Application of a growth-promoting bacteria for stable mass culture of three marine microalgae

The practical mass culture of marine microalgae, facesoccasionally unexpected problems or collapse. The effect of amarine bacterium, Flavobacterium sp., which was found topromote growth of a marine diatom Chaetoceros gracilisin the axenic culture condition, was examined on the masscultures of three marine microalgae.Three marine microalgae (C. gracilis, Isochrysisgalbana, and Pavlova lutheri) were mass cultured in 3 lflatbottom flasks (2.5 l capacity of culture medium), in anindoor culture room at a commercial pearl oyster hatchery. Themicroalgal cells and the bacterium were inoculated at the sametime, in the culture media. The specific growth rate andmaximal cell density were determined in treated cultures (withadded bacterial strain) and in controls (without addedbacterial strain). The specific growth rate of C.gracilis in treated cultures was significantly higher thanthat of control cultures, and the stationary growth phase inthe treated cultures lasted longer till the end of the cultureperiod. However, the bacterium had no apparent effect on theexponential growth phase of two phytoflagellates, I.galbana and P. lutheri, but kept longer the high celldensity in the stationary growth phases. The added bacterialstrain (Flavobacterium sp.) was the dominant species(more than 45%) among the bacterial flora during the cultureperiod.     

Feeding in adult females of Argyrodiaptomus furcatus (Sars,1901), Copepoda-Calanoida,of Lobo Reservoir (Broa), São Carlos,São Paulo,Brazil

The aim of this work was to study the feeding process of Argyrodiaptomus furcatus (Copepoda-Calanoida) in the Lobo Reservoir (São Carlos, SP, Brazil). Non-ovigerous adult females and the ¹⁴C technique were used to measure filtration and assimilation rates. The diet contained the following phytoplankton species: Chlamydomonas sp., Ankistrodesmus gracilis, Melosira italica, Scenedesmus quadricauda and Chlorella zoofingensis.The experiments were carried out using unialgal and mixed cultures during 2-, 4- and 6-h periods. The results of the filtration and assimilation rates were compared.The data obtained by statistical tests showed the highest assimilation rate in Argyrodiaptomus furcatus fed Chlamydomonas sp. in both culture types. However, Chlorella zoofingensis and Scenedesmus quadricauda were the most filtered species in unialgal and mixed cultures, respectively. A higher filtration rate was observed for the 2-h period than for the 4- and 6-h periods.Culture agent was also important. Higher assimilation and filtration rates were obtained during the log phase of Chlamydomonas sp. growth than during the stationary phase.     

Changes in the lipid composition and maximisation of the polyunsaturated fatty acid content of three microalgae grown in mass culture

Three species of microalgae were grown in mass culture to investigate the influence of culture technique and growth phase on the production of 20:5(n?3) and 22:6(n?3). These polyunsaturated fatty acids (PUFA) are considered to be essential in many marine animals diets for high growth and survival rates. The species of microalgae examined wereNannochloropsis oculata, Pavlova lutheri andIsochrysis sp. (clone T.Iso). All batch cultures (logarithmic and stationary phase) and semi-continuous cultures (logarithmic phase) examined contained high levels of the long-chain (n?3) PUFA, but production could be maximised by harvesting at specific times and growth phases. Maximum cellular content (pg cell^-1) of long-chain PUFA was found in logarithmic phase batch cultures ofN. oculata and in stationary phase cultures ofP. lutheri. The cellular content of PUFA in cultures ofIsochrysis sp. did not change significantly with culture technique or growth phase. Alternatively, stationary phase cultures of all three species showed increased proportions (%) and cellular contents of triacylglycerols, and saturated and monounsaturated fatty acids with correspondingly decreased proportions of polar lipids and most PUFA relative to logarithmic phase cultures. The exception was the proportion and cellular content of 22:6(n?3) inP. lutheri which increased with triacylglycerol content. The mass of long-chain (n?3) PUFA per volume of culture was significantly higher in stationary phase cultures due to the higher cell counts per volume. These findings indicate that the opportunity exists to maximise PUFA production by microalgae with the potential to improve animal growth and reduce production costs in mariculture operations and may be of use in the large scale culture and harvesting of microalgae for the biotechnology industry.     

VISCOUS POLYSACCHARIDE AND STARCH SYNTHESIS IN RHODELLA RETICULATA (PORPHYRIDIALES,RHODOPHYTA)1

Rhodella reticulata Deason, Butler and Rhyne produces copious amounts of a viscous polysaccharide (VP) during growth in batch cultures. The VPs accumulated on the cell surface and in the culture medium once cells ceased growth; starch concurrently accumulated within the cells. Light-saturated ¹⁴C-uptake declined steadily as the cells aged. Net synthesis rates for starch and mucilage were two- and four-fold lower, respectively, in non-growing cells than in growing cells, while the relative partitioning of newly-fixed carbon into these materials was not different. These data suggest that total photosynthetic loading, rather than partitioning into one specific pool, controls cellular synthesis rates. No preferential synthesis of VPs occurred during the stationary phase. The findings have important implications for the commercial production of VPs.     

Nutritional Regulation of Organelle Biogenesis in Euglena: REPRESSION OF CHLOROPHYLL AND NADP-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE SYNTHESIS

Nitrogen deficiency and the presence of specific organic carbon sources prevent chloroplast development in Euglena. In exponentially growing cultures, chlorophyll levels were low and independent of the nitrogen content of the growth medium. Chlorophyll levels increased in stationary phase and the amount of chlorophyll formed was proportional to the initial nitrogen content of the growth medium; the greater the concentration of nitrogen, the greater the amount of chlorophyll synthesized during stationary phase. Washing experiments demonstrated that the major nutritional factor inhibiting chlorophyll synthesis in stationary phase cultures grown on medium containing a high carbon to nitrogen ratio was the absence of nitrogen rather than the presence of utilizable organic carbon.     

Non-aerated cultivation ofHalobacterium cutirubrum and its effect on cellular squalenes

Summary Halobacterium cutirubrum was successfully cultivated under aerobic and microaerobic conditions. The early stationary phase of growth was obtained at 2.2 days and 45–55 days for aerated and non-aerated cultures, respectively. The dry cell yields were 0.7–1.2 gm/l in all preparations grown to early stationary growth phase. The cellular ratio of squalene to dihydro- and tetra-hydrosqualene decreased proportionately with decreased aeration rates.     

Lipid synthesis during reinitiation of growth from stationary phase cultures of Candida albicans

Lipid synthesis has been studied in the dimorphic fungus Candida albicans. ¹⁴C-acetate incorporation into lipid material was used to measure new lipid synthesis in two cultures in which either yeast or mycelial growth was initiated from stationary phase yeast cells. When resuspended in fresh medium at 37 °C, cells resume growth and change morphology while at 30 °C cells resume budding growth. When resuspended at the appropriate temperature, both yeast and germ tube cultures immediately incorporated ¹⁴C-acetate into lipid material. The labeled lipid was more or less evenly divided between neutral and phospholipid. Phosphatidyl choline was the major phospholipid fraction and along with phosphatidyl ethanolamine accounted for 60–65 % of the total phospholipid. Lipid synthesis during growth initiation of either morphology showed a similar pattern, with no significant differences observed in neutral or phospholipid or phospholipid components between yeast and mycelial forms.     

DIMETHYLSULFONIOPROPIONATE PRODUCTION BY PSYCHROPHILIC DIATOM ISOLATES1

Dimethylsulfoniopropionate (DMSP) production by psychrophilic diatom strains, Chaetoceros sp., Navicula sp., and Nitzschia sp., were experimentally analyzed. All strains showed rapid growth (0.3–0.5 d^?1) under cold culture conditions (1.7°C). DMSP concentrations (both as particulate, DMSP_p, and dissolved, DMSP_d) were negligible in cultures of Chaetoceros sp., whereas those for Navicula sp. and Nitzschia sp. increased toward the end of the 56‐day experiments. The ratio of DMSP_p:chl a of the latter two species was approximately 5 in the early exponential growth phase and decreased slightly toward the early stationary phase. During the stationary phase, when chl a and fluorescence remained constant, the ratios in both species increased linearly by up to approximately 6.5 times the value at the start of the stationary phase. This growth‐related DMSP production by diatoms may result in the low concentrations of DMSP_p observed in the early stage of diatom blooms under natural conditions.     

The ascorbic acid content of eleven species of microalgae used in mariculture

The ascorbic acid (vitamin C) concentrations in 11 species of microalgae commonly used in mariculture were determined. The species examined were 4 diatoms (Chaetoceros calcitrans (Paulsen) Takano,Chaetoceros gracilis Schütt,Skeletonema costatum (Greville) Cleve,Thalassiosira pseudonana (Hustedt, clone 3H) Hasle and Heimdal); 2 prymnesiophytes (Isochrysis sp. (clone T.ISO) Parke,Pavlova lutheri (Droop) Green); 1 prasinophyte (Tetraselmis suecica (Kylin) Butcher); 2 chlorophytes (Dunaliella tertiolecta Butcher,Nannochloris atomus Butcher); 1 eustigmatophyte (Nannochloropsis oculata (Droop) Green); and 1 cryptophyte (Chroomonas salina (Wislouch) Butcher). Duplicate cultures of each species were grown under defined conditions and analysed during both logarithmic and stationary phase of growth.Average values for ascorbic acid ranged from 9.4 fg cell^–1 (N. oculata, stationary phase) to 700 fg cell^–1 (S. costatum, stationary phase). This value was generally related to cell size. Levels of ascorbic acid cell^–1 increased during the stationary growth phase forS. costatum andD. tertiolecta and decreased forC. gracilis, T. pseudonana, C. salina andN. oculata. Levels did not change significantly for the remaining species.Average values for per cent ascorbic acid ranged from 0.11% (T. pseudonana, stationary phase) to 1.62% of dry weight (C. gracilis, logarithmic phase). The per cent ascorbic acid was not related to algal class. Also, the percentage between logarithmic and stationary phase cultures differed for many of the species, but differences were unrelated to algal class.Chaetoceros gracilis, T. pseudonana, N. oculata andIsochrysis sp. (T.ISO) had higher per cent ascorbic acid during the logarithmic phase, whereasD. tertiolecta andN. atomus contained more per cent ascorbic acid during the stationary phase.Despite the differences in the composition of the different microalgae (0.11–1.62% ascorbic acid), all species would provide a rich source of ascorbic acid for maricultured animals, which can require 0.003–0.02% of the vitamin in their diet.     

DAILY FLUCTUATIONS OF EXOPOLYMERS IN CULTURES OF THE BENTHIC DIATOMS CYLINDROTHECA CLOSTERIUM AND NITZSCHIA SP. (BACILLARIOPHYCEAE)1

Dynamics in the production of extracellular polymeric substances (EPS) were investigated for the benthic diatoms Cylindrotheca closterium (Ehrenberg) and Nitzschia sp. The effect of growth phase and light:dark conditions were examined using axenic cultures. Two EPS fractions were distinguished. Soluble EPS was recovered from the culture supernatant and represented polysaccharides that were only loosely associated with the cells. Bound EPS was extracted from the cells using warm (30° C) water and was more closely associated with the diatom aggregates. Concentrations of EPS exceeded internal concentrations of sugar throughout growth, indicating that EPS production is important in these organisms. Soluble and bound EPS revealed distinct differences in daily dynamics during the course of growth. Soluble EPS was produced continuously once cultures entered the stationary phase. During the stationary phase, chl a‐normalized EPS production rates equaled 6.4 and 3.4 d ^{? 1} for C. closterium and Nitzschia sp., respectively. In contrast, production of bound EPS occurred only in the light and was highest during the exponential phase. Up to 90% of the attached EPS that was produced in the light was degraded during the subsequent dark period. The monosaccharide distribution of EPS was constant during the course of the experiment. The soluble EPS consisted of high amounts of galactose and glucuronic acid, relative to rhamnose, glucose, xylose/mannose, and galacturonic acid. In contrast, glucose was the dominant monosaccharide present in the bound EPS. These differences suggest that the production of the two distinct EPS fractions is under different metabolic controls and probably serves different cellular functions.     

磷浓度对小环藻、大型溞和金鱼藻三者相互作用的影响

为了解磷浓度对水生植被恢复和生物操纵效果的影响, 分别用小环藻(Cyclotella sp.)、大型溞(Daphnia magna)和金鱼藻(Ceratophyllum demersum)代表浮游植物、浮游动物和大型沉水植物建立水生微宇宙模型, 在25℃、2600 lx光强和11 mg/L氮浓度条件下, 分别研究小环藻与大型溞、小环藻与金鱼藻、小环藻-大型溞-金鱼藻共培养时4种磷浓度(0.05、0.1、0.5和2 mg/L)下小环藻、大型溞、金鱼藻的增长率以及培养液中氮磷去除率的变化。结果表明: 小环藻与大型溞、小环藻与金鱼藻两两共培养时, 磷浓度为0.05-2 mg/L时, 金鱼藻和大型溞均生长良好, 小环藻受到明显抑制, 其密度保持较小幅度的正增长。在小环藻-大型溞-金鱼藻三者共培养时, 在0.05-2 mg/L的磷浓度范围内大型溞和金鱼藻生长良好, 与两两共培养相比, 小环藻则受到了更大程度的抑制, 在磷浓度为0.05-0.1 mg/L时藻密度呈现负增长. 这说明在水生态系统中, 大型浮游动物和沉水植物对浮游藻类的联合控制效果远好于各自单独的控制效果, 该控制效果随磷浓度的提高而减弱, 以0.1 mg/L的磷浓度为最佳。在实验结束后测定氮磷去除率发现, 在最低磷浓度(0.05 mg/L),即磷限制时, 水中磷去除率最高, 在最高磷浓度(2 mg/L), 即氮限制时, 水中氮去除率最高。     

Role of dissolution rate and solubility in biodegradation of aromatic compounds.

Strains of Moraxella sp., Pseudomonas sp., and Flavobacterium sp. able to grow on biphenyl were isolated from sewage. The bacteria produced 2.3 to 4.5 g of protein per mol of biphenyl carbon, and similar protein yields were obtained when the isolates were grown on succinate. Mineralization of biphenyl was exponential during the phase of exponential growth of Moraxella sp. and Pseudomonas sp. In biphenyl-supplemented media, Flavobacterium sp. had one exponential phase of growth apparently at the expense of contaminating dissolved carbon in the solution and a second exponential phase during which it mineralized the hydrocarbon. Phase-contrast microscopy did not show significant numbers of cells of these three species on the surface of the solid substrate as it underwent decomposition. Pseudomonas sp. did not form products that affected the solubility of biphenyl, although its excretions did increase the dissolution rate. It was calculated that Pseudomonas sp. consumed 29 nmol of biphenyl per ml in the 1 h after the end of the exponential phase of growth, but 32 nmol of substrate per ml went into solution in that period when the growth rate had declined. In a medium with anthracene as the sole added carbon source, Flavobacterium sp. converted 90% of the substrate to water-soluble products, and a slow mineralization was detected when the cell numbers were not increasing. Flavobacterium sp. and Beijerinckia sp. initially grew exponentially and then arithmetically in media with phenanthrene as the sole carbon source. Calculations based on the growth rates of these bacteria and the rates of dissolution of phenanthrene suggest that the dissolution rate of the hydrocarbon may limit the rate of its biodegradation.     

Role of dissolution rate and solubility in biodegradation of aromatic compounds

Strains of Moraxella sp., Pseudomonas sp., and Flavobacterium sp. able to grow on biphenyl were isolated from sewage. The bacteria produced 2.3 to 4.5 g of protein per mol of biphenyl carbon, and similar protein yields were obtained when the isolates were grown on succinate. Mineralization of biphenyl was exponential during the phase of exponential growth of Moraxella sp. and Pseudomonas sp. In biphenyl-supplemented media, Flavobacterium sp. had one exponential phase of growth apparently at the expense of contaminating dissolved carbon in the solution and a second exponential phase during which it mineralized the hydrocarbon. Phase-contrast microscopy did not show significant numbers of cells of these three species on the surface of the solid substrate as it underwent decomposition. Pseudomonas sp. did not form products that affected the solubility of biphenyl, although its excretions did increase the dissolution rate. It was calculated that Pseudomonas sp. consumed 29 nmol of biphenyl per ml in the 1 h after the end of the exponential phase of growth, but 32 nmol of substrate per ml went into solution in that period when the growth rate had declined. In a medium with anthracene as the sole added carbon source, Flavobacterium sp. converted 90% of the substrate to water-soluble products, and a slow mineralization was detected when the cell numbers were not increasing. Flavobacterium sp. and Beijerinckia sp. initially grew exponentially and then arithmetically in media with phenanthrene as the sole carbon source. Calculations based on the growth rates of these bacteria and the rates of dissolution of phenanthrene suggest that the dissolution rate of the hydrocarbon may limit the rate of its biodegradation.     

GROWTH AND DOMOIC ACID PRODUCTION BY PSEUDO‐NITZSCHIA SERIATA (BACILLARIOPHYCEAE) UNDER PHOSPHATE AND SILICATE LIMITATION1

Pseudo‐nitzschia seriata (Cleve) H. Peragallo isolated from Scottish west coast waters was studied in batch culture with phosphate (P) or silicate (Si) as the yield‐limiting nutrient at 15°C. This species produced the neurotoxin domoic acid (DA) when either nutrient was limiting but produced more when stressed by Si limitation during the stationary phase. Under P‐limiting conditions, exponential growth stopped after P was reduced to a low threshold concentration. Under Si‐limiting conditions, fast exponential growth was followed by a period of slower exponential growth, until Si became exhausted. A stationary phase was observed in the P‐limited but not the Si‐limited cultures, the latter showing a rapid decrease in cell density after the second exponential growth phase. Si‐limited cultures exhibited a further period of active metabolism (as indicated by increases in chl and carbon per cell) late in the experiment, presumably fueled by regenerated Si. DA production was low in exponential phase under both conditions. In P‐limited cultures, most DA was produced during the immediate postexponential phase, with little or no new DA produced during later cell senescence. In contrast, although a substantial amount of DA was produced during the slower exponential phase of the Si‐limited cultures, DA production was even greater near the end of the experiment, coincident with the period of chl synthesis and increase in carbon biomass. Comparison of the magnitude of toxin production in the two nutrient regimes indicated a greater threat of P. seriata‐generated amnesic shellfish poisoning events under Si rather than P nutrient limitation.     

Carbon metabolism and energy conversion of Synechococcus sp. PCC 7942 under mixotrophic conditions: comparison with photoautotrophic condition

To investigate the carbon metabolism and energy conversion efficiency of the cyanobacterium Synechococcus sp. PCC 7942 under mixotrophic conditions, we studied its growth characteristics in mixotrophic cultures with glucose and with acetate, respectively, and further discussed the carbon metabolism and energy utilization based on metabolic flux analysis. Results showed that both glucose and acetate could enhance the growth of Synechococcus sp. PCC 7942. The metabolic flux through the glycolytic pathway, tricarboxylic acid cycle, and mitochondrial oxidative phosphorylation was affected by the two organic substrates. Additionally, the cellular composition was also modulated by glucose and acetate. Under mixotrophic conditions, glucose exerts more significant impact on the diminishment of photochemical efficiency. Although the contribution of light energy was smaller, the cell yields based on total energy in mixotrophic cultures were higher compared with that of photoautotrophic one. On the basis of chlorophyll fluorescence analysis, the actual energy conversion efficiencies based on ATP synthesis in the photoautotrophic, glucose-mixotrophic, and acetate-mixotrophic cultures were evaluated to be 4.59%, 5.86%, and 6.60%, respectively.     

利用麻风树油驯化筛选高活性脂肪酶产生菌及其催化酯化反应

以1株分解麻风树油的脂肪酶产生菌Pseudomonas sp. LP-1为出发菌株, 通过麻疯树油定向驯化筛选获得1株酶活较高且产酶稳定的菌株P. sp. X-2-45, 其水解酶活为29.79 U/mL, 比原始菌株提高了288%。对P. sp. X-2-45生长与产酶特征、对植物油脂水解能力及在有机相中催化脂肪酸和有机醇间的酯化反应研究发现, 该菌株生长速率和产酶速率明显加快, 培养30 h时生物量和酶活达到最大, 稳定期延长, 培养过程中脂肪酶在培养基中的稳定性提高。以麻疯树油诱导合成的P. sp. X-2-45脂肪酶对麻疯树油的水解能力比原始菌株提高了378%, 说明采用麻风树油定向驯化可提高脂肪酶对相应底物的水解能力。X-2-45脂肪酶可以催化月桂酸与正丁醇、正辛醇、月桂醇和丙三醇之间, 棕榈酸、硬脂酸与甲醇、正辛醇、月桂醇和丙三醇之间, 油酸与甲醇、正丁醇、正辛醇、月桂醇和丙三醇之间发生酯化反应。     

Dynamics of Metabolic Activities and Gene Expression in the Roseobacter Clade Bacterium Phaeobacter sp. Strain MED193 during Growth with Thiosulfate

Metagenomic analyses of surface seawater reveal that genes for sulfur oxidation are widespread in bacterioplankton communities. However, little is known about the metabolic processes used to exploit the energy potentially gained from inorganic sulfur oxidation in oxic seawater. We therefore studied the sox gene system containing Roseobacter clade isolate Phaeobacter sp. strain MED193 in acetate minimal medium with and without thiosulfate. The addition of thiosulfate enhanced the bacterial growth yields up to 40% in this strain. Concomitantly, soxB and soxY gene expression increased about 8-fold with thiosulfate and remained 11-fold higher than that in controls through stationary phase. At stationary phase, thiosulfate stimulated protein synthesis and anaplerotic CO₂ fixation rates up to 5- and 35-fold, respectively. Several genes involved in anaplerotic CO₂ fixation (i.e., pyruvate carboxylase, propionyl coenzyme A [CoA], and crotonyl-CoA carboxylase) were highly expressed during active growth, coinciding with high CO₂ fixation rates. The high expression of key genes in the ethylmalonyl-CoA pathway suggests that this is an important pathway for the utilization of two-carbon compounds in Phaeobacter sp. MED193. Overall, our findings imply that Roseobacter clade bacteria carrying sox genes can use their lithotrophic potential to gain additional energy from sulfur oxidation for both increasing their growth capacity and improving their long-term survival.     

Riboflavin content of six species of microalgae used in mariculture

Concentrations of riboflavin — a vitamin essential for maricultured animals—were measured in six species of microalgae commonly used in mariculture. These were two diatoms (Chaetoceros gracilis, Thalassiosira pseudonana); two prymnesiophytes (Isochrysis sp. (clone T.ISO),Pavlova lutheri); one chlorophyte (Nannochloris atomus) and one eustigmatophyte (Nannochloropsis oculata). Cultures were analysed during both logarithmic and stationary growth phase.The proportions of riboflavin (µg g^-1 dry weight) during logarithmic growth-phase ranged from 20 (T. pseudonana) to 40 µg g^-1 (Isochrysis sp. T.ISO). With the onset of stationary phase, the proportion of riboflavin increased in all species; the increase was most dramatic in cultures ofC. gracilis, T. pseudonana andN. atomus (2- to 3-fold).Chaetoceros gracilis contained more riboflavin (106 µg g^-1) than all other species (48 to 61 µ g^-1).Despite the differences in the composition of the different microalgae, across both logarithmic and stationary growth-phases, all species should provide a rich source of riboflavin for maricultured animals.Author for correspondence     

聚球藻7942混养培养中碳代谢与能量利用

为了考察聚球藻7942在混养条件下的能量利用效率,分别以葡萄糖和乙酸为碳源开展了聚球藻7942的混养培养研究,并在此基础上利用代谢通量分析方法对聚球藻7942混养条件下的碳代谢和能量利用进行了探讨。结果表明:葡萄糖和乙酸均能促进藻细胞生长,且乙酸促进藻细胞生长的作用更为明显;葡萄糖利用可明显增加藻细胞糖酵解途径中碳代谢流量,而乙酸利用则导致糖酵解途径中碳代谢流量减小,两种有机碳源均增加了柠檬酸循环中碳代谢流量;有机碳源导致藻细胞光化学效率下降,而葡萄糖较之乙酸降低藻细胞光化学效率更为明显。虽然混养条件下光能的贡献率要小于光自养,但基于能量的细胞得率和能量转换率均高于光自养,光自养和以葡萄糖、乙酸为碳源的混养中基于ATP生成的能量转换效率分别为6.81%、7.43%和8.77%。