Marine microalgae as a potential source of single cell protein (SCP)

Summary The marine microalgaeTetraselmis suecica, Isochrysis galbana, Dunaliella tertiolecta andChlorella stigmatophora are good biological sources of single cell protein (SCP). Protein content accounts for 39.12%–54.20% of the dry matter,D. tertiolecta having the highest. Lysine values are between 3.67 and 4.52 g/100 g of protein, and thus are higher than those for freshwater species. The total nucleic acid content is less than 7% of the dry matter; this value is definitely lower than that for yeasts or bacteria, commonly used as SCP sources. Amino acid profiles of the four species are very similar and comparable to the FAO reference protein, buth with a low content of methionine and cystine and a high content of lysine. The MEAA indices are between 81 and 84.98, without significant differences among the four species. Marine microalgae can be used as a potential SCP source.     

Exopolysaccharide, pigment and protein production by the marine microalga Chroomonas sp. in semicontinuous cultures

The marine microalga Chroomonas sp. isolated from Venezuela was grown in semicontinuous culture in order to study the effect of renewal rate and nutrient concentration on alloxanthin, chlorophyll a, carotenoid, carbohydrate, exopolysaccharide, protein and cell productivity. Maximal cell productivity of 8.43 ± 1.8 and 8.81 ± 2.3 × 10⁹ cell l^–1 day^–1 were achieved with renewal rates of 30 and 40%. Maximal protein and chlorophyll productivity of 64.64 ± 2.3 and 2.72 ± 0.3 mg l^–1 day^–1 were obtained with renewal rate of 20 and 30%. Biochemical composition of Chroomonas sp. was influenced by renewal rate. Nutrient concentration seems not to affect cell, protein, chlorophyll and carotenoid productivity. However, carbohydrate and exopolysaccharide productivity of 7.56 ± 0.4 and 9.57 ± 1.2 mg l^–1 day^–1 were increased at 12 mM NaNO₃(P < 0.05). Also, alloxanthin and chlorophyll a production analysed by HPLC, were higher between 8 and 12 mM NaNO₃ at a renewal rate of 30%. Results demonstrated that a renewal rate of 30% and nutrient concentration at 8 mM NaNO₃ optimize the cell, protein, carbohydrate, chlorophyll a, and exopolysaccharide productivity in semicontinuous cultures of Chroomonas. This microalga, as biological source of commercially valuable compounds, shows high capacity for changing its productivity and biochemical composition in semicontinuous system on the basis of nutrient concentration and the renewal rate.     

Productivity and biochemical composition of cyclostat cultures of the marine microalga Tetraselmis suecica

Cyclostat, light/dark-synchronized cultures of the marine microalga Tetraselmis suecica were carried out with six nutrient concentrations: 0.5, 1, 2, 4, 8, and 16 mmol N/l. A renewal of 50% culture volume, equivalent to a dilution rate of 1 day^–1 was applied. Maximal steady-state cell density (2.5 × 10⁶ cells/ml) and dry-weight productivity (0.2 g l^–1 day^–1) were achieved with 4 mmol N/l. Maximal protein (94.3 pg/cell) and lipid contents (25.4 pg/cell) were achieved with8 mmol N/l. The high variability reported in chlorophyll cellular content invalidates any estimation of biomass or cell number based on chlorophyll concentration or fluorescence, especially when nutrient concentration is being varied. The total fatty acid cellular content increased with increasing nutrient concentration although the amount of eicosapentaenoic acid decreased. Results indicated that a different desaturation pathway may be present in this species. The change of the nutrient concentration in the cyclostat system is a powerful tool to manipulate the biochemical composition of microalgae regarding protein, lipid, carbohydrates and fatty acid content.     

Enhanced production of lutein in heterotrophic Chlorella protothecoides by oxidative stress

The fast growing unicellular green microalgae Chlorella protothecoides has attracted interest as a promising organism for commercial production of a high-value carotenoid, lutein, by heterotrophic fermentation. Effects of two oxidant-forming reactive oxygen species (ROS) on the biomass concentration, and yield and content of lutein in batch culture of heterotrophic Chlorella protothecoides were investigated in this study. The addition of 0.1 mmol/L H₂O₂ and 0.01 mmol/L NaClO plus 0.5 mmol/L Fe²⁺ to the culture led to the generation of ^·OH and enhanced the lutein content from 1.75 to 1.90 and 1.95 mg/g, respectively. The lutein content further increased to 1.98 mg/g when 0.01 mmol/L H₂O₂ and 0.5 mmol/L NaClO were added to generate ¹O₂. The maximum yield of lutein (28.5, 29.8 and 31.4 mg/L) and a high biomass concentration (15.0, 15.3 and 15.9 g/L) were also achieved through the above treatments. The results indicated that 1O2 could promote lutein formation and enhance lutein production in heterotrophic Chlorella protothecoides. Moreover, ¹O₂ produced from the reaction of H₂O₂ and NaClO was more effective in enhancing lutein production and reducing biomass loss than ^·OH from the reaction of H₂O₂ or NaClO plus Fe²⁺. Supported by the National Key Project of Sci & Tech Supporting Programs Funded by Ministry of Science & Technology of China (Grant No. 2006BAD27B03), Sci & Tech Project of Guangzhou (Grant No. 2005Z3-E0331) and Sci & Tech Project of Guangdong (Grant No. 20052050166)     

Responses of wild plants to nitrate availability

Summary Two annual species of Bromus, an invader (B. hordeaceus, ex B. mollis) and a non-invader (B. intermedius), were grown for 28 days in growth chambers, at 5 and 100 M NO ₃ ^- in flowing nutrient solution. No differences between the two species were observed at either NO ₃ ^- level, in terms of relative growth rate (RGR) or its components, dry matter partitioning, specific NO ₃ ^- absorption rate, nitrogen concentration, and other characteristics of NO ₃ ^- uptake and photosynthesis. The effects of decreasing NO ₃ ^- concentration in the solution were mainly to decrease the NO ₃ ^- concentration in the plants through decreased absorption rate, and to decrease the leaf area ratio through increased specific leaf mass and decreased leaf mass ratio. Organic nitrogen concentration varied little between the two treatments, which may be the reason why photosynthetic rates were not altered. Consequently, RGR was only slightly decreased in the 5-M treatment compared to the 100-M treatment. This is in contrast with other species, where growth is reduced at much higher NO ₃ ^- concentrations. These discrepancies may be related to differences in RGR, since a log-linear relationship was found between RGR and the NO ₃ ^- concentration at which growth is first reduced. In addition, a strong linear relationship was found between the RGR of these species and their maximum absorption rate for nitrate, suggesting that the growth of species with low maximum RGR may be partly regulated by nutrient uptake.     

绞股蓝提取液对蛋白核小球藻的化感效应及影响机理研究

以中草药植物绞股蓝[Gynostemma pentaphyllum(Thunb.) Makino]为化感供体材料,研究其不同浓度的提取液(0、5、10、25、50 g/L)对蛋白核小球藻(Chlorella pyrenoidesa)生长及生理生化特征的化感效应。结果表明:(1)绞股蓝提取液对蛋白核小球藻生长均具有抑制作用,其抑制作用随提取液质量浓度增大和培养时间延长均呈增强趋势,且25 g/L绞股蓝提取液培养15 d时的抑制率达到最大(79.41%)。(2)各浓度绞股蓝处理组蛋白核小球藻细胞内的叶绿素a含量均低于对照组,且随着提取液浓度升高以及处理时间延长叶绿素a含量较对照的降低量越多,表明蛋白核小球藻光合作用受到的影响也越大。(3)绞股蓝处理组蛋白核小球藻细胞的膜透性(吸光度OD_(264))显著高于对照,且膜透性随着提取液浓度增大而增强;高浓度提取液处理下,藻细胞内部的可溶性蛋白质(OD_(280))及核酸(OD_(260))含量均显著高于对照,且随着处理时间延长,细胞膜透性增大,细胞内部的可溶性蛋白质及核酸向胞外渗透增多。研究发现,绞股蓝提取液能够抑制蛋白核小球藻生长,并随着提取液质量浓度增大而增强;绞股蓝提取液能促进藻细胞叶绿素分解、增加细胞膜透性,引起可溶性蛋白质和核酸向胞外渗透量升高,导致藻细胞结构受损,代谢功能紊乱,从而达到化感抑制作用。     

Mixotrophic culture of Chlorella pyrenoidosa with olive-mill wastewater as the nutrient medium

With olive-mill wastewater (`alpechín') as the nutrient medium, theinfluence of specific rate of aeration and initial alpechín concentrationhave been analysed in cultures of Chlorella pyrenoidosa, exposed bothto continuous and intermittent illumination (12/12 h light/dark cycles). The stirring rate in the bioreactor, as well as pH and temperature werefixed previously at 180 rpm, 6.5 and 30 ^°C, respectively. Themaximum specific growth rate (_m) and biomass productivity(b) were determined as kinetic parameters. The chlorophyll, protein andcarbohydrate contents were evaluated, as well as the fatty-acid compositionof the lipid fraction. The experimental conditions most conducive to abalanced biomass composition with regard to proteins and lipids were: initial alpechín concentration of 10% (v/v), continuous illumination,and aeration rate of 1 L (litre cell suspension)^-1 min^-1. Under these conditions, the highest values of _m and b wereclose to 0.04 h^-1 and 1.4 10^-3 g L^-1 h^-1, respectively.     

Influence of culture density,pH, organic acids and divalent cations on the removal of nutrients and metals by immobilized Anabaena doliolum and Chlorella vulgaris

The potential of alginate-immobilized Anabaena doliolum and Chlorella vulgaris was assessed for removal of nutrients (NO _inf3 ^sup- and NH _inf4 ^sup+ ) and metals (Cr₂O _inf7 ^sup2- and Ni²⁺) at different biomass concentrations (0.05, 0.1, 0.25, 0.49 and 1.22 g dry wt l^-1) and pH values (4 to 10). Though uptake of all these substances was higher in concentrated algal beads (0.25, 0.49 and 1.22 g dry wt l^-1), their rate of uptake was significantly (P<0.001) lower than that of low (0.05 g dry wt l^-1) cell density beads. For A. doliolum, there was no significant difference in uptake rates for beads having densities of 0.05 and 0.1 g dry wt l^-1. Chlorella vulgaris, however, showed maximum efficiency at 0.1 g dry wt l^-1. Uptake of both the nutrients and the metals was maximal at pH 7 followed by pH 8, 6, 9, 10, 5 and 4. Of the different substances (organic acids and divalent cations) used, humic acid was most efficient in decreasing metal uptake. Mg²⁺ was, however, more efficient than Ca²⁺ in decreasing Ni²⁺ uptake. Immobilized algae with a cell density of 0.1 g dry wt l^-1 were the most efficient for nutrient and metal removal at pH 6 to 8.     

Cr3+和Cd2+对普通小球藻生长及抗氧化酶活性的影响

[目的] 为探究重金属对淡水绿藻生长的影响。[方法] 选取对水质检测具有明显指示作用的普通小球藻（Chlorella vulgaris）为实验材料,CdCl₂·2H₂O和CrCl₃·7H₂O提供重金属离子,探究不同浓度Cr³⁺和Cd²⁺在单一和复合胁迫下对藻细胞浓度、叶绿素a及相关抗氧化酶活性的影响。[结果] 随着Cr³⁺和Cd²⁺浓度不断增加,藻细胞浓度呈先增长后下降趋势;叶绿素a含量呈现先下降后升高再下降的现象,浓度为1 mg/L的单一和复合胁迫下有最大值,且毒性作用表现为Cr³⁺ < Cd²⁺ < Cr³⁺+Cd²⁺;与藻细胞膜相关的丙二醛（MDA）和过氧化氢（H₂O₂）含量随着重金属离子浓度的增大而增长;重金属离子浓度低于10 mg/L时对藻细胞内抗氧化酶系统中的超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和过氧化物酶（POD）表现为促进作用,而大于10 mg/L时具有抑制作用。[结论] 结果表明在单一或复合重金属胁迫下,普通小球藻会充分调动与抗逆性相关的酶来维持自身的正常生长。     

Production of microalgae from olive mill wastewater

With olive mill wastewater (alpechin) used as a nutrient medium, the influence of the aeration level and the composition of the culture medium were analysed in relation to the concentration of alpechin and KNO₃ added in a batch culture of the microalgae Chlorella pyrenoidosa and Scenedesmus obliquus. The pH and temperature of the culture were previously fixed at 6.5 and 30°C. As kinetic parameters, the maximum specific growth rate (μ_m) and the biomass productivity (P_max) were determined. The biomass composition obtained was evaluated for chlorophyll and crude protein content as well as fatty-acid composition corresponding to the lipid fraction. To achieve a balanced biomass composition in terms of proteins and lipids, the most suitable conditions included an alpechin concentration of 10%, without the addition of nitrates and with an aeration level of 1 v/v min. Under these conditions, the μ_m values reached with both microalgae were around 0.03h⁻¹.     

Assimilatory sulfur metabolism in marine microorganisms: Sulfur metabolism,growth, and protein synthesis of Pseudomonas halodurans and Alteromonas luteo-violaceus during sulfate limitation

Sulfate concentration in the growth medium exerted a strong influence on the sulfur content of protein in two marine bacteria, Pseudomonas halodurans and Alteromonasluteo-violaceus, but the distribution of sulfur in major biochemical fractions was not affected. 90% of the total cellular sulfur was contained in low molecular weight organic compounds and protein; inorganic sulfate was not an important component. The sulfur content of isolated protein and total cellular sulfur increased in proportion to the external sulfate concentration for both bacteria, reaching a maximum at about 100–250 M. The growth rate of P. halodurans only was dependent on the sulfate concentration.Sulfur starvation of cells labeled to equilibrium with ³⁵S-sulfate resulted in a rapid decrease in low molecular weight organic S with a concommitant increase in alcohol soluble (P. halodurans) or residue protein (A. luteo-violaceus). Although cell division was prevented, total protein increased in both bacteria, resulting in synthesis of sulfur-deficient protein. This effect was most pronounced in P. halodurans.Addition of ³⁵S-sulfate to sulfur-starved A. luteo-violaceus further demonstrated that sulfur metabolism was restricted primarily to the synthesis and utilization of sulfurcontaining protein precursors. The low molecular weight organic S pool was replenished rapidly, and the pool size per cell reached twice the normal value before cell division resumed. Incorporation into protein was very rapid.Abbreviations L.M.W. low molecular weight - TCA trichloroacetic acid     

The DNA,RNA and protein composition of the cyanobacterium Anacystis nidulans grown in light- and carbon dioxide-limited chemostats

The DNA, RNA and protein content of the cyanobacterium Anacystis nidulans was determined in light-limited and carbon dioxide-limited chemostat cultures over the dilution rate range, D=0.02 h^-1 to 0.19 h^-1. The macromolecular contents as a percentage of the dry weight and on a per cell basis varied significantly as a function of organism growth rate and the nature of the growth conditions. For both limitations the RNA content per cell increased [20–55 fg RNA (cell)^-1] with increasing dilution rate and also showed an increase as a percentage of the dry weight. The DNA content as a percentage of the dry weight showed a 2-fold decrease with increasing dilution rate over the range examined. On a per cell basis DNA reached a peak at D=0.1 h^-1 [4.5 fg DNA (cell)^-1] for light-limited organisms and at D=0.08 h^-1 [8.0 fg DNA (cell)^-1] for carbon dioxide-limited organisms. The q _RNA increased with increasing dilution rates over the complete growth rate range examined whilst q _DNA reached a maximum at D=0.09 to 0.10 h^-1. The protein content as a percentage of the dry weight was greater in CO₂-limited organisms than light-limited organisms but in both cultures declined as the dilution rate was increased above D=0.10 h^-1.     

Influence of different levels of ammonium concentrations on cell growth,RNA and protein production byCandida albicans

Candida albicans starved cells were incubated in minimal synthetic liquid media containing different concentrations of ammonium sulphate (0.00, 0.02, 0.05, 0.10, 0.03, 0.50 g/L). Culture growth was monitored by measuring daily the optical density and by evaluating RNA and protein cellular content after 48 and 96 hours from the inoculum. The environmental availability of ammonium ion influenced the biomass production, that was maximum when its concentration was 0.10 and 0.30 g/L. In addition, an effect on cell duplication time was observed, this was particularly evident when the (NH₄)₂SO₄ concentration was 0.10 g/L. The protein content increased in relation to the increase of ammonium ion availability, with a peak in correspondence to 0.30 g/L and a drop when the greatest concentrations were employed. RNA production was inversely proportional in respect to protein production. The optimal range of ammonium sulphate concentration forC. albicans growth was 0.10–0.30 g/L; over these concentrations there was an inhibitory effect. The rate of the protein and RNA syntheses seems to indicate the growth phase and the nitrogen nutritional conditions of the cultures, respectively.     

Effect of high nitrate concentrations on growth and nitrate uptake by free-living and immobilizedChlorella vulgaris cells

Growth and nitrate uptake were studied on free-living and immobilizedChlorella vulgaris cells cultivated in medium containing different nitrate concentrations. First, the effect of nitrate concentrations on growth indicated that cells can live in the presence of high concentrations as high as 97 mM. Although no lethal effect on cells was observed such concentration a slow down in growth and a decrease in biomass produced was observed. The rate of nitrate uptake increased with the nitrate concentration in the medium. The maximum uptake rate was reached in first days of culture in both free-living and immobilized cells. The rate dropped more rapidly for cells growing in 2 mM nitrate than for cells growing in higher nitrate concentration. The maximum rate was very much the same for free-living and immobilized and was within the order of 0.45 to 0.57 g NO₃ h^–1 10^–6 cells. Immobilization modified the changes of nitrate uptake rate for concentration higher than 2 mM.     

Steady-state kinetics of the photosystem I reaction in chloroplasts of Dunaliella which contain variable concentrations of plastocyanin

The endogenous plastocyanin (PC) concentrations of Dunaliella cultures were varied from 0.3 to 3.1 molecules per pigment 700 (P700) by decreasing the Cu⁺ supply of the nutrient. With these cultures the amount of PC which is sufficient for maximum photosynthesis in intact cells was determined to be about 1 to 1.5 PC/P700. Chloroplasts were also prepared from these cells and were employed in enzyme kinetic measurements of the PSI reaction from ascorbate reduced diaminodurene (DAD) to methylviologen/O₂. The k _m value for DAD in this reaction was 106 M. A decrease of the endogenous PC concentration caused no change of the k _m value but affected the V _max in the DAD-dependent reaction. A similar interference of the PC concentration on the maximum reaction rate could also be observed when the light intensity was varied.     

Cell cycle duration in antheridial filaments ofChara spp. (Characeae) with different genome size and heterochromatin content

The relationship between nuclear 1 C DNA content and cell cycle progression throughout successive stages of antheridial filaments were studied among five taxa ofChara: two dioecious species (n = 14):C. aspera (7.2 pg DNA),C. tomentosa (7.4 pg DNA), and three monoecious species (n = 28):C. vulgaris (13.5 pg DNA),C. fragilis (19.3 pg DNA), andC. contraria (19.6 pg DNA). With the use of double³H-thymidine labelling and morphometry a number of characteristics common to all of the investigated species were determined within the proliferative periods preceding spermiogenesis. These include: (1) simplified type of the cell cycle (S + G₂ + M), due to complete lack of G₁ intervals, (2) constant duration of S phase, (3) progressive shortening of G₂ + M periods, and (4) gradual reduction of cell lengths at successive mitotic divisions. Nucleotypic dependence was found between genome size and several time parameters estimated for consecutive stages of antheridial filaments: the higher the DNA C-value, the longer the cell cycles, their component phases, the total duration of the proliferative period, as well as the lower the rate of growth of interphase cells. Differential Giemsa staining of late G₂ phase nuclei revealed that the higher content of C-heterochromatin is connected with prolonged cell cycle durations in species with similar DNA C-values.     

Chelating Properties of Extracellular Polysaccharides from Chlorella spp

Chlorella stigmatophora LB 993 was grown in artificial seawater under controlled conditions. The production of cell wall polysaccharides attached to the cells and dissolved in the growth medium was monitored during algal growth. Preliminary characterization of the dissolved polysaccharides of C. stigmatophora and other Chlorella species is presented. The capacity of dissolved polysaccharides of C. stigmatophora to bind toxic heavy metals was also studied and compared with that of polysaccharides produced by other marine Chlorella species. The differences in metal-complexing capacity observed for dissolved polysaccharides obtained from various Chlorella species is attributable to differences in the composition of the polysaccharides, notably the uronic acids content.     

Sagebrush carbon allocation patterns and grasshopper nutrition: the influence of CO2 enrichment and soil mineral limitation

Summary Artemisia tridentata seedlings were grown under carbon dioxide concentrations of 350 and 650 l l^–1 and two levels of soil nutrition. In the high nutrient treatment, increasing CO₂ led to a doubling of shoot mass, whereas nutrient limitation completely constrained the response to elevated CO₂. Root biomass was unaffected by any treatment. Plant root/shoot ratios declined under carbon dioxide enrichment but increased under low nutrient availability, thus the ratio was apparently controlled by changes in carbon allocation to shoot mass alone. Growth under CO₂ enrichment increased the starch concentrations of leaves grown under both nutrient regimes, while increased CO₂ and low nutrient availability acted in concert to reduce leaf nitrogen concentration and water content. Carbon dioxide enrichment and soil nutrient limitation both acted to increase the balance of leaf storage carbohydrate versus nitrogen (C/N). The two treatment effects were significantly interactive in that nutrient limitation slightly reduced the C/N balance among the high-CO₂ plants. Leaf volatile terpene concentration increased only in the nutrient limited plants and did not follow the overall increase in leaf C/N ratio. Grasshopper consumption was significantly greater on host leaves grown under CO₂ enrichment but was reduced on leaves grown under low nutrient availability. An overall negative relationship of consumption versus leaf volatile concentration suggests that terpenes may have been one of several important leaf characteristics limiting consumption of the low nutrient hosts. Digestibility of host leaves grown under the high CO₂ treatment was significantly increased and was related to high leaf starch content. Grasshopper growth efficiency (ECI) was significantly reduced by the nutrient limitation treatment but co-varied with leaf water content.     

Response of Chlorella to Scandium and Potassium at Various pH

The response of Chlorella pyrenoidosa cells to moderate potassium deficiency and to the addition of various scandium amounts in the nutrient solution were studied at different pH. The effects on growth, chlorophyll content, oxygen consumption, and the ¹⁴CO₂ incorporation in photosynthesizing cells were measured. Considerable metabolic changes inside the cells were registered under the given nutrient conditions. The separated ¹⁴C-labelled compounds showed, at acid and neutral pH in complete nutrient solution, an increase in the relative content of sugar phosphates and a decrease in the relative content of sucrose, caused by increasing scandium concentrations. Moderate potassium deficiency caused a diminution of the relative content of sugar phosphates. The relative content of amino acids was increased by scandium added to complete nutrient solution, but it decreased under potassium deficiency.     

Nutritional properties of four marine microalgae for albino rats

The nutritive value of the marine microalgaeTetraselmis suecica, Isochrysis galbana, Dunaliella tertiolecta andChlorella stigmatophora was studied in diets given to rats. Biological assays were carried out in order to determine the Protein Efficiency Ratio (PER) and the Food Conversion Efficiency (FCE). Each dried microalga was fed to weaning Wistar albino rats as the sole protein source at a protein level of 12%. Control rats were given diets containing 12 % casein. Food consumption was similar in all groups. PER values obtained were 1.14 withT. suecica diet, 1.13 withI. galbana diet, 2.07 withD. tertiolecta diet and 1.13 withC. stigmatophora diet (casein, 2.50). FCE values followed a similar pattern. The data showed that the marine microalgaD. tertiolecta is a source of protein of good quality. Its PER is quite high, compared to vegetable and cereal proteins, and compares favourably with other microbial protein sources, such as yeasts or different freshwater microalgae. Haematological tests showed no significant differences among the groups in haemoglobin levels, red and white blood cell counts, differential count and mean corpuscular volume. Different blood parameters were also determined and a significant decrease in triglycerides levels appeared with all the microalgal diets, whereas a significant decrease in cholesterol appeared inD. tertiolecta andC. stigmatophora diets.