Researchers at Delft University of Technology, VU University Amsterdam and Radboud University in the Netherlands developed a new technique for monitoring biological rates in dynamic microbial systems using on-line measurements from a Thermo Scientific Prima BT benchtop mass spectrometer.1
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Making sense of microbial worlds
Microbial communities are complex, dynamic systems in which the behavior and survival of each strain is inexorably linked to every other strain.2 These complex microbial ecosystems can form intricate webs of metabolic activity in which the products of one strain’s metabolic activity serve as fuel – or poison – for countless other organisms.
One of the fundamental tools that scientists use to make sense of these convoluted communities is the enrichment study. In an enrichment study, a microbial community is exposed to certain selective pressures within a bioreactor. By controlling environmental factors such as temperature, light levels, pH or the availability of certain carbon sources; researchers can create conditions that are favorable to a certain strain within the culture. Over time, the culture will become “enriched” with whichever microbial strain is best adapted to the imposed conditions.3
Throughout the 20th and 21st centuries, enrichment studies have enabled the discovery and characterization of a huge number of new microorganisms. However, the majority of these enrichments have been carried out by imposing specific static conditions in either continuous or batch systems – holding the pH constant, for example, or inhibiting the availability of certain growth media. Recent work shows that imposing dynamic process conditions reveals additional microbial diversity compared to traditional chemostat or batch cultivation.4–6
The challenge of studying dynamic enrichment cultures
Done right, dynamic enrichments provide access to additional information on time-dependent evolution of the system, through analysis and comparison of each operational cycle.
But studying dynamic microbial systems is much more experimentally demanding than studying static ones. Cyclic changes in conditions (such as nutrient pulses) result in variable conditions within the bioreactor, and there is a certain response time before measurable parameters (such as dissolved oxygen concentration) respond to these changes. In order to gain insight into dynamic cultivation systems, researchers need to be able to examine the time-dependent properties of the system. This requires repeated measurement at short time intervals.
Enrichment studies often take place over the course of weeks or months, making short-interval manual sampling cumbersome and often impractical. Online data from liquid probes and off-gas analyzers is readily available – however, utilizing this data for detailed process characterization is often limited due to complicating physicochemical processes and inherent measurement noise.
A new mass spectrometry-based approach to on-line characterization of dynamic cultures
With a setup of eight 2.2 L bioreactors, the team of researchers demonstrated a novel technique for detailed, time-resolved analysis of dynamic cultures.
Each bioreactor in the study was equipped with four feed/effluent pumps and two acid/base pumps. pH and dissolved oxygen probes were placed in each reactor. At the heart of their measurement system, the researchers used a Prima BT process mass spectrometer for online monitoring of bioreactor in- and off-gas composition. Offering fast switching between sample streams without compromising sample quality, the Prima BT enabled the researchers to collect frequent measurements of gas composition: the in- and off-gas composition of all eight bioreactors was measured every three minutes.
Using mass spectrometry to measure continuously changing gas streams presents challenges. When gas composition remains constant, the Prima BT enables analysis of composition down to ppm levels. However, during dynamic bioreactor operation, the off-gas composition changes continuously – as a result, gas composition within the flow cell changes continuously throughout the measurement window. This typically requires a trade-off between accuracy (achieved by larger sample times) and analysis time. Because the Prima BT features a small flow cell, which is continuously analyzed at different mass/charge ratios, the researchers were able to achieve a strong compromise between the two: alternating between 10 seconds of stream flushing and 8 seconds of measurement per channel (2 seconds at 4 different mass/charge ratios), they were able to take measurements of each system in under 3 minutes with an accuracy of between 10-300 ppm depending on the gas compound mole fraction.
The team combined their rapid on-line mass spectrometry measurements with sophisticated automated data processing. Gas composition measurements were processed with a Particle Filter and kinetic process model, enabling accurate reconstruction of the dominant biological rates within the bioreactor.
This, in turn, enabled differentiation between the processes of storage compound production and biomass growth within the bioreactors.
The researchers’ methods allow for accurate and time-resolved assessment of the functional behavior of long-running enrichment cultures without needing off-line samples. This work paves the way for new insights into process dynamics with minimal experimental effort.
Utilizing scanning magnetic-sector technology, the Prima BT bench top mass spectrometer offers powerful and precise gas real-time gas analyses. Features including 16-port rapid multistream sampling (RMS) and six-port automatic calibration manifold make it the ideal MS solution for biotechnology research and process development.7 To learn more about the range of mass spectrometry systems available from Thermo Scientific, get in touch with us today.
References and Further Reading
- Stouten, G. R., Douwenga, S., Hogendoorn, C. & Kleerebezem, R. System characterization of dynamic biological cultivations through improved data analysis. (2021) doi:10.1101/2021.05.14.442977.
- Into the complexity of microbial communities. Instituto Gulbenkian de Ciência https://gulbenkian.pt/ciencia/news/into-the-complexity-of-microbial-communities/ (2021).
- Schlegel, H. G. & Zaborosch, C. General Microbiology. (Cambridge University Press, 1993).
- van Loosdrecht, M., Smolders, G., Kuba, T. & Heijnen, S. Metabolism of micro-organisms responsible for enhanced biological phosphorus removal from wastewater. Use of dynamic enrichment cultures. Antonie van Leeuwenhoek 71, pp. 109–116 (1997).
- Simultaneous storage and degradation of phb and glycogen in activated sludge cultures - ScienceDirect. https://www.sciencedirect.com/science/article/abs/pii/S0043135400005637.
- Microbial community development in a dynamic gut model is reproducible, colon region specific, and selective for Bacteroidetes and Clostridium cluster IX - PubMed. https://pubmed.ncbi.nlm.nih.gov/20562281/.
- Prima BT Bench Top Process Mass Spectrometer. https://www.thermofisher.com/order/catalog/product/PRIMABT.
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