Fast-track development of a lactase production process with Kluyveromyces lactis by a progressive parameter-control workflow.

The time-to-market challenge is key to success for consumer goods affiliated industries. In recent years, the dairy industry faces a fast and constantly growing demand for enzymatically produced lactose-free milk products, mainly driven by emerging markets in South America and Asia. In order to take advantage of this opportunity, we developed a fermentation process for lactase (ß-galactosidase) from Kluyveromyces lactis within short time. Here, we describe the process of stepwise increasing the level of control over relevant process parameters during scale-up that established a highly efficient and stable production system. Process development started with evolutionary engineering to generate catabolite-derepressed variants of the K. lactis wild-type strain. A high-throughput screening mimicking fed-batch cultivation identified a constitutive lactase overproducer with 260-fold improved activity of 4.4 U per milligram dry cell weight when cultivated in glucose minimal medium. During scale-up, process control was progressively increased up to the level of conventional, fully controlled fed-batch cultivations by simulating glucose feed, applying pH- and dissolved oxygen tension (DOT)-sensor technology to small scale, and by the use of a milliliter stirred tank bioreactor. Additionally, process development was assisted by design-of-experiments optimization of the growth medium employing the response surface methodology.

Online bioprocess data generation, analysis, and optimization for parallel fed-batch fermentations in milliliter scale.

Bioprocess development, optimization, and control in mini-bioreactor systems require information about essential process parameters, high data densities, and the ability to dynamically change process conditions. We present an integration approach combining a parallel mini-bioreactor system integrated into a liquid handling station (LHS) with a second LHS for offline analytics. Non-invasive sensors measure pH and DO online. Offline samples are collected every 20 min and acetate, glucose, and OD620 subsequently analyzed offline. All data are automatically collected, analyzed, formalized, and used for process control and optimization. Fed-batch conditions are realized via a slow enzymatic glucose release system. The integration approach was successfully used to apply an online experimental re-design method to eight Escherichia coli fed-batch cultivations. The method utilizes generated data to select the following experimental actions online in order to reach the optimization goal of estimating E. coli fed-batch model parameters with as high accuracy as possible. Optimal experimental designs were re-calculated online based on the experimental data and implemented by introducing pulses via the LHS to the running fermentations. The LHS control allows for various implementations of advanced control and optimization strategies in milliliter scale.

Toward Microbioreactor Arrays: A Slow-Responding Oxygen Sensor for Monitoring of Microbial Cultures in Standard 96-Well Plates.

In this study, a slow-responding chemo-optical sensor for dissolved oxygen (DO) integrated into a 96-well plate was developed. The slow response time ensures that the measured oxygen value does not change much during plate transport to the microplate reader. The sensor therefore permits at-line DO measurement of microbial cultures. Moreover, it eliminates the necessity of individual optical measurement systems for each culture plate, as many plates can be measured successively. Combined with the 96-well format, this increases the experimental throughput enormously. The novel sensor plate (Slow OxoPlate) consists of fluorophores suspended in a polymer matrix that were placed into u-bottom 96-well plates. Response time was measured using sodium sulfite, and a t90 value of 9.7 min was recorded. For application, DO values were then measured in Escherichia coli and Saccharomyces cerevisiae cultures grown under fed-batch-like conditions. Depending on the DO sensor's response time, different information on the oxygenation state of the culture plate was obtained: a fast sensor variant detects disturbance through sampling, whereas the slow sensor indicates oxygen limitation during incubation. A combination of the commercially available OxoPlate and the Slow OxoPlate enables operators of screening facilities to validate their cultivation procedures with regard to oxygen availability.

Robotic platform for parallelized cultivation and monitoring of microbial growth parameters in microwell plates.

The enormous variation possibilities of bioprocesses challenge process development to fix a commercial process with respect to costs and time. Although some cultivation systems and some devices for unit operations combine the latest technology on miniaturization, parallelization, and sensing, the degree of automation in upstream and downstream bioprocess development is still limited to single steps. We aim to face this challenge by an interdisciplinary approach to significantly shorten development times and costs. As a first step, we scaled down analytical assays to the microliter scale and created automated procedures for starting the cultivation and monitoring the optical density (OD), pH, concentrations of glucose and acetate in the culture medium, and product formation in fed-batch cultures in the 96-well format. Then, the separate measurements of pH, OD, and concentrations of acetate and glucose were combined to one method. This method enables automated process monitoring at dedicated intervals (e.g., also during the night). By this approach, we managed to increase the information content of cultivations in 96-microwell plates, thus turning them into a suitable tool for high-throughput bioprocess development. Here, we present the flowcharts as well as cultivation data of our automation approach.

Performance and stability of ethanologenic Escherichia coli strain FBR5 during continuous culture on xylose and glucose.

Escherichia coli FBR5 containing recombinant genes for ethanol production on plasmids that are also required for anaerobic growth was cultivated continuously on 50 g/l xylose or glucose in the absence of antibiotics and without the use of special measures to limit the entry of oxygen into the fermenter. Under chemostat conditions, stable ethanol yields of ca. 80-85% of the theoretical were obtained on both sugars over 26 days at dilution rates of 0.045/h (xylose) and 0.075/h (glucose), with average plasmid retention rates of 96% (xylose) and 97% (glucose). In a continuous fluidized bed fermenter, with the cells immobilized on porous glass beads, the extent of plasmid retention by the free cells fell rapidly, while that of the immobilized cells remained constant. This was shown to be due to diffusion of oxygen through the tubing used to recirculate the medium and free cells. A change to oxygen-impermeable tubing led to a stable high rate of plasmid retention (more than 96% of both the free and immobilized cells) with ethanol yields of ca. 80% on a 50 g/l xylose feed. The maximum permissible level of oxygen availability consistent with high plasmid retention by the strain appears to be of the order of 0.1 mmol per hour per gram dry biomass, based on measurements of the rate of oxygen penetration into the fermenters. Revertant colonies lacking the ethanologenic plasmid were easily detectable by their morphology which correlated well with their lack of ampicillin resistance upon transfer plating.