Getting Grip on Phosphorus: Potential of Microalgae as a Vehicle for Sustainable Usage of This Macronutrient.

Phosphorus (P) is an important and irreplaceable macronutrient. It is central to energy and information storage and exchange in living cells. P is an element with a "broken geochemical cycle" since it lacks abundant volatile compounds capable of closing the P cycle. P fertilizers are critical for global food security, but the reserves of minable P are scarce and non-evenly distributed between countries of the world. Accordingly, the risks of global crisis due to limited access to P reserves are expected to be graver than those entailed by competition for fossil hydrocarbons. Paradoxically, despite the scarcity and value of P reserves, its usage is extremely inefficient: the current waste rate reaches 80% giving rise to a plethora of unwanted consequences such as eutrophication leading to harmful algal blooms. Microalgal biotechnology is a promising solution to tackle this challenge. The proposed review briefly presents the relevant aspects of microalgal P metabolism such as cell P reserve composition and turnover, and the regulation of P uptake kinetics for maximization of P uptake efficiency with a focus on novel knowledge. The multifaceted role of polyPhosphates, the largest cell depot for P, is discussed with emphasis on the P toxicity mediated by short-chain polyPhosphates. Opportunities and hurdles of P bioremoval via P uptake from waste streams with microalgal cultures, either suspended or immobilized, are discussed. Possible avenues of P-rich microalgal biomass such as biofertilizer production or extraction of valuable polyPhosphates and other bioproducts are considered. The review concludes with a comprehensive assessment of the current potential of microalgal biotechnology for ensuring the sustainable usage of phosphorus.

Evaluation of Extraction Techniques for Recovery of Microalgal Lipids under Different Growth Conditions.

Microalgal lipids contain a wide array of liposoluble bioactive compounds, but lipid extraction remains a critical limitation for their commercial use. An accelerated solvent extraction (ASE) was used to extract lipids from Chlamydomonas reinhardtii, Arthrospira platensis (Spirulina), and Chlorella vulgaris grown under either standard or nitrogen depletion conditions. Under standard growth conditions, ASE using methanol:chloroform (2:1), methyl tert-butyl ether (MTBE):methanol:water, and ethanol at 100 °C resulted in the highest recovery of total lipids (352 ± 30, 410 ± 32, and 127 ± 15 mg/g biomass from C. reinhardtii, C. vulgaris, and A. platensis, respectively). Similarly, the highest total lipid and triacylglycerols (TAGs) recovery from biomass cultivated under nitrogen depletion conditions was found at 100 °C using methanol:chloroform, for C. reinhardtii (total, 550 ± 21; TAG, 205 ± 2 mg/g biomass) and for C. vulgaris (total, 612 ± 29 mg/g; TAG, 253 ± 7 mg/g biomass). ASE with MTBE:methanol:water at 100 °C yielded similar TAG recovery for C. reinhardtii (159 ± 6 mg/g) and C. vulgaris (200 ± 4 mg/g). Thus, MTBE:methanol:water is suggested as an alternative substitute to replace hazardous solvent mixtures for TAGs extraction with a much lower environmental impact. The extracted microalgal TAGs were rich in palmitic (C16:0), stearic (C18:0), oleic (C18:1,9), linoleic (C18:2n6), and α-linolenic (C18:3n3) acids. Under nitrogen depletion conditions, increased palmitic acid (C16:0) recovery up to 2-fold was recorded from the biomasses of C. reinhardtii and C. vulgaris. This study demonstrates a clear linkage between the extraction conditions applied and total lipid and TAG recovery.

Key Proteomics Tools for Fundamental and Applied Microalgal Research.

Microscopic, photosynthetic prokaryotes and eukaryotes, collectively referred to as microalgae, are widely studied to improve our understanding of key metabolic pathways (e.g., photosynthesis) and for the development of biotechnological applications. Omics technologies, which are now common tools in biological research, have been shown to be critical in microalgal research. In the past decade, significant technological advancements have allowed omics technologies to become more affordable and efficient, with huge datasets being generated. In particular, where studies focused on a single or few proteins decades ago, it is now possible to study the whole proteome of a microalgae. The development of mass spectrometry-based methods has provided this leap forward with the high-throughput identification and quantification of proteins. This review specifically provides an overview of the use of proteomics in fundamental (e.g., photosynthesis) and applied (e.g., lipid production for biofuel) microalgal research, and presents future research directions in this field.

Pathogens and predators impacting commercial production of microalgae and cyanobacteria.

Production of phytoplankton (microalgae and cyanobacteria) in commercial raceway ponds and other systems is adversely impacted by phytoplankton pathogens, including bacteria, fungi and viruses. In addition, cultures are susceptible to productivity loss, or crash, through grazing by contaminating zooplankton such as protozoa, rotifers and copepods. Productivity loss and product contamination are also caused by otherwise innocuous invading phytoplankton that consume resources in competition with the species being cultured. This review is focused on phytoplankton competitors, pathogens and grazers of significance in commercial culture of microalgae and cyanobacteria. Detection and identification of these biological contaminants are discussed. Operational protocols for minimizing contamination, and methods of managing it, are discussed.

Microalgae-based photosynthetic strategy for oxygenating avascularised mouse brain tissue - An in vitro proof of concept study.

Hypoxic brain injury is a leading cause of loss of quality of life globally for which there are currently no effective treatments. There has been increasing interest in incorporating photosynthesising agents into hypoxic tissue as a mechanism for in situ oxygen delivery, independent of vascular perfusion. To date this has not been tested in the brain. The oxygen production capacity of Chlamydomonas reinhardtii microalgal cultures was measured in artificial cerebrospinal fluid (aCSF) in benchtop assays and in cortical slices in situ. Cortical slice function was quantified by measuring the length, frequency and amplitude of seizure-like event (SLE) activity - in conventionally oxygenated aCSF, C. reinhardtii cultures, unoxygenated and deoxygenated aCSF. The possibility of direct toxic algal effects was investigated by exposing slices to cultures for 5 h. An oxygen level of 25 mg.L-1 was achieved with C. reinhardtii in no-Mg aCSF. Slice SLE function was preserved in C. reinhardtii, without the need for supplemental oxygen. In contrast, functional parameters deteriorated in unoxygenated and deoxygenated aCSF. In the former, there was a 66% reduction in SLE frequency and a 37% reduction in event amplitude. In the latter, SLE activity ceased completely. No toxic algae effects were seen in slices exposed to cultures for 5 h. These results confirm that C. reinhardtii oxygenation of aCSF can sustain cortical network activity - without tissue toxicity for the normal lifespan of an acute cortical slice. This study shows promise for the concept of photosynthesis as a mechanism for providing oxygen to rescue ischaemic avascularised brain tissue.

Responses of Chlamydomonas reinhardtii during the transition from P-deficient to P-sufficient growth (the P-overplus response): The roles of the vacuolar transport chaperones and polyphosphate synthesis.

Phosphorus (P) assimilation and polyphosphate (polyP) synthesis were investigated in Chlamydomonas reinhardtii by supplying phosphate (PO43- ; 10 mg P·L-1 ) to P-depleted cultures of wildtypes, mutants with defects in genes involved in the vacuolar transporter chaperone (VTC) complex, and VTC-complemented strains. Wildtype C. reinhardtii assimilated PO43- and stored polyP within minutes of adding PO43- to cultures that were P-deprived, demonstrating that these cells were metabolically primed to assimilate and store PO43- . In contrast, vtc1 and vtc4 mutant lines assayed under the same conditions never accumulated polyP, and PO43- assimilation was considerably decreased in comparison with the wildtypes. In addition, to confirm the bioinformatics inferences and previous experimental work that the VTC complex of C. reinhardtii has a polyP polymerase function, these results evidence the influence of polyP synthesis on PO43- assimilation in C. reinhardtii. RNA-sequencing was carried out on C. reinhardtii cells that were either P-depleted (control) or supplied with PO43- following P depletion (treatment) in order to identify changes in the levels of mRNAs correlated with the P status of the cells. This analysis showed that the levels of VTC1 and VTC4 transcripts were strongly reduced at 5 and 24 h after the addition of PO43- to the cells, although polyP granules were continuously synthesized during this 24 h period. These results suggest that the VTC complex remains active for at least 24 h after supplying the cells with PO43- . Further bioassays and sequence analyses suggest that inositol phosphates may control polyP synthesis via binding to the VTC SPX domain.

Nitrous oxide emissions during microalgae-based wastewater treatment: current state of the art and implication for greenhouse gases budgeting.

Microalgae can synthesise the ozone depleting pollutant and greenhouse gas nitrous oxide (N2O). Consequently, significant N2O emissions have been recorded during real wastewater treatment in high rate algal ponds (HRAPs). While data scarcity and variability prevent meaningful assessment, the magnitude reported (0.13-0.57% of the influent nitrogen load) is within the range reported by the Intergovernmental Panel on Climate Change (IPCC) for direct N2O emissions during centralised aerobic wastewater treatment (0.016-4.5% of the influent nitrogen load). Critically, the ability of microalgae to synthesise N2O challenges the IPCC's broad view that bacterial denitrification and nitrification are the only major cause of N2O emissions from wastewater plants and aquatic environments receiving nitrogen from wastewater effluents. Significant N2O emissions have indeed been repeatedly detected from eutrophic water bodies and wastewater discharge contributes to eutrophication via the release of nitrogen and phosphorus. Considering the complex interplays between nitrogen and phosphorus supply, microalgal growth, and microalgal N2O synthesis, further research must urgently seek to better quantify N2O emissions from microalgae-based wastewater systems and eutrophic ecosystems receiving wastewater. This future research will ultimately improve the prediction of N2O emissions from wastewater treatment in national inventories and may therefore affect the prioritisation of mitigation strategies.

Chlamydomonas reinhardtii, an Algal Model in the Nitrogen Cycle.

Nitrogen (N) is an essential constituent of all living organisms and the main limiting macronutrient. Even when dinitrogen gas is the most abundant form of N, it can only be used by fixing bacteria but is inaccessible to most organisms, algae among them. Algae preferentially use ammonium (NH4+) and nitrate (NO3-) for growth, and the reactions for their conversion into amino acids (N assimilation) constitute an important part of the nitrogen cycle by primary producers. Recently, it was claimed that algae are also involved in denitrification, because of the production of nitric oxide (NO), a signal molecule, which is also a substrate of NO reductases to produce nitrous oxide (N2O), a potent greenhouse gas. This review is focused on the microalga Chlamydomonas reinhardtii as an algal model and its participation in different reactions of the N cycle. Emphasis will be paid to new actors, such as putative genes involved in NO and N2O production and their occurrence in other algae genomes. Furthermore, algae/bacteria mutualism will be considered in terms of expanding the N cycle to ammonification and N fixation, which are based on the exchange of carbon and nitrogen between the two organisms.

The biosynthesis of nitrous oxide in the green alga Chlamydomonas reinhardtii.

Over the last decades, several studies have reported emissions of nitrous oxide (N2 O) from microalgal cultures and aquatic ecosystems characterized by a high level of algal activity (e.g. eutrophic lakes). As N2 O is a potent greenhouse gas and an ozone-depleting pollutant, these findings suggest that large-scale cultivation of microalgae (and possibly, natural eutrophic ecosystems) could have a significant environmental impact. Using the model unicellular microalga Chlamydomonas reinhardtii, this study was conducted to investigate the molecular basis of microalgal N2 O synthesis. We report that C. reinhardtii supplied with nitrite (NO2- ) under aerobic conditions can reduce NO2- into nitric oxide (NO) using either a mitochondrial cytochrome c oxidase (COX) or a dual enzymatic system of nitrate reductase (NR) and amidoxime-reducing component, and that NO is subsequently reduced into N2 O by the enzyme NO reductase (NOR). Based on experimental evidence and published literature, we hypothesize that when nitrate (NO3- ) is the main Nitrogen source and the intracellular concentration of NO2- is low (i.e. under physiological conditions), microalgal N2 O synthesis involves the reduction of NO3- to NO2- by NR followed by the reduction of NO2- to NO by the dual system involving NR. This microalgal N2 O pathway has broad implications for environmental science and algal biology because the pathway of NO3- assimilation is conserved among microalgae, and because its regulation may involve NO.

Nitrous oxide emissions from high rate algal ponds treating domestic wastewater.

This study investigated the generation of N2O by microcosms withdrawn from 7-L high rate algal ponds (HRAPs) inoculated with Chlorella vulgaris and treating synthetic wastewater. Although HRAPs microcosms demonstrated the ability to generate algal-mediated N2O when nitrite was externally supplied under darkness in batch assays, negligible N2O emissions rates were consistently recorded in the absence of nitrite during 3.5-month monitoring under 'normal' operation. Thereafter, HRAP A and HRAP B were overloaded with nitrate and ammonium, respectively, in an attempt to stimulate N2O emissions via nitrite in situ accumulation. Significant N2O production (up to 5685±363 nmol N2O/g TSS h) was only recorded from HRAP B microcosms externally supplied with nitrite in darkness. Although confirmation under full-scale outdoors conditions is needed, this study provides the first evidence that the ability of microalgae to synthesize N2O does not affect the environmental performance of wastewater treatment in HRAPs.