Synthetic hydrophilic polymer PVA was used to develop nanofibre with optimum size for encapsulating the beneficial microorganism Methylorubrumaminovorans. The results of fibre production revealed that quality fibres were developed with diameter size of 93.3 to 166.1, 164.8 to 218.2 and 194.4 to 303.2 nm at 7, 8 and 9 % respectively without formation of beads (Fig.1a-c). Due to higher viscosity which prohibits the beads formation and droplets than fibres at low concentrations (5 and 6 %) which had poor quality with more of beads because of low viscosity that persuaded the surface tension, which causes the breaking of entangled polymer chain in to fragments that leads to beads formation or beaded nanofibres. During electrospinning, a solution with low viscosity possesses a low viscoelastic force, which is not able to match the electrostatic and columbic repulsion forces that stretch the electrospinning jet. Under the effect of surface tension, the high numbers of free solvent molecules in the solution come together into a spherical shape causing formation of beads. Higher PVA concentration had increased the viscosity, which resulted increase in the chain entanglement that overcome the surface tension and ultimately results in beadles and uniform electrospun nanofibers development (Deitzel et al. 2001). Solution viscosity is major parameter in determining the fiber size and structure (Korycka et al. 2018). At higher viscosity, the developed fibers were free from beads while at lower viscosity the number of beads appeared to be more due to the influence of high surface tension, low charge density of the polymer resulting in formation of droplets or beads (Bhagure and Rao 2020). Higher concentration of polymer, overlapping of the polymer chains favours entanglement, which gives rise to a much stronger interaction and also leads to smooth fibres rather than particles (Yu et al. 2006).
In the microbial cells optimization study, the results of imprinting plating method showed the growth of microbial cells in all the blending proportions of PVA and microbial broth but the significant bacterial cells growth was observed at blending of 5 mL of microbial broth with 5 ml of 14 % polyvinyl alcohol (Fig. 2). Topography of electrospun fibre of microbial (Methylorubrum aminovorans) cells immobilized revealed that the diameter of fibre was increased due to encapsulation of microbial cells. Polyvinyl alcohol (PVA) at 7 % found to produce electrospun fibres with diameter ranges from 93.30 nm to 166.1 nm (Fig. 1a). The diameter of bacteria cells encapsulated electrospun fibre measured from 379.9 nm to 845.5 nm (Fig. 3). The TEM image further confirmed the loading of bacteria cells depicting rod shaped morphology with size ranges from 267.7 nm to 466.0 nm (Fig.4). This finding derive support from the report (Salalha et al. 2006) there in E. coli bacteria had encapsulated in PVA nanofibre where the SEM and TEM images proved the immobilization of microbial cells in polymer matrix. Gensheimer et al. (2011) had encapsulated the bacteria Micrococcus luteus in polylactic acid and polyvinyl alcohol electrospun nanofibre. The SEM morphology of microbial cells immobilized nanofibre confirmed the bacteria cells loading by showing increased size of the fibre. The Rhizobium bacterium was entrapped successfully in PVA polymeric nanofibre and the loading was recognized in SEM morphology (De Gregorio et al 2017). According to Theron et al. (2001) the SEM morphology of polyacrylonitrile nano-fibres was altered due to fortification of eugenol as the average diameter of fibre increased from 127 ± 21 nm to 212 ± 29 nm after loading.
The enumeration of PPFMs (Methylorubrum aminovorans) population immobilized in polyvinyl alcohol electrospun nanofibre exhibited that a total of 2640 single colonies were observed per 0.004 g of nanofibre, and computed value showed that a total of 6.6 x 105 CFU g-1 were observed out of 1 × 108 CFU initially loaded (Fig. 5). De Gregorio et al. (2017) found that a total of 2.25 × 105 CFU of Bradyrhizobium japonicum was enumerated in PVA nanofibre to the total of 1 × 108 CFU added initially in the blend of polymer and microbial cells solutions. In the viability test of Methylorubrum aminovorans cells entrapped in PVA nanofibre stored under normal room temperature, microbial cells viability decreased with advance of storage time. There was a total of 1.85 × 105 CFU g-1, 2.2 × 104 CFU g-1 and 1.2 × 104 CFU g-1 observed on 10, 20 and 30 days after storage, respectively (Fig.6).
Over all, the results indicated that viability of Methylorubrum aminovorans cells could be protected for more than 30 days under ambient environment when they are immobilized in polymeric electrospun nanofibre, and this might be due to polymeric matrix which acts as protective shell against environmental stress and dehydration of microbial cells. The microbes viz., E. coli, Zymomonas and Pseudomonas encapsulated in poly ethylene oxide (PEO) electrospun nanofibre of 100 to 300 nm prolonged the cell viability of microbes and precisely delivered at targeted site (Theron et al. 2001). Similarly, the cell viability of Escherichia coli, Staphylococcus albus and bacteriophage (Salalla et al. 2006), Lactobasillus acidophilus (Nagy et al. 2014), Bradyrhizobium japonicum (Damasceno et al. 2013), L. rhamnosus (Vejan et al. 2016), and Pantoea agglomerans (De Gregorio et al. 2017) immobilized in PVA nanofibre found to be prolonged while stored under ambient environment.
Microbial cells immobilized nanofibre seed invigoration on germination, seedling vigor and plant growth under in vitro conditions
The surface morphology of electrospun nano fibre coated seeds was depicted in figure 7a and 7b. In this study, the seeds inoculated with Methylorubrum aminovorans PVA electrospun nanofibres recorded higher germination (84 %), root (11.4 cm) & shoot length (16.3 cm), seedling vigor (2322) (figure 8) and dry matter production (3.67 g per 10 seedlings) while tested under in vitro conditions. The increase was 10 % in germination (Fig. 9), 15.1 % in root length, 12.4 % in shoot length, 28.8 % in vigor (Fig. 10) and 13.6 % in dry matter production over untreated control. The improved germination and seedling vigour might be attributed to secretion of phytohormones (Auxins, Gibberellins, Cytokinins and IAA) by the Methylobacterium. Moreover, the hydrophilic effect of polyvinyl alcohol that increases rate of water uptake and maintain higher moisture content around the germinating seeds resulting in improved germination and seedling growth. In addition, the nutrient property of PVA triggers the metabolic events that results enhanced germination and seedling growth. Damasceno et al. (2013), they demonstrated the seed coating with rhizobia loaded electrospun polyvinyl alcohol (PVA) nanofibres improving germination and seedling growth in soybean.
The pot culture study expressed that the seeds inoculated with microbial cells immobilized nanofibre registered higher seedling emergence (83 %), seedling root growth (9.60 cm), seedling shoot growth (18.08 cm) and seedling vigor (2294). There was 9.0 % increase in seedling emergence 15.1 % in seedling root growth, 26.6 % increase in seedling shoot growth and 36.3 % increase in seedling vigor noted at the initial growth (Table 2). The higher emergence and potential seedling growth in microbial cells encapsulated nanofibre coated seeds is due to the combined effect of PPFMs and polymer matrix. Methylotrophs excrete phytohormones (IAA & cytokinin) and mobilize the nutrient from the seed on germination that contributed to the enhanced seedling emergence and growth. The hydrophilic nature and nutrient content of PVA also helped in improved seedling growth under potculture. De Gregorio et al (2017) observed that seeds invigorated with rhizobia entrapped polyvinyl alcohol (PVA) nanofibres promoted seedling growth in soybean.
The growth parameters viz., plant height, plant biomass, root volume, nodules number and fresh weight of nodules were observed on 25 and 45 days after sowing. The outcome of this experiment exhibited that the microbial cells encapsulated nanofibre coated seeds have expedited the plant growth as it recorded higher plant height & plant biomass (Table 3), root volume (Table 4 and Fig.11), root nodules number and fresh weight (Table 5; Fig.12a&b) at 25 and 45 days after sowing as compared to the control. The positive impact of bacteria cells loaded nanofibre invigorated seeds are ascribed to contribution of effective microbial colonization in the roots (Fig. 13), which positively promoted the plant growth under potculture. This result undoubtedly proved the potentiality of electrospun nanofibre to encapsulate the beneficial microorganisms for targeted delivery. Damasceno et al (2013) demonstrated the seed coating with rhizobia loaded electrospun polyvinyl alcohol (PVA) nanofibres in improving germination, seedling growth, number of nodules and plant biomass of soybean. Further, seed coating with PVA nanofiber-immobilized rhizobacteria P. agglomerans ISIB55 and B. caribensis ISIB40 contributed to the successful colonization of both bacteria on the plant root resulting in increased germination, seedling length & dry weight of root and leaf number in soybean (De Gregorio et al. 2017).