Microbial film fermenters: Their present and future applications

The productivity of growth associated fermentations is dependent upon the quantity of biomass contained in the fermenter (microbial hold-up). In contrast, the performance characteristics of a fermenter are dominated by the form in which the microbial hold-up occurs, i.e., whether as films, flocs, or a combination of both. Industrial fermentations using biological films include biological waste-water treatment (trickling filter, rotating disc), the “quick” vinegar process, animal tissue culture, and bacterial leaching. Atkinson and Davies (Trans. Inst. Chem. Eng., 50 , 208 (1972)), proposed a completely mixed microbial film fermenter (CMMFF) based upon the fluidized bed principle, for application to continuous operation when using growth associated systems. The potential performance characteristics of this fermenter are reviewed and the construction of a laboratory scale unit capable ofaseptic operation is described (volume 9 liters, maximum throughput 15 liter/hr). The fermenter has been continuously used for periods up to 2500 hr without contamination when growing brewer's yeast (NCYC 1026 flocculating). The experimental performance characteristics have been interpreted with the aid of the theory for the CMMFF. The knowledge and experience gained to date is used as a basis for speculation as to the future role of microbial film fermenters in the biochemical process industries.     

Growth of Streptococcus faecalus in dense culture

A fermentation system was designed and constructed to study the growth characteristics of microorganisms at low and high cell concentrations. The technique used to develop high cell densities utilized a rotating microfiltration unit to permit the removal of cell-free product from the fermenter. The fermenter volume and the filter were contained in a single unit composed of a series of concentric cylinders. Annuli contained the fermenter volume while the second outermost cylinder supported a microfiltration membrane. Feed to the system was pumped at constant rates, and the internal pressure built up to a value, which would effect the required filtration rate. The system was operated batchwise and continuously with and without filtration. The anaerobie growth characteristics of Streptococcus faccalus were determined at 37°C and pH 7.0 for batch, continuous, and continuous with filtration modes of operation. The growth characteristics were unchanged when the cell density was increased. Changes in cell yield peer model of glucose consumed were clearly illustrated during thee course of single run by operating the fermenter in the unsteady state with filtration. No consumption of glucose for developed was 40% packed cell volume, a value 45 times larger than could be grown in simple batch culture.     

Physiological response ofCandida tropicalis grown on n-paraffin to mixing in a tubular closed loop fermenter

Summary A special tubular closed loop fermenter was used in order to simulate the particular mixing condition of a large scale recycle fermenter.Some mixing parameters of the system are characerized.During continuous cultivation ofCandida tropicalis on n-paraffin as a substrate the biomass yield with respect to carbon and oxygen increased, when a controlled oxygen limit was imposed on the culture.Mixing in the closed loop fermenter generates undamped short period oscillations in the respiration activity, in the dissolved oxygen tension and in the actual ATP content of the culture. These oscillations likely represent oscillations of allosteric feedback loops which manifest themselves by some synchronising action of the particular environmental transients in the closed loop fermenter.     

Continuous cultivation of rumen microorganisms,a system with possible application to the anaerobic degradation of lignocellulosic waste materials

Summary An in vitro continuous fermentation device is described which allows the maintenance of a mixed rumen microbial population under conditions similar to those in the rumen. The differences in flow rates of solids and liquids found in the rumen were established in vitro by means of a simple filter construction. A grass-grain mixture was used as a solid growth substrate. During a test period of 65 days the artificial rumen fermenter showed stable operation with respect to ciliate numbers, fibre degradation and volatile fatty acids production. Values obtained were comparable to those found in vivo. Optimal fibre degradation and volatile fatty acids production were maintained when hydraulic retention times (HRT) ranged from 11 to 14 h. At these HRT-values ciliate numbers were maintained at about 8.5×10⁴ cells per ml. Ciliate numbers declined drastically at HRT-values above 14h. A fermenter inoculated with a small volume of rumen fluid (1:100, v/v) reached normal protozoal numbers, fibre degradation and volatile fatty acids productions after a start up period of only 8 to 10 days. The possible application of rumen microorganisms for an efficient degradation of lignocellulosic waste material in an artificial rumen digester is discussed.     

Bioconversion of cellulose wastes to protein and sugar

A schematic representation of the variety of products which can be obtained by microbial conversion of cellulose is presented. Alkaline pre-treatment has been used after milling in all the experiments. Solka-floc or sugarcane bagasse was used as sources of cellulose. A cellulolytic strain of Aspergillus terreus (ATCC 30514) was cultivated in batch-, fed batch and continuous culture up to 7 liter stirred tank fermenter. The general growth characteristics were determined by growing on glucose. Results of experiments on the growth of A. terreus for production of biomass on Solka-floc or Sugarcane bagasse are given, also the ability of crude cellulases to produce sugar syrups by enzymatic hydrolysis of cellulose has been evaluated.     

The biofreezer,a new contained freezing equipment in biotechnology

Summary The Biofreezer has been designed for freezing biomass or cell-suspensions in the laboratory, pilot and large scale biotechnology under hygienic and biosafe conditions. The principle is based on the freezing of biomass drops on the surface of a liquid nitrogen bath to form small beads, which are harvested automatically. The application is presented as a part of the Roferon-A production (leukocyte interferon -2a Roche), representing a physically contained system extending from 1000 l fermenter through continuous centrifugation to the frozen biomass beads. The freezing process in the Biofreezer is extremely fast and complete and the beads can be thawed instantaneously. Comparisons with other freezing systems are described.     

Extractive fermentation-integrated reaction and product recovery

Summary Refinements have been made to a prototype process for the production of ethanol by extractive fermentation. The process is characterized by thein situ extraction of ethanol from a 7 L continuous stirred tank fermenter and the thermal recovery of ethanol from the extracting solvent, which is circulated in a closed loop through the process. Data are provided to show the efficacy and stability of the process under various operating conditions, and the near complete (>96%) continuous conversion of a 300 g/L glucose feed.     

Der Respirationsfermentor – ein Labormeßsystem zur Charakterisierung aerober mikrobiologischer Reaktionen

By use of a laboratory fermenter with a gastight circle aeration system all interesting values connected with the respiration of microorganisms can be determined. The advantage of the respiration fermenter is the simple control of the oxygen partial pressure with high accuracy. The aeration is carried out by inert gas-oxygen-mixtures. This system was used to avoid errors in oxygen-concentration measurement and regulation by oxygen sensitive sensors in fermentation fluids. For each selected oxygen partial pressure in the course of fermentation the following date in connection with all desired analytical values can be determined by manual or automatical means. Total oxygen consumption, momentary oxygen consumption rate, kinetics of oxygen consumption rate, kinetics of respiratory quotient. Several opportunities of measurement are presented and examples for application in various microbial systems are given.     

Laboratory glass fermenter on the basis of the airlift principle

A 1.5-1 minifermenter consisting of RASOTHERM glass on the basis of the airlift principle was constructed and tested in the Institute of Biotechnology. The development of this fermenter bridged the gap between shake flasks and lab-scale fermenters and provided a possibility for the continuous cultivation of extremely halophilic bacteria. It was demonstrated that the fermenter can be used for cultivating bacteria that are sensitive to shear stress. It can also be recommended for purposes of cultivation of plant and animal cells because of this advantage and the possibility of this fermenter to be sterilized.     

Fluidized-bed fermenters: A steady-state analysis

A new mathematical model is presented model is presented for use in the design and optimization of fluidized-bed fermenters. Unlike previous models, the biomass particle size is not a required input parameter, but is predicted as a consequence of the process by which the fermenter reaches a steady state. Both tower fermenters and supported-film bioreactors are included in the analysis. The differences between them are explained as a consequence of the different effects of added biomass on the particle settling velocity and the tendency of a fluidized bed to stratify. A detailed qualitative treatment of solids mixing allows the model to predict the varying biomass concentration through a tower fermenter and the more constant concentration in the supported film reactor. Other features of this analysis are the inclusion of an axial dispersion term to allow for different liquid mixing conditions, and the introduction of a variable transformation that eliminates the need for a computer solution. A sample design problem is included.     

Maximizing productivity of a continuous fermenter using nonlinear adaptive optimizing control

The control of a continuously operated fermenter at its maximum productivity level gives rise to a difficult control problem as the location of the optimum operating point changes due to the disturbances. In addition, the fermenter exhibits a change in the sign of the steady state gain near the optimum operating point. This study is aimed at developing an on-line optimizing control scheme that can track the changing location of the steady state optimum so as to maximize the fermenter productivity. A nonlinear Laguerre model, whose parameters are estimated on-line, is used for tracking the optimum operating point. The control at the optimum point is achieved using an adaptive nonlinear MPC strategy that uses the nonlinear Laguerre model for prediction. The efficiency of the proposed algorithm is demonstrated by simulating the control of a continuous fermenter that exhibits shift in the location of the optimum operating point in response to the changes in the maximum specific growth rate. The proposed on-line optimizing control strategy is shown to result in a considerable improvement in the closed loop performance even in the presence of measurement noise.     

Development of a two-phase combination fermenter for the conversion of cellulose to methane

Cellulose degradation to methane under continuous fermentation conditions was compared using fully mixed, fully mixed with solids return, sludge-blanket, and fixed-film fermenters. In fully mixed fermenters, a decrease in hydraulic retention time (HRT) of two weeks or less caused the wash out of anaerobes capable of converting volatile fatty acids to methane, while at increased feeding rates over 1 g/L day the rapid growth of cellulolytic anaerobes upset the balance between acid formation and its conversion to CH(4). Circulation of cellulose and difficulty in settling of cellulose with attached bacteria imposed problems in the use of other types of fermenters. On the basis of information obtained from this study, a fermenter which combined a fully mixed phase for cellulose degradation and a fixed-film phase with pre-immobilized bacteria for converting fatty acids to CH(4) in one vessel, was devised. Using this fermenter, a mixed culture converted cellulose to CH(4) at 4.8 g/L day at a HRT of six days as compared to 0.7 g/L day at a HRT of 28 days in the fully mixed fermenter.     

Low-pressure airlift fermenter for single cell protein production: II. Continuous culture of pichia yeast

Experiments using Pichia yeast grown on n-paraffins have been conducted in laboratory 10-L airlift fermenters and in a 640-L module of commercial scale. Results confirmed the design concept of combining oxygen transfer and fermenter cooling with low-pressure air. However, in the absence of mass transport constraints, the build up of toxic factors in the fermenter appeared to be a major variable limiting cell productivity. Foaming in the large fermenter also presented a serious problem, which must be solved before low-pressure airlift fermenters become practical.     

Multi-input multi-output control of a continuous fermenter using nonlinear model based controllers

Internal Model Control (IMC) and Model Predictive Control (MPC), the two most important members of model based controllers, are favourable alternatives for control of nonlinear processes. However, the performance of these controllers deteriorates drastically in the presence of substantial process-model mismatch. Hu and Rangaiah (1998) proposed feedback augmentation for nonlinear IMC (hence named Augmented IMC, AuIMC) for improving control in the presence of modelling errors, and demonstrated its success on a neutralization process. In the present study, IMC, MPC and AuIMC strategies are tested in a more difficult case of multi-input multi-output (MIMO) operation of a highly nonlinear continuous fermenter. A new control configuration is introduced as the conventional configuration is not applicable. Simulation results for different modelling errors show that IMC is better than MPC for fermenter control. The advantage of augmentation as in AuIMC manifests in the significantly improved regulatory control of the fermenter.     

Effect of multistream ethanol feeding on growth and physiological characteristics of Candida utilis in a multistage tower fermenter

The effect of using a multistream feed for carbon and energy supply on the growth and physiological activity of the yeast Candida utilis in a multistage tower fermenter has been studied. Measurements were made at steady states of continuous culture for single values of dilution rate, temperature and pH in all stages of the fermenter and with the same total ethanol supplied. A comparison of the results obtained with multistream and single-stream ethanol feeds revealed that the type of ethanol feed influences the cell growth rate, rate of ethanol dissimilation, biomass yield, productivity and the cell physiology in the individual stages of the fermenter. Multistream ethanol feeding eliminates the growth inhibition due to insufficient energy production from ethanol oxidation at higher partial pressure of oxygen in the aeration gas. Using the optimal type of ethanol feed, better process parameters for SCP production are achieved.     

The physiology of lactate production by Lactobacillus delbreuckii in a chemostat with cell recycle

The physiology of lactate production by Lactobacillus delbreuckii NRRL B-445 in a continuous fermenter with partial cell recycle has been studied and compared with that observed in a conventional chemostat. Partial cell recycle was achieved using a hollow-fiber ultrafiltration cartridge. The biomass growth yield was reduced in the recycle fermenter while culture viability and the cellular content of polysaccharide, protein, carbon, and nitrogen remained constant, suggesting an enlarged specific rate of glucose consumption for nonanabolic (e.g., maintenance) functions. The volumetric productivity of lactate was enhanced in the recycle fermenter due to the complete utilization of glucose. The yield of lactate from biomass and the molar product ratio, lactate: ethanol plus acetate, decreased with increasing recycle ratio. Enhanced formation of ethanol and acetate occurred in the recycle fermenter although lactate remained the major product. The change in product profile was due to glucose limitation. The specific activity of lactate dehydrogenase remained constant during recycle fermentation. These physiological observations have implications for the future application of cell recycle to production processes.     

Efficient cellulase production by Trichoderma reesei in continuous cultivation on lactose medium with a computer-controlled feeding strategy

A low-foaming hydrophobin II deletant of the Trichoderma reesei strain Rut-C30 was used for production of cellulases by continuous cultivation on lactose medium in a laboratory fermenter. The control paradigm of the addition of new medium to the continuous process was based on the growth dynamics of the fungus. A decrease in the rate of base addition to the cultivation for pH-minimum control was used as an indicator of imminent exhaustion of carbon source for growth and enzyme induction. When the amount of base added per 5 min computation cycle decreased below a given value, new medium was added to the fermenter. When base addition for pH control thereafter increased above the criterion value, due to increased growth, the medium feed was discontinued or decreased. The medium feeding protocol employed was successful in locking the fungus in the stage of imminent, but not actual, exhaustion of carbon source. According to the results of a batch cultivation of the same strain on the same medium, this is the phase of maximal enzyme productivity. The medium addition protocol used in this work resulted in a very stable continuous process, in which cellulase productivity was maintained for several hundred hours at the maximum level observed in a batch cultivation for only about 10 h. Despite a major technical disturbance after about 420 h, the process was restored to stability. When the cultivation was terminated after 650 h, the level of enzyme production was still maximal, with no signs of instability of the process.     

Rheologie von Fermentationslösungen

Rheological measurements can give interesting informations for the characterization of fermentation broth, especially concerning the depending of the oxygen transfer rate. Rheological measurements can report decisions for choosing the best reactor type or other process steps e.g. purification and separation. An advantageous method was used by the combination of a dynamic process-viscosimeter with the fermenter and the continuous measurement of medium density by X-ray absorption method.     

Control of fermenters – a review

Fermenter control has been an active area of research and has attracted more attention in recent years. This is due to the new developments in other related areas which can be exploited to overcome the inherent difficulties in fermenter control. Beginning with conventional regulatory control of operating variables such as temperature, pH and dissolved oxygen concentration, research in fermenter control has undergone significant changes including the recent neural network based approaches. The objective of the paper is to focus the attention of the researchers to the developments in the control of batch, fed-batch and continuous fermenters over the past few years.     

Induced flocculation of yeasts for use in the tower fermenter

Summary It has been demonstrated that it is possible to induce flocculation in a yeast normally classified as non-flocculent. Further, it has been possible to accumulate and retain a very high cell density of this yeast in an upflow floc (tower) fermenter and to rapidly and efficiently convert glucose to ethanol. The method outlined for inducing flocculation appears to greatly broaden the range of organisms that might be considered for use in the tower fermenter, thus greatly enhancing this simple mode of continuous operation.