Team:Duesseldorf/Implementation

Proposed Implementation

Phosphate is a non-substitutable and thus, essential compound for plants and therefore securing its availability, especially in an environmentally friendly manner, will be crucial for the next decades.

Current Phosphate Recovery Systems

Current systems for the recovery of phosphate from wastewater consist of chemical, physical and biological methods (McGrath and Quinn 2003), (Bunce et al. 2018), as well as hybrids in-between. Physicochemical methods require the use of salts to induce precipitation of phosphate containing sludge (McGrath and Quinn 2003), (Bunce et al. 2018). The problem herein lies in the costs and excessive amount of precipitants needed, as well as in the large quantity of phosphate containing sludge generated this way (McGrath and Quinn 2003), (Bunce et al. 2018). Moreover, the used chemical reagents are toxic on nature and in their product, iron or aluminum salts, and a high yield of phosphate necessitates the use of corrosive substances like potassium hydroxide (McGrath and Quinn 2003), (Bunce et al. 2018). As there are many parameters controlling this process, it creates a complexity applicable only to larger facilities.

Purely physical methods are still too novel and/or still unreliable like adsorption or ion-exchange (Bunce et al. 2018). Therefore, the focus is currently on biological methods (McGrath and Quinn 2003), (Bunce et al. 2018).

The two main types of biological phosphate removal are the Enhanced Biological Phosphate removal (EBPR) and Algae-based systems (Bunce et al. 2018). The former utilizes certain types of bacteria in a two-phased mechanism: In the first anaerobic step, polyphosphate-containing microorganisms break down their own polyphosphate reserves and use the energy to accumulate polyhydroxyalkanoates (PHA), at the same time orthophosphate is exported into the extracellular medium (McGrath and Quinn 2003), (Bunce et al. 2018). In the second aerobic phase the organisms then use their PHA-reserves to take up and accumulate phosphate and store it in the form of polyphosphate, this time to a much higher content then before (McGrath and Quinn, 2003), (Bunce et al. 2018). Variations and modifications of this method exist which further improve the basic design. EBPR seems to be a mostly reliable, cost-efficient and environmentally friendly method to remove both phosphorus as well as other components out of the wastewater (McGrath and Quinn 2003), (Bunce et al. 2018). However, it and especially, recent advancements with bioreactors still represent a somewhat complex and energy-intensive approach (Bunce et al. 2018), which is further complicated by difficulties in controlling the process (McGrath and Quinn 2003). Furthermore, the method suffers from being only reliably efficient at larger scales (Bunce et al. 2018).

Algal-based systems shine out through their simplicity and resilience (Bunce et al. 2018) in the design of the bioreactor type. Those systems are established well, however current systems fail at targeted phosphate removal with a removal efficiency of maximum 30% compared to 70 - 90 % of EBPR’s (Bunce et al. 2018).

This is where our plan comes into play.

Our approach

Our approach principally falls under algae-based systems, only that instead of algae, we use a genetically modified Physcomitrella patens. Our idea is to up-scale our approach by adding a new stage in wastewater facilities (WWF). The wastewater would pass through a bioreactor array, where the Physcomitrella patens can efficiently take up most of the phosphate and accumulate it in the form of polyphosphate. The wastewater is then hypotrophic, in respect, to the phosphate content and the phosphate-rich moss can be harvested. The harvested moss will be dried and can be used as phosphate-rich fertilizer. As the moss is dried, it can be safely released into nature and crop plant fields.

Our design keeps the simplicity of the main-type and further improves it via targeted genetic engineering of the host organism. Moreover, as even simple plants like mosses require their rhizoids to anchor to a substrate or alike, they can be controlled and contained more easily than microalgae.

This would be done by using the bioreactor design called “turf-scrubber”. A horizontal mesh which is submerged in the water serves as the ground for the moss and can be easily exchanged by a fresh one once the plants have reached their maximum capacity of phosphate. Knowing the exact time when the maximum capacity is reached within the organism and therefore the optimal time point to harvest, can be done by implementing a biosensor. This would not complicate the design of the bioreactor itself. The bioreactor type itself is also efficient enough on a lower scale (Nedbal 2020) in contrast to most of the other types discussed above, but at the same time is easy to up-scale, making it very flexible.

Fig. 1: A scheme of the production line for Mossphate. Wastewater is treated by running it through our photobioreactor, leading to the growth of moss rich in phosphate. This moss can be dried and processed, leading to a sustainable, natural fertilizer.

Safety / kill-switch

The main disadvantage of our design compared to all previously discussed is the utilization of GMO’s. Therefore, a kill-switch system is obligatory for any operation.

A rather facile device could see the use of reduced graphenoxide (rGO) embedded in a matrix of bacterial nanocellulose (BNC). Qisheng et al. reported excellent anti-biofouling properties of rGO/BNC for their use in ultrafiltration membranes (Jiang et al. 2018). rGO has unique properties as a photocatalyst and can in and of itself convert light into other forms of energy, in this context it is heat. While 60 °C is not the highest temperature, they are high enough to sterilize any organisms in long-term exposure. The BNC acts as a matric for the rGO and makes the assembly extraordinarily resilient to environmental stress, including pH and temperature (Jiang et al. 2018). If the mesh in the turf-scrubber would be covered with rGO/BNC, the mesh itself would act as a mechanical kill-switch, quickly eliminating any plants anchored to it. This is also useful for the final act of harvesting, as the moss rich in phosphate should be dried to increase the phosphate content per weight. And the chemical resilience of the rGO/BNC would give the mesh a large lifetime, so that only a handful are needed for each bioreactor that would be exchanged regularly.

To further ensure a biosafety of the GMO a biological kill-switch should be employed as well. iGEM 2013 Team Munich used red-light specific proteases that recombine upon illumination and cleave a membrane-bound nuclease, which is then translocated into the nucleus and degrades the genome (iGEM Munich 2013). The moss is normally shielded by a filter, but if it escapes into the environment, it is no longer protected and thus induces it’s self-destruction. Such a device would proof very effective while being rather simple. The kill-switch could further be enhanced by including a thermosensitive protease (possibly splitted under normal conditions as well) that would be activated by the heating of the rGO/BNC membrane of the mesh. This way the modified genome would no longer be a danger via horizontal gene transfer which could be an issue when the moss is simply put on the field again after harvest and drying. A slower and less targeted approach is to use classical breeding and mutagenesis techniques to generate a P. patens strain that accumulates phosphate. This strain would not be considered a GMO by the current legislature, so it could be used with minimal regulations. We prefer a targeted approach, as random mutagenesis changes the function of genes more unpredictably than genetic engineering and for us, a safe GMO is preferable to a non-GMO strain with unknown side-effects.

Current Wastewater-based fertilizers

Today, a variety of possible wastewater-based fertilizers do exist. They are urine and its derivatives like struvite (a precipitation product) and blackwater and compost - mixtures of urine, faeces and water (Winker et al. 2008). According to Clemens those types of fertilizers have the potential to cover as much as 20% of the fertilizer demand in Germany (Clemens 2007). While all of those fertilizers are good enough for the use on fields, they have the disadvantage that they contain significant amounts of organic pollutants (like drugs) and pathogens (Winker et al. 2008). Modifying the moss to express efflux carriers in its membrane would protect them from the adverse effects of those pollutants and at the same time create a rather clean fertilizer. The drying process during harvesting would also eliminate any other organisms or viruses adsorbed on the mesh and the plants and make the fertilizer contaminant-free on another level.

Our focus in our project is on phosphate. However as part of our goal is to further establish P. patens as a biotechnological chassis incorporating mechanisms to accumulate other nutrients would be easier in future approaches once our proof-of-concept works. For example polyphosphate has the advantage over orthophosphate as that it can sequestrate certain metals better (Lohry 2001). While this poses a problem of possible metal starvation during the “living phase” of the moss, inserting metal importers that are effective just before the harvest would create a fertilizer that is also rich in some macro and micronutrients.

Economics and Bureaucracy

The simple mechanism coupled with the low maintenance costs of the design would make our approach economically viable even for smaller wastewater treatment facilities. Larger facilities would benefit from the “environmental innovation program” of the “federal institute for environment, conservation of nature and reactor safety” (Everding et al. 2012).

Current approaches for phosphate recycling from wastewater fail economically due to the low amount of phosphate recovered compared to the sludge produced in the process (Kraus et al. 2019). Furthermore safety concerns due to chemical and biological contaminants pose a serious threat under the light of current laws and acts (Kraus et al. 2019). As discussed above, our design has the potential to overcome those problems and reach a necessary reduction of phosphate in the sludge by 50% which is mandated by law (“Article 5 of the act of sewage sludge”) (Kraus et al. 2019). Meeting those demands, it will also be possible to run a bioreactor array like proposed by us economically and sell the phosphate-rich moss on the market. In the future with further modifications/variations as discussed above as well, such a fertilizer could become a very competitive product on the market and will ensure the safety of food in the coming decades and beyond.

References
Bunce, J. T., Ndam, E., Ofiteru, I. D., Moore, A., & Graham, D. W. (2018). A review of phosphorus removal technologies and their applicability to small-scale domestic wastewater treatment systems. Frontiers in Environmental Science, 6, 8.

Clemens, J. (2007). Integrative Betrachtung von (Nähr) Stoffflüssen. Proceedings: Jahrestagung der Deutschen Gesellschaft für Pflanzenernährung, Berlin.

de Boer M.A., Wolzak L., Slootweg J.C. (2019) Phosphorus: Reserves, Production, and Applications. In: Ohtake H., Tsuneda S. (eds) Phosphorus Recovery and Recycling. Springer, Singapore. https://doi.org/10.1007/978-981-10-8031-9_5

Everding, W., Montag, D., Pinnekamp, J. (2012). Ergebnisse und Schlussfolgerungen der BMBF-Förderinitiative “Phosphorrecycling”. In Pinnekamp (Hrsg.), J., Kölling, V. (Bearb.), 45.

Essener Tagung für Wasser- und Abfallwirtschaft 'Wasserwirtschaft und Energiewende' (Seiten/Artikel-Nr: 20/1-20/15): 14. bis 16. März 2012 in der Messe Essen Ost / Institut und Lehrstuhl für Siedlungswasserwirtschaft der RWTH Aachen

Heckenmüller, C., Narita, D., Klepper, G. (2014). Global availability of phosphorus and its implications for global food supply: An economic overview. Retrieved from http://hdl.handle.net/10419/90630

iGEM TU Munich, retrieved from http://2013.igem.org/Team:TU-Munich/Project/Killswitch. Last access: 21.10.2020

Jiang, Q., Ghim, D., Cao, S., Tadepalli, S., Liu, K. K., Kwon, H., ... & Singamaneni, S. (2018). Photothermally active reduced graphene oxide/bacterial nanocellulose composites as biofouling-resistant ultrafiltration membranes. Environmental science & technology, 53(1), 412-421.

Kraus, F., Zamzow, M., Conzelmann, L., Remy, C., Kleyböcker, A., Seis, W., ... & Kabbe, C. (2019). Ökobilanzieller Vergleich der P-Rückgewinnung aus dem Abwasserstrom mit der Düngemittelprodukti-on aus Rohphosphaten unter Einbeziehung von Umweltfolgeschäden und deren Vermeidung.

Ladislav Nedbal, FZ Juelich, personal communication.

Lohry, R. (2001). Ortho vs. Poly. Fluid J, 35, 17-19.

McGrath, J. W., & Quinn, J. P. (2003). Microbial phosphate removal and polyphosphate production from wastewaters. Advances in applied microbiology, 52, 75-100.

Winker, M., Vinnerås, B., Arnold, U., Muskulos, A., & Clemens, J. (2008, June). New Fertilizers from Advanced Wastewater Treatment: Their potential Values and Risks. In 13th RAMIRAN International Conference (pp. 236-241).

Zhang, W., Ma, W., Ji, Y., Fan, M., Oenema, O., & Zhang, F. (2008). Efficiency, economics, and environmental implications of phosphorus resource use and the fertilizer industry in China. Nutrient Cycling in Agroecosystems, 80(2), 131-144