Interaction of cadmium and copper with surface-active organic matter and complexing ligands released by marine phytoplankton

The organic matter released by the marine phytoplankton species Dunaliella tertiolecta and its physico-chemical interaction with cadmium and copper ions were studied by electrochemical methods (differential pulse anodic stripping voltammetry (DPASV) and a.c. polarography). The interactions with cadmium and copper were studied at the model interface (mercury electrodesolution) and in the bulk phase by measuring the complexing ability of the released organic material.The axenic cultures were grown on different growth media, without and with trace metals and chelators. Culture media were analyzed 10 days after inoculation, containing 5 × 10⁵−1.2 × 10⁶ cells cm⁻³ when untreated or after separation of cells by gentle centrifugation.It was found that the content and type of the released surface-active material and complexing ligands depend on the initial composition of the growth media. In all cases, strong interaction of excreted organic substances with copper in the bulk phase and with cadmium at the model interface were observed.A rather high value of the complexing capacity, 9.5 × 10⁻⁷ mol Cu²⁺ dm⁻³, was found in the culture grown on medium without trace metals and chelators (medium I) whereas the surface activity of this culture was not high (0.2 mg dm⁻³ equivalent to Triton-X-100). Higher contents of surface-active material (0.8 and 1.0 mg dm⁻³) were found in cultures grown in media with trace metals and without chelators (II and III), accompanied by a high content of complexing ligands (5.8 × 10⁻⁷ and 9.5 × 10⁻⁷ mol Cu²⁺ dm⁻³). However, if the complexing capacity is calculated per cell the values obtained for cultures grown in media II and III (0.79 × 10⁻¹⁵ and 0.98 × 10⁻¹⁵ mol Cu²⁺ dm⁻³) are lower than for cultures grown on medium I (1.8 × 10⁻¹⁵ mol Cu²⁻ dm⁻³). The exceptional adsorption effects and the copper complexing capacity for medium 1, and the presence of cells with degenerative symptoms can be ascribed to stressed growth conditions, and, particularly, to deficiency of metals. A qualitatively similar behaviour has been observed in natural samples of estuarine waters, indicating the existence of stressed conditions during the mixing of fresh and saline waters.     

剧毒卡尔藻与东海原甲藻间的化感作用研究

为研究剧毒卡尔藻(Karlodinium veneficum)与东海原甲藻(Prorocentrum donghaiense)之间的化感作用,在f/2营养盐条件下,分别采用了混合培养及滤液培养的方法。结果表明:剧毒卡尔藻指数生长后期的无藻细胞滤液明显抑制了东海原甲藻的生长,而东海原甲藻滤液对剧毒卡尔藻的生长几乎无影响,化感作用作为重要的影响因素使剧毒卡尔藻获得竞争优势。将剧毒卡尔藻指数生长末期的无藻细胞滤液置于固相萃取(SPE) C18小柱,经吸附、不同比例的甲醇溶液洗脱,考察不同洗脱部分对东海原甲藻生长的影响,发现80%的甲醇洗脱部分明显抑制了东海原甲藻的生长。本研究为了解剧毒卡尔藻的竞争机制提供了重要的科学依据,今后将重点分析剧毒卡尔藻释放的有效抑制东海原甲藻生长的化感物质。     

Complexation of zinc by exudates from Skeletonema costatum grown in culture

Measurements have been made, by amperometric titration using anodic stripping voltammetry, of the zinc binding properties of organic material released by Skeletonema costatum in culture, after the stationary growth phase had been reached. Titration curves showed two inflection points, interpreted as indicating the presence of two analytically distinguishable ligand assemblages (L₁ and L₂). The complexing capacities of these assemblages increased with time and the associated conditional stability constants K₁^′ and K₂^′ decreased with time. This apparent reduction in stability constants could be accounted for by complex dissociation. Several methods for the calculation of complexing capacity and stability constant were employed and although the calculated concentrations of C_L varied, each method showed similar trends.Size fractionation of the organic moieties revealed that C_L, was composed of organic assemblages with apparent molecular weight greater than 3 × 10⁴. The ligands L₂ were of mixed size composition.     

Copper complexing capacity of phytoplanktonic cell exudates

Copper complexing capacity of cell exudates of Dunaliella salina in natural seawater culture medium was investigated in order to evaluate the influence of this organism on speciation of trace metals in seawater.Seawater samples were collected at 200 m and 2 miles off the coast and immediately filtered. Copper complexing capacity (CC_Cu) and stability constants (K′) of related cupric complexes were then measured. They were, respectively, 27.1 × 10⁻⁸ mol l⁻¹ and 0.56 × 10⁷ l mol⁻¹ for the samples collected at 200 m and 12.8 × 10⁻⁸ mol l⁻¹ and 6.10 × 10⁷ l mol⁻¹ for those collected 2 miles off the coast. A stock culture (20 ml, 10⁶ cells ml⁻¹) in log-phase was inoculated in 2 l of each sample of filtered natural seawater. The trend of cell influence was estimated on filtered culture medium by measuring CC_Cu and K′ after 1 h, 3 and 7 days. From the results it appears that CC_Cu increased with respect to time and this was related to the growth rate, indicating a certain relationship with cell metabolic activity.It can be concluded that a comparison between the culture referring to 200 m and 2 miles, respectively, shows that the former presents a CC_Cu two times higher than the latter while the K′ is ten times higher at 2 miles than that at 200 m.     

Iron availability and the release of iron-complexing ligands by Emiliania huxleyi

The ubiquitous algal species, Emiliania huxleyi, was incubated in sea water supplemented only with nitrate and phosphate (N and P) without chelating agents to control metal speciation. Growth was slow in a “low-iron” culture containing 1.3 nM iron and was found to be iron-limited, growth-accelerating when a 1-nM iron addition was made. The growth rate in a “high-iron” culture (5.4 nM iron) was greater, reaching 0.4 div day⁻¹ but this culture too was found to have become iron-limited when a 9-nM iron addition was made on day 17 of the incubation. Both cultures were found to release iron-complexing ligands in excess of the iron concentration, 6 nM in the low-iron culture, and 10 nM in the high-iron culture. More ligands were produced after the iron addition taking the ligand concentration to 11 nM in the low-iron culture. The data show that the ligands are released in response to the iron addition, when at least some of the iron had already been taken up. This type of release is contrary to the concept of a siderophore, which is supposed to be released in periods of lack of iron; however the increase in the ligand concentration is similar to that released by the natural community in response to the iron addition in the IRON-EX II experiment [Rue, E.L., Bruland, K.W., 1997. The role of organic complexation on ambient iron chemistry in the equatorial Pacific Ocean and the response of a mesoscale iron addition experiment. Limnol. Oceanogr. 42, 901–910]. The enhanced growth in the cultures when more iron was added indicated that the organically complexed iron present in the cultures was not immediately available to the organisms (or at least not at sufficiently high rate), and that the organisms responded to freshly added, inorganic, iron.     

Allelopathic growth inhibition of Heterosigma akashiwo by the three Ulva spcieces (Ulva Pertusa, Ulva Linza, Enteromorpha intestinalis) under laboratory conditions

Allelopathic effects of several concentrations of fresh tissue,dry powder and dry tissue of three bloom-forming green macroalgae Ulva pertusa,Ulva linza and Enteromopha intestinalis on the red tide microalga Heterosigma akashiwo were evaluated in microcosms systems.The effects of macroalgae culture medium filtrate were investigated on H.akashiwo using initial or semi-continuous filtrate addition.Preliminary studies on the algicidal effects of one aqueous and four organic solvent extracts from the macroalgae on the microalga were carried out to confirm the existence of allelochemicals in the tissue of these macroalgae.The dry powder of U.pertusa was extracted with methanol,and the methanol extracts were partitioned to petroleum ether phase,ethyl acetate phase,butanol phase and distilled phase by liquid-liquid fractionation.The bioassays of the activity of every fraction were carried out on H.akashiwo.The resultant microcosms assay showed that the growth of H.akashiwo was strongly inhibited by using fresh tissues,dry powder or dry tissue of these three macroalgae,while aqueous and methanol extracts of both macroalgae had strong inhibitory effects on the growth of H.akashiwo,and the EC 50 values for methanol extract of U.pertusa,U.linza or E.intestinalis were 0.016,0.028×10-12 or 0.033×10-12,respectively.While the other three organic solvent extracts（acetone,ether and chloroform） had no apparent effect on its growth,this suggests that the allelochemicals from these three macroalgae had relatively high polarities.The activity of petroleum ether phase,ethyl acetate phase,butanol phase and distilled phase of U.pertusa methanol extract was carried out on H.akashiwo indicating that petroleum ether phase and ethyl acetate phase had stronger algicidal effect on H.akashiwo.The inhibition effect of the ethyl acetate phase was not as strong as that of petroleum ether phase,and effective concentration of petroleum ether phase was 17 mg/L for H.akashiwo.However,no significant algicidal effects were observed on the butanol phase and distilled water phase.These three macroalgae＇s culture medium filtrate exhibited no apparent growth inhibitory effect on the microalga under initial filtrate addition whereas the growth of H.akashiwo was significantly inhibited under semi-continuous filtrate addition,which suggests that continuous release of small quantities of rapidly degradable allelochemicals from the fresh tissue of both macroalgae was effective in inhibiting the growth of H.akashiwo.     

Complexation of zinc by exudates of Thalassiosira fluviatilis grown in culture

Measurements have been made, by amperometric titration using anodic stripping voltammetry, of the zinc-binding properties of organic material released by Thalassiosira fluviatilis in culture over a period from the beginning of growth to senescence. After the onset of the stationary phase, titration curves showed two inflection points, interpreted as indicating the presence of two analytically distinguishable ligand assemblages (L₁ and L₂). The complexing capacities (C_L) of these assemblages changed with time, C_L1 increasing linearly from <0.5 × 10^?8 M to 2.0 × 10^?6 M into the stationary phase and then more rapidly during senescence, and C_L2 increasing after its initial appearance to a final value of 4.4 × 10^?6 M. These changes were accompanied by systematic decreases in the corresponding conditional stability constants (K′) calculated for 1 : 1 associations of the ligands with zinc. Values of log K′₁ decreased from 6.6 to 5.8, and those of log K′₂ from 7.2 to 6.2. The presence of the second ligand assemblage may be related to the release of intracellular material during senescence.     

Lagrangian observation of phytoplankton dynamics at an artificially enriched subsurface water in Sagami Bay,Japan

Phytoplankton dynamics in the lower euphotic zone were observed by tracking a subsurface water released at 20-m depth from Takumi, an artificial upwelling device. Takumi continually discharged seawater pumped up from a depth of 205 m: this water was mixed with 5-m depth water to adjust the density to that of 20-m depth water of Sagami Bay, Japan. The discharged water was pulse-labeled at Takumi with uranine and tracked for 63.9 h with a drifting buoy equipped with a drogue at 20-m depth. We present a simple model to estimate in situ phytoplankton net growth rates from temporal changes in phytoplankton abundance in the discharged water with correction for the influence of water exchange between the discharged water and neighboring layers. Lagrangian observation showed active growth of pico- and nanophytoplankton, especially cryptophytes and Synechococcus (Cyanobacteria), in the subsurface layer. In contrast, diatoms grew little in spite of micromolar concentrations of nutrients. The active growth of pico- and nanophytoplankton was in good agreement with shipboard serial dilution culture experiments. The low growth activity of diatoms was suggested to be related to low light availability in the subsurface layer.     

Contrasting complexing capacity of dissolved organic matter produced during the onset,development and decay of a simulated bloom of the marine diatom Skeletonema costatum

The capacity of natural dissolved organic matter (DOM) produced during the onset, development and decay of a simulated bloom of the marine diatom Skeletonema costatum to complex free copper has been followed for a 2 week period. Copper binding capacity of the culture was measured by anodic stripping voltammetry (ASV) with a hanging mercury drop electrode (HMDE). The concentration of dissolved organic carbon (DOC) and two fluorophores, M (humic-like, Ex/Em: 320 nm/410 nm) and T (protein-like, Ex/Em: 280 nm/350 nm), were followed during the course of the incubation. Models using DOC concentrations alone could not accurately predict the complexing capacity of the culture, especially at the end of the bloom, and better predictions were obtained when fluorescence measurements were considered. They were helpful in characterising two types of copper ligands produced in the culture. The first type, traced by the fluorescence of peak T, was related to labile DOC directly exuded by phytoplankton. The second type, traced by the fluorescence of peak M, was the refractory humic-like material presumably produced in situ as a by-product of the bacterial degradation of phytogenic materials. During the onset and development of the bloom (days 0 to 7), the fluorescence of peak T explains 60–80% of the total complexing capacity of the culture, suggesting that exuded “protein-like” compounds among other exuded complexing agents efficiently complexed free copper. On the contrary, during the decay (days 8 to 13), these ligands were replaced by humic substances as the complexing agent for copper.     

抗生素对浒苔形态及共附生细菌多样性的影响

本文研究了3种抗生素联合处理黄海绿潮浒苔(Ulva prolifera)形态及共附生细菌多样性的影响。将浒苔放散的配子加入到含有3种广谱抗生素(青霉素G、硫酸新霉素、多黏菌素B)的培养液中进行培养, 与对照组相比, 发现浒苔幼苗生长速度缓慢, 且细胞变小, 藻体无法形成正常形态。进一步采用16S rDNA高通量测序分析抗生素处理藻体前后的共附生细菌多样性, 发现抗生素处理后浒苔共附生细菌丰富度和多样性降低, 其中Maribacter丰度降低。将藻体与形态发生化合物thallusin(Maribacter spp. 提取物)共培养, 发现thallusin可促进藻体形态恢复和生长, 推测抗生素可通过抑制Maribacter等细菌从而影响浒苔形态和生长, 这将为浒苔共附生细菌功能的深入研究奠定一定基础。     

Organic complexation and its control of the dissolved concentrations of copper and zinc in the Scheldt estuary

Cathodic stripping voltammetry (CSV) is used to determine total (after UV-irradiation) and labile dissolved metal concentrations as well as complexing ligand concentrations in samples from the river Scheldt estuary. It was found that even at high added concentrations of catechol (1 m for copper and 0·4 m for iron) and of APDC (1 m for zinc) only part of the dissolved metal was labile (5–58% for copper, 34–69% for zinc, 10–38% for iron); this discrepancy could be explained by the low solubility of iron which is largely present as colloidal material, and by competition for dissolved copper and zinc by organic complexing ligands. Ligand concentrations varied between 28 and 206 n for copper and between 22 and 220 n for zinc; part of the copper complexing ligands could be sub-divided into strong complexing sites with concentrations between 23 and 121 n and weaker sites with concentrations between 44 and 131 n . Values for conditional stability constants varied between (logK′ values) 13·0 and 14·8 for strong and between 11·5 and 12·1 for weaker copper complexing ligands, whereas for zinc the values were between 8·6 and 10·6. The average products of ligand concentrations and conditional stability constants (a-coefficients) were 6 × 10² for zinc and 6 × 10⁶ for copper.The dissolved zinc concentration was found to co-vary with the zinc complexing ligand concentration throughout the estuary. It is argued that the zinc concentration is regulated, in this estuary at least, by interactions with dissolved organic complexing ligands. A similar relationship was apparent between the dissolved copper and the strong copper complexing ligand concentration. The total copper complexing ligand concentrations were much greater than the dissolved copper concentrations, suggesting that only strongly complexed copper is kept in solution.These results provide evidence for the first time that interactions of copper and zinc with dissolved organic complexing ligands determine the geochemical pathway of these metals.     

利用光生物反应器培养雨生红球藻的研究初探

用光生物反应器与光照培养箱两种培养容器培养雨生红球藻(Haematococcus pluvialis),对雨生红球藻在两种容器培养过程中细胞生长、pH值、溶解氧(DO)及虾青素积累情况进行比较,同时比较不同接种密度对光生物反应器培养效果的影响。实验表明,光生物反应器中藻细胞的调整期较短,接种1 d后即进入指数生长阶段,在胁迫阶段则仅需4 d即达到虾青素含量的峰值;将pH值控制在偏碱性条件下(7.75±0.10)有利于藻细胞更好生长;营养培养阶段DO相对饱和度上升至80%,而在胁迫阶段则迅速降低,最低值小于6%;较高的接种密度(2.3×104个/mL)具有较短的营养培养周期(7 d),且因接种密度变化对胁迫周期长短无明显影响(均为4 d),选用较高的接种密度可望降低工业生产成本。     

中肋骨条藻与微小亚历山大藻间的他感作用研究

在f/2全营养培养条件下,采用共培养以及滤液交叉培养的方法研究了中肋骨条藻(Skeletonema costatum)和微小亚历山大藻(Alexandrium minutum)之间的竞争作用,结果表明: 微小亚历山大藻指数生长后期的无藻细胞滤液对中肋骨条藻的生长没有抑制作用,但中肋骨条藻滤液明显抑制了微小亚历山大藻的生长,且抑制作用随着中肋骨条藻滤液比例的增大而增大,他感作用是影响这两种微藻间竞争的重要方式之一;通过向培养基中添加自他感物质标准品15S-hydroxy-5Z,8Z,11Z,13E,17Z-eicosapentaenoic acid (15(S)-HEPE)考察其对中肋骨条藻自身以及微小亚历山大藻生长的影响,发现15(S)-HEPE在中肋骨条藻滤液中并不是能够抑制微小亚历山大藻生长的他感物质,今后应进一步深入分析中肋骨条藻释放的抑制微小亚历山大藻生长的他感物质。     

兼养对雨生红球藻细胞生长的促进作用及藻株差异性

结合叶绿素荧光技术,对比研究了不同雨生红球藻藻株在兼养(光照+乙酸钠)培养过程中细胞生长和光合作用的共性与差异性反应。结果表明:兼养明显提高红球藻细胞密度和比生长速率;乙酸钠不仅为红球藻提供异养的有机碳源,而且还明显导致藻细胞光自养的光合综合性能指数发生改变,兼养对细胞生长促进作用的贡献在前期主要来自于其中的异养部分,而在后期却明显来自于自养部分;兼养对细胞生长的促进作用在藻株之间存在一定差异,不同藻株对乙酸钠的适应能力和最适质量浓度也存在明显差异,藻株H0可适应的乙酸钠质量浓度较高,而藻株H6对乙酸钠需求和适应性相对较低。上述结果意味着规模化培养红球藻过程中,针对不同藻株采用适宜乙酸钠兼养方式可有效缩短培养周期,提高生产效率。     

小角刺藻生长过程中溶解游离氨基酸含量在海水中的变化

于1986年12月-1987年2月在温度为25±1℃下培养小角刺藻，以HPLC法测定该藻在培养过程中海水的溶解游离氨基酸（DFAA）含量变化。结果表明，小角刺藻不仅是海水中DFAA的主要生产者，而且也是DFAA的消耗者。小角刺藻不仅在缺乏硝酸盐的时候要吸收DFAA，即使在硝酸盐充足的情况下也会吸收。在小角刺藻的生长初期，水体中DFAA的含量迅速降低，而在后期却又大大增高。在小角刺藻的不同生长阶段，水体中DFAA的组成也不同。这些都可以说明海洋中DFAA的含量、组成和地区分布是密切地同浮游植物群落的繁殖消亡过程相关联的。     

牙鲆苗种大规模培育

牙鲆Paralichthys olivaceus是鲽形目鱼中的一种经济鱼类,具有生长迅速、繁殖力强、肉质鲜美等优点,为我国北方人民所喜爱食用。目前日本已把它作为重要的养殖对象。 早在五十年代末期,我所脊椎动物研究室就开展了牙鲆人工育苗的研究,并取得实验性培苗的成功。 从1983年开始,我们在过去研究的基础上,开展了牙鲆苗种大规模培育试验。主要目     

无机碳与雨生红球藻(Haematococcus pluvialis)细胞调节物质

以单细胞雨生红球藻为材料，采用酸碱滴定和CO2加富通气培养微藻的方法，对旧液中HCO3^-和CO3^2-浓度变化以及对红球藻细胞生长的影响进行了研究。结果表明，旧液具有限制红球藻细胞生长和诱导细胞转化的作用。同时，旧液中无机碳离子浓度明显高于新液。培养液中富含CO3^2-时，各细胞数量与CO3^2-浓度呈正相关，相关系数为0．88。溶液中仅有HCO3^-时，各细胞数与HCO3^-浓度也呈正相关性。因此，排除了CO3^2-和HCO3^-作为旧液中的调节物质，限制红球藻细胞生长和诱导细胞转化的可能性。旧液乙酸乙酯提取物生物检测实验表明，在粗提取物中有降低细胞增长和诱导细胞转化的活性，表明调节物质能溶于有机相，也反过来证实无机碳离子不是旧液中的调节物质。DNA含量和倍性分析结果表明，红球藻游动细胞DNA复制可以加倍后不经过原生质分裂就可以再次进行，因此推测旧液中的调节物质对原生质分裂过程产生抑制作用，而不对DNA复制过程产生抑制作用。     

Aquaculture of Sarcothalia atropurpurea tetraspores in New Zealand for carrageenan production

The endemic red seaweed Sarcothalia atropurpurea is a potentially valuable source of carrageenan, but no information is available on growing this species as a crop in New Zealand. Tetraspores released from wild-harvested reproductive thalli were settled onto strings and grown initially in tanks under two different day-lengths. Seeded strings were transplanted to open water at 2, 6 and 10 weeks after spore release. Gametophytic biomass was harvested twice by pruning plants approximately six and nine months after spore release. Sporeling length was correlated with day length during tank culture but these differences were not apparent after a period of open-water culture. Spores cultured with medium day-length for 6–10 weeks before transfer to open water grew into harvestable plants that contained similar amounts and types of carrageenan to that from wild material. These results demonstrate for the first time that aquaculture of gametophytic S. atropurpurea from tetraspores is possible.     

微小亚历山大藻(Alexandrium minutum)的氮磷吸收、生长、产毒动态变化

采用浮游植物计数法、高效液相色谱串联质谱法、分光光度法等分析方法,探索了微小亚历山大藻(Alexandrium minutum)在批次培养过程中氮磷吸收、产毒、生物量、p H等参数的动态变化关系。结果表明,微小亚历山大藻对磷的吸收迅速,可以将磷储存于体内,待生长使用。该藻对氮的吸收相对缓慢,环境中氮缺乏时,产毒量不再增加,说明氮对产毒有着重要的调控作用。在第二对数期中出现了碳限制环境,导致叶绿素a在碳限制条件下无法表征藻的生物量,相反,叶绿素a和生物量呈负线性关系,可能是叶绿素转化成其它含碳物质,用于生长。毒素不仅存在于细胞体内,培养液中(胞外)也含有毒素,并且胞外毒素从稳定期开始逐渐升高。胞内毒素的组成中GTX1/4占据绝对优势,GTX2/3含量相对较少。生长延缓期和第一对数期,各种毒素组成比例相对稳定,而在随后的生长期内,GTX1/4在总毒素中的占比逐渐上升,GTX2/3占比逐渐下降,表明微小亚历山大藻毒素组成会随着生长周期的变化而发生变化。     

A high sensitivity, low volume HPLC method to determine soluble reactive phosphate in freshwater and saltwater

A reverse phase High Performance Liquid Chromatography (HPLC) method was developed to detect soluble reactive phosphate (SRP) in marine and freshwater samples. This technique is scalable over a range of sample volumes from 10 μl to 10 ml, requires minimal sample preparation, and responds linearly from 1 nM to 100 μM (R²>0.964). Standard Murphy–Riley molybdenum blue reagents were used to react with phosphate in samples for analysis. Solid phase extraction tubes were used to clean the mixed complexing reagent prior to combining the reagent with samples. The method was applied to freshwater samples collected in July 2001 along an Andean mountain stream located in the Eastern Cordillera (2400 m) of Peru and marine samples collected in January 2002 within the low marsh of the estuarine Nueces Delta System, Texas Coastal Bend. For method comparison, field samples were analyzed using a standard manual spectrophotometric method. Field sample phosphate concentrations produced from each method were nearly identical. This method offers the advantages of low sample volume (1 ml or less) with high sensitivity, precision, and potential automation.