(338f) Tools for Easy, Fast and Accurate Quantitative Characterization of the Methanotroph-Photoautotroph Coculture

Biogas is comprised primarily of methane (50%~70%) and carbon dioxide (30% ~50%). It can be produced under anaerobic conditions from various organic waste sources, including landfill waste, animal manure; wastewater sludge; and industrial, institutional, and commercial organic wastes. Recent studies have demonstrated that natural microbial communities have developed a highly efficient way to recover the energy and capture carbon from both CH₄ and CO₂ through interspecies coupling of methane oxidation to oxygenic photosynthesis [1-3]. In fact, multispecies associations are ubiquitous in nature as they provide key ecosystem services such as carbon, nutrient, and metal cycling. It has been recognized that a mixed culture, including methanotroph-photoautotroph cocultures, could offer a number of advantages over a conventional single-culture, such as complete utilization of substrate, better stability and robustness, higher product yield, higher growth rate, as well as the capability to carry out multistep transformation that would be impossible for a single organism. Despite these potential significant advantages, utilization of mixed cultures for biotechnological applications in bioenergy and related areas have been limited, partially due to the lack of effective tools to characterize the mixed culture accurately and frequently, and partially due to the lack of clearly understanding on the complex dynamics of coculture growth.

In our previous work, using the principles that drive the natural consortia [1-3], we have assembled several synthetic methanotroph-photoautotroph cocultures that exhibit stable growth under various substrate delivery and illumination regimes [4]. In order to develop coculture-based biotechnology for commercial biogas conversion, one of the key challenges is how to accurately characterize the function and composition of each species in the coculture. In this work we developed an experimental-computational protocol to deliver easy, fast and accurate quantitative characterization of various synthetic methanotroph-microalgae cocultures. Besides determining the individual biomass concentration of each organism in the coculture, the developed protocol can also obtain the individual consumption and production rates of O₂ and CO₂ for each strain (Figure 1 (a)). The accuracy and effectiveness of the developed protocol is demonstrated using two coculture pairs, one pair is Methylomicrobium alcaliphilum 20Z^R (methanotroph) - Synechococcus sp. PCC7002 (cyanobacterium) which prefer high salt high pH medium, another pair is Methylococcus capsulatus (methanotroph) - Chlorella sorokiniana (microalgae) which prefer low/no salt and neutral pH medium. The individual biomass concentrations for each strain obtained from the developed protocol were compared with that obtained from cell counting using flow cytometry. As shown in Figure 1 (b), the developed protocol delivers better accuracy in individual biomass concentration than the cell counting method, and doesn’t require any special equipment.

References:

1. Kip, N., van Winden, J.F., Pan, Y., Bodrossy, L., Reichart, G.-J., Smolders, A.J., Jetten, M.S., Damsté, J.S.S., den Camp, H.J.O., (2010). Global prevalence of methane oxidation by symbiotic bacteria in peat-moss ecosystems. Geosci. 3, 617–621.
2. Milucka, J., Kirf, M., Lu, L., Krupke, A., Lam, P., Littmann, S., Kuypers, M.M., Schubert, C.J., (2015). Methane oxidation coupled to oxygenic photosynthesis in anoxic waters. ISME J.
3. Raghoebarsing, A.A., Smolders, A.J., Schmid, M.C., Rijpstra, W.I.C., Wolters-Arts, M., Derksen, J., Jetten, M.S., Schouten, S., Damsté, J.S.S., Lamers, L.P., (2005). Methanotrophic symbionts provide carbon for photosynthesis in peat bogs. Nature 436, 1153–1156.
4. Badr K., Hilliard M., Roberts N., He Q.P., Wang J., (2019). “Photoautotroph-Methanotroph Coculture – A Flexible Platform for Efficient Biological CO2 – CH4 Co-utilization,” IFAC PapersOnLine, 52-1, 916–92.