Accelerated Bioprocess Development of Endopolygalacturonase-Production with Saccharomyces cerevisiae Using Multivariate Prediction in a 48 Mini-Bioreactor Automated Platform.

Mini-bioreactor systems enabling automatized operation of numerous parallel cultivations are a promising alternative to accelerate and optimize bioprocess development allowing for sophisticated cultivation experiments in high throughput. These include fed-batch and continuous cultivations with multiple options of process control and sample analysis which deliver valuable screening tools for industrial production. However, the model-based methods needed to operate these robotic facilities efficiently considering the complexity of biological processes are missing. We present an automated experiment facility that integrates online data handling, visualization and treatment using multivariate analysis approaches to design and operate dynamical experimental campaigns in up to 48 mini-bioreactors (8⁻12 mL) in parallel. In this study, the characterization of Saccharomyces cerevisiae AH22 secreting recombinant endopolygalacturonase is performed, running and comparing 16 experimental conditions in triplicate. Data-driven multivariate methods were developed to allow for fast, automated decision making as well as online predictive data analysis regarding endopolygalacturonase production. Using dynamic process information, a cultivation with abnormal behavior could be detected by principal component analysis as well as two clusters of similarly behaving cultivations, later classified according to the feeding rate. By decision tree analysis, cultivation conditions leading to an optimal recombinant product formation could be identified automatically. The developed method is easily adaptable to different strains and cultivation strategies, and suitable for automatized process development reducing the experimental times and costs.

Automated Cell Treatment for Competence and Transformation of Escherichia coli in a High-Throughput Quasi-Turbidostat Using Microtiter Plates.

Metabolic engineering and genome editing strategies often lead to large strain libraries of a bacterial host. Nevertheless, the generation of competent cells is the basis for transformation and subsequent screening of these strains. While preparation of competent cells is a standard procedure in flask cultivations, parallelization becomes a challenging task when working with larger libraries and liquid handling stations as transformation efficiency depends on a distinct physiological state of the cells. We present a robust method for the preparation of competent cells and their transformation. The strength of the method is that all cells on the plate can be maintained at a high growth rate until all cultures have reached a defined cell density regardless of growth rate and lag phase variabilities. This allows sufficient transformation in automated high throughput facilities and solves important scheduling issues in wet-lab library screenings. We address the problem of different growth rates, lag phases, and initial cell densities inspired by the characteristics of continuous cultures. The method functions on a fully automated liquid handling platform including all steps from the inoculation of the liquid cultures to plating and incubation on agar plates. The key advantage of the developed method is that it enables cell harvest in 96 well plates at a predefined time by keeping fast growing cells in the exponential phase as in turbidostat cultivations. This is done by a periodic monitoring of cell growth and a controlled dilution specific for each well. With the described methodology, we were able to transform different strains in parallel. The transformants produced can be picked and used in further automated screening experiments. This method offers the possibility to transform any combination of strain- and plasmid library in an automated high-throughput system, overcoming an important bottleneck in the high-throughput screening and the overall chain of bioprocess development.

Design of experiments-based high-throughput strategy for development and optimization of efficient cell disruption protocols.

Efficient and reproducible cell lysis is a crucial step during downstream processing of intracellular products. The composition of an optimal lysis buffer should be chosen depending on the organism, its growth status, the applied detection methods, and even the target molecule. Especially for high-throughput applications, where sample volumes are limited, the adaptation of a lysis buffer to the specific campaign is an urgent need. Here, we present a general design of experiments-based strategy suitable for eight constituents and demonstrate the strength of this approach by the development of an efficient lysis buffer for Gram-negative bacteria, which is applicable in a high-throughput format in a short time. The concentrations of four lysis-inducing chemical agents EDTA, lysozyme, Triton X-100, and polymyxin B were optimized for maximal soluble protein concentration and ß-galactosidase activity in a 96-well format on a Microlab Star liquid handling platform under design of experiments methodology. The resulting lysis buffer showed the same performance as a commercially available lysis buffer. The developed protocol resulted in an optimized buffer within only three runs. The established procedure can be easily applied to adapt the lysis buffer to other strains and target molecules.

Detection of growth rate-dependent product formation in miniaturized parallel fed-batch cultivations.

Saccharomyces cerevisiae is a popular expression system for recombinant proteins. In most cases, production processes are performed as carbon-limited fed-batch cultures to avoid aerobic ethanol formation. Especially for constitutive expression systems, the specific product formation rate depends on the specific growth rate. The development of optimal feeding strategies strongly depends on laboratory-scale cultivations, which are time and resource consuming, especially when continuous experiments are carried out. It is therefore beneficial for accelerated process development to look at alternatives. In this study, S. cerevisiae AH22 secreting a heterologous endo-polygalacturonase (EPG) was characterized in microwell plates with an enzyme-based fed-batch medium. Through variation of the glucose release rate, different growth profiles were established and the impact on EPG secretion was analyzed. Product formation rates of 200-400 U (gx h)-1 were determined. As a reference, bioreactor experiments using the change-stat cultivation technique were performed. The growth-dependent product formation was analyzed over dilution rates of D = 0.01-0.35 with smooth change of D at a rate of 0.003 h-2. EPG production was found to be comparable with a qp of 400 U (gx h)-1 at D = 0.27 h-1. The presented results indicate that parallel miniaturized fed-batch cultures can be applied to determine product formation profiles of putative production strains. With further automation and parallelization of the concept, strain characterization can be performed in shorter time.

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.

Lactose autoinduction with enzymatic glucose release: characterization of the cultivation system in bioreactor.

The lactose autoinduction system for recombinant protein production was combined with enzymatic glucose release as a method to provide a constant feed of glucose instead of using glycerol as a carbon substrate. Bioreactor cultivation confirmed that the slow glucose feed does not prevent the induction by lactose. HPLC studies showed that with successful recombinant protein production only a very low amount of lactose was metabolized during glucose-limited fed-batch conditions by the Escherichia coli strain BL21(DE3)pLysS in well-aerated conditions, which are problematic for glycerol-based autoinduction systems. We propose that slow enzymatic glucose feed does not cause a full activation of the lactose operon. However recombinant PDI-A protein (A-domain of human disulfide isomerase) was steadily produced until the end of the cultivation. The results of the cultivations confirmed our earlier observations with shaken cultures showing that lactose autoinduction cultures based on enzymatic glucose feed have good scalability, and that this system can be applied also to bioreactor cultivations.

Glucose-limited high cell density cultivations from small to pilot plant scale using an enzyme-controlled glucose delivery system.

The enzyme controlled substrate delivery cultivation technology EnBase(®) Flo allows a fed-batch-like growth in batch cultures. It has been previously shown that this technology can be applied in small cultivation vessels such as micro- and deep well plates and also shake flasks. In these scales high cell densities and improved protein production for Escherichia coli cultures were demonstrated. This current study aims to evaluate the scalability of the controlled glucose release technique to pilot scale bioreactors. Throughout all scales, that is, deep well plates, 3 L bioreactor and 150 L bioreactor cultivations, the growth was very similar and the model protein, a recombinant alcohol dehydrogenase (ADH) was produced with a high yield in soluble form. Moreover, EnBase Flo also was successfully used as a controlled starter culture in high cell density fed-batch cultivations with external glucose feeding. Here the external feeding pump was started after overnight cultivation with EnBase Flo. Final optical densities in these cultivations reached 120 (corresponding to about 40 g L(-1) dry cell weight) and a high expression level of ADH was obtained. The EnBase cultivation technology ensures a controlled initial cultivation under fed-batch mode without the need for a feeding pump. Because of the linear cell growth under glucose limitation it provides optimal and robust starting conditions for traditional external feed-based processes.

Microbial safety of air-dried and rewetted biosolids.

To assess microbial safety of treated sewage sludge (biosolids), we examined the inactivation of microbial indicators for potential bacterial, viral and protozoan pathogens. The levels of indicators were determined throughout the air-drying and storage phases of anaerobically digested sewage sludge. Samples were collected from two wastewater treatment plants (WWTPS) in Victoria, Australia. Established methods were applied for analysis of bacteria and coliphages, based on membrane filtration and layered plates, respectively. In the pan drying phase, the prevalence of Escherichia coli was reduced by >5 log10 compared with sludge entering the pan. Thus, after pan drying of 8-11 months at WWTP A and 15 months at WWTP B, the numbers of E. coli were reduced to below 10(2) cfu/g dry solids (DS). This level is acceptable for unrestricted use in agriculture in Australia (P1 treatment grade), the UK (enhanced treatment status) and the USA (Class A pathogen reduction). Coliphage numbers also decreased substantially during the air-drying phase, indicating that enteric viruses are also likely to be destroyed during this phase. Clostridium perfringens appeared to be an overly conservative indicator. Survival, but not regrowth, of E. coli or Salmonella was observed in rewetted biosolids (15-20% moisture content), after being seeded with these species, indicating a degree of safety of stored biosolids upon rewetting by rain.