Photogranules Shake Sewage Treatment up – PSST

Photogranulation, or the transformation of activated sludge into photogranules over the course of several days to three weeks, was first observed in small vials incubated under static conditions, i.e., in the absence of any kind of mixing or shaking. In this type of incubation, exactly one granule is formed per vial. Later, we were able to show that morphologically similar photogranules could be formed in well-mixed, laboratory scale sequencing batch reactors. Naturally, in this kind of setting, thousands of photogranules populate the reactors. Incubations under static conditions as well as in well-mixed reactors are used in the three experimental tasks of the project.
After comparative analyses of the spatial and community structure, we hypothesized that the general formation mechanisms in these two entirely different systems is comparable. Consequently, we may be able to observe formation and maturation of an individual granule using the static approach, extending our findings to the well-mixed environment in the bioreactors, where these observations are impossible. The link between the two incubation systems is made through the formalization of generalized formation mechanisms by mathematical modelling.
Formation kinetics are followed using time-lapse imaging coupled to automated image analysis. Throughout the project, 16S rRNA amplicon sequencing and metagenomic analyses of simplified communities will be used follow the microbial communities constituting oxygenic photogranules. Microelectrodes are used to visualize the development of physico-chemical gradients within the granule environment.
The experimental part of the project is accompanied by a sequentially refined Life Cycle Assessments (LCA), leading to a robust LCA of a matured photogranule bioprocess towards the end of the project. LCA allows us to identify early on bottlenecks of the prospective bioprocess and to focus our efforts on areas with the highest environmental impact.

We are happy to welcome in Fall 2017 Hicham Ouazaite and Esmee Joosten as PhD students in the photogranule team. Hicham is funded through ANR JCJC PSST and the INRA department MICA. Esmee receives funding from the graduate school Gaïa of the University of Montpellier.
Esmee works on the identification of minimal conditions for photogranulation using static incubations. She recently finalized a fully automated experimental set-up for monitoring granulation kinetics using time-lapse photography. To assess biological reasons for granulation failure, Esmee extracted DNA for 16S rRNA amplicon sequencing from failed and successfully formed photogranules of 11 experiments. Results are currently analyzed. Her preliminary findings raise the important question if seasonal variations in activated sludge may affect photogranulation.
Hicham focusses on microstructure development and modeling of photogranulation. He has finished setting up microelectrode equipment for measuring biochemical gradients in photogranules. Hicham currently uses his experimental results to calibrate his 1-D reaction-diffusion model of a phototrophic ecosystem. This model will form the physico-chemical basis of a 3-D individual based model.
We have completed the installation of four lab-scale sequencing batch reactors. Two of them have been operated for more than one year. We observed in these bioreactors that nitrogen removal is primarily achieved by biomass growth and not through nitrification/denitrification, confirming results obtained by LCA.
The preliminary LCA is finished with a manuscript in progress for Water Research. The unexpected results of the analysis, i.e., the comparably high electricity cost of lighting as well as the potentially high value of the generated biomass have introduced a dogma shift in our thinking way from zero-energy wastewater treatment towards resource recover. This result already at this early stage proves our coupled approach of fundamental research and LCA right.

Activities in our project have picked up substantially in Fall 2017 after recruiting our two PhD students. With the PhD students, the immediate implication of six researchers / engineers and technicians (Kim Milferstedt, Jérôme Hamelin, Elie Le Quémeneur, Arnaud Hélias, Michel Torrijos, Anaïs Bonnafous) and the supporting activities of one researcher (Jean-Philippe Steyer) and one technician (Philippe Sousbie), we have reached a critical mass of people with complementary expertise allowing us to move ahead as planned. Our first publications on photogranulation have generated an unexpectedly high attention of potential industrial partners. After consulting with our innovation and technology transfer team in the laboratory, we decided to put a stronger focus on the development of the bioprocess to obtain a more mature idea of reactor performance and to consolidate intellectual property before negotiating official collaborations with industrial partners. This phase will be finished this summer and may lead to project applications for upscaling the process, potentially with an industrial partner.
In the submitted ECOS-Nord project (May 2018) in collaboration with Germán Buitrón Méndez at Universidad Nacional Autónoma de México (UNAM), we investigate the use of oxygenic photogranules in the context of water treatment in small, decentralized units. While our principal contribution to the project is in microbial ecology and mathematical modelling, we will gain insight into a bioprocess application that could generate access to treated water and biofeedstock in less-developed regions.

Abouhend, A.S., McNair, A., Kuo-Dahab, W.C., Watt, C., Butler, C.S., Milferstedt, K., Hamelin, J., Seo, J., Gikonyo, G.J., El-Moselhy, K.M., Park, C., 2018. The Oxygenic Photogranule Process for Aeration-Free Wastewater Treatment. Environ. Sci. Technol. 52, 3503–3511. doi.org/10.1021/acs.est.8b00403

Milferstedt, K., Hamelin, J., Park, C., Jung, J., Hwang, Y., Cho, S.K., Jung, K.W., Kim, D.H., 2017a. Biogranules applied in environmental engineering. Int. J. Hydrogen Energy 42, 27801–27811. doi.org/10.1016/j.ijhydene.2017.07.176

Milferstedt, K., Joosten, E., Hamelin, J., 2018. Cyanobacterial abundance drives photogranulation, in: IWA Biofilms: Granular Sludge Conference. Delft, The Netherlands. March 18-21, 2018.

Milferstedt, K., Kuo-Dahab, W.C., Butler, C.S., Hamelin, J., Abouhend, A.S., Stauch-White, K., McNair, A., Watt, C., Carbajal-González, B.I., Dolan, S., Park, C., 2017b. The importance of filamentous cyanobacteria in the development of oxygenic photogranules. Sci. Rep. 7. doi.org/10.1038/s41598-017-16614-9

Effective sanitation of wastewater is without question a milestone in the development of mankind. But wastewater treatment comes at high economic and energetic costs, mostly caused by pumping air into activated sludge basins for the degradation of organic pollutants. It is in part these costs that prevent access to sanitation of wastewater in many parts of the world. Using our available resources more wisely is a challenge in wastewater treatment and may only be achieved by developing a next generation bioprocess where input energy is reduced and higher energy is recovered from waste products. Recent trends point towards using phototrophic technologies as potential alternatives for the classical activated sludge process, even though separability of phototrophic biomass from treated water is notoriously difficult.
Granulated biomass is easily separated from an aqueous phase, but phototrophic organisms are typically not known to form granules. In 2014, we initiated work on photogranules in our lab and have since then been successful to establish working cultivation conditions and a first characterization of granule communities. These granules could be the core of a novel bioprocess in which oxygen is produced by a dense outer layer of cyanobacteria at the surface of the granules. This oxygen is consumed by bacteria in the interior for the degradation and conversion of water pollutants. In addition, phototrophic growth increases the overall biomass yield of granules and therefore leads to higher energy recovery than activated sludge, e.g. as methane in anaerobic digestion.
In PSST, we extend our focus to investigating the formation of oxygenic phototrophic granules. We combine our fundamental research in microbial ecology with Life Cycle Assessment. Our project thus allows us to understand the ecological principles guiding granulation and to gain a thorough understanding of the environmental impact of the bioprocess as a whole. This unusual richness of information already in the proof of concept phase of a promising novel technology may enable us to recommend a process design with minimal environmental impact in which we engineer the microbial resource to our advantage.
Using a microbial ecological approach, we will (i) study functional characteristics of cyanobacteria responsible for granulating activated sludge, (ii) systematically test what kind of matrices other than activated sludge can be embedded, (iii) analyze potential failure of granulation using metagenomic approaches, and (iv) test the fate of engineered communities when exposed to a metacommunity of bacteria in raw wastewater. In terms of process engineering, data about pollution removal efficiency in continuous bioreactor will feed the Life Cycle Assessment of this promising technology.
The enormous potential of OPGs for wastewater treatment and the potential use of OPGs in other applications merit the investment in the promising research theme at INRA-LBE led by a young scientist.