Antonie van Leeuwenhoek63: 225-235, 1993.

© 1993KluwerAcademicPublishers. Printedin the Netherlands.

Nutrient-limited microbial growth kinetics: overview and recent advances

D.K. Button
Institute of Marine Science and Department of Chemistry, University of Alaska, Fairbanks, Alaska 99775, USA

Key words: kinetics, transport, collision frequency, growth

Abstract

Traditional concepts of nutrient uptake and growth kinetics as linked by cell yield are presented. Phenomena
affecting the kinetics are examined along with a discussion of those which lead to ambiguity. Concepts of flux
control are presented to help understand the distribution of material along metabolic pathways. Specific
affinity is described to relate nutrient accumulation rates to transporter density. It is shown to be a primary
kinetic constant and the best available index of nutrient collection ability. As an aid to understanding, specific
affinity is reexpressed in terms of membrane permeability. Formulations of nutrient transport rate as a func-
tion of cellular composition, particularly transporter and enzyme content and known as janusian kinetics, are
described as an improvement to specific affinity theory. Procedures for quantified unidirectional fluxes are
reviewed to identify the difference between gross and net transport rates of substrate. Collision frequency
theory is used to show that in addition to total biomass, cell size and transporter density should also be in-
cluded in rate equations describing microbial growth. Theory diversity suggests that one reason for microbial
metabolic is that the likelihood of additional collisions of substrate molecules with a cell surface, after an
initial collision, requires only a sparse distribution of transporter sites for maximal rate, leaving room for
additional transporters able to collect other substrate types.

Growth kinetics and nutrient transport
Formulations of the kinetics of microbial growth
began with Monod (1950). He observed that the
specific rate of growth rate increased in a hyperbol-
ic way with the an external concentration of limiting
nutrient. By analogy with enzyme kinetics the rela-
tionship is:
~maxA
g- KM+A
where gmaxis the maximal growth rate, A is sub-
strate concentration, and K Mis the Monod or half-
maximal rate associated concentration (see p. 234
for definitions). The relationship is empirical and
no theoretical base was proposed. The main defi-
ciencies are that nutrient limitation implies kinetic
control by transport, and the linkage between trans-
port and growth may be filtered through macro-
molecular processes having metabolic control that
varies with the specific rate of growth.
As techniques for measuring substrate uptake
developed, it was found that these kinetics were hy-
perbolic as well, and transport was also described
by the Michaelis Menten analogy:
VlnaxA
v- - - (2)
(1) Kv+A
where v and Vmax describe rates and maximal rates
of transport rather than growth and K a-is the associ-
ated Michaetis constant. Growth is then coupled to
transport by the relationship
vY
~=--2- (3)
226
where Y is the yield of cells of biomass X from sub-
strate which gives
(K-~--~A) (4)
The theoretical base was presumed to be that trans-
porters limited the rate of growth and behaved like
enzymes. Measurements of cell yield have shown
that Y is not a constant, but a term that varies with
the rate of growth. The change in yield with growth
rate has been explained by endogenous metabo-
lism: maintenance energy devoted to active trans-
port, macromolecule replacement, replacement of
leaked constituents, synthesis of materials to alle-
viate cell damage during starvation, and metabolic
control (Chesbro 1988). For carbon, this change in
yield, extrapolated to a growth rate of zero, is some-
times used to evaluate endogenous metabolism.
Yield is a strong function of growth rate for sub-
strates such as phosphate, silica, and vitamins where
the cells can continue to grow with reduced
amounts of the limiting nutrient in their biochem-
ical makeup. This observation led to the cell quota
concept which has growth rate a function of the
amount of limiting nutrient within the cells (Droop
1968). These formulations are inappropriate for
carbon limitation because much of the transported
carbon is excreted and usual changes in cell-carbon
per unit biomass with growth rate are not large. For
mineral nutrients such as phosphate, the change in
yield leads to a changing requirement for limiting
nutrient per unit mass of cells produced. In addi-
tion, the accumulation of phosphate from dilute so-
lutions often leads to an increase in tranporters for
the substrate which results in high transport capac-
ity (Vmax).The large Vmaxvalues lead to large values
for the transport constant K T (which depends on
Vm,x) relative to the Monod constant for growth K M
(which depends on ~l,max), Consequences include lin-
ear uptake with concentration up to the maximal
growth rate, and growth kinetic curves with inflec-
tions that lead to an apparent Michaelis constant
K Mthat is independent of saturation (Button et al.
1973). Other difficulties with these approaches to
growth kinetics are that
1. thresholds for growth are specified by the com-
mon assumption that endogenous requirements
of the cell must be satisfied before substrate can
be diverted to growth and, if true, render the ba-
sic form of most kinetic formulations insuffi-
cient,
2. metabolic costs of nutrient limitation can shift
with substrate concentration,
3. changes in transporter content can change the
rate of substrate transport at low concentration
while K T is unaffected because of a correspond-
ing change in Vmax which may mask important
changes in culture kinetics,
4. the whole-cell Michaelis constant K Mmay sim-
ply reflect the ratio of transporters to enzymes,
and
5. the relevant flux for describing growth in com-
plex dilute systems is the sum of those flows con-
tributing limiting material rather than from a
single substrate.
Saturation phenomena
As the permeases of microorganisms and other en-
zymes of metabolism become occupied with sub-
strate, saturation phenomena ensue. For aquatic
systems ambient substrate concentrations seldom
attain concentrations sufficient to saturate these
transporters, however experimental additions that
achieve saturation are useful to probe mechanisms.
In the case of simple enzymes, the shape of the ki-
netic curve is perfectly hyperbolic in theory and of-
ten in practice as well. For whole cells, departure
from this ideal shape has been used to evaluate a
number of phenomena such as natural substrate
levels (Wright & Hobbie 1965) and multiple trans-
porters (Azam & Hodson 1981). The kinetics for
each of two individual transporters with different
Michaelis constants for transport K 1and K 2 can be
evaluated as a simultaneous operation of a pair of
transporters:
{ ~ [Vmax2A~
v+ + AI +
If the curve is biphasic, the individual kinetic con-
stants can be extracted (Neal 1972). However the
resulting kinetics which show more gentle curva-
ture in the region of saturation, are similar to a corn-

bination of diffusion limitation and saturation ki-
netics (Martinez et al. 1992).
Flux through pathways
Linkage between nutrient transport rate and
growth rate may depend not only on nutrient accu-
mulation by transporters, but on the rate of utiliza-
tion of the accumulated nutrient through enzymatic
sequences as well. One might presume that overall
kinetics of microbial growth are controlled by the
kinetics of formation of a particular intermediate in
scant supply. Further that the formation rate of that
component is controlled by the slowest step in an
enzymatic sequence. Such control led to the idea of
flux-generating enzymes (Newsholme & Crabtree
1981), enzymes that control flow at the beginnings
or branch points of pathways and are particularly
receptive to metabolic control. Another view is that
flux control is broadly distributed among pathway
components (Kell & Westerhoff 1986). This sharing
is quantified by flux control coefficients C which re-
late the influence of each enzyme on total pathway
flux. Thus the conversion of substrate to product
with various rate constants
tl c, c2 c3
A --~ E t --~ E z --+ E 3 ---) P
I[J kl k2 k3
Scheme 1
leads to some steady state flux j across the mem-
brane. Without complexities such as those associ-
ated with the phosphotransferase mechanism, the
sum of the flux control coefficients Z C is often uni-
ty, according to the flux control summation theo-
rem, and the value of each coefficient here is 1/3
(Van Dam et al. 1993). It follows that individual en-
zymes in long pathways are less effective in control-
ling overall fluxj in a sequence than those in a short
pathway. These considerations reveal strategies for
the distribution of protein resources among path-
ways and ultimately affect properties such as DNA/
protein ratio and cell size.
Rate constants and overall kinetic constants for
227
complete pathways can be deduced from those of
component enzymes by the simplified procedures
of Cleland (1975). For example, if k 2 << kl, k 3 the
Michaelis constant is simply K A = k ] k l as usually
presumed. However, if k 3 << k 2 < k 1then K A = k3/kl,
and would (W.W. Cleland, pers. comm.) be analo-
gous to a situation where an oligotroph had many
transporters but only few molecules of catabolic en-
zymes. This leads to the conclusion that
1. kinetic constants for long pathways may reflect
the properties of a number of enzymes,
2. changing the kinetic behaviour of a whole orga-
nism is difficult to effect by mutational or other
adjustment of a single enzyme,
3. individual enzymes in short pathways are more
influential in controlling overall flux than in long
ones, and
4. large ratios of transporter to limiting metabolic
enzymes lead to small values of the transport
constant Kr.
Specific affinity theory
This theory was derived to express the concept that
the affinity (ability of organisms to sequester) nutri-
ents reflected the density of transporters on the cell
surface, and that the resulting rate of growth de-
pended on the associated mass of cells, i.e. the sur-
face to volume ratio, as well. Further considerations
showed that specific affinity, as formulated below,
rather than the Michaelis constant for transport KT,
along with maximal velocity for substrate accumu-
lation, V .... are the primary kinetic constants which
describe that ability. The general case is that sub-
strate uptake is given by the second-order rate
equation
v = aAX A (6)
which defines the specific affinity a a of the cells for
substrate A as the rate of substrate collection v/(X
A). As A approaches zero, a n approaches the limit
a0
A and is the initial slope of equation 2 as well as the
initial slope of any v/X vs A curve whether hyper-
bolic or not.
That specific affinity is a primary kinetic constant
228
can be seen by multiplying equation 2 by Vmax/KT
(Cleland 1987),
(Vraax/KT) . Vma x A
V -- Vmax q" (Vmax/KT) A (7)
For hyperbolic kinetics a0A is Vmax/KT, the initial
slope of the curve, i.e. the derivative of euqation 2 at
A = 0. Thus
a ° Vma x A
0A (8)
V = Vmax -[- aA
The Michaelis constant K T is now eliminated giving
uptake kinetics that are completely defined by Vmax
and a °.
Specific affinity compared to traditional affinity
Since Monod observed hyperbolic or saturation ki-
netics for sugar uptake by bacteria and likened the
phenomenon to an enzymatic reaction, microbial
uptake (transport) kinetics have been described by
the Michaelis or transport constant KT and maximal
v e l o c i t y Vma x of Michaelis and Menten (1913). While
widely used as a measure of the affinity of cells for
substrate, KT'S are ambiguous parameters. Since K T
is defined as the concentration of A at Vmax/2,it de-
pends directly on Vmax. Then if the number density
of transporters on the cell surface were to double, so
would Vmax. If the characteristics of these transpor-
ters remained constant, then rate at Vmax/2 would
double as well leaving K T unchanged and the new
ability of the cells to sequester substrate at low con-
centration unrecorded. However the specific affin-
ity Vmax/KT depends on both the activity of the indi-
vidual transporter assemblages and their number
would also double accurately, refecting the new
ability of the cells to sequester nutrients. Even if the
kinetics were not hyperbolic, the specific affinity
would remain the initial slope of the kinetic curve
and, measured as j/A at small substrate concentra-
tions, defines transporter activity. Thus a ° alone de-
scribes the ability of the cells to accumulate sub-
strate at small A. Unrelated to V . . . . a0Ais an unam-
biguous measure of the ability to capture substrate
with Vm,, a measure of the cell's ability to process it.
Induction
Exposure to nutrients is generally observed to in-
crease the transporter and associated metabolic
system content of receptive organisms and thus af-
fects kinetics in many species (Zimmerman et al.
1987). The linkage between substrate uptake rate
and induction depends on inducer concentration
and time. This linkage has been formulated for tolu-
ene (Robertson & Button 1987) using the empirical
relationship
/ / a max K
( v ) t--. t t . GA ~ _T~ . ) A
\ X
~ ] ] t (9)
where the maximum measured specific affinity
amaxA attained in time t sets the rate of uptake ac-
cording to an induction constant Kind- Equation 9
specifies rate following a particular time of induc-
tion as a function of substrate concentration. It can
be rearranged to coincide with the second-order
rate equation form of equation 6 (but becomes
more bulky). Full induction may take many days of
substrate-limited growth and result in specific affin-
ities an order of magnitude larger than those often
taken as normal for substrate-adapted cells (Egli et
al. 1993).
The membrane-permeability analogy of specific
affinity
While specific affinity may be the best available
measure of the substrate sequestering power of mi-
croorganisms, its use as a primary kinetic constant is
uncommon. Reformulation as transporter-assisted
permeability may therefore be helpful to the con-
cept. From Fick's first law the movement of mole-
cules n across a membrane of area ~, and thickness 1
is given by:
dn/dt = - D fi dAout/dl (10)
If the membrane thickness is specified, the diffusion
coefficient D is replaced by the permeability coeffi-
cient P. In the strict case where permeation is relat-
ed only to membrane thickness, P-- D/1. The influx j
of substrate into organisms of mass X can be written

j = P a (A-Apool) (10)
where j is amount of substrate transported per unit
mass of cells and A and Apool are the external and
internal substrate concentrations. From equation 6
uptake rate per unit biomass (v)x is
(V)x = a A A (12)
Since (V)x= j and aAA = P i~Aout,the specific affin-
ity a A becomes P ~. Thus the specific affinity is di-
rectly related to the ease of penetration of the sol-
ute into the cells as measured by D in diffusion the-
ory (Sen et al. 1980). Of course, in real microbial sys-
tems that penetration is facilitated by the
transporters in the cell surface where transport is
active and A << Apool. Multiplying equation 6 by the
yield of cells produced from substrate used gives the
specific growth rate as a function of both permeabil-
ity and diffusivity:
P hAY (~_~_7_)$ A Y (13)
g= X --
This demonstrates that a cell can increase its ability
to sequester nutrients by either increasing the den-
sity of transport sites in the cell surface or increasing
its surface to mass ratio which is conceptually equiv-
alent to increasing the diffusivity of the cell mem-
brane or decreasing its thickness.
Specific affinity and collision frequency
If specific affinity from equation 6 is taken as the
forward rate c o n s t a n t k I between substrate and
cells, the kl is given by the collision rate between
molecules and cells (Abbot & Nelsetuen 1988),
/4AD]
kl = \ 1-T0-0-O! n r x (14)
where r x is the radius of the cell. Both, substrate
concentration requirements and the maximal spe-
cific affinitycan thus be computed directly from col-
lision frequency. To do this one assumes that the cell
provides a perfectly absorbing surface. Then for a
typical marine bacterium that is approximately
spherical, 0.3 gm in diameter and growing on glu-
229
cose, diffusivity of 6.7 x 10-6 cm2s-1, k 1 is 1.2 x 10 -12
L/cell. s. For this organism to grow with a doubling
time of 13 h, specific growth rate g = ln2/13 = 0.05 h -1,
the required glucose uptake rate would be, at a yield
Y of 0.5, v = g/Y = 0.1 g glucose/g cells, h. The con-
centration of glucose necessary to supply this rate,
according to equation 6 written as v = k 1X A, is 0.31
gg/L. This requires a specific affinity of v/A = 3.2 ×
105 L/g cells, h which represents the theoretical lim-
it. Values of 104 are however approached (unpubl.,
this laboratory). The importance of small size for
growth in dilute systems is well known (Chisholm
1992) and quantitatively demonstrable by recom-
puting the glucose concentration required for a
more commonly cultured larger 0.4 gm 3cell, 1 gm in
diameter, to grow at the same rate. Minimum glu-
cose concentrations increase from 0.31 to 2.5 gg/L
and the theoretical upper limit for the specific affin-
ity is 40,000 L/g cells, h. This demonstrates one rea-
son why ultramicrobacteria dominate marine bio-
mass.
One might presume that the chosen model of a
perfectly absorbing cell might require complete
coverage with active sites. However, because of re-
collision the condition is thought to be satisfied with
attainable transporter densities. For example it is
estimated that cage effect increases the likelihood
of successive collisions of substrate with cell surface
by a factor of 1000, and that coverage of only 0.1%
of the cell surface with receptor sites reduces ab-
sorption by only a factor of 2 (Berg & Purcell 1977).
Transporter/enzyme ratio and janusian kinetics
Increased transporter content of cells increases up-
take as given by specific affinity theory. However,
down-stream enzymes can restrict flow if, for exam-
ple, k2E2 of Scheme 1 is small. In laboratory experi-
ments a decrease in uptake rate over time for both
natural populations and for the typical marine oli-
gobacterium isolate RM 1 was observed indicating
the importance of this phenomenon (unpubl. data,
this laboratory). To quantify this effect one may as-
sume that the immediate result of transport is in-
ternal or pool-nutrient concentration, that trans-
port is Michaelian, and that utilization is Michae-
230
lian as well. By specifying pool concentration as the
product of transport and a translation coefficient L,
the four system components, external substrate,
transporter content of the cell membranes, pool
concentrations, and internal enzymes using those
pools, can be related. Overall rate is given by
_ [ V1VzA
v = X~KcRcxLK° + (KcRcxL + VOA)
where VIV2are maximal velocities for the transpor-
ter and limiting enzyme, K o and Kc are their Mi-
chaelis constants, and Rcx relates the cytoplasmic
content of the cells to their biomass. Because of the
two-step process involved, transport and metabo-
lism, these formulations are known as janusian ki-
netics. The grouping in equation 15 is that of a typ-
ical hyperbolic rate equation which means that the
kinetic constants for the whole cell process can be
described from the amount and kinetics of the com-
ponent transporter and rate-limiting enzyme. Col-
lection of terms together with equation 18 below
gives all the kinetic constants, the specific affinity,
maximal uptake rate, and whole-cell Michaelis con-
stant in molecular terms (Button 1991). Further,
equation 15 shows that these constants depend on
the content and kinetic characteristics of the trans-
porters and rate-limiting enzymes. Numerical sim-
ulations show that increasing the limiting enzyme-
to-transporter ratio substantially increases the
whole cell Michaelis constant, and increasing the
transporter content increases the affinity. However,
significant cross correlations are also seen to occur.
Table 1. Effect of transporter density on substrate uptake.
Sparesely distributed transporters
Trr/X is small
T<<rcr x
v=kfAT
kf = (4 A D/1000) r r
kf changes with T
Site properties set transport rate
Effect of transporter density
Transporter density, as demonstrated above, affects
the specific affinity. However, internal interactive
effects may be caused by limited amounts of down-
stream enzyme and can reduce the specific affinity
as well (Button 1991). In addition the fundmental
nature of the controlling rate constants can change
(15)
with conditions. The forward rate c o n s t a n t kf for bi-
molecular collisions (Berg & Purcell 1977; Martinez
et al. 1992) is given by
[ [ ~A4 Dx ~[ T rTf(rs)
(16)
k f= ~ 1--]-ff0--O}rx f(rx)] \T rtf(rs)+ x Gf(rx)]
where A is Avogadro's number (other abbrevia-
tions defined below). The equation can be modified
to describe substrate A reacting with transporter
molecules having an active site radius r T and num-
bering T on the surface of a cell radius r x according
to
kr..
A +T TA (17)
~kr
Uptake rate is specified according to forward rate
constant kf for nutrient capture, not kl, because the
effective units change with cell size as shown below.
The first term in brackets describes the collision be-
tween particles while the second is the probability
that binding will occur. The function firs) corrects
the forward rate constant by reducing the apparent
transporter site radius to account for molecules in
the process of binding with the transporter, while
fiG) corrects for the portion of the substrate colli-
sions with the cell that result in entry into the cy-
toplasm. T is limited to the number of transporter
sites that are free. When large, it cancels with the
Densely distributed transporters
TrT/X is large
T>>xr x
v=kfAX
kf = (4 _AD/1000) r x
kf constant with T giving normal bimolecular kinetics and
collisional limit attained
Diffusion sets transport rate

first term of the denominator giving a cell-size de-
pendent rate constant. When small, it drops from
the equation giving the rate constant as a property
of the transporter site. System properties described
by the relationships (Table 1) show that the basic
kinetic formulations for whole cells are cell-size de-
pendent.
An inherent assumption in both specific affinity
theory and janusian kinetics is that the collision lim-
it does not apply because cell size is not accounted
for, only the number of transporters T. Thus if T/X
is small and T, which is constrained to the number of
free sites, is decreased by increasing substrate, then
kf increases with rate because the number of free
transporters is reduced. Reduction of the number
of available transporters can cause the kinetics to
revert to transporter-dependent rates. Because of
recollisions, maximal rates, called the collisional
limit, may be attained at relatively low site density
and rate can be formulated as a function of cell size.
Thus while transporters operate at maximal effi-
ciency at low density, the overall nutrient capture
rate is low. By increasing transporter density, the
rate is increased but diffusion time reduces the spe-
cific affinityfrom what it might otherwise have been
and increases KT. Experimental verification was
obtained by using an Escherichia coli system in
which both the porin and internal enzyme concen-
tration could be controlled (Martinez et al. 1992).
For this reason kinetic constants depended on
transporter and other enzyme content, which is in-
consistent with conventional theory, and curve
shapes became nonhyperbolic at high enzyme con-
centrations, presumably due to limitation by diffu-
sion. This helps to verify janusian kinetics and docu-
ments limitations of conventional theory.
Leakage and relaxation methods
Important aspects of cell-leakage include i) loss of
substrate actively deposited into the metabolic
pools, and ii) loss of substrate-derived metabolic
products from the pools to the environment either
due to failure of the cell membranes to completely
retain these metabolites over the steep concentra-
tion gradient produced, or inability of the cells to
231
utilize the products as fast as they are formed. Such
losses result in a difference between gross transport
rate as may be measured in short-term uptake ex-
periments and net transport which contributes
more directly to growth. Rate constants connecting
these unidirectional fluxes are shown in equation
set (18)
A kl" Apool (18)
hpool k3" Vpool
Vpool k5~ Vcell
k"-:-~-
k7...
Ppooi .k 8 P
The flux associated with the active transport of sub-
strate A is identified by Jl; that associated with sub-
strate leakage is J2. Pool products may be either used
for growth, Js, or leaked from the cell, JT-These uni-
directional fluxes can be determined in continuous
culture while maintaining nutrient-limited growth
at steady state (Button & Kinney 1987). For exam-
ple, to determine the rate of substrate leakage and
therefore net transport Jl-J2, isotope relaxation
methods can be employed. The continuous culture
reactor is supplied by two reservoirs of medium,
identical in all respects, except that the substrate in
one is radiolabeled. If the steady state is achieved
from the radiolabeled substrate, and the supply is
suddenly switched, radioactivity in the medium is
quickly lost. The loss rate depends on the biomass
in the reactor as controlled by the amount of sub-
strate A 0in the supply, and on the specific affinity of
the cells for the substrate. This rate of radioactivity
loss is known to the extent that the growth rate is
equal to the dilution rate, a central assumption of
continuous culture, and the accuracy with extracel-
lular substrate concentration A is measured. Such
concentrations, it turns out, can be quite difficult to
measure (Law et al. 1976). A small and known
amount of substrate is lost due to washout from the
reactor as well. If the specific affinity is large, A is
small, its residence time is short and of the order of
seconds, even if A 0 is small. But the residence time
232
of the cells is much longer, and equal to the rate of
dilution of the reactor. Thus the extracellular sub-
strate radioactivity soon depends almost entirely on
leakage Jr The set of equations (19) for determining
the rate of leakage of pool substrate, Apool , and pool
p r o d u c t , Ppool, formulated in terms of the specific
rate of growth, is given by
(19)
g = X(j~-j2)/(Ao-A)
g = - X(js-jT)/P
B = [01 + J4)-(J2 + J3)] R A
[.t - [03 + J6 + Js)-(J4 + J5 + J7)] R Ppool
g = (is-J6)/R Pcell
where the ratio R converts the mass of cells to the
fraction occupied by metabolic pools. For the trans-
port of phosphate by Rhodotorula rubra, pool or-
thophosphate substrate and organic phosphate
product leakage during phosphate-limited growth
was up to 5% and 10% of the phosphate transport
rate respectively, depending on the rate of growth
(Robertson and Button 1980). In the case of toluene
metabolism, leakage of metabolic products such as
2-hydroxy-6-oxo-heptadienoic acid can amount to
most of the substrate accumulated (Button & Ro-
bertson 1987), presumably because of inability of
the cells to either retain this amphipathic metabo-
lite or actively transport it back in. In such cases the
difference between the measured rate of transport
and the net substrate flux is quite large.
Table 2. Response of kinetic parameters to changes in composi-
tion.
Compositional parameter Kinetic parameter
responding
Transporter mass/cell mass
Limiting enzyme x Transporter content Wmax
Limiting enzyme/Transporter content
Biomass/Transporter content Allofunctional
overhead ~
Oligotrophic capacity log a0A/KT
1Biomass devoted to functions other than nutrient collection.
Use of kinetic data
Interpretation of kinetic data from microbial sys-
tems is more difficult than for enzymatic systems
because of additional constraints:
1. Rate limitation may involve diffusion either to
an organism buried in a viscous matrix or to a
limiting enzyme buried within the organism.
2. Limiting steps may shift with concentration. For
example at low concentrations, collision with
transporters may be limiting; at intermediate
concentrations, collisions of transported nutri-
ent with internal enzymes may be limiting; and at
high concentrations the turnover number of an
enzyme or the activity of a subcellular particle
such as a ribosome may control rate.
3. As shown above, the nature of the rate equation
may change with cell size and transporter densi-
ty.
4. Evaluation of the responsible microbial popula-
tion is necessary to give affinities for natural
populations a basis in biomass.
Difficulties in evaluating microbial biomass have
constrained environmental kinetics because micro-
bial populations and cell size have been hard to
measure. Flow cytometry is helpful for minimizing
this difficulty in the case of bacteria (Button et al.
1992), and good progress is underway for phyto-
plankton (Olson et al. 1985; Chisholm 1992). Mole-
cularbiological methods offer potential for helping
identify population components responsible for
particular activities (Schmidt et al. 1991).
Janusian kinetics allow the formulation of kinetic
constants for microbial processes in molecular
terms (Button 1991) which are useful for under-
standing morphological constraints. For example
they can be used to anticipate the effect of whole-
cell composition on kinetic properties (Table 2).
It can be seen, for example, that increasing the
transporter-to-biomass ratio might be useful for
collecting nutrients (line 1), but that accumulation
capacity would suffer (line 2). Simplifications ig-
nore the kinetic interactions discussed above as
well as the influences of metabolic control. How-
ever, it is suggested that kinetic constraints imposed
by quantities of catalytic components do reflect the
ability of organisms to collect and process nutrients,

and as such help describe the balance between dis-
solved nutrients and particulate material in the
form of microorganisms that process these nutri-
ents.
Microbial ecology
A central question is the extent of microbial diversi-
ty in the presence of a single major limiting nutrient.
We know from oligotrophic strategy that good oli-
gotrophs lie at the extreme end of a log a0A -log K r
plot (Button 1991). This can be seen from the defini-
tion of specific affinity where usual hyperbolic ki-
netics apply:
a ° = Vmax/KT (20)
and the assumption that the variation in Vmax is
small with respect to variation in K T. Logic of the
assumption is that required rates of major carbon/
energy source accumulation vary only approxi-
mately as much as the difference in maximal growth
rates among species. Then equation 19 specifies a
reciprocal relation between specific affinity and Mi-
chaelis constant and leads to a definition of oligo-
trophic capacity (Table 2). The interpretation is that
good oligotrophs are small and have large transpor-
ter/limiting-enzyme ratios for operation at small
limiting nutrient concentrations.
For example Vibrio $14 with a specific affinity of
563 liters/g-cells- h for glucose and a transport con-
stant of 9.9 x 10-5 g/L (Akagi & Taga 1989) has an
0
oligotrophic capacity, log aA/KT, of 6.7. On the other
hand Pseudomonas sp. RP-386, a 0A= 2.5 and K T= 6.4
x 10-4 (Albertson et al. 1990) has an oligotrophic ca-
pacity of only 3.5. Perhaps this pseudomonad de-
votes more material to the metabolism of glucose as
well as the metabolism of other substrates relative
to the number of glucose transporters formed.
An advantage of diversity in nutrient transport
ability may lie in the colision frequency equations.
Consider that once a nutrient molecule collides
with a cell surface, it remains in the vicinity and of
the order to 1000 additional collisions are likely as
discussed above. Thus a colliding molecule is
marked and distinct from others. If the density of
233
sites can reach the collisional limit when rather
sparesely distributed within the cell envelope, the
space may advantageously be used by other types of
sites. A diversity of sites (TA, TB, Tc.... ) may there-
fore collect a diversity of dilute substrates (A, B,
C...) faster than an equal number of total sites con-
strained to a single molecular species. Multiple
transporter types can lead to a diversity of species.
Since each species may focus on a particular combi-
nation of dilute substrates, the number of possible
combinations exceeds the total number of nutrients
collected. Multiple transporters must be connected
to multiple metabolic systems which are costly to
build. However multiple systems can lead to higher
nutrient-collection efficiency if the additional costs
of the extra transport and metabolic systems are
outweighed by increased nutrient flux. In addition
there can be cost advantage to transporting re-
quired biochemical monomers over de novo syn-
thesis. Other costs of transport site diversity include
small maximal growth rates from a single substrate,
pool imbalance, and substrate accelerated death
upon nutrient addition. However, these costs are
unlikely for many organisms in oligotrophic sys-
tems. When transporters are dilute, rates of trans-
port v of various substrates are given by their re-
spective forward rate constants, transporter con-
centations, and substrate concentrations
VA + VB + Vc = kATAA + k BTBB + k c TcC >
3kAA, 3kBB, 3kcC (21)
which leads to an overall average rate constant k s
for the total substrate concentration S. The inequal-
ity of equation 21 states the observed preference by
oligotrophic organisms for simultaneous multisub-
strate collection and the collision frequency analy-
sis gives a plausible reason. Namely, if additional ca-
tabolic system construction costs are outweighed by
the additional substrate collection rate of a diverse
cell surface, then a mixed diet is of substantial ad-
vantage.
234

Definitions oligotrophic and heterotrophic marine bacteria. Can. J. Mi-

crobiol. 26:454-459
Albertson H, Nystr~m T & Kjelleberg S (1990) Starvation in-
duced modulations in binding protein-dependent glucose
Symbol Definition Units
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aA Specific affinity for substrate
A; a °, base value; a max, Abbott AJ & Nelsetuen GL (1988) The eollisional limit: an im-
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Azam F & RE Hodson (1981) Multiphasic kinetics for D-glucose
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Prog. Ser. 6:213-222
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~, Avogadro's number
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C Flux control coefficient dimensionless
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D Diffusivity
2038
E Enzyme concentration g liter-1
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h)-~
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Button DK & Robertson BR (1987) Toluene induction and up-
Ko, outside concentration
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k Rate constant variable
611
1 Thickness cm
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n Number of molecules
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Environ. Microbiol. 58:243-25
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Chisholm SW (1992) Phytoplankton size. In: Falkowaski PG &
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Cleland WW (1975) Partition analysis and the concept of net rate
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V 1 for system g-substrate liter-~
3224
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