Kinetic models for growth and product formation on multiple substrates

Hydrolyzates from lignocellulosic biomass contain a mixture of simple sugars; the predominant ones being glucose, cellobiose and xylose. The fermentation of such mixtures to ethanol or other chemicals requires an understanding of how each of these substrates is utilized.Candida lusitaniae can efficiently produce ethanol from both glucose and cellobiose and is an attractive organism for ethanol production. Experiments were performed to obtain kinetic data for ethanol production from glucose, cellobiose and xylose. Various combinations were tested in order to determine kinetic behavior with multiple carbon sources. Glucose was shown to repress the utilization of cellobiose and xylose. However, cellobiose and xylose were simultaneously utilized after glucose depletion. Maximum volumetric ethanol production rates were 0.56, 0.33, and 0.003 g/L-h from glucose, cellobiose and xylose, respectively. A kinetic model based on cAMP mediated catabolite repression was developed. This model adequately described the growth and ethanol production from a mixture of sugars in a batch culture.     

Cell counting and carbon utilization velocities via microbial calorimetry

Bacteria rapidly metabolize sugars and produce heat accordingly (Escherichia coli, aerobic conditions, 25 degrees C). Two kinds of heat output are gotten: (1) from excess cells and limiting carbon, 2 x 10(9) to 5 x 10(9) cells, 5-50 nanomole glucose; (2) from limited cells and excess carbon, 0. 1 x 10(9)-1 x 10(9) bacteria and 200-600 nmol glucose. The thermograms from heat conduction calorimetry under the first conditions measure velocities of sugar uptake and initial metabolic throughput in 1-6-min time spans before a growth cycle possibly can occur. Under the second conditions with limited cells, power output plateaus to a steady state proportional to cell biomass and number of cells. In order to evaluate the calorimetric means for measuring number of cells, six independent means including spectrophotometry (turbidity) were compared: microkjeldahl nitrogen, biuret protein, dry weight, microscopy direct counting in Petroff-Hausser chambers, and viable colony counting. Using turbidity as a central standard, all methods including calorimetry under the second set of conditions agree within +/-18% of one another. Spectrophotometry is the most rapid method but is seriously interfered with by pigments that absorb and foreign particles that also scatter. Calorimetry requires 10-30 min but measures cell numbers in opaque samples impossible for optical means.     

Conversion of cellulose to sugars by resting cells of a mesophilic anaerobe, Bacteriodes cellulosolvens

During the growth of Bacteroides cellulosolvens in media containing cellulose, the accumulation of unutilized sugars in the culture broth occurred mainly during the stationary phase of growth. Cells harvested during the stationary phase of growth continued to convert both cellulose and hemicellulose to cellobiose, glucose, and xylose. These three sugars caused feedback inhibition. Continuous removal of these sugars during the incubation of cells with cellulose at pH 5 accumulated ca. 32 g/L of sugars as compared to ca. 17 g/ produced under batch conditions of growth. Sugar formation by resting cells also increased with increasing cell concentration and did not require any nutrient.     

Determination of the solubilizing activity of a cellulase complex with dyed substrates

When the solubilizing activity of a microbial cellulase complex (e.g.,Trichoderma viride) is determined with conventional methods based on formation of reducing sugars, the results depend on the concentration ratio of cellobiose and glucose in the reaction mixture and thus on the β-glucosidase present and on the type of measurement of reducing sugars. The use of dyed substrates is one way to avoid this problem. The release of coloured compounds from a dyed substrate is proportional to the solubilization.     

Comparative engineering of Escherichia coli for cellobiose utilization: Hydrolysis versus phosphorolysis

Microbial biocatalysts capable of cellobiose assimilation are of interest in bioconversion of cellulosic materials. This study provides a careful comparison in the two mechanisms of cellobiose assimilation, hydrolysis versus phosphorolysis, between two otherwise isogenic E. coli strains. Relative to cells assimilating cellobiose hydrolytically, phosphorolysis cells tolerated common inhibitors better under both anaerobic and aerobic conditions. Additionally, phosphorolysis cells were able to direct the favorable energy metabolism to recombinant protein production, resulting in up to five fold more recombinant proteins. In a mixed sugar fermentation (5% (w/v) cellobiose+5.0% (w/v) xylose), however, xylose utilization in phosphorolysis cells came to a complete halt after only about 60% consumption whereas the hydrolysis cells were able to ferment both sugars to near completion. These results provide insights into the new metabolic engineering strategy. To our best knowledge, this is the first comparison study in E. coli on the two cellobiose assimilation mechanisms.     

Utilization of organic substrates during mixotrophic and heterotrophic cultivation of algae

Pure cultures ofChlorella pyrenoidosa (82) andScenedesmus obliquus (125) were grown in the nutrient medium according to Benson in the presence of 0·05m sugars or 0·025m sodium salts of organic acids. The density of culture was measured throughout the course of growth. Satisfactory heterotrophic sources of nutrition forChlorella pyrenoidosa appear to be galactose, glucose and acetate, whereasScenedesmus utilizes glucose, cellobiose and acetate. The growth ofChlorella in the light is enhanced by galactose, glucose, fructose, cellobiose and maltose, that ofScenedesmus by glucose, fructose, cellobiose, galactose, maltose, acetate and pyruvate. Soluble starch suppresses growth of both cultures. The role of the substrates is discussed. It follows from the results that the growth-promoting sugars and organic acids can act not only as a source of carbon during general carbon shortage but also as ergastic material. The mechanism of utilization of some organic substrates will be taken up in a subsequent paper.     

Modelling of microbial growth for sequential utilization in a multisubstrate environment

Microbial growth in multisubstrate environments is posed as a problem of multivariable constraint optimization. The optimization aims at maximizing the instantaneous growth rate of cells. The model developed for microbial growth using this hypothesis involves simple representation of complex cell structure as an optimization function which regulates the interplay of cellular machinery. The model parameters are estimated using single substrate growth data. Model simulation fits very well with earlier published experimental data of bacterial growth of Klensiella oxytoca on a variety of sugar mixtures involving glucose, fructose, lactose, and xylose. Moreover, the model is also able to predict the diauxic growth of Saccharomyces cerevisiae on glucose and galactose. One of the interesting outcomes of the above representation is the ability to prove analytically that the growth on the mixture of two sugars will be diauxic if one of the substrates has a very low K_s value and a high μ_m value.     

Utilization of cellobiose and D-glucose by Clostridium thermocellum ATCC-27405

Cultures of Clostridium thermocellum ATCC-27405, maintained on cellulose and not adapted to grow on glucose utilize cellobiose preferentially over D-glucose, and are only able to initiate growth on D-glucose when the cellobiose has been exhausted from the growth medium. However, D-glucose is the carbon source preferentially utilized when cultures of this microorganism, previously adapted for growth on glucose, are transferred to a medium with equivalent concentrations of both sugars. One reason for the preferential utilization of glucose over that of cellobiose might be the competitive inhibition of cellobiose phosphorylase by intracellular glucose accumulation. When in the glucose-adapted cultures the pressure to grow on glucose as the sole carbon source is again released, both sugars can be simultaneously utilized.     

Study of cellulases from a newly isolated thermophilic and cellulolytic <Emphasis Type="Italic">Brevibacillus</Emphasis> sp. strain JXL

A potentially novel aerobic, thermophilic, and cellulolytic bacterium designated as Brevibacillus sp. strain JXL was isolated from swine waste. Strain JXL can utilize a broad range of carbohydrates including: cellulose, carboxymethylcellulose (CMC), xylan, cellobiose, glucose, and xylose. In two different media supplemented with crystalline cellulose and CMC at 57°C under aeration, strain JXL produced a basal level of cellulases as FPU of 0.02 IU/ml in the crude culture supernatant. When glucose or cellobiose was used besides cellulose, cellulase activities were enhanced ten times during the first 24 h, but with no significant difference between these two simple sugars. After that time, however, culture with glucose demonstrated higher cellulase activities compared with that from cellobiose. Similar trend and effect on cellulase activities were also obtained when glucose or cellobiose served as a single substrate. The optimal doses of cellobiose and glucose for cellulase induction were 0.5 and 1%. These inducing effects were further confirmed by scanning electron microscopy (SEM) images, which indicated the presence of extracellular protuberant structures. These cellulosome-resembling structures were most abundant in culture with glucose, followed by cellobiose and without sugar addition. With respect to cellulase activity assay, crude cellulases had an optimal temperature of 50°C and a broad optimal pH range of 6–8. These cellulases also had high thermotolerance as evidenced by retaining more than 50% activity at 100°C after 1 h. In summary, this is the first study to show that the genus Brevibacillus may have strains that can degrade cellulose.     

Role of catabolite regulatory mechanisms in control of carbohydrate utilization by the rumen anaerobic fungus Neocallimastix frontalis.

Neocallimastix frontalis PN-1 utilized the soluble sugars D-glucose, D-cellobiose, D-fructose, maltose, sucrose, and D-xylose for growth. L-Arabinose, D-galactose, D-mannose, and D-xylitol did not support growth of the fungus. Paired substrate test systems were used to determine whether any two sugars were utilized simultaneously or sequentially. Of the paired monosaccharides tested, glucose was found to be preferentially utilized compared with fructose and xylose. The disaccharides cellobiose and sucrose were preferentially utilized compared with fructose and glucose, respectively, an cellobiose was also the preferred substrate compared with xylose. Xylose was the preferred substrate compared with maltose. In further incubations, the fungus was grown on the substrate utilized last in the two-substrate tests. After moderate growth was attained, the preferred substrate was added to the culture medium. Inhibition of nonpreferred substrate utilization by the addition of the preferred substrate was taken as evidence of catabolite regulation. For the various combinations of substrates tested, fructose and xylose utilization was found to be inhibited in the presence of glucose, indicating that catabolite regulation was involved. No clear-cut inhibition was observed with any of the other substrate combinations tested. The significance of these findings in relation to rumen microbial interactions and competitions is discussed.     

Role of catabolite regulatory mechanisms in control of carbohydrate utilization by the rumen anaerobic fungus Neocallimastix frontalis

Neocallimastix frontalis PN-1 utilized the soluble sugars D-glucose, D-cellobiose, D-fructose, maltose, sucrose, and D-xylose for growth. L-Arabinose, D-galactose, D-mannose, and D-xylitol did not support growth of the fungus. Paired substrate test systems were used to determine whether any two sugars were utilized simultaneously or sequentially. Of the paired monosaccharides tested, glucose was found to be preferentially utilized compared with fructose and xylose. The disaccharides cellobiose and sucrose were preferentially utilized compared with fructose and glucose, respectively, an cellobiose was also the preferred substrate compared with xylose. Xylose was the preferred substrate compared with maltose. In further incubations, the fungus was grown on the substrate utilized last in the two-substrate tests. After moderate growth was attained, the preferred substrate was added to the culture medium. Inhibition of nonpreferred substrate utilization by the addition of the preferred substrate was taken as evidence of catabolite regulation. For the various combinations of substrates tested, fructose and xylose utilization was found to be inhibited in the presence of glucose, indicating that catabolite regulation was involved. No clear-cut inhibition was observed with any of the other substrate combinations tested. The significance of these findings in relation to rumen microbial interactions and competitions is discussed.     

Influence of the presence of Zymomonas anaerobia on the conversion of cellobiose, glucose, and xylose to ethanol by Clostridium saccharolyticum

To convert sugar mixtures containing cellobiose, glucose, and xylose to ethanol in a single step, the possibility of using a coculture consisting of Clostridium saccharolyticum and Zymomonas anaerobia was studied. In monoculture, C. saccharolyticum utilized all three sugars; however, it preferentially utilized glucose and produced acetic acid in addition to ethanol. The formation of acetic acid from the metabolism of glucose inhibited the growth of C. saccharolyticum and, consequently, the utilization of cellobiose and xylose. In monoculture, Z. anaerobia utilized glucose at a rate of 50 g/L day, but it did not ferment cellobiose or xylose. In coculture, Z. anaerobia converted most of the glucose to ethanol during the lag phase of growth of C. saccharolyticum, which then converted cellobiose and xylose to ethanol. The use of this coculture increased both the rate and the efficiency of the conversion of these three sugars to ethanol, and produced relatively small amounts of acetic acid.     

Utilization of glucose and cellobiose by the microflora of soil enriched with cellulose

The ability of soil microflora to utilize glucose or celloboise was found to depend on previous incubation of the soil with glucose, celloboise or cellulose. Glucose was utilized more rapidly than cellobiose in soil preincubated with glucose or cellobiose. The opposite situation was observed in soil preincubated with cellulose. In the presence of a mixture of both sugars the rate of utilization of one of them was decreased by the second and this decrease could be characterized as competitive inhibition. Glucose accumulated in the medium during utilization of cellobiose alone in soil preincubated with cellulose. This phenomenon was not observed during the utilization of cellobiose in soil preincubated with glucose or cellobiose.     

Engineered Escherichia coli capable of co-utilization of cellobiose and xylose

Natural ability to ferment the major sugars (glucose and xylose) of plant biomass is an advantageous feature of Escherichia coli in biofuel production. However, excess glucose completely inhibits xylose utilization in E. coli and decreases yield and productivity of fermentation due to sequential utilization of xylose after glucose. As an approach to overcome this drawback, E. coli MG1655 was engineered for simultaneous glucose (in the form of cellobiose) and xylose utilization by a combination of genetic and evolutionary engineering strategies. The recombinant E. coli was capable of utilizing approximately 6 g/L of cellobiose and 2 g/L of xylose in approximately 36 h, whereas wild-type E. coli was unable to utilize xylose completely in the presence of 6 g/L of glucose even after 75 hours. The engineered strain also co-utilized cellobiose with mannose or galactose; however, it was unable to metabolize cellobiose in the presence of arabinose and glucose. Successful cellobiose and xylose co-fermentation is a vital step for simultaneous saccharification and co-fermentation process and a promising step towards consolidated bioprocessing.     

Comparison of methods for quantifying the hydrolytic potential of cellulase enzymes

Summary The adequacy of -glucosidase activity in various cellulase enzyme mixtures was assessed by monitoring the accumulation of cellobiose in the reaction mixture. The influence of accumulated glucose and cellobiose on a filter paper (FP) assay indicated the relative susceptibility of the different enzyme preparations to end-product inhibition. An HPLC analysis of the profile of sugars released also provided a better means of predicting the hydrolytic potential of the various cellulase mixtures. An accurate prediction of the hydrolytic potential of a cellulase preparation could not be based on the conventional FP assay alone. The hydrolytic potential of a Celluclast/Novozym mixture was superior to that of Trichoderma harzianum even when the latter system was supplemented with increased concentrations of -glucosidase (Novozym).     

Kinetic modeling of microbial growth,enzyme activity,and gene deletions: An integrated model of β-glucosidase function in Cellvibrio japonicus

Understanding the complex growth and metabolic dynamics in microorganisms requires advanced kinetic models containing both metabolic reactions and enzymatic regulation to predict phenotypic behaviors under different conditions and perturbations. Most current kinetic models lack gene expression dynamics and are separately calibrated to distinct media, which consequently makes them unable to account for genetic perturbations or multiple substrates. This challenge limits our ability to gain a comprehensive understanding of microbial processes towards advanced metabolic optimizations that are desired for many biotechnology applications. Here, we present an integrated computational and experimental approach for the development and optimization of mechanistic kinetic models for microbial growth and metabolic and enzymatic dynamics. Our approach integrates growth dynamics, gene expression, protein secretion, and gene-deletion phenotypes. We applied this methodology to build a dynamic model of the growth kinetics in batch culture of the bacterium Cellvibrio japonicus grown using either cellobiose or glucose media. The model parameters were inferred from an experimental data set using an evolutionary computation method. The resulting model was able to explain the growth dynamics of C. japonicus using either cellobiose or glucose media and was also able to accurately predict the metabolite concentrations in the wild-type strain as well as in β-glucosidase gene deletion mutant strains. We validated the model by correctly predicting the non-diauxic growth and metabolite consumptions of the wild-type strain in a mixed medium containing both cellobiose and glucose, made further predictions of mutant strains growth phenotypes when using cellobiose and glucose media, and demonstrated the utility of the model for designing industrially-useful strains. Importantly, the model is able to explain the role of the different β-glucosidases and their behavior under genetic perturbations. This integrated approach can be extended to other metabolic pathways to produce mechanistic models for the comprehensive understanding of enzymatic functions in multiple substrates.     

New patterns of mixed-substrate utilization during batch growth of Escherichia coli K12

Microbial growth on mixtures of substrates is of considerable engineering and biological interest. Most of the work until now has dealt with microbial growth on binary mixtures of sugars or polyols. In these cases, it is often found that no matter how the inoculum is precultured, only one of the two substrates is consumed in the first growth phase, leading to the diauxic growth pattern. The goal of the experiments reported here is to investigate growth on mixtures containing at least one organic acid. These experiments show that the substrate utilization patterns in such mixtures are qualitatively different from the diauxic growth pattern. For instance, during growth of Escherichia coli K12 on certain binary mixtures of organic acids, the two substrates are utilized simultaneously, and the mixed-substrate maximum specific growth rate exceeds the single-substrate maximum specific growth rate on either one of the two constituent substrates. Furthermore, the very same mixed-substrate maximum specific growth and substrate uptake rates are observed no matter how the inoculum is precultured. On the other hand, in a mixture of glucose and pyruvate, the maximum specific growth rate seems to depend on the preculturing conditions, thus suggesting the existence of multiple physiological quasi-steady states. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 747-757, 1997.     

Glucose suppression of beta-glucosidase activity in a chloramphenicol-producing strain of Streptomyces venezuelae

beta-Glucosidase activity was induced in Streptomyces venezuelae during growth on cellobiose, gentiobiose, salicin, methyl beta-glucoside, and p-nitrophenyl beta-D-glucopyranoside. Activity in cell extracts was separated by DEAE-cellulose chromatography into two fractions differing in substrate preference. One component showed higher activity with, and was more strongly induced by, cellobiose; the other showed greater activity and inducibility with salicin. Addition of glucose to cultures severely depressed induction of beta-glucosidase activity by cellobiose but not by salicin. Acetate and several amino acids inhibited induction by either substrate. The action of glucose was not reversed by cyclic AMP. Cultures of S. venezuelae using glucose, cellobiose, or a mixture of the two saccharides as their carbon source produced chloramphenicol during growth. In contrast with its effect on the induction of cellobiose activity, glucose did not suppress chloramphenicol production, indicating that the control mechanisms that establish carbon source preferences are not linked to those that regulate antibiotic biosynthesis in this organism.     

A kinetic model for production of glucose by hydrolysis of levoglucosan and cellobiosan from pyrolysis oil

Anhydro sugars, produced during wood pyrolysis, can by hydrolyzed to sugars under acidic conditions. The acid hydrolysis of two common anhydro sugars in wood pyrolysis oils, levoglucosan (1,6-anhydro-beta-D-glucopyranose) and cellobiosan (beta-D-glucopyranosyl-(1-->4)-1,6-anhydro-D-glucopyranose), was investigated. Levoglucosan hydrolysis to glucose follows a first-order reaction, with an activation energy of 114 kJ mol(-1). For cellobiosan hydrolysis, 44% of the cellobiosan is hydrolyzed initially via the beta-(1-->4) glycosidic bond to form levoglucosan and glucose. The remaining cellobiosan is hydrolyzed initially at the 1,6 anhydro bond to form cellobiose. Both reactions are first order with respect to cellobiosan, with an activation energy of 99 kJ mol(-1). The intermediate levoglucosan and cellobiose are hydrolyzed to glucose.