Introduction

Cyanobacteria are photosynthetic prokaryotes with a range of morphological diversity, i.e., unicellular, colonial, filamentous unbranched, and with branching [1]. Three types of cells, i.e., vegetative, heterocyst, and spores are present in filamentous cyanobacteria. Heterocyst and spore perform the special function of nitrogen fixation and perennation, respectively [2,3,4]. Cyanobacteria have a worldwide distribution, including extreme habitats. Cyanobacteria play an important role in maintaining the structure and fertility of the soil by increasing nitrogen content, carbon content, phosphorus solubilization, and through various secretions [1, 5]. Cyanobacteria induce plant growth commonly by secretion of growth hormone, siderophore production, phosphate solubilization, and release of fixed nitrogen. These properties of cyanobacteria make them eco-friendly biofertilizers. The heterocyst is the main site for nitrogen fixation [3]. Most nitrogen fixed by cyanobacteria is not available for immediate use to plants. Nitrogen fixed by cyanobacteria is only available to plants on autolysis and mineralization of dead cyanobacteria [6]. A very small amount of fixed nitrogen is released by most nitrogen-fixing cyanobacteria during the growth period. The rate of release of fixed nitrogen was increased tremendously in cyanobacteria treated with glutamine synthetase inhibitor, i.e., L-methionine-DL-sulfoximine [7]. Aulosira fertilissima, Nostoc muscorum, Anabaena variabilis, and Tolypothrix Tenuis are being used in algal biofertilizer technology [8]. Indigenous cyanobacterial strains are suitable for algalisation [9]. A list of indigenous cyanobacteria with the potential to excrete ammonia is needed. Hence, an attempt was made in the present investigation to study on isolation, characterization, and ammonia excretion potential of Nostoc sp. NDUPC007.

Material and Methods

Cyanobacterium was isolated as described by Mishra et al. [10] and purified by repeated streaking method [11]. It was grown in BG-11 medium in a culture room temperature maintained at 28° ± 2 °C and illuminated by the fluorescent lamp (12 wm–2) with a 14:10 light and dark cycle.

Identification was done based on morphological as well as molecular parameters as followed by Mishra et al. [10]. The morphological parameters of strain, i.e., nature of filament, shape, and size of the vegetative cell, heterocyst, and spore were studied at 400 × and 1000 × using an Olympus 21Xi microscope. Dimensions of morphological parameters were measured with the help of Magnus PRO Micromeasurement & Image analysis software. The strain was assigned to cyanobacterial species following taxonomic descriptions provided in the literature [12, 13]. Molecular identification was done based on 16 s rRNA gene sequence analysis. Protocol devised by Singh et al. [14] was followed to isolate genomic DNA. For_5/-GAGTT(CT)GATCCTGGCTCAGGA-3/ and Rev_5/-TCCAGCCGCACCTTCCAGTA-3/ primers were used to amplify the 16S rRNA gene [14]. Sequencing of the purified PCR product was done at IIVR, Varanasi, India by the automated capillary sequencer (ABI 3130 Genetic Analyser, Applied Biosystems, Foster City, CA, USA). Partial 1371 base pair was obtained. NCBI-BLASTn program was used to search for similarity of sequence with other cyanobacterial strains.

Growth was monitored by measuring chlorophyll-a content at 24 h intervals up to 20 days. Total chlorophyll was measured by the method of Myers and Kratz [15]. Extracellular ammonia release was measured by the Phenol hypochlorite method [16]. Added 2 ml of phenol solution, 2 ml of sodium nitroprusside solution, and 5 ml of the oxidizing reagent to 50 ml of the cyanobacterial extract with thorough mixing after each addition. The blue color developed after one hour at room temperature. Optical density was noted at 640 nm and the amount of ammonia was calculated through the standard graph of ammonia. Glutamine synthetase activity was measured by the method of Shapiro and Stadtman [17] and expressed as mM γ- glutamyl hydroxamate formed min−1 mg chl−1.

Heterocyst frequency was calculated by counting the number of heterocysts per 100 vegetative cells of strains with 100 replicates.

$$ {\text{Heterocyst frequency }}\left( \% \right) = \frac{{{\text{No}}.{\text{ of heterocyst}}}}{{{\text{Total no}}.{\text{ of vegetative cells}}}} \times 100 $$

Two sets of autoclaved Petri plates were partially filled with autoclaved sand, and both were moistened with 25 ml of autoclaved soil extract. A total of 10 seeds (sterilized) of rice (Kaveri variety) were placed in each set and placed in the incubator for 36 h at 30 °C. One set was algalised with 1 ml of cyanobacterial culture and both sets were placed in a growth chamber maintained at 30 °C, illuminated with 14:10 light and dark duration. The growth of rice seedlings was measured after 10 days of growth.

Results and Discussion

Cyanobacterium is filamentous, unbranched and heterocystous (Fig. 1). Trichome is blue-green to violet. Cells are cylindrical and barrel-shaped, constricted at the cross-wall with 4.16–6.58 µm length and 3.45–4.44 µm breadth (Fig. 1). Apical cells are rounded. Heterocysts are apical as well as intercalary, spherical, subspherical, and barrel-shaped with 3.42–10.39 µm length and 4.31–6.53 µm breadth (Fig. 1). There is a frequent occurrence of multiple contiguous heterocysts (Fig. 1). Spore formation always starts away from the heterocyst. Spores are formed in the chain and arranged in a zig-zag manner. Spores are elliptical, smooth hyaline with 7.91–12.15 µm length and 4.11–5.93 µm breadth (Fig. 1). Morphological features closely matched with cyanobacterial genera Nostoc [12]. The presence of knot-like structures and cell dimensions do not match with known species of Nostoc [12], hence this strain may be a new one but further characterization is needed. Hence, this cyanobacterium is being identified as Nostoc sp. with strain as NDUPC007. A genus of strain was further confirmed by molecular method. 16S rRNA of strain was sequenced. Partial 1371 base pair was obtained and deposited to NCBI with accession no. KM281209.1. BLASTin search of the sequence showed a maximum 96% similarity with different strains of Nostoc. The deposition of strain at NBAIM, Mau, India is under process.

Fig. 1
figure 1

Photomicrograph of Nostoc sp. NDUPC007 (Scale bar = 5.1 µm). A, filament with apical cell. B, Filament with apical heterocyst. C, Filament with intercalary heterocyst. D, Filament with apical heterocyst and knot-like structure. E, Filament with apical multiple contiguous heterocysts. F, Filament with intercalary multiple contiguous heterocysts. G, Spores in the chain

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The growth of cyanobacterium was measured by monitoring total chlorophyll-a content at 24 h intervals for 20 days. Cyanobacterium reached a stationary phase after 13 days of growth (Fig. 2). Total chlorophyll-a was 17.6 µg/ mg dry weight in the stationary phase of growth. The heterocyst frequency of both single and contiguous multiple heterocysts was 2.14%. The number of heterocysts in multiple contiguous heterocyst chains ranged from two to seven (Fig. 1). Ammonia secretion in culture media was monitored from 6 h of culture to 20 days. Ammonia content in culture media increased up to the 9th day of growth and then remained approximately constant (2.34–2.43 µg/ ml) up to the monitored period (Fig. 3). Glutamine synthetase activity increased up to the 5th day of growth (maximum rate of 5.36 mM γ-glutamyl hydroxamate/ mg chl/ min). The rate of glutamine synthetase became approximately constant (3.21 mM γ-glutamyl hydroxamate/ mg chl/ min) after the 9th day of growth (Fig. 4).

Fig. 2
figure 2

Growth behavior of Nostoc sp. NDUPC007. Values are mean of triplicate ± S.D., bars indicate standard deviation

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Fig. 3
figure 3

Ammonia excretion by Nostoc sp. NDUPC007. Values are mean of triplicate ± S.D., bars indicate standard deviation

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Fig. 4
figure 4

Glutamine synthetase activity of Nostoc sp. NDUPC007. Values are mean of triplicate ± S.D., bars indicate standard deviation

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Cyanobacteria are known to promote plant growth through the secretion of growth regulators, fixed nitrogen and phosphate solubilization, etc. Algalisation of rice fields with cyanobacteria increased the nitrogen content by up to 14% [18, 19]. Kaushik, [8] reported 15–53 kg Nitrogen (N)/hectare (h)/year(y) fixation by cyanobacteria in our country. N2-fixing cyanobacteria are the main contributors to the nitrogen economy of paddy fields [20]. Most of the fixed nitrogen of cyanobacteria is made available to plants by autolysis and mineralization after the death of cyanobacteria [6]. Nitrogen fixed by free-living cyanobacteria is released in insignificant quantities during the growth period. Treatment of cyanobacteria with mutagen L-methionine-DL-sulfoximine, a highly specific, irreversible inhibitor of glutamine synthetase, increased the rate of ammonia release several times [7]. Heterocyst is the main site of nitrogen fixation in cyanobacteria [3] though some non-heterocystous form, i.e., Trichodesmium sp. [21], Plectonema boryanum [22], Gleoecapsa [23] also fix nitrogen. Nitrogen fixed in heterocysts is assimilated to amino acid glutamine by GS-GOGAT pathway [24]. Glutamine is transported to surrounding vegetative cells [25]. Excess ammonia not assimilated by GS-GOGAT pathway is released by heterocyst by simple diffusion so the rate of ammonia assimilation is the key determinant of ammonia release. Low GS activity has been observed in some ammonia secreting cyanobacterial mutants [26, 27]. Nostoc sp. NDUPC007 is continuously secreting ammonia in culture medium as well as in Petri plates used for bioassay. The frequent presence of multiple contiguous heterocysts, appears to be responsible for surplus nitrogen fixation. GS activity of strain is not enough at all stages of growth to fully assimilate the nitrogen fixed by cyanobacteria and consequently the continuous release of ammonia. Heterocyst differentiation and pattern formation are regulated by the HetR gene [28]. PatS gene also plays role in heterocyst pattern formation [29]. Reported causes for multiple contiguous heterocysts are inactivity of PatS gene [29], multiple copies of HetR genes, inactivation of calcium sequestering proteins [30], overexpression of HetF gene [30], etc. The multiple contiguous heterocysts of this cyanobacterium might be due to these reasons. Ammonia release and pattern of heterocyst were monitored for five years.

An increase in plant growth was observed in a set algalised with cyanobacterium (Fig. 5). Length of radical and plumule was 2.6–3.1 cm and 14.3–17.1 cm, respectively in algalised plant, whereas it was 1.9–2.3 cm and 8.9–9.4 cm in non-algalised plants (Fig. 5). 2.13 µg/ ml ammonia was noted in the algalised set and no ammonia was observed in non-algalised even 10 days after algalisation. Ammonia is a well-known utilizable form of nitrogen and is necessary for the growth of plants. Ammonia in the Petri plate is inducing the growth of rice seedlings (Fig. 5). Ammonia secreting strains of cyanobacteria are being searched as suitable inocula for algalisation of rice fields. This cyanobacterium has shown the capacity to continuously release ammonia and is indigenous to rice fields of Varanasi, India. Hence, Nostoc sp. NDUPC007 may prove as suitable inocula for algalisation of rice fields of Varanasi, U.P., India.

Fig. 5
figure 5

Effect of algalisation on rice seedling growth. A, Non- algalised seedling. B, Algalised seedling

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Conclusion

Cyanobacterium Nostoc sp. NDUPC007 was isolated from the agricultural field of Varanasi, India. It was characterized based on morphological as well as molecular parameters. The frequent occurrence of contiguous multiple heterocysts was noted. The number of heterocysts in multiple contiguous heterocyst chains ranged from two to seven. It has shown the potential for continuous excretion of ammonia in the culture medium. Algalisation by this cyanobacterium induced the growth of rice seedlings. Indigenous cyanobacterium with biofertilizer potentials are sought for algalisation. Hence, Nostoc sp. NDUPC007 might prove as potential inocula for algalisation of rice fields of Varanasi, India.