could help determine the potential physiological

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capability of diazotrophs and their ecological relevance in various environments. The Arabian Sea is recommended as a unique region for studying the nitrogen budget (Capone et

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al., 1998), which has a diverse assemblage of diazotrophs that may well fix nitrogen at varying rates

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(Ahmed et al., 2017). The knowledge about their diversity, activity and ecological role is limited, which

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warrants further studies from this region. In a previous study, we have reported the potential of various

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Bacillus sp. to fix nitrogen in the marine and estuarine environment (Yousuf et al., 2017). A recent report

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by Thajudeen et al. (2017) also summarized the prevalence of diverse α, β and γ-Proteobacterial

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diazotrophs in the study area. However, the cell-specific potential of the above organisms to fix nitrogen

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has not been studied. Hence, the objective of the present study is to quantify and compare the nitrogen-

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fixing potential of diverse Gram-positive and Gram-negative diazotrophs in the estuarine and coastal

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environment of Southeastern Arabian Sea (SEAS).

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2. Materials and methods

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2.1. Study area and sampling

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The study was carried out in the Cochin estuary (CE) and adjacent coastal environments along the

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SEAS in 2012. The CE is considered as largest tropical estuary in south India which is highly productive

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and supports rich fish and shellfish resources. The sampling was carried out at 8 stations from the CE and

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4 stations from the coastal region (Fig. 1). The water and sediment samples from the estuary were

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collected on board RV Kingfisher. Water samples were collected using a 2 litre Niskin water sampler

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(General Oceanic, USA) and sediment samples using Van Veen Grab (Hydrobios, Germany), and stored

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in sterile polyethylene bottles. The coastal samples were collected on-board FORV Sagar Sampada

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(Cruise No. 311) using Niskin sampler for water and Smith McIntyre Grab for sediments.

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2.2. Isolation and screening of diazotrophic strains

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The screening of heterotrophic diazotrophic bacteria from each sample of water and sediments

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was carried out by the spread plate technique on a nitrogen-free medium (Norris Glucose Nitrogen free

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medium, NGNF; HIMEDIA-M712). The seeded plates were incubated in dark at 28±1°C for 7-14 days. 4

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After incubation, the morphological features of the colonies were recorded and well-separated colonies

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with different morphology were picked up using a sterile inoculation loop. These isolates were re-

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streaked to ensure purity and maintained on the sterile NGNF agar slants at 4°C. These isolates were

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subjected to Gram staining, spore staining (in case of Gram-positive rods) and examined by light

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microscopy (Olympus CX21i) to determine the cell morphology. Based on the differences in color,

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morphology and good quality growth on NGNF media, various Gram-negative (n=12) and Gram-positive

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(n=8) isolates were selected for further study. These isolates were stored at -20°C in NGNF liquid

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medium containing 20% glycerol for further analysis.

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2.3. Polymerase Chain Reaction amplification of the 16S rRNA and nifH gene

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The selected isolates were grown in Luria-Bertani broth (Hi-media, India) at 28°C for 48h.

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Genomic DNA was isolated from cultures as per the standard Proteinase-K digestion method (Sambrook

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et al., 1989). Using the extracted DNA as template (Bosshard et al., 2000) polymerase chain reaction

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(PCR) was used to amplify the bacterial 16S rRNA gene with the help of universal primer set (27F: 5′-

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AGA GTT TGATC TGG CTC AG-3′ and 1492R: 5′-GGT TAC CTT GTT ACG ACT T-3′).

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Amplification conditions were as follows: 2min of initial denaturation at 95°C, followed by 30 cycles of

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denaturation at 95°C for 2min, annealing at 58°C for 1min, and extension at 72°C for 2min. A final

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extension was carried out at 72°C for 10min.

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A partial sequence (360bp) of the dinitrogenase reductase nifH gene was amplified using

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previously designed degenerate oligonucleotide primers Zehr-nifHf (5′-TGYGAYCCNAARGCNGA-3′)

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and Zehr-nifHr (5′-ADNGCCATCATYTCNCC-3′) (Zehr and McReynolds, 1989) as per the procedure of

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Zehr et al. (1998), with slight modification. The PCR conditions for the amplification of the nifH gene

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were as follows: initial denaturation at 94°C for 5min, followed by 40 cycles of denaturation at 94°C for

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1min, annealing at 57°C for 1min and extension at 72°C for 2min., followed by final extension at 72°C

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for 10min. All the PCR amplification was carried out in a total volume of 25μl reaction mixtures

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consisting of sterile MilliQ water (15.5μl), 10X PCR buffer (2μl), primer (1μl each), dNTP mix (1μl,

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200mM), template (4μl) and Taq DNA polymerase (0.5μl). After amplification, the nifH gene fragments

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were checked by ultra-pure agarose gel (1.5%) electrophoresis. The size of the resolved bands in the gel

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was confirmed by comparing with 100bp nucleotide marker.

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2.4. Sequencing and phylogenetic analysis

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The 16S rRNA and nifH gene amplicons were purified using the Promega PCR clean-up system

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(Nucleospin, MN, Duren, Germany) as per the manufacturer’s instructions and sequenced using an ABi

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3730 XL Genetic Analyzer (Applied Biosystems, USA) at Scigenome Pvt Ltd., Cochin. The obtained

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gene sequences were subjected to Basic Local Alignment Search Tool (BLAST) sequence similarity

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search (http://blast.ncbi.nlm.nih.gov/BLAST) in the National Centre for Biotechnology Information

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(NCBI) GenBank database to identify the nearest taxa. The phylogenetic tree based on 16S rRNA and

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nifH gene sequences were constructed using two tree making algorithms such as maximum likelihood

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(ML) and Neighbor-joining (NJ) methods using MEGA version 7.0 software package (Tamura et al.,

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2013). The multiple alignments were performed using ClustalW analysis. The bootstrap calculations of

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1000 runs were also carried out to authenticate the reliability of the branching pattern.

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2.5. Nitrogen fixation activity

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The nitrogen fixation activity of bacterial isolates was quantified by acetylene reduction assay,

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which estimated the rate of acetylene (C2H2) reduction to ethylene (C2H4) by nitrogenase enzyme (Stewart

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et al., 1967; Kifle and Laing, 2016). The C2H4 production by the bacterial cultures was measured by gas

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chromatography equipped with flame ionization detector (FID). The detailed method of estimation and

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operating conditions of gas chromatography was reported in Yousuf et al. (2017). The mean values of

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each experiment along with standard deviation were calculated and expressed as nmol C2H4/mg

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protein/day.

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2.6. Statistical data analysis

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The data were evaluated with the ‘Statistical Package for the Social Sciences (SPSS)’ version 20.

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The Mann-Whitney U test was performed for comparison of nitrogen fixation activity by each group. All

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the experiments were tested at 1% level of significance. 6

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3. Results

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3.1. Isolation and identification of diazotrophic bacterial strains The diazotrophic bacteria were isolated on solid nitrogen-free media, NGNF. Most of the

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colonies obtained on the NGNF media were very small, clear round, convex and gummy, with watery

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dewdrop like appearance (Supplementary Fig. S1). Some of the colonies were found submerged in the

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medium and a very few colonies showed pigmentation. A total of 20 morphologically different bacterial

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colonies (12 from the estuary and 8 from coastal regions) were isolated and characterized from the study

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area (Table 1). All the isolates were able to grow well on nitrogen-free agar medium and were identified

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as diazotrophs. Phylogenetic diversity of the selected heterotrophic diazotrophs revealed that 12 isolates

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belonged to the phylum Proteobacteria, while remaining 8 belonged to Firmicutes.

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Out of the 20 strains, 12 isolates belonged to various Gram-negative Proteobacterial species such

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as α-Proteobacteria (Nitratireducter kimnyogensis and Rhizobium rosettiformans), β-proteobacteria

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(Alcaligenes faecalis) and γ-proteobacteria (Klebsiella pneumonia, K. quasipneumonia, K. variicola,

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Rheinheimera aquimaris, Acinetobacter johnsonii, Halomonas meridiana, Pseudomonas flavescens and

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two strains of Enterobacter cloacae). The remaining 8 isolates belonged to Gram-positive Firmicutes,

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which were identified as Bacillus aryabhattai, B. aerius, B. amyloliquefaciens, Oceanobacillus iheyencis,

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Exiguobacterium profundum, Staphylococcus caprae, S. warneri and S. arlettae. The phylogenetic tree

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was constructed based on 16S rRNA gene sequences obtained from the present study and the reference

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sequences retrieved from the GenBank (Fig. 2.) All the sequences retrieved from this study were

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submitted to NCBI database under the accession numbers KT868878 to KT868887, MF795413 to

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MF795417, MH045570, KT833390, MF795419, MF795420 and MF795422 (Table 1).

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3.2. Amplification of nifH gene

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Presence of nifH gene in all the selected bacterial strains was determined by the PCR method. All the

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strains except three Gram-positive strains namely B. amyloliquefaciens CES SD11, S. arlettae CCS Z-31

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and S. caprae CCS Z-36; showed a positive amplification of nifH gene (Fig. 3). The result reconfirmed

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the genetic potential of these bacterial strains to fix atmospheric nitrogen to a usable form. The nifH gene

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(360bp) previously amplified and sequenced in our lab (B. flexus CESM15-54, nifH gene sequence

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accession number: MH248365; Supplementary data S2) was taken as the reference for the sequencing

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analysis. The phylogenetic tree was constructed using the obtained nifH gene sequences and represented

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in the supplementary Fig. S3.

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3.3. Nitrogen fixation activity of the isolates

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All the isolates that showed positive amplification of the nifH gene, as well as the isolates which

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failed to amplify the nifH gene, were tested for their ability to fix nitrogen. The nitrogen fixation activity

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is estimated by acetylene reduction assay. The results revealed that all the isolates showed varying levels

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of nitrogenase activity (Fig. 4). The highest nitrogen-fixing activity was observed in the α-Proteobacteria

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N. kimnyogensis CCS Z-27 (190.3±4.55 nmol C2H4/mg protein/day) which was isolated from the coastal

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sediment. Other strains which showed relatively good nitrogen fixing activity included K. variicola CCW

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62C (151.78±1.80 nmol C2H4/mg protein/day), K. pneumonia CEW 61S (134.92±2.68 nmol C2H4/mg

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protein/day) and H. meridiana CES SD3 (143.94±2.75 nmol C2H4/mg protein/day). The K.

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quasipneumonia CEW W7 and P. flavescens CCW M-40 also exhibited fairly good nitrogen-fixing

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activity (>110 nmol C2H4/mg protein/day). The nitrogen-fixing capability of above strains was superior to

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the nitrogen-fixing potential of reference species B. flexus CES M15-54 (76.95±2.4 nmol C2H4/mg

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protein/day) (Fig. 4) (Yousuf et al., 2017). Among the Gram-negative strains which were evaluated for

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nitrogen fixing potential, Al. faecalis showed the lowest activity (21.05±0.44 nmol C2H4/mg protein/day).

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Among the Gram-positive strains, B. aryabhattai CES SD6, S. caprae CCS Z-36, O. iheyensis

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CCW 38C, and Ex. profundum CEW 26MY14 have shown good nitrogen fixation, while relatively low

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activity was recorded in B. aerius CES SD5 and S. arlettae CCS Z-31 (10.48±0.63 and 14.48±1.99 nmol

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C2H4/mg protein/day respectively). The results were also subjected to statistical analysis to understand the

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disparity in the nitrogen-fixing activity among various isolates belonging to the dissimilar genera. The

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result revealed that the potential to fix nitrogen was significantly high (p<0.01) among Gram-negative

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diazotrophs when compared to that of Gram-positive diazotrophs (Fig. 5).

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4. Discussion The widespread distribution of non-cyanobacterial diazotrophs in the oceanic waters highlights

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the importance of heterotrophic nitrogen fixation (Delmont et al., 2018). Heterotrophic diazotrophs play a

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key role in the marine nitrogen cycle and provide a considerable amount of fixed nitrogen (Bombar et al.,

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2016) which in turn could have significant effect on primary production. However, the knowledge

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regarding their ecological role in the water column is not understood. Therefore it is necessary to identify

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the distribution and activity of heterotrophic diazotrophs in order to understand their influence on the

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aquatic nitrogen cycle. Due to the difficulty in obtaining these isolates in culture, very little is known

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about their physiological characteristics, ecological significance as well as genetic heterogeneity of

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nitrogen fixation and their cell-specific potential to fix nitrogen (Farnelid, 2013; Martinez-Perez et al.,

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2018). Bentzon-Tilia et al. (2014) suggested that the culture based approaches are also more relevant than

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in-situ measurements of nitrogen fixation because field studies were found unsuccessful to connect

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nitrogen fixation rate to species-specific cells. Hence the focus of the present study was to understand the

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diversity and nitrogen fixation potential of various cultivable heterotrophic diazotrophs from the estuarine

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and coastal environments of SEAS.

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We have observed that the colony morphology of most of the isolates that developed on nitrogen-free

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media were gummy and viscous in nature; which might be due to any of the self-protection mechanisms

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of those species to regulate physiological functions. Sabra et al. (2000) reported that the gumminess of

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diazotrophic culture may be due to the alginate production by the organisms, which thereby trims down

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the oxygen transfer rate. According to Reddy et al. (1993) and Berman-Frank et al. (2007), the

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cyanobacterial diazotrophs avoid oxygen inhibition through heterocyst formation or temporal segregation

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of photosynthetic oxygen production and nitrogen fixation. It was reported that the high respiratory

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activity of some heterotrophic diazotrophs such as Azotobacter helps to protect the nitrogenase enzyme

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(Phillips and Johnson, 1961), however the exact mechanisms by which the heterotrophic diazotrophs

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protect their nitrogenase enzyme from oxygen is still not clear.

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The acetylene reduction assay is the most common laboratory procedure used for the determination of the

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specific activity of the enzyme nitrogenase (Stewart et al., 1967; Bentzon-Tilia et al., 2015; Kifle and

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Laing, 2016). All the tested strains (n=20) showed nitrogen fixation potential and results indicated that

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various species of heterotrophic diazotrophs showed a high degree of variation in nitrogease activity

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(ranging from 10 to 190 nmol C2H4/mg protein/day). Park et al. (2005) successfully isolated a variety of

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diazotrophic bacteria from rhizosphere with varying nitrogenase activities such as B. fusiformis, P.

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fluorescens, Stenotrophomonas maltophilia etc. (14 to 3677 nmol C2H4/mg protein/h). Yousuf et al.

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(2017) also studied the diazotrophic potential of various Bacillus strains from the SEAS where they

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observed considerable variation in nitrogen fixation potential among different Bacillus strains (3 to 210

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nmol C2H4/mg protein/day). Martinez et al. (1996) and Baskar and Prabhakaran (2015) reported that the

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potential of enzymatic activity among the diazotrophs may not be the same as it may depend on various

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environmental factors.

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Nitratireducter kimnyeogencis CCS Z-27 was identified as the putative α-Proteobacterial

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diazotroph prevailing in the study area. The study confirmed the presence of the nifH gene in this

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bacterium which exhibited relatively high nitrogen fixing activity compared to all other tested strains.

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Though the presence of diverse diazotrophic α-Proteobacteria were reported in the estuarine and marine

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environments (Moisander et al., 2008; Thajudeen et al., 2017; 2018, Martinez-Perez et al., 2018) the

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nitrogenase activity of the species N. kimnyogensis is not yet reported. This could be first demonstration

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of the identification of nifH gene and nitrogen fixation potential in N. kimnyogensis. We have also

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observed that the various species of Klebsiella (K. pneumonia, K. quasipneumonia, and K. variicola) were

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also capable of fixing atmospheric nitrogen in to usable form. Klebsiella sp. encountered in the present

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study could quickly grow on NGNF media and fix atmospheric nitrogen at environmentally relevant in-

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vitro conditions suggesting that they might be very active in marine environments. Our findings agree

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with previous observations (Schmitz et al., 2002; Feng et al., 2018) that various species of Klebsiella

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could be considered as one of the endowed γ-Proteobacterial diazotrophs capable of fixing atmospheric

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nitrogen.

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The results of the present study are consistent with the previous reports from terrestrial environments

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regarding the nitrogenase potential of Halomonas (Llmas et al., 2006), Pseudomonas (Eckford et al.,

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2002; Bentzon-Tilia et al., 2015), Rhizobium rosettiformans (Burbano et al., 2011) and Alcaligenes

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faecalis (Mazumda and Deka, 2013), though our isolates are from aquatic ecosystems. Furthermore, the

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current study also revealed the innovative report from the SEAS regarding the nitrogen fixation potential

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of Oceanobacillus, Exiguobacterium and various genera of Staphylococcus. The major highlight of the

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present study was that we were successful in cultivating the heterotrophic diazotrophs, which would allow

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the ecophysiological and molecular characterization of them in the future.

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The primers used in this study have been successfully used to amplify the nifH gene from distantly related

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diazotrophs (Zehr and McReynolds, 1989; Tan et al., 2003). Even in the absence of the nifH gene, the

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species like B. amyloliquefaciens CES SD11 and S. caprae CCS Z-36 has shown relatively high nitrogen-

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fixing activity (50.16 ± 6.16 and 63.29±1.25 nmol C2H4/mg protein/day) than the nifH gene amplified Al.

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faecalis CCS Z-1 and S. warneri CCW 69C (Fig. 4). Such inconsistencies between nifH gene

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amplification and nitrogen fixation activity are reported by several researchers (Achouak et al., 1999;

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Bostrom et al., 2007; Islam et al., 2010; Mirza and Rodrigues, 2012; Yousuf et al., 2017). It is also

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reported that the unsuccessful amplification of nifH gene does not mean that the species is incapable of

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nitrogen fixation, as there could be a diverse nifH gene nucleotide at inter or intra species level (Zehr et

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al., 2003). We assume that the presence of mutant variety of nifH gene could be the most probable reason

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for the unsuccessful amplification of nifH gene. Hence a combination of tools is recommended for the

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identification of active diazotrophic bacteria.

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Statistical analysis of the results revealed that Gram-negative Proteobacterial strains were more

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efficient in nitrogen fixation (p<0.01) than Gram-positive Firmicutes (Fig. 5) suggesting that Gram-

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negative groups could be an integral part of the diazotrophic bacterial community in the study area. 11

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Soares et al. (2006) suggested that free-living heterotrophic diazotrophs belonging to Gram-negatives

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play an important role in nitrogen fixation. An exact measurement strategy is needed to determine the

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activity of heterotrophic diazotrophs from the natural environments (Postgate, 1988; Bostrom et al.,

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2007). The current study proved the proficient enzymatic capability and genetic evidence related to

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nitrogen fixation among the heterotrophic diazotrophs and their significant contribution to the aquatic

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nitrogen fixation. These findings also imply that we still have much to learn about the major as well as a

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diverse array of heterotrophic microorganisms which can apparently perform this significant process in

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the nitrogen cycle.

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5. Conclusion

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The current study revealed that heterotrophic diazotrophs contributed to nitrogen fixation in the

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coastal and estuarine ecosystems of SEAS. We also documented that diverse diazotrophs such as

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Firmicutes and Proteobcateria have potential ability to fix atmospheric nitrogen.

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potential among these organisms emphasizes the need to reconsider their ecological relevance in the

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aquatic environments. Most of the Gram-negative strains identified from our study area, especially

298

Nitratireducter kimnyeongencis and various species of Klebsiella are found to be powerful diazotrophs,

299

which contribute significantly to heterotrophic nitrogen fixation. This report also suggests further

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microbiological and ecological monitoring of heterotrophic diazotrophs in various estuarine and marine

301

environments. The sustained use of cultivation methods will definitely lead to the discovery of more

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heterotrophic diazotrophic organisms from the study area. It also provides a direct means to learn further

303

about the functions and activity of heterotrophic diazotrophs in the aquatic nitrogen cycle.

304

Acknowledgments

The nitrogenase

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This work was supported by the Ministry of Earth Science (MoES), Government of India under

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the Sustained Indian Ocean and Biochemical and Ecological Research programme (SIBER -

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MoES/36/001S/SIBER/07). The author Mrs. Jesmi Yousuf gratefully acknowledge the UGC-Maulana

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Azad National Fellowship (F1 – 17.1/ 2013/MANF-2013-14-MUS-KER-25319/ (SA-III website) dated

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06-Feb-2014) for their financial support. We would like to express our gratitude to the Head, Department

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of Marine Biology, Microbiology and Biochemistry, Cochin University of Science and Technology for

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providing facilities to carry out the work. The authors would like to express gratitude to the anonymous

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reviewers for their helpful and productive comments that greatly contributed to improving the manuscript.

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List of Tables and Figures

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Table 1. The details of diazotrophic bacterial strains isolated from the study area and the identification of nifH gene. GenBank Accession number

Strain

From estuarine environment

MF795414

α-Proteobacteria

+ve

2

Klebsiella pneumonia CEW 61S*

KT868880

γ-Proteobacteria

+ve

3

Enterobacter cloacae CEW M15/24*

MH045570

γ-Proteobacteria

+ve

4

Enterobacter cloacae CEW M15/1*

KT868887

γ-Proteobacteria

+ve

5

Klebsiella quasipneumonia CEW W7*

MF795413

γ-Proteobacteria

+ve

6

Rheinheimera aquimaris CEW W8*

MF795422

γ-Proteobacteria

+ve

7

Acinetobacter johnsonii CEW W11*

MF795415

γ-Proteobacteria

+ve

8

Halomonas meridiana CES SD3#

MF795416

γ-Proteobacteria

+ve

9

Exiguobacterium profundum CEW 26MY14* KT868879

Firmicutes

+ve

10

Bacillus aryabhattai CES SD6#

MF795419

Firmicutes

+ve

11

Bacillus aerius CES SD5#

MF795417

Firmicutes

+ve

12

Bacillus amyloliquefaciens CES SD11#

MF795420

Firmicutes

-ve

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Rhizobium rosettiformansCEW W9*

Nitratireducter kimnyeogencis CCS Z-27#

KT868884

α-Proteobacteria

+ve

14

Alkaligenes faecalis CCS Z-1#

KT868883

β-Proteobacteria

+ve

15

Klebsiella variicola CCW 62C*

KT868881

γ-Proteobacteria

+ve

16

Pseudomonas flavescens CCW M-40*

KT868878

γ-Proteobacteria

+ve

17

Oceanobacillus iheyencis CCW 38C*

KT833390

Firmicutes

+ve

18

Staphylococcus warneri CCW 69C*

KT868882

Firmicutes

+ve

19

Staphylococcus caprae CCS Z-36#

KT868886

Firmicutes

-ve

20

Staphylococcus arlettae CCS Z-31#

KT868885

Firmicutes

-ve

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13

*

475

Identification of nifH gene

1

From coastal environment

474

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Sl. No

isolated from water; # isolated from sediment

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Fig. 1. Location map showing the study area of Cochin estuary and adjacent coastal waters.

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20

481 482 483 484 485 486

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Fig. 2. Neighbour-joining phylogenetic tree based on the 16S rRNA gene sequence, showing the relationship of tested strains to closely related representatives of related taxa retrieved from GenBank. Pink and blue mark represents the Gram-negative and Gram-positive strains isolated from the study area with GenBank accession numbers. 21

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Fig. 3. The agarose gel image showing the amplification of nifH gene (~360bp). Lane 1 to 8 represents the 1 kb DNA ladder, B. flexus (nifH+ve reference strain), K. pneumonia, R. rosettiformans, H. meridiana, N. kimnyeogencis, O. iheyencis and P. flavescens respectively.

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B. flexus CES M15/54 (reference strain) H. meridiana CES SD3 A. johnsonii CEW W11 R. rosettiformans CEW W9 Rh. aquimaris CEW W8 K. quasipneumonia CEW W7

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E. cloacae CEW M15/1 E. cloacae CEW M15/24 Tested strains

K. pneumonia CEW 61S P. flavescens CCW M-40 K. variicola CCW 62C

N. kimnyeongencis CCS Z-27 A. faecalis CCS Z-1 S. warneri CCW 69C S. arlettae CCS Z-31

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S. caprae CCS Z-36 B. amyloliquefaciens CES SD11 B. aerius CES SD5 B. aryabhattai CES SD6 Ex. profundum CEW 26MY14

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O. iheyencis CCW 38C

0

492

200

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Fig. 4. Nitrogen fixation potential of various heterotrophic diazotrophs isolated from the study area by acetylene reduction assay.

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493 494 495

20 40 60 80 100 120 140 160 180 Nitrogen fixing activity (nmol C2H4 /mg protein/day)

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100 80 60 40 20 0

39.53

pro of

Nitrogen Fixing Activity (nmol C2H4/mg protein/day)

120

99.71

Gram-positive

Gram-negative

Strains

497

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Fig. 5. Variation in the average nitrogenase activity between Gram-positive and Gram-negative strains.

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503 504

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Sub: RSMA_2018_669, Conflict of Interest reg. Title: Diversity and activity of culturable nitrogen fixing heterotrophic bacteria from estuarine and coastal environments of Southeastern Arabian Sea (SEAS) Authors: Jesmi Yousuf, Jabir Thajudeen, Aneesa P.A, Ajith Joseph, Divya P.S, Abin

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Varghese, and Mohamed Hatha A.A

CONFLICT OF INTEREST

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We the undersigned declare that this manuscript is original, has not been published before and is not currently being considered for publication elsewhere. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property.

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We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). She is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Author and which has been configured to accept emails from the Editorial Office of Regional Studies in Marine Science.

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Jabir Thajudeen

Aneesa P.A

Ajith Joseph

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Abin Varghese

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Mohamed Hatha A.A

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