Lipid Production In Microalgae Research Articles

Optimation of the cell density, biomass production, lipid, and carbohydrate content of Nannochloropsis oculata with fluorescein and pH manipulation

Nannochloropsis oculata is a potential marine microalga. However, the mass production of N. oculata encounters several challenges, such as the high costs of resources, contamination, and light absorption optimization. These problems can be solved by pH manipulation and fluorescein induction on culture media. Various studies have shown that pH values manipulation can cause fluctuations in cell density, dry biomass, and carbohydrate and lipid production of microalgae. On the other hand, fluorescein can act as a molecular antenna that improves light absorption. This research is novel because of its focus on its unique attempt to utilize pH manipulation combined with fluorescein induction to enhance the growth and metabolite production of N. oculata. The implications of this research may provide cost-effective, viable, and sustainable development of the algal industry in general. In this research, cultivation was carried out for seven days with four levels of pH (7 – 10) combined with three variations of fluorescein addition (0, 0.15, and 0.3 mL). Every treatment combination had three replications. Cell density and biomass as growth analysis parameters were calculated during the cultivation using the Haemocytometer and gravimetric methods. Bligh & Dyer and phenol sulfate methods were used to analyze lipid and carbohydrate content. Based on the result, the best treatment combination, pH 9 with 0.15 mL of fluorescein, increases cell density and carbohydrate content of N. oculata by 31.45 and 4.4%, albeit not statistically significant.

New insights into rare earth element-induced microalgae lipid accumulation: Implication for biodiesel production and adsorption mechanism

A coupling technology for lipid production and adsorption of rare earth elements (REEs) using microalgae was studied in this work. The microalgae cell growth, lipid production, biochemical parameters and lipid profiles were investigated under different REEs (Ce3+, Gd3+and La3+). The results showed that the maximum lipid production was achieved at different concentrations of REEs, with lipid productivities of 300.44, 386.84 and 292.19 mg L−1 d−1 under treatment conditions of 100 μg L−1 Ce3+, 250 μg L−1 Gd3+ and 1 mg L−1 La3+, respectively. Moreover, the adsorption efficiency of Ce3+, Gd3+ and La3+exceeded 96.58 %, 93.06 % and 91.3 % at concentrations of 25–1000 μg L−1, 100–500 μg L−1 and 0.25–1 mg L−1, respectively. In addition, algal cells were able to adsorb 66.2 % of 100 μg L−1 Ce3+, 48.4 % of 250 μg L−1 Gd3+ and 59.9 % of 1 mg L−1 La3+. The combination of extracellular polysaccharide and algal cell wall could adsorb 25.2 % of 100 μg L−1 Ce3+, 44.5 % of 250 μg L−1 Gd3+ and 30.5 % of 1 mg L−1 La3+, respectively. These findings indicated that microalgae predominantly adsorbed REEs through the intracellular pathway. This study elucidates the mechanism of effective lipid accumulation and adsorption of REEs by microalgae under REEs stress conditions. It establishes a theoretical foundation for the efficient microalgae lipid production and REEs recovery from wastewater or waste residues containing REEs.

Regulation effects of indoleacetic acid on lipid production and nutrient removal of Chlorella pyrenoidosa in seawater-containing wastewater

The utilization of seawater supplemented with wastewater nutrients for microalgae cultivation represents a promising and cost-effective approach that combines the benefits of wastewater treatment and microalgal resource recovery. However, the high salt content in seawater poses a significant challenge, hindering microalgal growth and reducing the removal of nitrogen and phosphorus on a large scale. The phytohormone indoleacetic acid (IAA) was used in this study to enhance stress resistance and lipid production of Chlorella pyrenoidosa grown in seawater-wastewater medium. Compared to the control groups involving regular wastewater and seawater-containing wastewater without IAA, Chlorella pyrenoidosa cultivated in the seawater-containing wastewater supplemented with IAA exhibited remarkable outcomes. Specifically, microalgae in IAA-enhanced seawater-containing wastewater achieved the highest lipid productivity (22.67 mg L−1 d−1) along with impressive nitrogen (99.3 %) and phosphorus (97.3 %) removal rates. Moreover, their cell sedimentation ratio reached 76.6 %, indicating enhanced settling properties. Additionally, the physiological mechanism changes after exposure to seawater stress and IAA were revealed based on the changes in antioxidant enzymes, endogenous hormones, and fatty acid saturation. Furthermore, the transcriptomic analysis elucidated the molecular mechanisms underlying microalgal lipid synthesis and their response to antioxidant stress when exposed to seawater. The supplementation of IAA under seawater stress stimulated energy metabolism and the antioxidant response in microalgal cells, effectively mitigating the adverse effects of seawater stress and promoting overall algal lipid productivity. Overall, this study unveiled the potential of exogenous plant hormones, particularly IAA, in enhancing stress resistance and lipid productivity of microalgae grown in seawater-wastewater medium, which significantly contributed towards the efficient use of seawater resources for microalgae cultivation and biofuel production.

Strategies to enhance lipid productivity in microalgae: Role of cell disruption and media composition

The major bottleneck in biofuel production from microalgae is the optimization of biomass productivity, lipid productivity, and the lipid extraction process. The diverse cell structure of microalgae results in variation in algal growth and the yield of intracellular content. Therefore, it is quintessential to study the algae thoroughly under varied pretreatment and extraction conditions in order to make downstream processing affordable and cost-effective in large-scale production. In this study, Chaetophora sp., Microchaete sp., and Nostoc sp., isolated from a freshwater pond, were subjected to various physical and chemical disruption techniques to extract lipid. Further, the effects of nitrate and phosphate concentrations on biomass and lipid productivity were statistically assessed. Lipid yield was higher when algal cells were subjected to sonication and microwave disruption followed by extraction using a modified Folch’s method. The BBM supplemented with 0.125 g l−1 of sodium nitrate, 37.5×10−3 g l−1 of dipotassium phosphate, and 87.5×10−3 g l−1 of potassium dihydrogen phosphate was found to be ideal for cultivating Chaetophora sp. for the purpose of biofuel. While Microchaete sp. and Nostoc sp. showed better biomass and lipid productivity in BG11 media supplemented with 7.5 g l−1 of sodium nitrate and 37.5×10−3 g l−1 of potassium phosphate.

Characterization of oil body-associated proteins obtained from oil bodies with different sizes in oleaginous diatom Fistulifera solaris

Oil body-associated proteins from the oleaginous diatom Fistulifera solaris were identified by proteomic analysis of oil bodies of various sizes (small, middle, and large) by time-dependent culturing upon nutrient-starvation at 36, 96 and 168h. This diatom strain has the capability to accumulate neutral lipids and triacylglycerol. Liquid chromatography-tandem mass spectrometry analysis revealed 662 proteins in all oil body sizes. Among these, 132 proteins were predicted to be localized to the endoplasmic reticulum. Seventeen proteins that exhibited a positive correlation with gene expression and the oil body size were selected as novel candidates for oil body-associated proteins. Among the 17 protein candidates, two proteins encoded by fso:g8246 and fso:g10200 were confirmed to be localized on the surface of the oil body and endoplasmic reticulum. A protein encoded by fso:g2514, which is involved in sterol biosynthesis, was also identified. This protein was likely to localize to mitochondria; however, inhibitor assays suggested that it might play a role in lipid degradation. Our work provides new insights into the proteomics of microalgae and provides a valuable strategy for boosting lipid productivity in microalgae.

Effects of Iron Valence on the Growth, Photosynthesis, and Fatty Acid Composition of Phaeodactylum tricornutum

Iron is a limiting factor that controls the phytoplankton biomass of the ocean and plays an important role in the lipid production of microalgae. Elucidating the effects of different iron valences on microalgae is helpful for their commercial production. We investigated the growth, photosynthesis, and fatty acid profile of the model diatom Phaeodactylum tricornutum cultured with depleted Fe, Fe2+, Fe2+/Fe3+, and Fe3+. Samples were taken every 24 h for 8 days, and their cell density, photosynthetic pigment content, chlorophyll fluorescence, total fatty acid content, and fatty acid composition were analyzed. The cell densities of the Fe2+ and Fe2+/Fe3+ groups were significantly higher than those of the control and Fe3+ groups (p < 0.05). They were 1.26 times and 1.23 times higher than those in the Fe-depleted group. The contents of chlorophyll a and c in the Fe2+ group were significantly higher than those in the Fe-depleted group (p < 0.05). The chlorophyll fluorescence results show that Fe2+ enhanced the photosynthesis of P. tricornutum to a greater extent than Fe3+. On the eighth day of harvest, Fv/Fm and Y(II) in the Fe2+ group were 0.672 and 0.476, respectively, being 1.10 and 1.19 times greater than those in the Fe3+ group and 1.15 and 1.33 times greater than those in the Fe-depleted group, respectively. Compared with the control group, the levels of saturated fatty acids of the Fe2+/Fe3+ and Fe3+ groups were significantly higher (p < 0.05) at 21.36 ± 1.24% and 21.20 ± 0.13%, respectively. The levels of polyunsaturated fatty acids of the Fe2+/Fe3+ group were significantly lower (p < 0.05) at 29.82 ± 2.75%. Our results show that P. tricornutum exhibited physiological plasticity, including changes in photosynthetic activities and shifts in fatty acid composition, in response to different iron valences and that Fe2+ was more beneficial to the biomass production of this species than Fe3+. These findings are applicable to the production of biomass and polyunsaturated fatty acids.

Insights into the Effect of Rhamnolipids on the Anaerobic Fermentation and Microalgae Lipid Production of Waste Activated Sludge: Performance and Mechanisms

In the last few years, the production of waste activated sludge (WAS) has increased dramatically, and the treatment and disposal have attracted widespread attention. In this study, rhamnolipids were used to improve the efficiency of hydrogen production from WAS, and the fermentation solution was used as a substrate by lipid-producing microalgae to recover resources and generate clean energy. Results showed that rhamnolipids had the best effect among several surfactants in promoting hydrogen production, and the hydrogen production of WAS added with 0.4 g/g TS rhamnolipids reached 178.48 ± 6.07 mL/g TS, which improved by 13.53 times compared to the control group. The content of SCOD, soluble protein, and soluble polysaccharide in WAS fermentation solution with the addition of 0.4 g/g TS rhamnolipid increased by 9.19, 11.46, and 42.28 times, respectively, indicating that rhamnolipid could improve the hydrolysis of WAS, which led to an increase in the efficiency of hydrogen production. And three-dimensional excitation–emission matrix fluorescence spectroscopic analysis showed that the intensity of dissolved organic matter increased, suggesting a rise in soluble microbial metabolites. The analysis of the composition of volatile fatty acids in the fermentation solution showed that the type of hydrogen production was mainly butyric acid-type fermentation hydrogen production with high concentrations of n-butyric acid (9330.14 ± 525.33 mg/L) and acetic acid (6324.07 ± 80.67 mg/L). In addition, analysis of the microbial community structure of the WAS fermentation hydrogen production system showed that the dominant genus was Clostridium sensu stricto. Microalgae can utilize acetic acid, ethanol, NH4+-N, PO43–-P, dissolved proteins, and dissolved polysaccharides in the fermentation solution to grow and produce lipids. The biomass and lipid productivity of microalgae cultured by fermentation solution reached 3.16 ± 0.58 g/L and 356.86 ± 4.91 mg/L/day, respectively. The microalgae contained 84.57 ± 4.33% saturated fatty acid content, which was more favorable for biodiesel production. This study not only successfully utilized rhamnolipids to promote hydrogen production from WAS but also coupled microalgae lipid production system, further laying the foundation for realizing the resource utilization of waste resources and clean energy production.

Efficient coupling of sulfadiazine removal with microalgae lipid production in a membrane photobioreactor

This study explored the feasibility of a coupled system for antibiotic removal and biofuel production through microalgae cultivation. Initial, batch culture experiments demonstrated that sulfadiazine (SDZ) had an inhibitory effect on Chlorella sp. G-9, and 100.0 mg L-1 SDZ completely inhibited its growth. In order to improve SDZ removal efficiency by microalgae, three membrane photobioreactors (MPBRs) with different hydraulic retention times (HRTs) were established for continuous microalgae cultivation. The efficient coupling of SDZ removal and microalgal lipid production was achieved through the gradual increment of influent SDZ concentration from 0 to 100.0 mg L-1. The reduction in SDZ ranged between 57.8 and 89.7%, 54.7–91.7%, and 54.6–93.5% for the MPBRs with HRT of 4 d, 2 d, and 1 d, respectively. Chlorella sp. Was found to tolerate higher concentrations of SDZ in the MPBR system, and the resulting stress from high concentrations of SDZ effectively increased the lipid content of microalgae for potential biodiesel production. With the increase of influent SDZ concentration from 0 to 100.0 mg L-1, the lipid content of microalgae increased by 43.5%. Chlorophyll content, superoxide dismutase activity, and malondialdehyde content of microalgae were also evaluated to explore the mechanism of microalgae tolerance to SDZ stress in MPBR.

The growth and lipid accumulation of Scenedesmus quadricauda under nitrogen starvation stress during xylose mixotrophic/heterotrophic cultivation.

In order to conquer the block of high cost and low yields which limit to realize the commercialization of microalgal biodiesel, the mixotrophic and heterotrophic cultivation of Scenedesmus quadricauda FACHB-1297 fed on xylose was separately studied employing six forms of media: phosphorus sufficient, phosphorus restricted, and phosphorus starvation were combined with nitrogen sufficient and nitrogen starvation conditions. The maximum lipid content (about 41% of dry weight) was obtained on the 5th day (heterotrophic cultivation) and 8th day (mixotrophic cultivation) under the nitrogen starved and phosphorus sufficient (N0&P) conditions, which was about twofold in comparison to the final lipid content on the sufficient nitrogen condition (control). Under mixotrophic and heterotrophic modes, the highest lipid production was achieved in the N0&P trial, with the value of 274.96mg/L and 193.77mg/L, respectively. Xylose utilization rate of 30-96% under heterotrophic modes was apparently higher than that of 20-50% in mixotrophic modes. In contrast, phosphorus uptake rate of 100% under mixotrophic cultivation was significantly more than that of 60-90% in heterotrophic cultivation. Furthermore, under the condition of heterotrophic cultivation using xylose as a carbon source, the phosphorus had a positive impact on microalgae cell synthesis and the lipid content enhanced with the augmentation in phosphorus concentrations. We suggested that sufficient phosphorus should be supplied for obtaining higher microalgal lipid production in the lack of nitrogen under xylose heterotrophic/mixotrophic condition. This was a highly effective way to obtain efficient microalgae lipid production.

The p-hydroxybenzoic acid enhanced lipid accumulation of Chlorella under antibiotic stress

Phytohormones are widely known to regulate plant growth under environmental stress, however, their roles of microalgae are not clear during the antibiotic wastewater treatment. A hypothesis was proposed that phytohormones were capable to relieve the inhibition of microalgae growth and improve the bioresources recovery from antibiotic wastewater. Chlorella and enrofloxacin (ENR) were utilized to simulate the antibiotic wastewater treatment by microalgae, and explored the roles of p-hydroxybenzoic acid (pHBA). With 10 mg/L ENR and 1 μM pHBA condition, the Chlorella biomass increased by 23%. Moreover, lipid production increased by 30%. Trace amounts of pHBA improved microalgae growth and lipid accumulation by up-regulating photosynthesis, glycolysis, and TCA cycle. Furthermore, pHBA stimulated the signal transduction pathways of microalgae to improve antioxidant defense capability. Therefore, pHBA enhanced tolerance of ENR and improved microalgae lipid production. This study provided a new clue into using microalgae to recover high-value bioresources from antibiotic wastewater.

Microalgae Biomass and Lipids as Feedstock for Biofuels: Sustainable Biotechnology Strategies

Microalgae exhibit remarkable potential as a feedstock for biofuel production compared with other sources, owing to their high areal productivity, low environmental effect, and negligible influence on food security. However, the primary obstacle to the commercialization of algae-based biofuels is the high economic cost due to the low-yield lipid content in the microalgae biomass. Maximizing biomass and lipid production is crucial to improve the economic viability of microalgae for biofuels. Identifying appropriate algal strains, particularly from indigenous environments, and developing those ‘platform strains’ using mutagenesis and genetic-engineering techniques is preferable. The provided discussion of conventional methods to increase microalgae’s biomass and lipid productivity mostly entailed adjusting environmental (such as temperature, light, and salinity) and nutritional (such as nitrogen and phosphorus) parameters. This review illustrated a comprehensive overview of biotechnological approaches and the recent strategies to enhance the lipid productivity of microalgae. The research also emphasized the need to streamline engineering strategies with the aid of recent advancements in DNA-manipulation techniques to hinder the existing biological intricacies in lipogenesis. This review also discussed the current economic and commercialization of this algal biorefinery along with the drawbacks.

Growth behavior, phenol removal and lipid productivity of microalgae in mixotrophic and heterotrophic conditions under synergistic effect of phenol and bicarbonate for biodiesel production

Growth behavior, phenol removal and lipid productivity of microalgae in mixotrophic and heterotrophic conditions under synergistic effect of phenol and bicarbonate for biodiesel production

Effects of high-strength landfill leachate effluent on stress-induced microalgae lipid production and post-treatment micropollutant degradation

This research investigates the effects of landfill leachate effluent concentrations from moving bed biofilm reactor (MBBR) on stress-induced Chlorella vulgaris and Scenedesmus armatus lipid production and post-treatment micropollutant degradation. The effluent concentrations were varied between 25%, 50%, 75%, and 100% (v/v). The landfill leachate influent was treated using two-stage moving bed biofilm reactor under 24 h and 18 h hydraulic retention time (HRT). The results indicated that the effluent concentration was positively correlated with the stress-induced microalgae lipid production in the post-treatment of residual micropollutants. C. vulgaris and S. armatus completely remove residual micropollutants in the effluent. The superoxide dismutase and peroxidase activity were positively correlated with the cellular lipid content. The lipid content of C. vulgaris and S. armatus cultivated in the 18 h HRT effluent were 31–51% and 51–64%, while those in the 24 h HRT effluent were 15–16% and 5–19%. The optimal condition of microalgae cultivation for the post-treatment of residual micropollutants was 50–75% (v/v) effluent concentrations under 18 h HRT, achieving the highest lipid production of 113–116 mg/L for C. vulgaris and 74–75 mg/L for S. armatus. Essentially, the MBBR landfill leachate effluent holds promising potential as a substrate for microalgae lipid production.

Nano magnetite assisted flocculation for efficient harvesting of lutein and lipid producing microalgae biomass

For commercial scale algal biorefining, harvesting cost is a major bottleneck. Thus, a cost-effective, less-energy intensive, and efficient harvesting method is being investigated. Among several harvesting methods, magnetic flocculation offers the benefits of modest operation, energy savings and quick separation. This study aims to develop novel magnetite-(Fe3O4) nanoparticles (MNPs) of 20 nm average size and their high reusability potential to reduce the harvesting cost of microalgae biomass. The MNPs were synthesized and characterized using FTIR, Zeta analyzer, and SEM before performing on Chlorella sorokiniana Kh12 and Tu5. For maximum harvesting efficiency >99%, the optimal culture pH, MNPs concentration, and agitation speed were 3, 200 mg/L, and 450 rpm, respectively. Subsequently, MNPs were recovered via alkaline treatment and reused up to 5 cycles as they retained their reactivity and harvesting efficiency. The studied MNPs-based harvesting method could be adopted at a commercial scale for cost-effective algae biorefinery in the future.

A microfluidic droplet array demonstrating high-throughput screening in individual lipid-producing microalgae

Microalgae are a group of photoautotrophic microorganisms which could use carbon dioxide for autosynthesis. They have been envisioned as one of the most prospective feedstock for renewable oil. However, great endeavors will still be needed to increase their economic feasibility. The screening of competitive species and suitable culture conditions are such issues. To greatly accelerate these rather laborious steps and also improve their experimental lump-sum-manner, we developed a microfluidic droplet-based 2 × 103 resolution “identification card”, which allowed high throughput real-time monitoring of individual algae among population. A novel fluid-blocking-based droplet generating and trapping performance were integrated in the platform which made it excellent in operational simplicity, rapidity and stability and full of the potentials in single-cell-isolation/screening. The developed platform was successfully used to screen three unicellular algae, namely, Isochrysis zhanjiangensis, Platymonas subcordiformis and Platymonas helgolandica var. tsingtaoensis. In situ bioassays of the lipid accumulation and cell proliferation at single cell level for interspecies comparison were possible. Furthermore, lipid-producing inhomogeneity was demonstrated among cells in the same specie and batch. Nitrogen stress condition can be identified that induce positive-skewed frequency distribution of lipid content, even among individual inhomogeneous cells over the typically used culture condition.

Screening of the heterotrophic microalgae strain for the reclamation of acid producing wastewater

For the sustainable development of the environment, to reduce the high cost and low productivity of microalgae biofuel, nine microalgae strains were screened to study the growh and nutrient removal properties under heterotrophic culture by using the waste carbon source of volatile fatty acids (VFAs). Chlorella sorokiniana (C.sorokiniana) was selected as the best strain with the highest biomass concentration of 0.77 g L−1, specific growth rate of 0.25 d−1, biomass productivity of 91.43 mg L−1 d−1, total nitrogen removal efficiency of 95.96% and total phosphorus removal efficiency of 93.42%. To study the utilization potential of acid-producing wastewater by heterotrophic microalgae, actual acid-producing wastewater was recycled three times for the utilization of C.sorokiniana. After the three utilization cultivation, the removal rates of COD, total nitrogen, ammonia nitrogen, and total phosphorus were 74.44%, 88.05%, 79.08%, and 82.69%, respectively. The total utilization rates of acetic acid, propionic acid, and butyric acid were 58.99%, 70.54%, and 81.52%, respectively. In addition, the highest lipid content of 39.15% and protein content of 42.43% achieved at the third cultivation. After the first cultivation, the composition and diversity of the microbial community structure changed dramatically, with Protebacteria, Bacteroidota, Hydrogenophaga, and Algoriphagus becoming enriched. These results showed a promising way of coupling wastewater treatment with biomass production for long-term sustainability of microalgae lipid production.

Technoeconomic Evaluation of Microalgae Oil Production: Effect of Cell Disruption Method

Microalgae have a high capacity to capture CO2. Additionally, biomass contains lipids that can be used to produce biofuels, biolubricants, and other compounds of commercial interest. This study analyzed various scenarios for microalgae lipid production by simulation. These scenarios include cultivation in raceway ponds, primary harvest with three flocculants, secondary harvest with pressure filter (and drying if necessary), and three different technologies for the cell disruption step, which facilitates lipid extraction. The impact on energy consumption and production cost was analyzed. Both energy consumption and operating cost are higher in the scenarios that consider bead milling (8.79–8.88 kWh/kg and USD 41.06–41.41/kg), followed by those that consider high-pressure homogenization (HPH, 5.39–5.46 kWh/kg and USD 34.26–34.71/kg). For the scenarios that consider pressing, the energy consumption is 5.80–5.88 kWh/kg and the operating cost is USD 27.27–27.88/kg. The consumption of CO2 in scenarios that consider pressing have a greater capture (11.23 kg of CO2/kg of lipids). Meanwhile, scenarios that consider HPH are the lowest consumers of fresh water (5.3 m3 of water/kg of lipids). This study allowed us to develop a base of multiple comparative scenarios, evaluate different aspects involved in Chlorella vulgaris lipid production, and determine the impact of various technologies in the cell disruption stage.

The impact of auxin analogues on microalgal intracellular component accumulation and nutrient removal for mariculture wastewater treatment basing on bacterial-algal coupling technology

Acidogenic fermentation coupled with microalgae cultivation is a promising approach for mariculture wastewater (MW) treatment. However, the low biomass and lipid production of microalgae restrict its practical application. In this study, auxin analogues including α-naphthylacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and indomethacin were applied to the cultivation of Chlorella pyrenoidosa under mixotrophic condition. The effects of auxin analogues on microalgal growth, intracellular component accumulation and nutrient removal were evaluated. The optimal growth and intracellular composition accumulation of Chlorella pyrenoidosa were obtained at 2 mg L−1 NAA, 1 mg L−1 indomethacin, and 1 mg L−1 2,4-D. NAA was more efficient than indomethacin and 2,4-D for lipid production, and the maximum lipid accumulation reached 52% with 2 mg L−1 NAA. Auxin analogues enhanced the uptake of volatile fatty acids, ammonium and phosphate from acidogenic fermentation effluent, attributed to the improvement of microalgal growth. This study provided a new insight for enhancing mariculture wastewater treatment with mixotrophic microalgae.

Measurement of dielectric properties of cells at single-cell resolution using electrorotation.

Dielectric properties of a cell are biophysical properties of high interest for various applications. However, measuring these properties accurately is not easy, which can be exemplified by the large variations in reported dielectric properties of the same cell types. This paper presents a method for measuring the dielectric properties of cells at high frequency, especially lipid-producing microalgae, at single-cell resolution, by integrating an electrorotation-based dielectric property measurement method with a negative dielectrophoretic (nDEP) force-based single-cell trapping method into a single device. In this method, a four-electrode nDEP structure was used to trap a single cell in an elevated position in the center of another four-electrode structure that can apply electrorotational force. By measuring the speed of cell rotation under different applied electrorotation frequencies and fitting the results into a theoretical core-shell cell model, the dielectric properties of cells, including membrane capacitance and cytoplasm conductivity, could be obtained. This system was applied to measure the dielectric properties of lipid-accumulating microalga Chlamydomonas reinhardtii strain Sta6 by applying an electrorotation signal of up to 100MHz. By utilizing a broad frequency range and expanding the measurement spectra to a high frequency region, increased accuracy in fitting the dielectric parameters to a theoretical model was possible, especially the cytoplasm conductivity. The developed method can be used in various applications, such as screening microalgae based on their lipid production capabilities, separating cells of different dielectric properties, identifying different cell types, as well as conducting basic biophysical analyses of cellular properties.

Separation of Heterotrophic Microalgae Crypthecodinium cohnii by Dielectrophoresis.

Microalgae constitute an abundant source of poly-unsaturated fatty acids which are applied in various biotechnological fields such as pharmaceuticals and food supplement. Separating microalgae cells with respect to their lipid content would establish a relevant at-line analytical technique. The present study demonstrates an electrical approach for the separation of the lipid-producing microalgae Crypthecodinium cohnii using the effect of dielectrophoresis (DEP) in a microfluidic flow cell. Microalgae were cultivated for 8 days, while cell growth was characterized by optical density, dry cell weight, glucose concentration and lipid content via fluorescence microscopy. The size distribution of cells during cultivation was thoroughly investigated, since the DEP force scales with cell volume, but also depends on lipid content via cell electrophysiological constants. Thus, the challenge was to deconvolute one separation effect from the other, while the electrical cell constants of C. cohnii are not known yet. The DEP-dependent separation was realized by slanted top-bottom electrodes with the flowing cell suspension between them. Turning on the voltage deflected the cells from their initial path as determined by the streaming and thus changed their direction of flow. The separation efficiency of DEP was tested for various electrical field strengths and its performance was determined by quantitative analysis of optical and fluorescence videos. It could be shown for all size groups that the most lipid-containing cells were always subject to DEP separation and that the method is thus not only suitable for process analysis, but also for strain selection of the most productive cell lines.