Azotobacter chroococcum  encapsulated Chitosan/PVA composite nanofiber coated seeds for enhancing germination, seedling vigor and growth of green gram (Vigna radiata)

doi:10.21203/rs.3.rs-2433418/v1

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Azotobacter chroococcum encapsulated Chitosan/PVA composite nanofiber coated seeds for enhancing germination, seedling vigor and growth of green gram (Vigna radiata)

https://doi.org/10.21203/rs.3.rs-2433418/v1

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Seed treatment with plant growth promoting rhizobacteria (PGPR) is an idyllic tool for improving germination, growth and productivity of crop. However, the maintenance of viable microorganisms in biofertilizers is a major issue during the seed and soil application. In this work, a superlative methodology was made for encapsulation of Azotobacter chroococcum in Chitosan/Polyvinyl alcohol (PVA) composite nanofiber by electrospin technique and evaluated its effect in improving green gram seed quality. The morphology and functional group of microbes loaded in Chitosan/PVA nanofibers were characterized and confirmed by Scanning Electron Microscope and FT-IR. The results of microbial plating technique showed that nanofiber has maintained the viability of microbes for prolonged time. In addition, Exopolysaccharide and IAA production by Azotobacter chroococcum loaded in nanofiber was estimated and found with constructive outcomes. The bio-efficacy studies exposed that the seeds coated with Azotobacter chroococcum loaded nanofiber, improved the germination percentage, seedling vigor, plant height, plant root length, plant biomass, nodule count and nodules fresh weight. This study concluded that immobilizing PGPR bacteria in nanofiber could improve prolonged shelf-life of microbes and as a potential seed invigoration technique for smart delivery of microbes, and efficient colonization at rhizosphere for better emergence and growth.

Azotobacter chroococcum

Chitosan

PVA

nanofiber

green gram

encapsulation

Biofertilizers (bioinoculants) are the organic preparation of beneficial microorganisms for crop improvement in agriculture. Biofertilizers can be applied on seeds, seedling root and in soil as plant growth promoting rhizobacteria (PGPR), which colonize over the roots to promote plant growth directly and indirectly (Saharan and Nehra 2011; Vejan et al. 2016). PGPR acts as alternative to harmful chemical fertilizers and pesticides. PGPR have massive beneficial characteristics, such as nitrogen fixation (Figueiredo et al. 2008), regulating and increasing nutrient uptake of plants through solubilization of phosphorous (Oteino et al. 2015), production of siderophores (Sayyed et al. 2005) and phytohormones (Ahmed and Hasnain 2014); and induce resistance against wide range of pests and pathogens in plants (Beneduzi et al. 2012). Recently, agricultural sector turned towards the application of PGPR for its diversity and sustainability (Bhattacharyya and Jha 2012; Di Benedetto et al. 2017).

Nitrogen fixation is the important process of microbial activity as it recycles nitrogen from atmosphere to earth’s surface, helps in improving soil fertility and crop production. Azotobacter is a free living and nitrogen fixing gram negative aerobic bacteria. They form cysts at dormant stage to resist against unfavorable environmental conditions. They are polymorphic in nature having 2 to 10 µm length and 1 to 2 µm width. Azotobacter genus was first recognized as free living aerobic nitrogen fixer, by Dutch microbiologist Beijerinck and his co-workers in 1901. During cellular protein synthesis of Azotobacter, it fixes large amount nitrogen in the form of ammonia to soil by exploiting atmospheric nitrogen. Some studies recommend that Azotobacter can be an alternative for mineral N fertilizers, as it approximately fix 20 kg N/ha/year and upgraded the crop production (Kizilkaya 2009; Esmailpour et al. 2013). It also produces the many stimulatory growth substances and natural hormones which helps in improving physiological and biochemical activity of plants. A single strain of Azotobacter chroococcum can produce three gibberellin compounds, five cytokinins and auxins like IAA (Brown 1968; Wani et al. 2013). In addition to the above profits, Azotobacter sp. can remove oil contamination (Piperidou et al. 2000) and degrade pesticides like 2,4- dichlorophenol, 2, 4, 6- trichlorophenol and pendimethalin (Wu et al. 2004; Kole et al. 1994). Azotobacter chroococcum also suppresses the plant disease incidence of nematode Meloidogyne incognita (Akram et al. 2016) and induce solid antagonistic effect against fungus Macrophomina phaseolina and Fusarium oxysporum (Maheshwari et al. 2012). Eventhough Azotobacter offers numerous benefits, maximum potential has not been conveyed to plant productivity as its population is very low in the soil.

The application of microbial inoculants can be done to seed and soil. Land application require large quantity of inoculums for even distribution of bacteria, whereas seed treatment reduces the application cost and found effective in the rhizosphere. Due to the direct delivery of inoculum in seed, high density of viable microbe colonizes over the emerging roots of seedling (Deaker et al. 2012). Though several studies demonstrated the capacity of commercial bioinoculants in improving crop production, the major problem with them is maintaining viability of microorganisms during seed treatment and storage (O'Callaghan et al. 2012). As a solution for improving viability of microbes, the polymers have the ability to encapsulate beneficial microbes and protecting them from external environment and control gradual release. In addition, polymers can guard and maintain the viability of bacteria for long period when dry stored at room temperature (Bashan et al. 2002). Bacteria can be encapsulated in micro and macro polymer beads for seed coating. However, these polymer beads have drawbacks in application such as, (i) inoculant beads mixed with seeds may detach and face competition with native microbes, (ii) additional adhesive required for preventing detachment of micro beads from seeds, and (iii) requiring additional treatment incurs extra cost and time (Bashan et al. 2014; Bashan et al. 2002; John et al. 2011). Therefore, a versatile carrier should be designed for finest encapsulation of inoculums and ensuring its prolonged viability in all environmental conditions.

The field nanotechnology fashioned a nanocarrier for microbe encapsulation is electrospin nanofiber. In electrospinning, a high voltage has applied to reduce surface tension and stretch the polymer solution into nanofiber (Kusumastuti et al. 2016). The nanofiber has copious unique characteristics, such as high porosity, large surface area, small diameter, unique morphology, easy fabrication and composition bids several advantages for agriculture. Currently, nanofiber is used for loading chemical and biological substances that elevate plant production and protection (Castañeda et al. 2014). Few successful studies are conducted practically in nanofiber encapsulation of microbe for improving yield parameter. (De Gregorio et al. 2017) immobilized Pantoea agglomerans and Burkholderia caribensis inoculants in PVA nanofiber for seed coating and reported that the viability of bacterial strains has been improved. Further, nanofiber seed coating for effective localized and targeted delivery of supplements needed for improvement of germination percentage and seedling vigor of black gram (Latha et al. 2022), rice (Castañeda et al. 2014) and groundnut (Raja et al. 2017). There are several polymers available, which having high biocompatibility, biodegradability, good physical and chemical properties suitable for the nanofiber fabrication and microbial encapsulation. Encapsulation of microbes in biodegradable PVA nanofiber could protect the viability by providing advantageous environment around the cells (Damasceno et al. 2013).

In this work, Azotobacter chroococcum has selected for its potential benefits and encapsulated in Chitosan/PVA composite nanofiber. These polymers have chosen for their excellent properties like non toxicity, biocompatibility, biodegradability and fiber forming capability. PVA is a synthetic water-soluble polymer whereas chitosan is partially water soluble. On blending and fabricating composite nanofiber using these polymers, ensure the controlled release of inputs. The hypothesize is that the immobilization of bacteria in nanofiber by electrospinning can preserve its viability for long time. Thus, the objectives of this study have been: i) evaluating morphology of nanofiber encapsulated Azotobacter chroococcum, ii) testing the bacterial viability, iii) exopolysaccharide (EPS) and Indole Acetic Acid (IAA) production by nanofiber encapsulated Azotobacter chroococcum, iv) green gram seed invigoration with nanofiber encapsulated Azotobacter chroococcum.

Green gram seeds (variety CO8) with high genetic and physical purity were purchased from Department of Pulses, Tamil Nadu Agricultural University, Coimbatore. Partially hydrolyzed water-soluble PVA polymer (Molecular weight 1,60,000 kDa) was purchased from HiMedia. Medium molecular weight chitosan, glacial acetic acid, nutrient broth and nutrient agar medium used in the present study was purchased from Sigma Aldrich Pvt, Ltd. Azotobacter Chroococcum bacterial culture was obtained from MTCC (CSIR-Institute of Microbial Technology, Chandigarh, India).

Encapsulation of Azotobacter chroococcum in chitosan/PVA composite nanofiber and characterization

Preparation of bacteria and chitosan/PVA composite solution cocktail

All the experiments were carried out under sterile condition to avoid cross contamination.

PVA 10% solution was prepared by dissolving PVA powder in distilled water and stirred continuously using a magnetic stirrer at 70^oC until the complete dissolution. Chitosan 2% was prepared by mixing chitosan in 1% of glacial acetic acid and allowed for continuous stirring at 200rpm until complete dissolution occurred. PVA and chitosan solution were mixed at 9:1 ratio and stirred for 6 hours at 300rpm to obtain the homogenous composite solution and autoclaved for 20 minutes at 121^oC.

Azotobacter chroococcum was inoculated in 300 ml of nutrient broth and incubated at 37^oC for 24 hours with constant shaking of 100rpm. Then the bacterial cells were centrifuged at 4000rpm for 20 minutes and washed thrice with phosphate buffer solution. The collected bacterial cells were mixed in Chitosan/PVA composite solution and stirred at 80rpm for 3 hours under room temperature to obtain homogenous distribution of microbes in the solution.

Fabrication of Azotobacter chroococcum loaded chitosan/PVA composite nanofiber (E-spin method)

E-spin mixture contains Azotobacter chroococcum cells and composite polymer solution; and filled into the syringe for electrospinning (monoaxial loading). The needle tip of syringe was supplied with high voltage and cathode connected to the nanofiber collector lined with aluminum foil. Spinning parameters were set as follows: flow rate, 0.6mL hr^-1; tip to collector distance, 15cm; Voltage, 17kV. After electrospinning, microbe loaded nanofiber was collected and characterized for morphology and functional groups through SEM (FEI QUANTA 250) and FTIR spectrophotometer-6800 type A (M/s. Jasco, Japan). The viability of Azotobacter chroococcum loaded in nanofiber was evaluated by microbial plating method.

Positive measures of immobilizing Azotobacter chroococcum in nanofiber

The observations like viability, exopolysaccharide and IAA production of Azotobacter chroococcum loaded in nanofiber were taken from two different storage conditions (4^oC and ambient temperature) for six months by monthly assessment.

Viability of Azotobacter chroococcum loaded in nanofiber

The total colony count of Azotobacter chroococcum mixed in the polymer solution was approximately 1*10¹⁶ CFUs and encapsulated in the composite nanofiber. The initial microbial loading in the nanofiber was recorded immediately after electrospinning. Then the cell viability of Azotobacter chroococcum in nanofiber was assessed by approving microbial plating technique. The number of colonies forming units were denoted as log₁₀CFU.gm^-1 of nanofiber.

Exopolysaccharide (EPS) production

Exopolysaccharide production by Azotobacter chroococcum was determined quantitatively before and after loading in nanofiber. Microbe loaded nanofiber was dissolved in nutrient broth and incubated at 37^oC for 5 days to attain maximum lag phase growth. The contents were centrifuged at 10,000xg for 20 minutes and the culture supernatant was collected. To the one-part supernatant, three parts of ethanol was added slowly along the side wall of conical flask and allowed to stand for 20 minutes at 4^oC for the precipitation of exopolysaccharides. The weight of the EPS was measured after drying at 85^oC (Lee et al. 2002). Exopolysaccharide production was expressed in mg per gram of bacteria loaded nanofiber.

Indole Acetic Acid (IAA)

The IAA production of Azotobacter chroococcum loaded in nanofiber was measured by the modified method of (Loper and Schroth 1986). Microbes loaded nanofiber was dissolved in nutrient broth and incubated for 72 hours at 37^oC. The fully grown culture was centrifuged at 10000xg for 15 minutes. To the 2ml of culture supernatant, 2 drops of ortho-phosphoric acid and 4ml of Salkouski reagent was added. The sample was incubated at room temperature for 25 minutes. The pink color development was observed and measured the optical density at 530nm by using UV-Visible spectrophotometer. The concentration of IAA production by bacteria in nanofiber was measured with the help of standard graph of IAA in range of 1 to 100 and expressed in µg per ml.

Seed coating with microbes loaded nanofiber

Green gram seeds were surface sterilized with 4.5% sodium hypochlorite and washed with sterile distilled water followed by 30 min shade drying. Then the seeds were placed in the petridishes with aluminium foil as base. The dishes were subjected to electrospinning for 10 min with Azotobacter chroococcum-polymer mix. The coated seeds were characterized with SEM.

Effect of Azotobacter chroococcum loaded nanofiber on green gram seed invigoration at laboratory condition

The green gram seeds were invigorated with Azotobacter chroococcum (T₂), nanofiber (T₃) and nanofiber loaded with Azotobacter chroococcum (T₄) and uncoated seeds as control (T₁). The experiment was carried out using roll towel method and each treatment have five replications. Then, these were incubated at 28^oC for seven days and the readings were recorded. The coated and uncoated seeds were evaluated for germination percentage, root length, shoot length (IRST 2013), and vigor index (Abdul‐Baki and Anderson 1973) under laboratory condition.

Effect of Azotobacter chroococcum loaded nanofiber on green gram seed invigoration under pot culture at invitro condition

The impact of Azotobacter chroococcum loaded nanofiber on green gram plant growth parameters were evaluated under pot culture along with untreated seeds. The field soil was filled in 30 x 30 cm sized pots and seeds were sown at 2.5 cm depth and supplied with water as per the requirement. The plant growth parameters like seedling length, root volume, root nodules and plant biomass were recorded at 25DAS and 45DAS.

Statistical analysis

The experimental design adopted was simple Completely Randomized Block design with five replications for each treatment. The statistical analysis software Agdata and Agres was used for data analysis. The critical difference (CD) value was calculated at probability level of 5% and the percentage data were transformed by Arcsine transformation into angular values.

Immobilization of Azotobacter chroococcum in nanofiber

Electrospinning is a versatile technique used for the fabrication of one-dimensional nanostructure with exclusive properties such as high surface area, porosity and safety in comparison with other nanomaterials (Nurfaizey et al. 2012). The polymer solution from the nozzle was charged with high voltage, which counteracts the surface tension by electrostatic repulsion and stretched the polymer droplet into nanofiber by forming Taylor cone (Zeleny 1914). The fabrication of nanofiber is significantly influenced by various aspects viz., concentration and molecular weight of polymer, solvent, viscosity, applied voltage, flow rate and tip to collector distance (Haider et al. 2018). PVA was an additive polymer having non-toxic, biodegradable, biocompatible, transparent properties and possibility of creating hydrogen bond with chitosan molecule (Li and Hsieh 2006; Askari et al. 2014; Matsumura et al. 1993). In present study, the electrospinning of nanofiber was carried out as per the parameters stated. Primarily, the various proportions viz., 5:5, 6:4, 7:3, 8:2 and 9:1 of chitosan/PVA solution were investigated in nanofiber formation (Supplementary material). The results revealed that the gradual decrease of beaded structure in nanofiber were found from 5:5 to 8:2 polymer ratios. The beaded nanofiber formation was due to the instability in surface tension, absence of suitable chain entanglement, imbalance of electrical and repulsive force among polycations of polymer solution (Li and Hsieh 2006; Ding et al. 2006). Electrospinning of chitosan/PVA at 9:1 ratio was executed as per the parameters (Voltage,17 kV; Flow rate,0.6 mL/hr; Tip to collector distance,15cm). The beadless nanofiber was fabricated in 9:1 ratio. The fine nanofiber fabrication was a result of proper hydrogen bonding among the amino or hydroxyl group of chitosan with PVA molecule, which enhanced the proper mixing, homogenization and stable jet formation of these polymers (Li and Hsieh 2006; Askari et al. 2014; Habiba et al. 2016; Zhang et al. 2007). To achieve the formation microbes loaded nanofiber, 1*10¹⁶CFUs cells were added to 9:1 ratio polymer solution and electrospinned as per the parameters depicted. The nanofiber encapsulation can protect the microorganisms by maintaining the temperature and humidity more favorable for microbial survival(Deaker et al. 2012). The successful blending of bacteria in chitosan/PVA composite solution, and fabrication of microbes loaded nanofiber using electrospinning were investigated.

Characterization of Azotobacter Chroococcum loaded nanofiber

Morphological analysis

In fig.1a, SEM micrograph of Azotobacter chroococcum was presented and measured the size, which ranges from 1160 µm to 3090 µm in length and 670 µm to 1434 µm in width. The average size of the nanofiber ranging between 150 and 250nm and had a uniform thickness without formation of beads for 9:1 ratio (Fig. 1b). After immobilizing bacteria in nanofiber, the size of nanofiber was increased in both length and width, respectively. Azotobacter chroococcum cells were distributed along the nanofiber and clearly understood that the bacteria embedded entirely in polymeric matrix and forming a local widening and aligned longitudinally along nanofiber axis which was shown in (Fig. 1c). Similarly, Escherichia coli incorporated in PVA nanofiber was characterized by HRSEM and found increase in nanofiber thickness, which ranged from 160 nm to 10 µm after bacteria loaded in nanofiber (Salalha et al. 2006).

Stuctural analysis by FTIR

The chemical bonds present in the sample was identified by producing infrared absorption spectrum using FTIR. The FTIR spectra of Azotobacter chroococcum, nanofiber and microbe loaded nanofiber was shown in (Fig. 2). In general, cell wall of gram negative bacteria contains phospholipids, proteins, lipopolysaccharides and lipoproteins which comprises important functional gropus like hydroxyl, carboxyl, phosphate, amide and polymer molecules (Beveridge 1981). FTIR spectra of Azotobacter chroococcum had the transmittance peaks at 1645 cm^-1 and 1510 cm^-1 due to the strong stretching of C=O in (amide I) and less intensity N-H bending and C-N stretching in (amide II). Likely, the polysaccharide groups comprising C-C, C-OH and C-H exhibited sharp peak at 930 cm^-1, 1020 cm^-1, and 1083 cm^-1 respectively for bacteria. The phosphate group of Azotobacter chroococcum was confirmed by the stretching of P=O bonds at around 1210 cm^-1 (Fig.2a). (Jiang et al. 2004) confirmed the bacterial sample of Bacillus subtilis by FTIR spectral graph, which showed the stretching of functional groups like, amide I and II, phosphate group and saccharide groups at 1651 cm^-1, 1545 cm^-1, 1215 cm^-1 and 1050 to 1110 cm^-1, respectively.

The characteristic FTIR Spectra of chitosan polymer have major peaks of amide, C-O and saccharide groups at 1638 cm^-1, 1055 cm^-1 and 844 cm^-1 (Don et al. 2006). Whereas PVA have functional groups like O-H which stretch between 3550 cm^-1 and 3200 cm^-1, C=O peaks at 1730 cm^-1 and 1680 cm^-1, and typical vibrational band between 2840 cm^-1 and 3000 cm^-1 due to C-H stretch (Wang et al. 2004; Shigemasa et al. 1996; Mansur et al. 2008). Similar group of peaks were resembled in fabricated nanofiber. FTIR spectra of nanofiber showed resonance bands of saccharide groups between 1055 cm^-1and 869 cm^-1 which were present in both polymers. Chitosan/PVA nanofiber have peaks at 1633 cm^-1and 1528 cm^-1 due to presence of chitosan’s amide functional molecule. In addition to those peaks in nanofiber, stretching of O-H and PVA’s characteristics alkyl group existed at 3337 cm^-1 and 2903 cm^-1 (Fig.2b). FTIR spectra confirmed the presence of charatcteristic peaks of PVA and chitosan in the nanofiber.

After loading of Azotobacter chroococcum in nanofiber, few additional peaks were found between 1078 cm^-1 and 823 cm^-1, due to saccharide groups of bacteria. In microbe loaded nanofiber, a strong intense sharp peak at 1237 cm^-1was due to existence of phosphate group which present in Azotobacter chroococcum. FTIR showed less intensity and deformation of amide I and II at 1638 cm^-1 and 1538 cm^-1due to full entrenching of bacteria in nanofiber. The carboxylic group of nanofiber has been shifted from 1752 cm^-1to 1724 cm^-1 in microbes loaded nanofiber and deeped with high intensity. Due to presence of typical alkyl group in both Azotobacter chroococcum and PVA, intensity of this group was increased and stetched from 2903 cm^-1 to 2945 cm^-1 and also intensified O-H peak was observed at 3356 cm^-1 in microbe loaded nanofiber (Fig. 2c). Likewise, (Wai Chun et al. 2021) found microbial composite flims have extra functional groups like C-C, C-OH, C-H at 800 cm^-1 and 662 cm^-1, when compared to FTIR spectra of sodium alginate film. In this regard, FTIR spectra characterized the positive loading of bacteria in nanofiber, by showing additional existence of phosphate group and deformation of amide (I and II) group in bacteria loaded when compared to non loaded nanofiebr.

Positive measures of immobilizing Azotobacter chroococcum in nanofiber

Viability of bacteria

On the basis ofpreviousresults obtained, polymers were excellent carrier sources for immobilization of bacteria and supporting material for viability of bacteria (Fung et al. 2011; Damasceno et al. 2013). Polymers control and protect the innoculant cell integrity from external environment. The PVA and chitosan were selected for their forementioned properties and has high oxygen barrier during dry condition without distressing the bacterial properties. The exposure of Azotobacter chroococcum to the spinning solution had no effect in its viability, even it suspended in solution for several days. The average value of viable cells were represented in log₁₀CFU.gm^-1±standard error. The concentration of bacteria in the electrospinning solution was1*10¹⁶ CFU cells (Fig.3a). After electrospinning, 92.8% of Azotobacter chroococcum cells were loaded in the nanofiber, which was 7*10¹⁴ Colony forming units (Fig.3b). The loss of 7.2% bacterial cells was due to mechanical stress during nanofiber fabrication and rapid evaporation of solvent creates a pressure over bacteria lead to the cell death. The bacteria remain viable after electrospinning, as the amount of applied voltage travelled low to the bacteria and did not affect the cell integrity (Hülsheger and Niemann 1980).

After embedding Azotobacter chroococcum in nanofiber, it was stored at room temperature and 4^oC; and the viability was tested for six months. On accessing the viability of Azotobacter chroococcum in nanofiber stored at 4^oC (Fig. 3c & 3d) and normal temperature (Fig. 3e & 3f), there was a total of (13.26±0.06, 12.7±0.26, 12.5±0.26,11.36±0.25, 11.26±0.8 and 11.04± 0.23) log10 CFU and(13.15±0.11, 12.58±0.23, 11.15±0.12, 10.46±0.15, 9.04±0.09 and 7.45± 0.09) log₁₀ CFU per gramof nanofiber, which were observed on 1^st, 2^nd, 3^rd, 4^th, 5^th , and 6^th month, respectively. Towards the end of 6^th month, gradual decrease of cell count was observed for 4^oC accounted from 89.3% to 74.3% (Fig.4a). But in ambient storage, viability declined hugely from 88.5% to 50.1% due to unstable environment around the nanofiber which hindered the bacterial survival (Fig. 4b). Exposure of heat to bacteria in nanofiber was comparitively low for 4^oC than ambient storage, as the heat transfer through nanofiber to bacteria was less. At the end of sixth month refrigerator storage recorded the high range of viable colony count. The results were in correlation with studies of(Pradeep Prasanna and Charalampopoulos 2019)who studied the viability of probiotics in nanofiber was more than 9log cfu/g at the end of 6^th month, when stored at 5^oC. From the results, it is probable to store many organisms at lower temperature more efficiently in this dry form.

Exopolysacharide production

Certain group of bacteria produce extracellular polysaccharides, which was comprised of sugars, proteins, nucleic acids and lipid material. EPS around the bacteria subsidize the shape and rigidity to cell stucture. EPS has substantial number of functions like cell protection against antimicrobial compounds and environmental stress, water retaining capability, entrapment of nutrienets and carbon storage (Sandhya and Ali 2015; Wang et al. 2015). The efficient nitrogen fixing ability of Azotobacter in soil was directly proportional to its viability and EPS production under stress condition. During unfavourable conditions, metabolically active Azotobacter cells produce high level of protective coating around the cells by secreting EPS to prevent the cell dehydration (Gacesa 1998). Like EPS, nanofiber avoided the cell dehydration and protect Azotobacter chroococcum from external environment. EPS produced by Azotobacter chroococcum was expressed in mg/g of nanofiber with standard error.

The survivability and habitability of Azotobacter chroococcum was conditioned by secreting microstucture around its cell wall during extreme heat as well as ice cold condition. During the encapsulation of bacteria, a polymer flim was produced over the cell to protect and bind inside the nanofiber (Zupančič et al. 2019). EPS produced by 1*10¹⁶CFU Azotobacter chroococcum was 147±3.7 mg before loading in nanofiber. After loading, the cell count was reduced to 7*10¹⁴ CFU per gram of nanofiber and EPS production too reduced from 147±3.7 mg to 133±2.4 mgg^-1. From the table 1, it was proved that EPS production directly influenced by temperature differences and cell count in nanofiber. In 4^oC storage, bacteria produced 124±2.7 mg/g at first month, which was subsequently decreased to 101±1.5 mg/g at the sixth month according to the ranges of colony forming units. At the refrigerator condition, bacteria produce excess EPS to protect it from ice crystallization(Krembs et al. 2011). The decrease in temperature than optimal level for bacterial growth favoured the reduction in cell wall poylmer synthesis and made more available form of minerals for the EPS biosynthesis(Sutherland 1972; Gassem et al. 1995; Kimmel et al. 1998). Meanwhile at ambient storage, the EPS production was radically reduced to 67±0.6 mg/g from 120±0.5 mg/g due to temperature fluctuations. Storing the bacteria at ambient condition, the nutriments were fully utilized for cell growth and there was a lack of essenial minerals for EPS production(Gorret et al. 2001). At 4^oC storage, the temperature was maintained stable and dehydration of bacteria was well shielded by nanofiber, but ambient condition have the temperature fluctuations which disequilibrate the hydro content of polymer and leaded to subsequent cell death. The study meticulously clarified the importance of temperature influence over the EPS production for the bacterial viability maintanance.

Table 1 Exopolysaccharide production byAzotobacter chroococcum loaded in the nanofiber

Bacteria-polymer mix solution EPS production (mg)	Month	EPS production of microbe loaded in nanofiber (mg/g)
	Month	(4^oC)	(Ambient condition)
	1	124±2.7	120±0.5
147±3.7	2	117±0.3	115±2.9
Bacteria loaded nanofiber EPS production (mg/g)	3	113±1.5	103±0.9
	4	106±0.5	93±2.1
	5	103±0.8	86±1.5
133±2.4	6	101±1.5	67±0.6

IAA production by Azotobacter chroococcum immobilized on nanofiber

IAA is the physiologically active auxin, which took part in physiological processes comprising, root and shoot development, embryogenesis, organogenesis, fruit development and trophic growth of the plant. The present work deals with the quantification of IAA produced by Azotobacter chroococcum encapsulated in nanofiber. At first, the standard graph of IAA was prepared by plotting concentration against the Optical density at 530nm. The standard graph showed straight line indicating direct proportional concentration of Indole acetic acid (Fig. 5). The validity of graph was showed by R² value, which was found to be 0.9903.

IAA produced by bacteria was varied with colony forming unit count, species, growth stage, culture condition and substrate availability (Sridevi and Veera Mallaiah 2007). IAA produced by 1*10¹⁶CFUs was 97±2 µg/ml. After loading in nanofiber, the cell count was reduced and IAA too decreased from 97±2 µg/ml to 89±0.5 µg/ml. In the Fig. 6, IAA production was presented at different time period and decrease in IAA production was observed in both storage conditions with respect to decrease in colony forming units. The IAA production was directly proportional to number of active cells and utilize the mineral nutrient for the synthesis and release of IAA on the culture broth. At 4^oC storage, bacterial cells were well stockpiled and produced IAA of 79±0.5 µg/ml, which reduced to 66±0.6 µg/ml at the 6^th month observation.

Similar trend of reduced IAA production was detected at ambient storage of Azotobacter chroococcum loaded in nanofiber. The results were analogous to the findings of (De Gregorio et al. 2017), in which found that IAA production was reduced after the encapsulation of Pantoea agglomerans in nanofiber according to the decrease in cell count.

Effect of Azotobacter chroococcum loaded nanofibers seed invigoration under laboratory condition

The surface morphology of untreated seeds, Azotobacter chroococcum inoculated seeds, nanofiber coated seeds, and Azotobacter chroococcum loaded nanofiber coated seeds was depicted in (Fig. 7a-d). Fig. 7d confirms Azotobacter chroococcum loaded nanofiber adhered to the green gram seed and Fig. 7b showed that the microbial presence in the Azotobacter chroococcum inoculated seed surface. Supplementary fig.1a-d, showed the green gram seedling vigor due to effect of Azotobacter chroococcum loaded nanofiber at seventh day of observation in laboratory condition. The laboratory study showed that the seeds coated with Azotobacter chroococcum loaded nanofiber recorded significantly higher germination percentage (85.2%), seedling root length (14.7cm), seedling shoot length (19.1cm), and seedling vigor (2670), when compared to seeds invigorated with Azotobacter chroococcum (T₂), nanofiber (T₃) and untreated control (T₁). There was an increase in seedling emergence of (13%), shoot length (36.1%), root length (25.2%) and seedling vigor index (36.6%) observed in Azotobacter chroococcum loaded nanofiber coated seeds, when compared to untreated control seeds (Table 2).

This higher potential seed invigoration in T₄ is due to the combined effect of Azotobacter chroococcum and polymer mix. In addition, this vigorous seedling growth was due to the secretion of plant growth regulating hormones (Gibberellin, cytokinin and auxin) by the Azotobacter chroococcum (Wani et al. 2013). And the polymer mix (chitosan and PVA) has the property of improving seed water absorption capacity and use efficiency by maintaining the higher moisture content around the seed. Moreover, the polymer contents catalyze the metabolic activities of seed, which are essential for potential germination and seedling vigor.

Table 2. Effect ofAzotobacter chroococcum loaded nanofiber seed invigoration on germination percentage, shoot length, root length and vigour index of green gram var. CO8 under laboratory condition.

Treatments	Germination (%)	Shoot length (cm)	Root length (cm)	Vigour index
T₁	72.2±1.16 (58.05)	12.2±0.38	11.0±0.62	1693±20.98
T₂	77.2±1.46 (58.69)	16.8±0.49	12.6±0.76	2234±71.29
T₃	75.8±1.47 (57.42)	17.9±0.46	14.0±0.56	2479±43.32
T₄	85.2±1.31 (71.57)	19.1±0.33	14.7±0.99	2670±131.02
CD (P=0.05)	4.067	1.252	2.254	234.950

(Figure in parenthesis are arcsine values)

T₁ – Untreated seeds, T₂ - Azotobacter chroococcum inoculated seeds, T₃ - Nanofiber coated seeds and T₄ - Nanofiber loaded with Azotobacter chroococcum coated seeds

Effect of Azotobacter chroococcum loaded nanofiber seed invigoration under pot culture at invitro condition

The plant growth parameters like plant height, root length, plant biomass, nodule count and fresh weight of nodules were recorded on 25 and 45 days after sowing
(Supplementary fig. 2a-d and fig 3a-d). The results of pot culture experiment showed that the Azotobacter chroococcum loaded nanofiber coated seeds have higher plant height, root length and plant biomass (Table 3), nodule count and nodule fresh weight (Table 4) at 25 and 45 DAS, when compared to untreated control. This positive approach in plant promotion by microbes loaded nanofiber is due to the colonization of microbes over the root region of green gram. These results proved the positive effect of electrospun nanofiber in encapsulating plant growth promoting rhizobacteria for the targeted delivery. The encapsulation of microbes in the polymer protects and improves the viability of microbes (Semnani et al. 2018), and increased the plant growth parameters viz., germination percentage, plant height, root length, root nodules and plant biomass in soyabean seeds, coated with Rhizobium loaded nanofiber (Damasceno et al. 2013). According to (Raja et al. 2021), the blackgram seeds coated with GA₃ and IAA loaded PVA nanofiber increased the germination percentage and seedling vigor.

Table 3. Effect ofAzotobacter chroococcum loaded nanofiber seed invigoration on plant height and root length of green gram var. CO8 under pot culture

Treatments	Plant height (cm)		Root length (cm)		Plant biomass (mg/plant)
Treatments	25 DAS	45 DAS	25 DAS	45 DAS	25 DAS	45 DAS
T₁	18.8±0.4	33.1±1.3	7.6±0.3	12.4±0.5	456.6±13.7	475.8±23.3
T₂	23.1±0.9	36.4±1.4	9.8±0.4	15.6±0.6	497.7±17.6	552.6±19.6
T₃	21.5±0.5	35.6±1.3	9.4±0.4	14.1±0.6	478.6±16.9	520.5±18.4
T₄	29.6±0.7	43.9±1.7	12.1±0.5	19.8±0.8	553.9±19.6	606.5±21.5
CD (P=0.05)	1.979	4.441	1.173	1.874	51.277	62.612

T₁ – Untreated seeds, T₂ - Azotobacter chroococcum inoculated seeds, T₃ - Nanofiber coated seeds and T₄ - Nanofiber loaded with Azotobacter chroococcum coated seeds

Table 4. Effect of Azotobacter chroococcum loaded nanofiber seed invigoration on nodule count and nodule fresh weight of green gram var. CO8 under pot culture

Treatments	Nodule count per plant		Fresh weight of nodules (mg/plant)
Treatments	25 DAS	45 DAS	25 DAS	45 DAS
T₁	14.4±0.5	23.7±0.8	131.8±8.7	236.4±15.6
T₂	15.8±0.6	28.7±1.0	141.7±9.3	287.4±18.9
T₃	15.3±0.5	25.9±0.9	138.8±9.1	272.0±17.9
T₄	19.4±0.7	31.4±1.1	175.5±10.6	301.7±19.9
CD (P=0.05)	1.691	2.881	24.551	49.782

T₁ – Untreated seeds, T₂ - Azotobacter chroococcum inoculated seeds, T₃ - Nanofiber coated seeds and T₄ - Nanofiber loaded with Azotobacter chroococcum coated seeds

Nanofiber encapsulation was proved to be the premium and effective carrier material for the delivery of Azotobacter chroococcum as agricultural input. In this study, the methodology for encapsulation of bacteria in nanofiber was disclosed and the successful impregnation was characterized by analysing nanofiber morphology and functional groups under SEM and FTIR. Nanofiber stored the viability of bacteria without affecting its properties for long period with less reduction in cell counts. Likewise, the EPS and IAA production was reasonably reduced at the end of sixth month for both storage conditions with respect to the viability of Azotobacter chroococcum in nanofiber. The suitable polymer composite preserves the microbe’s viability and improve its efficacy in successful colonization over green gram rhizosphere, resulting in improved germination percentage, seedling vigor and plant growth. This is a preliminary invention in targeted delivery of microbes for potential seed invigoration and several improvements should be carried out in this methodology for large scale industrial production.

Supplementary Material: The online version comprises supplementary material at (https://www.researchtrend.net/bfij/pdf/62%20Fabrication%20and%20Characterization%20of%20PVAChitosan%20Composite%20Nanofiber%20for%20Encapsulation%20of%20Active%20Ingredients%20M.S.%20Kumuthan.pdf)

Acknowledgement

The authors thankfully acknowledge the practice of services and facilities provided by Department of Nano Science and Technology, TNAU, Coimbatore. We also wish to thank the funding agency GoI, SERB (DST) for providing financial support (EMR/2016/008061) in the scheme entitled, “Development of electrospun fibre nanomatrix to encapsulate beneficial microbes for smart delivery and sustainable productivity”.

Author contributions

MSK- Investigation; Writing; Original Draft; AL- Conceptualization; Supervision; KGS- Resources; Validation; KSS- Review & Editing; ML- Data Curation; Writing; Review; KR- Review & Editing; MG- Resources; Validation.

Declarations

Conflict of interest All the authors declare that they have no conflict of interest.

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Azotobacter chroococcum encapsulated Chitosan/PVA composite nanofiber coated seeds for enhancing germination, seedling vigor and growth of green gram (Vigna radiata)

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