Final Report Summary - FUEL MAKING ALGAE (Real-time non-invasive characterization and selection of oil-producing microalgae at the single-cell level)
In the “Fuel making algae” research programme we proposed to address two aspects in the analysis of algal samples, namely:
(i) Characterization of the physiological state of individual microalgal cells - the characterization of the nutrient status of microalgae for rapid monitoring of nutrient dynamics and metabolism of algae on the single-cell level (in-vivo, and in real-time). The determination of the quantitative information on the degree of unsaturation (iodine value) of the bio-oil of individual microalgal cells.
(ii) Identifying and optimizing microalgal species for biofuel production - algae production (optimization of the growth process, cell density, and lipid contents variations) for selected species and selection of the cells according to the metabolic parameters for cultivation in the photobioreactor.
In order to achieve the above goals, we exploited an experimental technique based on the combination of optical micromanipulation with Raman microspectroscopy – so-called Raman tweezers. In our approach, the analyzed cells were spatially confined in suspension using an optical trap and, simultaneously, their chemical composition corresponding to the cell metabolic state has been studied by means of the Raman spectroscopy.
Following the spectroscopic analysis, the studied cells are sorted according to their metabolic state/degree of unsaturation using microfluidic systems. Raman spectroscopy is capable to measure nutrient dynamics and metabolism in vivo, in real-time, and label free making it possible to monitor/evaluate population variability.
Also, as mentioned above, degree of unsaturation of the algae oil (iodine value) can be measured using Raman spectra obtained from single microalgae. In conjuction with a multivariate analysis package it was feasible to develop a real-time identification procedure, which excels over other analytical techniques (such as time-consuming GC/MS) not lending themselves for real-time, in-vivo single-cell analysis.
Chemometric - multivariate analysis package developed in-house has been applied on the obtained Raman spectra of cells including Principal component analysis (PCA) for data visualization and dimensionality reduction. These routines were written in Matlab programming environment including sub-routines.
Microfluidic channels - experimental arrangements of microfluidic systems enabling cell confinement and delivery have been designed and produced at ISI. For microfluidic channel production soft lithography was the method of choice.
Using the Raman technique we designed and tested a novel analytical setup for microalgae biotechnology. In particular a photobioreactor analytical performance could be accompanied by Raman tweezers measurements at the dimensional level of individual cells. Using this strategy nutrient dynamics and metabolism of algae on the single-cell level (in-vivo, and in real-time) can be monitored. Thus, this techniques that can be employed for enhancing lipid production of microalgae (e.g. by applying physiological stress).
The success of our studies has resulted in estimation of algal lipids iodine values which can be monitored/followed so that optimum nutritional or cultivation conditions can be reached in order to produce algae with high bio-oil yield. This means that „fine tuning“ of the conditions concerning the kinetics and dynamics of algae cells (e.g. light and carbon uptake) can be achieved to advance photobioreactor performance. Moreover within the frame of this project we have developed and tested the algal sorting device based on the microfluidic chip and Raman tweezers.
(i) Characterization of the physiological state of individual microalgal cells - the characterization of the nutrient status of microalgae for rapid monitoring of nutrient dynamics and metabolism of algae on the single-cell level (in-vivo, and in real-time). The determination of the quantitative information on the degree of unsaturation (iodine value) of the bio-oil of individual microalgal cells.
(ii) Identifying and optimizing microalgal species for biofuel production - algae production (optimization of the growth process, cell density, and lipid contents variations) for selected species and selection of the cells according to the metabolic parameters for cultivation in the photobioreactor.
In order to achieve the above goals, we exploited an experimental technique based on the combination of optical micromanipulation with Raman microspectroscopy – so-called Raman tweezers. In our approach, the analyzed cells were spatially confined in suspension using an optical trap and, simultaneously, their chemical composition corresponding to the cell metabolic state has been studied by means of the Raman spectroscopy.
Following the spectroscopic analysis, the studied cells are sorted according to their metabolic state/degree of unsaturation using microfluidic systems. Raman spectroscopy is capable to measure nutrient dynamics and metabolism in vivo, in real-time, and label free making it possible to monitor/evaluate population variability.
Also, as mentioned above, degree of unsaturation of the algae oil (iodine value) can be measured using Raman spectra obtained from single microalgae. In conjuction with a multivariate analysis package it was feasible to develop a real-time identification procedure, which excels over other analytical techniques (such as time-consuming GC/MS) not lending themselves for real-time, in-vivo single-cell analysis.
Chemometric - multivariate analysis package developed in-house has been applied on the obtained Raman spectra of cells including Principal component analysis (PCA) for data visualization and dimensionality reduction. These routines were written in Matlab programming environment including sub-routines.
Microfluidic channels - experimental arrangements of microfluidic systems enabling cell confinement and delivery have been designed and produced at ISI. For microfluidic channel production soft lithography was the method of choice.
Using the Raman technique we designed and tested a novel analytical setup for microalgae biotechnology. In particular a photobioreactor analytical performance could be accompanied by Raman tweezers measurements at the dimensional level of individual cells. Using this strategy nutrient dynamics and metabolism of algae on the single-cell level (in-vivo, and in real-time) can be monitored. Thus, this techniques that can be employed for enhancing lipid production of microalgae (e.g. by applying physiological stress).
The success of our studies has resulted in estimation of algal lipids iodine values which can be monitored/followed so that optimum nutritional or cultivation conditions can be reached in order to produce algae with high bio-oil yield. This means that „fine tuning“ of the conditions concerning the kinetics and dynamics of algae cells (e.g. light and carbon uptake) can be achieved to advance photobioreactor performance. Moreover within the frame of this project we have developed and tested the algal sorting device based on the microfluidic chip and Raman tweezers.