Cryobiology 47 (2003) 21–29

www.elsevier.com/locate/ycryo

Optimisation of initial cell concentration enhances

freeze-drying tolerance of Pseudomonas chlororaphisq
Johan Palmfeldt, Peter R

adstr€
om, and B€ agerdal*
arbel Hahn-H€
Department of Applied Microbiology, University of Lund, P.O. Box 124, SE-22 100 Lund, Sweden
Received 5 February 2003; accepted 2 June 2003

Abstract

The freeze-drying tolerance of Pseudomonas chlororaphis, an antifungal bacterium used as biocontrol agent was
investigated. P. chlororaphis is freeze-drying sensitive and the viability drops more than 3 log units in the absence of
protective freeze-drying medium. Of the freeze-drying media tested, lactose, sucrose, trehalose, glutamate, sucrose with
glutamate, skimmed milk, and skimmed milk with trehalose, skimmed milk gave the lowest survival (0.6  0.2%) and
sucrose the highest (6.4  1.2%). Cellular accumulation of sucrose from the freeze-drying medium and the protective
effect of sucrose were dependent on sucrose concentration. The effect of initial cell concentration, from 1  107 to
5  1010 CFU/ml, on survival after freeze-drying was studied for carbon starved cells with sucrose as freeze-drying
medium. The highest freeze-drying survival values, 15–25%, were obtained for initial cell concentrations between
1  109 and 1  1010 CFU/ml. For cell concentrations outside this window more than 10 times lower survival values
were observed. P. chlororaphis was cultivated to induce stress response that could confer protection against freeze-
drying inactivation. Carbon starvation and, to a lesser extent, heat treatment enhanced freeze-drying tolerance. By
combining optimal cell concentration, optimal sucrose concentration and carbon starvation the survival after freeze-
drying was 26  6%.
Ó 2003 Elsevier Inc. All rights reserved.

Keywords: Freeze-drying; Gram-negative bacteria; Sucrose; Cryoprotectant; Pseudomonas chlororaphis; Stress; Storage; Lyophilization

Freeze-drying is a commonly used method to
preserve bacteria, in research as well as in industry.
Freeze-drying is suitable for production of con-
centrated bacterial cultures, with the advantage
q
This work was partly funded by The Swedish Agency for
Innovation Systems.
*
Corresponding author. Fax: +46-46-222-4203.
E-mail address: Barbel.Hahn-Hagerdal@tmb.lth.se (B.
Hahn-H€ agerdal).
that the dried material can be stored at ambient
temperatures [26]. However, freeze-drying dam-
ages the cells, which results in loss of viability
during processing as well as during subsequent
storage [8,19,24,28]. Different species display dif-
ferent degree of freeze-drying survival [11], with
Gram-negative bacteria often showing lower sur-
vival than Gram-positive bacteria [19]. In addition
to species, freeze-drying tolerance also depends on
freeze-drying medium [11,20], the physiological

0011-2240/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved.
doi:10.1016/S0011-2240(03)00065-8
22 J. Palmfeldt et al. / Cryobiology 47 (2003) 21–29
state of the cells [6], freezing rate [22], freeze-dry-
ing parameters, rehydration conditions [9], and
initial cell concentration [4,9].
Different physiological states are obtained by
harvesting the cells in different growth phases or
by transiently creating non-optimal growth con-
ditions. Freeze-drying survival is commonly found
to be higher for stationary phase cells than for
exponentially growing cells [24,26]. Stationary
phase, induced by carbon starvation, triggers a
general stress response, which involves induction
of a wide range of stress proteins [14]. Also tem-
perature stressed cells have been shown to have
higher freeze-drying survival than cells growing at
optimal temperature [6]. Temperature stress in-
duces general stress proteins, although a different
set compared with carbon starvation [3,12].
The freeze-drying medium should prevent freeze-
drying damage, improve storage stability, and fa-
cilitate rehydration. Commonly used freeze-drying
media contain protective solutes such as non-
reducing disaccharides, sugar alcohols, polysac-
charides, amino acids, proteins, and complex
mixtures such as skimmed milk [9,11,28]. The pro-
tective properties of the non-reducing disaccharides
sucrose and trehalose have been ascribed to inter-
action with proteins and membranes [10,18]. Fur-
thermore, the percentage freeze-drying survival has
been reported to increase with increasing initial cell
concentration up to concentrations of 1011 CFU/ml
[4,9,27]. As a minimal concentration 107 CFU/ml
has been recommended [25].
In the present study Pseudomonas chlororaphis,
a Gram-negative soil bacterium used as biocontrol
agent against seed-borne diseases [16,17], was in-
vestigated. P. chlororaphis was found to be sensi-
tive to freeze-drying and is therefore a suitable
model organism for investigations of parameters
that influence freeze-drying tolerance. Different
freeze-drying media, the concentration of protec-
tive solute in the freeze-drying medium, the phys-
iological state of the cells and the initial cell
concentration were investigated. Initial cell con-
centrations between 107 and 5  1010 CFU/ml were
studied at different levels of sucrose in the freeze-
drying medium. The importance of initial cell
concentration for freeze-drying tolerance is de-
scribed.
Materials and methods
Organism and growth
Pseudomonas chlororaphis MA 100, was kindly
provided by BioAgri, Uppsala Sweden. P. chloro-
raphis was cultivated in Tryptic Soy Broth, TSB
(Difco, MD) at 25 °C. Precultures of 50 ml were
grown in baffled Erlenmeyer flask at 200 rpm in a
rotary shaker (St
alprodukter, Uppsala, Sweden).
The cells were harvested by centrifugation, 5500g,
for 5 min at 20 °C (Sigma Laborzentrifugen 3K15,
Osterode am Herz, Germany), resuspended in
10 ml TSB, and inoculated into the fermenter cul-
tivation. The fermenter (B. Braun Biotech Inter-
national, Melsungen, Germany) had a working
volume of 1 L and was equipped with an electrical
heating jacket and temperature sensor. Aeration
was carried out by sparging with air at an initial
gas flow of 0.2 L/min, which was stepwise in-
creased to maximally 1.0 L/min. The ingoing gas
flow was controlled by mass flowmeters (El-Flow,
Insat, Switzerland). Air saturation was controlled
to 50% by automatic regulation of the stirring
speed, which was in the range 10–700 rpm, de-
pending on oxygen consumption rate. Cells were
harvested by centrifugation, 5500g, for 5 min at
20 °C, washed in pH 7 saline buffer containing
150 mM NaCl (Merck, Darmstadt, Germany) and
10 mM Pipes (Sigma–Aldrich, Steinheim, Ger-
many), and subsequently suspended in 1 ml freeze-
drying medium or in Millipore water as control.
The volume of the cell pellet was considered when
adjusting the solute concentration of the freeze-
drying medium in the final cell suspension.
Preparation of freeze-drying media
Lactose, sucrose, trehalose, glutamate (from
ICN Biomedicals, Aurora, OH), and skimmed
milk (Difco) were suspended in Millipore water
and autoclaved for 15 min at 1.2 bar overpressure.
Analyses
Cell growth was monitored as optical density
(OD) at 620 nm with water as reference (spectro-
photometer model Hitachi U1100, Tokyo, Japan).
 J. Palmfeldt et al. / Cryobiology 47 (2003) 21–29
The composition of the outgoing gas was contin-
uously monitored by a carbon dioxide and oxygen
monitor (Model 1308, Br€ uel & Kjær, Copenhagen,
Denmark) using photoacoustic and magnetoa-
coustic detection for CO2 and O2 , respectively. The
water activity of the dried material was measured
by a Water Activity Meter (Aqualab, Decagon,
WA). The water content of the dried material was
determined by heating it from 20 to 90 °C over
silica gel and letting it dry until constant weight.
Psysiological states
Exponentially growing cells were harvested at
OD620 1.0.
Carbon starved cells were harvested 30 min after
onset of stationary phase, which was caused by
carbon starvation. The onset of stationary phase
was detected when the level of carbon dioxide in
the outgoing gas no longer increased and when the
culture had reached its maximum optical density,
approximately OD620 2.5.
The type of limiting nutrient in the TSB growth
medium was investigated by addition of the car-
bon source glucose or the nitrogen source ammo-
nium sulphate to a stationary phase culture.
Optical density was subsequently monitored dur-
ing 4 h.
Heat treatment was conducted on exponentially
growing cells in a 1-L fermenter when the culture
had an OD620 of 1.0. The stirring rate was above
200 rpm to ensure a sufficient heat transfer. The
culture was heated from 25 to 34.5 °C in 8 min and
held for 15 min at 34.5 °C. The cells were harvested
directly after the heat treatment.
Maximum growth temperature for P. chloro-
raphis in TSB medium was determined in shake
flask cultures as logarithmic increase of optical
density for several generations.
Freezing, freeze-drying, and storage
Aliquots of 1 ml cell suspension were frozen in
15-ml plastic tubes (Sarstedt, N€ umbrecht, Ger-
many) at )80 °C, with an average cooling rate of
4 °C/min. The frozen samples were freeze-dried for
17 h at 0.3 mbar on a manifold freeze-dryer
(Hetosics, Birkerod, Denmark). The time for pri-
23
mary drying was about 6 h, the condenser tem-
perature )50 °C and the final sample temperature
20 °C. For storage tests the samples were dried in
plastic coated aluminium bags, which were sealed
at normal atmosphere and stored at +8 °C.
Determination of cell viability
The number of viable cells before freezing, after
freezing, and after freeze-drying was determined as
colony forming units (CFU). Prior to CFU de-
termination, freeze-dried samples were rehydrated
by addition of 10 ml, pH 7, saline buffer (150 mM
NaCl and 10 mM Pipes) followed by 15-min in-
cubation at 25 °C. Saline buffer (10 ml) was used as
rehydration medium instead of distilled water to
get normalized rehydration conditions indepen-
dently of the freeze-drying medium used. The cell
suspension was diluted in pH 7-saline buffer and
100 ll was thereafter plated on TSB agar. For
freeze–thaw survival analysis, the sample was
thawed at room temperature, which took 15 min,
incubated for another 15 min, and then diluted.
The diluted cell suspension was distributed on the
agar by using five spherical glass beads (flint glass
with 6-mm diameter, from Merck, Darmstadt,
Germany) per plate and gently moving the plate
until the cell suspension was distributed and ab-
sorbed by the agar. The number of colonies was
counted after 48-h incubation at 25 °C. Survival
rate was calculated as colony forming units per
milliliter after freeze-drying divided by colony
forming units per milliliter before freezing.
Estimation of intracellular sucrose concentrations
To determine intracellular sucrose accumula-
tion, cells were resuspended in [U-14 C]sucrose
(Amersham Pharmacia Biotech, Buckinghamshire,
UK). Solutions with 50, 25, 10, and 5 g/L sucrose
were prepared by dilution of a stock solution with
100 g/L sucrose and 400 counts per minute (CPM)/
nmol. Cell suspensions with 5  109 CFU/ml were
incubated at 25 °C for 5 min, the time it normally
takes to resuspend the samples, in sucrose solu-
tions labeled with [U-14 C]sucrose. The samples
were subsequently filtered through a glass micro-
fibre filter with a diameter of 25 mm (Whatman
24 J. Palmfeldt et al. / Cryobiology 47 (2003) 21–29

international, Maidstone, UK) and washed three ing was determined (Table 1). For sucrose and
times with 2 ml unlabeled sucrose solution with the skimmed milk samples the final water activity was
same concentration as in the incubation mixture. 0.13  0.02 and 0.08  0.01, respectively. The
Blanks were analyzed in the same way but instead moisture content was about 6.4% for the sucrose
of incubation they were immediately diluted 50 samples and 3.3% for the skimmed milk samples.
times and filtered. The filters, in duplicate for both The survival in 200 g/L skimmed milk (0.6%) was
blank and sample, were counted on a Rackbeta very much lower than the survival in 100 g/L of the
Liquid Scintillation Counter 1219 (LKB Wallac, three disaccharides investigated (4.2–6.4%). Mix-
Turku, Finland). The value of the blank was tures of protective solutes in the freeze-drying
subtracted from the value of the sample. From medium did not result in higher freeze-drying
knowing the cell concentration and assuming a cell survival than sole disaccharides did. Sucrose so-
volume of 1 fl, intracellular sucrose concentrations lutions were chosen as freeze-drying medium for
could be calculated. The volume of 1 fl is the vol- the following experiments.
ume of a cylinder with radius 0.33 lm and a length The effect of sucrose concentration on freeze-
of 3 lm, which is in accordance with the size of drying survival was investigated for concentrations
P. chlororaphis MA 100 that was measured in light up to 300 g/L (Fig. 1). Up to 50 g/L, increased
microscope. sucrose concentration resulted in increase in
freeze-drying protection. The highest survival rates
Sucrose metabolism were obtained with sucrose concentrations in the
range 50–130 g/L sucrose. At 300 g/L sucrose the
Experiments were carried out to determine survival was lower than with 50–130 g/L sucrose.
whether the sucrose in the freeze-drying medium The structure of the freeze-dried material was
could be metabolized or not. different for the 300 g/L sucrose sample compared
Extracellular degradation of sucrose was in- to the ones with lower sucrose concentrations. The
vestigated for cells resuspended in 100 g/L sucrose. 300 g/L sample had a continuous structure with
After 10 min incubation cells were removed by bubbles and was difficult to resuspend, whereas the
5-min centrifugation at 5500g and 20 °C. The cell
supernatant was incubated at 25 °C and samples Table 1
were taken during 2 h and analyzed by HPLC. Survival of exponentially growing cellsa after freeze-drying
Separation was carried out on an Aminex HPX- when different freeze-drying media were used
87H-column (BioRad, Hercules, USA) at 45 °C Freeze-drying Concentration Survival after
and detection with a refractometer (Shimadzu medium (g/L) freeze-drying (%)b
RID-6A, Kyoto, Japan). The mobile phase was Controlc — 0.08  0.03
5 mM sulfuric acid, 0.6 ml/min. Lactose 100 4.2  0.9
Growth of P. chlororaphis on sucrose was in- Sucrose 100 6.4  1.2
vestigated by adding sucrose to a concentration of Trehalose 100 4.7  1.3
10 g/L to a carbon-starved culture. Optical density Glutamate 100 1.0  0.3
Skimmed milk 200 0.60  0.2
was subsequently monitored during 2 h.
Sucrose + 100
glutamate 41 2.4  0.5

Results Skimmed milk + 150

trehalose + 50 4.3  0.5
ascorbic acid 1
Effect of freeze-drying medium
a
Initial cell concentrations were in the range 3–
5  109 CFU/ml.
When P. chlororaphis was freeze-dried without b
Averages and standard deviations were calculated from at
protective agents the viability dropped more than least three independent freeze-drying analyses.
3 log units. Cells were freeze-dried in different c
Control sample contained cells suspended in Millipore
freeze-drying media and survival after freeze-dry- water.
 J. Palmfeldt et al. / Cryobiology 47 (2003) 21–29 25

present at rehydration did not influence the sur-

vival.

Effect of heat treatment and carbon starvation

The influence of non-optimal growth conditions

on freeze-drying survival was investigated by ex-
posing P. chlororaphis to sublethal heat treatment
and carbon starvation. P. chlororaphis was able to
grow at 34 °C but not at 36 °C. Based on this a
Fig. 1. Freeze-drying survival versus sucrose concentration. sublethal heat treatment at 34.5 °C was chosen for
The cells come from carbon starved cultures. Initial cell con-
centrations were in the range 3–7  109 CFU/ml. Bars represent
heat treatment experiments. In TSB medium sta-
standard deviation of three independent cultivations with each tionary phase was induced by carbon starvation,
three freeze-drying analyses. since the growth was restored by glucose addition
but not by addition of ammonium.
Exponentially growing cells, carbon starved
others had a porous structure. When pure sucrose
cells and heat treated cells, were suspended in
solutions were freeze-dried, 136 g/L yielded the
100 g/L sucrose, freeze-dried, and cell survival was
porous structure whereas 150 g/L yielded the
determined (Table 2). The lowest survival (8.7%)
structure with bubbles.
was obtained for exponentially growing cells
[U-14 C]Sucrose was used to estimate intracel-
without heat treatment. With heat treatment ex-
lular sucrose accumulation. The intracellular su-
ponentially growing cells had 15.8% survival.
crose concentration increased with increasing
Carbon starved cells, with and without heat
sucrose concentration in the suspension (Fig. 2).
treatment, gave the highest survival, 25 and 26%,
Sucrose was not hydrolyzed by extracellular en-
respectively. The survival of exponentially grow-
zymatic activity as determined by HPLC in culture
ing, heat-treated, and carbon starved cells was also
supernatant. Neither was P. chlororaphis able to
determined for cells resuspended in 200 g/L skim-
use sucrose as carbon source, since addition of
med milk, which resulted in 0.62  0.23, 0.71 
sucrose to a carbon starved culture did not restore
0.58, and 5.9  1.5 % survival, respectively. Thus,
growth.
also with skimmed milk as freeze-drying medium
To clarify if the sucrose concentration also in-
carbon starved cells had the highest survival
fluenced rehydration, samples were freeze-dried in
value.
a solution with high sucrose content (100 g/L) and
subsequently rehydrated at low concentration
Effect of initial cell concentration
(5 g/L) and vice versa. The sucrose concentration
Samples with cell concentrations ranging from
1  107 to 5  1010 CFU/ml were freeze-dried to

Table 2
Percentage survival after freeze-drying for different combina-
tions of physiological statea;b
No heat Heat
Exponentially growing 8.7  1.9 15.8  6.6
Carbon starved 26.0  6.0 25.1  5.5
a
Initial cell concentrations were in the range 3–
6  109 CFU/ml. The freeze-drying medium was a 100 g/L su-
crose solution.
b
Fig. 2. Estimated intracellular concentrations of sucrose as a Averages and standard deviations were calculated from six
function of sucrose in cell suspension. values; from two cultivations with each three analyses.
26 J. Palmfeldt et al. / Cryobiology 47 (2003) 21–29
Fig. 3. Freeze-drying survival versus initial cell concentration.
The cells come from carbon starved cultures. Symbols: r 10, j
30, m 50, and  100 g/L sucrose, respectively. Bars represent
standard deviation of three independent freeze-drying analyses.
determine the influence of the initial cell concen-
tration on survival rate (Fig. 3). This was carried
out for four different sucrose concentrations: 10,
30, 50, and 100 g/L. The same pattern, with max-
imum survival rates at colony forming units per
milliliter between 1  109 and 1  1010 , was seen for
all sucrose concentrations. However, the lowest
sucrose concentration protected at most 2% of the
cells whereas the highest sucrose concentration
protected up to 25% of the cells. For all sucrose
concentrations a drop in survival was seen when
the colony forming units per milliliter exceeded
1  1010 CFU/ml.
Survival after freeze–thaw challenge
The freezing step is part of the freeze-drying
procedure and the viability after freezing and
subsequent thawing was also assessed. When the
cells were frozen in saline buffer without protective
substance the freezing step caused a viability drop
of 2 log units. Thus P. chlororaphis is not only
sensitive to freeze-drying but also to freezing.
However, the freezing step had only little effect on
viability when skimmed milk (200 g/L), sucrose,
lactose, or trehalose (100 g/L) was present and the
initial cell concentration was higher than
5  108 CFU/ml. Under these conditions expo-
nentially growing cells had survival rates between
55 and 80% and carbon starved cells had survival
Fig. 4. Effect of freeze-drying and storage on survival of
P. chlororaphis. Storage was carried out at 8 °C, in dark bags
sealed at normal atmosphere. Bars represent standard deviation
of three independent freeze-drying analyses.
rates between 77 and 100%. At initial cell con-
centrations below 5  108 CFU/ml and sucrose
concentrations of 30–100 g/L the survival values
after freezing and thawing were between 18 and
38%.
Storage test
For the storage tests the samples were prepared
under conditions favoring freeze-drying survival:
5  109 CFU/ml, 100 g/L sucrose and carbon-
starved cells. The viability after freeze-drying was
24%. After 50 days storage at +8°C the viability
was reduced to 6% and after 200 days to 2%
(Fig. 4).
Discussion
It has previously been observed that low initial
cell concentration is detrimental for freeze-drying
survival [4,9] and the present study confirms that
this is the case also for P. chlororaphis. The opti-
mal initial cell concentration was between 1  109
and 1  1010 CFU/ml when sucrose was used as
protective solute. The sucrose concentration in-
fluenced the optimal cell concentration, so that the
highest survival at 30 g/L sucrose occurred at
higher cell concentration than at 100 g/L sucrose.
The maximum freeze-drying survival occurred in a
rather narrow window of initial cell concentration
and this has to the best of our knowledge not been
described previously. The practical implication of
 J. Palmfeldt et al. / Cryobiology 47 (2003) 21–29
these results is that an exaggeration of the cell
concentrating procedure may lead to decreased
yield of viable cells after freeze-drying. The ob-
served phenomenon might be species-dependent,
since Lactococcus lactis has a survival of 100%
when freeze-dried at an initial cell concentration as
high as 2.5  1011 CFU/ml [27].
Also for freeze-thawing the survival was reduced
for samples with low cell concentrations (<5 
108 CFU/ml). This indicates that the freezing pro-
cess is different at low cell concentrations compared
with intermediate (5  108 –5  1010 CFU/ml) and
high (>1010 CFU/ml) cell concentration, where the
freeze–thaw survival was in the same range. For
Escherichia coli cells the percentage surviving
freeze-thawing is increased with an increase in cell
concentration, which was attributed to protective
substances released from lyzed cells [5].
The protective effect of disaccharides on freeze-
drying survival has been attributed to their ca-
pacity to hydrate biological structures, such as
proteins and membranes, referred to as the water
replacement hypothesis [2,10]. Thus a protective
solute must be present also inside the microbial cell
to offer protection. Using [14 C]sucrose we could
show that intra- and extracellular sucrose con-
centrations for P. chlororaphis were similar, so that
sucrose accessed the cytoplasmic matrix before the
cells were frozen. P. chlororaphis MA100 did not
use sucrose as a carbon source for growth and did
not hydrolyze sucrose to monosaccharides extra-
cellularly. Thus it seems like sucrose is taken up in
the disaccharide form and that the cells do not
hydrolyze the sucrose further. In Lactobacillus
plantarum the intracellular and extracellular su-
crose concentrations are in equilibrium [13] and
Pseudomonas mendocina is able to take up sucrose
as a compatible solute when exposed to high su-
crose concentrations [23]. The present results sug-
gest that also P. chlororaphis uses sucrose as a
compatible solute.
When increasing the sucrose concentration up
to 50 g/L the survival increased (Fig. 1). Similar
results have been obtained for the yeast Candida
sake, which in 10, 50, and 100 g/L sucrose had
survival values of 0.7, 6.2, and 11.4%, respectively
[1]. Sucrose has also been used in freeze-drying
media at concentrations of 36 g/L for E. coli and
27
Bacillus thuringiensis [18], at 100 g/L for Pantoea
agglomerans [9] and at 120 g/L by The American
Type Culture Collection [25]. For P. chlororaphis
the highest sucrose concentration (300 g/L) re-
sulted in lower survival rate. These samples had a
structure with bubbles, indicating sample melting
followed by sucrose crystallization. For trehalose
crystallization during sublimation has been shown
to diminish the availability of the sugar as pro-
tective agent during freeze-drying [7].
Pseudomonas chlororaphis was exposed to car-
bon starvation and heat treatment prior to freezing
and freeze-drying to induce stress response aimed
at protecting the cells during freezing and freeze-
drying. Both carbon starvation and heat treatment
increased the freeze-drying survival when exercised
separately. However, when carbon-starved cells
were heat-treated the freeze-drying survival did
not increase further. At temperatures close to the
maximum growth temperature a heat shock re-
sponse is induced, which protects vital cellular
functions [6,12]. Heat treated L. lactis cultures
have higher freeze-drying survival than growing
cultures have [6]. For Pseudomonas putida a heat
shock response protects the cells against heat as
well as against ethanol, H2 O2 , and osmotic stress
[12]. However, carbon starvation in P. putida in-
duces a more pronounced protective stress re-
sponse than heat treatment does.
Besides carbon starvation and heat treatment,
P. chlororaphis was also exposed to cold treat-
ment, 3 °C for 40 min, and osmotic stress, 0.5 M
NaCl for 15 min, although without improved
freeze-drying survival (data not shown). All the
stress inducing conditions—carbon starvation,
heat treatment, cold treatment, and osmotic
stress—resulted in growth arrest of P. chlororaphis,
but only carbon starvation and to some extent
heat treatment had positive effect on freeze-drying
survival.
Freeze-drying survival data for micro-organ-
isms show great variation, which reflects the nu-
merous factors influencing this process: species
[19], cell concentration [4,15], freeze-drying me-
dium [9,11,20], physiological state [6,26], freezing
rate [22], freeze-drying parameters [26], and rehy-
dration [9]. However, industrially used bacteria,
such as lactic acid bacteria, typically show survival
28 J. Palmfeldt et al. / Cryobiology 47 (2003) 21–29
values above 50% when dried in the presence of a
protective solute [11,21,28] and above 1% when
dried in the absence of protective solute [18,20,28].
The survival values of 26% in the presence of a
protective solute and below 0.1% in its absence
show that P. chlororaphis is rather sensitive to ly-
ophilization.
In conclusion, the present study shows that
freeze-drying medium, carbon starvation, and the
initial cell concentration contribute to enhanced
freeze-drying survival and if one of them is sub-
optimal the survival decreases. Initial cell concen-
tration outside the window between 5  108 and
1  1010 CFU/ml was shown to be especially det-
rimental.
Acknowledgments
We thank Bioagri, Uppsala, Sweden for sup-
plying us with the strain Pseudomonas chlororaphis
MA 100 and Marco Paese for excellent technical
assistance.
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