Development of an immobilized cell reactor for the production of 1,3-propanediol by Citrobacter freundii

Citrobacter freundii DSM 30040 immobilized on modified polyurethane carrier particles PUR 90/16 was used for continuous glycerol fermentation in an anaerobic fixed bed reactor with effluent recycle and pH control (fixed bed loop reactor). The fermentor was run with buffered mineral medium under growth conditions resulting in the permanent renewal of active biomass. The effects of glycerol concentration in the feed, dilution rate (D), pH and temperature (T) were investigated to optimize the process. With 400 mm glycerol in the feed, pH 6.9, T = 36°C and D = 0.5 h^–1 the maximum productivity could be determined as 8.2 g/l per hour of 1,3-propanediol.     

Fermentative production of 1,3-propanediol from glycerol by Clostridium butyricum up to a scale of 2m3

Summary The conversion of glycerol to 1,3-propanediol (PD) by Clostridium butyricum DSM 5431 was studied in anaerobic culture. Growth and product formation were optimal at pH = 7.0 and T = 35° C, while aeration rate and stirrer speed were found to have no significant influence. As increasing amounts of initial glycerol led to inhibition of growth, cultivations were done in fed-batch operation. Comparative cultivations were carried out in an air-lift (ALR) and a stirred-tank reactor (STR) having equal working volumes (V _L = 30 l) and no difference in product formation was found. The process was scaled up to reactor sizes of 1.2 m³ (ALR) and 2.0 m³ (STR). The same results were obtained irrespective of reactor volume as well as reactor type (STR/ALR). PD concentrations of approximately 50–58 g·l^–1 and overall productivities of 2.3–2.9 g·l^–1 ·h^–1 could be reached. Offprint requests to: W.-D. Deckwer     

1,3-Propanediol production in a two-step process fermentation from renewable feedstock

In this work, the production of 1,3-propanediol from glucose and molasses was studied in a two-step process using two recombinant microorganisms. The first step of the process is the conversion of glucose or other sugar into glycerol by the metabolic engineered Saccharomyces cerevisiae strain HC42 adapted to high (>200 g l⁻¹) glucose concentrations. The second step, carried out in the same bioreactor, was performed by the engineered strain Clostridium acetobutylicum DG1 (pSPD5) that converts glycerol to 1,3-propanediol. This two-step strategy led to a flexible process, resulting in a 1,3-propanediol production and yield that depended on the initial sugar concentration. Below 56.2 g l⁻¹ of sugar concentration, cultivation on molasses or glucose showed no significant differences. However, at higher molasses concentrations, glycerol initially produced by yeast could not be totally converted into 1,3-propanediol by C. acetobutylicum and a lower 1,3-propanediol overall yield was observed. In our hand, the best results were obtained with an initial glucose concentration of 103 g l⁻¹, leading to a final 1,3-propanediol concentration of 25.5 g l⁻¹, a productivity of 0.16 g l⁻¹ h⁻¹ and 1,3-propanediol yields of 0.56 g g⁻¹ glycerol and 0.24 g g⁻¹ sugar, which is the highest value reported for a two-step process. For an initial sugar concentration (from molasses) of 56.2 g l⁻¹, 27.4 g l⁻¹ of glycerol were produced, leading to 14.6 g l⁻¹ of 1.3-propanediol and similar values of productivity, 0.15 g l⁻¹ h⁻¹, and overall yield, 0.26 g g⁻¹ sugar.     

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)     

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.     

Microbial fed-batch production of 1,3-propanediol by Klebsiella pneumoniae under micro-aerobic conditions

The microbial production of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae under micro-aerobic conditions was investigated in this study. The experimental results of batch fermentation showed that the final concentration and yield of 1,3-PD on glycerol under micro-aerobic conditions approached values achieved under anaerobic conditions. However, less ethanol was produced under microaerobic than anaerobic conditions at the end of fermentation. The batch micro-aerobic fermentation time was markedly shorter than that of anaerobic fermentation. This led to an increment of productivity of 1,3-PD. For instance, the concentration, molar yield, and productivity of 1,3-PD of batch micro-aerobic fermentation by K. pneumoniae DSM 2026 were 17.65 g/l, 56.13%, and 2.94 g l^–1 h^–1, respectively, with a fermentation time of 6 h and an initial glycerol concentration of 40 g/l. Compared with DSM 2026, the microbial growth of K. pneumoniae AS 1.1736 was slow and the concentration of 1,3-PD was low under the same conditions. Furthermore, the microbial growth in fed-batch fermentation by K. pneumoniae DSM 2026 was faster under micro-aerobic than anaerobic conditions. The concentration, molar yield, and productivity of 1,3-PD in fed-batch fermentation under micro-aerobic conditions were 59.50 g/l, 51.75%, and 1.57 g l^–1 h^–1, respectively. The volumetric productivity of 1,3-PD under microaerobic conditions was almost twice that of anaerobic fed-batch fermentation, at 1.57 and 0.80 g l^–1 h^–1, respectively.     

Physiological aspects of l-lysine production: effect of nutritional limitations on a producing strain of Corynebacterium glutamicum

The effect of nutritional limitations, such as phosphorus and carbon, on the production of l-lysine by Corynebacterium glutamicum was studied in continuous culture. We observed that phosphate-limited cultures at low growth rates were favourable to l-lysine production. l-Lysine was produced when a culture at low dilution rate (0.03 h^–1) was established. A dilution rate of about 0.04 h^–1 should be maintained in order to assure good productivity and an l-lysine yield of 0.53 g/g. Under carbon-limiting conditions the maintenance energy and growth yield of 0.03 g/g·g^–1·h^–1 and 0.41 g/g, respectively, have been obtained. Under these limiting conditions the l-lysine production was not favoured even at lower dilution rates.Correspondence to: N. Coello     

Production of 1,3-Propanediol by Clostridium butyricum VPI 3266 in continuous cultures with high yield and productivity

The effects of dilution rate and substrate feed concentration on continuous glycerol fermentation by Clostridium butyricum VPI 3266, a natural 1,3-propanediol producer, were evaluated in this work. A high and constant 1,3-propanediol yield (around 0.65 mol/mol), close to the theoretical value, was obtained irrespective of substrate feed concentration or dilution rate. Improvement of 1,3-propanediol volumetric productivity was achieved by increasing the dilution rate, at a fixed feed substrate concentration of 30, 60 or 70 g l⁻¹. Higher 1,3-propanediol final concentrations and volumetric productivities were also obtained when glycerol feed concentration was increased from 30 to 60 g l⁻¹, at D=0.05–0.3 h⁻¹, and from 60–70 g l⁻¹, at D=0.05 and 0.1 h⁻¹·30 g l⁻¹ of 1,3-propanediol and the highest reported value of productivity, 10.3 g l⁻¹ h⁻¹, was achieved at D=0.30 h⁻¹ and 60 g l⁻¹ of feed glycerol. A switch to an acetate/butyrate ratio higher than one was observed for 60 g l⁻¹ of feed glycerol and a dilution rate higher than 0.10 h⁻¹; moreover, at D=0.30 h⁻¹ 3-hydroxypropionaldehyde accumulation was observed for the first time in the fermentation broth of C. butyricum.     

Conversion of glycerol to 1,3-propanediol by a newly isolated thermophilic strain

Of 60 different thermophilic enrichment cultures, 16 converted glycerol anaerobically to 1,3-propanediol. Two PD-forming strains were further enriched, isolated, and characterised. For the most active strain, AT1, the optimal cultivation parameters for pH and temperature were determined as 5.8 to 6.0 and 58°C, respectively. In batch-fermentations with AT1, 6.4 g propanediol per litre was formed with a productivity of 0.17 g l^–1 h^–1.     

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     

Synthesis of l-dopa by escherichia intermedia cells immobilized in a polyacrylamide gel

Summary Escherichia intermedia cells were immobilized by entrapment in a polyacrylamide gel and used for l-dopa synthesis from pyrocatechol, pyruvate and ammonia. An immobilized cell preparation containing 75 mg cells/g gel retained 45%–50% of the activity of free cells. The effect of temperature, pH and substrate concentration of the initial rate of l-dopa synthesis was very similar for free and immobilized cells. Substrate inhibition was observed for pyrocatechol, pyruvate and ammonia. In a batch reactor, 5.4 g·l^-1 l-dopa was obtained, with 100% conversion yield of pyrocatechol and l-dopa productivity of 0.18 g·l^-1·h^-1. The use of a pyrocatechol-borate complex decreased by-product formation and catalyst inactivation.     

Nitrite uptake by Chlamydomonas reinhardtii cells immobilized in calcium alginate

When an initial cell loading of about 30–40 µg chlorophyll (Chl)·g^–1 gel and alginate suspension of 3% (w/v) were used for immobilization of Chlamydomonas reinhardtii, the resulting cell beads showed optimum nitrite uptake rate, at 30° C and pH 7.5, of 14 µmol NO _inf2 ^sup– ·mg^–1 Chl·h^–1, the photosynthetic and respiratory activities being about 120 µmol O₂ produced·mg^–1 Chl·h^–1, and 40 µmol O₂ consumed ·mg^–1 Chl·h^–1, respectively. The nitrite uptake activity required CO₂ in the culture and persisted after 8 days of cells immobilization, or in the presence of 0.2 mm ammonium in the medium. Our data indicate that alginate-entrapped C, reinhardtii cells may provide a stable and functional system for removing nitrogenous contaminants from waste-waters.Correspondence to: C. Vílchez     

An improved screening method for microorganisms able to convert crude glycerol to 1,3-propanediol and to tolerate high product concentrations

A new screening method was developed and established to find high-performance bacteria for the conversion of crude glycerol to 1,3-propanediol. Three soil samples from palm oil-rich habitats were investigated using crude glycerol of a German biodiesel plant. Nine promising 1,3-propanediol producers could be found. Because of a special pH buffer system, a fast evaluation on microscale and high 1,3-propanediol concentrations up to 40 g L⁻¹ could be achieved. Three strains demonstrated very high product tolerance and were identified as Clostridium butyricum. Two strains, AKR91b and AKR102a, grew and produced 1,3-propanediol in the presence of 60 g L⁻¹ initial 1,3-propanediol, the strain AKR92a even in the presence of 77 g L⁻¹ 1,3-propanediol. The strains AKR91b and AKR102a tolerated up to 150 g L⁻¹ crude glycerol and produced 80% of the 1,3-propanediol attained from pure glycerol of the same concentration. Further criteria for the choice of a production strain were the pathogenicity (risk class), ability to grow on low-cost media, e.g., with less yeast extract, and robustness, e.g., process stability after several bioconversions. Overall, the strain C. butyricum AKR102a was chosen for further process optimization and scale-up due to its high productivity and high final concentration in a pH-regulated bioreactor.     

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     

Biotransformation of phenylpyruvic acid to l-phenylalanine using a strain of Pseudomonas fluorescens ATCC 11250 with high transaminase activity

The rate of l-phenylalanine production from phenylpyruvic acid by whole cells of Pseudomonas fluorescens strain ATCC 11250 was greater than 3 g·l^-1 h^-1. Synthesis of transaminase was constitutive but activity was greatest in medium containing d- or l- phenylalanine as sole nitrogen source. Maximum conversion was observed at 34–40° C and at alkaline pH, with over six times initial rate of conversion at pH 12 than at pH 5. The optimum catalyst (cell) concentration was between 10–20 mg ml^-1 dry weight. The initial rate of conversion was directly proportional to phenylpyruvate concentration, up to 4%, but the conversion yield steadily decreased between 2% and 4% substrate concentration. The rate of conversion, as expected, increased as the concentration of glutamate increased. Whole cells were still capable of over 63% conversion after 40 days providing reactions were supplemented with pyridoxal phosphate. Immobilisation of cells in calcium alginate and operation of a packed bed bioreactor enabled the continuous production of l-phenylalanine in concentrations greater than 15 g·l^-1 after 60 days operation.     

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     

Inhibition of Clostridium butyricum by 1,3-propanediol and diols during glycerol fermentation

1,3-Propanediol inhibition during glycerol fermentation to 1,3-propanediol by Clostridium butyricum CNCM 1211 has been studied. The initial concentration of the 1,3-propanediol affected the growth of the bacterium more than the glycerol fermentation. μ _max was inversely proportional to the initial concentration of 1,3-propanediol (0–65 g l⁻¹). For glycerol at 20 g l⁻¹, the growth and fermentation were completely stopped at an initial 1,3-propanediol concentration of 65 g l⁻¹. However, for an initial 1,3-propanediol concentration of 50 g l⁻¹ and glycerol at 70 g l⁻¹, the final concentration (initial and produced) of 1,3-propanediol reached 83.7 g l⁻¹(1.1 M), with complete consumption of the glycerol. Therefore, during the fermentation, the strain tolerated a 1,3-propanediol concentration higher than the initial inhibitory concentration (65 g l⁻¹). The addition of 1,2-propanediol or 2,3-butanediol (50 g l⁻¹) in the presence of glycerol (50–100 g l⁻¹), showed that 2-diols reduced the μ _max in a similar way to 1,3-propanediol. The measurement of the osmotic pressure of glycerol solutions, diols and diol/glycerol mixtures did not indicate any differences between these compounds. The hypothesis of diol inhibition was discussed. Taking into account the strain tolerance of highly concentrated 1,3-propanediol during fermentation, the fermentation processes for optimising production were considered. Received: 15 November 1999 / Revision received: 1 February 2000 / Accepted: 4 February 2000     

Purification and characterization of a high molecular mass serine carboxypeptidase from <Emphasis Type="Italic">Monascus pilosus</Emphasis>

Two serine carboxypeptidases, MpiCP-1 and MpiCP-2, were purified to homogeneity from Monascus pilosus IFO 4480. MpiCP-1 is a homodimer with a native molecular mass of 125 kDa composed of two identical subunits of 61 kDa, while MpiCP-2 is a high mass homooligomer with a native molecular mass of 2,263 kDa composed of about 38 identical subunits of 59 kDa. This is unique among carboxypeptidases and distinguishes MpiCP-2 as the largest known carboxypeptidase. The two purified enzymes were both acidic glycoproteins. MpiCP-1 has an isoelectric point of 3.7 and a carbohydrate content of 11%, while for MpiCP-2 these values were 4.0 and 33%, respectively. The optimum pH and temperature were around 4.0 and 50°C for MpiCP-1, and 3.5 and 50°C for MpiCP-2. MpiCP-1 was stable over a broad range of pH between 2.0 and 8.0 at 37°C for 1 h, and up to 55°C for 15 min at pH 6.0, but MpiCP-2 was stable in a narrow range of pH between 5.5 and 6.5, and up to 50°C for 15 min at pH 6.0. Phenylmethylsulfonylfluoride strongly inhibited MpiCP-1 and completely inhibited MpiCP-2, suggesting that they are both serine carboxypeptidases. Of the substrates tested, benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu) was the best for both enzymes. The K_m, V_max, K_cat and K_cat/K_m values of MpiCP-1 for Z-Tyr-Glu at pH 4.0 and 37°C were 1.33 mM, 1.49 mM min^–1, 723 s^–1 and 545 mM^–1 s^–1, and those of MpiCP-2 at pH 3.5 and 37°C were 1.55 mM, 1.54 mM min^–1, 2,039 s^–1 and 1,318 mM^–1 s^–1, respectively.     

Effect of growth rate on the eicosapentaenoic acid and docosahexaenoic acid content of Isochrysis galbana in chemostat culture

An isolate of Isochrysis galbana rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) has been grown as a chemostat culture at 20° C and pH 8.00 controlled by CO₂ injection. From a low dilution rate (D) of 0.0024 h^–1 to 0.0377 h^–1, close to maximum growth, a decrease in EPA content from 5.21% dry weight (d.w.) to 2.80% d.w. was observed, although the percentage of EPA in the total fatty acids increased. Lipids were fractionated, EPA being the major fatty acid found in the glycolipid fraction, whereas in the neutral lipid fraction were mainly palmitic and palmitoleic acids. At the same time, the biomass concentration also decreased from 1015 mg·l^–1 to 202 mg·l^–1 over the range of Ds mentioned. Nonetheless, EPA productivity had a maximum value of 15.26 mg·l^–1·day^–1 at D=0.0208 h^–1.Correspondence to: E. Molina Grima     

Oxygen consumption in Tardigrada from Spitsbergen

Summary Oxygen consumption was measured in seven species of Tardigrada (Doryphoribius smreczynskii, Diphascon spitzbergensis, Macrobiotus islandicus, M. echinogenitus, M. harmswothi, M. spectabilis and M. dispar) from the Spitzbergen tundar. The metabolic rate was measured at 2°, 6° and 10°C. At 6°C it ranged from 0.055 (D. smreczynskii) to 0.101 mm³·10^-3/g·10^-6h (M. harmswothi). In D. smreczynskii the phenomenon of relative thermal independence was observed at a temperature range of 2°–6°C. The dependence of oxygen consumption (R in mm³·10^-3·individ^-1·h^-1) on body weight (W in g·10^-6) for the latter species at 2°C was R=0.088·W^0.82.