JOURNAL OF BACTEIUOLOOY, Apr. 1969, p. 232-237
`Copyriaht ® 1969 Amerk:all Society for MicrobioiOSY
`
`Vol. 98, No. 1
`Printed In U.S.A.
`
`Effect of Decreasing Growth Temperature on Cell
`Yield of Escherichia coli
`HENRY NGl
`Meat Laboratory, Eastern Utilization Research and Development Division, Agricultural Research Service,
`U.S. Department of Agriculture, Beltsville, Maryland 20705
`
`Received for publication 8 October 1968
`
`Studies of the relationship between yield coefficient and growth rate, as affected
`by temperature of growth, in Escherichia coli have shown that, over a wide range of
`temperature, yield is relatively constant until the specific growth rate falls below about
`0.2 hr1, at which point the yield begins to fall off precipitously. No intermediates
`of glucose metabolism in a form utilizable at higher temperatures could be found in
`the medium, and no toxic product was produced which limited growth. At 10 C,
`37% of the carbon from glucose-UL-14C was assimilated into cellular material,
`whereas, at 30 C, 53% was assimilated. Cells grown at 10 C contained more carbo(cid:173)
`hydrate than did cells grown at 37 C, and the glycogen-to-protein ratio of cells
`grown at 10 C was approximately three times higher than that of cells grown at 37 C.
`Adenosine triphosphatase activities of cells grown at 10 and 35 C were similar.
`Growth rates on glucose, glycerol, and succinate were quite similar at 10 C, but at
`35 C growth was most rapid on glucose and slowest on succinate. The data suggest
`that the decrease in yield with decrease in temperature is a result of uncoupling of
`energy production from energy utilization.
`
`glucose continues to increase with increases in
`temperature and does not reach a maximum until
`42 C. He interpreted these data to mean that, at
`the higher temperature, biosynthesis is proceed(cid:173)
`ing at a slower rate than is catabolism, with
`resultant energy uncoupling, a condition in which
`energy is being made available faster than it can
`be utilized.
`This paper provides evidence to show that the
`yield of E. coli decreases if growth temperature
`is decreased below the temperatures at which
`the growth rates deviate from the Arrhenius
`equation (15). It is suggested that the decrease
`in yield may be the result of an energy uncoupling.
`
`For many years after the report of Graham(cid:173)
`Smith (4), it was generally accepted that yields
`of cells were greater at temperatures below those
`at which most rapid growth occurred. More
`recently, however, Sinclair and Stokes (20)
`demonstrated that, when oxygen is not a limiting
`factor, maximal cell numbers produced by se(cid:173)
`lected mesophiles and psychrophiles are as high
`at 30 C as they are at 10 C. Besides oxygen limi(cid:173)
`tation, other factors which undoubtedly have led
`to confusion in interpreting the effect of tem(cid:173)
`perature on growth have been (i) the use of cell
`numbers instead of cell mass as a measure of
`yield (20), (ii) the use of chemically undefined
`media (13, 20), and (iii) the different methods
`used for calculating and reporting yield (1, 5, 11,
`13, 14, 18).
`Monod (11) found that, in aerated cultures of
`Escherichia coli incubated between 20 and 37 C,
`the yield was between 0.26 and 0.25 g of cells
`(dry weight) per g of glucose. At 39 and 41 C,
`however, the yields dropped to 0.21 and 0.20,
`respectively. Senez (18) observed a similar de(cid:173)
`crease in yield with Aerobacter aerogenes above
`37 C. The rate of growth of A. aerogenes is
`maximum at 37 C, but the rate of respiration of
`
`• Present address: Western Utilization Research and De(cid:173)
`velopment Division, U.S. Department of Asrlculture, Albany,
`Calif. 94710.
`
`MATERIALS AND METHODS
`Organism. The organism used throughout this
`study was Escherichia coli ML 30, obtained from
`Jacques Monod.
`Media. The basal medium used was medium 56 of
`Monod, Cohen-Bazire, and Cohn (12). Glucose,
`glycerol, or sodium succinate was sterilized by filtra(cid:173)
`tion through a membrane filter (Millipore Corp.,
`Bedford, Mass.).
`Chemicals. The uniformly labeled uc-glucose was
`purchased from New England Nuclear Corp., Boston,
`Mass. The scintillation mixture was "Liqueftuor,"
`obtained from Nuclear-Chicago Corp., Des Plaines,
`Ill., and the hydroxide of hyamine was purchased
`from Packard Instrument Co., Inc., La Grange, Ill.
`232
`
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`Page 1
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`

`
`VoL. 98, 1969
`
`GROWTH TEMPERATURE AND YIELD OF E. COLI
`
`233
`
`Other chemicals were of reagent grade from the usual
`commercial sources.
`Cultural conditions and measurement of growtb.
`Cultures were grown, their growth was measured, and
`the specific growth rate, k, was calculated as described
`previously (15) with the following exceptions: cells
`for preparation of standard curves were obtained
`from cultures growing exponentially at 35 C, and a
`Spectronic-20 (Bausch & Lomb, Inc., Rochester,
`N.Y.) and model DB spectrophotometer (Beckman
`Instruments, Inc., Fullerton, Calif.) were used for
`measurements of optical density. Generation time,
`g, was computed by the formula g = 0.69/k.
`Determination of yield. Two independent methods
`were used to determine yield. Method A consisted of
`inoculating 5 ml of a starter culture, grown at the
`same temperature, into 145 ml of minimal medium
`lacking a carbon source, and then allowing the cul(cid:173)
`ture to exhaust the carried-over substrate before
`adding glucose (Fig. 1). At zero-time, sterile glucose
`was added aseptically to give a final concentration of
`250 pg of glucose/mi. Turbidity measurements were
`made on the culture until growth ceased. Glucose
`determinations were made at the beginning and end
`of the growth cycle. The yield coefficient, K, was
`computed from the equation K = (G - Go)/C,
`where G and Go are cell mass [estimated as 1-18 (dry
`weight)ml] at the end of the experiment and at zero(cid:173)
`time, respectively, and Cis the glucose concentration
`in pgrams/milliliter at zero-time.
`Method B was as follows. Minimal medium con(cid:173)
`taining 250 1-18 of glucose per ml was placed in a side(cid:173)
`arm flask adapted for a Klett colorimeter and was
`inoculated with a washed suspension of cells from an
`exponentially growing culture (10% inoculum). The
`flask was incubated on a water-bath shaker at the
`desired temperature. The turbidity of the culture was
`followed on a Klett colorimeter until a constant
`reading was obtained. Turbidity readings were made
`at the beginning and at the end of the growth phase
`on the Beckman Model DB spectrophotometer to
`permit estimation of the cell mass. Glucose determi(cid:173)
`nations also were made on the filtered medium. The
`yield coefficient was then computed as described for
`Method A. The results obtained with either method
`were in good agreement.
`Utilization of 14C-glucose. The procedure was
`essentially the same as that described for determining
`yield by method A except that uniformly labeled
`glucose was used as the substrate. After removing a
`sample for the determination of turbidity, glucose,
`and radioactivity, the vessel was stoppered and con(cid:173)
`nected to two test tubes in series containing 0.1 N
`NaOH to trap the respiratory COt, which was swept
`through by sparging with air.
`After growth had ceased, the radioactivity in(cid:173)
`corporated into cellular material was determined by
`liquid scintillation counting of cells collected and
`dried on membrane filters; the radioactivity remaining
`in the culture fluid also was determined. The bound
`COt dissolved in the culture fluid was released into a
`closed container by 10 N HtSO, and, then, was ab(cid:173)
`sorbed by hyamine after overnight incubation. The
`value for co,, thus determined, was subtracted from
`
`.... 100
`E
`.....
`C>
`3
`1- 50
`:I: 40
`52
`w
`3: 30
`>-
`0::
`0
`
`20
`
`I 0 .....__..___...___.,L.__......_ _ _.
`-50
`50
`100
`150 200
`0
`TIME (minutes)
`
`Flo. 1. Typical experiment for determining yield by
`means of method A. Arrow denotes the time at which
`sterile glucose was added aseptically to give a final
`concentration of 250 p.gfml. The temperature was 30 C.
`G represents the increment of growth resulting from the
`glucose atltkd.
`
`the total radioactivity in the culture fluid. The COs
`removed by the alkali traps was determined in a
`similar manner, and this value was added to that
`obtained as bound COs in the supernatant fluid to
`give total COs. All aqueous samples (0.1 ml) were
`made miscible with 10 ml of "Liquefluor" by the
`addition of 3 ml of absolute ethyl alcohol. Radio(cid:173)
`activity was determined in a Nuclear-Chicago scintil(cid:173)
`lation spectrometer at the ambient temperature of an
`air-conditioned room maintained at about 20 C.
`Counting efficiency, as determined by the channels
`ratio method, was approximately SO%.
`Respiratory rate. Uptake of oxygen was deter(cid:173)
`mined by conventional Warburg techniques (22) on
`samples from exponentially growing cultures at the
`temperature of growth of the cultures. Cells were
`resuspended in 0.1 M phosphate buffer (pH 7.4) to a
`density of 600 to 800 1-18 (dry weight)/ml. Each War(cid:173)
`burg vessel contained 2.5 ml of washed cell suspension,
`0.3 ml of buffer in the main compartment, and 0.2
`ml of 1% (w/v) glucose in the side arm, which was
`tipped in after temperature equilibration. The center
`well contained 0.2 ml of 20% KOH to absorb re(cid:173)
`spiratory COs. Two sets of data were obtained; one
`set was corrected for endogenous respiration, whereas
`the other was not. QOs is defined as pliters of Os
`taken up per milligram of protein per hour
`Protein and carbohydrate determinations. Protein
`was determined by the method of Lowry et al. (7).
`The total carbohydrate was determined by the an(cid:173)
`throne method as modified by Fales (2). Glucose was
`
`BEQ 1045
`Page 2
`
`

`
`234
`
`NO
`
`J. BACI'J!RIOL.
`
`o.ss.--------------------,
`
`18
`0
`
`0
`35
`
`0
`30
`
`... 0.50
`ifi
`0
`IE
`8
`9
`!!! >
`
`10
`
`SPECIFIC GROWTH RATE (Hour-1 )
`
`FIG. 2. Yield of Escherichia coli ML 30 as a func(cid:173)
`tion of specific growth rate. Numbers above the experi(cid:173)
`mental points indicate the temperature of growth that
`gives the growth rates shown on the abscissa. The 30,
`13, and 10 C points are averages of six different experi(cid:173)
`ments in which both methods A and B were used,· the
`other points represent single determinations.
`
`TABLE 1. Ability of the supernatant fluid from a
`culture grown at 10 C to support additional
`growth at 30 ca
`
`Cell density (pgfml)
`
`Inoculum
`
`No glucose at
`hour
`
`Glucose added
`(0.1%) at hour
`
`determined by the enzymatic Glucostat reagent
`(Worthington Biochemical Corp., Freehold, N.J.).
`Preparation of cell extract. Cells grown in a glucose
`minimal medium at 35 C or at 10 C were resuspended
`in 0.1 M
`tris(hydroxymethyl)aminomethane
`(Tris)
`buffer (pH 8.0) to a density equivalent to about
`160 pg of protein/mi. The resuspended cells were
`treated for 5 min at the full power of a 10-kc MSE
`sonicator
`(Measuring and Scientific Equipment,
`Ltd., London, England) by use of the large probe.
`The sample was kept cool in an ice bath during sonic
`treatment.
`Adenosine triphosphatase assay. The method of
`assay used was that of Marsh and Militzer (8). The
`incubation mixture consisted of 0.5 ml of 0.2 M Tris
`buffer (pH 8.0), 0.3 ml of adenosine triphosphate
`(ATP)-Mg mixture (0.032 M ATP and 0.015 M MgCll),
`and 0.5 ml of cell extract. The mixture was incubated
`for 30 min at 35 C. After the reaction was stopped
`with 0.2 ml of SO% trichloroacetic acid, the pre(cid:173)
`cipitate was removed by centrifugation. The inor(cid:173)
`ganic phosphate liberated was determined on a 0.5-ml
`sample of the supernatant liquid by use of the Fiske(cid:173)
`SubbaRow procedure (22). Phosphat liberatede by a
`boiled enzyme extract was subtracted to correct for
`instability of ATP. One unit of enzyme is defined as
`that amount which liberates 1 pg of phosphorus per
`hr under these conditions.
`
`RFSULTS
`Yield of cells at various temperatures. Figure 2
`shows the relationship between yield coefficient
`and specific growth rates as affected by tempera(cid:173)
`ture of growth. The yield is quite constant over
`a wide range of growth rates until rates fall below
`about 0.2 hr1, a point corresponding to a tem(cid:173)
`perature of about 18 C, at which the yield de(cid:173)
`creases precipitously. For example, at 30 C,
`the average growth rate in this medium is about
`0.55 hr1, and the average yield coefficient is
`about 0.5; i.e., 0.5 g of cells (dry weight) is pro(cid:173)
`duced from 1 g of glucose. At 10 C, however, the
`growth rate decreases to about 0.04 hr1, and
`the yield coefficient drops to 0.39.
`Growth at 10 C on culture supernatant Huid.
`To explore the possibility that intermediates of
`glucose metabolism may have accumulated which
`could not be metabolized at 10 C, the supernatant
`liquid from a culture at 10 C that had ceased
`growing was examined for its ability to support
`growth at 30 C. This supernatant liquid failed
`to support growth of either cells grown at 10
`or 30 C and subsequently incubated at 30 C
`(Table 1). Thus, no intermediates are excreted
`into the medium in a form that can be utilized
`at a higher temperature. The fact that even cells
`grown at 30 C cannot grow eliminates the pos(cid:173)
`sibility that cells may not have been adapted at
`10 C to use the intermediate. The growth ob(cid:173)
`tained when 0.1% glucose was added indicates
`
`0
`
`Cells grown at
`10 c ........ 53
`Cells grown at
`30 c ........ 115
`None ..........
`2
`
`0
`20
`4
`4
`20
`- - - - - - - - --
`so
`101
`2
`
`35
`
`53 106 213
`
`2SO
`85 117 287
`3
`1
`2 211
`
`" Incubated at 30 C on shaking water bath.
`
`that cessation of growth at 10 C was due not to a
`toxic condition but rather to a lack of a carbon
`and energy source. The growth in the uninocu(cid:173)
`lated glucose-supplemented medium after 20
`hr results from the incomplete removal of cells
`by the centrifugation.
`Carbon balance of E. coli growing on glucose.
`The proportion of carbon from glucose-UL-14C
`going into cellular material, into C02, and re(cid:173)
`maining in the culture Huid is presented in Table
`
`BEQ 1045
`Page 3
`
`

`
`VoL. 98, 1969
`
`GROWTH TEMPERATURE AND YIELD OF E. COLI
`
`235
`
`TABLE 2. Percent of 14C present in cells, COs, and
`culture supernatant fluid at 10 and 30 C.
`
`&:pt
`
`Temp
`
`1
`
`2
`
`Avg
`
`c
`10
`30
`
`10
`30
`
`10
`30
`
`Super-
`natant
`ftuld
`
`Cells
`
`co.
`- - - - - -
`36.6
`42.8
`22.7
`52.7
`21.6
`29.2
`
`38.0
`53.5
`
`37.3
`53.1
`
`38.3
`33.2
`
`40.6
`31.2
`
`20.9
`18.0
`
`21.8
`19!8
`
`Total
`
`102.1
`103.5
`
`97.2
`104.7
`
`99.7
`104.1
`
`2. At 10 C, only about 37% of the glucose carbon
`is assimilated into cellular material, whereas at
`30 C, 53% is recovered as cells. These values
`are in good agreement with the yield based on
`dry weight. The lower rate of conversion of
`glucose carbon into cellular material at 10 C is
`concomitant with an increased recovery of carbon
`in the form of C02 at 10 C, 41% as compared to
`only 31% at 30 C. The percentage of carbon
`remaining in the culture supernatant fluid at
`both temperatures is about the same.
`Respiratory rate and growth rate at various
`temperatures. The data in Fig. 3 are in the
`form of an Arrhenius plot, in which the logarithm
`of the specific growth rate and the logarithm of
`the respiratory rate are plotted as functions of
`the reciprocal of the absolute temperature.
`This figure shows that this function for the
`respiratory rate is linear throughout the range of
`temperatures studied, whereas the corresponding
`function for the growth rate deviates from lin(cid:173)
`earity at temperatures below about 20 C.
`Respiration of glucose at 35 C by ceUs grown
`at 10 C. Cells grown at 10 C respired glucose at
`35 Cat a faster rate than do cells grown at 35 C;
`the former had a Q02 (corrected for endogenous)
`of 180 as compared to only 159 for the latter.
`Furtheimore, the rate of endogenous respiration
`was higher for the cells grown at 10 C than for
`those grown at 35 C, and a longer period of
`endogenous metabolism was required to deplete
`the endogenous reserve of cells grown at 10 C.
`Carbohydrate-to-protein ratio of ceUs grown at
`10 and 37 C. The data of Table 3 confirm the
`finding that cells grown at 10 C are richer in
`carbohydrate (22% of dry weight) than are cells
`grown at 37 C (8.4% of dry weight). The protein
`content of cells grown at 10 C is slightly lower
`than that of cells grown at 37 C; the carbohy(cid:173)
`drate-to-protein ratio was 0.37 at 10 C as com(cid:173)
`pared to 0.13 at 37 C. There was no significant
`difference in optical densities at 420 nm of suspen-
`
`I.O.-----.,o:::--r--..,..--,----,--~11,1100
`100
`100
`
`400
`8
`0
`zoo
`
`..
`,.; ..
`= ,.
`;
`..
`::10.1
`u ;;:
`u ... ... .,.
`
`0"01 '--"-'T35.----.301n--~z5c----.zll-o --7.15;:---+.lo.-'
`TEMPERATURE, •c
`3.40
`110001"111
`
`3.20
`
`3.30
`TEMPERATURE,
`
`3.50
`
`FIG. 3. Arrhenius plot of growth rates and respira(cid:173)
`tory rates of E. coli ML 30. The circles are specific
`growth rates (k), and the triangles are the rates of
`oxygen uptake (QOz). Open and closed symbols indi(cid:173)
`cate two different sets of data.
`
`sions each containing an equivalent dry weight
`of cells grown at the two temperatures.
`Adenosine
`triphosphatase activity of cells
`grown at 10 and at 35 C. The lower yield of cells
`at 10 C could be the result of higher adenosine
`triphosphatase activity in the cells. However,
`the data in Table 4 show that the specific activity
`of adenosine triphosphatase in cells grown at
`10 C may be slightly lower than that in those
`grown at 35 C.
`Growth rate on various carbon sources at high
`and low temperatures. In Table 5, growth rates of
`E. coli on glucose, glycerol, and succinate media
`are shown. These data show that, at the higher
`temperatures, growth is most rapid on glucose,
`slowest on succinate, and
`intermediate on
`glycerol. When temperature is low (13 C), how(cid:173)
`ever, the rate of growth is approximately equal
`on all three sources.
`
`DISCUSSION
`The results of this study demonstrate that,
`over a wide range of temperature, cell yield of
`E. coli is independent of growth rate until the
`rates fall below the temperature which approxi(cid:173)
`mately coincides with that at which growth rate
`begins to deviate from the Arrhenius equation
`(Fig. 3). Senez (18) has found that growing
`A. aerogenes at temperatures above the optimal
`growth temperature, 37 C, also results in lower
`cell yields. He has postulated that biosynthetic
`reactions at the high temperatures do not keep
`pace with catabolic reactions, which results in a
`
`BEQ 1045
`Page 4
`
`

`
`236
`
`NG
`
`]. BACTEIUOL.
`
`TABLE 3. Carbohydrate and protein content in E. coli grown at 10 and at 37 C
`
`Growth temp
`
`Dry weight
`
`Optical density" at
`420nm
`
`Proteinb
`
`Carbohydrateb
`
`Carbohydrate(
`protein (w/w
`
`c
`10
`37
`
`p.g/ml
`1,068
`1,094
`
`0.303
`0.323
`
`635(59.5)
`718 (65.5)
`
`234(21.9)
`92(8.4)
`
`0.37
`0.13
`
`" Determined in a 1: 10 dilution of cell suspension.
`b Expressed as micrograms per milliliter. Values in parentheses represent per cent (dry weight).
`
`TABLE 4. Adenosine triphosphatase activity in cells
`of E. coli grown at 10 and at 35 C
`
`dogenous respiration of cells grown at 10 C.
`The observation that growth rates on glucose
`and succinate differ considerably at 37 C but are
`similar at 13 C suggests that biosynthesis limits
`growth at low temperatures, whereas catabolism
`of the carbon source is the limiting factor at
`higher temperatures. Glucose, being more readily
`utilized than succinate or glycerol, will allow a
`faster growth rate. Marr, Ingraham, and Squires
`(9) also concluded that growth at low tempera(cid:173)
`tures results in a higher level of catabolic re(cid:173)
`pression of tJ-galactosidase in E. coli.
`O'Donovan, Kearney, and Ingraham (17) have
`isolated a variety of conditional lethal mutants
`which can grow at low temperatures only when
`supplemented with one or more amino acids,
`whereas the parent strain is capable of growing
`without supplementation. One of these cold(cid:173)
`sensitive mutants, E. coli strain K-11-27, requiring
`histidine for growth at 20 C, has been analyzed
`by O'Donovan and Ingraham (16), and the
`lesion has been traced to an enzyme in the
`is ex(cid:173)
`histidine-synthesizing pathway which
`tremely sensitive to allosteric (feedback) inhibi(cid:173)
`tion at low temperatures. Thus, a preferential
`inhibition, at low temperatures, of allosteric
`enzymes involved in biosynthetic reactions could
`bring about an uncoupling of energy utilization
`from energy production. A related observation
`is that uncoupled growth caused decreased yield
`and increased glycogen content for a leaky
`threonine auxotrophic E. coli growing under
`threonine limitation (19). Similar findings have
`been reported for A. aerogenes growing linearly
`in the presence of an amino acid analogue,
`p-ftuorophenylalanine (19), and for E. coli
`growing in the presence of selenate in the place
`of sulfate (6). All these conditions lead to restric(cid:173)
`tion of protein synthesis.
`Finally, the decreased permeability of bac(cid:173)
`terial cells to sugars at low temperature has often
`been suggested as a possible basis for the exist(cid:173)
`ence of a minimal temperature for growth of
`bacteria (3). The fact that my results show that
`the respiratory rate at low temperature is well
`in excess of that required for growth renders this
`explanation unlikely. Stokes and Larkin (21)
`
`Growth
`temp
`
`Phosphorus liberated
`in30 min"
`
`Net
`
`Adeno-
`Protein Specific
`sine
`in
`triphos-
`activity
`Un-
`P~!r5 extract
`heated Heated
`activity
`ex-
`ex-
`tract"
`tract
`- - - - - - - - - - - - - -
`,.,
`c
`unils/p.g
`p.g/ml
`p.g
`p.g
`nils/ml
`10
`48.0 15.2 32.8 65.6
`164.6 0.40
`55.8 13.6 42.2 84.4
`35
`171.6 0.49
`
`" Heated extract was boiled for 5 min.
`b Units of activity are defined as micrograms of
`phosphorus liberated per hour per milliliter.
`
`TABLE 5. Effect of temperature on specific growth
`rates of E. coli ML 30 on three carbon sources
`
`Growth
`temp
`
`c
`37
`35
`13
`
`Glucose
`
`Glycerol
`
`Succinate
`
`0.993
`
`0.099
`
`0.722
`0.088
`
`0.621
`
`0.105
`
`condition he terms energy uncoupling. On the
`basis of evidence provided in this paper, this
`hypothesis may apply equally well at low tem(cid:173)
`peratures.
`It was demonstrated that, whereas the rate of
`growth at low temperature is slower than pre(cid:173)
`dicted by the Arrhenius equation, respiratory
`rate seems to obey the equation at least down to
`10 C. The inability of biosynthesis (anabolism)
`to keep pace with catabolism should result in
`the diversion of energy from catabolism into
`forms of storage, e.g., glycogen, and this is
`reflected by the higher carbohydrate-to-protein
`ratio in cells grown at 10 C as compared to those
`grown at 37 C. That catabolic reactions predomi(cid:173)
`nate over biosynthetic reactions is also suggested
`by the fact that cells grown at 10 C respire glucose
`at 35 C at a rate in excess of that of cells grown at
`35 C; furthermore, a longer period of endogenous
`respiration is required to reduce the rate of en-
`
`BEQ 1045
`Page 5
`
`

`
`VoL. 98, 1969
`
`GROWTH TEMPERATURE AND YIELD OF E. COLI
`
`237
`
`have recently shown that the extract of a psy(cid:173)
`chrophilic bacillus is capable of oxidizing glucose
`at low temperature (5 C) faster than is the ex(cid:173)
`tract of a mesophilic bacillus. Therefore, the
`failure of some bacteria to grow at low tempera(cid:173)
`ture is probably not due to a limitation in per(cid:173)
`meability of cell membrane to substrates.
`
`ACKNOWLEDGMENT
`
`I thank Iohn A. Alford for his critical review of the manu·
`acript.
`
`LITERATURE CITED
`
`I. Bauchop, T., and S. R. Bladen. 1960. The growth of micro(cid:173)
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