Biology subjects

Ballottari, M.

Publications and source records attributed to Ballottari, M..

8 recordsLinked to original sources

bioRxiv2026

Agricultural digestate ultrafiltrate outperforms synthetic media for microalgal biomass production while enabling biostimulant generation

Livestock digestate ultrafiltrate (UF), obtained through membrane treatment of anaerobic digestate, is a nutrient-rich stream whose valorization remains largely unexplored. By removing suspended solids while retaining dissolved nutrients, ultrafiltration reduces turbidity and overcomes major limitations associated with using digestate for microalgal cultivation. This study evaluated UF as a nutrient source for Chlorella sorokiniana and Nannochloropsis oceanica, coupling ammonium recovery and biomass production with biomass valorization as a plant biostimulant. Given the high ammonium content of UF, ammonium tolerance was assessed in both species using ammonium bicarbonate as the nitrogen source. Increasing UF concentrations were then tested in laboratory photobioreactors and compared with synthetic media. Biomass productivity, nutrient removal, biomass composition, and biostimulant activity were evaluated. Both species tolerated ammonium-rich conditions and grew efficiently in UF-based media. UF significantly outperformed synthetic media, increasing biomass accumulation by up to 104% in N. oceanica and 133% in C. sorokiniana, while ammonium removal exceeded 99% and 98%, respectively. C. sorokiniana maintained high productivity when UF was diluted exclusively with water, indicating that UF supplied most nutrients required for growth. The enhanced productivity could not be explained solely by ammonium availability, suggesting a contribution from the complex nutrient composition retained during ultrafiltration. UF-derived biomass exhibited biostimulant activity, promoting tomato root development and increasing root and shoot biomass under reduced nitrogen availability. Digestate-derived UF therefore acts as both an efficient nutrient-recovery stream and a growth-promoting microalgal medium, enabling the conversion of membrane-treated digestate into biostimulant biomass within a circular biorefinery framework.

bioengineering↗

bioRxiv2026

Photoprotection comes at a cost: non-photochemical quenching shapes growthproductivity trade-offs in Haematococcus lacustris

Non-photochemical quenching (NPQ) dissipates excess absorbed light energy and protects photosynthetic organisms from photodamage, but its role in regulating the balance between growth, stress tolerance and astaxanthin accumulation in Haematococcus lacustris remains unclear. Here, we investigated how enhanced NPQ affects photosynthetic performance, stress-induced differentiation, and productivity in this astaxanthin-producing microalga. We isolated and characterized an NPQ-enhanced mutant line, A116, using cultivation assays under different stress conditions, analysis of photosynthetic parameters, pigment profiling, and whole-genome resequencing. A116 displayed stronger and faster NPQ induction, driven by increased LHCSR accumulation, resulting in decreased photosynthetic electron transport and lower photochemical efficiency under moderate-to-high light. Enhanced NPQ delayed the transition to astaxanthin-rich cysts under high light, allowing greater biomass accumulation under CO2-limiting conditions. However, under high CO2 availability, where carbon fixation relieved excitation pressure supporting efficient photosynthesis, the enhanced NPQ phenotype reduced growth and astaxanthin productivity compared with the wild type. These results show that NPQ modulates a context-dependent trade-off between photoprotection and productivity in Haematococcus lacustris. Increased energy dissipation can improve high-light tolerance under carbon limitation, but becomes detrimental when absorbed light can be efficiently used for carbon assimilation. Thus, optimal algal productivity requires tuning photoprotective capacity to environmental conditions rather than maximizing NPQ.

plant biology↗

bioRxiv2025

Heterologous expression of the cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase in Chlamydomonas reinhardtii causes increased cell size and biomass productivity in mixotrophic conditions

The Calvin-Benson-Bassham (CBB) cycle is the metabolic pathway responsible for CO assimilation in oxygenic photosynthetic organisms. Two key rate-limiting steps in this cycle are catalyzed by the enzymes fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase), making them promising targets for genetic enhancement to improve carbon fixation. A potential strategy involves overexpressing a cyanobacterial dual-function FBP/SBPase, which catalyzes both reactionsOverexpression of this enzyme in tobacco plants or in other plants led to an increase in growth rate and biomass accumulation. Here, the overexpression of the same enzyme was achieved in Chlamydomonas reinhardtii. The recombinant cyanobacterial FBP/SBPase isolated from C. reinhardtii exhibited the expected catalytic activity, being Mg2+ dependent and strongly activated in the presence of a reducing agent. The FBP/SBPase expressing lines exhibited an increased photosynthetic activity at the cell level and decreased production of singlet oxygen upon exposure to high irradiances, suggesting improved capacity to manage high excitation pressure of the photosynthetic apparatus. Increased cell volume was measured in FBP/SBPase-expressing lines under different growth conditions. However, increased growth and biomass productivity were observed only in mixotrophy when light and CO2 were limiting, leading to increased starch, protein, and lipid content on a cellular basis. This phenotype caused an increased sedimentation rate in the transformant lines: the expression of FBP/SBPase enzyme could thus be considered as a strategy to improve the cell harvesting process. These findings provide new insights into carbon metabolism in microalgae, and could, in the future, support improved biomass accumulation, paving the way for effective domestication and industrial use.

plant biology↗

bioRxiv2024

Abiotic stress-induced chloroplast and cytosolic Ca2+ dynamics in the green alga Chlamydomonas reinhardtii

Calcium (Ca2+)-dependent signalling plays a well-characterized role in the perception and response mechanisms to environmental stimuli in plant cells. In the context of a constantly changing environment, it is fundamental to understand how crop yield and microalgal biomass productivity are affected by external factors. Ca2+ signalling is known to be important in different physiological processes in microalgae but many of these signal transduction pathways still need to be characterized. Here, the role of compartment-specific Ca2+ signalling was investigated in Chlamydomonas reinhardtii in response to environmental stressors such as nutrient availability, osmotic stress, temperature fluctuations and carbon sensing. An in vivo single-cell imaging approach was adopted to directly visualize signalling processes at the level of specific subcellular compartments, using Chlamydomonas reinhardtii lines expressing a genetically encoded ratiometric Ca2+ indicator. Hyper-osmotic shock caused cytosolic and chloroplast Ca2+ elevations, whereas high temperature and inorganic carbon availability primarily induced Ca2+ transients in the chloroplast. In contrast, hypo-osmotic stress only induced Ca2+ elevations in the cytosol. The results herein reported show that compartment-specific signalling pathways are likely to play an important role in the response of Chlamydomonas to these stimuli providing new understanding of the mechanisms exploited by microalgae to respond to specific natural conditions.

plant biology↗

bioRxiv2022

Photosystem II monomeric antenna CP26 has a key role in Non-Photochemical Quenching in Chlamydomonas reinhardtii

O_LIThermal dissipation of the excitation energy harvested in excess, named non-photochemical quenching (NPQ), is one of the main photoprotective mechanisms evolved in oxygenic photosynthetic organisms. Here, the specific function in photoprotection and light harvesting of the monomeric Photosystem II antenna CP26, was investigated in Chlamydomonas, model organism for green algae C_LIO_LICRISPR/Cas9 genome editing and complementation strategies were applied to generate new cp26 knock-out mutants (named k6#) that differently from previous findings, did not negatively affected CP29 accumulation, allowing to compare mutants specifically deprived of CP26, CP29 or both C_LIO_LIThe absence of CP26 partially affected Photosystem II activity causing a reduced growth at low or medium light but not at high irradiances. However, the main phenotype observed in k6# mutants was a more than 70% reduction of NPQ compared to wild-type. This NPQ phenotype could be fully rescued by genetic complementation demonstrating that [~]50% of CP26 content compared to wild-type was sufficient to restore the NPQ capacity. C_LIO_LIOur findings demonstrate a pivotal role for CP26 in NPQ induction while CP29 has a crucial function for Photosystem II activity. The genetic engineering of these two proteins could be a promising strategy to regulate photosynthetic efficiency of microalgae under different light regimes. C_LI

plant biology↗

bioRxiv2021

CO2 supply modulates lipid remodelling, photosynthetic and respiratory activities in Chlorella species

Microalgae represent potential solutions to reduce the atmospheric CO2 level through photosynthesis. To boost CO2 fixation by microalgae it is essential to understand physiologic and metabolic responses at the base of CO2 assimilation and carbon flow. In this work two Trebouxiophyceae species, Chlorella sorokiniana and Chlorella vulgaris, were investigated for their metabolic responses to high and low CO2 (air level) availability. High CO2 availability resulted in an increase in biomass accumulation in both species but with a different chloroplast and mitochondrial responses. In C. sorokiniana we observed increased polar lipids and protein amount and a balanced NADPH redox state and a similar total respiration in the two conditions analysed. In contrast, in C. vulgaris high CO2 level caused an increase in TAG accumulation and a higher NADPH consumption suggesting a CO2 dependent increase of reducing power consumption in the chloroplast, which in turn influences the redox state of the mitochondria by lowering total dark respiration. Several rearrangements of the photosynthetic machinery were observed in both species, which differ from those described for the model organism Chlamydomonas reinhardtii. In the case of C. reinhardtii, adaptation of the photosynthetic apparatus to different CO2 availability relies on the translational repressor NAB1. NAB1 homologous protein could be identified only in C. vulgaris but lacked the regulation mechanisms previously described in C. reinhardtii. These findings highlight that the acclimation strategies to cope with a fluctuating inorganic carbon supply are diverse among green microalgae and point to new biotechnological strategies to boost CO2 fixation. One sentence summaryHigh/low CO2 availability induces cell responses as lipids remodelling, adaptations of the photosynthetic apparatus and modulation of mitochondrial respiration not conserved among green algae

plant biology↗

bioRxiv2021

A synthetic switch based on orange carotenoid protein to control blue light responses in chloroplasts

ABSTRACTSynthetic biology approaches to engineer light-responsive system are widely used, but their applications in plants are still limited, due to the interference with endogenous photoreceptors. Cyanobacteria, such as Synechocystis spp., possess a soluble carotenoid associated protein named Orange Carotenoid binding Protein (OCP) that, when activated by blue-green light, undergoes reversible conformational changes that enable photoprotection of the phycobilisomes. Exploiting this system, we developed a new chloroplast-localized synthetic photoswitch based on a photoreceptor-associated protein-fragment complementation assay (PCA). Since Arabidopsis thaliana does not possess the prosthetic group needed for the assembly of the OCP2 protein, we implemented the carotenoid biosynthetic pathway with a bacterial {beta}-carotene ketolase enzyme (crtW), to generate keto-carotenoids producing plants. The novel photoswitch was tested and characterized in Arabidopsis protoplasts with experiments aimed to uncover its regulation by light intensity, wavelength, and its conversion dynamics. We believe that this pioneer study establishes the basis for future implementation of plastid optogenetics to regulate organelle responses, such as gene transcription or enzymatic activity, upon exposure to specific light spectra. One-sentence summaryInspired by the light-driven conformational transitions of orange carotenoid proteins of cyanobacteria, we generated a molecular device able to switch its dimeric state in response to blue light.

synthetic biology↗

bioRxiv2019

Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii.

The green alga Chlamydomonas reinhardtii does not synthesize high-value ketocarotenoids like canthaxanthin and astaxanthin, however, a {beta}-carotene ketolase (CrBKT) can be found in its genome. CrBKT is poorly expressed, contains a long C-terminal extension not found in homologues and likely represents a pseudogene in this alga. Here, we used synthetic re-design of this gene to enable its constitutive overexpression from the nuclear genome of C. reinhardtii. Overexpression of the optimized CrBKT extended native carotenoid biosynthesis to generate ketocarotenoids in the algal host causing noticeable changes the green algal colour to a reddish-brown. We found that up to 50% of native carotenoids could be converted into astaxanthin and more than 70% into other ketocarotenoids by robust CrBKT overexpression. Modification of the carotenoid metabolism did not impair growth or biomass productivity of C. reinhardtii, even at high light intensities. Under different growth conditions, the best performing CrBKT overexpression strain was found to reach ketocarotenoid productivities up to 4.5 mg L-1 day-1. Astaxanthin productivity in engineered C. reinhardtii shown here is competitive with that reported for Haematococcus lacustris (formerly pluvialis) which is currently the main organism cultivated for industrial astaxanthin production. In addition, the extractability and bio-accessibility of these pigments was much higher in cell wall deficient C. reinhardtii than the resting cysts of H. lacustris. Engineered C. reinhardtii strains could thus be a promising alternative to natural astaxanthin producing algal strains and may open the possibility of other tailor-made pigments from this host.

plant biology↗