Acetone butanol ethanol (ABE) production from concentrated substrate: reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping

Acetone butanol ethanol (ABE) was produced in an integrated fed-batch fermentation-gas stripping product-recovery system using Clostridium beijerinckii BA101, with H₂ and CO₂ as the carrier gases. This technique was applied in order to eliminate the substrate and product inhibition that normally restricts ABE production and sugar utilization to less than 20 g l^–1 and 60 g l^–1, respectively. In the integrated fed-batch fermentation and product recovery system, solvent productivities were improved to 400% of the control batch fermentation productivities. In a control batch reactor, the culture used 45.4 g glucose l^–1 and produced 17.6 g total solvents l^–1 (yield 0.39 g g^–1, productivity 0.29 g l^–1 h^–1). Using the integrated fermentation-gas stripping product-recovery system with CO₂ and H₂ as carrier gases, we carried out fed-batch fermentation experiments and measured various characteristics of the fermentation, including ABE production, selectivity, yield and productivity. The fed-batch reactor was operated for 201 h. At the end of the fermentation, an unusually high concentration of total acids (8.5 g l^–1) was observed. A total of 500 g glucose was used to produce 232.8 g solvents (77.7 g acetone, 151.7 g butanol, 3.4 g ethanol) in 1 l culture broth. The average solvent yield and productivity were 0.47 g g^–1 and 1.16 g l^–1 h^–1, respectively.     

Efficient conversion of lactic acid to butanol with pH-stat continuous lactic acid and glucose feeding method by Clostridium saccharoperbutylacetonicum

In order to achieve high butanol production by Clostridium saccharoperbutylacetonicum N1-4, the effect of lactic acid on acetone–butanol–ethanol fermentation and several fed-batch cultures in which lactic acid is fed have been investigated. When a medium containing 20 g/l glucose was supplemented with 5 g/l of closely racemic lactic acid, both the concentration and yield of butanol increased; however, supplementation with more than 10 g/l lactic acid did not increase the butanol concentration. It was found that when fed a mixture of lactic acid and glucose, the final concentration of butanol produced by a fed-batch culture was greater than that produced by a batch culture. In addition, a pH-controlled fed-batch culture resulted in not only acceleration of lactic acid consumption but also a further increase in butanol production. Finally, we obtained 15.5 g/l butanol at a production rate of 1.76 g/l/h using a fed-batch culture with a pH-stat continuous lactic acid and glucose feeding method. To confirm whether lactic acid was converted to butanol by the N1-4 strain, we performed gas chromatography–mass spectroscopy (GC-MS) analysis of butanol produced by a batch culture during fermentation in a medium containing [1,2,3-¹³C₃] lactic acid as the initial substrate. The results of the GC-MS analysis confirmed the bioconversion of lactic acid to butanol.     

A heuristic approach to fed-batch optimisation of streptokinase fermentation

Previous studies have shown that the rate of formation of streptokinase, a secondary metabolite, in batch fermentation is proportional to the specific growth rate of the biomass, which in turn is inhibited by its substrate and the primary product (lactic acid). These kinetics suggest the suitability of fed-batch operation to increase the yield of streptokinase. A near-optimal feed policy has been calculated by the chemotaxis algorithm, and it shows a substrate feed rate decreasing nonlinearly and vanishing after 11 hours. This is followed by batch fermentation for a further 8 hours, at the end of which 12% more streptokinase is generated than by purely batch fermentation. Further improvements in productivity are possible.List of Symbols k _dh^–1 decay constant for active cells - k _ph^–1 decay constant for streptokinase - K _Igl^–1 inhibition constant for lactic acid - K_S gl^–1 inhibition constant for substrate - M gl^–1 lactic acid concentration - P gl^–1 streptokinase concentration - Q 1h^–1 substrate feed rate - S gl^–1 substrate concentration - S _ingl^–1 inlet concentration of substrate - t h time - t _bh end-point of batch fermentation - t _fh end-point of fed-batch fermentation - V l volume of broth in fermenter - V ₀ l initial value of V (at t=0) - V _ml maximum value of V - X gl^–1 total biomass concentration - X _agl^–1 concentration of active biomass - Y _MX yield coefficient for lactic acid from biomass - Y _PX yield coefficient for streptokinase from biomass - Y _XS yield coefficient for biomass from substrate Greek Letters h^–1 specific growth rate of biomass - _mh^–1 maximum specific growth rate     

Fed-batch propionic acid production by Propionibacterium acidipropionici

The batch fermentations were conducted using lactose as the substrate at pH 6.5 and temperature 30°C. Average batch kinetic data was eventually used to develop an unstructured mathematical model. The kinetic parameters of the model were determined by non-linear regression technique using the batch experimental results. Parametric sensitivity analysis showed the maximum specific substrate consumption rate (r_S^max) and the maintenance energy constant (m_S) to be the most sensitive parameters. The experimental observations in batch fermentation were close to the model predictions. The batch model was extrapolated to identify nutrient feeding strategies, which were tested successfully for two different fed-batch fermentations. It demonstrated enhanced propionic acid productivity. The developed model was found suitable for the design of feeding strategies to increase propionic acid production in fed-batch mode of reactor operation.     

Microbial fed-batch production of 1,3-propanediol by Klebsiella pneumoniae under micro-aerobic conditions

The microbial production of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae under micro-aerobic conditions was investigated in this study. The experimental results of batch fermentation showed that the final concentration and yield of 1,3-PD on glycerol under micro-aerobic conditions approached values achieved under anaerobic conditions. However, less ethanol was produced under microaerobic than anaerobic conditions at the end of fermentation. The batch micro-aerobic fermentation time was markedly shorter than that of anaerobic fermentation. This led to an increment of productivity of 1,3-PD. For instance, the concentration, molar yield, and productivity of 1,3-PD of batch micro-aerobic fermentation by K. pneumoniae DSM 2026 were 17.65 g/l, 56.13%, and 2.94 g l^–1 h^–1, respectively, with a fermentation time of 6 h and an initial glycerol concentration of 40 g/l. Compared with DSM 2026, the microbial growth of K. pneumoniae AS 1.1736 was slow and the concentration of 1,3-PD was low under the same conditions. Furthermore, the microbial growth in fed-batch fermentation by K. pneumoniae DSM 2026 was faster under micro-aerobic than anaerobic conditions. The concentration, molar yield, and productivity of 1,3-PD in fed-batch fermentation under micro-aerobic conditions were 59.50 g/l, 51.75%, and 1.57 g l^–1 h^–1, respectively. The volumetric productivity of 1,3-PD under microaerobic conditions was almost twice that of anaerobic fed-batch fermentation, at 1.57 and 0.80 g l^–1 h^–1, respectively.     

Batch and fed-batch production of coenzyme Q10 in recombinant Escherichia coli containing the decaprenyl diphosphate synthase gene from Gluconobacter suboxydans

Coenzyme Q₁₀ (CoQ₁₀) is a quinine consisting of ten units of the isoprenoid side-chain. Because it limits the oxidative attack of free radicals to DNA and lipids, CoQ₁₀ has been used as an antioxidant for foods, cosmetics and pharmaceuticals. Decaprenyl diphosphate synthase (DPS) is the key enzyme for synthesis of the decaprenyl tail in CoQ₁₀ with isopentenyl diphosphate. The ddsA gene coding for DPS from Gluconobacter suboxydans was expressed under the control of an Escherichia coli constitutive promoter. Analysis of the cell extract in recombinant E. coli BL21/pACDdsA by high performance liquid chromatography and mass spectrometry showed that CoQ₁₀ rather than endogenous CoQ₈ was biologically synthesized as the major coenzyme Q. Expression of the ddsA gene with low copy number led to the accumulation of CoQ₁₀ to 0.97 mg l^–1 in batch fermentation. A high cell density (103 g l^–1) in fed-batch fermentation of E. coli BL21/pACDdsA increased the CoQ₁₀ concentration to 25.5 mg l ^–1 and its productivity to 0.67 mg l^–1 h^–1, which were 26.0 and 6.9 times higher than the corresponding values for batch fermentation.     

Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Sweet sorghum juice supplemented with 0.5% ammonium sulphate was used as a substrate for ethanol production by Saccharomyces cerevisiae TISTR 5048. In batch fermentation, kinetic parameters for ethanol production depended on initial cell and sugar concentrations. The optimum initial cell and sugar concentrations in the batch fermentation were 1 × 10⁸ cells ml⁻¹ and 24 °Bx respectively. At these conditions, ethanol concentration produced (P), yield (Y _ps) and productivity (Q _p ) were 100 g l⁻¹, 0.42 g g⁻¹ and 1.67 g l⁻¹ h⁻¹ respectively. In fed-batch fermentation, the optimum substrate feeding strategy for ethanol production at the initial sugar concentration of 24 °Bx was one-time substrate feeding, where P, Y _ps and Q _p were 120 g l⁻¹, 0.48 g g⁻¹ and 1.11 g l⁻¹ h⁻¹ respectively. These findings suggest that fed-batch fermentation improves the efficiency of ethanol production in terms of ethanol concentration and product yield.     

Glycerol inhibition of growth and dihydroxyacetone production byGluconobacter oxydans

The evidence, kinetic aspects, and modelization of the inhibitory effect of glycerol on dihydroxyacetone (DHA) production byGluconobacter oxydans have been studied. The comparison of the maximal productivities and specific rates evaluated for initial concentrations of 31, 51, 76, 95, and 129 g L^–1 of substrate showed that glycerol exerts an inhibitory effect both on growth and DHA production: decrease of the growth-specific rate and of the specific rate of DHA production with increase of the initial glycerol content. The inhibition phenomenon was attributed to an immediate effect of glycerol on the biological activity. It was also established that the presence of glycerol at high concentration induces an increase in the time necessary for the cells to reach their maximal level of specific rates. This result tends to show that glycerol brings into play on the biological system the capacity to reach its optimal range of activity. The main models found in the literature dealing with substrate inhibition phenomena were then tested on experimental data. The exponential model describes at best the glycerol inhibition on growth (=0.53e^(–S/93.6)) and on DHA production (q_P=7e^(–S/76.7)). The kinetic study and modelization of the inhibition effect of glycerol on DHA production allows one, therefore, to fill the gap in the fundamental knowledge of this industrial fermentation, to show the maladjustment of the classical fermentation process used (batch), and to reconsider the conception for the optimization of the production (proposition of more adapted process like fed-batch and/or biphasic systems).     

Modeling of <Emphasis Type="Italic">Fusarium redolens</Emphasis> Dzf2 mycelial growth kinetics and optimal fed-batch fermentation for beauvericin production

Beauvericin (BEA) is a cyclic hexadepsipeptide mycotoxin with notable phytotoxic and insecticidal activities. Fusarium redolens Dzf2 is a highly BEA-producing fungus isolated from a medicinal plant. The aim of the current study was to develop a simple and valid kinetic model for F. redolens Dzf2 mycelial growth and the optimal fed-batch operation for efficient BEA production. A modified Monod model with substrate (glucose) and product (BEA) inhibition was constructed based on the culture characteristics of F. redolens Dzf2 mycelia in a liquid medium. Model parameters were derived by simulation of the experimental data from batch culture. The model fitted closely with the experimental data over 20–50 g l⁻¹ glucose concentration range in batch fermentation. The kinetic model together with the stoichiometric relationships for biomass, substrate and product was applied to predict the optimal feeding scheme for fed-batch fermentation, leading to 54% higher BEA yield (299 mg l⁻¹) than in the batch culture (194 mg l⁻¹). The modified Monod model incorporating substrate and product inhibition was proven adequate for describing the growth kinetics of F. redolens Dzf2 mycelial culture at suitable but not excessive initial glucose levels in batch and fed-batch cultures.     

In-situ recovery of butanol during fermentation

End-product inhibition in the acetone-butanol fermentation was reduced by using extractive fermentation to continuously remove acetone and butanol from the fermentation broth. In situ removal of inhibitory products from Clostridium acetobutylicum resulted in increased reactor productivity; volumetric butanol productivity increased from 0.58 kg/(m³h) in batch fermentation to 1.5 kg/(m³h) in fed-batch extractive fermentation using oleyl alcohol as the extraction solvent. The use of fed-batch operation allowed glucose solutions of up to 500 kg/m³ to be fermented, resulting in a 3.5- to 5-fold decrease in waste water volume. Butanol reached a concentration of 30–35 kg/m³ in the oleyl alcohol extractant at the end of fermentation, a concentration that is 2–3 times higher than is possible in regular batch or fed-batch fermentation. Butanol productivities and glucose conversions in fed-batch extractive fermentation compare favorable with continuous fermentation and in situ product removal fermentations.List of Symbols C _g kg/m³ concentration of glucose in the feed - C _w dm³/m³ concentration of water in the feed - F(t) cm³/h flowrate of feed to the fermentor at time t - V(t) dm³ broth volume at time t - V _i dm³ initial broth volume - V _si dm³ volume of the i-th aqueous phase sample - effective fraction of water in the feed Part 1. Bioprocess Engineering 2 (1987) 1–12     

Bioconversion of acid-hydrolyzed poplar hemicellulose to acetic acid byClostridium thermoaceticum

Summary Clostridium thermoaceticum was used to ferment carbohydrate released from pretreated oat splet xylan and hemicellulose isolated from hybrid poplar. Hydrolysis with dilute sulfuric acid (2.5% (v/v) for oat spelt xylan and 4.0% (v/v) for poplar hemicellulose) at 100°C for 60 min was found to release the highest concentration of fermentable substrate.C. thermoaceticum, when grown in non-pH controlled batch culture at 55°C under a headspace of 100% CO₂, typically produced 14gl^–1 acetic acid during a 48 h fermentation in medium containing 2% xylose. In fed-batch fermentations this organism was able to produce 42gl^–1 acetic acid after 116h when the concentration of xylose was maintained at approximately 2% and the pH was controlled at 7.0.     

Kinetics of Bifidobacterium longum ATCC 15707 fermentations: effect of the dilution rate and carbon source

The effect of the dilution rate on biomass and product synthesis in fermentations of glucose, fructose and a commercial mixture of fructooligosaccharides (FOS) by Bifidobacterium longum ATCC 15707 was studied. Kinetic parameters (maximum specific growth rate, Monod constant, maintenance, and yield coefficients) in the mathematical model of the fermentation were estimated from experimental data. In the FOS mixture fermentations, approximately 12% of the total reducing sugars (mainly fructose) in the feed were not metabolized by the bacterium. In fermentations of fructose and the FOS mixture, biomass concentration increased as the dilution rate increased and, once maximum values were reached [3.90 (D=0.20 h^–1) and 2.54 g l^–1 (D=0.15 h^–1), respectively], decreased rapidly as the culture was washed out. Formic acid was detected at low dilution rates in glucose and fructose fermentations. The main products in fermentations of the three carbon sources were lactic and acetic acids. Average values of the molar ratio between acetic and lactic acids of 1.18, 1.21 and 0.83 mol mol^–1 were obtained in glucose, fructose and FOS mixture fermentations, respectively. In batch fermentations carried out without pH control this molar ratio was lower than 1.5 only when fructose was used as the carbon source.     

Batch and fed-batch fermentation of Bacillus thuringiensis using starch industry wastewater as fermentation substrate

Bacillus thuringiensis var. kurstaki biopesticide was produced in batch and fed-batch fermentation modes using starch industry wastewater as sole substrate. Fed-batch fermentation with two intermittent feeds (at 10 and 20 h) during the fermentation of 72 h gave the maximum delta-endotoxin concentration (1,672.6 mg/L) and entomotoxicity (Tx) (18.5 × 10⁶ SBU/mL) in fermented broth which were significantly higher than maximum delta-endotoxin concentration (511.0 mg/L) and Tx (15.8 × 10⁶ SBU/mL) obtained in batch process. However, fed-batch fermentation with three intermittent feeds (at 10, 20 and 34 h) of the fermentation resulted in the formation of asporogenous variant (Spo−) from 36 h to the end of fermentation (72 h) which resulted in a significant decrease in spore and delta-endotoxin concentration and finally the Tx value. Tx of suspended pellets (27.4 × 10⁶ SBU/mL) obtained in fed-batch fermentation with two feeds was the highest value as compared to other cases.     

Fed-batch versus batch cultures of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> for γ-decalactone production from methyl ricinoleate

Constant medium feeding rate and intermittent fed-batch fermentation strategies were investigated aiming to increase the yields of γ-decalactone production by Yarrowia lipolytica, using methyl ricinoleate as substrate and ricinoleic acid source. The accumulation of another compound, 3-hydroxy-γ-decalactone, was also analyzed since it derives from the direct precursor of γ-decalactone thereby providing information about the enzymatic activities of the pathway. Both strategies were compared with the traditional batch mode in terms of overall productivity and yield in respect to the substrate. Although the productivity of γ-decalactone was considerably higher in the batch mode (168 mg l⁻¹ h⁻¹), substrate conversion to lactone (73 mg γ-decalactone g⁻¹) was greater in the intermittent fed-batch giving 6.8 g γ-decalactone l⁻¹. This last strategy therefore has potential for γ-decalactone production at an industrial level.     

A repeated batch process for cultivation of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis>

A repeated batch process was performed to culture Bifidobacterium longum CCRC 14634. An on-line device, oxidation-reduction potential (ORP), was used to monitor cell growth and uptake of nutrients in the culture. The ORP of the culture medium decreased substantially during fermentation until nutrients were depleted. Six cycles of batch fermentation using ORP as a control parameter were successfully carried out. As soon as ORP remained constant or increased, three-quarters of the broth was removed, and the same volume of fresh medium was fed to the fermenter for a new cycle of cultivation. Average cell concentrations of 1.9×10⁹ and 3.4×10⁹ cfu ml^–1 for repeated batch fermentation in MRS (Lactobacilli MRS broth) and WY (containing whey hydrolyzates, yeast extract, l-cysteine) medium, respectively, were achieved. Cell mass productivities for batch, fed-batch and repeated batch fermentation using MRS medium were 0.51, 0.41, and 0.64 g l^–1 h^–1, respectively, and those for batch and repeated batch using WY medium were 0.76, 0.99 g l^–1 h^–1, respectively. The results indicate a possible industrial process to culture Bifidobacteria sp.     

Production of acetic acid by Acetogenium kivui

Summary The formation of acetic acid by the thermophilic nonsporeforming homoacetogenic bacterium Acetogenium kivui was studied under various conditions. In pH-controlled batch fermentation at pH 6.4 this bacterium was able to produce up to 625 mM of acetic acid from glucose within 50–60 h. The value of _max obtained was about 0.17 h^-1, the yield was about 2.55 mol of acetic acid per mol of glucose utilized. In continuous fermentation both substrate concentration and dilution rate (D) influenced the yield of acetate and the stationary concentration: a glucose concentration of 67 mM at D=0.09 h^-1 resulted in 2.82 mol acetate/mol glucose and 190 mM acetate at a production rate of 17.1 mM/1 h. When the dilution rate was increased the production rate reached a maximal value of 43.2 mM/1 h at D=0.32 h^-1. At a glucose concentration of 195 mM the dependence of yield upon dilution rate followed a similar pattern and an acetate concentration of 420 mM could be obtained. Enzymatic studies indicate that in A. kivui pyruvate ferredoxin-oxidoreductase and acetate kinase are inhibited at acetate concentrations higher than 800 mM. Based on these results a fed-batch fermentation was developed, which allowed to produce more than 700 mM acetic acid within 40–50 h.Dedicated to Prof. Dr. H. J. Rehm on the occasion of his 60th birthday     

Experimental bioenergetics ofSaccharomyces cerevisiae in respiration and fermentation

Aerobic growth of Saccharomyces cerevisiae on glucose was investigated, focusing on the heat evolution as it relates to biomass and ethanol synthesis. “Aerobic fermentation” and “aerobic respiration” were established respectively in the experimental system by performing batch and fed-batch experiments. “Balanced growth” batch cultivations were carried out with initial sugar concentrations ranging from 10 to 70 g/L, resulting in different degrees of catabolite repression. The fermentative heat generation was continuously monitored in addition to the key culture parameters such as ethanol production rate, CO₂ evolution rate, O₂ uptake rate, specific growth rate, and sugar consumption rate. The respective variations of the above quantities reflecting the variations in the catabolic activity of the culture were studied. This was done in order to evaluate the microbial regulatory system, the energetics of microbial growth including the rate of heat evolution and the distribution of organic substrate between respiration and fermentation. This study was supported by closing C, energy, and electron balances on the system. The comparison of the fractions of substrate energy evolved as heat (δ_h) with the fraction of available electrons transferred to oxygen (?_O2) indicated equal values of the two (0.46) in the aerobic respiration (fed-batch cultivation). However, the glucose effect in batch cultivations resulted in smaller ?_O2 than δ_h, while both values decreased in their absolute values. The evaluation of the heat energetic yield coefficients, together with the fraction of the available electrons transferred to O, contributed to the estimation of the extent of heat production through oxidative phosphorylation.     

Production of pig liver esterase in batch fermentation of E. coli Origami

The establishment of a fermentation process for the production of pig liver esterase (PLE) in high yields is necessary for industrial applications. In our previous studies, we reported the recombinant expression of PLE in Escherichia coli Origami™ (DE3) in shake flask. Only a coexpression with chaperones GroEL/ES allowed the production of soluble and active enzyme. The optimization of the cultivation conditions, such as temperature, inducer concentrations, or media compositions to increase enzyme yield in a fermentation process is described here. Using fed-batch fermentation cell densities up to OD = 50 were obtained, but almost no active enzyme was expressed. Only batch fermentation was found suitable for production of active pig liver esterase and cell densities between OD = 7–13 and activities of 300–400 U L⁻¹ for isoenzyme PLE-1 (γPLE) and 1,400 U L⁻¹ for PLE-5 were obtained after 22 h total cultivation time or 18 h after induction of PLE expression, respectively.     

Methane fermentation of xylan by mesophilic and thermophilic methane sludges

The degradation of xylan during methane fermentation proceeded as a first-order reaction. The rate constants were calculated to be 0.40–0.09 day^–1 at 37° C and 0.341 day^–1 at 55° C. From calculations based on the experimental data, K _A and C _A values in the expression of the velocity of xylose consumption changed as the fermentation progressed. In the mesophilic fermentation, the degradation of xylan slowed down after 2 days of incubation, but the rate of consumption of xylose increased between days 3 and 4 of incubation and slow again at the 5th day of incubation. In the thermophilic fermentation, the degradation of xylan proceeded at a constant rate and the rate of consumption of xylose increased slightly on the 3rd day of incubation. When the velocity of gas evolution was determined, the C _G value for acetate at 55° C was about 1.8 times larger than the value at 37° C.     

Switching the mode of sucrose utilization by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H⁺ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures.