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Microalgae have been shown as a potential bioresource for food, biofuel, and pharmaceutical products. During the growth phases with corresponding environmental conditions, microalgae accumulate different amounts of various metabolites. We quantified the neutral lipids accumulation and analyzed the swimming signatures (speed and trajectories) of the motile green alga, Dunaliella primolecta, during the lag–exponential–stationary growth cycle at different nutrient concentrations. We discovered significant changes in the neutral lipid content and swimming signatures of microalgae across growth phases. The timing of the maximum swimming speed coincided with the maximum neutral lipid content and both maxima occurred under nutrient stress at the stationary growth phase. Furthermore, the swimming trajectories suggested statistically significant changes in swimming modes at the stationary growth phase when the maximum intracellular neutral lipid content was observed. Our results provide the potential exploitation of microalgal swimming signatures as possible indicators of the cultivation conditions and the timing of microalgal harvest to maximize the lipid yield for biofuel production. The findings can also be implemented to explore the production of food and antibiotics from other microalgal metabolites with low energy costs.     

Influence of nitrogen stress on Isochrysis galbana strain U4, a candidate for biodiesel production

Lipid accumulation has been investigated in numerous microalgal species to assess their potential with respect to biodiesel production. The present work determines the effect of nitrogen stress on physiological and ultrastructural changes in Isochrysis galbana U4. This study is unique in showing the correlations between growth, lipid production, pigmentation and ultrastructural changes in Isochrysis cells undergoing nitrogen starvation. The continuation of algal growth after the complete depletion of external nitrogen was shown to be supported by internal nitrogen stores, possibly in the pyrenoid. Cell growth ceased and lipid accumulation was initiated after the internal store of nitrogen had become exhausted. The depletion of intracellular nitrogen reservoirs to critical thresholds initiated the onset of the stationary phase, a decline in chlorophyll content and the initiation of lipid and carotenoid accumulation. The most notable ultrastructural changes, upon nitrogen stress, were the accumulation of plastidial and cytoplasmic lipid bodies and the dismantling of the chloroplast. The size of the pyrenoid when external nitrogen became depleted was found to decrease significantly, up to four‐fold. This was attributed to the remobilization of nitrogen from Rubisco. The level of expression of heterochromatin was found to increase when cells were nitrogen starved. This is thought to favor long‐term dormancy in this species because aging cells have been noted to recover rapidly when returned to conditions favorable for growth. The observations of this study are consistent with the hypothesis that the responses of Isochrysis cells to nitrogen starvation are regulated by the internal reserves of nitrogen, and the depletion of these reserves is an important trigger for lipid accumulation in this species. The findings of this study also indicate that Isochrysis galbana U4 is a promising candidate for biodiesel lipid production.     

Elevated acetyl‐CoA by amino acid recycling fuels microalgal neutral lipid accumulation in exponential growth phase for biofuel production

Microalgal neutral lipids [mainly in the form of triacylglycerols (TAGs)], feasible substrates for biofuel, are typically accumulated during the stationary growth phase. To make microalgal biofuels economically competitive with fossil fuels, generating strains that trigger TAG accumulation from the exponential growth phase is a promising biological approach. The regulatory mechanisms to trigger TAG accumulation from the exponential growth phase (TAEP) are important to be uncovered for advancing economic feasibility. Through the inhibition of pyruvate dehydrogenase kinase by sodium dichloroacetate, acetyl‐CoA level increased, resulting in TAEP in microalga Dunaliella tertiolecta. We further reported refilling of acetyl‐CoA pool through branched‐chain amino acid catabolism contributed to an overall sixfold TAEP with marginal compromise (4%) on growth in a TAG‐rich D. tertiolecta mutant from targeted screening. Herein, a three‐step α loop‐integrated metabolic model is introduced to shed lights on the neutral lipid regulatory mechanism. This article provides novel approaches to compress lipid production phase and heightens lipid productivity and photosynthetic carbon capture via enhancing acetyl‐CoA level, which would optimize renewable microalgal biofuel to fulfil the demanding fuel market.     

Scanning electron microscopic analysis of intramyocellular lipid droplets in an animal model of type 2 diabetes

Objective: To evaluate the accumulation pattern of intramyocellular lipids (IMCLs) in striated muscle during the development and progression of diabetes, using a novel scanning electron microscopic method. Methods and Procedures: Hyperglycemia was induced by feeding diabetes‐prone (DP) Psammomys obesus a high‐energy (HE) diet. Lipid accumulation within gastrocnemius muscle fibers was assessed in formalin‐fixed muscle samples during the development of hyperglycemia using high resolution imaging in a scanning electron microscope. We evaluated the temporal relationship between changes in IMCL quantity and morphology and the altered glucose metabolism and assessed the effect of reversal of hyperglycemia on IMCL level and morphology. Diabetes‐resistant (DR) P. obesus served as controls. Results: Lipid accumulation in the muscle fibers of DP animals was increased with the development of hyperglycemia. This was characterized by increased lipid density as well as by an abundance of large lipid droplets. Reversal of the phenotype resulted in the disappearance of large lipid droplets. The IMCL level and the distribution of lipid droplet size were similar in muscles of both the normoglycemic DR and DP animals, with an abundance of small lipid droplets. This profile was changed following a HE diet only in the DP animals. Discussion: Lipid accumulation in the muscle of P. obesus during the development of hyperglycemia is characterized by increased quantity and accumulation of large lipid droplets. These changes were reversible upon normalization of blood glucose. The evaluated methodology is a useful tool for the study of the dynamics of lipid accumulation in different metabolic conditions.     

Enrichment as a screening method for a high-growth-rate microalgal strain under continuous cultivation system

Microalgae are a promising feedstock for renewable biodiesel production. High productivity of biodiesel production from microalgae is directly related to growth rate as well as lipid content of cells. In the present study, an enrichment process in a continuous cultivation system was developed to screen a high-growth-rate microalga from a mixed culture of microalgal species; Chlorella vulgaris, Chlorella protothecoides, and Chlamydomonas reinhardtii were used as test organisms for our experiments. The time-dependent washout of mixed microalgal pool was executed to successfully enrich the C. reinhardtii, which exhibits the higher growth rate than C. vulgaris and C. protothecoides under turbidostat conditions within 75 h. The domination of C. reinhardtii in the mixed culture was validated by on-line monitoring of growth rate and flowcytometric analysis. For the time-efficient production of microalgal biomass, this screening process has a high potential to segregate the fast-growing microalgal strains from the pool of various uncharacterized microalgal species and random mutants.     

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.     

Growth,lipid content,productivity, and fatty acid composition of tropical microalgae for scale‐up production

Biomass and lipid productivity, lipid content, and quantitative and qualitative lipid composition are critical parameters in selecting microalgal species for commercial scale‐up production. This study compares lipid content and composition, and lipid and biomass productivity during logarithmic, late logarithmic, and stationary phase of Nannochloropsis sp., Isochrysis sp., Tetraselmis sp., and Rhodomonas sp. grown in L1‐, f/2‐, and K‐medium. Of the tested species, Tetraselmis sp. exhibited a lipid productivity of 3.9–4.8 g m^?2 day^?1 in any media type, with comparable lipid productivity by Nannochloropsis sp. and Isochrysis sp. when grown in L1‐medium. The dry biomass productivity of Tetraselmis sp. (33.1–45.0 g m^?2 day^?1) exceeded that of the other species by a factor 2–10. Of the organisms studied, Tetraselmis sp. had the best dry biomass and/or lipid production profile in large‐scale cultures. The present study provides a practical benchmark, which allows comparison of microalgal production systems with different footprints, as well as terrestrial systems. Biotechnol. Bioeng. 2010;107: 245–257. © 2010 Wiley Periodicals, Inc.     

Screening of biocompatible organic solvents for enhancement of lipid milking from Nannochloropsis sp.

To extract the microalgal lipid in situ, biocompatible solvents were screened for lipid milking of Nannochloropsis sp. in an aqueous–organic system. The effects of organic solvents on the microalgal growth, the lipid extractability, the dehydrogenases activity and the cell membrane integrity were investigated by UV–visible spectrophotometer, FT-IR spectroscopy, 2,3,5-triphenyltetrazolium chloride (TTC) and Evans Blue stain method, respectively. The results showed that alkane solvents with log P > 5.5 were biocompatible while the hydrophilic solvents with log P < 5.5 were toxic to Nannochloropsis sp. due to the deactivated dehydrogenase and increased cell membrane permeability. As 10% (v/v) hexadecane was used to establish biphasic system, the total lipid production of Nannochloropsis sp. was increased by 28.9% compared to the control. The screened biocompatible solvent hexadecane enhanced not only the algal growth but also the lipid accumulation, showing an effective way to facilitate the process for in situ lipid milking from Nannochloropsis sp.     

A quantitative analysis of microalgal lipids for optimization of biodiesel and omega‐3 production

The lipid characteristics of microalgae are known to differ between species and change with growth conditions. This work provides a methodology for lipid characterization that enables selection of the optimal strain, cultivation conditions, and processing pathway for commercial biodiesel production from microalgae. Two different microalgal species, Nannochloropsis sp. and Chlorella sp., were cultivated under both nitrogen replete and nitrogen depleted conditions. Lipids were extracted and fractionated into three major classes and quantified gravimetrically. The fatty acid profile of each fraction was analyzed using GC–MS. The resulting quantitative lipid data for each of the cultures is discussed in the context of biodiesel and omega‐3 production. This approach illustrates how the growth conditions greatly affect the distribution of fatty acid present in the major lipid classes and therefore the suitability of the lipid extracts for biodiesel and other secondary products. Biotechnol. Bioeng. 2013; 110: 2096–2104. © 2013 Wiley Periodicals, Inc.     

Functional group richness: implications of biodiversity for light use and lipid yield in microalgae

Currently, very few studies address the relationship between diversity and biomass/lipid production in primary producer communities for biofuel production. Basic studies on the growth of microalgal communities, however, provide evidence of a positive relationship between diversity and biomass production. Recent studies have also shown that positive diversity–productivity relationships are related to an increase in the efficiency of light use by diverse microalgal communities. Here, we hypothesize that there is a relationship between diversity, light use, and microalgal lipid production in phytoplankton communities. Microalgae from all major freshwater algal groups were cultivated in treatments that differed in species richness and functional group richness. Polycultures with high functional group richness showed more efficient light use and higher algal lipid content with increasing species richness. There was a clear correlation between light use and lipid production in functionally diverse communities. Hence, a powerful and cost‐effective way to improve biofuel production might be accomplished by incorporating diversity related, resource‐use‐dynamics into algal biomass production.     

Bioprospecting for hyper-lipid producing microalgal strains for sustainable biofuel production

Global petroleum reserves are shrinking at a fast pace, increasing the demand for alternate fuels. Microalgae have the ability to grow rapidly, and synthesize and accumulate large amounts (approximately 20-50% of dry weight) of neutral lipid stored in cytosolic lipid bodies. A successful and economically viable algae based biofuel industry mainly depends on the selection of appropriate algal strains. The main focus of bioprospecting for microalgae is to identify unique high lipid producing microalgae from different habitats. Indigenous species of microalgae with high lipid yields are especially valuable in the biofuel industry. Isolation, purification and identification of natural microalgal assemblages using conventional techniques is generally time consuming. However, the recent use of micromanipulation as a rapid isolating tool allows for a higher screening throughput. The appropriate media and growth conditions are also important for successful microalgal proliferation. Environmental parameters recorded at the sampling site are necessary to optimize in vitro growth. Identification of species generally requires a combination of morphological and genetic characterization. The selected microalgal strains are grown in upscale systems such as raceway ponds or photobireactors for biomass and lipid production. This paper reviews the recent methodologies adopted for site selection, sampling, strain selection and identification, optimization of cultural conditions for superior lipid yield for biofuel production. Energy generation routes of microalgal lipids and biomass are discussed in detail.     

Growth Rate of the Microalga Tetraselmis suecica Changes the Biochemical Composition of Artemia Species

The impact of different microalgal semicontinuous cultures on growth and biochemical composition in the next link of the food chain was tested using the filter feeder Artemia species as a model. The marine microalga Tetraselmis suecica was cultured semicontinuously with renewal rates between 10% and 50% and used to feed Artemia. Microalgal cultures maintained with a low renewal rate that had biochemical composition similar to that of the stationary-phase cultures commonly used in aquaculture produced poor growth and survival and low food-conversion efficiency compared to cultures maintained with a high renewal rate. Changes in the renewal rate in microalgal cultures also resulted in important changes in the gross biochemical composition of the filter feeder. The gross biochemical composition of the Artemia resembled that of the microalgae used as food except for total lipid content. The percentage of protein in the organic fraction of Artemia increased from 45% to 65% of the organic weight with increasing renewal rates in the microalgal cultures, while the carbohydrate percentage decreased under the same conditions. Higher renewal rates resulted in higher lipid percentages in the microalga, but in Artemia the percentage of lipids decreased from 19% of the organic weight with a renewal rate of 10%, to 13% with a renewal rate of 50%. The percentage of all polyunsaturated fatty acids in Artemia, including 20:5n-3, increased slightly with increasing renewal rates in the microalgal cultures. Results emphasize the importance of controlling microalgal nutritional value for the success of aquaculture food chains in which filter feeders are involved. Received October 15, 2000; accepted December 29, 2000.     

Molecular Identification and Comparative Evaluation of Tropical Marine Microalgae for Biodiesel Production

Marine microalgae have emerged as important feedstock for liquid biofuel production. The identification of lipid-rich native microalgal species with high growth rate and optimal fatty acid profile and biodiesel properties is the most challenging step in microalgae-based biodiesel production. In this study, attempts have been made to bio-prospect the biodiesel production potential of marine and brackish water microalgal isolates from the west coast of India. A total of 14 microalgal species were isolated, identified using specific molecular markers and based on the lipid content; seven species with total lipid content above 20% of dry cell weight were selected for assessing biodiesel production potential in terms of lipid and biomass productivities, nile red fluorescence, fatty acid profile and biodiesel properties. On comparative analysis, the diatoms were proven to be promising based on the overall desirable properties for biodiesel production. The most potential strain Navicula phyllepta MACC8 with a total lipid content of 26.54 % of dry weight of biomass, the highest growth rate (0.58 day^?1) and lipid and biomass productivities of 114 and 431 mgL^?1 day^?1, respectively, was rich in fatty acids mainly of C16:0, C16:1 and C18:0 in the neutral lipid fraction, the most favoured fatty acids for ideal biodiesel properties. The biodiesel properties met the requirements of fuel quality standards based on empirical estimation. The marine diatoms hold a great promise as feedstock for large-scale biodiesel production along with valuable by-products in a biorefinery perspective, after augmenting lipid and biomass production through biochemical and genetic engineering approaches.     

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.     

Monitoring cell‐specific neutral lipid accumulation in Phaeodactylum tricornutum (Bacillariophyceae) with Nile Red staining – a new method for FlowCAM

With the fluorescent stain Nile Red (NR), phytoplankton lipid accumulation can be monitored quickly and in situ. In the light of recent results in phytoplankton diversity research, there is also a need for cell‐ and species‐specific lipid measurement techniques. The objective of this work was to investigate whether cell‐specific phytoplankton lipid accumulation could be monitored with the image‐based particle analyzer FlowCAM? and NR staining. Applying Phaeodactylum tricornutum as a model species, we compared the FlowCAM method to two established lipid quantification methods: spectrofluorometric NR fluorescence measurement and total lipid analysis by gas chromatography. The experiment was carried out in batch cultures under nitrogen limitation to induce lipid accumulation. We showed significant correlation between the three different lipid quantification methods confirming the applicability of the novel FlowCAM method in cell‐specific and near real‐time lipid quantification. Furthermore, with the method described here, the lipid content of taxonomically distinguished cells can eventually be measured from multispecies cultures, opening several new possibilities to study species‐specific responses to stress conditions and the complementarity effect.     

Rapid estimation of lipid content in an Antarctic ice alga (Chlamydomonas sp.) using the lipophilic fluorescent dye BODIPY505/515

Chlamydomonas sp. ICE-L, isolated from Antarctic coastal marine environments, was selected as a high lipid producer, which may be useful for biodiesel production. The lipophilic fluorescent dye BODIPY505/515 was used to determine the algal lipid content. Lipid bodies stained with BODIPY505/515 have a characteristic green fluorescence, and their volumes were determined using the sphere volume formula. In this study, lipid accumulation by Chlamydomonas ICE-L was analyzed under different cultivation conditions (nitrogen deficiency and UV-B radiation). The results demonstrated that nitrogen deficiency and UV-B radiation could significantly promote the accumulation of lipid content per cell. The highest yields of total lipid content (reaching 84?μL?L^?1) were obtained in full Provasoli medium after 12?days of cultivation, but not in the nitrogen-deficient medium. The inoculum used in this experiment was obtained from the late-exponential growth phase. The main reason was that the cell numbers in nitrogen-deficient medium had not increased and total lipid contents were offset by the lower growth rate. Considering the high lipid content in Chlamydomonas sp. ICE-L, this alga might be a promising alternative species for production of microalgal oil for the production of renewable biodiesel in the future.     

Effects of carbon source and light intensity on the growth and total lipid production of three microalgae under different culture conditions

We attempted to enhance the growth and total lipid production of three microalgal species, Isochrysis galbana LB987, Nannochloropsis oculata CCAP849/1, and Dunaliella salina, which are capable of accumulating high content of lipid in cells. Low nitrogen concentration under photoautotrophic conditions stimulated total lipid production, but a decreasing total lipid content and an increasing biomass were observed with increasing nitrogen concentration. Among the different carbon sources tested for heterotrophic cultivation, glucose improved the growth of all three strains. The optimal glucose concentration for growth of I. galbana LB987 and N. oculata CCAP849/1 was 0.02 M, and that of D. salina was 0.05 M. Enhanced growth occurred when they were cultivated under heterotrophic or mixotrophic conditions compared with photoautotrophic conditions. Meanwhile, high total lipid accumulation in cells occurred when they were cultivated under photoautotrophic or mixotrophic conditions. During mixotrophic cultivation, biomass production was not affected significantly by light intensity; however, both chlorophyll concentration and total lipid content increased dramatically with increasing light intensity up to 150 µmol/m²/s. The amount and composition ratio of saturated and unsaturated fatty acids in cells were different from each other depending on both species and light intensity. The highest accumulation of total fatty acid (C16–C18) among the three strains was found from cells of N. oculata CCAP849/1, which indicates that this species can be used as a source for production of biodiesel.     

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.     

Continuous microalgae cultivation in a photobioreactor

New biomass sources for alternative fuels has become a subject of increasing importance as the nation strives to resolve the economic and strategic impacts of limited fossil fuel resources on our national security, environment, and global climate. Algae are among the most promising non‐food‐crop‐based biomass feedstocks. However, there are currently no commercially viable microalgae‐based production systems for biofuel production that have been developed, as limitations include less‐than optimal oil content, growth rates, and cultivation techniques. While batch studies are critical for determining basic growth phases and characteristics of the algal species, steady‐state studies are necessary to better understand and measure the specific growth parameters. This study evaluated the effects of dilution rate on microalgal biomass productivity, lipid content, and fatty acid profile under steady‐state conditions with continuous illumination and carbon dioxide supplemention for two types of algae. Continuous cultures were conducted for more that 3 months. Our results show that the productivity of Chlorella minutissima varied from 39 to 137 mg/L/day (dry mass) when the dilution rate varied from 0.08 to 0.64 day^?1. The biomass productivity of C. minutissima reached a maximum value (137 mg/L/day) at a dilution rate of 0.33 day^?1, while the productivity of Dunaliella tertiolecta varied from 46 to 91 mg/L/day at a dilution rate of 0.17 to 0.74 day^?1. The biomass productivity of D. tertiolecta reached a maximum value of 91 mg/L/day at a dilution rate of 0.42 day^?1. Moreover, the lipid content had no significant change with various dilution rates. Biotechnol. Bioeng. 2012; 109: 2468–2474. © 2012 Wiley Periodicals, Inc.