Microfluidic biolector—microfluidic bioprocess control in microtiter plates

In industrial‐scale biotechnological processes, the active control of the pH‐value combined with the controlled feeding of substrate solutions (fed‐batch) is the standard strategy to cultivate both prokaryotic and eukaryotic cells. On the contrary, for small‐scale cultivations, much simpler batch experiments with no process control are performed. This lack of process control often hinders researchers to scale‐up and scale‐down fermentation experiments, because the microbial metabolism and thereby the growth and production kinetics drastically changes depending on the cultivation strategy applied. While small‐scale batches are typically performed highly parallel and in high throughput, large‐scale cultivations demand sophisticated equipment for process control which is in most cases costly and difficult to handle. Currently, there is no technical system on the market that realizes simple process control in high throughput. The novel concept of a microfermentation system described in this work combines a fiber‐optic online‐monitoring device for microtiter plates (MTPs)—the BioLector technology—together with microfluidic control of cultivation processes in volumes below 1 mL. In the microfluidic chip, a micropump is integrated to realize distinct substrate flow rates during fed‐batch cultivation in microscale. Hence, a cultivation system with several distinct advantages could be established: (1) high information output on a microscale; (2) many experiments can be performed in parallel and be automated using MTPs; (3) this system is user‐friendly and can easily be transferred to a disposable single‐use system. This article elucidates this new concept and illustrates applications in fermentations of Escherichia coli under pH‐controlled and fed‐batch conditions in shaken MTPs. Biotechnol. Bioeng. 2010;107: 497–505. © 2010 Wiley Periodicals, Inc.     

Room and low temperature brewing with yeast immobilized on gluten pellets

A biocatalyst prepared by the immobilization of a cryotolerant strain of Saccharomyces cerevisiae on gluten pellets was used for batch and continuous fermentation at low temperatures. The immobilized yeast showed important operational stability in repeated batch fermentations without a decrease of activity even at 0 and 5°C. Repeated batch fermentations using the biocatalyst resulted in improvement of ethanol productivity in comparison with bottom brewing fermentation and free cells using the same yeast strain. At 0 and 10°C, the fermentation rate was four and seven times higher than that of free cells, respectively. For immobilized yeast, diacetyl and polyphenol contents were lower and the alcohol concentration higher at low temperatures (0–7°C) when compared to free cells. Fine clarity was also observed in the beers. Continuous brewing using gluten-supported biocatalyst had an operational stability of 3 months with relatively high productivity and without contamination. Polyphenol and bitterness contents were lower in the continuous process than those of batch fermentations, but at low temperature (5°C) they were higher. The diacetyl content was higher than in batch fermentations and beers had a fine aroma and taste.     

Simultaneous fructose and ethanol production from sucrose,usingZymomonas mobilis 2864 co-immobilised with invertase

Summary Fructokinase negativeZymomonas mobilis UQM 2864, was co-immobilised with invertase in alginate and incubated on sucrose-based media in batch and fedbatch culture. The highest fructose concentration achieved was 138 g/l using fed-batch cultivation with sugar-cane syrup-simultaneously producing 79.9 g/l or 10.1% (v/v) ethanol in less than 24 hours. The ethanol and fructose yields were 95 and 84% respectively. Co-immobilisation resulted in faster fermentation times, particularly for the batch fermentations, and complete utilisation of substrate.     

Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae

Fermentation of sugar by Saccharomyces cerevisiae, for production of ethanol in an immobilized cell reactor (ICR) was successfully carried out to improve the performance of the fermentation process. The fermentation set-up was comprised of a column packed with beads of immobilized cells. The immobilization of S. cerevisiae was simply performed by the enriched cells cultured media harvested at exponential growth phase. The fixed cell loaded ICR was carried out at initial stage of operation and the cell was entrapped by calcium alginate. The production of ethanol was steady after 24 h of operation. The concentration of ethanol was affected by the media flow rates and residence time distribution from 2 to 7 h. In addition, batch fermentation was carried out with 50 g/l glucose concentration. Subsequently, the ethanol productions and the reactor productivities of batch fermentation and immobilized cells were compared. In batch fermentation, sugar consumption and ethanol production obtained were 99.6% and 12.5% v/v after 27 h while in the ICR, 88.2% and 16.7% v/v were obtained with 6 h retention time. Nearly 5% ethanol production was achieved with high glucose concentration (150 g/l) at 6 h retention time. A yield of 38% was obtained with 150 g/l glucose. The yield was improved approximately 27% on ICR and a 24 h fermentation time was reduced to 7 h. The cell growth rate was based on the Monod rate equation. The kinetic constants (K(s) and mu(m)) of batch fermentation were 2.3 g/l and 0.35 g/lh, respectively. The maximum yield of biomass on substrate (Y(X-S)) and the maximum yield of product on substrate (Y(P-S)) in batch fermentations were 50.8% and 31.2% respectively. Productivity of the ICR were 1.3, 2.3, and 2.8 g/lh for 25, 35, 50 g/l of glucose concentration, respectively. The productivity of ethanol in batch fermentation with 50 g/l glucose was calculated as 0.29 g/lh. Maximum production of ethanol in ICR when compared to batch reactor has shown to increase approximately 10-fold. The performance of the two reactors was compared and a respective rate model was proposed. The present research has shown that high sugar concentration (150 g/l) in the ICR column was successfully converted to ethanol. The achieved results in ICR with high substrate concentration are promising for scale up operation. The proposed model can be used to design a lager scale ICR column for production of high ethanol concentration.     

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.     

Inhibition of yeast by lactic acid bacteria in continuous culture: nutrient depletion and/or acid toxicity?

Lactic acid was added to batch very high gravity (VHG) fermentations and to continuous VHG fermentations equilibrated to steady state with Saccharomyces cerevisiae. A 53% reduction in colony-forming units (CFU) ml^–1 of S. cerevisiae was observed in continuous fermentation at an undissociated lactic acid concentration of 3.44% w/v; and greater than 99.9% reduction was evident at 5.35% w/v lactic acid. The differences in yeast cell number in these fermentations were not due to pH, since batch fermentations over a pH range of 2.5–5.0 did not lead to changes in growth rate. Similar fermentations performed in batch showed that growth inhibition with added lactic acid was nearly identical. This indicates that the apparent high resistance of S. cerevisiae to lactic acid in continuous VHG fermentations is not a function of culture mode. Although the total amount of ethanol decreased from 48.7 g l^–1 to 14.5 g l^–1 when 4.74% w/v undissociated lactic acid was added, the specific ethanol productivity increased ca. 3.2-fold (from 7.42×10^–7 g to 24.0×10^–7 g ethanol CFU^–1 h^–1), which indicated that lactic acid stress improved the ethanol production of each surviving cell. In multistage continuous fermentations, lactic acid was not responsible for the 83% (CFU ml^–1) reduction in viable S. cerevisiae yeasts when Lactobacillus paracasei was introduced to the system at a controlled pH of 6.0. The competition for trace nutrients in those fermentations and not lactic acid produced by L. paracasei likely caused the yeast inhibition.     

A new immobilized cell system with protection against toxic solvents

A new immobilized cell system providing protection against toxic solvents was investigated so that normal fermentations could be carried out in a medium containing toxic solvents. The system consists of immobilized growing cells in Ca-alginate gel beads to which vegetable oils, which are inexpensive absorbents of solvents, had been added. The ethanol fermentation of Saccharomyces cerevisiae ATCC 26603 was used as a model fermentation to study the protection afforded by the system against solvent toxicities. The fermentation was inhibited by solvents such as 2-octanol, benzene, toluene, and phenol. Ethanol production of one batch was not finished even after 35 h using immobilized growing yeast cells in conventional Ca-alginate gel beads in an ethanol production medium (5% glucose) containing 0.1% 2-octanol, which is used as a solvent for liquid-liquid extraction and is one of the most toxic solvents in our experiments. With the new immobilized growing cell system using vegetable oils, however, four repeated batch fermentations were completed in 35 h. Castor oil provided even more protection than soy bean, olive, and tung oils, and it was possible to complete six repeated batches in 35 h. The immobilized cell system with vegetable oils also provided protection against other toxic solvents such as benzene and toluene. A possible mechanism for the protective function of the new immobilized cell system is discussed.     

The occurrence of bacteriophages in spirit vinegar fermentation

Summary Bacteriophages in concentrations of approximately 10⁸–10⁹ particles/ml were demonstrated in culture lysates of industrial submerged spirit vinegar fermentations running in different European vinegar factories. Besides frequently found fragments of phages, two types of phages could be described. The frequent type I seems to belong to Bradley-group A; type II, with a remarkably larger head, may belong to group C. For simulation in the laboratory a phage-contaminated industrial culture was kept over 102 charges (time of cycle 24->240 h) in a semicontinuously operated 8-1 fermentor. A close relationship between spontaneous breakdowns and phage occurrence exists. Discrimination of breakdowns caused by lack of ethanol could not be made. Attempts to establish a bioassay for the phages failed presumably because of the instability of the phages. Continuing cultivation and waiting for secondary growth finally results in stable and productive fermentation.     

Ethanol production from seaweed extract

Extracts from Laminaria hyperborea could possibly be fermented to ethanol commercially. In particular, seaweed harvested in the autumn contains high levels of easily extractable laminaran and mannitol. Four microorganisms were tested to carry out this fermentation, one bacterium and three yeasts. Only Pichia angophorae was able to utilise both laminaran and mannitol for ethanol production, and its substrate preferences were investigated in batch and continuous cultures. Laminaran and mannitol were consumed simultaneously, but with different relative rates. In batch fermentations, mannitol was the preferred substrate. Its share of the total laminaran and mannitol consumption rate increased with oxygen transfer rate (OTR) and pH. In continuous fermentations, laminaran was the preferred substrate at low OTR, whereas at higher OTR, laminaran and mannitol were consumed at similar rates. Optimisation of ethanol yield required a low OTR, and the best yield of 0.43 g ethanol (g substrate)⁻¹ was achieved in batch culture at pH 4.5 and 5.8 mmol O₂ l⁻¹ h⁻¹. However, industrial production of ethanol from seaweed would require an optimisation of the extraction process to yield a higher ethanol concentration. Journal of Industrial Microbiology & Biotechnology (2000) 25, 249–254. Received 25 February 2000/ Accepted in revised form 05 August 2000     

Repeated-batch production of kojic acid in a cell-retention fermenter using Aspergillus oryzae M3B9

A cell-retention fermenter was used for the pilot-scale production of kojic acid using an improved strain of Aspergillus oryzae in repeated-batch fermentations. Among the various carbon and nitrogen sources used, sucrose and yeast extract promoted pellet morphology of fungi and higher kojic acid production. Repeated-batch culture using a medium replacement ratio of 75% gave a productivity of 5.3 g L^–1 day^–1 after 11.5 days of cultivation. While batch culture in shake-flasks resulted in a productivity of 5.1 g L^–1 day^–1, a productivity of 5 g L^–1 day^–1 was obtained in a pilot-scale fermenter. By converting the batch culture into repeated batches, the non-productive downtime of cleaning, filling and sterilizing the fermenter between each batch were eliminated, thereby increasing the kojic acid productivity.     

Comparative metabolomic analysis on industrial continuous and batch ethanol fermentation processes by GC-TOF-MS

The intracellular metabolic profile characterization of Saccharomyces cerevisiae throughout industrial ethanol fermentation was investigated using gas chromatography coupled to time-of-flight mass spectrometry. A total of 143 and 128 intracellular metabolites in S. cerevisiae were detected and quantified in continuous and batch fermentations, respectively. The two fermentation processes were both clearly distinguished into three main phases by principal components analysis. Furthermore, the levels of some metabolites involved in central carbon metabolism varied significantly throughout both processes. Glycerol and phosphoric acid were principally responsible for discriminating seed, main and final phases of continuous fermentation, while lactic acid and glycerol contributed mostly to telling different phases of batch fermentation. In addition, the levels of some amino acids such as glycine varied significantly during both processes. These findings provide new insights into the metabolomic characteristics during industrial ethanol fermentation processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A novel parallel shaken bioreactor system for continuous operation

A novel continuous bioreactor system was developed as a shaken culture vessel for the investigation of the growth kinetics and product formation of microorganisms in milliscale. The novel bioreactor system mainly consists of a specially designed 250-mL shake flask with two inlets, one for gas supply and one for medium supply, and one combined outlet on the side of flask for exhaust gas and culture liquid. As a result of the circulating motion of the fermentation broth in the shake flask, the maximum liquid height reaches the edge of the outlet and the fermentation broth is accelerated into the outlet by centrifugal force. Additionally, the excess fermentation broth leaving the culture vessel is continuously driven by the exhaust gas. Because of the small scale and the simple handling it is possible to operate many of these shaken bioreactor vessels simultaneously. By using parallel vessels operated at different dilution rates on the same shaker, the data for a complete biomass over dilution rate (X-D) diagram of a biological culture can be evaluated in an efficient manner, thus saving money, materials, and time. Continuous fermentations of the yeast Saccharomyces cerevisiae H1022 (ATCC 32167) in the shaken bioreactor system and in a conventional stirred tank fermentor showed very similar results.     

Fermentation of cellodextrins to ethanol using mixed-culture fermentations

The potential for enhancing ethanol production from cellodextrins by employing mixed-culture (Candida wickerhamii-Saccharomyces cerevisiae) fermentations was investigated. Initially, ethanol production was monitored in fermentation medium containing 50 g/L glucose plus 45 g/L cellobiose. Inoculum levels and times of inoculum addition were varied. Of the conditions tested, the most rapid rates of ethanol formation occurred in fermentations in which either C. wickerhamii and S. cerevisiae were coinoculated at a ratio of 57 : 1 cell/mL or in fermentations in which a 10-fold-greater S. cerevisiae inoculum was added to a pure culture C. wickerhamii fermentation after 1 day incubation. These conditions were used to attempt to enhance fermentations in which cellodextrins produced by trifluoroacetic acid hydrolysis of cellulose served as the sole carbon source. Cellodextrins that were not further purified after cellulose hydrolysis contained compounds that were slightly inhibitory to C. wickerhamii. In this case the mixed-culture fermentations produced 12-45% more ethanol than a pure culture C. wickerhamii fermentation. However, if the substrate was treated with Darco G-60 charcoal, the toxic materials were apparently removed and the pure culture C. wickerhamii fermentations performed as well as the mixed-culture fermentations.     

Repeated batch production of citric acid from sugarcane molasses using recycled solid-state surface culture ofAspergillus niger

Summary The recycled solid-state surface fermentation (SSF) culture ofAspergillus niger KCU520 was used for repeated batch production of citric acid from sugarcane molasses. The rate of citric acid production was doubled, reducing the fermentation time to half, compared to the normal single cycle batch submerged or surface fermentation process. About 80% sugar was converted to citric acid in five-day batch fermentation and three batches were carried out with the same fungal mat without any significant loss of productivity.     

Cultivation and preservation of vinegar bacteria

Ten strains of acetic acid bacteria were investigated for their characteristics of growth and metabolism. The strains were identified as those presently in use for industrial vinegar production in southern Germany. At the time of isolation from industrial acetators the total concentrations, i.e. acetic acid (w/v) plus ethanol (v/v), of the fermenting vinegars were 6.1–14.9%. The applicability of a previously described method for starter preparation was examined for the various isolates as well as for the type strains of species of the genera Gluconobacter and Acetobacter. Isolates from cider or wine vinegar fermentations grew readily in RAE-medium to total counts of >1×10⁹ cells ml⁻¹. For the cultivation of strains isolated from spirit vinegar fermentations AE-medium proved most suitable. Cultures of these strains exhibited lag phases of 2–5 days and grew up to total counts of <1×10⁹ cells ml⁻¹. All type strains could be grown on RAE-agar. The use of 20% malt extract as cryo-protectant was effective for the preservation of all strains. Upon revitalization the cultures were suitable as inoculum for starting fermentations in pilot acetators. 16S rRNA-targeted oligonucleotide probes were constructed which were species specific for Gluconobacter oxydans or Acetobacter aceti or group specific for Acetobacter europaeus/Acetobacter xylinum. The probes hybridized with the DNA of the respective type strains. Four isolates were allotted to A. europaeus/A. xylinum applying the group specific probe. The DNA of six of the Acetobacter sp. hybridized with none of the probes.     

Fermentation of deproteinized cheese whey powder solutions to ethanol by engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: effect of supplementation with corn steep liquor and repeated-batch operation with biomass recycling by flocculation

The lactose in cheese whey is an interesting substrate for the production of bulk commodities such as bio-ethanol, due to the large amounts of whey surplus generated globally. In this work, we studied the performance of a recombinant Saccharomyces cerevisiae strain expressing the lactose permease and intracellular ß-galactosidase from Kluyveromyces lactis in fermentations of deproteinized concentrated cheese whey powder solutions. Supplementation with 10 g/l of corn steep liquor significantly enhanced whey fermentation, resulting in the production of 7.4% (v/v) ethanol from 150 g/l initial lactose in shake-flask fermentations, with a corresponding productivity of 1.2 g/l/h. The flocculation capacity of the yeast strain enabled stable operation of a repeated-batch process in a 5.5-l air-lift bioreactor, with simple biomass recycling by sedimentation of the yeast flocs. During five consecutive batches, the average ethanol productivity was 0.65 g/l/h and ethanol accumulated up to 8% (v/v) with lactose-to-ethanol conversion yields over 80% of theoretical. Yeast viability (>97%) and plasmid retention (>84%) remained high throughout the operation, demonstrating the stability and robustness of the strain. In addition, the easy and inexpensive recycle of the yeast biomass for repeated utilization makes this process economically attractive for industrial implementation.     

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.     

Improvement of very high gravity ethanol fermentation by media supplementation using Saccharomyces cerevisiae

The final ethanol concentration achieved was increased by 17% (to 103 g ethanol/l) when excess assimilable nitrogen was added to the batch very high gravity (VHG) ethanolic fermentations by Saccharomyces cerevisiae. The supplementation of the media with 12 g yeast extract l–1, 0.3 g cell walls l–1, 3 g glycine l–1 and 20 g soya flour l–1 led to halving reduction of the fermentation time to 28 h. The ethanol productivity was enhanced by more than 50% (to achieved value 3.3 g l–1 h–1).     

Proof-of-concept of a novel micro-bioreactor for fast development of industrial bioprocesses

The experimental performance of a novel micro-bioreactor envisaged for parallel screening and development of industrial bioprocesses has been tested in this work. The micro-bioreactor with an internal volume of 4.5 mL is operated under oscillatory flow mixing (OFM), where a controllable mixing and mass transfer rates are achieved under batch or continuous laminar flow conditions. Several batch fermentations with a flocculent Saccharomyces cerevisiae strain were carried out at initial glucose concentrations (S(0)) range of approximately 5-20 g/L and compared to yeast growth kinetics in a stirred tank (ST) bioreactor. Aerobic fermentations were monitored ex situ in terms of pH, DO, glucose consumption, and biomass and ethanol production (wherever applicable). An average biomass production increase of 83% was obtained in the micro-bioreactor when compared with the ST, with less 93.6% air requirements. It also corresponded to a 214% increase on biomass production when compared with growth in a shaken flask (SF) at S(0) = 20 g/L. Further anaerobic fermentations at the same initial glucose concentration ranges gave the opportunity to use state-of-the-art fiber optics technology for on-line and real-time monitoring of this bioprocess. Time profiles of biomass concentration (measured as optical density (OD)) were very similar in the ST bioreactor and in the micro-bioreactor, with a highly reproducible yeast growth in these two scale-down platforms.     

Ethanol production from nonsterilized carob pod extract by free and immobilized Saccharomyces cerevisiae cells using fed-batch culture

The production of ethanol from carob pod extract by free and immobilized Saccharomyces cerevisiae cells in batch and fed-batch culture was investigated. Fed-batch culture proved to be a better fermentation system for the production of ethanol than batch culture. In fed-batch culture, both free and immobilized S. cerevisiae cells gave the same maximum concentration (62 g/L) of final ethanol at an initial sugar concentration of 300 g/L and F = 167 mL/h. The maximum ethanol productivity (4.4 g/L h) was obtained with both free and immobilized cells at a substrate concentration of 300 g/L and F = 334 mL/h. In repeated fed-batch culture, immobilized S. cerevisiae cells gave a higher overall ethanol concentration compared with the free cells. The immobilized S. cerevisiae cells in Ca-alginate beads retained their ability to produce ethanol for 10 days. (c) 1994 John Wiley & Sons, Inc.