Comparison of free and immobilizedCephalosporium acremonium for β-lactam antibiotic production

Summary Cephalosporium acremonium cells were immobilized in calcium alginate beads. Immobilized cells were used to produce -lactam antibiotics in rest medium under various oxygen concentrations, and the results were compared with free cell performance. Cell growth rate of immobilized cells was 35% of the growth rate of free cells. -Lactam antibiotic production rate of immobilized cells was also limited by mass transfer of oxygen. -Lactam antibiotic production rate of immobilized cells was 70% of that of free cells at oxygen saturation condition (i.e., 0.27 mM O₂). Specific antibiotic production of immobilized cells was about 200% of that of free cells at 0.27 mM O₂.     

Production of tylosin and nikkomycin by immobilized Streptomyces cells

Summary Mycelia of Streptomyces sp. T 59-235 and Streptomyces tendae Tü 901 (producing the antibiotics tylosin and nikkomycin, resp.) were immobilized in different carriers. With both organisms best antibiotic production was observed in calcium alginate gel.Influence of aeration, cell density and flow rate on antibiotic production was investigated in batch fermentation and in a continuous system (air-bubbled reactor).In batch fermentation, immobilization prolongued the production phase from 72 to 120 h (Streptomyces T 59-235) and from 72 to 96 h (S. tendae). The relative productivity of immobilized cells was 40 to 50% compared to that of free mycelia in both cases.In continuous tylosin fermentation highest production rate was observed in a medium nearly saturated with oxygen.Nikkomycin production by immobilized S. tendae could be maintained for longer than 350 h in a continuous system. The production rate depended on cell density and flow rate of the medium. The maximum specific productivity was 100% compared to that of free mycelium in batch culture.     

Citric acid production by Aspergillus niger immobilized on polyurethane foam

Summary Citric acid was produced using Aspergillus niger immobilized on polyurethane foam in a bubble column reactor. Most of the adsorbed cells remained on the support and, as a result, high oxygen tension was maintained during the reactor operation. However, uncontrolled growth of the pellets made continuous reactor operation difficult. The citric acid productivity obtained from 15 vol.% foam particles containing immobilized cells was 0.135 g/l per hour. This productivity of immobilized cells was almost the same as that of free cells. The oxygen level dropped to half saturation in 5 days in the immobilized cell culture in contrast to 2 days in the free cell culture.     

Fermentation kinetics of free and immobilized Penicillium raistrickii able perform the 15α-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione

Growing Penicillium raistrickii i 477 cells immobilized by microencapsulation, entrapment in calcium alginate beads and photopolymerization were used for the 15α-hydroxylation of 13-ethyl-gon-4-en-3,17-dione (I) to 15α-hydroxy-13-ethyl-gon-4-en-3,17-dione (II). The immobilized cells had lower maximum specific growth rates and yield coefficients when cultivated on the carbon source glucose than the non-immobilized cells, which leads to lower volumetric productivities than the use of nonimmobilized cells. However, the cells immobilized by microencapsulation and calcium alginate entrapment showed a specific productivity equal to that of the respective non-immobilized cells based on product formation per dry biomass and time. Photommobilized cells were not able to grow in the presence of the steroid because the substrate concentrations within the polymer reached inhibiting amounts for growth and product formation. In the absence of the steroid, the growing photoimmobilized cells showed a prolonged lag-phase in comparison with the free cells.     

Can exploiting natural genetic variation in leaf photosynthesis contribute to increasing rice productivity? A simulation analysis

Rice productivity can be limited by available photosynthetic assimilates from leaves. However, the lack of significant correlation between crop yield and leaf photosynthetic rate (A) is noted frequently. Engineering for improved leaf photosynthesis has been argued to yield little increase in crop productivity because of complicated constraints and feedback mechanisms when moving up from leaf to crop level. Here we examined the extent to which natural genetic variation in A can contribute to increasing rice productivity. Using the mechanistic model GECROS, we analysed the impact of genetic variation in A on crop biomass production, based on the quantitative trait loci for various photosynthetic components within a rice introgression line population. We showed that genetic variation in A of 25% can be scaled up equally to crop level, resulting in an increase in biomass of 22–29% across different locations and years. This was probably because the genetic variation in A resulted not only from Rubisco (ribulose 1,5‐bisphosphate carboxylase/oxygenase)‐limited photosynthesis but also from electron transport‐limited photosynthesis; as a result, photosynthetic rates could be improved for both light‐saturated and light‐limited leaves in the canopy. Rice productivity could be significantly improved by mining the natural variation in existing germ‐plasm, especially the variation in parameters determining light‐limited photosynthesis.     

Optimization of fermentation conditions for gluconic acid production using Aspergillus niger immobilized on cellulose microfibrils

The optimisation of gluconic acid fermentation using immobilized Aspergillus niger on a highly porous cellulose support is described. Experimental results showing the effects of variations in oxygen partial pressure, glucose concentration and biomass concentration have been obtained with a continuous recirculation reactor. Levels of dissolved oxygen and glucose concentrations during fermentation significantly affect the production and fermentation time. The optimum biomass requirement on a porous cellulose support has been estimated to be 0.234 mg cm⁻² for efficient bioconversion. Increasing the quantum of biomass beyond this value resulted in an overgrown biofilm, which affected productivity adversely. Morphological characteristics of immobilized A. niger have also been investigated.     

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.     

Alcoholysis of olive oil for producing wax esters by intracellular lipase in immobilized fungus cells

Summary Alcoholysis of olive oil was carried out with lauryl and palmityl alcohols for producing wax esters. The reaction can be catalyzed efficiently by cell-bound lipase of Rhizopus niveous fungus cells immobilized within cellulose biomass support particles. Influences of reaction conditions such as water content, temperature, and substrate concentrations on reaction rates and yields were investigated.     

Immobilization of Penicillium chrysogenum: spore growth on Celite

Summary Penicillium chrysogenum spores have been immobilized by adsorption on two grades of wet or dry diatomaceous earth particles, Chromosorb-W and Celite R-633. Almost 90% of the spores were adsorbed within 2 h and those remaining in suspension were removed by washing to minimise the growth of free mycelia. After germination the immobilized biomass was almost independent of the spore loading on the particles and whether or not the spore suspension was added to wet or dry particles. The free biomass obtained was less than 5% of the immobilized biomass.     

Oxygen supply to immobilized cells

Summary Oxygen supply is a critical point in technical processes when aerobic cells are used in immobilized preparations. In this study p-benzoquinone is used as a substitute for oxygen in the oxidation of glycerol to dihydroxyacetone by immobilized Gluconobacter oxydans cells. The reaction rate was much higher when p-benzoquinone was used compared to when oxygen was used. In an experiment with free cells p-benzoquinone gave a rate more than four times that of oxygen, and with immobilized cells the difference was even greater. p-benzoquinone is more effective than oxygen because it gives a higher maximal reaction rate (the reason for this fact is discussed) and because it is more soluble in water than oxygen. The operational stability of the process is comparatively good. In one experiment the productivity decreased from 60 to 10 mmol/h·g over an 8-day period when p-benzoquinone was used. When oxygen was used in a similar experiment the productivity decreased from 14 to 6 mmol/h·g. The byproduct formed from p-benzoquinone, hydroquinone, can be oxidized to p-benzoquinone which can be re-used. Seven succesive regenerations of p-benzoquinone were performed without any loss of efficiency.     

Influence of agitation and aeration on growth and antibiotic production by <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis>

The effect of agitation and aeration on the growth and antibiotic production by Xenorhabdus nematophila YL001 grown in batch cultures were investigated. Efficiency of aeration and agitation was evaluated through the oxygen mass transfer coefficient (K _L a). With increase in K _L a, the biomass and antibiotic activity increased. Activity units of antibiotic and dry cell weight were increased to 232 U ml⁻¹ and 19.58 g l⁻¹, respectively, productivity in cell and antibiotic was up more than 30% when K _L a increased from 115.9 h⁻¹ to 185.7 h⁻¹. During the exponential growth phase, DO concentration was zero, the oxygen supply was not sufficient. So, based on process analysis, a three-stage oxygen supply control strategy was used to improved the DO concentration above 30% by controlling the agitation speed and aeration rate. The dry cell weight and activity units of antibiotic were further increased to 24.22 g l⁻¹ and 249 U ml⁻¹, and were improved by 24.0% and 7.0%, compared with fermentation at a constant agitation speed and a constant aeration rate (300 rev min⁻¹, 2.5 l min⁻¹).     

Production of high-quality edible protein from Candida yeast grown in continuous culture

Candida utilis NRRL Y-900 was grown in aerobic continuous culture with cane molasses as the source of the growth-limiting carbon. At 1% reducing sugar in the chemostal (10 liter working volume) feed medium, addition of Zn (25μM) to a minimal salts medium resulted in an increase in the biomass productivity of the chemostat from 1.7 to 2.6 g/liter/hr with a growth yield of 0.55 g dry biomass/g reducing sugar utilized at D_max. On the average, the yeast biomass was 50–55% protein. At S_R > 2% sugar, the biomass productivity was limited by the oxygen supply. With O₂-supplemented aeration (at S_R = 4.2%)the maximum biomass productivity Was 7.25 g/liter/hr. Aerobic ethanol production was not observed. A highquality undenatured protein fraction was isolate from the yeast homogenate by isoelectric precipitation at pH 4.5. Contaminating nucleic acid was removed as an insoluble complex by chelation with an organic cation (cetavlon). The final protein product contained about 3% RNA (DWB) and was suitable for use as a food additive.     

Effects of metabolic inhibitors on the alcoholic fermentation by several yeasts in batch or in immobilized cell systems

Summary In previous papers it was shown that the bacterium Zymomonas mobilis might be an interesting alternative for industrial alcohol production from sugar, compared to Saccharomyces bayanus. Factors that might increase the glucose to ethanol conversion efficiency and which are in favour of the bacterium, are the production of less biomass and less by-products such as glycerol, succinic acid, butanediol, acetoin, and acetic acid. In order to reduce the synthesis of biomass three metabolic inhibitors were now studied: dinitrophenol, azide and arsenate. Their effects on the alcoholic fermentation in batch and in immobilized cell system were investigated, using three yeasts: Saccharomyces bayanus, Schizosacharomyces pombe, and Saccharomyces diastaticus. It was found that dinitrophenol in 0.1 mM concentration was effective in increasing the conversion of glucose to ethanol especially with Saccharomyces bayanus while azide in 0.1 mM concentration was better with Schizosaccharomyces pombe. In immobilized systems high steady state ethanol production from 15% glucose media was obtained by inclusion into the media of dinitrophenol or azide. Arsenate had less effect at the concentration used. Arsenate had less effect at the concentrations used. As a result ethanol productivity in g·l^-1·h^-1 was increased from around 70 in the absence of inhibitor to around 74 in the presence of dinitrophenol with Saccharomyces bayanus. With Schizosaccharomyces pombe the productivity was increased from around 65 in the absence of inhibitor to around 74 in the presence of azide. The specific ethanol productivity expressed as g ethanol formed per hour and per g viable cells was increased from 0.87 to 1.37 for Schizosaccharomyces pombe and from 1.02 to 1.66 for Saccharomyces bayanus.     

Chlortetracycline production with immobilized Streptomyces aureofaciens

Summary The semicontinuous production of chlortetracycline by immobilized cells of Streptomyces aureofaciens ATCC 10762 was compared with that of free cells. Immobilized cells transferred repeatedly to a new production medium, showed a fourfold increase in the half life time of antibiotic production.In an air bubble column a high chlortetracycline productivity was obtained with a high aeration rate.A semicontinuous production of chlortetracycline by immobilized S. aureofaciens could be improved by varying the fermentation conditions.For continuous chlortetracycline production by immobilized cells, no improvement was detected.     

Production of gluconic acid by immobilized in pumice stones mycelium of Aspergillus niger using unconventional oxygenation of culture

Gluconic acid was produced in repeated batch processes with Aspergillus niger AM-11, immobilized in pumice stone particles using an unconventional oxygenation of culture media based on the addition of H₂O₂, decomposed by catalase to O₂ and water. The highest gluconic acid productivity of 8.2 g l^–1 h^–1 was reached with 30 g immobilized mycelium per 150 ml, 10% (w/v) glucose, at 24 °C and pH 6.5, with O₂ at 100% saturation. The immobilized mycelium was successfully reused up to 8 times in 1-h batches with only a slight loss (11%) of gluconic acid productivity.     

Impact of the Invasive Alien Plant Solidago Giganteaon Primary Productivity, Plant Nutrient Content and Soil Mineral Nutrient Concentrations

Invasion by alien plants can alter ecosystem processes and soil properties. In this study, we compared aboveground productivity, nutrient pools in standing biomass and topsoil (0–0.10 m) mineral nutrient concentrations between plots invaded by Early Goldenrod (Solidago gigantea) and adjacent, uninvaded, vegetation at five sites in Belgium. The five sites were characterised by a resident perennial herbaceous vegetation and spanned a wide range in soil fertility level and floristic composition. Invaded stands consistently had higher (2–3-fold) aboveground productivity and lower mineral element concentrations in standing phytomass. Nutrient pools (calculated as concentration × phytomass) was ca. twice higher in invaded plots, suggesting that S. gigantea might enhance nutrient cycling rates. Impacts on topsoil chemistry were surprisingly modest, with slightly higher nutrient concentrations under the invader. A noticeable exception was phosphorus, which showed higher concentrations of ammonium acetate-extractable fraction in invaded plots in four of five sites. It appears that S. gigantea does not significantly contribute to nutrient uplift from deep soil layers to topsoil, possibly because it does not root much deeper compared to resident vegetation.Equally contributing authors: S. Vanderhoeven, N. Dassonville     

Substrate effects in tower loop reactors

Summary Hansenula polymorpha was cultivated in a bubble column loop bioreactor employing ethanol and/or glucose as substrates. By varying the substrate concentration, the cultivations were carried out in non-limited, substrate limited and oxygen transfer limited growth ranges. The influence of the transitions from one range to another on reactor performance (OTR,k _L a, a) and cell productivity () were investigated. When employing ethanol as a substrate, the concentration considerably influences the fluid dynamics, mass transfer phenomena and cell productivity. When employing glucose as a substrate, glucose repression occurs. At high glucose concentrations no transition into the oxygen transfer limited growth is possible. The ethanol produced during the glucose repression influences the fluid dynamics, mass transfer phenomena and productivity. With decreasing glucose concentration the glucose repression can be gradually eliminated.     

Effect of Dissolved Oxygen on Continuous Production of Methionine

Methionine production by a mutant strain of Corynebacterium lilium was studied at different dilution rates and different dissolved oxygen concentrations, based on a statistical central composite design. The effect of dissolved oxygen levels between 2 % and 60 % and dilution rate levels between 0.04 to 0.29 on continuous production of methionine was studied. The process is described by the Leudeking‐Piret model. The experimentally obtained maxima for methionine and biomass productivities, observed at the same dilution rate of 0.17 h^–1, also corresponds to the theoretical prediction based on this model. This experimental observation is further supported by the surface response prediction of a dilution rate of 0.16 h^–1 for maximum methionine productivity and 0.15 h^–1 for cell mass productivity. Also, beyond the critical value of 30 % dissolved oxygen, the apparent benefits of oxygen transfer become less significant.     

Continuous production ofl-lactic acid from whey permeate by immobilizedLactobacillus casei subsp.casei

Summary The production of l-lactic acid from whey permeate, a waste product of the dairy industry, by fermentation with the lactic acid bacterium Lactobacillus casei subsp. casei was investigated. A fermentation medium consisting of permeate and supplements, which enables exponential growth of the organisms, was developed. A fast method for determination of free and immobilized biomass in solid-rich media, based on measurement of cellular ATP, was evolved. Continuous fermentations in a stirred tank reactor (STR) and in a fluidized bed reactor (FBR) with immobilized biomass were compared. In the STR a volumetric productivity of 5.5 g/l per hour at 100% substrate conversion [dilution rate (D) = 0.22 h^–1] was determined. In the FBR porous sintered glass beads were used for immobilization and a maximum biomass concentration of 105 g/kg support was measured. A productivity of 10 g/l per hour was obtained at D = 0.4 h^–1 (substrate conversion 93%) and of 13.5 g/l per hour at D = 1.0 h^–1 (substrate conversion 50%). Offprint requests to: W. Krischke     

Plasmid inheritability and biomass production: comparison between free and immobilized cell cultures of Escherichia coli BZ18(pTG201) without selection pressure.

Maintenance of the plasmid pTG201 in Escherichia coli BZ18 was studied for both free and immobilized cells during chemostat culture, in the absence of the antibiotic against which resistance was plasmid encoded. Electron microscopic observations of immobilized proliferant cells within carrageenan gel beads showed high cell concentrations and growth into distinct cavities. The plasmid which coded for the catechol 2,3-dioxygenase activity was stably maintained during 80 generations in the case of immobilized cells. A theoretical analysis founded on the compartmentalization resulting from the immobilized growth conditions was described. However, the model still showed a plasmid stability inferior to that determined experimentally. Hypotheses dealing with physiological changes of immobilized cells were presented. In addition, the high cell concentrations obtained in the outer 50 microns of the carrageenan gel beads gave a biomass productivity within this useful volume which was 20 times higher than in free-cell cultures.