Genome Engineering by RNA-Guided Transposition for Anabaena sp. PCC 7120.

In genome engineering, the integration of incoming DNA has been dependent on enzymes produced by dividing cells, which has been a bottleneck toward increasing DNA insertion frequencies and accuracy. Recently, RNA-guided transposition with CRISPR-associated transposase (CAST) was reported as highly effective and specific in Escherichia coli. Here, we developed Golden Gate vectors to test CAST in filamentous cyanobacteria and to show that it is effective in Anabaena sp. strain PCC 7120. The comparatively large plasmids containing CAST and the engineered transposon were successfully transferred into Anabaena via conjugation using either suicide or replicative plasmids. Single guide (sg) RNA encoding the leading but not the reverse complement strand of the target were effective with the protospacer-associated motif (PAM) sequence included in the sgRNA. In four out of six cases analyzed over two distinct target loci, the insertion site was exactly 63 bases after the PAM. CAST on a replicating plasmid was toxic, which could be used to cure the plasmid. In all six cases analyzed, only the transposon cargo defined by the sequence ranging from left and right elements was inserted at the target loci; therefore, RNA-guided transposition resulted from cut and paste. No endogenous transposons were remobilized by exposure to CAST enzymes. This work is foundational for genome editing by RNA-guided transposition in filamentous cyanobacteria, whether in culture or in complex communities.

Harnessing photosynthetic microorganisms for enhanced bioremediation of microplastics: A comprehensive review.

Mismanaged plastics, upon entering the environment, undergo degradation through physicochemical and/or biological processes. This process often results in the formation of microplastics (MPs), the most prevalent form of plastic debris (<1 mm). MPs pose severe threats to aquatic and terrestrial ecosystems, necessitating innovative strategies for effective remediation. Some photosynthetic microorganisms can degrade MPs but there lacks a comprehensive review. Here we examine the specific role of photoautotrophic microorganisms in water and soil environments for the biodegradation of plastics, focussing on their unique ability to grow persistently on diverse polymers under sunlight. Notably, these cells utilise light and CO2 to produce valuable compounds such as carbohydrates, lipids, and proteins, showcasing their multifaceted environmental benefits. We address key scientific questions surrounding the utilisation of photosynthetic microorganisms for MPs and nanoplastics (NPs) bioremediation, discussing potential engineering strategies for enhanced efficacy. Our review highlights the significance of alternative biomaterials and the exploration of strains expressing enzymes, such as polyethylene terephthalate (PET) hydrolases, in conjunction with microalgal and/or cyanobacterial metabolisms. Furthermore, we delve into the promising potential of photo-biocatalytic approaches, emphasising the coupling of plastic debris degradation with sunlight exposure. The integration of microalgal-bacterial consortia is explored for biotechnological applications against MPs and NPs pollution, showcasing the synergistic effects in wastewater treatment through the absorption of nitrogen, heavy metals, phosphorous, and carbon. In conclusion, this review provides a comprehensive overview of the current state of research on the use of photoautotrophic cells for plastic bioremediation. It underscores the need for continued investigation into the engineering of these microorganisms and the development of innovative approaches to tackle the global issue of plastic pollution in aquatic and terrestrial ecosystems.

Improved lipid production and component of mycosporine-like amino acids by co-overexpression of amt1 and aroB genes in Synechocystis sp. PCC6803.

Implementing homologous overexpression of the amt1 (A) and aroB (B) genes involved in ammonium transporter and the synthesis of mycosporine-like amino acids (MAAs) and aromatic amino acids, respectively, we created three engineered Synechocystis sp. PCC6803 strains, including Ox-A, Ox-B, and Ox-AB, to study the utilization of carbon and nitrogen in cyanobacteria for the production of valuable products. With respect to amt1 overexpression, the Ox-A and Ox-AB strains had a greater growth rate under (NH4)2SO4 supplemented condition. Both the higher level of intracellular accumulation of lipids in Ox-A and Ox-AB as well as the increased secretion of free fatty acids from the Ox-A strain were impacted by the late-log phase of cell growth. It is noteworthy that among all strains, the Ox-B strain undoubtedly spotted a substantial accumulation of glycogen as a consequence of aroB overexpression. Additionally, the ammonium condition drove the potent antioxidant activity in Ox strains with a late-log phase, particularly in the Ox-B and Ox-AB strains. This was probably related to the altered MAA component inside the cells. The higher proportion of P4-fraction was induced by the ammonium condition in both Ox-B and Ox-AB, while the noted increase of the P1 component was found in the Ox-A strain.

Sustainable production of photosynthetic isobutanol and 3-methyl-1-butanol in the cyanobacterium Synechocystis sp. PCC 6803.

BACKGROUND: Cyanobacteria are emerging as green cell factories for sustainable biofuel and chemical production, due to their photosynthetic ability to use solar energy, carbon dioxide and water in a direct process. The model cyanobacterial strain Synechocystis sp. PCC 6803 has been engineered for the isobutanol and 3-methyl-1-butanol production by introducing a synthetic 2-keto acid pathway. However, the achieved productions still remained low. In the present study, diverse metabolic engineering strategies were implemented in Synechocystis sp. PCC 6803 for further enhanced photosynthetic isobutanol and 3-methyl-1-butanol production. RESULTS: Long-term cultivation was performed on two selected strains resulting in maximum cumulative isobutanol and 3-methyl-1-butanol titers of 1247 mg L-1 and 389 mg L-1, on day 58 and day 48, respectively. Novel Synechocystis strain integrated with a native 2-keto acid pathway was generated and showed a production of 98 mg isobutanol L-1 in short-term screening experiments. Enhanced isobutanol and 3-methyl-1-butanol production was observed when increasing the kivdS286T copy number from three to four. Isobutanol and 3-methyl-1-butanol production was effectively improved when overexpressing selected genes of the central carbon metabolism. Identified genes are potential metabolic engineering targets to further enhance productivity of pyruvate-derived bioproducts in cyanobacteria. CONCLUSIONS: Enhanced isobutanol and 3-methyl-1-butanol production was successfully achieved in Synechocystis sp. PCC 6803 strains through diverse metabolic engineering strategies. The maximum cumulative isobutanol and 3-methyl-1-butanol titers, 1247 mg L-1 and 389 mg L-1, respectively, represent the current highest value reported. The significantly enhanced isobutanol and 3-methyl-1-butanol production in this study further pave the way for an industrial application of photosynthetic cyanobacteria-based biofuel and chemical synthesis from CO2.

Novel concepts and engineering strategies for heterologous expression of efficient hydrogenases in photosynthetic microorganisms.

Hydrogen is considered one of the key enablers of the transition towards a sustainable and net-zero carbon economy. When produced from renewable sources, hydrogen can be used as a clean and carbon-free energy carrier, as well as improve the sustainability of a wide range of industrial processes. Photobiological hydrogen production is considered one of the most promising technologies, avoiding the need for renewable electricity and rare earth metal elements, the demands for which are greatly increasing due to the current simultaneous electrification and decarbonization goals. Photobiological hydrogen production employs photosynthetic microorganisms to harvest solar energy and split water into molecular oxygen and hydrogen gas, unlocking the long-pursued target of solar energy storage. However, photobiological hydrogen production has to-date been constrained by several limitations. This review aims to discuss the current state-of-the art regarding hydrogenase-driven photobiological hydrogen production. Emphasis is placed on engineering strategies for the expression of improved, non-native, hydrogenases or photosynthesis re-engineering, as well as their combination as one of the most promising pathways to develop viable large-scale hydrogen green cell factories. Herein we provide an overview of the current knowledge and technological gaps curbing the development of photobiological hydrogenase-driven hydrogen production, as well as summarizing the recent advances and future prospects regarding the expression of non-native hydrogenases in cyanobacteria and green algae with an emphasis on [FeFe] hydrogenases.

Overexpressing Carotenoid Biosynthetic Genes in Synechocystis sp. PCC 6803 Improved Intracellular Pigments and Antioxidant Activity, Which Can Decrease the Viability and Proliferation of Lung Cancer Cells In Vitro.

In the antioxidant system in cyanobacteria, non-enzymatic antioxidants, such as carotenoids, are considered good candidates for coping with oxidative stress, particularly light stress, and pharmaceutical therapeutic applications. A significant amount of carotenoid accumulation has been recently improved by genetic engineering. In this study, to achieve higher carotenoid production with higher antioxidant activity, we successfully constructed five Synechocystis sp. PCC 6803 strains overexpressing (OX) native genes related to the carotenoids biosynthetic pathway, including OX_CrtB, OX_CrtP, OX_CrtQ, OX_CrtO, and OX_CrtR. All of the engineered strains maintained a significant quantity of myxoxanthophyll, while increasing zeaxanthin and echinenone accumulation. In addition, higher components of zeaxanthin and echinenone were noted in all OX strains, ranging from 14 to 19% and from 17 to 22%, respectively. It is worth noting that the enhanced echinenone component responded to low light conditions, while the increased ß-carotene component contributed to a high light stress response. According to the higher antioxidant activity of all OX strains, the carotenoid extracts presented lower IC50 in lung cancer cell lines H460 and A549, with values less than 157 and 139 µg/mL, respectively, when compared with those of WTc, particularly OX_CrtR and OX_CrtQ. A higher proportion of zeaxanthin and ß-carotene in OX_CrtR and OX_CrtQ, respectively, may considerably contribute to the ability to treat lung cancer cells with antiproliferative and cytotoxic effects.

Recent developments in the production and utilization of photosynthetic microorganisms for food applications.

The growing use of photosynthetic microorganisms for food and food-related applications is driving related biotechnology research forward. Increasing consumer acceptance, high sustainability, demand of eco-friendly sources for food, and considerable global economic concern are among the main factors to enhance the focus on the novel foods. In the cases of not toxic strains, photosynthetic microorganisms not only provide a source of sustainable nutrients but are also potentially healthy. Several published studies showed that microalgae are sources of accessible protein and fatty acids. More than 400 manuscripts were published per year in the last 4 years. Furthermore, industrial approaches utilizing these microorganisms are resulting in new jobs and services. This is in line with the global strategy for bioeconomy that aims to support sustainable development of bio-based sectors. Despite the recognized potential of the microalgal biomass value chain, significant knowledge gaps still exist especially regarding their optimized production and utilization. This review highlights the potential of microalgae and cyanobacteria for food and food-related applications as well as their market size. The chosen topics also include advanced production as mixed microbial communities, production of high-value biomolecules, photoproduction of terpenoid flavoring compounds, their utilization for sustainable agriculture, application as source of nutrients in space, and a comparison with heterotrophic microorganisms like yeast to better evaluate their advantages over existing nutrient sources. This comprehensive assessment should stimulate further interest in this highly relevant research topic.

Photobiohydrogen Production and Strategies for H2 Yield Improvements in Cyanobacteria.

Hydrogen gas (H2) is one of the potential future sustainable and clean energy carriers that may substitute the use of fossil resources including fuels since it has a high energy content (heating value of 141.65 MJ/kg) when compared to traditional hydrocarbon fuels [1]. Water is a primary product of combustion being a most significant advantage of H2 being environmentally friendly with the capacity to reduce global greenhouse gas emissions. H2 is used in various applications. It generates electricity in fuel cells, including applications in transportation, and can be applied as fuel in rocket engines [2]. Moreover, H2 is an important gas and raw material in many industrial applications. However, the high cost of the H2 production processes requiring the use of other energy sources is a significant disadvantage. At present, H2 can be prepared in many conventional ways, such as steam reforming, electrolysis, and biohydrogen production processes. Steam reforming uses high-temperature steam to produce hydrogen gas from fossil resources including natural gas. Electrolysis is an electrolytic process to decompose water molecules into O2 and H2. However, both these two methods are energy-intensive and producing hydrogen from natural gas, which is mostly methane (CH4) and in steam reforming generates CO2 and pollutants as by-products. On the other hand, biological hydrogen production is more environmentally sustainable and less energy intensive than thermochemical and electrochemical processes [3], but most concepts are not yet developed to production scale.

Enhancing microalgal lipid accumulation for biofuel production.

Microalgae have high lipid accumulation capacity, high growth rate and high photosynthetic efficiency which are considered as one of the most promising alternative sustainable feedstocks for producing lipid-based biofuels. However, commercialization feasibility of microalgal biofuel production is still conditioned to the high production cost. Enhancement of lipid accumulation in microalgae play a significant role in boosting the economics of biofuel production based on microalgal lipid. The major challenge of enhancing microalgal lipid accumulation lies in overcoming the trade-off between microalgal cell growth and lipid accumulation. Substantial approaches including genetic modifications of microalgal strains by metabolic engineering and process regulations of microalgae cultivation by integrating multiple optimization strategies widely applied in industrial microbiology have been investigated. In the present review, we critically discuss recent trends in the application of multiple molecular strategies to construct high performance microalgal strains by metabolic engineering and synergistic strategies of process optimization and stress operation to enhance microalgal lipid accumulation for biofuel production. Additionally, this review aims to emphasize the opportunities and challenges regarding scaled application of the strategic integration and its viability to make microalgal biofuel production a commercial reality in the near future.

Enhanced productivity of extracellular free fatty acids by gene disruptions of acyl-ACP synthetase and S-layer protein in Synechocystis sp. PCC 6803.

BACKGROUND: Based on known metabolic response to excess free fatty acid (FFA) products, cyanobacterium Synechocystis sp. PCC 6803 preferentially both recycles via FFA recycling process and secrets them into medium. Engineered cyanobacteria with well growth and highly secreted FFA capability are considered best resources for biofuel production and sustainable biotechnology. In this study, to achieve the higher FFA secretion goal, we successfully constructs Synechocystis sp. PCC 6803 mutants disrupting genes related to FFA recycling reaction (aas gene encoding acyl-acyl carrier protein synthetase), and surface layer protein (encoded by sll1951). RESULTS: Three Synechocystis sp. PCC 6803 engineered strains, including two single mutants lacking aas (KA) and sll1951 (KS), and one double mutant lacking both aas and sll1951 (KAS), significantly secreted FFAs higher than that of wild type (WT). Certain increase of secreted FFAs was noted when cells were exposed to nitrogen-deficient conditions, BG11-half N and BG11-N conditions, with the exception of strain KS. Under BG11-N condition at day 10, strain KAS strikingly secreted FFAs products up to 40%w/DCW or 238.1 mg/L, with trace amounts of PHB. Unexpectedly, strain KS, with S-layer disruption, appeared to have endured longer in BG11-N growth medium. This strain KS significantly acclimated to the BG11-N environment by accumulating a greater glycogen pool with lower FFA production, whereas strain KA favored higher PHB and intracellular lipid accumulations with moderate FFA secretion. CONCLUSIONS: Mutations of both aas and sll1951 genes in Synechocystis sp. PCC 6803 significantly improved the productivity of secreted FFAs, especially under nitrogen deprivation.

Expressing 2-keto acid pathway enzymes significantly increases photosynthetic isobutanol production.

BACKGROUND: Cyanobacteria, photosynthetic microorganisms, are promising green cell factories for chemical production, including biofuels. Isobutanol, a four-carbon alcohol, is considered as a superior candidate as a biofuel for its high energy density with suitable chemical and physical characteristics. The unicellular cyanobacterium Synechocystis PCC 6803 has been successfully engineered for photosynthetic isobutanol production from CO2 and solar energy in a direct process. RESULTS: Heterologous expression of α-ketoisovalerate decarboxylase (KivdS286T) is sufficient for isobutanol synthesis via the 2-keto acid pathway in Synechocystis. With additional expression of acetolactate synthase (AlsS), acetohydroxy-acid isomeroreductase (IlvC), dihydroxy-acid dehydratase (IlvD), and alcohol dehydrogenase (Slr1192OP), the Synechocystis strain HX42, with a functional 2-keto acid pathway, showed enhanced isobutanol production reaching 98 mg L-1 in short-term screening experiments. Through modulating kivdS286T copy numbers as well as the composition of the 5'-region, a final Synechocystis strain HX47 with three copies of kivdS286T showed a significantly improved isobutanol production of 144 mg L-1, an 177% increase compared to the previously reported best producing strain under identical conditions. CONCLUSIONS: This work demonstrates the feasibility to express heterologous genes with a combination of self-replicating plasmid-based system and genome-based system in Synechocystis cells. Obtained isobutanol-producing Synechocystis strains form the base for further investigation of continuous, long-term-photosynthetic isobutanol production from solar energy and carbon dioxide.

Differential catalase activity and tolerance to hydrogen peroxide in the filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Anabaena sp. PCC 7120.

Photoautotrophic cyanobacteria often confront hydrogen peroxide (H2O2), a reactive oxygen species potentially toxic to cells when present in sufficiently high concentrations. In this study, H2O2 tolerance ability of filamentous cyanobacteria Nostoc punctiforme ATCC 29133 (Nostoc 29133) and Anabaena sp. PCC 7120 (Anabaena 7120) was investigated at increasing concentrations of H2O2 (0-0.5 mM). In Nostoc 29133, 0.25 and 0.5 mM H2O2 caused a reduction in chlorophyll a content by 12 and 20%, respectively, whereas with similar treatments, a total loss of chlorophyll a was detected in Anabaena 7120. Further, Nostoc 29133 was able to maintain its photosystem II performance in the presence of H2O2 up to a concentration of 0.5 mM, whereas in Anabaena 7120, 0.25 mM H2O2 caused a complete reduction of photosystem II performance. The intracellular hydroperoxide level (indicator of oxidative status) did not increase to the same high level in Nostoc 29133, as compared to in Anabaena 7120 after H2O2 treatment. This might be explained by that Nostoc 29133 showed a 20-fold higher intrinsic constitutive catalase activity than Anabaena 7120, thus indicating that the superior tolerance of Nostoc 29133 to H2O2 stems from its higher ability to decompose H2O2. It is suggested that difference in H2O2 tolerance between closely related filamentous cyanobacteria, as revealed in this study, may be taken into account for judicious selection and effective use of strains in biotechnological applications.

Current advances in engineering cyanobacteria and their applications for photosynthetic butanol production.

Cyanobacteria are natural photosynthetic microbes which can be engineered for sustainable conversion of solar energy and carbon dioxide into chemical products. Attempts to improve target production often require an improved understanding of the native cyanobacterial host system. Valuable insights into cyanobacterial metabolism, biochemistry and physiology have been steadily increasing in recent years, stimulating key advancements of cyanobacteria as cell factories for biochemical, including biofuel, production. In the present review, we summarize the current progress in engineering cyanobacteria and discuss the achieved and potential utilization of these advances in cyanobacteria for the production of the bulk chemical butanol, specifically isobutanol and 1-butanol.

Overexpression of lipA or glpD_RuBisCO in the Synechocystis sp. PCC 6803 Mutant Lacking the Aas Gene Enhances Free Fatty-Acid Secretion and Intracellular Lipid Accumulation.

Although engineered cyanobacteria for the production of lipids and fatty acids (FAs) are intelligently used as sustainable biofuel resources, intracellularly overproduced FAs disturb cellular homeostasis and eventually generate lethal toxicity. In order to improve their production by enhancing FFAs secretion into a medium, we constructed three engineered Synechocystis 6803 strains including KA (a mutant lacking the aas gene), KAOL (KA overexpressing lipA, encoding lipase A in membrane lipid hydrolysis), and KAOGR (KA overexpressing quadruple glpD/rbcLXS, related to the CBB cycle). Certain contents of intracellular lipids and secreted FFAs of all engineered strains were higher than those of the wild type. Remarkably, the KAOL strain attained the highest level of secreted FFAs by about 21.9%w/DCW at day 5 of normal BG11 cultivation, with a higher growth rate and shorter doubling time. TEM images provided crucial evidence on the morphological changes of the KAOL strain, which accumulated abundant droplets on regions of thylakoid membranes throughout the cell when compared with wild type. On the other hand, BG11-N condition significantly induced contents of both intracellular lipids and secreted FFAs of the KAOL strain up to 37.2 and 24.5%w/DCW, respectively, within 5 days. Then, for the first time, we shone a spotlight onto the overexpression of lipA in the aas mutant of Synechocystis as another potential strategy to achieve higher FFAs secretion with sustainable growth.

Doing synthetic biology with photosynthetic microorganisms.

The use of photosynthetic microbes as synthetic biology hosts for the sustainable production of commodity chemicals and even fuels has received increasing attention over the last decade. The number of studies published, tools implemented, and resources made available for microalgae have increased beyond expectations during the last few years. However, the tools available for genetic engineering in these organisms still lag those available for the more commonly used heterotrophic host organisms. In this mini-review, we provide an overview of the photosynthetic microbes most commonly used in synthetic biology studies, namely cyanobacteria, chlorophytes, eustigmatophytes and diatoms. We provide basic information on the techniques and tools available for each model group of organisms, we outline the state-of-the-art, and we list the synthetic biology tools that have been successfully used. We specifically focus on the latest CRISPR developments, as we believe that precision editing and advanced genetic engineering tools will be pivotal to the advancement of the field. Finally, we discuss the relative strengths and weaknesses of each group of organisms and examine the challenges that need to be overcome to achieve their synthetic biology potential.

Photosynthetic hydrogen production: Novel protocols, promising engineering approaches and application of semi-synthetic hydrogenases.

Photosynthetic production of molecular hydrogen (H2 ) by cyanobacteria and green algae is a potential source of renewable energy. These organisms are capable of water biophotolysis by taking advantage of photosynthetic apparatus that links water oxidation at Photosystem II and reduction of protons to H2 downstream of Photosystem I. Although the process has a theoretical potential to displace fossil fuels, photosynthetic H2 production in its current state is not yet efficient enough for industrial applications due to a number of physiological, biochemical, and engineering barriers. This article presents a short overview of the metabolic pathways and enzymes involved in H2 photoproduction in cyanobacteria and green algae and our present understanding of the mechanisms of this process. We also summarize recent advances in engineering photosynthetic cell factories capable of overcoming the major barriers to efficient and sustainable H2 production.

Photoautotrophic production of renewable ethylene by engineered cyanobacteria: Steering the cell metabolism towards biotechnological use.

Ethylene is a volatile hydrocarbon with a massive global market in the plastic industry. The ethylene now used for commercial applications is produced exclusively from nonrenewable petroleum sources, while competitive biotechnological production systems do not yet exist. This review focuses on the currently developed photoautotrophic bioproduction strategies that enable direct solar-driven conversion of CO2 into ethylene, based on the use of genetically engineered photosynthetic cyanobacteria expressing heterologous ethylene forming enzyme (EFE) from Pseudomonas syringae. The emphasis is on the different engineering strategies to express EFE and to direct the cellular carbon flux towards the primary metabolite 2-oxoglutarate, highlighting associated metabolic constraints, and technical considerations on cultivation strategies and conditional parameters. While the research field has progressed towards more robust strains with better production profiles, and deeper understanding of the associated metabolic limitations, it is clear that there is room for significant improvement to reach industrial relevance. At the same time, existing information and the development of synthetic biology tools for engineering cyanobacteria open new possibilities for improving the prospects for the sustainable production of renewable ethylene.

Biotechnological Production of the Sunscreen Pigment Scytonemin in Cyanobacteria: Progress and Strategy.

Scytonemin is a promising UV-screen and antioxidant small molecule with commercial value in cosmetics and medicine. It is solely biosynthesized in some cyanobacteria. Recently, its biosynthesis mechanism has been elucidated in the model cyanobacterium Nostoc punctiforme PCC 73102. The direct precursors for scytonemin biosynthesis are tryptophan and p-hydroxyphenylpyruvate, which are generated through the shikimate and aromatic amino acid biosynthesis pathway. More upstream substrates are the central carbon metabolism intermediates phosphoenolpyruvate and erythrose-4-phosphate. Thus, it is a long route to synthesize scytonemin from the fixed atmospheric CO2 in cyanobacteria. Metabolic engineering has risen as an important biotechnological means for achieving sustainable high-efficiency and high-yield target metabolites. In this review, we summarized the biochemical properties of this molecule, its biosynthetic gene clusters and transcriptional regulations, the associated carbon flux-driving progresses, and the host selection and biosynthetic strategies, with the aim to expand our understanding on engineering suitable cyanobacteria for cost-effective production of scytonemin in future practices.

NordAqua, a Nordic Center of Excellence to develop an algae-based photosynthetic production platform.

NordAqua is a multidisciplinary Nordic Center of Excellence funded by NordForsk Bioeconomy program (2017-2022). The research center promotes Blue Bioeconomy and endeavours to reform the use of natural resources in a environmentally sustainable way. In this short communication, we summarize particular outcomes of the consortium. The key research progress of NordAqua includes (1) improving of photosynthetisis, (2) developing novel photosynthetic cell factories that function in a "solar-driven direct CO2 capture to target bioproducts" mode, (3) promoting the diversity of Nordic cyanobacteria and algae as an abundant and resilient alternative for less sustainable forest biomass and for innovative production of biochemicals, and (4) improving the bio-based wastewater purification and nutrient recycling technologies to provide new tools for integrative circular economy platforms.