Modeling substrate inhibition of microbial growth

This article presents a general equation for substrate inhibition of microbial growth using a statistical thermodynamic approach. Existing empirical models adapted from enzyme kinetics, for example, the Haldane-Andrews equation, often criticized for not being physically based for microbial growth, are shown to derive from the general equation in this article, and their empirical parameters are shown to be well defined physically. Three sets of experimental data from the literature are used to test the modeling abilities of the general equation to represent experimental data. The results are compared with those obtained by fitting the same data set to a widely used empirical model existing in the literature. The general equation is found to represent all three experimental data sets better than the alternative model tested. In addition, a graphical method existing in enzyme kinetics is successfully adapted and further developed to determine the number of inhibition sites of a basic functional unit of a bacterial cell. (c) 1996 John Wiley & Sons, Inc.     

Oxygen-absorption rate-controlled feeding of substrate into aerobic microbial cultures

A system of automatic control of substrate inflow into an aerated culture of microorganisms which depend on oxygen-absorption rate (OAR) has been devised and tested. As the control variable, dissolved oxygen concentration (DOC), which shows the equilibrium between OAR and oxygen-uptake rate in the microbial culture, was chosen. If the equilibrium is disturbed by changes in OAR, then the oxygen-uptake rate is changed by substrate limitation. The DOC is measured by means of a Clark-type polarographic electrode, and the signal is used to actuate the substrate inflow valve or pump. The oxygen-uptake rate changes of microorganisms, after the addition or exhaustion of substrate in the medium, are so rapid that they can be used for this type of control. Fundamental equations were derived and graphical solutions for the control system parameters were suggested for the steady-state relations between DOC, oxygen-uptake rate, specific growth rate, substrate concentration, K_La, and concentration of microorganisms. The system is stable in the entire range of the uptake rates up to nearly the maximum attainable in unlimited substrate conditions, and can be operated in batch feed or continuous flow modifications. It was experimentally tested with the yeast Saccharomyces cerevisiae. The complete utilization of aeration-system capacity of the fermentor was achieved with high yeast yield and no alcohol formation. The quality of the product was excellent.     

Amino acid limited growth of starter cultures in milk

The specific growth rates of several Streptococcus cremoris strains were 10–40% lower in milk than in other growth in media. The growth rates in milk increased when an amino acid mixture or casein was added, whereas, when milk was diluted, the specific growth rate of the streptococci decreased. This decrease could be overcome by bringing the casein concentration in the diluted milk back to the normal value (3%). This indicates that casein-hydrolysis proceeded at a rate too low for the streptococci to reach their potential maximum specific growth rates in milk so that growth in milk is essentially amino acid-limited. This was subsequently demonstrated for S. cremoris by continuous cultivation in media with low casein concentrations. At a low dilution rate casein hydrolysis was fast enough to supply the cells with enough amino acids and lactose was growth-limiting, whereas at higher dilution rates amino acids became growth-limiting. In cultures exponentially growing in milk the concentration of free amino acids was measured to determine which amino acid(s) was(were) absent and could possibly limit growth. A number of essential amino acids (leucine, methionine, glutamate and in some cases phenylalanine) were not detected and addition of these, together, stimulated the growth of S. cremoris in milk. The amino acids leucine and phenylalanine appeared to play a particularly important role in this stimulation. These two are, supposedly, the first amino acids that become limiting during growth in milk. The effect of competition for casein and amino acids by different organisms was studied in continuous cultures. At different dilution rates different strains became dominant in these mixed cultures, suggesting that differences in apparent affinity constants (K_S) for casein, leucine and glutamate existed between the strains.     

Macrokinetic basis for the model of microbial growth in a limited volume under constant conditions with a single leading substrate

Within the framework of the macrokinetic approach and continuum and chemical/biochemical gross reaction conceptions, an equation describing the complete dynamics of microbial growth and decline as function of a variable concentration of the leading substrate was deduced. This equation allows us to distinguish quantitatively and qualitatively the stages of microbial growth and the intervals of microbial tolerance to the initial concentration of the leading substrate. Adequacy of the model was confirmed by comparison with experimental dynamics of aerobic microorganisms in the samples of groundwater collected from a region polluted with uranium.     

Processivity of cellobiohydrolases is limited by the substrate

Processive cellobiohydrolases (CBHs) are the key components of fungal cellulase systems. Despite the wealth of structural data confirming the processive mode of action, little quantitative information on the processivity of CBHs is available. Here, we developed a method for measuring cellulase processivity. Sensitive fluorescence detection of enzyme-generated insoluble reducing groups on cellulose after labeling with diaminopyridine enabled quantification of the number of reducing-end exo-mode and endo-mode initiations. Both CBHs TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium employed reducing-end exo- and endo-mode initiation in parallel. Processivity values measured for TrCel7A and PcCel7D on cellulose hydrolysis were more than an order of magnitude lower than the values of intrinsic processivity that were found from the ratio of catalytic constant (k(cat)) and dissociation rate constant (k(off)). We propose that the length of the obstacle-free path available for a processive run on cellulose chain limits the processivity of CBHs on cellulose. TrCel7A and PcCel7D differed in their k(off) values, whereas the k(cat) values were similar. Furthermore, the k(off) values for endoglucanases (EGs) were much higher than the k(off) values for CBHs, whereas the k(cat) values for EGs and CBHs were within the same order of magnitude. These results suggest that the value of k(off) may be the primary target for the selection of cellulases.     

An automatic turbidimeter for studies of microbial growth in aerated cultures

Summary An automatic turbidimeter has been described which permits registration of turbidity in 24 culture tubes permanently aerated by means of mechanical agitation.     

Determination of the Monod substrate saturation constant for microbial growth

Abstract Methods used to determine the Monod substrate saturation constant for microbial growth are surveyed. The preferred and most accurate method is to assay the concentrations of growth rate-limiting nutrients in steady-state continuous cultures. But, this is not always possible due to the lack of sufficiently sensitive assay methods or due to high nutrient fluxes in rapidly growing cultures. It is suggested that an acceptable and simple alternative method for aerobic microorganisms is to measure initial oxygen uptake rates during growth in the presence of different initial concentrations of growth rate-limiting nutrient. It is important in this method that the microbial cells are taken from rapidly growing cultures and are suspended in a medium permitting growth.     

Modeling linear and variable growth in phosphate limited suspension cultures of Opium poppy

In examining the growth kinetics of cell suspensions of opium poppy (Papaver somniferum), the increase in biomass with time was observed to be linear over the entire batch growth period of up to 20 days. Although batch growth profiles were reproducible utilizing the same inoculum, growth rates varied tremendously when experiments were inoculated with cells from different flasks. Both of these phenomena are difficult to explain with conventional batch growth models. In a series of a experiments, phosphate was determined to be the growth-rate-limiting substrate. By expressing growth rate in terms of the intracellular reserves of phosphorus, a growth model which expresses kinetics in terms of the intracellular phosphorus contents of the cells is shown to predict both linear growth character and inoculum dependent variability in growth. The stationary phase phosphate content of seven plant suspension cultures of different plant species was found to be comparable to phosphorus levels of phosphate-starved poppy cells, which suggests that phosphate limitation may be common for plant tissue culture. The applicability of this model to other biological systems which display similar batch growth patterns when subjected to inorganic nutrient deprivation is discussed.     

Production of polyhydroxyalkanoates by mixed microbial cultures

Polyhydroxyalkanoates (PHAs) are biodegradable bioplastics formed from renewable resources, like sugars, with similar characteristics of polypropylene. These bioplastics are industrially produced by pure cultures using expensive pure substrates. These factors lead to a much higher selling price of PHAs compared to petroleum-based plastics, like polypropylene. The use of mixed cultures and cheap substrates (waste materials) can reduce costs of PHA production by more than 50%. Storage of PHAs by mixed populations occurs under transient conditions mainly caused by discontinuous feeding and variation in the electron donor/acceptor presence. In the last years the mechanisms of storage, metabolism and kinetics of mixed cultures have been studied. The maximum capacity of PHA storage and production rate is dependent on the substrate and on the operating conditions used. In this paper an overview and discussion of various mechanisms and processes for PHA production by mixed cultures is presented.     

An optimal strategy to model microbial growth in a multiple substrate environment

A comprehensive model is developed based on an optimal strategy describing varied microbial growth phenomenon involving sequential and simultaneous utilization of substrate. The model mimics the complex regulatory process of a cell which results in diverse growth process with the help of simple multi-variable constrained optimization, which aims at maximizing the specific cell growth. The metabolic processes of a cell are represented by simple flux balance equations. The different growth phenomenon exhibited by a microorganism are attributed to different levels of control present inside the cell. Provision is made in the model for these controls, in the form of constraints in the optimization formulation. The model prediction matches well with the experimental data of simultaneous growth of E. coli K12 on a mixture of glucose and organic acids like lactate, pyruvate, and acetate. Moreover, the model predictions are well in agreement with earlier published experimental data for the growth of E. coli K12 on other organic acids like fumarate, alpha-ketoglutarate, and succinate. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 635-644, 1997.     

Modeling conserved nutrient limited growth: Effects of a bound substrate pool

Summary An extension to the model previously presented by Brown et al. (1988) is developed for the case of a conserved substrate that is incorporated into the cell structure. It is shown that the model can predict both an inflection point and a period of essentially linear growth. Effects of the adjustable parameters and the ability to predict experimental results are determined.     

The relationship between kinetics of substrate-limited transitions and steady-state growth in continuous cultures ofAquaspirillum autotrophicum limited by pyruvate

Heterotrophic growth at steady state and during transient states caused by the sudden change of the concentration of the limiting factor in the feed medium was investigated experimentally for continuous cultures ofAquaspirillum autotrophicum limited by pyruvate. A model for describing the growth at steady state was selected from three unstructured models after statistical tests of the data. This model postulates that the growth yield increases linearly with the growth rate. Growth during transitions where the substrate remained limiting at all times was fitted with first-order kinetics. Theoretical predictions of these kinetics were derived from the unstructured models used to describe steady state. The predicted rate coefficients of the transients were compared to the experimental coefficients. It appeared that the model which best described steady-state growth also provided the best predictions for growth during the transient state. It is a widespread opinion that unstructured models are adequate to describe growth under steady-state conditions but not to predict transitions in continuous culture. However, for the particular case studied here, no higher degree of complexity was required to describe transitions, provided the growth of the culture was always limited by the substrate.     

Studies on microbial chromate reduction by Pseudomonas Sp. In aerobic continuous suspended growth cultures

Reduction of hexavalent chromium was studied in three bench-scale continuous stirred tank reactors. The inoculum was a culture of Pseudomonas sp., capable of giving 83% to 87% chromate reduction in 72-h batch assays with 60 mg Cr(VI) L(-1) in synthetic medium. The continuous culture studies were conducted for about 100 days using synthetic feed containing different levels of chromate (5 to 124 mg L(-1)) at 28 degrees to 30 degrees C and pH 6.8. The feed rate was varied over the range 0.5 to 1 L d(-1) to obtain hydraulic retention time of 36 to 72 h. Chromate reduction efficiency was 81% to 91% and 100% for influent Cr(VI) concentrations of 15 to 124 and 5 mg L(-1), respectively, with a hydraulic retention time of 72 h. (c) 1994 John Wiley & Sons, Inc.     

Response of sweet pepper to phenols accumulated in greenhouse substrate

Prolonged cucumber cultivation in the same substrates leads to accumulation of phytotoxic phenolic compounds. Introduction of sweet pepper as an aftercrop eliminates substrate phytotoxicity. The aim of the study was to examine whether in sweet pepper detoxication of substrate phenols occurs by means of the glucosylation process. The examined materials were substrates differing in phytotoxicity level, and sweet pepper plants grown on these substrates. Substrate phytotoxicity was obtained by means of either repeated cucumber cultivation or by phenolic acid addition. During the vegetative growth phase of sweet pepper, the phytotoxicity and phenolic compound levels in the substrate, and the glucosylated phenol contents in above-ground plant parts were determined. Results showed that sweet pepper responds to an increased presence of phenols in the substrate by an intensified glucosylation.     

Cholesterol is accumulated by mycobacteria but its degradation is limited to non-pathogenic fast-growing mycobacteria

In this report we show that fast-growing non-pathogenic mycobacteria degrade cholesterol from liquid media, and are able to grow on cholesterol as a sole carbon source. In contrast, slow-growing mycobacteria, including pathogenic Mycobacterium tuberculosis and bacillus Calmette-Guérin (BCG), do not degrade and use cholesterol as a carbon source. Nevertheless, pathogenic mycobacteria are able to uptake, modify, and accumulate cholesterol from liquid growth media, and form a zone of clearance around a colony when plated on solid media containing cholesterol. These data suggest that cholesterol may have a role in mycobacterial infection other than its use as carbon source.     

Exopolysaccharide production by free and immobilized microbial cultures

The batch production of different exopolysaccharides (alginate, xanthan, pullulan, dextran) by free and immobilized microbial cultures was investigated. First, conventional free-cell cultures were performed to obtain control fermentation parameters and macromolecular characteristics of exopolysaccharides. Then microbial cultures were immobilized in composite agar layer/microporous membrane structures and tested for polysaccharide production. The immobilized-cell system proved unsuitable for xanthan and pullulan production. Owing to the fouling of the microporous membrane by the polysaccharide, dextran production by immobilized Leuconostoc mesenteroides also was inefficient. More promising results have been obtained with immobilized Azotobacter vinelandii cultures. The amount of alginate produced by immobilized A. vinelandii represented about 60% of that recovered from a free-cell culture, whereas the polysaccharide yield reached 35% instead of 9% for the free counterpart. These results are compared to the macromolecular characteristics of exopolysaccharides.     

Degradation of 4-chlorophenol by enriched mixed cultures utilizing phenol and glucose as added growth substrate

Two mixed cultures, phenol-oxidizing (PO) and glucose-oxidizing (GO), were cultivated in two parallel chemostat reactors. The PO culture was enriched on phenol, and the GO culture was enriched on glucose. Batch biodegradation experiments were conducted to examine the degradation of 4-chlorophenol (4-CP) under various substrate conditions. The results indicate that in the absence of added growth substrate, 4-CP transformation by PO culture was complete at S _c ^o /X _o (initial 4-CP concentration/initial biomass concentration) 0.27 and that by GO culture was complete at S _c ^o /X _o = 0.09. In the presence of 5–500 mg phenol/l, the phenol dosage required to achieve the complete transformation of 4-CP was 60 mg/l at S _c ^o /X _o = 1, increasing to 120 mg/l at S _c ^o /X _o = 2, and to 180 mg/l at S _c ^o /X _o = 5. As glucose was added to the GO culture at a concentration of over 5–500 mg chemical oxygen demand (COD)/l, 4-CP was not completely transformed at S _c ^o /X _o = 5 [S _c ^o = 50 mg/l, X _o = 10 mg/l volatile suspended solids (VSS)]. These two cultures in utilizing added growth substrate were easily switched between glucose and phenol. Overall, the capacity of PO culture to degrade 4-CP, expressed as T _c (4-CP mass consumed /biomass inactivated, having unit of mg 4-CP/mg VSS), was 0.15–0.80, which compares with T _c values of 0.05–0.26 for GO culture. This work shows that adding phenol as a growth substrate is preferable over adding glucose, as it enhances 4-CP transformation, but a final choice should take into account both degradation efficiency and the risk of phenol toxicity.