Azotobacter vinelandii and Bacillus licheniformis in Phaseolus vulgaris Growth at 50% NH₄NO₃ With Multi- Wall Carbon Nanotubes to Reduce N₂O Release Volume 60- Issue 3

Juan Luis Ignacio-de la Cruz and Juan Manuel Sánchez-Yáñez*

• Environmental Microbiology Laboratory, Institute of Chemical Biological Research, B-B3, University City, Universidad Michoacana de San Nicolas de Hildalgo, México

Received: January 16, 2024; Published: February 11, 2025

*Corresponding author: Juan Manuel Sánchez-Yáñez, Environmental Microbiology Laboratory, Institute of Chemical Biological Research, B-B3, University City, Universidad Michoacana de San Nicolas de Hildalgo. Francisco J Mujica S/N, Col Felicitas del Rio ZP 58030, Morelia, Michoacán, Mexico, syanez@umich.mx

DOI: 10.26717/BJSTR.2025.60.009466

Abstract PDF

In Phaseolus vulgaris production it is important to regulate the application of NH₄NO₃, by inoculation with Azotobacter vinelandii and Bacillus licheniformis plus multi-wall carbon nanotubes (MWCN), to decrease the release N₂O from NH₄NO₃ a greenhouse gas, according to soil physical and chemical properties under P. vulgaris cultivation. The objective of this work was to analyze the response of P. vulgaris with A. vinelandii/B. licheniformis with NH₄NO₃ at 50% plus MWCN. For this, P. vulgaris seeds were treated with a mixture of A. vinelandii and B. licheniformis and 50% NH₄NO₃ plus 10 or 20 ppm of MWCN, by the response variables: germination percentage, phenology and seedling biomass, the experiment was carried out under a random block: P. vulgaris fed with 100% NH₄NO₃ uninoculated, non-MWCN or relative control; P. vulgaris non NH₄NO₃, uninoculated, irrigated only water or absolute control; the treatments: P. vulgaris with A. vinelandii and B. licheniformis 50% NH₄NO₃ plus 10 or 20 ppm of MWCN, individually and in mixture. The experimental data were analyzed by ANOVA Tukey (P<0.05). The results showed that P. vulgaris with A. vinelandii/B. licheniformis with 50% NH₄NO₃ plus MWCN, germinated better and faster, positive response of P. vulgaris was also observed on the phenology and biomass of P. vulgaris, compared to P. vulgaris plus 100% NH₄NO₃, uninoculated. The results support that the positive effect of A. vinelandii /B. licheniformis, inside the root system of P. vulgaris due to transforming organic compounds, from root metabolisms into phytohormones, to enhance 50% NH₄NO₃ uptake. This root activity of P. vulgaris was improved by combination of A. vinelandii /B. licheniformis by the MWCN, that accelerated and optimized 50% NH₄NO₃ uptake, consequently there was not NH₄NO₃ remaining, in that sense, to reduce greenhouse gas generation compared to 100% NH₄NO₃ uptake in uninoculated P. vulgaris, when non-uptake of NH₄NO₃ caused release of NO3- with subsequent generation of N₂O, reason is why to decrease NH₄NO₃ with A. vinelandii /B. licheniformis plus MWCN are, important to mitigate the impact of greenhouse gases on P. vulgaris production, also to preserve soil fertility and prevent surface and groundwater pollution by overfertilization.

Keywords: Soil; Overfertilization; Loss of Fertility; Beneficial Plant Bacteria; Carbon Nanoparticles; NH₄NO₃ Optimization; Greenhouse Effect Mitigation

Currently, the generation of greenhouse gases in the production of Phaseolus vulgaris causes global warming, due to over nitrogen fertilization with NH₄NO₃,that caused N₂O from NO₃ under common environmental conditions during P. vulgaris cropping [1], reason is why strategies are proposals, that combine the reduction of the NH₄NO₃ dose, without risk of compromising healthy plant growth, as well as to inoculate P. vulgaris seed with Azotobacter vinelandii with B. licheniformis, genera of endophytic plant growth bacteria [2,3], that optimize nitrogen fertilizer to the maximum, through the synthesis of phytohormones, to induce maximum activity of the root system that uptakes NH₄NO₃ action [4,5], that can be improved by applying multi-wall carbon nanotubes or MWCN [6], non-toxic for plants, animals or human beings [7], that accelerate the action of bacterial phytohormones [8], so that NH₄NO₃ is consumed in such a way, that there is no remaining NH₄NO₃ [9], that generates N₂O from NO₃ greenhouse gas that causes global warming [1]. There is evidence that the combination of seed with genus of plant growth promoting bacteria genera, mixing with MWCN can prevent the loss of soil fertility, due to excess NH₄NO₃ non uptake by the root vegetal system [9-15]. Therefore, the objective of this work was to analyze the response of P. vulgaris with A. vinelandii and B. licheniformis, NH₄NO₃ at 50% plus MWCN to reduce N

The genera of plant growth-promoting endophytic bacteria: A. vinelandii and B. licheniformis are part of the bacterial collection of the Environmental Microbiology Laboratory of the Research Institute, and were isolated from the interior of roots of Zea mays var mexicana (teocintle); A. vinelandii was grown in Burk broth while B. licheniformis was grown in nutrient broth to inoculate a dose of 1.0 x 10⁶ CFU/ml/0.5 g of P. vulgaris seeds individually and in a mixture of 0.5 ml of each to have the dose 1.0 x 10⁶/ml/0.5 g of P. vulgaris seed [15]. The seeds of P. vulgaris var canario were obtained from the Secretariat of Livestock and Hydraulic Resources and Environment, Mexican government delegation, Michoacán, México. The MWCN were acquired from Sigma-Adrich; were suspended in saline solution (0.85% NaCl and 0.01% commercial detergent, sterile by autoclave), applied to the P. vulgaris seed at doses of 10 and 20 ppm [16]. The response variables used to evaluate the response of P. vulgaris with 50% NH₄NO₃ with A. vinelandii and/or B. licheniformis, plus carbon nanotubes were: germination percentage, seedling phenology: plant height (PH), root length (RL), fresh and dry aerial and root weight (AFW/RFW/ADW/RDW). The experiment was carried out under a random block design with P. vulgaris fed with 100% NH₄NO₃ uninoculated or relative control, P. vulgaris non NH₄NO₃ irrigated only with uninoculated water or absolute control and the treatments: P. vulgaris with A. vinelandii and B. licheniformis 50% NH₄NO₃ with 10 and 20 ppm of MWCN, individually and in mixture with and without MWCN, P. vulgaris with 50% NH₄NO₃ uninoculated with 10 ppm of MWCN. The experimental data were analyzed by ANOVA Tukey [17]. On Figure1 show scheme of the experimental design to evaluate the response of P. vulgaris with 50% NH₄NO₃ with A. vinelandii and/or B. licheniformis plus MWCN.

Figure 1

Table 1 shows the effect of A. vinelandii and B. licheniformis on the germination of P. vulgaris seeds, with 50% NH₄NO₃ and 10 or 20 ppm of MWCN. A germination percentage of 71.43% was registered in P. vulgaris, irrigated with water or AC alone, a statistically different numerical value compared to 90.47% of P. vulgaris with 100% NH₄NO₃, non- MWCN, in contrast to the value of 83.33% germination in P. vulgaris with A. vinelandii, 50% NH₄NO₃ plus 10 ppm of MWCN: numerical value with statistical difference compared to 76.19% in P. vulgaris with 50% NH₄NO₃, uninoculated plus 10 ppm of MWCN; by germination percentage, that was also statistically different compared to the 90.19 % in P. vulgaris, with A. vinelandii / B. licheniformis and NH₄NO₃ at 50% plus 10 ppm of MWCN, in evident contrast with the germination percentage registered for P. vulgaris and A. vinelandii/B. licheniformis at 50% NH₄NO₃, plus 20 ppm of MWCN, with a value of 66.66%. According to this results, 3 fundamental facts can be inferred. First, that the germination of P. vulgaris with NH₄NO₃ at 50% alone, was equal to germination of P: vulgaris at 100% NH₄NO₃, with the combination of A. vinelandii / B. licheniformis, plus 10 ppm of MWCN [18]. Second to increase germination that P. vulgaris was only possible with A. vinelandii and B. licheniformis plus 50% NH₄NO₃ [19]; because individually neither of the two bacterial genera reached a higher percentage of germination, including at seedling stage (data not shown). Third that although MWCN increase and accelerate the production of phytohormones associated with germination [20], only if the concentration is not greater or less than 10 ppm, because as it was not registered when 20 ppm was applied germination was reduced, due to negative effect both on the seed of P. vuglaris and on the beneficial phytohormonal activity of A. vinelandii / B. licheniformis [21,22]. Table 2 shows the effect of A. vinelandii and B. licheniformis on P. vulgaris with 50% NH₄NO₃ plus 20 ppm MWCN, on the phenology and biomass of P. vulgaris at the seedling level, where a plant height (PH) of 33.08 cm was registered, a statistically different value compared to 28.42 cm HP of P. vulgaris uninoculated, with 100% NH₄NO₃ or RC, a numerical value with statistical difference compared, to 35.67 cm HP of P. vulgaris with A. vinelandii/B. licheniformis at 50% NH₄NO₃, plus 10 ppm MWCN. This value was statistically different compared to 31.83 cm PH of P. vulgaris at 50% NH₄NO₃, A. vinelandii/B. licheniformis non MWCN, compared to the PH of P. vulgaris used as relative control (RC), also with the 32.92 cm of uninoculated P. vulgaris, non NH₄NO₃ or neither MWCN. While the root length (RL) of P. vulgaris was: 18.33 cm with A. vinelandii and B. licheniformis plus 10 ppm of MWCN, a statistically different value compared to the 15 cm RL of P. vulgaris with A. vinelandii / B. licheniformis 50% NH₄NO₃, with 20 ppm of MWCN and the 14. 66 cm RL of P. vulgaris with A. vinelandii / B. licheniformis, 50% NH₄NO₃ non- MWCN, a statistically different value compared to the 16.92 cm RL o0f P. vulgaris non NH₄NO₃, uninoculated, or neither MWCN, used as absolute control (AC).

Table 1: Effect of A. vinelandii/B.licheniformis at 50% NH₄NO₃ plus MWCN+ on the germination of Phaseolus vulgaris var Canario.

Note: *n= 50 **Values with different letters had statistical difference (P<0.05) according to ANOVA-Tukey. +MWCN= multi-wall carbon nanotubes.

Table 2: Effect of Azotobacter vinelandii and Bacillus licheniformis on growth of Phaseolus vulgaris at seedling stage at 50% NH₄NO₃ plus multi-wall carbon nanotubes (MWCN)+.

Note: *n = 6 **Values with different letters had statistical difference (P<0.05) according to ANOVA-Tukey.

In relation to biomass such as fresh and dry aerial and radical weight (AFW/RFW/ADW/RDW), the highest was registered in; P. vulgaris with A. vinelandii /B. licheniformis and NH₄NO₃ at 50% plus 10 ppm of MWCN, were registered 1.59 g of AFW 0.57 g of RFW, 0.17 g of ADW and 0.05 g of RDW values with statistical difference compared to the values of P. vulgaris with NH4NO3 at 100% uninoculated or neither MWCN with: 1.38 g of FAW, 0.52 g of RFW, 0.14 g of ADW and 0.04 g of RDW; these values were statistically different from those registered in P. vulgaris with A. vinelandii / B. licheniformis with NH₄NO₃ at 50% non- MWCN with: 1.39 g of AFW, 0.53 g of RFW, with 0.14 of ADW and 0.04 of RDW, these values were also statistically different to P. vulgaris uninoculated at 50% NH₄NO₃ plus 10 ppm MWCN, 1.34 g AFW, 0.50 g RFW with 0.13 g ADW and 0.03 g RDW. It was evident that all these numerical values were different from those registered in P. vulgaris uninoculated, neither NH₄NO₃, non- MWCN irrigated only with water with 1.21 g AFW, 0.34 g RFW as well as 0.11 ADW and 0.02 RDW.

These results support several obvious facts. First is that the uptake capacity of NH₄NO₃ by P. vulgaris roots for the 100% dose is limited, or excessive, because even 50% of NH₄NO₃ is required for the beneficial phytohormonal activity, in P. vulgaris roots of A. vinelandii in synergic action with B. licheniformis to be optimized (4,5:10,11), and avoid the release of NO₃ with the possibility of generating N₂O (1,2:12,13). Second is that MWCN have a positive effect, on the water and mineral uptake mechanism that initiates germination and favors the uptake of NH₄NO₃ at 50%[14,16,23], according to the concentration of the MWCN a positive effect better with 10 ppm than with 20 ppm MWCN [23-25], that is associated with the possible toxicity in the metabolism of seeds and roots of P. vulgaris [21,22]. Third is that if the seeds are inoculated with A. vinelandii and B. licheniformis it is induced, the growth promotion effect on the germination of P. vuglaris, is accelerated and improved, allowing the optimization of the 50% NH₄NO₃ dose [26-28], to prevent the release of NO₃ into the soil to avoid the generation of N₂O, as well as the loss of organic matter, as well as the contamination of surface water and groundwater [1,2,10].The results registered on the germination and growth of P. vulgaris with 50% NH₄NO₃, support the potential of MWCN, to solve problems associated with the dynamics of NH₄NO₃ in plant roots [29,30], as well as the real possibility that MWCN, can be used as part of an integral strategy for sustainable agriculture with plant growth promoting bacteria [26,27].

It is concluded that the combination of A. vinelandii and B. licheniformis, at dose of 50% NH₄NO₃ in P. vulgaris, can be improved with MWCN, under the premise of finding the minimum concentration sufficient, to optimize the uptake of NH₄NO₃, without risk compromising the healthy growth of the legume, or the environment by preventing the loss of soil fertility, the contamination of surface water and groundwater, as well as the release of N₂O to mitigate global warming due to cultivation of P. vulgaris.

To the Coordinación de Investigación Científica de la UMSNH “Aislamiento y selección de microorganismos endófitos promotores de crecimiento vegetal para la agricultura y biorecuperacion de suelos” from the Research Project 2.7 (2025), Universidad Michoacana de San Nicolas de Hidalgo, Morelia, Michoacán, México.

“Field Test of a Living Biofertilizer for Crop Growth in México” from Harvard University, Cambridge, Ma, USA (2021-2024) with support of Rockefeller fund, and to Phytonutrimentos de México and BIONUTRA S, A de CV, Maravatío, Michoacán, México for the P. vulgaris seeds and verification of greenhouse tests. To Jeaneth Caicedo Rengifo for her help in the development of this research project.

The authors declare no conflicts of interest.

1. Abebe Z, Deressa H (2017) The effect of organic and inorganic fertilizerson the yield of two contrasting soybean varieties and residual nutrient effects on a subsequent finger millet crop. Agronomy 27(2): 42.2
2. Backer R, Rokem JS, Ilangumaran G, Lamont J, Praslickova D, et al. (2018) Plant growth-promoting rhizobacteria: context, mechanisms of action, and roadmap to commercialization of biostimulants for sustainable agriculture. Front Plant Sci 9: 1473.
3. Barrera-Ortíz S, Mezo-Villalobos M, Aranda-Ocampo S, Guevara-García AA, Galindo E, et al. (2020) Bacillus velezensis 83 a bacterial strain from mango phyllosphere, useful for biological control and plant growth promotion. Amb. Express 10: 163.
4. Khan MS, Gao J, Chen X, Zhang M, Yang F, et al. (2020) The endophytic bacteria Bacillus velezensis Lle-9, isolated from Lilium leucanthum, harbors antifungal activity and plant growth-promoting effects. J Microbiol Biotechnol 30: 668-680.
5. Wang C, Zhao D, Qi G, Mao Z, Hu X, et al. (2020) Effects of Bacillus velezensis FKM10 for promoting the growth of Malus hupehensis and inhibiting Fusarium verticillioides. Front Microbiol10: 2889.
6. Vithanage M, Seneviratne M, Ahmad M, Sarkar B, Ok YS (2017) Contrasting effects of engineered carbon nanotubes on plants: a review. Environ Geochem Health 39(6):1421-1439.
7. Chichiriccò G, Poma A (2015) Penetration and Toxicity of Nanomaterials in Higher Plants. Nanomaterials (Basel) 5(2):851-873.
8. Khodakovskaya MV, Kim BS, Kim JN, Alimohammadi M, Dervishi E, et al. (2013) Carbon nanotubes as plant growth regulators: effects on tomato growth, reproductive system, and soil microbial community. Small 9(1):115-123.
9. Sanzari I, Leone A, Ambrosone A (2019) Nanotechnology in Plant Science: To Make a Long Story Short. Front Bioeng Biotechnol 7: 120.
10. Santos SN, Kavamura VN, da Silva JL (2010) Plant growth promoter rhizobacteria in plants inhabiting harsh tropical environments and its role in agricultural improvements. In Plant Growth and Health Promoting Bacteria 4: 251-272.
11. Egamberdieva D, Wirth SJ, Alqarawi AA, Elsayed F Abd_Allah, Abeer Hashem (2017) Phytohormones and beneficial microbes: essential components for plants to balance stress and fitness. Front Microbiol 8: 2104.
12. Vasconcelos MW, Grusak MA, Pinto E (2020) The biology of legumes and their agronomic, economic, and social impact. In The Plant Family Fabaceae, p. 3-25.
13. Huang R, McGrath SP, Hirsch PR, Ian M Clark, Jonathan Storkey, et al. (2019) Plant–microbe networks in soil is weakened by century‐long use of inorganic fertilizers. Microb Biotechnol 12(6):1464-
14. Khodakovskaya M, Dervishi E, Mahmood M, Xu Y, Li Z, et al. (2008) Carbon nanotubes are able to penetrate plant seed coat and dramatically affect seed germination and plant growth. ACS Nano 3(10): 3221-3227.
15. Sánchez-Yáñez JM (2007) Breve Tratado de Microbiología Agrícola, teoría y práctica, Instituto de Investigaciones Químico-Bioló Universidad Michoacana de San Nicolás de Hidalgo. Corporativo de investigación sustentable, SA de CV, Centro de Investigación y Desarrollo del Estado de Michoacán, SEDAGRO. Morelia, Michoacán, México.
16. Zaytseva O, Neumann G (2016) Carbon nanomaterials: production, impact on plant development, agricultural and environmental applications. Chem Biol Technol Agric 3:1-26.
17. Walpole ER, Myers R, Myers LS (2007) Probability & Statistics for Engineering & Sciences. Ed Pearson 13: 978-970-26-0936.
18. Hatami M, Ghorbanpour M, Salehiarjomand H (2014) Nano-anatase TiO2 modulates the germination behavior and seedling vigority of some commercially important medicinal and aromatic plants. Journal of Environment Biological 8(22): 53-59.
19. Safdar M, Kim W, Park S, Gwon Y, Kim YO, et al. (2022) Engineering plants with carbon nanotubes: a sustainable agriculture approach. J Nanobiotechnology 20(1): 275.
20. Vithanage M, Seneviratne M, Ahmad M, Sarkar B, Ok YS (2017) Contrasting effects of engineered carbon nanotubes on plants: a review. Environ Geochem Health. 39(6):1421-1439.
21. Chung H, Son Y, Yoon TK, Kim S, Kim W (2011) The effect of multi-walled carbon nanotubes on soil microbial activity. Ecotoxicol Environ Saf 74(4): 569-575.
22. Chichiriccò G, Poma A (2015) Penetration and Toxicity of Nanomaterials in Higher Plants. Nanomaterials (Basel) 5(2): 851-873.
23. Safdar M, Kim W, Park S, Gwon Y, Kim YO, et al. (2016) Carbon nanotube plant engineering: a sustainable agriculture approach. J Nanobiotechnol 20: 1-30.
24. Rahimi D, Kartoolinejad D, Nourmohammadi K, Naghdi R (2016) Increasing drought resistance of Alnus subcordata CA Mey. Seeds using a nano priming technique with multi-walled carbon nanotubes. J For Sci 62: 269–278.
25. Shojaei TR, Salleh MAM, Tabatabaei M, Mobli H, Aghbashlo M, et al. (2019) Applications of nanotechnology and carbon nano-particles in agriculture, in Synthesis, Technology and Applications of Carbon Nanomaterials. Elsevier, Amsterdam, pp. 247-277.
26. Khodakovskaya MV, Kim BS, Kim JN, Alimohammadi M, Dervishi E, et al. (2013) Carbon nanotubes as plant growth regulators: effects on tomato growth, reproductive system, and soil microbial community. Small 9(1):115-123.
27. Sanzari I, Leone A, Ambrosone A (2019) Nanotechnology in Plant Science: To Make a Long Story Short. Front Bioeng Biotechnol 7: 120.
28. Cañas JE, Long M, Nations S, Vadan R, Dai L, et al. (2008) Effects of functionalized and non functionalized single-walled carbon nanotubes on root elongation of select crop species. Environ Toxicol Chem Int J 27: 1922-1931.
29. Mukherjee A, Majumdar S, Servin AD, Pagano L, Dhankher OP, et al. (2016) Carbon nanomaterials in agriculture: a critical review. Front Plant Sci 7: 172.
30. Lahiani MH, Chen J, Irin F, Puretzky AA, Green MJ, et al. (2015) Interaction of carbon nanohorns with plants: uptake and biological effects. Carbon 81: 607-619.