Samples collection
Soil samples and pea seedlings were collected in Panevezys region, Lithuania. Soil samples were collected from field where wheat grew. The pea seedlings were uprooted from the pea field, seedlings were picked randomly. Isolation of microorganisms was performed the same day when the samples were collected. The experimental research on plants, including the collection of plant material, complied with relevant institutional, national, and international guidelines and legislation. The appropriate permissions and/or licenses for collection of plant were obtained for the study.
Isolation of diazotrophic microorganisms
During isolation of diazotrophic soil microorganisms 1 g of soil sample was added to 10 mL of sterile deionized water and suspended. The soil suspension was incubated for 20 minutes in a shaking incubator at 30°C temperature. After incubation suspension was diluted in 10-3; 10-4; 10-5; 10-6 series according to the serial dilution method and 100 µL of each dilution was plated onto solid NF media using spread plate method. Plates were incubated in bacteriological incubator at 30°C for 48 hours 17. Each isolate was tested for the growth on Ashby’s Mannitol 18, Winogradsky’s 19, NF 20, NFB 21 nitrogen-free agar media.
During isolation of diazotrophic microorganisms from pea root nodules, roots were washed under running tap water for 10 minutes, then using tweezers pink colour nodules were carefully taken from the roots. The nodules surface disinfected for 30 s in 70% ethanol solution and 5 times washed with sterile deionized water. Then nodules were treated for 1 min. in 3 % sodium hypochlorite solution and 10 times washed with sterile deionized water 22. Disinfected nodules were transferred to a tube with 5 mL sterile deionized water and homogenized using a sterile glass rod. Prepared bacterial suspension was diluted in 10-3; 10-4; 10-5; 10-6 series according to the serial dilution method and 100 µL of each dilution was plated onto solid NF media using spread plate method. Plates were incubated in bacteriological incubator at 30°C for 48 hours. Each isolate was tested for the growth on Ashbys Mannitol, Winogradsky’s, NF and NFB nitrogen-free agar media.
Endospore formation test
The ability of bacteria to form endospores was evaluated microscopically and by standard plating procedure. Suspensions of all strains were incubated in laboratory water bath at 75 ° C for 15 min and 1 mL of the heat-treated bacterial suspension was plated onto MPA agar medium and incubated in bacteriological incubator for 48 hours at 30 ° C. After incubation bacterial colonies were counted and phenotype traits were tested 23.
Molecular identification of isolated microorganisms
Isolates identification were carried out using partial 16S rDNA sequence analysis. All partial 16S rDNA sequences were determined by PCR with primers 8 F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) 24 and compared to the GenBank database in the National Center for Biotechnology Information (NCBI) using BLAST program. To determine the phylogenetic relationships phylogenetic tree was constructed using MEGA 5.0 software.
Vegetative growth promotion in wheat plants by Paenibacillus spp.
The vegetative pot experiment was conducted in 2019-2020, under regulated greenhouse conditions. Soil was used from organically managed field from the top 0–20 cm. The soil was a loamy Endocalcaric Epigleyic Cambisol (Drainic, Loamic) CM-can.glp-dr.lo 25 and was characterised as high fertility level with 3.9 % humus, 151 mg N kg-1, 93 mg P kg-1 and 156 mg K kg-1. The soil (5 kg dry weight pot-1) was filled in 8.5 l PVC pots. Two microbiological products of Paenibacillus sp. S1 and Paenibacillus sp. S7 were spread on the top soil in the form of water solution containing 400 mL H2O and 38 µL suspension of bacteria. It was 1.5 × 108 cfu per pot. Solution was applied two times: in the beginning and 3 weeks later. Only water applied on the control treatment. The spring wheat "Collada" was sown in pots, in five replications, 10 plants per each pot. Growth conditions in the greenhouse during the experiment were controlled: 16/8 h light/dark; photosynthetically active radiation at canopy level: 600 mol m2 s-1; temperature 20°C day and 16°C night time; irrigation of 200-400 mL per pot, 2 times a week.
Soil and plant analysis
The ammonium (N-NH4) in the soil was measured spectrophotometrically (with Cary 60 UV-Vis, USA) 4 days, 2 weeks and 2 months after first Paenibacillus spp. application. Mineral nitrogen (Nmin) content measured after 2 months as the sum of N–NO3 and NH4. Spring wheat in the pots were analysed during BBCH 37 (SPAD) and BBCH 87 growing stages for the grain yield per plant, thousand kernels weight (TKW), proteins in grain and kernels per spike. The concentration of protein and N yield in grain was measured using Kjeldahl method 26.
According to results of soil nitrogen changes and spring wheat experiments, accession number MT900581 and strain name MVY-024 for Paenibacillus sp. S7 in NCBI nucleotide bank were assigned.
Optimization of carbon and nitrogen sources for Paenibacillus sp. MVY-024 cultivation
Optimization of carbon and nitrogen sources is performed in shake-flask experiment using AF medium which was also used for inoculum preparation. Glucose, sucrose, glycerol, mannitol, molasses and starch were selected as a carbon sources and soybean, peptone, urea, ammonium sulphate, casein peptone, agropeptone, yeast extract and meat extract were used as a nitrogen source (Table 1). Taking into account that 100 g of cane molasses contains around 70 g of sugars, the molasses concentration was recalculated. During optimization of carbon source, the nitrogen source was selected yeast extract.
Table 1
Nitrogen and carbon sources optimization.
Carbon source optimization | Nitrogen source optimization |
Carbon source, g L-1 | Nitrogen source, g L-1 | Nitrogen source, g L-1 | Carbon source, g L-1 |
Control | 0 | Yeast extract | 0 | Control | 0 | Molasses | 0 |
Glucose | 40 | Yeast extract | 10 | Soybean peptone | 10 | Molasses | 57 |
Sucrose | 40 | Yeast extract | 10 | Carbamide | 10 | Molasses | 57 |
Glycerol | 40 | Yeast extract | 10 | Ammonium suphate | 10 | Molasses | 57 |
Mannitol | 40 | Yeast extract | 10 | Casein peptone | 10 | Molasses | 57 |
Molasses | 57 | Yeast extract | 10 | Agropeptone | 10 | Molasses | 57 |
Starch | 40 | Yeast extract | 10 | Yeast extract | 10 | Molasses | 57 |
| | | | Meat extract | 10 | Molasses | 57 |
Molasses concentration optimization | Yeast extract concentration optimization |
Control | 0 | Yeast extract | 0 | Control | 0 | Molasses | 0 |
Molasses | 25 | Yeast extract | 10 | Yeast extract | 5 | Molasses | 0 |
Molasses | 50 | Yeast extract | 10 | Yeast extract | 10 | Molasses | 0 |
Molasses | 100 | Yeast extract | 10 | Yeast extract | 15 | Molasses | 0 |
Molasses | 200 | Yeast extract | 10 | Yeast extract | 20 | Molasses | 0 |
Under sterile conditions 200 mL of sterile medium was poured to a 1 L Erlenmeyer flask and 2 mL of fresh Paenibacillus sp. MVY-024 inoculum was inoculated to the flask. The concentration of Paenibacillus sp. MVY-024 cells in the initial inoculum was 5.0 × 108 cfu mL-1. All samples using different carbon or nitrogen sources were incubated for 24 hours at 30 ºC, 130 rpm in a shaking incubator. After incubation, the number of bacterial cells in suspensions were determined on solid MPA (20 g meat extract, 5 g glucose, 10 g agropeptone, 20 g agar in 1000 mL deionized water, pH 6,6-7,0) medium according to the serial dilution-spread plate method. Plates were incubated in bacteriological incubator at 30°C for 48 hours.
After determination that sugarcane molasses and yeast extract are the best carbon and nitrogen sources for Paenibacillus sp. MVY-024 biomass production, the same shake-flask experiment was performed using different concentrations (Table 1).
pH and temperature optimization
To determine the optimum initial pH for Paenibacillus sp. MVY-024 biomass production, the pH of the medium was adjusted to the desired pH by adding 1 M HCl and 1 M NaOH before sterilization. pH values of AF medium: 6.0, 6.5, 7.0, 7.5, 8.0. 200 mL of sterile medium was poured to a 1 L Erlenmeyer flask and 2 mL of fresh Paenibacillus sp. MVY-024 inoculum was inoculated. All samples using different pH values were incubated for 24 hours at 30 ºC, 130 rpm in a shaking incubator. After incubation, the number of bacteria cells in suspensions was obtained on solid MPA medium according to the serial dilution-spread plate method. Plates were incubated in bacteriological incubator at 30°C for 48 hours.
After determination of optimal pH for biomass production, the same shake-flask experiment was repeated in the same conditions but using different temperature values: 28°C, 30°C, 32°C, 34°C, 36°C. Samples were diluted in serial dilutions, plated on MPA solid medium and were incubated in bacteriological incubator at 30°C for 48 hours. The number of bacterial colonies was counted.
Optimization of air flow
Paenibacillus sp. MVY-024 air flow optimization was performed in a fermenter (EDF 5.4_1). Cells culturing was performed in AF medium, using molasses and yeast extract as carbon and nitrogen sources in determined optimal concentrations. Based on the results of temperature and pH optimization, biomass culturing was performed at 32°C when the pH value was 7.0 ± 0.5. During fermentation process pH value of medium was adjusted by using automatic titration with 2 M NaOH and 2 M H2SO4 and antifoam was used to reduce foaming. Feeding was started after 8 hours of fermentation and was fed in to the bioreactor for 5 hours. Every hour 60 mL of feeding was used. During air flow optimization, the same partial pressure of oxygen and different air flow rates were selected. The partial pressure of oxygen in the medium 20±2, air flow rates: 0.1 vvm, 0.2 vvm, 0.4 vvm, 0.8 vvm, 1.2 vvm, 1.6 vvm and 2.0 vvm. The air to the bioreactor was supplied through a 0.2 µm pore size filter. The stirrer was set to automatic mode, from 45 to 800 rpm. 200 mL of inoculum and 3 L of NF medium were used for fermentation. During the process, parameters such as temperature, pH, agitation rate and partial pressure of oxygen were monitored and recorded. The optimal cell culturing time in bioreactors was about 70 hours, during this time all fermentable bacterial cells had to form endospores.
Statistical analysis
All statistical analyses were performed using SAS software version 9.4 (SAS Institute Inc., Copyright © 2002–2010). Graphical representation of data was performed using Microsoft Office 2013 software package. Homogeneity and normality were verified using Bartlett’s test. Experimental data were analysed by one-way analysis of variance (ANOVA) and mean comparisons between treatments were performed using Duncan’s mean separation test. The smallest significant difference R05 was calculated using a probability level of p<0.05.