Epoxidation of 1,7‐octadiene by Pseudomonas oleovorans in a membrane bioreactor

A growing cell culture of Pseudomonas oleovorans was used to biotransform 1,7‐octadiene to 1,2‐epoxy‐7,8‐octene in a continuous‐flow bioreactor with an external membrane module. A dense silicone rubber membrane was used to contact an organic phase, containing both the reactant (1,7‐octadiene) and the growth substrate (heptane), with an aqueous biomedium phase containing the biocatalyst. Heptane and octadiene delivery to the aqueous phase, and epoxide extraction into the solvent, occurred by diffusion across the dense membrane under a concentration‐driving force. In addition, a liquid feed of heptane and octadiene was pumped directly into the bioreactor to increase the rate of delivery of these compounds to the aqueous phase. In this system 1,2‐epoxy‐7,8‐octene accumulated in a pure solvent phase, thus, product recovery problems associated with emulsion formation were avoided. Furthermore, no phase breakthrough of either liquid across the membrane was observed. In this system, the highest volumetric productivity obtained was 30 U.L⁻¹, and this was achieved at a dilution rate of 0.07 h⁻¹, 70 m².m⁻³ of membrane area, and a steady‐state biomass concentration of 2.5 g.L⁻¹. The system was stable for over 1250 h. Decreasing the dilution rate led to an increased biomass concentration, however, the specific activity was significantly reduced, and therefore, an optimal dilution rate was determined at 0.055 h⁻¹. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 601–611, 1999.     

Synthesis of enantiopure (5R)-hydroxyhexane-2-one with immobilised whole cells of Lactobacillus kefiri

Whole-cell reduction of (2,5)-hexanedione to yield highly enantiopure (5R)-hydroxyhexane-2-one (enantiomeric excess >99%) with Lactobacillus kefiri DSM 20587 was investigated. Cell immobilisation with sodium cellulose sulphate was chosen as the most suitable encapsulation matrix, giving an immobilisation yield of 40%. Despite the lowered biocatalytic activity from cell immobilisation, the bioreduction process was vastly improved with the help of reaction engineering techniques (batch to a plug flow reactor set-up). High selectivity (95%) and space–time yield (87 g L⁻¹ day⁻¹) were achieved in the plug flow reactor. The biocatalyst remained active (68% residual activity) after 6 days of operation.     

Biotransformation of cladribine using a stabilized biocatalyst in calcium alginate beads

Cladribine is a nucleoside analogue widely used in the pharmaceutical industry for the treatment of several neoplasms, including hairy-cell leukemia among others. This compound has also shown efficacy in the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. In this work, a green bioprocess for cladribine biosynthesis using immobilized Arthrobacter oxydans was developed. The microorganism was stabilized by entrapment immobilization in the natural matrix alginate. Different reaction parameters were optimized obtaining a biocatalyst able to achieve cladribine bioconversion values close to 85% after 1 hr, the shortest reaction times reported so far. The developed bioprocess was successfully scaled-up reaching a productivity of 138 mg L⁻¹ hr⁻¹. Also, the biocatalyst was stable for 5 months in storage and in 96 hr at operational conditions.     

Microscale process evaluation of recombinant biocatalyst libraries: application to Baeyer–Villiger monooxygenase catalysed lactone synthesis

Microscale processing techniques are rapidly emerging as a cost- effective means for parallel experimentation and hence the evaluation of large libraries of recombinant biocatalysts. In this work, the potential of an automated microscale process is demonstrated in a linked sequence of operations comprising fermentation, enzyme induction and bioconversion using three whole-cell biocatalysts each expressing cyclohexanone monoxygenase (CHMO). The biocatalysts, Escherichia coli TOP 10 [pQR239], E. coli JM107 and Acinetobacter calcoaceticus NCIMB 9871, were first produced in 96-deep square well fermentations at various carbon source concentrations (10 and 20 g L⁻¹ glycerol). Following induction of CHMO activity biomass concentrations of up to 6 g_DCW L⁻¹ were obtained. Cells from each fermentation were subsequently used for the Baeyer–Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one, cyclohexanone and cyclopentanone. Each bioconversion was performed at two initial substrate concentrations (0.5 and 1.0 g L⁻¹) in order to simultaneously explore both substrate specificity and inhibition. The microscale process sequences yielded quantitative and reproducible data for each biocatalyst on maximum growth rate, biomass yield, initial rate of lactone formation, specific biocatalyst activity and bioconversion yield. E. coli TOP 10 [pQR239] was demonstrated to be an efficient biocatalyst showing substrate specificities and substrate inhibition effects in line with previous studies. Finally, in order to show that the data obtained with E. coli TOP 10 [pQR239] at microwell scale (1,000 μL) could be related to larger scales of operation, the process was performed in a 2-L stirred-tank bioreactor. Using conditions designed to enable microwell kinetic measurements under none oxygen-limited conditions, the fermentation and bioconversion data obtained at the two scales showed good quantitative agreement. This study therefore confirms the potential of automated microscale experimentation for the whole-process evaluation of recombinant biocatalyst libraries and the specification of pilot and process scale operating conditions.     

Solid support membrane‐aerated catalytic biofilm reactor for the continuous synthesis of (S)‐styrene oxide at gram scale

Catalytic biofilms minimize reactant toxicity and maximize biocatalyst stability in selective transformations of chemicals to value‐added products in continuous processes. The scaling up of such catalytic biofilm processes is challenging, due to fluidic and biological parameters affording a special reactor design affecting process performance. A solid support membrane‐aerated biofilm reactor was optimized and scaled‐up to yield gram amounts of (S)‐styrene oxide, a toxic and instable high value chemical synthon. A sintered stainless steel membrane unit was identified as an optimal choice as biofilm substratum and for high oxygen mass transfer. A stable expanded polytetrafluoroethylene (ePTFE) membrane was best suited for in situ substrate delivery and product extraction. For the verification of scalability, catalytic biofilms of Pseudomonas sp. strain VLB120ΔC produced (S)‐styrene oxide to an average concentration of 390 mM in the organic phase per day (equivalent to 24.4 g L_aq^–1 day^–1). This productivity was gained by efficiently using the catalyst with an excellent product yield on biomass of 13.6 g_product g_biomass^–1. This product yield on biomass is in the order of magnitude reported for other continuous systems based on artificially immobilized biocatalysts and is fulfilling the minimum requirements for industrial biocatalytic processes. Overall, 46 g of (S)‐styrene oxide were produced and isolated (purity: 99%; enantiomeric excess [ee]: >99.8%. yield: 30%). The productivity is in a similar range as in comparable small‐scale biofilm reactors highlighting the large potential of this methodology for continuous bioprocessing of bulk chemicals and biofuels.     

Biocatalytic conversion of cycloalkanes to lactones using an in‐vivo cascade in Pseudomonas taiwanensis VLB120

Chemical synthesis of lactones from cycloalkanes is a multi‐step process challenged by limitations in reaction efficiency (conversion and yield), atom economy (by‐products) and environmental performance. A heterologous pathway comprising novel enzymes with compatible kinetics was designed in Pseudomonas taiwanensis VLB120 enabling in‐vivo cascade for synthesizing lactones from cycloalkanes. The respective pathway included cytochrome P450 monooxygenase (CHX), cyclohexanol dehydrogenase (CDH), and cyclohexanone monooxygenase (CHXON) from Acidovorax sp. CHX100. Resting (non‐growing) cells of the recombinant host P. taiwanensis VLB120 converted cyclohexane, cyclohexanol, and cyclohexanone to ?‐caprolactone at 22, 80–100, and 170 U g_CDW^?1, respectively. Cyclohexane (5 mM) was completely converted with a selectivity of 65% for ?‐caprolactone formation in 2 hr without accumulation of intermediate products. Promiscuity of the whole‐cell biocatalyst gave access to analogous lactones from cyclooctane and cyclodecane. A total product concentration of 2.3 g L^?1 and a total turnover number of 36,720 was achieved over 5 hr with a biocatalyst concentration of 6.8 g_CDW L^?1.

     

The response of Vallisneria spinulosa (Hydrocharitaceae) to different loadings of ammonia and nitrate at moderate phosphorus concentration: a mesocosm approach

1. While phosphorus (P) is often considered the most important growth limiting factor for plants in lakes, recent studies of shallow lakes indicate that nitrogen (N) may be of greater importance than realized hitherto and that submerged macrophytes may be lost when the N concentration exceeds a certain threshold, as long as the concentration of P is sufficiently high. 2. We studied the effects of different loadings of NH₄‐N and NO₃‐N on chlorophyll a and on a macrophyte tolerant of eutrophication, Vallisneria spinulosa (Hydrocharitaceae). In outdoor mesocosms we used water from a pond as control and created four levels of NH₄‐N and NO₃‐N (approximately 2.5, 5, 7.5 and 10 mg L⁻¹) by dosing with NH₄Cl and NaNO₃, respectively. After the experiment, the plants were transferred back to a holding pond to study their recovery. In contrast to previous research, we used a low background concentration of phosphorus (TP 0.024 ± 0.003 mg L⁻¹) so we could judge whether any effects of N were apparent when P is in short supply. 3. Chlorophyll a increased significantly with N dosing for both forms of N, but the increase was highest in the NH₄‐N dosed mesocosms (maximum 58 μg L⁻¹ versus 42 μg L⁻¹ in the NO₃‐N mesocosms), probably due to a higher total inorganic N concentration (part of the added ammonia was converted to nitrate during the experiment). 4. Although the number of ramets of V. spinulosa was not affected by the N treatment, the biomass increased up to concentrations of 7.5 mg L⁻¹, while biomass at 10 mg L⁻¹ remained at the control level for both N ions treatments. A similar pattern was apparent for the content of N and soluble sugar of the plant, while there were no differences in the plant P content among treatments. Five months after transplantation back to the pond no difference was found in the number of ramets or in biomass, except that the biomass of plants grown at 10 mg N L⁻¹ during the experiment was greater than that in the control, while the N and P contents of plants were similar to those of the controls. 5. Nitrogen concentration had little influence on the growth of the eutrophication tolerant submerged macrophyte at moderately low concentrations of phosphorus. Moreover, the two N ions showed no toxic effects, suggesting that loss of macrophytes observed in other studies, run at higher phosphorus concentrations, was probably related to enhanced shading by periphyton and/or phytoplankton rather than to any toxic effects of N.     

Enhanced biocatalytic production of <Emphasis Type="SmallCaps">l</Emphasis>-cysteine by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. B-3 with in situ product removal using ion-exchange resin

Bioconversion of dl-2-amino-Δ²-thiazoline-4-carboxylic acid (dl-ATC) catalyzed by whole cells of Pseudomonas sp. was successfully applied for the production of l-cysteine. It was found, however, like most whole-cell biocatalytic processes, the accumulated l-cysteine produced obvious inhibition to the activity of biocatalyst and reduced the yield. To improve l-cysteine productivity, an anion exchange-based in situ product removal (ISPR) approach was developed. Several anion-exchange resins were tested to select a suitable adsorbent used in the bioconversion of dl-ATC for the in situ removal of l-cysteine. The strong basic anion-exchange resin 201 × 7 exhibited the highest adsorption capacity for l-cysteine and low adsorption for dl-ATC, which is a favorable option. With in situ addition of 60 g L^?1 resin 201 × 7, the product inhibition can be reduced significantly and 200 mmol L^?1 of dl-ATC was converted to l-cysteine with 90.4 % of yield and 28.6 mmol L^?1 h^?1 of volumetric productivity. Compared to the bioconversion without the addition of resin, the volumetric productivity of l-cysteine was improved by 2.27-fold using ISPR method.     

Bioreaction Engineering Leading to Efficient Synthesis of L‐Glyceraldehyd‐3‐Phosphate

Enantiopure L‐glyceraldehyde‐3‐phosphate (L‐GAP) is a useful building block in natural biological and synthetic processes. A biocatalytic process using glycerol kinase from Cellulomonas sp. (EC 2.7.1.30) catalyzed phosphorylation of L‐glyceraldehyde (L‐GA) by ATP is used for the synthesis of L‐GAP. L‐GAP has a half‐life of 6.86 h under reaction conditions. The activity of this enzyme depends on the Mg²⁺ to ATP molar ratio showing maximum activity at the optimum molar ratio of 0.7. A kinetic model is developed and validated showing a 2D correlation of 99.9% between experimental and numerical data matrices. The enzyme exhibits inhibition by ADP, AMP, methylglyoxal and Ca²⁺, but not by L‐GAP and inorganic orthophosphate. Moreover, equal amount of Ca²⁺ exerts a different degree of inhibition relative to the activity without the addition of Ca²⁺ depending on the Mg²⁺ to ATP molar ratio. If the Mg²⁺ to ATP molar ratio is set to be at the optimum value or less, inorganic hexametaphosphate (PPi6) suppresses the enzyme activity; otherwise PPi6 enhances the enzyme activity. Based on reaction engineering parameters such as conversion, selectivity and specific productivity, evaluation of different reactor types reveals that batchwise operation via stirred‐tank reactor is the most efficient process for the synthesis of L‐GAP.     

Syngas fermentation to biofuel: Evaluation of carbon monoxide mass transfer coefficient (kLa) in different reactor configurations

Lignocellulosic biomass such as agri‐residues, agri‐processing by‐products, and energy crops do not compete with food and feed, and is considered to be the ideal renewable feedstocks for biofuel production. Gasification of biomass produces synthesis gas (syngas), a mixture primarily consisting of CO and H₂. The produced syngas can be converted to ethanol by anaerobic microbial catalysts especially acetogenic bacteria such as various clostridia species.One of the major drawbacks associated with syngas fermentation is the mass transfer limitation of these sparingly soluble gases in the aqueous phase. One way of addressing this issue is the improvement in reactor design to achieve a higher volumetric mass transfer coefficient (k_La). In this study, different reactor configurations such as a column diffuser, a 20‐μm bulb diffuser, gas sparger, gas sparger with mechanical mixing, air‐lift reactor combined with a 20‐μm bulb diffuser, air‐lift reactor combined with a single gas entry point, and a submerged composite hollow fiber membrane (CHFM) module were employed to examine the k_La values. The k_La values reported in this study ranged from 0.4 to 91.08 h^?1. The highest k_La of 91.08 h^?1 was obtained in the air‐lift reactor combined with a 20‐μm bulb diffuser, whereas the reactor with the CHFM showed the lowest k_La of 0.4 h^?1. By considering both the k_La value and the statistical significance of each configuration, the air‐lift reactor combined with a 20‐μm bulb diffuser was found to be the ideal reactor configuration for carbon monoxide mass transfer in an aqueous phase. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

A chiral,porous, organic cage‐based,enantioselective potentiometric sensor for 2‐aminobutanol

A new enantioselective potentiometric sensor containing R‐type chiral porous organic cage CC9 as the chiral selector was designed for the assay of 2‐aminobutanol. Optimized membrane electrodes displayed a linear dynamic range from 10⁻³ ~ 10⁻¹ mol·L⁻¹ with a detection limit of 2.5 × 10⁻⁴ mol·L⁻¹ and a Nernstian response of 27 ± 0.5mV·decade⁻¹ toward S‐2‐aminobutanol within the pH range 7.0–10.0. The potentiometric enantioselectivity coefficient ( ) of this sensor was −1.333, indicating that the porous organic cage‐based electrode exhibited good discrimination toward S‐2‐aminobutanol over R‐2‐aminobutanol.     

Production of 3‐Fucosyllactose in Engineered Escherichia coli with α‐1,3‐Fucosyltransferase from Helicobacter pylori

3‐Fucosyllactose (3‐FL), one of the major oligosaccharides in human breast milk, is produced in engineered Escherichia coli. In order to search for a good α‐1,3‐fucosyltransferase, three bacterial α‐1,3‐fucosyltransferases are expressed in engineered E. coli deficient in β‐galactosidase activity and expressing the essential enzymes for the production of guanosine 5′‐diphosphate‐l ‐fucose, the donor of fucose for 3‐FL biosynthesis. Among the three enzymes tested, the fucT gene from Helicobacter pylori National Collection of Type Cultures 11637 gives the best 3‐FL production in a simple batch fermentation process using glycerol as a carbon source and lactose as an acceptor. In order to use glucose as a carbon source, the chromosomal ptsG gene, considered the main regulator of the glucose repression mechanism, is disrupted. The resulting E. coli strain of ?LP‐YA+FT shows a much lower performance of 3‐FL production (4.50 g L^?1) than the ?L‐YA+FT strain grown in a glycerol medium (10.7 g L^?1), suggesting that glycerol is a better carbon source than glucose. Finally, the engineered E. coli ?LW‐YA+FT expressing the essential genes for 3‐FL production and blocking the colanic acid biosynthetic pathway (?wcaJ) exhibits the highest concentration (11.5 g L^?1), yield (0.39 mol mol^?1), and productivity (0.22 g L^?1 h) of 3‐FL in glycerol‐limited fed‐batch fermentation.     

Pathway Engineering of Bacillus subtilis for Enhanced N‐Acetylneuraminic Acid Production via Whole‐Cell Biocatalysis

N‐acetylneuraminic acid (NeuAc) is a common sialic acid that has a wide range of applications in nutraceuticals and pharmaceuticals. However, low production efficiency and high environmental pollution associated with traditional extraction and chemical synthesis methods constrain the supply of NeuAc. Here, a biological approach is developed for food‐grade NeuAc production via whole‐cell biocatalysis by the generally regarded as safe (GRAS) bacterium Bacillus subtilis (B. subtilis). Promoters for controlling N‐acetylglucosamine 2‐epimerase (AGE) and NeuAc adolase (NanA) are optimized, yielding 32.84 g L^?1 NeuAc production with a molar conversion rate of 26.55% from N‐acetylglucosamine (GlcNAc). Next, NeuAc production is further enhanced to 46.04 g L^?1, which is 40.2% higher than that of the strain with promoter optimization, by expressing NanA from Staphylococcus hominis instead of NanA from Escherichia coli. To enhance the expression level of ShNanA, the N‐terminal coding sequences of genes with high expression levels are fused to the 5′‐end of the ShNanA gene, resulting in 56.82 g L^?1 NeuAc production. Finally, formation of the by‐product acetoin from pyruvate is blocked by deleting the alsS and alsD genes, resulting in 68.75 g L^?1 NeuAc production with a molar conversion rate of 55.57% from GlcNAc. Overall, a GRAS B. subtilis strain is demonstrated as a whole‐cell biocatalyst for efficient NeuAc production.     

Maximizing the productivity of catalytic biofilms on solid supports in membrane aerated reactors

A new solid support membrane aerated biofilm reactor was designed for the synthesis of enantiopure (S)‐styrene oxide utilizing Pseudomonas sp. strain VLB120ΔC growing in a biofilm as biocatalyst. In analogy to traditional packed bed systems, maximizing the volumetric oxygen mass transfer capability (k_La) was identified as the most critical issue enabling a consistent productivity, as this parameter was shown to directly influence biofilm growth and biotransformation performance. A microporous ceramic unit was identified as an ideal microenvironment for biofilm growth and for efficient oxygen transfer. A uniform and dense biofilm developed on this matrix. Due to this dual function, the reactor configuration could be significantly simplified by eliminating additional packing materials, as used in traditional packed bed reactors. Up to now, a maximum productivity of 28 g L day^?1 was achieved by integrating an in situ substrate feed and an in situ product recovery technique based on a silicone membrane. The system was stable for more than 30 days before it was actively terminated. Biotechnol. Bioeng. 2010;106: 516–527. © 2010 Wiley Periodicals, Inc.     

Metabolic engineering of Escherichia coli for the production of cadaverine: A five carbon diamine

A five carbon linear chain diamine, cadaverine (1,5‐diaminopentane), is an important platform chemical having many applications in chemical industry. Bio‐based production of cadaverine from renewable feedstock is a promising and sustainable alternative to the petroleum‐based chemical synthesis. Here, we report development of a metabolically engineered strain of Escherichia coli that overproduces cadaverine in glucose mineral salts medium. First, cadaverine degradation and utilization pathways were inactivated. Next, L ‐lysine decarboxylase, which converts L ‐lysine directly to cadaverine, was amplified by plasmid‐based overexpression of the cadA gene under the strong tac promoter. Furthermore, the L ‐lysine biosynthetic pool was increased by the overexpression of the dapA gene encoding dihydrodipicolinate synthase through the replacement of the native promoter with the strong trc promoter in the genome. The final engineered strain was able to produce 9.61 g L⁻¹ of cadaverine with a productivity of 0.32 g L⁻¹ h⁻¹ by fed‐batch cultivation. The strategy reported here should be useful for the bio‐based production of cadaverine from renewable resources. Biotechnol. Bioeng. 2011; 108:93–103. © 2010 Wiley Periodicals, Inc.     

Production of d-proline from l-arginine using Pseudomonas aeruginosa

Several microorganisms that can use (S)-5-[(amino-iminomethyl) amino]-2-chloropentanoic acid (l-Cl-arginine) as a nitrogen source have been isolated, the most interesting of which is a spontaneous mutant of Pseudomonas aeruginosa PAO1 (DSM 10581). In a fermenter, this unique biocatalyst hydrolysed l-Cl-arginine to (S)-5-amino-2-chloropentanoic acid (l-Cl-ornithine), which spontaneously converted to d-proline with inversion of configuration at an apparent average rate of 0.12 mmol ^−l h⁻¹ OD⁻¹. The enzyme, for which we suggest the name Cl-arginine amidinohydrolase, was best induced by using the substrate l-Cl-arginine as inducer and l-arginine as nitrogen source. The results presented here describe a new route for the production of d-proline from l-arginine, involving a chemical step and a biocatalytic step followed by a spontaneous chemical cyclisation.     

Integrated two‐liquid phase bioconversion and product‐recovery processes for the oxidation of alkanes: Process design and economic evaluation

Pseudomonas oleovorans and recombinant strains containing the alkane oxidation genes can produce alkane oxidation products in two‐liquid phase bioreactor systems. In these bioprocesses the cells, which grow in the aqueous phase, oxidize apolar, non‐water soluble substrates. The apolar products typically accumulate in the emulsified apolar phase. We have studied both the bioconversion systems and several downstream processing systems to separate and purify alkanols from these two‐liquid phase media. Based on the information generated in these studies, we have now designed bioconversion and downstream processing systems for the production of 1‐alkanols from n‐alkanes on a 10 kiloton/yr scale, taking the conversion of n‐octane to 1‐octanol as a model system. Here, we describe overall designs of fed‐batch and continuous‐fermentation processes for the oxidation of octane to 1‐octanol by Pseudomonas oleovorans, and we discuss the economics of these processes. In both systems the two‐liquid phase system consists of an apolar phase with hexadecene as the apolar carrier solvent into which n‐octane is dissolved, while the cells are present in the aqueous phase. In one system, multiple‐batch fermentations are followed by continuous processing of the product from the separated apolar phase. The second system is based on alkane oxidation by continuously growing cultures, again followed by continuous processing of the product. Fewer fermentors were required and a higher space‐time‐yield was possible for production of 1‐octanol in a continuous process. The overall performance of each of these two systems has been modeled with Aspen software. Investment and operating costs were estimated with input from equipment manufacturers and bulk‐material suppliers. Based on this study, the production cost of 1‐octanol is about 7 US$kg⁻¹ when produced in the fed‐batch process, and 8 US$kg⁻¹ when produced continuously. The comparison of upstream and downstream capital costs and production costs showed significantly higher upstream costs for the fed‐batch process and slightly higher upstream costs for continuous fermentation. The largest cost contribution was due to variable production costs, mainly resulting from media costs. The organisms used in these systems are P. putida alk+ recombinants which oxidize alkanes, but cannot oxidize the resulting alkanols further. Hence, such cells need a second carbon source, which in these systems is glucose. Although the continuous process is about 10% more expensive than the fed‐batch process, improvements to reduce overall cost can be achieved more easily for continuous than for fed‐batch fermentation by decreasing the dilution rate while maintaining near constant productivity. Improvements relevant to both processes can be achieved by increasing the biocatalyst performance, which results in improved overall efficiency, decreased capital investment, and hence, decreased production cost. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 84: 459–477, 1999.     

A Two‐stage CSTR Cascade for Studying the Effect of Inhibitory and Toxic Substances in Bioprocesses

In some biotechnological processes like wastewater treatment and biotransformation, substances are involved which are inhibitory or even toxic to the microorganisms. Their presence changes the cell physiology or even acts lethal on the cells so that the process breaks down completely. For studying such processes, a two‐stage continuous‐flow stirred tank reactor (CSTR) cascade was developed where the toxic substance is only supplied to the second reactor. Mathematical modeling of the system showed that identical steady‐state conditions can be established in both bioreactors of the two‐stage CSTR cascade when the dilution rate of the second reactor is twice as high as the dilution rate in the first reactor, provided that both reactors are fed with the same culture medium and possess an identical working volume. The theoretically derived concept was verified by cultivating Saccharomyces cerevisiae CBS 8066 under glucose‐limited aerobic conditions. Independently of the dilution rates established (D₁ in the range of 0.26 to 0.38 h^–1 and D₂ = 2·D₁), the steady‐state values of the biomass, glucose and ethanol concentration were almost identical in both reactors. Moreover, the dynamic behavior after each stepwise change of the dilution rates was also identical in both reactors, which was detected by dissolved‐oxygen measurements. Finally, the system was applied to the whole‐cell biotransformation of ethyl 2‐chloro‐3‐oxo‐butanoate as an example.     

Effects of light history on primary productivity in a phytoplankton community dominated by the toxic cyanobacterium Cylindrospermopsis raciborskii

1. The effects of instantaneous irradiance and short‐term light history on primary production were determined for samples from a subtropical water reservoir dominated by the toxic cyanobacterium Cylindrospermopsis raciborskii. ¹⁴C‐bicarbonate uptake incubations were conducted on water samples from the reservoir, for irradiance (photosynthetically active radiation) ranging from 0 to 1654 μmol quanta m⁻² s⁻¹. Prior to the ¹⁴C incubations, cells were pre‐treated at irradiance levels ranging from 0 to 1006 μmol quanta m⁻² s⁻¹. 2. The average irradiance experienced by cells during the 2–2.5 h pre‐treatment incubations affected the productivity–irradiance (P–I) parameters: exposure to high light in pre‐treatment conditions caused a substantial decrease in maximum rate of primary production P_max and the photoinhibition parameter β when compared to cells pre‐treated in the dark. 3. While the data collected in this study were not sufficient to develop a full dynamic model of C. raciborskii productivity, P_max and β were modelled as a function of pre‐treatment irradiance, and these models were applied to predict the rate of primary production as a function of both instantaneous and historical irradiance. The results indicated that while cells with a history of exposure to high irradiance will be the most productive in high irradiance, production rates will be highest overall for dark‐acclimated cells in moderate irradiance. 4. Our results may explain why optically‐deep mixing favours C. raciborskii. If the mixing depth z_m exceeds the euphotic depth z_eu, cells will be dark‐acclimated, which will increase their rate of production when they are circulated through the euphotic zone. These results also predict that production rates will be higher during morning hours than for the same irradiance in the afternoon, which is consistent with other phytoplankton studies. 5. Since the rate of production of C. raciborskii‐dominated systems cannot be described by a single P–I curve, accurate estimates of production rates will require measurements over the daily light cycle.