Modeling and static optimization of the ethanol production in a cascade reactor. I. Modeling

Let us consider the modeling of a cascade reactor for the production of ethanol. The rates of reaction in alcoholic fermentation are modeled so that it can assume both ethanol and substrate inhibition, in relation to the observed results.A nonstructured model, based on biomass, substrate, and ethanol concentrations, is developed and identified. It is a complex model, this being due to the nonlinearity between the specific rate of ethanol production and the growth rate and, on the other hand, the study of the static optimization of ethanol fermentation is performed.     

Rapid ethanol fermentation of cellulose hydrolysate. II. Product and substrate inhibition and optimization of fermentor design

High concentrations of both ethanol and sugar in the fermentation broth inhibit the growth of yeast cells and the rate of product formation. Inhibitory effects of ethanol on the yeast strain Saccharomyces cerevisiae NRRL-Y-132 were studied in batch and continuous chemostat cultures. Growth was limited by either glucose or ethanol. Feed medium was supplemented with different ethanol concentrations. Ethanol was found to inhibit growth and the activity of yeast to produce ethanol in a noncompetitive manner. A linear kinetic pattern for growth and product formation was observed according to μ = μ_m (1 – P/P_m) and v = v_m (1 – P/P_m′), where μ_m is the maximum specific growth rate at P = 0 (hr^?1); P_m is the maximum specific product formation rate at P = 0 (hr^?1); P_m is the maximum ethanol concentration above which cells do not grow (g/liter); P_m′ is the maximum ethanol concentration above which cells do not produce ethanol (g/liter). Substrate inhibition studies were carried out using short-time experimental techniques under aerobic and anaerobic condition. The degree of substrate inhibition was found to be higher than that has been reported for ethanol fermentation of pure sugar. The kinetic relationships thus obtained were used to compute growth, substrate utilization, and alcohol production patterns and have been discussed with reference to batch and continuous fermentation of enzymatically produced bagasse hydrolysate.     

Modeling and advanced control of recombinant Zymomonas mobilis fed-batch fermentation

This work presents the development of an unstructured kinetic model incorporating the differing degrees of product, substrate, and pH inhibition on the kinetic rates of ethanol fermentation by recombinant Zymomonas mobilis CP4:pZB5 for growth on two substrates. Product inhibition was observed to start affecting the specific growth rate at an ethanol concentration of 20 g/L and the specific productivity at about 35-40 g/L. Specific growth rate was also shown to be more sensitive to inhibition by lowered pH as well. A model for the inhibition of two competing substrates' cellular uptake via membrane transport is proposed. Inhibition functions and model parameters were determined by fitting experimental data to the model. The model was utilized in a nonlinear model predictive control (NMPC) algorithm to control the product concentration during fed-batch fermentation to offset the inhibitory effects of product inhibition. Using the optimal feeding policy determined online, the volumetric productivity of ethanol was improved 16.6% relative to the equivalent batch operation when the final ethanol concentration was reached.     

Steady state analysis of the enhancement in ethanol productivity of a continuous fermentation process employing a protein-phospholipid complex as a protecting agent

A continuous cascade fermentation process comprising eight tanks in series, employing a protein-phopholipid complex as a protective agent (PA) was performed for ethanol production from glucose. An increase of 58.4% in fermenter productivity was obtained due to the addition of PA. A kinetic model including product and substrate inhibition effects is proposed. Parameters appearing in the kinetic model were estimated by using the method of least squares. It is found that the product inhibition effect dominates over the substrate inhibition effect for the range of concentrations studied in our fermentation system. Upon addition of PA, both inhibitory effects are reduced to as little as about one quarter of that without PA. It was also found that the use of PA primarily protected the cells against ethanol inhibition rather than substrate inhibition. A steady state criterion is also discussed.     

Optimal substrate feeding policy for a fed batch fermentation with substrate and product inhibition kinetics

The optimal substrate feeding policy for the fed batch fermentation which is governed by product and substrate inhibited kinetics is presented. The conjunction point between nonsingular and singular arcs and the feeding policy along the singular arc are derived analytically in terms of the concentrations of substrate and product and the liquid volume. Thus, it is possible to determine the feeding rate by monitoring the state variables (i.e., closed loop control). As a specific example, an optimization study of the fed batch fermentation for ethanol production by Saccharomyces cerevisiae is presented. It is shown that the optimal feeding patterns are heavily dependent upon the initial conditions. The point selectivity provides the guideline for predicting the optimal feeding patterns and explaining the results of rigorous mathematical analysis.     

Effects of high product and substrate inhibitions on the kinetics and biomass and product yields during ethanol batch fermentation

In ethanol fermentation, instantaneous biomass yield of the yeast Saccharmoyces cerevisiae was found to decrease (from 0.156 to 0.026) with increase in ethanol concentration (from 0 to 107 g/L), indicating a definite relationship between biomass yield and product inhibition. A suitable model was proposed to describe this decrease which incorporates the kinetic parameters of product inhibition rather than pure empirical constants. Substrate inhibition was found to occur when substrate concentration is above 150 g/L. A similar definite relationship was observed between substrate inhibition and instantaneous biomass yield. A simple empirical model is proposed to describe the declines in specfic growth rate and biomass yield due to substrate inhibition. It is observed that product inhibition does not have any effect on product yield whereas substrate inhibition significantly affects the product yield, reflecting a drop in overall product yield from 0.45 to 0.30 as the initial substrate concentration increases from 150 to 280 g/L. These results are expected to have a significant influence in formulating optimum fermentor design variables and in developing an effective control strategy for optimizing ethanol producitivity.     

Ethanol production by thermophilic bacteria: metabolic control of end product formation in Thermoanaerobium brockii.

Specific changes in the chemical and microbial composition of Thermoanaerobium brockii fermentations were compared and related to alterations of process rates, end product yields, and growth parameters. Fermentation of starch as compared with glucose was associated with significant decreases in growth rate and intracellular fructose-1,6-bisphosphate concentration and with a dramatic increase in the ethanol/lactate product ratio. Glucose or pyruvate fermentation in the presence of acetone was correlated with increased substrate consumption, growth (both rate and yield), acetate yield, and quantitative reduction of acetone to isopropanol in lieu of normal reduced fermentation products (i.e., H2, ethanol, lactate). Acetone altered pyruvate phosphoroclastic activity of cell extracts in that H2, lactate, and ethanol levels decreased, whereas the acetate concentration increased. Glucose fermentation in the presence of exogenous hydrogen was associated with inhibition of endogenous H2 production and either increased ethanol/acetate product ratios and decreased growth at less than 0.5 atm (51 kPa) of H2 or total growth inhibition at 1.0 atm (102 kPA). The effects of exogenous hydrogen on glucose fermentation were totally reversed by the addition of acetone. Glucose fermentation in coculture with Methanobacterium thermoautotrophicum correlated with increased growth (both rate and yield), acetate yield, and the formation of methane in lieu of monoculture reduced products. In coculture, but not monoculture, T. brockii grew on ethanol as the energy source, and acetate and methane were the end products as a direct consequence of hydrogen consumption by the methanogen.     

Inhibierungserscheinungen bei der alkoholischen Gärung,II. Einfluß der Substratkonzentration auf die spezifische Ethanolbildungsgeschwindigkeit von Saccharomyees cerevisiae

The influence of sucrose concentration on the specific ethanol production rate was studied during batch processes using the yeast strain Saccharomyces cerevisiae Hansen Sc 5. From experimental data a model could be derived for the simulataneous effect of substrate and product inhibition. It was found that both the decreases of fermentation activity of the cells caused by sucrose and ethanol have an additional relation to each other. This model also takes into consideration the fact that the maximum ethanol concentration P′ can't be realized at high substrate concentrations in a batch process. Compared to it sucrose concentrations below 100 g/l did not inhibit the ethanol production by the strain used in this investigation.     

An immobilized cell reactor with simultaneous product separation. I. Reactor design and analysis

A four-phase reactor-separator (gas, liquid, solid, and immobilized catalyst) is proposed for fermentations characterized by a volatile product and nonvolatile substrate.In this reactor, the biological catalyst is immobilized onto a solid column packing and contacted by the liquid containing the substrate.A gas phase is also moved through the column to strip the volatile product into the gas phase. The Immobilized Cell Reactor-Separator (ICRS) consists of two basic gas-liquid flow sections: a cocurrent "enricher" followed by a countercurrent-"stripper".In this article, an equilibrium stage model of the reactor is developed to determine the feasibility and important operational variables of such a reactor-separator. The ICRS concept is applied to the ethanol from whey lactose fermentation using some preliminary immobilized cell reactor performance data. A mathematical model for a steady-state population based on an adsorbed monolayer of cells is also developed for the reactor. The ICRS model demonstrated that the ICRS should give a significant increase in reactor productivity as compared to an identically sized Immobilized Cell Reactor (ICR) with no separation. The gas-phase separation of the product also allows fermentation of high inlet substrate concentrations. The model is used to determine the effects of reactor parameters on ICRS performance including temperature, pressure, gas flow rates, inlet substrate concentration, and degree of microbial product inhibition.     

Continuous production of ethanol by yeast "immobilized" in a membrane-contained fermentor

A dialysate-feed, immobilized-cell dialysis continuous fermentation system was investigated as a method of relieving product inhibition in the conversion of glucose to ethanol by cells of Saccharomyces cerevisiae ATCC 4126. The substrate was fed into a continuous dialysate circuit and then into a batch fermentor circuit via diffusion through the microporous membranes of an intermediate dialyzer. Simultaneously, product was withdrawn from the fermentor circuit through the dialyzer membranes into the dialysate circuit and out in the effluent. Since the fermentor was operated without an effluent, the cells essentially were immobilized and converted substrate to product by maintenance metabolism. Contrary to prior results with this novel system for the continuous fermentation of lactose to lactate by lactobacillus cells, a steady state of yeast cells in the fermentor did not occur initially but was obtained by the depletion of medium nitrogen and the prevention of cell breakage, although the substrate and product concentrations then became unsteady. The inherent advantages of the system was offset in the ethanol fermentation by relatively low productivity, which appeared to be limited by membrane permeability.     

Ethanol and lactic acid production from sugar and starch wastes by anaerobic acidification

Anaerobic conversion of carbohydrates can generate various end‐products. Besides physical parameters such as pH and temperature, the types of carbohydrate being fermented influences the fermentation pattern. Under uncontrolled pH, microbial mixed cultures from activated sludge and anaerobic digester sludge anaerobically produced ethanol from glucose while producing lactic acid from starch conversion. This trend was not only observed in batch trials. Also, continuous chemostat operation of anaerobic digester sludge resulted in the reproducible predominance of ethanol fermentation from glucose solution and lactic acid production from starch. Different feeding regimes and substrate availability (shock load versus continuous feeding) in glucose fermentation under non‐controlled pH did not affect the ethanol production as the major end product. Shifts in feed composition from glucose to starch and vice versa result in an immediate change of fermentation end products formation.     

A substrate feeding strategy--using an oxystat for L-phenylalanine fermentation by Corynebacterium glutamicum

Summary A substrate feeding strategy using an oxystat was first successfully applied to a fed-batch phenylalanine fermentation. The control method allowed the fermentation to be under low dissolved oxygen tension, which was favourable phenylalanine formation, and to be from substrate inhibition during the course of fed-batch operation. The final product concentration was 3 times higher than in a batch culture.     

Glycerol fermentation by (open) mixed cultures: a chemostat study

Glycerol is an important byproduct of bioethanol and biodiesel production processes. This study aims to evaluate its potential application in mixed culture fermentation processes to produce bulk chemicals. Two chemostat reactors were operated in parallel, one fed with glycerol and the other with glucose. Both reactors operated at a pH of 8 and a dilution rate of 0.1 h(-1). Glycerol was mainly converted into ethanol and formate. When operated under substrate limiting conditions, 60% of the substrate carbon was converted into ethanol and formate in a 1:1 ratio. This product spectrum showed sensitivity to the substrate concentration, which partly shifted towards 1,3-propanediol and acetate in a 2:1 ratio at increasing substrate concentrations. Glucose fermentation mainly generated acetate, ethanol and butyrate. At higher substrate concentrations, acetate and ethanol were the dominant products. Co-fermentations of glucose-glycerol were performed with both mixed cultures, previously cultivated on glucose and on glycerol. The product spectrum of the two experiments was very similar: the main products were ethanol and butyrate (38% and 34% of the COD converted, respectively). The product spectrum obtained for glucose and glycerol fermentation could be explained based on the general metabolic pathways found for fermentative microorganisms and on the metabolic constraints: maximization of the ATP production rate and balancing the reducing equivalents involved.     

Substrate and product inhibition kinetics in succinic acid production by Actinobacillus succinogenes

The inhibition of substrate and products on the growth of Actinobacillus succinogenes in fermentation using glucose as the major carbon source was studied. A. succinogenes tolerated up to 143 g/L glucose and cell growth was completely inhibited with glucose concentration over 158 g/L. Significant decrease in succinic acid yield and prolonged lag phase were observed with glucose concentration above 100 g/L. Among the end-products investigated, formate was found to have the most inhibitory effect on succinic acid fermentation. The critical concentrations of acetate, ethanol, formate, pyruvate and succinate were 46, 42, 16, 74, 104 g/L, respectively. A growth kinetic model considering both substrate and product inhibition is proposed, which adequately simulates batch fermentation kinetics using both semi-defined and wheat-derived media. The model accurately describes the inhibitory kinetics caused by both externally added chemicals and the same chemicals produced during fermentation. This paper provides key insights into the improvement of succinic acid production and the modelling of inhibition kinetics.     

Applied in situ product recovery in ABE fermentation

The production of biobutanol is hindered by the product's toxicity to the bacteria, which limits the productivity of the process. In situ product recovery of butanol can improve the productivity by removing the source of inhibition. This paper reviews in situ product recovery techniques applied to the acetone butanol ethanol fermentation in a stirred tank reactor. Methods of in situ recovery include gas stripping, vacuum fermentation, pervaporation, liquid–liquid extraction, perstraction, and adsorption, all of which have been investigated for the acetone, butanol, and ethanol fermentation. All techniques have shown an improvement in substrate utilization, yield, productivity or both. Different fermentation modes favored different techniques. For batch processing gas stripping and pervaporation were most favorable, but in fed‐batch fermentations gas stripping and adsorption were most promising. During continuous processing perstraction appeared to offer the best improvement. The use of hybrid techniques can increase the final product concentration beyond that of single‐stage techniques. Therefore, the selection of an in situ product recovery technique would require comparable information on the energy demand and economics of the process. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:563–579, 2017     

A study of ethanol tolerance in yeast

The ethanol tolerance of yeast and other microorganisms has remained a controversial area despite the many years of study. The complex inhibition mechanism of ethanol and the lack of a universally accepted definition and method to measure ethanol tolerance have been prime reasons for the controversy. A number of factors such as plasma membrane composition, media composition, mode of substrate feeding, osmotic pressure, temperature, intracellular ethanol accumulation, and byproduct formation have been shown to influence the ethanol tolerance of yeast. Media composition was found to have a profound effect upon the ability of a yeast strain to ferment concentrated substrates (high osmotic pressure) and to ferment at higher temperatures. Supplementation with peptone-yeast extract, magnesium, or potassium salts has a significant and positive effect upon overall fermentation rates. An intracellular accumulation of ethanol was observed during the early stages of fermentation. As fermentation proceeds, the intracellular and extracellular ethanol concentrations become similar. In addition, increases in osmotic pressure are associated with increased intracellular accumulation of ethanol. However, it was observed that nutrient limitation, not increased intracellular accumulation of ethanol, is responsible to some extent for the decreases in growth and fermentation activity of yeast cells at higher osmotic pressure and temperature.     

IN SITU SEPARATION OF ETHANOL WITH AQUEOUS TWO-PHASE SYSTEM AND ASSESSMENT OF KLa FOR YEAST GROWTH IN BATCH CULTIVATION

In the fermentation process, the separation of product and its purification is the most difficult and exigent task in the ground of biochemical engineering. Another major problem that is encountered in the fermentation is product inhibition, which leads to low conversion and low productivities. Extractive fermentation is a technique that helps in the in situ removal of product and better performance of the fermentation. An aqueous two-phase system was employed for in situ ethanol separation since the technique was biofriendly to the Saccharomyces cerevisiae and the ethanol produced. The two-phase system was obtained with polyethylene glycol 4000 (PEG 4000) and ammonium sulfate in water above critical concentrations, with the desire that the ethanol moves to the top phase while cells rest at the bottom. The overall mass transfer coefficient (K_La) was also estimated for the yeast growth at different rpm. The concentration and yield of ethanol were determined for conventional fermentation to be around 81.3% and for extractive fermentation around 87.5% at the end of the fermentation. Based on observation of both processes, extractive fermentation was found to be the best.     

Batch ethanol production with dual organisms

Summary The concept of improving ethanol productivity in batch fermentation by utilising two organisms with different substrate and product inhibition characteristics was examined. The inocula consisted of two yeasts chosen because of their different inhibition properties at high sugar and high ethanol concentrations respectively. Improved productivity was found in the dual system. A numerical analysis which incorporates the non-linear nature of the inhibition demonstrates the level of improvement which might be attained in such systems.     

A general inhibition kinetics model for ethanol production using a novel carbon source: sodium gluconate

In this study, sodium gluconate was applied as a novel carbon source for the fuel ethanol production using an engineered Escherichia coli strain KO11 in batch fermentations. Ethanol and acetic acid were produced as two major products as well as small amount of lactic acid during the fermentation. Compared to the conventional carbon source glucose, the bioconversion of sodium gluconate possessed two distinct advantages: faster utilization rate of sodium gluconate (1.66 g/L per h) compared to glucose (0.996 g/L per h) and no requirement for pH control during fermentation. A general inhibition model including both substrate and products inhibitory effects was proposed, which adequately simulated batch fermentation kinetics at various concentrations of sodium gluconate. All of the products showed inhibitory effects on cell growth. The order of the inhibitory strength of all products and substrate was for the first time clarified in this study. Acetic acid was the most inhibitory product mitigating the cell growth, followed by ethanol and lactic acid. Sodium gluconate stimulated cell growth when its concentration was below 16 g/L, while it inhibited the cell growth when the concentration was above this concentration. It completely inhibited the cell growth when the concentration was 325 g/L. The high value of both the coefficient of determination (R ²) and the adjusted R ² verified the good fit of the model. This paper provides key insights into further engineering these strains to improve ethanol production.     

Ethanol production by thermophilic bacteria: biochemical basis for ethanol and hydrogen tolerance in Clostridium thermohydrosulfuricum.

The metabolic and enzymatic bases for growth tolerance to ethanol (4%) and H2 (2 atm [1 atm = 101.29 kPa]) fermentation products in Clostridium thermohydrosulfuricum were compared in a sensitive wild-type strain and an insensitive alcohol-adapted strain. In the wild-type strain, ethanol (4%) and H2 (2 atm) inhibited glucose but not pyruvate fermentation parameters (growth and end product formation). Inhibition of glucose fermentation by ethanol (4%) in the wild-type strain was reversed by addition of acetone (1%), which lowered H2 and ethanol production while increasing isopropanol and acetate production. Pulsing cells grown in continuous culture on glucose with 5% ethanol or 1 atm of H2 significantly raised the NADH/NAD ratio in the wild-type strain but not in the alcohol-adapted strain. Analysis of key oxidoreductases demonstrated that the alcohol-adapted strain lacked detectable levels of reduced ferredoxin-linked NAD reductase and NAD-linked alcohol dehydrogenase activities which were present in the wild-type strain. Differences in the glucose fermentation product ratios of the two strains were related to differences in lactate dehydrogenase and hydrogenase levels and sensitivity of glyceraldehyde 3-phosphate dehydrogenase activity to NADH inhibition. A biochemical model is proposed which describes a common enzymatic mechanism for growth tolerance of thermoanaerobes to moderate concentrations of both ethanol and hydrogen.