Optimizing Shelf-life of Pseudomonas fluorescens after Freeze Drying

Abstract

The excess use of chemical fertilizers diminishes soil fertility and yield from crops gradually. To regain and enhance our soil nutrients to get more yields, it is mandatory to rely on soil microbes. Some beneficial microbes’ termed as bio-fertilizers especially rhizosphere bacteria have well contribution in increasing plant growth, and yield without any toxins. It is a very natural process of interaction between plant and some microbes to increase the assimilation of nutrients and it helps plants to enhance better production. In this research, we showed the use of microbes especially Pseudomonas fluorescens survival in the soil. We applied different carriers such as dextrose, talc, and peat with freeze-dried P. fluorescens and studied the shelf-life of the P. fluorescens. Among different carrier’s peat with centrifuged cell suspension survived up to 60 days with significant CFU’s 2×107/gm CFU’s, our research will be a prospective to make new formulations and to increase the shelf-life and survival rate of soil microbes.

1. Introduction

The world observes so many challenges in the food and agriculture sector especially in consuming quality and nontoxic foods. The excessive use of chemicals and chemical fertilizers is to enhance food production causes soil crust, infertile, acidification, and heavy metals accumulation in soil. As a consequence, long-term effects have been created in the land such as soil erosion and environmental deterioration. The agricultural yield, production, and food security are being globally hampered hugely by the climate change [1]. Therefore, the world needs a natural sustainable solution that can encourage environment-friendly approach with boosting up sound ecology, biodiversity, and resilient form of agriculture [2]. Currently, agro-experts and researchers are looking for the solution with the implementation of local crops cultivation, varieties, and indigenous strategies. Some beneficial microbes’ termed as bio-fertilizers especially rhizosphere bacteria have well contribution in increasing plant growth, and yield without any toxins. It is a very natural process of interaction between plants and some microbes increase the assimilation of nutrients and it helps plants to enhance better production.

Bio-fertilizers are microbial-based fertilizers that provide essential NPK (Nitrogen, Phosphorous, and Potassium) nutrients to plants and reestablish the soil integrity. Various microorganisms are utilized as bio-fertilizers around the world such as Rhizobium, Bacillus spp., Mycorrhizae, Azotobacter, and Pseudomonas [3, 4].

Among other microbes, Pseudomonas spp is one of the notable bacteria which acts as bio-fertilizer as well as biocontrol agents. They play a dual role on crops; together they improve the plant growth, production, and resist the growth of pathogenic microorganisms. Alongside, they promote the establishment of other rhizosphere bacteria with roots [5]. Off all species of different Pseudomonas spp, researchers found P. fluorescens solubilize the insoluble phosphate source, mobilize them and increase the availability of phosphorus to plants [6]. They also enhance to assimilate nitrogen and produce phytohormones that encourage the vegetative growth of plants. P. fluorescens is well-known bio-fertilizer in agro-science, which has the ability to stimulate the plants growth that lives in contact with roots [7]. Researchers also proved that P. fluorescens resist the growth of pathogenic microorganisms of crop plants.

Using microbes as bio-fertilizers has a challenge for farmers due to their viability and survival in the soil. Many researchers investigated the viability and survival of the microbes which is used in organic farming. The researchers studied on various liquid- and solid-based microbial formulations as bio-fertilizers and their sustainability with significant results. Previous study showed that P. fluorescens bacteria can survive up to 6 months when glycerol was given in liquid formulation and it maintained the huge number of cells and viability well [8]. It was also found adding glycerol in nutrient broth media increased the survival of Pseudomonas cells which later showed efficacy against Fusarium oxysporum f. sp. cubense and Helicotylenchus multicinctus at multiple banana plantations [9]. A liquid formulation using humic acid for P. fluorescens has been developed which showed long shelf-life. The mixed formulation of humic acid along with P. fluorescens was used for crop protection and enhanced production [10].

The disadvantages of liquid bio-fertilizers are: it easily evaporates and diminishes their mode of action by sunlight. Solid bio-fertilizers (microbes with organic carrier materials) stabilize in sunlight and the chance of evaporation is less. Currently, there are different carriers such as peat, talc, corn flour, alginates, vermicomposte, and other organic materials. The solid bio-fertilizers are made by adding liquid colonies on to carrier. The difficulties of these carriers are they possess high moisture and humidity which causes microbial cell death.

Freeze drying or lyophilization of microbes and mixing with carrier may resolve this problem. So, here our research is intended to detect appropriate carrier particle and suitable technique to enhance the viability and sustainability of the microbes and to increase the shelf-life.

Although, P. fluorescens works very well as bio-fertilizers and has a good role in sustainable agriculture management, but the formulation and carrier materials are not fixed for long-time survival for solid formulation. Therefore, our focus also includes P. fluorescens enhanced production (more CFUs), to make suitable formulation and viability checking.

2. Materials and methods

Tryptone Type-1(Casitose Type-I) from Hi-Media laboratories, India; Yeast extract powder from Hi-Media laboratories, India; Sodium chloride from Merck, India; Dimethyl sulfoxide (DMSO) from Daejung, South Korea; skim milk powder (DANO) from Bangladesh; Agar powder from Merck, India; Dextrose from Hi-Media laboratories, India. And all other culture media were procured from Hi-Media laboratories, India and the reagents and chemicals from Merck, USA.

2.1 Isolation of Pseudomonas fluorescens

Soil sample was collected from rhizosphere of maize plant roots and brought to the lab. Then cetrimide agar media was prepared (pH 7.0 ± 0.2), autoclaved, poured, and 1.0 g soil was diluted in 0.9% saline solution. Diluted soil samples were spread on cetrimide agar plate and incubated for 72 hours. Vigorous growth colonies were picked up and subculture on LB plate. Light yellowish colonies were selected for the further process [11]. The strains were confirmed further by biochemical and 16 s rRNA genomic analysis.

2.2 Media preparation

Luria-Bertani (LB) a common media for microbial growth in the laboratory was used. The composition of media (g/l) is Tryptone 10 g, Yeast extract powder 5 g, and NaCl 10 g. pH was adjusted to 7.0 ± 0.2. LB Media was sterilized at 121°C, 15p si for 15 min. P. fluorescens strain was grown at 30°C in LB broth media [12, 13, 14] and was also maintained in paraffin at -86°C for further process.

2.3 Mother inoculum preparation

P. fluorescens (FLU-L) strain was routinely maintained in Luria-Bertani (LB) media. The broth media is used to prepare the mother inoculum for lab-scale production [14]. The 2% of previous cells were used as inoculum for lab-scale production. QC checked and recorded the CFUs.

2.4 PBS buffer (1X solution)

PBS buffer was prepared with the following composition (Table 1) and adjusted the pH to 7.0 Autoclaved the PBS buffer solution and cooled and stored at 4°C [15].

Reagents	g/l
NaCl	8 g
KCl	0.2 g
Na2HPO4	1.44 g
KH2PO4	0.24 g
Water	1 L
pH	7.0

Table 1.

Composition of the PBS buffer.

2.5 Lab-scale production

LB broth media was used for lab-scale production of inoculum [16]. We prepared 5 L for lab-scale production media, autoclaved at 121°C, 15 psi for 15 min, cooled and added 2% mother inoculum, and incubated at 30°C, 160 rpm, and 22 h at the shaker incubator. The media pH was kept at 7.0 ± 0.2. The growth of bacteria and CFU was analyzed.

2.6 Centrifugation

After incubation, the 5 L bacteria culture was aseptically transferred to centrifuge tubes for centrifugation. The centrifuge (CENLEE, Model: CFLR8, made in China) was precooled at 4°C and centrifugation was done at 4000 rpm for 5 mins [9]. After centrifugation, the pellet was collected and the supernatant was discarded. Repeated the same procedure twice with PBS buffer. The QC analysis was done by checking CFU of the pellet.

2.7 Pre-freezing

Different ingredients can be used as cryoprotectant and give good survival of bacteria. Here, we used 10% Skim milk and 1% Dimethyl sulfoxide (DMSO) as cryoprotectant [17]. The cell pellets and cryoprotectant ratio was maintained equally and mixed gently. Subsequently, pre-freezing of mixed cell pellets was initiated at 4°C for 2 hrs in the refrigerator, 4 hrs in-20°C and at last it was kept overnight at -86°C.

2.8 Lyophilization

Freeze drying process was completed in a lyophilizer machine [18]. The pre-freeze cell pellet from the −86°C was freeze dried in the lyophilizer (Bioevopeak, model: LYO80V-2S), made in China). Running time can be variable on the basis of sample amount. The freeze drying was done for 50 hrs. After 50 hours, the dry pellets were collected and stored at 4°C for further process. The QC analysis was done by checking CFU of the dried cells.

2.9 Formulation

Formulations of the lyophilized dried (LP) cells were mixed with different carrier particles such as dextrose, peat soil, and talc and kept it for shelf-life analysis. The CFU of the formulated products was checked initially and frequently with time intervals.

2.10 Dextrose formulation

The 5% and 10% freeze-dried microbes were mixed with 95% and 90% dextrose, respectively, and kept it for shelf-life analysis at room temperature.

2.11 Peat formulation

We used two different methods, that is, solid and liquid to make peat formulations. For solid formulations, we mixed 5% lyophilized microbes and 95% peat (with less than 5% moisture) and kept it at room temperature for shelf-life analysis. We also made liquid formulations by using cell pellets suspended in the following solution (Table 2) and mixed with the peat powder. Then 20 ml liquid pellet suspension was mixed with 70 g peat soil fine powder. Initially, the peat was grinded into fine powder, and pH was adjusted to pH 7 with calcium carbonate (CaCO3).

5% Dextrose	0.5 g
1% Tryptone	0.1 g
1% PVP	0.1 g
Pellet	10 g
Water	100 ml

Table 2.

Pellet solution preparation.

2.12 Talc formulation

The 5% and 10% freeze-dried microbes were mixed with 95% and 90% talc, respectively, and kept it for shelf-life analysis at room temperature.

2.13 Shelf-life analysis

After different formulations, we analyzed the finished products quality and its life span. We assayed our products periodically at 0, 7, 14, 30, 60, 90, 120, 150, and 180 days interval.

3. Results

3.1 Pseudomonas spp. morphology

Colony morphology and microscopic analysis of P. fluorescens were observed and recorded as shown below (Figure 1). P. fluorescens showed pale yellowish cream color with smooth moderate shape colonies. In microscopic, it showed a small rod shape, gram-negative bacteria.

Figure 1.

a) Pseudomonas fluorescens colonies in LB plate; b) Pseudomonas fluorescens showed gram-negative rod shape bacilli in gram staining.

3.2 Mother inoculum

P. fluorescens (FLU-L) showed yellowish turbid growth after 22 hrs incubation. We observed that the mother culture CFU was 1.0×10⁸/ml (Figure 2).

Figure 2.

CFU of mother inoculum.

3.3 Lab-scale production

The 5 L P. fluorescens grown flasks in LB media were observed for CFU and showed 1.0×10⁸/ml. Culture formed a yellowish appearance in LB broth (Figure 3).

Figure 3.

a) Broth culture in flask; b) broth culture in centrifuged tube; c) CFU of production broth.

3.4 Centrifugation

After centrifugation, the yield of the cells was 0.7–0.8% pellet from 5 L LB broth. Pellets were pinkish in color with smooth cream (Figure 4). The pellets CFUs showed 1.0×10¹⁰/ml.

Figure 4.

(a) Picture of pellet; b) CFU of pellet.

3.5 Lyophilization

The lyophilized microbial powder (LP) was light brownish color and it showed 6% moisture during harvesting. The CFU of LP was recorded as 1.0×10⁹ (Figure 5).

Figure 5.

(a) Lyophilization is running; (b) Lyophilized powder (LP); (c) CFU of LP.

3.6 Formulation

We analyzed dextrose formulations in two configurations, 5% and 10%. Both compositions showed 4.0×10⁷/gm (Figure 6) and 5.0×10⁷/gm (Figure 7), respectively, on the formulation day (Tables 3 and 4).

Figure 6.

Colonies in LB plate from 95% dextrose formulation.

Figure 7.

Colonies in LB plate from 90% dextrose formulation.

Formulation	Days	pH	Moisture	CFU/gm
5% LP + 95% dextrose	0	≈7.00	7.10	4.0 × 10^7

Table 3.

Dextrose formulation.

Formulation	Days	pH	Moisture	CFU/gm
10% LP + 90% dextrose	0	≈7.00	6.98	5.0 × 10^7

Table 4.

Dextrose formulation.

3.7 Solid peat formulation

The mixture of 5% LP and 95% peat was also resembled dextrose results. It showed 4.0×10⁷ /gm CFU’s (Table 5 and Figure 8).

Formulation	Days	pH	Moisture	CFU/gm
5% LP +95% peat	0	≈7.0	6.11	4.0 × 10^7

Table 5.

Solid peat formulation.

Figure 8.

Colonies in LB plate from solid peat formulation.

3.8 Talc formulation

Our results showed that the mixture of LP and talc as a carrier was void. There was no CFU’s recorded even after formulation (Table 6).

Formulation	Days	pH	Moisture	CFU/gm
5% LP +95% Talc	0	≈7.0	6.99	00.00

Table 6.

Talc formulation.

3.9 Liquid peat formulation (with pellet suspension)

The pellets mixed with suspension formulations and added with peat showed us increasing CFU’s 5.0×108/gm (Table 7 and Figure 9) than other formulations on the formulation day.

Formulation	Days	pH	Moisture	CFU/gm
70% peat+30% CaCO3 + 20 ml pellet suspension	0	≈7.0	17.48	5.0 × 10^8

Table 7.

Peat formulation with pellet suspension.

Figure 9.

Colonies in LB plate from liquid peat formulation.

3.10 Shelf-life analysis

In this research, we also revealed the shelf-life of LP microbes with different formulations such as dextrose, peat, and talc. Peat with centrifuged liquid cell suspension showed significant shelf-life up to 30 days. It showed 1.0×10⁷/gm CFU’s. Rather the 5% (Table 8 and Figure 10) and 10% LP (Table 9 and Figure 11) and 95% and 90% dextrose showed no colonies on 07 days’ shelf-life analysis. The LP and the peat mixture survived for 14 days with 4.0×10⁴/gm CFU’s (Table 10 and Figure 12). Previously, we showed that the talc formulations were not survived even a day and this proves that centrifuged pellet liquid suspension mixed with peat gives us the 60-day survival significant CFU’s (Table 11 and Figure 13).

Formulation	Days	pH	Moisture	CFU/gm
5% LP + 95% dextrose	0	≈7.00	7.10	4.0×10^7
	7	≈7.00	4.83	00.00
	14	≈7.00	6.91	00.00

Table 8.

Dextrose formulation shelf-life.

Figure 10.

Shelf-life colonies in LB plate.

Formulation	Days	pH	Moisture	CFU/gm
10% LP + 90% dextrose	0	≈7.00	6.98	5.0 × 10^7
	7	≈7.00	5.08	00.00
	14	≈7.00	8.44	00.00

Table 9.

Dextrose formulation shelf-life.

Figure 11.

Shelf-life colonies of 10% LP + 90% dextrose in LB plate.

Formulation	Days	pH	Moisture	CFU/gm
5% LP +95% peat	0	≈7.0	6.11	4.0 × 10^7
	07	≈7.0	6.29	2.0 × 10^5
	14	≈7.0	7.75	4.0 × 10^4
	30	≈7.0	5.83	00.00

Table 10.

Solid peat formulation shelf-life.

Figure 12.

Shelf-life colonies of 95% peat+5% LP in LB plate.

Formulation	Days	pH	Moisture	CFU/gm
70% peat+30% CaCO3 + 20 ml pellet suspension	0	≈7.0	17.48	5.0 × 10^8
	7	≈7.0	18.60	6.0 × 10^7
	14	≈7.0	20.22	4.0 × 10^7
	30	≈7.0	22.13	1.0 × 10^7
	60	≈7.0	18.96	2 × 107

Table 11.

Peat formulation with pellet suspension shelf-life.

Figure 13.

Shelf-life of 70% peat+30% CaCO3 + 20 ml pellet suspension in LB plate.

4. Discussion

The inclusive results of our experiments showed that P. fluorescens could survive initially with dextrose, solid peat, and talc carriers at room temperature. But P. fluorescens was not viable in dextrose carrier up to 1 week. Peat formulation was found as a good carrier for formulating product of P. fluorescens. When LP was mixed with peat powder, it was observed cell number 4×10⁴ per gram till 14 days. The best significant result was observed when peat was mixed with centrifuged cell pellet suspension; we observed 2×10⁷ CFUs per gram surviving more than 60 days. Peat is very common used carrier material globally and has high content of organic matter, water holding capacity, and simply obtainable [19]. In previous study, it was found that peat showed good efficacy in formulation, and help colonization in plant roots when peat was used for bio formulation of P. fluorescens [20]. Nakkeeran et al. also observed similar potential results of peat-based formulation for P. chlororaphis shelf-life [21]. Therefore, we also reveal that peat with less moisture is the suitable carrier for centrifuged cells suspension. Our research will be prospective to make new formulations and to increase the shelf-life and survival rate of soil microbes especially P. fluorescens.

5. Conclusion

From our results, we can conclude that centrifuged cells suspended in peat survived for long duration. Applying peat with suspended P. fluorescens may not hamper soil fertility, since peat is a universal bio carrier and sustains with all soil conditions. Even though we tried different carriers such as dextrose and talc, peat termed to be a suitable carrier for P. fluorescens.

In conclusion, although different types of carrier can be mixed with P. fluorescens for bioformulation, peat-based formulation showed good viability at room temperature.

Acknowledgments

We express our gratitude and thanks to our Managing Director and Director, Apex bio-fertilizers and bio-pesticides Ltd. for supporting and funding this research. We also extend our thanks to our product development lab members and their associates.