The rotorfermentor. II. Application to ethanol fermentation.

The kinetics of microbial growth and product formation are described as applied to the high cell concentration scheme of the rotorfermentor. A bench scale pilot plant was designed and built in order to demonstrate the operational feasibility of the rotorfermentor. The fermentation of glucose to ethanol by Saccharomyces cerevisiae ATCC 4126 was used. When the rotorfermentor was used with a glucose feed concentration of 104 g/liter almost 100% glucose utilization was obtained and the ethanol productivity rate was 27.3 g ethanol/liter hr which was found to be about 10 times greater than the ethanol productivity obtained from an ordinary continuous stirred tank (CST) fermentor. The ethanol experimental results obtained from the rotorfermentor and an ordinary CST fermentor were used as a basis to assess the economic feasibility of the rotorfermentor. The economics of an industrial scale ordinary CST fermentor with and without cell recycle is compared with a rotorfermentor unit for the same ethanol production throughput. For the process conditions considered in this case, calculations showed that the rotorfermentor may replace both a CST fermentor and cell centrifuge resulting in lower capital equipment costs and lower power consumption requirements.     

Studies on continuous ethanol fermentation of sugar cane molasses

Summary A new laboratory system for continuous fermentation is described. It is well suited for fermenting concentrated substrates such as moderately dilute molasses. A rotating microporous filter, which is annexed to the fermentor vessel, allows the free escape of metabolic products while retaining yeast in the fermentor.The slop is recirculated after removal of ethanol by distillation leading to a build-up of non-fermentables. The concentration of these and of yeast cells is checked by a controlled bleed. The described system is a useful tool for small-scale experiments on continuous ethanol fermentation.     

Mathematical model of microporous hollow-fiber membrane extractive fermentor

A mathematical model is presented for a microporous hollow-fiber membrane extractive fermentor (HFEF). The model is based on the continuous flow of the aqueous nutrient phase and cells through the shell space of the fermentor where the fermentation reaction occurs. The product diffuses from the shell space through the hollow-fiber membrane where it is continuously removed by solvent flowing concurrently through the fiber lumen. Results for ethanol production show that the HFEF has a volumetric productivity significantly higher than that possible using conventional methods. The model predicts the existence of an optimum volume fraction of hollow fibers in the fermentor that maximizes the total volumetric productivity. This optimum is the result of a classic trade-off between the volume fraction of the fermentor required for fermentation and that required for efficient removal of the ethanol product to minimize 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.     

A simple capillary viscosity meter for the on-line measurement and control of the biomass concentration in high-density cultures

A capillary viscosity meter was used for the on-line determination of the biomass concentration in a fermentation broth. At high cell densities the viscosity of the broth increases, which can be measured as the pressure drop over a capillary. Calibration aspects of this viscosity meter are presented, and the use of the device for the control of the biomass concentration in a membrane recycle fermentor is demonstrated.     

Optimum operating mode for a class of fermentation

This paper is concerned with optimization of the operating mode of a fermentor. Combining the various modes of operation—batch, semibatch, and continuous—the operating pattern which maximizes the desired metabolic product in a single fermentor is determined by using Kelley's transformation method with Pontryagin's maximum principle. Kelley's transformation method is a device which avoids the singular situation which occurs when the usual procedure of selecting the optimal control function by the maximum principle breaks down. This is the case in the problem considered in this paper. For lysine fermentation, the best operating mode depends on the fermentor capacity and operating time. The results of this study are summarized thus: (i) when the operating time is “long enough,” optimal conditions require that continuous operation follows either semibatch and/or batch operation, and (ii) when the fermentor capacity becomes “large enough,” semibatch operation becomes important.     

Cultivation of a filamentous mold in a glass pilot-scale airlift fermentor

Results of pilot plant studies using a glass airlift fermentation device (55 liter fermentation volume) have proven the relative merits of such a system in the fermentation of a filamentous mold, Monascus purpureus, on 4% (w/w) starch media. The resultant overall yield of cell mass (Y_x/s) of 0.38 was an appreciable increase over the 0.32 obtained with a pilot scale stirred tank fermentor previously studied. Power requirements of the airlift fermentor were approximately 50% of those for the mechanically agitated system. The lack of mechanical shear in the airlift system provides a more gentle environment or the cultivation of organisms than does the high degree of shear prevalent in the mechanically agitated vessels. Mass transfer of oxygen to the aqueous phase of the fermentation volume is improved significantly through use of the airlift device. Mass transfer coefficients in the range of 200 reciprocal hr were obtained to approximately 80 reciprocal hr in the stirred tank fermentor.     

Theoretical analysis of the effect of cell recycling on recombinant cell fermentation processes.

A cell recycle system is studied for two-stage continuous fermentation. Cell recycle around the second stage provides higher cell concentrations than processes without recycle and a longer residence time of the cell, which is necessary for inducible products, especially in recombinant cell fermentation. Residence time distribution of the cell in the fermentor is important for the optimization of inducible products. The residence time distributions are studied for the cases with and without significant cell growth in the second stage. With cell growth in the second stage, three cases are considered. These are the cases of (1) zero residence time for two daughter cells after the cell division, (2) zero residence time of one daughter cell after the cell division and inherited residence time for the other daughter cell from the mother cell after the cell division, and (3) two daughter cells having the residence time of the mother cell after the cell division.     

Rotating drum fermentor for plant cell suspension cultures

A rotating drum fermentor designed for plant cell suspension cultures was constructed and tested. The oxygen transfer coefficient (k(L)a) and power requirements in the fermentor were determined with the water system under various conditions and the relationship between them in the fermentor was clarified. Also, the relationship between k(L)a and the apparent viscosity in the fermentor was investigated in the cell suspension system. The rotating drum fermentor was found to be superior to the mechanically agitated fermentor in the capacity of oxygen supply under high viscosity and low hydrodynamic stress conditions. This finding was also confirmed by the experiments with plant cell suspension cultures.     

Dialysis Continuous Process for Ammonium-Lactate Fermentation of Whey: Experimental Tests

Laboratory experiments were conducted to validate theoretical predictions describing a dialysis continuous process for the fermentation of whey lactose to ammonium lactate, in which the fermentor contents are poised at a constant pH by adding ammonia solution and dialyzed through a membrane against water. Dried sweet-cheese whey was rehydrated to contain 230 mg of lactose per ml, supplemented with 8 mg of yeast extract per ml, charged into a 5-liter fermentor without sterilization, adjusted in pH (5.3) and temperature (44°C), and inoculated with Lactobacillus bulgaricus. The fermentor and dialysate circuits were connected, and steady-state conditions were established. A series of such conditions was managed nonaseptically for 94 days to study the process and to demonstrate efficiency and productivity. As time progressed, the fermentation remained homofermentative and increased in conversion efficiency, although membrane fouling necessitated dialyzer cleaning about every 4 weeks. With a retention time of 19 h, 97% of the substrate was converted into products. Relative to nondialysis continuous or batch processes for the fermentation, the dialysis continuous process enabled the use of more concentrated substrate, was more efficient in the rate of substrate conversion, and additionally produced a second effluent of less concentrated but purer ammonium lactate.     

Process engineering for a membrane recycle fermentor

Membrane recycle fermentors are used successfully on laboratory scale to increase the efficiency of fermentation processes. The design of a process on larger scale however is obstructed by the lack of relevant data in literature. Compared to a stand-alone fermentor a membrane recycle fermentor presents new features which must be considered in the design. These features include the use of high density cultures, the additional volume in the membrane section and the circulation of the broth. In this theoretical study these aspects are analyzed with the characteristic time concept, in case of an ethanol fermentation integrated with microfiltration. The analysis shows that depending on the reactor configuration used concentration gradients can be expected. These gradients may decrease the efficiency of the fermentation, or can be advantageous, for example by letting the substrate conversion approach completion in the membrane section.     

Dialysis Continuous Process for Ammonium-Lactate Fermentation: Improved Mathematical Model and Use of Deproteinized Whey

Separate terms for substrate limitation and product inhibition were incorporated into an equation describing the rate of cell growth for the steady-state fermentation of lactose to lactic acid with neutralization to a constant pH by ammonia. The equation was incorporated into a generalized mathematical model of a dialysis continuous process for the fermentation, developed previously, in which the substrate is fed into the fermentor and the fermentor contents are dialyzed through a membrane against water. The improved model was used to simulate the fermentation on a digital computer, and the results agreed with previous experimental tests using whole whey as the substrate. Further simulations were then made to guide experimental tests using deproteinized whey as the substrate. Dried cheese-whey ultrafiltrate was rehydrated with tap water to contain 242 mg of lactose per ml, supplemented with 8 mg of yeast extract per ml, charged into a 5-liter fermentor without sterilization, adjusted in pH (5.5) and temperature (44°C), and inoculated with an adapted culture of Lactobacillus bulgaricus. The fermentor and dialysate circuits were connected, and a series of steady-state conditions was managed nonaseptically for 71 days. The fermentation of deproteinized whey relative to whole whey, with both highly concentrated, resulted in similar extents of product accumulation but at a lesser rate.     

Ethanol production in a microporous hollow-fiber-based extractive fermentor with immobilized yeast

Microporous-membrane-based extractive product recovery in product-inhibited fermentations allows in situ recovery of inhibitory products in a nondispersive fashion. A tubular bioreactor with continuous strands of hydrophobic microporous hollow fibers having extracting solvent flowing in fiber lumen was utilized for yeast fermentation of glucose to ethanol. Yeast was effectively immobilized on the shell side in small lengths of chopped microporous hyrophilic hollow fibers. The beneficial effects of in situ dispersion-free solvent ex (oleyl alcohol and dibutyl phthalate) were demonstrated for a 300 g/L glucose substrate feed. Outlet glucose concentration dropped drastically from 123 to 41 g/L as solvent/ substrate flow ratio was increased from 0 to 3 at 9 mL/h of substrate flow rate with oleyl alcohol as extracting solvent. The significant productivity increase with in situ solvent extraction became more evident as solvent/ substrate flow ratio increased. A model of the locally integrated extractive bioreactor describes the observed fermentor performance quite well.     

Hollow fiber supported gas membrane for in situ removal of ammonium during an antibiotic fermentation

To study the influence of ammonium on an antibiotic cultivation, mass transfer measurements of ammonium through microporous hydrophobic membranes using different stripping methods were carried out and compared. The higher overall mass transfer coefficients for ammonium were obtained with an acid stripping solution compared to water, vacuum, or sweeping air. A hollow fiber module for in situ removal of ammonium during cultivation was designed and operated in an external bypass to a 10-L fermentor. Compared to a control fermentation, the cell dry mass could be increased 2.6 times and the antibiotic concentration 8 times, if the in situ ammonium removal was in operation.     

Ethanol production from starch in a pervaporation membrane bioreactor using Clostridium thermohydrosulfuricum

To attain both high productivity and efficient recovery of ethanol from broth, a membrane bioreactor consisting of a jar fermentor and a pervaporation system was applied to the direct production of ethanol from uncooked starch with a thermophilic anaerobic bacterium, Clostridium thermohydrosulfuricum. From four types of ethanol-selective membranes tested, microporous polytetrafluoroethylene (PTFE) membrane, the pores of which are impregnated with silicone rubber, was chosen for its large flux, high ethanol selectivity, and high stability. During fed-batch fermentation with pervaporation in the membrane bioreactor, ethanol was continuously extracted and concentrated in two traps with concentrations at 5.6%-6.2% (w/w) in trap 1 (20 degrees C) and 27%-32% (w/w) in trap 2 (liquid N(2)), while the ethanol concentration in the broth was maintained at 0.85-0.9% (w/w). Due to the low ethanol concentration in the broth, and the immobilization of bacterial cells by the membrane, the number of viable cells, and, eventually, the ethanol productivity, increased in the membrane bioreactor.     

基因工程菌生产重组人组织因子的发酵研究

根据表达重组人组织因子基因工程菌的自身特点,在30L发酵罐上,通过控制发酵pH、葡萄糖浓度、诱导物磷酸浓度、搅拌速度及采用分批补料等方法,对重组人组织因子基因工程菌高密度发酵和高效表达的条件进行了研究。实验结果表明:诱导物终浓度低于0.1mmol/L,菌体密度OD60014,发酵周期10.5h,基因工程菌批发酵平均产量为37.41g/L,重组人组织因子表达量为6.56 mg/L。关键词:基因工程菌;高密度发酵;组织因子     

Production of rosmarinic acid from perfusion culture ofAnchusa officinalis in a membrane-aerated bioreactor

Summary In this study, a perfusion fermentation ofAnchusa officinalis was carried out in a stirred tank bioreactor integrated with an internal cross-flow filter. Bubble-free aeration via microporous membrane fibers was used to provide oxygen. A two-stage culture was successfully conducted in this reactor without filter fouling. In a 17 day fermentation, a cell density of 26 g dw/I and a rosmarinic acid productivity of 94 mg/l day were achieved. This productivity is three times that obtained in a batch culture.     

In vivo NMR system for evaluating oxygen-dependent metabolic status in microbial culture

To optimize an appropriate microbial culture in a fermentor, precise control of the medium's dissolved oxygen tension (DOT) is crucial. In particular, to study the effect of DOT on cellular metabolic status by using in vivo nuclear magnetic resonance (NMR) measurements, the set-up of the experiment must be optimized to maintain DOT in the culture. In the conventional method, DOT is monitored by a sensor inside a fermentor and is controlled by changing the agitation rate. Here, we report a novel and accurate system that minimizes time lag by an automated aeration flow control device, allowing an NMR spectrometer to monitor representative metabolites in real-time. To fulfill these two objects, the fermentor was composed of a fermentation vessel and two outer tubes, through which the medium was circulated by rotary pumps. One tube monitored DOT in via a sensor, and at the same time the other tube monitored metabolites via an NMR spectrometer.In this study, we used this system to analyze the responses of Escherichia coli cells under various oxygen conditions. The results validated the use of this system in the study of microbial metabolism.     

Studies on on-line bioreactor identification. IV. Utilization of pH measurements for product estimation

Relationships between the total rate of biomass growth and the rate of ammonia addition to a fermentor for pH control are presented. These equations make use of the concept of reaction invariants and provide the additional information needed for bioreactor identification. They are especially useful when the RQ measurement is not sufficient for this purpose, such as when sensitivities arise with the measured values of the respiratory quotient or when fermentation products are formed. The cases of batch, fed-batch and continuous fermentations, forming products with or without acidic/basic properties are considered. The derived relationships were successfully tested with nonbiological acid-base continuous flow reaction systems and subsequently applied to the identification of the continuous yeast fermentation of glucose to ethanol. Results of these experimental studies are also presented.     

Monitoring the aroma production during wine-must fermentation with an electronic nose.

This work discusses the feasibility of using the electronic nose for the on-line and real-time monitoring of the production of a complex aroma profile during a bioconversion process. As a case study, the formation of the muscatel aroma during the wine-must fermentation was selected. During wine-must fermentation, aroma compounds responsible for the organoleptic character are produced in the ppm range, while simultaneously one of the main metabolic products, ethanol, is produced in much higher quantities (up to 10% wt). Because the sensors of the electronic nose array are cross-selective to different volatile compounds, it was investigated in detail how far the electronic nose was able to evaluate the aroma profile along the fermentation. This article discusses and evaluates subsequently the integration of a membrane separation process-organophilic pervaporation-for selectively enriching aroma compounds relative to ethanol, to improve sample discrimination.