Three step flow focusing enables image-based discrimination and sorting of late stage 1 Haematococcus pluvialis cells.
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ABSTRACT:
SUBMITTER: Kraus D
PROVIDER: S-EPMC8007022 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Haematococcus pluvialis is a freshwater unicellular green microalga belonging to the class Chlorophyceae and is of commercial interest for its ability to accumulate massive amounts of the red ketocarotenoid astaxanthin (3,3'-dihydroxy-?,?-carotene-4,4'-dione). Using confocal Raman microscopy and multivariate analysis, we demonstrate the ability to spectrally resolve resonance-enhanced Raman signatures associated with astaxanthin and ?-carotene along with chlorophyll fluorescence. By mathematically isolating these spectral signatures, in turn, it is possible to locate these species independent of each other in living cells of H. pluvialis in various stages of the life cycle. Chlorophyll emission was found only in the chloroplast whereas astaxanthin was identified within globular and punctate regions of the cytoplasmic space. Moreover, we found evidence for ?-carotene to be co-located with both the chloroplast and astaxanthin in the cytosol. These observations imply that ?-carotene is a precursor for astaxanthin and the synthesis of astaxanthin occurs outside the chloroplast. Our work demonstrates the broad utility of confocal Raman microscopy to resolve spectral signatures of highly similar chromophores in living cells.
| S-EPMC3167842 | biostudies-literature
Project description:The operation of cell disruption is indispensable but cost much in microalgae industry. To be simplified, two different reaction mechanisms await in the cell to respond to moderated or stressed environment. The physical and chemical changes of enzyme and turgor pressure of cell in this conversion play an important role in the enhancement of biomass and metabolites. Repeated turgor pressure (based on the structure and mechanics of cell wall) and converted enzyme system (based on photosynthesis) were used to loosen cell wall and then repeated cultivation of Haematococcus pluvialis for astaxanthin extraction was proposed. There was no significant difference of extraction yield between the broken cell (94.75?±?3.13%) and non-broken cell (92.32?±?3.24%) treated by the repeated cultivation. Meanwhile, fed-batch culture according to the relationship among pH and nutrient concentration was used to enhance the biomass of Haematococcus pluvialis with the dry cell weight of 1.63?±?0.07?g/L.
| S-EPMC4738327 | biostudies-literature
Project description:Astaxanthin derived from Haematococcus pluvialis is a valuable metabolite applied in a wide range of products. Its extraction depends on a sophisticated series of downstream process steps, including harvesting, disruption, drying, and extraction, of which some are dependent on each other. To determine the processes that yield maximum astaxanthin recovery, bead milling, high-pressure homogenization, and no disruption of H. pluvialis biomass were coupled with spray-drying, vacuum-drying, and freeze-drying in all possible combinations. Eventually, astaxanthin was extracted using supercritical CO2. Optimal conditions for spray-drying were evaluated through the design of experiments and standard least squares regression (feed rate: 5.8 mL/min, spray gas flow: 400 NL/h, inlet temperature: 180 °C). Maximal astaxanthin recoveries were yielded using high-pressure homogenization and lyophilization (85.4%). All combinations of milling or high-pressure homogenization and lyophilization or spray-drying resulted in similar recoveries. Bead milling and spray-drying repeated with a larger spray-dryer resulted in similar astaxanthin recoveries compared with the laboratory scale. Smaller astaxanthin recoveries after the extraction of vacuum-dried biomass were mainly attributed to textural changes. Evaluation of these results in an economic context led to a recommendation for bead milling and spray-drying prior to supercritical CO2 extraction to achieve the maximum astaxanthin recoveries.
| S-EPMC9105871 | biostudies-literature
Project description:Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final committed step in triacylglycerol biosynthesis in eukaryotes. In microalgae, the copy number of DGAT genes is extraordinarily expanded, yet the functions of many DGATs remain largely unknown. This study revealed that microalgal DGAT can function as a lysophosphatidic acyltransferase (LPAAT) both in vitro and in vivo while losing its original function as DGAT. Among the five DGAT-encoding genes identified and cloned from the green microalga Haematococcus pluvialis, four encoded HpDGATs that showed triacylglycerol synthase activities in yeast functional complementation analyses; the exception was one of the type II DGAT encoding genes, HpDGTT2. The hydrophobic recombinant HpDGTT2 protein was purified in soluble form and was found to function as a LPAAT via enzymatic assay. Introducing this gene into the green microalga Chlamydomonas reinhardtii led to retarded cellular growth, enlarged cell size, and enhanced triacylglycerol accumulation, identical to the phenotypes of transgenic strains overexpressing CrLPAAT. This study provides a framework for dissecting uncharacterized DGATs, and could pave the way to decrypting the structure-function relationship of this large group of enzymes that are critical to lipid biosynthesis.
| S-EPMC7853605 | biostudies-literature
Project description:BackgroundMicroalgae-derived extracellular vesicles (EVs), which transfer their cargos to the extracellular environment to affect recipient cells, play important roles in microalgal growth and environmental adaptation. And, they are also considered as sustainable and renewable bioresources of delivery nanocarrier for bioactive molecules and/or artificial drug molecules. However, their molecular composition and functions remain poorly understood.ResultsIn this study, isolation, characterization, and functional verification of Haematococcus pluvialis-derived EVs (HpEVs) were performed. The results indicated that HpEVs with typical EV morphology and size were secreted by H. pluvialis cells during the whole period of growth and accumulated in the culture medium. Cellular uptake of HpEVs by H. pluvialis was confirmed, and their roles in regulation of growth and various physiological processes of the recipient cells were also characterized. The short-term inhibition of HpEV secretion results in the accumulation of functional cellular components of HpEVs, thereby altering the biological response of these cells at the molecular level. Meanwhile, continuously inhibiting the secretion of HpEVs negatively influenced growth, and fatty acid and astaxanthin accumulation in H. pluvialis. Small RNA high-throughput sequencing was further performed to determine the miRNA cargoes and compelling details in HpEVs in depth. Comparative analysis revealed commonalities and differences in miRNA species and expression levels in three stages of HpEVs. A total of 163 mature miRNAs were identified with a few unique miRNAs reveal the highest expression levels, and miRNA expression profile of the HpEVs exhibited a clear stage-specific pattern. Moreover, a total of 12 differentially expressed miRNAs were identified and their target genes were classified to cell cycle control, lipid transport and metabolism, secondary metabolites biosynthesis and so on.ConclusionIt was therefore proposed that cargos of HpEVs, including miRNA constituents, were suggested potential roles in modulate cell physiological state of H. pluvialis. To summarize, this work uncovers the intercellular communication and metabolism regulation functions of HpEVs.
| S-EPMC10823724 | biostudies-literature
Project description:The green microalga Haematococcus pluvialis can synthesize high amounts of astaxanthin, which is a valuable antioxidant that has been utilized in human health, cosmetics, and aquaculture. To illustrate detailed molecular clues to astaxanthin yield, we performed PacBio HIFI along with Hi-C sequencing to construct an improved chromosome-level haplotypic genome assembly with 32 chromosomes and a genome size of 316.0 Mb. Its scaffold N50 (942.6 kb) and contig N50 (304.8 kb) have been upgraded remarkably from our previous genome draft, and a total of 32,416 protein-coding genes were predicted. We also established a high-evidence phylogenetic tree from seven representative algae species, with the main aim to calculate their divergence times and identify expanded/contracted gene families. We also characterized genome-wide localizations on chromosomes of some important genes such as five BKTs (encoding beta-carotene ketolases) that are putatively involved in astaxanthin production. In summary, we reported the first chromosome-scale map of H. pluvialis, which provides a valuable genetic resource for in-depth biomedical investigations on this momentous green alga and commercial astaxanthin bioproduction.
| S-EPMC10400597 | biostudies-literature
Project description:Haematococcus pluvialis is a freshwater species of Chlorophyta, family Haematococcaceae. It is well known for its capacity to synthesize high amounts of astaxanthin, which is a strong antioxidant that has been utilized in aquaculture and cosmetics. To improve astaxanthin yield and to establish genetic resources for H. pluvialis, we performed whole-genome sequencing, assembly, and annotation of this green microalga. A total of 83.1 Gb of raw reads were sequenced. After filtering the raw reads, we subsequently generated a draft assembly with a genome size of 669.0 Mb, a scaffold N50 of 288.6 kb, and predicted 18,545 genes. We also established a robust phylogenetic tree from 14 representative algae species. With additional transcriptome data, we revealed some novel potential genes that are involved in the synthesis, accumulation, and regulation of astaxanthin production. In addition, we generated an isoform-level reference transcriptome set of 18,483 transcripts with high confidence. Alternative splicing analysis demonstrated that intron retention is the most frequent mode. In summary, we report the first draft genome of H. pluvialis. These genomic resources along with transcriptomic data provide a solid foundation for the discovery of the genetic basis for theoretical and commercial astaxanthin enrichment.
| S-EPMC6330051 | biostudies-literature
Project description:Droplet-based microfluidic devices hold immense potential in becoming inexpensive alternatives to existing screening platforms across life science applications, such as enzyme discovery and early cancer detection. However, the lack of a predictive understanding of droplet generation makes engineering a droplet-based platform an iterative and resource-intensive process. We present a web-based tool, DAFD, that predicts the performance and enables design automation of flow-focusing droplet generators. We capitalize on machine learning algorithms to predict the droplet diameter and rate with a mean absolute error of less than 10 μm and 20 Hz. This tool delivers a user-specified performance within 4.2% and 11.5% of the desired diameter and rate. We demonstrate that DAFD can be extended by the community to support additional fluid combinations, without requiring extensive machine learning knowledge or large-scale data-sets. This tool will reduce the need for microfluidic expertise and design iterations and facilitate adoption of microfluidics in life sciences.
| S-EPMC7782806 | biostudies-literature
Project description:The PII signaling protein is a key protein for controlling nitrogen assimilatory reactions in most organisms, but little information is reported on PII proteins of green microalga Haematococcus pluvialis. Since H. pluvialis cells can produce a large amount of astaxanthin upon nitrogen starvation, its PII protein may represent an important factor on elevated production of Haematococcus astaxanthin. This study identified and isolated the coding gene (HpGLB1) from this microalga. The full-length of HpGLB1 was 1222 bp, including 621 bp coding sequence (CDS), 103 bp 5' untranslated region (5' UTR), and 498 bp 3' untranslated region (3' UTR). The CDS could encode a protein with 206 amino acids (HpPII). Its calculated molecular weight (Mw) was 22.4 kDa and the theoretical isoelectric point was 9.53. When H. pluvialis cells were exposed to nitrogen starvation, the HpGLB1 expression was increased 2.46 times in 48 h, concomitant with the raise of astaxanthin content. This study also used phylogenetic analysis to prove that HpPII was homogeneous to the PII proteins of other green microalgae. The results formed a fundamental basis for the future study on HpPII, for its potential physiological function in Haematococcus astaxanthin biosysthesis.
| S-EPMC5666412 | biostudies-literature
Project description:Background:Zinc oxide nanoparticles (ZnO NPs) are widely used in household and cosmetic products which imply an increased releasing of these particles into the environment, especially aquatic ecosystems, resulting in the need of assessing the potential toxic effects of ZnO NPS on the aquatic organisms, particularly on microalgae which form the base for food chain of aquatic biota. The present study has investigated the dose- and time-dependent cellular accumulation and the corresponding cytotoxic effects of increasing concentrations of ZnO NPs from 10-200 ?g/mL on microalga Haematococcus pluvialis at an interval of 24 h for 96 h. Methods:The scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDX) was used to qualitatively detect the cellular accumulation of ZnO NPs in algal cells, while inductively coupled plasma optical emission spectrometry (ICP OES) was performed to quantify the cell associated-zinc in algal cells. The percentage of cell death, reduction in algal biomass, and loss in photosynthetic pigments were measured to investigate the cytotoxic effects of ZnO NPs on H. pluvialis. Extracellular and intracellular changes in algal cells resulted from the treatment of ZnO NPs were demonstrated through optical, scanning, and transmission electron microscopic studies. Results:SEM-EDX spectrum evidenced the accumulation of ZnO NPs in algal biomass and ICP OES results reported a significant (p < 0.05) dose- and time-dependent accumulation of zinc in algal cells from 24 h for all the tested concentrations of ZnO NPs (10-200 ?g/mL). Further, the study showed a significant (p < 0.05) dose- and time-dependent growth inhibition of H. pluvialis from 72 h at 10-200 ?g/mL of ZnO NPs. The morphological examinations revealed substantial surface and intracellular damages in algal cells due to the treatment of ZnO NPs. Discussion:The present study reported the significant cellular accumulation of ZnO NPs in algal cells and the corresponding cytotoxic effects of ZnO NPs on H. pluvialis through the considerable reduction in algal cell viability, biomass, and photosynthetic pigments together with surface and intracellular damages.
| S-EPMC6765357 | biostudies-literature