APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1986, p. 838-841 Vol. 52, No.

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0099-2240/86/100838-04$02.00/0
Copyright © 1986, American Society for Microbiology

Large-Scale Production of Rhizobium meliloti on Wheyt

N. BISSONNETTE,* R. LALANDE, AND L. M. BORDELEAU
Research Station, Agriculture Canada, Sainte-Foy, Quebec, Canada GI V 2J3
Received 2 May 1986/Accepted 25 July 1986

Whey, a by-product of the cheese industry, can sustain the growth of fast-growing rhizobia. To avoid any
latency of growth, rhizobial inoculum must be prepared under inducing conditions. In unsupplemented whey,
the number of cells of Rhizobium meliloti Balsac reached 5 x 109 CFU/ml in 48 h of incubation. This is
comparable to the yield obtained with yeast-mannitol broth, the standard medium for the growth of rhizobia.

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In raw whey supplemented with yeast extract (1.0 g/liter) and phosphate (0.5 g/liter), the number of cells
reached 1010 CFU/ml in 48 h of incubation. This is a twofold increase compared with the population normally
obtained in industrial production. Whey represents a relatively inexpensive and efficient substrate medium for
the large-scale production of fast-growing rhizobia.

The nutritional diversity of carbon utilization by rhizobia
has been the subject of numerous investigations. Different
media have been proposed for growing cultures of rhizobia
(5). The standard medium is composed of a carbon source
(mannitol, sucrose, or glycerol), a source of nitrogen and
growth factors (yeast extract), and different minerals such as
potassium phosphate, magnesium sulfate, and sodium chlo-
ride. Mannitol is the conventional carbon source used in
routine laboratory media, although the growth of slow grow-
ers on this sugar is variable (15). The most satisfactory
carbohydrate is glycerol, on which slow growers have
shorter generation times than on glucose, mannitol, galac-
tose, or sucrose (1). Yeast extract can be used as the sole
carbon and nitrogen source (12); however, high concentra-
tions cause cell distortion (17) and even growth inhibition of
Rhizobium trifolii, which can be counteracted by calcium
(16).
The first step in the production of legume inoculant is
massive production of a selected Rhizobium species in liquid
medium. The economy of such a process is largely governed
by the price and availability of a suitable carbon source.
Corn-steep liquor (5), proteolyzed pea husks (9), malt
sprouts (2), and industrial-grade yeast extract (12) have been
proposed as media for Rhizobium biomass production at the
industrial level. All of these materials are industrial by-prod-
ucts which contain growth factors, nitrogen, and carbon.
The cheese industry produces large amounts of whey,
presenting a major problem for pollution control and a
substantial challenge to feed and food technologists. The
present study shows that whey is a suitable, inexpensive
medium for the massive production of Rhizobium meliloti
cells.
MATERIALS AND METHODS
Whey composition. Lactose was determined by the method
of Marier and Boulet (11). Total nitrogen was analyzed as
described by Bremner (4), and minerals such as calcium,
magnesium, phosphorus, potassium, and sodium were deter-
mined as described by Chapman (7).
*
Corresponding author.
t Contribution 295 of the Research Station, Agriculture Canada,
Sainte-Foy, Quebec.
838
Rhizobial strain and fermentation media. R. meliloti A2 (3),
commercialized under the name of Balsac, which is a very
effective strain, was used throughout this study. Cultures
were maintained at 4°C on slants of mannitol agar (19). Cell
production was studied in different liquid media, based
either on standard medium (19) or on whey, The standard
medium contained the following constituents (in grams per
liter): K2HPO4, 0.5; MgSO4 7H20, 0.2; NaCl, 0.1; and
yeast extract (Difco Laboratories, Detroit, Mich.), 1.0; to
this base 10.0 g of either mannitol, sucrose, or glycerol was
added per liter. The whey-based media contained 22.0 g of
dehydrated cheese whey (a by-product of the manufacture of
mild cheddar cheese) per liter, either alone or supplemented
with yeast extract (1.0 or 5.0 g/liter) or yeast extract (1.0
g/liter) and phosphate (K2HPO4; 0.5 g/liter). In all cases, the
pH was adjusted to 7.0 after the medium was autoclaved at
121°C for 60 min.
Preparation of inoculum. The inoculum was prepared
under inducing conditions by growing strain A2 in 250-ml
Erlenmeyer flasks containing 100 ml of the fermentation
medium. The flasks were incubated at 30°C for 48 h on a
rotary shaker at 150 rpm.
Growth in fermentor. The incubations were done in a
5-liter Microferm fermentor (New Brunswick Scientific Co.,
Inc., Edison, N.J.) containing 3.5 liters of culture medium
with 2% (vol/vol) inoculum. The head plate was equipped
with a hooded sampler device, an oxygen electrode (series
900, model M 1016-5001; New Brunswick Scientific), and a
pH electrode (model 465-K9; Ingold Electrodes, Inc., Wil-
mington, Mass.). The pH was either maintained at 7.0 with
an Automatic pH Controller (model 40; New Brunswick
Scientific) by using 2.0 M CH3COOH or KOH or was
recorded at each sampling time. The temperature was set at
30°C, the agitation was set at 800 rpm, and the aeration rate
was set at 2 liters/min, which assured 95 to 100% dissolved
oxygen saturation throughout the incubation. At 24-h inter-
vals, samples (25 ml) were removed for enumeration of CFU
and spread on yeast-mannitol agar supplemented with congo
red (19) after appropriate serial dilutions in a peptone phos-
phate buffer (pH 6.85) containing the following (in grams per
liter): peptone, 1.0; K2HPO4, 1.21; and KH2PO4, 0.34. At the
end of the fermentation, a plant infection enumeration was
performed by the most probable number technique (19) with
alfalfa (Medicago sativa cv. Saranac) seeds sown in plastic
VOL. 52, 1986 LARGE-SCALE PRODUCTION OF R. MELILOTI ON WHEY
10
0
0 CFU/ml. Raw whey supplemented with yeast extract and
E Infectivity of cells grown in whey medium. With cells grown
QL
C-
0 24 48 72
TIME(h)
FIG. 1. Growth ( ) and pH of media (-) supporting growth
of R. meliloti on mannitol standard mineral medium (O) and
nonsupplemented cheese whey (a) in a 3.5-liter fermentor. The
inoculation level was 2% (vol/vol) with induced cells.
growth pouches (21). These infection tests were done with
cells obtained from fermentations with whey supplemented
with yeast extract (1.0 g/liter) and phosphate (0.5 g/liter).
Statistical analysis of the results was performed by calcula-
tion of the least significant difference. Each experiment was
repeated three times.
RESULTS
Composition of whey. The composition of the mild-
cheddar-cheese whey was 73.54% lactose, 1.83% total nitro-
gen, 2.37% potassium, 0.77% sodium, 0.65% calcium, 0.55%
phosphorus, and 0.14% magnesium. These are percentages
based on dry weight.
Influence of carbon source on change of pH. Incubations
were performed on standard medium with mannitol or on
whey; the pH was adjusted to 7.0 after sterilization. pH
values obtained at the end of the incubations were 6.6 and
8.0 for the mannitol medium and whey, respectively (Fig. 1).
In the mannitol medium, the cell population reached a
maximum after 24 h of incubation and leveled off thereafter
at 4.8 x 109 CFU/ml; with whey, it took 48 h for the
population to reach a plateau, and after 72 h the population
was 6.3 x 109 CFU/ml.
Influence of carbon source on yield of R. meliloti A2. The
growth curves for R. meliloti A2 on standard medium sup-
plemented with different carbon sources under controlled
pH in the fermentor are shown in Fig. 2A. Similar yields
were obtained when the medium contained mannitol or
sucrose, and the lowest yield was obtained with glycerol.
The yield obtained with raw whey was higher, although not
significantly different, on media containing the sugars tested
(Fig. 2B), but the maximum cell number was attained after
48 h of incubation; with the standard medium, it was attained
within 24 h. Significantly higher yields (compared with those
in the standard medium) were obtained after 72 h of incuba-
839
tion when whey was supplemented with 1.0 g of yeast
extract per liter, but they were not significantly different
compared with yields on raw whey alone. Whey supple-
mented with 5.0 g of yeast extract per liter yielded, after 24
h of incubation, a cell population equivalent to that obtained
in mannitol or sucrose (5.0 x 109 CFU/ml), and after 48 h of
incubation, the yield obtained was approximately 1010
phosphate also yielded this high number of cells after 48 h of
incubation.
in whey medium supplemented with yeast extract (1.0 g/liter)
and phosphate (0.5 g/liter), no difference was found in the
numbers of CFU obtained with plate-count and most-
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probable-number techniques. The mean CFU per milliliter in
whey medium with the plate-count technique was 0.99 x
1010 (Fig. 2B), and with the most-probable-number technique
it was 1.05 x 1010 (least significant difference, 0.32 x 1010 [P
= 0.05]).
DISCUSSION
Dairy whey, a by-product of cheese manufacture, is
considered a process effluent. It is a major disposal problem
because its biological oxygen demand is very high (14). For
a long time regarded as a substance of little economic value,
whey is now processed into several useful commercial
products for animal and human nutrition (13). However, the
elimination of the millions of tons of whey produced annu-
ally remains a challenge requiring the introduction of new
processes. Its use as a growth medium for fast-growing
rhizobia is therefore attractive.
Whey is rich in major chemicals usually included in
synthetic media and contains them at concentrations suffi-
cient to sustain rhizobial growth (20). Cerbulis et al. (6)
indicated that lactose, the major dissolved solid in whey,
represents 70 to 75% of the total solids; total nitrogen values
range between 1.82 and 2.40%, of which a great proportion is
hydrolyzable into amino acids. The fast-growing rhizobia
grow well on disaccharides because they have an uptake
mechanism and catabolic enzymes for their metabolism (18).
In R. meliloti, the lactose uptake system is an active process,
and it is inducible (8). The catabolism of lactose involves
cleavage to monosaccharides by ,-galactosidase (EC
3.2.1.23), the synthesis and activity of which are strain
dependent, being constitutive or inducible (8). To avoid any
latency of growth in our studies, R. meliloti inoculum was
grown under inducing conditions, i.e., in the presence of the
particular disaccharides.
The results presented in this paper indicate that on me-
dium containing only whey as the carbon, vitamin, and
nitrogen source, induced cells of fast-growing R. meliloti
grow readily and yield a population comparable to that
obtained with an industrial medium such as mannitol-yeast
extract medium (Fig. 1). Raw whey supplemented with 1.0 g
of yeast extract per liter yielded 1.25 times more cells than
were obtained with mannitol or sucrose broth (Fig. 2). With
a higher concentration of yeast extract (5.0 g/liter), the yield
increased 2.5-fold; at this concentration, the bacteria may
utilize the yeast extract as a carbon and nitrogen source (12).
This would explain the fast growth rate obtained for the first
24 h of incubation on this medium. A value of 2.4 x 109 CFU
of Rhizobium leguminosarum per ml and a significant in-
crease in the pH of the medium were obtained when indus-
trial-grade yeast extract (5.0 g/liter) was used as the sole
carbon and nitrogen source (12). In this work, the pH of the
840 BISSONNETTE ET AL.
1 0.0
0 9.0
0
c
C.)
IL
8.0
0 24 48
FIG. 2. Growth of R. meliloti on carbon-supplemented standard mineral media (A) and on whey alone or supplemented (B) in a 3.5-liter
fermentor. The inoculation level was 2% (vol/vol) with induced cells. The pH was automatically controlled at 7.0. The vertical bars indicate
least significant differences (P = 0.05) between growth means at 72 h of incubation. Y.E., Yeast extract.
whey medium significantly increased during incubation, pos-
sibly limiting growth (Fig. 1). Automatic pH control did not
significantly change the yield. However, increasing the phos-
phate content resulted in an increase in the yield (Fig. 2B);
this confirms the findings of Meade et al. (12), who obtained
a threefold increase in yield by increasing the phosphate
concentration from 0.6 to 0.9 glliter. This was expected
because in sweet wheys the largest part of the calcium
phosphate remains bound to the casein curd formed through
the action of rennin (10); it is not liberated into the whey,
which becomes limited in phosphorus. Moreover, utilization
of the phosphorus by the Rhizobium species may liberate the
calcium of the tetracalcium phosphate complex, thus in-
creasing the alkalinity of the medium in a fermentation with
uncontrolled pH (Fig. 1).
Yeast extract may be used as a carbon and nitrogen source
by rhizobia (12), but a high concentration (higher than
0.35%) produces distorted cells in some Rhizobium strains
and depresses the viabilty of these cells (17). Moreover,
yeast extract is an expensive nitrogen and carbon source;
thus, high concentrations of it may not be generally suitable
for the commercial growth of Rhizobium spp.
The productivity obtained with raw whey moderately
supplemented with yeast extract and phosphate was 1010
CFU/ml, with no impairment of the infectivity of the
rhizobia cells. This is a twofold increase in yield compared
APPL. ENVIRON. MICROBIOL.
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72 0 24 48 72
TIME (h)
with the normal limit in industrial productions (5). From a
commercial point of view, whey represents a relatively
inexpensive and efficient growth substrate for the large-scale
production of fast-growing Rhizobium spp.
ACKNOWLEDGMENTS
We thank Jacques Goulet for helpful discussions and the Rosell
Institute, Inc., for providing the cheese whey.
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