Fed-batch Mode Research Articles

A novel nitrilase from Ralstonia eutropha H16 and its application to nicotinic acid production.

A novel aliphatic nitrilase, REH16, was found in Ralstonia eutropha H16 and overexpressed in Escherichia coli BL21(DE3), and its enzymatic properties were studied. The temperature and pH optima were 37°C and 6.6, respectively, and the best thermostability of the nitrilase was observed at 25°C, which preserved 95% of activity after 120h of incubation. REH16 has a broad hydrolytic activity toward aliphatic and heterocyclic nitriles and showed high tolerance of 3-cyanopyridine; this enzyme could hydrolyze as high as 100mM 3-cyanopyridine completely. To improve the 3-cyanopyridine conversion efficiency in an aqueous reaction system, water-miscible organic solvents were tested, and ethanol (10% v/v) was chosen as the optimal co-solvent. Finally, under optimized conditions, using the fed-batch reaction mode, total of 1050mM 3-cyanopyridine was hydrolyzed completely in 20.8h with eight substrate feedings, yielding 129.2g/L production of nicotinic acid and thus showing a potential for industrial application.

Dextran Utilization During Its Synthesis by Weissella cibaria RBA12 Can Be Overcome by Fed-Batch Fermentation in a Bioreactor.

Weissella cibaria RBA12 produced a maximum of 9mg/ml dextran (with 90% efficiency) using shake flask culture under the optimized concentration of medium components viz. 2% (w/v) of each sucrose, yeast extract, and K2HPO4 after incubation at optimized conditions of 20°C and 180rpm for 24h. The optimized medium and conditions were used for scale-up of dextran production from Weissella cibaria RBA12 in 2.5-l working volume under batch fermentation in a bioreactor that yielded a maximum of 9.3mg/ml dextran (with 93% efficiency) at 14h. After 14h, dextran produced was utilized by the bacterium till 18h in its stationary phase under sucrose depleted conditions. Dextran utilization was further studied by fed-batch fermentation using sucrose feed. Dextran on production under fed-batch fermentation in bioreactor gave 35.8mg/ml after 32h. In fed-batch mode, there was no decrease in dextran concentration as observed in the batch mode. This showed that the utilization of dextran by Weissella cibaria RBA12 is initiated when there is sucrose depletion and therefore the presence of sucrose can possibly overcome the dextran hydrolysis. This is the first report of utilization of dextran, post-sucrose depletion by Weissella sp. studied in bioreactor.

3‐Hydroxypropionic acid production by recombinant Escherichia coli ZJU‐3HP01 using glycerol–glucose dual‐substrate fermentative strategy

3-Hydroxypropionic acid (3-HP) is an important platform synthesis block for sets of chemicals, but the relatively low production of 3-HP from biological sources presented major barriers for its industrial applications. In this study, a dual-substrate fermentative strategy by glycerol and glucose was proposed, and the aim was to evaluate the effect of different substrate addition strategies on the fermentation process. The results indicated that the optimal cosubstrate was glucose (20 g/L), and the enzymatic activity of aldehyde dehydrogenase (AldH) could be improved 3.5-fold as compared with no glucose addition. Continuous fed-batch fermentation at a constant speed displayed better 3-HP production of 17.20 g/L and highest specific 3-HP productivity of 1.79 mmol/(g cell·H) than the other fed-batch mode. The addition of glucose could greatly reduce the imbalance of the activity between glycerol dehydratase and AldH and provide a feasible method for improving 3-HP production. These results would be helpful in developing the 3-HP fermentation process.

Bioelectrochemical precipitation system for removal of scale-forming ions from seawater using two different buffers

Removal of scale-forming ions (e.g., Ca2+, Mg2+) from seawater using a bioelecrochemical system (BES) was investigated in a fed-batch and a continuous mode. In the fed-batch mode, Ca2+ removal with different anolyte buffers at different external resistances (10Ω and 1kΩ) was evaluated. As a result, Ca2+ was effectively removed with PBS (phosphate buffer solution) and PIPES (piperazine-N,N′-bis(2-ethanesulfonic acid) buffer solution. However, the removal of Ca2+ with PBS was not solely due to current generated from the anode but also from precipitation induced by combining Ca2+ with phosphate ions transferred from the anode chamber while the removal of Ca2+ with PIPES entirely relies on a cathodic reaction. Continuous removal of Ca2+ and Mg2+ with PIPES was more effective than the fed-batch mode with removal efficiencies of >97±2% (Ca2+ removal) and 98±1% (Mg2+ removal). The XRD spectra revealed that the precipitates were mainly composed of brucite (Mg(OH)2), calcite, and aragonite (CaCO3). These results indicate that BES could be a promising way to remove scale-forming ions that can promote inorganic fouling in membrane-based processes.

Medium optimization for pyrroloquinoline quinone (PQQ) production by Methylobacillus sp. zju323 using response surface methodology and artificial neural network–genetic algorithm

ABSTRACTMethylobacillus sp. zju323 was adopted to improve the biosynthesis of pyrroloquinoline quinone (PQQ) by systematic optimization of the fermentation medium. The Plackett–Burman design was implemented to screen for the key medium components for the PQQ production. CoCl2 · 6H2O, ρ-amino benzoic acid, and MgSO4 · 7H2O were found capable of enhancing the PQQ production most significantly. A five-level three-factor central composite design was used to investigate the direct and interactive effects of these variables. Both response surface methodology (RSM) and artificial neural network–genetic algorithm (ANN–GA) were used to predict the PQQ production and to optimize the medium composition. The results showed that the medium optimized by ANN–GA was better than that by RSM in maximizing PQQ production and the experimental PQQ concentration in the ANN–GA-optimized medium was improved by 44.3% compared with that in the unoptimized medium. Further study showed that this ANN–GA-optimized medium was also effective in improving PQQ production by fed-batch mode, reaching the highest PQQ accumulation of 232.0 mg/L, which was about 47.6% increase relative to that in the original medium. The present work provided an optimized medium and developed a fed-batch strategy which might be potentially applicable in industrial PQQ production.

Different fermentation processes produced variants of an anti-CD52 monoclonal antibody that have divergent in vitro and in vivo characteristics.

The anti-CD52 antibody has already been approved for the treatment of patients with resistant chronic lymphocytic leukemia, relapsing-remitting multiple sclerosis, and has demonstrable efficacy against stem cell transplantation rejection. A CHO cell line expressing a humanized anti-CD52 monoclonal antibody (mAb-TH) was cultivated in both fed-batch and perfusion modes, and then purified. The critical quality attributes of these mAb variants were characterized and the pharmacokinetics (PK) properties were investigated. Results showed that the perfusion culture achieved higher productivity, whereas the fed-batch culture produced more aggregates and acid components. Additionally, the perfusion culture produced similar fucose, more galactose and a higher proportion of sialic acid on the anti-CD52 mAb compared to the fed-batch culture. Furthermore, the perfusion process produced anti-CD52 mAb had higher complement-dependent cytotoxicity (CDC) efficacy than that produced by the fed-batch culture, a result probably linked to its higher galactose content. However, antibody produced by fed-batch and perfusion cultures showed similar PK profiles in vivo. In conclusion, perfusion is a more efficient method than fed-batch process in the production of functional anti-CD52 monoclonal antibody. Product quality variants of anti-CD52 mAb were found in different cell culture processes, which demonstrated different physiochemical and biological activities, but comparable PK properties. Whether these observations apply to all mAbs await further investigation.

Strategies for improved isopropanol\u2013butanol production by a Clostridium strain from glucose and hemicellulose through consolidated bioprocessing

BackgroundHigh cost of traditional substrates and formation of by-products (such as acetone and ethanol) in acetone–butanol–ethanol (ABE) fermentation hindered the large-scale production of biobutanol. Here, we comprehensively characterized a newly isolated solventogenic and xylanolytic Clostridium species, which could produce butanol at a high ratio with elimination of ethanol and conversion of acetone to more value-added product, isopropanol. Ultimately, direct butanol production from hemicellulose was achieved with efficient expression of indigenous xylanase by the novel strain via consolidated bioprocessing.ResultsA novel wild-type Clostridium sp. strain NJP7 was isolated and characterized in this study, which was capable of fermenting monosaccharides, e.g., glucose into butanol via a fermentative acetone–isopropanol–butanol pathway. With enhancement of buffering capacity and alcohol dehydrogenase activities, butanol and isopropanol titer by Clostridium sp. strain NJP7 was improved to 12.21 and 1.92 g/L, respectively, and solvent productivity could be enhanced to 0.44 g/L/h. Furthermore, with in situ extraction with biodiesel, the amount of butanol and isopropanol was finally improved to 25.58 and 5.25 g/L in the fed-batch mode. Meanwhile, Clostridium sp. strain NJP7 shows capability of direct isopropanol–butanol production from hemicelluloses with expression of indigenous xylanase. 2.06 g/L of butanol and 0.54 g/L of isopropanol were finally achieved through the temperature-shift simultaneous saccharification and fermentation, representing the highest butanol production directly from hemicellulose.ConclusionThe co-production of isopropanol with butanol by the newly isolated Clostridium sp. strain NJP7 would add on the economical values for butanol fermentation. Furthermore, the high isopropanol-butanol production with in situ extraction would also greatly enhance the economic feasibility for fermentative production of butanol–isopropanol in large scale. Meanwhile, its direct production of butanol–isopropanol from polysaccharides, hemicellulose through secretion of indigenous thermostable xylanase, shows great potential using lignocellulosic wastes for biofuel production.

Microbial fuel cell operation using nitrate as terminal electron acceptor for simultaneous organic and nutrient removal

A double-chambered biocathode microbial fuel cell with carbon felt employed as electrodes was developed for wastewater treatment and bioelectricity generation simultaneously. The system was operated in fed-batch mode for over eight batches. The effect of circuit connections on organic and nitrate reduction was investigated. The maximum power density recorded was 21.97 mW/m2 at current density of 88.57 mA/m2. The Coulombic efficiency and internal resistance of the system were 5% and 100 Ω. Up to 89.9 ± 5.9% of chemical oxygen demand reduction efficiency achieved with an influent of 1123 ± 28 mg/L. There was no significant difference in the chemical oxygen demand reduction when system operated in either open or closed circuit. This study clearly showed that higher nitrate reduction efficiency obtained in closed circuit (74.7 ± 7.0%) due to bio-electrochemical denitrification compared to only 41.7% in the open circuit. The result also successfully demonstrated nitrate as terminal electron acceptor for the cathodic nitrate reduction.

Using 3D Composite Electrode Materials for Electricity Generation from Swine Wastewater in a Dual-Chamber Microbial Fuel Cells

Swine wastewater has a high concentration of organic matter, suspended solids, and higher ammonia nitrogen, odor, complex polluting ingredients, and large emissions. A two-chambered cubic microbial fuel cell (MFC) was used to evaluate the effect of a novel three-dimensional (3D) electrode made of 3D iron composites and 3D stainless composites on the electricity generation. Swine wastewater with a total chemical oxygen demand (TCOD) of 3688 ± 300 mg/L was used as the feedstock in the anode chamber. The MFC reactor was incubated with an initial pH of 7.0 in an air shaker with a temperature of ~35°C and 100 rpm in the fed-batch mode. A fixed external resistance (R) of 100 Ω was connected between the electrodes, and the closed-circuit potentials of the MFCs were recorded every 5 min. The results showed that using an iron–carbon fiber composite 3D electrode resulted in a peak electricity generation of 321 mV on the first 2 days and maintained a stable voltage of 163 mV during the second to sixth days. The COD removal efficiency could reach 75%. Using a stainless–carbon fiber 3D electrode could generate a peak voltage of only 29.5 mV and a stable voltage of 15.2 mV with a COD removal efficiency of 54%.

High-pressure thermophilic electromethanogenic system producing methane at 5 MPa, 55°C

Toward applications of bio-electrochemical systems in industrial processes and extreme environments, electromethanogenesis under high-pressure conditions was examined. Stainless-steel single-chamber reactors specifically designed to examine bio-electrochemical reactions under pressurized conditions were inoculated with thermophilic microorganisms originated from an oilfield formation water. The reactors were incubated at 5MPa, 55°C in fed-batch operational mode with an applied voltage of 0.7V. In the first few fed-batch cycles, hydrogen was mainly produced. After the third cycle, however, the reactors produced only methane simultaneously with current generation. The methane-production rate of the reactors showed an applied-voltage dependence and increased from 34.9 to 168.4mmolm-2day-1 with an increase in the applied voltage from 0.4 to 0.9V. The efficiency of capturing electrons in the produced methane on average exceeded 70% with the applied voltage of 0.4V or higher. Cyclic voltammetry further confirmed abilities of the bioelectrodes to catalyze electrochemical reactions at 5MPa. Performance of the electromethanogenesis system was not altered under lower pressure conditions (1.2 and 2.5MPa). An exoelectrogenic bacterium affiliated with the genus Thermincola and a methanogen belonging to the genus Methanothermobacter were detected as the dominant species in the bioanode and biocathode microbiotas, respectively. Thus, our results indicated that electromethanogenesis systems could be developed and operated under highly-pressurized conditions, suggesting that applications of the bio-electrochemical system in high-pressure environments (including high-temperature subsurface reservoirs) can be technically feasible.

Evaluation of methanogenic microbial electrolysis cells under closed/open circuit operations

ABSTRACTThe present study investigated the independent roles of biofilm and external power supply for volatile fatty acids degradation and methane production in the microbial electrolysis cell. Reactors were operated in fed-batch mode in the presence of graphite felt (GF) or titanium rod (Ti) as electrodes, in open circuit (OC, without applied voltage) or closed circuit (CC) conditions, i.e. R1 (Ti + CC), R2 (GF + CC) and R3 (GF + OC). The first-order kinetic analysis of acetate degradation indicated that the presence of GF biofilm and applied voltage in R2 improved the degradation rate of acetate by 23% as compared to R1, while it was only a 7% increment in R3 with GF biofilm. The degradation of butyrate was accelerated by 12% in the first 24 h, whereas there was no enhancement of the propionic acid digestion. Generally, methane yields from the three reactors followed the sequence: R2 > R1 > R3, indicating the positive effect of external power supply on methane generation. High-throughput sequencing revealed that Geobacter sp. could be enriched on conductive GF even without electric stimulation. The clustered Geobacter and Methanosarcina in R2 presented the potential to promote interspecies electron transfer and accelerate substrate utilization and methane production.

Enhancement of recombinant BmK AngM1 production in Pichia pastoris by regulating gene dosage, co-expressing with chaperones and fermenting in fed-batch mode

The scorpion peptide BmK AngM1 was reported to exhibit evident analgesic effect, but its yield by extraction from scorpion venom limits the research and application. The heterologous expression of BmK AngM1 was achieved in Pichia pastoris in our previous study. In order to realize high-level expression of recombinant BmK AngM1 (rBmK AngM1), the gene dosage of BmK AngM1 was optimized in engineered strains. The yield of rBmK AngM1 in the four-copy strain reached up to 100 mg/L, which was further enhanced to 190 mg/L by co-expressing with chaperones of PDI, BiP, and HAC1. Moreover, the yield of rBmK AngM1 was up to 1200 mg/L by high-density fermentation in 10 L fermenter. Finally, 360 mg rBmK AngM1 was purified from 1 L cultures by a two-step purification method. The efficient and convenient techniques presented in this study could facilitate further scale-up for industrial production of rBmK AngM1.

Design study of an AnSBBR for hydrogen production by co-digestion of whey with glycerin: Interaction effects of organic load, cycle time and feed strategy

An anaerobic sequencing batch biofilm reactor (AnSBBR) treating a mixture of dairy industry wastewater and biodiesel production wastewater (co-digestion of whey with glycerin) was applied to hydrogen production. The influence of fed-batch and batch mode, cycle time and interactions effects between influent concentration and cycle time (2, 3 and 4 h) over the organic loading rate were assessed in order to obtain a sensitivity analysis for important operational variables to the reactor. It was possible to find an optimal cycle time of 3 h with an influent concentration of 7000 mgCOD L−1 (molar productivity 129.0 molH2 m−3 d−1 and yield 5.4 molH2 kgCOD−1). Reactor operation in fed-batch mode allowed higher hydrogen production rates. Increasing the influent concentration (with a constant cycle time) was better for the hydrogen production process than decreasing the cycle length (with a constant influent concentration), which means that these two parameters have different weights in the organic loading rate. The best operational conditions produce hydrogen via acetic, butyric and valeric acids similarly. The system is able to produce 1.3 kJ per gram of COD applied.

Anode air exposure during microbial fuel cell operation inoculated with marine sediment

Microbial fuel cells (MFC) have been a viable alternative for wastewater treatment and power generation with low environmental impact. Different architectures, materials and operational modes have been extensively studied to reduce operational constraints and improve energy efficiency. The goal of this study is to establish the biological process of electricity generation from natural marine sediment that can tolerate oxygen in the anode chamber. For this study, an MFC with a total volume of 2.5L was operated continuously for 280days in both sequential fed-batch and continuous modes over 24 runs. During operation, a maximum voltage of 562.67V/m3 was achieved. When 20% of the total area of the anode electrodes was exposed to air, there was a maximum voltage decrease of 35% in the first run. This value decreased to below 10% at the end of operation. During Run 19, the percentage of the anode electrodes exposed to air was increased to 40%, 60%, 80% and 100% of the total area and a linear relationship between the exposed area and the inhibition phenomenon was observed.

Pilot Scale System of Two Horizontal Rotating Bioreactors for Bioethanol Production from Household Food Waste at High Solid Concentrations

Household food wastes (HFW) a complex biomass containing soluble sugars, lipids, proteins, cellulose, starch was used for bioethanol production in a newly designed pilot scale system consisting of two horizontal rotating bioreactors (HRRs) operating at high solids content under non-isothermal simultaneous saccharification and fermentation (NSSF) in fed-batch mode. Operational conditions were determined in lab-scale experiments. More specifically, enzymes including cellulases, α-amylase and glucoamylase at different loadings were tested one at the time for the pre-hydrolysis and subsequent fermentation by Saccharomyces cerevisiae of the pretreated HFW. The highest ethanol production (42.74 g/L, corresponding to 72.33% of the maximum theoretical) was obtained when cellulases (at 60 FPU/g cellulose) and glucoamylase (60 FPU/g starch) were used. Fed-batch experiments were conducted in a 20 L bioreactor. Increasing batch additions resulted in a higher ethanol titer. Ethanol production of 60.69 g/L (corresponding to 69.77% of the maximum theoretical) was achieved when three additions were made. Implementation of the NSSF process operating in fed-batch mode in the pilot scale facility resulted in ethanol production of 53.90 g/L (corresponding to 73.26% of maximum theoretical yield). The pilot scale facility of the present study can produce 188 L of ethanol from one ton of dry HFW.

Kinetic insights into ϵ-caprolactone synthesis: Improvement of an enzymatic cascade reaction.

A computational approach for the simulation and prediction of a linear three-step enzymatic cascade for the synthesis of ϵ-caprolactone (ECL) coupling an alcohol dehydrogenase (ADH), a cyclohexanone monooxygenase (CHMO), and a lipase for the subsequent hydrolysis of ECL to 6-hydroxyhexanoic acid (6-HHA). A kinetic model was developed with an accuracy of prediction for a fed-batch mode of 37% for substrate cyclohexanol (CHL), 90% for ECL, and >99% for the final product 6-HHA. Due to a severe inhibition of the CHMO by CHL, a batch synthesis was shown to be less efficient than a fed-batch approach. In the fed-batch synthesis, full conversion of 100 mM CHL was 28% faster with an analytical yield of 98% compared to 49% in case of the batch synthesis. The lipase-catalyzed hydrolysis of ECL to 6-HHA circumvents the inhibition of the CHMO by ECL enabling a 24% higher product concentration of 6-HHA compared to ECL in case of the fed-batch synthesis without lipase. Biotechnol. Bioeng. 2017;114: 1215-1221. © 2017 Wiley Periodicals, Inc.

Detoxification of 1,1,2-trichloroethane to ethene in a bioreactor co-culture of Dehalogenimonas and Dehalococcoides mccartyi strains

1,1,2-Trichloroethane (1,1,2-TCA) is a non-flammable organic solvent and common environmental contaminant in groundwater. Organohalide-respiring bacteria are key microorganisms to remediate 1,1,2-TCA because they can gain metabolic energy during its dechlorination under anaerobic conditions. However, all current isolates produce hazardous end products such as vinyl chloride, monochloroethane or 1,2-dichloroethane that accumulate in the medium. Here, we constructed a syntrophic co-culture of Dehalogenimonas and Dehalococcoides mccartyi strains to achieve complete detoxification of 1,1,2-TCA to ethene. In this co-culture, Dehalogenimonas transformed 1,1,2-TCA via dihaloelimination to vinyl chloride, whereas Dehalococcoides reduced vinyl chloride via hydrogenolysis to ethene. Molasses, pyruvate, and lactate supported full dechlorination of 1,1,2-TCA in serum bottle co-cultures. Scale up of the cultivation to a 5-L bioreactor operating for 76d in fed-batch mode was successful with pyruvate as substrate. This synthetic combination of bacteria with known complementary metabolic capabilities demonstrates the potential environmental relevance of microbial cooperation to detoxify 1,1,2-TCA.

Process development of itaconic acid production by a natural wild type strain of Aspergillus terreus to reach industrially relevant final titers.

Itaconic acid is a promising organic acid and is commercially produced by submerged fermentation of Aspergillus terreus. The cultivation process of the sensitive filamentous fungus has been studied intensively since 1932, with respect to fermentation media components, oxygen supply, shearing rate, pH value, or culture method. Whereas increased final titers were achieved over the years, the productivity has so far remained quite low. In this study, the impact of the pH on the itaconic acid production was investigated in detail. The pH during the growth and production phase had a significant influence on the final itaconic acid concentration and pellet diameter. The highest itaconic acid concentration of 160g/L was achieved at a 1.5-L scale within 6.7days by raising and controlling the pH value to pH 3.4 in the production phase. An ammonia solution and an increased phosphate concentration were used with an itaconic acid yield of 0.46 (w/w) and an overall productivity of 0.99g/L/h in a fed-batch mode. A cultivation with a lower phosphate concentration resulted in an equal final concentration with an increased yield of 0.58 (w/w) after 11.8days and an overall productivity of 0.57g/L/h. This optimized process was successfully transferred from a 1.5-L scale to a 15-L scale. After 9.7days, comparable pellet morphology and a final concentration of 150g/L itaconic acid was reached. This paper provides a process strategy to yield a final titer of itaconic acid from a wild-type strain of A. terreus which is in the same range as the well-known citric acid production.

Biofuel production from birch wood by combining high solid loading simultaneous saccharification and fermentation and anaerobic digestion

Inhibitors generated during pretreatment of lignocellulosic biomass may affect the subsequent biochemical conversion to biofuels. In the present study, we tested 6 different reducing agents for their ability to detoxify steam-exploded birch used for ethanol production in a simultaneous saccharification and fermentation process. Cysteine, which was the most efficient detoxifying agent, increased both ethanol productivity and ethanol yield from 0.10 (non-detoxified) to 0.91g/L/h and from 0.17 (non-detoxified) to 0.46g/g, respectively. Gradual fed-batch feeding mode with a final total solid loading of 35% (w/w) resulted in an ethanol titer of 53.2g/L within 72h and a final ethanol concentration of 83.2g/L after prolonged incubation. Moreover, residual waste (stillage) remaining after bioethanol production was subsequently used for biogas production to make the process more economically feasible. The methane yield from the stillage was 188.1mL/g volatile solids (VS). The microbial community at the end of the bio-methane process was characterized by 16S rRNA analysis. The phyla Firmicutes and Bacteroidetes were dominant members of the bacterial community, whereas the archaeal communities were dominated by methanogenic Euryarchaeota belonging to the families Methanobacteriaceae and Methanosaetaceae.

Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris: functional characterisation of a novel promoter

As Pichia pastoris (syn. Komagataella sp.) yeast can secrete pure recombinant proteins at high rates, it is a desirable production system. The function of a novel synthetic variant of the AOX1 promoter was characterised comprehensively using a strain secreting Candida antarctica lipase B (CALB) as a model. A new time-saving approach was introduced to determine, in only one experiment, the hitherto unknown relationship between specific product formation rate (qp) and specific growth rate (μ). Tight control of recombinant protein formation was possible in the absence of methanol, while using glycerol as a sole carbon/energy source. CALB was not synthesised during batch cultivation in excess glycerol (>10 g l−1) and at a growth rate close to μmax (0.15 h−1). Between 0.017 and 0.115 h−1 in glycerol-limited fedbatch cultures, basal levels of qp > 0.4 mg g−1 h−1 CALB were reached, independent of the μ at which the culture grew. At μ > 0.04 h−1, an elevated qp occurred temporarily during the first 20 h after changing to fedbatch mode and decreased thereafter to basal. In order to accelerate the determination of the qp(μ) relationship (kinetics of product formation), the entire μ range was covered in a single fedbatch experiment. By linearly increasing and decreasing glycerol addition rates, μ values were repeatedly shifted from 0.004 to 0.074 h−1 and vice versa. Changes in qp were related to changes in μ. A rough estimation of μ range suitable for production was possible in a single fedbatch, thus significantly reducing the experimental input over previous approaches comprising several experiments.