Making sense of the complex metabolic activity of microbial communities is no mean feat. Understanding the stoichiometry and reaction kinetics of this activity typically requires precise quantification of reactants, metabolic products, and the composition of the microbial community itself. Researchers at the universities of Delft and Wageningen in the Netherlands used mass spectrometry, gas chromatography and fluorometry systems from Thermo Scientific to do just this; helping them to gain insight into the effects of pH and product inhibition on chain-elongating microorganisms in sequencing batch bioreactors.1
Image Credit: RattiyaThingdumhyu
Sequencing batch reactors
Sequencing batch reactors are one of the simplest types of bioreactors, each consisting of a single tank in which a full cycle of anaerobic processing is carried out by a community of microorganisms. This cycle consists of four sequential steps (hence “sequencing” bioreactor): feeding, reacting, settling, and decanting.2 Sequencing batch reactors are predominantly used to process wastewater from both municipal and industrial sources. In this capacity, sequencing batch reactors are a relatively new technology – however, their simple configuration and high efficiency in removing suspended solids and reducing biological oxygen demand (BOD) makes them a promising low-cost approach to the effective treatment of dangerous wastewater.
Processing wastewater using sequencing batch reactors has benefits beyond removing contaminants from wastewater and making it safe to return to the water cycle. By harnessing the metabolic power of microorganisms in sequencing batch reactors, researchers are hopeful that it may be possible to convert complex industrial and agricultural waste effluent into recoverable microbial “products”. In the transition away from a petroleum-based society, the microbial production of useful chemicals from renewable resources in this way has gained significant interest.3,4
Such techniques would have potentially enormous ecological and economic benefits. However, the influence of environmental conditions on the stoichiometry and kinetics of many types of reaction that occur in sequencing batch bioreactors remain elusive.
Investigating microbial carboxylic acid chain-lengthening reactions
A team of researchers from the universities of Delft and Wageningen in the Netherlands set out to investigate the effects of environmental factors on a specific type of reaction in a sequencing batch bioreactor: the elongation of short-chain fatty acids through reversed β-oxidation. Processed by microorganisms (in particular Clostridium kluyveri related species), the carbon “backbones” of short chain fatty acids such as butanoic acid, present in complex wastewater streams, are elongated. Product of this metabolic processing is medium-chain carboxylic acids such as hexanoic acid and octanoic acid (also known as caproic acid and caprylic acid, respectively).
Many medium-chain carboxylic acids are valuable compounds. Hexanoic acid, for example, can be used as an antimicrobial agent, as a corrosion inhibitor, and as a precursor for a number of flavors, fragrances, solvents and fuels.5
By tuning the conditions within a reactor, the composition and behavior of microbial communities can be changed in order to suppress or enhance certain reaction types. The team of researchers aimed to explore the influence of environmental factors (in particular, pH and product inhibition) on carboxylic acid chain-lengthening reactions within a sequencing batch reactor in order to help uncover the fundamental aspects of these reactions.
A window into the microbial realm
Sequencing batch bioreactors typically play host to a dynamic community of competing (and cooperating) microbial species. Untangling the complex metabolic processes within a bioreactor and quantifying a particular class of reactions can be challenging.
At the least, it’s generally necessary to keep a detailed inventory of the reactor’s inputs and outputs, and to monitor the presence of specific reactants and products in solution within the reactor. In addition, characterizing the microbial population itself can provide valuable insight into the roles of various microbial species.
Rather than processing real waste streams, the team of researchers created an experimental setup whereby process parameters and microbial conversions could be closely monitored within two 1L batch reactors, each operating for 48 days.
A Thermo Scientific PRIMA BT benchtop mass spectrometer was used to carry out in- and off-gas analysis of the reactors. Designed for process development laboratories, the PRIMA BT uses scanning magnetic-sector technology to deliver accurate, precise and stable on-line gas analysis. Physicochemical modeling enabled off-gas measurements to be converted into bioreactor-specific respiration rates of H2, N2, CO2, and CH4.
Concentrations of butanoic acid (a short chain fatty acid), hexanoic acid (a medium chain carboxylic acid) and ethanol (an electron donor for chain-lengthening reactions) were all measured using a Thermo Scientific Trace 1300 gas chromatograph equipped with an injector maintained at 180 °C. The trace 1300 series is a compact GC system designed to deliver extremely fast results at a low cost of ownership. The unique modular design enables researchers to build only the configuration they need, minimizing costs compared to traditional GC systems.
The team processed samples from the bioreactor into microbial pellets in order to carry out genomic analysis. Prior to DNA sequencing, a Thermo Scientific Qubit 4 fluorometer was used to provide rapid quantification of DNA within the pellets, ensuring that sufficient DNA was present before sending samples off for amplicon sequencing. Providing results in under 3 seconds and requiring as little as 1 µL of sample, the Qubit 4 is unparalleled for simple and rapid DNA/RNA quantification.
Through careful control of process parameters and precise monitoring of reactants, products and microbial populations; the researchers were able to elucidate the effects of varying pH and product inhibition on chain-lengthening reactions within the bioreactors. Their results provided insight into the ways microorganisms deal with energy losses associated with product inhibition, showing that the chain-elongating reactions rely on a balance between substrate uptake and product inhibition.
Thermo Scientific develops world-leading analytical solutions which enable researchers to solve complex challenges in biotechnology. To find out more about the Thermo Scientific systems used in this research – or any of our other analytical systems – get in touch with Thermo Scientific today.
References and Further Reading
- Allaart, M. T., Stouten, G. R., Sousa, D. Z. & Kleerebezem, R. Product Inhibition and pH Affect Stoichiometry and Kinetics of Chain Elongating Microbial Communities in Sequencing Batch Bioreactors. Front. Bioeng. Biotechnol. 9, 693030 (2021).
- Anderson, K., Sallis, P. & Uyanik, S. Anaerobic treatment processes. in Handbook of Water and Wastewater Microbiology 391–426 (Elsevier, 2003). doi:10.1016/B978-012470100-7/50025-X.
- Guerra-Rodríguez, S., Oulego, P., Rodríguez, E., Singh, D. N. & Rodríguez-Chueca, J. Towards the Implementation of Circular Economy in the Wastewater Sector: Challenges and Opportunities. (2020).
- Gherghel, A., Teodosiu, C. & De Gisi, S. A review on wastewater sludge valorisation and its challenges in the context of circular economy. Journal of Cleaner Production 228, pp. 244–263 (2019).
- Biological formation of caproate and caprylate from acetate: fuel and chemical production from low grade biomass - Energy & Environmental Science (RSC Publishing). https://pubs.rsc.org/en/content/articlelanding/2011/ee/c0ee00282h.
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