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giving curvilinear growth. It had previously
been shown that the cell crops obtained
increased in the order: slowly fed cultures <
from batch cultures < from rapidly fed
cultures. The radiochemical results confirmed this
observation and also showed that more of the
glucose is oxidized to C1402 in slowly fed than in
batch, which in turn is greater than in rapidly fed
cultures. The reverse order was found for C14
assimilated by the cells. It is not entirely clear
why the batch cultures should lie between the fast
and slow feeding of energy source, but some ex-
planations may be offered. During exponential
growth in batch culture, the rate of growth is not
limited by glucose concentration and it appears
that glucose is utilized less efficiently; e.g., it is
not oxidized to CO2 immediately, possibly owing
to a limitation of oxygen concentration. Conse-
141VOL. 28, 1964
quently, growth may cease and the stationary-
phase cells oxidize the accumulated intermediates
to CO2. Lower growth yields in batch than in
continuous or nutrient-limited cultures were also
observed by Pirt (61).
The energy required for turnover of macro-
molecules appears to account for a larger propor-
tion of the energy source that is not utilized for
growth in E. coli (53), but these workers could
not demonstrate that accumulation of methyl-
thiogalactoside was responsible for any signifi-
cant amount of energy expenditure, although
Kepes (40) noted that addition of this compound
to E. coli suspensions resulted in a doubling of the
rate of endogenous metabolism.
The earlier ideas concerning the concept of
energy of maintenance were excellently sum-
marized by Mallette (51) and McGrew and
Mallette (55). They indicated that lack of sensi-
tive techniques was among the reasons for the
difficulty of demonstration of the energy of main-
tenance. To overcome this problem, they used a
high cell density in relation to a low concentration
of carbon and energy source to study the mainte-
nance requirement of E. coli. Low concentrations
of glucose were fed to suspensions of E. coli in an
otherwise complete growth medium, and the tur-
bidity changes were recorded. When very small
additions of glucose were made, the extinction did
not alter appreciably from control cultures after
a standard time. Higher concentrations of glucose
permitted growth to occur. Thus, they demon-
strated that a threshold concentration of glucose
is required before growth can occur. Cell suspen-
sions starved with respect to the carbon and
energy source showed rapid decreases in turbidity
for 5 days, at which time only 15% of the cells
were viable, and then remained constant while
the viability continued to fall. A small addition
of glucose at 6-hr intervals, sufficient to maintain
the turbidity at a constant value, also suppressed
the rate of loss of viability; e.g., after 5 days only
20% of the cells had died. Glucose additions that
permitted a very slow growth (20% increase in
extinction over 10 days) did not prevent death of
the cells. (After 5 days, approximately 10% had
died.) Thus, it would seem that small amounts of
glucose can provide energy to maintain the cell
without allowing growth to occur. The loss of
viability that occurs during the slow growth may
be due to the interval method (6 hr) of feeding,
as pointed out by Marr et al. (53); i.e., some
breakdown of cellular material occurs before the
next glucose supplement, and this is insufficient
to allow reclamation of the lost cell materials. A
criticism which may be leveled at this type of ex-
periment is that growth may be occurring al-
though it is not revealed by turbidity measure-
ments. The number of cells dying may be such
that the turbidity undergoes no net change as
growth occurs. The influence of higher cell con-
centrations on these phenomena is not known,
and regrowth (28) may assume special impor-
tance; perhaps open systems should be considered
in experimental design.
The early literature concerning the effects of
starvation upon the survival of microorganisms
was critically reviewed by Postgate and Hunter
(62). They also draw attention to the pitfalls and
difficulties that may be encountered during the
estimation of viabilities. The cleanliness of labo-
ratory ware and purity of chemicals is considered
to be critical, as trace impurities may either per-
mit growth of otherwise starving organisms (23)
or kill the cells. The growth of bacteria at the
expense of their companions has been termed
cryptic growth (70), cannibalism (28), and re-
growth (81). This growth is a function of cell
density and can considerably influence the sur-
vival behavior of starving suspensions, since a
"population turnover" may occur.
Apart from the phenomenon of regrowth, the
initial cell density of starving suspensions also
affects their death rate. Harrison (28) first showed
the relationship between cell density and death
rate of starving suspensions of A. aerogenes, and
an optimal density for survival was demon-
strated. He concluded that an interaction be-
tween individual cells favors survival, and the
work of Postgate and Hunter (62) removes any
doubt about the possibility of regrowth occurring.
The latter authors made a very thorough study
of many factors that influence the survival of
starving suspensions of A. aerogenes. They elimi-
nated ambiguity that would arise from cryptic
growth, growth on impurities, and toxicity of
suspending fluids, by a suitable choice of cell
density [20 ,ug (dry weight) of cells per ml] and
suspending fluid [saline-tris(hydroxymethyl)-
aminomethane buffer-EDTA solution]. High illu-
mination, high temperatures, high pH values, and
high potassium ion concentrations increased the
death rates of starving suspensions. For example,
A. aerogenes survives better at 20 C than at 30,
40, or 10 C. Anaerobiosis accelerated death, and this
was attributed to the acid conditions produced.
We also have observed that anaerobically starved
E. coli die faster than the aerobically starved sus-
pensions (18); however, the pH value during
anaerobiosis fell only from 7.2 to 6.8 with our
more strongly buffered suspensions (unpublished
Various nutrients, or the previous history of
suspensions of A. aerogenes, markedly affected
the death rates. Ca2+, Mg2+, and to a lesser extent
Fe2+, when added to the saline-buffer, prolonged
the life of the cells. The slower the rate of growth
of the bacteria, the greater was their death rate
upon fasting. This applied to organisms whose
growth rate was limited by C, N, P, and S, but
with Mg2+-limited growth the reverse was true
and the cells that had most rapidly proliferated
died fastest when starved. The effect of nutrient
additives upon death rates is, however, more com-
plex and depends upon the previous history of the
cells. Thus, Postgate and Hunter (63) observed
a general phenomenon of substrate-accelerated
death, in which the addition of the growth-limit-
ing nutrient to starving suspensions increased the
death rate. Glycerol-limited cells of A. aerogenes
showed glycerol-accelerated death (metabolites
of glycerol, e.g., pyruvate, also accelerate death);
NH4+-limited cells are killed by NH4+ additions
but not by glycerol, which is slightly protective.
Phosphate-limited cells behaved similarly, as did
other carbon source-limited cells. Sulfate-limited
cells were not killed quickly by sulfate, but in-
stead showed glycerol-accelerated death. Mg2+-
limited cells were another exception, in that addi-
tion of Mg2+ actually prolonged the life of these
cells, as it does of other cells, a feature quite inde-
pendent of the nutrient that limited their growth.
Harrison and Lawrence (30) also noted that the
effect of nutrient additions to starving suspen-
sions is influenced