A novel membrane-integrated fermentation reactor system: application to pyruvic acid production in continuous culture by Torulopsis glabrata

This paper describes the performance of a novel bio-reactor system, the membrane-integrated fermentation reactor (MFR), for efficient continuous fermentation. The MFR, equipped with an autoclavable polyvinylidene difluoride membrane, has normally been used for biological wastewater treatment. The productivity of the MFR system, applied to the continuous production of pyruvic acid by the yeast Torulopsis glabrata, was remarkably high. The volumetric productivity of pyruvic acid increased up to 4.2 g/l/h, about four times higher than that of batch fermentation. Moreover, the membrane was able to filter fermentation broth for more than 300 h without fouling even though the cell density of the fermentation broth reached 600 as OD₆₆₀. Transmembrane pressure, used as an indicator of membrane fouling, remained below 5 kPa throughout the continuous fermentation. These results clearly indicate that the MFR system is a simple and highly efficient system that is applicable to the fermentative production of a range of biochemicals.     

Membrane-integrated fermentation system for improving the optical purity of D-lactic acid produced during continuous fermentation

This report describes the production of highly optically pure D-lactic acid by the continuous fermentation of Sporolactobacillus laevolacticus and S. inulinus, using a membrane-integrated fermentation (MFR) system. The optical purity of D-lactic acid produced by the continuous fermentation system was greater than that produced by batch fermentation; the maximum value for the optical purity of D-lactic acid reached 99.8% enantiomeric excess by continuous fermentation when S. leavolacticus was used. The volumetric productivity of the optically pure D-lactic acid was about 12 g/L/h, this being approximately 11-fold higher than that obtained by batch fermentation. An enzymatic analysis indicated that both S. laevolacticus and S. inulinus could convert L-lactic acid to D-lactic acid by isomerization after the late-log phase. These results provide evidence for an effective bio-process to produce D-lactic acid of greater optical purity than has conventionally been achieved to date.     

Reactors for continuous primary beer fermentation using immobilised yeast

A continuous multistage column bioreactor with fluidised beds and continuous gas-lift bioreactor system with immobilised yeast Saccharomyces cerevisiae was developed for the first step of wort fermentation. The system of gas-lift reactor with yeast entrapped in calcium pectate beads was stable for 5 weeks by the optimal residence time of 12.75h and produced beer with a composition and flavour profile similar to that of beer produced by batch fermentation. Concentration of diacetyl was less than 0.1mg/l.     

Continuous alcohol fermentation in an immobilized cell rotating disk reactor

The increasing interest in alcohol fermentation over these last years because of the energy crisis has been demonstrated by an increase in scientific research. After a brief analysis of the main results of the literature in the field of alcohol fermentation reactors, the use of a new type of immobilized cell reactor [the rotating biological surface (RBS) reactor] was studied. As is well known, the RBS reactor is a form of fixed-film reactor and can be described as a dynamic trickling filter. Our experimental apparatus employed a spongy material to trap the yeast cells on the disks. The results of fermentations carried out in the RBS reactor working in batch, in continuous with cell support, and in continuous without cell support have been presented in order to compare the different productivities and to assess the performance of the RBS immobilized cell reactor. An ethanol productivity of 7.1 g/L h was achieved in the RBS-ICR at a dilution rate of 0.3 h(-1), 2.5 times higher than the maximum productivity obtained in the RBS reactor without support at a lower dilution rate. The adoption of a spongy material as a cell immobilizer, combined with the use of the RBS reactor, enhances the particular advantages of both systems.     

Biological sludge reduction and enhanced nutrient removal in a pilot-scale system with 2-step sludge alkaline fermentation and A2O process

To enhance nutrient removal performance and reduce disposal amount of waste activated sludge (WAS), a pilot-scale continuous system consisting of a 2-step sludge alkaline fermentation process and an A²O reactor was proposed. The feasibility of WAS reducing and resourcing by alkaline fermentation was investigated. Volatile fatty acids (VFA) yield was higher under alkaline condition than that under acidic condition. Through 2-step alkaline fermentation, substantial VFA was accumulated, and then elutriated out continuously from an up-flow column by domestic wastewater. The results showed that 38.2% of sludge was hydrolyzed, 19.7% was finally acidified into VFA, and as high as 42.1% of WAS was reduced. Moreover, after introducing the fermentation liquids with higher proportion of acetic acid and propionic acid into the A²O reactor, the total nitrogen and phosphorus removal efficiencies reached to 80.1% and 90.0%, respectively. Sludge reduction and enhanced nutrient removal could be achieved simultaneously in the proposed system.     

Design and evaluation of a continuous semipartition bioreactor for in situ liquid‐liquid extractive fermentation

Extractive fermentation (or in situ product removal (ISPR)) is an operational method used to combat product inhibition in fermentations. To achieve ISPR, different separation techniques, modes of operation and physical reactor configurations have been proposed. However, the relative paucity of industrial application necessitates continued investigation into reactor systems. This article outlines a bioreactor designed to facilitate in situ product extraction and recovery, through adapting the reaction volume to include a settler and solvent extraction and recycle section. This semipartition bioreactor is proposed as a new mode of operation for continuous liquid‐liquid extractive fermentation. The design is demonstrated as a modified bench‐top fermentation vessel, initially analysed in terms of fluid dynamic studies, in a model two‐liquid phase system. A continuous abiotic simulation of lactic acid (LA) fermentation is then demonstrated. The results show that mixing in the main reaction vessel is unaffected by the inserted settling zone, and that the size of the settling tube effects the maximum volumetric removal rate. In these tests the largest settling tube gave a potential continuous volumetric removal rate of 7.63 ml/min; sufficiently large to allow for continuous product extraction even in a highly productive fermentation. To demonstrate the applicability of the developed reactor, an abiotic simulation of a LA fermentation was performed. LA was added to reactor continuously at a rate of 33ml/h, while continuous in situ extraction removed the LA using 15% trioctylamine in oleyl alcohol. The reactor showed stable LA concentration of 1 g/L, with the balance of the LA successfully extracted and recovered using back extraction. This study demonstrates a potentially useful physical configuration for continuous in situ extraction.     

Influence of alginate properties and gel reinforcement on fermentation characteristics of immobilized yeast cells

The effect of alginate composition, gel concentration, gelation method, cell loading and surface area on fermentation characteristics of immobilized yeast cells have been investigated. Molecular weight and G/M ratio had only little effect on fermentation velocity and gel strength, while increasing the alginate concentration caused a sizeable decrease in fermentation velocity and an increase in gel strength. The internally gelled immobilizates generally showed a higher fermentation velocity for the same gel strength and no decrease in gel strength was seen during fermentation. With high initial cell loadings, the fermentation velocity per g of immobilizate was higher, but the productivity per cell was lower than with low initial cell loadings. The difference decreased with time. Specific surface area (surface/volume) was shown to be an important factor for the observed productivity per gram of immobilizate, with high S/V ratios giving the highest productivity. Gel shape had no influence on fermentation velocity for a given S/V ratio. Gelation behaviour of externally gelled beads was determined by estimating the amount of cells liberated during gel formation through measurement of invertase activity (yeast-bound) in the gelling solution. A method for reinforcement of internally gelled alginate slabs with a nylon mesh was developed and utilized for production of a continuous fermentation reactor with reinforced gels.     

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.     

Acetone-butanol-ethanol (ABE) fermentation in an immobilized cell trickle bed reactor

Acetone-butanol-ethanol (ABE) fermentation was successfully carried out in an immobilized cell trickle bed reactor. The reactor was composed of two serial columns packed with Clostridium acetobutylicum ATCC 824 entrapped on the surface of natural sponge segments at a cell loading in the range of 2.03-5.56 g dry cells/g sponge. The average cell loading was 3.58 g dry cells/g sponge. Batch experiments indicated that a critical pH above 4.2 is necessary for the initiation of cell growth. One of the media used during continuous experiments consisted of a salt mixture alone and the other a nutrient medium containing a salt mixture with yeast extract and peptone. Effluent pH was controlled by supplying various fractions of the two different types of media. A nutrient medium fraction above 0.6 was crucial for successful fermentation in a trickle bed reactor. The nutrient medium fraction is the ratio of the volume of the nutrient medium to the total volume of nutrient plus salt medium. Supplying nutrient medium to both columns continuously was an effective way to meet both pH and nutrient requirement. A 257-mL reactor could ferment 45 g/L glucose from an initial concentration of 60 g/L glucose at a rate of 70 mL/h. Butanol, acetone, and ethanol concentrations were 8.82, 5.22, and 1.45 g/L, respectively, with a butanol and total solvent yield of 19.4 and 34.1 wt %. Solvent productivity in an immobilized cell trickle bed reactor was 4.2 g/L h, which was 10 times higher than that obtained in a batch fermentation using free cells and 2.76 times higher than that of an immobilized CSTR. If the nutrient medium fraction was below 0.6 and the pH was below 4.2, the system degenerated. Oxygen also contributed to the system degeneration. Upon degeneration, glucose consumption and solvent yield decreased to 30.9 g/L and 23.0 wt %, respectively. The yield of total liquid product (40.0 wt %) and butanol selectivity (60.0 wt %) remained almost constant. Once the cells were degenerated, they could not be recovered.     

In situ extractive fermentation of acetone and butanol

The productivity of the acetone-butanol fermentation was increased by continuously removing acetone and butanol from the fermentation broth during fed-batch culture. Whole broth containing viable cells of Clostridium acetobutylicum was cycled to a Karr reciprocating plate extraction column in which acetone and butanol were extracted into oleyl alcohol flowing counter-currently through the column. By continuously removing these toxic metabolites from the broth, end product inhibition was reduced, and a concentrated feed solution containing 300 g/L glucose was fermented at an overall butanol productivity of 1.0 g/L h, 70% higher than the productivity of normal batch fermentation. The continuous extraction process provides flexible operation and lends itself to process scale-up.     

Continuous butanol fermentation and feed starch retrogradation: butanol fermentation sustainability using Clostridium beijerinckii BA101

Use of starch solution as feed for butanol bioconversion processes employing Clostridium beijerinckii BA101 may have added economic advantage over the use of glucose. Acetone butanol ethanol (ABE) was produced from 30 gL(-1) starch solution using a continuous process. The bioreactor was fed at a dilution rate of 0.02 h(-1) and starch solution/feed volume (3 L) was replaced every 72 h. The continuous reactor fed with cornstarch solution (feed temperature 19 degrees C) produced approximately 6.0 gL(-1) total ABE. Increasing the feed storage temperature to 37 degrees C improved ABE production to 7.2 gL(-1) suggesting that retrogradation was occurring more rapidly at 19 degrees C. In both these cases the fermentation drifted toward acid production after approximately 260 h, consistent with the retrogradation of starch overtime. The use of soluble starch, which is less prone to retrogradation, resulted in the production of 9.9 gL(-1) ABE at 37 degrees C feed storage temperature, as compared to 7.2 gL(-1) ABE when cornstarch was used. It should be noted that gelatinized starch retrogradation takes place after sterilization and prior to use of the feed medium, and does not occur during long-term storage of the raw corn material in the months leading up to processing. The degree of hydrolysis of gelatinized starch decreased from 68.8 to 56.2% in 3 days when stored at 37 degrees C. Soluble starch which does not retrograde demonstrated no change in the degree of hydrolysis.     

Optimal process/solvent design for ethanol extractive fermentation with cell recycling

In this study, the computer-aided process/solvent design is introduced to find an optimal biocompatible solvent and to maximize the ethanol production rate simultaneously for the single- or double-stage extractive fermentation process with cell recycling. Such a process/solvent design problem is formulated as a mixed-integer nonlinear programming problem that is solved by mixed-integer hybrid differential evolution in order to obtain a global design. The double-stage process can use a smaller amount of fresh solvent to increase ethanol productivity compared with that of the single-stage process, but it will also decrease overall conversion. Comparing the case studies, the simultaneous process/solvent design could yield higher overall ethanol productivity than that of the process design. The maximum ethanol production rate for the double-stage extractive fermentation with cell recycling was about 10-fold higher than that of continuous fermentation and about twofold higher than that of continuous fermentation with cell recycling.     

Extractive fermentation for butyric acid production from glucose by Clostridium tyrobutyricum

A novel extractive fermentation for butyric acid production from glucose, using immobilized cells of Clostridium tyrobutyricum in a fibrous bed bioreactor, was developed by using 10% (v/v) Alamine 336 in oleyl alcohol as the extractant contained in a hollow-fiber membrane extractor for selective removal of butyric acid from the fermentation broth. The extractant was simultaneously regenerated by stripping with NaOH in a second membrane extractor. The fermentation pH was self-regulated by a balance between acid production and removal by extraction, and was kept at approximately pH 5.5 throughout the study. Compared with conventional fermentation, extractive fermentation resulted in a much higher product concentration (>300 g/L) and product purity (91%). It also resulted in higher reactor productivity (7.37 g/L. h) and butyric acid yield (0.45 g/g). Without on-line extraction to remove the acid products, at the optimal pH of 6.0, the final butyric acid concentration was only approximately 43.4 g/L, butyric acid yield was 0.423 g/g, and reactor productivity was 6.77 g/L. h. These values were much lower at pH 5.5: 20.4 g/L, 0.38 g/g, and 5.11 g/L. h, respectively. The improved performance for extractive fermentation can be attributed to the reduced product inhibition by selective removal of butyric acid from the fermentation broth. The solvent was found to be toxic to free cells in suspension, but not harmful to cells immobilized in the fibrous bed. The process was stable and provided consistent long-term performance for the entire 2-week period of study.     

Enhanced acetone-butanol fermentation using repeated fed-batch operation coupled with cell recycle by membrane and simultaneous removal of inhibitory products by adsorption

A novel acetone-butanol production process was developed which integrates a repeated fed-batch fermentation with continuous product removal and cell recycle. The inhibitory product concentrations of the fermentation by Clostridium acetobutylicum were reduced by the simultaneous extraction process using polyvinylpyridine (PVP) as an adsorbent. Because of the reduced inhibition effect, a higher specific cell growth rate and thus a higher product formation rate was achieved. The cell recycle using membrane separation increased the total cell mass density and, therefore, enhanced the reactor productivity. The repeated fed-batchoperation overcame the drawbacks typically associated with a batch operation such as down times, long lag period, and the limitation on the maximum initial substrate concentration allowed due to the substrate inhibition. Unlike a continuous operation, the repeated fed-batch operation could beoperated for a long time at a relatively higher substrate concentration without sacrificing the substrate loss in the effluent. As a result, the integrated process reached 47.2 g/L in the equivalent solvent concentration (including acetone, butanol, and ethanol) and 1.69 g/L . h in the fermentor productivity, on average, over a 239.5-h period. Compared with a controlled traditional batch acetone-butanol fermentation, the equivalent solvent concentration and the tormentor productivity were increased by 140% and 320%, respectively. (c) 1995 John Wiley & Sons Inc.     

Ethanol production from biomass by repetitive solid-state fed-batch fermentation with continuous recovery of ethanol

To save cost and input energy for bioethanol production, a consolidated continuous solid-state fermentation system composed of a rotating drum reactor, a humidifier, and a condenser was developed. Biomass, saccharifying enzymes, yeast, and a minimum amount of water are introduced into the system. Ethanol produced by simultaneous saccharification and fermentation is continuously recovered as vapor from the headspace of the reactor, while the humidifier compensates for the water loss. From raw corn starch as a biomass model, 95 ± 3, 226 ± 9, 458 ± 26, and 509 ± 64 g l⁻¹ of ethanol solutions were recovered continuously when the ethanol content in reactor was controlled at 10–20, 30–50, 50–70 and 75–85 g kg-mixture⁻¹, respectively. The residue showed a lesser volume and higher solid content than that obtained by conventional liquid fermentation. The cost and energy for intensive waste water treatment are decreased, and the continuous fermentation enabled the sustainability of enzyme activity and yeast in the system.     

Continuous immobilized yeast reactor system for complete beer fermentation using spent grains and corncobs as carrier materials

Despite extensive research carried out in the last few decades, continuous beer fermentation has not yet managed to outperform the traditional batch technology. An industrial breakthrough in favour of continuous brewing using immobilized yeast could be expected only on achievement of the following process characteristics: simple design, low investment costs, flexible operation, effective process control and good product quality. The application of cheap carrier materials of by-product origin could significantly lower the investment costs of continuous fermentation systems. This work deals with a complete continuous beer fermentation system consisting of a main fermentation reactor (gas-lift) and a maturation reactor (packed-bed) containing yeast immobilized on spent grains and corncobs, respectively. The suitability of cheap carrier materials for long-term continuous brewing was proved. It was found that by fine tuning of process parameters (residence time, aeration) it was possible to adjust the flavour profile of the final product. Consumers considered the continuously fermented beer to be of a regular quality. Analytical and sensorial profiles of both continuously and batch fermented beers were compared.     

Continuous hydrogen production during fermentation of alpha-cellulose by the thermophillic bacterium Clostridium thermocellum

Continuous hydrogen (H2) production during fermentation of alpha-cellulose was established using the thermophillic, anaerobic bacterium Clostridium thermocellum ATCC 27405. The objectives of this work were to characterize growth of C. thermocellum, quantify H2 production and determine soluble end-product synthesis patterns during fermentation of a cellulosic substrate under continuous culture conditions. A 5 L working volume fermentor was established and growth experiments were maintained for over 3,000 h. Substrate concentrations were varied from 1 to 4 g/L and the feed was introduced with continuous nitrogen gas sparging to prevent clogging of the feed-line. The pH and temperature of the reactor were maintained at 7.0 and 600 degrees C, respectively, throughout the study. At concentrations above 4 g/L, the delivery of alpha-cellulose was impaired due to feed-line clogging and it became difficult to maintain a homogenous suspension. The highest total gas (H2 plus CO2) production rate, 56.6 mL L(-1) h(-1), was observed at a dilution rate of 0.042 h(-1) and substrate concentration of 4 g/L. Under these conditions, the H2 production rate was 5.06 mmol h(-1). Acetate and ethanol were the major soluble end-products, while lactate and formate were greatly reduced compared to production in batch cultures. Concentrations of all metabolites increased with increasing substrate concentration, with the exception of lactate. Despite a number of short-term electrical and mechanical failures during the testing period, the system recovered quickly, exhibiting substantial robustness. A carbon balance was completed to ensure that all end-products were accounted for, with final results indicating near 100% carbon recovery. This study shows that long-term, stable H2 production can be achieved during direct fermentation of an insoluble cellulosic substrate under continuous culture conditions.     

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.     

Continuous acetone-butanol-ethanol (ABE) fermentation using immobilized cells of Clostridium acetobutylicum in a packed bed reactor and integration with product removal by pervaporation

An integrated solvent (ABE) fermentation and product removal process was investigated. A stable solvent productivity of 3.5 g/L h was achieved by using cells of Clostridium acetobutylicum immobilized onto a packed bed of bonechar, coupled with continuous product removal by pervaporation. Using a concentrated feed solution containing lactose at 130g/L, a lactose value of 97.9% was observed. The integrated fermentation and product removal system, with recycling of the treated fermentor effluent containing only low amount of solvents (/but lactose and acids), leads to only low acid losses. Therefore, most of the acids are converted to solvents, and this results in a high solvent yield of 0.39 g solvents/g lactose utilized. The pervaporation system provided a high product removal rate even at low solvent concentrations. A solvent membrane flux of 7.1 g/m(2) h with a selectivity of 5 was achieved during these investigations. The system proved to be very reliable.     

Effect of air supplement on the performance of continuous ethanol fermentation system

For the purpose of improving ethanol productivity, the effect of air supplement on the performance of continuous ethanol fermentation system was studied. The effect of oxygen supplement on yeast concentration, cell yield, cell viability, extracellular ethanol concentration, ethanol yield, maintenance coefficient, specific rates of glucose assimilation, ethanol production, and ethanol productivity have been evaluated, using a high alcohol tolerant Saccharomyces cerevisiae STV89 strain and employing a continuous fermentor equipped with an accurate air metering system in the flow rate range 0-11 mL air/L/h. It was found that, when a small amount of oxygen up to about 80mu mol oxygen/L/h was supplied, the ethanol productivity was significantly enhanced as compared to the productivity of the culture without any air supplement. It was also found that the oxygen supplement improved cell viability considerably as well as the ethanol tolerance level of yeast. As the air supply rate was increased, from 0 to 11 mL air/L/h while maintaining a constant dilution rate at about 0.06 h(-1), the cell concentration increased from 2.3 to 8.2 g/L and the ethanol productivity increased from 1.7 to 4.1 g ethanol/L/h, although the specific ethanol production rate decreased slightly from 0.75 to 0.5 g ethanol/g cell/h. The ethanol yield was slightly improved also with an increase in air supply rate, from about 0.37 to 0.45 ethanol/g glucose. The maintenance coefficient increased by only a small amount with the air supplement. This kind of air supplement technique may very well prove to be of practical importance to a development of a highly productive ethanol fermentation process system especially as a combined system with a high density cell culture technique.