External and internal glucose mass transfers in succinic acid fermentation with stirred bed of immobilized Actinobacillus succinogenes under substrate and product inhibitions

This paper is dedicated to the study on the external and internal mass transfers of glucose for succinic acid fermentation under substrate and product inhibitions using a bioreactor with stirred bed of immobilized Actinobacillus succinogenes cells. By means of the substrate mass balance for a single particle of biocatalysts, considering the kinetic model adapted for both inhibitory effects, specific mathematical models were developed for describing the profiles of the substrate concentration in the outer and inner regions of biocatalysts and for estimating the substrate mass flows in the liquid boundary layer surrounding the particle and inside the particle. The values of the mass flows were significantly influenced by the internal diffusion velocity and rate of the biochemical reaction of substrate consumption. These cumulated influences led to the appearance of a biological inactive region near the particle center, its magnitude varying from 0 to 5.3% of the overall volume of particles.     

Ethanol fermentation in a magnetically fluidized bed reactor with immobilized Saccharomyces cerevisiae in magnetic particles

Ethanol fermentation by immobilized Saccharomyces cerevisiae cells in magnetic particles was successfully carried out in a magnetically stabilized fluidized bed reactor (MSFBR). These immobilized magnetic particles solidified in a 2 % CaCl(2) solution were stable and had high ethanol fermentation activity. The performance of ethanol fermentation of glucose in the MSFBR was affected by initial particle loading rate, feed sugar concentration and dilution rate. The ethanol theoretical yield, productivity and concentration reached 95.3%, 26.7 g/L h and 66 g/L, respectively, at a particle loading rate of 41% and a feed dilution rate of 0.4 h(-1) with a glucose concentration of 150 g/L when the magnetic field intensity was kept in the range of 85-120 Oe. In order to use this developed MSFBR system for ethanol production from cheap raw materials, cane molasses was used as the main fermentation substrate for continuous ethanol fermentation with the immobilized S. cerevisiae cells in the reactor system. Molasses gave comparative ethanol productivity in comparison with glucose in the MSFBR, and the higher ethanol production was observed in the MSFBR than in a fluidized bed reactor (FBR) without a magnetic field.     

Modeling of heat and mass transfer for solid state fermentation process in tray bioreactor

A mathematical model is developed to simulate oxygen consumption, heat generation and cell growth in solid state fermentation (SSF). The fungal growth on the solid substrate particles results in the increase of the cell film thickness around the particles. The model incorporates this increase in the biofilm size which leads to decrease in the porosity of the substrate bed and diffusivity of oxygen in the bed. The model also takes into account the effect of steric hindrance limitations in SSF. The growth of cells around single particle and resulting expansion of biofilm around the particle is analyzed for simplified zero and first order oxygen consumption kinetics. Under conditions of zero order kinetics, the model predicts upper limit on cell density. The model simulations for packed bed of solid particles in tray bioreactor show distinct limitations on growth due to simultaneous heat and mass transport phenomena accompanying solid state fermentation process. The extent of limitation due to heat and/or mass transport phenomena is analyzed during different stages of fermentation. It is expected that the model will lead to better understanding of the transport processes in SSF, and therefore, will assist in optimal design of bioreactors for SSF.     

Succinic acid production with metabolically engineered <Emphasis Type="Italic">E. coli</Emphasis> recovered from two-stage fermentation

Escherichia coli AFP111 cells recovered from spent two-stage fermentation broth were investigated for additional production of succinic acid under anaerobic conditions. Recovered cells produced succinic acid in an aqueous environment with no nutrient supplementation except for glucose and MgCO₃. In addition, initial glucose concentration and cell density had a significant influence on succinic acid mass yield and productivity. Although the final concentration of succinic acid from recovered cells was lower than from two-stage fermentation, an average succinic acid mass yield of 0.85 g/g was achieved with an average productivity of 1.81 g/l h after three rounds of recycling, which was comparable to two-stage fermentation. These results suggested that recovered cells might be reused for the efficient production of succinic acid.     

Succinic acid production with reduced by-product formation in the fermentation of Anaerobiospirillum succiniciproducens using glycerol as a carbon source

Succinic acid was produced by fermentation of Anaerobiospirillum succiniciproducens using glycerol as a carbon source. When cells were anaerobically cultured in a medium containing 6.5 g/L glycerol, a high succinic acid yield (133%) was obtained while avoiding the formation of by-product acetic acid. The gram ratio of succinic acid to acetic acid was 25.8:1, which is 6.5 times higher than that obtained using glucose (ca. 4:1) as a carbon source. Therefore, succinic acid can be produced with much less by-product formation by using glycerol as a carbon source, which will facilitate its purification. When glucose and glycerol were cofermented with the increasing ratio of glucose to glycerol, the gram ratio of succinic acid to acetic acid and succinic acid yield decreased, suggesting that glucose enhanced acetic acid formation irrespective of the presence of glycerol. Glycerol consumption by A. succiniciproducens required unidentified nutritional components present in yeast extract. By intermittently feeding yeast extract along with glycerol, a high succinic acid yield (160%) could be obtained while still avoiding acetic acid formation. This resulted in the highest ratio of succinic acid to acetic acid (31.7:1).     

Propionic acid fermentation by Propionibacterium acidipropionici: effect of growth substrate

Summary Batch propionic acid fermentations by Propionibacterium acidipropionici with lactose, glucose, and lactate as the carbon source were studied. In addition to propionic acid, acetic acid, succinic acid and CO₂ were also formed from lactose or glucose. However, succinic acid was not produced in a significant amount when lactate was the growth substrate. Compared to fermentations with lactose or glucose at the same pH, lactate gave a higher propionic acid yield, lower cell yield, and lower specific growth rate. The specific fermentation or propionic acid production rate from lactate was, however, higher than that from lactose. Since about equimolar acid products would be formed from lactate, the reactor pH remained relatively unchanged throughout the fermentation and would be easier to control when lactate was the growth substrate. Therefore, lactate would be a preferred substrate over lactose and glucose for propionic acid production using continuous, immobilized cell bioreactors. Correspondence to: S. T. Yang     

Immobilized cells of recombinant Escherichia coli strain for continuous production of L-aspartic acid

For L-aspartic acid biosynthesis, high production cells of Escherichia coli mutant B-715 and P1 were immobilized in chitosan gel using a technique developed in our laboratory. The immobilization process reduced initial activity of the intact cells, however, the biocatalyst produced was very stabile for long-term use in multi-repeated batch or continuous processes. Temperature influence on the conversion of ammonium fumarate to L-aspartic acid was investigated. In long-term experiments, over 603 hours, the temperature 40 degrees C was found to be the best for both biocatalyst stability and high conversion rate. The optimum substrate concentration was 1.0 M. Continuous production of L-aspartic acid was investigated in three types of column bioreactors characterized by different volumes as well as different high to biocatalyst bed volume rations (Hz/Vz). The highest conversion rate, 99.8%, and the productivity 6 g/g/h (mass of L-aspartic acid per dry mass of cells in biocatalyst per time unit) was achieved in the bioreactor with the highest value Hz/Vz = 3.1, and liquid hour space velocity value of 5.2, defined as the volume of feeding substrate passed per volume of catalyst in bioreactor per one hour.     

Countercurrent multistage fluidized bed reactor for immobilized biocatalysts: II. Operation of a laboratory-scale reactor

In Part I of this series,(1) we derived a model and made simulations for a multistage fluidized bed reactor (MFBR). It was concluded that the MFBR can be an attractive alternative for a fixed bed reactor when operated with a deactivating biocatalyst. In Part II of this series, the design of a laboratory-scale MFBR and its evaluation to investigate the practical feasibility of this reactor type, will be described. Experiments with a duration as long as 10 days were carried out successfully using immobilized glucose isomerase as a model reaction system. The results predicted by the model are in good agreement with the measured glucose concentration and biocatalyst activity gradients, indicating perfect mixing of the particles in the reactor compartments.The diameters of the biocatalyst particles used in the experiments showed a large spread, with the largest being 1.7 times the smallest. Therefore, an additional check was carried out, to make sure that the particles were not segregating according to size. Particles withdrawn from the reactor compartments were investigated using an image analyzer. Histograms of particle size distribution do not indicate segregation and it is concluded that the particles used have been mixed completely within the compartments. As a result, transport of biocatalyst is nearly plug flow.     

Lactic acid fermentation in a recycle batch reactor using immobilized Lactobacillus casei

Lactic acid production by recycle batch fermentation using immobilized cells of Lactobacillus casei subsp. rhamnosus was studied. The culture medium was composed of whey treated with an endoprotease, and supplemented with 2.5 g/L of yeast extract and 0.18 mM Mn(2+) ions. The fermentation set-up comprised of a column packed with polyethyleneimine-coated foam glass particles, Pora-bact A, and connected with recirculation to a stirred tank reactor vessel for pH control. The immobilization of L. casei was performed simply by circulating the culture medium inoculated with the organism over the beads. At this stage, a long lag period preceded the cell growth and lactic acid production. Subsequently, for recycle batch fermentations using the immobilized cells, the reducing sugar concentration of the medium was increased to 100 g/L by addition of glucose. The lactic acid production started immediately after onset of fermentation and the average reactor productivity during repeated cycles was about 4.3 to 4.6 g/L . h, with complete substrate utilization and more than 90% product yield. Sugar consumption and lactate yield were maintained at the same level with increase in medium volume up to at least 10 times that of the immobilized biocatalyst. The liberation of significant amounts of cells into the medium limited the number of fermentation cycles possible in a recycle batch mode. Use of lower yeast extract concentration reduced the amount of suspended biomass without significant change in productivity, thereby also increasing the number of fermentation cycles, and even maintained the D-lactate amount at low levels. The product was recovered from the clarified and decolorized broth by ion-exchange adsorption. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:841-853, 1997.     

利用固定化米根霉在三相流化床中发酵生成L-乳酸

用聚氨酯泡沫吸附固定米根霉菌丝,在三相流化床中对葡萄糖、木糖以及木糖渣的纤维素酶解液等不同碳源进行Ｌ－乳酸发酵研究,并对游离菌丝和固定化菌丝发酵Ｌ－乳酸进行了比较。结果表明,聚氨酯泡沫是米根霉的良好载体,具有经济、高效等特点。实验条件下,不同碳源的乳酸转化率分别为：葡萄糖,８２．５％;木糖,５３．８％;木糖渣酶水解液,７１．９％。三相流化床中固定化米根霉产酸速率（对葡萄糖）为１９．１ｇ．ｈ＾－１．     

Salting-out extraction and crystallization of succinic acid from fermentation broths

In this study, salting-out extraction (SOE) and crystallization were combined to recover succinic acid from fermentation broths. Of the different SOE systems investigated, the system consisting of organic solvents and acidic salts appeared to be more favorable. A system using acetone and ammonium sulfate was investigated to determine the effect of phase composition and pH. The highest partition coefficient (8.64) and yield of succinic acid (90.05%) were obtained by a system composed of 30% (w/w) acetone and 20% (w/w) ammonium sulfate at a pH of 3.0. Additionally, 99.03% of cells, 90.82% of soluble proteins, and 94.89% of glucose could be simultaneously removed from the fermentation broths. Interestingly, nearly 40% of the pigment was removed using the single-step salting-out extraction process. The analysis of the effect of pH on salting-out extraction indicates that a pH lower than the pK of succinic acid is beneficial for the recovery of succinic acid in an SOE system. Crystallization was performed for the purification of succinic acid at 4 °C and pH 2.0. By combining salting-out extraction with crystallization, an identical total yield (65%) and a higher purity (97%) of succinic acid were obtained using a synthetic fermentation broth compared with the actual fermentation broth (65% and 91%, respectively).     

Countercurrent multistage fluidized bed reactor for immobilized biocatalysts: I. Modeling and simulation

For the application of immobilized enzymes, fixed bed reactors are used almost exclusively. Fixed bed reactors have specific disadvantages, especially for processes with a deactivating catalyst. Therefore, we have studied a novel reactor type with continuous transport of the immobilized biocatalyst. Flow of biocatalyst is countercurrent to the substrate solution. Because of a stagewise reactor design, back-mixing of biocatalyst is very limited and transport is nearly plug flow. The reactor operates at a constant flow rate and conversion, due to constant holdup of catalytic activity. The reactor performance is compared with a configuration of fixed bed reactors. For reactions in the first-order regime, enzyme requirements in this new reactor are slightly less than for fixed bed processes. The multistage fluidized bed appears to be an attractive reactor design to use biocatalyst to a low residual activity. However, nonuniformity of the particles might affect plug flow transport of the biocatalyst. A laboratory scale reactor and experiments are described in Part II(1) of this series. Hydrodynamic design aspects of a multistage fluidized bed are discussed in more detail in Part III.(2).     

Indirect estimation of cell mass and substrate concentration using a computer-coupled mass spectrometer

On-line estimation of cell mass and substrate concentration based on exhaust gas analysis was developed. The O₂, CO₂, H₂O, and N₂ contents at the inlet and outlet of fermentor, analyzed by a computer-coupled quadrupole mass spectrometer, were used to calculate the oxygen uptake rate and carbon dioxide evolution rate, and these rates were further used to evaluate cell mass and substrate concentration in a recombinant Escherichia coli fermentation. Cell mass, glucose concentration, specific growth rate, and specific consumption rate of glucose were well estimated by this method; the oxygen uptake rate gave more accurate estimates for these state variables than did the carbon dioxide evolution rate.     

Adaptive evolution for fast growth on glucose and the effects on the regulation of glucose transport system in Clostridium tyrobutyricum

Laboratory adaptive evolution of microorganisms offers the possibility of relating acquired mutations to increased fitness of the organism under the conditions used. By combining a fibrous-bed bioreactor, we successfully developed a simple and valuable adaptive evolution strategy in repeated-batch fermentation mode with high initial substrate concentration and evolved Clostridium tyrobutyricum mutant with significantly improved butyric acid volumetric productivity up to 2.25 g/(L h), which is the highest value in batch fermentation reported so far. Further experiments were conducted to pay attention to glucose transport system in consideration of the high glucose consumption rate resulted from evolution. Complete characterization and comparison of the glucose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) were carried out in the form of toluene-treated cells and cell-free extracts derived from both C. tyrobutyricum wide-type and mutant, while an alternative glucose transport route that requires glucokinase was confirmed by the phenomena of resistance to the glucose analogue 2-deoxyglucose and ATP-dependent glucose phosphorylation. Our results suggest that C. tyrobutyricum mutant is defective in PTS activity and compensates for this defect with enhanced glucokinase activity, resulting in the efficient uptake and consumption of glucose during the whole metabolism.     

Modeling of batch fermentation kinetics for succinic acid production by Mannheimia succiniciproducens

Kinetic models are proposed for the batch production of succinic acid from glucose by Mannheimia succiniciproducens MBEL55E. The models include terms accounting for both substrate and product inhibitions. Experimental data collected from a series of batch fermentations with different initial glucose concentrations were used to estimate parameters and also to validate the models proposed. The optimal values of the parameters were approximated by minimizing the discrepancy between the model predictions and corresponding experimental data. The growth of M. succiniciproducens could be expressed by a modified Monod model incorporating inhibitions of glucose and organic acids accumulated in the culture broth. The Luedeking–Piret model was able to describe the formation of organic acids as the fermentation proceeded, in which succinic, acetic, and formic acids followed a mixed-growth-associated pattern. However, unexpectedly, lactic acid fermentation by M. succiniciproducens was nearly nongrowth-associated. In all cases, the model simulation matched well with the experimental observations, which made it possible to elucidate the fermentation characteristics of M. succiniciproducens during efficient succinic acid production from glucose. These models thus can be employed for the development and optimization of biobased succinic acid production processes.     

基于pH的反馈补料方法在谷氨酸发酵中的应用

为简化谷氨酸发酵补料工艺,提出了一种新型的基于pH的补料方式。考察谷氨酸发酵过程中氨消耗量 (x) 和糖消耗量 (y) 发现,两者之间存在较好的线性关系 (y=7.4744x,R2=0.9989),以此为pH反馈补料工艺中补料液中葡萄糖与氨的混合比例,能较好地将谷氨酸发酵过程中葡萄糖浓度稳定在12~21 g/L。比较恒定葡萄糖浓度补料工艺与pH反馈补料工艺发现,采用pH反馈补料工艺进行发酵,葡萄糖转化率、谷氨酸产酸速率分别提高了9.06％和17.5％左右,同时发酵周期缩短2 h以上。     

Continuos IBE fermentation by immobilized growing Clostridium beijerinckii cells in a stirred-tank fermentor

The potential of continuous isopropanol-butanol-ethanol (IBE) fermentation by Ca-alginate-immobilized Clostridium beijerinckii cells in a continuous stirred-tank reactor is investigated. A mathematical model is presented to describe steady-state reactor performance. It appeared to be possible to use the biocatalyst particles repeatedly for successive fermentations (at least three times for a total duration of two months). Reactor productivity was 6-16 times higher than that of a batch fermentation (free cells), while the solvents yield was also increased. Measurements of substrate, product, and biomass concentrations were only partially in agreement with the model; however, a solid basis for further technological developement of the process has been laid.     

Kinetic study of the conversion of different substrates to lactic acid using Lactobacillus bulgaricus

Lactic acid fermentation includes several reactions in association with the microorganism growth. A kinetic study was performed of the conversion of multiple substrates to lactic acid using Lactobacillus bulgaricus. Batch experiments were performed to study the effect of different substrates (lactose, glucose, and galactose) on the overall bioreaction rate. During the first hours of fermentation, glucose and galactose accumulated in the medium and the rate of hydrolysis of lactose to glucose and galactose was faster than the convesion of these substrates. Once the microorganism built the necessary enzymes for the substrate conversion to lactic acid, the conversion rate was higher for glucose than for galactose. The inoculum preparation was performed in such a way that healthy young cells were obtained. By using this inoculum, shorter fermentation times with very little lag phase were observed. The consumption patterns of the different substrates converted to lactic acid were studied to determine which substrate controls the overall reaction for lactic acid production. A mathematical model (unstructured Monod type) was developed to describe microorganism growth and lactic acid production. A good fit with a simple equation was obtained. It was found experimentally that the approximate ratio of cell to substrate was 1 to 10, the growth yield coefficient (Y(XS)) was 0.10 g cell/g substrate, the product yield (Y(PS)) was 0.90 g lactic acid/g substrate, and the alpha parameter in the Luedeking-Piret equation was 9. The Monod kinetic parameters were obtained. The saturation constant (K(S)) was 3.36 g/L, and the specific growth rate (microm ) was 1.14 l/h.     

An engineering Escherichia coli mutant with high succinic acid production in the defined medium obtained by the atmospheric and room temperature plasma

Escherichia coli BA002, the ldhA and pflB deletion strain, cannot utilize glucose in nutrient-rich or minimal media anaerobically. Co-expression of heterologous pyruvate carboxylase and nicotinic acid phosphoribosyltransferase in BA002 resulted in a significant increase in cell mass and succinic acid production. Nevertheless, the resultant strain, BA016, still could not grow in a defined medium without tryptone and yeast extract. To solve the problem, a novel atmospheric and room temperature plasma mutation method was employed to generate mutants which can grow in the defined medium. A mutant designated as LL016 was observed to be the best strain that regained the capacity of cell growth and glucose utilization in a defined medium anaerobically. After 120 h of fermentation in the defined medium, 35.0 g/L of glucose was consumed to generate 25.2 g/L of succinic acid. Furthermore, with the highest glucose consumption rate in the dual-phase fermentation, the yield of succinic acid in LL016 reached 0.87 g/g, which was higher than that observed in other strains. From an industrial standpoint, the defined medium is much cheaper than LB medium, which shows a great potential usage for the economical production of succinic acid by LL016.