Using fluorochemical as oxygen carrier to enhance the growth of marine microalga Nannochloropsis oculata

The commercial value of marine Nannochloropsis oculata has been recognized due to its high content of eicosapentaenoic acid (>50 % w/w). To make it as a profitable bioresource, one of the most desirable goals is to develop a quality-controlled, cost-effective, and large-scale photobioreactor for N. oculata growth. Generally, closed culture system can offer many advantages over open system such as small space requirement, controllable process and low risk of contamination. However, oxygen accumulation is often a detrimental factor for enclosed microalgal culture that has seriously hampered the development of microalga-related industries. In this study, we proposed to use fluorochemical as oxygen carrier to overcome the challenge where four liquid fluorochemicals namely perfluorooctyl bromide, perfluorodecalin, methoxynonafluorobutane, and ethoxynonafluorobutane were investigated separately. Our results showed that the microalgal proliferation with different fluorinated liquids was similar and comparable to the culture without a fluorochemical. When cultured in the photobioreactor with 60 % oxygen atmosphere, the N. oculata can grow up in all the fluorochemical photobioreactors, but completely inhibited in the chamber without a fluorochemical. Moreover, the perfluorooctyl bromide system exhibited the most robust efficacy of oxygen removal in the culture media (perfluorooctyl bromide > perfluorodecalin > methoxynonafluorobutane > ethoxynonafluorobutane), and yielded a >3-fold increase of biomass production after 5 days. In summary, the developed fluorochemical photobioreactors offer a feasible means for N. oculata growth in closed and large-scale setting without effect of oxygen inhibition.     

Light/dark cycles in the growth of the red microalga porphyridium sp

The effect of light/dark cycles on the growth of the red microalga Porphyridium sp. was studied in a tubular loop bioreactor with light/dark cycles of different frequencies and in two 35-L reactors: a bubble column reactor and an air-lift reactor. Photon flux densities were in the range of 50 to 300 μE m-2 s-1, and flow rates were 1 to 10 L min-1. Under conditions of low illumination and high flow rates, similar results were obtained for the bubble column and air-lift reactors. However, higher productivities-in terms of biomass and polysaccharide-were recorded in the air-lift reactor under high light intensity and low gas flow rates. The interactions of both photosynthesis and photoinhibition with the fluid dynamics in the bioreactors was taken as the main element that allowed us to interpret the differences in performance of the bubble column and the air-lift reactor. It is suggested that the cyclic distribution of dark periods in the air-lift reactor facilitates better recovery from the photoinhibition damage suffered by the cells. Copyright 1998 John Wiley & Sons, Inc.     

Effect of oxygen concentration on the growth of Nannochloropsis sp. at low light intensity

In large-scale microalgal production in tubular photobioreactors, the build-up of O(2) along the tubes is one of the major bottlenecks to obtain high productivities. Oxygen inhibits the growth, since it competes with carbon dioxide for the Rubisco enzyme involved in the CO(2) fixation to generate biomass. The effect of oxygen on growth of Nannochloropsis sp. was experimentally determined in a fully controlled flat-panel photobioreactor operated in turbidostat mode using an incident photon flux density of 100?μmol photons m(-2) s(-1) and with only the oxygen concentration as variable parameter. The dissolved oxygen concentration was varied from 20 to 250% air saturation. Results showed that there was no clear effect of oxygen concentration on specific growth rate (mean of 0.48?±?0.40?day(-1)) upon increasing the oxygen concentration from 20% to 75% air saturation. Upon further increasing the oxygen concentration, however, a linear decrease in specific growth rate was observed, ranging from 0.48?±?0.40?day(-1) at a dissolved oxygen concentration of 75% air saturation to 0.18?±?0.01?day(-1) at 250% air saturation. In vitro data on isolated Rubisco were used to predict the quantum yield at different oxygen concentrations in the medium. The predicted decrease in quantum yield matches well with the observed decrease that was measured in vivo. These results indicate that the effect of oxygen on growth of Nannochloropsis sp. at low light intensity is only due to competitive inhibition of the Rubisco enzyme. At these sub-saturating light conditions, the presence of high concentrations of oxygen in the medium induced slightly higher carotenoid content, but the increased levels of this protective antioxidant did not diminish the growth-inhibiting effects of oxygen on the Rubisco.     

Suboptimal growth temperatures enhance the biological activity of cultured cyanobacterium Gloeocapsa sp.

The cytotoxic, antibacterial, and antifungal activities of cyanobacterium Gloeocapsa sp. strain Gacheva 2007/R-06/1 were investigated and the possibility for an enhancement of these activities by changing the culture conditions evaluated. Fatty acids of this cyanobacterium were found to be active against Streptococcus pyogenes. Exopolysaccharides inhibited the growth of both Gram-positive and Gram-negative bacteria and the fungus Candida albicans. Both exopolysaccharides and fatty acid mixtures also significantly decreased the viability of human cervical carcinoma cells, HeLa. Greater biological activities were exhibited by Gloeocapsa sp., cultured at suboptimal temperatures (15–26°C) than at optimal and supraoptimal ones. In comparison with higher light intensity, the low-light cultivation stimulated the cytotoxicity of the fatty acids. In general, low temperatures decreased the growth of Gloeocapsa sp., but promoted its biological activity. Prolonged cultivation also had a beneficial impact on the bioactivity. Compared to 4 days, the 17-day cultivation resulted in fourfold higher antibacterial and antifungal activities of exopolysaccharides and more than twice increases in their cytotoxicity. The study revealed that this cyanobacterial isolate is a new source of natural products with potential for pharmacological and medical applications.     

半导体激光辐照对拟微绿球藻的生物学效应

目的:研究半导体激光对拟微绿球藻的生物学效应,进而利用激光诱变筛选高产优质藻株.方法:采用半导体激光辐照拟微绿球藻,辐照时间为l 0min、20min、30min.结果:诱变后藻的代谢物产量均有提高.结论:l0min、20min、30min辐照条件下,对拟微绿球藻细胞生长及代谢产物均有不同程度的促进作用,其中10min辐照组有利于拟微绿球藻的生长及色素的积累,分别比对照组提高了45.06％、86.59％、56.06％;但在20min剂量下更有利于胞外多糖、总多糖、蛋白质及油脂含量的积累,分别比对照组提高了67.7％、51.89％、19.16％、69.78％.     

Effects of initial population density (IPD) on growth and lipid composition of Nannochloropsis sp.

The microalga Nannochloropsis sp. was cultured under different initial population densities (IPDs) ranging from 0.11 to 9.09 g L^?1. The IPD affected the biomass and lipid accumulation significantly. The algal cultured with higher IPD resulted higher biomass concentration (up to 13.07 g L^?1) in 10 days growth. The biomass productivity with 0.98 g L^?1 IPD was 0.75 g L^?1 d^?1 which was higher than that of other IPDs. For IPDs ranging from 0.11 to 0.98 g L^?1, with the increase of IPD, the biomass productivity increased, while for IPD over 0.98 g L^?1, the biomass productivity decreased. Lipid content of the algal culture started with 0.11 g L^?1 IPD reached to 42 % of dry weight. But with the increase of IPD, the lipid content decreased. Lipid composition was analyzed using thin layer chromatography coupled with flame ionization detection (TLC/FID). Seven lipid classes were identified and quantified. The main reserve lipid, triacylglyceride (TAG), accumulated under all different IPD conditions. However, with the increasing IPD values, TAG content decreased from 59.1 to 23.5 % of the total lipids. Based on these results, to obtain the maximal biomass productivity and lipid productivity of Nannochloropsis sp. in mass cultivation systems, it is necessary to select an appropriate IPD.     

叉鞭金藻固定化培养的初步研究

以海藻酸钙为载体,初步考察了CaCl2浓度、胶球大小、密度及初始细胞密度等条件：对叉鞭金藻固定化培养的影响,确定了该藻固定化生长的优化条件;当藻细胞密度大于10^6cells/ml,CaCl2浓度为0.15mol/L,在50ml培养液中加入150个微藻胶球时,藻细胞的生长量最大。与游离的叉鞭金藻相比,固定化叉鞭金藻生长速度慢,但生长周期长。     

Isolation and characterization of poly- and oligosaccharides from the red microalga Porphyridium sp.

The current study forms part of an ongoing research effort focusing on the elucidation of the chemical structure of the sulfated extracellular polysaccharide of the red microalga Porphyridium sp. (UTEX 637). We report here on the chemical structure of a fraction separated from an acidic crude extract of the polysaccharide, as investigated by methylation analysis, carboxyl reduction-methylation analysis, desulfation-methylation analysis, partial acid hydrolysis, Smith degradation, together with 1D and 2D ¹H and ¹³C NMR spectroscopy. This fraction with a molar mass of 2.39 × 10⁵ g mol⁻¹ comprised d- and l-Gal, d-Glc, d-Xyl, d-GlcA, and sulfate groups in a molar ratio of 1.0:1.1:2.1:0.2:0.7. The almost linear backbone of the fraction is composed of (1→2)- or (1→4)-linked d-xylopyranosyl, (1→3)-linked l-galactopyranosyl, (1→3)-linked d-glucopyranosyl, and (1→3)-linked d-glucopyranosyluronic acid and comprises a possible acidic building unit:

[(2 or 4)-β-d-Xylp-(l→3)]_m-α-d-Glcp-(1→3)-α-d-GlcpA-(1→3)-l-Galp(l→

Attached to the backbone are sulfate groups and nonreducing terminal d-xylopyranosyl and galactopyranosyl residues, which occur at the O-6 positions of Glc-derived moieties in the main chain.     

Response of growth and fatty acid compositions of Nannochloropsis sp. to environmental factors under elevated CO2 concentration

Nannochloropsis sp. was grown with different levels of nitrate, phosphate, salinity and temperature with CO₂ at 2,800 μl l⁻¹. Increased levels of NaNO₃ and KH₂PO₄ raised protein and polyunsaturated fatty acids (PUFAs) contents but decreased carbohydrate, total lipid and total fatty acids (TFA) contents. Nannochloropsis sp. grew well at salinities from 22 to 49 g l⁻¹, and lowering salinity enhanced TFA and PUFAs contents. TFA contents increased with the increasing temperature but PUFAs contents decreased. The highest eicosapentaenoic acid (EPA, 20:5ω3) content based on the dry mass was above 3% under low N (150 μM NaNO₃) or high N (3000 μM NaNO₃) condition. Excessive nitrate, low salinity and temperature are thus favorable factors for improving EPA yields in Nannochloropsis sp.     

Purification and Characterization of a Light-Harvesting Chlorophyll-Protein Complex from the Marine Eustigmatophyte Nannochloropsis sp.

Light-harvesting chlorophyll-protein was purified from thylakoidmembranes of the marine unicellular alga Nannochloropsis sp.(Eustigmatophyceae), which contains neither chlorophyll b norchlorophyll c. Solubilization of thylakoid membranes with octyl-ß-D-glucopyranosideor with digitonin followed by separation on sucrose densitygradient yielded a chlorophyll-protein complex composed of anapoprotein of 26 kDa and an average of 9 chlorophyll a and 4violaxanthin molecules per apoprotein. Excitation spectra ofchlorophyll a fluorescence for the algal thylakoid membranesindicated energy transfer from the xanthophylls; however, anyattempt to solubilize the membranes greatly decreased energytransfer which was further reduced as the purification proceeded.The 26 kDa polypeptide of the isolated light-harvesting complexdid not cross-react with polyclonal antibodies raised againstanalogous proteins from higher plants and chlorophyll a/c alga.The N-terminus amino acid sequence of the apoprotein shows significantstructural similarity to the N-termini of the mature light harvestingfucoxanthin, chlorophyll a/c proteins from the diatom Phaeodactylumtricornutum, but not with the N-termini of light-harvestingproteins from chlorophyll a/b containing organisms. (Received June 25, 1992; Accepted July 28, 1992)     

Regulation of inorganic carbon acquisition by nitrogen and phosphorus levels in the Nannochloropsis sp.

Nannochloropsis sp. was grown to the exponential phase and transferred to the high CO₂ (2,800 μl l⁻¹) and irradiance (100 μmol photons m⁻² s⁻¹) condition with different levels of nitrate and phosphate for 72 h, then the photosynthetic activity and inorganic carbon acquisition of the alga were measured. The apparent photosynthetic efficiency (α) of Nannochloropsis sp. decreased with increasing NO₃ ⁻ concentration from 150 to 3,000 μM, and the high nitrate-grown cells showed the lowest levels of light-saturated photosynthetic rate (P _m), while the low nitrate-grown cells showed the highest levels of dark respiration rate (R _d). The maximal light-saturated photosynthetic rate and the minimal dark respiration rate were seen under the middle nitrate condition. When the nitrate concentration ranged from 150 to 3,000 μM, the affinity for inorganic carbons of Nannochloropsis sp. increased sharply with the increasing NO₃ ⁻ concentration to 300 μM and then decreased significantly. The middle phosphate-grown cells exhibited the highest light-saturated photosynthetic rate and apparent photosynthetic efficiency, however, the affinity for inorganic carbons of Nannochloropsis sp. was the maximum under the low phosphate condition. It was shown that the appropriate nitrogen and phosphorus levels were of vital importance to the photosynthesis of cells.     

CO2对微拟球藻(Nannochloropsis sp.)利用有机碳和光合作用的影响

CO2浓度提高时,微拟球藻吸收醋酸钠的速率增加2倍。混养生长的藻细胞最大光合作用速率、光合作用效率、无机碳半饱和常数和无机碳饱和的光合作用速率均显著低于光自养条件下生长的。     

The phytoplankton Nannochloropsis oculata enhances the ability of Roseobacter clade bacteria to inhibit the growth of fish pathogen Vibrio anguillarum

Background

Phytoplankton cultures are widely used in aquaculture for a variety of applications, especially as feed for fish larvae. Phytoplankton cultures are usually grown in outdoor tanks using natural seawater and contain probiotic or potentially pathogenic bacteria. Some Roseobacter clade isolates suppress growth of the fish pathogen Vibrio anguillarum. However, most published information concerns interactions between probiotic and pathogenic bacteria, and little information is available regarding the importance of phytoplankton in these interactions. The objectives of this study, therefore, were to identify probiotic Roseobacter clade members in phytoplankton cultures used for rearing fish larvae and to investigate their inhibitory activity towards bacterial fish pathogens in the presence of the phytoplankton Nannochloropsis oculata.

Methodology/Principal Findings

The fish pathogen V. anguillarum, was challenged with 6 Roseobacter clade isolates (Sulfitobacter sp. (2 strains), Thalassobius sp., Stappia sp., Rhodobacter sp., and Antarctobacter sp.) from phytoplankton cultures under 3 different nutritional conditions. In an organic nutrient-rich medium (VNSS), 6 Roseobacter clade isolates, as well as V. anguillarum, grew well (10⁹ CFU/ml), even when cocultured. In contrast, in a phytoplankton culture medium (ESM) based on artificial seawater, coculture with the 6 isolates decreased the viability of V. anguillarum by approximately more than 10-fold. Excreted substances in media conditioned by growth of the phytoplankton N. oculata (NCF medium) resulted in the complete eradication of V. anguillarum when cocultured with the roseobacters. Autoclaved NCF had the same inhibitory effect. Furthermore, Sulfitobacter sp. much more efficiently incorporated ¹⁴C- photosynthetic metabolites (¹⁴C-EPM) excreted by N. oculata than did V. anguillarum.

Conclusion/Significance

Cocultures of a phytoplankton species and Roseobacter clade members exhibited a greater antibacterial effect against an important fish pathogen (V. anguillarum) than roseobacters alone. Thus, cooperation of N. oculata, and perhaps other phytoplankton species, with certain roseobacters might provide a powerful tool for eliminating fish pathogens from fish-rearing tanks.     

Growth promotion of Chlorella ellipsoidea by co-inoculation with Brevundimonas sp. isolated from the microalga

Eight bacterial strains identified as P1, P2, Y1, Y2, W1, W2, G, and R were isolated from a long-term laboratory culture of the green alga Chlorella ellipsoidea. Although it is unknown how these bacterial strains have been maintained with the C. ellipsoidea culture, all appeared to promote the growth of C. ellipsoidea. Co-inoculation of each bacterial strain with C. ellipsoidea resulted in 0.5–3 times greater algal growth than that of C. ellipsoidea alone. The most effective bacterium (i.e., strain P1) was selected and further characterized. Biochemical analysis and transmission electron microscopy revealed that strain P1 is closely related to the genus Brevundimonas. Sequence analysis of the 16S rRNA of strain P1 showed 99.9 and 99.4% nucleotide sequence identity to that of B. nasdae and B. vesicularis, respectively. In addition to the growth promotion of C. ellipsoidea by strain P1, the growth of strain P1 was also significantly enhanced by co-culturing with C. ellipsoidea, indicating a symbiotic relationship between the bacterium and alga. Scanning electron microscopy showed the direct adhesion of strain P1 cells to the surface of C. ellipsoidea cells, as well as the development of abundant crinkles on the surface of co-cultured C. ellipsoidea cells. Handling editor: J. Padisak     

CO_2激光辐照拟微绿球藻的当代生物学效应研究

采用CO_2激光(波长10.6μm,功率10 w,光束长74 cm)辐照拟微绿球藻(Nannochloropsis sp.YW0980),辐照时间为30 s、60 s、90 s,通过测定藻色素、多糖、蛋白质及油脂含量,研究CO_2激光对藻的生物学效应。结果表明:30 s、60 s、90 s辐照条件下,对拟微绿球藻细胞生长及代谢产物均有一定的促进作用,其中CO_2激光60 s处理组有利于拟微绿球藻的生长及色素的积累,但30 s剂量下更有利于多糖、蛋白质及油脂含量的积累,分别比对照组提高了51.05%(胞外多糖),289.45%(总多糖)、37.05%、172.16%。     

Metabolic activity of cadmium-stressed and/or starvedVibrio sp.

Starvation ofVibrio sp. strain S14 cells for at least 2.5 h induced an enhanced resistance to subsequently applied cadmium stress. Bacterial cultures starved for a shorter time (0–2 h) exhibited a decreased ability to incorporate glucose when exposed to Cd²⁺. Cyclic increase and decrease in protein synthetic activity of stressed vibrios reflect stages of starvation-induced protiens expression. Vibrio cells pre-stressed by Cd²⁺ addition or by starvation responded in many aspects similarly to the next stress challenge. The presence of 100 mg/L chloramphenicol significantly lowered cell resistance against the secondary stress. Proteins synthesized due to the primary stress provideVibrio S14 with an enhanced probability to survive in unfavourable environment.     

Rheological behaviour of the culture medium during growth of the microalga Botryococcus braunii

A non-axenic strain of the microalga Botryococcus braunii Kützing, isolated from a small lake in Portugal, when cultured at 25°C in mineral medium and under continuous illumination, showed a poor production of hydrocarbons (5% of the dry biomass) but excreted remarkably high quantities of an exopolysaccharide (4–4·5 g litre⁻¹) into the medium. The production of the soluble polysaccharide, which contains galactose, fucose and uronic acid residues, occurs mainly after the exponential phase of growth.The rheological properties of broth during growth were studied. The increase of polysaccharide concentration as a consequence of its continuous biosynthesis, changes the medium behaviour from Newtonian to non-Newtonian with a flow characterized by a power-law equation. This behaviour becomes Newtonian again, when the culture is maintained for a longer period of time.     

Multiple approaches to enhance the cultivability of bacteria associated with the marine sponge Haliclona (gellius) sp

Three methods were examined to cultivate bacteria associated with the marine sponge Haliclona (gellius) sp.: agar plate cultures, liquid cultures, and floating filter cultures. A variety of oligotrophic media were employed, including media with aqueous and organic sponge extracts, bacterial signal molecules, and siderophores. More than 3,900 isolates were analyzed, and 205 operational taxonomic units (OTUs) were identified. Media containing low concentrations of mucin or a mixture of peptone and starch were most successful for the isolation of diversity, while the commonly used marine broth did not result in a high diversity among isolates. The addition of antibiotics generally led to a reduced diversity on plates but yielded different bacteria than other media. In addition, diversity patterns of isolates from agar plates, liquid cultures, and floating filters were significantly different. Almost 89% of all isolates were Alphaproteobacteria; however, members of phyla that are less commonly encountered in cultivation studies, such as Planctomycetes, Verrucomicrobia, and Deltaproteobacteria, were isolated as well. The sponge-associated bacteria were categorized into three different groups. The first group represented OTUs that were also obtained in a clone library from previously analyzed sponge tissue (group 1). Furthermore, we distinguished OTUs that were obtained from sponge tissue (in a previous study) but not from sponge isolates (group 2), and there were also OTUs that were not obtained from sponge tissue but were obtained from sponge isolates (group 3). The 17 OTUs categorized into group 1 represented 10 to 14% of all bacterial OTUs that were present in a large clone library previously generated from Haliclona (gellius) sp. sponge tissue, which is higher than previously reported cultivability scores for sponge-associated bacteria. Six of these 17 OTUs were not obtained from agar plates, which underlines that the use of multiple cultivation methods is worthwhile to increase the diversity of the cultivable microorganisms from sponges.     

Effects of nitrogen starvation on the photosynthetic physiology of a tropical marine microalga Rhodomonas sp. (Cryptophyceae)

The effects of nitrogen starvation on biomass composition and photosynthetic function were examined in the marine cryptophyte Rhodomonas sp. Batch-cultured cells in N-sufficient medium showed a 2.5-fold increase in total carbohydrate content, and a 33% increase in cell volume when the cultures reached the stationary growth phase. These cultures also increased the ratio of phycoerythrin (PE)/hydrosoluble proteins from 6 to 22% by the 4th and 10th day of culture, respectively. In contrast, light-saturated photosynthetic activity (P_m) progressively decreased, and the value obtained at the beginning of the stationary phase was about 45% of that obtained for cells in the late exponential growth phase. Transfer to N-lacking medium caused a 3.2-fold increase in cell volume. N starvation also triggered a rapid decline in N-containing compounds such as hydrosoluble proteins and photosynthetic pigments, causing an almost complete loss of PE. The ratio of PE/hydrosoluble proteins decreased from 6 to 1% after 6 d of N deprivation. Furthermore, the PSII fluorescence capacity declined under N-starved conditions, which caused a pronounced decrease in both the P_m (circa 90%) and the apparent photosynthetic efficiency (circa 55%). Under these conditions, photosynthetically fixed carbon was used to synthesize large amounts of carbohydrates. We suggest that, in addition to the role of phycoerythrin as a light-harvesting pigment, Rhodomonas sp. responds to N-depleted conditions by mobilizing combined nitrogen from biliproteins.