Growth kinetics of suspended microbial cells: from single-substrate-controlled growth to mixed-substrate kinetics

Abstract

Growth kinetics, i.e., the relationship between specific growth rate and the concentration of a substrate, is one of the basic tools in microbiology. However, despite more than half a century of research, many fundamental questions about the validity and application of growth kinetics as observed in the laboratory to environmental growth conditions are still unanswered. For pure cultures growing with single substrates, enormous inconsistencies exist in the growth kinetic data reported. The low quality of experimental data has so far hampered the comparison and validation of the different growth models proposed, and only recently have data collected from nutrient-controlled chemostat cultures allowed us to compare different kinetic models on a statistical basis. The problems are mainly due to (i) the analytical difficulty in measuring substrates at growth-controlling concentrations and (ii) the fact that during a kinetic experiment, particularly in batch systems, microorganisms alter their kinetic properties because of adaptation to the changing environment. For example, for Escherichia coli growing with glucose, a physiological long-term adaptation results in a change in KS for glucose from some 5 mg liter-1 to ca. 30 microg liter-1. The data suggest that a dilemma exists, namely, that either "intrinsic" KS (under substrate-controlled conditions in chemostat culture) or micromax (under substrate-excess conditions in batch culture) can be measured but both cannot be determined at the same time. The above-described conventional growth kinetics derived from single-substrate-controlled laboratory experiments have invariably been used for describing both growth and substrate utilization in ecosystems. However, in nature, microbial cells are exposed to a wide spectrum of potential substrates, many of which they utilize simultaneously (in particular carbon sources). The kinetic data available to date for growth of pure cultures in carbon-controlled continuous culture with defined mixtures of two or more carbon sources (including pollutants) clearly demonstrate that simultaneous utilization results in lowered residual steady-state concentrations of all substrates. This should result in a competitive advantage of a cell capable of mixed-substrate growth because it can grow much faster at low substrate concentrations than one would expect from single-substrate kinetics. Additionally, the relevance of the kinetic principles obtained from defined culture systems with single, mixed, or multicomponent substrates to the kinetics of pollutant degradation as it occurs in the presence of alternative carbon sources in complex environmental systems is discussed. The presented overview indicates that many of the environmentally relevant apects in growth kinetics are still waiting to be discovered, established, and exploited.

FIG. 1

Kinetic processes which affect microbial growth, specified with respect to compartment, kinetic model, and biodegradability characteristics. Ranges of definition for the most important aspects of microbial growth and degradation kinetics are given.

FIG. 2

Experimentally determined and predicted steady-state glucose concentrations for E. coli ML 30 in glucose-controlled chemostat cultures at 17.4, 28.4, 37, and 40°C, as a function of dilution (growth) rate. Bars indicate the steady-state substrate concentrations (indicating vertically the standard deviation of the steady-state glucose concentrations determined as a mean of about 10 measurements and horizontally the approximate variation in D); lines indicate predictions of steady-state glucose concentrations by different models (adapted from reference 34) as follows:  (34),  (266), (195), and  (166).

FIG. 3

Kinetic properties of E. coli reported in the literature for glucose-controlled growth at 37°C. Experimental data from batch (□) and chemostat (■) cultures are given. The numerical values and references are listed in Table 2. “Ideal” intrinsic kinetic properties (indicated by arrows) cannot be determined experimentally at the same time, because cells can be cultivated only in such a way that they exhibit either the intrinsic K_s (in chemostat culture) or the intrinsic μ_max (in batch culture). The shaded area represents an approximation of the extant kinetic properties that E. coli can potentially exhibit.

FIG. 4

Time courses (in hours) of the residual glucose concentration in the initial phase of glucose (carbon)-controlled continuous cultures of E. coli ML 30 operated at different dilution rates. The experiment was performed so that at time zero a batch culture of E. coli, growing exponentially at μ_max = 0.92 h⁻¹, was switched to chemostat mode at the dilution rate indicated. Data for a culture operated at D = 0.2 h⁻¹ is shown (▵); data for independent continuous cultures are shown for D = 0.6 h⁻¹ (■ and □) and D = 0.3 h⁻¹ (• and ○). Adapted from reference .

FIG. 5

Mixed-substrate kinetics during growth of E. coli in carbon-limited culture. (a) Growth with mixtures of glucose, fructose, and galactose at a dilution rate of 0.3 h⁻¹. Data from reference . (b) Growth with mixtures of glucose and 3-PPA at a dilution rate (D) of 0.6 h⁻¹. All the mixtures were designed in such a way that the total biomass concentration was always approximately 45 mg liter⁻¹ (dry weight). Data from reference . Adapted from reference .

FIG. 6

Effect of enzyme regulation on the relationship between specific substrate consumption rate and steady-state substrate concentration (note the link between panels a and d, panels b and e, and panels c and f). (a) Steady-state concentrations of glucose and galactose in chemostat cultures of E. coli growing under carbon-controlled conditions at a constant dilution rate (D = 0.3 h⁻¹) with different glucose-galactose mixtures in the feed. The total sugar concentration in the inflowing medium was always 10 mg liter⁻¹, and the composition of glucose-galactose is given in weight proportions. Data from reference . (b) Regulated catabolic enzyme level. Concentrations of 3-PPA during growth of E. coli in carbon-controlled chemostat cultures with different mixtures of 3-PPA and glucose at constant dilution rate are shown (D = 0.3 h⁻¹). The shaded area indicates the range from 0 mg of 3-PPA liter⁻¹ up to the apparent threshold concentration below which the 3-PPA was not utilized (i.e., the same residual concentrations [□] as those in medium feed were measured). Once induced, 3-PPA was utilized down to concentrations (■) that were lower than those required to trigger induction. Data from reference . (c) Steady-state methanol concentration (■) and specific activity of alcohol oxidase (□) in the yeast Kloeckera sp. strain 2201 during simultaneous utilization of glucose-methanol mixtures in carbon-controlled chemostat culture at a constant dilution rate (D = 0.14 h⁻¹). The specific activity of alcohol oxidase is given in micromoles per milligram of protein per minute. Adapted from references and . (d to f) Different enzyme expression patterns (the details of the meaning of the numbers are discussed in the text). It is assumed that the consumption kinetics of a microbial culture for a substrate can be described by a Monod-type relationship. q_max(ind) and q_max(rep) are the maximum specific substrate consumption rates under fully induced and repressed conditions, respectively.

FIG. 7

Influence of the molar ratio of glucose to ammonium in the feed medium on the steady-state concentration of glucose (○) and ammonium (■) in chemostat cultures of Klebsiella pneumoniae at dilution rates of 0.2 h⁻¹ (a) and 0.4 h⁻¹ (b). The shaded area indicates the dual carbon- and nitrogen-limited zone. Adapted from reference .

FIG. 8

Model predictions concerning the mineralization of a pollutant in the presence of various constant concentrations of uncharacterized DOC, assuming that both are utilized simultaneously. The calculation was performed numerically with following assumptions: (i) both the pollutant and uncharacterized DOC supported the growth of the organism and the initial biomass concentration was always 0.01 mg liter⁻¹ (dry weight); (ii) the initial pollutant concentration was always 1 mg liter⁻¹, whereas the available DOC concentration was assumed to remain constant at 0, 0.01, 0.5, 1, 10, 100, or 1,000 mg liter⁻¹ (as indicated) due to a continuous supply of DOC by hydrolysis of particulate organic matter; (iii) growth of the microorganism was described by the Monod model (equation 1) with the parameters K_s = 1 mg liter⁻¹ (for the pollutant), K_s = 0.1 mg liter⁻¹ (for DOC), and μ_max = 0.3 h⁻¹ (for both substrates); and (iv) the substrate consumption was proportional to growth via a constant yield factor Y_X/c = 1.1 (for both pollutant and DOC) (equation 2).

FIG. 9

Regulation strategies involved in NTA degradation in WWTP and rivers. Labeled curves indicate the isolines of constant catabolic capacity (equation 10), which represents the number of active degraders (here, the number of immunopositive cells) multiplied by their degree of induction (here, the concentration of NTA monooxygenase component A). Due to enrichment of NTA degraders in the WWTP at Glatt, the catabolic capacity changed from a (in the river water above the plant) to b (in the activated sludge of the WWTP) and subsequently to c (in the effluent of the WWTP). The catabolic capacity of NTA degraders in the WWTP at Säntis changed mainly due to induction from d (periods before adaptation of the activated sludge to high NTA concentrations in the wastewater) to e (microorganisms adapted to the high NTA concentrations). The directions of the two main regulation strategies are also indicated by the arrows. Data from reference .