The tubular loop fermentor: Oxygen transfer,growth kinetics and design

Oxygen transfer measurements using a dynamic method and evaluated with an appropriate mathematical model have been made on a tubular loop bioreactor. Correlations of the type used in tank systems are used to describe the influence of power and aeration rate on the mass transfer coefficient. Yeast cultures grown on hydrocarbon and glucose substrates show growth characteristics similar to conventional tank results. Model considerations for large-scale tubular fermentors allow for the prediction of the steady-state oxygen profiles and maximum reactor length. Combination with two-phase flow and oxygen transfer correlations yields a design procedure for commercial scale tubular loop fermentors.     

Crossflow filtration of Saccharomyces cerevisiae using an unsteady jet

This paper deals with the influence of a new flow unsteadiness on the permeate flux in crossflow filtration of microbial suspension during fermentation. A pneumatically controlled valve generates intermittent jets from the main flow, leading to the formation of large vortices moving downstream along the tubular membrane. The unsteadiness does not affect the cell behaviour during fermentation and the resulting permeate flux is found twice higher than for usual filtration process.     

Design of a novel automated methanol feed system for pilot-scale fermentation of Pichia pastoris

Large-scale fermentation of Pichia pastoris requires a large volume of methanol feed during the induction phase. However, a large volume of methanol feed is difficult to use in the processing suite because of the inconvenience of constant monitoring, manual manipulation steps, and fire and explosion hazards. To optimize and improve safety of the methanol feed process, a novel automated methanol feed system has been designed and implemented for industrial fermentation of P. pastoris. Details of the design of the methanol feed system are described. The main goals of the design were to automate the methanol feed process and to minimize the hazardous risks associated with storing and handling large quantities of methanol in the processing area. The methanol feed system is composed of two main components: a bulk feed (BF) system and up to three portable process feed (PF) systems. The BF system automatically delivers methanol from a central location to the portable PF system. The PF system provides precise flow control of linear, step, or exponential feed of methanol to the fermenter. Pilot-scale fermentations with linear and exponential methanol feeds were conducted using two Mut(+) (methanol utilization plus) strains, one expressing a recombinant therapeutic protein and the other a monoclonal antibody. Results show that the methanol feed system is accurate, safe, and efficient. The feed rates for both linear and exponential feed methods were within ± 5% of the set points, and the total amount of methanol fed was within 1% of the targeted volume.     

Feedback-mediated dynamics in a model of coupled nephrons with compliant thick ascending limbs

The tubuloglomerular feedback (TGF) system in the kidney, a key regulator of glomerular filtration rate, has been shown in physiologic experiments in rats to mediate oscillations in thick ascending limb (TAL) tubular fluid pressure, flow, and NaCl concentration. In spontaneously hypertensive rats, TGF-mediated flow oscillations may be highly irregular. We conducted a bifurcation analysis of a mathematical model of nephrons that are coupled through their TGF systems; the TALs of these nephrons are assumed to have compliant tubular walls. A characteristic equation was derived for a model of two coupled nephrons. Analysis of that characteristic equation has revealed a number of parameter regions having the potential for differing stable dynamic states. Numerical solutions of the full equations for two model nephrons exhibit a variety of behaviors in these regions. Also, model results suggest that the stability of the TGF system is reduced by the compliance of TAL walls and by internephron coupling; as a result, the likelihood of the emergence of sustained oscillations in tubular fluid pressure and flow is increased. Based on information provided by the characteristic equation, we identified parameters with which the model predicts irregular tubular flow oscillations that exhibit a degree of complexity that may help explain the emergence of irregular oscillations in spontaneously hypertensive rats.     

串联填充管式反应系统中高浓度乙醇连续发酵

在一套由搅拌罐和管式反应器串联而成的组合式反应系统中,利用酿酒酵母进行连续发酵生产高浓度乙醇。后续管式反应器内通过装填聚氨酯颗粒和木块对酵母细胞进行吸附固定化,在乙醇抑制造成细胞活性大幅降低的情况下,通过大幅提高细胞浓度保证发酵效率,在稀释速率0.02h^-1和280g/L葡萄糖的条件下,系统的终点乙醇浓度为15.4 % (v/v)。研究表明在一定稀释速率之下,应该通过增加反应器的级数来降低稀释速率,以达到提高终点乙醇浓度,如简单地降低进料速率则可能增加整个系统所受的乙醇抑制,对提高终点乙醇浓度效果不显著。     

Design of scraped tubular fermentors

Conventional stirred-tank fermentors are inefficient in carrying out certain fermentation processes because of one or more of the following constraints: media backmix–flow, solids wall–deposits, microbial growth–disruption. Two series of novel design of aerated scraped tubular fermentors have been developed to over come the deleterious effects of these constraints. One design is based on a horizontal tube fitted with an internal mechanical wall-scraper that also promotes media segregation; the other design is based on a vertical array of vessels interconnected by small gas-jetting orifices that promote media segregation and clean-surface operation. Tests with cultures of Trichdorma viride (for single-cell protein production) and Candida lipolytica (for lipase production) have been carried out. It is shown that these novel fermentors can minimize the effects of catabolite repression inherent in both cultures and of wall growth in the former.     

Semicontinuous diffusion fermentation of fodder beets for fuel ethanol and cubed protein feed production

A novel, semicontinuous diffusion fermentation system was used to produce fuel ethanol and a cubed protein feed (CPF) from fodder beets at an intermediate scale. In the process, fodder beet cubes were augered diagonally upward against a flow of 0.26N H(2)SO(4) and yeast in a tubular fermentor. Exiting one end of the fermentor was CPF, while fermented beer [6-9% (v/v) ethanol] exited the other end. Retention times for beer and CPF were 264 and 72 h, respectively. Contamination was controlled by maintaining the fermentation pH between 2.1 and 2.6 using H(2)SO(4). Production costs for a greatly scaled-up (times 1400) conceptual version of this system (using a continuous rather than a semicontinuous processing mode) were projected by calculation to be $0.529/L for 95% ethanol (net of a $0.112/L credit for CPF). The calculated energy balance (energy output-energy input ratio) was estimated to be 3.04. In calculating the energy balance, the output energy of the CPF and input energy for growing the fodder beets were not included. A design for the scaled-up plant is provided.     

Rheometry of fermentation liquids

This article is concerned with the measurements of rheological properties of nonhomogeneous fermentation liquids. In order to determine the flow curves of such liquids, an impeller measuring system of own design was used, and the method of calibration of the system is presented. The experimental verification was carried out using samples of fermentation broth of Aureobasidium pullulans and it has been shown that the applied measuring technique gives internally consistent results. The sensitivity of the technique proves its applicability for monitoring and control purposes in fermentation technology.     

Aqueous two-phase: the system of choice for extractive fermentation

Extractive fermentation in aqueous two-phase systems is a meaningful approach to overcome low product yield in a conventional fermentation process, and by proper design of the two-phase system it is possible to obtain the product in a cell-free stream. The characteristics of an aqueous two-phase system, various polymers used for forming an aqueous two-phase system, the physicochemical parameters of the aqueous two-phase system, partitioning of biomolecules and cell mass and the effect of individual phase forming polymers on cell growth and product formation are reviewed in this article. The various extractive fermentation processes are also summarised here. At the end, the economic viability and scope of aqueous two-phase fermentation are briefly discussed in relation to the wider application of this topic. Received: 16 June 1999 / Received revision: 29 December 1999 / Accepted: 4 January 2000     

An evaluation of a ceramic monolith as an enzyme support material

Catalase was immobilized on commercially available monolithic catalyst supports and also on participate support obtained by crushing the monolith. The kinetics of the monolith- and particulate-supported enzymes were analyzed in a continuous tubular reactor system and pressure drop was also monitored. Analysis of the results indicates that the monolith-supported system presents very little resistance to flow which results in a considerably smaller pressure drop than is obtained in flow through particulate-supported systems under comparable conversion conditions. Ceramic monoliths thus appear to be very suitable for use as enzyme supports in continuous tubular reactor applications, particularly where high pressure drops might be expected.     

On-line monitoring of monoclonal antibody formation in high density perfusion culture using FIA

An automated flow injection system for on-line analysis of proteins in real fermentation fluids was developed by combining the principles of stopped-flow, merging zones flow injection analysis (FIA) with antigen-antibody reactions. IgG in the sample reacted with its corresponding antibody (a-IgG) in the reagent solution. Formation of insoluble immunocomplexes resulted in an increase of the turbidity which was determined photometrically. This system was used to monitor monoclonal antibody production in high cell density perfusion culture of hybridoma cells. Perfusion was performed with a newly developed static filtration unit equipped with hydrophilic microporous tubular membranes. Different sampling devices were tested to obtain a cell-free sample stream for on-line product anlysis of high molecular weight (e.g., monoclonal antibodies) and low molecular weight (e.g., glucose, lactate) medium components. In fermentation fluids a good correlation (coefficient: 0.996) between the FIA method and an ELISA test was demonstrated. In a high density perfusion cultivation process mAb formation was succesfully monitored on-line over a period of 400 h using a reliable sampling system. Glucose and lactate were measured over the same period of time using a commercially available automatic analyser based on immobilized enzyme technology.Abbreviations TIA Turbidimetric immunoassay - mAb Monoclonal Antibody     

Clean-in-place systems for industrial bioreactors: Design,validation and operation

Summary Guidelines for design, validation and operation of clean-in-place systems for industrial fermentation plant are presented. Design of vessels, surface finishes, materials of construction, types and locations of valves are some of the considerations addressed. Requisite levels of turbulence for cleaning of pipes and vessels are discussed as well as typical cleaning sequences. Recommendations for validation of cleaning are presented and the significance of design of cleaning systems in ensuring satisfactory validation is pointed out. To the extent possible, validation of cleaning should be carried out with real process soil or soil closely simulating actual fermentation broths.     

Review: Ethanol production at elevated temperatures and alcohol concentrations: Part I – Yeasts in general

There are a number of process advantages which could be exploited through the use of thermophilic microorganisms for ethanol production. Energy savings through reduced cooling costs, higher saccharification and fermentation rates, continuous ethanol removal and reduced contamination have stimulated a search for routes to thermophilic or thermotolerant yeasts. These routes have included screening existing culture collections, temperature adaptation, mutagenesis and molecular techniques and finally isolating new strains. Varying success has been achieved, however, the most thermotolerant yeasts have come from fresh isolations from environments which experience high temperatures. Thermotolerant yeasts have been investigated for the following potential applications: simultaneous saccharification and fermentation of cellulose, where the high fermentation temperature allows more rapid and efficient enzymatic cellulose hydrolysis; whey fermentation, where high salt and low fermentable substrate concentrations make conditions difficult; and fermentation of D-xylose and cellobiose, which is essential for efficient conversion of woody biomass to ethanol. Ethanol and temperature tolerance are important characteristics for commercial yeast strains. Both characteristics are interactive and generally decrease with increasing temperature and ethanol concentration. Considerable research has been directed towards investigation of fatty acid composition changes in response to these stresses and the role of heat shock proteins in tolerance mechanisms. If thermotolerant yeasts are to be used in commercial processes, bioreactor configuration will play an important part in the design of production processes. Batch and fed-batch systems have been shown to be useful in some circumstances as have continuous flow systems, however, some of the newly isolated thermotolerant yeasts such as Kluyveromyces marxianus do not show the high growth rate under anaerobic conditions that is characteristic of Saccharomyces cerevisiae. Various immobilization techniques appear to offer a means of presenting and maintaining high biomass in anaerobic continuous flow reactors.     

Photobioreactor design: Mixing, carbon utilization, and oxygen accumulation

Photobioreactor design and operation are discussed in terms of mixing, carbon utilization, and the accumulation of photosynthetically produced oxygen. The open raceway pond is the primary type of reactor considered; however small diameter (1-5 cm) horizontal glass tubular reactors are compared to ponds in several respects. These are representative of the diversity in photobioreactor design: low capital cost, open systems and high capital cost, closed systems. Two 100-m(2) raceways were operated to provide input data and to validate analytical results. With a planktonic Chlorella sp., no significant difference in productivity was noted between one pond mixed at 30 cm/s and another mixed from 1 to 30 cm/s. Thus, power consumption or CO(2) outgassing limits maximal mixing velocities. Mixing power inputs measured in 100-m(2) ponds agreed fairly well with those calculated by the use of Manning's equation. A typically configured tubular reactor flowing full (1 cm diameter, 30 cm/s) consumes 10 times as much energy as a typical pond (20 cm deep flowing at 20 cm/s). Tubular reactors that flow only partially full would be limited by large hydraulic head losses to very short sections (as little as 2 m length at 30 cm/s flow) or very low flow velocities. Open ponds have greater CO(2) storage capacity than tubular reactors because of their greater culture volume per square meter (100-300 L/m(2) vs. 8-40 L/m(2) for 1-5-cm tubes). However, after recarbonation, open ponds tend to desorb CO(2) to the atmosphere. Thus ponds must be operated at higher pH and lower alkalinity than would be possible with tubular reactors if cost of carbon is a constraint. The mass transfer coefficient, K(L), for CO(2) release through the surface of a 100-m(2) pond was determined to be 0.10 m/h. Oxygen buildup would be a serious problem with any enclosed reactor, especially small-diameter tubes. At maximal rates of photosynthesis, a 1-cm tubular reactor would accumulate 8-10 mg O(2)/L/min. This may result in concentrations of oxygen reaching 100 mg/L, even with very frequent gas exchange. In an open pond, dissolved oxygen rises much more slowly as a consequence of the much greater volume per unit surface area and the outgassing of oxygen to the atmosphere. The maximum concentration of dissolved oxygen is typically 25-40 mg/L. The major advantage of enclosed reactors lies in the potential for aseptic operation, a product value which justifies the expense. For most products of algal mass cultivation, open ponds are the only feasible photobioreactor design capable of meeting the economic and operating requirements of such systems, provided desirable species can be maintained.     

Gaseous concentration gradients in tray type solid state fermentors — Effect on yields and productivities

In aerobic solid state fermentation systems, interaction of mass transfer effects with bioreaction plays an important role on the yields and productivities of the bioreactors. Experimental observations on the oxygen and carbon dioxide concentration gradients in a tray type solid state fermentation system are reported in this paper. Steep gradients are experienced in deep beds making large portions of the bioreactor ineffective. The results are useful in the design of the bioreactor in terms of efficient mass transfer as well as critical thickness of the substrate bed to be used.     

Immobilized electric eel acetylcholinesterasemii. II. Flow kinetics of acetylcholinesterase chemically attached to nylon tubing.

Acetylcholinesterase has been attached covalently to the inner surface of nylon tubing. An experimental study has been carried out on the flow kinetics; solutions of acetylthiocholine at various concentrations were passed through tubing at various flow rates, and measurements made of the rates of formation of product. The results were analyzed in the light of the theoretical treatment of Kobayashi and Laidler, four different methods of analysis being employed. It is found that at lower substrate concentrations and flow rates the reactions are largely diffusion controlled. The Km(app) values are substantially higher than the Km value for diffusion-free conditions, but approach it as the flow rate is increased, when the diffusion layer becomes less important. The results are entirely consistent with the Kobayaski-Laidler theory, and provide guidelines for the design of open tubular heterogeneous enzyme reactors, both for industrial and analytic purposes.     

Parameter oscillation attenuation and mechanism exploration for continuous VHG ethanol fermentation

A bioreactor system composed of a stirred tank and three tubular bioreactors in series was established, and continuous ethanol fermentation was carried out using a general Saccharomyces cerevisiae strain and a very high gravity medium containing 280 g L(-1) glucose, supplemented with 5 g L(-1) yeast extract and 3 g L(-1) peptone. Sustainable oscillations of glucose, ethanol, and biomass were observed when the tank was operated at the dilution rate of 0.027 h(-1), which significantly affected ethanol fermentation performance of the system. After the tubular bioreactors were packed with 1/2' Intalox ceramic saddles, the oscillations were attenuated and quasi-steady states were achieved. Residence time distributions were studied for the packed bioreactors by the step input response technique using xylose as a tracer, which was added into the medium at a concentration of 20 g L(-1), indicating that the backmixing alleviation assumed for the packed tubular bioreactors could not be established, and its contribution to the oscillation attenuation could not be verified. Furthermore, the role of the packing's yeast cell immobilization in the oscillation attenuation was investigated by packing the tubular bioreactors with packings with significant difference in yeast cell immobilization effects, and the experimental results revealed that only the Intalox ceramic saddles and wood chips with moderate yeast cell immobilization effects could attenuate the oscillations, and correspondingly, improved the ethanol fermentation performance of the system, while the porous polyurethane particles with good yeast cell immobilization effect could not. And the viability analysis for the immobilized yeast cells illustrated that the extremely lower yeast cell viability within the tubular bioreactors packed with the porous polyurethane particles could be the reason for their inefficiency, while the yeast cells loosely immobilized onto the surfaces of the Intalox ceramic saddles and wood chips could be renewed during the fermentation, guaranteeing their viability and making them more efficient in attenuating the oscillations. The packing Raschig rings without yeast cell immobilization effect did not affect the oscillatory behavior of the tubular bioreactors, further supporting the role of the yeast cell immobilization in the oscillation attenuation.     

Data model for the elimination of matrix effects in enzyme-based flow-injection systems

This contribution presents a new conceptional enzyme-based flow injection analysis (FIA) system for the process and quality control of food processing and biotechnological systems. It provides the determination of different analytes in distinct process media on the base of a common experimental set-up. In contrast to known comparable systems, analysis is performed without the commonly used sample preparation and dilution steps. Instead, the adaptation to the necessary measurement range is realized by optimization of intrinsic system parameters. The central principle of the work presented is the elimination of occurring interferences by the heterogeneous matrix of the process sample. Based on a particular injection mode, the application of dehydrogenases as indicator enzymes and a specially developed data model using cognitive methods, cross sensitivities of the detector as well as disturbed reaction rates of the enzymes could be almost completely compensated. Two applications are presented, the analysis of ethanol in non-alcoholic beer and the online determination of D-/L-lactate during a lactic acid fermentation, which reveal the advantage of the developed system.     

Determination of division rates of rCHO cells in high density and immobilized fermentation systems by flow cytometry

As most high density and immobilized fermentation systems do not allow the direct quantitative determination of cell density, two flow cytometric methods (the determination of incorporation of bromodeoxyuridine into newly synthesized DNA and the increase in mitotic cells by colchicine blockage) were evaluated as to their suitability to measure true division rates of cells in bioreactors. The BrdU method gave division rates identical to the growth rates measured by cell count, while the colchicine block method gave values that were lower and varied with the cell line. This is due to the cytotoxicity of colchicine and makes a calibration of the method for each cell line necessary. Both methods have been successfully used to measure division rates of rCHO cells immobilized in an alginate matrix as well as in macroporous carriers in a fluidised bed system and in dialysis culture.