Rapid and comprehensive evaluation of microalgal fatty acids via untargeted gas chromatography and time‐of‐flight mass spectrometry

Due to their high triacylglyceride content, microalgae are intensively investigated for bio‐economy and food applications. However, lipid analysis is a laborious task incorporating extraction, transesterification and typically gas chromatographic measurement. Co‐elution induces a significant risk of fatty acid misidentification and thus, additional purification steps like thin layer chromatography are needed. Contrary to database matching approaches, solely targeted analysis is facilitated as compound identification is driven by matching retention times or indices with standard substances. In this context, a rapid workflow for the analysis of algal fatty acids is presented. In‐situ transesterification was used to simplify sample preparation and conditions were optimized towards fast processing. If results are needed at the very day of sampling, direct processing without a preceding drying step is feasible to obtain a rough estimate about the occurrence of the major compounds. Coupling gas chromatography and time‐of‐flight mass spectrometry enables untargeted analysis. Unknown compounds may be identified by structural reconstruction of their respective fragmentation patterns and by database matching to routinely avoid mismatches by co‐elution of disturbing agents. The developed workflow was successfully applied to derive the exact stereochemistry of all fatty acids from Chlorella vulgaris and a systematic shift depending on physiological state of the cells was confirmed.     

起始生物量比对3种海洋微藻种间竞争的影响

为深入了解饵料微藻与赤潮微藻间的种间竞争关系,通过微藻共培养的方法,研究了起始生物量比(1:4、1:1和4:1)对3种海洋微藻(塔玛亚历山大藻、蛋白核小球藻和湛江等鞭金藻)两两之间种间竞争的影响,并对其作用机制进行了探讨。结果表明:①3种海洋微藻表现出种间竞争的相互抑制效应;②在与塔玛亚历山大藻(简称A)的种间竞争中,蛋白核小球藻(简称C)和湛江等鞭金藻(简称I)均在竞争中占优势,蛋白核小球藻随自身起始生物量比的提高,其竞争优势越加明显,湛江等鞭金藻在A:I=1:1时竞争优势最为明显;在蛋白核小球藻和湛江等鞭金藻的种间竞争中,当C:I=1:4时,湛江等鞭金藻在竞争中占优势,C:I=1:1时,初期湛江等鞭金藻占竞争优势,随蛋白核小球藻的迅速生长,后期蛋白核小球藻占竞争优势,C:I=4:1时,蛋白核小球藻占绝对竞争优势;③由种间竞争抑制参数比较得出:3种微藻的种间竞争强弱依次为蛋白核小球藻>湛江等鞭金藻>塔玛亚历山大藻。蛋白核小球藻和湛江等鞭金藻在起始比例C:I=1:1时,可共培养利用,在海产经济动物育苗中可对其进行适时采收投喂;两种饵料藻对塔玛亚历山大藻具有明显的抑制作用,可为开发利用饵料藻进行赤潮生物防控提供一定的科学依据。     

利用Chlorella vulgaris C9-JN2010去除蓝藻-猪粪沼液废水中氮磷的研究

利用普通小球藻Chlorella vugaris C9-JN2010处理蓝藻-猪粪沼液废水,以实现废水无害化利用。实验考察了氮磷比和沼液浓度对小球藻生长及处理废水效果的影响,结果表明:在氮磷比(20:1)和沼液浓度(5%)条件下培养小球藻,藻细胞生长和废水处理效果最佳,最高细胞干重及生产强度分别为900.1 mg·L~(-1)和85.1 mg·L~(-1)·d~(-1),废水中总氮、总磷、氨氮的去除率分别为84.6%、95.9%和90.5%,对应去除强度分别为5.43 mg·L~(-1)·d~(-1)、0.30 mg·L~(-1)·d~(-1)和4.75 mg·L~(-1)·d~(-1)。利用小球藻可较彻底的去除蓝藻-猪粪沼液废水中氮、磷等营养,达到污水处理效果。     

不同光质对小球藻光自养培养积累油脂的影响

研究了5种光质对小球藻(Chlorella vulgaris)M209256生长和产油的影响。结果表明:蓝光为小球藻的最适生长和产油光质;与其他光质相比,蓝光培养的小球藻生物量和油脂含量均较高,为2.40×107个/mL和28%;红光培养的小球藻生长最慢且油脂含量最低,为1.32×107个/mL和15.13%,表现出明显的"红降"现象。在GCMS分析的基础上,对油脂甲酯化后的十六烷值进行评估,结果发现:蓝光的十六烷值最高;5种光质培养的小球藻所产油脂,甲酯十六烷值均在47以上。因此,小球藻油脂所制备的生物柴油具有较好的燃烧性能。     

Intron Conservation in a UV-Specific DNA Repair Gene Encoded by Chlorella Viruses

Large dsDNA-containing chlorella viruses encode a pyrimidine dimer-specific glycosylase (PDG) that initiates repair of UV-induced pyrimidine dimers. The PDG enzyme is a homologue of the bacteriophage T4-encoded endonuclease V. The pdg gene was cloned and sequenced from 42 chlorella viruses isolated over a 12-year period from diverse geographic regions. Surprisingly, the pdg gene from 15 of these 42 viruses contain a 98-nucleotide intron that is 100% conserved among the viruses and another 4 viruses contain an 81-nucleotide intron, in the same position, that is nearly 100% identical (one virus differed by one base). In contrast, the nucleotides in the pdg coding regions (exons) from the intron-containing viruses are 84 to 100% identical. The introns in the pdg gene have 5′-AG/GTATGT and 3′-TTGCAG/AA splice site sequences which are characteristic of nuclear-located, spliceosomal processed pre-mRNA introns. The 100% identity of the 98-nucleotide intron sequence in the 15 viruses and the near-perfect identity of an 81-nucleotide intron sequence in another 4 viruses imply strong selective pressure to maintain the DNA sequence of the intron when it is in the pdg gene. However, the ability of intron-plus and intron-minus viruses to repair UV-damaged DNA in the dark was nearly identical. These findings contradict the widely accepted dogma that intron sequences are more variable than exon sequences. Received: 13 May 1999 / Accepted: 20 August 1999     

THE HOST RANGE OF THE CHLORELLAVOROUS BACTERIUM ("VAMPIROVIBRIO CHLORELLVORUS")

The chlorellavorous bacterium . "Vampirovibrio chlorellavorus" Gromov et Mamkaeva, grows only by killing and consuming the cell contents of species of the green alga Chlorella Beijerinck. Of the 76 algal strains examined in this study, the bacterium attacks all 31 strains of the species C. vulgaris. C. sorokiniana, and C. kessleri, but attacks only two of 39 strains of nine other Chlorella species. Neither of two Prototheca strains was susceptible to attack. This narrow host specificity may be related to cell surface properties .     

Protective role of 20-hydroxyecdysone against lead stress in Chlorella vulgaris cultures

Treatment of cultured C. vulgaris cells with 10(-6)-10(-4) M lead decreased their growth and chemical composition during the first 48 h of cultivation. However, at concentrations above 10(-4) M, lead is cytotoxic to Chlorella vulgaris cells, resulting in cellular fragmentation and lysis. In contrast, at concentrations below 10(-6) M lead had no influence on the growth and metabolism of C. vulgaris cells. 20-Hydroxyecdysone (20E) (10(-10)-10(-8) M) increased growth and chemical composition of C. vulgaris cells over a concentration range. Levels per cell of chlorophylls, protein, sugars are all increased by 20E treatment, when compared to non-treated control cells. However, the cultures treated with 20E and lead show a lower stimulation than the cultures treated with 20E alone. The effects of 20E mixed with lead on the growth and the level of cellular lead, chlorophyll, sugar and protein in C. vulgaris are also reported. The decreased growth and composition of C. vulgaris cells treated with lead was restored by the 20E. Application of 20E to C. vulgaris cultures reduced the impact of lead stress on growth, prevented chlorophyll, sugar and protein loss and increased phytochelatins synthesis. Furthermore, 20E did not restore toxic effect of lead on C. vulgaris cells. The combined treatment with lead and 20E appeared to have a stimulatory effect on the above parameters during the 48 h of cultivation, as compared to the control. 20E reduced the toxicity of lead and the growth recovered to the level of cells treated with 20E alone. Concentration-dependent stimulation was observed with increasing concentration of 20E and decreasing concentration of lead.     

Possible evidence for novel metabolism of nitrogen gas by a green alga

We report the isolation of a cukaryotic green alga ( Chlorella , strain WPI-2) which accumulates large stores of nitrogen (N) during growth in N-free medium and seems to incorporate¹⁴N₂, yet does not reduce acetylene to ethylene. Total N accumulation during growth on N-free medium and in gases free of combined N was measured by three methods: Kjeldahl, oxidative pyrolysis via chemiluminescence (Antek N analyzer), and Dumas (Coleman N analyzer). Increases in N ranging from 22–64%± 1% were observed. Isotope dilution studies using cells labelled with ¹⁵NO ₃- and then shifted to ¹⁴N₂ in N-free medium showed dilution of the ¹⁵N isotope by ¹⁴N from 5.67 to 5.32%± 0.05%. Using a variety of conditions, we were unable to demonstrate the reduction of acctylene to ethylene by WPI-2, although diazotrophic cyanobacteria gave positive results. Although the data on WPI-2 are not conclusive in establishing this alga as a diazotroph, the data do suggest that within the Chlorophyceae there may exist a novel form of nitrogen gas metabolism.     

Development of an immunoassay to detect insect contamination of microalgal products

While the insect fragment count is currently the primary test used for assessing insect contamination of food products, this technique is very problematical for assaying microalgal materials. An account is given of a new immunoassay technique,which is based on an enzyme-linked immunosorbent assay(ELISA) detection of insect myosin and which provides a rapid and convenient means of quantitatively determining the amount of insect contamination in algal product samples with a high degree of replicability. Up to 30 samples can be tested in duplicate in 2.5–3 h. Experiments were carried out with a variety of common contaminant insects of algal products, using various life stages, including Corixidae, Ephydridaeand Chironomidae using both Spirulina (Arthrospira) and Chlorella as typical algal materials. As little as one insect per 50 g sample can readily be detected, with excellent correlation (r² = 0.99) between the number of insects present and the color produced. A matrix analysis to determine the ruggedness of the immunoassay was carried out following the protocols of the AOAC International and established that minor departures in seven variables from the standard assay resulted in no substantial differences. The insect myosin assay offers a quantitative and reliable means for assessing insect contamination of algal materials and should be considered for adoption as a standard method for this type of product. This revised version was published online in August 2006 with corrections to the Cover Date.     

Exogenous 24-epibrassinolide (EBL) facilitates cell growth of Chlorella pyrenoidosa under high temperatures by enhancing the photosynthetic energy utilization and alleviating oxidative damage

The microalga Chlorella pyrenoidosa is cultivated extensively for its constituents, which are of significant economic worth. Large-scale growth of C. pyrenoidosa in outdoor environments is subject to various stressors such as elevated temperature. The purpose of this study was to assess the protective effects of exogenous 24-epibrassinolide (EBL) on C. pyrenoidosa under high-temperature conditions. Compared to a temperature of 30°C, increasing the temperature to 43°C reduced the enzymatic capacity for carbon assimilation and resulted in the buildup of reactive oxygen species (ROS), thus reducing photosynthesis and proliferation. It was observed that exogenous EBL protected C. pyrenoidosa cells against high temperatures, with an optimal EBL concentration of 100 nM, resulting in enhanced capacity for photosynthetic carbon assimilation with a notable reduction in the imbalance between the absorption of light and energy used under high-temperature conditions. The addition of 100 nM EBL resulted in a 25.4% increase in cell density when exposed to elevated temperatures for 7 days. In addition, exogenous EBL reduced ROS production and increased the activities of critical antioxidant enzymes. This, in turn, mitigated heat-induced oxidative damage, resulting in advantageous outcomes in terms of cellular development and maintenance.