From frozen cell bank to product assay: high-throughput strain characterisation for autonomous Design-Build-Test-Learn cycles.

BACKGROUND: Modern genome editing enables rapid construction of genetic variants, which are further developed in Design-Build-Test-Learn cycles. To operate such cycles in high throughput, fully automated screening, including cultivation and analytics, is crucial in the Test phase. Here, we present the required steps to meet these demands, resulting in an automated microbioreactor platform that facilitates autonomous phenotyping from cryo culture to product assay. RESULTS: First, an automated deep freezer was integrated into the robotic platform to provide working cell banks at all times. A mobile cart allows flexible docking of the freezer to multiple platforms. Next, precultures were integrated within the microtiter plate for cultivation, resulting in highly reproducible main cultures as demonstrated for Corynebacterium glutamicum. To avoid manual exchange of microtiter plates after cultivation, two clean-in-place strategies were established and validated, resulting in restored sterile conditions within two hours. Combined with the previous steps, these changes enable a flexible start of experiments and greatly increase the walk-away time. CONCLUSIONS: Overall, this work demonstrates the capability of our microbioreactor platform to perform autonomous, consecutive cultivation and phenotyping experiments. As highlighted in a case study of cutinase-secreting strains of C. glutamicum, the new procedure allows for flexible experimentation without human interaction while maintaining high reproducibility in early-stage screening processes.

Robotic integration enables autonomous operation of laboratory scale stirred tank bioreactors with model-driven process analysis.

Given its geometric similarity to large-scale production plants and the excellent possibilities for precise process control and monitoring, the classic stirred tank bioreactor (STR) still represents the gold standard for bioprocess development at a laboratory scale. However, compared to microbioreactor technologies, bioreactors often suffer from a low degree of process automation and deriving key performance indicators (KPIs) such as specific rates or yields often requires manual sampling and sample processing. A widely used parallelized STR setup was automated by connecting it to a liquid handling system and controlling it with a custom-made process control system. This allowed for the setup of a flexible modular platform enabling autonomous operation of the bioreactors without any operator present. Multiple unit operations like automated inoculation, sampling, sample processing and analysis, and decision making, for example for automated induction of protein production were implemented to achieve such functionality. The data gained during application studies was used for fitting of bioprocess models to derive relevant KPIs being in good agreement with literature. By combining the capabilities of STRs with the flexibility of liquid handling systems, this platform technology can be applied to a multitude of different bioprocess development pipelines at laboratory scale.

A fully automated pipeline for the dynamic at-line morphology analysis of microscale Aspergillus cultivation.

BACKGROUND: Morphology, being one of the key factors influencing productivity of filamentous fungi, is of great interest during bioprocess development. With increasing demand of high-throughput phenotyping technologies for fungi due to the emergence of novel time-efficient genetic engineering technologies, workflows for automated liquid handling combined with high-throughput morphology analysis have to be developed. RESULTS: In this study, a protocol allowing for 48 parallel microbioreactor cultivations of Aspergillus carbonarius with non-invasive online signals of backscatter and dissolved oxygen was established. To handle the increased cultivation throughput, the utilized microbioreactor is integrated into a liquid handling platform. During cultivation of filamentous fungi, cell suspensions result in either viscous broths or form pellets with varying size throughout the process. Therefore, tailor-made liquid handling parameters such as aspiration/dispense height, velocity and mixing steps were optimized and validated. Development and utilization of a novel injection station enabled a workflow, where biomass samples are automatically transferred into a flow through chamber fixed under a light microscope. In combination with an automated image analysis concept, this enabled an automated morphology analysis pipeline. The workflow was tested in a first application study, where the projected biomass area was determined at two different cultivation temperatures and compared to the microbioreactor online signals. CONCLUSIONS: A novel and robust workflow starting from microbioreactor cultivation, automated sample harvest and processing via liquid handling robots up to automated morphology analysis was developed. This protocol enables the determination of projected biomass areas for filamentous fungi in an automated and high-throughput manner. This measurement of morphology can be applied to describe overall pellet size distribution and heterogeneity.

Microbioreactor-assisted cultivation workflows for time-efficient phenotyping of protein producing Aspergillus niger in batch and fed-batch mode.

In recent years, many fungal genomes have become publicly available. In combination with novel gene editing tools, this allows for accelerated strain construction, making filamentous fungi even more interesting for the production of valuable products. However, besides their extraordinary production and secretion capacities, fungi most often exhibit challenging morphologies, which need to be screened for the best operational window. Thereby, combining genetic diversity with various environmental parameters results in a large parameter space, creating a strong demand for time-efficient phenotyping technologies. Microbioreactor systems, which have been well established for bacterial organisms, enable an increased cultivation throughput via parallelization and miniaturization, as well as enhanced process insight via non-invasive online monitoring. Nevertheless, only few reports about microtiter plate cultivation for filamentous fungi in general and even less with online monitoring exist in literature. Moreover, screening under batch conditions in microscale, when a fed-batch process is performed in large-scale might even lead to the wrong identification of optimized parameters. Therefore, in this study a novel workflow for Aspergillus niger was developed, allowing for up to 48 parallel microbioreactor cultivations in batch as well as fed-batch mode. This workflow was validated against lab-scale bioreactor cultivations to proof scalability. With the optimized cultivation protocol, three different micro-scale fed-batch strategies were tested to identify the best protein production conditions for intracellular model product GFP. Subsequently, the best feeding strategy was again validated in a lab-scale bioreactor.

Parallelized microscale fed-batch cultivation in online-monitored microtiter plates: implications of media composition and feed strategies for process design and performance.

Limited throughput represents a substantial drawback during bioprocess development. In recent years, several commercial microbioreactor systems have emerged featuring parallelized experimentation with optical monitoring. However, many devices remain limited to batch mode and do not represent the fed-batch strategy typically applied on an industrial scale. A workflow for 32-fold parallelized microscale cultivation of protein secreting Corynebacterium glutamicum in microtiter plates incorporating online monitoring, pH control and feeding was developed and validated. Critical interference of the essential media component protocatechuic acid with pH measurement was revealed, but was effectively resolved by 80% concentration reduction without affecting biological performance. Microfluidic pH control and feeding (pulsed, constant and exponential) were successfully implemented: Whereas pH control improved performance only slightly, feeding revealed a much higher optimization potential. Exponential feeding with µ = 0.1 h-1 resulted in the highest product titers. In contrast, other performance indicators such as biomass-specific or volumetric productivity resulted in different optimal feeding regimes.

A closer look at Aspergillus: online monitoring via scattered light enables reproducible phenotyping.

BACKGROUND: Filamentously growing microorganisms offer unique advantages for biotechnological processes, such as extraordinary secretion capacities, going along with multiple obstacles due to their complex morphology. However, limited experimental throughput in bioprocess development still hampers taking advantage of their full potential. Miniaturization and automation are powerful tools to accelerate bioprocess development, but so far the application of such technologies has mainly been focused on non-filamentous systems. During cultivation, filamentous fungi can undergo remarkable morphological changes, creating challenging cultivation conditions. Depending on the process and product, only one specific state of morphology may be advantageous to achieve e.g. optimal productivity or yield. Different approaches to control morphology have been investigated, such as microparticle enhanced cultivation. However, the addition of solid microparticles impedes the optical measurements typically used by microbioreactor systems and thus alternatives are needed. RESULTS: Aspergillus giganteus IfGB 0902 was used as a model system to develop a time-efficient and robust workflow allowing microscale cultivation with increased throughput. The effect of microtiter plate geometry, shaking frequency and medium additives (talc and calcium chloride) on homogeneity of culture morphology as well as reproducibility were analyzed via online biomass measurement, microscopic imaging and cell dry weight. While addition of talc severely affected online measurements, 2% (w v-1) calcium chloride was successfully applied to obtain a highly reproducible growth behavior with homogenous morphology. Furthermore, the influence of small amounts of complex components was investigated for the applied model strain. By correlation to cell dry weight, it could be shown that optical measurements are a suitable signal for biomass concentration. However, each correlation is only applicable for a specific set of cultivation parameters. These optimized conditions were used in micro as well as lab-scale bioreactor cultivation in order to verify the reproducibility and scalability of the setup. CONCLUSION: A robust workflow for A. giganteus was developed, allowing for reproducible microscale cultivation with online monitoring, where calcium chloride is an useful alternative to microparticle enhanced cultivation in order to control the morphology. Independent of the cultivation volume, comparable phenotypes were observed in microtiter plates and in lab-scale bioreactor.

Rapid and comprehensive evaluation of microalgal fatty acids via untargeted gas chromatography and time-of-flight mass spectrometry.

Due to their high triacylglyceride content, microalgae are intensively investigated for bio-economy and food applications. However, lipid analysis is a laborious task incorporating extraction, transesterification and typically gas chromatographic measurement. Co-elution induces a significant risk of fatty acid misidentification and thus, additional purification steps like thin layer chromatography are needed. Contrary to database matching approaches, solely targeted analysis is facilitated as compound identification is driven by matching retention times or indices with standard substances. In this context, a rapid workflow for the analysis of algal fatty acids is presented. In-situ transesterification was used to simplify sample preparation and conditions were optimized towards fast processing. If results are needed at the very day of sampling, direct processing without a preceding drying step is feasible to obtain a rough estimate about the occurrence of the major compounds. Coupling gas chromatography and time-of-flight mass spectrometry enables untargeted analysis. Unknown compounds may be identified by structural reconstruction of their respective fragmentation patterns and by database matching to routinely avoid mismatches by co-elution of disturbing agents. The developed workflow was successfully applied to derive the exact stereochemistry of all fatty acids from Chlorella vulgaris and a systematic shift depending on physiological state of the cells was confirmed.

Differential transcriptomic analysis reveals hidden light response in Streptomyces lividans.

Recently, a comprehensive screening workflow for the filamentous bacterium Streptomyces lividans, a highly performant source for pharmaceutically active agents was introduced. This framework used parallelized cultivation in microtiter plates to efficiently accelerate early upstream process development. Focusing on growth performance, cultivation was successfully scaled-up to 1 L stirred tank reactors. However, metabolic adaptation was observed on the transcriptomic level as among others, several genes incorporated in light response were upregulated during bioreactor cultivation. Despite it was assumed that this was attributed to the fact that reactor cultivations were performed in glass vessels exposed to daylight and artificial room light, this setup did not allow distinguishing exclusively between light and other effects. Upon that, the present study directly investigates the influence of light by defined illumination of microtiter plate cultures. Almost identical growth performance was observed for cultures grown in the dark or with illumination. Transcriptomics revealed the upregulation of seven genes of which 6 have previously been described to be relevant for carotenoid synthesis and its regulation. These pigments are effective quenchers of reactive oxygen species. The seventh transcript coded for a photo-lyase incorporated in UV-damage repair of DNA further confirming induced light response. However, this was fully compensated by metabolic adaptation on the transcriptomic level and overall process performance was maintained. Consequently, environmental conditions need extremely careful control and evaluation during in-depth omics analysis of bioprocesses. Otherwise metabolic adaptation induced by such issues can easily be misinterpreted, especially during studies addressing cultivation system comparisons. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:287-292, 2018.

Laboratory-scale photobiotechnology-current trends and future perspectives.

Phototrophic bioprocesses are a promising puzzle piece in future bioeconomy concepts but yet mostly fail for economic reasons. Besides other aspects, this is mainly attributed to the omnipresent issue of optimal light supply impeding scale-up and -down of phototrophic processes according to classic established concepts. This MiniReview examines two current trends in photobiotechnology, namely microscale cultivation and modeling and simulation. Microphotobioreactors are a valuable and promising trend with microfluidic chips and microtiter plates as predominant design concepts. Providing idealized conditions, chip systems are preferably to be used for acquiring physiological data of microalgae while microtiter plate systems are more appropriate for process parameter and medium screenings. However, these systems are far from series technology and significant improvements especially regarding flexible light supply remain crucial. Whereas microscale is less addressed by modeling and simulation so far, benchtop photobioreactor design and operation have successfully been studied using such tools. This particularly includes quantitative model-assisted understanding of mixing, mass transfer, light dispersion and particle tracing as well as their relevance for microalgal performance. The ultimate goal will be to combine physiological data from microphotobioreactors with hybrid models to integrate metabolism and reactor simulation in order to facilitate knowledge-based scale transfer of phototrophic bioprocesses.

A framework for accelerated phototrophic bioprocess development: integration of parallelized microscale cultivation, laboratory automation and Kriging-assisted experimental design.

BACKGROUND: Even though microalgae-derived biodiesel has regained interest within the last decade, industrial production is still challenging for economic reasons. Besides reactor design, as well as value chain and strain engineering, laborious and slow early-stage parameter optimization represents a major drawback. RESULTS: The present study introduces a framework for the accelerated development of phototrophic bioprocesses. A state-of-the-art micro-photobioreactor supported by a liquid-handling robot for automated medium preparation and product quantification was used. To take full advantage of the technology's experimental capacity, Kriging-assisted experimental design was integrated to enable highly efficient execution of screening applications. The resulting platform was used for medium optimization of a lipid production process using Chlorella vulgaris toward maximum volumetric productivity. Within only four experimental rounds, lipid production was increased approximately threefold to 212 ± 11 mg L-1 d-1. Besides nitrogen availability as a key parameter, magnesium, calcium and various trace elements were shown to be of crucial importance. Here, synergistic multi-parameter interactions as revealed by the experimental design introduced significant further optimization potential. CONCLUSIONS: The integration of parallelized microscale cultivation, laboratory automation and Kriging-assisted experimental design proved to be a fruitful tool for the accelerated development of phototrophic bioprocesses. By means of the proposed technology, the targeted optimization task was conducted in a very timely and material-efficient manner.

Comparative evaluation of phototrophic microtiter plate cultivation against laboratory-scale photobioreactors.

Extended cultivation times, rendering phototrophic bioprocess development time inefficient, resulted in the recent development of micro-photobioreactors enabling accelerated process development. However, especially for laboratory photobioreactors, only little is known concerning the influence of design on process performance. Thus, the aim of the present investigation was to evaluate the scalability of a microtiter plate-based parallelized micro-photobioreactor against a representative set of established laboratory photobioreactors. Lipid production by Chlorella vulgaris was used as a model system. During exponential growth, the microtiter plate cultures achieved maximal growth rates of ca. 1.44 ± 0.02 day-1 being in good agreement with the larger systems. Moreover, cultures in the micro-photobioreactor could be kept in the exponential phase up to the highest biomass concentrations most probably due to the beneficial light supply at this scale. Compared to the shake flask and test tube cultures, microtiter plate cultivation achieved an equivalent biomass yield, lipid content, and lipid fingerprint. In contrast, the flat-panel process resulted only in marginal productivity due to insufficient light supply. Thus, microtiter plates showed good scalability to the investigated laboratory photobioreactors as overall differences were rather small taking the differing scales into account.

Design and validation of a parallelized micro-photobioreactor enabling phototrophic bioprocess development at elevated throughput.

Microalgae offer great potential for the industrial production of numerous compounds, but most of the currently available processes fail on economic aspects. Due to the lack of appropriate microcultivation systems, especially screening and early stage laboratory process characterization limit throughput in process development. Consequently, a demand for high throughput photobioreactors has recently been identified upon which some prototype systems emerged. However, compared to microbial microbioreactors, the systems so far introduced suffer from at least one of several drawbacks, that is, inhomogeneous conditions, poor mixing or excessive evaporation. In this context, a microtiter plate based micro-photobioreactor was developed enabling 48-fold parallelized cultivation. Strict control of the process conditions enabled a high comparability between the distinct wells of one plate (±5% fluctuation in biomass formation). The small scale, resulting in a beneficial surface to volume ratio, as well as the fast mixing due to rigorous orbital shaking, ensured an excellent light supply of the cultures. Moreover, non-invasive online biomass quantification was implemented via a scattered light analyzer that is capable of biomass measurements during continuous illumination of the cultures. The system was shown to be especially qualified for parallelized laboratory screening applications like for instance media optimization. Easy automation via integration into a liquid handling platform is given by design. Thereby, the presented micro-photobioreactor system significantly contributes to improving the time efficiency during the development of phototrophic bioprocesses. Biotechnol. Bioeng. 2017;114: 122-131. © 2016 Wiley Periodicals, Inc.

Automation of a Nile red staining assay enables high throughput quantification of microalgal lipid production.

BACKGROUND: Within the context of microalgal lipid production for biofuels and bulk chemical applications, specialized higher throughput devices for small scale parallelized cultivation are expected to boost the time efficiency of phototrophic bioprocess development. However, the increasing number of possible experiments is directly coupled to the demand for lipid quantification protocols that enable reliably measuring large sets of samples within short time and that can deal with the reduced sample volume typically generated at screening scale. To meet these demands, a dye based assay was established using a liquid handling robot to provide reproducible high throughput quantification of lipids with minimized hands-on-time. RESULTS: Lipid production was monitored using the fluorescent dye Nile red with dimethyl sulfoxide as solvent facilitating dye permeation. The staining kinetics of cells at different concentrations and physiological states were investigated to successfully down-scale the assay to 96 well microtiter plates. Gravimetric calibration against a well-established extractive protocol enabled absolute quantification of intracellular lipids improving precision from ±8 to ±2 % on average. Implementation into an automated liquid handling platform allows for measuring up to 48 samples within 6.5 h, reducing hands-on-time to a third compared to manual operation. Moreover, it was shown that automation enhances accuracy and precision compared to manual preparation. It was revealed that established protocols relying on optical density or cell number for biomass adjustion prior to staining may suffer from errors due to significant changes of the cells' optical and physiological properties during cultivation. Alternatively, the biovolume was used as a measure for biomass concentration so that errors from morphological changes can be excluded. CONCLUSIONS: The newly established assay proved to be applicable for absolute quantification of algal lipids avoiding limitations of currently established protocols, namely biomass adjustment and limited throughput. Automation was shown to improve data reliability, as well as experimental throughput simultaneously minimizing the needed hands-on-time to a third. Thereby, the presented protocol meets the demands for the analysis of samples generated by the upcoming generation of devices for higher throughput phototrophic cultivation and thereby contributes to boosting the time efficiency for setting up algae lipid production processes.