Viability of bacterial dried cells

Details Viability of bacterial dried cells Viability of bacterial dried cells

1995-05-12

1998-03-17

Wityshyn, Michael G.

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Preserving or maintaining micro-organism

424 9347, 435243, 4352521, 4352533, A01N 6300, C12N 100, C12N 104

Patent

active

057285748

DESCRIPTION:

BRIEF SUMMARY
This invention relates to processes for production of a biological control agent.
Various biological organisms (including bacteria and fungi) are known to possess general or specific anti-bacterial, anti-fungal (including anti-yeast), insecticidal and/or herbicidal activity. Such organisms have enormous potential as alternatives or additions to chemical agrochemicals, and are known as biological control agents or BCAs. Numerous naturally-occurring or genetically modified biological control agents are known, and more may be discovered or developed in the future. These BCAs may be mass-produced by means of large-scale fermentation.
An obstacle to the successful commercial exploitation of biological control agents (BCAs) is their formulation, which ideally involves desiccation. Drying bacteria (such as a Pseudomonas fluorescens BCA) in a commercial situation leads to losses in viability which can exceed 99%. One challenge to formulation technologists is to minimise these losses.
In work leading to the present invention, we have shown that the choice of fermentation parameters can affect the eventual success of the whole formulation process.
According to the present invention, there is provided a method of improving the viability of a dried bacterial cell mass comprising maintaining a culture of bacteria in stationary phase for a period of time and thereafter drying the bacteria.
The period of time for which the culture should be maintained in stationary phase to achieve maximum viability after drying may be determined by simple testing. A period the region of six hours is generally suitable.
Preferably also the culture is conducted in a medium deficient in nitrogen.
It is also advantageous that the culture medium has a higher than normal osmotic potential.
Viability after drying may be further improved by subjecting the culture to heat shock prior to drying. Exposure to a temperature of around 37.degree. C. for a period of from five to 15 minutes is generally suitable.
Preferably the bacterium is a Pseudomonas, more preferably Pseudomonas fluorescens. One particular strain of interest in this invention is Pseudomonas fluorescens, strain 54/96 (NCIB 40186). A culture of Pseudomonas fluorescens, strain 54/96 was deposited on Sep. 1, 1989 under the terms of the Budapest Treaty with the National Collection of Industrial and Marine Bacteria Limited, 23 St. Marcer Road, Aberdeen AB2 lRY United Kingdom, under the Accession Number 40186.
The production process of the invention pre-conditions the BCA to withstand drying processes that are an integral part of formulation of the product. The enhanced viability of the BCA improves the efficiency and effectiveness of the formulation process.
One or more of the following fermentation parameters are required for preconditioning the cells of the biological control agent:


(1) GROWTH PHASE

Drying survival depends on culture age. Maximum survival is achieved with stationary phase cultures (i.e. when the cells are starved of some nutrient). There is a dramatic improvement in survival at the critical point when cells enter stationary phase and an optimum survival after some period within the stationary phase (for example, at around 6 hours into stationary phase).


(2) NUTRIENT STARVATION

Nitrogen starved cells survive drying better than carbon starved cells.


(3) HEAT SHOCK

Cultures subjected to a short period of heat shock immediately prior to drying survive better. This increase in survival is particularly marked in log phase cultures but also occurs in stationary phase cultures.


(4) OSMOADAPTATION

Cells grown in media of high osmotic potential (for example, TSB with added NaCl, 0.5M) are shown to accumulate the sugar trehalose. Trehalose is known to have a protectant effect against desiccation and heat damage in many biological systems. Cells grown in this way have improved thermotolerance which may give an advantage in a drying process.
It is possible to apply one of more of the treatments listed in (1) to (4) above synergistically in a fermentation process to incre

REFERENCES:
patent: 3261761 (1966-07-01), Anderson et al.
patent: 5288634 (1994-02-01), Harman et al.
"The Stablizing Effect of Compatible Solutes on Drying and Storage of Escherichia Coli, " 6th European Congress on Biotechnology, Firenze, 13-17 Jun. 1993, Abs. vol. 1.
"Production of Conidial Biomass of Trichoderma harzianum for Biological Control," Biological Control, 1, p. 23 (1991).
"Endogenous reserves and survival of blastospores of Beauveria bassiana harvested from carbon- and nitrogen-limited batch cultures," Mycol. Res., 95(7), p. 821 (1991).
"Liquid Fermentation Technology for Experimental Production of Biocontrol Fungi," Phytopathology, 74(10), p. 1171 (1984).
Anders Persson et al "Physiological . . . ", Applied Env & Microb., Mar. 1990, pp. 686-692.

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