Direct alcoholic fermentation of starchy biomass using amylolytic yeast strains in batch and immobilized cell systems

Summary Direct alcoholic fermentation of dextrin or soluble starch with selected amylolytic yeasts was studied in both batch and immobilized cell systems. In batch fermentations, Saccharomyces diastaticus was capable of fermenting high dextrin concentrations much more efficiently than Schwanniomyces castellii. From 200 g·l^–1 of dextrin S. diastaticus produced 77 g·l^–1 of ethanol (75% conversion efficiency). The conversion efficiency decreased to 59% but a higher final ethanol concentration of 120 g·l^–1 was obtained with a medium containing 400 g·l^–1 of dextrin. With a mixed culture of S. diastaticus and Schw. castellii 136 g·l^–1 of ethanol was produced from 400 g·l^–1 of dextrin (67% conversion efficiency). S. diastaticus cells attached well to polyurethane foam cubes and a S. diastaticus immobilized cell reactor produced 69 g·l^–1 of ethanol from 200 g·l^–1 of dextrin, corresponding to an ethanol productivity of 7.6g·l^–1·h^–1. The effluent from a two-stage immobilized cell reactor with S. diastaticus and Endomycopsis fibuligera contained 70 g·l^–1 and 80 g·l^–1 of ethanol using initial dextrin concentrations of 200 and 250 g·l^–1 respectively. The corresponding values for ethanol productivity were 12.7 and 9.6 g·l^–1·h^–1. The productivity of the immobilized cell systems was higher than for the batch systems, but much lower than for glucose fermentation.     

The production of 2,3-butanediol by fermentation of high test molasses

Summary Klebsiella oxytoca fermented 199 g·l^–1 high test or invert molasses using batch fermentation with substrate shift to produce 95.2–98.6 g 2,3-butanediol·l^–1 and 2,4–4.3 g acetoin·l^–1 with a diol yield of 96–100% of the theoretical value and a diol productivity of 1.0–1.1 g·l^–1·h^–1. Fermentation was performed numerous times with molasses in repeated batch culture with cell recovery. Such repeated batch fermentation, in addition to a high product yield, also showed a very high product concentration. For example, 118 g 2,3-butanediol·l^–1 and 2.3 g acetoin·l^–1 were produced from 280 g·l^–1 of high test molasses. The diol productivity in this fermentation amounted to 2.4 g·l^–1·h^–1 and can undoubtedly be further increased by increasing the cell concentration. Because the Klebsiella cultures ferment 2,3-butanediol at an extremely high rate once the sugar has been consumed, the culture was inhibited completely by the addition of 15 g ethanol·l^–1 and switching off aeration. Offprint requests to: A. S. Afschar     

Ethanol formation from cellulose by thermophilic bacteria

Summary A wild coculture of obligately thermophilic bacteria, including only a single cellulolytic species Clostridium, ferments 2% crystalline cellulose and produces 4.6–5.1 g·l^–1 of ethanol at 55°–60° C; that is, 0.96–1.1 moles of ethanol from 1 mole of glucose equivalent of cellulose degraded. However, the ethanol yield decreases with increasing cellulose concentration. Ethanolacetic acid ratio varies around 1 and cannot be influenced by substrate concentration. However, this ratio can be influenced by changing pH and temperature. For the ethanol production from cellulose, neutral and weekly alkaline media with a pH of 7.0–8.0 and a temperature of 55° C are optimal. Experiments in which the coculture was subjected to high ethanol concentrations showed that higher concentrations of added ethanol (up to 20 g·l^–1) suppress cellulose degradation by 50% and inhibit the actual production of ethanol.     

Alcoholic fermentation of glucose and xylose by Pichia stipitis,Candida shehatae,Saccharomyces cerevisiae and Zymomonas mobilis: oxygen requirement as a key factor

Summary To investigate simultaneous alcoholic fermentation of glucose and xylose derived from lignocellulosic material by separate or co-culture processes, the effect of oxygen transfer rate (OTR) on the fermentation of 50 g/l xylose by Pichia stipitis NRRL Y 7124 and Candida shehatae ATCC 22984, and the fermentation of 50 g/l glucose by Saccharomyces cerevisiae CBS 1200 and Zymomonas mobilis ATCC 10988 was carried out in batch cultures. The kinetic parameters of the xylose-fermenting yeasts were greatly dependent on the OTR. The optimum OTR values were found to be 3.9 and 1.75 mmol·1^–1·h^–1 for C. shehatae and P. stipitis, respectively. By contrast the fermentative parameters of S. cerevisiae were poorly affected by the OTR range tested (0.0–3.5 mmol·l^–1·h^–1) Under these conditions the ethanol yields ranged from 0.41 g·g^–1 to 0.45 g·g^–1 and the specific ethanol productivity was around 0.70 g·g^–1·h^–1. Z. mobilis gave the highest fermentative performance under strictly anaerobic conditions (medium continually flushed with nitrogen): under these conditions, the ethanol yield was 0.43 g·g^–1 and the average specific ethanol productivity was 2.3 g·g^–1·h^–1. Process considerations in relation to the effect of OTR on the fermentative performance of the tested strains are discussed. Offprint requests to: J. P. Delgenes     

Continuous ethanol fermentation using flocculent yeast entrapped in Horizontal Parallel Flow bioreactor system

Summary Continuous ethanol fermentations were conducted in single-stage and three-stage Horizontal Parallel Flow (HOPAF) bioreactor systems. Biological entrapment of yeast could be achieved by virtue of its growth and flocculence in reusable porous stainless steel fiber sheets. Twenty-five g·l^–1·h^–1 productivity was obtained in three-stage system. Distributions of ethanol and glucose in reactors were examined.     

Studies on the variables and kinetics of SCP production from nopal fruit juice

Summary Submerged batch cultivation under controlled environmental conditions of pH 3.8, temperature 30°C, and K_La200 h^–1 (above 180 mMO₂ l ^–1 h^–1 oxygen supply rate) produced a maximum (12.0 g·l ^–1) SCP (Candida utilis) yield on the deseeded nopal fruit juice medium containing C/N ratio of 7.0 (initial sugar concentration 25 g·l ^–1) with a yield coefficient of 0.52 g cells/g sugar. In continuous cultivation, 19.9 g·l ^–1 cell mass could be obtained at a dilution rate (D) of 0.36 h^–1 under identical environmental conditions, showing a productivity of 7.2 g·l ^–1·h^–1. This corresponded to a gain of 9.0 in productivity in continuous culture over batch culture. Starting with steady state values of state variables, cell mass (C_X–19.9 g·l ^–1), limiting nutrient concentration (C_ln–2.5 g·l ^–1) and sugar concentration (C_S–1.5 g·l ^–1) at control variable conditions of pH 3.8, 30°C, and K_La 200 h^–1 keeping D=0.36 h^–1 as reference, transient response studies by step changes of these control variables also showed that this pH, temperature and K_La conditions are most suitable for SCP cultivation on nopal fruit juice. Kinetic equations obtained from experimental data were analysed and kinetic parameters determined graphically. Results of SCP production from nopal fruit juice are described.Nomenclature C_ln concentration of ammonium sulfate (g·l ^–1) - C_S concentration of total sugar (g·l ^–1) - C_X cell concentration (g·l ^–1) - D dilution rate (h^–1) - K_ln Monod's constant (g·l ^–1) - m maintenance coefficient (g ammonium sulfate cell^–1 h^–1) - m_(S) maintenance coefficient (g sugar g cell^–1 h^–1) - t time, h - Y yield coefficient (g cells/g ammonium sulfate) - Y_m maximum of Y - Y_S yield coefficient based on sugar consumed (g cells · g sugar^–1) - Y_S(m) maximum value of Y_S - ^µm maximum specific growth rate constant (h^–1)     

Nutritional and hormonal requirements of Ginkgo biloba embryo-derived callus and suspension cell culture

Calli were obtained from Ginkgo biloba embryos grown on Murashige and Skoog (MS) medium. The G. biloba cells could grow on either MS or Gamborg B5 mineral salt medium supplemented with sucrose (3% and 2%, respectively) and naphthaleneacetic acid (NAA) and kinetin (K) in concentrations ranging from 0.1 to 2.0 mg·L^–1. Best growth and maintenance of callus cultures were achieved using MS medium supplemented with 2 mg·L^–1 NAA and 1 mg·L^–1 K (N₂K₁MS). Light was required to maintain healthy growth of the callus tissue.In both MS and B5 based media, sucrose was hydrolyzed extracellularly before being taken up by Ginkgo cell suspension cultures. Specific growth rates of 0.13 d^–1 and 0.08 d^–1 were obtained in MS medium supplemented with 1 mg·L^–1 NAA, 0.1 mg·L^–1 K and 30 g·L^–1 sucrose (N₁K_0.1MS) and B5 medium supplemented with the same growth regulator regime and 20 g·L^–1 sucrose (N₁K_0.1B5) respectively. Complete phosphate and ammonium uptake was observed in 11 days when cultured in MS medium and 10 days and 4 days respectively when cultured in B5 medium. During the culture, G. biloba cells consumed only 64% and 29% of the nitrate content of N₁K_0.1MS and N₁K_0.1B5 media respectively. Maximum dry biomass concentrations were 13.4 g·L^–1 and 7.9 g·L^–1, and yields on carbohydrate were 0.39 and 0.45 in N₁K_0.1MS and N₁K_0.1B5 media respectively. The better performance of MS cultures came from the higher sucrose and nitrogen salts concentrations of this medium.Abbreviations B5 Gamborg mineral salt medium - d.w. Dry weight - K Kinetin - MS Murashige and Skoog mineral salt medium - N or NAA Naphthaleneacetic acid - N_iK_jMS i and j are the respective concentrations (mg·L^–1) of NAA and K - n Number of experimental points - r Linear regression correlation coefficient     

Evaluation of solvents for extractive butanol fermentation with Clostridium acetobutylicum and the use of poly(propylene glycol) 1200

Summary In an effort to improve the viability of acetone-butanol-ethanol fermentation by extractive fermentation, 63 organic solvents, including alkanes, alcohols, aldehydes, acids, and esters, were experimentally evaluated for biocompatibility with Clostridium acetobutylicum by observing gas evolution from cultures in contact with candidate solvents. Thirty-one of these solvents were further tested to determine their partition coefficient for butanol in fermentation medium. The biocompatible solvent with the highest partition coefficient for butanol (4.8), was poly(propylene glycol) 1200, which was selected for fermentation experiments. This is the highest partition coefficient reported to date for a biocompatible solvent. Extractive fermentations using concentrated feeds were observed to produce up to 58.6 g·l^–1 acetone and butanol in 202 h, the equivalent of three control fermentations in a single run. Product yields (based on total solvent products and glucose consumed) of 0.234 g·g^–1 to 0.311 g·g^–1 and within run solvent productivities of 0.174 g·l^–1·h^–1 to 0.290 g·l^–1·h^–1 were consistentwith conventional fermentations reported in the literature. The extended run-time of the fermentation resulted in an overall improvement in productivity by reducing the fraction of between-run down-time for fermentor cleaning and sterilization.     

Factors which influence the sedimentation characteristics of Torulopsis glabrata after growth in a recirculation system

Summary Some environmental affects on cell aggregation described in the literature are briefly summarized. By means of a biomass recirculation culture (Contact system), using the yeast Torulopsis glabrata, the aggregation behavior of cells in static and in dynamic test systems is described. Sedimentation times required to obtain 50 g · l^–1 yeast dry matter in static systems were always higher than in dynamic ones.In addition to, influencing the biomass yield, the specific growth rate of the yeast also affected cell aggregation. The specific growth rate and therefore the aggregation could be regulated by the biomass recirculation rate as well as by the sedimenter volume.Abbreviations f_o Overflow flow rate (l·h^–1) - f_R Recycle flow rate (l·h^–1) - f_t0t Total flow rate through the fermenter (l·h^–1) - g Gram - h Hour - D_R Fermenter dilution rate due to recycle (h^–1) - D_S Fermeter dilution rate due to substrate (h^–1) - D_tot Total fermenter dilution rate (h^–1) - l Liter - Specific growth rate (h^–1) - P_F Fermenter productivity (g·l^–1·h^–1) - P_FS Overall productivity (g·l^–1·h^–1) - R_pM Rates per minute - RS Residual sugar content in the effluent with respect to the substrate concentration (%) - Y Yield of biomass with respect to sugar concentration (%) - Sed 50 Sedimentation time to reach a YDM of 50 g·l^–1 (min) - V Volume (l) - V_F Fermenter volume (l) - V_Sed Sedimenter volume (l) - VVM Volumes per volume and minute - X_F YDM in the fermenter (g·l^–1) - X_F YDM in the recycle (g·l^–1) - X_S Yeast dry matter due to substrate concentration (g·l^–1) - YDM Yeast dry matter (g·l^–1)     

Continuous ethanol production by Zymomonas mobilis in a fluidized bed reactor. Part II: Process development for the fermentation of hydrolysed B-starch without sterilization

A continuous fluidized bed reactor operation system has been developed for ethanol production by Zymomonas mobilis using hydrolysed B-starch without sterilization. The operation system consists of two phases. In the first phase macroporous glass carriers in a totally mixed fluidized bed reactor were filled up totally with a monoculture of Z. mobilis by fast computer-controlled colonization, so that in the subsequent production phase no contaminants, especially lactic-acid bacteria, could penetrate into the carrier beads. In the production phase the high concentration of immobilized Z. mobilis cells in the fluidized bed reactor permits unsterile fermentation of hydrolysed B-starch to ethanol at short residence times. This results in wash-out conditions for contaminants from the substrate. Long-term experimental studies (more than 120 days) of unsterile fermentation of hydrolysed B-starch in the laboratory fluidized bed reactor (2.2 l) demonstrated stable operation up to residence times of 5 h. A semi-technical fluidized bed reactor plant (cascade of two fluidized bed reactors, each 55 l) was operated stably at a mean residence time of 4.25 h. Glucose conversion of 99% of the unsterile hydrolysed B-starch was achieved at 120 g glucose/l^–1 in the substrate, resulting in an ethanol concentration of 50 g·l^–1 and an ethanol space-time yield of 13 g·l^–1·h^–1. This is a factor of three compared to ethanol fermentation of hydrolysed B-starch with Z. mobilis in a continuous stirred tank reactor, which can only be operated stably under sterile conditions. Correspondence to: D. Weuster-Botz     

Production of acetone,butanol and ethanol (ABE) from potato wastes: fermentation with integrated membrane extraction

The technical possibilities of the microbial production of acetone, butanol and ethanol (ABE) from potato waste using in-line solvent recovery, are evaluated. Clostridium acetobutylicum DSM 1731 produces up to 20 g·l^–1 of solvents when grown on a medium containing 14% (w/v) potato powder. Using a polypropylene perstraction system and a oleyl alcohol/decane mixture as the extractant, the product yield (based on total solvents and potato dry weight) increased from 0.13 g·g^–1 to 0.23 g·g^–1. The recovery system worked well for 50 h, after which membrane fouling frustrated proper operation. In the second system a microfiltration step was incorporated whereas the solvents were extracted through a hydrophilic membrane using fatty acid methyl esters from sunflower oil as an extractant. This process configuration resulted in a comparable increase of ABE production. Correspondence to: G. Eggink     

The role of acids on the production of acetone and butanol by Clostridium acetobutylicum

Summary The production of solvent by Clostridium acetobutylicum was studied, using fed-batch fermentations. Different specific rates of carbohydrate utilisation were obtained by variations in feeding rates of sugar. At slow catabolic rates of sugar, addition of acetic acid or butyric acid, alone or together, increased the rate of the metabolic transition by a factor 10 to 20, the amount of solvents by a factor 6 and the percentage of fermented glucose to solvents by a factor 3. The same results were obtained with both glucose and xylose fermentations. Depending on the rates of growth, butanol production began at acid levels of 3–4 g·l^-1 for fast metabolism and at acid levels of 8–10 g·l^-1 for slow metabolism. Associated with slow metabolism, reassimilation of acids required values as high as 6.5 g·l^-1 of acetic acid and 7.5 g·l^-1 of butyric acid. At a high rate of metabolism, acetic and butyric acids were reassimilated at concentrations of 4.5 g·l^-1.     

Production of ψ-linolenic acid by the fungus Mucor rouxii on cheap nitrogen and carbon sources

Summary The production of -linolenic acid (GLA) by the fungus Mucor rouxii CBS 416.77 was studied on low budget nitrogen and carbon sources, i.e. rape meal, cocos expeller and two types of yeast extract (nitrogen sources), and starch, starch hydrolysate, beet molasses and cocos expeller (carbon sources). As references, Difco yeast extract and glucose were used. In flask cultivations the three yeast extracts were fully interchangeable, while the Difco yeast extract (the most expensive of those tested) gave a higher productivity of GLA in fermentor cultures (14 mg·l^–1·h^–1). The yield of lipids and GLA were increased in the order yeast extract < rape meal < cocos expeller. Thus the amount of lipid increased from 0.56 to 2.8 g·l^–1, and that of GLA from 0.15 to 0.33 g·l^–1. Use of beet molasses or cocos expeller as carbon sources gave poor growth. Starch and starch hydrolysate resulted in better productivity of GLA than glucose (4.7 and 4.9 compared to 3.4 mg·l^–1·h^–1). Offsprint requests to: A.-M. Lindberg     

Application of a membrane recycle bioreactor for continuous ethanol production

Summary A series of continuous fermentations were carried out with a production strain of the yeast Saccharomyces cerevisiae in a membrane bioreactor. A membrane separation module composed of ultrafiltration tubular membranes retained all biomass in a fermentation zone of the bioreactor and allowed continuous removal of fermentation products into a cell-free permeate. In a system with total (100%) cell recycle the impact of fermentation conditions [dilution rate (0.03–0.3 h^–1); substrate concentration in the feed (50–300 g·1^–1); biomass concentration (depending on the experimental conditions)] was studied on the behaviour of the immobilized cell population and on ethanol formation. Maximum ethanol productivity (15 g·1^–1·h^–1) was attained at an ethanol concentration of 81 g·1^–1. The highest demands of cells for maintenance energy were found at the maximum feed substrate concentration (300 g·1^–1) and at very low concentrations of cells in the broth.     

Oxygen availability for bacterial growth with a flow microcalorimeter

Summary The enthalpy change associated with aerobic growth of E. coli K₁₂ on minimal media with succinic acid as sole carbon and energy source, determined by flow microcalorimetry (with aerobic mixing cell) was 733.01±15.32 kJ·mol^–1. Molar growth yield was 39.6±1.2 g·mol^–1. When the microcalorimetric growth was limited by oxygen supply, the power-time curve was altered and the total heat evolved was less than the enthalpy change. The maximum thermal output corresponding to a fully aerobic growth in the calorimetric cell was 1.89×10^–3 W·ml^–1. Thus, the oxygen uptake rate was about 0.39 ml O₂·h^–1·ml^–1.     

Continuous ethanol production by Zymomonas mobilis in a fluidized bed reactor. Part I. Kinetic studies of immobilization in macroporous glass beads

A model has been developed to calculate the ethanol production in a well-mixed fluidized bed reactor. This model takes into account diffusion and the reaction inside porous glass beads and the activity of suspended cells in the fluidized bed reactor. The associated model parameters have been determined from the literature and by kinetic studies with Zymomonas mobilis in a continuous stirred tank reactor. The model permits good predictions of steady-state data in a fluidized bed reactor at residence times longer than 1–1.5 h. The immobilization of Z. mobilis in a fluidized bed reactor results in high ethanol space-time yields of more than 50 g·^–1·h^–1 at a glucose conversion of 80% (glucose in substrate: 120 gl^–1). At 99% conversion a space-time yield of 30 g·^–1·^–1 can be achieved when two fluidized bed reactors operate as cascade.     

Seasonal changes in sea-water tolerance of Arctic charr (Salvelinus alpinus)

Summary Groups of Arctic charr,Salvelinus alpinus, which had been acclimated to water with a salinity of 7 g·l^–1 and natural temperature and photoperiod, were exposed to water with different salinities and temperatures in June, September and February. At a salinity of 15 g·l^–1, plasma osmolality, plasma Na⁺, Cl^–, Mg²⁺ concentrations and the activity of gill Na-K-ATPase were stable, irrespective of temperature and season. In June, the charr were able to regulate blood plasma ionic levels within narrow limits when exposed to a salinity of 34 g·l^–1 (sea water) and a temperature of 8°C. The hypo-osmoregulatory capacity was less, but sufficient if the temperature was only 1°C during the seawater exposure. At the start of the experiment, the gill Na-K-ATPase activity was significantly higher in June than corresponding enzyme activities in September and February. Furthermore, an increase in gill Na-K-ATPase activity during the seawater exposure (8°C) was seen in June. Irrespective of ambient temperature and salinity, no fish died during the June experiments. In September and February, exposure to sea water produced marked increases in plasma osmolality and plasma ion concentrations. There were no changes in gill Na-K-ATPase activity. Consequently, the fish became dehydrated and were moribund after a short period of seawater exposure. Highest mortality was recorded when charr were exposed to winter sea conditions (34 g·l^–1 and 1°C) in February. The results indicate that an increase in daylength induce a hypo-osmoregulatory capacity in the Arctic charr during summer. In fall and winter, however, reduced daylength are accompanied by poor hypo-osmoregulatory capacity. This leads to high mortality as a result of increased electrolyte levels and dehydration.     

Cell recovery by continuous flotation

Summary Cell recovery by means of continuous flotation of the Hansenula polymorpha cultivation medium without additives was investigated as a function of the cultivation conditions as well as of the flotation equipment construction and flotation operational parameters. The cell enrichment and separation is improved at high liquid residence times, high aeration rates, small bubble sizes, increasing height of the aerated column, and diameter of the foam column. Increasing cell age and cultivation with nitrogen limitation reduce the cell separation.Symbols C_P cell mass concentration in medium g·l^–1 - C_R cell mass concentration in residue g·l^–1 - C_S cell mass concentration in foam liquid g·l^–1 - V equilibrium foam volume cm³ - V gas flow rate through the aerated liquid column cm³·s^–1 - V_F feed rate to the flotation column ml/min - ¹ V _S/V foaminess s - mean liquid residence time in the column s     

Isolation and physiological study of an amylolytic strain of Lactobacillus plantarum

Summary An amylolytic lactic acid bacterium identified as Lactobacillus plantarum was isolated from cassava roots (Manihot esculenta var. Ngansa) during reting. The amylolytic enzyme synthesized was an extracellular -amylase with an optimum pH of 5.0 and an optimum temperature of 55° C. Cultured on starch, the strain displayed a growth rate of 0.43 h^–1, a biomass yield of 0.19 g·g^–1 and a lactate yield of 0.81 g·g^–1. The growth kinetics were similar on starch and glucose. Sufficient enzyme was synthesized and starch hydrolysis was not a limiting factor for growth. Biosynthesis of the enzyme was observed when the glucose concentration was less than 6.7 g·l^–1 and reached up to 4 IU·ml^–1 at the end of the fermentation. Offprint requests to: M. Raimbault     

Fermentation of glycerol to 1,3-propanediol in continuous cultures of Citrobacter freundii

The conversion of glycerol to 1,3-propanediol by Citrobacter freundii DSM 30040 was optimized in single- and two-stage continuous cultures. The productivity of 1,3-propanediol formation was highest under glycerol limitation and increased with the dilution rate (D) to a maximum of 3.7 g·l^–1·h^–1. Glycerol dehydratase seemed to be the rate-limiting step in 1,3-propanediol formation. Conditions for the two-stage fermentation process were as follows: first stage, glycerol limitation (250mM), pH 7.2, D=0.1 h^–, 31° C; second stage, additional glycerol, pH 6.6, D=0.05 h^–1, 28° C. Under these conditions 875mM glycerol were consumed, the final 1,3-propanediol concentration was 545mM, and the overall productivity 1.38 g·1^–1·h^–1. Correspondence to: G. Gottschalk