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
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S2352-4855(18)30699-6 https://doi.org/10.1016/j.rsma.2019.100973 RSMA 100973
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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.
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Diversity and activity of culturable nitrogen fixing heterotrophic bacteria from estuarine and
coastal environments of Southeastern Arabian Sea (SEAS)
Jesmi Yousuf1*, Jabir Thajudeen2#, Aneesa P.A2, Ajith Joseph2, Divya P.S2, Abin Varghese1, and
Mohamed Hatha A.A2*
University of Science and Technology, Cochin, 682016, Kerala, India.
School of Environmental Sciences, Mahatma Gandhi University, Kottayam, 686560, Kerala, India. Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin
8 9 10 11
#Present Address: National Centre for Polar and Ocean Research, Headland Sada, Vasco-da-Gama, Goa.403 804, India
School of Environmental Sciences
Mahatma Gandhi University
Kottayam, Kerala, India
Pin Code -686560
Dr. A. A. Mohamed Hatha
Dept. of Marine Biology, Microbiology and Biochemistry
School of Marine Sciences
Cochin University of Science and Technology
Kochi, Kerala, India
Compliance with ethical standards
Conflicts of interest
Authors declare that they have no conflicts of interest.
Abstract Nitrogen fixation by diazotrophic bacteria serve as an important source of fixed nitrogen in an
aquatic ecosystem and thereby directly influence the carbon flux and primary production. Currently there
is little information about the cultivable heterotrophic diazotrophs and the eco-physiological roles of
nitrogen fixing bacteria in aquatic environments. The focus of the present study was to understand the
diversity of cultivable heterotrophic diazotrophs and to evaluate their nitrogen fixation capability in the
estuarine and coastal environments of the Southeastern Arabian Sea. The heterotrophic diazotrophic
bacteria were isolated on nitrogen-free media and the potential activity of nitrogen fixation was estimated
by acetylene reduction assay. The molecular basis of the nitrogen fixation capability among the above
isolates was analyzed testing for the presence of dinitrogenase reductase (nifH) gene. The 16S rRNA gene
based identity revealed that the cultivable heterotrophic diazotrophs belonged to α, β, γ-Proteobacteria
and Firmicutes. The results also revealed that α-Proteobacterium Nitratireducter kimnyeongensis was
found to be a potential diazotroph (190.3±4.55 nmol C2H4/mg protein/day), which was isolated from the
coastal ecosystem. Various strains of γ-Proteobacteria such as Klebsiella pneumonia, Klebsiella
quasipneumonia and Klebsiella variicola also exhibited relatively high nitrogen-fixing activity compared
to that of Pseudomonas flavescens and Halomonas meridiana. Other cultivable diazotrophs encountered
in the study area were Oceanobacillus iheyencis, Bacillus aerius, Exiguobacterium profundum,
Staphylococcus warneri, Bacillus amyloliquefaciens, Staphylococcus arlettae and Staphylococcus caprae.
The results revealed that Gram-negative bacterial strains possessed relatively superior nitrogen fixation
activity over Gram-positive isolates (p<0.01). In the light of our observations, we hypothesize that
heterotrophic diazotrophs play key role in nitrogen fixation process and contribute to primary production
in estuarine and coastal environments of Southeastern Arabian Sea.
Acetylene reduction assay; Biological nitrogen fixation; Cultivable bacteria; Heterotrophic diazotrophs;
1. Introduction Biological nitrogen fixation is the process by which a specialized group of microorganisms
(called diazotrophs) enzymatically transform atmospheric nitrogen into biologically available ammonium
(Zehr et al., 2003) with the help of the enzyme nitrogenase. The diazotrophs include a wide range of
archaeal and bacterial lineages, which possess the enzyme nitrogenase (Cobo-Diaz, 2015). This enzyme
consists of multiple subunits that are encoded by various genes such as nifH, nifD and nifK (Rubio and
Ludden, 2002) in which the nifH gene (encoding the dinitrogenase reductase subunit) is highly-conserved.
Hence, this gene is widely used as a molecular marker for tracing the active diazotrophic organisms in the
natural environment (Raymond et al., 2004; Zehr and Paerl, 2008; Gaby and Buckley, 2012).
While the role of cyanobacteria as the most important group carrying out nitrogen fixation in the
marine ecosystem is well documented, knowledge about non-cyanobacterial diazotrophs remains sparse.
However, very recently reports have emerged highlighting the potential nitrogen fixing activity of
heterotrophic bacteria and archaea (Bombar et al., 2016; Delmont et al., 2018). It is reported that nitrogen
fixing capability of several heterotrophic diazotrophic bacteria such as Azotobacter, Bacillus, Clostridium
and Klebsiella is significant because they provide considerable amount of fixed nitrogen into the
biosphere (Emmyrafedziawati and Stella, 2018).
Although the presence of potential heterotrophic diazotrophs was previously reported in the
pelagic water column and sediments (Farnelid et al., 2013; Thajudeen et al., 2018), the activity and
importance these heterotrophic nitrogen fixers needs to be determined (Riemann et al., 2010; Mirza and
Rodrigues, 2012; Moisander et al., 2017). The majority of recent studies on heterotrophic nitrogen
fixation mainly targeted the diversity of nifH gene and composition of the bacterial communities by
molecular cloning and next-generation sequencing (Tai et al., 2013; Thajudeen et al., 2017). However, the
expression of nif gene cannot be linked to nitrogen fixation rates without evidence of cell-specific activity
(Moisander et al., 2017). A culture-dependent analysis could help determine the potential physiological
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
al., 1998), which has a diverse assemblage of diazotrophs that may well fix nitrogen at varying rates
(Ahmed et al., 2017). The knowledge about their diversity, activity and ecological role is limited, which
warrants further studies from this region. In a previous study, we have reported the potential of various
Bacillus sp. to fix nitrogen in the marine and estuarine environment (Yousuf et al., 2017). A recent report
by Thajudeen et al. (2017) also summarized the prevalence of diverse α, β and γ-Proteobacterial
diazotrophs in the study area. However, the cell-specific potential of the above organisms to fix nitrogen
has not been studied. Hence, the objective of the present study is to quantify and compare the nitrogen-
fixing potential of diverse Gram-positive and Gram-negative diazotrophs in the estuarine and coastal
environment of Southeastern Arabian Sea (SEAS).
2. Materials and methods
2.1. Study area and sampling
The study was carried out in the Cochin estuary (CE) and adjacent coastal environments along the
SEAS in 2012. The CE is considered as largest tropical estuary in south India which is highly productive
and supports rich fish and shellfish resources. The sampling was carried out at 8 stations from the CE and
4 stations from the coastal region (Fig. 1). The water and sediment samples from the estuary were
collected on board RV Kingfisher. Water samples were collected using a 2 litre Niskin water sampler
(General Oceanic, USA) and sediment samples using Van Veen Grab (Hydrobios, Germany), and stored
in sterile polyethylene bottles. The coastal samples were collected on-board FORV Sagar Sampada
(Cruise No. 311) using Niskin sampler for water and Smith McIntyre Grab for sediments.
2.2. Isolation and screening of diazotrophic strains
The screening of heterotrophic diazotrophic bacteria from each sample of water and sediments
was carried out by the spread plate technique on a nitrogen-free medium (Norris Glucose Nitrogen free
medium, NGNF; HIMEDIA-M712). The seeded plates were incubated in dark at 28±1°C for 7-14 days. 4
After incubation, the morphological features of the colonies were recorded and well-separated colonies
with different morphology were picked up using a sterile inoculation loop. These isolates were re-
streaked to ensure purity and maintained on the sterile NGNF agar slants at 4°C. These isolates were
subjected to Gram staining, spore staining (in case of Gram-positive rods) and examined by light
microscopy (Olympus CX21i) to determine the cell morphology. Based on the differences in color,
morphology and good quality growth on NGNF media, various Gram-negative (n=12) and Gram-positive
(n=8) isolates were selected for further study. These isolates were stored at -20°C in NGNF liquid
medium containing 20% glycerol for further analysis.
2.3. Polymerase Chain Reaction amplification of the 16S rRNA and nifH gene
The selected isolates were grown in Luria-Bertani broth (Hi-media, India) at 28°C for 48h.
Genomic DNA was isolated from cultures as per the standard Proteinase-K digestion method (Sambrook
et al., 1989). Using the extracted DNA as template (Bosshard et al., 2000) polymerase chain reaction
(PCR) was used to amplify the bacterial 16S rRNA gene with the help of universal primer set (27F: 5′-
AGA GTT TGATC TGG CTC AG-3′ and 1492R: 5′-GGT TAC CTT GTT ACG ACT T-3′).
Amplification conditions were as follows: 2min of initial denaturation at 95°C, followed by 30 cycles of
denaturation at 95°C for 2min, annealing at 58°C for 1min, and extension at 72°C for 2min. A final
extension was carried out at 72°C for 10min.
A partial sequence (360bp) of the dinitrogenase reductase nifH gene was amplified using
previously designed degenerate oligonucleotide primers Zehr-nifHf (5′-TGYGAYCCNAARGCNGA-3′)
and Zehr-nifHr (5′-ADNGCCATCATYTCNCC-3′) (Zehr and McReynolds, 1989) as per the procedure of
Zehr et al. (1998), with slight modification. The PCR conditions for the amplification of the nifH gene
were as follows: initial denaturation at 94°C for 5min, followed by 40 cycles of denaturation at 94°C for
1min, annealing at 57°C for 1min and extension at 72°C for 2min., followed by final extension at 72°C
for 10min. All the PCR amplification was carried out in a total volume of 25μl reaction mixtures
consisting of sterile MilliQ water (15.5μl), 10X PCR buffer (2μl), primer (1μl each), dNTP mix (1μl,
200mM), template (4μl) and Taq DNA polymerase (0.5μl). After amplification, the nifH gene fragments
were checked by ultra-pure agarose gel (1.5%) electrophoresis. The size of the resolved bands in the gel
was confirmed by comparing with 100bp nucleotide marker.
2.4. Sequencing and phylogenetic analysis
The 16S rRNA and nifH gene amplicons were purified using the Promega PCR clean-up system
(Nucleospin, MN, Duren, Germany) as per the manufacturer’s instructions and sequenced using an ABi
3730 XL Genetic Analyzer (Applied Biosystems, USA) at Scigenome Pvt Ltd., Cochin. The obtained
gene sequences were subjected to Basic Local Alignment Search Tool (BLAST) sequence similarity
search (http://blast.ncbi.nlm.nih.gov/BLAST) in the National Centre for Biotechnology Information
(NCBI) GenBank database to identify the nearest taxa. The phylogenetic tree based on 16S rRNA and
nifH gene sequences were constructed using two tree making algorithms such as maximum likelihood
(ML) and Neighbor-joining (NJ) methods using MEGA version 7.0 software package (Tamura et al.,
2013). The multiple alignments were performed using ClustalW analysis. The bootstrap calculations of
1000 runs were also carried out to authenticate the reliability of the branching pattern.
2.5. Nitrogen fixation activity
The nitrogen fixation activity of bacterial isolates was quantified by acetylene reduction assay,
which estimated the rate of acetylene (C2H2) reduction to ethylene (C2H4) by nitrogenase enzyme (Stewart
et al., 1967; Kifle and Laing, 2016). The C2H4 production by the bacterial cultures was measured by gas
chromatography equipped with flame ionization detector (FID). The detailed method of estimation and
operating conditions of gas chromatography was reported in Yousuf et al. (2017). The mean values of
each experiment along with standard deviation were calculated and expressed as nmol C2H4/mg
2.6. Statistical data analysis
The data were evaluated with the ‘Statistical Package for the Social Sciences (SPSS)’ version 20.
The Mann-Whitney U test was performed for comparison of nitrogen fixation activity by each group. All
the experiments were tested at 1% level of significance. 6
3.1. Isolation and identification of diazotrophic bacterial strains The diazotrophic bacteria were isolated on solid nitrogen-free media, NGNF. Most of the
colonies obtained on the NGNF media were very small, clear round, convex and gummy, with watery
dewdrop like appearance (Supplementary Fig. S1). Some of the colonies were found submerged in the
medium and a very few colonies showed pigmentation. A total of 20 morphologically different bacterial
colonies (12 from the estuary and 8 from coastal regions) were isolated and characterized from the study
area (Table 1). All the isolates were able to grow well on nitrogen-free agar medium and were identified
as diazotrophs. Phylogenetic diversity of the selected heterotrophic diazotrophs revealed that 12 isolates
belonged to the phylum Proteobacteria, while remaining 8 belonged to Firmicutes.
Out of the 20 strains, 12 isolates belonged to various Gram-negative Proteobacterial species such
as α-Proteobacteria (Nitratireducter kimnyogensis and Rhizobium rosettiformans), β-proteobacteria
(Alcaligenes faecalis) and γ-proteobacteria (Klebsiella pneumonia, K. quasipneumonia, K. variicola,
Rheinheimera aquimaris, Acinetobacter johnsonii, Halomonas meridiana, Pseudomonas flavescens and
two strains of Enterobacter cloacae). The remaining 8 isolates belonged to Gram-positive Firmicutes,
which were identified as Bacillus aryabhattai, B. aerius, B. amyloliquefaciens, Oceanobacillus iheyencis,
Exiguobacterium profundum, Staphylococcus caprae, S. warneri and S. arlettae. The phylogenetic tree
was constructed based on 16S rRNA gene sequences obtained from the present study and the reference
sequences retrieved from the GenBank (Fig. 2.) All the sequences retrieved from this study were
submitted to NCBI database under the accession numbers KT868878 to KT868887, MF795413 to
MF795417, MH045570, KT833390, MF795419, MF795420 and MF795422 (Table 1).
3.2. Amplification of nifH gene
Presence of nifH gene in all the selected bacterial strains was determined by the PCR method. All the
strains except three Gram-positive strains namely B. amyloliquefaciens CES SD11, S. arlettae CCS Z-31
and S. caprae CCS Z-36; showed a positive amplification of nifH gene (Fig. 3). The result reconfirmed
the genetic potential of these bacterial strains to fix atmospheric nitrogen to a usable form. The nifH gene
(360bp) previously amplified and sequenced in our lab (B. flexus CESM15-54, nifH gene sequence
accession number: MH248365; Supplementary data S2) was taken as the reference for the sequencing
analysis. The phylogenetic tree was constructed using the obtained nifH gene sequences and represented
in the supplementary Fig. S3.
3.3. Nitrogen fixation activity of the isolates
All the isolates that showed positive amplification of the nifH gene, as well as the isolates which
failed to amplify the nifH gene, were tested for their ability to fix nitrogen. The nitrogen fixation activity
is estimated by acetylene reduction assay. The results revealed that all the isolates showed varying levels
of nitrogenase activity (Fig. 4). The highest nitrogen-fixing activity was observed in the α-Proteobacteria
N. kimnyogensis CCS Z-27 (190.3±4.55 nmol C2H4/mg protein/day) which was isolated from the coastal
sediment. Other strains which showed relatively good nitrogen fixing activity included K. variicola CCW
62C (151.78±1.80 nmol C2H4/mg protein/day), K. pneumonia CEW 61S (134.92±2.68 nmol C2H4/mg
protein/day) and H. meridiana CES SD3 (143.94±2.75 nmol C2H4/mg protein/day). The K.
quasipneumonia CEW W7 and P. flavescens CCW M-40 also exhibited fairly good nitrogen-fixing
activity (>110 nmol C2H4/mg protein/day). The nitrogen-fixing capability of above strains was superior to
the nitrogen-fixing potential of reference species B. flexus CES M15-54 (76.95±2.4 nmol C2H4/mg
protein/day) (Fig. 4) (Yousuf et al., 2017). Among the Gram-negative strains which were evaluated for
nitrogen fixing potential, Al. faecalis showed the lowest activity (21.05±0.44 nmol C2H4/mg protein/day).
Among the Gram-positive strains, B. aryabhattai CES SD6, S. caprae CCS Z-36, O. iheyensis
CCW 38C, and Ex. profundum CEW 26MY14 have shown good nitrogen fixation, while relatively low
activity was recorded in B. aerius CES SD5 and S. arlettae CCS Z-31 (10.48±0.63 and 14.48±1.99 nmol
C2H4/mg protein/day respectively). The results were also subjected to statistical analysis to understand the
disparity in the nitrogen-fixing activity among various isolates belonging to the dissimilar genera. The
result revealed that the potential to fix nitrogen was significantly high (p<0.01) among Gram-negative
diazotrophs when compared to that of Gram-positive diazotrophs (Fig. 5).
4. Discussion The widespread distribution of non-cyanobacterial diazotrophs in the oceanic waters highlights
the importance of heterotrophic nitrogen fixation (Delmont et al., 2018). Heterotrophic diazotrophs play a
key role in the marine nitrogen cycle and provide a considerable amount of fixed nitrogen (Bombar et al.,
2016) which in turn could have significant effect on primary production. However, the knowledge
regarding their ecological role in the water column is not understood. Therefore it is necessary to identify
the distribution and activity of heterotrophic diazotrophs in order to understand their influence on the
aquatic nitrogen cycle. Due to the difficulty in obtaining these isolates in culture, very little is known
about their physiological characteristics, ecological significance as well as genetic heterogeneity of
nitrogen fixation and their cell-specific potential to fix nitrogen (Farnelid, 2013; Martinez-Perez et al.,
2018). Bentzon-Tilia et al. (2014) suggested that the culture based approaches are also more relevant than
in-situ measurements of nitrogen fixation because field studies were found unsuccessful to connect
nitrogen fixation rate to species-specific cells. Hence the focus of the present study was to understand the
diversity and nitrogen fixation potential of various cultivable heterotrophic diazotrophs from the estuarine
and coastal environments of SEAS.
We have observed that the colony morphology of most of the isolates that developed on nitrogen-free
media were gummy and viscous in nature; which might be due to any of the self-protection mechanisms
of those species to regulate physiological functions. Sabra et al. (2000) reported that the gumminess of
diazotrophic culture may be due to the alginate production by the organisms, which thereby trims down
the oxygen transfer rate. According to Reddy et al. (1993) and Berman-Frank et al. (2007), the
cyanobacterial diazotrophs avoid oxygen inhibition through heterocyst formation or temporal segregation
of photosynthetic oxygen production and nitrogen fixation. It was reported that the high respiratory
activity of some heterotrophic diazotrophs such as Azotobacter helps to protect the nitrogenase enzyme
(Phillips and Johnson, 1961), however the exact mechanisms by which the heterotrophic diazotrophs
protect their nitrogenase enzyme from oxygen is still not clear.
The acetylene reduction assay is the most common laboratory procedure used for the determination of the
specific activity of the enzyme nitrogenase (Stewart et al., 1967; Bentzon-Tilia et al., 2015; Kifle and
Laing, 2016). All the tested strains (n=20) showed nitrogen fixation potential and results indicated that
various species of heterotrophic diazotrophs showed a high degree of variation in nitrogease activity
(ranging from 10 to 190 nmol C2H4/mg protein/day). Park et al. (2005) successfully isolated a variety of
diazotrophic bacteria from rhizosphere with varying nitrogenase activities such as B. fusiformis, P.
fluorescens, Stenotrophomonas maltophilia etc. (14 to 3677 nmol C2H4/mg protein/h). Yousuf et al.
(2017) also studied the diazotrophic potential of various Bacillus strains from the SEAS where they
observed considerable variation in nitrogen fixation potential among different Bacillus strains (3 to 210
nmol C2H4/mg protein/day). Martinez et al. (1996) and Baskar and Prabhakaran (2015) reported that the
potential of enzymatic activity among the diazotrophs may not be the same as it may depend on various
Nitratireducter kimnyeogencis CCS Z-27 was identified as the putative α-Proteobacterial
diazotroph prevailing in the study area. The study confirmed the presence of the nifH gene in this
bacterium which exhibited relatively high nitrogen fixing activity compared to all other tested strains.
Though the presence of diverse diazotrophic α-Proteobacteria were reported in the estuarine and marine
environments (Moisander et al., 2008; Thajudeen et al., 2017; 2018, Martinez-Perez et al., 2018) the
nitrogenase activity of the species N. kimnyogensis is not yet reported. This could be first demonstration
of the identification of nifH gene and nitrogen fixation potential in N. kimnyogensis. We have also
observed that the various species of Klebsiella (K. pneumonia, K. quasipneumonia, and K. variicola) were
also capable of fixing atmospheric nitrogen in to usable form. Klebsiella sp. encountered in the present
study could quickly grow on NGNF media and fix atmospheric nitrogen at environmentally relevant in-
vitro conditions suggesting that they might be very active in marine environments. Our findings agree
with previous observations (Schmitz et al., 2002; Feng et al., 2018) that various species of Klebsiella
could be considered as one of the endowed γ-Proteobacterial diazotrophs capable of fixing atmospheric
The results of the present study are consistent with the previous reports from terrestrial environments
regarding the nitrogenase potential of Halomonas (Llmas et al., 2006), Pseudomonas (Eckford et al.,
2002; Bentzon-Tilia et al., 2015), Rhizobium rosettiformans (Burbano et al., 2011) and Alcaligenes
faecalis (Mazumda and Deka, 2013), though our isolates are from aquatic ecosystems. Furthermore, the
current study also revealed the innovative report from the SEAS regarding the nitrogen fixation potential
of Oceanobacillus, Exiguobacterium and various genera of Staphylococcus. The major highlight of the
present study was that we were successful in cultivating the heterotrophic diazotrophs, which would allow
the ecophysiological and molecular characterization of them in the future.
The primers used in this study have been successfully used to amplify the nifH gene from distantly related
diazotrophs (Zehr and McReynolds, 1989; Tan et al., 2003). Even in the absence of the nifH gene, the
species like B. amyloliquefaciens CES SD11 and S. caprae CCS Z-36 has shown relatively high nitrogen-
fixing activity (50.16 ± 6.16 and 63.29±1.25 nmol C2H4/mg protein/day) than the nifH gene amplified Al.
faecalis CCS Z-1 and S. warneri CCW 69C (Fig. 4). Such inconsistencies between nifH gene
amplification and nitrogen fixation activity are reported by several researchers (Achouak et al., 1999;
Bostrom et al., 2007; Islam et al., 2010; Mirza and Rodrigues, 2012; Yousuf et al., 2017). It is also
reported that the unsuccessful amplification of nifH gene does not mean that the species is incapable of
nitrogen fixation, as there could be a diverse nifH gene nucleotide at inter or intra species level (Zehr et
al., 2003). We assume that the presence of mutant variety of nifH gene could be the most probable reason
for the unsuccessful amplification of nifH gene. Hence a combination of tools is recommended for the
identification of active diazotrophic bacteria.
Statistical analysis of the results revealed that Gram-negative Proteobacterial strains were more
efficient in nitrogen fixation (p<0.01) than Gram-positive Firmicutes (Fig. 5) suggesting that Gram-
negative groups could be an integral part of the diazotrophic bacterial community in the study area. 11
Soares et al. (2006) suggested that free-living heterotrophic diazotrophs belonging to Gram-negatives
play an important role in nitrogen fixation. An exact measurement strategy is needed to determine the
activity of heterotrophic diazotrophs from the natural environments (Postgate, 1988; Bostrom et al.,
2007). The current study proved the proficient enzymatic capability and genetic evidence related to
nitrogen fixation among the heterotrophic diazotrophs and their significant contribution to the aquatic
nitrogen fixation. These findings also imply that we still have much to learn about the major as well as a
diverse array of heterotrophic microorganisms which can apparently perform this significant process in
the nitrogen cycle.
The current study revealed that heterotrophic diazotrophs contributed to nitrogen fixation in the
coastal and estuarine ecosystems of SEAS. We also documented that diverse diazotrophs such as
Firmicutes and Proteobcateria have potential ability to fix atmospheric nitrogen.
potential among these organisms emphasizes the need to reconsider their ecological relevance in the
aquatic environments. Most of the Gram-negative strains identified from our study area, especially
Nitratireducter kimnyeongencis and various species of Klebsiella are found to be powerful diazotrophs,
which contribute significantly to heterotrophic nitrogen fixation. This report also suggests further
microbiological and ecological monitoring of heterotrophic diazotrophs in various estuarine and marine
environments. The sustained use of cultivation methods will definitely lead to the discovery of more
heterotrophic diazotrophic organisms from the study area. It also provides a direct means to learn further
about the functions and activity of heterotrophic diazotrophs in the aquatic nitrogen cycle.
This work was supported by the Ministry of Earth Science (MoES), Government of India under
the Sustained Indian Ocean and Biochemical and Ecological Research programme (SIBER -
MoES/36/001S/SIBER/07). The author Mrs. Jesmi Yousuf gratefully acknowledge the UGC-Maulana
Azad National Fellowship (F1 – 17.1/ 2013/MANF-2013-14-MUS-KER-25319/ (SA-III website) dated
06-Feb-2014) for their financial support. We would like to express our gratitude to the Head, Department
of Marine Biology, Microbiology and Biochemistry, Cochin University of Science and Technology for
providing facilities to carry out the work. The authors would like to express gratitude to the anonymous
reviewers for their helpful and productive comments that greatly contributed to improving the manuscript.
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List of Tables and Figures
Table 1. The details of diazotrophic bacterial strains isolated from the study area and the identification of nifH gene. GenBank Accession number
From estuarine environment
Klebsiella pneumonia CEW 61S*
Enterobacter cloacae CEW M15/24*
Enterobacter cloacae CEW M15/1*
Klebsiella quasipneumonia CEW W7*
Rheinheimera aquimaris CEW W8*
Acinetobacter johnsonii CEW W11*
Halomonas meridiana CES SD3#
Exiguobacterium profundum CEW 26MY14* KT868879
Bacillus aryabhattai CES SD6#
Bacillus aerius CES SD5#
Bacillus amyloliquefaciens CES SD11#
Rhizobium rosettiformansCEW W9*
Nitratireducter kimnyeogencis CCS Z-27#
Alkaligenes faecalis CCS Z-1#
Klebsiella variicola CCW 62C*
Pseudomonas flavescens CCW M-40*
Oceanobacillus iheyencis CCW 38C*
Staphylococcus warneri CCW 69C*
Staphylococcus caprae CCS Z-36#
Staphylococcus arlettae CCS Z-31#
Identification of nifH gene
From coastal environment
isolated from water; # isolated from sediment
Fig. 1. Location map showing the study area of Cochin estuary and adjacent coastal waters.
481 482 483 484 485 486
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
487 488 489 490
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.
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
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
S. caprae CCS Z-36 B. amyloliquefaciens CES SD11 B. aerius CES SD5 B. aryabhattai CES SD6 Ex. profundum CEW 26MY14
O. iheyencis CCW 38C
Fig. 4. Nitrogen fixation potential of various heterotrophic diazotrophs isolated from the study area by acetylene reduction assay.
493 494 495
20 40 60 80 100 120 140 160 180 Nitrogen fixing activity (nmol C2H4 /mg protein/day)
100 80 60 40 20 0
Nitrogen Fixing Activity (nmol C2H4/mg protein/day)
Fig. 5. Variation in the average nitrogenase activity between Gram-positive and Gram-negative strains.
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
Varghese, and Mohamed Hatha A.A
CONFLICT OF INTEREST
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