Scale-up of whey fermentation in a pilot-scale fermenter

Summary The scale-up of a whey fermentation byKluyveromyces fragilis was carried out in order to reproduce on a larger scale (100-l fermenter) the results obtained on a smaller scale (15-l fermenter).Using a standard procedure for inoculum development and medium pasteurization, the effects of mixing and lactose concentration on yeast growth, lactose consumption, COD reduction and dissolved oxygen have been studied.The most successful operation for this fermentation was found to be associated with high stirring rates and low lactose concentrations, since the process was controlled by both oxygen and lactose concentrations.     

Scale-up and operation of a pilot-scale ammonia fiber expansion reactor

Pretreatment and densification of agricultural residues at regional depots can simplify feedstock supply logistics for the production of biofuels in commercial biorefineries. We have previously reported the performance of a laboratory-scale (5 L) packed-bed ammonia fiber expansion (AFEX) reactor system, which showed significant promise for biomass pretreatment at distributed depots. In this paper, we describe the performance of a 90-fold larger pilot-scale packed-bed AFEX-reactor system, used to produce over 1,500 batches (~36 tons) of pretreated crop residues over a 5-year period. Virtually all unreacted ammonia was successfully removed from the biomass, and 76% of the ammonia was recycled and reused. Pretreatment performance at pilot scale was comparable to laboratory-scale, averaging 74% glucose and 75% xylose yield in a standard test compared with 71% and 73%, respectively. Other operating and maintenance aspects are also discussed.     

Development and application of a membrane cyclone reactor for in vivo NMR spectroscopy with high microbial cell densities

A new bioreactor system has been developed for in vivo NMR spectroscopy of microorganisms under defined physiological conditions. This cyclone reactor with an integrated NMR flow cell is continuously operated in the magnet of a 400-MHz wide-bore NMR spectrometer system. The residence times of medium and cells are decoupled by a circulation-integrated cross-flow microfiltration module to achieve higher cell densities as compared to continuous fermentations without cell retention (increase in cell density up to a factor of 10 in steady state). Volumetric mass transfer coefficients k(L)a of more than 1.0 s(-1) are possible in the membrane cyclone reactor, ensuring adequate oxygen supply [oxygen transfer rate >15,000 mg O(2) .(L h)(-1)] of high cell densities. With the aid of the membrane cyclone reactor we were able to show, using continuous in vivo (31)P NMR spectroscopy of anaerobic glucose fermentation by Zymomonas mobilis, that the NMR signal intensity was directly proportional to the cell concentration in the reactor. The concentration profiles of intracellular inorganic phosphate, NAD(H), NDP, NTP, UDP-sugar, a cyclic pyrophosphate, two sugar phosphate pools, and extracellular inorganic phosphate were recorded after a shift from one steady state to another. The intracellular cyclic pyrophosphate had not been detected before in in vitro measurements of Zymomonas mobilis extracts due to the high instability of this compound. Using continuous in vivo (13)C NMR spectroscopy of aerobic glucose utilization by Corynebacterium glutamicum at a density of 25 g(cell dry weight) . L(-1), the membrane cyclone reactor served to measure the different dynamics of labeling in the carbon atoms of L-lactate, L-glutamate, succinate, and L-lysine with a time resolution of 10 min after impressing a [1-(13)C]-glucose pulse.     

Scale-up of monoclonal antibody purification processes

Mammalian cell culture technology has improved so rapidly over the last few years that it is now commonplace to produce multi-kilogram quantities of therapeutic monoclonal antibodies in a single batch. Purification processes need to be scaled-up to match the improved upstream productivity. In this chapter key practical issues and approaches to the scale-up of monoclonal antibody purification processes are discussed. Specific purification operations are addressed including buffer preparation, chromatography column sizing, aggregate removal, filtration and volume handling with examples given.     

木聚糖酶发酵工艺在50L罐中的放大

自制的酵母水解液成功替代有机N源酵母浸膏被应用于木聚糖酶发酵,大大降低了原料成本。在此基础上,于50 L罐中进行发酵工艺放大,得到最佳发酵条件:搅拌转速220 r/min、空气流量23 L/min、初始pH 5.5、温度30℃、罐压0.04 MPa,最终发现产酶水平可达到2 864 U/mL,用箭叶圆盘涡轮搅拌桨代替上层平叶圆盘涡轮搅拌桨,产酶水平无显著变化,搅拌功率节约11%。     

Scale-up of purine nucleoside fermentation from a shaking flask to a stirred-tank fermentor

Scaling-up purine nucleoside fermentation by a mutant strain of Bacillus subtilis from a shaking flask to a stirred-tank fermentor was attempted. The dimensions and the operating conditions of the stirred tank were determined in order to satisfy the optimum conditions of O₂ transfer and power consumption per unit volume for the shaking flask. When the purine nucleoside fermentation was carried out in the stirred-tank fermentor under these conditions, in which the temperature simulated that in the shaking flask, the total amount of purine nucleosides produced was almost the same as that in the shaking flask, but the accumulation ratio of guanosine to total nucleotides was different from that in the flask. Since urea could not be utilized so efficiently in the stirred-tank fermentor, the NH^p+ _f4 concentration and the pH of the culture broth were lower than those in the shaking-flask culture during fermentation. The activity of inosine monosphosphate dehydrogenase and the accumulation ratio were significantly affected by the NH^p+ _f4 concentration. When the pH of the stirred-tank culture was maintained at 6.9 by ammonia water to keep the NH^p+ _f4 level higher, the ratio was improved to the same level as that observed in the shaking-flask culture. The fermentation heat calculated from the shaking-flask data and its pattern of change were similar to those in the stirred-tank fermentor. Correspondence to: Y. Sumino     

Scale-up synthesis of lipase-catalyzed palm esters in stirred-tank reactor

Lipase-catalyzed production of palm esters by alcoholysis of palm oil with oleyl alcohol in n-hexane was performed in 2L stirred-tank reactor (STR). Investigation on the performance of reactor operation was carried out in batch mode STR with single impeller mounted on the centrally located shaft. Rushton turbine (RT) impellers provide the highest reaction yield (95.8%) at lower agitation speed as compared to AL-hydrofoil (AL-H) and 2-bladed elephant ear (EE) impellers. Homogenous enzyme particles suspension was obtained at 250 rpm by using RT impeller. At higher impeller speed, the shear effect on the enzyme particles caused by agitation has decreased the reaction performance. Palm esters reaction mixture in STR follows Newtons' law due to the linear relation between the shear stress (tau) and shear rate (dupsilon/dy). High stability of Lipozyme RM IM was observed as shown by its ability to be repeatedly used to give high percentage yield (79%) of palm esters even after 15 cycles of reaction. The process was successfully scale-up to 75 L STR (50 L working volume) based on a constant impeller tip speed approach, which gave the yield of 97.2% after 5h reaction time.     

Dynamic modeling of immobilized cell reactor: application to ethanol fermentation

A mathematical model has been developed for the unsteady-state operation of an immobilized cell reactor. The substrate solution flows through a mixed-flow reactor in which cells immobilized in gel beads are retained. The substrate diffuses from the external surface of the gel beads to some internal location where reaction occurs. The product diffuses from the gel beads into liquid medium which flows out of the reactor. The model combines simultaneous diffusion and reaction, as well as cell growth, and it can predict how the rates of substrate consumption, product formation, and cell growth vary with time and with initial conditions. Ethanol fermentation was chosen as a representative reaction in the immobilized cell reactor, and numerical calculations were carried out. Excellent agreement was observed between model predictions and experimental data available in the literature.     

Scale-up of upstream and downstream operations for the production of glucosamine using microbial fermentation

Glucosamine is used to treat osteoarthritis or as a nutritional supplement. The synthesis, isolation, and purification of glucosamine play a crucial role in its industrial application. This work presents the production of glucosamine from microbial fermentation, and discusses the production problems at both the upstream and downstream operations when the fermentation process is scaled up. The cost evaluation of process design was used to analyze the feasibility of using microbial fermentation for the production of glucosamine. The calculated result shows that the cost of the production of glucosamine should be designed to be approximately between US$200 and 300/kg for the project to be viable.     

A near-infrarod spectroscopy technique for the control of fermentation processes: An application to lactic acid fermentation

A near-infrared (NIR) spectroscopy technique for the control of lactic acid fermentation process has been proposed. Lactic acid, glucose, and biomass concentrations were determined by the NIR spectroscopy method. The three parameters examined were closely correlated to the results obtained with classical laboratory procedures. Moreover, the conditions for the on-line utilization of the NIR spectroscopy measurement system were pointed out. The great versatility of the NIR spectroscopy should permit its use for other fermentation processes. (c) 1994 John Wiley & Sons, Inc.     

Scale-up of biopesticide production processes using wastewater sludge as a raw material

Studies were conducted on the production of Bacillus thuringiensis (Bt)-based biopesticides to ascertain the performance of the process in shake flasks, and in two geometrically similar fermentors (15 and 150 l) utilizing wastewater sludge as a raw material. The results showed that it was possible to achieve better oxygen transfer in the larger capacity fermentor. Viable cell counts increased by 38–55% in the bioreactor compared to shake flasks. As for spore counts, an increase of 25% was observed when changing from shake flask to fermentor experiments. Spore counts were unchanged in bench (15 l) and pilot scale (5.3–5.5 e⁺⁰⁸ cfu/ml; 150 l). An improvement of 30% in the entomotoxicity potential was obtained at pilot scale. Protease activity increased by two to four times at bench and pilot scale, respectively, compared to the maximum activity obtained in shake flasks. The maximum protease activity (4.1 IU/ml) was obtained in pilot scale due to better oxygen transfer. The Bt fermentation process using sludge as raw material was successfully scaled up and resulted in high productivity for toxin protein yield and a high protease activity.     

Continuous pullulan fermentation in a biofilm reactor

Biofilm is a natural form of cell immobilization in which microorganisms attach onto solid support. In this study, a pigment-reduced pullulan-producing strain, Aureobasidium pullulans (ATCC 201253), was used for continuous pullulan fermentation in a plastic composite support (PCS) biofilm reactor. Optimal conditions for the continuous pullulan production were determined by evaluating the effects of the feeding medium with various concentrations of ammonium sulfate and sucrose and dilution rate. Pullulan concentration and production rate reached maximum (8.3 g/l and 1.33 g/l/h) when 15 g/l of sucrose, 0.9 g/l of ammonium sulfate, and 0.4 g/l of yeast extract were applied in the medium, and the dilution rate was at 0.16 h⁻¹. The purity of produced pullulan was 93.0%. The ratio of hyphal cells of A. pullulans increased when it was grown on the PCS shaft. Overall, the increased pullulan productivity can be achieved through biomass retention by using PCS biofilm reactor.     

Self-cycling fermentation in a stirred tank reactor

Self-cycling fermentations (SCFs) were conducted in a stirred tank apparatus using Bacillus subtilis and Acinetobacter calcoaceticus. The systems were very stable and the experiments lasted through many cycles. The variation of parameters such as biomass and doubling time from cycle to cycle was small. The stirred tank reactor (STR) allowed a much better control of the working volume in the fermentor from cycle to cycle, compared to the cyclone column, and it was not necessary to make periodic corrections.The production of surfactin from B. subtilis was achieved without extending the cycle time. The harvested broth at the end of each cycle was allowed to remain in a secondary vessel, at ambient temperature, before being collected. It is exhaustion of the limiting nutrient which causes an increase in dissolved oxygen (DO). At this point, the computer, which constantly monitors the DO, triggered the harvesting sequence to end the cycle. Thus, the mature culture in the secondary vessel experienced appropriate conditions for the production of the secondary metabolite. Meanwhile, the next batch of cells was being grown in the primary reactor.The response of a gas analyzer on the effluent paralleled that of the DO measurements in the fermentor. These data for oxygen and carbon dioxide exhibited less noise than the DO readings. Either would be a more reliable parameter for feedback control of the SCF because the problem of fouling of the DO probe after extended runs of many cycles would be eliminated. (c) 1993 John Wiley & Sons, Inc.     

Software sensors for fermentation processes

Four software sensors based on standard on-line data from fermentation processes and simple mathematical models were used to monitor a number of state variables in Escherichia coli fed-batch processes: the biomass concentration, the specific growth rate, the oxygen transfer capacity of the bioreactor, and the new R _O/S sensor which is the ratio between oxygen and energy substrate consumption. The R _O/S variable grows continuously in a fed-batch culture with constant glucose feed, which reflects the increasing maintenance demand at declining specific growth rate. The R _O/S sensor also responded to rapid pH shift-downs reflecting the increasing demand for maintenance energy. It is suggested that this sensor may be used to monitor the extent of physiological stress that demands energy for survival.     

Scale-up of dialysis fermentation for high cell density cultivation of Escherichia coli.

In dialysis fermentations inhibiting metabolites can be removed from cell suspensions resulting in a prolonged exponential growth phase and higher production yields. Because of successful high cell density cultivations of Escherichia coli in a laboratory dialysis reactor, a scale-up of the process was investigated. To provide sufficient membrane area for dialysis in a technical scale fermenter, an external membrane module was used, that was also applied for oxygen supply to the culture in the external loop. Cultivations with recombinant E. coli K12, with and without induction, in 2- and 300-l reactors were carried out using external modules. Cell densities exceeding 190 g l(-1), previously obtained in laboratory dialysis fermentation, were also produced with external dialysis modules. Protein concentration in a 300-l reactor was increased to the 3.8-fold of industrial fed-batch-fermentations.     

Mass transfer characteristics of a fermentation broth in a reactor: co-current downflow contacting reactor

Gas holdups, dispersion height, volumetric mass transfer coefficients and k_La of water and a yeast fermentation broth were studied in a co-current downflow contacting reactor. k_La and gas holdup increased with increasing superficial gas velocity and there was a parallelism between k_La and gas holdup. The values of k_La gas holdup, and dispersion height measured in the air/fermentation broth system were all lower than those in air/water system.     

Scale-up of microbial processes: impacts, tools and open questions

Industrial biotechnological production is developing rapidly worldwide. Consequently, more and novel bioprocesses need to perform optimally not only in small lab- but also in large production scales. This article shortly reviews typical impacts found when cells are exposed to micro environmental heterogeneities typically occurring in poorly mixed large scale production reactors. The current state-of-the-art of tool development is presented for analyzing these phenomena. Finally, still open questions are formulated and needs for future research are outlined to further support the expansion of biotech industries by successful research results.     

A database system for fermentation processes

Data base management is needed in the whole industries, particularly in the fermentation industry, whose jobs are tedious yet require carefulness. The most important problem in the database system is not how to collect many informations, but how to handle the meaningful ones.The authors have recently developed an on-line monitoring and control system for the fermentation processes in co-operation with Fuji Facom Co. Ltd. and Komatsugawa Chemical Engineering Co. Ltd.This system enables us to measure directly those concentrations in fermentation systems which have been measured by offline so far, such as cell mass, substrate and metabolic products. The physiological activities of a microorganism, such as specific rate of cellular growth, that of substrate consumption, that of metabolites production, etc., became estimable precisely by eliminating the effect of noises.By enlarging the function of our monitoring and control system, we have developed a database system which is applicable in job scheduling not only in the laboratory but also in the production line, in automatic resource allocation and fault analyses of the fermentation processes.