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Feedstock Cyanobacteria

Feedstock Cyanobacteria 101 – How to get from wastewater to PHB PlastoCyan is a project developing an eco-innovative technology for the production of bioplastics in cyanobacteria that uses nutrients from municipal wastewater and wastewater from the dairy industry. Biodegradable plastics produced by microorganisms such as polyhydroxyalkanoates (PHA) are among the best solutions to replace conventional petroleum-based plastics to protect the environment. Despite the non-comparable price and productivity with conventional plastics, scientists are looking for a way to make them more feasible, economically, ecologically friendly, and sustainable. In the production of PHB by bacteria, up to 50 % cost is precursor substrate material, especially the carbon source. Most cyanobacteria naturally produce 20 % PHB of their cell mass. Cyanobacteria cannot compete with chemotrophic bacteria in terms of PHB content or biomass growth rate [1]. However, since cyanobacteria are photosynthesizing organisms, they can metabolize CO 2 . Unlike bacterial PHB producers, photoautotrophic cyanobacteria do not require the addition of substrate and are not dependent on crops, making them a more sustainable alternative production system. Production of PHB by cyanobacteria is a complex phenomenon that needs to be fully described. (Cyano) bacteria synthesize PHB under different stress conditions and form inclusion bodies in their cells, which are accumulated as intracellular energy and reserve carbon source. Mainly nitrogen starvation induces so-called chlorosis, a survival process in which cyanobacteria degrades photosynthetic machinery and accumulate biopolymers such as glycogen or PHB. Two stages of cyanobacterial growth are necessary to obtain PHB from bacteria for the biotechnology industry: 1. growth of biomass in nutrient-rich conditions and 2. accumulation of PHB during nutrient-stress conditions. Culture of Synechocystis growing in urban wastewater in a stage of chlorosis with specific orange colour. The thin-layer raceway pond was placed in a greenhouse with a cultivation area of 5 m 2 and a working volume of 100 – 600 l, mixing is provided by a paddle wheel. The project emphasizes establishing a cyanobacteria cultivation process where wastewater is used as a substrate for mixotrophic growth. Wastewater from a municipal treatment plant and wastewater from dairy products are tested as potential substrates. The Plastocyan project joins three institutes – The Institute of Microbiology of the Czech Academy of Sciences – Algatech Centre in Třebon, in cooperation with the Technical University of Vienna and the University of Applied Sciences in Wels in Upper Austria. The Institute of Microbiology, a project lead partner, focuses on growing cyanobacteria on a pilot scale using wastewater as a source of nutrients. The aim is to develop sustainable remediation and valorisation of wastewater for a zerocarbon circular economy. Bacteria and protozoa are usually used in secondary treatment to remove biodegradable organic matter by producing biomass. However, this biomass has no further use and often ends up in landfills. Recently, a new idea came up using specific microorganisms which can produce valuable biomass. This would also address the demands of a circular economy, resulting in the bioconversion of waste streams from dairy industries. When processing milk, approximately three litres of cleaning water are necessary per litre of milk. Those wastewaters are excellent substrates containing a large amount of nitrogen and phosphorus, the primary nutrients required for the growth of cyanobacteria. Tomáš Grivalský, the lead project coordinator, says: “Microalgae, including cyanobacteria, produce a fantastic diversity of metabolites. The main issue is scale-up. They grow very well in a laboratory under defined conditions, but if you start to grow them in tens of litres, you will encounter completely new problems, such as grazers or predators, which can completely devastate the culture in a matter of hours. When we started a pilotscale cultivation, we had a similar problem. The culture was 24 bioplastics MAGAZINE [06/22] Vol. 17

By: Tomáš Grivalský Institute of Microbiology, CAS, Centre Algatech, Třebon, Czech Republic Julian Kopp Technical University of Vienna, Vienna, Austria attacked overnight by predators. We searched the literature to find a solution. The only recommendation was to adjust the pH. During the subsequent cultivation, as soon as the predators appeared, we changed the pH to highly alkaline, and the cyanobacteria continued to grow without predators”. In the project, we use a strain generated by UV mutagenesis at the Technical University of Vienna with higher PHB production [2]. The advantage of random mutagenesis is that it is not subject to the standards for genetically modified organisms. The strain achieved 37 % PHB cell dry weight (CDW) under laboratory conditions on a small scale in the optimal growth substrate, which is more than 20 % higher in PHB content per cell compared to the wild-type. From Science & Research In the PlastoCyan project, the strain was tested in an outdoor cultivation unit called a thin layer hybrid raceway pond in a volume of 100 litres using urban wastewater and high alkaline pH, reaching biomass content of 2 – 2.5 g/L and 23 % PHB per CDW which makes it a promising result. The eco-friendly approach is still ongoing after wastewater remediation and cyanobacterial biomass production. The group led by Oliver Spadiut at Technical University in Vienna is developing an extraction procedure based on ionic liquids (ILs). Conventionally PHB extraction is performed with chloroform; a well-established method referred to in the literature. However, the project also deals with developing an ecological and economically friendly alternative of PHB extraction from biomass using ionic liquids, belonging to the category of green solvents. The ILs, chosen for this study were highly corroding, can dissolve biomass, are very polar (so PHB should not be soluble), and are partially able to cleave ester bonds. As PHB should not dissolve due to its high polarity, the IL-biomass mixture can be separated from the resulting PHB via centrifugation/filtration. However, the biomass-IL mixture has currently such a high viscosity that a complete separation from the precipitating PHB is impossible. Therefore, cosolvents obtaining similar Kamlett Taft parameters are currently tested to decrease the viscosity. Once the viscosity issue to separate the dissolved biomass IL mixture from the PHB pellet is addressed, nothing should stand in the way of a suitable standard operation procedure for a green dissolution of PHB in ionic liquids (IL). The project is also dedicated to generating a cyanobacterial strain that would be able to process the lactose present in dairy wastewater. Having such a strain would be fascinating not only for biotechnologies but also for the scientific community. However, cyanobacteria can metabolize glucose; they do not have a system to break down more complex sugars such as lactose. This is the part where scientists from the University of Applied Sciences in Wels, Austria, are modifying the genome to improve the strain. Additionally, they attempt to enhance the metabolic pathway for PHB A 30 l annular photobioreactor with with adjustable internal LED lighting. The culture is mixed with air through tubes located at the bottom of the photobioreactor. This culture of Synechocystis is growing in dairy waste. production. This can lead to more than 60 % PHB per cell biomass production under nutrient-limited conditions, as referred by Koch et al [3]. The project PlastoCyan (ATCZ260) is funded by the Interreg V-A Austria-Czech Republic programme. In 2022, it was awarded by the Austrian Federal Ministry of Education, Science and Research with the “Sustainability award” for second place in the “Research” category. www.alga.cz/en/a-106-centre-algatech.html www.tuwien.at/en/ www.fh-ooe.at/en/ References [1] B. Drosg, I. Fritz, F. Gattermayr, L. Silvestrini, Photo-autotrophic production of poly(hydroxyalkanoates) in cyanobacteria, Chem. Biochem. Eng. Q. 29 (2015) 145–156. https://doi.org/10.15255/CABEQ.2014.2254. [2] D. Kamravamanesh, T. Kovacs, S. Pflügl, I. Druzhinina, P. Kroll, M. Lackner, C. Herwig, Increased poly-Β-hydroxybutyrate production from carbon dioxide in randomly mutated cells of cyanobacterial strain Synechocystis sp. PCC 6714: Mutant generation and characterization, Bioresour. Technol. 266 (2018) 34–44. https://doi.org/10.1016/j. biortech.2018.06.057. [3] M. Koch, J. Bruckmoser, J. Scholl, W. Hauf, B. Rieger, K. 5 Forchhammer, Maximizing PHB content in Synechocystis sp. PCC 2 6803: development of a new photosynthetic 3 overproduction strain. 4, BioRxiv. (2020) 2020.10.22.350660. https://doi.org/10.1101/2020.10.22.350660. bioplastics MAGAZINE [06/22] Vol. 17 25

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