Structured kinetic model to represent the utilization of multiple substrates in complex media during rifamycin B fermentation

Industrial fermentations typically use media that are balanced with multiple substitutable substrates including complex carbon and nitrogen source. Yet, much of the modeling effort to date has mainly focused on defined media. Here, we present a structured model that accounts for growth and product formation kinetics of rifamycin B fermentation in a multi-substrate complex medium. The phenomenological model considers the organism to be an optimal strategist with an in-built mechanism that regulates the sequential and simultaneous uptake of the substrate combinations. This regulatory process is modeled by assuming that the uptake of a substrate depends on the level of a key enzyme or a set of enzymes, which may be inducible. Further, the fraction of flux through a given metabolic branch is estimated using a simple multi-variable constrained optimization. The model has the typical form of Monod equation with terms incorporating multiple limiting substrates and substrate inhibition. Several batch runs were set up with varying initial substrate concentrations to estimate the kinetic parameters for the rifamycin overproducer strain Amycolatopsis mediterranei S699. Glucose and ammonium sulfate (AMS) demonstrated significant substrate inhibition toward growth as well as product formation. The model correctly predicts the experimentally observed regulated simultaneous uptake of the substitutable substrate combinations under different fermentation conditions. The modeling results may have applications in the optimization and control of rifamycin B fermentation while the modeling strategy presented here would be applicable to other industrially important fermentations.     

Measurement of the proton-motive stoichiometry of the respiratory chain of rat liver mitochondria: the effect of N-ethylmaleimide

The apparent proton-motive stoichiometry as measured by the oxygen-pulse technique in KCl medium is depressed by the rapid uptake of inorganic phosphate, unless endogenous phosphate is depleted or uptake is inhibited. In sucrose or choline chloride media, where the internal pH is more acid than in KCl media, uptake may be greatly diminished. In the absence of significant phosphate uptake, the observed stoichiometry of around 8, obtained with no added substrate or respiratory inhibitors, appears to be characteristic of NADH oxidation without significant participation of the proton-translocating NAD(P) transhydrogenase. A mechanistic stoichiometry of at least 8 is indicated.     

Fed-batch fermentation dealing with nitrogen limitation in microbial transglutaminase production by Streptoverticillium mobaraense

In the later stages of a batch fermentation for microbial transglutaminase production by Streptoverticillium mobaraense the availability of a nitrogen source accessible to the microorganism becomes critical. Fed-batch fermentation is investigated with the aim of avoiding this substrate limitation. When peptone is used as a nitrogen source in the feed, no significant improvement of growth and transglutaminase production is observed. This is probably due to crosslinking of the nitrogen source by the transglutaminase produced. Using an inorganic nitrogen source alone does not give satisfactory growth and production. A fed-batch fermentation method has thus been developed to deal with this problem. In the batch phase of the fermentation, an initial medium containing peptone, designed on the basis of the stoichiometric requirements of the microorganism, is used to ensure optimal growth. In the feeding phase, ammonium sulphate is used instead to avoid the crosslinking effect. The feed composition, mainly the amount of nitrogen and carbon source, is also based on the stoichiometric requirements of the organism, taking into account the replacement of peptone by ammonium sulphate. By using this fed-batch fermentation technique, cell-mass dry weight and transglutaminase production could be increased by 33% and 80% respectively, compared to those in a batch fermentation. Received: 10 July 1997 / Received revision: 24 October 1997 / Accepted: 24 October 1997     

A novel feeding strategy for enhanced plasmid stability and protein production in recombinant yeast fedbatch fermentation

A novel feeding strategy in fedbatch recombinant yeast fermentation was developed to achieve high plasmid stability and protein productivity for fermentation using low-cost rich (non-selective) media. In batch fermentations with a recombinant yeast, Saccharomyces cerevisiae, which carried the plasmid pSXR125 for the production of beta-galactosidase, it was found that the fraction of plasmid-carrying cells decreased during the exponential growth phase but increased during the stationary phase. This fraction increase in the stationary phase was attributed to the death rate difference between the plasmid-free and plasmid-carrying cells caused by glucose starvation in the stationary phase. Plasmid-free cells grew faster than plasmid-carrying cells when there were plenty of growth substrate, but they also lysed or died faster upon the depletion of the growth substrate. Thus, pulse additions of the growth substrate (glucose) at appropriate time intervals allowing for significant starvation period between two consecutive feedings during fedbatch fermentation should have positive effects on stabilizing plasmid and enhancing protein production. A selective medium was used to grow cells in the initial batch fermentation, which was then followed with pulse feeding of concentrated non-selective media in fedbatch fermentation. Both experimental data and model simulation show that the periodic glucose starvation feeding strategy can maintain a stable plasmid-carrying cell fraction and a stable specific productivity of the recombinant protein, even with a non-selective medium feed for a long operation period. On the contrary, without glucose starvation, the fraction of plasmid-carrying cells and the specific productivity continue to drop during the fedbatch fermentation, which would greatly reduce the product yield and limit the duration that the fermentation can be effectively operated. The new feeding strategy would allow the economic use of a rich, non-selective medium in high cell density recombinant fedbatch fermentation. This new feeding strategy can be easily implemented with a simple IBM-PC based control system, which monitors either glucose or cell concentration in the fermentation broth.     

Proton release during successive oxidation steps of the photosynthetic water oxidation process: stoichiometries and pH dependence.

Flash-induced absorption changes of pH-indicating dyes were investigated in photosystem II enriched membrane fragments, in order to retrieve the individual contributions to proton release of the successive transitions of the Kok cycle. These stoichiometric coefficients were found to be, in general, noninteger and to vary as a function of pH. Proton release on the S0----S1 step decreases from 1.75 at pH 5.5 to 1 at pH 8, while, on S1----S2 the stoichiometry increases from 0 to 0.5 in the same pH range and remains close to 1 for S2----S3. These findings are analyzed in terms of pK shifts of neighboring amino acid residues caused by electrostatic interactions with the redox centers involved in the two first transitions. The electrochromic shift of a chlorophyll, associated with the S transitions, responding to local electrostatic effects was investigated under similar conditions. The pH dependence of this signal upon the successive transitions was found correlated with the titration of the proton release stoichiometries, expressing the electrostatic balance between the oxidation and deprotonation processes.     

Mass production of thiostrepton by fed-batch culture of Streptomyces laurentii with pH-stat modal feeding of multi-substrate

Summary High concentration production of an antibiotic, thiostrepton, was achieved by the fed-batch culture of Streptomyces laurentii. To produce thiostrepton efficiently, the pH of the medium had to be maintained in a very narrow range between 6.0 and 6.2. As the substrates, not only glucose but also natural nutrients such as defatted soy bean meal and corn steep liquor were demanded. All of these substrates (multi-substrate) had to be supplied during the cultivation. pH was used as the indicator to detect the deficiency of substrates. When the glucose in the medium had been exhaustively consumed, the pH increased immediately; the multi-substrate solution (MS-solution) was then supplied. The composition of the feeding solution was determined from data obtained in batch cultures. During the fed-batch culture, glucose concentration was kept lower than 5 g/l by regulating the feed amount of MS-solution with this pH-stat modal control. By this control strategy a high concentration of thiostrepton, 10.5 g/l, was obtained, while total cell mass concentration reached 157 g/l. The productivity of thiostrepton was greatly increased compared with the conventional batch culture.     

Detection of phase shifts in batch fermentation via statistical analysis of the online measurements: a case study with rifamycin B fermentation

Industrial production of antibiotics, biopharmaceuticals and enzymes is typically carried out via a batch or fed-batch fermentation process. These processes go through various phases based on sequential substrate uptake, growth and product formation, which require monitoring due to the potential batch-to-batch variability. The phase shifts can be identified directly by measuring the concentrations of substrates and products or by morphological examinations under microscope. However, such measurements are cumbersome to obtain. We present a method to identify phase transitions in batch fermentation using readily available online measurements. Our approach is based on Dynamic Principal Component Analysis (DPCA), a multivariate statistical approach that can model the dynamics of non-stationary processes. Phase-transitions in fermentation produce distinct patterns in the DPCA scores, which can be identified as singular points. We illustrate the application of the method to detect transitions such as the onset of exponential growth phase, substrate exhaustion and substrate switching for rifamycin B fermentation batches. Further, we analyze the loading vectors of DPCA model to illustrate the mechanism by which the statistical model accounts for process dynamics. The approach can be readily applied to other industrially important processes and may have implications in online monitoring of fermentation batches in a production facility.     

Fermentation of kraft black liquor for the production of citric acid by Candida tropicalis

Summary The production of citric acid by batch fermentation with the yeast strain Candida tropicalis ATCC 20240 was chosen as a potential process for the valorization of kraft black liquor. The effect of nitrogen concentration was studied and direct bioconversion of acetate to citrate was achieved when no nitrogen was supplemented to the medium. The use of kraft black liquor's acetate as a potential substrate for citric acid production was investigated. The acid precipitated liquor was highly inhibitory when its concentration was above 25% of the fermentation broth content. The yields of citric acid at low concentrations of kraft black liquor (5% and 15%) were the same as those recorded in synthetic acetate medium. Other organic acids present in the liquor may affect the yields and rates of citric acid production over acetate. Substrate uptake rates and product formation rates were lower, however, in comparison to synthetic media. The utilization of immobilized biomass improved the process parameters on kraft black liquor and enhanced the fermentation capabilities.     

Fed-batch fermentation of glucose to acetate by an improved strain ofClostridium thermoaceticum

The fed-batch approach to the production of acetate from glucose by an improved strain ofClostridium thermoaceticum resulted in better performance than the batch fermentation, especially in media containing an excess (3X) of nutrients and trace salts. At pH 6.6, 46 g/l acetic acid was produced in 192 hours with 93% substrate utilization. In contrast, batch fermentation under similar conditions resulted in a maximum of 35 g/l acetic acid with less than 82% substrate utilization.     

Hierarchical amino acid utilization and its influence on fermentation dynamics: rifamycin B fermentation using Amycolatopsis mediterranei S699, a case study

Background  

Industrial fermentation typically uses complex nitrogen substrates which consist of mixture of amino acids. The uptake of amino acids is known to be mediated by several amino acid transporters with certain preferences. However, models to predict this preferential uptake are not available. We present the stoichiometry for the utilization of amino acids as a sole carbon and nitrogen substrate or along with glucose as an additional carbon source. In the former case, the excess nitrogen provided by the amino acids is excreted by the organism in the form of ammonia. We have developed a cybernetic model to predict the sequence and kinetics of uptake of amino acids. The model is based on the assumption that the growth on a specific substrate is dependent on key enzyme(s) responsible for the uptake and assimilation of the substrates. These enzymes may be regulated by mechanisms of nitrogen catabolite repression. The model hypothesizes that the organism is an optimal strategist and invests resources for the uptake of a substrate that are proportional to the returns.     

Stoichiometric modeling of Clostridium acetobutylicum fermentations with non-linear constraints.

A stoichiometric model of Clostridium acetobutylicum and related strains has been previously derived. The stoichiometric matrix of the model contains a singularity which has prevented the calculation of a unique set of fluxes which describe the primary metabolic activity. To resolve the singularity, we have developed a non-linear constraint relating the acetate and butyrate uptake fluxes. Subsequently, we developed a software package utilizing a model independent heuristic global optimization approach to solve the resultant non-linear problem. We have validated the use of the non-linear constraint by correlating calculated butyrate production pathway flux profiles with measured intracellular pH profiles. Finally, we examined a controlled batch fermentation to determine that the acid formation pathways play critical roles throughout solventogenesis. The broader usefulness of reformulating the stoichiometric model as a constrained minimization problem is discussed.     

Continuous hydrogen production during fermentation of alpha-cellulose by the thermophillic bacterium Clostridium thermocellum

Continuous hydrogen (H2) production during fermentation of alpha-cellulose was established using the thermophillic, anaerobic bacterium Clostridium thermocellum ATCC 27405. The objectives of this work were to characterize growth of C. thermocellum, quantify H2 production and determine soluble end-product synthesis patterns during fermentation of a cellulosic substrate under continuous culture conditions. A 5 L working volume fermentor was established and growth experiments were maintained for over 3,000 h. Substrate concentrations were varied from 1 to 4 g/L and the feed was introduced with continuous nitrogen gas sparging to prevent clogging of the feed-line. The pH and temperature of the reactor were maintained at 7.0 and 600 degrees C, respectively, throughout the study. At concentrations above 4 g/L, the delivery of alpha-cellulose was impaired due to feed-line clogging and it became difficult to maintain a homogenous suspension. The highest total gas (H2 plus CO2) production rate, 56.6 mL L(-1) h(-1), was observed at a dilution rate of 0.042 h(-1) and substrate concentration of 4 g/L. Under these conditions, the H2 production rate was 5.06 mmol h(-1). Acetate and ethanol were the major soluble end-products, while lactate and formate were greatly reduced compared to production in batch cultures. Concentrations of all metabolites increased with increasing substrate concentration, with the exception of lactate. Despite a number of short-term electrical and mechanical failures during the testing period, the system recovered quickly, exhibiting substantial robustness. A carbon balance was completed to ensure that all end-products were accounted for, with final results indicating near 100% carbon recovery. This study shows that long-term, stable H2 production can be achieved during direct fermentation of an insoluble cellulosic substrate under continuous culture conditions.     

The consequence of stimulating glucose dehydrogenase activity by the addition of PQQ on metabolite production by Agrobacterium radiobacter NCIB 11883

Summary Agrobacterium radiobacter NCIB 11 883 does not produce gluconate under conditions of glucose excess in batch or continuous culture. However, the addition of micromolar concentrations of pyrrolo quinoline quinone (PQQ) to fermentation media resulted in rapid excretion of gluconate by batch and continuous cultures. This rapid dehydrogenation of glucose was found in cells grown under carbon and nitrogen limitation and is constitutive which suggests that the only reason why this activity is not normally expressed is due to the inability of the organism to synthesize the prosthetic group (PQQ) of the glucose dehydrogenase enzyme.Although the addition of PQQ to batch and continuous cultures caused a very rapid specific rate of gluconate production (0.6–1.1 g gluconate g^-1 dry wt. h^-1) the rate of exopolysaccharide production remained unaltered. Indeed, when the rates of substrate and oxygen uptake are corrected for the rate of gluconate production in the presence of PQQ there appears to be little physiological consequence as a result of this oxidation.     

Biodegradation of di-n-butyl phthalate (DnBP) in bioaugmented bioslurry phase reactor

Bioremediation of di-n-butyl phthalate (DnBP) in soil was studied with various concentrations in a bioslurry phase batch reactor operated in sequenting batch mode (bioaugmented with effluent treatment plant (ETP) microflora) for a total cycle period of 96h. Process performance during the reactor operation was assessed by monitoring DnBP concentration and biochemical process parameters viz., pH, dissolved oxygen (DO), colony forming units (CFU) and oxygen uptake rate (OUR), during the sequence phase operation. The degradation rate was observed to be rapid at lower substrate concentrations and found to be slow as the substrate concentration increased. The potent bacterial strain was also isolated from the slurry phase reactor. Metabolites formed during the degradation of DnBP in the slurry phase reactor were identified. Studies on the kinetics and half-life of the reaction revealed that the degradation process followed zero-order kinetic model.     

毕赤酵母高密度发酵工艺的研究

高密度发酵是毕赤酵母提高蛋白表达量的一种重要策略,发酵工艺是高密度发酵的一个重要因素。采用下列措施均可以有效地提高表达水平：调节基础培养基,采用变pH和变温发酵,提高DO,选择最适的诱导前菌体密度和比生长速率并降低甘油初始浓度和采用分段式指数流加进行调控。选择合适的甲醇补料策略：甲醇限制补料（MLFB）、氧气限制补料（OLFB）、甲醇不限制补料（MNLFB）和温度限制补料（TLFB）。采用两种方式调控补料：诱导阶段菌体生长时,甲醇比消耗速率(qMeOH)为0.02-0.03gg-1h-1,而菌体不生长时,qMeOH采用较高值。     

A comparison of carbon/energy and complex nitrogen sources for bacterial sulphate-reduction: potential applications to bioprecipitation of toxic metals as sulphides

Detailed nutrient requirements were determined to maximise efficacy of a sulphate-reducing bacterial mixed culture for biotechnological removal of sulphate, acidity and toxic metals from waste waters. In batch culture, lactate produced the greatest biomass, while ethanol was more effective in stimulating sulphide production and acetate was less effective. The presence of additional bicarbonate and H₂ only marginally stimulated sulphide production. The sulphide output per unit of biomass was greatest using ethanol as substrate. In continuous culture, ethanol and lactate were used directly as efficient substrates for sulphate reduction while acetate yielded only slow growth. Glucose was utilised following fermentation to organic acids and therefore had a deleterious effect on pH. Ethanol was selected as the most efficient substrate due to cost and efficient yield of sulphide. On ethanol, the presence of additional carbon sources had no effect on growth or sulphate reduction in batch culture but the presence of complex nitrogen sources (yeast extract or cornsteep) stimulated both. Cornsteep showed the strongest effect and was also preferred on cost grounds. In continuous culture, cornsteep significantly improved the yield of sulphate reduced per unit of ethanol consumed. These results suggest that the most efficient nutrient regime for bioremediation using sulphate-reducing bacteria required both ethanol as carbon source and cornsteep as a complex nitrogen source.     

Oxygen transfer kinetics of riboflavin fermentation by Ashbya gossypii in agitated fermentors

Effects of agitation and aeration rates on volumetric oxygen transfer coefficient and oxygen uptake rate of a riboflavin broth containing Ashbya gossypii were investigated in three batch, sparged, and agitated fermentors having the working volumes of 0.42, 0.85, and 2.5 l. The change of oxygen uptake rate with time at 250 rev min⁻¹ stirring and vvm aeration rates was shown. The volumetric oxygen transfer coefficients and maximum oxygen uptake rates obtained have been correlated to mechanical power inputs per unit volume of the fermentation broth and the superficial air velocities.     

Influence of pH and malate-glucose ratio on the growth kinetics of Leuconostoc oenos

Summary Growth, substrate utilization and product formation were studied in batch cultures of a Leuconostoc oenos strain. The effect of various culture conditions, i.e. pH-control at different values and various initial concentrations of malate and glucose, on growth and metabolism were investigated. Addition of malate resulted in a marked stimulation of growth, with only a slight increase in final biomass but a high conversion yield of glucose. Under pH control this stimulation was much greater than could be accounted for from changes in pH profile resulting from malate utilization. The specific rate of malate utilization was maximal at pH 4.0 whereas the specific rate of glucose consumption was highest at pH 5.5. During co-metabolism of malic acid and glucose, substrate utilization and product formation agreed with the stoichiometric relationships of the malo-lactic reaction and the heterolactic fermentation of glucose. Offsprint requests to: A. Pareilleux     

N:P stoichiometric changes via species turnover in arid versus saline desert environments

1. Aridity and salinity have a key role in driving physiological and ecological processes in desert ecosystems. However, how community‐scale foliar nutrients respond to aridity and salinity, and how these responses might vary with community composition along aridity and salinity gradients is unclear. We hypothesize that the response will be a shift in community stoichiometric values resulting from nutrient variability of shared species and unique species (site‐specific species), but little research has addressed the relative contribution of either component.
2. We analyzed the community‐scale stoichiometric response of a desert community of perennial plants along an aridity and salinity transect by focusing on foliar nitrogen (N) and phosphorous (P) concentrations and N:P ratios. After evaluating the shared and unique species variability, we determined their relative contribution to the community stoichiometric response to aridity and salinity, reflected by changes in nonweighted and weighted community‐average values.
3. Community‐scale stoichiometry decreased significantly under aridity and salinity, with significantly consistent changes in nonweighted and weighted community‐average stoichiometry for most shared and unique species measurements. The relative contribution of unique species shifts to the changes in community stoichiometry was greater (15%–77%) than the relative contribution of shared species shifts (7%–45%), excluding the change in weighted P concentration under aridity. Thus, the shifts of unique species amplified the community stoichiometric response to environmental changes.
4. Synthesis. These results highlighted the need for a more in‐depth consideration of shared and unique species variability to understand and predict the effects of environmental change on the stoichiometry of plant communities. Although variation in community stoichiometry can be expected under extreme aridity and salinity conditions, changes of unique species could be a more important driver of the stoichiometric response of plant communities.