Control of yeast fed-batch process through regulation of extracellular ethanol concentration

At high growth rates, the biomass yield of baker's yeast (Saccharomyces cerevisiae) decreases due to the prodn. of ethanol. For this reason, it is std. industrial practice to use a fed-batch process whereby the specific growth rate, m, is fixed at a level below the point of ethanol prodn., i.e., mcrit. Optimally, growth should be maintained at mcrit, but in practice, this is difficult because mcrit is dependent upon strain and culture conditions. In this work, growth was maintained at a point just above mcrit by regulating ethanol concn. in the bioreactor. The models used for control design are shown, as are the exptl. results obtained when this strategy was implemented. This technique should be applicable to all microorganisms that exhibit an "overflow" type metab. [on SciFinder (R)]

Fuel bioethanol production from whey permeate

François-Nicolas de Glutz

The objective of this thesis was to evaluate the feasibility for the production of fuel bioethanol from whey permeate. The identification of the limiting factors of the process revealed that the stabilization of the substrate was critical to the use of whey as a fermentation substrate and that the conversion yields and ethanol tolerance limited the process. A comparison of the possible scenarios for the production of bioethanol from whey was first performed. It was demonstrated that fresh whey should be concentrated at the production site before being transported to a centralized treatment plant, since transporting dilute material or producing ethanol at the dairy would result in too high operating costs. An economic comparison of a treatment plant, which (i) "directly" ferments lactose into ethanol using Kluyveromyces fragilis and (ii) "indirectly" ferments hydrolyzed whey using Saccharomyces cerevisiae, resulted in similar production costs of 1.35 CHF/LEtOH and 1.32 CHF/LEtOH respectively. Whey stabilization was investigated by testing the addition of chemical compounds to whey. These were evaluated according to their ability to prevent the growth of lactic acid bacteria which were identified as being mainly responsible for whey instability. Formic acid (50 mM) or hydrogen peroxide (100 mM) were shown to extend the stability from 2-3 days, at 4°C to 21 days at ambient temperature. However, such concentrations of preservative also inhibit the growth of yeasts, therefore they must be removed prior to fermentation. Of the compounds tested, formic acid was preferred due to the high level of bacterial growth inhibition at pH 4, and its non-toxicity for yeast at neutral pH. This would enable whey to be stored at room temperature for a three-week period without negatively influencing the subsequent yeast fermentation. Global productivity of the initial fermentation cultures was considerably reduced as a result of a long non-productive lag phase. In order to improve the understanding of the principle factors which, not only influence the duration of the lag phase, but also the biomass produced during a pre-culture period of 24h and the maximum growth rate in fermentation cultures, six pre-culture parameters were tested alone or in combination on two ethanol-producing yeasts, K. marxianus CBS 5795 and CBS 397, using whey permeate as substrate through the application of design of experiment procedures. The key parameters identified through this strategy were: influence of temperature, type of sugar, culture mode, initial biomass concentration and initial sugar concentration. Optimum ethanol productivity was achieved by cultivating the pre-culture anaerobically on medium containing lactose, which resulted in an improvement of the productivity by 10-11% compared to an aerobic pre-culture with glucose. The principal organism studied for ethanol fermentation from whey permeate was K. fragilis due to its ability to directly ferment lactose. However, such direct fermentation yeasts generally suffer from low conversion yields and poor tolerance to ethanol (2-3% v/v). An alternative is to utilize indirect fermentation yeasts, such as S. cerevisiae, which show considerably better ethanol fermentation performance but has the disadvantage that an expensive enzymatic hydrolysis step is required prior to fermentation. In this study both types of process have been characterized involving eight ethanol producing yeasts. The culture conditions were optimized for each strain using a design of experiment methodology. Highest conversion yield and alcohol tolerance were achieved with S. cerevisiae Ethanol Red (YP/S= 0.662 C-mol/C-mol, cEtOHmax= 148 g/L), of the indirect fermentation yeasts, and with K. marxianus CBS 5795 (YP/S= 0.660 C-mol/C-mol, cEtOHmax= 79 g/L) of the direct fermentation yeasts studied. Introducing the data obtained from cultures with these yeasts to former economic evaluations of both scenarios, showed that direct fermentation should be preferred for fermenting whey permeate to ethanol. A maximum volumetric productivity of 6.24 g/(L·h) at 37°C and pH 4 was achieved with K. marxianus CBS 5795. Fermentation of non-sterile whey permeate with a consortium (CEKI) of K. marxianus (S1), I. orientalis (S2) and E. faecalis (S3), isolated from spontaneous cultures, was then studied. Maximal ethanol yield of 0.65 C-mol/C-mol, as highest ethanol concentration of 55 g/L, was achieved with CEKI, at 37°C and pH 4, compared to isolated cultures of these organisms. The results also suggest that E. faecalis exhibits a protective effect against lactic acid bacteria. Specific productivity of CEKI was 0.21 gEtOH/(gbiomass·h). Finally, a novel in-situ product recovery method, based on capsular perstraction with an organic solvent, was developed for ethanol extraction in batch fermentation systems using CEKI, which utilizes microcapsules. The production of capsules of 2 mm diameter that contained a hydrophobic core of laurinaldehyde and an alginate-based membrane enabled (i) to reduce the toxicity of the solvent for the growing cultures, and (ii) make the separation of the organic phase easier for a subsequent ethanol recovery. For the produced capsules mass transfer was determined by the solvent layer (0.27·10-5 cm2/s), which resulted in a maximum specific ethanol recovery of 3.17 gEtOH/(gsolvent·s).

EPFL2009

Optimal Control of Fed-Batch Fermenters

Dominique Bonvin, Christopher Cannizzaro, Martin Rhiel, Sergio Valentinotti, Urs von Stockar

Optimal control of fed-batch fermenters S. Valentinotti† C. Cannizzaro‡ M.Rhiel‡ U. Holmberg† U. von Stockar‡ D. Bonvin† †Institut d’Automatique, EPFL, 1015 Lausanne, Switzerland ‡Institut de Genie Chimique, EPFL, 1015 Lausanne, Switzerland Fermentors are often run in a fed-batch manner to avoid the formation of overﬂow metabolites. At a high growth rate, the most eﬃcient metabolic pathway(s) of certain microorganisms become saturated resulting in overﬂow metabolite production. These byproducts are undesirable since their accumulation in the reactor may be inhibitory and the productivity of biomass and growth-associated products is reduced. The ideal way to run such fed-batch fermentation is to grow the cells in the reactor at the critical growth rate, i.e., the point at which overﬂow metabolite production begins. However, since this value changes from run to run, or even during a given fermentation, its identiﬁcation is not trivial. A simple way to overcome this diﬃculty is to maintain a very small, but constant overﬂow metabolite concentration in the reactor, ensuring that most of the substrate is consumed eﬃciently. However due to exponential cell growth, standard controllers can maintain a constant concentration only for a limited time period. In this work an adaptive control strategy to maintain a constant overﬂow metabolite concentration in fed-batch fermentation is presented. The proposed approach requires the knowledge of only two system parameters: the yield coeﬃcient, expressing the relation between overﬂow metabolite and substrate, and the instantaneous concentration of the overﬂow metabolite. Baker’s yeast fed-batch experiments were performed with the ob jective of maximizing biomass productivity and minimizing ethanol production. Mid-infrared spectroscopy was used to measure the ethanol concentration that was provided on-line to the controller. The results from numerous experiments have demonstrated the eﬀectiveness of the proposed control strategy. The speciﬁc growth rate was maintained constant, at a value close to the critical point, until oxygen transfer limitation occurred. Then, the controller automatically reduced the feed rate to prevent excess ethanol production. The biomass increased from 0.5 to 65 grams per liter during the exponential growth phase. Simulation results based on this control strategy show its applicability to other overﬂow metabolite organisms, such as Escherichia coli.

2000

Comprehensive in-situ Bioreactor Monitoring and Control Based on a Mid-Infrared Spectroscopic Sensor System

Christopher Cannizzaro, Ian William Marison, Martin Rhiel, Sergio Valentinotti, Urs von Stockar

Comprehensive in-situ bioreactor monitoring and control based on a midinfrared spectroscopic sensor system M. Rhiel1, C. Cannizzaro1, S. Valentinotti2, I. Marison1, U. von Stockar1 1 Institute of Chemical Engineering, 2Institute of Automatic Control Swiss Federal Institute of Technology (EPFL), CH- 1015 Lausanne, Switzerland Abstract A mid-infrared spectroscopic sensor system consisting of a spectrometer, a single attenuated total reflectance (ATR) probe immersed in the bioreactor, and a software package for multivariate spectra analysis was used to monitor glucose, ethanol, glycerol, acetic acid, sulfate, phosphate, PPG 2000 antifoam, and biomass in Saccharomyces cerevisiae cultures. The provided ethanol concentration was used to control the feed of a concentrated glucose solution to maintain maximum oxidative growth. Introduction: Bioprocess monitoring and control rely on the use of appropriate sensors. Ideally, these sensors should be in situ and measure simultaneously the concentrations of all major bioprocess metabolites with sufficient accuracy and long-term stability under minimum maintenance. Spectroscopic sensors utilizing the mid-infrared (MIR) electromagnetic range offer many advantages, including simultaneous multi-analyte determinations, in situ sterilizability, low maintenance during operation, and enhanced information about most biologically important species compared to near-infrared (NIR) spectroscopic sensors. bioprocess monitoring. Overlapping absorbance features, however, require the application of advanced chemometric analysis methods. This paper discusses the simultaneous in-situ monitoring of glucose, ethanol, glycerol, acetic acid, biomass, pH, temperature, and bioreactor volume in batch and fed-batch cultures of Saccharomyces cerevisiae with a Therefore, they seem to be good candidates for single-probe mid-infrared spectroscopic sensor (ReactIRTM, ASI Applied Systems). Calibration models for each analyte were established with partial least-squares regression (PLSR) implemented for immediate process analysis (QuantIRTM, ASI Applied Systems). Concentration data was available every two minutes and allowed the implementation of a fed-batch control strategy to maximize biomass production. Materials and Methods: A ReactIRTM 1000 mid-infrared spectrometer (ASI Applied Systems, Millersville, MD) equipped with a diamond ATR immersion probe (DiCompTM , ASI Applied Systems) and QuantIRTM software (ASI Applied Systems) was used for in situ data acquisition and analysis. Saccharomyces cerevisiae CBS426 and S. cerevisiae W303-1A PVD32 cells were grown on defined medium in a 16L stirred tank bioreactor (Bioengineering AG, Wald, Switzerland). A customized bioprocess management and control environment (BioOPT) was developed with LabVIEWTM (National Instruments, Austin, TX). Results: Quantitative spectra analysis was performed by establishing PLSR models for each of the analytes: glucose, ethanol, glycerol, acetic acid, sulfate, phosphate, PPG 2000 antifoam, and biomass. The calibration data set consisted of 60 in situ collected pure component spectra and 40 in situ collected reaction spectra. Maintaining the ethanol concentration reported by the mid-infrared spectroscopic sensor system around 0.7 g/L resulted in an exponential growth rate of 0.255 1/h. Exponential growth was sustained until oxygen, supplied by sparging air, became limiting. Thereafter, the controller adapted automatically and linear growth occurred up to a cell density of 60 g/L.

2000

Fuel bioethanol production from whey permeate

François-Nicolas de Glutz

EPFL2009

Optimal Control of Fed-Batch Fermenters

Dominique Bonvin, Christopher Cannizzaro, Martin Rhiel, Sergio Valentinotti, Urs von Stockar

2000

Comprehensive in-situ Bioreactor Monitoring and Control Based on a Mid-Infrared Spectroscopic Sensor System

Christopher Cannizzaro, Ian William Marison, Martin Rhiel, Sergio Valentinotti, Urs von Stockar

2000