A pulsing device for packed-bed bioreactors: II. Application to alcoholic fermentation

When the immobilized cells are employed in packed-bed bioreactors several problems appear. To overcome these drawbacks, a new bioreactor based on the use of pulsed systems was developed [1]. In this work, we study the glucose fermentation by immobilized Saccharomyces cerevisiae in a packed-bed bioreactor. A comparative study was then carried out for continuous fermentation in two packed-bed bioreactors, one of them with pulsed flow. The determination of the axial dispersion coefficients indicates that by introducing the pulsation, the hydraulic behaviour is closer to the plug flow model. In both cases, the residence time tested varied from 0.8 to 2.6 h. A higher ethanol concentration and productivity (increases up to 16%) were achieved with the pulsated reactors. The volumes occupied by the CO₂ were 5.22% and 9.45% for fermentation with/without pulsation respectively. An activity test of the particles from the different sections revealed that the concentration and viability of bioparticles from the two bioreactors are similar. From the results we conclude that the improvements of the process are attributable to a mechanical effect rather than to physiological changes of microorganisms.List of Symbols D m²/s dispersion coefficient - K _is l/g inhibition substrate constant - K _ip l/g inhibition ethanol constant - K _s g/l Apparent affinity constant - P g/l ethanol concentration - q _p g/(gh) specific ethanol productivity - Q _p g/(lh) overall ethanol productivity - q _s g/(gh) specific glucose consumption rate - Q _s g/(lh) glucose consumption rate - S g/l residual glucose concentration - S(in0) g/l initial glucose concentration - V _max g/(lh) maximum rate - Y _p/s g/g yield in product     

Continuous ethanol fermentation at 45 °C using Kluyveromyces marxianus IMB3 immobilized in Calcium alginate and kissiris

The thermotolerant ethanol-producing yeast strain Kluyveromyces marxianus IMB3 was immobilized in calcium alginate and a 1:1 mixture of calcium alginate and the porous volcanic mineral, kissiris. Immobilized preparations were placed in fixed-bed column bioreactors and continuous ethanol production by systems containing both immobilized preparations was examined at 45?°C with a 100?g/l glucose feed. The effect of residence time on product concentration, bioreactor efficiency and volumetric productivities have been examined and these were all higher in systems containing the alginate/kissiris mixed immobilization matrix. Maximum ethanol concentrations produced by the continuous system ranged between 46 and 48?g/l representing efficiencies of 90–94%.     

Citric acid production by immobilized Aspergillus niger in a fluidized bed reactor

Summary Citric acid production by immobilized of Aspergillus niger in a fluidized bed reactor was performed, evaluating the productivity and the stability of the process when pulsing device was used. The application of a pulsing flow to fluidized bed reactor and the feed nitrogen limited allow to control of bioparticles morphology avoiding bed compactation. When operated at optimum pulsation frequency (0.3 s^–1) the stability of the bioreactor was maintained for more than 30 days, increasing the citric acid production in more than 52.2%.     

Continuous ethanol production from molasses at 45 °C using alginate-immobilized Kluyveromyces marxianus IMB3 in a continuous-flow bioreactor

The thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus IMB3 was immobilized in calcium alginate and used in a continuous flow bioreactor to produce ethanol from molasses at 45?°C. The molasses was diluted to yield a number of final sugar concentrations and the effect of molasses sugar concentration on ethanol production by the continuous system was examined. Although maximum ethanol concentrations were obtained using sugar concentrations of 140?g/l, within 10?h of introducing the feed to the column bioreactors, those ethanol concentrations subsequently decreased to lower levels over a 48?h period. Examination of viable yeast cell number within the immobilization matrix indicated a dramatic reduction over this time period. At lower molasses concentrations, ethanol production by the continuous flow system remained relatively constant over this time period. In addition, the effect of residence time on ethanol production by the continuous flow bioreactor was examined at a fixed molasses sugar concentration (120?g/l) and a residence time of 0.66?h was found to be optimal on the basis of volumetric productivity. Efficiencies of the continuous flow bioreactor configuration used in these studies ranged from 31–76%.     

Economic improvement of continuous pharmaceutical production via the optimal control of a multifeed bioreactor

Projections on the profitability of the pharmaceutical industry predict a large amount of growth in the coming years. Stagnation over the last 20 years in product development has led to the search for new processing methods to improve profitability by reducing operating costs or improving process productivity. This work proposes a novel multifeed bioreactor system composed of independently controlled feeds for substrate(s) and media used that allows for the free manipulation of the bioreactor supply rate and substrate concentrations to maximize bioreactor productivity and substrate utilization while reducing operating costs. The optimal operation of the multiple feeds is determined a priori as the solution of a dynamic optimization problem using the kinetic models describing the time‐variant bioreactor concentrations as constraints. This new bioreactor paradigm is exemplified through the intracellular production of beta‐carotene using a three feed bioreactor consisting of separate glucose, ethanol and media feeds. The performance of a traditional bioreator with a single substrate feed is compared to that of a bioreactor with multiple feeds using glucose and/or ethanol as substrate options. Results show up to a 30% reduction in the productivity with the addition of multiple feeds, though all three systems show an improvement in productivity when compared to batch production. Additionally, the breakeven selling price of beta‐carotene is shown to decrease by at least 30% for the multifeed bioreactor when compared to the single feed counterpart, demonstrating the ability of the multifeed reactor to reduce operating costs in bioreactor systems. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:902–912, 2017     

Enhanced production of L-(+)-lactic acid in chemostat by Lactobacillus casei DSM 20011 using ion-exchange resins and cross-flow filtration in a fully automated pilot plant controlled via NIR

Due to the lack of suitable in-process sensors, on-line monitoring of fermentation processes is restricted almost exclusively to the measurement of physical parameters only indirectly related to key process variables, i.e., substrate, product, and biomass concentration. This obstacle can be overcome by near infrared (NIR) spectroscopy, which allows not only real-time process monitoring, but also automated process control, provided that NIR-generated information is fed to a suitable computerized bioreactor control system. Once the relevant calibrations have been obtained, substrate, biomass and product concentration can be evaluated on-line and used by the bioreactor control system to manage the fermentation. In this work, an NIR-based control system allowed the full automation of a small-scale pilot plant for lactic acid production and provided an excellent tool for process optimization. The growth-inhibiting effect of lactic acid present in the culture broth is enhanced when the growth-limiting substrate, glucose, is also present at relatively high concentrations. Both combined factors can result in a severe reduction of the performance of the lactate production process. A dedicated software enabling on-line NIR data acquisition and reduction, and automated process management through feed addition, culture removal and/or product recovery by microfiltration was developed in order to allow the implementation of continuous fermentation processes with recycling of culture medium and cell recycling. Both operation modes were tested at different dilution rates and the respective cultivation parameters observed were compared with those obtained in a conventional continuous fermentation. Steady states were obtained in both modes with high performance on lactate production. The highest lactate volumetric productivity, 138 g L(-1) h(-1), was obtained in continuous fermentation with cell recycling.     

High solids fermentation of hydrolysates of wheat starch B in a continuous dynamic immobilized biocatalyst bioreactor

Summary A simple and efficient method of conversion of wheat starch B to ethanol was investigated. Employing a two-stage enzymatic saccharification process, 95% of the wheat starch was converted to fermentable sugars in 40 h. From 140 g/l total sugars in the feed solution, 63.6 g/l ethanol was produced continuously with a residence time of 3.3 h in a continuous dynamic immobilized biocatalyst bioreactor by immobilized cells ofSaccharomyces cerevisiae. The advantages and the application of this bioreactor to continuous alcoholic fermentation of industrial substrates are presented.     

Continuous production of gluconic acid by immobilized Gluconobacter oxydans cell bioreactor

Summary Living Gluconobacter oxydans cells were attached on fibrous nylon carrier. Free gluconic acid was directly continuously produced in an aerated tubular immobilized-cell bioreactor for at least 6 months, with a volumetric productivity of at least 5 g/lh at 100 g/l substrate glucose and about 80 g/l product gluconic acid concentrations. The highest volumetric productivity in respect to glucose concentration was obtained with 175 g/l glucose, with about 120 g/l product gluconic acid level. With self-directing optimization procedure in respect to maximum product gluconic acid level, productivities as high as about 12–15 g/lh were obtained at relatively high substrate feed rate of 0.166 l/lh and relatively low aeration rate of 0.5 l/lmin. The highest glucose conversion of about 96% was obtained with a long residence time, at the lowest substrate feed rate used at a relatively low aeration rate, resulting however in a significant increase in ketogluconic acid production.     

Tower fermentation of sugar cane juice

Summary Sugar cane juice, of feed fermentables concentration of 100–180g/l, was simply and rapidly fermented to ethanol in a tower fermenter, using a naturally flocculant yeast. Stable cell densities of 65–80g dry wt/l were obtained. Conversion and ethanol productivity data are given as a function of throughput and initial feed concentration. The limitation to fermenter performance with this yeast was imposed by carryover of non-fermented sugars, principally fructose, rather than washout of the yeast flocs under high hydraulic loadings (residence times of less than 1.5 hours). Further improvement of the fermenter performance may be achieved by strain selection.     

Scale up of fuel ethanol production from sugar beet juice using loofa sponge immobilized bioreactor

Production of fuel ethanol from sugar beet juice, using cells immobilized on loofa sponge was investigated. Based on ethanol productivity and ease of cell immobilization, a flocculating yeast strain, Saccharomyces cerevisiae IR2 was selected for ethanol production from sugar beet juice. It was found that raw sugar beet juice was an optimal substrate for ethanol production, requiring neither pH adjustment nor nitrogen source supplement. When compared with a 2 l bubble column bioreactor, mixing was not sufficient in an 8 l bioreactor containing a bed of sliced loofa sponges and consequently, the immobilized cells were not uniformly distributed within the bed. Most of the cells were immobilized in the lower part of the bed and this resulted in decreased ethanol productivity. By using an external loop bioreactor, constructing the fixed bed with cylindrical loofa sponges, dividing the bed into upper, middle and lower sections with approximately 1 cm spaces between them and circulating the broth through the loop during the immobilization, uniform cell distribution within the bed was achieved. Using this method, the system was scaled up to 50 l and when compared with the 2 l bubble column bioreactor, there were no significant differences (P > 0.05) in ethanol productivity and yield. By using external loop bioreactor to immobilize the cells uniformly on the loofa sponge beds, efficient large scale ethanol production systems can be constructed.     

On-line monitoring and controlling system for fermentation processes

A personal computer-based on-line monitoring and controlling system was developed for the fermentation of microorganism. The on-line HPLC system for the analysis of glucose and ethanol in the fermentation broth was connected to the fermenter via an auto-sampling equipment, which could perform the pipetting, filtration and dilution of the sample and final injection onto the HPLC through automation based on a programmed procedure. The A/D and D/A interfaces were equipped in order to process the signals from electrodes and from the detector of HPLC, and to direct the feed pumps, the motor of stirrer and gas flow-rate controller. The software that supervised the control of the stirring speed, gas flow-rate, pH value, feed flow-rate of medium, and the on-line measurement of glucose and ethanol concentration was programmed by using Microsoft Visual Basic under Microsoft Windows. The signal for chromatographic peaks from on-line HPLC was well captured and processed using an RC filter and a smoothing algorithm. This monitoring and control system was demonstrated to be effective in the ethanol fermentation of Zymomonas mobilis operated in both batch and fed-batch modes. In addition to substrate and product concentrations determined by on-line HPLC, the biomass concentration in Z. mobilis fermentation could also be on-line estimated by using the pH control and an implemented software sensor. The substrate concentration profile in the fed-back fermentation followed well the set point profile due to the fed-back action of feed flow-rate control.     

Ethanol from Whey: Continuous Fermentation with a Catabolite Repression-Resistant Saccharomyces cerevisiae Mutant

An alternative method for the conversion of cheese whey lactose into ethanol has been demonstrated. With the help of continuous-culture technology, a catabolite repression-resistant mutant of Saccharomyces cerevisiae completely fermented equimolar mixtures of glucose and galactose into ethanol. The first step in this process was a computer-controlled fed-batch operation based on the carbon dioxide evolution rate of the culture. In the absence of inhibitory ethanol concentrations, this step allowed us to obtain high biomass concentrations before continuous fermentation. The continuous anaerobic process successfully incorporated a cell-recycle system to optimize the fermentor productivity. Under conditions permitting a low residual sugar concentration (≤1%), maximum productivity (13.6 g liter⁻¹ h⁻¹) was gained from 15% substrate in the continuous feed at a dilution rate of 0.2 h⁻¹. Complete fermentation of highly concentrated feed solutions (20%) was also demonstrated, but only with greatly diminished fermentor productivity (5.5 g liter⁻¹ h⁻¹).     

High ethanol productivities using small Ca-alginate beads of immobilized cells of Zymomonas mobilis

Summary Zymomonas mobilis cells were immobilized into small 1 mm diameter beads of Ca-alginate in order to minimize mass transfer limitations and maximize immobilized cell activity. A combination of small bead size with a high cell concentration of 58 g dry wt. cell per lit. bead volume resulted in high ethanol productivities using a newly designed packed bed bioreactor system. Steady-state dilution rates ranging from 0.4 h^-1 to 3.9 h^-1 were run resulting in a maximum productivity of 102 g ethanol/l/h for an inlet substrate concentration of 100 g glu/l and 87% conversion. The bioreactor was run continuously at a fixed dilution rate for 384 h and short intermittent treatment of the beads with CaCl₂ temporarily increased ethanol productivity to a maximum of 116 g ethanol/l/h.     

Biological hydrogen production by immobilized cells of Clostridium tyrobutyricum JM1 isolated from a food waste treatment process

A fermentative hydrogen-producing bacterium, Clostridium tyrobutyricum JM1, was isolated from a food waste treating process using 16S rRNA gene sequencing and amplified ribosomal DNA restriction analysis (ARDRA). A fixed-bed bioreactor packed with polyurethane foam as support matrix for the growth of the isolate was operated at different hydraulic retention time (HRT) to evaluate its performance for hydrogen production. The reactor achieved the maximal hydrogen production rate of 7.2 l H(2)l(-1)d(-1) at 2h HRT, where hydrogen content in biogas was 50.0%, and substrate conversion efficiency was 97.4%. The maximum hydrogen yield was 223 ml (g-hexose)(-1) with an influent glucose concentration of 5 g l(-1). Therefore, the immobilized reactor using C. tyrobutyricum JM1 was an effective and stable system for continuous hydrogen production.     

Monitoring and control of Gluconacetobacter xylinus fed-batch cultures using in situ mid-IR spectroscopy

A partial least-squares calibration model, relating mid-infrared spectral features with fructose, ethanol, acetate, gluconacetan, phosphate and ammonium concentrations has been designed to monitor and control cultivations of Gluconacetobacter xylinus and production of gluconacetan, a food grade exopolysaccharide (EPS). Only synthetic solutions containing a mixture of the major components of culture media have been used to calibrate the spectrometer. A factorial design has been applied to determine the composition and concentration in the calibration matrix. This approach guarantees a complete and intelligent scan of the calibration space using only 55 standards. This calibration model allowed standard errors of validation (SEV) for fructose, ethanol, acetate, gluconacetan, ammonium and phosphate concentrations of 1.16 g/l, 0.36 g/l, 0.22 g/l, 1.54 g/l, 0.24 g/l and 0.18 g/l, respectively. With G. xylinus, ethanol is directly oxidized to acetate, which is subsequently metabolized to form biomass. However, residual ethanol in the culture medium prevents bacterial growth. On-line spectroscopic data were implemented in a closed-loop control strategy for fed-batch fermentation. Acetate concentration was controlled at a constant value by feeding ethanol into the bioreactor. The designed fed-batch process allowed biomass production on ethanol. This was not possible in a batch process due to ethanol inhibition of bacterial growth. In this way, the productivity of gluconacetan was increased from 1.8 x 10(-3) [C-mol/C-mol substrate/h] in the batch process to 2.9 x 10(-3) [C-mol/C-mol substrate/h] in the fed-batch process described in this study.     

On-line control of fed-batch fermentation of dilute-acid hydrolyzates

Dilute-acid hydrolyzates from lignocellulose are, to a varying degree, inhibitory to yeast. In the present work, dilute-acid hydrolyzates from spruce, birch, and forest residue, as well as synthetic model media, were fermented by Saccharomyces cerevisiae in fed-batch cultures. A control strategy based on on-line measurement of carbon dioxide evolution (CER) was used to control the substrate feed rate in a lab scale bioreactor. The control strategy was based solely on the ratio between the relative increase in CER and the relative increase in feed rate. Severely inhibiting hydrolyzates could be fermented without detoxification and the time required for fermentation of moderately inhibiting hydrolyzates was also reduced. The feed rate approached a limiting value for inhibiting media, with a corresponding pseudo steady-state value for CER. However, a slow decrease of CER with time was found for media containing high amounts of 5-hydroxymethyl furfural (HMF). The success of the control strategy is explained by the conversion of furfural and HMF by the yeast during fed-batch operation. The hydrolyzates contained between 1.4 and 5 g/l of furfural and between 2.4 and 6.5 g/l of HMF. A high conversion of furfural was obtained (between 65-95%) at the end of the feeding phase, but the conversion of HMF was considerably lower (between 12-40%).     

On-line estimation of sugar concentration for control of fed-batch fermentation of lignocellulosic hydrolyzates by Saccharomyces cerevisiae

A feed control strategy, based on estimated sugar concentrations, was developed with the purpose of avoiding severe inhibition of the yeast Saccharomyces cerevisiae during fermentation of spruce hydrolyzate. The sum of the fermentable hexose sugars, glucose and mannose, was estimated from on-line measurements of carbon dioxide evolution rate and biomass concentration by use of a simple stoichiometric model. The feed rate of the hydrolyzate was controlled to maintain constant sugar concentration during fed-batch fermentation, and the effect of different set-point concentrations was investigated using both untreated and detoxified hydrolyzates. The fed-batch cultivations were evaluated with respect to cellular physiology in terms of the specific ethanol productivities, ethanol yields, and viability of the yeast. The simple stoichiometric model used resulted in a good agreement between estimated sugar concentrations and off-line determinations of sugar concentrations. Furthermore, the control strategy used made it possible to maintain a constant sugar concentration without major oscillations in the feed rate or the sugar concentration. For untreated hydrolyzates the average ethanol productivity could be increased by more than 130% compared to batch fermentation. The average ethanol productivity was increased from 0.12 to 0.28 g/g h. The productivity also increased for detoxified hydrolyzates, where an increase of 16% was found (from 0.50 to 0.58 g/g h).     

Ethanol fermentation by flocculating yeast: Performance and stability dependence on a critical fermentation rate

Summary A system coupling fermentor and decantor permitted strong accumulation of yeast flocs that were homogeneously suspended in the reactional volume. At 100–190 g/l glucose feed practically total substrate conversion was attained. At 130 g/l glucose feed the highest productivity (18.4 g.l.h) and the highest ethanol yield (90.6%) were reached with biomass levels of 80–90 g/l. We observed that the stability of this system is limited when a critical fermentation rate (D.S_o) close to 39–40 g/l.h (with corresponding ethanol productivities of 19–20 g/l.h) is reached. Higher fermentation rates provoked de-flocculation and lost of biomass.Symbols D dilution rate (h^–1) - E ethanol (g/l) - S_r residual substrate (g/l) - S_o substrate in the feed (g/l) - X biomass (g/l) - ethanol yield (%) - DS_o fermentation rate (g/l.h) (for S_r0) - P_E ethanol productivity (g/l.h)     

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.     

Continuous conversion of D-xylose to ethanol by immobilized pachysolen tannophilus

The yeast Pachysolen tannophilus was entrapped in calcium alginate beads to ferment D-xylose on a continous basis in the presence of high cell densities. Experimental operating variables included the feed D-xylose concentration, the dilution rate, and the fermentor biomass concentration. Under favorable operating conditions, cultures retained at least 50% of their initial productivity after 26 days of operation. The specific ehanol production rate was dependent on the substrate level in the fermentor, passing through an optimum when the D-xylose concentration was between 28 and 35 g/L. Consequently, reactor productivity increased with dilution rate and feed D-xylose concentration until a maximum was reached. The ethanol content of the effluent always decreased with increasing dilution rate, but excessive dilution rates diminished the ethanol content without increasing productivity. Unlike production rate, ethanol yield declined monotonically from 0.35 g/g as the fermentor substrate concentration increased. The yield was 69% of that theoretically possible when the D-xylose concentration was near zero, as opposed to 42% when it was in the range supporting the optimum specific rate of ethanol production. As long as D-xylose was supplied to cells faster than they could consume it, productivity increased with the mass of cells immobilized. The effectiveness factor associated with the calcium alginte beads used in this system was 0.4, indicating that only 40% of the entrapped biomass was effective in converting D-xylose to ethanol because of diffusion limitations.