Enzymatic Preparation of Aromatic Ethylamines from Aromatic L-Amino Acids

The characteristic mode for the herbicidal action of 2-amino-3-chloro-1,4-naphthoquinone (ACN) was investigated by using autotrophic green microalgae (Scenedesmus acutus). The effects of ACN on the growth, chlorophyll content, protoporphyrin-IX accumulation, and ethane production in Scenedesmus cells were measured in comparison with three reference herbicides, i.e., ioxynil, dinoseb, and chlorophthalim. ACN appeared to have a different mechanism for action from these three herbicides. S. acutus cells grown with ACN produced ethane, but the mechanism for its production is considered to have been different from that of chlorophthalim. ACN may inhibit porphyrin biosynthesis at a different stage from that by chlorophthalim.     

Growth and lipid synthesis promotion in mixotrophic Neochloris oleoabundans (Chlorophyta) cultivated with glucose

In the recent years, the studies concerning the cultivation of Neochloris oleoabundans for biofuel purposes have increased, in relation to its capability to accumulate lipids when grown under nutrient starvation. Unfortunately, this cultivation mode does not allow to reach high biomass densities, which are required to improve the feasibility of the process. Increasing knowledge of the microalgal physiology is necessary to obtain new useful information for the improvement of culture performance in the perspective of large-scale cultivation. In this work, the mixotrophic cultivation of N. oleoabundans in a brackish medium added with different glucose concentrations has been tested under shaking, with the aim of stimulating growth alongside lipid accumulation inside cells. Cell morphology, glucose consumption, photosynthetic pigment content and photosynthetic efficiency were also investigated. Among all tested glucose concentrations (0–30 g L^?1), it was observed that 2.5 g L^?1 was the optimal concentration, allowing to obtain the best compromise between glucose supplement, biomass production and lipid accumulation. Growth was highly enhanced in mixotrophic cultures, linked to the release of cells from sporocysts. A unique feature characterising mixotrophy in N. oleoabundans was the promotion of the maximum quantum yield of Photosystem II. Moreover, when mixotrophic cells entered the stationary phase, high lipid accumulation was induced. This study shows that the addition of glucose to N. oleoabundans remarkably increases the production of biomass enriched in lipids and represents an advancement for the cultivation of this microalga for applied purposes.     

Triacylglycerol content,productivity and fatty acid profile in Scenedesmus acutus PVUW12

A detailed lipid characterization of Scenedesmus acutus PVUW12, with emphasis on the evaluation of triacylglycerols (TAGs) as a biodiesel feedstock, is presented. When algal cells were grown in nitrogen-free medium (N stress), a lipid increase was detected that was mainly due to TAG accumulation. In situ fluorescence measurements allowed the kinetics and extent of neutral lipid accumulation to be followed. Under N stress, the productivity of total lipids and TAGs increased significantly (80.99 and 63.74 mg L^?1 day^?1, respectively) compared with controls (29.51 and 16.23 mg L^?1 day^?1, respectively). Monounsaturated fatty acids were the major fraction and increased further (49.74 %) in stressed cells, with oleic acid as the most abundant compound (46.97 %). The polyunsaturated fatty acid composition of this algal oil appears to meet the European Standard EN 14214. These results indicate that S. acutus oil meets the requirements for its use as a biodiesel feedstock. Since this strain was also proposed for wastewater bioremediation, this opens up the possibility of its use in an integrated system combined with biofuel production.     

“Density equilibrium” method for the quantitative and rapid in situ determination of lipid,hydrocarbon, or biopolymer content in microorganisms

The work provides a simple method, based on a direct density equilibrium measurement, for the rapid in situ estimation of total lipid, hydrocarbon or biopolymer content in a variety of prokaryotic and eukaryotic samples. The method can be readily applied to live microalgae and photosynthetic bacteria, single‐celled or colonial microorganisms, as well as cellular fractions and isolated subcellular compartments or components. In this approach, the absolute lipid, hydrocarbon, or biopolymer content of the cells can be readily calculated. This method is especially useful for tracking the oil or polymer content of strains of microalgae and other microorganisms, whose lipid, hydrocarbon or biopolymer content may change with cultivation conditions and/or time, as the case would be in microorganism lipid‐induction industrial processes. The method is also useful for the direct in situ measurement of storage polymer accumulation in live cells, such as starch in microalgae and polyhydroxybutyrate, or other polyhydroxyalkanoates, in photosynthetic and non‐photosynthetic bacteria. Biotechnol. Bioeng. 2009;102: 1406–1415. © 2008 Wiley Periodicals, Inc.     

Temporal and metabolic overlap between lipid accumulation and programmed cell death due to nitrogen starvation in the unicellular chlorophyte Chlamydomonas reinhardtii

Lipid accumulation due to nitrogen depletion has been studied extensively in Chlamydomonas reinhardtii and the metabolic changes that lead to triacylglycerol biosynthesis have been of particular interest to researchers in the biodiesel industry. The induction of programmed cell death (PCD) in response to nitrogen starvation has also been documented in related chlorophytes. Here, we examined the temporal and metabolic overlap of lipid accumulation and PCD in response to nitrogen starvation in the important model organism C. reinhardtii. Nitrogen starvation induced physiological stress, measured by the progressive decline in chlorophyll a fluorescence, reduced photosynthetic efficiency and decreased growth. In keeping with previous reports, cells accumulated lipids reaching a peak after 2–3 days. At the same time, DNA nicking and caspase‐like protease activity was observed in a proportion of cells, and ultrastructural observations confirmed that death was via PCD. Our results demonstrate that DNA nicking and caspase‐like activity are observed during PCD in C. reinhardtii in response to nitrogen starvation, and that death occurs at the same time as lipid biosynthesis. Microalgal lipid production due to nitrogen depletion in C. reinhardtii is limited by the decrease in culture growth and knowing that the loss of culture density is, at least in part, due to PCD is important for the biotechnology industry.     

硫素营养水平对产油尖状栅藻光合生理及生化组成的影响

以产油尖状栅藻(Scenedesmus acuminatus)为实验材料, 在持续300 μmol photons/(m2·s)光照条件下, 选用3种不同初始Na₂SO₄浓度(2.0S、1.0S对照、0.25S)的改良BG-11培养基, 在Φ3.0 cm×60 cm光生物反应器中进行通气培养, 研究分析硫素营养水平与尖状栅藻产油过程光合生理和生化组成的关系。实验结果表明, 初始硫素浓度对尖状栅藻生长有显著的影响(P<0.05), Na₂SO₄初始浓度为2.0S实验组的生物量最高, 为7.47 g/L, 显著高于1.0S组(6.43 g/L)和0.25S组(4.17 g/L)(P<0.05), 说明加富硫素营养可促进藻细胞的生长。尖状栅藻细胞的叶绿素a、b以及总类胡萝卜素含量变化均与培养基中初始硫素营养水平呈正相关。在培养初期低硫营养有利于藻细胞快速积累碳水化合物, 0.25S实验组碳水化合物含量最高, 占干重的44.37%, 比1.0S和2.0S组分别高出14.43%和13.78%, 培养后期总碳水化合物和蛋白含量均发生不同程度的降低, 转向大量累积油脂, 0.25S实验组的总脂含量最高, 达55.15% DW, 显著高于1.0S和2.0S组(P<0.05)。藻细胞的光合放氧速率、PSⅡ最大光能转化效率(F_v/F_m)、实际光能转换效率(Yield)以及相对电子传递效率(ETR)均与培养液的初始硫素浓度呈正相关, 在整个培养周期中呈先上升后下降的趋势。77 K低温荧光显示, 尖状栅藻在培养初期2个光系统之间存在光能调配现象。上述结果说明, 尖状栅藻细胞的生长、油脂积累和光合生理状况与硫素营养水平直接相关。     

The effects of cultivation depth,areal density,and nutrient level on lipid accumulation of <Emphasis Type="Italic">Scenedesmus acutus</Emphasis> in outdoor raceway ponds

Lipid accumulation is critical in the production of biodiesel from microalgae. However, little work has been done on the assessment of lipid accumulation during nitrogen stress in large research-scale outdoor raceways during different seasons; most values for lipid accumulation are assumptions based on work completed in laboratory settings or outdoor photobioreactors. This study focused on the use of raceway ponds operated in batch cultivation mode with an area of 30.37 m² to determine the impacts of nitrate-nitrogen concentration and cultivation depth on the ability of Scenedesmus acutus strain LB 0414 to accumulate lipids. A concentration of less than 60 mg N-NO₃ ^??L^?1 was required for removal of nitrogen in the cultivation medium within 8 days to stimulate lipid accumulation and increase lipid productivity. When nitrate concentrations were increased to prevent nitrogen depletion, lipid productivity decreased, which demonstrates that stressing is needed to induce lipid accumulation for increased lipid productivity. Additionally, decreasing cultivation depth below 9 cm, compared to raceways operated at a depth of 20–24 cm, increased lipid productivity by 62 % in December 2014 and 38 % in February 2015. More desirable environmental conditions, mainly increased sunlight and temperature, in February, increased biodiesel productivity for all raceways and account for the decrease in productivity differences. This research highlights increased lipid productivity found by reducing cultivation depth and nitrogen concentrations in outdoor raceways and provides insight into the optimal conditions for large-scale biodiesel production.     

Growth, morphology and photosynthetic responses of Neochloris oleoabundans during cultivation in a mixotrophic brackish medium and subsequent starvation

The green microalga Neochloris oleoabundans is able to grow in both low and high salinity media and is largely studied for its capability to accumulate lipids under starvation. Moreover, N. oleoabundans is a mixotrophic alga, and then organic carbon addition can promote its growth. This research aims to study the morpho-physiological aspects, with a particular attention on the photosynthetic response, both during mixotrophic growth and starvation in brackish media, more sustainable than freshwater cultivation. In the first step, the alga was cultivated mixotrophically in a brackish medium added with an apple waste product; in the second one, cells were starved also to verify lipid induction. Results indicate that growth is highly promoted during the first week of mixotrophic cultivation, while photosynthetic pigments and lipids are over-produced during the following three weeks of cultivation. In parallel, in mixotrophic cultures the maximum PSII quantum yield was enhanced during the exponential phase of growth. Interesting changes affected the mixotrophic cultures with respect to the partitioning of absorbed light energy. Starvation of both 7-day-grown mixotrophic and autotrophic cultures caused growth inhibition, pigments and photosynthesis downshifting, and concomitantly promoted evident lipid synthesis.     

EFFECT OF PHOSPHATE CONCENTRATION ON THE FINE STRUCTURE OF THE CYANOBACTERIUM,MICROCYSTIS AERUGINOSA KÜTZ. EMEND. ELENKIN

SUMMARY

Microcystis aeruginosa toxic strain UV-006 stored a fixed amount of polyphosphate in spherical granules located in the centroplasm. Twenty four hours of phosphate starvation induced use of stored polyphosphate, manifested by reduction in granule numbers. Reintroduction of 2, 4 or 8 mg l^?1 K₂HPO₄ resulted in redeposition of polyphosphate in a critical number of centroplasmic polyphosphate granules. Growth rate was unaffected by phosphate concentrations, although the final cell yield was slightly lower at 8 mg l ^?1

Continued starvation decreased photosynthetic rate and growth ceased. Cells appeared senescent. Cyanophycin and polyglucoside reserves apparently increased in these cells, whilst thylakoids were reduced in number and reorientated away from. the cell wall and polyhedral bodies were lost. After the initial decrease, centroplasmic polyphosphate bodies increased to about half of the maximum numbers stored in cells grown in the presence of phosphate, suggesting that translocation of phosphorus from other areas in the phosphate-starved cell occurred.

Two further polyphosphate deposition areas were observed. DNA fibrils may have represented nucleation sites for developing polyphosphate granules. Intrathylakoidal deposits were rare.     

Factors affecting lipid accumulation by <Emphasis Type="Italic">Nannochloropsis oculata</Emphasis> in a two-stage cultivation process

Reserve lipids of microalgae are promising for biodiesel production. However, optimization of cultivation conditions for both biomass yield and lipid production of microalgae is a contradictory problem because required conditions for both targets are different. In this study, a two-stage cultivation strategy is proposed to enhance lipid production of the microalga Nannochloropsis oculata. Biomass growth and lipid production were carried out in two separate and non-interacting stages. In first-stage cultivation, microalgae were cultivated in optimal conditions for cell growth. Then, microalgae were harvested and transferred into a growth-limited environment, thus enhancing lipid production of microalgae. Here, optimization of the lipid production stage (second stage) with respect to different levels of inoculum concentration, salinity of culture broth, and intensity of irradiance was performed. The results show that irradiance exhibits a significant influence on lipid production. The highest lipid productivity of 0.324 g L⁻¹ day⁻¹ was obtained with an inoculum concentration of 2.3 g L⁻¹, a salinity of 35 g L⁻¹, and an irradiance of 500 μmol photons m⁻² s⁻¹. The final yield of lipid obtained from the two-stage process was 2.82-times higher than that from traditional single-stage batch cultivation systems.     

Lipid dynamics and feeding of dominant Antarctic calanoid copepods in the eastern Weddell Sea in December

Lipid content, fatty acid composition, and feeding activity of the dominant Antarctic copepods, Calanoides acutus, Calanus propinquus, and Metridia gerlachei, were studied at a quasi-permanent station in the eastern Weddell Sea in December 2003. During 3 weeks of the spring phytoplankton development, total lipid levels of females and copepodite stages V (CVs) of C. acutus were almost doubled. Meanwhile, only a slight increase in total lipid content occurred in M. gerlachei, and no clear trend was observed in lipids of C. propinquus females. The pronounced increase of lipids in C. acutus was due to an accumulation of wax esters. The proportion of wax esters in the lipids of M. gerlachei was clearly lower, while triacylglycerols played a more important role. In C. propinquus, triacylglycerols were the only neutral lipid class. There were no pronounced changes in the feeding activity of M. gerlachei, whereas the feeding activity of C. acutus had rapidly increased with the development of the phytoplankton bloom in December, which explains its rapid lipid accumulation. The combination of gut content and fatty acid trophic marker analyses showed that C. acutus was feeding predominantly on diatoms. The typical diatom fatty acid marker, 16:1(n-7), slightly decreased and the tracer for flagellates, 18:4(n-3), increased in females and CVs of C. acutus. This shift indicates the time, when the significance of flagellates started to increase. The three copepod species exhibited different patterns of lipid accumulation in relation to their trophic niches and different duration of their active phases. The investigations filled a crucial data gap in the seasonal lipid dynamics of dominant calanoid copepods in the Weddell Sea in December and support earlier hypotheses on their energetic adaptations and life cycle strategies.     

Ultrahigh-cell-density heterotrophic cultivation of the unicellular green microalga Scenedesmus acuminatus and application of the cells to photoautotrophic culture enhance biomass and lipid production

Although production of biodiesels from microalgae is proved to be technically feasible, a commercially viable system has yet to emerge. High-cell-density fermentation of microalgae can be coupled with photoautotrophic cultivation to produce oils. In this study, by optimizing culturing conditions and employing a sophisticated substrate feed control strategy, ultrahigh-cell-density of 286 and 283.5 g/L was achieved for the unicellular alga Scenedesmus acuminatus grown in 7.5-L bench-scale and 1,000-L pilot-scale fermenters, respectively. The outdoor scale-up experiments indicated that heterotrophically grown S. acuminatus cells are more productive in terms of both biomass and lipid accumulation when they are inoculated in photobioreactors for lipid production as compared to the cells originally grown under photoautotrophic conditions. Technoeconomic analysis based on the pilot-scale data indicated that the cost of heterotrophic cultivation of microalgae for biomass production is comparable with that of the open-pond system and much lower than that of tubular PBR, if the biomass yield was higher than 200 g/L. This study demonstrated the economic viability of heterotrophic cultivation on large-scale microalgal inocula production, but ultrahigh-productivity fermentation is a prerequisite. Moreover, the advantages of the combined heterotrophic and photoautotrophic cultivation of microalgae for biofuels production were also verified in the pilot-scale.     

Statistical evaluation and modeling of cheap substrate-based cultivation medium of Chlorella vulgaris to enhance microalgae lipid as new potential feedstock for biolubricant

Chlorella vulgaris (C. vulgaris) microalga was investigated as a new potential feedstock for the production of biodegradable lubricant. In order to enhance microalgae lipid for biolubricant production, mixotrophic growth of C. vulgaris was optimized using statistical analysis of Plackett–Burman (P-B) and response surface methodology (RSM). A cheap substrate-based medium of molasses and corn steep liquor (CSL) was used instead of expensive mineral salts to reduce the total cost of microalgae production. The effects of molasses and CSL concentration (cheap substrates) and light intensity on the growth of microalgae and their lipid content were analyzed and modeled. Designed models by RSM showed good compatibility with a 95% confidence level when compared to the cultivation system. According to the models, optimal cultivation conditions were obtained with biomass productivity of 0.123 g L^?1 day^?1 and lipid dry weight of 0.64 g L^?1 as 35% of dry weight of C. vulgaris. The extracted microalgae lipid presented useful fatty acid for biolubricant production with viscosities of 42.00 cSt at 40°C and 8.500 cSt at 100°C, viscosity index of 185, flash point of 185°C, and pour point of ?6°C. These properties showed that microalgae lipid could be used as potential feedstock for biolubricant production.     

Isolation and characterization of a Scenedesmus acutus strain to be used for bioremediation of urban wastewater

Green microalgae, due to their short growth cycle and to their ability to photosynthetically fix carbon dioxide producing an oil-rich biomass, have been proposed as an attractive alternative feedstock for the production of “second generation” biofuels. However, it has been anticipated that owing to their ability to colonize very different environments characterized by high levels of nitrogen, they can also be good candidates for bioremediation, thus integrating environmental protection with sustainable biomass production. We have isolated a strain belonging to Scenedesmus genus from urban wastewater. This isolate, Scenedesmus acutus PVUW12, was tested for its ability to grow and actively deplete eutrophicating inorganic molecules present in wastewater. In order to test its biomass productivity, the PVUW12 strain was grown in a vertical-column photobioreactor using standard growth medium obtaining a maximal productivity of 0.3?g dry weight L^?1?d. When the same strain was grown in the photobioreactor filled with wastewater collected from the final step of the local urban purifier plant containing 18.8?mg?L^?1 nitrate, we observed complete nitrogen removal coupled with a biomass production of about 0.74?g dry weight L^?1 within 3 days. After 10?days, the recovered biomass was analyzed for triglyceride content which was found to be 9.3% of the dry biomass. However, when algal cells were left for additional 10?days in static conditions the triglyceride content increased to 28.8%. These data show that this Scenedesmus strain can be used for wastewater bioremediation producing a biomass suitable for energy production.     

Morphophysiological analyses of Neochloris oleoabundans (Chlorophyta) grown mixotrophically in a carbon-rich waste product

Neochloris oleoabundans is considered one of the most promising oil-rich microalgae because of its ability to store lipids under nitrogen starvation. However, high biomass densities, required for applications on medium to large scale, are not reached in this condition of growth. As previous studies on other microalgae have shown that mixotrophy allows to obtain higher biomass in comparison to autotrophic cultures, we performed morphophysiological analyses in order to test the mixotrophic growth capability of N. oleoabundans. A carbon-rich manure derived from the apple vinegar production (AWP) was added to the medium. Cells were also cultivated under nutrient starvation (tap water), to observe the expected lipids accumulation, and combining AWP to water, to test the potential of this waste in a low-cost culture system. The results highlighted that AWP in the medium allowed to obtain the highest final cell density. Moreover, starch granules were stored inside chloroplast at the beginning of the experiment. The presence of AWP did not induce variations on light harvesting complex II (LHCII)–photosystem II (PSII) assembly, even if an interesting promotion of pigment synthesis in cells was observed. On the other hand, in starved cells, chloroplast degeneration, pigment content decrease, altered LHCII–PSII assembly and accumulation of high amount of lipid globules were observed, irrespective of the presence of AWP. The results suggest that mixotrophy promotes growth in N. oleoabundans and open up the possibility of using waste products from agri-food industries for this purpose. After growth, cells could be transferred under nutrient starvation to induce lipid accumulation.     

High temperature enhances lipid accumulation in nitrogen-deprived <Emphasis Type="Italic">Scenedesmus obtusus</Emphasis> XJ-15

This study investigated the changes in lipid and starch contents, lipid fraction, and lipid profile in the nitrogen-starved Scenedesmus obtusus XJ-15 at different temperatures (17, 25, and 33 °C). The optimal temperature for both growth and lipid accumulation under nitrogen-sufficient condition was found to be 25 °C. However, under nitrogen deprivation, the total and neutral lipids increased with increasing temperature, and achieved the highest lipid content of 47.60 % of dry cell weight and the highest TAG content of 79.66 % of total lipid at 33 °C. In the meantime, the stored cellular starch content decreased with the increasing temperature. Thus, high temperature induced carbon flux from starch toward TAG accumulation in microalgae during nitrogen starvation. In addition, the decreased polar lipids may also serve for TAG synthesis under high temperature, and high temperature further reduced the degree of the fatty acid unsaturation and favored a better biodiesel production. These results suggested that high-temperature stress can be a good strategy for enhancing biofuel production in oleaginous microalgae during nitrogen deficiency.     

Microalgae Improve the Photosynthetic Performance of Rice Seedlings (<i>Oryza sativa</i> L.) under Physiological Conditions and Cadmium Stress

The aim of this study was to assess the impact of the microalgae Chlorella vulgaris on the rice seedlings at physiological conditions and under cadmium (Cd) stress. We examined the effects of C. vulgaris in the nutrient solution on rice seedlings grown hydroponically in the presence and the absence of 150 μM CdCl₂, using the low (77 K) temperature and pulse amplitude modulated (PAM) chlorophyll fluorescence, P700 photooxidation measurements, photochemical activities of both photosystems, kinetic parameters of oxygen evolution, oxidative stress markers (MDA, H₂O₂ and proline), pigment content, growth parameters and Cd accumulation. Data revealed that the application C. vulgaris not only stimulates growth and improves the functions of photosynthetic apparatus under physiological conditions, but also reduces the toxic effect of Cd on rice seedlings. Furthermore, the presence of the green microalgae in the nutrient solution of the rice seedlings during Cd exposure, significantly improved the growth, photochemical activities of both photosystems, the kinetic parameters of the oxygen-evolving reactions, pigment content and decreased lipid peroxidation, H₂O₂ and proline content. Data showed that the alleviation of Cd-induced effects in rice seedlings is a result of the Cd sorption by microalgae, as well as the reduced Cd accumulation in the roots and its translocation from the roots to the shoots.     

Nitrogen Starvation Induced Oxidative Stress in an Oil-Producing Green Alga Chlorella sorokiniana C3

Microalgal lipid is one of the most promising feedstocks for biodiesel production. Chlorella appears to be a particularly good option, and nitrogen (N) starvation is an efficient environmental pressure used to increase lipid accumulation in Chlorella cells. The effects of N starvation of an oil-producing wild microalga, Chlorella sorokiniana C3, on lipid accumulation were investigated using thin layer chromatography (TLC), confocal laser scanning microscopy (CLSM) and flow cytometry (FCM). The results showed that N starvation resulted in lipid accumulation in C. sorokiniana C3 cells, oil droplet (OD) formation and significant lipid accumulation in cells were detected after 2 d and 8 d of N starvation, respectively. During OD formation, reduced photosynthetic rate, respiration rate and photochemistry efficiency accompanied by increased damage to PSII were observed, demonstrated by chlorophyll (Chl) fluorescence, 77K fluorescence and oxygen evolution tests. In the mean time the rate of cyclic electron transportation increased correspondingly to produce more ATP for triacylglycerols (TAGs) synthesis. And 0.5 d was found to be the turning point for the early stress response and acclimation of cells to N starvation. Increased level of membrane peroxidation was also observed during OD formation, and superoxide dismutase (SOD), peroxide dismutase (POD) and catalase (CAT) enzyme activity assays suggested impaired reactive oxygen species (ROS) scavenging ability. Significant neutral lipid accumulation was also observed by artificial oxidative stress induced by H₂O₂ treatment. These results suggested coupled neutral lipid accumulation and oxidative stress during N starvation in C. sorokiniana C3.     

Differential effects of nitrogen and sulfur deprivation on growth and biodiesel feedstock production of Chlamydomonas reinhardtii

Biodiesel production from microalgae is a promising approach for energy production; however, high cost of its process limits the use of microalgal biodiesel. Increasing the levels of triacylglycerol (TAG) levels, which is used as a biodiesel feedstock, in microalgae has been achieved mainly by nitrogen starvation. In this study, we compared effects of sulfur (S) and nitrogen (N) starvation on TAG accumulation and related parameters in wild-type Chlamydomonas reinhardtii CC-124 mt(-) and CC-125 mt(+) strains. Cell division was interrupted, protein and chlorophyll levels rapidly declined while cell volume, total neutral lipid, carotenoid, and carbohydrate content increased in response to nutrient starvation. Cytosolic lipid droplets in microalgae under nutrient starvation were monitored by three-dimensional confocal laser imaging of live cells. Infrared spectroscopy results showed that relative TAG, oligosaccharide and polysaccharide levels increased rapidly in response to nutrient starvation, especially S starvation. Both strains exhibited similar levels of regulation responses under mineral deficiency, however, the degree of their responses were significantly different, which emphasizes the importance of mating type on the physiological response of algae. Neutral lipid, TAG, and carbohydrate levels reached their peak values following 4 days of N or S starvation. Therefore, 4 days of N or S starvation provides an excellent way of increasing TAG content. Although increase in these parameters was followed by a subsequent decline in N-starved strains after 4 days, this decline was not observed in S-starved ones, which shows that S starvation is a better way of increasing TAG production of C. reinhardtii than N starvation.