Immobilization of beneficial microbe Methylobacterium aminovorans in electrospun nanofibre as potential seed coatings for improving germination and growth of groundnut Arachis hypogaea

Seed inoculation with microbial cells is one of the potential invigouration techniques for enhancing the emergence and growth of plants. Herein, we approached a new localized delivery of beneficial microbial cells (Methylobacterium) by invigorating seeds with electrospun Polyvinyl alcohol (PVA) nanofibre containing microbial cells. Methylobacterium is a growth promoting bacteria that has recently drawn attention in agriculture, particularly for drought management. PVA was used in this research because of its electrospinnability and biodegradability. Encapsulation study shows effective immobilization of bacteria cells (Methylorubrum aminovorans) in PVA nanofibre. SEM and TEM characterization further confirmed the entrapment of microbial cells. The microbial plating enumeration reveals 6.6 × 105 CFU g−1 of nanofibre to the initial loading population of 1 × 108 CFU. Viability of nanofibre encapsulated bacterial cells under ambient environment found 1.85 × 105 CFU g−1, 2.2 × 104 CFU g−1 and 1.2 × 104 CFU g−1 on 10, 20 and 30 days after storage, respectively. In vitro bio-efficacy study exhibits that the seeds coated by PVA nanofibres containing M. aminovorans recorded higher germination, root & shoot length, seedling vigor, drymatter production, plant biomass, plant root volume, nodule numbers and fresh weight of nodules. The study concludes that microbial cells could be immobilized in electrospun nanofibre for extended shelf-life of microbial cells and as an effective seed coating for localized delivery.


Introduction
Plant growth promoting beneficial soil bacteria that cause a positive effect on plants through direct and indirect mechanisms. The beneficial effects of bacteria are increased plant nutrient uptake, nitrogen fixation and siderophore production. Inoculants applied to seeds with plant growth promoting microbes, is a viable tool to deliver beneficial microorganisms to the soil where they may colonize emerging plant roots and improve plant development. Nanofibers technology is currently practiced for seed treatment with chemical/biological substances/ microbes encapsulation that improves seedling establishment and promotes plant growth. Electrospinning is an advanced technology wherein liquid polymer is converted as fibre ranges from a few nano to micro-meter in diameter with the high surface area exposed to high voltage current (Li and Xia 2004;Bhardwaj and Kundu 2010;Sullivan et al. 2014). The high surface area to volume ratio and easy incorporation of active molecules has impelled researchers to investigate the value of using electro-spun nanofibers in agricultural application such as nanosensors for pesticide residues detection (Ding et al. 2009), protective clothing for farm labourers and agrochemical inputs (Lee and Lee 2012). Recently, seed invigoration with nanofibres developed from various biodegradable polymers was explored as an alternate approach for smart delivery of inputs (Krishnamoorthy et al. 2016;Krishnamoorthy and Rajiv 2017;Hussain et al. 2019). Further, e-spun fibre has Communicated by Shubhpriya Gupta. been investigated as effective seed coating for localized and targeted delivery of inputs for improving germination and seedling growth of rice (Castaneda et al. 2014), black gram (Raja et al. 2020) and groundnut (Raja et al. 2020(Raja et al. , 2017, and also for pathogen control in soybean (Farias et al. 2019). The formation of fibre from polymer in electrospinning depends on various properties like solution parameters, solvent composition, and processing conditions (Yadav et al. 2019). There are many biodegradable polymers tested for production of fibres among which Polyvinyl alcohol (PVA) is one which has excellent physical and chemical properties (Damasceno et al. 2013). PVA is a semi-crystalline polymer that is biocompatible with high thermal and chemical stability (Mojaveri et al. 2020).
Generally, growth promoting bacteria are inoculated in plants either by direct application of liquid or solid based formulation to soil or coating over seeds. The carrier based beneficial bacterial cells loaded commercial formulation when applied over the seed, the viability of microbial cells is reduced due to toxic exudates of seed coat and increased environmental temperatures around the seed during inoculation and sowing. This focuses on developing an alternative, viable and cost-effective technique. Encapsulation of microbes in biodegradable polymer e-spun nanofibre improves the shelf-life, and has been proposed as an alternative emerging and viable technique over the last half decades. Encapsulation of microbial cells in PVA nanofiber, would be an innovative one with technique that could protect the microbial cells by providing favourable environment. Preserving, storing, and maintaining the biologically active materials in the fiber offer various advantages in agricultural research. Encapsulation of bacteria in electrospun PVA nanofibre extended the shelf life period and survivability for three months without further loss (Semnani et al. 2018). Damasceno et al. (2013) demonstrated the successful rhizobial encapsulation in PVA electrospun nanofibre to protect the microbes and increase the shelf-life besides improved germination, seedling emergence, number of root nodules, plant biomass and growth in soybean seeds invigorated with rhizobium loaded nanofibres. Herein, we focused to entrap Methylotrophs, they are gram negative bacteria belonging to the genus Methylobacterium capable of colonizing nodules and other plant tissue by using their ability to utilize single carbon substrates as a competitive advantage (Sy et al. 2005), fixing nitrogen, improving germination and growth (Meenakumari and Shehkar 2012) by producing plant growth regulators like zeatin, cytokinins and auxins (Ivanova et al. 2005). Seed inoculation with this microbial cells improves germination and growth even under limited water availability. Considering the potentiality of e-spun nanofibre, the present investigation immobilization of bacterial cells (Methylorubrum aminovorans) in electrospun PVA polymer matrix for extending the viability, survivability and as an effective seed coating in groundnut for enhanced germination, seedling growth and plant productivity.

Materials and methods
Genetically and physically pure seeds of groundnut variety TMV13, Polyvinyl alcohol (PVA) having molecular weight of 1,15,000 g/mole purchased from M/s. Sigma Aldrich Ltd, and Methylorubrum aminovorans obtained from Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore.

Development and characterization of PVA nanofibre for effective microbial immobilization
Polyvinyl alcohol (PVA) prepared at various concentrations viz., 4, 5, 6, 7, 8 and 9% (w/v), subjected for electrospinning at constant voltage of 15 kV and flow rate of 0.6 ml per hour with constant distance between collector and tip of the needle. The fibres were characterized for morphological feature using Scanning Electron Microscope (SEM-Quanta 250, FEI, Netherlands).

Optimization of microbial cell concentration for nanofibre encapsulation
Microbial cell concentration for nanofibre encapsulation was standardized by adding the microbial cells obtained from cell shrinking technique and PVA polymer at 7%. For this, PVA of 14% solution was prepared and blended with equal volume of microbial inoculums containing 10 8 CFU mL −1 cells as detailed below, and the blend run for electrospinning and microbial cell concentration was optimized by observing the bacteria growth in electrospun nanofibre through imprinting method (Table 1).

Assessment of nanofibre encapsulated microbial viability
E-spin mixture (Microbial inoculums & PVA polymer blend) of 10 mL containing microbial load of 10 8 CFU mL −1 was prepared and immobilized in PVA nanofibre as detailed earlier. The microbial cells loaded nanofibre was divided into equal strips of 1 × 1 cm size each and calculated the initial microbial cells loading (James 1958). Then a total of 30 strips were selected

PPFMs (Methylorubrum aminovorans) immobilized nanofibre seed invigouration on germination and seedling growth
The pure seeds of groundnut var. TMV 13 with initial viability of 74% were surface sterilized with 0.2% mercury chloride, and invigorated with Methylorubrum aminovorans (T 2 ), Polyvinyl alcohol nanofibre (T 3 ) and Methylorubrum aminovorans immobilized PVA nanofibre (T 4 ). The E-spun microbial cells encapsulated nanofibres along with PVA fibres (without bacteria cells) were applied electrostatically to the seed surface in electrospinning unit at the flow rate 0.6 mL/h and voltage of 15 kV with constant distance between needle tip and collector plate. The nanofibre invigorated seeds along with microbial cells alone (T 2 ) and uncoated seeds (T 1 ) seeds were evaluated for germination and seedling growth (IRST 2013), vigor index (Abdul-Baki and Anderson 1973) and drymatter production (Gupta 1993) under controlled laboratory conditions.

Effect of PPFMs (Methylorubrum aminovorans) immobilized nanofibre seed invigoration on plant growth parameters under pot culture
Nanofibre and microbial cells invigorated along with untreated seeds were evaluated for their impact on plant growth parameters under pot culture experiment in in vitro conditions. Pots with 30 × 30 cm size filled with field soil, and seeds were sown in equal distance at a depth of 2.5 cm, and watered as per requirement. Observation on emergence, seedling length, plant growth, root volume, root nodules number and fresh weight and plant biomass was recorded.

Statistical analysis
The experiment was designed in simple Completely Randomized Block Design (CRD) with five replications by adopting the techniques (Panse and Sukhatme 1967) and the data obtained from various experiments were analyzed statistically using Agdata and Agres software. The critical differences (CD) were calculated at 5% probability level. The data recorded in percentage were transformed into angular values (Arcsine transformation) wherever needed.  Fig. 1a-c). Because of higher viscosity, which prevents the formation of beads and droplets, compared to fibres at low concentrations (5 and 6%), which had poor quality with more beads due to low viscosity, which persuaded the surface tension, causing the breaking of entangled polymer chains into fragments, resulting in 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. High numbers of free solvent molecules in the solution come together into a spherical shape causing formation of beads due to higher surface tension. 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 nanofiber development (Deitzel et al. 2001;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).

Results and discussion
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 loaded with microbial (Methylorubrum aminovorans) cells revealed that the diameter of fibre was increased due to encapsulation of microbial cells. Polyvinyl alcohol (PVA) at 7% was found to be produced electrospun fibres with diameter ranging from 93.30 to 166.1 nm (Fig. 1a). The diameter of bacteria cells encapsulated electrospun fibre measured from 379.9 to 845.5 nm (Fig. 3a, b). The TEM image indicated the loading of bacteria cells depicting rod-shaped morphology with size ranging from 267.7 to 466.0 nm (Fig. 4a,  b). This finding is supported by a study (Salalha et al. 2006) in which E. coli bacteria were encapsulated in PVA nanofibre and the immobilization of microbial cells in polymer matrix was demonstrated using SEM and TEM images. Micrococcus luteus was encapsulated in polylactic acid and polyvinyl alcohol electrospun nanofibre by Gensheimer et al. (2011). 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 to 212 ± 29 nm after loading.
The enumeration of PPFMs (Methylorubrum aminovorans) population immobilized in polyvinyl alcohol electrospun nanofibre exhibited a total of 2640 single colonies observed per 0.004 g of nanofibre, and computed value showed that a total of 6.6 × 10 5 CFU g −1 were observed out of 1 × 10 8 CFU initially loaded (Fig. 5). De Gregorio et al. (2017) found that a total of 2.25 × 10 5 CFU of Bradyrhizobium japonicum was enumerated in PVA nanofibre to the total of 1 × 10 8 CFU added initially in the blend of polymer and microbial cell 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 × 10 5 CFU g −1 , 2.2 × 10 4 CFU g −1 and 1.2 × 10 4 CFU g −1 observed on 10, 20 and 30 days after storage, respectively (Fig. 6).
Overall, 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 were 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 control, PPFMs alone inoculated seeds, PVA nano fibre alone coated seeds and Bacterial cells loaded PVA nanofibre coated seeds was depicted in Figs. 7 and 8. The image in Fig. 8d confirms the nanofibre carrying microbial cells which is coated over the groundnut kernel and supplementary Fig. 3b demonstrates the bacterial cells invigorate in to seeds and colonize in the rhizosphere. The pot culture study showed (supplementary Fig. 1a-d) that the seeds inoculated with microbial cells immobilized nanofibre registered significantly higher seedling emergence (83%), seedling root growth (9.60 cm), seedling shoot growth (18.08 cm) and seedling vigor (2294) as compared to seed invigourated with Methylorubrum aminovorans (T 2 ), Polyvinyl alcohol nanofibre (T 3 ) and untreated (T 3 ) (Positive control). 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. The improved germination and seedling vigor 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. 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 (supplementary Fig. 2a-c and Fig. 3a, b). The outcome of this experiment exhibited that the microbial cells encapsulated nanofibre coated seeds have significantly expedited the plant growth as it recorded higher plant height & plant biomass (Table 3), root volume (Table 4), root nodules number and fresh weight (Table 5) 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 (supplementary Fig. 4a-c), which positively promoted the plant growth under pot culture. This result undoubtedly proved the potentiality of electrospun nanofibre to encapsulate the beneficial microorganisms for targeted delivery. According to Damasceno et al (2013) the viability of Rhizobium increases while immobilizing the bacterial cells in PVA e-spun nanofibres and they reported that soybean seeds coated with microbial cells entrapped nanofibres found to be recorded higher seedling emergence, plant biomass and yield. Further, seed coating with hormones (GA 3 and IAA) loaded PVA nanofiber has increased germination and seedling vigor in blackgram (Raja et al. 2020).

Conclusions
The study clearly evidenced the successful encapsulation of pink pigmented facultative methylotrophs (Methylorubrum aminovorans) in electrospun nanofibre for extended shelf-life of microbes and applied to the seeds for effective and targeted delivery of microbes, which enhanced microbial colonization in the roots, resulting in improved germination, seedling vigor and plant growth. This is a preliminary nanotechnological intervention to evolve innovative seed invigoration techniques for improving seed quality. Hence, it needs further fine tuning, large scale testing compared with the existing recommended seed treatment(s) for groundnut before reaching the farm gate.