High‐throughput fluorescence‐activated cell sorting for lipid hyperaccumulating Chlamydomonas reinhardtii mutants

The genetically tractable microalga Chlamydomonas reinhardtii has many advantages as a model for renewable bioproducts and/or biofuels production. However, one limitation of C. reinhardtii is its relatively low‐lipid content compared with some other algal species. To overcome this limitation, we combined ethane methyl sulfonate mutagenesis with fluorescence‐activated cell sorting (FACS) of cells stained with the lipophilic stain Nile Red to isolate lipid hyperaccumulating mutants of C. reinhardtii. By manipulating the FACS gates, we sorted mutagenized cells with extremely high Nile Red fluorescence signals that were rarely detected in nonmutagenized populations. This strategy successfully isolated several putative lipid hyperaccumulating mutants exhibiting 23% to 58% (dry weight basis) higher fatty acid contents than their progenitor strains. Significantly, for most mutants, nitrogen starvation was not required to attain high‐lipid content nor was there a requirement for a deficiency in starch accumulation. Microscopy of Nile Red stained cells revealed that some mutants exhibit an increase in the number of lipid bodies, which correlated with TLC analysis of triacyglycerol content. Increased lipid content could also arise through increased biomass production. Collectively, our findings highlight the ability to enhance intracellular lipid accumulation in algae using random mutagenesis in conjunction with a robust FACS and lipid yield verification regime. Our lipid hyperaccumulating mutants could serve as a genetic resource for stacking additional desirable traits to further increase lipid production and for identifying genes contributing to lipid hyperaccumulation, without lengthy lipid‐induction periods.     

Hyperspectral imaging techniques for the characterization of Haematococcus pluvialis (Chlorophyceae)

A hyperspectral imaging camera was combined with a bright‐field microscope to investigate the intracellular distribution of pigments in cells of the green microalga Haematococcus pluvialis, a synonym for H. lacustris (Chlorophyceae). We applied multivariate curve resolution to the hyperspectral image data to estimate the pigment contents in culture and revealed that the predicted values were consistent with actual measurements obtained from extracted pigments. Because it was possible to estimate pigment contents in every pixel, the intracellular distribution of the pigments was investigated during various life‐cycle stages. Astaxanthin was localized specifically at the eyespot of zoospores in early culture stages. Then, it became widely distributed in cells, but subsequently localized differently than the chl. Integrated with our recently developed image‐processing program “HaematoCalMorph,” the hyperspectral imaging system was useful for monitoring intracellular distributions of pigments during culture as well as for studying cellular responses under various conditions.     

Evaluation of closed photobioreactor types and operation variables for enhancing lipid productivity of <Emphasis Type="Italic">Nannochloropsis sp.</Emphasis> KMMCC 290 for biodiesel production

Previously, we have successfully produced biodiesel using the marine microalga Nannochloropsis sp. KMMCC 290 cultivated in a raceway open pond. Here, we investigated the effects of closed photobioreactors and operating variables on cell concentration and lipid content of the microalga to increase its lipid productivity. The flatplate photobioreactor (FPP) showed higher performance than bubble column and air-lift photobioreactors. Among the variables evaluated, light intensity, aeration rate, and carbon dioxide feeding significantly influenced cell concentration, whereas a simultaneous increase in light intensity and aeration rate, as well as carbon dioxide feeding noticeably increased the lipid content. The lipid productivity in the FPP was 26.7 × 10^-3 g/L/day, which was 16.6 times higher than that produced by the microalga cultivated in the raceway pond, 4.8 times higher than that from the simple flask-grown control culture, and 2.1 times higher than that from the FPP under initial conditions.     

In-situ root extent measurements by electrical capacitance methods

A conceptual model is presented that provides a rational basis for using plant root capacitance as an in-situ measurement for assessing plant root development. This method is based on measuring the electricla capacitance of an equivalent parallel resistance-capacitance circuit formed by the interface between soil-water and the plant root surface. Nutrient solution studies using tomato (Lycopersicon esculentum Mill.) showed a good correlation between plant root capacitance and root mass. Stage of development studies showed plant root capacitance measurements capable of detecting root development rate and suggested the method to be sensitive to root function. Soil water content was shown to have a significant effect on plant root capacitance measurement. The possibility of using this technique to assess relative root function is discussed. Positioning of the plant shoot electrode was shown to also have a significant effect on measurement of plant root capacitance, demonstrating the need for using consistent measurement techniques. The electrical capacitance method shows considerable promise. More research is needed before it can be used routinely.     

Multi-parameter flow cytometry as a tool to monitor heterotrophic microalgal batch fermentations for oil production towards biodiesel

Multi-parameter flow cytometry was used to monitor cell intrinsic light scatter, viability, and lipid content of Chlorella protothecoides cells grown in shake flasks. Changes in the right angle light scatter (RALS) and forward angle light scatter (FALS) were detected during the microalgal growth, which were attributed to the different microalgal cell cycle stages. The proportion of cells not stained with PI (cells with intact cytoplasmic membrane) was high (> 90%) during the microalgal growth, even in the latter stationary phase, suggesting that the microalgal cells built-up storage materials which allowed them to survive under nutrient starvation, maintaining their cytoplasmic membranes intact. A high correlation between the Nile Red fluorescence intensity measured by flow cytometry and total lipid content assayed by the traditional lipid extraction method was found for this microalga, making this method a suitable and quick technique for the screening of microalgal strains for lipid production, optimization of biofuel production bioprocesses, and scale-up studies. The highest oil content (∼28% w/w dry cell weight, estimated by flow cytometry) was observed in the latter stationary phase. In addition, C. protothecoides oil also depicted the adequate fatty acid methyl ester composition for biodiesel purposes at this growth phase, suggesting that the microalgal oil produced during the latter stationary phase could be an adequate substitute for diesel fuel. Medium growth optimization for enhancement of microalgal oil production is now in progress, using the multi-parameter approach.     

The mixed culture of microalgae Chlorella pyrenoidosa and yeast Yarrowia lipolytica for microbial biomass production

Microbial biomass which mostly generated from the microbial processes of bacteria, yeasts, and microalgae is an important resource. Recent concerns in microbial biomass production field, especially microbial lipid production for biofuel, have been focused towards the mixed culture of microalgae and yeast. To more comprehensive understanding of the mixed culture for microbial biomass, mono Chlorella pyrenoidosa, mono Yarrowia lipolytica and the mixed culture were investigated in the present work. Results showed that the mixed culture achieved significantly faster cell propagation of microalga and yeast, smaller individual cell size of yeast and higher relative chlorophyll content of microalga. The mixed culture facilitated the assimilation of carbon and nitrogen and drove the carbon flow to carbohydrate. Besides higher lipid yield (0.77 g/L), higher yields of carbohydrates (1.82 g/L), protein (1.99 g/L) and heating value (114.64 kJ/L) indicated the microbial biomass harvested from the mixed culture have more potential utilization in renewable energy, feedstuff, and chemical industry.

     

Effects of carbon and nitrogen sources on fatty acid contents and composition in the green microalga, Chlorella sp. 227

In order to investigate and generalize the effects of carbon and nitrogen sources on the growth of and lipid production in Chlorella sp. 227, several nutritional combinations consisting of different carbon and nitrogen sources and concentrations were given to the media for cultivation of Chlorella sp. 227, respectively. The growth rate and lipid content were affected largely by concentration rather than by sources. The maximum specific growth was negatively affected by low concentrations of carbon and nitrogen. There is a maximum allowable inorganic carbon concentration (less than 500~1,000 mM bicarbonate) in autotrophic culture, but the maximum lipid content per gram dry cell weight (g DCW) was little affected by the concentration of inorganic carbon within the concentration. The lipid content per g DCW was increased when the microalga was cultured with the addition of glucose and bicarbonate (mixotrophic) at a fixed nitrogen concentration and with the lowest nitrogen concentration (0.2 mM), relatively. Considering that lipid contents per g DCW increased in those conditions, it suggests that a high ratio of carbon to nitrogen in culture media promotes lipid accumulation in the cells. Interestingly, a significant increase of the oleic acid amount to total fatty acids was observed in those conditions. These results showed the possibility to induce lipid production of high quality and content per g DCW by modifying the cultivation conditions.     

Mixed culture of oleaginous yeast Rhodotorula glutinis and microalga Chlorella vulgaris for lipid production from industrial wastes and its use as biodiesel feedstock

A mixed culture of oleaginous yeast Rhodotorula glutinis and microalga Chlorella vulgaris was performed to enhance lipid production from industrial wastes. These included effluent from seafood processing plant and molasses from sugar cane plant. In the mixed culture, the yeast grew faster and the lipid production was higher than that in the pure cultures. This could be because microalga acted as an oxygen generator for yeast, while yeast provided CO(2) to microalga and both carried out the production of lipids. The optimal conditions for lipid production by the mixed culture were as follows: ratio of yeast to microalga at 1:1; initial pH at 5.0; molasses concentration at 1%; shaking speed at 200 rpm; and light intensity at 5.0 klux under 16:8 hours light and dark cycles. Under these conditions, the highest biomass of 4.63±0.15 g/L and lipid production of 2.88±0.16 g/L were obtained after five days of cultivation. In addition, the plant oil-like fatty acid composition of yeast and microalgal lipids suggested their high potential for use as biodiesel feedstock.     

High quality biodiesel production from a microalga Chlorella protothecoides by heterotrophic growth in fermenters

The aim of the study was to obtain high quality biodiesel production from a microalga Chlorella protothecoids through the technology of transesterification. The technique of metabolic controlling through heterotrophic growth of C. protothecoides was applied, and the heterotrophic C. protothecoides contained the crude lipid content of 55.2%. To increase the biomass and reduce the cost of alga, corn powder hydrolysate instead of glucose was used as organic carbon source in heterotrophic culture medium in fermenters. The result showed that cell density significantly increased under the heterotrophic condition, and the highest cell concentration reached 15.5 g L(-1). Large amount of microalgal oil was efficiently extracted from the heterotrophic cells by using n-hexane, and then transmuted into biodiesel by acidic transesterification. The biodiesel was characterized by a high heating value of 41 MJ kg(-1), a density of 0.864 kg L(-1), and a viscosity of 5.2 x 10(-4) Pa s (at 40 degrees C). The method has great potential in the industrial production of liquid fuel from microalga.     

Active extracellular substances of Bacillus thuringiensis ITRI‐G1 induce microalgae self‐disruption for microalgal biofuel

Microalgal cultures are a clean and sustainable means to use solar energy for CO₂ fixation and fuel production. Microalgae grow efficiently and are rich in oil, but recovering that oil is typically expensive and consumes much energy. Therefore, effective and low‐cost techniques for microalgal disruption and oil or lipid extraction are required by the algal biofuel industry. This study introduces a novel technique that uses active extracellular substances to induce microalgal cell disruption. A bacterium indigenous to Taiwan, Bacillus thuringiensis, was used to produce the active extracellular substances, which were volatile compounds with high thermal stability. Approximately 74% of fresh microalgal cells were disrupted after a 12‐h treatment with the active extracellular substances. Algal lipid extraction efficiency was improved and the oil extraction time was decreased by approximately 37.5% compared with the control treatment. The substances effectively disrupted fresh microalgal cells but not dehydrated microalgal cells. An analysis of microalgal DNA from fresh cells after disruption treatment demonstrated typical DNA laddering, indicating that disruption may have resulted from programmed cell death. This study revealed that biological treatments are environmentally friendly methods for increasing microalgal lipid extraction efficiency, and introduced a microalgal cell self‐disruption mechanism.     

An optical fibre photobioreactor for enhanced production of the marine unicellular alga Isochrysis aff. galbana T-Iso (UTEX LB 2307) rich in docosahexaenoic acid

We have screened six species of marine microalga for their ability to produce the important dietary lipid docosahexaenoic acid (DHA). Isochrysis aff. galbana T-Iso (UTEX LB 2307), which produced DHA in the highest quantities (5.4 mg-g^–1), was grown in a new type of closed photobioreactor in which efficient light distribution was achieved using light-diffusing optical fibres. The optimal temperature and light intensity for DHA production were determined and a maximal DHA production of 4.3 mg-l was achieved, twofold greater than that obtained using conventional culture methods. In addition, the DHA content could be enhanced by low temperature or dark incubation of the culture after growth. Correspondence to: T. Matsunaga     

IDENTIFICATION AND PHYSIOLOGICAL ASPECTS OF A NOVEL CAROTENOID‐ENRICHED,METAL‐RESISTANT MICROALGA ISOLATED FROM AN ACIDIC RIVER IN HUELVA (SPAIN)1

A heavy‐metal‐resistant, carotenoid‐enriched novel unicellular microalga was isolated from an acidic river in Huelva, Spain. The isolated ribosomal 18S subunit rDNA sequence showed homology with known sequences from green microalgae, the closest sequence (98% homology) belonging to the genus Coccomyxa. The isolated microalga therefore was an up to now uncultured microalga. The microalga was isolated from Tinto River area (Huelva, Spain), an acidic river that exhibits very low pH (1.7–3.1) with high concentrations of sulfuric acid and heavy metals, including Fe, Cu, Mn, Ni, and Al. Electron micrographs show that the microalga contains a large chloroplast with a presence of lipid droplets, an increased number of starch bodies as well as electron‐dense deposits and plastoglobules, the last observed only in iron‐exposed cells. Unlike other acidophile microalgae, the isolated microalga showed high growth rates when cultivated photoautotrophycally (up to 0.6 d^?1) in a suitable culture medium prepared at our laboratory. The growth was shown to be iron dependent. When the microalga is grown in fluidized bed reactors, the high growth rates resulted in unexpectedly high productivities for being a microalga that naturally grows in acidic environments (0.32 g·L^?1·d^?1). The microalga also grows optimally on reduced carbon sources, including glucose and urea, and at an optimal temperature of 35°C. The alga pigment profile is particularly rich in carotenoids, especially lutein, suggesting that the microalga might have potential for antioxidant production, namely, xanthophylls.     

Observation of non-linear biomass-capacitance correlations: reasons and implications for bioprocess control

Electrical capacitance has been discussed as a real time measure for living biomass concentration in technical bioreactors such as brewery (fermentation) tanks. Commonly, a linear correlation between biomass concentration and capacitance is assumed. While following the growth and subsequent lipid formation of the yeast Arxula adeninivorans we observed non-linearity between biomass concentration and capacitance. Capacitance deviation from linearity coincided with incipient lipid formation and depended on the intracellular lipid content. As the extent of deviation between capacitance and biomass concentration was proportional to the lipid concentration, it was considered as a quantitative measure of intracellular product formation. The correlation between shifts in dielectric relaxation (summarized as characteristic frequency of the Cole-Cole equation) and lipid content could not be explained by interfacial polarization on the lipid droplets alone. However, the parameters of the Cole-Cole equation were found to be a clear indicator for different phases of growth and lipid production. Integrating all results in a redundancy analysis (RDA), we were able to accurately describe the formation of cellular lipid inclusions. Our measurements are thus potentially valuable as components of future bioprocess control strategies targeting intracellular products such as proteins or biopolyesters.     

<Emphasis Type="Italic">Dunaliella salina</Emphasis> (Chlorophyta) as a Test-Object for Assessment of Detergent Pollution of a Marine Environment

The dynamics of the number of the microalga Dunaliella salina, depending on the age of the matrix culture, the number of cells, and the time of toxicant administration in the culture medium and on oxygen production as a parameter of the functional condition of the dunaliella, was studied in solutions of sodium dodecylsulfate (SDS) containing 0.1, 1, and 10 mg/l of the detergent. The parameters at which the application of the considered test-object allowed determination of the most exact information on the environment quality and toxicity of substances were determined. It was shown that the SDS in concentrations of 0.1 and 1 mg/l did not affect significantly the growth of the microalga, and an inhibiting effect was recorded at a toxicant content of 10 mg/l.Original Russian Text Copyright ¢ 2005 by Biologiya Morya, Markina, Aizdaicher.     

Determination of antibody content in live versus dead hybridoma cells: analysis of antibody production in osmotically stressed cultures

Hybridomas are known to exhibit increased specific antibody production rated when subjected to environmental stress. Under these conditions, viability is low so that population-average measurements do not properly reflect the state of viable cells. Even for flow cytometry, which gives a population distribution, special techniques must be used to discriminate between viable and nonviable cells. We describe the use of the vital stain ethidium monoazide (EMA) for independent measurement of intracellular antibody content in live and dead cells via flow cytometry. EMA is shown to be superior to light scattering techniques in identifying dead cells. We apply this technique to show that, in control batch culture, the specific antibody prodution rate and antibody content in live cells are constant during exponential growth, but decrease as cells enter the stationary phase. Antibody is retained in dead cells, but at a lower level than in live cells. We further show that, under hyperosmotic stress, the specific antibody production rate and antibody content in live both remain high during death phase. (c) 1992 John Wiley & Sons, Inc.     

UVA‐induced reset of hydroxyl radical ultradian rhythm improves temporal lipid production in Chlorella vulgaris

We report for the first time that the endogenous, pseudo‐steady‐state, specific intracellular levels of the hydroxyl radical (si‐OH) oscillate in an ultradian fashion (model system: the microalga, Chlorella vulgaris), and also characterize the various rhythm parameters. The ultradian rhythm in the endogenous levels of the si‐OH occurred with an approximately 6 h period in the daily cycle of light and darkness. Further, we expected that the rhythm reset to a shorter period could rapidly switch the cellular redox states that could favor lipid accumulation. We reset the endogenous rhythm through entrainment with UVA radiation, and generated two new ultradian rhythms with periods of approximately 2.97 h and 3.8 h in the light phase and dark phase, respectively. The reset increased the window of maximum lipid accumulation from 6 h to 12 h concomitant with the onset of the ultradian rhythms. Further, the saturated fatty acid content increased approximately to 80% of total lipid content, corresponding to the peak maxima of the hydroxyl radical levels in the reset rhythm. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:673–680, 2014     

A novel approach for medium formulation for growth of a microalga using motile intensity

Motile intensity of the cells, defined as the specific mean kinetic energy, was measured by image analysis and used to formulate a suitable medium for the cultivation of a motile microalga. Nitrogen source at 60 mg/L was used as the target component. The cells grown in a medium containing urea showed the highest motile intensities during cultivation when compared to the cells grown with NH(4)Cl or (NH(4))(2)SO(4) as the nitrogen source. The urea culture gave the highest biomass yield and lipid content of 1.06+/-0.12 g/L and 22.34+/-2.56%, respectively after 150 h of cultivation. These results indicated a strong dependence of motile intensity on the nitrogen source used in the growth medium. This concept, which requires only a small droplet of the sample, can find potential application in the design and optimization of culture media.     

Effect of a salt-osmotic upshock on the edaphic microalga Neochloris oleoabundans

Abstract. The edaphic microalga Neochloris oleoabundans (isolated from desert soil) was subjected to a salt-osmotic shock from 0 to 0·6 kmol m⁻³ NaCl. The effect of the osmotic upshock on the cell composition was determined. The cell dry weight and the lipid, glycerol and soluble amino acids contents remained unchanged during 5d of osmotic upshock. The protein content increased after 2d of initial osmotic shock, and it appears to be a long-term haloadaptation process of the cells. The most important short-term effects of salt osmotic upshock were a decrease in polysaccharide content and an increase in the soluble carbohydrate content of Neochloris oleoabundans cells. Within the first 4h after the initial shock, there was a transfer of carbon units from polysaccharides to sucrose which was independent of photosynthesis. The increase of intracellular concentration of sucrose contributed to cell osmoregulation.     

Glucose‐6‐Phosphate Dehydrogenase from the Oleaginous Microalga Nannochloropsis Uncovers Its Potential Role in Promoting Lipogenesis

Microalgae have long been considered as potential biological feedstock for the production of wide array of bioproducts, such as biofuel feedstock because of their lipid accumulating capability. However, lipid productivity of microalgae is still far below commercial viability. Here, a glucose‐6‐phosphate dehydrogenase from the oleaginous microalga Nannochloropsis oceanica is identified and heterologously expressed in the green microalga Chlorella pyrenoidosa to characterize its function in the pentose phosphate pathway. It is found that the G6PD enzyme activity toward NADPH production is increased by 2.19‐fold in engineered microalgal strains. Lipidomic analysis reveals up to 3.09‐fold increase of neutral lipid content in the engineered strains, and lipid yield is gradually increased throughout the cultivation phase and saturated at the stationary phase. Moreover, cellular physiological characteristics including photosynthesis and growth rate are not impaired. Collectively, these results reveal the pivotal role of glucose‐6‐phosphate dehydrogenase from N. oceanica in NADPH supply, demonstrating that provision of reducing power is crucial for microalgal lipogenesis and can be a potential target for metabolic engineering.     

Responses of the Antarctic microalga Koliella antarctica (Trebouxiophyceae,Chlorophyta) to cadmium contamination

Ultrastructural and physiological effects of exposure to 1 ppm and 5 ppm of cadmium (Cd) on cultured cells of Koliella antarctica, a green microalga from Antarctica, were investigated. The amount of Cd in the alga rose with the increase of the metal concentration in the growth medium and most Cd remained outside the cells, bound to the components of the cell walls. The increase of Cd in the microalga was concomitant with the decrease of other elements, mainly calcium (Ca). Exposure to 1 ppm Cd slowed culture growth by inhibiting cell division and also caused the development of some misshapen cells with chloroplast showing disordered thylakoids. However, this concentration did not substantially affect the chlorophyll (Chl) content or photosystem (PS) activity. At 5 ppm, Cd cell growth suddenly stopped and some cells lysed. After a week of Cd contamination, the cells were enlarged and severely damaged. The chloroplasts showed great ultrastructural alterations and a reduced Chl content. Cd exposure negatively affected PSII, whose activity was almost completely lost after four days.