Nutrient stoichiometry (N:P) controls nitrogen fixation and distribution of diazotrophs in a tropical eutrophic estuary

Marine Pollution Bulletin 151 (2020) 110799

Contents lists available at ScienceDirect

Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul

Nutrient stoichiometry (N:P) controls nitrogen fixation and distribution of diazotrophs in a tropical eutrophic estuary

T

Thajudeen Jabira, ,1, Puthiya Veettil Vipindasa,1, Yousuf Jesmia,b, Sudheesh Valliyodana,e, ⁎ Prabhakaran Meethal Parambathc, Arvind Singhd, Mohamed Hatha Abdullaa, ⁎

a

Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and Technology, Fine Arts Avenue, Kochi 682016, Kerala, India b School of Environmental Sciences, Mahatma Gandhi University, Kottayam, Kerala 686560, India c Kerala University of Fisheries and Ocean Studies, Panangad, Kochi, Kerala 682506, India d Physical Research Laboratory, Ahmedabad 380 009, India e Centre for Marine Living Resources and Ecology, LNG Rd, Puthuvype, Kochi, Kerala, India 682508

ARTICLE INFO

ABSTRACT

Keywords: Cochin estuary Diversity N:P ratio Monsoon Nitrogen fixation rate

Nitrogen fixation and its ecological regulation are poorly understood in the tropical estuaries, which are highly influenced by anthropogenic disturbances. In this study, we investigated the role of nutrient stoichiometry in the diversity, abundance and activity of N2-fixing bacterial community and their seasonal variations in the water column of a tropical eutrophic estuary (Cochin estuary). The N2 fixation rates in the estuary ranged from 0.1 to 2.0 nmol N2 l−1 h−1, with higher activity during post-monsoon and lower during monsoon. The rates are appeared to be primarily controlled by dissolved inorganic nitrogen and phosphorous (N:P) ratio. Clone library analysis of nitrogenase (nifH) gene revealed that the major N2 fixing phylotypes belong to Cluster I and Cluster III diazotrophs. The overall findings of this study suggest that monsoon induced seasonal changes in nutrient stoichiometry control the distribution and activity of diazotrophs in a tropical estuary.

1. Introduction Biological N2 fixation, microbial conversion of molecular nitrogen (N2) to ammonium, is fundamental to productivity in aquatic environments (Bentzon-Tilia et al., 2015). N2 fixation is vital in offsetting the deficit in bioavailable nitrogen (N) relative to phosphorus (P) in aquatic ecosystems, and in contributing to the P-limiting status of such systems (Černá et al., 2009, Voss et al., 2011). Though N regulates biological productivity in most marine environments, it is essential to understand processes that control its availability (Singh et al., 2013). N2 fixation is a common trait of autotrophic and heterotrophic bacteria and both the organisms use nitrogenase enzyme complex for this process (Hoffman et al., 2014). Traditionally cyanobacteria were thought to be the only diazotrophs in the marine environment and heterotrophic diazotrophs were ignored to be a significant contributor in marine N2fixation. However, recent studies in nitrogenase reductase gene (nifH) community composition revealed that heterotrophic diazotrophs are also prevalent in marine environment and can significantly contribute to the N2 fixation (Sohm

et al., 2011; Shiozaki et al., 2014; Kumar et al., 2017; Moisander et al., 2017). At the same time, the knowledge of their ecological role in estuarine system especially in the water column is limited and the factors which control their diversity and distribution in space and time scale are not clearly understood. Estuaries receive a large amount of anthropogenic nutrients from different sources such as atmosphere, river and land runoff, which include industrial, agricultural and sewage wastes. Excess nutrients entering into the coastal ecosystem as organic and inorganic N and P compounds pose a threat to the ecological stability of this environment (Downing, 1997; Galloway et al., 2004). Over the past few decades many estuarine systems shifted drastically from ‘nutrient-limiting’ to ‘nutrient-surplus’ condition due to the increased anthropogenic inputs of nutrients which lead to eutrophication (Nixon et al., 1996). Unbalanced loading of any one of the nutrients (N or P) can lead to changes in the nutrient ratio, either to P-limitation or to N-limitation (Blomqvist et al., 1994; Conley et al., 2009). Bioavailability of N and P are critical in regulating N2 fixation rates. High P content may induce N2 fixation that maintains nutrient stoichiometry of estuaries (Howarth

Corresponding authors. E-mail addresses: [email protected] (T. Jabir), [email protected] (M.H. Abdulla). 1 Present address: Cryobiology Laboratory, National Centre for Polar and Ocean Research, Vasco-da-Gama, Goa 403804, India. ⁎

https://doi.org/10.1016/j.marpolbul.2019.110799 Received 2 August 2019; Received in revised form 26 November 2019; Accepted 3 December 2019 Available online 29 January 2020 0025-326X/ © 2019 Elsevier Ltd. All rights reserved.

Marine Pollution Bulletin 151 (2020) 110799

T. Jabir, et al.

and Marino, 2006; Paerl, 2017). Despite the extensive studies on environmental and biogeochemical changes in the tropical estuaries, the role of N2 fixing organisms in the production of bioavailable N is still poorly understood (Veettil et al., 2015; Bhavya et al., 2016). The present study was carried out in the Cochin estuary (CE), one of the largest tropical wetland ecosystems along the south-west coast of India, located in close proximity to the south-eastern Arabian Sea. The release of untreated industrial wastes (104 × 103 m3 d−1) and domestic sewage (260 m3 d−1) into the CE has led to deleterious changes in the ecosystem (Menon et al., 2000), such as alteration in the nutrient stoichiometry and even changes in the microbial activity (Martin et al., 2008, Miranda et al., 2008). Hence, the major objectives of the present study were to 1) understand the diversity of N2 fixers and the rate of N2 fixation in the water column of the CE, 2) elucidate the distribution of diazotrophs in the CE, and 3) study the role of nutrient stoichiometry in regulating the distribution and activity of diazotrophs.

water quality analyzer (Systronics - 371; precision: ± 0.01) calibrated with standard sea water (Greenberg, 1992). Dissolved Oxygen (DO) was analyzed by Winkler's method (Grasshoff and Ehrhardt, 1999) with a detection limit of 0.05 ml l−1. Samples for nutrients (phosphate, nitrate, nitrite and ammonium) were analyzed spectrophotometrically (Hitachi - 2J1-0004, Japan) within 6 h of collection using the protocol of Grasshoff and Ehrhardt (1999). The analytical precision (standard deviation) was ± 0.01, 0.01, 0.05 and 0.07 μM for phosphate, nitrite, nitrate and ammonium, respectively. The samples for culture based microbiological analysis were collected in sterile plastic bottles, transported in an ice box and were analyzed within 4 h. The water samples for DNA isolation were filtered through 0.2 μm pore size polycarbonate membrane filter (1000 ml, Triplicates; Millipore; GTTP2500) and kept in −20 °C until analysis. 2.4. DNA extraction and PCR amplification of the nifH gene Genomic DNA from water samples was extracted following method of Boström et al. (2004) with minor modification. The filters (0.2 μm pore size) were transferred to lysis buffer (NaCl 400 mM, sucrose 750 mM, EDTA 20 mM, and Tris-HCl 50 mM) containing 1 mg/ml lysozyme and incubated at 37 °C for 1 h. Thereafter, 1% of sodium dodecyl sulphate (SDS) and proteinase K (100 μmg/l) were added to the solution and allowed to incubate overnight at 55 °C. To this, isopropanol was added and kept at −20 °C for 60 min. Precipitated DNA pellet was washed copiously with 70% ethanol and dissolved in TE buffer. The nifH sequences were amplified from the sample with high-efficiency DNA polymerase (Takara Bio Inc., India) on a Thermal Cycler (Applied Biosystem Inc., California). A nested PCR reaction was used, as previously described (Zehr et al., 1998) with minimum modification, 50 μl PCR reaction mixture was amplified for 30 cycles (1 min at 98 °C, 45 s at 57 °C, 1 min at 72 °C), first with the outer nifH PCR primers (Zani et al., 2000), followed by amplification with the inner nifH PCR primers (nifHf 5′-TGYGAYCCNAARGCNGA-3′, nifHr 5′-ADNGCCATCATYTCNCC-3′) (Zehr and McReynolds, 1989). To check the possibility of contaminant in the reagents (Zehr et al., 2003), negative controls were run with every PCR reaction. To avoid potential sample bias and to obtain adequate PCR products for cloning, triplicate amplifications were carried out for each sample. PCR conditions comprised of 30 cycles at 94 °C (1 min), 56 °C (1 min) and 72 °C (2 min), with a final extension at 7 min at 72 °C after the final cycle. The amplification products were purified using the PCR clean-up kit (Nucleospin PCR clean up kit, MN, Germany) and inserted into pGEM-T Easy Vector system kit (Promega, USA) according to the manufacturer's instructions. The PCR products with the inserts were isolated and plasmid extracted and clones were sequenced. The sequencing was carried out from SciGenom Labs Pvt. Ltd. Kochi, India.

2. Materials and methods 2.1. Study area The Cochin Estuary (CE) (~256 km2 between 9° 40′–10° 12′ N and 76° 15′–76° 25′ E) is a complex micro-tidal tropical estuary situated along the south-west coast of India. The CE has an average depth of 2 to 3 m, and the shipping channel into the Cochin harbor maintains a depth of 10 to 13 m by regular dredging (Qasim, 2003). The estuary stretches parallel to the coast and is linked to the Arabian Sea by two permanent openings, one at Cochin (450 m wide) and the other at Munambam (250 m wide). Six major rivers, their tributaries and a network of canals discharge ~2 × 1012 m3 of fresh water into the estuary annually (Srinivas et al., 2003). The summer monsoon, which accounts for about 70% of annual rainfall, and its associated run off significantly contribute to this freshwater discharge. The CE shows high flushing rate during the monsoon (1 to 2.5 days) and relatively low flushing rate during the non-monsoonal period (8.7 days) (Janardanan et al., 2015). CE is affected by fresh water discharge from river and land run-off during monsoon, whereas in non-monsoonal period tidal influence results in intrusion of sea water up to 20 km towards the estuary from the Barmouth (Martin et al., 2011) making the CE a dynamic estuarine system with strong seasonal variability in the hydrographic conditions. 2.2. Sampling Study area was selected based on its geographic importance and characteristics of point sources (Fig. 1). Station 1 (Kadakkara, 2 m depth) was located in an area where saline water from both the inlets (Munambam and Cochin) meet during the high tide which results in weak transport of nutrients. Station 2 (Near Bolghatty, 2 m depth) is located at the mouth of the Periyar River and receives nutrients from many small and large industries located upstream and untreated sewage from the human settlements around it. Station 3 (Marine Drive, 2 m depth) is the region situated at the center point of the CE and is affected by sewage effluents from Cochin city and nearby settlements. The station 4 (Barmouth, 12 m depth) is the meeting point of estuarine and coastal waters and is largely affected by Cochin harbor activities as well as dredging. Station 5 (Marine Science Jetty, 3 m depth) situated at the center point of the estuary and is polluted by ship ballast and sewage outflow from the city of Cochin. Station 6 (Arookutty, 6 m depth) is located at the southern part of estuary and the meeting point of the Muvattupuzha river to the main estuarine system.

2.5. Phylogenetic analysis The partial nifH gene sequences obtained were checked for the presence of vector sequences contamination using the Vec Screen program of NCBI and it removed using BIOEDIT v. 7.0.0 (http://www. mbio.ncsu.edu/Bioedit/bioedit.html). Then the chimeric sequences detected using DECIPHER (http://www.decipher.cee.wisc.edu/ FindChimeras.html) were also removed from the dataset. The remaining sequences were then clustered into OTUs with 3% distance cutoff using MOTHUR. The nifH sequences were compared with GenBank database using the BLAST-N algorithm and were aligned by CLUSTAL X software package (Thompson et al., 1997). The phylogenetic trees were constructed using the neighbor-joining and maximum parsimony methods using MEGA 6.0 (Tamura et al., 2013). Bootstrap re-sampling analysis for 1000 replicates was performed to estimate the confidence of the tree topologies (Tamura et al., 2013). The sequences were reported from the study area were submitted into NCBI database

2.3. Analysis of physico-chemical parameters Water samples were collected seasonally from subsurface using 2 L Niskin sampler (General Oceanic, USA). Salinity was measured using 2

Marine Pollution Bulletin 151 (2020) 110799

T. Jabir, et al.

Fig. 1. Location map of the Cochin Estuary showing sampling locations and two inlets into the estuary from the Arabian Sea (Small back arrows indicate the shipping channel and large curved arrows indicate the inlet of water enter into CE from AS).

under the accession number MH881569 - MH881599 and MH881625 MH881669.

of nifH gene using nifH primers (Zehr and McReynolds, 1989). The representative sequences were submitted into NCBI GenBank data base KT921128, KT848794, KT833388, KT764106, KT868879, KT868887, MF795414, KT868880, MH045570, KT868887, MF795413, MF795422 and MF795415.

2.6. Abundance of heterotrophic diazotrophs (HD) Heterotrophic diazotrophs (HD) were isolated using N free media (Norris Glucose Nitrogen Free Medium, HIMEDIA-M712). In this media the organisms capable for N2 fixation only grow and form colonies on plates (Ranganayaki and Mohan, 1981). The plates were incubated at 28 ± 1 °C for two weeks and then the colonies were counted and the counts were expressed as colony forming units (CFU) per ml. Based on the morphological characteristics, individual colonies were picked up and purified by re-streaking on fresh medium and maintained on agar slants for further analysis. Polymerase chain reaction (PCR) amplification of bacterial 16SrRNA was performed by using the universal primer set (27F: 5′-AGA GTT TGATCC TGG CTC AG-3′ and 1492R: 5′-GGT TAC CTT GTTACG ACT T-3′) with the extracted DNA as the template. The PCR products were sequenced using an ABi 3730 XL Genetic Analyzer (Applied Biosystems, USA) at Scigenome Pvt. Ltd., Cochin. The 16SrRNA gene sequences obtained was subjected to Basic Local Alignment Search Tool (BLAST) sequence similarity search (http://blast. ncbi.nlm.nih.gov/BLAST) to identify the nearest taxa. The presence of nifH gene in these isolates was also confirmed by the PCR amplification

2.7. N2 fixation rate N2fixation rates were estimated using acetylene (C2H2) reduction assay method (Stewart et al., 1968; Wilson et al., 2012). The water samples were transferred to acid washed 300 ml crimp sealed vials after thorough rinsing. Bottles were carefully closed using septum closure caps (Sigma-Aldrich) with no headspace and inverted 10–15 times. Subsequently, 30 ml of C2H2 (commercial grade) was injected into serum bottles using gas-tight syringe (Hamilton) through the septum cap, which replaced an equal amount of water sample that came out through the second needle fitted on the cap and formed headspace gas. Replicate (n = 3) samples were immediately analyzed after the introduction of C2H2 into serum bottle to measure C2H4 concentration at time zero. Control samples (n = 3) consisted of 0.2 μm filtered estuarine water and processed the same way as the samples. Experimental and control treatments were incubated on flowing surface estuarine water in situ. The incubation period was 4 h at local noon (from 10 am 3

Marine Pollution Bulletin 151 (2020) 110799

T. Jabir, et al.

to 2 pm). To measure the dissolved ethylene (C2H4) concentration, a head space analysis technique was used after incubation (Wilson et al., 2012). One hundred microlitres of the headspace gas was injected (using gas tight syringe Hamilton 100 μl) (through a split mode) into the flame ionization detector equipped gas Chromatograph (Clarus 580, Perkin Elmer) having PLOT 50 m length capillary column (Elite Alumina/KCl column Product. No. N9316544). The analytical conditions of gas chromatograph consisted of oven temperature at 80 °C; the injection at 200 °C and the detection temperature maintained at 200 °C. The flow of N2 gas was maintained at 7 ml/min, H2 at a flow rate of 45 ml/ min and air at a flow rate of 450 ml/min. The standard curve used for quantification of C2H4 was generated by an absolute calibration method using a series of absolute concentrations (1, 2, 3, 4, 5 and 10 μl) of standard C2H4 gas (Sigma Aldrich, 295329). Representative peak retention times for C2H4 gas and C2H2 were 3.7 min and 9.9 min, respectively with an analytical precision of ± 5 pmol/L. The mean values of each experiment were calculated and expressed as nmol N2 l−1 h−1 (Gothwal et al., 2008). The rate of production of C2H4 concentration was converted into N2 fixation rate by a conversion factor of 3C2H4:1N2 (Hardy et al., 1968; Bertics et al., 2013).

monsoon (μ ± σ: 31.4 ± 1.0 °C). The pH values ranged from 6.6 to 8.6 during the study period. The water was slightly acidic during premonsoon and alkaline during monsoon. Salinity showed wide fluctuation during the study period and ranged from 0 to 26.3 (μ ± σ: 13.8 ± 11.6). Most of the stations showed zero salinity during monsoon except station 4 (Barmouth) and intermediate during post-monsoon (range, μ ± σ: 8–32, 16.5 ± 8.8). Average dissolved oxygen (DO) was 5.9 ± 0.8, 4.1 ± 0.8 and 4.0 ± 1.0 mg/ L during premonsoon, monsoon and post-monsoon, respectively. Dissolved nutrient concentration showed seasonal variability. Dissolved inorganic nitrogen (DIN) - (sum of nitrate, nitrite and ammonium) ranged from 4.3–125 μM, with higher values during monsoon. Ammonium (NH4+) contributed 60% to DIN in the estuary and varied from 2.3 to 116 μM. Higher concentration of NH4+ was observed during monsoon (37 ± 44.7 μM) followed by post-monsoon (6.3 ± 3.6 μM) and minimum during pre-monsoon (4.0 ± 1.7 μM). Dissolved inorganic phosphorous (DIP) concentration varied from 0.4 to 4.8 μM. High concentration of DIP was prevalent in the post-monsoon (2.2 ± 1.6 μM), compared to monsoon (1.4 ± 1.0 μM) and premonsoon (0.8 ± 0.4 μM). DIN:DIP ratio was lowest during postmonsoon (5 ± 3), whereas, it was higher during monsoon (37 ± 15 μM) and pre-monsoon (19 ± 9 μM). One-way ANOVA showed that most of the environmental variables, except pH and DIP, showed significant seasonal variations, whereas they did not show any significant spatial variation (Table 2). (See Table 3.)

2.8. Statistical analysis One-away ANOVA was performed to find out the significance in the spatio-temporal variations of both environmental and biological parameters in the study (IBM SPSS Statistics 22). Pearson correlation analysis (significant level α = 0.05 & 0.01) was performed to find out the influence of environmental variables on the distribution and activity of nitrogen fixers in the CE. Clone library coverage was determined by C = [1 – (n/N)] × 100, where n is the number of unique OTUs and N is the total number of clones in the clone library (Good, 1953). Various diversity indices such as Shannon Weiner (H), Simpson (D) and evenness (J′) were calculated based on OTU data using Primer 6 software (Quest Research Limited).

3.2. Nitrogen fixation rates Spatial and temporal variations were observed in the N2fixation rates in the CE and it varied from 0.1 to 2.0 nmol N2 l−1 h−1 (μ ± σ: 1.0 ± 0.7 nmol N2 l−1 h−1). Highest N2fixation rate was observed in Stn 2 and lowest occurred in Stn 1 (Fig. 2). The higher N2fixation rate was observed during the post-monsoon (μ = 1.39 nmol N2 l−1 h−1) and pre-monsoon μ = 1.35 nmol N2 l−1 h−1) and lower during monsoon (μ = 0.41 nmol N2 l−1 h−1).

3. Results

3.3. Diversity of nitrogen fixing bacteria

3.1. Environmental characteristics of the CE

Clone libraries were constructed to understand the seasonal variations in the diazotrophic community distribution. The DNA samples were pooled from all the stations during pre-monsoon, monsoon and post-monsoon. Total three clone libraries were constructed and the details were mentioned in the Table 4 and Fig. 3. Thirty seven OTUs were obtained from the three hundred and forty two clones. The results

Substantial seasonal variations were observed for environmental variables in the CE (Table 1). Surface water temperature was lower during monsoon (average ± standard deviation, μ ± σ = 27.6 ± 0.8 °C) compared to pre-monsoon (μ ± σ: 33.2 ± 0.7 °C) and post-

Table 1 Spatio-temporal variations in the physico-chemical and nutrients parameters along the CE. Stations Stn.1 Stn.2 Stn.3 Stn.4 Stn.5 Stn.6 Stn.1 Stn.2 Stn.3 Stn.4 Stn.5 Stn.6 Stn.1 Stn.2 Stn.3 Stn.4 Stn.5 Stn.6

Pre-monsoon

Monsoon

Post-monsoon

Temp (°C)

pH

Salinity (psu)

DO (mg/L)

Ammonia (μM)

Nitrite (μM)

Nitrate (μM)

DIN (μM)

DIP (μM)

N/P Ratio

32.7 33.5 33.5 32.5 34.2 32.8 26.1 28 27.5 27.7 28 28.5 32.5 32.5 32 30.5 31 30

7.69 7.74 7.93 6.55 7.82 7.7 8.4 8.3 7.3 7.7 7.8 7.9 8.6 8.21 7.81 7.9 7.7 7.9

14.22 0.21 16.63 26.31 25.3 0 0 0 0 2 0 0 4.5 3.5 2.6 5.7 4.3 2.8

7.2 5.6 4.8 5.6 6 6 3.2 4.01 3.63 5.27 3.63 4.84 12 8 15 32 21 11

6.21 3.01 2.26 3.01 3.59 5.91 8.38 19.09 115.91 65.64 5.12 8.84 3.18 8.81 12.29 3.37 5.89 4.15

0.01 0.14 3.66 0.36 0.57 0.16 0.21 1.98 0.52 0.52 0.31 0.31 0.23 0.75 0.28 0.3 0.21 0.57

6.91 3.77 11.80 3.68 5.79 19.96 9.01 5.42 8.65 11.42 4.38 13.62 0.9 2.42 2.65 0.74 1.71 0.64

13.13 6.92 17.73 7.05 9.96 26.04 17.59 26.49 125.08 77.58 9.81 22.77 4.31 11.98 15.22 4.41 7.81 5.36

0.42 0.95 0.82 0.67 0.45 1.4 0.32 1.62 2.91 1.9 0.2 1.48 1.35 4.77 2.22 0.55 0.87 3.25

31.27 7.29 21.63 10.53 22.15 18.6 54.97 21.91 42.98 40.83 49.05 15.39 3.19 2.51 6.86 8.02 8.98 1.65

4

Marine Pollution Bulletin 151 (2020) 110799

T. Jabir, et al.

Proteobacterial OTUs was detected which is affiliated with Dechloromonas sp. The α-Proteobacterial OTUs included uncultured Bradyrhizobium and Sinorhizobium sp., Rheinheimera hassiensis and uncultured clones from Marginal Sea. Eight OTUs of Cluster III diazotrophs were also detected, the OTUs were affiliated with Desulfobacter latus and Desulfovibrio alkaliphilus and uncultured bacterial clones from the Bohai Sea, Cochin estuary, Chesapeake Bay and Pearl river estuary. The results also exhibited that the Cluster III proteobacterial OTUs were dominant in the post-monsoon and monsoon season and Cluster I cyanobacterial OTUs were dominant in the pre-monsoon season. Biodiversity index value (Table 4) indicated that the nifH gene was highly diverse throughout the season and their richness and evenness varied seasonally. The higher diversity delineated that the sequences in the nifH clone libraries represent the dominant putative OTUs of diazotrophs in the Cochin estuary.

Table 2 Results of One-way ANOVA of major environmental and biological parameters in the Cochin estuary (* - p < 0.05, ** - p < 0.01). (DIN - dissolved inorganic nitrogen, NFR - nitrogen fixation rate, HD - heterotrophic diazotrophs). Parameters

F value Temporal spatial

Temperature Salinity Dissolved oxygen pH Ammonia Nitrite Nitrate DIN DIP N/P NFR Abundance of HD

65.03** 6.35** 8.06** 2.3 3.06 0.34 5.91* 3.65* 2.3 15.5** 6.41** 11.06**

0.06 1.2 0.64 1.58 0.58 0.42 0.85 0.81 1.58 0.58 0.76 0.58

3.4. Culture dependent N2 fixing bacterial diversity The nitrogen fixing heterotrophic bacteria were isolated on bioavailable nitrogen free media. The colonies were very small, colorless and translucent. Diverse isolates were selected based on the morphological characteristics. The 16S rRNA gene was sequenced to evaluate the phylogenetic diversity of the heterotrophic N2-fixing bacteria isolated from the estuary. Based on the 16S rRNA and the presence of nifH gene sequence analysis, major heterotrophic diazotrophic bacteria were identified with NCBI GenBank data base, which revealed maximum homology with phyla of Firmicutes and γ-Proteobacteria. Major N2fixing isolates from the study area are Bacillus cereus, B. safencis, B. megaterium, B. licheniformis, Exiguobacterium profundum, Enterobacter cloacae, Rhizobium rosettiformans, Klebsiella pneumonia, Klebsiella quasipneumonia, Rheinheimera aquimaris, Acinetobacter johnsonii.

Table 3 Results of Pearson correlation analysis showing the influence of various environmental parameters on the activity and distribution of nitrogen fixers (* p < 0.05, ** - p < 0.01).

Temperature Salinity Dissolved oxygen pH Ammonia Nitrite Nitrate DIN DIP N/P

Nitrogen fixation rate (NFR)

Abundance of heterotrophic diazotrophs (HD)

0.68** 0.16 −0.01 0.03 −0.48* −0.1 0.14 −0.46 0.24 −0.80**

−0.09 0.02 −0.53* 0.39 −0.15 −0.49 −0.07 −0.22 0.62** −0.53*

3.5. Abundance of heterotrophic diazotrophs (HD) Abundance of heterotrophic diazotrophs varied from 5.0 × 102 to 7.1 × 104 CFU ml−1 during the study period (Fig. 2). The lowest abundance of HD (5.0 × 102–1.4 × 103 CFU ml−1; μ = 1.0 × 103 ± 4.0 × 102 CFU ml−1) was recorded during premonsoon while it was highest during post-monsoon (1.8 × 104 to 7.1 × 104 CFU ml−1; μ = 3.5 × 104 ± 2.0 × 104 CFU ml−1). During monsoon, the abundance of HD ranged from 2.3 × 103 to CFU ml−1 with an average of 2.3 × 104 1.2 × 104 ± 8.5 × 103 CFU ml−1.

revealed that the diazotrophs belong to cluster I and III (Chien & Zinder, 1996; Zehr et al., 2003; Jayakumar et al., 2012) (Supplementary Table 1). The Cluster I diazotrophs showed cyanobacterial and proteobacterial phylotypes. Twelve OTUs of cyanobacterial diazotrophs were observed in the estuary. The major phylotypes were affiliated with Trichodesmium sp., Lyngbya majuscula, uncultured Chroococcales, Synechocystis sp., Leptolyngbya sp., Oscillatoria sancta, Phormidium sp., Calothrix sp., Anabaena variabilis, two of the cyanobacterial OTUs were affiliated with clones from eastern tropical Atlantic Ocean. Seventeen OTUs of Cluster I Proteobacteria diazotrophs were detected in the estuary, in which nine OTUs of γ-Proteobacteria, one OTU of β-Proteobacteria and remaining seven OTUs of α-Proteobacteria. The γ-Proteobacterial OTUs were identical to Acinetobacter sp., and Methylobacter luteus, Pseudomonas stutzeri, uncultured clones from Red Sea, Arabian Sea, Gulf of Guinea and sediments of Cochin estuary. Only one β-

3.6. Role of environmental variables on abundance and activity of nitrogen fixers Environmental variables influenced N2 fixation rates and abundance of heterotrophic diazotrophs in the CE (Fig. 4). Pearson correlation analysis showed that the N2 fixation rate in the estuary was negatively

Fig. 2. Seasonal variations of nitrogen fixation rate and abundance of nitrogen fixing bacteria in the various stations of Cochin estuary. 5

Marine Pollution Bulletin 151 (2020) 110799

T. Jabir, et al.

Fig. 3. Phylogenetic tree of nifH phylotypes constructed based on a nifH sequences with neighbor-joining method. The alignment was performed using ClustalX (version 1.8). Phylogenetic distances (distance options according to the Jukes–Cantor model) and clustering with the neighbor-joining method were determined using bootstrap values based on 1000 replications. The scale bar indicates nucleotide substitution per site. Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013).

influenced by DO (p ≤ 0.05), except during monsoon. However, the abundance of HD was not influenced by the DO concentration. Among the nutrients analyzed in the study, DIN showed significant negative correlation with N2 fixation rate (p ≤ 0.05) irrespective of seasons. However, the DIN concentration did not show any influence on the abundance of HD, whereas the DIP concentration showed significant positive correlation with the abundance of HD (p ≤ 0.01) and not with N2 fixation rate. N:P ratio was negatively correlated with both N2 fixation rate (p ≤ 0.01) and abundance of HD (p ≤ 0.05). Other environmental variables did not show any significant influence on the N2 fixation rate and abundance of HD in the CE.

Table 4 Details of clone library, biodiversity indices and predicted richness of the nifH gene sequences obtained from the various seasons of Cochin estuary (PreM Pre-monsoon, Mon - Monsoon, PostM - Post-monsoon).

No. of clones No. of unique sequences No. of OTUs Coverage (%) Shannon-Weiner (H) diversity index Simpson dominance index (D) Evenness index (J') S (chao1)

PreM

Mon

PostM

114 40 25 64.91 4.4 5.05 0.94 25.33

83 25 22 69.87 4.26 4.75 0.95 34.33

145 35 29 75.86 4.5 5.6 0.93 36

Fig. 4. Pearson correlation analysis a) relationship between N:P ratios and nitrogen fixation rate b) relationship between N/P ratio and nitrogen fixing heterotrophic bacteria c) relationship between nitrogen fixation rate and nitrogen fixing heterotrophic bacteria. 6

Marine Pollution Bulletin 151 (2020) 110799

T. Jabir, et al.

4. Discussion

water column of the Red Sea (Foster et al., 2009) and the Arabian Sea (Bird et al., 2005). A unique OTU of beta proteobacteria were closely identical to sequences Dechloromonas sp. and the sequences from South China Sea. These sequences were previously reported in Cochin estuarine sediments (Thajudeen et al., 2017) and the gut from lucinid bivalve (Koning et al., 2016). Cluster 1 α-proteobacterial sequence showed close similarity to the nifH sequence of previously reported Bradyrhizobium sp. (Newell et al., 2016), Rheinheimera hassiensis (Sueraz et al., 2016) and Beijerinckia derxii (Rao, 1988). Cluster I proteobacterial sequences were observed throughout the season. The Cluster III proteobacterial diazotrophs were prevalent in the post-monsoon season. The sequences were closely similar to Desulfovibrio alkaliphilus and Desulfobacter latus, which were consistent with the presence of these bacteria in the sediments of CE (Thajudeen et al., 2017). The higher nutrient discharges during monsoon and postmonsoon could have led to the formation of hypoxic condition in the estuary, which might have stimulated the proliferation of Cluster III diazotrophs in this season. Dominance of Cluster III diazotrophs has also been reported from other estuaries like Neuse estuary, North Carolina USA (Affourtit et al., 2001) and Mekong River plume system (Moisander et al., 2008), New England estuary (Newell et al., 2016). The higher abundance of HD was observed in this study with noticeable spatio-temporal variations. The average abundance was higher during post-monsoon and lower during pre-monsoon. Relatively high abundance of HD observed in the southern part of the estuary (Station 6), which has higher fresh water influence due to river discharge. Station 3, which receives higher amount of nutrient inputs from the Cochin city showed the minimum abundance of heterotrophic diazotrophs. Recent studies also indicated that the heterotrophic N2 fixers were more abundant in estuaries and coastal areas than in open oceans and contributed more nitrogen through N2 fixation in these environments (Farnelid et al., 2013; Bentzon-Tilia et al., 2015; Yousuf et al., 2017).

The CE receives large amounts of anthropogenic organic inputs from various sources like industrial effluents, agricultural wastes, domestic sewage, port activities and leisure craft tourism (Jyothibabu et al., 2006; Renjith et al., 2016). The shallow depth of the estuary promotes organic matter mineralization, yet another major source of DIN and DIP in the estuary from surface sediment. Navigational activities in the lower reaches of the estuary further support this remineralization through the production of suspended sediments with high organic load (Gupta et al., 2009, Thottathil et al., 2008, Martin et al., 2011). Along with these sources, the low saline regions of the estuary also receive high organic matter (7–11 times) as a parallel input through rivers (Gupta et al., 2009). Though the nutrient concentrations were always higher in the CE, its peak values were observed during the onset of summer monsoon. This implies that monsoon rainfall and associated run-off plays an important role in regulating the nutrient dynamics in the estuary. N2 fixation rate in the Cochin estuary was relatively high during the study period with significant seasonal variations. Higher N2 fixation rate was observed during post-monsoon (μ = 1.39 nmol N2 l−1 h−1) and pre-monsoon (μ = 1.30 nmol N2 l−1 h−1) compared to that during monsoon. In Mandovi and Zuari estuaries, another tropical estuarine systems along the Arabian Sea, where, Ahmed et al. (2017) recently reported similar average N2 fixation rate of 1.91 nmol N2 l−1 h−1. The current observation is consistent with the N2 fixation rate reported from other world estuaries like Roskilde Fjord and the Great Belt strait (temperate estuarine systems, Denmark) (Bentzon-Tilia et al., 2015). Sediments of CE also witnessed higher N2 fixation activity ranging from 0.25 to 0.74 nmol N2 g−1 h−1 (Thajudeen et al., 2017). In this study, most of the nifH sequences from the Cochin estuary were associated with Cluster I and affiliated with putative diazotrophs which belongs to Proteobacteria and Cyanobacteria (Zehr et al., 2003; Turk-Kubo et al., 2014; Jayakumar et al., 2017). A large number of the sequences were affiliated with cyanobacteria. A major nitrogen fixing Cyanobacterium Trichodesmium sp. were also encountered in the clone library, which previously reported from the Arabian Sea. Intense blooms of Trichodesmium were reported in the Arabian Sea during premonsoon (Gandhi et al., 2011; Jayakumar et al., 2013; Jabir et al., 2018; Jyothibabu et al., 2017), which reach off Cochin estuary due to the tidal influence from the Arabian Sea. The potential ability of N2 fixation by Trichodesmium in the Cochin coastal waters was reported by Bhavya et al., (2016). The Trichodesmium sp. may be one of the major contributor for N2 fixation in the pre-monsoon season in the Cochin estuary. Also N2 fixing unicellular cyanobacteria clones were also reported from the Cochin estuary (Jabir et al., unpublished data, 2019). Recent studies revealed that unicellular cyanobacteria are also a major source of N2 fixation in the marine environments (Zehr et al., 2017; Harding et al., 2018). Other N2 fixing Cyanobacterial OTUs such as Calothrix, Anabaena, Lyngbya and Oscillatoria were also reported as N2 fixers from various marine environments (Bergmann et al., 1997; Issa et al., 2014; Singh et al., 2016). These different types of proteobacterial diazotrophs, observed widely in coastal and estuarine environments, were encountered in the CE, (Bird et al., 2005, Jayakumar et al., 2013, Shiozaki et al., 2018). Most of the nifH gene sequences were related to known bacterial diazotrophs (Supplementary Table 1). Proteobacterial sequences were dominant in the post-monsoon. Major group of proteobacterial sequences were under Cluster I diazotrophs. The gamma-proteobacterial subcluster has sequences related to Azotobacter vinelandii, methylobacter luteus, Pseudomonas stutzeri, and Vibrio diazotrophicus. These were detected in this study and were also reported in the oxygen minimum zones of the Arabian Sea and eastern tropical North Pacific (Jayakumar et al., 2013, 2017), and estuarine water column of Baltic Sea (). Several gamma-proteobacterial clones were closely related to sequences derived from Cochin estuarine sediments (Thajudeen et al., 2017), the

4.1. Influence of environmental variables on distribution and activity of diazotrophs Higher N2fixation rates were observed when the stations reached intermediate salinity range (5–15) during pre-monsoon and post-monsoon. During monsoon, large amount of fresh water enters into the estuary through the monsoonal rainfall and associated runoff that reduce the salinity to close to zero in the entire CE (except at station 4) leading to lower N2 fixation. Similar results were observed in temperate estuaries such as Roskilde Fjord and the Great Belt (Bentzon-Tilia et al., 2015). At the same time, it was reported that greater salinity (above 30) may reduce the activity of N2 fixing bacteria in estuaries (Howarth et al., 1988; Herbst, 1998). However, statistical analysis did not show any significant correlation between salinity and N2 fixation rate. Obviously this lower N2 fixation is not only due to the reduction in salinity but also due to the changes in other environmental variables associated with monsoon. Since the CE is a tropical estuary, the temperature was always in a narrow range of 27–32 °C and it did not show any significant influence on the abundance and activity of N2 fixers. Dissolved nutrients such as DIN and DIP can also act as critical environmental variables that influence the N2 fixation rate and abundance of heterotrophic diazotrophs. During the study, DIN showed significant negative correlation with N2 fixation rates irrespective of seasons (n = 18; p ≤ 0.05). A major fraction of DIN in the CE was ammonium, which is known to control the rates of N2 fixation by suppressing the synthesis of new nitrogenase enzyme (Horne and Goldman, 1972; Rees et al., 2006). DIN did not show any significant influence on the abundance of HD, at the same time, DIP concentration in the CE showed significant positive correlation with an abundance of HD only. Positive correlation between N2 fixation rates and i abundance of N2 fixing bacteria with DIP concentration is reported from other estuaries and coastal environments worldwide (Howarth et al., 1988; 7

Marine Pollution Bulletin 151 (2020) 110799

T. Jabir, et al.

Rees et al., 2006; Hayn et al., 2014). These results suggest that the abundance of HD is more dependent on DIP concentration than on DIN concentration in the CE, whereas N2 fixation rates were more influenced by DIN and not by the DIP. The Influence of increased input of nutrients from autochthonous and allochthonous sources lead to a deviation from the Redfield ratio of nitrogen and phosphorus (molar N:P = 16:1 in the inorganic and organic nutrients) (Frigstad et al., 2011; Paulo et al., 2011). N2 fixation was more prominent when the N:P ≤ 16:1 in nutrients. During premonsoon season, average N:P ratio in the CE was slightly above the Red field ratio (18:1), whereas the N2 fixation rate was also higher. During monsoon, due to the heavy anthropogenic load in to the estuary, N:P ratio was increased to the maximum (38:1) and it significantly reduced the N2 fixation rates. N:P ratio was far below the Redfield ratio (5.2:1) during post-monsoon and was associated with enhanced N2 fixation rates. N:P ratio is considered to be one of the key determinants regulating the abundance of N2 fixers (Soto-Pinto et al., 2010, Miller et al., 2005). The negative correlation between N2 fixation rate and DO concentration supports the adverse effect of anthropogenic overloading on N2 fixation rate in the CE. The abundance of HD also showed significant inverse correlation with N:P ratios with its higher abundance during post-monsoon. A detailed study is required to understand the contribution of HD to the N2 fixation rates in the CE. Nonetheless, the higher abundance of HD indicates the possibility of their significant contribution towards the process. Increased nutrient loads in the estuary stimulate primary productivity, which could alter the structure and function of biological communities within the estuary, particularly when producer groups respond differently to changes in nutrient availability (Woodland et al., 2015). Anthropogenic activities alter estuarine biogeochemistry by modifying both its absolute quantities and by altering N:P ratios through nutrients from atmospheric nitrogen deposition, run-off treatment and fertilizer application (Melillo et al., 2003; Weyhenmeyer et al., 2007). This study supports the hypothesis that the N2 fixation rates in the CE depend more on N:P ratio than on individual DIN and DIP concentrations (Bhavya et al., 2016), as evident from the significant negative correlation between the N:P ratio and N2 fixation rate (p ≤ 0.01) (Fig. 4).

Author contribution Thajudeen Jabir, Puthiya Veettil Vipindas, Yousuf Jesmi, Sudheesh Valliyodan designed and performed the experiments, analyzed the data. Mohamed Hatha Abdulla gave technical support and conceptual advice. Prabhakaran Meethal Parambath, Arvind Singh interpreted results. All authors discussed results and implications. Thajudeen Jabir and Vipindas Puthiyaveetil wrote the manuscript. All authors commented on the manuscript at all stages. Declaration of competing interest The authors declare that they have no conflict of interest. References Affourtit, J., Zehr, J.P., Paerl, H.W., 2001. Distribution of nitrogen-fixing microorganisms along the Neuse River Estuary, North Carolina. Microb. Ecol. 41, 114–123. Ahmed, A., Gauns, M., Kurian, S., Bardhan, P., Pratihary, A., Naik, H., Shenoy, D.M., Naqvi, S.W.A., 2017. Nitrogen fixation rates in the eastern Arabian Sea. Estuar. Coast. Shelf Sci. 191, 74–83. Bentzon-Tilia, M., Severin, I., Hansen, L.H., Riemann, L., 2015. Genomics and ecophysiology of heterotrophic nitrogen-fixing bacteria isolated from estuarine surface water. MBio 6, e00929-15. Bentzon-Tilia, M., Severin, I., Hansen, L.H., Riemann, L., 2015. Genomics and ecophysiology of heterotrophic nitrogen-fixing bacteria isolated from estuarine surface water. MBio 6 (4), e00929-15. Bergman, B., Gallon, J.R., Rai, A.N., Stal, L.J., 1997. N2 fixation by non-heterocystus cyanobacteria. FEMS Microbiol. Rev. 19, 139–185. Bertics, V.J., Löscher, C.R., Salonen, I., Dale, A.W., Gier, J., Schmitz, R.A., Treude, T., 2013. Occurrence of benthic microbial nitrogen fixation coupled to sulfate reduction in the seasonally hypoxic Eckernförde Bay, Baltic Sea. Biogeosciences 10, 1243–1258. https://doi.org/10.5194/bg-10-1243-2013. Bhavya, P.S., Kumar, S., Gupta, G.V.M., Sudheesh, V., Sudharma, K.V., Varrier, D.S., Dhanya, K.R., Saravanane, N., 2016. Nitrogen uptake dynamics in a tropical eutrophic estuary (Cochin, India) and adjacent coastal waters. Estuar. Coasts 39, 54–67. Bird, C., Martinez, J.M., O’Donnell, A.G., Wyman, M., 2005. Spatial distribution and transcriptional activity of an uncultured clade of planktonic diazotrophic γProteobacteria in the Arabian Sea. Appl. Environ. Microbiol. 71, 2079–2085. Blomqvist, P., Petterson, A., Hyenstrand, P., 1994. Ammonium-nitrogen: a key regulatory factor causing dominance of non-nitrogen-fixing cyanobacteria in aquatic systems. Arch. für Hydrobiol. 132, 141–164. Boström, K., Simu, K., Hagström, Å., Riemann, L., 2004. Optimization of DNA extraction for quantitative marine bacterioplankton community analysis. Limnol. Oceanogr. Methods 2, 365–373. Černá, B., Rejmánková, E., Snyder, J.M., Šantrůčková, H., 2009. Heterotrophic nitrogen fixation in oligotrophic tropical marshes: changes after phosphorus addition. Hydrobiologia 627, 55–65. Chien, Y.T., Zinder, S.H., 1996. Cloning, functional organization, transcript studies, and phylogenetic analysis of the complete nitrogenase structural genes (nifHDK2) and associated genes in the archaeon Methanosarcina barkeri 227. J. Bacteriol. 178 (1), 143–148. Conley, D.J., Paerl, H.W., Howarth, R.W., Boesch, D.F., Seitzinger, S.P., Karl, E., Karl, E., Lancelot, C., Gene, E., Gene, E., 2009. Controlling eutrophication: nitrogen and phosphorus. Science (80) 123, 1014–1015. Downing, J.A., 1997. Marine nitrogen: phosphorus stoichiometry and the global N:P cycle. Biogeochemistry 37, 237–252. Farnelid, H., Bentzon-Tilia, M., Andersson, A.F., Bertilsson, S., Jost, G., Labrenz, M., Jürgens, K., Riemann, L., 2013. Active nitrogen-fixing heterotrophic bacteria at and below the chemocline of the central Baltic Sea. ISME J 7, 1413–1423. Foster, R.A., Paytan, A., Zehr, J.P., 2009. Seasonality of N2 fixation and nifH gene diversity in the Gulf of Aqaba (Red Sea). Limnol. Oceanogr. 54, 219–233. Frigstad, H., Andersen, T., Hessen, D.O., Naustvoll, L.J., Johnsen, T.M., Bellerby, R.G.J., 2011. Seasonal variation in marine C: N: P stoichiometry: can the composition of seston explain stable Redfield ratios? Biogeosciences 8, 2917–2933. Galloway, J.N., Dentener, F.J., Capone, D.G., Boyer, E.W., Howarth, R.W., Seitzinger, S.P., Asner, G.P., Cleveland, C.C., Green, P.A., Holland, E.A., 2004. Nitrogen cycles: past, present, and future. Biogeochemistry 70, 153–226. Gandhi, N., Singh, A., Prakash, S., Ramesh, R., Raman, M., Sheshshayee, M.S., Shetye, S., 2011. First direct measurements of N2 fixation during a Trichodesmium bloom in the eastern Arabian Sea. Glob. Biogeochem. Cycles 25, GB4014. Good, I.J., 1953. The population frequencies of species and the estimation of population parameters. Biometrika 40, 237–264. Gothwal, R.K., Nigam, V.K., Mohan, M.K., Sasmal, D., Ghosh, P., 2008. Screening of nitrogen fixers from rhizospheric bacterial isolates associated with important desert plants. Appl Ecol Eviron Res 6, 101–109. Grasshoff, K.K.K., Ehrhardt, M., 1999. Methods of Sea Water Analysis, Third, Completely Revised and Extenden edition. WILEY-VCH Verlag GmbH (D). Greenberg, A.E., 1992. Standard Methods for the Examination of Water and Wastewater/ Prepared and Published Jointly by American Public Health Association, American Water Works Association, Water Pollution Control Federation, 18th ed. (Washington, DC, USA). Gupta, G.V.M., Thottathil, S.D., Balachandran, K.K., Madhu, N.V., Madeswaran, P., Nair, S., 2009. CO 2 supersaturation and net heterotrophy in a tropical estuary (Cochin,

5. Conclusion Nutrient stoichiometry played a major role in modulating the nitrogen transformation in the Cochin estuary (CE). Relatively high N2 fixation rates occurred in the CE when the N:P ratio in the estuary was close or lower than the canonical Redfield ratio. Increased N:P ratio suppressed the N2 fixation rates and abundance of heterotrophic diazotrophs in the CE. Major nifH phylotypes Clusters I and III diazotrophs. Cluster I cyanobacterial diazotrophs were dominant in pre-monsoon while Cluster III diazotrophs were prevalent in post-monsoon, which significantly contributed to the N2 fixation rates in the Cochin estuary. These findings have implications in enhancing our understanding of nitrogen cycling in anthropogenically perturbed tropical ecosystems. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.marpolbul.2019.110799. Acknowledgements This work was supported by the Ministry of Earth Sciences (MoES), Government of India, under the Sustained Indian Ocean Biogeochemical and Ecological Research (SIBER) program. The authors are thankful to the Head, Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and Technology (CUSAT), Kochi, for providing necessary facilities for carrying out this work. We are grateful to Prof. Jacob Chacko, Dept. of Chemical Oceanography, CUSAT, for constructive comments and corrections in this manuscript. 8

Marine Pollution Bulletin 151 (2020) 110799

T. Jabir, et al. India): influence of anthropogenic effect. Ecosystems 12 (7), 1145–1157. Harding, K., Turk-Kubo, K.A., Sipler, R.E., Mills, M.M., Bronk, D.A., Zehr, J.P., 2018. Symbiotic unicellular cyanobacteria fix nitrogen in the Arctic Ocean. Proc. Natl. Acad. Sci. 115 (52), 13371–13375. Hardy, R.W., Holsten, R.D., Jackson, E.K., Burns, R.C., 1968. The acetylene-ethylene assay for N2 fixation: laboratory and field evaluation. Plant Physiol. 43, 1185–1207. Hayn, M., Howarth, R., Marino, R., Ganju, N., Berg, P., Foreman, K.H., Giblin, A.E., McGlathery, K., 2014. Exchange of nitrogen and phosphorus between a shallow lagoon and coastal waters. Estuar. Coasts 37, 63–73. Herbst, D.B., 1998. Potential salinity limitations on nitrogen fixation in sediments from Mono Lake, California. Int. J. Salt Lake Res. 7, 261–274. Hoffman, B.M., Lukoyanov, D., Yang, Z.-Y., Dean, D.R., Seefeldt, L.C., 2014. Mechanism of nitrogen fixation by nitrogenase: the next stage. Chem. Rev. 114, 4041–4062. Horne, A.J., Goldman, C.R., 1972. Nitrogen fixation in Clear Lake, California. I. Seasonal variation and the role of heterocysts. Limnol. Oceanogr. 17, 678–692. Howarth, R.W., Marino, R., 2006. Nitrogen as the limiting nutrient for eutrophication in coastal marine ecosystems: evolving views over three decades. Limnol. Oceanogr. 51, 364–376. Howarth, R.W., Marino, R., Cole, J.J., et al., 1988. Nitrogen fixation in freshwater, estuarine, and marine ecosystems. 2. Biogeochemical controls. Limnol. Oceanogr. 33, 688–701. Issa, A.A., Abd-Alla, M.H., Ohyama, T., 2014. In: Ohyama, T. (Ed.), Nitrogen fixing cyanobacteria: future prospect, in: Advances in Biology and Ecology of Nitrogen Fixation. European Union, Croatia, pp. 26 Rejika: In Tech. Thajudeen, J., Yousuf, J., Veetil, V.P., Varghese, S., Singh, A., Mohamed Hatha, A.A., 2017. Nitrogen fixing bacterial diversity in a tropical estuarine sediments. World J. Microb. Biotech. 33 (2), 41. Jabir, T., Jesmi, Y., Vipindas, P.V., Mohamed Hatha, A., 2018. Diversity of nitrogen fixing bacterial communities in the coastal sediments of southeastern Arabian Sea (SEAS). Deep. Res. Part II Top. Stud. Oceanogr. https://doi.org/10.1016/j.dsr2.2018.09.010. Janardanan, V., Amaravayal, S., Revichandran, C., Manoj, N.T., Muraleedharan, K.R., Jacob, B., 2015. Salinity response to seasonal runoff in a complex estuarine system (Cochin estuary, West coast of India). J. Coast. Res. 31, 869–878. Jayakumar, A., Al-Rshaidat, M.M., Ward, B.B., Mulholland, M.R, 2012. Diversity, distribution, and expression of diazotroph nifH genes in oxygen-deficient waters of the Arabian Sea. FEMS Microbiol. Ecol. 82 (3), 597–606. Jayakumar, A., Peng, X., Ward, B.B., 2013. Community composition of bacteria involved in fixed nitrogen loss in the water column of two major oxygen minimum zones in the ocean. Aquat. Microb. Ecol. 70 (3), 245–259. Jayakumar, A., Chang, B.X., Widner, B., Bernhardt, P., Mulholland, M.R., Ward, B.B., 2017. Biological nitrogen fixation in the oxygen-minimum region of the eastern tropical North Pacific ocean. The ISME Journal 11 (10), 2356. Jyothibabu, R., Madhu, N.V., Jayalakshmi, K.V., Balachandran, K.K., Shiyas, C., Martin, G.D., Nair, K.K.C., 2006. Impact of freshwater influx on microzooplankton mediated food web in a tropical estuary (Cochin backwaters – India). Estuar. Coast. Shelf Sci. 69, 505–518. https://doi.org/10.1016/j.ecss.2006.05.013. Jyothibabu, R., Karnan, C., Jagadeesan, L., Arunpandi, N., Pandiarajan, R.S., Muraleedharan, K.R., Balachandran, K.K., 2017. Trichodesmium blooms and warmcore ocean surface features in the Arabian Sea and the Bay of Bengal. Mar. Pollut. Bull. 121 (1-2), 201–215. Kumar, P.K., Singh, A., Ramesh, R., Nallathambi, T., 2017. N2 fixation in the eastern Arabian Sea: probable role of heterotrophic diazotrophs. Front. Mar. Sci. 4, 80. König, S., Le Guyader, H., Gros, O., 2015. Thioautotrophic bacterial endosymbionts are degraded by enzymatic digestion during starvation: Case study of two lucinids C. odakia orbicularis and C. orbiculata. Microsc. Res. Tech. 78 (2), 173–179. Martin, G.D., Nisha, P. a, Balachandran, K.K., Madhu, N.V., Nair, M., Shaiju, P., Joseph, T., Srinivas, K., Gupta, G.V.M., 2011. Eutrophication induced changes in benthic community structure of a flow-restricted tropical estuary (Cochin backwaters), India. Environ. Monit. Assess. 176, 427–438. https://doi.org/10.1007/s10661-010-1594-1. Melillo, J.M., Field, C.B., Moldan, B., 2003. Interactions of the Major Biogeochemical Cycles: Global Change and Human Impacts. Island Press, Washington, DC, USA. Menon, N.N., Balchand, A.N., Menon, N.R., 2000. Hydrobiology of the Cochin backwater system-a review. Hydrobiologia 430, 149–183. Miller, T.E., Burns, J.H., Munguia, P., Walters, E.L., Kneitel, J.M., Richards, P.M., Mouquet, N., Buckley, H.L., 2005. A critical review of twenty years’ use of the resource-ratio theory. Am. Nat. 165, 439–448. Miranda, J., Balachandran, K.K., Ramesh, R., Wafar, M., 2008. Nitrification in Kochi backwaters. Estuar. Coast. Shelf Sci. 78, 291–300. https://doi.org/10.1016/j.ecss. 2007.12.004. Moisander, P.H., Beinart, R.A., Voss, M., Zehr, J.P., 2008. Diversity and abundance of diazotrophic microorganisms in the South China Sea during intermonsoon. ISME J 2, 954. Moisander, P.H., Benavides, M., Bonnet, S., Berman-Frank, I., White, A.E., Riemann, L., 2017. Chasing after non-cyanobacterial nitrogen fixation in marine pelagic environments. Front. Microbiol. 8, 1736. Newell, S.E., Pritchard, K.R., Foster, S.Q., Fulweiler, R.W., 2016. Molecular evidence for sediment nitrogen fixation in a temperate New England estuary. PeerJ 4, e1615. Nixon, S.W., Ammerman, J.W., Atkinson, L.P., Berounsky, V.M., Billen, G., Boicourt, W.C., Boynton, W.R., Church, T.M., Ditoro, D.M., Elmgren, R., 1996. The fate of nitrogen and phosphorus at the land-sea margin of the North Atlantic Ocean. Biogeochemistry 35, 141–180. Paerl, H., 2017. The cyanobacterial nitrogen fixation paradox in natural waters. F1000Research 6, 244. Paulo, J.G., Montes, M. de J.F., Santos, A.C., Batista, T.N.F., Travassos, R.K., do Nascimento Filho, G.A., Feitosa, F.A., Gaspar, F.L., Pitanga, M.E., 2011. Allochthonous and autochthonous organic matter in an urban tropical estuarine area of northeastern Brazil. J. Coast. Res. 1798. Qasim, S.Z., 2003. Indian Estuaries. Allied publication Pvt. Ltd, Heriedia Marg, Ballard

estate, Mumbai. Ranganayaki, S., Mohan, C., 1981. Effect of sodium molybdate on microbial fixation of nitrogen. Z. Allg. Mikrobiol. 21, 607–610. Rao, N.S.S., 1988. Ten decades of research on biological nitrogen fixation. Curr. Sci. 57, 710–713. Rees, A.P., Law, C.S., Woodward, E.M.S., 2006. High rates of nitrogen fixation during an in-situ phosphate release experiment in the eastern Mediterranean Sea. Geophys. Res. Lett. 3333, L10607. Renjith, K.R., Mary, J.M., Kumar, C.S.R., Manju, M.N., Chandramohanakumar, N., 2016. Nutrient distribution and bioavailability in a tropical microtidal estuary, Southwest India. J. Coast. Res. 32, 1445–1455. Shiozaki, T., Ijichi, M., Kodama, T., Takeda, S., Furuya, K., 2014. Heterotrophic bacteria as major nitrogen fixers in the euphotic zone of the Indian Ocean. Glob. Biogeochem. Cycles 28, 1096–1110. Singh, A., Lomas, M.W., Bates, N.R., 2013. Revisiting N2 fixation in the North Atlantic Ocean: significance of deviations from the Redfield Ratio, atmospheric deposition and climate variability. Deep Sea Res Part II Top Stud Oceanogr 93, 148–158. Singh, J.S., Kumar, A., Rai, A.N., Singh, D.P., 2016. Cyanobacteria: a precious bio-resource in agriculture, ecosystem, and environmental sustainability. Front in microbiol 7, 529. https://doi.org/10.3389/fmicb.2016.00529. Sohm, J.A., Webb, E.A., Capone, D.G., 2011. Emerging patterns of marine nitrogen fixation. Nat. Rev. Microbiol. 9, 499–508. Soto-Pinto, L., Anzueto, M., Mendoza, V.J., Jiménez Ferrer, G., de Jong, B., 2010. Carbon sequestration through agroforestry in indigenous communities of Chiapas, Mexico. Agrofor. Syst. 78, 39–51. Srinivas, K., Revichandran, C., Maheswaran, P.A., Asharaf, T.T.M., Murukesh, N., 2003. Propagation of tides in the Cochin estuarine system, southwest coast of India. Indian J. Mar. Sci. 32, 14–24. Stewart, W.D., Fitzgerald, G.P., Burris, R.H., 1968. Acetylene reduction by nitrogen-fixing blue-green algae. Arch. Mikrobiol. 62 (4), 336–348. Suarez, C., Ratering, S., Kramer, I., Schnell, S., 2014. Cellvibrio diazotrophicus sp. nov., a nitrogen-fixing bacteria isolated from the rhizosphere of salt meadow plants and emended description of the genus Cellvibrio. Int. J. Syst. Evol. Microbiol. 64 (2), 481–486. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. Thajudeen, J., Yousuf, J., Veetil, V.P., Varghese, S., Singh, A., Abdulla, M.H., 2017. Nitrogen fixing bacterial diversity in a tropical estuarine sediments. World J. Microbiol. Biotechnol. 33 (2), 41. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882. Thottathil, S.D., Balachandran, K.K., Gupta, G.V.M., Madhu, N.V., Nair, S., 2008. Influence of allochthonous input on autotrophic–heterotrophic switch-over in shallow waters of a tropical estuary (Cochin Estuary), India. Estuar. Coast. Shelf Sci. 78, 551–562. https://doi.org/10.1016/j.ecss.2008.01.018. Turk-Kubo, K.A., Karamchandani, M., Capone, D.G., Zehr, J.P., 2014. The paradox of marine heterotrophic nitrogen fixation: abundances of heterotrophic diazotrophs do not account for nitrogen fixation rates in the E astern T ropical S outh P acific. Environ. Microbiol. 16 (10), 3095–3114. Veettil, V.P., Abdulaziz, A., Chekidhenkuzhiyil, J., Ramkollath, L.K., Hamza, F.K., Kalam, B.K., Ravunnikutty, M.K., Nair, S., 2015. Bacterial domination over archaea in ammonia oxidation in a monsoon-driven tropical estuary. Microb. Ecol. 69, 544–553. Voss, M., Baker, A., Bange, H.W., Conley, D., Deutsch, B., Engel, A., Heiskanen, A.-S., Jickells, T., Lancelot, C., McQuatters-Gollop, A., 2011. Nitrogen Processes in Coastal and Marine Ecosystems. Cambridge University Press, UK. Weyhenmeyer, G.A., Jeppesen, E., Adrian, R., Arvola, L., Blenckner, T., Jankowski, T., Jennings, E., Nỡges, P., Noges, T., Straile, D., 2007. Nitrate-depleted conditions on the increase in shallow northern European lakes. Limnol. Oceanogr. 52, 1346–1353. Wilson, S.T., Böttjer, D., Church, M.J., Karl, D.M., 2012. Comparative assessment of nitrogen fixation methodologies, conducted in the oligotrophic North Pacific Ocean. Appl. Environ. Microbiol. 78, 6516–6523. Woodland, R.J., Thomson, J.R., Mac Nally, R., Reich, P., Evrard, V., Wary, F.Y., Walker, J.P., Cook, P.L.M., 2015. Nitrogen loads explain primary productivity in estuaries at the ecosystem scale. Limnol. Oceanogr. 60, 1751–1762. Shiozaki, T., Fujiwara, A., Ijichi, M., Harada, N., Nishino, S., Nishi, S., Nagata, T., Hamasaki, K., 2018. Diazotroph community structure and the role of nitrogen fixation in the nitrogen cycle in the Chukchi Sea (western Arctic Ocean). Limnol. Oceanogr. 63 (5), 2191–2205. Yousuf, J., Thajudeen, J., Rahiman, M., Krishnankutty, S., P Alikunj, A., Abdulla, A., Mohamed, H., 2017. Nitrogen fixing potential of various heterotrophic Bacillus strains from a tropical estuary and adjacent coastal regions. J. Basic Microbiol. 57, 922–932. Zani, S., Mellon, M.T., Collier, J.L., Zehr, J.P., 2000. Expression of nifH genes in natural microbial assemblages in Lake George, New York, detected by reverse transcriptase PCR. Appl. Environ. Microbiol. 66, 3119–3124. Zehr, J.P., McReynolds, L.A., 1989. Use of degenerate oligonucleotides for amplification of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii. Appl. Environ. Microbiol. 55, 2522–2526. Zehr, J.P., Shilova, I.N., Farnelid, H.M., del Carmen Muñoz-Marín, M., Turk-Kubo, K.A., 2017. Unusual marine unicellular symbiosis with the nitrogen-fixing cyanobacterium UCYN-A. Nat. Microbiol. 2 (1), 16214. Zehr, J.P., Mellon, M.T., Zani, S., 1998. New nitrogen-fixing microorganisms detected in oligotrophic oceans by amplification of nitrogenase (nifH) genes. Appl. Environ. Microbiol. 64, 3444–3450. Zehr, J.P., Jenkins, B.D., Short, S.M., Steward, G.F., 2003. Minireview Nitrogenase gene diversity and microbial community structure: a cross-system comparison. Environ. Microbiol. 5, 539–554.

9