Secondary carotenoids formation by the green alga Chlorococcum sp.

The effects of temperature, pH and oxygen level onsecondary carotenoids (SC) accumulation in Chlorococcum sp. were investigated. The optimaltemperature, pH and oxygen level for the yields ofsecondary carotenoids and astaxanthin were35 ^°C, pH 8 and 10% (v/v), respectively. Whilethe ratio (R) of products containing hydroxyl group(s) to those containing keto group (s) increased withthe increase of temperature and pH, R value decreasedwith the level of oxygen. These results indicate thathigher temperature and pH favor the introduction ofhydroxyl group to the corresponding substrates,however, under high oxygen level keto group wasrelatively easy to be added compared to the additionof hydroxyl.     

Composition and biosynthetic pathways of carotenoids in the astaxanthin-producing green alga Chlorococcum sp.

The composition of the secondary carotenoids in the astaxanthin-producing green alga Chlorococcum sp. was analyzed. Eight types of carotenoids were identified by a variety of spectroscopic techniques. Canthaxanthin, 3-hydroxyechinenone, adonirubin and adonixanthin comprised, respectively, 32%, 23%, 12% and 9% of total carotenoids. The results imply that Chlorococcum sp. synthesized astaxanthin from -carotene through various pathways which are different from other astaxanthin-producing microorganisms.     

Physiological response of the unicellular green alga Chlorococcum submarinum to rapid changes in salinity

Cell volumes and intracellular concentrations of major solutes of Chlorococcum submarinum were determined before and after salinity shocks. Cells were found to shrink in size by about 30% following changes from 0.1 to 0.5 M NaCl, there was a transitory increase in sodium concentration and more permanent increases in concentrations of potassium, proline and glycerol (the major osmolyte). Conversely, cells doubled in size after the reciprocal downshock, there was rapid loss of about 70% of the cells' glycerol to the medium, a much smaller loss of cellular potassium and a steady disappearance of proline from the cells. The respiratory and photosynthetic responses to salinity fluctuations were also studied. Salinity downshocks stimulated respiration by 30% and inhibited photosynthesis by 16% within 5 min, but within 2 h these rates were identical to control rates. Upshocks caused a slight inhibition of respiration, but decreased photosynthesis by 40% within 5 min and recovery took 2 h. Downshocks had little effect on chlorophyll fluorescence, however, Fo strongly increased and both Fm and Fv/Fm declined within 5 min of salinity increases. This is consistent with a decrease in efficiency of PS2. Ecological and metabolic implications of the results are discussed.Abbreviations DMSO dimethyl sulphoxide - Hepes N-[2-hydroxyethyl]piperazine-N-2-ethane sulphonic acid - TCA trichloroacetic acid - Tris tris[hydroxymethyl]aminoethane     

Non-photochemical quenching of chlorophyll fluorescence in the green alga Dunaliella

The relaxation of the non-photochemical quenching of chlorophyll fluorescence has been investigated in cells of the green alga Dunaliella following illumination. The relaxation after the addition of DCMU or darkening was strongly biphasic. The uncoupler NH₄Cl induced rapid relaxation of both phases, which were therefore both energy-dependent quenching, qE. The proportion of the slow phase of qE increased at increasing light intensity. In the presence of the inhibitors rotenone and antimycin the slow phase of qE was stabilised for in excess of 15 min. NaN₃ inhibited the relaxation of almost all the qE. The implications of these results are discussed in terms of the interpretation of the non-photochemical quenching of chlorophyll fluorescence in vivo and the mechanism of qE.Abbreviations PS II Photosystem II - qQ photochemical quenching of chlorophyll fluorescence - qNP non-photochemical quenching of chlorophyll fluorescence - qE energy-dependent quenching of chlorophyll fluorescence - F _m maximum level of chlorophyll fluorescence for dark adapted cells - F _m level of fluorescence at any time when qQ is zero     

Induction of astaxanthin formation by reactive oxygen species in mixotrophic culture of Chlorococcum sp.

The induction of astaxanthin formation by reactive oxygen species in mixotrophic culture of Chlorococcum sp. was investigated. H₂O₂ (0.1 mM) enhanced the total astaxanthin formation from 5.8 to 6.5 mg g^–1 cell dry wt. Fe²⁺ (0.5 mM) added to the medium with H₂O₂ (0.1 mM) further promoted astaxanthin formation to 7.1 mg g^–1 cell dry wt. Similarly, Fe²⁺ (0.5 mM) together with methyl viologen (0.01 mM) promoted astaxanthin formation to 6.3 mg g^–1 cell dry wt. In contrast, an addition of KI (1 mM), a specific scavenger for hydroxyl radicals (OH), together with H₂O₂ (0.1 mM) and Fe²⁺ (0.5 mM), to the medium decreased astaxanthin formation to 1.8 mg g^–1 cell dry wt. KI (1 mM) also inhibited the enhancement of carotenogenesis by superoxide anion radicals (O₂ ^–), with a decrease of astaxanthin formation to 1.7 mg g^–1 cell dry wt. This suggested that O₂ ^– might be transformed to OH before promoting carotenogenesis in Chlorococcum sp.     

Chlorophyll fluorescence quenching in the alga Euglena gracilis

When far red light preincubated cells of Euglena gracilis are transferred to dark or light, chlorophyll fluorescence (F₀ and F_m) decreases. Non-photochemical quenching in the dark is suggested to be induced partly by chlororespiration and partly by changes in the distribution of excitation energy between the photosystems. Depending on the light intensities it was possible to resolve the non-photochemical quenching into at least three different components. The slowest relaxation phase of non-photochemical quenching occurred only after exposure to high light and was assigned to photoinhibition. The other two components were an energy-dependent quenching (qE), and the one which we attribute to a spill over mechanism. We suggest that both photosystems use a common antenna system consisting of LHC I and LHC II proteins. In contrast to higher plants, qE in Euglena gracilis is independent of the xanthophyll cycle and an aggregation of LHC II. This revised version was published online in June 2006 with corrections to the Cover Date.     

Determination of intracellular volume and internal solute concentrations of the green alga Chlorococcum submarinum

A method is described for measuring the cell volume of the unicellular green alga Chlorococcum submarinum, which depends on measurements of bromide concentration before and after disruption of the cells by ammonium hydroxide. Simultaneous equations are derived, which along with direct determination of cell water weight, allow the calculation of the intracellular volume in three different ways. The volumes calculated are in agreement indicating the validity of the method. The cell volumes and internal concentrations of glycerol, proline, potassium and sodium were determined for algae adapted to three salinities, 0.1, 0.5 and 1.0 M NaCl. The results showed that glycerol was the major internal solute and that the total measured solutes balanced the external osmotic pressure at all three salinities.Abbreviations DMSO dimethyl sulphoxide - Hepes N-[2-hydroxyethyl]piperazine-N-2-ethane sulfonic acid - TCA trichloroacetic acid - Tris tris[hydroxymethyl]aminoethane     

Variations in biochemical parameters of Heterocapsa sp. and Olisthodiscus luteus grown in 12:12 light:dark cycles II. Changes in pigment composition

Photosynthetic pigment composition was studied in batch cultures of Heterocapsa sp. and Olisthodiscus luteus growing exponentially in a 12:12 light:dark cycle. Both species divided in the dark. The synthesis of pigments was continuous for both species. However for chlorophyll c and peridinin, in Heterocapsa sp., and chlorophyll c and fucoxanthin, in O. luteus, (pigments belonging to light harvesting complexes) the synthesis was significantly higher during the light period. Concentrations per total cell volume (TCV) of chlorophyll a, chlorophyll c, peridinin and diadinoxanthin in Heterocapsa sp., and chlorophyll a, chlorophyll c, fucoxanthin and violaxanthin in O. luteus, showed a maximum at the onset of light and decreased during the light period. The values of the chlorophyll a:chlorophyll c, chlorophyll a:peridinin and chlorophyll a:fucoxanthin ratios are compared with data reported in the literature.     

Photon irradiance required to support optimal growth and interrelations between irradiance and pigment composition in the green alga Haematococcus pluvialis

The aim of the work was to find the optimal photon irradiance for the growth of green cells of Haematococcus pluvialis and to study the interrelations between changes in photochemical parameters and pigment composition in cells exposed to photon irradiances between 50 and 600?µmol?m^?2?s^?1 and a light:dark cycle of 12:12?h. Productivity of cultures increased with irradiance. However, the rate of increase was higher in the range 50–200?µmol?^?2?s^?1. The carotenoid content increased with increasing irradiance, while the chlorophyll content decreased. The maximum quantum yield of PSII (F_v/F_m) gradually declined from 0.76 at the lowest irradiance of 50?µmol?^?2?s^?1 to 0.66 at 600?µmol?^?2?s^?1. Photosynthetic activity showed a drop at the end of the light period, but recovered fully during the following dark phase. A steep increase in non-photochemical quenching was observed when cultures were grown at irradiances above 200?µmol?^?2?s^?1. A sharp increase in the content of secondary carotenoids also occurred above 200?µmol?m^?2?s^?1. According to our results, with H. pluvialis green cells grown in a 5-cm light path device, 200?µmol?^?2?s^?1 was optimal for growth, and represented a threshold above which important changes in both photochemical parameters and pigment composition occurred.     

Influence of nitrogen deficiency on biochemical composition of the green alga <Emphasis Type="Italic">Botryococcus</Emphasis>

A study has been carried out to investigate the influence of nitrogen deficiency on intracellular lipid composition, including total fatty acid composition of lipids, polar lipids, and triacylglycerols, of the alga Botryococcus braunii Kütz IPPAS H-252 in batch culture. Under nitrogen limitation, the alga accumulates lipids as triacylglycerols and the total fatty acid (FA) composition changes: trienoic acids decrease (from 52.8–57.2 to 19.5–24.7% of the total FAs) and the oleic acid increases (from 1.1–1.2 to 17.1–24.4%) as does the saturated acids (from 23.7–26 to 32.9–46.1%). A similar rearrangement in the FA spectrum occurs at later times in the control culture, but it is less pronounced. Under nitrogen limitation, considerable changes in the polar lipid FAs are registered at day 13: saturated acids increase (from 28.6–35.5 to 76.8%) and all polyenoic acids markedly decrease (from 56.9–64.1 to 6.8%). Changes in the triacylglycerol fatty acid spectrum are seen on day 7: the oleic acid increases (from 14.7 to 34.2%) and remains at a high level till the end of the culture. In the control, triacylglycerols with large contents of oleic acid are detected at day 13, the total lipids and triacylglycerols still remaining unchanged.     

Photosynthetic acclimation to photon irradiance and its relation to chlorophyll fluorescence and carbon assimilation in the halotolerant green alga Dunaliella viridis

This work describes the long-term acclimation of the halotolerant microalga Dunaliella viridis to different photon irradiance, ranging from darkness to 1500 μmol m⁻² s⁻¹. In order to assess the effects of long-term photoinhibition, changes in oxygen production rate, pigment composition, xanthophyll cycle and in vivo chlorophyll fluorescence using the saturating pulse method were measured. Growth rate was maximal at intermediate irradiance (250 and 700 μmol m⁻² s⁻¹). The increase in growth irradiance from 700 to 1500 μmol m⁻² s⁻¹ did not lead to further significant changes in pigment composition or EPS, indicating saturation in the pigment response to high light. Changes in Photosystem II optimum quantum yield (F_v/F_m) evidenced photoinhibition at 700 and especially at 1500 μmol m⁻² s⁻¹. The relation between photosynthetic electron flow rate and photosyntetic O₂ evolution was linear for cultures in darkness shifting to curvilinear as growth irradiance increased, suggesting the interference of the energy dissipation processes in oxygen evolution. Carbon assimilation efficiencies were studied in relation to changes in growth rate, internal carbon and nitrogen composition, and organic carbon released to the external medium. All illuminated cultures showed a high capability to maintain a C:N ratio between 6 and 7. The percentage of organic carbon released to the external medium increased to its maximum under high irradiance (1500 μmol m⁻² s⁻¹). These results suggest that the release of organic carbon could act as a secondary dissipation process when the xanthophyll cycle is saturated. This revised version was published online in June 2006 with corrections to the Cover Date.     

Non-photochemical quenching of chlorophyll fluorescence in Chlorella fusca acclimated to constant and dynamic light conditions

Non-photochemical quenching of chlorophyll fluorescence (NPQ) involves dissipation of light energy in the photosynthetic apparatus via a number of physiologically distinct processes. The relationships among NPQ, the (de)epoxidation state of the xanthophyll cycle pigments and state transitions was studied in the green alga Chlorella fusca, acquired from six differently light-acclimated continuous cultures. A 10 h light and 14 h darkness, periodicity was obeyed in all cultures. Three cultures received a high total daily irradiance, three others a low one. High and low irradiances were each dosed in three different modes at constant supply, with sine shape intensity modulation, or as a sine with superimposed oscillations. In the constant supply mode, but not for the sine and oscillating modes, high-light rendered a three-fold higher xantophyll cycle pigment content than low-light. Dynamic interconversion of xantophyll cycle pigments was restricted to high-light cultures. NPQ followed the kinetics of the light supply mode and was highest in high light cultures. In low-light cultures, NPQ correlated mainly to state transitions. These observations were supported by experiments with dithiothreithol-treated samples. The relative impact of xantophyll cycle operation and state transitions on NPQ in green algae from different light climates will be discussed with reference to higher plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

一株红球菌属细菌LV4在高盐条件下的吡啶降解特性

由微生物介导的吡啶降解技术是解决高盐吡啶环境污染的经济有效方法之一,开发具有吡啶降解性能且能够耐受高盐分的微生物是该类研究的重要前提。本研究从山西太原钢铁公司焦化废水处理厂活性污泥中分离培养了一株耐盐吡啶降解菌,通过菌落形态和16S rDNA基因系统发育分析,鉴定其为红球菌属(Rhodococcus sp.)的细菌。耐盐性实验结果表明,菌株LV4能够在0%–6%盐度范围内生长,并完全降解初始浓度为500 mg/L的吡啶;但当盐度高于4%时,菌株LV4因其生长变缓而导致吡啶完全降解时间明显延长。扫描电镜结果显示,高盐环境会使菌株LV4的菌体细胞分裂变慢,诱导细胞表面分泌更多的颗粒状胞外聚合物(extracellular polymeric substance, EPS)。当盐度不高于4%时菌株LV4主要依靠EPS中蛋白含量的增加来响应高盐环境的冲击。单因素实验优化发现,菌株LV4在盐度为4%的高盐环境中降解吡啶的最佳条件为温度30℃、pH 7.0、转速为120 r/min (DO 10.30 mg/L)。最优条件下菌株LV4对于初始浓度为500 mg/L的吡啶,在经过12 h的适应期后,...     

光强及氮浓度对丝状绿藻双星藻生长及生化组成的影响

[背景]环境因子和营养因子对微藻的生长和生化组成都有显著的影响,其中光强和氮浓度是最重要的两个条件。[目的]研究不同光强和初始氮浓度对丝状绿藻-双星藻(Zygnema sp.)生长及生化组成的影响。[方法]采用改良的BBM培养基,设置了两组光强[100μmol/(m²·s)和300μmol/(m²·s)]和6种初始氮浓度(3、6、9、12、15和18 mmol/L)在柱状光生物反应器中对双星藻进行培养。[结果]在高光强条件下[300μmol/(m²·s)],12 mmol/L初始氮浓度最有利于双星藻生物质的积累,其最高生物量可以达到6.60 g/L,而初始低氮浓度(3 mmol/L)则促进了油脂和脂肪酸的积累,油脂最高含量占干重的32.13%,且脂肪酸组成主要包括棕榈酸(C_16:0)、油酸(C_18:1)、亚油酸(C_18:2)和亚麻酸(C_18:3),其中油酸含量最高达到总脂肪酸含量的55.01%;在低光强条件下[100μmol/(m     

Nitrogen and carbon fixation byAnabaena sp. isolated from a rice paddy and grown under P and light limitations

Anabaena sp., isolated from a rice paddy, was investigated for its nitrogen fixation as measured by acetylene reduction activity (ARA) in P-limited continuous and light-limited semi-continuous cultures. Growth rate (μ) under P limitation was a function of cell P content (q _p). Both the photosynthetic capacity (P_max) and photosynthetic efficiency (α) increased with μ when expressed per cell, but not per unit chla. The ARA of steady-state cells under P limitation increased with μ and was linearly related to C-fixation rate. This was apparently a consequence of the control of C-fixation by P limitation. In light-limited cells, steady state ARA, both at the culture light intensity and in the dark, increased asymptotically with μ, but the activity in the dark was only about 51% of that in the light. When the light level of steady-state cells grown at a high in intensity was switched to a low level, ARA decreased exponentially with time. Dark ARA activity also showed a similar decline, but at much lower levels. Thus, ARA depended not only on light history, but also immediate photosynthesis. Steady-state ARA at the ambient intensity or in the dark showed a strong correlation with¹⁴C-fixation rate. ARA of light-limited cells showed the same light-saturation characteristics as their¹⁴C-fixation, with the same initial saturation intensity,I _k. The ratios of P_max to the maximum ARA (ARA_max), and α to the slope of ARA (α_ara) were identical. A comparison of gross to net photosynthesis and N₂ fixation suggested that there was little leakage or excretion of fixed C or N.     

Effects of nitrogen source and concentration on growth rate and fatty acid composition of Ellipsoidion sp. (Eustigmatophyta)

In order to study the effects of different nitrogen source and concentrationon the growth rate and fatty acid composition, a marine microalga Ellipsoidion sp. with a high content of eicosapentaenoic acid (EPA) wascultured in media with different nitrogen sources and concentrations.During the pre-logarithmic phase, the alga grew faster with ammoniumas N source than with nitrate, but the reverse applied during thepost-logarithmic phase. The alga grew poorly in N-free mediumor medium with urea as the sole N source. In the same growth phase,ammonium medium resulted in higher yield of total lipid, but the EPA yielddid not differ significantly different from that using nitrate medium. Themaximum growth rate occurred in medium containing 1.28 mmolL^-1 sodium nitrate, while maximum EPA and total lipid contents werereached at 1.92 mmol L^-1, when EPA accounted for 27.9% totalfatty acids. The growth rate kept stable when NH₄Cl ranged from0.64 to 2.56 mmol L^-1, and the maximum content of total lipidand EPA occurred in the medium with 2.56 mmol L^-1NH₄Cl. The EPA content was higher in the pre- thanpost-logarithmic phase, though the total lipid content was lower. Thehighest EPA content expressed as percent total fatty acid was 27.9% innitrate medium and and 39.0% in ammonium medium.     

Influence of temperature and salinity on carbon and nitrogen content in Dunaliella viridis teodoresco under nitrogen sufficiency

Cell carbon and nitrogen in D. viridis are strongly dependent on the culturing conditions. Both elements increase with increasing salinity. At 31°C cell carbon is maximum and cell nitrogen minimum. This temperature was described previously (Jiménez, C., Niell, F. X. & Fernandez, J. A. (1990). Hydrobiologia, 197, 165-72) as the optimal one for achieving the maximum oxygen evolution. These results point out a possible competence for the reducing power during carbon and nitrogen assimilation processes, and under conditions of high photosynthesis (carbon assimilation) there is a partial inhibition of nitrate reduction, making C:N ratio maximum under conditions of maximum net photosynthesis.The study of cell glycerol, nitrate, structural proteins and free amino acids indicates that all of these solutes accumulate in the cells as a result of the high salinity adaptation.     

Light-path length and population density in photoacclimation of Nannochloropsis sp. (Eustigmatophyceae)

Photoacclimation in the marine eustigmatophyte Nannochlropsis sp., used extensively as a food chaincomponent in aquaculture, was studied both in thelaboratory and outdoors. Cell-chlorophyll andcarotenoids were used as markers to assessphotoacclimation to strong light, as well as todecreasing growth irradiance due to cellproliferation. Focusing on practical aspects involvedin mass cultivation, three different approaches wereused as follows: (a) cultures initially exposed to lowlight (150 mol photon m^-2 s^-1) thentransferred to strong light (1000 to 3000 molphoton m^-2 s^-1); (b) initially low celldensity cultures grown in reactors of differentlight-paths, exposed to strong PFD, in the laboratoryand outdoors; (c) initially low or high cell densitycultures exposed to strong light. As has already beenestablished in many reports, cell-chlorophyllrepresented a sensitive parameter in assessing cellresponse to changes in the intensity of the lightsource as well as to modifications in the light regimeto which the cells were exposed. Cell-chlorophyllconcentration sharply decreased initially upontransferring the culture from low PFD cell^-1 tohigh PFD cell^-1 due to either culture dilution(i.e. decrease in cell density and mutual shading) orto an increase in PFD. After some 7 days ofphotoacclimating to 2000 and 3000 mol photonm^-2 s^-1, chlorophyll a content began to riseat a much faster rate than cell number, which alsoincreased in response to the higher irradiance.Cell-chlorophyll in the culture exposed to 2000mol photon m^-2 s^-1 increased afteracclimation earlier and at a faster rate than in theculture exposed to 3000 mol photon m^-2s^-1, indicating the later irradiance affected astronger stress. The length of the reactor's lightpath exerted a decisive effect on cell response tostrong light through its influence on the light regimein the culture. Upon a sharp increase in PFD,carotenoids in the 1-cm reactor increased in muchhigher rate than chlorophyll, compared with the 3-cmlight path reactors. This marked difference in cellresponse to a shift-up in light was attributed to thevast variations in the light regime associated withdifferences in the length of the light path and areal density. Growth oflow cell density cultures ceased temporarily upontransfer to strong light, in contrast with high celldensity cultures transferred to strong light, whichcontinued growth without a lag.     

Xanthophyll cycle and energy-dependent fluorescence quenching in leaves from pea plants grown under intermittent light

The possible role of zeaxanthin formation and antenna proteins in energy-dependent chlorophyll fluorescence quenching (qE) has been investigated. Intermittent-light-grown pea (Pisum sativum L.) plants that lack most of the chlorophyll a/b antenna proteins exhibited a significantly reduced qE upon illumination with respect to control plants. On the other hand, the violaxanthin content related to the number of reaction centers and to xanthophyll cycle activity, i.e. the conversion of violaxanthin into zeaxanthin, was found to be increased in the antenna-protein-depleted plants. Western blot analyses indicated that, with the exception of CP 26, the content of all chlorophyll a/b-binding proteins in these plants is reduced to less than 10% of control values. The results indicate that chlorophyll a/b-binding antenna proteins are involved in the energy-dependent fluorescence quenching but that only a part of qE can be attributed to quenching by chlorophyll a/b-binding proteins. It seems very unlikely that xanthophylls are exclusively responsible for the qE mechanism.Abbreviations CAB chlorophyll a/b-binding - Chl chlorophyll - F_V variable fluorescence - IML intermittent light - LHC light harvesting complex - PFD photon flux density - qP photochemical quenching of chlorophyll fluoresence - qN non-photochemical quenching - qE energy-dependent quenching - qI photoinhibitory quenching - qT quenching by state transition