Viability of bacterial dried cells

The viability of bacterial cells after drying is improved by aging the culture in the stationary phase for periods of around six hours prior to drying. Further improvement may be obtained by culturing in a nitrogen deficient medium. The bacterial cells accumulate trehalose to protect the cells against drying out and other cell damage and the medium is further defined as having a higher than normal osmotic potential.

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 increase drying 
survival. For example, the BCA may be grown in a nitrogen-limited medium 
until in stationary phase (preferably well into stationary phase, for 
example six hours) and then heat shocked for 5-15 minutes prior to drying: 
this should give maximum drying survival. 
By way of example only, we describe a process for production by 
fermentation of a Pseudomonas fluorescens strain for use as a biological 
control agent (BCA). The following Examples use the biological control 
agent Pseudomonas fluorescens strain 54/96 (NCIMB 40186) as described in 
International Application Publication Number WO91/05475. 
Experiments investigating the effect of fermentation parameters have been 
carried out. Cells were grown in fermenters on tryptone soya broth (TSB) 
with and without various defined supplements and subjected to sublethal 
stresses before air drying on glass beads. 
All experiments were done using laboratory scale (131 volume) fermentation. 
The base medium was Tryptone Soya Broth (TSB). TSB contains in 1 liter: 17 
g tryptone (pancreatic digest of casein); 3 g soytone (papaic digest of 
soybean meal); 2.5 g dextrose; 5 g sodium chloride; 2.5 g dipotassium 
phosphate. TSB (C-limited) was supplemented with various media components 
to achieve increased drying survival. 
Drying survival was measured primarily using a glass bead test, although 
this is not a commercial process. Some data is presented on the effect of 
preconditioning using nitrogen starvation on the survival of P fluorescens 
in a laboratory scale spray dryer. 
Stationary phase cells survived better than growth phase cells; a sharp 
increase was observed at the transition point with optimum survival six 
hours into stationary phase. 
Stationary phase cells grown in N-limiting media showed 5-20 fold better 
survival than stationary phase cells grown in TSB which is C-limiting 
(both on the bead test and in a laboratory spray dryer). 
Short periods of heat shock (37.degree. C. for 5-10 minutes) resulted in 
enhanced survival although this was more pronounced in the otherwise 
susceptible log phase cultures (95 fold increase) than in stationary phase 
cultures (3 fold increase). 
These data suggest that a fermentation process giving maximum drying 
survival would involve growth of P fluorescens in a medium of TSB 
supplemented for nitrogen limitation (recipe below) until 6 hours into 
stationary phase followed by heat shock for 5-10 minutes prior to drying. 
It will be apparent to one skilled in the art that the processes described 
herein by way of example must be scaled-up for use in the commercial 
production of BCAs. This will involve a certain degree of optimisation to 
determine the exact parameters suitable for large-scale fermentation. 
However, the general principles of the invention (conditions or treatments 
which cause physiological adaptation such that cells are more able to 
withstand drying) are clearly applicable to small- or large-scale 
fermentation. 
In a preferred embodiment, therefore, the present invention provides a 
method of producing a dried viable cell mass of Pseudomonas fluorescens 
comprising culturing the said P. fluorescens in stationary phase in a 
nitrogen-deficient medium, isolating the cultured cells and drying same. 
Preferably the culture is subjected to heat shock prior to drying.

EXAMPLE 1 
DRYING AND CELL VIABILITY 
Cell samples were dried by coating onto glass beads in a dish which was 
left static for up to 5 days at 20.degree. C. Beads were sampled over a 5 
day period, washed in sterile distilled water (SDW), diluted serially, and 
plated on Tryptone soya agar (TSA) using spread plate or Spiral Plater. 
Plates were incubated in the dark at 28.degree. C. for 24 h. 
EXAMPLE 2 
FERMENTATION 
Fermenters used were Braun Biostat E, with a total volume of 18.3 l. 
Standard fermenter parameters were: Temperature controlled at 25.degree. 
C.; Dissolved Oxygen Tension (DOT) controlled at 50% by stirrer speed and 
valve setting; pH controlled at 7.0 using 2M H.sub.2 SO.sub.4 and 4M NaOH; 
antifoam controlled by additions of polypropylene glycol MW2025 (PPG 
2025). Medium was Tryptone Soya Broth (TSB) except where stated. Working 
volume was 13 l and a 24 h shake flask culture was used to inoculate at 
0.1%. 
2.1 GROWTH 
Biomass was routinely measured as optical density at 550 nm wavelength 
(OD.sub.550)in a Corning 258 spectrophotometer. The growth curve in a 
Braun Biostat was defined (FIG. 1): maximum specific growth rate 
(.mu..sub.max) estimated as 0.87; Doubling time(t.sub.d)=47 m; Growth 
yield=12 ODs. 
2.2 BCA ACTIVITY 
BCA activity of fermenter grown culture applied as a drench was compared to 
shake flask culture in a standard pot test for BCA activity. 11 h (log 
phase) and 35 h (stationary phase) fermenter cultures plus 24 h shake 
flask cultures were compared. No significant difference in control of 
Pythium ultimum was observed. 
EXAMPLE 3 
TREHALOSE ACCUMULATION AND LOSS 
Cell samples were extracted for sugar analysis. Shake flask samples were 
spun down (15 mins, 3,000 rpm, 4.degree. C. in Sorval RT6000B) washed in 
an isotonic NaCl solution spun again and re-suspended in 70% ethanol. This 
suspension was sonicated (24 microns in a Soniprep 150) until total cell 
disruption and the cellular matter removed by centrifugation. 
Analysis of intracellular extracts was by HPLC. Samples were deionised on 
Amberlite MB-3, dried by vacuum centrifugation (3,000 rpm, overnight in a 
Uniscience Univap). Dried samples were resuspended in deionised water 
filtered through 0.45 .mu.m PVDF filters (Gelman Acrodisc). 
Samples were analysed by HPLC (Waters Differential Refractometer Sugar 
Analyzer). Samples were run through filtered deionised water, with or 
without 5 mg/l Ca.sup.2+ EDTA, at 80.degree. C. with a flow rate of 0.5 
ml/min. The column was an Aminex HPX-42C (Biorad) and peak integration was 
on an IBM VG Data Systems-Minichrom v1.62) 
The predominant intracellular sugar was trehalose. In TSB cultures 
trehalose accumulated to a maximum of 2% of cell dry weight. In TSB 
amended with 0.5M NaCl trehalose accumulated to 11% of cell dry weight. 
Maximum trehalose content was found towards the end of the growth phase 
with a decline during stationary and decline phase (FIGS. 2A and 2B). 
Cells harvested from TSB+NaCl were resuspended in either water or isotonic 
NaCl solution. At intervals after resuspension cells were rapidly 
separated by centrifugation and both cells and supernatant analysed for 
trehalose. Cells washed in deionised water rapidly lost trehalose to the 
supernatant. 
3.4. THERMOTOLERANCE OF TREHALOSE ACCUMULATING CELLS 
Cell samples grown under osmotic stress were incubated at 50.degree. C. 
before and after washing in sterile distilled water. Unwashed `stressed` 
cells displayed greater thermotolerance than washed `stressed` cells. It 
is not clear from the current data whether stressed cells are 
significantly more thermotolerant than unstressed cells. 
Cells cultured to accumulate trehalose retained their via ability better in 
an accelerated shelf-life study. 
Cells were grown in TSB with and without NaCl. These cells were vacuum 
dried in the presence of formulation additives and varying concentration 
of NaCl. Samples of formulated material were stored at 37.degree. C. and 
tested for viability over a period of 76 days. 
Referring to FIG. 4, samples formulated using cells grown in TSB (nos. 1, 2 
and 3) showed a more rapid decrease in viability over 76 days. Samples 
formulated using cells grown in TSB+0.25M NaCl (7, 8 and 9) to accumulate 
trehalose showed a slower decrease in viability. Samples 1,4 and 7 were 
formulated with additives in water: samples 2, 5 and 8 were formulated 
with additives in 0.25M NaCl: samples 3, 6 and 9 were formulated in 0.5M 
NaCl. 
EXAMPLE 4 
HEAT SHOCK 
4.1 TEMPERATURE PROFILE 
Growth rates were determined over a temperature range of 20.degree. C. to 
37.degree. C. in TSB shake flasks. Optimum growth temperature was 
estimated at 28.degree.-29.degree. C. (.mu.=0.72). Growth rate at 
37.degree. C. was slow (.mu.=0.13); P fluorescens is known not to grow at 
41.degree. C., a diagnostic characteristic. Rapid cell death occurs at 
50.degree. C. 
4.2 HEAT TREATMENT OF GROWING CELLS 
Fermenter cultures were allowed to grow to late log phase at 21.degree. C. 
The temperature was raised rapidly (-3.degree. C. min.sup.-1) to 
37.degree. C. and held there. Samples were taken immediately prior to the 
temperature shift and at intervals throughout. Samples were tested for 
drying survival. 
Heat-shocked samples showed increases in survival up to 95.times. the 
pre-shock sample. The effect of heat-shock duration under these conditions 
has been examined in a number of experiments. The data shows an optimum 
exposure of 10-15 mins with some variation between experiments. There is 
evidence that the more severe the drying (i.e. longer incubation on the 
beads and hence lower moisture content) the smaller the effect. There is 
also some evidence to suggest that this decline in effect can be off-set 
to some extent by increasing the heat exposure. 
4.3 HEAT TREATMENT OF STARVED (STATIONARY PHASE) CELLS 
Cells in stationary phase survive drying much better than those in the 
growth phase; this will be dealt with below. Thus stationary phase 
cultures were heat shocked and tested for drying survival. The conditions 
were the same as in 4.2 except the cultures were 20-24 h old (a few hours 
into stationary phase). 
Some reproducible but limited increase in survival could be achieved using 
short periods of heat exposure (5-10 mins) with mild drying (24 h). The 
maximum effect was a 3-fold increase. This effect is reduced with longer 
drying times. Also, longer exposure to the heat-shock has a detrimental 
effect on survival. 
4.4 GEL ANALYSIS OF HEAT SHOCK PROTEINS(HSPs) 
Cell samples from the heat-shock experiments were analysed for 
intracellular protein content by polyacrylamide gel electrophoresis 
(PAGE). 
At least 6 different proteins were shown to accumulate during heat-shock of 
growing cells. There was no detectable protein accumulation in 
heat-shocked stationary phase cells. 
EXAMPLE 5 
STARVATION 
5.1 COMPLEX MEDIA 
Drying survival was related to growth phase of cultures growing on TSB in 
fermenters. Samples taken at time intervals during the fermentation were 
subjected to the bead test. There was a dramatic increase in survival rate 
at the transition between growth phase and stationary phase. Optimum 
survival occurred 6 hours into stationary phase (FIG. 3). 
5.2 NITROGEN LIMITATION 
Growth of P fluorescens in TSB is carbon limiting (C-limiting) i.e. 
stationary phase cells are starved of carbon. TSB was supplemented with 
varying concentrations of glucose, KH.sub.2 PO.sub.4 and MgSO.sub.4 in a 
constant ratio of 25:5:1 in an attempt to achieve N-starvation. 
Supplementation with 20 g/l glucose showed clear increases (2/3.times.) in 
bead test survival at all incubation times over a TSB control. Cultures 
were not apparently N-limited (no increase in OD after addition of 
NH.sub.4 Cl). 
With the higher levels of supplementation (40 g/l glucose), in the Biolab 
fermenters, N-limitation was achieved with a concomitant 5-8 fold increase 
in survival over the control (PS/073,074). An attempt to repeat this in 
Biostat fermenters failed to show an increase in survival on the bead 
test. This material was also spray dried with, again, no difference from 
the control (PS/091,092). Problems with the N-limited fermentation were 
identified as a possible cause of failure including temporary fall in pH 
to pH6, temporary fall in DOT and addition of excessive antifoam. 
Confirmation of this result was achieved in the 20 liter fermenters Drying 
survival on beads was clearly better with the N-limited culture. The data 
suggests improvements in % survival of 5-20 (FIG. 3) fold although the 
variability was high. Harvest time was more clearly defined than in 
previous experiments and was estimated at 4-5 hours into stationary phase 
for both fermentations. NB: it was more difficult to estimate this in the 
supplemented culture because the transition from growth to stationary was 
more gradual--this is typical. 
Washed and unwashed samples were spray dried. There was no significant 
difference in total CFUs recovered. However approximately 65% of unwashed 
N-limited cells and 35% of the washed was lost in the spray dryer due to 
sticking. Cell viability in terms of CFUs/g powder from 20 g wet weight 
starting material was of the order of 3-fold more for the N-limited 
samples. However recovery was low, although there was some evidence that 
this was improved by washing in SDW. 
TABLE 1 
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Product 
Viability 
Recovery 
Fermenter Treatment (CFU/g) (grams) 
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C-limited Unwashed 1.1 .times. 10.sup.10 
20.5 
Washed 2.7 c 10.sup.10 
17.9 
N-limited Unwashed 7.3 .times. 10.sup.10 
8.5 
Washed 5.6 .times. 10.sup.10 
13.2 
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