Chloroplasts Isolation from Chlamydomonas reinhardtii under Nitrogen Stress.

Triacylglycerols are produced in abundance through chloroplast and endoplasmic reticulum pathways in some microalgae exposed to stress, though the relative contribution of either pathway remains elusive. Characterization of these pathways requires isolation of the organelles. In this study, an efficient and reproducible approach, including homogenous batch cultures of nitrogen-deprived algal cells in photobioreactors, gentle cell disruption using a simple custom-made disruptor with mechanical shear force, optimized differential centrifugation and Percoll density gradient centrifugation, was developed to isolate chloroplasts from Chlamydomonas reinhardtii subjected to nitrogen stress. Using this approach, the maximum limited stress duration was 4 h and the stressed cells exhibited 19 and 32% decreases in intracellular chlorophyll and nitrogen content, respectively. Chloroplasts with 48 - 300 µg chlorophyll were successfully isolated from stressed cells containing 10 mg chlorophyll. These stressed chloroplasts appeared intact, as monitored by ultrastructure observation and a novel quality control method involving the fatty acid biomarkers. This approach can provide sufficient quantities of intact stressed chloroplasts for subcellular biochemical studies in microalgae.

Coordinated response of photosynthesis, carbon assimilation, and triacylglycerol accumulation to nitrogen starvation in the marine microalgae Isochrysis zhangjiangensis (Haptophyta).

The photosynthetic performance, carbon assimilation, and triacylglycerol accumulation of Isochrysis zhangjiangensis under nitrogen-deplete conditions were studied to understand the intrinsic correlations between them. The nitrogen-deplete period was divided into two stages based on the photosynthetic parameters. During the first stage, carbon assimilation was not reduced compared with that under favorable conditions. The marked increase in triacylglycerols and the variation in the fatty acid profile suggested that triacylglycerols were mainly derived from de novo synthesized acyl groups. In the second stage, the triacylglycerol content continued increasing while the carbohydrate content decreased from 44.0% to 26.3%. These results indicated that the intracellular conversion of carbohydrates to triacylglycerols occurred. Thus, we propose that sustainable carbon assimilation and incremental triacylglycerol production can be achieved by supplying appropriate amounts of nitrogen in medium to protect the photosynthetic process from severe damage using the photosynthetic parameters as indicators.

Identification of carbohydrates as the major carbon sink of the marine microalga Isochrysis zhangjiangensis (Haptophyta) and optimization of its productivity by nitrogen manipulation.

Microalgae represent a potential feedstock for biofuel production. During cultivation under nitrogen-depleted conditions, carbohydrates, rather than neutral lipids, were the major carbon sink of the marine microalga Isochrysis zhangjiangensis (Haptophyta). Carbohydrates reached maximum levels of 21.2 pg cell(-1) on day 5, which was an increase of more than 7-fold from day 1, while neutral lipids simultaneously increased 1.9-fold from 4.0 to 7.6 pg cell(-1) during the ten-day nitrogen-depleted cultivation. The carbohydrate productivity of I. zhangjiangensis was improved by optimization of the nitrate supply mode. The maximum carbohydrate concentration was 0.95 g L(-1) under batch cultivation, with an initial nitrogen concentration of 31.0 mg L(-1), which was 2.4-fold greater than that achieved under nitrogen-depleted conditions. High performance liquid chromatography (HPLC) analysis showed that the accumulated carbohydrate in I. zhangjiangensis was composed of glucose. These results show that I. zhangjiangensis represents an ideal carbohydrate-enriched bioresource for biofuel production.

Salinity manipulation as an effective method for enhanced starch production in the marine microalga Tetraselmis subcordiformis.

Microalgal starch is considered a promising feedstock for bioethanol production. The biomass and starch accumulation in the marine microalga Tetraselmis subcordiformis were characterized under different salinities in response to nitrogen repletion (+N) or depletion (-N) at high irradiance (HI) or low irradiance (LI). Under favorable nutritional conditions (HI+N), biomass accumulation was seldom affected under 20% normal salinity, though starch accumulation were somewhat reduced. Increased salinity impaired overall biomass and starch accumulation, though it led to a temporary starch accumulation at initial cultivation phase. Under nitrogen deprivation, decreased salinity strengthened biomass and starch accumulation regardless of irradiance. The highest starch content of 58.2% dry weight and starch productivity of 0.62 g L(-1) d(-1) were obtained under HI-N with 20% normal salinity. Decreased salinity combined with -N generated moderate stress to facilitate starch accumulation. Salinity manipulation can be effectively applied for enhanced starch production in marine microalgae.

Characterization of cell growth and starch production in the marine green microalga Tetraselmis subcordiformis under extracellular phosphorus-deprived and sequentially phosphorus-replete conditions.

Microalgal starch is a potential feedstock for biofuel production. Nutrient stress is widely used to stimulate starch accumulation in microalgae. Cell growth and starch accumulation in the marine green microalga Tetraselmis subcordiformis were evaluated under extracellular phosphorus deprivation with initial cell densities (ICD) of 1.5, 3.0, 6.0, and 9.0×106 cells mL⁻¹. The intracellular stored phosphorus supported cell growth when extracellular phosphorus was absent. The maximum starch content of 44.1% was achieved in the lowest ICD culture, while the maximum biomass productivity of 0.71 g L⁻¹ day⁻¹, starch concentration of 1.6 g L⁻¹, and starch productivity of 0.30 g L⁻¹ day⁻¹ were all obtained in the culture with the ICD of 3.0×106 cells mL⁻¹. Appropriate ICD could be used to regulate the intracellular phosphorus concentration and maintain adequate photosynthetic activity to achieve the highest starch productivity, along with biomass and starch concentration. The recovery of phosphorus-deprived T. subcordiformis in medium containing 0.5, 1.0, or 6.0 mM KH2PO4 was also tested. Cell growth and starch accumulation ability could be recovered completely. A phosphorus pool in T. subcordiformis was shown to manipulate its metabolic activity under different environmental phosphorus availability. Though lower starch productivity and starch content were achieved under phosphorus deprivation compared with nitrogen- or sulfur-deprived conditions, the higher biomass and starch concentration make T. subcordiformis a good candidate for biomass and starch production under extracellular phosphorus deprivation.

Deoxyuridines from the marine sponge associated actinomycete Streptomyces microflavus.

One new nucleoside derivative, named 3-acetyl-5-methyl-2'-deoxyuridine (1), along with two known compounds 3,5-dimethyl-2'-deoxyuridine (2) and 3-methyl-2'-deoxyuridine (3), were isolated from the cultures of Streptomyces microflavus. This strain was an associated actinomycete isolated from the marine sponge Hymeniacidon perlevis collected from the coast of Dalian (China). Their structures were elucidated by detailed NMR and MS spectroscopic analysis as well as comparison with literature data.

Cellular localization of debromohymenialdisine and hymenialdisine in the marine sponge Axinella sp. using a newly developed cell purification protocol.

Sponges (Porifera), as the best known source of bioactive marine natural products in metazoans, play a significant role in marine drug discovery and development. As sessile filter-feeding animals, a considerable portion of the sponge biomass can be made of endosymbiotic and associated microorganisms. Understanding the cellular origin of targeted bioactive compounds from sponges is therefore important not only for providing chemotaxonomic information but also for defining the bioactive production strategy in terms of sponge aquaculture, cell culture, or fermentation of associated bacteria. The two alkaloids debromohymenialdisine (DBH) and hymenialdisine (HD), which are cyclin-dependent kinase inhibitors with pharmacological activities for treating osteoarthritis and Alzheimer's disease, have been isolated from the sponge Axinella sp. In this study, the cellular localization of these two alkaloids was determined through the quantification of these alkaloids in different cell fractions by high-performance liquid chromatography (HPLC). First, using a differential centrifugation method, the dissociated cells were separated into different groups according to their sizes. The two bioactive alkaloids were mainly found in sponge cells obtained from low-speed centrifugation. Further cell purifications were accomplished by a newly developed multi-step protocol. Four enriched cell fractions (C1, C2, C3, and C4) were obtained and subjected to light and transmission electron microscopy, cytochemical staining, and HPLC quantification. Compared to the low concentrations in other cell fractions, DBH and HD accounted for 10.9% and 6.1%, respectively, of dry weight in the C1 fraction. Using the morphological characteristics and cytochemical staining results, cells in the C1 fraction were speculated to be spherulous cells. This result shows that DBH and HD in Axinella sp. are located in sponge cells and mostly stored in spherulous cells.