Journal of Applied Bacteriology 1985, 58, 517-524 1564/04/84

Production and storage of Rhizobium leguminosarum cell

concentrates for use as inoculants

J . MEADE,P. H I C G I N S& F. O'GARADepartment of Microbiology, University College,

Cork, Ireland

Received 18 April 1984 and accepted 8 October 1984

M E A D E ,J . , HIGGINS,P. & O'GARA,F . 1985. Production and storage of

Rhizobium leguminosarum cell concentrates for use as inoculants. Journal of Applied
Bacteriology 58, 517-524.
Recovery of Rhizobium leguminosurum cells by centrifugation after growth in an
industrial fermenter was 100-fold higher when cells were grown on yeast extract (5
g/l) as sole source of carbon and nitrogen when compared with the yields recovered
when cells were grown in standard mannitol-yeast extract medium. Methods of
storing concentrated suspensions of R. leguminosarum were investigated. Freeze-
drying caused a marked decrease in viable cell numbers. Viable cell numbers of
bacterial concentrates stored in peat decreased steadily from 10"-10'* cfu/g to lo9
cfu/g or less during 26 weeks storage at room temperature or at 4C. Cell concen-
trates stored in 40% glycerol at -20C maintained viable numbers higher than
10" cfu/ml during a 76 week storage period.

Leguminous plants can be rendered self-
sufficient for some or all of their nitrogen
requirements by symbiotic association with root
nodule bacteria. Soils may contain no rhizobia
or high proportions of inefficient or non-
nitrogen fixing rhizobia (Peterson & Loynachan
1981). In these circumstances, in order to derive
maximum benefit from symbiotic nitrogen fix-
ation, inoculation of seed or soils with efficient
natural or genetically engineered rhizobial
strains is desirable.
Successful inoculant production requires the
use of competitive nitrogen-fixing strains and
also the most efficient use of available pro-
duction technology. The maintenance of high
viable numbers of Rhizobium between pro-
duction and application is also an important
factor in the successful use of inoculants.
Current practice involves the addition of rhizo-
bial cells to protective carriers, usually peat,
which prolong rhizobial viability during storage
(Roughley & Vincent 1967; Strijdom & Des-
chodt 1976). Such preparations generally
contain a minimum of 109cfu/g on manufacture
(Thompson 1980). A concentrated culture for
making inoculants which maintains constant
viable numbers over long periods, would be
desirable for several reasons. Concentrated
preparations of cells can be produced well in
advance of seasonal demands and cell counts
standardized (Porter 1969). Stocks of concen-
trated cells can be stored and transported easily
from the fermentation plant to the site of inocu-
lant manufacture or application. They can be
used in the manufacture of different types of
inoculants, e.g. soil inoculants (both granular
and liquid), seed inoculants, peat based, freeze
dried or liquid. Frozen concentrates of Rhizo-
bium strains have been used in the preparation
of inoculants particularly in North America
(Porter 1969). Considering the advantages they
offer, however, there is little information avail-
able on the survival of concentrated rhizobial
suspensions.
In this study, the industrial-scale production
and concentration of an inoculant strain of Rhi-
zobium leguminosarum was investigated and the
feasibility of storing rhizobial concentrates (i) by
freeze drying, (ii) in peat and (iii) in glycerol at
- 20C was assessed.
518 J . Meade et al.
Materials and methods
RHIZOBIAL STRAIN
A streptomycin (100 Fg/ml) resistant derivative
of R. leyuminosarum C53 (obtained from D.
Phillips, University of California, Davis, USA)
was used in all experiments.
MEDIA
The basal salts medium of OGara & Shanmu-
gam (1976) was used in all growth experiments.
Cultures were routinely grown on the above
medium (MSY) containing (g/l): mannitol, 1 ;
and yeast extract (Difco) 1. The final pH of the
medium was 6.8. Agar was added (15 g/l) for a
solid medium.
PRELIMINARY GROWTH EXPERIMENTS
Agar cultures were used to inoculate liquid
media (10 ml) in screw capped bottles (25 ml)
which were incubated at 30C in a rotary incu-
bator for 48 h. In different experiments, the
mannitol concentration was increased (to 5 g/l)
or was replaced by industrial grade molasses
sugar at concentrations of (v/v) 0.1%,0.5% or
1.00/;,. Industrial grade yeast extract (1 g/1 or 5
g/l) served as sole source of carbon and nitrogen
in some experiments.
Extracellular polysaccharide production was
assessed visually using cultures streaked on the
various test media (15 g/l agar) and incubated
for 3 d at 30C.
Growth was followed by measuring the
increase in absorbance at 420 nm in liquid cul-
tures (100 ml) inoculated from freshly grown
MSY cultures (2% inoculum). Viable numbers
(after 45 h growth) were determined by spread-
plating serial dilutions of culture samples on
MSY agar containing streptomycin (100 pg/ml).
Colonies were counted following incubation for
3 d at 30C.
G R O W T H IN AN INDUSTRIAL FERMENTER
( 2 0 0 1)
Rhizohium leguminosarum was grown in basal
salts medium containing mannitol (5 g/l) and
industrial grade yeast extract (1 g/l). In some
experiments, industrial grade yeast extract (5 g/l)
served as sole source of carbon and nitrogen.
The phosphate buffer concentration of the
medium was increased in some experiments
from (g/l) KH,PO,, 0.3 to 0.45; Na,HPO,, 0.3
to 0.45.The medium was autoclaved in situ in
the fermenter. The final pH of all media was
66-6.8. The inoculum (2%) was grown in MSY
or yeast extract (YE) for 3 d at 30C. Aeration
in the fermenter was at the rate of 0.5 l/l/min.
Cultures were harvested after 40 h at 30C.
RECOVERY O F CELLS
Celis were recovered from the growth medium
in laboratory experiments by centrifugation at
7000 g for 15 min using a Beckmann model
J-21C centrifuge. Cultures from the 200 1 fer-
menter were harvested in a continuous centri-
fuge (Sharples). The resulting suspension was
further centrifuged in the laboratory and
resuspended in 500 ml of MSY. The viable cell
density was estimated by plate count.
-
F R E E Z E D R Y I N G EX P E R I M E N I
Cells grown in yeast extract medium (1 1) were
harvested and resuspended in different freeze
drying media (ca 5 ml) as follows: (1) MSY;
MSY + 0.5 g peat, MSY + 40% glycerol, skim
milk (10%) reconstituted in distilled water.
Samples of each suspension were serially diluted
and viable numbers determined. The suspen-
sions were freeze dried for 24 h in a Modulyo
freeze drier (Edwards, UK). The freeze dried
concentrates were resuspended immediately and
viable cell numbers were determined.
STORAGE IN PEAT
Peat, obtained as fine dust, was brought to p H
6 5 by addition of Ca (OH), and autoclaved for
2 h at 121C (1c-30 g lots). Larger lots were
autoclaved for a total of 4 h in covered beakers
or screw capped cans. To make peat concen-
trates, concentrated suspensions of YE grown
cells (200 ml) from the fermenter were mixed
with peat dust (100 g). The resulting paste with
an initial moisture level of 60% (w/w) was dis-
tributed in polyethylene bags which were heat
sealed and stored at 4C.
Peat inoculants were made by harvesting
laboratory grown cultures, resuspending in
MSY and adding 20 ml of suspension to 10 g of
peat dust. Inoculants were incubated in tinfoil at
 Rhizobium inoculant Concentrates
30C for 3-5 weeks, resulting in a moisture level
of 40--45% (w/w), and subsequently stored in
sealed bags or screw capped bottles at 4C or
room temperature. Samples were taken at
appropriate intervals from separate inoculant
preparations and viable numbers were deter-
mined.
STORAGE I N GLYCEROL
Laboratory scale cultures were harvested and
concentrated in MSY medium (5 ml). Glycerol
was added to give a final concentration of 40%
(v/v). The suspensions were stored at 4C and
sampled at approximate two-weekly intervals.
In experiments with fermenter grown cells (YE),
concentrated suspensions (100 ml) in MSY were
mixed with glycerol to give a final glycerol con-
centration of 40% (v/v). These suspensions were
stored in screw capped polypropylene bottles at
- 20C. Samples taken from individual concen-
trates at suitable intervals were diluted (1 : 100)
in MSY. Subsequent ten-fold dilutions were
made in Ringer's solution and viable numbers
were determined.
P L A N T TLSTS
Seeds of Pisum sativurn were surface sterilized by
washing in alcohol (95%) for 5 min followed by
calcium hypochlorite (7 g/100 ml, filtered) for 15
min. Seeds were rinsed 8 times in sterile distilled
water and germinated on plant medium agar in
the dark. The plant medium composition was as
follows (g/l): KCl, 0.125; K H z P 0 4 , 0.05;
MgSO, .7H,O, 0.05; CaSO,, 2 H z 0 , , 0.05; and
trace elements as for rhizobial basal salts
medium. The medium was adjusted to pH 7 and
autoclaved. Germinated seedlings (two per pot)
were added to pots of autoclaved sand and
perlite (1 : 1 ratio by weight).
A suspension of R. leguminosarum cells in
glycerol (40?41),which had been held at -20C
for 18 weeks, was harvested and the cells
washed in plant medium. Pots were inoculated
to give cu 10'" cells per pot. A fresh MSY
culture was harvested and the cells washed in
plant medium and used to inoculate a separate
set of pots (cu 10'" cells per pot) in a control
experiment. The pots were placed under artifi-
cial light (14 h illumination, 10 h darkness) and
watered with sterile plant medium. After 7
weeks, plants were assessed for nodulation and
519
nitrogenase activity by the standard acetylene
reduction assay. In a separate experiment, a
peat based inoculant which had been held for 20
weeks at 4C was similarly tested.
Results
P R O D u c T Io N O F Rhizobium leguminosarum
I N O C U L U M CONCENTRATES
The growth of R. leguminosarum in an industrial
fermenter (200 1) and the subsequent recovery of
cells were studied to determine optimum condi-
tions for the production of cell concentrates for
use in inoculant manufacture. Mannitol-yeast
extract broth (MSY) is a standard growth
medium for rhizobia and growth experiments in
the fermenter indicated that MSY supported
high growth yields of R. leguminosarum (3 x lo9
cfu/ml) over a 40 h incubation period. The
recovery yield of viable cells from the culture
medium, using a continuous centrifugation
system, was very low, however, with less than
1% of the cells being recovered (Table 1). In
contrast, high recovery yields were obtained
when the same centrifugation conditions were
employed to harvest either Bacillus or Luclo-
bacillus cells. Attempts to recover MSY-grown
R. leguminosarurn cells from the growth medium
by filtration were also unsuccessful and resulted
in extensive clogging of the filtration apparatus.
Rhizobium species are known to produce large
amounts of extracellular polysaccharide under
certain growth conditions (Humphrey &
Vincent 1959; Ghai et 01. 1981). The appearance
of R. leguminosarurn colonies on MSY agar indi-
cated that this strain produced large amounts of
polysaccharide. In addition, culture filtrates
were viscous and this may have contributed to
the inability to recover R . leguminosurum cells
by centrifugation. Attempts were made, there-
fore, to find alternative growth substrates
capable of supporting high growth yields of R.
leguminosarum without significant poly-
saccharide production and which would also be
cost-effective. When molasses was substituted
for mannitol in a range of concentrations (1-10
g/l), high cell yields ( > 1 x lo9 cfuiml) were
obtained. Observation of cultures grown on
molasses-yeast extract agar indicated, however,
that considerable amounts of polysaccharide
were produced. In contrast, no evidence of poly-
saccharide production was observed in agar oul-
5 20 J . Meade et al.
Table 1. Etrect of growth medium on growth yield, final pH and recovery
yield of Rhizobium leguminosarum cultured in a fermenter (200 1)
Growth yield Recovery yield
Growth medium (cfu/ml) Final pH* ("/.I
Mannitol yeast extract 3 109 5.5 0.6
Yeast extract 2.4 109 8.5 70 10
(0.6 g/l phosphate) ( k 0.4)
Yeast extract 8.1 x 109 8.0 63 + 12
(0.9 ail Dhowhate) ( + 1.9)

Values for mannitol yeast extract medium are the results of one experi-
ment. Every other value is the mean of two individual experiments. Three
dilutions of each sample were plated in duplicate.
* Initial pH of the medium was 6.8.

tures where yeast extract (5 g/l) served as the
sole source of carbon and nitrogen. Growth
experiments using a small scale fermenter (5 1)
indicated that high cell yields could be obtained
with yeast-extract medium (YE) after 40 h of
incubation. Observation of culture samples cen-
trifuged in the laboratory (7000 g for 15 min)
indicated that R. leguminosarum cells could be
pelleted effectively when grown in this medium.
Therefore, it was decided to test the yeast-
extract medium under industrial-scale condi-
tions. As expected, the YE medium supported
high growth yields in the 200 1 fermenter and
the majority of the cells were recoverable from
the culture medium using the continuous cen-
trifugation system (Table 1). In contrast to the
MSY condition, there was an approximate 100-
fold improvement in recovery yields in repeated
experiments. A significant increase in the pH of
the YE culture occurred during growth in the
200 I fermenter. Laboratory experiments indi-
cated that increasing the phosphate buffer con-
centration in the growth medium resulted in
higher cell yields. When the phosphate buffer
concentration of YE medium was increased
(from 0.6 g/l to 0.9 g/l) in the 200 1 fermenter, a
three fold increase in growth yield occurred
(Table 1).
S T O R A G E O F Rhizobium leguminosarum
INOCU LU M CON CENTRATES
The survival of high cell densities (5 x 10"
cfu/ml) of R. leguminosarum was examined in
order to find a method of prolonging the 'shelf-
life' of inoculum concentrates. Firstly, the effect
of freeze drying on concentrated suspensions of
R. leguminosarum was investigated (Table 2). A
considerable drop (100 fold) in viable cell
numbers was observed upon reconstitution of
suspensions freeze dried in MSY medium. The
use of additives such as peat (10 w/v) or glycerol
(40 v/v) in the suspending medium during freeze
drying aided higher recovery of cells. The best
results were obtained when MSY was replaced
by skim-milk (10% w/v) as the suspending
medium, when only a seven-fold decrease in
viable cell numbers was observed.

Table 2. The effect of freeze-drying in different media on viable cell numbers of Rhizo-
bium leauminosarum
~~

Viable numbers (cfuiml) Decrease in

viable numbers
Freeze drying medium Pre-freeze drying On reconstitution (n-fold)
Mannitol yeast extract 9.6 x 10" 9.2 x lo8 104
(k0.2) ( f 0.4)
Mannitol yeast extract 6.1 x 10" 3.7 x 109 16.5
+ Peat (10%) (k0.5) (kO.3)
Mannitol yeast extract 6.3 x 10" 2.2 109 28.6
+ glycerol (40%,) ( k 0.4) (ko.3)
Skim-milk (10%) 6.3 x 10" 8.8 x lo9 7.2
(f 0 3 lf0.5) ~

Each value is a mean value obtained by plating each of two samples in duplicate.
 Rhizobium inoculant concentrates
Peat is well known as a suitable carrier of
rhizobial cells (Strijdom & Deschodt 1976). It
was decided to assess the effect of storage in
peat on the survival of very high cell densities
(> 10" cfu/g) of R. leguminosarum. When peat
and suspensions of fermenter grown cells were
mixed and stored at 4C in sealed containers at
a n initial moisture level of 60%, it wss found
that viability declined steadily from 10"-10'2
cfu/g to 107-10s cfu/g in different experiments
over 26 weeks of storage (Fig. la). Peat concen-
trate dried at 30C to a moisture content
\0
8
rsI
\
*
c maintained constant viable numbers (2 x lo9
a 7- I I I I I I
Storage time (weeks)
Fig. 1. Survival of Rhizobium leguminosarum in peat
.,
at (a) 4C: 0 , peat inoculants containing initially
9.1 x 10" cfu/g; A, inoculants containing initial cell
densities of 9.0 x lo9 cfu/g; (b) room temperature: 0 ,
inoculants containing 5.0 x 10" cfu/g initially; A,
inoculants containing 6 x 10" cfu/g initially; ino-
culants containing 1.5 x lo7 cfu/g initially. Values are
the mean of duplicate experiments and standard
errors of mean values were less than 10%.
between 4&45% and stored at room tem-
perature also showed a similar decline in viable
cell numbers over a 26 week period (Fig. Ib).
Thus, the protective effect on rhizobial viability
usually associated with peat was not apparent
when cell densities greater than 10'l cfu/g were
521
employed. In subsequent experiments it was
shown that peat supported the growth and sur-
vival of R . leguminosarum when low initial cell
densities were used, indicating that the peat
itself was not toxic to the cells. When a suspen-
sion of cells was mixed with peat to give an
initial cell density of 1.5 x lo7 cfu/g, cell
numbers increased to 1.5 x lo9 cfu/g during a
10 d period at 30C. During subsequent storage
at room temperature, viable cell numbers
declined slowly from 1.5 x 10' cfu/g to 1 x lo8
cfu/g over 36 weeks (Fig. lb). Viable cell
numbers of 2.7 x lo7 cfu/g were observed in
these suspensions after one year at room tem-
perature. There was no net increase in viable
numbers in peat suspensions containing initial
viable numbers of 8.6 x lo9 cfu/g and 4 x 10"
cfu/g during incubation at 30C. The survival
rate over 35 weeks at room temperature was
good and a 10 fold decrease in cell numbers
from 2 x 10" cfu/g to 1.5 x lo9 cfu/g was
observed (Fig. lb). Peat suspensions held at 4C
cfu/g) over 35 weeks.
Glycerol, a t high concentrations is often used
as a protective agent in the frozen storage of
liquid culture stocks. The effect of glycerol (400/,
v/v) on rhizobial viability at two temperatures,
4C and -2o"C, was assessed. Broth cultures
( 2 x lo9 cfu/ml) of R. leguminosarum generally
begin to show a decrease in viable cell numbers
after storage for 4 d. It was found that cell sus-
pensions containing glycerol (40%) retained
constant viable numbers ( 2 x 10" cfu/ml) for 14
d after which a million-fold decrease occurred
over an eight week period (Fig. 2). However,
when high cell densities (1.2 x 10" cfu/ml) were
stored at -20C in glycerol (40%) viable
numbers remained constant for 32 weeks and
only decreased ten fold during a further period
of 42 weeks. Thus cell numbers declined from
1.2 x 10'' cfu/ml to 3.2 x 10" cfu/ml over a
period of one and a half years at - 20C.
N O D U L A T I O N A N D NITROGEN FIXATION
BY STORED Rhizobium leguminosarum
CELLS
It is essential that the important characters of
nodule formation and nitrogen fixation should
not be altered by storage of rhizobia destined
for inoculant manufacture. Therefore nodu-
lation tests were done using pea seedlings culti-
522 J . Meade et al.
12
/ I
2 study, however, yeast extract was used suc-
$ 10
c
3
V
g 9
0
> R . leyuminosurum. Yeast extracts generally
i
g 8
_1
7 Elkan 1971) that Rhizohium spp. are capable of
6 Therefore, it appears that R . leguminosurum can
5 for growth from amino acids supplied as an
Storage time (weeks)
Fig. 2. Survival of Rhizobium leguminosarum in sus-
pensions containing glycerol (40% v/v) at -20C ( 0 )
and 4C (A).Values are the mean of two individual
experiments. Three dilutions of each sample were
plated in duplicate. Standard errors of mean values
were less than 10%.
vated under controlled conditions. As expected,
peat storage did not affect nodulation and nitro-
gen fixation of rhizobia when used as a storage
carrier at 4C. Glycerol concentrates which had
maintained constant viable numbers (ca 10
cfu/ml) for 18 weeks at -20C were assessed for
nodulation and nitrogen fixation. Glycerol-
stored cells were inoculated into pots containing
pea seedlings. A suspension of freshly grown
(MSY) cells of similar cell density was used as
an inoculum in a control experiment. The
average number of nodules per plant for peas
inoculated with stored R. leguminosarum cells
was 51 18 (0.16 f 0.04 g fresh wt per plant)
compared with 56 f 30 (0.14 f 0.04 g fresh wt
per plant) nodules per plant for peas inoculated
with freshly grown cells. Rates of nitrogen fix-
ation, measured by acetylene reduction, were
not impaired in nodules formed by glycerol
stored cells (7.2 f 4.5 pmol C,H, reduced/h/g
fresh wt) when compared to rates obtained with
nodules formed by freshly-grown cells (3.7 & 2
pmol C2H, reduced/h/g fresh wt). Thus storage
in glycerol (40% v/v) at -20C for 18 weeks did
not cause any significant loss of symbiotic activ-
ity in cells of R. leguminosarum under the labor-
atory conditions used.
Discussion
Yeast extract is generally used as the source of
nitrogen, vitamins and growth factors for rhizo-
bial growth (Date 1972; Burton 1979). In this
cessfully, both in laboratory culture and in an
industrial fermenter, as the sole source of
carbon, energy and nitrogen for the growth of
contain a variety of amino acids (Burton 1979)
and it has been known for some time (Lillich &
utilizing amino acids as an energy source.
obtain all its carbon and energy requirements
undefined mixture but this substrate does not
favour polysaccharide production. From a com-
mercial viewpoint, yeast extract represents a
relatively inexpensive but efficient growth sub-
strate for the large-scale production of R . leyu-
minosarum. It supports high viable numbers and
favours efficient separation of cells from the
culture medium. It has been reported, however,
that some yeast extract preparations in concen-
trations greater than 0.35% depressed viability
and produced distorted cells in some Rhizobium
strains (Skinner et al. 1977). Thus high concen-
trations of yeast extract may not be generally
suitable for the commercial growth of some Rhi-
zohium spp.
The production of extracellular poly-
saccharides from sugars is known to occur in
Rhizohium spp. (Ghai et al. 1981) and results in
the formation of mucoid colonies on agar
medium. It has been noted, in this study and
elsewhere (Fraser 1975), that the production of
polysaccharides increases the viscosity of culture
media. The increased viscosity leads to ineff-
cient separation of cells from culture media by
centrifugation (Elsworth 1962). This probably
accounts for the failure to recover cells grown
on MSY, which supported polysaccharide pro-
duction, and for the considerable improvement
in recovery yields when yeast extract alone was
the growth medium. Freeze-drying has been
used successfully in the long term storage of
stock cultures (Dye 1982) and in the production
of inoculants (McLeod & Roughley 1961;
Kremer & Peterson 1983). It is not widely used
for this purpose, however, although it has the
advantage of producing a concentrated inocu-
 Rhizobium inoculant concentrates
lant with a long shelf-life particularly at higher
temperatures. Results obtained in this study
indicate that freeze-drying produces an imme-
diate and significant decrease in cell numbers.
As much as 99% (MSY) of cells may be lost
between freeze-drying and reconstitution. The
use of protective agents in the suspending
medium favours the survival of high cell
numbers, though this protection varies with the
particular medium used. Skim milk seemed to
offer best protection of the media tested. Closer
investigation of factors such as the type of sus-
pending medium, conditions of storage and
most favourable cell densities may further
improve the feasibility of preserving cell concen-
trates by freeze-drying. It should be noted,
however, that freeze-dried inoculants have not
has widespread success and are poor inoculants
in less than ideal conditions.
Peat is still the carrier of choice in the major-
ity of legume inoculants currently produced
(Strijdom & Deschodt 1976). A good quality
inoculant would contain ca lo9 cfu/g (Thomson
1980). The growth of R. leguminosarum in peat
observed in this study when low initial cell den-
sities were used, is in agreement with the obser-
vations of others (Roughley & Vincent 1967,
Burton 1979). Survival rates of R. leyumino-
sarum in peat at both room temperature and
4C were also as expected. The peat used in this
study, did not apparently contain any substance,
such as large amounts of sodium chloride
(Steinborn & Roughley 1974), which would
impede rhizobial growth or accelerate cell death
during storage. But high cell densities declined
very rapidly both at room temperature and at
4 C. It may be that there is a maximum cell
density which can be accommodated in peat
carriers and that very high cell densities cannot
avail of the protective effect exerted by peat. The
protective action of peat is greater when cells
are grown in it. Based on these results, peat does
not appear to represent a suitable carrier for
rhizobial concentrates.
Glycerol is well known as a cryoprotective
agent (Precht et al. 1973), effective in the frozen
storage of micro-organisms. It has been used
successfully in the storage of bacteria, though
not in concentrated form, at -196" in liquid
nitrogen (Breese & Sharpe 1980) and at - 76C
(Feltham et a!. 1978). Glycerol has also been
used with some success in storing concentrates
of lactic streptococci at -21C and at -30C
523
(Stadhouders et al. 1971). Glycerol concentra-
tions in the region of 10-15% were mentioned
in these reports. It has been postulated that
damage to cells by freezing is probably caused
by an increase in the electrolyte concentration
in the cell when water is removed as ice (Smith
1961). Glycerol seems to prevent or to minimize
this process. The results of this study show that
R . leguminosarum concentrates can be stored
successfully at -20C in glycerol (40%). Cells
stored in this way can be resuspended easily and
retain their symbiotic properties in laboratory
tests under controlled conditions. It is accepted
in the case of inoculation studies that there may
be inherent limitations in extrapolating results
of laboratory plant tests to practical field situ-
ations. Preliminary results from field inoculation
experiments, however, indicate that there is no
significant decrease in the establishment rate of
R. leguminosarum in nodules when glycerol con-
centrates are used in place of an inoculum of
fresh cells (unpublished data). Furthermore, it is
accepted that storage of Rhizobium strains as
glycerol stocks does not result in the detectable
loss of the symbiotic plasmid(s) which harbour
the nodulation and nitrogenase genes, (e.g.
Tichy & Lotz 1981). In practical terms the widc
acceptance of glycerol concentrates would be
dependent on the successful testing of such
preparations using a variety of Rhizobium
strains and a range of environmental field con-
ditions. From a commercial viewpoint, it is easy
to achieve a temperature of -20C using
mechanical cooling and freezing procedures and
that would cost considerably less than, for
example, storage in liquid nitrogen. It therefore
seems that storage at -20C in glycerol rep-
resents a feasible method for holding large
batches of R. leguminosarum concentrates for
long periods without loss of viability. Further
study is required to determine if other rhizobial
strains behave similarly under these conditions.
This work was supported in part by NBST
grants 72/79, 74/81, HEIC 23/80 and EEC con-
tract No. GB1.5.030 EIR.
References
BREESE,M.D. & SHARP,R.J. 1980 Storage of E . coli
strains containing plasmid DNA in liquid nitrogen.
Journul of Applied Bacteriology 48,63-68.
BURTON,J.C. 1979 Rhizobiurn species. In Microbial
Technology. 2nd ed. Vol. 1 ed. Peppler, H.J. &
Perlman, D. pp. 29-58. New York: Academic Press.
524 J. Meade et al.
DATE, R.A. 1972 Sources and quantities of yeast
extract for growth of rhizobia. Journal of Applied
Bacteriology 35, 379-387.
DYE, M. 1982 A note on some factors affecting the
survival of Rhizobium cultures during freeze drying
and subsequent storage. Journal of Applied Bacte-
riology 52, 461-464.
ELSWORTH, R. 1962 Phase Separation of Suspensions
by Centrificge. New Jersey: New Brunswick Sci. Co.
FELTHAM, R.K.A.. POWER,A.K., PELL,P.A. & SNEATH,
P.H.A. 1978 A simple method of storage of bacteria
at -76C. Journal of Applied Bacteriology 44, 313-
316.
FRASER,M.E. 1975 A method of culturing Rhizobium
meliloti on porous granules to form a pre-inoculant
for lucerne seed. Journal of Applied Bacteriology 39,
345-351.
GHAI,S.K., HISAMATSU, M., AMENURA, A. & HARADA,
T. 1981 Production and chemical composition of
extracellular polysaccharides of Rhizobium. Journal
of General Microbiology 122, 33-40.
HUMPHREY, B.A. & VINCENT, J.M. 1959 Extracellu!ar
polysaccharides of Rhizobium. Journal of General
Microbiology 21,477484.
KREMER, R.J. & PETERSON, H.L. 1983 Effects of carrier
and temperature on survival of Rhizobium spp. in
legume inocula: development of an improved type
of inoculant. Applied and Environmental Micro-
biology 45, 179Cb1794.
LILLICH, T.T. & ELKAN,G.H. 1971 The biosynthesis
of aspartic, glutamic acid and alanine in Rhizobium
japonicum. Canadian Journal of Microbiology 17,
683-68 8.
MCLEOD,R.W. & ROUGHLEY, R.J. 1961 Freeze dried
cultures as commercial legume inoculants. Aus-
tralian Journal of Experimental Agriculture and
Animal Husbandry 1,29-33.
OGARA,F. & SHANMUGAN, K.T. 1976 Regulation of
nitrogen fixation by rhizobia: Export of fixed N, as
NH,. Biochemica Biophysica Acta 437,313-321.
PETERSON, H.L. & LOYNACHAN, T.E. 1981 The signifi-
cance and application of Rhizobium in agriculture.
International Review of Cytology Supplement 13,
311-331.
PORTER,F.E. 1969 Status report on the preparation of
alfafa preinoculated by the Noculized process. In
Developments in Industrial Microbiology. Vol. 10,
pp. 88-100. Washington: American Institute of Bio-
logical Sciences.
PRECHT, B.H., CHRISTOPHERSEN, J., HEUSEL,H. &
LARBER,W. 1973 Temperature and Life. pp. 3-85.
Berlin: Springer Verlag.
ROUGHLEY,R.J. & VINCENT, J.M. 1967 Growth and
survival of Rhizobium spp. on peat culture. Journal
of Applied Bacteriology 30, 362-376.
SKINNER, F.A., ROUGHLEY, R.J. & CHANDLER, M.R.
1977 Effect of yeast extract concentration on via-
bility and cell distortion in Rhizobium spp. Journal
of Applied Bacteriology 43, 287-297.
SMITH,A.U. 1961. Biological Eflects of Freezing and
Supercooling. Monograph number 9 of the Physio-
logical Society. pp. 1 1 s 13 1. London : Edward
Arnold.
STADHOUDERS, J., HUP, G. & JANSEN,L.A. 1971 A
study of the optimum conditions of freezing and
storing concentrated mesophilic starters. Nether-
lands Milk Dairy Journal 25,229-239.
STEINBORN, J. & ROUGHLEY, R.J. 1974 Sodium chlo-
ride as a cause of low numbers of Rhizobium in
legume inoculants. Journal of Applied Bacteriology
37,93-99.
STRIJDOM, B.W. & DESCHODT, C.C. 1976 Carriers of
rhizobia and the effects of prior treatment on the
survival of rhizobia. In Symbiotic Nitrogen Fixation
in Plants ed. Nutman, P.S. pp. 151-168. Cambridge:
The University Press.
TICHY,H.V. & LOTZ,W. 1981 Identification and char-
acterization of large plasmids in newly isolated
strains of Rhizobium leguminosarum. FEMS Micro-
biology Letters 10,203-207.
THOMSON, J.A. 1980 Production and quality control of
legume inoculants. In Methods for Evaluating Bio-
logical Nitrogen Fixation ed. Bergerson, F.J. pp.
489-534. New York: John Wiley & Sons.