Optimizing Shelf-life of *Pseudomonas fluorescens* after Freeze Drying

*Nirmal Chandra Barman, Mohammad Sharif Sarker, Mahir Ahmed, Zahur Ahmed and Sankar Ramachandran*

### **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.

**Keywords:** *Pseudomonas fluorescens*, centrifugation, lyophilization, dextrose formulation, peat formulation, talc formulation, shelf-life

#### **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.
