Growth relationships of a lipid-producing Chlorella-alga with common microalgae in laboratory co-cultures

Co-existence growth relationships were studied in communities consisting of a lipid-producing alga Chlorella sp. HQ, another green alga and one cyanobacterium: I Scenedesmus obliquus and Microcystis aeruginosa; II Chlamydomonas reinhardtii and Anabaena flos-aquae; III Selenastrum capricornutum and Microcystis wesenbergii. The cyanobacteria and green algae except for Chlorella sp. HQ were commonly detected in Chinese reservoir and wastewater. The rate of increase of apparent cell number difference with other algae (k _app), inhibition/stimulation ratio (ISR) and the parameters of logistic model and co-existence model were determined for Chlorella. Chlorella strains were the most competitive in Combination I, and were stimulated during 75% of the cultivation time for all three combinations. Anabaena growth exceeded those of Chlorella and Chlamydomonas on the 5th cultivation day under 1 : 1 : 1 inoculum ratio. Scenedesmus colonies consisted of fewer cells, whose average length significantly shortened after the 5th cultivation day under 1 : 1 : 1 inoculum ratio. The developed co-existence model can identify the concrete growth inhibitor or stimulator among three species compared with the single method of cell number monitoring. Good correlation was found between transformed and non-transformed co-existence model through a _mn and b _mn values. Allelopathy and nutrient competition are both possible mechanisms in the above growth relationships.     

Study of the growth and development ofChlorella populations in the culture as a whole

Colour tests for study of the nuclear apparatus ofChlorella cells are described. It can be concluded from the results thatChlorella cell nucleus contains a relatively large nucleolus; the peripheral layers of the nucleus are rich in chromatin only in the nuclear division phase. The nucleus divides by a simplified form of mitosis, characterized morphologically by simple constriction.     

The eurythermal adaptivity and temperature tolerance of a newly isolated psychrotolerant Arctic <Emphasis Type="Italic">Chlorella</Emphasis> sp.

A new strain of Chlorella sp. (Chlorella-Arc), isolated from Arctic glacier melt water, was found to have high specific growth rates (μ) between 3 and 27 °C, with a maximum specific growth rate of 0.85 day^?1 at 15 °C, indicating that this strain was a eurythermal strain with a broad temperature tolerance range. To understand its acclimation strategies to low and high temperatures, the physiological and biochemical responses of the Chlorella-Arc to temperature were studied and compared with those of a temperate Chlorella pyrenoidosa strain (Chlorella-Temp). As indicated by declining F _v/F _m, photoinhibition occurred in Chlorella-Arc at low temperature. However, Chlorella-Arc reduced the size of the light-harvesting complex (LHC) to alleviate photoinhibition, as indicated by an increasing Chl a/b ratio with decreasing temperatures. Interestingly, Chlorella-Arc tended to secrete soluble sugar into the culture medium with increasing temperature, while its intracellular soluble sugar content did not vary with temperature changes, indicating that the algal cells might suffer from osmotic stress at high temperature, which could be adjusted by excretion of soluble sugar. Chlorella-Arc accumulated protein and lipids under lower temperatures (<15 °C), and its metabolism switched to synthesis of soluble sugar as temperatures rose. This reflects a flexible ability of Chlorella-Arc to regulate carbon and energy distribution when exposed to wide temperature shifts. More saturated fatty acids (SFA) in Chlorella-Arc than Chlorella-Temp also might serve as the energy source for growth in the cold and contribute to its cold tolerance.     

Interactive Effects of Temperature and UV Radiation on Photosynthesis of Chlorella Strains from Polar,Temperate and Tropical Environments: Differential Impacts on Damage and Repair

Global warming and ozone depletion, and the resulting increase of ultraviolet radiation (UVR), have far-reaching impacts on biota, especially affecting the algae that form the basis of the food webs in aquatic ecosystems. The aim of the present study was to investigate the interactive effects of temperature and UVR by comparing the photosynthetic responses of similar taxa of Chlorella from Antarctic (Chlorella UMACC 237), temperate (Chlorella vulgaris UMACC 248) and tropical (Chlorella vulgaris UMACC 001) environments. The cultures were exposed to three different treatments: photosynthetically active radiation (PAR; 400–700 nm), PAR plus ultraviolet-A (320–400 nm) radiation (PAR + UV-A) and PAR plus UV-A and ultraviolet-B (280–320 nm) radiation (PAR + UV-A + UV-B) for one hour in incubators set at different temperatures. The Antarctic Chlorella was exposed to 4, 14 and 20°C. The temperate Chlorella was exposed to 11, 18 and 25°C while the tropical Chlorella was exposed to 24, 28 and 30°C. A pulse-amplitude modulated (PAM) fluorometer was used to assess the photosynthetic response of microalgae. Parameters such as the photoadaptive index (E_k) and light harvesting efficiency (α) were determined from rapid light curves. The damage (k) and repair (r) rates were calculated from the decrease in ΦPSII_eff over time during exposure response curves where cells were exposed to the various combinations of PAR and UVR, and fitting the data to the Kok model. The results showed that UV-A caused much lower inhibition than UV-B in photosynthesis in all Chlorella isolates. The three isolates of Chlorella from different regions showed different trends in their photosynthesis responses under the combined effects of UVR (PAR + UV-A + UV-B) and temperature. In accordance with the noted strain-specific characteristics, we can conclude that the repair (r) mechanisms at higher temperatures were not sufficient to overcome damage caused by UVR in the Antarctic Chlorella strain, suggesting negative effects of global climate change on microalgae inhabiting (circum-) polar regions. For temperate and tropical strains of Chlorella, damage from UVR was independent of temperature but the repair constant increased with increasing temperature, implying an improved ability of these strains to recover from UVR stress under global warming.     

Antioxidant responses of two microalgae, Selenastrum capricornutum and Chlorella sp., to estradiol and ethinylestradiol

The cellular characteristics and antioxidant responses including reactive oxygen species (ROS), esterase activity, superoxide dismutase, peroxidase (POD), glutathione (GSH), and malondialdehyde (MDA) content of two microalgal species, Selenastrum capricornutum and Chlorella sp., exposed to single and mixed estradiol (E2) and ethinlyestradiol (EE2) at a final concentration of 200 μg L^?1 were investigated. The growth, autofluorescence, cell complexity, and cell size of S. capricornutum and Chlorella sp. were not affected by single and mixed estrogen treatments, but the temporal trends of these two species were different. The temporal changes of the esterase activity and ROS level in these two species were also very different, with a continuous increase with time in Chlorella but peak on Day 2 in S. capricornutum. The esterase activity of S. capricornutum was not affected by estrogen treatments; however, the activity in treated Chlorella decreased significantly from Day 2 onwards. The single EE2 and mixed E2 and EE2 treatments induced more ROS of S. capricornutum than the single E2 treatment and control on Day 4, but no other difference was found between treatments and control of Chlorella on that day. The other oxidative damage responses of S. capricornutum were not affected by estrogen treatments, except MDA increased in EE2 treatment, on the other hand, POD and GSH of Chlorella increased in both single and mixed estrogen treatments on Day 4. These results suggested that microalgae species were tolerant to E2 and EE2, but some species-specific changes occurred to combat the oxidative stress posed by estrogens.     

Inorganic carbon and pH effect on growth and lipid productivity of Tetraselmis suecica and Chlorella sp (Chlorophyta) grown outdoors in bag photobioreactors

There has been considerable interest in cultivation of green microalgae (Chlorophyta) as a source of lipid that can alternatively be converted to biodiesel. However, almost all mass cultures of algae are carbon-limited. Therefore, to reach a high biomass and oil productivities, the ideal selected microalgae will most likely need a source of inorganic carbon. Here, growth and lipid productivities of Tetraselmis suecica CS-187 and Chlorella sp were tested under various ranges of pH and different sources of inorganic carbon (untreated flue gas from coal-fired power plant, pure industrial CO₂, pH-adjusted using HCl and sodium bicarbonate). Biomass and lipid productivities were highest at pH 7.5 (320?±?29.9 mg biomass L^?1 day^?1and 92?±?13.1 mg lipid L^?1 day^?1) and pH 7 (407?±?5.5 mg biomass L^?1 day^?1 and 99?±?17.2 mg lipid L^?1 day^?1) for T. suecica CS-187 and Chlorella sp, respectively. In general, biomass and lipid productivities were pH 7.5?>?pH 7?>?pH 8?>?pH 6.5 and pH 7?>?pH 7.5?=?pH 8?>?pH 6.5?>?pH 6?>?pH 5.5 for T. suecica CS-187 and Chlorella sp, respectively. The effect of various inorganic carbon on growth and productivities of T. suecica (regulated at pH?=?7.5) and Chlorella sp (regulated at pH?=?7) grown in bag photobioreactors was also examined outdoor at the International Power Hazelwood, Gippsland, Victoria, Australia. The highest biomass and lipid productivities of T. suecica (51.45?±?2.67 mg biomass L^?1 day^?1 and 14.8?±?2.46 mg lipid L^?1 day^?1) and Chlorella sp (60.00?±?2.4 mg biomass L^?1 day^?1 and 13.70?±?1.35 mg lipid L^?1 day^?1) were achieved when grown using CO₂ as inorganic carbon source. No significant differences were found between CO₂ and flue gas biomass and lipid productivities. While grown using CO₂ and flue gas, biomass productivities were 10, 13 and 18 %, and 7, 14 and 19 % higher than NaHCO₃, HCl and unregulated pH for T. suecica and Chlorella sp, respectively. Addition of inorganic carbon increased specific growth rate and lipid content but reduced biomass yield and cell weight of T. suecica. Addition of inorganic carbon increased yield but did not change specific growth rate, cell weight or content of the cell weight of Chlorella sp. Both strains showed significantly higher maximum quantum yield (F_v/F_m) when grown under optimum pH.     

Biohydrogen production by immobilized Chlorella sp. using cycles of oxygenic photosynthesis and anaerobiosis

Hydrogen production was studied using immobilized green alga Chlorella sp. through a two-stage cyclic process where immobilized cells were first incubated in oxygenic photosynthesis followed by anaerobic incubation for H₂ production in the absence of sulfur. Chlorella sp. used in this study was capable of generating H₂ under immobilized state in agar. The externally added glucose enhanced H₂ production rates and total produced volume while shortened the lag time required for cell adaptation prior to H₂ evolution. The rate of hydrogen evolution was increased as temperature increased, and the maximum evolution rate under 30 mM glucose was 183 mL/h/L and 238 mL/h/L at 37 °C and 40 °C, respectively. In order to continue repeated cycles of H₂ production, at least two days of photosynthesis stage should be allowed for cells to recover H₂ production potential and cell viability before returning to H₂ production stage again.     

Water permeability of chlorella cell membranes by nuclear magnetic resonance: measured diffusion coefficients and relaxation times

Measurement by two nuclear magnetic resonance (NMR) techniques of the mean residence time τ_a of water molecules inside Chlorella vulgaris (Beijerinck) var. “viridis” (Chodot) is reported. The first is the Conlon and Outhred (1972 Biochim Biophys Acta 288: 354-361) technique in which extracellular water is doped with paramagnetic Mn²⁺ ions. Some complications in application of this technique are identified as being caused by the affinity of Chlorella cell walls for Mn²⁺ ions which shortens the NMR relaxation times of intra- and extracellular water. The second is based upon observations of effects of diffusion on the spin echo of intra- and extracellular water. Echo attenuation of intracellular water is distinguished from that of extracellular water by the extent to which diffusive motion is restricted. Intracellular water, being restricted to the cell volume, suffers less echo attenuation. From the dependence of echo amplitude upon gradient strength at several values of echo time, the mean residence time of intracellular water can be determined. From the mean residence time of intracellular water, the diffusional water permeability coefficient of the Chlorella membrane is calculated to be 2.1 ± 0.4 × 10⁻³ cm sec⁻¹.     

Effect of cell movement by random mixing between the surface and bottom of photobioreactors on algal productivity

Effect of algae movement, as a result of random mixing, between the surface and bottom zones of shallow, moderately deep and deep photobioreactors (incident light intensities per unit volume were 8125, 4062 and 2031 μmol·m⁻³·s⁻¹, respectively) on the reactor productivity was investigated. The results showed that at low cell concentrations, movement of cells between the surface and bottom zones of shallow and moderately deep reactors had no significant effect on Chlorella pyrenoidosa C-212 growth and productivity. However, as the cell concentration in the reactors increased, cell movement between the two zones resulted in increased productivity of the shallow reactor but decreased productivity of the moderately deep reactor. On the other hand, in the deep reactor, random movement of cells between the two zones resulted in decreased Chlorella growth rate regardless of the cell concentration. This may be attributed to the fact that at high cell concentration or in a deep reactor, if the cells move between the surface and bottom of the reactor, they spend too long a time in the dark part of the reactor where there is no cell growth, and endogenous respiration as well as cell death may lead to a decrease in cell concentration. When Spirulina platensis M-135 cells were cultivated in the deep reactor, even at high cell concentration, movement of cells between the surface and bottom zones of the reactor led to an increase in the reactor productivity. The reasons for the difference in the results obtained with these two strains of algae could be attributed to the difference in their light requirements since it was found that the saturation light intensity and specific decrease in cell concentration when incubated in the dark were lower for Spirulina than for Chlorella cells.     

Immediate effects of pyridazinone herbicides on photosynthetic electron transport in algal systems

Pyridazinone herbicides, SANDOZ 9785 (4-chloro-5-dimethylamino2-phenyl-3-(2H) pyridazinone), SANDOZ 9789 (4-chloro-5 (methylamino)-2-(α, α, α-trifluoro-m-tolyl-3-(2H) pyridazinone) and SANDOZ 6706 (4-chloro-5-(methylamino)-2-(α, α, α-trifluoro-m-tolyl-3-(2H) pyridazinone) inhibited photosystem II electron transport inChlorella protothecoides, when the herbicides were added to the assay medium. The inhibitory eficiency varied with the algal species and the nature of substitution of pyridazinones. Using 3 algal systemsviz., Chlorella, Scenedesmus andAnacystis, the I₅₀ value of for the inhibition of photosynthesis of 3 substituted pyridazinones (SANDOZ 9785, SANDOZ 6706 and SANDOZ 9789) were determined. SANDOZ 9789 was found to be the weakest inhibitor of photosystem II electron transport (H₂O→ benzoquinone) as compared to SANDOZ 9785 and SANDOZ 6706. In general, the order of inhibition could be given as SANDOZ 6706 >- SANDOZ 9785 > SANDOZ 9789. The I₅₀ value of photosynthetic particles obtained fromChlorella cells was similar to that of whole cells, suggesting that the cell wall ofChlorella did not act as a barrier for the herbicide action. Studies on the light intensity dependence of SANDOZ 9785 inhibition of electron transport (H₂O→ benzoquinone) showed that the light-dependent portion of the curve was more sensitive than the light independent portion of the curve. It is suggested that the site of action was on the reducing side of photosystem II.     

Productivity and heat generation of fermentation under oxygen limitation

The elemental balance equation of microbial growth on carbon substrate of generalized composition is given. Yield of dried bio-mass per oxygenY _o is calculated. Yield per oxygenY _o is found to be determined by two factors—carbon yieldy and the reducing power of substrate γ _s . The mode of dependence ofY _o on these two quantities is studied. The energetic interpretation ofy and γ _s is given. The dependence ofY _o ony and γ _s is shown to be equivalent to the dependence on a single factor, the energetie yield of growth η. Fermentor productivity increases with growth of η, the increase being directly proportional if η is not large (up to 25%) and becoming steeper if η is larger. The restrictions on a range of workable carbon yields during growth on various substrates are found. Metabolic heat generation of fermentor is shown to be proportional to oxygen consumption and to average 3.38 kcal per gram of O₂ irrespective of substrate and microorganism used.     

Enzyme activities of the carbon reduction cycle in some photosynthetic organisms

Profile analyses of the enzymes comprising the photosynthetic carbon reduction cycle have been performed in extracts of dark grown and greening Euglena gracilis var. bacillaris. Chlorella pyrenoidosa grown photoautotrophically, in the light with glucose or in the dark with glucose, Tolypothrix tenuis, Chromatium and leaves of spinach. Amounts of activity are compared with the level of photosynthetic CO₂ fixation. Only in Chromatium were all enzyme activities sufficient to support the in vivo rate of CO₂ fixation. In organisms other than Chromatium, some enzymes and particularly fructose 1,6-phosphatase and ribulose 1.5-diphosphate carboxylase appeared to be present in insufficient amounts to support the photosynthetic rate of the intact cell. Developmental studies with Euglena and growth studies with Chlorella led to the conclusion that these enzymes were associated with the cycle. Suppression of CO₂ fixation in heterotrophically grown Chlorella was accompanied by a striking decrease in the same enzymes whose activities increased in greening Euglena.     

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.     

Cyclic Photophosphorylation in Vivo and its Relation to Photosynthetic CO(2)-Fixation

Salicylaldoxime (2 × 10⁻³m and less) inhibits cyclic photophosphorylation in intact Chlorella cells severely whereas photosynthetic O₂-evolution and ¹⁴CO₂-fixation is hardly affected. Cyclic photophosphorylation in vivo was measured by following anaerobic light dependent glucose uptake. A similar difference in susceptibility has been observed with carbonylcyanide-p-trifluoromethoxyphenylhydrazone. Various controls exclude the possibility that the difference in inhibition was caused by differing experimental conditions or, in the case of glucose assimilation, by an inhibition of a reaction other than photophosphorylation.     

Long-term outdoor growth and lipid productivity of Tetraselmis suecica, Dunaliella tertiolecta and Chlorella sp (Chlorophyta) in bag photobioreactors

There has been considerable interest on cultivation of green microalgae (Chlorophyta) as a source of lipid that can alternatively be converted to biodiesel. The ideal microalga characteristics are that it must grow well even under high cell density and under varying outdoor environmental conditions and be able to have a high biomass productivity and contain a high oil content (~25–30 %). The main advantage of Chlorophyta is that their fatty acid profile is suitable for biodiesel conversion. Tetraselmis suecica CS-187 and Chlorella sp. were grown semi-continuously in bag photobioreactors (120 L, W?×?L?=?40?×?380 cm) over a period of 11 months in Melbourne, Victoria, Australia. Monthly biomass productivity of T. suecica CS-187 and Chlorella sp. was strongly correlated to available solar irradiance. The total dry weight productivity of T. suecica and Chlorella sp. was 110 and 140 mg L^?1 d^?1, respectively, with minimum 25 % lipid content for both strains. Both strains were able to tolerate a wide range of shear produced by mixing. Operating cultures at lower cell density resulted in increasing specific growth rates of T. suecica and Chlorella sp. but did not affect their overall biomass productivity. On the other hand, self shading sets the upper limit of operational maximum cell density. Several attempts in cultivating Dunaliella tertiolecta CS-175 under the same climatic conditions were unsuccessful.     

Characterization of Nitrate Reductase Deficient Mutants of Chlorella sorokiniana

After x-ray irradiation, 13 mutants of Chlorella sorokiniana incapable of using NO₃⁻ as N source were isolated using a pinpoint method. Using immunoprecipitation and Western blot assays, no nitrate reductase was found in five strains while in eight mutants the enzyme was detected. The latter strains contained different patterns of nitrate reductase partial reactions. All isolates were of the nia-type as indicated by the inducibility of purine hydroxylase I and by complementation of nitrate reductase activity in the Neurospora crassa mutant Nit-1. A restoration of NADP-nitrate reductase in Nit-1 was also obtained with NH₄⁺-grown cells indicating that Mo-cofactor is constitutive in Chlorella. Complementation experiments among the Chlorella mutants resulted in restoration of NADH-nitrate reductase activity. The characteristics of some of the Chlorella mutants are discussed in view of an improper orientation of Mo-cofactor in the residual nitrate reductase protein.     

Evaluation of antioxidant capacities of green microalgae

Three strains of green microalgae, Chlorococcum sp.C53, Chlorella sp. E53, and Chlorella sp.ED53 were studied for their antioxidant activities. Crude extracts of these microalgae in hot water and in ethanol were examined for their total phenolic contents and for their antioxidant capacities. In order to determine their phenolic contents, the Folin–Ciocalteu method was used. As for the determination of their antioxidant capacities, four different assays were used: (1) total antioxidant capacity determination; (2) DPPH radical scavenging assay; (3) ferrous ion chelating ability assay; and (4) inhibition of lipid peroxidation (using thiobarbituric acid reactive substance). For all the strains we have studied, their ethanolic extract showed more antioxidant activities than their hot water extract. Categorically, the ethanolic extract of Chlorella sp.E53 exhibited both the highest total phenolic content of 35.5?±?0.14 mg gallic acid equivalent (GAE) g^?1 dry weight and the highest DPPH radical scavenging of 68.18?±?0.38 % at 1.4 mg mL^?1 (IC₅₀ 0.81 mg mL^?1), whereas Chlorella sp.ED53 showed both the highest ferrous ion chelation activity of 42.78?±?1.48 % at 1 mg mL^?1 (IC₅₀ 1.23 mg mL^?1) and the highest inhibition of lipid peroxidation of 87.96?±?0.59 % at 4 mg mL^?1. This high level of inhibition is comparable to 94.42?±?1.39 % of butylated hydroxytoluene, a commercial synthetic antioxidant, at the same concentration.     

Comparative studies onChlorella cell walls: Induction of protoplast formation

Among 12 strains ofChlorella ellipsoidea, C. vulgaris, andC. saccharophila tested, 4 strains (1,C. ellpsoidea; 2,C. vulgaris; 1,C. saccharophila) formed osmotically labile protoplasts after treatment with mixtures of polysaccharide degrading enzymes. The relationship between enzymatical digestibility and structure or composition ofChlorella cell walls were studied by electron microscopy and staining techniques with some specific dyes. The cell wall structures of the 12Chlorella strains were grouped into three types: (1) with a trilaminar outer layer, (2) with a thin outer monolayer, and (3) without an outer layer. Protoplasts were formed only from the strains with a cell wall of Type 2. In the strains with a cell wall of Type 1, the outer layer protected the inner major microfibrillar layer against enzymatic digestion. The cell wall of Type 3 was totally resistant to the enzymes; the chemical composition of the cell wall would be somewhat different from that of other types.     

Kinetics of water loss and the likelihood of intracellular freezing in mouse ova

To avoid intracellular freezing and its usually lethal consequences, cells must lose their freezable water before reaching their ice-nucleation temperature. One major factor determining the rate of water loss in the temperature dependence of the water permeability,L _p (hydraulic conductivity). Because of the paucity of water permeability measurements at subzero temperatures, that temperature dependence has usually been extrapolated from above-zero measurements. The extrapolation has often been based on an exponential dependence ofL _p on temperature. This paper compares the kinetics of water loss based on that extrapolation with that based on an Arrhenius relation betweenL _p and temperature, and finds substantial differences below ?20 to ?25°C. Since the ice-nucleation temperature of mouse ova in the cryoprotectants DMSO and glycerol is usually below ?30°C, the Arrhenius form of the water-loss equation was used to compute the extent of supercooling in ova cooled at rates between 1 and 8°C/min and the consequent likelihood of intracellular freezing. The predicted likelihood agrees well with that previously observed. The water-loss equation was also used to compute the volumes of ova as a function of cooling rate and temperature. The computed cell volumes agree qualitatively with previously observed volumes, but differ quantitatively.