Tris-acetate-phosphate Medium Research Articles

Assessment of Nitrate Removal Capacity of Two Selected Eukaryotic Green Microalgae

Eutrophication is a leading problem in water bodies all around the world in which nitrate is one of the major contributors. The present study was conducted to study the effects of various concentrations of nitrate on two eukaryotic green microalgae, Chlamydomonas sp. MACC-216 and Chlorella sp. MACC-360. For this purpose, both microalgae were grown in a modified tris-acetate-phosphate medium (TAP-M) with three different concentrations of sodium nitrate, i.e., 5 mM (TAP-M5), 10 mM (TAP-M10) and 15 mM (TAP-M15), for 6 days and it was observed that both microalgae were able to remove nitrate completely from the TAP-M5 medium. Total amount of pigments decreased with the increasing concentration of nitrate, whereas protein and carbohydrate contents remained unaffected. High nitrate concentration (15 mM) led to an increase in lipids in Chlamydomonas sp. MACC-216, but not in Chlorella sp. MACC-360. Furthermore, Chlamydomonas sp. MACC-216 and Chlorella sp. MACC-360 were cultivated for 6 days in synthetic wastewater (SWW) with varying concentrations of nitrate where both microalgae grew well and showed an adequate nitrate removal capacity.

Lipid accumulation on optimized condition through biomass production in green algae

Microalgae, mainly green algae, are well known as sources of lipids for food, feed, and energy. As our institution owned microalgae collections, we performed microalgae screening to find candidate/s, producing high lipid content. In this research, microalgae were screened by various media, and then the medium was optimized. Larger scale cultivation was done in order to find out the yield of the biomass production and lipid content. The condition with phosphate buffer was applied to increase the lipid accumulation. The lipid content was analyzed by Nile red staining. As a result, Chlamydomonas reinhardtii and Chlorella vulgaris showed high biomass production in the Tris-Acetate-Phosphate (TAP) medium. Chlamydomonas reinhardtii at four times concentration and C. vulgaris at the initial concentration of TAP medium demonstrated the most increased biomass production. The maximum biomass production and lipid content was 0.9 g/l and 31% for C. reinhardtii and 1.7 g/l and 27% for C. vulgaris, respectively. Under starvation, the lipid content was increased up to 52% for C. reinhardtii and 34% for C. vulgaris, which were higher than their control. From this study, the TAP medium was found to have the most increased biomass production, and starvation enhanced lipid accumulation.

Role of autophagy in triacylglycerol biosynthesis in Chlamydomonas reinhardtii revealed by chemical inducer and inhibitors

Autophagy mediates degradation and recycling of cellular components and plays an important role in senescence and adaptive responses to biotic and abiotic stresses. Nutrient deprivation has been shown to trigger triacylglycerol (TAG) accumulation and also induces autophagy in various green algae. However, the functional relationship between TAG metabolism and autophagy remains unclear. To gain preliminary evidence supporting a role of autophagy in TAG synthesis, Chlamydomonas reinhardtii CC-2686 was grown in Tris-acetate phosphate medium with or without nitrogen and treated with an autophagy inducer (rapamycin) or inhibitors (wortmannin, 3-methyladenine, and bafilomycin A1). Fluorescence microscopic analysis of Nile red-stained cells following 72-h treatments showed that rapamycin induced accumulation of subcellular lipid droplets which are storage sites of TAG. Rapamycin treatment in combination with nitrogen starvation led to a greater abundance of lipid droplets. Wortmannin and bafilomycin A1, but not 3-methyladenine, inhibited lipid droplet accumulation in rapamycin-treated cells and to a less extent in nitrogen-depleted cells. These results suggested that autophagy contributes to TAG synthesis in C. reinhardtii, but is not a necessary process. Autophagy induction may also be used to further enhance TAG accumulation in microalgae under nutrient deprivation.

Hydrogen production by unicellular green alga chlorella sp. LSD-W2 isolated from seawater in Thailand

Green algae are able to convert the unlimited sunlight energy to produce hydrogen via photosynthesis. In seawater, several kinds of m arine microalgae are widespread and abundant and have been shown to tolerate and survive under the extreme salt concentrations . This work aimed to study the screening of high H 2 producing marine green algal strains isolated from the Gulf of Thailand and the Andaman Sea, and the selection of the highest H 2 producing strain. Its H 2 production was investigated under photoheterotrophic cultivation. The result revealed that among 20 marine green algal strains, the green alga Chlorella sp. LSD-W2 gave the highest H 2 production rate in both light and dark anaerobic conditions. During photoheterotrophic cultivation Chlorella sp. LSD-W2 was rapidly grown in TAP (Tris-Acetate-Phosphate) medium and reached the stationary growth phase after 36 h of cultivation. The highest photohydrogen production rate was found in cells incubated in NH 4 Cl-deprived TAP medium. It was approximately 20-fold higher than H 2 production rate of cells in a normal TAP medium.

Enhancement of lipid production and fatty acid profiling in Chlamydomonas reinhardtii, CC1010 for biodiesel production

Lipid from microalgae is one of the putative oil resources to facilitate the biodiesel production during this era of energy dissipation and environmental pollution. In this study, the key parameters such as biomass productivity, lipid productivity and lipid content were evaluated at the early stationary phase of Chlamydomonas reinhardtii, CC1010 cultivated in nutrient starved (nitrogen, phosphorous), glucose (0.05%, 0.1%, 0.15% and 0.2%) and vitamin B12 supplementation (0.001%, 0.002% and 0.003%) in Tris-Acetate-Phosphate (TAP) medium. The lipid content in nitrogen starved media was 61% which is 2.34 folds higher than nutrient sufficient TAP medium. Glucose supplementation has lead to proportional increase in biomass productivity with the increasing concentration of glucose whereas vitamin B12 supplementations had not shown any influence in lipid and biomass production. Further, fatty acid methyl ester (FAME) profiling of C. reinhardtii, CC 1010 has revealed more than 80% of total SFA (saturated fatty acid) and MUFA (mono unsaturated fatty acid) content. Quality checking parameters of biodiesel like cetane number, saponification value, iodine number and degree of unsaturation were analyzed and the biodiesel fuel properties were found to be appropriate as per the international standards, EN 14214 and ASTM D6751. Conclusively, among all the treatments, nitrogen starvation with 0.1% glucose supplementation had yielded high lipid content in C. reinhardtii, CC 1010.

Flow cytometry pulse width data enables rapid and sensitive estimation of biomass dry weight in the microalgae Chlamydomonas reinhardtii and Chlorella vulgaris.

Dry weight biomass is an important parameter in algaculture. Direct measurement requires weighing milligram quantities of dried biomass, which is problematic for small volume systems containing few cells, such as laboratory studies and high throughput assays in microwell plates. In these cases indirect methods must be used, inducing measurement artefacts which vary in severity with the cell type and conditions employed. Here, we utilise flow cytometry pulse width data for the estimation of cell density and biomass, using Chlorella vulgaris and Chlamydomonas reinhardtii as model algae and compare it to optical density methods. Measurement of cell concentration by flow cytometry was shown to be more sensitive than optical density at 750 nm (OD750) for monitoring culture growth. However, neither cell concentration nor optical density correlates well to biomass when growth conditions vary. Compared to the growth of C. vulgaris in TAP (tris-acetate-phosphate) medium, cells grown in TAP + glucose displayed a slowed cell division rate and a 2-fold increased dry biomass accumulation compared to growth without glucose. This was accompanied by increased cellular volume. Laser scattering characteristics during flow cytometry were used to estimate cell diameters and it was shown that an empirical but nonlinear relationship could be shown between flow cytometric pulse width and dry weight biomass per cell. This relationship could be linearised by the use of hypertonic conditions (1 M NaCl) to dehydrate the cells, as shown by density gradient centrifugation. Flow cytometry for biomass estimation is easy to perform, sensitive and offers more comprehensive information than optical density measurements. In addition, periodic flow cytometry measurements can be used to calibrate OD750 measurements for both convenience and accuracy. This approach is particularly useful for small samples and where cellular characteristics, especially cell size, are expected to vary during growth.

Acetate Versus Sulfur Deprivation Role in Creating Anaerobiosis in Light for Hydrogen Production by Chlamydomonas reinhardtii and Spirulina platensis: Two Different Organisms and Two Different Mechanisms

This work was devoted to separate acetate role in creating anaerobiosis from that of sulfur deprivation. Chlamydomonas reinhardtii grown in TAP (Tris-acetate-phosphate) medium was resuspended in sulfur-replete or -deprived medium in sealed or nonsealed cultures. Sulfur deprivation was substantial for starch accumulation and hydrogen evolution; however, acetate induced anaerobiosis in the presence or absence of sulfur in only sealed cultures. In nonsealed cultures, Chlamydomonas did not lose its photosynthetic activity; however, it was arrested in anoxia with no photosynthetic activity as long as the culture was sealed. The sealed cultures resumed photosynthesis upon unsealing overnight unless the cells died by anoxia at late stage of the experiment. These results indicate that the enhanced oxygen consumption for the enormous acetate respiration and inhibition of the external oxygen supply in sealed cultures of Chlamydomonas are the main reasons for the steady anaerobic conditions. Although acetate was substantial for creating anaerobiosis in Chlamydomonas, sulfur deprivation alone could create anaerobiosis in Spirulina platensis grown autotrophically. Hydrogen evolution and glycogen accumulation were induced under such conditions. Severely reduced phycocyanin, chlorophyll and photosynthesis, while respiration had increased, induced anaerobiosis in Spirulina. This study reports for the first time anaerobiosis under autotrophic conditions in a cyanobacterium.

A newly isolated green alga, Tetraspora sp. CU2551, from Thailand with efficient hydrogen production

A novel unicellular hydrogen-producing green alga was isolated from fresh water pond in Pathumthani province, Thailand. Under light microscope, this alga was identified as belonging to the genus Tetraspora. Phylogenetic analysis of 18S rRNA sequence revealed that the green alga, identified as Tetraspora sp. CU2551, is closely related to other unicellular green algal species. Tetraspora sp. CU2551 had the shortest doubling time when grown in Tris-acetate-phosphate (TAP) medium under a light intensity of 48–92 μE/m 2/s and a temperature of 36 °C. Hydrogen production increased with increasing pH from 5.75 to 9.30; however, almost no production was observed at a pH of 5.25. Addition of 0.5 mM β-mercaptoethanol to the TAP medium stimulated hydrogen production about two-fold. During the hydrogen production phase, the use of TAP medium lacking both nitrogen and sulfur resulted in about 50% increase in the hydrogen production. This was in contrast to only a small increase in the production when either nitrogen or sulfur was omitted in TAP medium. The stimulation of hydrogen production by 0.5 mM β-mercaptoethanol under nitrogen- and sulfur-deprived conditions occurred only when the cells were grown at a light intensity lower than 5 μE/m 2/s with no effects at higher intensities. Maximal calculated hydrogen production, 17.3–61.7 μmol/mg Chl a/h, is a very high production rate compared to other green algae and makes Tetraspora sp. CU2551 an interesting model strain for photobiological hydrogen production.

PGRL1 Participates in Iron-induced Remodeling of the Photosynthetic Apparatus and in Energy Metabolism in Chlamydomonas reinhardtii

PGRL1 RNA and protein levels are increased in iron-deficient Chlamydomonas reinhardtii cells. In an RNAi strain, which accumulates lower PGRL1 levels in both iron-replete and -starved conditions, the photosynthetic electron transfer rate is decreased, respiratory capacity in iron-sufficient conditions is increased, and the efficiency of cyclic electron transfer under iron-deprivation is diminished. Pgrl1-kd cells exhibit iron deficiency symptoms at higher iron concentrations than wild-type cells, although the cells are not more depleted in cellular iron relative to wild-type cells as measured by mass spectrometry. Thiol-trapping experiments indicate iron-dependent and redox-induced conformational changes in PGRL1 that may provide a link between iron metabolism and the partitioning of photosynthetic electron transfer between linear and cyclic flow. We propose, therefore, that PGRL1 in C. reinhardtii may possess a dual function in the chloroplast; that is, iron sensing and modulation of electron transfer.

A Cation-regulated and Proton Gradient-dependent Cation Transporter from Chlamydomonas reinhardtii Has a Role in Calcium and Sodium Homeostasis

The CrCAX1 gene encoding a Ca2+/H+ and Na+/H+ exchanger was cloned and characterized from the unicellular green alga Chlamydomonas reinhardtii to begin to understand the mechanisms of cation homeostasis in this model organism. CrCAX1 was more closely related to fungal cation exchanger (CAX) genes than those from higher plants but has structural characteristics similar to plant Ca2+/H+ exchangers including a long N-terminal tail. When CrCAX1-GFP was expressed in Saccharomyces cerevisiae, it localized at the vacuole. CrCAX1 could suppress the Ca2+-hypersensitive phenotype of a yeast mutant and mediated proton gradient-dependent Ca2+/H+ exchange activity in vacuolar membrane vesicles. Ca2+ transport activity was increased following N-terminal truncation of CrCAX1, suggesting the existence of an N-terminal auto-regulatory mechanism. CrCAX1 could also provide tolerance to Na+ stress when expressed in yeast or Arabidopsis thaliana because of Na+/H+ exchange activity. This Na+/H+ exchange activity was not regulated by the N terminus of the CrCAX1 protein. A subtle tolerance by CrCAX1 in yeast to Co2+ stress was also observed. CrCAX1 was transcriptionally regulated in Chlamydomonas cells grown in elevated Ca2+ or Na+. This study has thus uncovered a novel eukaryotic proton-coupled transporter, CrCAX1, that can transport both monovalent and divalent cations and that appears to play a role in cellular cation homeostasis by the transport of Ca2+ and Na+ into the vacuole.

Identification of Precise Electrostatic Recognition Sites between Cytochrome c6 and the Photosystem I Subunit PsaF Using Mass Spectrometry

The reduction of the photo-oxidized special chlorophyll pair P700 of photosystem I (PSI) in the photosynthetic electron transport chain of eukaryotic organisms is facilitated by the soluble copper-containing protein plastocyanin (pc). In the absence of copper, pc is functionally replaced by the heme-containing protein cytochrome c6 (cyt c6) in the green alga Chlamydomonas reinhardtii. Binding and electron transfer between both donors and PSI follows a two-step mechanism that depends on electrostatic and hydrophobic recognition between the partners. Although the electrostatic and hydrophobic recognition sites on pc and PSI are well known, the precise electrostatic recognition site on cyt c6 is unknown. To specify the interaction sites on a molecular level, we cross-linked cyt c6 and PSI using a zero-length cross-linker and obtained a cross-linked complex competent in fast and efficient electron transfer. As shown previously, cyt c6 cross-links specifically with the PsaF subunit of PSI. Mass spectrometric analysis of tryptic peptides from the cross-linked product revealed specific interaction sites between residues Lys27 of PsaF and Glu69 of cyt c6 and between Lys23 of PsaF and Glu69/Glu70 of cyt c6. Using these new data, we present a molecular model of the intermolecular electron transfer complex between eukaryotic cyt c6 and PSI.

Alanine racemase from the green alga Chlamydomonas reinhardtii

Chlamydomonas reinhardtii, a unicellular green microalga, could grow to a stationary phase having optical density of 2.0-2.5 at 750 nm in Tris-acetate-phosphate (TAP) medium containing 0.1% D-alanine. D-alanine has no inhibitory effect on growth and induced alanine racemase activity 130-fold more than without D-alanine in the green alga. Although C. reinhardtii cultured in the TAP medium showed alanine racemase activity, the content of free D-alanine was only 0.14%. The enzyme was partially purified by ammonium sulfate fractionation followed by three kinds of liquid chromatography using DEAE Toyopearl, Phenyl Sepharose, and TSK G3000 SWXL columns. The specific activity for L-alanine of the partially purified alanine racemase was 3.8 micromol/min/mg. The molecular weight of the enzyme was determined to be approximately 72,000 by gel filtration. The enzyme showed a maximum activity at 45 degrees C and pH 8.4 and requires pyridoxal 5'-phosphate as a coenzyme.

Modeling and Optimization of Photosynthetic Hydrogen Gas Production by Green Alga Chlamydomonas reinhardtii in Sulfur-Deprived Circumstance

Biological hydrogen production by the green alga Chlamydomonas reinhardtii under sulfur-deprived conditions has attracted great interest due to the fundamental and practical importance of the process. The photosynthetic hydrogen production rate is dependent on various factors such as strain type, nutrient composition, temperature, pH, and light intensity. In this study, physicochemical factors affecting biological hydrogen production by C. reinhardtii were evaluated with response surface methodology (RSM). First, the maximum specific growth rate of the alga associated with simultaneous changes of ammonium, phosphate, and sulfate concentrations in the culture medium were investigated. The optimum conditions were determined as NH(4+) 8.00 mM, PO(4)(3-) 1.11 mM, and SO(4)(2-) 0.79 mM in Tris-acetate-phosphate (TAP) medium. The maximum specific growth rate with the optimum nutrient concentrations was 0.0373 h(-1). Then, the hydrogen production rate of C. reinhardtii under sulfur-deprivation conditions was investigated by simultaneously changing two nutrient concentrations and pH in the medium. The maximum hydrogen production was 2.152 mL of H(2) for a 10-mL culture of alga with density of 6 x 10(6) cells mL(-1) for 96 h under conditions of NH(4)(+) 9.20 mM, PO(4)(3-) 2.09 mM, and pH 7.00. The obtained hydrogen production rate was approximately 1.55 times higher than that with the typical TAP medium under sulfur deficiency.

Extracellular deamination of l-amino acids by Chlamydomonas reinhardtii cells

When grown in the light and in a Tris-acetate phosphate medium, cells of Chlamydomonas reinhardtii Dang. can use the following L-amino acids as a sole nitrogen source: asparagine, glutamine, arginine, lysine, alanine, valine, leucine, isoleucine, serine, methionine, histidine, and phenylalanine, whereas, in the absence of acetate, the cells only used L-arginine. The utilization system in the acetate medium consisted of an extracellular deaminating activity induced by L-amino acids; it took between 10 to 30 h before the system appeared in cells previously grown with ammonium. This deaminase activity was nonspecific, required an organic carbon source for its de-novo synthesis, and was sensitive to high ammonium concentration and light deprivation.