​Optimized Bioprocess for the Formulation of Liquid Microbial Inoculants for Forage Cowpea and Insight of Plant-microbe Interaction by SEM

Plant growth promoting rhizobacteria have the potential for sustainable plant growth promotion in this neoteric era. This journey was started with Rhizobium but now it has broadened its horizon and includes plethora of microorganisms which are not only being exploited for nutrient mobilization or plant growth promotion but also playing a pertinent role in plant protection (PP). Nevertheless, in spite of good going, the actual utilization is nowhere near the potential. Actually, vast variety of the research work was done in the field of strains development, mechanism of action and on exaggerating the science behind their potentiality. Very little work has been done to convert the science into technology (Yadav and Chandra, 2014). To assist high cell numbers and increased survival of microorganisms in soil, preparation of microbial cultures is essential. However, solid carrier based preparations generally suffer from (i) short shelf-life, (ii) poor quality, (iii) high contamination and (iv) low and unpredictable field performances (Vendan and Thangaraju, 2006). To overcome these problems, the liquid carrier based formulations have been introduced (Albareda et al., 2008). Liquid bioinoculants are special liquid formulations containing not only the desired microorganisms and their nutrients, but also the special cell protectants or substances that encourage the longer shelf life and tolerance to adverse conditions (Vora et al., 2008). It has observed that it is possible to make bacteria survive in liquid microbial inoculants more than six months with the help of additives/cell protectants such as methyl cellulose, gum arabic, alginate, polyacrylamide (Bashan et al., 2002). The selection of ideal polymer is based upon several properties like cell adhesion to seed, stabilizer and non-toxicity.
               
There is a tremendous scope for increasing the yield potential of cowpea by use of microbial inoculants thereby enhancing the nutrient availability and better plant growth (Ramya and Pandove, 2021). The scanning electron micrographs can furnish brilliant resolution and exact localization of microorganisms in connection to the root structure and can further strengthen plant (host) and plant growth promoting rhizobacteria (PGPRs) interactions. Thereupon, the main objective of the present study was to formulate liquid microbial inoculants with enhanced shelf life and appraisal of liquid microbial inoculants on roots surface of forage cowpea under axenic conditions by scanning electron microscopic (SEM) studies to study plant-microbe interaction.

Procurement of test cultures
 
Three bacterial cultures were evaluated in this study. Burkholderia seminalis an endophytic bacteria was isolated from the root nodules of the vegetable cowpea [Vigna unguiculata (L.) Walp] plants and obtained from Electron Microscopy and Nanoscience Laboratory, Department of Soil Science, PAU, Ludhiana, India. The other bacteria, Burkholderia sp., was isolated from the decomposed paddy straw and obtained from the Department of Microbiology, Punjab Agricultural University, Ludhiana, Punjab, India. The molecular identification of this culture showed similarity with Burkholderia sp. STM1441 based on nucleotide homology and phylogenetic analysis. The reference bacterial culture, Bradyrhizobium sp. (NAIMCC-B-00260) was obtained from National Agriculturally Important Microbial Culture Collection from ICAR-National Bureau of Agriculturally Important Microorganisms (ICAR-NBAIM), Kushmaur, Mau Nath Bhanjan, Uttar Pradesh.
 
Bioprocess optimization for liquid microbial inoculants formulation and shelf life studies
 
Mother culture preparation
 
A loopful of pure cultures of Burkholderia seminalis, Burkholderia sp. and Bradyrhizobium sp. was inoculated in 100 ml of autoclaved nutrient broth dispensed in 500 ml flask respectively and incubated at 27± 1°C for 48 hours. Further, the growing cells of log phase were used for the further inoculation for each culture respectively.
 
Optimization of the growth medium for liquid microbial inoculants
 
This experiment was conducted to determine the optimum concentration of additives to be added to the basal medium for obtaining the highest population density of the bacteria. The freshly prepared mother culture of B. seminalis, Burkholderia sp. and Bradyrhizobium sp. were inoculated @ 1% to the flask containing 100 ml of autoclaved basal medium with different additives, designated from T₁ to T₁₇ respectively. There were a total of seventeen liquid microbial inoculants formulations for each test cultures. The treatments in detail were as in the Table 1.
 

        
Unamended basal medium was used as standard control. All the above inoculated flasks were kept on the shaker at 29+1°C for 48 hours and later shifted to stationary phase at room temperature for each culture respectively. One ml sample from each treatments were drawn on 5^th, 10^th, 20^th and 30^th days of storage for total viable counts by dilution pour plate method and plates were incubated at 28±l2°C for three days for B. seminalis and Burkholderia sp. and for five days for Bradyrhizobium sp.
 
Liquid microbial inoculants preparation for shelf life studies
 
For the preparation of liquid microbial inoculants of B. seminalis, Burkholderia sp. and Bradyrhizobium sp. The mother culture of each test culture was transferred @ 5% to each 250 ml flask containing 100 ml of sterilized standardised growth medium respectively and each 250ml flask containing 100 ml of sterilized basal medium without any additive and inoculated with respective test culture was used as control. The inoculated flasks were now kept on shaker at 28±1°C for 24 hours. They were then transferred into the sterilized 100 ml capacity plastic vials under aseptic conditions (Fig 1).
 

 
Preparation of charcoal carrier based microbial inoculants
 
Two sets of carrier based inoculants were prepared (Autoclaved and non-autoclaved charcoal) for each test culture. The charcoal carrier was sterilized by tyndalization process in an autoclave at 15 psi pressure and at temperature of 121°C for 20 minutes, three times on three succeeding days. The charcoal carrier based formulations were prepared by mixing the broth cultures of respective culture with charcoal powder at 1: 2.5 ratios (Fig 1). Broth cultures were prepared by inoculating a loopful inoculums of pure culture of B. seminalis, Burkholderia sp. and Bradyrhizobium sp. in sterilized 200 ml of nutrient broth dispensed in 500 ml flask respectively. The broth culture of respective bacterium was incubated until it reaches maximum cell population count.
 
Periodical monitoring of shelf life of liquid microbial inoculants and charcoal carrier based formulations
 
The plastic vials containing liquid microbial inoculants of B.seminalis, Burkholderia sp. and Bradyrhizobium sp. respectively were stored at room temperature. One ml of each sample was drawn aseptically at 0^th, 30^th, 60^th, 90^th, 120^th, 150^th and 180^th day for total viable count by dilution spread plate method and incubated at 28±2°C for three days for B. seminalis and Burkholderia sp. and five days for Bradyrhizobium sp. Similarly for charcoal carrier based formulations. The total viable count in the charcoal carrier based microbial inoculants was determined by adding 10 grams of inoculant to 90 ml of the sterilized distilled water and making a 10-fold dilution series. Then, one ml aliquots of the appropriate dilutions were spread plated on nutrient agar medium aseptically at 0^th, 30^th, 60^th, 90^th, 120^th, 150^th and 180^th day. Colonies were counted after incubation (28°C) of three days for B.seminalis and Burkholderia sp. and five days for Bradyrhizobium sp.
 
Insight of liquid microbial inoculant colonization of roots surface of forage cowpea under gnotobiotic condition by scanning electron microscopic studies
 
The roots of forage cowpea were examined for colonization by liquid microbial inoculants of B. seminalis, Burkholderia sp. and Bradyrhizobium sp. respectively by Scanning electron microscopic studies.
        
The cowpea seeds of variety CL-367 were first surface-sterilized with 2.5% sodium hypochlorite solution for five minutes, followed by 70% ethanol for five minutes and then rinsed continuously with sterilized water for eight times. Sterilized seeds were then treated with liquid microbial inoculants of B.seminalis, Burkholderia sp. and Bradyrhizobium sp. respectively and allowed to sit for 30-45 min in the sterilized petriplates. The treated seeds were placed aseptically in the test tubes containing 0.85% agar gel (0.85 g in 100 ml) using sterilized forceps respectively for each treatments i.e B.seminalis, Burkholderia sp. and Bradyrhizobium sp., In one set of the tubes sterilized untreated seeds were placed and it was used as control. Further for each treatment, a total of 5 test tubes were maintained. The tubes were then incubated at room temperature under light for 2 weeks. To further strengthen the impact of liquid microbial inoculants, periodical observation of root length was taken at 5 days interval.
 
Scanning electron microscopy (SEM)
 
Fresh fine root samples were fixed in 2.5-3.0% gluteraldehyde for 4 hours and washed in 0.1M sodium cacodylate buffer (3 changes) for one hour. Then, the roots were dehydrated by dipping in the increasing concentrations of acetone in water and finally washed in 100% acetone. The samples were dried using the tetra-methyl silane (TMS) method in which the specimens were immersed in TMS for 5-10 min for two changes at 4°C followed by drying at room temperature (25-26°C). The root samples were mounted on brass or aluminium stubs and coated using silver or gold (35 nm) thick before being examined and digested under SEM.

Bioprocess optimization for the formulation of liquid microbial inoculants
 
In the present study to enhance the shelf life of liquid formulation of microbial inoculants, additives like Poly vinyl alcohol, Trehalose, Poly ethylene glycol and Glycerol were exploited to standardise liquid formulation of microbial inoculants. The viability of all the three test cultures were evaluated for one month in the medium amended with different concentration of polyvinyl alcohol (PVA) (0.5%, 1%, 2% and 3%), trehalose (5 mM, 10 mM, 15 mM and 20 mM), polyethylene glycol (PEG) (0.5%, 1%, 2% and 3%) and glycerol (1%, 3%, 5% and 7%).
        
For B. seminalis, at 5^th day and 30^th day maximum growth and survival was observed in the basal medium amended with 5 mM trehalose (Fig 2). In the entire amended basal medium, B. seminalis showed superior growth over the control (without additives). Similarly, Burkholderia sp. and Bradyrhizobium sp. showed maximum count i.e 10.32 log ₁₀ no of viable cells/ml and 10.41 log ₁₀ no. of viable cells/ml respectively in basal medium amended with 5mM trehalose at 30^th day at room temperature (Fig 3 and 4). This might be attributed to the capability of trehalose in enhancing cell tolerance to desiccation, osmotic pressure and temperature stress (Streeter, 2003) and stabilizing both enzymes and cell membranes (Fillinger et al., 2001).
 

 

 

 
Periodical monitoring of shelf life of liquid microbial inoculant of B. seminalis at room temperature
 
The data pertaining to survivability of B. seminalis in liquid microbial inoculant without trehalose 5 mM (BST₁), liquid inoculant with trehalose 5 mM  (BT₂), autoclaved charcoal carrier based inoculant (BST₃) and non-autoclaved charcoal carrier based inoculant (BST₄) at room temperature has been presented in Table 2. Highest viable count of B. seminalis was observed in BST₂ followed by BST₃ and least vial count was observed in BST₁. However, microbial inoculants prepared using non-autoclaved charcoal carrier (BST₄) showed contamination throughout the study.
 
        
Initially liquid microbial inoculant with trehalose 5 mM (BST₂) showed 10.49 log₁₀ no. of viable cells which increased to 10.88 log₁₀ no. of cells at 90^th day (with increase of 3.71 per cent) and further decreased to 9.21 no. of log₁₀ cells at 180^th day (with decrease of 12.2 per cent) at room temperature. In autocalved charcoal based carrier, initial count was 10.31 log₁₀ no. of cells which reduced to 8.20 log₁₀ no. of cells at 180^th day (20.46% decrease) where as in liquid inoculant without trehalose (BST₁) initial count of 10.16 log₁₀ no. of viable cells were observed which decreased to 4.23 log₁₀ no. of cells at 180^th day (decrease of 58.36%) at room temperature. Microbial inoculants prepared using non-autoclaved charcoal carrier showed contaminated throughout the study period.
        
The results are supported by the findings of Soni et al., (2017) who reported that trehalose as additive maintained the viable counts of Burkholderia cepia (10.933- 6.77 log CFU/ml) throughout the year at room temperature.
 
Periodical monitoring of shelf life of liquid microbial inoculants of Burkholderia sp. at room temperature
 
The data pertaining to survivability of Burkholderia sp. In liquid microbial inoculant without trehalose 5 mM (BT₁), liquid inoculant with trehalose 5 mM  (BT₂), autoclaved charcoal carrier based inoculant (BT₃) and non-autoclaved charcoal carrier based inoculant (BT₄) at room temperature has been presented in Table 2. Highest viable count of Burkholderia sp. was observed in BT₂ followed by BT₃ and least vial count was observed in BT₁. Microbial inoculants prepared using non-autoclaved charcoal carrier (BT₄) showed contamination throughout the study.
 

        
Initially 10.62 log₁₀ no. of viable cells were observed which reduced to 9.28 log₁₀ no. of cells at 180^th day (with the decrease of 12.61 per cent) in liquid microbial inoculant amended with trehalose 5 mM (BT₂). In charcoal based carrier, initial count was 10.16 log₁₀ no. of cells which reduced to 8.24 log₁₀ no. of cells at 180^th day (18.89% decrease) where as in liquid microbial inoculant without trehalose (BT₁) initial count was 10.30 log₁₀ no. of cells which further decreased to 3.68 log10 no. of cells at 180^th day at room temperature (64.27% decrease).
            
These results were well corroborated with the results that indicate the growth of bacteria with trehalose in the medium resulted in 294% increase in survival (Streeter 2003).

Periodical monitoring of shelf life of liquid microbial inoculants of Bradyrhizobium sp. at room temperature
 
The perusal of the data in Table 2 indicated that at room temperature, the liquid microbial inoculant of Bradyrhizobium sp. amended with trehalose 5 mM (BRT₂) supported better viability as compared to liquid broth without trehalose 5 mM (BRT₁), charcoal based carrier (BRT₃) and non-autoclaved charcoal based carrier (BRT₄). Initially 10.23 log₁₀ no. of viable cells were observed in liquid microbial inoculant without trehalose 5 mM (BRT₁) which decreased to 4.11 log₁₀ no. of cells at 180^th day at room temperature (with decrease of 59.82 per cent). This may be due the stationary phase achievement and desiccation of cells. The liquid inoculant amended with trehalose 5 mM (BRT₂) initially showed 10.29 log₁₀ no. of viable cells which increased to 10.65 log₁₀ no. of cells at 90^th day which further decreased to 9.39 no. of log₁₀ cells at 180^th day (with decrease of 8.75 per cent) followed by autoclaved charcoal based carrier microbial inoculant of Bradyrhizobium sp. (BRT₃) with  10.18 log₁₀ no. of cells initially which decreased to 8.16 log₁₀ no. of cells (with decrease of 19.84 per cent) at 180^th day. Further, microbial inoculant prepared using non-autoclaved charcoal showed contamination throughout the study period.
            
Overall our findings in the present study were in line with Streeter, (2003) who proved feasible to add trehalose to the culture medium containing B. japonicum where trehalose was found accumulated in the cells during growth and further added that trehalose concentration in cells could be a useful predictor of survival during desiccation.
        
Hence, technology for the preparation of liquid microbial inoculants using trehalose as additive would be an attractive alternative to prepare microbial inoculants with enhanced shelf life.
 
Insight of root colonization of forage cowpea by liquid microbial inoculants under gnotobiotic conditions by scanning electron microscopic studies
 
In the present study, surface sterilized forage cowpea seeds were treated with liquid microbial inoculants of Burkholderia seminalis, Burkholderia sp. and Bradyrhizobium sp. and placed in agar gel assembly, incubated at 25±2°C for 15 days (Fig 5). After two weeks of incubation, the root surface of the germinated cowpea seedlings from each treatment was observed under scanning electron microscope following processing (Fig 5c, 5d and 5e).
 

        
Micrographs of SEM from root explant of seedling at 15^th day after inoculation with Burkholderia seminalis, Burkholderia sp. and Bradyrhizobium sp. showed root surface colonization of primary and lateral roots, root hair zone, lateral root junction, in root tissue crevices, elongation and discrimination zones of the root tips (Fig 6). Burkholderia seminalis colonized the root surface of cowpea seedlings non-uniformly occurring as aggregates or groups (arrows in Fig 6a). Root surfaces from Burkholderia seminalis inoculated seedlings were associated with fibrillar material, which resulted in the formation of “microcolonies” (Fig 6d). The nature of this fibrillar materials might be anticipated as secretion and deposition of the extracellular polymeric substances comprised of primarily polysaccharides and glycoproteins. Haas and Defago (2005) have also demonstrated that endophytes secrete secondary metabolites that allow them to colonize internal host tissue. Further, chronologically endophytes can be detected first on root surfaces followed by their presence in the internal root tissues. Further, colonization of root surfaces of forage cowpea seedlings by Burkholderia sp. using electron microscopy has provided topographical and morphological images of the colonization features of rhizobacteria. Scanning electron microscopy showed that Burkholderia sp. randomly colonized the root surface of cowpea seedlings as aggregates or as doublets and triplet cells (Fig 6b and 6e). These micrographs also revealed that Burkholderia sp. cells were densely distributed throughout the surface of the roots of the cowpea seedlings. Further, plant roots release plethora of root exudates (sugars, amino acids, organic acids and phenolic fractions) and result in significant stimulation of the microbial growth and activity.
 

        
Similarly, roots from 15 days old seedling inoculated with Bradyrhizobium sp. observed through SEM showed that the Bradyrhizobium sp. were able to colonize the surface of the roots (Fig 6c and 6f). SEM micrographs showed less number of rod shaped bacterial cells of Bradyrhizobium sp. on the root surface. The production of extracellular polysaccharide substance (EPS) or cell aggregation might have favoured the colonization process.
        
Root seedlings free of inoculant bacteria typically revealed no colonization of epidermal root surface (Fig 6g and 6h). Furthermore, the microflora associated with the roots affects plant growth and health. The ability to colonize plant roots can lead to increased biological nitrogen fixing activity for the plant). This study demonstrated that plant growth can be augmented by inoculation with liquid microbial inoculants of Burkholderia seminalis, Burkholderia sp. and Bradyrhizobium sp. On the 15^th day, maximum root length of forage cowpea was showed by Burkholderia seminalis (10.2 cm) followed by Burkholderia sp. (8.3 cm) and Bradyrhizobium sp. (7.6 cm) and uninoculated control (6.8). This might also be due to the production of phytohormones by the inoculants.
               
There upon, SEM has provided a comprehensive understanding of microbial inoculant colonization on the root surface of plants.

Liquid microbial inoculants (LMI) formulated in the present study is a viable and stable technology with improved shelf stability. Further, the scanning electron micrographs revealed the insight of plant-microbe interaction. Therefore, LMI of forage cowpea can play pertinent role in improving the yield and quality of forage cowpea in the neoteric era of bio evolution.