Parallel cultivation platform

The parallel cultivation platform located at Delft University of Technology consists of 40 bioreactors units that allow for cultivation and investigation of most chemotrophic and phototrophic microbial communities in extremely well-defined conditions. The flexible arrangement of analytical equipment enables the investigation of virtually all redox conditions of interest in microbial ecology, ranging from aerobic to strictly anaerobic conditions. High resolution cultivation and characterization studies are facilitated with non-defined microbial communities (reductionist enrichment approach) as well as defined mixtures of microorganisms (synthetic ecology approach), that typically have been established in the Biodiscovery platform of UNLOCK. To our knowledge there is worldwide no other platform that facilitates cultivation and analysis of microbial communities at the scale and resolution of the UNLOCK parallel cultivation platform.

Figure 1 – Close up photograph of 5 parallel operated bioreactors during a long-term enrichment study under dynamic conditions.

Platform benefits

The parallel cultivation platform is specifically suitable for investigating the impact of dynamic process conditions on the functional and molecular development of microbial communities. Dynamic process conditions are typical for many natural and man-made ecosystems (day-night cycles, seasonal variations, variable substrate supply rates etc.) but to date largely unexplored in microbial ecology. Since supply of both gaseous and liquid substrates and removal of effluent is conducted with computer controlled systems, we can impose a wide variation of feeding regimes to the system (as indicated as the influent variations in Figure 2). The combination of detailed on-line measurements with a dynamic feeding regime provides an unprecedented high resolution of functional system information, specifically suitable for dynamic process modelling and analysis. This evidently does not mean that bioreactors cannot be operated as chemostat or Sequencing Batch Reactor (SBR), but we have repeatedly demonstrated that inclusion of (periodic) dynamics in operation results in superior systems characterization and process identification (Johnson el al., 2009; Kleerebezem & van Loosdrecht, 2008). Another specific feature included in the experimental setup proposed, is the ability to uncouple the liquid and solid (biomass) retention time in the process through a distinct channel for removal of the mixed fermentation broth and liquid effluent removal via an ultrafiltration (UF) membrane. User-friendly data interpretation software is under development that allows for direct analysis and inter-reactor comparison of all data generated in the experiments.

Figure 2 – Detailed overview of the experimental setup of reactors in the parallel cultivation platform at TUD. This is shown to exemplify the conceptual set-up of the different reactor types included in UNLOCK.

Platform Technical details

The parallel cultivation platform consists of temperature controlled stirred bioreactor units that can be operated at 0.5 to 2.0 liter liquid volume, and are equipped with an extensive set of on- and off-line analytical facilities. There are two types of experimental units that are different in terms of the intensity of process analysis:

·     The standard experimental setup (30 units) is equipped with the on-line analytical equipment shown in red in Figure 2: Liquid electrode measurements include pH, Dissolved Oxygen (DO) concentration, Oxidation Reduction Potential (ORP), and ion specific electrode measurements (ISE, not shown). Off-gas analysis after drying of the gas is conducted with a multi-channel Mass Spectrometer (MS). 

·     The intensive experimental setup (10 units, with extra analytical facilities shown in green) is equipped with an ultrafiltration (UF) sampling unit for a continuous supply of solid-free fermentation broth for periodic sampling and on-line analysis by High Pressure Liquid Chromatrography (HPLC), Gas Chromatography (GC), and/or Ion Chromatography (IC). Liquid samples are available for off-line analysis through a wide range of methods, e.g., ICP-MS, LC-MS, GC-MS.

The standard and intensive experimental setups serve a different experimental objective. The low-maintenance standard experimental setup is specifically suitable for medium to long-term experiments (1 to 12 months) with the objective to identify the functional development of a microbial community in well-defined conditions over many generations. When working with non-defined microbial communities these experiments typically concern microbial competition and succession studies, whereas for defined communities these systems are specifically suitable for directed evolution studies. On-line analysis of crucial process variables via e.g.-gas measurements allow for direct identification of major changes in functional process properties that may suggest that extra analyses are worthwhile to be taken. The intensive experimental setup is more suitable for short-term experiments (1 to 10 weeks) that aim to assess the effect of a specific process variable (e.g. pH, Temperature, influent composition) on the functional and molecular development of a specific microbial community. A high intensity experiment provides a high-resolution comparative analysis of the response of the system to the change imposed through detailed on-line liquid analysis. 

Platform Integration

Integration of the parallel cultivation platform in the UNLOCK facility enables a direct link with the Wageningen platforms. The biodiscovery platform can provide defined synthetic communities, and in combination with the sample processing platform facilitates a wide range of molecular analyses of samples from the parallel cultivation platform. Herewith the link between functional and molecular system properties can be investigated which is of crucial importance for this field of research. The cloud-based FAIR data platform facilitates storage and accessibility of both functional and molecular data obtained in the experiments conducted at the parallel cultivation platform. The standard operating procedures (SOPs) used in the various steps of the experiments and the data handling, facilitates inter-experimental comparison of data and simplifies the procedures needed for data analysis and translation of data into knowledge.

A working example: Linking microbial community dynamics and functionality 

As a working example we describe the work performed in the recently published manuscript: “Temperature as competitive strategy determining factor in pulse-fed aerobic bioreactors.” The ISME Journal (2019) by Stouten, G. R., Hogendoorn, C., Douwenga, S., Kilias, E. S., Muyzer, G., & Kleerebezem, R. (2019). In this work we investigated to which extent intermediate storage polymer production (Figure 3) is a temperature independent microbial competition determining factor. Eight parallel bioreactors were operated in the temperature range of 20°C to 40°C. The use of parallel-operated reactors combined with online data collection with community structure analysis, this study furthermore strived to align the structural and functional development in time of the microbial community as a function of temperature.

Figure 3 – Microscopy picture of microbial cells with PHA inclusion bodies, able to store up to 90% of the dry weight as PHA

The sequencing batch bioreactors were operated for over 100 days and were online monitored with state-of-the-art equipment. Online data was used to reconstruct functional development of the enrichment cultures, as was verified by offline measurements (Figure 3). 

Figure 4 – Online and offline data of a single bioreactor cycle, where the online data could be used to a large extent to describe the functional behaviour of the enrichment culture. The gradual enrichment of hoarding biomass in the bioreactors could be monitored over the 200+ consecutive cycles.

By utilizing all online data, it was possible to gain insights in the functional development of the enrichment cultures over time. A new data processing pipeline was developed to improve data handling and data interpretation by converting online measurements to real time microbial activity. For each operational cycle of each bioreactor the microbial functionality was captured as a mathematic depiction of the microbial activity. Three characteristic indicators were derived that showed strong correlations with microbial community structure development and offline verified measurements, as reflected in Figure 4 forone bioreactor system. 

Figure 2 – Detailed overview of the experimental setup of reactors in the parallel cultivation platform at TUD. This is shown to exemplify the conceptual set-up of the different reactor types included in UNLOCK.

Major benefits for this case study

Major benefits of the UNLOCK parallel research platform for this research included the ability to operate 8 bioreactors in parallel from the same inoculum at high resolution. The online data processing tools, combined with the community structure analysis by 16s rRNA gene amplicon sequencing allowed for a novel experimental design and aided in the understanding of competitive strategies in microbial communities.

Figure 2 – Detailed overview of the experimental setup of reactors in the parallel cultivation platform at TUD. This is shown to exemplify the conceptual set-up of the different reactor types included in UNLOCK.