Growth In Continuous Culture Research Articles

Acid adaptation alters Streptococcus mutans drug susceptibility profile

The ability of Streptococcus mutans, the primary causative agent of dental caries, to generate and tolerate an acidic environment is a leading factor in its capacity to outcompete other organisms within the oral cavity. The acid tolerance response (ATR) involves coordination of multiple pathways to upregulate macromolecule repair, maintenance of cytosolic pH, and restructuring the membrane. In this study, we asked if the changes associated with adaptation to a low pH environment would result in detectable changes to drug susceptibility. Accordingly, we screened using the Selleck library of 853 FDA approved drugs for agents that exhibit antimicrobial activity toward planktonic cultures of S. mutans or disrupt biofilm formation in growth medium titrated to pH 5 or in the presence of hydrogen peroxide. Results revealed that a total of 182 compounds displayed antimicrobial properties toward the test strain in at least one screening condition. Our findings provide evidence that the S. mutans ATR is characterized by a concerted shift in drug susceptibility. The presence of an intact ATR, associated with growth in continuous culture at pH 5, leads to increased sensitivity to 22 compounds and decreased sensitivity to a separate set of 20 compounds. Disruption of the ATR in a mutant lacking the fabM gene resulted in a susceptibility profile that was distinct from the result of our results. This suggests that loss of unsaturated fatty acids produces a physiological change that is not only measurable, but perhaps more significant than the process of ATR. Additional tests revealed that S. mutans exhibits a distinct drug susceptibility profile compared to a closely related, non-cariogenic bacterium, S. gordonii. Overall, shifts in S. mutans susceptibility patterns based on pH of growth medium or type of culture (batch vs. continuous culture), indicate that the ATR can be tracked using changes in drug susceptibility profile. Furthermore, our results provide clues that compounds could be useful in treatment of dental caries as well as providing molecular tools to study shifts in physiology of disease-associated bacteria.

Microcystin aids in cold temperature acclimation: Differences between a toxic Microcystis wildtype and non-toxic mutant

For Microcystis aeruginosa PCC 7806, temperature decreases from 26 °C to 19 °C double the microcystin quota per cell during growth in continuous culture. Here we tested whether this increase in microcystin provided M. aeruginosa PCC 7806 with a fitness advantage during colder-temperature growth by comparing cell concentration, cellular physiology, reactive oxygen species damage, and the transcriptomics-inferred metabolism to a non-toxigenic mutant strain M. aeruginosa PCC 7806 ΔmcyB. Photo-physiological data combined with transcriptomic data revealed metabolic changes in the mutant strain during growth at 19 °C, which included increased electron sinks and non-photochemical quenching. Increased gene expression was observed for a glutathione-dependent peroxiredoxin during cold treatment, suggesting compensatory mechanisms to defend against reactive oxygen species are employed in the absence of microcystin in the mutant. Our observations highlight the potential selective advantages of a longer-term defensive strategy in management of oxidative stress (i.e., making microcystin) vs the shorter-term proactive strategy of producing cellular components to actively dissipate or degrade oxidative stress agents.

Producción de acetato de isoamilo durante el cultivo continuo de Pichia fermentans

Pichia fermentans ITD-00165 is a yeast with a high potential to produce isoamyl acetate, but no previous works report its growth in continuous culture. A bioreactor containing a chemically defined medium allowed continuous cultures to develop three dilution rates (0.25, 0.30 y 0.35 h-1), measuring the isoamyl acetate production. Steady-state isoamyl acetate concentration was measured using an entrapping system based on a bubbling column with n-decane. Results demonstrate that the entrapping system showed a linear correlation between the isoamyl acetate concentration into the culture medium and the absorption rate into the column. Airstream for oxygen supply was used to continuously feed isoamyl alcohol to the culture to diminish the inhibitory effect of the precursor. Finally, it was demonstrated that the yeast used the precursor to produce isoamyl acetate (0.144 mg L-1).

Molecular Logic of Prokaryotic Surface Layer Structures.

Most prokaryotic cells are encased in a surface layer (S-layer) consisting of a paracrystalline array of repeating lattice-forming proteins. S-layer proteins populate a vast and diverse sequence space, performing disparate functions in prokaryotic cells, including cellular defense, cell-shape maintenance, and regulation of import and export of materials. This article highlights recent advances in the understanding of S-layer structure and assembly, made possible by rapidly evolving structural and cell biology methods. We underscore shared assembly principles revealed by recent work and discuss a common molecular framework that may be used to understand the structural organization of S-layer proteins across bacteria and archaea.

Modeling microalgae growth in continuous culture: Parameters analysis and temperature dependence

Microalgae are considered to be a promising biofuel resource for carbon dioxide fixation in the future. Modeling the growth of microalgae is an effective method to study the growth performance of microalgae, which is also helpful to control the cultivation of microalgae in artificial bioreactor. In continuous culture, dilution rate and influent inorganic nitrogen concentration have shown significant effects on the growth of microalgae. In this paper, the common and overall influences of dilution rate and influent inorganic nitrogen concentration on the microalgae growth have been investigated. Based on numerical simulation, this paper plots the results of total and unit time particulate carbon concentration in 3D figures to expose the varied tendency. It is found that the value of points on the ridge line increases with the dilution rate decreasing and influent inorganic nitrogen concentration increasing for both the total particulate carbon concentration and the particulate carbon concentration per day. The influence of several typical temperatures has been modeled and calculated. The value of biomass productivity will be further increased when the dilution rate is getting closer to 0. This paper will go a step further for the parameters design of continuous culture photo-bioreactor (PBR) for later study.

Influence of light on microalgae growth in continuous culture of low density

A mathematical model of the dependence of the specific growth rate of microalgae on the light intensity for a continuous culture of low optical density is proposed. In the basis of the mechanism of light-limited growth is the thesis on photosynthesis having light and dark phases. The product of the first phase is carbohydrates, which are used in the dark phase as a source of energy and carbon skeletons for the synthesis of all structural components of cells .Rates of the processes are described by linear splines. The equations describing the dependence of the specific growth rate on the intensity of the acting light are obtained.

Analysis of the Genes Involved in Thiocyanate Oxidation during Growth in Continuous Culture of the Haloalkaliphilic Sulfur-Oxidizing Bacterium Thioalkalivibrio thiocyanoxidans ARh 2T Using Transcriptomics.

Thiocyanate (N=C-S-) is a moderately toxic, inorganic sulfur compound. It occurs naturally as a by-product of the degradation of glucosinolate-containing plants and is produced industrially in a number of mining processes. Currently, two pathways for the primary degradation of thiocyanate in bacteria are recognized, the carbonyl sulfide pathway and the cyanate pathway, of which only the former has been fully characterized. Use of the cyanate pathway has been shown in only 10 strains of Thioalkalivibrio, a genus of obligately haloalkaliphilic sulfur-oxidizing Gammaproteobacteria found in soda lakes. So far, only the key enzyme in this reaction, thiocyanate dehydrogenase (TcDH), has been purified and studied. To gain a better understanding of the other genes involved in the cyanate pathway, we conducted a transcriptomics experiment comparing gene expression during the growth of Thioalkalivibrio thiocyanoxidans ARh 2T with thiosulfate with that during its growth with thiocyanate. Triplicate cultures were grown in continuous substrate-limited mode, followed by transcriptome sequencing (RNA-Seq) of the total mRNA. Differential expression analysis showed that a cluster of genes surrounding the gene for TcDH were strongly upregulated during growth with thiocyanate. This cluster includes genes for putative copper uptake systems (copCD, ABC-type transporters), a putative electron acceptor (fccAB), and a two-component regulatory system (histidine kinase and a σ54-responsive Fis family transcriptional regulator). Additionally, we observed the increased expression of RuBisCO and some carboxysome shell genes involved in inorganic carbon fixation, as well as of aprAB, genes involved in sulfite oxidation through the reverse sulfidogenesis pathway. IMPORTANCE Thiocyanate is a moderately toxic and chemically stable sulfur compound that is produced by both natural and industrial processes. Despite its significance as a pollutant, knowledge of the microbial degradation of thiocyanate is very limited. Therefore, investigation of thiocyanate oxidation in haloalkaliphiles such as the genus Thioalkalivibrio may lead to improved biotechnological applications in wastewater remediation.

Assembly of a Mini-Chemostat Array.

Here, we describe instructions for the assembly of an array of miniature (20-mL) chemostats or "ministats" built from relatively inexpensive off-the-shelf parts. In experiments with yeast cultures, we have observed reproducibility in cellular physiology, gene expression patterns, and evolutionary outcomes with different ministats as well as between ministats and commercial large-volume platforms. Growth in continuous culture is a primary means for the characterization of yeast steady-state physiology, competition between strains, and long-term evolution experiments. We hope that these relatively inexpensive and high-throughput devices make the advantages of continuous culture growth more accessible to researchers.

Generation and Characterization of Acid Tolerant Fibrobacter succinogenes S85

Microorganisms are key components for plant biomass breakdown within rumen environments. Fibrobacter succinogenes have been identified as being active and dominant cellulolytic members of the rumen. In this study, F. succinogenes type strain S85 was adapted for steady state growth in continuous culture at pH 5.75 and confirmed to grow in the range of pH 5.60–5.65, which is lower than has been reported previously. Wild type and acid tolerant strains digested corn stover with equal efficiency in batch culture at low pH. RNA-seq analysis revealed 268 and 829 genes were differentially expressed at pH 6.10 and 5.65 compared to pH 6.70, respectively. Resequencing analysis identified seven single nucleotide polymorphisms (SNPs) in the sufD, yidE, xylE, rlmM, mscL and dosC genes of acid tolerant strains. Due to the absence of a F. succinogenes genetic system, homologues in Escherichia coli were mutated and complemented and the resulting strains were assayed for acid survival. Complementation with wild-type or acid tolerant F. succinogenes sufD restored E. coli wild-type levels of acid tolerance, suggesting a possible role in acid homeostasis. Recent genetic engineering developments need to be adapted and applied in F. succinogenes to further our understanding of this bacterium.

Heat shock and prolonged heat stress attenuate neurotoxin and sporulation gene expression in group I Clostridium botulinum strain ATCC 3502.

Foodborne pathogenic bacteria are exposed to a number of environmental stresses during food processing, storage, and preparation, and in the human body. In order to improve the safety of food, the understanding of molecular stress response mechanisms foodborne pathogens employ is essential. Many response mechanisms that are activated during heat shock may cross-protect bacteria against other environmental stresses. To better understand the molecular mechanisms Clostridium botulinum, the causative agent of botulism, utilizes during acute heat stress and during adaptation to stressfully high temperature, the C. botulinum Group I strain ATCC 3502 was grown in continuous culture at 39°C and exposed to heat shock at 45°C, followed by prolonged heat stress at 45°C to allow adaptation of the culture to the high temperature. Growth in continuous culture was performed to exclude secondary growth phase effects or other environmental impacts on bacterial gene transcription. Changes in global gene expression profiles were studied using DNA microarray hybridization. During acute heat stress, Class I and III heat shock genes as well as members of the SOS regulon were activated. The neurotoxin gene botA and genes encoding the neurotoxin-associated proteins were suppressed throughout the study. Prolonged heat stress led to suppression of the sporulation machinery whereas genes related to chemotaxis and motility were activated. Induced expression of a large proportion of prophage genes was detected, suggesting an important role of acquired genes in the stress resistance of C. botulinum. Finally, changes in the expression of a large number of genes related to carbohydrate and amino acid metabolism indicated remodeling of the cellular metabolism.

An Immortalized Human Blood-Nerve Barrier Endothelial Cell Line for In Vitro Permeability Studies

Solute and macromolecular transport studies may elucidate nutritional requirements and drug effects in healthy and diseased peripheral nerves. Endoneurial endothelial cells are specialized microvascular cells that form the restrictive blood-nerve barrier (BNB). Primary human endoneurial endothelial cells (pHEndECs) are difficult to isolate, limiting their widespread availability for biomedical research. We developed a simian virus-40 large T-antigen (SV40-LTA) immortalized human BNB cell line via stable transfection of low passage pHEndECs and observed continuous growth in culture for >45 population doublings. As observed with pHEndECs, the immortalized BNB endothelial cells were Ulex Europaeus agglutinin-1-positive and endocytosed low density lipoprotein, but lost von Willebrand factor expression. Glucose transporter-1, P-glycoprotein (P-gp), γ-glutamyl transpeptidase (γ-GT), large neutral amino acid transporter-1 (LAT-1), creatine transporter (CRT), and monocarboxylate transporter-1 (MCT-1) mRNA expression were retained at all passages with loss of alkaline phosphatase (AP) expression after passages 16-20. Compared with an SV40-LTA immortalized human blood-brain barrier endothelial cell line, there was increased γ-GT protein expression, equivalent expression of organic anion transporting polypeptide-C (OATP-C), organic anion transporter 3 (OAT-3), MCT-1, and LAT-1, and reduced expression of AP, CRT, and P-gp by the BNB cell line at passage 20. Further studies demonstrated lower transendothelial electrical resistance (~181 vs. 191 Ω cm(2)), equivalent permeability to fluoresceinated sodium (4.84 vs. 4.39 %), and lower permeability to fluoresceinated high molecular weight (70 kDa) dextran (0.39 vs. 0.52 %) by the BNB cell line. This cell line retained essential molecular and biophysical properties suitable for in vitro peripheral nerve permeability studies.

Caspase-8 deficiency facilitates cellular transformation in vitro

Caspase-8 is frequently deficient in several kinds of human tumors, suggesting that certain effects of this enzyme restrict tumor development. To examine the nature of the cellular function whose regulation by caspase-8 contributes to its antitumor effect, we assessed the impact of caspase-8 deficiency on cell transformation in vitro. Caspase-8-deficient mouse embryonic fibroblasts immortalized with the SV40 T antigen did not survive when cultured in soft agar, and were nontumorogenic in nude mice. However, the rate of transformation of these cells during their continuous growth in culture, as reflected in the observed emergence of cells that do grow in soft agar and are able to form tumors in nude mice, was far higher than that of cells expressing caspase-8. These findings indicate that caspase-8 deficiency can contribute to cancer development in a way that does not depend on the enzyme's participation in killing of the tumor cells by host immune cytotoxic mechanisms, or on its involvement in the cell-death process triggered upon detachment of the cells from their substrate, but rather concerns cell-autonomous mechanisms that affect the rate of cell transformation.

Passive optical separation within a 'nondiffracting' light beam

A passive, optical cell sorter is created using the light pattern of a 'nondiffracting' beam-the Bessel beam. As a precursor to cell sorting studies, microspheres are used to test the resolution of the sorter on the basis of particle size and refractive index. Variations in size and, more noticeably, refractive index, lead to a marked difference in the migration time of spheres in the Bessel beam. Intrinsic differences (size, refractive index) between native (unlabeled) cell populations are utilized for cell sorting. The large difference in size between erythrocytes and lymphocytes results in their successful separation in this beam pattern. The intrinsic differences in size and refractive index of other cells in the study (HL60 human promyelocytic leukaemic cells, murine bone marrow, and murine stem/progenitor cells) are not large enough to induce passive optical separation. Silica microsphere tags are attached to cells of interest to modify their size and refractive index, resulting in the separation of labeled cells. Cells collected after separation are viable, as evidenced by trypan blue dye exclusion, their ability to clone in vitro, continued growth in culture, and lack of expression of Caspase 3, a marker of apoptosis.

Global gene expression in Escherichia coli K-12 during short-term and long-term adaptation to glucose-limited continuous culture conditions

Microarray technology was used to study the cellular events that take place at the transcription level during short-term (physiological) and long-term (genetic) adaptation of the faecal indicator bacterium Escherichia coli K-12 to slow growth under limited nutrient supply. Short-term and long-term adaptation were assessed by comparing the mRNA levels isolated after 40 or 500 h of glucose-limited continuous culture at a dilution rate of 0.3 h(-1) with those from batch culture with glucose excess. A large number of genes encoding periplasmic binding proteins were upregulated, indicating that the cells are prepared for high-affinity uptake of all types of carbon sources during glucose-limited growth in continuous culture. All the genes belonging to the maltose (mal/lamB) and galactose (mgl/gal) operons were upregulated. A similar transcription pattern was observed for long-term cultures except that the expression factors were lower than in the short-term adaptation. The patterns of upregulation were confirmed by real-time RT-PCR. A switch from a fully operational citric acid cycle to the PEP-glyoxylate cycle was clearly observed in cells grown in glucose-limited continuous culture when compared to batch-grown cells and this was confirmed by transcriptome analysis. This transcriptome analysis confirms and extends the observations from previous proteome and catabolome studies in the authors' laboratory.

Effects of 2-Hydroxy-4-(methylthio) Butanoic Acid (HMB) on Microbial Growth in Continuous Culture

2-Hydroxy-4-(methylthio) butanoic acid (HMB) positively affects milk composition and yield, potentially through ruminal actions. Four continuous culture fermenters were used to determine the optimal concentration of HMB for digestibility of organic matter (OM), neutral detergent fiber (NDF), acid detergent fiber (ADF), and hemicellulose and synthesis of microbial N. A highly degradable mix of hay and grain was used as a basal diet to simulate a typical lactation diet. Three concentrations of HMB (0, 0.055, and 0.110%) and one concentration of dl-Met (0.097%) were infused into the fermenters according to a 4×4 Latin square design. Digesta samples were collected during the last 3 d of each of the four 10-d experimental periods. Digestibility of OM, hemicellulose, and NDF was largely insensitive to treatment. Digestibility of ADF showed a quadratic effect to supplementation of HMB, with 0.055% having lower digestibility than 0 or 0.110%. Total production of VFA was not influenced by HMB supplementation, but differences in concentration and production of individual VFA were seen. Isobutyrate increased linearly with increasing HMB supplementation. Propionate concentration decreased linearly with increased HMB supplementation, but propionate production showed a quadratic trend (P=0.13). A higher concentration of acetate was detected for dl-Met compared with the highest HMB concentration. There were trends (P<0.15) for dl-Met to decrease the production of isobutyrate and to lower the concentration of butyrate when compared with HMB. Microbial efficiency was not different among treatments. The proportion of bacterial N produced from NH3-N decreased linearly with increasing HMB, and bacteria receiving dl-Met synthesized more N from NH3-N than those receiving HMB. These data suggest that supplementation of HMB may have a sparing effect on branched chain volatile fatty acids because the fatty acids are not needed to provide carbon for synthesis of valine, isoleucine and leucine with ammonia. Comparisons of bacterial community structure in the fermenter effluent samples using PCR amplicons containing the ribosomal intergenic spacer region and its flanking partial 16S ribosomal RNA gene showed no distinct banding patterns, though treatments tended to group together. Both Met and HMB affect the rumen microbial population, but Met supplied as dl-Met does not act identically to that supplied as HMB.

Kinetic parameters of continuous cultures of Bacillusthermoleovorans sp. A2 degrading phenol at 65 °C

In this paper, we report on the kinetics of phenol degradation and cell growth in continuous cultures of suspended cells of Bacillus thermoleovorans sp. A2 at 65 °C. A high yield coefficient of Y x/s=0.84 g cell dry weight g −1 phenol was measured at a dilution rate of 0.5 h −1. At the same dilution rate the coefficient for maintenance metabolism (m s) was determined to be 0.045 g phenol g −1 cell dry weight h −1. The maximal growth rate (wash-out) determined at a phenol inlet concentration of 188 mg l −1 was 0.9 h −1. Up to 7 g phenol l −1 per day were degraded in a continuously operated 2-l stirred tank reactor with suspended cells (feed concentration 660 mg l −1). Additionally, yield coefficients for oxygen and ammonium are reported.

Use of a dynamic in vitro attachment and invasion system (DIVAS) to determine influence of growth rate on invasion of respiratory epithelial cells by group B Streptococcus

Expression of capsular polysaccharide (CPS) and some surface proteins by group B Streptococcus (GBS) is regulated by growth rate. We hypothesized that precise control of GBS growth, and thus surface-expressed components, could modulate the ability of GBS to invade eukaryotic cells. To test this hypothesis, a dynamic in vitro attachment and invasion system (DIVAS) was developed that combines the advantages of bacterial growth in continuous culture with tissue culture. Tissue culture flasks were modified with inlet and outlet ports to permit perfusion of GBS. Encapsulated type III GBS strains M781 and COH1 and strains COH1-11 and COH1-13 (transposon mutants of COH1 that express an asialo CPS or are acapsular, respectively) were grown in continuous culture in a chemically defined medium at fast mass doubling time (t(d) = 1.8 h) and slow (t(d) = 11 h) growth rates, conditions previously shown to induce and repress, respectively, type III CPS expression. Encapsulated GBS strains invaded A549 respiratory epithelial cells 20- to 700-fold better at the fast than at the slow growth rate, suggesting a role for CPS. However, unencapsulated GBS were also invasive but only when cultured at the fast growth rate, which indicates that GBS invasion is independent of CPS expression and can be regulated by growth rate. Growth rate-dependent invasion occurred when GBS was grown in continuous culture under glucose-defined, thiamine-defined, and undefined nutrient limitations. These results suggest a growth rate-dependent regulation of GBS pathogenesis and demonstrate the usefulness of DIVAS as a tool in studies of host-microbe interactions.

Continuous cultures limited by a gaseous substrate: Development of a simple, unstructured mathematical model and experimental verification with Methanobacterium thermoautotrophicum

This article presents a simple, unstructured mathematical model describing microbial growth in continuous culture limited by a gaseous substrate. The model predicts constant gas conversion rates and a decreasing biomass concentration with increasing dilution rate. It has been found that the parameters influencing growth are primarily the gas transfer rate and the dilution rate. Furthermore, it is shown that, for correct simulation of growth, the influence of gaseous substrate consumption on the effective gas flow through the system has to be taken into account.Continuous cultures of Methanobacterium thermoautotrophicum were performed at three different gassing rates. In addition to the measurement of the rates of biomass production, product formation, and substrate consumption, microbial heat dissipation was assessed using a reaction calorimeter. For the on-line measurement of the concentration of the growth-limiting substrate, H(2), a specially developed probe has been used. Experimental data from continuous cultures were in good agreement with the model simulations. An increase in gassing rate enhanced gaseous substrate consumption and methane production rates. However, the biomass yield as well as the specific conversion rates remained constant, irrespective of the gassing rate. It was found that growth performance in continuous culture limited by a gaseous substrate is substantially different from "classic" continuous culture in which the limiting substrate is provided by the liquid feed. In this report, the differences between both continuous culture systems are discussed.

The Effect of the Growth Medium on the Composition and Metal Binding Behaviour of the Extracellular Polymeric Material of a Metal-Accumulating Citrobacter sp.

Cells of a heavy metal-accumulating Citrobacter sp. appeared electron-opaque under the electron microscope when pre-grown in medium containing lactose and inorganic phosphate as the respective carbon and phosphorus sources, compared to cells supplied with glycerol and glycerol 2-phosphate. This was attributable to a greater proportion of bound metal cations, as analysed using proton induced X-ray emission analysis of extracted extracellular polymeric material (EPM). This was associated with a greater content of EPM-associated phosphorus and also an enhanced activity of isolated EPM to accumulate uranyl ion, to 23% of the polymer dry weight. EPM from glycerol 2-phosphate-grown cells, analysed using 31P nuclear magnetic resonance spectroscopy, gave several major phosphorus resonances between −1 and 2 ppm, in accordance with published spectra for enterobacterial lipopolysaccharide. Addition of Cd2+ promoted a shift downfield of the P resonances to a new, common, resonance at 3 ppm which was similar to that observed in native, Cd-unchallenged EPM obtained from inorganic phosphate-pregrown cells. The two types of polymer were morphologically distinct, with a shift to a more defined structure on addition of Cd2+ to the EPM from glycerol phosphate-grown cells. The two types of 31P spectra of the EPM were influenced by the pregrowth phosphorus source, and not by the carbon source. In contrast, analysis of the monosaccharide content of the EPM by gas chromatography and high performance liquid chromatography showed that the main components, rhamnose, glucose and ribose, were unaffected by the pre-growth phosphate source but cells pre-grown in glycerol medium had a higher proportion of glucose residues (ratios of glucose:rhamnose of 4:1 and 2.5:1 in glycerol and lactose-pregrown cells, respectively), and a higher content of EPM monosaccharides, overall, in the lactose-grown cultures. Restriction of carbon or nitrogen source by growth in continuous culture and analysis of the EPM gave major 31P resonances in the region of −1 to 3 ppm, while growth under phosphate-limiting continuous culture showed only one major species, at 1 ppm. The data illustrate that the composition of the cell surface polymers, and the ability of these to sorb heavy metal, are functions of the environment in which the cells are grown. This has implications in the application of biosorption techniques to the removal of heavy metals from wastes.

Analysis of the optimal model for substrate substitutability in continuous microbial cultures

A comprehensive model for microbial growth in multiple substrate environment developed by Venkatesh et al. (1997, Biotechnol. Bioengng, 56, 635–644) is applied to growth in continuous cultures and analyzed at steady state. Analytical expressions are derived for substrate concentrations in terms of cellular and reactor parameters. These expressions are seen to describe the substrate utilization pattern and to identify regions of steady state and wash-out for a given feed concentration. These expressions also give the nature of the isoclines just based on simple manipulation of the model equations. The model predictions were compared with experimental data and were found to match reasonably well. Simulations were also performed to get steady-state concentrations of substrates with varying dilution rate. It was seen that in mixed substrate cultures, the system washes out at higher dilution rates than when they were growing on single substrates. The model was extended for microbial growth on various substrate pairs and also on a mixture containing three substrates.