Characterization of Nitrogen-Fixing Cyanobacterial Consortia Isolated from the Rhizosphere of Carica papaya

Abstract

Nitrogen-fixing cyanobacterial consortia are an alternative to the indiscriminate use of chemical fertilizers that affect the environment, fix atmospheric nitrogen, and can therefore be used as plant growth promoters, synthesize various substances such as auxins, vitamins, and total proteins, and fix atmospheric biofertilizers and soil conditioners. The present study aimed to obtain and develop, by biotechnological means, two consortia of cyanobacteria isolated from the root and rhizosphere of Carica papaya grown in sandy loam soil. The culture was carried out in Blue Green Medium without modified nitrogen (BG11₀), with aeration of 0.32 L min⁻¹, at a light intensity of 48.83 μEm⁻²s⁻¹, and a temperature of 22 ± 2 °C. Two consortia consisting of Nostoc commune, Aphanothece minutissima, Planktothrix sp. (C1), Nostoc commune, Calothrix sp., and Aphanothece minutissima (C2) were isolated and morphologically identified. The effective development of these consortia was verified at the laboratory level by obtaining biomass in dry weight as well as photosynthetic pigments, proteins, carbohydrates, and lipids. Germination parameters were determined in seeds of Cucumis sativus L. var. Market plus treated with the nitrogen-fixing cyanobacteria consortia, obtaining a higher germination percentage (>90%), greater root length (>6 cm), and higher vigour index I (513), II (13.02) for the C2 consortium. This broadens the spectrum of rhizosphere-derived microorganisms with potential as growth biostimulators.

1. Introduction

It is estimated that global agricultural food production would have to double to feed a worryingly growing population [1]. It is, therefore, necessary to look for alternatives to reduce dependence on agrochemicals (chemical fertilizers and pesticides) [2]. It is essential to explore and understand what happens in the mutualistic interactions between plant roots and rhizosphere microbiota [3]. When this potential is fully harnessed in the agricultural scenario, it will be possible to move towards a truly sustainable and safe agriculture [4].

Emerging solutions for improving crop yields, particularly biotechnological ones, were suggested as they can revolutionize agricultural systems and contribute to solving current and future problems [5,6]. The use of such biological and renewable products that stimulate plant growth through different mechanisms is already a well-established reality for the cultivation of a variety of crops [7]. Soil conditioners, organic fertilizers, biofertilizers, and biostimulants are emerging environmentally friendly solutions. Biostimulants have the potential to naturally promote plant growth, increase soil fertility, and enhance soil microbial activity [8].

The search for new species of rhizosphere microorganisms with potential for plant growth stimulation has been a subject that has been approached from various angles, ranging from the diversity of crops and plant species used as a source of microorganism isolation, the agricultural ecosystems explored, as well as the variety of microbial species [9].

Among these microorganisms, there are several species of cyanobacteria, a very diverse group of photoautotrophic prokaryotic organisms belonging to the Bacteria domain that synthesize chlorophyll a and phycobilin and are capable of carrying out some vitally important processes such as oxygenic photosynthesis [10]. Cyanobacteria have a wide ecological distribution, spanning a wide range of habitats, from aquatic to terrestrial systems, and even extreme environments such as arid deserts and hot springs [11,12].

These organisms have been reported to be beneficial for soil fertility and crop production because of their ability to fix atmospheric nitrogen, solubilize phosphate, and produce plant growth regulators [13]. This type of bacteria releases varied amounts of phytohormones (auxins, gibberellins, cytokinin), polypeptides, amino acids, polysaccharides, and siderophores for the growth and development of plants, together with ammonia and small nitrogenous polypeptides during active cell growth, as well as other secondary metabolites after death and decomposition [14]. These types of substances have been described as important factors with stimulating effects on plants [15].

The use of cyanobacteria as biofertilizers for crops is then positioned as an ecologically positive and sustainable alternative since this type of biofertilizer can be used in non-potable water, organic leachates, atmospheric CO₂, and developing crops in small spaces or even on unproductive land, in photobioreactor systems [16,17]. This study aimed to evaluate the effects of two consortia of nitrogen-fixing cyanobacteria isolated from the root and rhizosphere of Carica papaya on the germination of Cucumis sativus L. var. Market more.

2. Materials and Methods

2.1. Culture Growth and Maintenance

Consortia of nitro-fixing cyanobacteria were isolated from the root and rhizosphere of Carica papaya crops from the Santiago de Cuba Biofactory and are kept in stock at the Laboratory of Ecotoxicology and Environmental Services [18] of the National Centre of Applied Electromagnetism (CNEA), Cuba.

Nitro-fixing cyanobacterial colonies were obtained from a subculture in a Petri dish of rhizosphere soil and root of Carica papaya plants, which was performed in triplicate and incubated in BG11₀ medium, a selective medium for cyanobacteria [19] for 30 days at a temperature of 22 ± 2 °C under a continuous light regime at an intensity of 48.83 μEm⁻²s⁻¹ (Daylight, Phillips, Amsterdam, The Netherlands, 40 W), thus obtaining the primary culture. From the initial detection of growth, cyanobacteria were present. Species were morphologically identified and classified whenever possible in all primary cultures obtained, using various dichotomous keys and taxonomic criteria [20,21,22].

From the primary growths, solid subcultures and isolates are developed in BG11₀ liquid medium, using 10 mL screw-capped test tubes and Petri dishes. In this way, a pre-inoculation is obtained for the establishment of natural consortia.

For the development of the cultures, the modified nitrogen-free Blue Green culture medium (BG11₀) was used, which is formulated to promote nitrogen fixation by cyanobacteria [19]. Modifications were made to the original formulation, and it was prepared per stock in concentrations of gL⁻¹ as follows:

Stock I: K₂HPO₄·3H₂O (0.04), Mg SO₄·7 H₂O (0.075), CaCl₂ H₂O (0.036), and C₆H₈O₇ (0.006). Stock II: H₃BO₃ (0.143), and Zn SO₄·7 H₂O (0.0111). Stock III: Algal (0.0186). EDTA (39.80%), Fe (10.20%), Zn (0.53%), Mn (0.44%), Mo (0.56%), Co (0.46%), Cu (0.49%), Thiamin (0.162%), Biotin (0.006%), Cyanocobalamin (0.008%), and Excipients (0.390%). Stock IV: Na₂CO₃ (0.02).

The pH of the BG11₀ medium was adjusted to 7.4 u. To prepare 1 L of BG11₀ medium, 10 mL of stock solutions I and II and 1 mL of stock solutions III and IV were added. The culture medium was then autoclaved at 120 °C for 25 min.

The cultures were grown in triplicate cylindrical glass flasks with a capacity of 1 L, establishing discontinuous cultures from a 100 mL inoculum. The temperature conditions in the culture lab were stable at 20 ± 2 °C. A 40 W fluorescent lamp was used to keep the cultures illuminated under continuous light. The light intensity was adjusted daily with a traceable light meter (TP LM 8000 4 in 1, China, error: ±8 lx), maintaining the cultures with an illumination of 48.83 µE m⁻²s⁻¹. Cultures were aerated by bubbling filtered air through 0.20 µm glass microfiber syringe filters. The device to aerate the cultures was an air pump for aquariums and ponds (SERA air 550 R plus, Germany) with an airflow capacity of 0.32 Lmin⁻¹.

Morphological Identification

Observations of the cyanobacteria were made under the light microscope (Motic B3, CE). Distinctive taxonomic features were identified in relation to shape, the presence of heterocysts, hormogonial arrangement, and size. Subsequently, identification to species level was carried out using different dichotomous keys and taxonomic criteria [20,21,23,24]. The following characteristics were used to identify the cyanobacteria studied: colony morphology, length of vegetative cells, absence or presence of a sheath, length and arrangement of trichomes in colonies, heterocysts, acinetos, and filament shape.

2.2. Growth Kinetics Assessed by Determining Pigment Concentration and Dry Weight of the Cultures

Dry weight determination: dry weight was carried out every other day for 21 days, according to the technique proposed by Tredici et al. [25] and Mishra & Pabbi [26]. For this purpose, 10 mL aliquots of culture were taken and filtered through cellulose membranes of 5 μm pore diameter (Millipore^®, Burlington, MA, USA), previously dried and weighed. An oven (MCW WS200, GRR, Grand Rapids, MI, USA) was used, subjecting the samples to 70 °C for 24 h. Weighing was performed on a digital analytical balance (OHAUS PA114C, Pioneer, Long Beach, CA, USA). Three replicates per sample were performed. Pigment concentration values are adjusted with these results and expressed in gL⁻¹.

On the other hand, the productivity and specific growth rate (μ, d⁻¹) were calculated following Equations (1) and (2).

P(Xt − X₀)/(t − t₀)

(1)

where Xt and X₀ are the biomass concentrations (CDW, g L⁻¹) at times t_t and t₀, respectively, and t₀ and t_t are the first and last days of cell growth. The CDW parameter was calculated by the biomass concentration per dry weight on the first day of cultivation and at 21 days of cultivation.

μ = (ln (Xt) − ln (X₀))/(t_t − t₀)

(2)

The percentage of chlorophyll a and carotenoids in each consortium was evaluated, for which 5 mL of culture were taken, after homogenization with a (WiseMix VM-10 vortex, Seoul, Republic of Korea), performing six replicates per experiment. The samples were centrifuged in a Sigma 3-16KL centrifuge (Taufkirchen, Germany) at 10,000× g for 15 min. The supernatant was then discarded using a Pasteur pipette. From the pellet, extraction was performed by adding 2 mL of 95% methanol (Quality Chemicals, Barcelona, Spain). This procedure was carried out for 24 h in the dark at −4 °C to avoid oxidation of the chlorophylls.

After measuring the optical density, the samples were clarified by centrifugation at 10,000× g for 5 min. Measurements of the supernatant were made in a Thermo Genesys 10S UV-Vis spectrophotometer (Thermo Fisher Scientific, Shanghai, China) at 480 and 665 nm, diluted appropriately, after reading in the spectrophotometer.

The concentration of photosynthetic pigments was obtained from the equations proposed by Marker [27] and Strickland and Parsons [28]:

Chlorophyll a (μgmL⁻¹) = (A665 × 12.7) × VE ÷ VM

(3)

For the determination of the carotenoid pigments, the equation proposed by Strickland and Parsons [28] was used.

Carotenoid (μgmL⁻¹) = (A480 × 4) × VE ÷ VM, proposed by Murialdo et al. [29], where VE is the volume of the extract and VM is the volume of the sample (5 mL).

For the extraction of the phycobiliproteins: phycocyanin (PC), allophycocyanin (AFC), and phycoerythrin (FE), 5 mL of culture were taken, after homogenization, performing six replicates per experiment, according to the previous description. Each sample was centrifuged at 10,000× g for 15 min, using a Sigma 3-16KL centrifuge (Sigma Aldrich, Steinheim, Germany).

The supernatant was discarded, and 4 drops of glycerol (BDH PROLABO^®, Ecuador, South America) were added to the pellet. The extraction was carried out in the dark at −4 °C for 24 h. Cell disruption was induced with the application of the freeze–thaw process of the cyanobacterial biomass, in addition to the osmotic effect with glycerol, for the release of the water-soluble pigments of protein nature, both phycoerythrin and phycocyanin [30], by performing two successive freeze–thaws.

Once the sample was removed from the freezer, 2 mL of distilled water was added and shaken vigorously, homogenizing with the aid of a vortex. Subsequently, the sample was centrifuged at 10,000× g for 15 min. The supernatant was used to measure the optical density at 562, 615, and 652 nm.

The formulae proposed by Bennett and Bogorad were used to calculate the phycobiliprotein concentration [31].

Phycocyanin (mg mL⁻¹) =DO615 − (DO652 × 0.474) ÷ 5.34 × VE ÷ VM

(4)

Allophycocyanin (mg mL⁻¹) = (DO652 − (DO615 × 0.208) ÷ 5.09 × VE ÷ VM

(5)

Phycoerythrin (mg mL⁻¹) = (DO562 − (FC × 2.41) − (AFC × 0.849) ÷ 5.34 × VE ÷ VM (NC:827)

(6)

where VE is the volume of the extract (4 mL) and VM is the volume of the sample (5 mL).

2.3. Biochemical Profile Evaluation

Total protein, total carbohydrates, and total lipids were determined following the protocols described by Moheimani et al. [32]. After 21 days of culture, 10 mL of each sample was taken and vacuum filtered with 0.45 µm microfibre filters. The filters with the samples were then washed with Milli Q water and frozen at −20 °C until further use. Before each analysis, the samples were thawed, and 2 mL of liquid nitrogen was added to facilitate cell disruption.

Protein total determination was performed by the Lowry method, and carbohydrate determination was performed by the phenol-sulphuric colorimetric method [32]. Lipid determination was performed according to the Folch method [32], using methanol and chloroform as extraction solvents [32].

2.4. Determination of Nitrates in Culture Medium

Supernatants were collected from the cultures of the different consortia at days 7, 14, and 21 days after centrifugation at 15,000× g for 10 min at 200 °C with a refrigerated centrifuge (Sigma 3-16KL, Germany). Results were compared between days and with cultures aged more than 30 days. Samples were refrigerated and sent to the Laboratory of the National Analysis and Technical Services Company. Determinations were performed by the selective electrode method [32,33].

2.5. Evaluation of the Biostimulant Potential of Consortia in Seed Germination and Seedling Growth

For the development of these experiments, certified seeds (OSRO/CUB/01/CHA) of Cucumis sativus L. were used. For germination, 100 seeds were previously placed in a solution of formaldehyde (1.6 mL in 100 of distilled water) and agitated with a magnetic stirrer (RETOMED AM.04, Cuba) for 30 min. Subsequently, three successive washes with distilled water were carried out, and they were left in immersion in distilled water for 24 h.

They were then placed in a solution of boric acid in distilled water (0.05 gmL⁻¹) for 24 h under constant stirring in a shaker (WiseShake SHO-2D, Witeg, Germany) and washed with distilled water.

The fresh biomass of the consortium was obtained by sedimentation, after switching off the aeration system of the culture. Subsequently, the liquid supernatant was decanted several times until only the biomass was obtained. A total of 20 mL of each culture was taken 21 days after the aeration had been removed. The sedimented biomass was volumetric in a glass bottle to 100 mL [34].

Sterile Petri dishes were prepared with 9 cm diameter filter paper (HAO HF) with 4 replicates for each treatment. In each plate, 25 seeds of the evaluated species (Cucumis sativus L.) were placed equidistantly and pre-treated by adding 3 mL of each of the consortia. The plates with the seeds were incubated at a temperature of 23 °C and a light intensity of 58.59 µEm⁻²s⁻¹ [35].

The germinated seeds were counted for seven days. Radicle length was measured with a 20 cm ruler. In addition, fresh and dry weights were determined using an analytical balance (OHAUS PA114C, Parsippany, NJ, USA). For dry weight, an oven (MCW WS200, GRR) was used at 70 °C for 24 h, and the corresponding measurements were made.

The germination percentage and vigor index were calculated using the following formulas [36]:

% germination = (No. of seeds germinated /No. of total seeds sown) × 100

(7)

Vigour Index I = Average radicle length + average shoot length × % germination

(8)

Vigor index II = % germination × dry weight (root and stem)

(9)

2.6. Statistical Analysis

Statistical analysis was performed using a completely randomized design with Kolmogorov–Smirnov normality test with a simple ranked analysis of variance (ANOVA), a Student’s t-test, and a Tukey’s test, according to the corresponding experiments and with a probability of 95%. The benefit of cyanobacteria consortia obtained in comparison to the control in the measured variables can be recommended as biostimulants.

3. Results

The cyanobacterial consortia were obtained naturally from subcultures and isolates from the root and rhizosphere of Carica papaya. Figure 1 shows the appearance of the colonies obtained in the primary culture. They are gelatinous in appearance, with an intense green color and irregular edges.

3.1. Cyanobacteria Species in Consortia

The natural consortia identified and isolated from the root and rhizosphere of Carica papaya consisted of Nostoc commune, Aphanothece minutissima, and Planktothrix sp. (C1) (Figure 2A), and consortia (C2) Nostoc commune, Calothrix sp., and Aphanothece minutissima (Figure 2B).

3.2. Biochemical Characterization of Consortia

As shown in

Table 1. Growth parameters: biomass productivity (P), specific growth rate (µ), and pigment concentration of cyanobacterial consortia C1 and C2.

Parameter	C1	C2
Dry weigth (gL−1)	0.13 ± 0.04	0.24 ± 0.04
Specific growth rate µ (d)	0.16 ± 0.15 *	0.06 ± 0.003
Productivity (gL−1d−1)	0.02 ± 0.02	0.01 ± 0.003
Chlorophyll a (µg mL−1)	0.19 ± 0.07 *	0.16 ± 0.002
Carotenoids (µg mL−1)	0.07 ± 0.03	0.10 ± 0.02
Phycocyanin (mg mL−1)	5.50 ± 1.11 *	4.50 ± 0.22
Allophycocyanin (mg mL−1)	7.00 ± 1.8	6.50 ± 0.22
Phycoerythrin (mg mL−1)	9.50 ± 2.01 *	8.00 ± 0.45

Legend: asterisks (*) indicate significant statistical differences (p < 0.05).

, the parameters evaluated for growth at 21 days of culture showed a statistically significant difference (p < 0.05) in the growth rate of the C1 consortium compared to the C2 consortium.

That is to say, with only the addition of the BG11₀ culture medium, devoid of nitrogen sources, an increase in biomass is obtained, reaching a dry weight at the end of the test of 0.13 ± 0.04 gL⁻¹ for the C1 consortium and 0.24 ± 0.04 gL⁻¹ for the C2 consortium. Obtaining a productivity of 0.02 ± 0.02, and 0.01 ± 0.003 gL⁻¹d⁻¹, respectively. There was no statistically significant difference in biomass concentration or productivity.

On the other hand, for the growth rate, there was a statistically significant difference (p < 0.05) for the C1 consortium compared to the C2 consortium.

For the concentration of photosynthetic pigment chlorophyll a, there was a statistically significant difference (p < 0.05) in the C1 consortium with respect to the C2 consortium; however, for the concentrations of carotenoids, there was no statistically significant difference between the two consortia, although the C1 consortium presented a higher value than the C2 consortium. For phycocyanin and phycoerythrin, there was a statistically significant difference (p < 0.05) in the concentration of both pigments in the C1 consortium compared to the C2 consortium. However, there was no statistically significant difference in the concentration of allophycocyanin.

3.3. Protein, Carbohydrate, and Lipid Concentration

Figure 3 shows the total protein, total carbohydrate, and total lipid concentration in the biomass of the C1 and C2 consortia at 21 days of culture. A statistically significant increase was obtained for protein concentration and carbohydrate (p < 0.05) between the C2 consortium and the C1 consortium. This was not the case for lipid concentration, which showed a statistically significant decrease (p < 0.05) in the C1 consortium compared to the C2 consortium.

Regarding the biochemical composition, it was observed that the concentration of total protein and total carbohydrate of the C2 consortium was higher, with a concentration of (31.2 ± 5.6%) and (47.22 ± 5.03%), respectively, and for the C1 consortium of (13.76 ± 1.92% and (38.36 ± 2.46%), respectively. However, the total lipid concentration was higher for the C1 consortium at (37.34 ± 7.17%) and for the C2 consortium at (13.92 ± 2.09%).

Figure 4 shows the nitrate concentration in the medium of both consortia determined at 7, 14, and 21 days of culture. There was a statistically significant difference (p < 0.05) in the nitrate concentration on days 14 and 21 in the C1 consortium compared to the C2 consortium.

The concentration of nitrate in the culture medium for the C1 consortium increased steadily throughout the experiment, and by the end of the experiment, after 21 days, the highest concentration was obtained. This was not the case for the C2 consortium, where the concentration decreased throughout the day. The quantified assimilable forms of nitrogen are those produced by N₂ fixation are NH⁴⁺ (which are assimilated, producing amino acids) and not NO³⁻.

3.4. Evaluation of the Biostimulant Potential of Consortia on Germination

Table 2. Effect of C1 and C2 consortia on Cucumis sativus L. seed germination.

Treatment	Germination (%)	Dry Weight (g)	Shoot Length (cm)			Vigor Index
			Coleoptile	Radicle	Total	I	II
Control	85.00 ± 0.63 b	0.10 ± 0.01	0.49 ± 0.18 b	2.05 ± 0.93 b	2.54 ± 1.09 b	181.63 ± 1.09 c	6.92 ± 0.09 c
Consortium C1	86.00 ± 0.65 b	0.16 ± 0.01	0.57 ± 0.20 b	5.50 ± 2.66 a	6.07 ± 2.84 a	435.47 ± 2.84 b	11.69 ± 0.01 b
Consortium C2	90.00 ± 1.44 a	0.18± 0.01	0.83 ± 0.12 a	6.11 ± 0.52 a	6.94 ± 0.57 a	513.19 ± 0.57 a	13.02 ± 0.04 a

Different letters indicate a statistically significant difference (p < 0.05).

shows the mean values of the germination parameters of Cucumis sativus L. seeds with the application of solutions of the C1 and C2 consortium cultures. These showed statistically significant differences (p < 0.05) in the root length variable, with the highest values in the C1 and C2 consortiums compared to the control.

The variable vigor indexes I and II were higher for the C2 consortium, with statistically significant differences (p < 0.05) compared to the C1 consortium and the control.

The seed vigor index indicates that the seeds can germinate according to the properties of the seed type, which determines the activity and performance of plant growth.

These results corroborate that the C2 consortium presents two species of nitro-fixing cyanobacteria, which allow a better response to germination and seedling growth, which is related to its biochemical profile, specifically its high content of proteins and carbohydrates.

The germination percentage showed statistically significant differences (p < 0.05) in consortium C2 with respect to C1 and control. Furthermore, for dry weight, no statistically significant differences were obtained (p < 0.05); however, from the biological point of view, the highest values were obtained in the C1 and C2 consortia compared to the control group.

4. Discussion

Cyanobacteria and microalgae are indispensable components of the soil microbiota and live in symbiosis with multiple microorganisms in the rhizosphere environment, indicating that this association may be important for their existence [37]. Each species shows a particular capacity to produce metabolites involved in fertilization, phytostimulation, and photoprotection strategies, so the synergy between different species can lead to better results when microbial consortia are applied, when compared to the application of axenic crops.

The cyanobacteria that were isolated and identified taxonomically and could be cultured under laboratory conditions were Nostoc commune, Aphanothece minutissima, and Planktothrix sp., which form the first consortium (C1), the first two being nitrofixing cyanobacteria, with heterocysts and without heterocysts. The second consortium (C2) is composed of the genera Nostoc commune, Calothrix sp., and Aphanothece minutissima, all nitrofixing, the last one being without heterocyst.

The results obtained in the evaluation of the growth of the consortium for dry weight confirmed the diazotrophic growth capacity of the cyanobacteria [38].

This difference in growth rate is due to the nitrogen fixation process of the cyanobacteria, because they must first fix nitrogen and then use it for their metabolic activities [39]. With the nitrogen provided by aeration supplied through aeration equipment for culture agitation and as a carbon source, such growth was obtained. In addition, the BG11₀ medium is selective exclusively for cyanobacteria with nitrogen-fixing capacity, not allowing the growth of other microorganisms dependent on other sources of nitrogen.

When comparing growth as a function of culture age, it is observed that growth increases significantly over time for the C2 consortium due to the tendency of the cells to increase in size and accumulate metabolites in the stationary phase, which is generally accompanied by a decrease in growth rate [40].

According to Betancourt Fernández [41], under conditions of nitrogen deficiency, the synthesis of chlorophyll a decreases and cell content begins to decline, while carotenoid pigments can continue to be synthesized for longer and then decline at a much slower rate than chlorophyll. Thus, an elevated carotenoid/chlorophyll ratio in algal cultures reflects a nutrient deficiency, which may be correlated with a reduction in the photosynthetic capacity of the cells.

Phycobiliproteins have multiple biological activities against human and animal pathogens, such as bacteria, fungi, and viruses [42,43], which makes them attractive tools for the development of new biological products in different fields. Both consortia obtained higher concentrations of phycobiliproteins than those obtained by Reghini et al. [44], in which different concentrations (0.6–4.8 mg mL⁻¹) of phycobiliproteins from Anabaena minutissima were applied to tomato seeds to study their effect against the soil pathogen Rhizoctonia solani and to promote plant growth. PBPs increased seedling emergence and vigor, showed activity against root rot (67%), and improved plant dry weight, length, and height [44].

On the other hand, protein, carbohydrate, and lipid concentrations were similar to concentrations reported in other research [45,46], in which it is explained, that according to genus, protein percentages of 17.62% to 23.94% are presented.

Likewise, similar lipid values have been reported in previous studies [47,48]. Lipids are essential chemical compounds in cyanobacteria that can be used as a source of animal feed and biodiesel [49,50,51]. According to Sinensky [52], the ability to modify the type and amount of cellular lipids is one of the reasons that may explain why cyanobacteria are used as a source of food, feed, and biodiesel [49,50,51]. The fact that cyanobacteria manage to survive in diverse and extreme conditions (e.g., extremophilic species in the Antarctic and hot springs).

Proteins and carbohydrates are related to dry weight, with proteins representing up to 50% of dry weight and carbohydrates reaching up to 40% in the consortium species. Concentrations similar to those obtained in this study are reported by other authors [46]; these authors refer to the fact that, according to the gender present in the consortium, percentages of 30 to 50% of proteins may be present.

The proximal composition of these metabolites in cyanobacteria is mainly composed of a protein > carbohydrate > lipid ratio (% of dry weight). Their proportions will depend on the characteristics of each species and its culture or growth conditions [53].

Cyanobacteria are an excellent source of proteins, either as a food supplement or as an input to increase the concentration of this nutrient in food. They can be applied as biostimulants or biofertilizers, animal feed, or to produce human nutrient-enriched food [50].

Many cyanobacteria are known to contain high amounts of carbohydrates in the form of intracellular monosaccharides, polymeric reserve α-glucans, and structurally complex extracellular polysaccharides. The potential use of cyanobacterial polysaccharides as foliar biostimulants was recently reported [54,55]. The polysaccharides extracted or released from cyanobacteria (A. platensis) and microalgae (Dunaliella salina and Porphyridium sp.) showed growth-promoting effects in tomato and pepper [54,55].

This may be related to the species composition, since in the first consortium there are two nitro-fixing cyanobacteria, one without heterocyst and the other with heterocyst, as well as Planktothrix sp., which grows through symbiosis, in which it uses the nitrogen fixed by the other two cyanobacteria.

In cyanobacteria, heterocyst development is triggered in response to the external signal of depletion or lack of a combined nitrogen source in the medium [56], which is important to note. While it is strongly inhibited in the presence of ammonium, during this stage it starts the specific degradation of some proteins. This leads to the loss of several functions, such as photosystem II activity, as well as the suppression and destruction of parts of the CO₂ fixation machinery [57].

Although the formation of the proheterocyst involves profound cellular changes, it is a reversible process: after the addition of combined nitrogen (nitrate or ammonium) to the medium, the proheterocyst reverts to the vegetative cell [57].

Roots are one of the most important organs of the plant because it is through them that the different nutrients and minerals are absorbed and participate in the metabolic processes of the plant [58]. These results are in agreement with those found by Jose et al. [59], in whose work a Nostoc strain was able to increase the root/shoot ratio in cucumber seedlings by 20% compared to control plants.

Seed vigor is a very important parameter since it allows the identification of differences between germination and emergence in the field, especially when field conditions can cause stress. The practical usefulness of seed vigor tests includes their use in genetic improvement programs for the development of cultivars with better seed performance [38], or as in the case of this research, in which the biostimulant properties of a homogenate obtained from a consortium of cyanobacteria isolated from the natural environment are evaluated. It also has applications in the study of aspects of seed production, harvesting, conditioning, and storage procedures [60].

Seed vigor is the biological potential that favors rapid and uniform establishment under even unfavorable field conditions [61]. Seed shows the greatest vigor and germination potential when it reaches physiological maturity, as long as no factors are limiting its viability. Biostimulants can contribute to germination due to the presence of metabolites such as proteins and carbohydrates, which normally increase during maturity, conferring greater vigor and even making seeds more viable.

All the parameters evaluated in both the C1 and C2 consortia had a positive influence on the germination process, growth, and development of the crop. Similar results were obtained in Zea mays (maize) [62] and in other plant species [63].

The germination process consists of three phases: (i) imbibition of water; (ii) activation of metabolism, synthesis of proteins and carbohydrates, and degradation of reserves; (iii) development of the embryo and rupture of the testes, through which the emergence of the radicle and later the plumule or stem is observed [64]. Cyanobacterial consortia act mainly on the second phase, due to their macromolecular composition and nitrogen fixation, which is a process with other benefits for the seedlings.

The C1 and C2 consortia obtained have a positive influence on the germination of Cucumis sativus seeds. The presence of these cyanobacteria could be positive under cultivation conditions because they are involved in establishing symbiotic or near-rhizosphere relationships, improving the overall soil environment through CO₂ sequestration, nitrogen fixation, pollutant removal by bioabsorption, and nutrient turnover [65]. They could also contribute to the growth and development of the species through the direct or indirect transfer of nitrogen, which improves soil quality and nutrient status by solubilizing plant-available phosphates. The production of hormones and vitamins that promote plant growth should also be considered.

Rhizosphere cyanobacteria occupy an important place in the soil microbiota, forming symbiotic associations with plants due to their ability to carry out biological fixation of molecular nitrogen as well as producing biostimulant compounds. The main studies on nitro-fixing cyanobacteria with biofertilizing properties have been carried out on species of agri-food interest such as rice, maize, and wheat, among others. In general, fruit crops have been little or almost unexplored; hence, the research is a novel study with practical value, obtaining quality biostimulant consortia from the rhizosphere of C. papaya.

5. Conclusions

The isolation and subsequent development of dense cultures yielded two natural consortia, C1 (Nostoc commune, Aphanothece minutissima, Planktothrix sp.) and C2 (Nostoc commune, Calothrix sp., Aphanothece minutissima), with the presence of nitro-fixing cyanobacteria. The biochemical profile of the consortia differs, highlighting the higher presence of proteins and carbohydrates in C2 and lipids in C1; also, C1 allows higher extracellular nitrate concentrations than C2 due to the presence of nitrofixing cyanobacteria with different strategies for nitrofixation. The biostimulant potential of both consortia in the germination of C. sativus seeds is confirmed, which would support their agricultural application length and vigor index.