Kinetics of microbial processes: General principles

NS Panikov, Chemistry and Chemical Biology Department, Northeastern University, Boston, MA, United States
© 2023 Elsevier Ltd. All rights reserved.

Introduction 169
State variables and growth parameters 170
Amount of microorganisms 170
The concentration of limiting substrate 171
Growth yield 171
Maintenance energy 172
Energy overflow 172
Cell quota. Variation of yield on anabolic substrates 173
Products 173
Kinetic models of microbial bioprocesses 174
Chemical and enzyme kinetics 174
Exponential growth 175
Logistic growth 175
Monod model 175
Cell quota model 176
Structured models 176
Genome-scale models (GEM) 176
Models of intermediate complexity 178
Growth dependence on environmental factors 180
Hierarchy of factors, the primary significance of the nutrient resources 180
Effects of pH 180
Effects of temperature 182
The special case of near-zero growth kinetics 183
Concluding comments 184
References 184
Further reading 185

Abstract

Kinetics describes rates and mechanisms of processes. Microbiological kinetics studies all dynamic behaviors, such as growth,
survival, death, mutations, adaptations, product formation, cell cycles, using a combination of experiments and mechanistic
mathematical simulations. This chapter introduces the basic terms, variables, and concepts moving from chemical and enzyme
kinetics, to unstructured growth models (logistic, Monod, cell quota), structured models simulating self-adaptive microbial behavior
(cybernetic, SCM), and to modern genome-scale reconstructions. Effects of environmental factors are illustrated as an interaction of
variable pH and temperature with nutrient concentration. The final topic is the near-zero growth of microbial populations prevailing
in the biosphere.

Key points
• Microbiological kinetics aims to elucidate the underlying mechanisms of all dynamic manifestations of microbial life by
using mechanistic mathematical models. This covers growth, survival and death, mutations, differentiation of cells into
forms with contrasting function or activity, product formation, adaptations of metabolism to changing environments, cell
cycling and interactions with other organisms.
• The relationship between microbial biomass and cell numbers or length of mycelium is not straightforward, because the
mass of individual cells or width of hyphae vary among different organisms and even those of the same species depending
on growth conditions.
• As all potentially available nutrients cannot normally be assessed, the focus is on one or few individual compounds or
classes of molecules representing the limiting nutrient substrate, i.e., the nutrients that control the growth and activity of the
microorganisms investigated.
• Kinetic models are either deterministic (clear, determined and regular processes) or stochastic (dealing with random
variables participating in biological processes). The main variables used in deterministic models (concentrations of cell
mass, substrates, and products) are the same as in chemical kinetics, whereas stochastic models consider variables such as
probabilities, frequency distributions, variance.

168 Encyclopedia of Soils in the Environment, Second Edition https://doi.org/10.1016/B978-0-12-822974-3.00260-3

 SOIL BIOLOGY | Kinetics of microbial processes: General principles 169

• Under natural conditions in the soil, microbial growth is mostly continuous and very slow because of severe nutrient
limitations. Even in eutrophic habitats (e.g., the rhizosphere) microorganisms divide no more than 1–2 times per day,
whereas in mesotrophic and oligotrophic habitats the doubling time can be months and years.

Nomenclature
D¼F/V Chemostat dilution rate
F Medium feeding rate
Ks Saturation constant, at s¼Ks, m¼0.5mm
m Maintenance term, the catabolic rate qs!m, as m!0
NZG Near-zero growth under chronic starvation
ODE Ordinary differential equation
p Product concentration
PPA Plant photosynthetic activity
qp¼(1/x)dp/dt Specific product formation rate (SPR)
qs¼(1/x)ds/dt Specific substrate uptake rate (SUR)
ROS Reactive oxygen species
s Limiting nutrient substrate concentration
SCM Synthetic Chemostat Model
SGR Specific growth rate
t Time
V Volume of cell culture kept constant in the chemostat
VBNC Viable but nonculturable cells
x Cell mass concentration
Y¼−dx/ds Cell yield per unit of consumed substrate
m¼(1/x)dx/dt SGR calculation
mm Maximum SGR, m!mm as s!1

Introduction

Kinetics (from the Greek kinetiwοs, forcing to move) is a branch of natural science that deals with the rates and mechanisms of any
processes, physical, chemical, or biological. Microbiological kinetics studies all dynamic manifestations of microbial life: including
growth itself, survival and death, mutations, adaptive reconfigurations of metabolism to the changeable environment, products
formation, cell cycles, interactions with other microorganisms or microbial hosts (humans, animals, plants), the functional
differentiation of cells into active-or-dormant, motile-or-sessile, resistant-or-susceptible. Contrary to descriptive monitoring of
process dynamics, kinetic studies attempt to elucidate the underlying mechanisms by using mechanistic mathematical models.
The epithet ‘mechanistic’ implies that the respective model translates the postulated essential point of the studied processes into the
mathematical equations with variables and coefficients having clear physical, chemical, or biological meaning (contrary to, say, the
polynomial regression equations). Then the comparison of observation and the model’s prediction allows researchers to discard
incorrect hypotheses or modify them to resolve the interpretive contradictions.
A hypotheses-driven approach to acquiring knowledge is a common part of any scientific method established in science since at
least the 17th century. Testing of hypotheses can be done against a wide range of data and be based on any revealed inconsistencies.
The distinctive feature of kinetics is the added strength of the failed dynamic simulation as inconsistency, at the same time typical
kinetic study requires an experimental component of high standard, that should be based on reliable quantitative dynamic data, such
as precise rates measurements under fully controlled experimental conditions or observations of process dynamics over time,
preferentially with continuous logging of a set of data interconnected by cause-and-effect relationships. Descriptive information that
cannot be expressed numerically is generally not acceptable.
Microbial kinetics is tightly linked to the growth stoichiometry (from the Ancient Greek stοiweῖοn ‘element’ and mέtrοn
‘measure’), which is the quantitative relationship between substrates and the products of the bioprocess. In practical terms,
stoichiometry addresses mainly problems of a static nature, like “how much?” and “in what proportion?” while kinetics considers
the dynamics and deals with questions like “how fast?” and “by which mechanism?”
There could be reluctance in applying such a biokinetic approach to environmental and biospheric studies, including soil
biology, on the basis that these natural systems are too complex for precise rate measurements and fully controlled experimental
conditions. This is not true. The complexity of soil as a part of nature does not prevent its deeper understanding by using precise rate
measurements and other quantitative methods. Moreover, the combination of mechanistic mathematical simulations with field
170 SOIL BIOLOGY | Kinetics of microbial processes: General principles

observations, laboratory soil incubations, and planted soil microcosms should be highly beneficial for a better understanding of
biospheric processes including on-going global climatic changes. It could even be highly beneficial in the practical developments
of the mitigation strategies and novel environmental biotechnologies aimed at establishing sustainable land use.
The first chapter in this two-parts series has the objective of introducing the reader to the basic terms, variables and principles of
microbiological kinetics that apply equally to industrial bioreactors, laboratory cultures and soil communities. The second chapter
focuses on specific applications of the general principles to soil biology. We recommend that an interested reader read both parts,
although the sequence order is not critical!

State variables and growth parameters

There are two types of kinetic models, deterministic and stochastic. The former describes clear, determined, and regular processes. The
latter models deal with random variables participating in bioprocesses. The main variables used in deterministic models are the
same as in chemical kinetics - concentrations of cell mass, substrates, and products - while stochastic models consider instead
probabilities, frequency distributions, variance, etc. For example, a deterministic model can describe the process of N2-fixation as a
function of energy and nutrient supply to the rhizosphere, while a stochastic model considers the probability of rhizobia infecting
legume roots for a given frequency distribution of bacteria across the environmental space. Any real biospheric process has both
deterministic and stochastic components, so ideally the kinetic model should be a ‘hybrid’ and contain a deterministic core with a
stochastic interface, generating probability distributions and confidence corridors rather than single values or curves.
The major state variables of kinetic models describing microbial processes in nature are summarized in Table 1 and described in
detail below.

Amount of microorganisms
Quantitatively, the abundance of microbes can be expressed as biomass (x) or cell numbers (N) of unicellular organisms (bacteria,
yeasts, protozoa, fungal spores) per unit of soil or water mass, volume, or surface area. Filamentous organisms (fungi, actinobac-
teria) are characterized by the length (L) of mycelium and the number of hyphal tips, N0 , initiated by hyphal branching. Note that N0
is equivalent to N when branching is equivalent to cell division.
The relationship between x and N or L is not straightforward because the mass of individual cells and width of hyphae vary
among different organisms and even for the same species depending on growth conditions. Generally, a plentiful supply of
nutrients fosters the formation of bigger cells or wider filaments, whereas starvation causes cells to shrink. To estimate microbial
biomass from direct microscopic counting, one should generate several arrays of data: the number N ¼ [N1, N2, . . ., Nn] of cells
with specifying biovolume V ¼ [V1, V2, . . ., Vn], where Ni is the number of cells in the ith volume class Vi and n is the number of
such classes, the volumes being calculated from the geometry of all detected cell shapes (spherical, ellipsoid, rod-shaped, irregular).
For filamentous microorganisms, one should have arrays of hyphal length L ¼ [L1, L2, . . ., Lm] and corresponding hyphal diameters
D ¼ [D1, D2, . . ., Dm]. The total biomass, x, is calculated as a sum of unicellular and mycelial soil microorganisms:

x ¼ V  N  ð1 − yÞ + ¥ pD2  L  ð1 − yÞ (1)
where y is the cellular water content.
Advanced microscopy (epifluorescence, electron- and atomic force microscopy) and computer-aided image-analysis techniques
make cell counting and sizing more accurate and less tedious. Additionally, Fluorescence In Situ Hybridization (FISH) allows
targeting of specific taxonomic or functional groups based on the use of RNA-oligonucleotide probes. The total microbial biomass,
x, can be estimated by extracting from environmental samples one of the following unique cell constituents: (a) DNA, (b) ATP,
(c) membrane phospholipids, (d) microbial cell wall components (chitin for fungi and muramic acid for bacteria), or (e) the sum of
labile or extractable C released after natural sample fumigation with chloroform or drying-rewetting (Blagodatskiy et al., 1987).
Selection of either biomass x or cell number N or mycelium length L depends on the specific objectives of the study. Biomass, x,
has the obvious advantage in studies of carbon and nutrient cycling, while cell number, N, is preferred in population studies when,
say, mutation or plasmid transfer is crucial for the understanding of the studied natural processes.
A large microbial biomass does not necessarily imply a fast growth rate or vice versa, because low abundance could be caused by
intensive elimination (e.g., washout, chemotactic movement, grazing, and lysis by parasites, see discussion below) of growing cells
while high abundance could be due to migration of cells from other habitats or accumulation of dormant cells. In the absence of

Table 1 The major dynamic variables used in deterministic kinetic models.

Variable Symbol Dimension (examples)

Concentration of cell biomass x mg cell mass (g soil)−1, g cell mass m−2

Cell number N count (g soil)−1
Mycelium length L m (g soil)−1
Hyphal tips number n count (g soil)−1
Concentration of limiting substrate s mg (g soil)−1, mmol (g soil)−1
Concentration of products (CO2, CH4,..) p mg (g soil)−1, mmol (g soil)−1
 SOIL BIOLOGY | Kinetics of microbial processes: General principles 171

elimination, the microbial growth rate can be found by recording one of the used quantities (x, N, L, ATP content, etc.) over time t.
The absolute growth rate is calculated as a simple slope of cell mass x versus time:
dx Dx x2 − x1
 ¼ (2)
dt Dt t2 − t1
This slope normalized to the biomass represents the specific growth rate (SGR) of microorganisms, m:
1 dx 1 Dx 1 x2 −x1 x + x1
m¼  ¼  , where x ¼ 2 (3)
x dt x Dt x t 2 −t 1 2
In the case of explicit or hidden elimination of microorganisms accompanied microbial growth, the formulae above are no longer
valid and the calculated from Eq. (3) SGR should be referred to as the apparent growth rate.

The concentration of limiting substrate

Normally we cannot assess all potentially available nutrients and consequently we focus on one or few individual compounds or
classes of molecules representing the limiting nutrient substrate, i.e., such nutrient(s) that control the growth and activity of
microorganisms in question. Typically, the growth of chemoorganotrophic microorganisms in soil and subsoil is limited by available
organic compounds, while photosynthetic microbiota is limited either by light or sources of P, N, or Fe.
The absolute rate of substrate consumption by microorganisms is measured as a time derivative (−ds/dt) of the substrate
depletion curve, s(t), while the specific rate of substrate consumption, q is calculated by normalizing the absolute rate to the instant
cell mass concentration, x:
1 ds s
q¼− ¼Q (4)
x dt Ks + s
Eq. (4) is similar to the Michaelis–Menten equation established in enzymology with parameters Q standing for maximum uptake
rate and saturation constant Ks numerically equal to such substrate concentration at which uptake rate q ¼ 0.5Q. SGR m is related to
q by a mass balance Eq. (6) (see below) and therefore can be derived from (4) as follows:
s
m ¼ Yq ¼ mm (5)
Ks + s
where mm ¼ YQ is the maximal SGR. This expression is known as the Monod equation. Note that both Eqs. (4) and (5) are empirical
and should be applied with caution. The substrate uptake rate not only depends on its concentration but also on other
environmental factors (temperature, tonicity, pH, etc.) and the physiological state of cells (macromolecular cell composition,
proteome expression profile, and other cell qualities). Even more complex is the intracellular machinery responsible for growth (see
Section “Models of intermediate complexity”). Therefore Eqs. (4) and (5) should be applied only to relatively simple experiments
with s as the only variable and under conditions in which studied cells preserve the same physiological state at all tested values of s.
There are two groups of microbial nutrient substrates: (1) catabolic substrates which are sources of energy, and (2) anabolic or
conserved substrates which are sources of biogenic elements forming cellular material. Examples of catabolic substrates are H2 for
hydrogen-oxidizing microorganisms, NH4 + and NO2 − for nitrifying bacteria, S0 for sulfur-oxidizing bacteria, and oxidizable or
fermentable organic substances for diverse heterotrophic species. Their consumption is accompanied by oxidation and dissipation
of chemical substances into waste products, which are no longer reusable as an energy source (H2O, NO3− , SO42−, CO2, etc.). The
anabolic substrates are consumed and incorporated into de novo synthesized cell components, being conserved in biomass; that
is why they are referred to as the conserved substrates. Contrary to catabolic substrates, the conserved substrates can be reabsorbed
after excretion or cell lysis. They include nearly all non-carbon sources of biogenic elements (N, P, K, Mg, Fe, and trace elements),
CO2 for autotrophs, as well as amino acids and growth factors for respective auxotrophic species.

Growth yield
Two variables, cell mass x and substrate concentration s, are linked via stoichiometric parameter growth yield:
dx Dx
Y¼−  − (6)
ds Ds
where Dx is the increase in microbial biomass consequent on utilization of the amount Ds of the substrate. Dividing both parts
of (6) by xdt gives the relationship between SGR and substrate production:
dx 1 dx 1 ds m
Y¼− ¼ : ¼− (7)
ds x dt x dt q
The reason for Y variation is different for catabolic and anabolic substrates.
172 SOIL BIOLOGY | Kinetics of microbial processes: General principles

Maintenance energy
In the case of energy sources, some fraction of the total substrate flux is diverted from growth per se to meet so-called maintenance
requirements. Specific maintenance functions are resynthesis of self-degrading macromolecules (proteins, nucleic acids, cell wall),
cell motility, osmoregulation, leakage, proofreading, ROS detoxification, etc. The energy balance is described by the following
equation:

total energy source uptake consumption for growth consumption for maintenance
q ¼ m=Y max
+ m (8)
max
where m is the maintenance coefficient and Y is the hypothetic maximum yield that would be observed with zero maintenance,
m ¼ 0.
To account for the maintenance requirements, Eq. (4) should be modified as follows:

m s s −s ∗
q¼ +m¼Q ∴m ¼ mm (9)
Y max
Ks + s Ks + s
where s ¼ YmaxKsm is the threshold substrate concentration below which cells are not expected to sustain growth, if s < s ,
then m < 0.
If m and Ymax are constants, then the plot of q versus m should be a straight line with y-intercept equal to m. Such a plot is the only
possible experimental way of finding the maintenance term m. In practice, a chemostat culture of the tested microorganism is run
sequentially at several dilution rates until steady-state (D ¼ m) is established; at each D steady-state biomass and some catabolic rate
(respiration, heat production, uptake of the energy source, etc.) are monitored and q plotted versus D ¼ m as illustrated in Fig. 1.
Note that the described technique is indirect and time-consuming but unfortunately there are no direct ways to estimate the
maintenance as a part of cellular energy balance. Numerous maintenance processes (osmoregulation, resynthesis, and repair of
damaged polymers, stress-response) are mixed up with the growth-associated processes and could not be accurately translated into
the energetic cost. The only manifestation of the maintenance is a decrease of apparent cell yield at low SGR. Under very deep
nutrient limitation typical for the soil environment, the maintenance term m can no longer be considered as a constant, so the
Eq. (9) must be replaced by more advanced kinetic models, see the Section “Near-zero growth kinetics.”

Energy overflow
Maintenance requirements should be distinguished from other non-productive use of energy called wasteful respiration or energy-
spilling reactions. They are observed under several special circumstances: (i) when growth is energy-sufficient but limited by anabolic
substrates (sources of N, P, K, Fe, vitamins, etc.); (ii) over extended lag-phase of batch culture; (iii) in transient experiments when
starving cells are brought to rich medium and (iv) when cells are exposed to uncoupling inhibitors. A common feature of all the
listed processes is so-called energy overflow: the energy flux significantly exceeds the level required for biosynthetic processes
constrained by inhibitors or a depleted pool of ribosomes or by other than energy source nutrients. As a result, the diversion of
energy consumption from growth is much greater than expected from the basic maintenance functions observed in the
energy-limited chemostat culture. Note that the energy overflow has been observed in laboratory experiments; this phenomenon
has not been demonstrated in situ.

Fig. 1 Two ways of representing the maintenance term. (A) The Monod-Pirt model assumes the constancy of the maintenance term. The tested microorganisms
are grown at several dilution rates D in a chemostat limited by the energy source. The specific catabolic rates qs (respiration, catabolic substrate uptake) are plotted
vs D ¼ m, and fitted to Eq. (9) giving 1/Ymax (slope) and the maintenance coefficient, m (intercept and the dotted line). (B) The same plot is simulated by a more
realistic SCM model, which allows adaptive self-regulation of the maintenance term. Note a progressive decrease in maintenance with slowing growth. Modified
from Panikov NS (1995) Microbial Growth Kinetics. The Netherlands: Springer. 378 p.
 SOIL BIOLOGY | Kinetics of microbial processes: General principles 173

Cell quota. Variation of yield on anabolic substrates

Cell yield on anabolic substrates varies mainly as a result of alterations in biomass chemical composition as expressed by the parameter
ss, the intracellular content of a deficient element, or cell quota. For example, the variation in N content in bacteria from 5% to 15% gives
the ss range 0.05–0.15 g N (g cell mass)−1. For most known cases, the quota ss increases parallel to growth acceleration, because the
higher growth rate requires higher intracellular content of proteins and RNA (contain N, P, S) as well as K+, Mg2+ and vitamins
participating in primary metabolic reactions. The yield and cell quota are inversely related to each other, e.g., small N-content
(sN ¼ 0.05 g N/g) corresponds to large cell yield (YN ¼ 1/sN ¼ 20 g cell per g of N utilized), large N-content in rapidly growing cells
can be attained only with a small cell yield (YN ¼ 1/0.15 ¼ 6.67 g cell/g N) (Fig. 2). The principal difference between Y and inverse
s (1/s) is that the first stoichiometric parameter is subjected to instantaneous changes as dependent on s level in various transient
shift-up or shift-down processes, while the second parameter is essentially slower required deep reconfiguration in cellular composition.

Products
There could be intermediate and end metabolic products accompanying microbial growth. The typical intermediates of the glycolytic
and the Entner–Doudoroff pathways are:

• glucose ! 2 pyruvate−+2H+;
• glucose ! gluconic acid ! pyruvate;
• pyruvate ! acetate + CO2
biological activity due to the wide availability of gas analyzers (GC, IR- and MS). For the case of aerobic decomposition, the
measured CO2 evolution is a significant fraction of the C budget as described by the following mass-balance equation:
The only major end product of aerobic microbial decomposition is CO2. Under anaerobic conditions, the spectrum of products
is much wider and includes several organic acids, alcohols, and ketones, H2, CO2, CH4, and various reduced inorganic compounds
(S2−, Fe2+. . .). CO2 evolution rate is probably the most popular parameter to characterize soil

Total carbon consumed C incorporated into cell C oxidized to CO2 C in extracellular products
DC-s ¼ DC-x + DC-CO2 + DC-p (10)

Division by time increment dt gives:

Uptake of C-substrate Microbial growth Respiration Products formation

ds dx d½CO2  dp
¼ + + (11)
dt dt dt dt

Thus, microbial consumption of C-substrate is the sum of growth, respiration, and product formation, if all four variables are
expressed in the same mass-C units, e.g., mg C/g soil. Based on such a C-balance, we can estimate with confidence some variables
that are hard to measure directly. For example, cell mass, x in a laboratory soil incubation study can be calculated as the difference
between the amount of initially added substrate s0 and the time-dependent qualities of residual substrate s and products CO2 + p.
However, it would be incorrect to identify the CO2 evolution rate alone with total soil decomposition, as some degraded C is
incorporated into soil organisms or their extracellular products.

Fig. 2 Variation of the cell mass yield on catabolic (A) and anabolic (B) substrates related to SGR. All curves were calculated using SCM or its simplified versions.
Modified from Panikov NS (1995) Microbial Growth Kinetics. The Netherlands: Springer, 378 p.
174 SOIL BIOLOGY | Kinetics of microbial processes: General principles

Kinetic models of microbial bioprocesses

Chemical and enzyme kinetics
Microbial growth is a complex bioprocess that includes numerous enzymatic and chemical reactions. The basic equations of
chemical and enzymatic kinetics are presented in Table 2. There are notions of molecularity (the number of molecules that are altered
in the reaction) and kinetic order, which should not be confused. According to the law of mass action, the rate of the chemical
reaction is directly proportional to the product of the activities or concentrations of the reactants:
ds
¼ −kn sn (12)
dt
where n is the kinetic order, and kn is a rate constant. For a simple single-step reaction, the order is generally the same as the
molecularity. For a complex reaction involving a sequence of unimolecular and bimolecular steps, the molecularity is not the same
as its order. Reactions of molecularity greater than 2 are common, but reactions of order greater than 2 are very rare. For instance, a
trimolecular reaction, such as A + B + C ! P, as a rule proceeds through two elementary steps, A + B ! X and X + C ! P, each of
which are of the second or first order. Table 2 gives expressions for three typical cases (zero, first and second order), the left column
shows reaction rate as a function of reactant concentration (s), the right column presents results of integration under the initial
conditions: s ¼ so at time t ¼ 0.
A unique feature of all enzymatic reactions is reversible formation of the intermediate enzyme-substrate complex
k1
ES:E + S ! ES ! E + P, where k−1 and k2 are the first order and k1 is the second order rate constants. Under steady-state assumption
k2

k −1

Table 2 Basic equations of chemical and enzyme kinetics.

 SOIL BIOLOGY | Kinetics of microbial processes: General principles 175

(constancy of ES concentration over time), this mechanism is translated into the Michaelis-Menten equation (Table 2, the bottom
line), predicting a mixed reaction order (the first order at small s and zero order at large s), a unique kinetic feature of reactions
driven by enzymes. Based on experimental data including the reactant depletion curve s(t), we can distinguish abiotic vs enzymatic
reactions and identify the reaction kinetic order.

Exponential growth
It is the simplest growth pattern displayed by all microorganisms (binary dividing bacteria, budding yeasts, fungal mycelium).
Exponential growth is observed temporarily under favorable physicochemical conditions before depletion of nutrient substrates
and accumulation of self-inhibitory products. The basic assumption is that the current growth rate is proportional to the instant cell
mass, x, the quotient m remaining constant:
dx
¼ mx (13)
dt
The integration of (13) at initial condition, x ¼ x0 at time t ¼ 0, gives the exponential equation:

x ¼ x0 emt or ln x ¼ ln x0 + mt (14)
Any metabolic rate, such as intensity of methane production, the evolution of CO2, rate of sulfate reduction recorded in microbial
culture or nutrient-amended soil samples, is proportional to the growth rate of microorganisms:
Metabolic rate Stoichiometric factor Cell growth
dp 1 dx x x0
¼  ¼ m ¼ m 0 emt ¼ Aemt (15)
dt Y0 dt Y0 Y

where p is the concentration of metabolic product expressed for simplicity in the same carbon units as biomass, mg C per g sample,
and Y0 is the cell mass yield per mass unit of the metabolic product. Integration of (15) under initial condition p ¼ 0 at t ¼ 0 gives:
x0 mt
p¼ ðe −1Þ ¼ Bðemt −1Þ (16)
Y
Comparing (14) and (16) we see that x and p time courses are not identical, although both imply exponential cell growth, the p(t)
curve deviates from the exponential law, especially at small t. In particular, the “ln x – t” plot is linear, but the plot of ln p vs t is not.
As t increases, the difference progressively decreases. However, the rate of product formation, e.g., soil respiration measured as mg
CO2-C/h/g soil, follows the exponential pattern (Eq. 14).

Logistic growth
In contrast to the exponential growth equation described above, the logistic equation established by Verhulst in 1838 sets an upper
limit for microbial growth by assuming that SGR is dependent on the microbial density, x:

  
dx x x
¼ mðxÞx ¼ ðm − aÞx 1 − ¼ rx 1 − (17)
dt xm K

where xm is the upper limits of cell mass. In ecological literature, xm is called the carrying capacity of the ecosystem, K, and the
difference between true growth rate, m, and mortality rate, a, is presented as apparent growth rate, r. This equation mimics the
S-shaped growth curve frequently observed in nature. However, it represents nothing more than an empirical relationship ignoring
real ecological mechanisms and interactions; note that parameters of logistic equation r and K cannot be predicted before actual
observation.

Monod model
This model was developed by a famous French scientist in 1941 but it is still very popular due to its elegant simplicity. There are two
basic premises: (a) the SGR is dependent on the concentration of limiting substrate (Eq. 4); and (b) biomass formation is linked to
substrate uptake by mass-conservation condition (Eq. 6):
dx s
¼ mðsÞx ¼ mm x
dt Ks + s
(18)
ds 1 dx
¼−
dt Y dt
Equation set (18) contains three parameters: yield Y, maximal SGR mm, and saturation constant Ks, which can be thought of as ‘ID’
for individual organisms and used to predict their growth dynamics. Remarkably, this model was used to develop a chemostat
theory before actual experiments with continuous culture had been undertaken, one of the very rare events in the history of
mathematical biology! For this purpose, the equation set (18) was modified as follows:
176 SOIL BIOLOGY | Kinetics of microbial processes: General principles

dx s
¼ ðm ¼ DÞx, m ¼ mm
dt Ks + s
(19)
ds 1 F
¼ Dðsr − sÞ − mx, D ¼ ,
dt Y V
where sr is the concentration of limiting substrate in the fed fresh medium delivered with pump from a reservoir, F is the pumping
rate (cm3/h), V is culture volume (cm3), and D is the dilution rate defined as the ratio D¼F/V, h−1. The model predicted several
counter-intuitive features of the chemostat, e.g., that SGR can be set up by an experimentalist by changing the medium flow at any
values between 0 and mm (before specific growth was believed to be a constant mm) and that growth rates depend on s (the residual
nutrient concentration in bioreactor) rather than on sr (the nutrient concentration in the feed medium).
However, the Monod model fails to explain many dynamic phenomena observed experimentally, namely: lag-phase, death of
starving cells, product formation, and any kind of adaptive changes in the microbial population, such as induction-repression of
enzymes, yield variation, changes in the cell RNA content, etc. These gaps were intended to be filled by the structured models.

Cell quota model

The model is based on the empirical observation that SGR is proportional to the intracellular content of a deficient element s, such
as N, P, Fe, and other conserved nutrient substrates (Droop, 1973). As applied to the chemostat culture, it contains the same two
variables (x and s) as Eq. (19), but a different expression for specific growth rate:
dx
¼ mx − Dx
dt
ds
¼ Dðsr − sÞ −mxs (20)
dt
mm sm ðs − s0 Þ
m¼
sðsm − s0 Þ
It was the first attempt to express the relationship between SGR and cell quota, the variable characterizing the chemical composition
of the cell biomass. However, it is wrong to consider cell quota as an independent variable. As a matter of fact, s itself is a function of
the environmental factors, such as the concentration of respective sources of biogenic elements in situ.

Structured models
These models explicitly describe variations in cell composition (Fredrickson, 1976), they include mass balance equations not only
for external substrates and products concentrations but also several intracellular components, C1, C2, . . ., Cn, such as DNA, RNA,
proteins, cell wall constituents, reserved polysaccharides, ATP-pool, etc. For each variable Ci a differential equation is written which
accounts for all sources, r+, and sinks, r−, as well as its dilution due to cell mass expansion (growth),
dCi
¼ r + ðs, C1 , . . . , Cn Þ − r − ðs, C1 , . . . , Cn Þ − mCi (21)
dt
The earliest structured models contained just 2–3 highly aggregated internal variables, e.g., the total content of cellular proteins,
RNA, and DNA. More advanced models covered up to 50 real cellular constituents and the lumped variables such as pools of amino
acids and nucleotides, rNTP and dNTP, cell wall precursors, stable (rRNA), and short-lived RNAs (mRNA and tRNA), regulatory
metabolite ppGpp, glycogen, peptidoglycan. These structured models produced modest results and never gained a wide recognition
blamed by biochemists for oversimplification of real microbial metabolism and by modelers for extreme complexity. Two of their
main disadvantages were (i) a subjective selection of internal variables, and (ii) a problem of experimental verification of the
aggregated data.

Genome-scale models (GEM)

The ambitious goal of GEM is the accurate prediction of the phenotype from the respective genotype under specified environmental
conditions. High throughput genome sequencing makes it possible to screen nearly all cultivable microorganisms of whatsoever
medical, industrial, or environmental significance, including soil isolates. Presently, NCBI lists more than 300,000 completed
whole-genome projects including 7500 sequenced fungal species and more than 31,000 prokaryotic (archaeal and bacterial)
species. Soil microbial communities are still underrepresented in culture collections because most soil inhabitants resist isolation.
However, the metagenomic approach (shotgun sequencing of the DNA directly extracted from the soil) potentially allows in silico
reconstruction of phenotypes for any microorganisms, well-known or mysterious uncultivable species. We are still far away from
this ultimate goal, but in the case of success, GEMs would be able to reconstruct their growth dynamics under a specified state of the
environment.
 SOIL BIOLOGY | Kinetics of microbial processes: General principles 177

Fig. 3 Simplified view on the bacterial genome structure through the prism of GEM-modeling.

The progress of GEM development is impressive. The first GEMs were constructed for the simplest parasitic bacteria and E. coli in
1995–1997, and then the number of GEM publications has risen exponentially. GEM is the metabolic network reconstruction.
Contrary to the structured models above, GEM uses real individual intracellular metabolites and metabolic reactions extracted from
genomic data.
GEMs cover only the part of the genome, mostly up to 35%. From a modeling perspective, there are four distinct parts of
the genome (Fig. 3): (i) junk material and sequencing errors needed to be removed (blue background), (ii) the M-matrix (M for
metabolism) standing for genes encoding enzymes catalyzing the entire intermediary metabolism, which is about one-third in
prokaryotes and smaller fraction in eukaryotes; (iii) the E-matrix (Е for expression) accommodating genes encoding enzymes and
RNAs responsible for proteins synthesis, transcription, and translation; (iv) finally, the O-matrix (O for operon) containing the
transcriptional regulatory network, a part of global regulatory machinery orchestrating cellular functions via transcription factors,
TFs. Respective GEMs are called M-, E- and O-models. Reconstruction of metabolic networks (M-models) is now а well-established
process. The E-models have recently been started by using a similar approach and are produced as combined ME-models. Both
models convert genomic data into stoichiometric matrices of intermediate (M-matrix) and biosynthetic reactions (E-matrix) that are
made interactive. Unfortunately, there is no way to predict in theory or based on sequence similarity the functionality of the
O-matrix, ‘the brain’ of the metabolic network. Instead, GEMs apply the optimality principle, assuming that natural regulation of
metabolic network is performed in such a way that maximizes the growth rate of microorganisms under given growth conditions.
There could be other objective functions, e.g., the minimizing cost of protein biosynthesis, minimizing the ATP-pool size or the
energy consumption.
The most popular is the M-models called the Flux Balance Analysis (FBA). It seeks only the steady-state solutions for metabolic
fluxes and uses constraints to define a closed solution space for the flux vectors by setting up the low and high boundaries for each
flux. Additional constraints are coming from the reaction thermodynamics as well as from the metabolomic, transcriptomic, or
proteomic data.
The starting point of FBA is the annotated genome sequence. The product of each gene is annotated by homology searches to
identify all the metabolic enzymes. It happens frequently that some enzymes in a known pathway are missing, so other reactions are
added to close the mass balance. In addition to enzymatic reactions, all transport mechanisms must be accounted for, such as
diffusion, active or facilitated transport through membrane, and secretion of exometabolites. In the next step, a dynamic mass
balance is derived for all the metabolites using a matrix notation:
dX
¼Sv (22)
dt
where X is the vector of cellular metabolites, S is the stoichiometric matrix and v is the matrix of the fluxes.
The main FBA advantage is its computational efficiency: the COBRA toolbox in Matlab or COBRApy in Python installed on a
regular laptop executes full-size M-models (thousands of metabolic reactions) in a few seconds. The FBA is not designed for
dynamic simulations and cannot present growth as a time series of x, s, and p to demonstrate the agreement. Instead, it produces the
metabolic map with fluxes for each metabolic reaction. It correctly reproduces specific rates of unlimited growth (observed in the
batch culture exponential phase), metabolic reconfigurations after switching from one C-source to another, from aerobic to anoxic
growth conditions; it also accurately predicts genes essentiality and the impact of the gene knockdown on metabolic flows including
the exometabolic products that is especially valuable for bioengineering.
The dynamic FBA (dFBA) applies the quasi-steady-state approximation. The metabolic fluxes can be differentiated into slow
and fast categories as dependent on reaction volume. The exchange reactions between cell and environment (nutrient uptake and
product release) take place in the volume range 100–104 cm3. The intracellular metabolic reactions occur in a much smaller volume
of a single cell, 10−8–10−9 cm3. The volumetric difference 108–1013 times implies that intracellular reactions can be safely assumed
to be at a steady-state, and therefore the flux distribution can be resolved by the FBA. Few remaining slow exchange variables
are to be numerically solved as a set of ordinary differential equations (ODEs). Numerous dFBA models were developed to
simulate growth dynamics of the model (Saccharomyces cerevisiae and E. coli) and industrially important organisms, some of which
(e.g., Pseudomonas putida) are used for soil remediation.
178 SOIL BIOLOGY | Kinetics of microbial processes: General principles

The ME-models and the whole-cell models aimed to simulate a self-regulatory behavior of microorganisms under fluctuating
environment have been constructed for smallest parasitic prokaryotes as well as for the two best studied organisms, E. coli and baker
yeasts. Unfortunately, they remain too cumbersome for execution and too modest in discovery of the emergent systems qualities of
the cell (Panikov, 2021). Indisputably, GEMs are the most promising tools in biokinetic studies, but their practical implementation
to environmental sciences is still not possible.

Models of intermediate complexity

Several growth models occupy an intermediate position between oversimplified Monod-type equations and gigantic genome-scale
metabolic reconstructions operating with thousands of variables. In real life, we face a necessity to have a ‘fundamentally correct’
and compact representation of microbial dynamics that can be instantly solved with regular computers and standard software.
A perfect equivalent would be the ideal gas law in physical chemistry, the simplified but dependable model reproduces correctly
the behavior of many gases under many conditions. It would be wrong to simplify GEM by selecting a limited subset of genes
(see above a failure of the structured models), a more promising option would be to rely on compact models able to reproduce the
behavior of an ideal cell in a real variable environment. A loose analogy with ideal gas (having zero intermolecular attractions)
allows us to picture such a cell as free from numerous specific details and having only two essential functional qualities – to reproduce
itself and adapt to the fluctuating environment by conditional expression of subcellular structures. From the kinetic perspective, both qualities
are unique to cells and absent in enzymes, which do not proliferate or change their composition. There is no problem with
reproduction, it is manifested kinetically as the exponential x increases with the constant SGR under permanently optimal
conditions (Eqs. 13 and 14). Under fluctuating conditions, we typically observe increases or decreases of SGR parallel to changes
in the macromolecular composition of cells (MMCC), including proteomic and transcriptomic profiles, the composition of lipids,
metabolic intermediates, and other results of the omics molecular inventories. We apply here the term ‘models of intermediate
complexity’ to those non-GEM growth models that make a very important step forwards to the genome-scale level by attempting to
relate the macroscopic growth kinetics (SGR, yield, respiration, maintenance, etc.) with MMCC and variable environmental
conditions. There are two types of such models: (i) the cybernetic model and (ii) the synthetic chemostat model (SCM). Both
reached some success in the simulation of the self-regulated behavior of cells in response to environmental changes.
The cybernetic models (Kompala et al., 1986) were introduced initially as simple structured models, the word ‘cybernetic’
implying a parallel between a living cell and electronic gadgets, both dealing with circular causalities and feedback. Two cybernetic
variables were incorporated as Boolean ON/OFF switches into the conventional Monod-style growth models applied to situations
when the cell must take a decision, e.g., select the preferential substrate in the mixture of glucose and lactose:
dx X2 mi
¼ x i¼1 mi ni − Dx; ni ¼
dt max ðmi Þ


dsi mn
¼ Dðsri − si Þ − x i i + mi nmi (23)
dt Yi
dei mi X2 si
¼ qi ui − mei ; ui ¼ ; m ¼ m e
i¼1 mi i K si + si
dt m
Here the elements of MMCC are two transporters e1 and e2 specializing on uptake of glucose and lactose respectively. The cybernetic
variables regulate transcription (u) and uptake rates (n) as dependent on the difference between the current values of m1 and m2, the
n n
SGR on each of the competing substrates: n1 ¼ 10,, ifif mm2 <m
m
1
; n2 ¼ 10,, ifif mm2m
<m1
2 1 2 1:

Not surprisingly, the model successfully predicted a correct choice of glucose as a preferential substrate with the following
diauxic pattern, unfortunately only in the simplest setup of the canonic exponential phase with constant SGR and MMCC. The
modern cybernetic models use similar v and u cybernetic variables but are scaled up to the genomic level (Kim et al., 2008; Song and
Ramkrishna, 2011). Their value as a self-dependent subtype of GEM is questionable because of the artificial nature of these
operational variables. So far, the cybernetic models have not been applied to complex microbial biodynamics, such as non-steady
state and unbalanced growth in bioreactors or in situ.
The SCM (Panikov, 1995) has been built upon all the chemostat models listed above, including the Monod model and its
modifications, cell quota and structured models (that explains the epithet ‘synthetic’). The basic SCM contains three differential
equations for the limiting substrate, s, cell biomass, x, and the r-variable compactly characterizing the MMCC (the ‘quality’ of cells):
ds s
¼ Dðsr − sÞ − qs x, qs ¼ rQ (24)
dt Ks + s
dx
¼ mx − Dx, m ¼ Y ðqs − mÞ (25)
dt


dr s
¼m −r (26)
dt Kr + s

The r-variable (Eq. 26) has been introduced as follows. There are at least two functional groups of cellular constituents: the content
of P-components correlates with SGR because they participate in the Primary metabolic reactions (ribosomes, key biosynthetic
 SOIL BIOLOGY | Kinetics of microbial processes: General principles 179

enzymes). Other conditionally-expressed cell constituents are associated with the secondary metabolism responsible for cell
sUrvival (U-components) under stresses, and their content negatively correlates with SGR. Examples of U-components are enzymes
involved in drug resistance and synthesis of antibiotics, protective pigments, reserved polymers, high-affinity transporters, etc. The
first and second groups are combined respectively into vectors P and U satisfying conservation condition P + U ¼ 1.0 if each Pi and
Uj are normalized to cell dry mass (the contribution of small molecules is neglected for clarity). The constitutive (unchangeable)
parts are denoted as Pmin
i and Umin
j while Pmax
i and Umax
j are the upper limits respectively. The SCM makes three assumptions: (1) all
P-components change in response to environmental signal synchronously and in the same direction (otherwise sustainable growth
would be impossible); (2) the main environmental signal controlling the vectors P and U expression is the concentration of limiting
substrate, s that is sensed by cells; (3) the expression degree is approximated by a hyperbolic function of s:
 min   min   max   max 
P  U  P min  U min 
 1   1   1 − P1   1 − U1 
       
P + U ¼  ⋯  +  ⋯  + r  ⋯  + ð1 − r Þ ⋯ ; r ¼ s
  Kr + s (27)
       
 P min   U min   P max − P min   U max − U min 
n m n n m m
Constitutive part Changeable P part Changeable U part Steady state r

where r has been introduced as a common quotient to all elements of the vector P. It is a scalar function of s, which varies between 0
(long-term starvation) and 1.0 (established unlimited growth) and expresses the quality of cells: the higher is r, the higher is the
content of P-components. These assumptions and expression 27 agree with available chemostat data on the growth rate driven
changes in the composition of cells (Fig. 4A) and the observed patterns in gene expression (Panikov, 2021) as well as with the
well-established knowledge on the master role of transcription factors orchestrating simultaneous transcription of the long list
(100–1000) genes responsible for stress resistance (Hengge-Aronis, 2002; Gasch et al., 2000). Thus, gene expression is likely to vary
in a well-coordinated manner and SCM accurately mimics this trend although unable to specify individual conditionally expressed
macromolecules. Nonetheless, the combining kinetic terms with MMCC improves significantly the predictive power of the SCM.
The model can reproduce and predict before the actual experiment, the time course of batch culture (Fig. 4B) starting from the initial
lag phase to the following growth phases leading eventually to starvation death of cells in depleted culture. To run the simulation,

Fig. 4 Simulation success of the SCM model. (A) Cell composition of A. aerogenes as dependent on dilution rate in a NH4+-limited chemostat culture (Tempest
et al., 1967). (B) Batch growth of Schwanniomyces vanrijiae on the minimal glucose–mineral medium. VBNC stands for the viable-but-not-cultivable cells (Panikov,
2019). (C) The shift-up experiment in a chemostat culture of Aerobacter aerogenes limited by glycerol; at time zero, the dilution rate was changed from 0.004 to
0.24 h−1 (Tempest et al., 1967). With permission from Panikov NS (2019) 1.18-Microbial growth dynamics, In: Comprehensive Biotechnology (3rd edn.) Moo-Young
M. Oxford: Pergamon. p. 231–273. Copyright (2019) Elsevier.
180 SOIL BIOLOGY | Kinetics of microbial processes: General principles

one needs to know the initial conditions (xo, so, ro) and the basic growth parameters as a set of constant coefficients specific to every
cultivable microorganism. Further, the SCM successfully simulates the steady-state and transient growth in various continuous
bioprocesses (chemostat, turbidostat, pH-stat, retentostat and dialysis culture), including the most challenging transient shift-up
and shift-down regimes (Fig. 4C). This model will be mostly used below for analysis of the effect of environmental factors on
microbial growth, as well as in the linked chapter, where it is devoted to soil applications.

Growth dependence on environmental factors

The factors affecting growing cells in culture and in situ can be physical (temperature, tonicity, moisture content, texture,
conductivity, gas exchange rates); chemical (pH, Eh, ionic strength, surface chemistry of particulate matter, medium or soil solution
composition); and biological, e.g., the positive, or negative interactions with other microorganisms or a host.

Hierarchy of factors, the primary significance of the nutrient resources

In the ecological literature, all factors are viewed as important, their priority is dependent on local circumstances. For example, the
radiation regime and temperature are supposed to be the key environmental factors in polar ecosystems, water availability in
deserts, metals toxicity in areas affected by industrial pollution, salinity in salt marsh soil, and so on. However, from the biokinetic
perspective, the nutritional factor stands alone as a primary factor under any circumstances, while the rest of the modifying factors
(temperature, pH, Eh, pressure, precipitation, moisture content, and many others) can accelerate, slow or stop a microbial process
but can never produce new cell mass. The state variables representing the nutritional factor, such as s, the limiting substrate
concentration, must be present in any kinetic model of microbial growth. The variables characterizing the modifying factors
are incorporated into specialized kinetic models focusing on a particular problem. The following three arguments justify the stated
difference between the primary and modifying factors. First, the nutrients participate in growth stoichiometry, they are reactants
initiating the entire cellular metabolic network eventually producing new cells and products. The modifying factors do not
participate in growth stoichiometry or mass-balance of microbial growth. Second, contrary to most modifying factors that
characterize the state of the environment, nutrients represent the (actually or potentially) limited consumable resources shared
between various competitors: coexisting species in communities, subpopulations of the same species (e.g., the spontaneous mutant
vs wild parental cells), alternative pathways of the cellular metabolic network. It makes the nutritional factor especially influential
in the simulations of complex systems aimed at the selection of the most probable scenario from numerous alternative options.
Third, the temperature and other modifying environmental factors are important; however, simulation of their action can be
meaningful only in combination with the nutritional factors. The opposite is not true: the effect of nutrients can be fruitfully
explored separately from the modifying factors, provided they remain constant. The reason is that microbial response to any
environmental factor heavily depends on the metabolic status of cells, gene expression profile, and proteome – all internal
characteristics being shaped by the nutritional status. Specifically, the stress response to heat, cold, or drugs, is mediated by
chaperones and other reparation biomolecules that require energy and biosynthetic precursors. Interestingly, the maximal stress
resistance is observed not under plentiful nutrient supply but rather under deep substrate limitations (Panikov et al., 2015), a
phenomenon simulated and explained by SCM.
Below we demonstrate how two archetype modifying factors (pH and temperature) interact with the nutritional factor in the
characterization of growth kinetics. The final example is a special case of the exceptionally deep nutrient limitation neglected in
laboratory studies and widely occurring in nature responsible for so-called, near-zero microbial growth (NZG).

Effects of pH
In enzymology, the effect of pH is explained by the ionization of several functional groups within the active center. The first
assumption is that an enzyme is the dibasic acid H2E, which undergoes dissociation:
K1 K2
H2 E $ HE − + H+ $ E2− + H+
 2−  +
½HE − ½H+  E ½H  (28)
K1 ¼ , K2 ¼
½H2 E ½HE − 
The second assumption is that only the HE− form is active, it cuts the reaction rate by the pH-dependent factor y ¼ [HE−]/([H2E]
+ [HE−] + [E2−]), where all three concentrations can be expressed via [H+], K1 and K2. The final assumption is that all cellular
enzymes have similar pH profiles, then the proposed mechanism can be applied to the SGR:

K 1 ½H+ 
mpH ¼ mm y ¼ mm + 2
(29)
½H  + K 1 ð½H+  + K 2 Þ
Eq. (29) does not account other than pH restriction factors. So, in the case of nutrient limitation and non-optimal temperature, we
should add respectively Eqs. (18), (19), (24)–(26) and (36) (see below) in a multiplicative way. Eq. (29) reproduces the
experimentally observed symmetrical bell-shaped curve with a maximum at (pK1 + pK2)/2 (Fig. 5). There could be other
 SOIL BIOLOGY | Kinetics of microbial processes: General principles 181

Fig. 5 Effect of pH on microbial growth, Eq. (28).

mechanisms involved at the cellular level. However, for practical purposes, Eq. (29) works reasonably well in finding the optimal
pH, especially useful when data points are rare and scattered.
Contrary to the most enzymatic reactions, microbial growth is accompanied by dramatic pH changes. A widely spread
misconception is that growth is predetermined to release acidic metabolites (acetic, pyruvic, citric acids) produced in glycolysis
and TCA cycles. This idea is not accurate, microbial growth can be associated with either metabolic acidification or alkalization and
is dependent on media composition; acidic metabolites are not the only reason for pH drop, it is better to state that growth is
accompanied by uptake and release of ionized species which can cause the acid-base disbalance. Very often, the main reason for
disbalance is the intensive uptake of the N-source rather than release of organic acids. Let us identify where the problem lies.
The starting point in the understanding and prediction of the acid-base disbalance, is to draft the stoichiometric equation of
bacterial growth. Cell biomass is represented as a pseudo-compound with the empirical formula CHhOoNn derived from the
average elemental composition (below the E. coli data are used) and normalized to the C-atoms. The lower-case indexes h, o, and n
indicate the amount (very rarely the integer as in individual chemical compounds) of respective elements H, O, and N per one C
atom. Other macro-elements (P, K, S, Ca, Mg, . . .) and trace elements are ignored because of their minor effect on the ionic status
of a typical microbial culture. The following three stoichiometric equations are just examples. They are complete analogs of
well-known chemical equations, each side must represent the same number of atoms, and the same holds true for the total electric
charge (condition of neutrality) through conversion of three ‘reactants’ (O2, C- and N-sources) into de novo cell mass, CO2, and
H2O with assumed cell yield 0.4 g cell-C per g glucose-C:

Conditions C-source Oxygen N-source Cells Carbon dioxide Water Acid-base disbalance

Glucose + NH4Cl: CH2O + 0.593 O2 + 0.096 NH4+ ! 0.4 CH1.77O0.49N0.24 + 0.600 CO2 + 0.790 H2O + 0.096 H+ + 0 OH− (30)
Glucose +NaNO3: CH2O + 0.401 O2 + 0.096 NO3− ! 0.4 CH1.77O0.49N0.24 + 0.600 CO2 + 0.598 H2O + 0 H + 0.096 OH− (31)
+

Glucose +urea: CH2O + 0.593 O2 + 0.048 CON2H4 ! 0.4 CH1.77O0.49N0.24 + 0.648 CO2 + 0.742 H2O + 0 H+ + 0 OH− (32)

To comply with the conservation conditions, these Eqs. (29)–(31) produce either H+ (NH4Cl as N-source) or OH− (KNO3 as
N-source) that disturb the acid-base balance. The uptake of urea, an uncharged N-source, does not change the ionic status of cultural
liquid, its only effect is slight stimulation of the CO2 release.
The next interesting case is the complex protein-rich media of uncertain composition, which is the most popular type of media in
laboratory practice. We selected the casein as a standardized commercial product with known elemental composition serving as a
combined source of C- and N:

CH1:54 O0:48 N0:27 + 0:536 O2 ! 0:4 CH1:77 O0:49 N0:24 + 0:6 CO2 0:018 H2 O + 0:174 NH4 + + 0:174 OH − (33)
It turns out that casein is the strongest among the tested nutrient sources alkalization agent, nearly two times stronger than nitrate.
Other widely used alkalization nutrients are the sodium and potassium salts of organic and amino acids.
The next biokinetic objective is the reconstruction of the pH time course in real batch culture or it could be the reconstruction of
the auto-titration dynamics aimed at neutralizing metabolic acidification or alkalization. To fulfill this task, we need to specify not
only the growth stoichiometry (Eqs. 30–32) but also the buffering capacity of cultural or environmental liquid, and to reconstruct
the growth dynamics, expressed as a set of ODEs for cell biomass, nutrient substrates consumption and products formation. The
182 SOIL BIOLOGY | Kinetics of microbial processes: General principles

parameters required for coupling the growth of microorganisms with their degree of acid-base disbalance can be found again in
Eqs. (30)–(32). For example, we can easily convert the cell ‘moles’ of these equations into the conventional dry weight of cell
biomass and compare it with the moles of produced acidity or alkalinity. For example, the production of one gram of cells on
glucose + NH4Cl requires the cumulative use of 7.8 mmol of NaOH to neutralize the metabolic acidity. We can also state a
stoichiometric yield-like parameter Yn ¼ 7.8 mmol (g cell)−1, stating that production of one gram of cell biomass causes metabolic
acidification equivalent to release of 7.8 mmol of strong acid. Such data can be fruitfully used for the planning purposes, e.g., to
calculate in advance the composition of nutrient medium (amount of energy, C- and N-sources, buffer molarity and respective
buffering capacity) required for cultivation without instrumental pH control. Another more creative biotech solution would be to
revise the medium composition in such a way that minimizes acid-base disbalance.

Effects of temperature
In chemical kinetics, the dependence of the reaction rate on temperature is explained by the Eyring transition-state theory. It is based
on the use of thermodynamics and principles of quantum mechanics. The reaction proceeds through a continuum of energy states
and must surpass the state of maximum energy when a transient activated complex is formed. Then, the dependence of the reaction
rate constant k on the absolute temperature, T, is expressed as follows:

d ln k DH ⁎ + RT
¼ (34)
dT RT 2
where R is the gas constant and DH⁎ is the enthalpy of activated complex formation. The more popular Arrhenius equation is
obtained by integration of Eq. (34) and a simplified substitution DH⁎ +RT  DH ¼ Ea:
Ea
ln k ¼ ln A − or k ¼ Ae −Ea =RT (35)
RT
where Ea is the activation energy and A is the integration constant, interpreted as the frequency of molecular collisions.
The Arrhenius equation fits well the biokinetic data under a narrow temperature range from zero to 20–60 C as dependent on
tolerance to the thermal denaturation (minimal in psychrophiles, maximal in thermophiles). Let us assume that denaturation is
reversible with the equilibrium constant KT ¼ [E0 ]/[E], where E and E0 stand for the active and denatured molecules respectively.
Then, the combination of Eq. (35) with the van Hoff expression for KT (−RT ln KT ¼ DGo ¼ DHo − TDSo) results in the following
updated Arrhenius equation and its exponential approximation in Celsius temperature:
Ea Ea
Ae −RT Ae −RT AeaT c
mm ¼ ¼  (36)
1 + eBð1 − T Þ 1 + eBð1 − 273+T c Þ
DSo DHo
−
1 + e R RT
C C

where DGo, DSo, and DHo are the standard Gibbs free energy, enthalpy, and entropy of the denaturation reaction, respectively,
aggregated into constants B and C. In this equation, we made substitution of the reaction rate k for the maximum SGR.
The best experimental setup for verification and estimation of kinetic parameters is the exponential phase of the batch culture or
turbidostat and pH-auxostat. The chemostat is not appropriate because of SGR dependence on two strong factors, temperature
and limiting substrate concentration. In the case of soil incubation studies, soil community should have either continuous supply
of nutrients, or experiments must be a short-term to avoid the effect of a hidden change of growth rate. In the example (Fig. 6), we
measured the intensity of methane production by stabilized methanogenic community characterized by low SGR, that was
continuously supplied by gaseous substrate (H2: CO2).

Fig. 6 The effect of temperature on microbial activity in a methanogenic bioreactor (Panikov, unpublished). Experimental conditions: the Sphagnum peat core was
continuously flushed with gas mixture N2:CO2:H2 ¼ 90:5:5 and off-gas mass-spectrometric recording of methane. After stabilizing at 3 C for 2 days, the incubation
temperature was programmed to rise linearly with the rate of 1.0 deg. C h−1 from 3 to 50 C (panel A) or to cycle between 1 and 30 C as shown by the red
line (panel B insert). (A): The 10–min averaged CH4 production rates plotted versus temperature fitted to Eq. (36). (B) The hysteresis in microbial response to
temperature variation, red arrows indicate the temporal progression, and the insert shows the time series of temperature (red curve) and methane formation rate.
Note different symbols (unfilled, grey, and black-filled circles) for three parts of the temperature gradient.
 SOIL BIOLOGY | Kinetics of microbial processes: General principles 183

Within the biologically meaningful range of temperatures (0–60 C), the exponential approximation does not deviate from the
more accurate Arrhenius equation (Fig. 6A) but is more stable in solving the inverse problem. Contrary to chemical reactions,
microorganisms demonstrate significant hysteresis in their response to cyclic temperature changes (Fig. 6B). In other words, the effect
of temperature depends on how it varied in the past. It is explained by the fact that adaptation to temperature extremes (too cold,
too warm) involves conditionally expressed macromolecules, e.g., the heat-shock proteins, desaturating enzymes, and chaperones
(Strocchi et al., 2006). The SCM is capable of simulating the process after the update of Eq. (26): the r-variable should be multiple
functions of s and T.

The special case of near-zero growth kinetics

Laboratory cultivation mainly occurs under an excess of nutrients with SGR close to the maximum, mm. In nature, microbial growth
follows diverse patterns, remaining mostly continuous and very slow (m mm) because of severe nutrient limitations. Even in
relatively rare eutrophic habitats, such as a plant rhizosphere or soil invertebrate gut, microorganisms divide no more than 1–2 times
per day, while in mesotrophic and oligotrophic habitats (plant-free soil, subsoils, sediments) the doubling time can be months and
years. Here we call such a low-intensity process a near-zero growth (NZG) and summarize below its specific features. Formally, any
growth model, simple or structured gives a prediction of how microorganisms should react to extremely low s. According to the
unstructured models, the maintenance term should play a crucial role: if we ignore the maintenance (m ¼ 0 as in the original
Monod model), the NZG is nothing more than a smooth growth deceleration (m ! 0 as s ! 0), however, if we accept the non-zero
maintenance (m > 0, Eq. 9), then NZG is predicted to stop at certain threshold substrate concentration, s ¼ YmKsm, below which m
becomes negative implying net death of starving cells (Fig. 7B).
The laboratory reproduction of continuous NZG is a challenging problem. Batch culture at low starting substrate concentration
(s0) is not adequate because, at any s0 producing a measurable amount of cells, the growth initially is inevitably too intensive and
then abruptly stops and becomes negative after substrate depletion. In theory, a chemostat is a perfect option as the dilution rate, D,
can be set at any level. However, the NZG range requires an unrealistically long stabilization period. For example, a chemostat
culture would need to be operated uninterrupted for at least 4 to 5 months to establish a steady division frequency of once per
month. Over this time period, there is a high probability of accidental equipment malfunction, wall growth, mutations, or
contamination invalidating experimental results.

Fig. 7 Laboratory reproduction of the near-zero microbial growth (NZG) typical for soil environment (Panikov et al., 2015). (A) Schematic of continuous cultivation
with cell retention (CCR). Medium is continuously delivered from the reservoir, while the outflow is passed through the tangential-flow filter, returning cells to the
bioreactor. (B) The effect of the energy source concentration on microbial SGR. The non-structured Monod-Pirt model assuming a constant maintenance rate
predicts cell death (negative m) at s < s . The more advanced SCM model predicts an adjustable maintenance term allowing the positive NZG. (C) Experimental data
on the NZG growth dynamics of Pseudomonas putida fitted to the SCM. (D) The hypothetical mechanism explaining the loss of reproductive capacity in the VBNC
(viable-but-not-cultivable) cells caused by an uneven distribution of ribosomes during the cell division.
184 SOIL BIOLOGY | Kinetics of microbial processes: General principles

Continuous culture with cell retention (CCR) by using a dialysis membrane or tangential-flow filtration (Fig. 7A), provides a
better practical alternative, bringing microbial cells to a monthly division frequency in only 1–2 weeks. The Monod chemostat
model with constant positive m as applied to CCR (Eq. 9 combined with 19) predicts attaining of the so-called state of maintenance,
the transition of microbial cells to the non-proliferating status (m ¼ 0) preserving relatively high the maintenance catabolic activity.
Such a hypothetical state has never been confirmed experimentally (Fig. 7). Instead, it was found (Panikov et al., 2015) that under
chronic starvation in CCR, microorganisms undergo deep metabolic reconfiguration (about 40% of proteome replaced as
compared with the cells grown at conventional SGR range). An immediate result of such adaptive changes was the progressive
decrease of the maintenance term allowing a near linear increase of cell biomass concentration over time (Fig. 7C). The second
conclusion was that a stable long-term NZG requires the differentiation of starving cells into growing and non-replicating VBNC
(viable-but-not-nonculturable cells) forms, the latter preserving measurable catabolic activity. Over cultivation time, the prolifer-
ating cells eventually attain a steady state, their slow growth being balanced by VBNC production. Since growth deceleration is
always accompanied by a dramatic decrease in the rRNA content, it was hypothesized that VBNC cells are produced because of
asymmetry in the distribution of ribosomal particles between chronically starving mother and daughter cells (Fig. 7D). It is
interesting that extremely slowly growing cells acquiring broad resistance to stresses other than starvation, including suboptimal
temperature, toxic compounds, desiccation, and osmotic stress. Apart from the described epigenetic changes, there is a strong
possibility of spontaneous mutations and selection of the GASP (Growth Advantage of Stationary Phase) phenotypes.

Concluding comments

The chapter introduces the basic terms, variables, and concepts established in microbiological growth kinetics. There is a wide range
of models that present different degrees of perception of bioprocesses. It is a normal phenomenon, there is no perfectly correct
model, and all of them including the simplest formulation could be helpful in scientific exploration provided their selection has
been appropriately justified. The choice of biokinetic models should be based on available data and depends on the objectives of a
particular study. Specifically, a soil scientist should estimate the degrees of involvement in a process of soil microorganisms and
other soil biotas. We can envisage several situations:
1) The short-term (the time scale minutes-hours) response of the soil community to some specific factor. The process duration is
too short to induce a measurable growth of soil microorganisms. In this case, the process kinetics should follow the equations
listed in Table 1. It gives basic information about the kinetic order of reactions that could be very useful. Any growth models are
not applicable.
2) The time scale of biomonitoring is extended to a few days, and there is a good chance of observing the initial growth of soil
microorganisms but without deep changes in the community structure and accompanying deep shifts in environmental
conditions. A typical example is soil incubation with easily degradable organic compounds, inducing intensive exponential
growth. Such growth can be categorized as simple biodynamics with constant SGR and MMCC. Such growth can obey the simplest
growth models (Monod, cell quota, logistic equation) or requires more advanced models (SCM, GEM) in the case of clearly
expressed lag-phase indicating metabolic reconfiguration during the complex biodynamics.
3) The next step is the issue of microbial heterogeneity; how diverse the acting community is. If the studied bioprocess is carried out by
a mixture of species with similar biokinetic characteristics, then the SCM could be still well appropriate to explore the dynamic
patterns and help its better mechanistic understanding. Otherwise, researchers must address the multispecies interacting models.
That topic will be touched on in the 2nd chapter of this series.

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Further reading
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