厌氧条件下小球藻细胞的异养转化和乳酸发酵(简报)

某些藻类植物,如小球藻——一种单细胞绿藻,不但能利用无机碳源通过光合作用进行自养生长,还能利用有机碳源转化为异养生长,这种转化又是可逆的。迄今为止,对于人工控制下藻细胞向异养转化的代谢和调控机理尚不清楚。本文通过分析小球藻细胞异养转化过程对氧气的依赖性,进一步研究它们在厌氧条件下的生长和乳酸发酵特征,探讨可异养转化的单细胞藻类(作为特殊的实验生物系统)在发酵工程和细胞工程领域里应用的可能性。     

小球藻两个品系在自养与异养条件下的生长、能荷与色素差异

Chlorella protothecoides中国品系(C品系)与美国品系(A品系)在自养培养基中生长正常。在高浓度葡萄糖、低浓度有机氮的异养生长条件下,C品系仍为绿色,细胞生长与繁殖缓慢,指数期生长速率降低,细胞内腺苷酸能荷值(AEC)下降,但细胞仍保留叶绿素;A品系细胞转变成乳黄色,细胞生长与繁殖加快,指数期生长速率是自养细胞的2倍,细胞内腺苷酸能荷值仍保持高水平,向异养条件转换后约24h,细胞内叶绿素基本消失,重新向自养条件转换后细胞内叶绿素又逐渐恢复。AEC值是判别微藻在异养条件下能量代谢与生理状态的良好指标。同一种小球藻不同品系间的上述差异可能与细胞内基因调控的差异相关联。     

不同营养条件下原始小球藻对蒽的富集和降解研究

研究了自养与异养条件下原始小球藻对蒽的降解和富集能力 .结果表明 ,自养条件下 ,浓度为1 0mg·L-1的蒽有 48.18%被降解 ,其中 2 8.81%属于自然光降解 ,仅有 19.37%被原始小球藻降解 .而异养条件下的原始小球藻对浓度为 2 .5mg·L-1的蒽降解率达到 33.5 3%,说明异养原始小球藻不仅能耐受高浓度蒽 ,而且表现出比自养原始小球藻更强的蒽降解能力 .两种条件下 ,80 %以上残留的蒽都被富集到藻细胞中 .虽然自养条件下原始小球藻对蒽的生物富集系数达 90 6 4,远大于异养条件下的生物富集系数(1899) ,但异养条件下藻对蒽的绝对富集量 (2 0 2 .2 9μg)远远高于自养条件下的 6 9.6 87μg .     

叶绿素缺失和异养生长对蓝藻Synechocystis sp.PCC 6803藻胆蛋白含量的影响

通过遮黑培养缺失ｆｒｘＣ基因的蓝藻Ｓｙｎｅｃｈｏｃｙｓｔｉｓｓｐ．ＰＣＣ６８０３突变工程株,获得了叶绿素缺失的藻细胞,吸收光谱测定及数学计算表明,藻细胞中叶绿素缺失后藻胆蛋白含量增加,藻蓝蛋白和别藻蓝蛋白含量分别为相同条件下野生株对照组的４倍和６倍。野生株遮黑培养时,细胞进行异养生长,藻胆蛋白含量下降,藻蓝蛋白和别藻蓝蛋白含量分别为光照培养条件下自养生长的野生株细胞的３４．５％和２５３％。另外,缺失ａｐｃＥ基因的突变工程株细胞的藻胆蛋白含量也少于对照野生株,表明ａｐｃＥ基因的编码蛋白ＬＣＭ与藻胆蛋白的含量相关。     

PHOTOSYNTHETIC LIGHT-HARVESTING FUNCTION OF VIOLAXANTHIN IN NANNOCHLOROPSIS SPP. (EUSTIGMATOPHYCEAE)1

Whole cell absorption spectra of the Eustigmatophycean algae Nannochloropsis salina Bourrelly and Nannochloropsis sp. reveal the presence of a distinct absorption peak at 490 nm. The lack of chlorophylls b and c in these species indicates that this peak must be attributed to carotenoid absorption. In vivo fluorescence excitation spectra for chlorophyll a emission show a corresponding maximum at 490 nm. This peak is more clearly resolved than carotenoid maxima in other algal classes due to the absence of accessory chlorophylls. The carotenoid composition of the two Nannochloropsis species shows that violaxanthin and vaucheriaxanthin are the main contributors to 490 nm absorption. Violaxanthin accounts for approximately 60% of the total carotenoid in both clones. We conclude that light absorption by violaxanthin, and possibly by vaucheriaxanthin, is coupled in energy transfer to chlorophyll a and that violaxanthin is the major light-harvesting pigment in the Eustigmatophyceae. This is the first report of the photosynthetic light-harvesting function of this carotenoid.     

真眼点藻类色素的提取与测定方法

分别采用甲醇、乙醇和丙酮3种有机溶剂提取7种真眼点藻的色素,比较3种有机溶剂提取色素的效果,测定3种有机溶剂色素提取液的吸收光谱,利用分光光度法计算藻的叶绿素a和类胡萝卜素的含量,并比较甲醇和乙醇色素提取液在A₄₇₀和A₆₆₆的最大吸收峰。结果表明:使用乙醇比甲醇和90%丙酮操作更简便、快捷并且毒害低。3种有机溶剂色素提取液的叶绿素a和类胡萝卜素的含量均无显著性差异(P>0.05),提取率基本一致。色素在3种有机溶剂中的吸收光谱相似,甲醇和乙醇的色素提取液在A₄₇₀和A₆₆₆的最大吸收峰并无显著性差异(P>0.05)。乙醇色素提取液可使用Lichtenthaler的公式计算色素含量。     

PHOTOSYNTHETIC LIGHT-HARVESTING FUNCTION OF VIOLAXANTHIN IN NANNOCHLOROPSIS SPP. (EUSTIGMATOPHYCEAE)1

Whole cell absorption spectra of the Eustigmatophycean algae Nannochloropsis salina Bourrelly and Nannochloropsis sp. reveal the presence of a distinct absorption peak at 490 nm. The lack of chlorophylls b and c in these species indicates that this peak must be attributed to carotenoid absorption. In vivo fluorescence excitation spectra for chlorophyll a emission show a corresponding maximum at 490 nm. This peak is more clearly resolved than carotenoid maxima in other algal classes due to the absence of accessory chlorophylls. The carotenoid composition of the two Nannochloropsis species shows that violaxanthin and vaucheriaxanthin are the main contributors to 490 nm absorption. Violaxanthin accounts for approximately 60% of the total carotenoid in both clones. We conclude that light absorption by violaxanthin, and possibly by vaucheriaxanthin, is coupled in energy transfer to chlorophyll a and that violaxanthin is the major light-harvesting pigment in the Eustigmatophyceae. This is the first report of the photosynthetic light-harvesting function of this carotenoid.     

Relation between the Light-Harvesting Chlorophyll a-Protein Complex LHCPa and Photosystem I in the Alga Chlamydobotrys stellata

The light-harvesting chlorophyll protein system of the alga Chlamydobotrys stellata consists of an as yet uncharacterized algal chlorophyll a-protein, called LHCPa, and a common photosystem II-related chlorophyll a/b-protein, called LHCPb (Brandt, Kaiser-Jarry, Wiessner 1982 Biochim Biophys Acta 679: 404-409). For further characterization, this LHCPa was isolated from the organism by polyacrylamide isoelectrofocusing and reelectrophoresis. It contains only chlorophyll a and has only one apoprotein (32,000 daltons). When separated from autotrophically grown cells, its absorption peak is at 674 nm and its isoelectric point at 5.3. Photoheterotrophic cultivation of the algae shifts the absorption maximum of LHCPa to 679 nm and its isoelectric point to 4.8. This LHCPa is a component of photosystem I particles. In relation to the total chlorophyll a content, the amount of LHCPa is low in autotrophic algae, but increases under photoheterotrophic growth conditions, where the organisms do not have the ability to assimilate CO₂ photosynthetically.     

Sequential heterotrophic/autotrophic cultivation – An efficient method of producing Chlorella biomass for health food and animal feed

Sequential heterotrophic/autotrophic cultivation method was investigated for production of high concentration of Chlorella biomass with high cellular protein and chlorophyll contents. By using autotrophic growth medium, which contains glucose as organic carbon source, for heterotrophic culture, the protein and chlorophyll contents of the cells could be increased by simply illuminating the culture broth and aerating with CO₂-enriched air at the end of the heterotrophic culture. A system was then constructed for continuous sequential heterotrophic/autotrophic production of algal biomass. The system was composed of the conventional mini-jar fermentor for the heterotrophic phase and a tubular photobioreactor for the autotrophic phase. The exhaust gas from the heterotrophic phase was used for aeration of the autotrophic phase in order to reduce the CO₂ emission into the atmosphere. With this system, it was possible to produce high Chlorella biomass concentration (14 g L-¹) containing 60.1% protein and 3.6% chlorophyll continuously for more than 640 h. During the steady state, about 27% of the CO₂ produced in the heterotrophic phase was re-utilized in the autotrophic phase. When the tubular photobioreactor was replaced with a 3.5-L internally illuminated photobioreactor, the productivity increased from 2 g L-¹ d-¹ to 4 g L-¹ d-¹. However, the chlorophyll content of the cells was lower due to the lower light supply coefficient of the photobioreactor. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of cultural conditions on the sterols and fatty acids of green algae

Six species of green algae were grown autotrophically, photohetrotrophically, and heterotrophically and their fatty acid and sterol compositions determined. Sterol composition was higher in autotrophic than in heterotrophic plants by a factor of from 2 to over 20 in five of the six species studied. Relative amounts of various sterols did not change significantly with cultural conditions. In five of the species studied, autotrophic growth produced a significant increase in the relative proportion of linolenic acid compared to that in heterotrophic or photoheterotrophic growth. This increase was usually accompanied by a corresponding decrease in oleic or linoleic acids or both.     

Distinct roles of alternative oxidase pathway during the greening process of etiolated algae

The vital function of mitochondrial alternative oxidase(AOX) pathway in optimizing photosynthesis during plant de-etiolation has been well recognized. However, whether and how AOX impacts the chloroplast biogenesis in algal cells remains unclear. In the present study, the role of AOX in regulating the reassembly of chloroplast in algal cells was investigated by treating Auxenochlorella protothecoides with salicylhydroxamic acid(SHAM), the specific inhibitor to AOX, in the heterotrophy to autotrophy transition process. Several lines of evidences including delayed chlorophyll accumulation, lagged reorganization of chloroplast structure, altered PSI/PSII stoichiometry, and declined photosynthetic activities in SHAM treated cells indicated that the impairment in AOX activity dramatically hindered the development of functioning chloroplast in algal cells. Besides, the cellular ROS levels and activities of antioxidant enzymes were increased by SHAM treatment, and the perturbation on the balance of NAD~+/NADH and NADP~+/NADPH ratios was also observed in A. protothecoides lacking AOX activity, indicating that AOX was essential in promoting ROS scavenging and keeping the redox homeostasis for algal chloroplast development during greening. Overall, our study revealed the essentiality of mitochondrial AOX pathway in sustaining algal photosynthetic performance and provided novel insights into the physiological roles of AOX on the biogenesis of photosynthetic organelle in algae.     

Detection and quantification of snow algae with an airborne imaging spectrometer.

We describe spectral reflectance measurements of snow containing the snow alga Chlamydomonas nivalis and a model to retrieve snow algal concentrations from airborne imaging spectrometer data. Because cells of C. nivalis absorb at specific wavelengths in regions indicative of carotenoids (astaxanthin esters, lutein, beta-carotene) and chlorophylls a and b, the spectral signature of snow containing C. nivalis is distinct from that of snow without algae. The spectral reflectance of snow containing C. nivalis is separable from that of snow without algae due to carotenoid absorption in the wavelength range from 0.4 to 0.58 microm and chlorophyll a and b absorption in the wavelength range from 0.6 to 0.7 microm. The integral of the scaled chlorophyll a and b absorption feature (I(0.68)) varies with algal concentration (C(a)). Using the relationship C(a) = 81019.2 I(0.68) + 845.2, we inverted Airborne Visible Infrared Imaging Spectrometer reflectance data collected in the Tioga Pass region of the Sierra Nevada in California to determine algal concentration. For the 5.5-km(2) region imaged, the mean algal concentration was 1,306 cells ml(-1), the standard deviation was 1,740 cells ml(-1), and the coefficient of variation was 1.33. The retrieved spatial distribution was consistent with observations made in the field. From the spatial estimates of algal concentration, we calculated a total imaged algal biomass of 16.55 kg for the 0.495-km(2) snow-covered area, which gave an areal biomass concentration of 0.033 g/m(2).     

Lactate dehydrogenase from autotrophic and heterotrophic cells of the marine diatom Cylindrotheca fusiformis Reimann & Lewin.

Cultures of Cylindrotheca furisormis grown either autotrohpically or heterotrophically on lactate contained significant amounts of NAD-dependent L(+)-lactate dehydrogenase (EC 1.1.1.27). Polyacylamide gel electrophoresis of crude enzyme extracts revealed a single band which was indistinguishable between autotrohpic and heterotrohpic cells. The Km for lactate of partially purified preparations was lower under heterotrophic conditions. The specific activity in crude extracts was higher under autotrophic than heterotrophic conditions; it dropped precipitously when autotrophic cells were transferred to the dark, increasing again only in the presence of lactate. These and related observations suggest that this enzyme has at most only a minor role in the assimilation of lactate during heterotrophic growth on lactate.     

叶绿素缺失和异养生长对盐藻Synechocysitis Sp PCC6803藻胆蛋白含量影响

通过遮黑培养缺失frxC基因的蓝藻Synechocystis sp.PCC 6803突变工程株,获得了叶绿素缺失的藻细胞,吸收光谱测定及数学计算表明,藻细胞中叶绿素缺失后藻胆蛋白含量增加,藻蓝蛋白和别藻蓝蛋白含量分别为相同条件下野生株对照组的4倍和6倍。野生株遮黑培养时,细胞进行异养生长, 藻胆蛋白含量下降,藻蓝蛋白和别藻蓝蛋白含量分别为光照培养条件下自养生长的野生株细胞的34.5％和25.3％。另外,缺失apcE基因的突变工程株细胞的藻胆蛋白含量也少于对照野生株,表明apcE基础因的编码蛋白Lcm与藻胆蛋白的含量相关。     

The impact of coral bleaching on the pigment profile of the symbiotic alga, Symbiodinium

Bleaching of corals by loss of symbiotic dinoflagellate algae and/or photosynthetic pigments is commonly triggered by elevated temperatures coupled with high irradiance, and is a first-order threat to coral reef communities. In this study, a high-resolution high-performance liquid chromatography method integrated with mass spectrometry was applied to obtain the first definitive identification of chlorophyll and carotenoid pigments of three clades of symbiotic dinoflagellate algae (Symbiodinium) in corals, and their response to experimentally elevated temperature and irradiance. The carotenoids peridinin, dinoxanthin, diadinoxanthin (Dn), diatoxanthin (Dt) and beta-carotene were detected, together with chlorophylls a and c2, and phaeophytin a, in all three algal clades in unstressed corals. On exposure to elevated temperature and irradiance, three coral species (Montastrea franksi and Favia fragum with clade B algae, and Montastrea cavernosa with clade C) bleached by loss of 50-80% of their algal cells, with no significant impact to chlorophyll a or c2, or peridinin in retained algal cells. One species (Agaricia sp. with clade C) showed no significant reduction in algal cells at elevated temperature and irradiance, but lost substantial amounts of chlorophyll a and carotenoid pigments, presumably through photo-oxidative processes. Two coral species (Porites astreoides and Porites porites both bearing clade A algae) did not bleach. The impact of elevated temperature and irradiance on the levels of the photoprotective xanthophylls (Dn + Dt) and beta-carotene varied among the corals, both in pool size and xanthophyll cycling, and was not correlated to coral bleaching resistance.     

High yield of hydrocarbon gases resulting from pyrolysis of yellow heterotrophic and bacterially degradedChlorella protothecoides

Four kinds of cells ofChlorella protothecoides, green autotrophic cells, bacterially degraded green autotrophic cells, yellow heterotrophic cells and bacterially degraded yellow heterotrophic cells, were used to simulate thermal degradation and gas formation by heating without oxygen at 300°C for 100 h. The yield of pyrolysed hydrocarbon gases in yellow heterotrophic cells with bacterial degradation was 8.5 times higher than that of green autotrophic cells without bacterial degradation. The use of bacterially degraded yellow heterotrophic cells resulted in relatively more lipid and less protein. The results suggest that the hydrocarbon-producing potential of microplanktonic algae in nature may be greater than previously thought based on studies of green autotrophic cells.     

Growth kinetics of algal populations exsymbiotic from Paramecium bursaria by flow cytometry measurements.

BACKGROUND: The ciliate Paramecium bursaria normally exists as a green paramecium system because each animal cell carries several hundred, unicellular, green, algal cells in its cytoplasm. One of the remarkable and poorly understood pecularities of this system is the steady state in the number of algae per protozoan cell. A major point in the study of mechanisms governing the persistence of symbiont numbers is adequate understanding of the algal life cycle. METHODS: Asynchronously growing cell populations of several algal strains (SA-1, SA-3, and SA-9) exsymbiotic from P. bursaria were characterized by flow cytometry. Algal endogenous chlorophyll and DNA contents were monitored to analyze cell growth kinetics at logarithmic and stationary culture phases. Cell sorting visualized the morphology of algae corresponding to the hyperhaploid (2C and 4C) DNA peaks. RESULTS: Cell-division cycle-dependent changes in chlorophyll and DNA content distributions were most dramatic in logarithmically growing algal populations (an increase in the number of S-phase cells and cells with more chlorophyll), which are thought to be associated with accelerated DNA and chlorophyll metabolism in log-phase algal cultures. Upon reaching the stationary phase of growth, algal populations distinctly showed, in addition to one haploid (1C) DNA peak, two hyperhaploid peaks (2C and 4C) corresponding mainly to cells with two and four nuclei, respectively. CONCLUSIONS: Growth characteristics of algae exsymbiotic from P. bursaria monitored by flow cytometry provide valuable information for the analysis of the algal life cycle, which is important for understanding the regulation mechanisms of symbiont numbers.     

An investigation of the heterotrophic culture of the green algaTetraselmis

The marine PrasinophyteTetraselmis may be cultured under both mixotrophic (photoheterotrophic) and heterotrophic conditions. The growth rate was slightly lower, and pigment levels and lipid composition were radically affected on heterotrophic culture in 1 L fermenters. Total chlorophyll levels of dark grown cultures were less than 1% of those observed in mixotrophically grown cells, the chlorophylla : b ratio also decreased as did the carotenoid content. In addition, the total amounts of lipids including polyunsaturated fatty-acids were also lower in heterotrophically cultured cells: 6.4 mg g^–1 (dried alga) and 0.35 mg g^–1 (dried alga); as compared to 37.1 mg g^–1 (dried alga) and 18.5 mg g^–1 (dried alga), for cells grown in the light. However, gross morphology and final yield (>16 g l^–1) were relatively unaffected. The algae produced were spray-dried and tested for their suitability as an aquaculture feed.Address for correspondence