Two-stage heterotrophic and phototrophic culture strategy for algal biomass and lipid production

A two-stage heterotrophic and phototrophic culture strategy for algal biomass and lipid production was studied, wherein high density heterotrophic cultures of Chlorellasorokiniana serve as seed for subsequent phototrophic growth. The data showed growth rate, cell density and productivity of heterotrophic C.sorokiniana were 3.0, 3.3 and 7.4 times higher than phototrophic counterpart, respectively. Hetero- and phototrophic algal seeds had similar biomass/lipid production and fatty acid profile when inoculated into phototrophic culture system. To expand the application, food waste and wastewater were tested as feedstock for heterotrophic growth, and supported cell growth successfully. These results demonstrated the advantages of using heterotrophic algae cells as seeds for open algae culture system. Additionally, high inoculation rate of heterotrophic algal seed can be utilized as an effective method for contamination control. This two-stage heterotrophic phototrophic process is promising to provide a more efficient way for large scale production of algal biomass and biofuels.     

Comparison of biomass production and total lipid content of freshwater green microalgae cultivated under various culture conditions

The growth and total lipid content of four green microalgae (Chlorella sp., Chlorella vulgaris CCAP211/11B, Botryococcus braunii FC124 and Scenedesmus obliquus R8) were investigated under different culture conditions. Among the various carbon sources tested, glucose produced the largest biomass or microalgae grown heterotrophically. It was found that 1 % (w/v) glucose was actively utilized by Chlorella sp., C. vulgaris CCAP211/11B and B. braunii FC124, whereas S. obliquus R8 preferred 2 % (w/v) glucose. No significant difference in biomass production was noted between heterotrophic and mixotrophic (heterotrophic with light illumination/exposure) growth conditions, however, less production was observed for autotrophic cultivation. Total lipid content in cells increased by approximately two-fold under mixotrophic cultivation with respect to heterotrophic and autotrophic cultivation. In addition, light intensity had an impact on microalgal growth and total lipid content. The highest total lipid content was observed at 100 μmol m^?2s^?1 for Chlorella sp. (22.5 %) and S. obliquus R8 (23.7 %) and 80 μmol m^?2s^?1 for C. vulgaris CCAP211/11B (20.1 %) and B. braunii FC124 (34.9 %).     

Microalgal Biomass and Lipid Production on Dairy Effluent Using a Novel Microalga, <Emphasis Type="Italic">Chlorella</Emphasis> sp. Isolated from Dairy Wastewater

The present study focused on cost-effective production of microalgal biomass and lipid production on dairy effluent. The novel microalga, Chlorella sp. isolated from the dairy effluent showed high growth and lipid production on the undiluted and two-fold diluted dairy effluent which were four to five times higher than those of Chlorella vulgaris (control). The high growth of Chlorella sp. was thought to be possibly due to its heterotrophic growth capacity, high turbidity, COD, nutrients and trace elements. In contrast, C. vulgaris showed poor heterotrophic and photoautotrophic growth under the highly turbid conditions of dairy effluent. Both Chlorella sp. and C. vulgaris showed similar total FAME (mg FAME/g algal cells). The fatty acid composition analysis revealed that both Chlorella sp. and C. vulgaris possessed major C18 and C20 fatty acids which will be used for biodiesel production. Overall, the novel microalga, Chlorella sp. isolated from the dairy effluent showed high potential for cost-effective algal cultivation and lipid production on dairy effluent without any modification of process.     

OCCURRENCE AND ABUNDANCE OF GREEN-FLUORESCING DINOFLAGELLATES IN SURFACE WATERS OF THE NORTHWEST ATLANTIC AND NORTHEAST PACIFIC OCEANS1

We have cultured green fluorescing heterotrophic dinoflagellates whose continuous green fluorescence is due to an unidentified compound, probably a flavin, that excites with blue (～460 nm) light and emits green (～535 nm) light. No evidence of bioluminescence was found, but we note that compounds with similar fluorescence characteristics have been associated with bioluminescence in other taxa. These cells, all naked gymnodinoids, are widespread and abundant in the Northwest Atlantic and Northeast Pacific Oceans (10³–10⁵ L^?1). They comprise 4–100% of the total heterotrophic dinoflagellate component which, in turn, is usually equivalent magnitude to the phototrophic naked dinoflagellate component of the phytoplankton community.     

MANOMETRIC MEASUREMENTS OF PHOTOSYNTHESIS IN THE MARINE ALGA GIGARTINA

A manometric method for measuring photosynthesis in marine algae is described. Photosynthesis in the red alga Gigartina harveyana is shown to be similar in all important respects to photosynthesis in Chlorella and other Chlorophyceae.     

Which are fatty acids of the green alga Chlorella?

Fatty acid composition of three species of Chlorella were studied under conditions of photoautotrophic and heterotrophic cultivation, nitrogen starvation, and outdoor in a photobioreactor. The composition 14:0, 16:0, 16:1, 16:2, 16:3, 18:0, 18:1, 18:2, α-18:3 is confirmed for Chlorella. Fatty acids with 20 carbon atoms and four or five double bonds are considered not originating from Chlorella. Other exceptions of this composition are interpreted as mixed algal culture, bacterial contamination or impurities.     

CHARACTERIZATION OF TWO CHLORELLA VULGARIS (CHLOROPHYCEAE) STRAINS ISOLATED FROM WASTEWATER OXIDATION PONDS1

We report here on the characterization and isolation of two ecotypes of Chlorella vulgaris Beyerinck that coexist in wastewater reservoirs. One ecotype (C1) contains high amounts of chlorophyll b, is capable of autotrophic growth, and can utilize only a few organic solutes for growth. The second ecotype (C2) contains low amounts of chlorophyll b, requires vitamin B₁₂, and can support its growth with a broad range of organic compounds. Of the two ecotypes, the latter showed slower growth rates when light was the sole source of energy. Cells of C2-type Chlorella attained higher photosynthetic activities than C1-type cells at saturating irradiances. However, their low chlorophyll b content and lower light utilization efficiency suggest that C2-type Chlorella contains relatively low amounts of light-harvesting antennae, a disadvantage in severely light-limited ecosystems like wastewater reservoirs. We hypothesize that the two Chlorella types coexist by adopting different lifestyles: C1-type cells rely largely on their photosynthetic potential for energy conservation and growth, whereas C2-type cells may exploit their heterotrophic properties for this purpose.     

STUDIES WITH CYANIDIUM CALDARIUM : I. The Fine Structure and Systematic Position of the Organism

The fine structure of Cyanidium caldarium, as seen in thin sections of KMnO₄-fixed cells examined with the electron microscope, is described. This organism, whose taxonomic position among algae is undetermined, contains a single well defined chloroplast, a nucleus, and mitochondria. Studies, with the electron microscope, of Chlorella pyrenoidosa and Nostoc are also reported. Structural differences within cells of Cyanidium, chlorella, and Nostoc are discussed. It is concluded that if Nostoc can be taken as a typical Cyanophyte and Chlorella as a representative Chlorophyte and if the items of fine structure examined are diagnostic, then Cyanidium is certainly not a Cyanophyte and, while it has numerous features in common with Chlorella, is not a green alga similar to Chlorella. Comparisons are also made between Cyanidium and other algae whose fine structure has been described by others.     

Growth of Chlorella sorokiniana at Hyperbaric Oxygen Pressures

The growth rate of Chlorella sorokiniana decreased in a linear fashion as the partial pressure of oxygen was increased from 711 to 1,478 mm of Hg. Under two atmospheres of oxygen pressure, growth ceased after 10 to 12 hr. This cessation of growth was not due to any permanent injury, as growth resumed when oxygen partial pressure was reduced to ambient levels. The inhibition occurred under both autotrophic and heterotrophic growth conditions and was not accompanied by an increase in cell size. The results indicated that the tolerance of Chlorella cells to elevated oxygen pressures was not an absolute immunity, and that inhibition of growth at very high oxygen pressures cannot be accounted for by an inhibition of photosynthesis alone.     

TEMPERATURE CHARACTERISTICS FOR THE METABOLISM OF CHLORELLA : III. THE CATALYTIC DECOMPOSITION OF HYDROGEN PEROXIDE BY CHLORELLA PYRENOIDOSA

The decomposition of hydrogen peroxide by intact Chlorella cells follows a first order course at very low temperatures, but at higher temperatures gives falling first order constants. Between 0.6° and 20°C. the value of µ is 10,500 calories.     

FREEZE ETCHING OF CELLS WITHOUT CRYOPROTECTANTS

The technique of spray-freeze etching was applied to unicellular organisms. The superior freezing rates obtainable with this method gave excellent cryofixation on Chlorella, Euglena, and spermatozoa without the use of antifreeze agents, and cell damage due to ice crystal formation was never observed. In many instances the resultant morphology differed significantly from that obtained from glycerol-treated, freeze-etched cells. Furthermore, viability studies of spray-frozen Chlorella compared favorably with cells frozen by other methods.     

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.     

Isolation and characterization of a Chlorella mutant producing high amounts of chlorophyll and carotenoids

Mutants of Chlorella that form dark green colonies under heterotrophic conditions were generated from the parental Chlorella regularisS-88 by MNNG mutagenesis. The mutant Y-21 was the highest in chlorophyll and carotenoids contents among these isolates. Y-21 grew a little slower than its parental S-88 under heterotrophic and photoautotrophic conditions. Although chloroplasts were generated in both S-88 and Y-21 during glucose starvation and under illumination, the chlorophyll and carotenoid contents of Y-21 were approximately 1.5–1.8 times those of S-88 under both growth conditions, suggesting that relative size of chloroplast is larger in Y-21 than in S-88. Consistent with these results, photosynthesis genes on chloroplast DNA were more highly up-regulated in Y-21 than in S-88 during glucose starvation and under illumination. These results lead us a model in which chloroplast generation is sugar-repressed and photoinduced in Chlorella. Y-21 appears to be a mutant in which sugar repression of chloroplast generation is weak.     

Auto-/heterotrophic endosymbiosis evolves in a mature stage of ecosystem development in a microcosm composed of an alga,a bacterium and a ciliate

We investigate an ecological mechanism by which endosymbiotic associations evolve, with a particular focus on the relationship between the evolution of endosymbiosis between auto- and heterotrophic organisms, and the stages of ecosystem development. For this purpose we conducted a long-term microcosm culture composed of three species, a green alga (Chlorella vulgaris), a bacterium (Escherichia coli), and a ciliated protozoan (Tetrahymena thermophila) for 3 years. During this culture T. thermophila cells harboring Chlorella cells emerged by phagocytotic uptake, and increased in frequency, reaching ca. 80–90%. This level was maintained in the late stage of ecosystem dynamics. Analysis of the ecosystem dynamics in the microcosm revealed that a complex causal process through direct/indirect interactions among ecosystem components led to reduction in dissolved O₂ and food (E. coli) available to the T. thermophila, which gave a selective advantage to the organisms in the endosymbiotic association. This result suggests that the endosymbiosis evolves in a mature stage of ecosystem development, where reproduction and survival of prospective partner organisms is highly resource-limited and density-dependent, favoring efficient matter/energy transfers among participating organisms due to physical proximity. Consequently, a complex web of interactions and pathways of matter/energy flow in ecosystem evolves from an initially simple one.     

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.     

Interspecific protection against oxidative stress: green algae protect harmful cyanobacteria against hydrogen peroxide

Oceanographic studies have shown that heterotrophic bacteria can protect marine cyanobacteria against oxidative stress caused by hydrogen peroxide (H₂O₂). Could a similar interspecific protection play a role in freshwater ecosystems? In a series of laboratory experiments and two lake treatments, we demonstrate that freshwater cyanobacteria are sensitive to H₂O₂ but can be protected by less-sensitive species such as green algae. Our laboratory results show that green algae degrade H₂O₂ much faster than cyanobacteria. Consequently, the cyanobacterium Microcystis was able to survive at higher H₂O₂ concentrations in mixtures with the green alga Chlorella than in monoculture. Interestingly, even the lysate of destructed Chlorella was capable to protect Microcystis, indicating a two-component H₂O₂ degradation system in which Chlorella provided antioxidant enzymes and Microcystis the reductants. The level of interspecific protection provided to Microcystis depended on the density of Chlorella. These findings have implications for the mitigation of toxic cyanobacterial blooms, which threaten the water quality of many eutrophic lakes and reservoirs worldwide. In several lakes, H₂O₂ has been successfully applied to suppress cyanobacterial blooms. Our results demonstrate that high densities of green algae can interfere with these lake treatments, as they may rapidly degrade the added H₂O₂ and thereby protect the bloom-forming cyanobacteria.     

Characterization of the terminal inverted repeats and their neighboring tandem repeats in the Chlorella CVK1 virus genome

A unique group of large icosahedral viruses that infect a unicellular green alga (Chlorella sp. NC64A) were isolated from freshwater sources in Japan. These viruses contain a linear double-stranded DNA (dsDNA) genome with hairpin ends. A physical map was constructed for the genomic DNA of CVK1 (Chlorella virus isolated in Kyoto, no. 1) by pulsed-field gel electrophoresis of restriction fragments. The nucleotide sequences around both termini of the CVK1 DNA revealed the presence of inverted terminal repeats (ITR) of approximately 1.0 kb. Adjacent to the ITR, unique sequence elements of 10 to 20 by were directly repeated 20 to 30 times in tandem array. Several copies of these repeat elements were deleted in virus mutants that were occasionally generated from Chlorella cells that were in a putative CVK1 carrier state. These repeats might represent a hot spot of rearrangement in the CVK1 genome.