Multiplex fluorometric assessment of nutrient limitation as a strategy for enhanced lipid enrichment and harvesting of Neochloris oleoabundans

Detailed in this study are the results of fluorometric assays used to assess the impact of gradual nutrient limitation versus punctuated nitrate limitation on the lipid content and morphology of Neochloris oleoabundans cells in batch culture. Punctuated nitrate limitation was imposed during pre‐log,...

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Published in:Biotechnology and bioengineering 2012-10, Vol.109 (10), p.2503-2512
Main Authors: Davis, Ryan W., Volponi, Joanne V., Jones, Howland D.T., Carvalho, Benjamin J., Wu, Huawen, Singh, Seema
Format: Article
Language:English
Subjects:
Algae
algal harvesting
algal morphology
batch culture
Cell culture
cell cycle
Chlorophyll
Chlorophylls
Culture Media - chemistry
Flow Cytometry
Fluorescence
Fluorometry - methods
Lipid Metabolism
Lipids
Lipids - analysis
MCR
Morphology
Multiplexing
multivariate analysis
Neochloris oleoabundans
Nile Red fluorescence
nitrate limitation
Nitrates
Nitrates - metabolism
Nutrients
Phases
Temporal logic
triacylglycerol
Volvocida - chemistry
Volvocida - metabolism
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Summary:Detailed in this study are the results of fluorometric assays used to assess the impact of gradual nutrient limitation versus punctuated nitrate limitation on the lipid content and morphology of Neochloris oleoabundans cells in batch culture. Punctuated nitrate limitation was imposed during pre‐log, log, late‐log, stationary, and senescent growth phases, and the cells were analyzed by bulk fluorescence emission, flow cytometry, and hyperspectral fluorescence imaging. In addition to intrinsic spectroscopic signatures provided by scatter and endogenous fluorescence, Nile Red staining was employed to monitor relative changes in lipid concentration. Analysis of the fluorescence images and temporal data sets was performed using multivariate curve resolution and fitting to logistic growth models to extract parameters of interest. The spectral components independently isolated from the image and temporal data sets showed close agreement with one another, especially relating to chlorophylls and Nile Red in polar and neutral lipid fractions, respectively. The fastest accumulation and highest total neutral lipid per cell and per chlorophyll were obtained with punctuated nitrate limitation during log phase growth on day 4 of culture. The presence of unbound chlorophyll in the resulting lipid bodies supports a membrane recycling TAG accumulation mechanism mediated by chloropolast–ER lipid exchange. Furthermore, an increase in cell size, indicated by forward scatter, was also found to correlate with increased neutral lipid, providing a size selection mechanism for passive harvest of algal cells at peak lipid enrichment. Biotechnol. Bioeng. 2012; 109: 2503–2512. © 2012 Wiley Periodicals, Inc. The combination of single‐cell and population‐based fluorometric studies revealed correlated dynamics among changes in lipid accumulation, lipid speciation, and cellular morphology as a result of nitrate limitation. The results suggest that actively imposed nitrate limitation during log‐phase growth maximizes total lipid and elicits a membrane recycling mechanism involving direct exchange of membrane components between plastids and the endoplasmic reticulum. Furthermore, changes in cell size corresponding to lipid‐enriched states may be exploited for selective and reduced energy harvesting.
ISSN:0006-3592
1097-0290
DOI:10.1002/bit.24517