Production of astaxanthin from Haematococcus in open pond by two-stage growth one-step process

We have developed a two-stage growth one-step process for cultivation of Haematococcus using a self-designed system that mimics an open pond in the natural environment. The characteristics of this process are green vegetative cell growth and cysts transformation and pigment accumulation that proceed spontaneously and successively in one open photobioreactor. Four strains of Haematococcus (H. pluvialis 26; H. pluvialis 30; H. pluvialis 34; H. pluvialis WZ) were cultured in this imitation system for a duration of 12 days. The changes in cell density and medium pH were closely monitored, and the astaxanthin content and yield of the four Haematococcus strains were measured at the end of 12 days of cultivation. Two of the strains, H. pluvialis 26 and H. pluvialis WZ, were selected as strains suitable for mass culture, resulting in the astaxanthin yield of 51.06 and 40.25 mg L-1 which are equivalent to 2.79 and 2.50% of their dry biomass respectively. Based on the laboratory work, 6 batch cultures of H. pluvialis WZ were conducted successfully to produce astaxanthin in two 100 m(2) open race-way pond by two-stage growth one-step process. The astaxanthin content ranged from 1.61 to 2.48 g 100 g(-1) dry wt., with average astaxanthin content of 2.10 g 100 g(-1) dry wt. Compared with the one-stage production of astaxanthin based on continuous culture, the superiority of our process is that it can accumulate much more astaxanthin in red cysts. Compared with two-stage production of astaxanthin, the advantage of our process is that it does not need to divide the production process into two parts using two bioreactors. The presented work demonstrates the feasibility for producing astaxanthin from Haematococcus using a two-stage growth one-step process in open pond, culture systems that have been successfully used for Spirulina and Chlorella mass culture. The future of Haematococcus astaxanthin production has been also discussed. (C) 2009 Elsevier B.V. All rights reserved.

Identification of a novel DNA methyltransferase 2 from the brine shrimp, Artemia franciscana

DNA methyltransferase 2 (Dnmt2) is a dual-specificity DNA methyltransferase, which contains a weak DNA methyltransferase and novel tRNA methyltransferase activity. However, its biological function is still enigmatic. To elucidate the expression profiles of Dnmt2 in Artemia franciscana, we isolated the gene encoding a Dnmt2 from A. franciscana and named it as AfDnmt2. The cDNA of AfDnmt2 contained a 1140-bp open reading frame that encoded a putative Dnmt2 protein of 379 amino acids exhibiting 32%similar to 39% identities with other known Dnmt2 homologs. This is the first report of a DNA methyltransferase gene in Crustacean. By using semi-quantitative RT-PCR, A)Dnmt2 was found to be expressed through all developmental stages and its expression increased during resumption of diapause cysts development. Southern blot analysis indicated the presence of multiple copies of AfDnmt2 genes in A. franciscana. (C) 2007 Published by Elsevier Inc.

The encysted dormant embryo proteome of Artemia sinica

The possibility of the brine shrimp Artemia to produce dormant embryo (cysts) in diapause is a key feature in its life history. In the present study, we obtained a proteomic reference map for the diapause embryo of Artemia sinica using two-dimensional gel electrophoresis with a pH range of 4-7 and a molecular weight range of 10-100 kDa. Approximately 233 proteins were detected, and 60 of them were analyzed by capillary liquid chromatography tandem mass spectrometry (LC-MS/MS). Of these, 39 spots representing 33 unique proteins were identified, which are categorized into functional groups, including cell defense, cell structure, metabolism, protein synthesis, proteolysis, and other processes. This reference map will contribute toward understanding the state of the diapause embryo and lay the basis and serve as a useful tool for further profound studies in the proteomics of Artemia at different developmental stages and physiological conditions.

雨生红球藻Rubisco在虾青素积累过程中的表达分析

虾青素因其具有极强的抗氧化性及优越的着色作用，被广泛应用于营养保健和水产养殖中，备受国内外研究者的关注。红球藻是目前虾青素的生物来源中较有优势的一条途径。 我们选取雨生红球藻作为研究材料，收集其生长过程中四个不同的阶段，分别为绿色游动细胞阶段，绿色不动细胞阶段，绿褐色不动细胞阶段（开始积累虾青素），以及红色不动孢子阶段。本研究中提取了核酮糖-1,5-二磷酸羧化/加氧酶（Rubisco）的粗提液，并测定了酶活。其编码基因rbcL的mRNA表达量也被测定。另外，光合速率与呼吸速率的比值（P/R）通过测定与计算得出，各个阶段藻细胞中虾青素的含量由分析得出。本研究还应用了叶绿素荧光测定方法，确定了光系统II潜在最大活性（Fv / Fm），光系统II实际活性（ΦPSII），电子传递速率（ETR）和非光化学淬灭参数（NPQ）。 结果表明，绿色游动细胞的生长状态最佳，其P/R、Fv / Fm、ΦPSII均为最大，NPQ为最小。这说明在此状态的细胞中，光系统II的活性最强；但是其Rubisco活性与rbcL表达量均为最小。相比之下，在绿褐色不动细胞中，P/R和NPQ的值较低，Fv / Fm、ΦPSII和ETR值都最小，但Rubisco活性与rbcL表达量均为最高。 结合工业生产虾青素的方法，我们认为，Rubisco或许参与了虾青素的合成，而非Calvin循环为色素合成提供前体和能量。因此，在生产过程中适当加入碳源，比如CO2，可以有效增大虾青素的产量。

红毛菜发育过程及其生理基础

红毛菜（Bangia Lyngb.）属于红藻门，与紫菜属同属红毛菜科，其味道和营养都优于紫菜。目前红毛菜栽培产业已在我国福建莆田展开，但栽培技术还有待提高。海藻栽培技术的发展和成熟依赖于对其生长发育过程的认识。本研究针对红毛菜发育过程及相关光合生理展开，并初步探讨了一采自山西娘子关泉淡水红毛菜群体（FWB）的系统地位。 色素突变标记的壳孢子萌发特征表明最初两次分裂产生的4细胞决定了完整植株的形态建成。成熟植株，为雌雄异体。雌性生殖器果胞的标志性分化结构为原始受精丝，环境因子是促发原始受精丝发展的外部因素，其膨大程度随受精的延迟而增大。原孢子是主要的无性生殖孢子类型，在不良环境中，藻体也会形成内生孢子或休眠包囊，或者藻体断裂后重新形成完整的植株。 红毛菜的生长发育很大程度上受环境因子的控制。高温不利于配子体的发育，15-20 ºC比较适宜。红毛菜无性繁殖的最适温度-光照组合为20 ºC-8 h，有性繁殖为15 ºC-12h。 不同发育阶段，PSII实际光合效率（Y(II)）与细胞的健康状况以及光合器官完整性及其在细胞内的分布有关，而与细胞的类型关系不大。健康的假根细胞、已分化未成熟的精子以及果孢子细胞均具有很高的Y(II)。色素体由中间位变为围周位，中央大液泡（营养藻丝）和大小纤维囊泡（成熟孢子与精子）的产生，使得细胞Y(II)降低。刚放散的壳孢子Y(II)很低，说明在壳孢子由贝壳基质释放到自由水体过程，光合作用受到一定程度抑制；而2h后，Y(II)开始恢复，rbcL的转录水平非常高，为孢子的萌发储备物质和能量需求。 在失水和低盐胁迫下，藻体均维持较高的Y(II)。干出处理至藻体重量不再变化，复水后Y(II)可回复初始水平。海生红毛菜在100％淡水培养基中（约20ºC）培养7天后，部分雄性藻体依然活着。从而体现了红毛菜位居高潮带的生理优势。 FWB终生行无性繁殖，藻体形态与发生以及染色体数目（4条）与海生群体没有区别。而rbcL-rbcS Spacer序列显示，红毛菜海生群体（无性和有性）具有完全相同的序列，而FWB与它们有5bp差异，但是与欧洲、北美地区的淡水群体仅1bp不同，初步说明所有淡水红毛菜群体具有共同的原始起源。

Recovery and fate of three species of marine dinoflagellates after yellow clay flocculation

The recovery and fate of three species of dinoflagellates, Alexandrium tamarense, Cochlodinium polykrikoides and Scrippsiella trochoidea, after having been sedimented by yellow clay, were investigated in the laboratory. The effect of burying period in yellow clay pellet and mixing on the recovery of settled algal cells were studied. The morphological changes of algal cells in yellow clay pellet were also tracked. Results showed that there was almost no recovery for A. tamarense and C. polykrikoides, and the cells decomposed after 2-3 days after visible changes in morphology and chloroplasts. There was some recovery for S. trochoidea. Moreover, S. trochoidea cysts were formed in clay pellet during the period of about 14 days, with the highest abundance of 87 000 cysts g(-1) clay and the incidence of cyst formation of 6.5%, which was considered as a potential threat for the further occurrence of algal blooms. S. trochoidea cysts were isolated from yellow clay and incubated to test their viability, and a germination ratio of more than 30% was obtained after incubation for 1 month. These results showed the species specificity of the mitigation effect of yellow clay. It is suggested that cautions be taken for some harmful species and thorough risk assessments be conducted before using this mitigation strategy in the field.

Ocean urea fertilization for carbon credits poses high ecological risks

The proposed plan for enrichment of the Sulu Sea, Philippines, a region of rich marine biodiversity, with thousands of tonnes of urea in order to stimulate algal blooms and sequester carbon is flawed for multiple reasons. Urea is preferentially used as a nitrogen source by some cyanobacteria and dinoflagellates, many of which are neutrally or positively buoyant. Biological pumps to the deep sea are classically leaky, and the inefficient burial of new biomass makes the estimation of a net loss of carbon from the atmosphere questionable at best. The potential for growth of toxic dinoflagellates is also high, as many grow well on urea and some even increase their toxicity when grown on urea. Many toxic dinoflagellates form cysts which can settle to the sediment and germinate in subsequent years, forming new blooms even without further fertilization. If large-scale blooms do occur, it is likely that they will contribute to hypoxia in the bottom waters upon decomposition. Lastly, urea production requires fossil fuel usage, further limiting the potential for net carbon sequestration. The environmental and economic impacts are potentially great and need to be rigorously assessed. (C) 2008 Elsevier Ltd. All rights reserved.

Descriptions of Protospathidium serpens (Kahl, 1930) and P-fraterculum n. sp (Ciliophora, Haptoria), two species based on different resting cyst morphology

Protospathidium serpens (Kahl, 1930) is frequent in semiterrestrial and terrestrial habitats worldwide. Conventionally, all populations are considered as conspecific because they have very similar overall morphologies and morphometrics. We studied in detail not only the morphology of the vegetative cells but also the resting cysts using live observation, protargol impregnation, and scanning electron microscopy. These revealed a cryptic diversity and biogeographic pattern in details of the dorsal brush and cyst wall morphology. The cyst wall is spiny in the Austrian specimens, while smooth in the South African and Antarctic populations. Accordingly, P. serpens consists of at least two species: P. serpens (with spiny cyst wall) and P. fraterculum n. sp. (with smooth cyst wall); the latter is probably composed of two distinct taxa differing by the absence (South African)/presence (Antarctic) of a monokinetidal bristle tail in brush row 3, the number of dikinetids comprising brush row 1 (seven versus three), and the total number of brush dikinetids (29 versus 17). Protospathidium serpens is neotypitied with the new population from Austria. The significance of resting cyst morphology is discussed with respect to alpha-taxonomy and overall ciliate diversity.