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Chlorella pyrenoidosa was found to grow rapidly in tap water. Peak growth was reached after 2 to 3 days. Chlorine and bromine, added to such water, were shown to be effective inhibitors of algal growth. Bromine and bromamine were primarily algicidal, whereas chlorine and chloramines were mainly algistatic. It is assumed that the mechanisms of action of these halogens on Chlorella are not the same.
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.
The growth-inhibiting behaviour of abacterial, liquid pure cultures made up of three water algae (Hydrodictyon reticulatum, Chlorella vulgaris, Aphanothece nidulans) which were made to grow profusely in special culture containers under constant exposure to light and ventilation was examined in ten different species of microorganisms during a period of contact of 4 days both in the light and in the dark. Subjected to the test were the 5 pathogenic species Staphylococcus aureus, Klebsiella aerogenes, Pseudomonas aeruginosa, Salmonella typhimurium and Candida albicans as well the 5 bacterial contamination indicators E. coli (faecal indicator), Streptococcus faecalis (enterococci), Enterobacter aerogenes ("coliforms"), Staphylococcus epidermidis (dermic germ) and Bacillus subtilis ("contamination germ"). It was found that --Hydrodictyon reticulatum and Aphanothece nidulans exert a strong antibacterial effect, while Chlorella vulgaris provides no indication of a bacterial growth-inhibiting effect. --this antibiosis is linked with the assimilative activity of the algal cultures, as in the dark no antibacterial action is discernible --the "antibiotic principle" must be liable to transitory or rapid disintegration because the algal cultures are ineffective in the dark and also culture filtrates of 5-days old algal cultures exercise no growth-inhibiting effect. With respect to the sanitation of waters it is important to state that a profuse growth of algae produces a certain, but hardly reliable "biological water disinfection" and renders difficult the assessment of the quality of a water-course as a result of the elimination of the usual contamination indicators. Therefore, additional microbiological quality parameters must be used for assessment of pollution of swimming pools with strong algal growth.
The endosymbiotic unit green Paramecium shows a strong specificity of its partners. The aposymbiotic Paramecium bursaria forms a stable symbiotic unit only with a special strain of Chlorella sp. Algae suitable for symbiosis formation are enclosed in individual perialgal vacuoles whereas unsuitable algae are sequestered into food vacuoles. It is probable that algae are recognized by the ciliate because of specific surface structures rather than by their physiological properties. Experiments with synchronized algae demonstrate that autospores are taken up into perialgal vacuoles to a higher degree than mother cells, which have a different surface structure as shown by immunological techniques. Symbiotic algae treated with cellulase and pectinase or having been coated with specific antibodies or with lectins (concanavalin A or Ricinus communis agglutinin) are usually not recognized as suitable and are mostly sequestered into food vacuoles although they show the same physiological properties as untreated algae. These results indicate the participation of carbohydrate structures at the recognition sites of symbiotic chlorellae in Paramecium bursaria which interact during infection with special receptor molecules in the membrane of the ingestion vacuole of the ciliate.
The basic requirements for high-density photoautotrophic microalgal cultures in enclosed photobioreactors are a powerful light source and proper distribution of light, efficient gas exchange, and suitable medium composition. This article introduces the concept of balancing the elemental composition of growth medium with biomass composition to obtain high-density cultures. N-8 medium, commonly used for culturing Chlorella vulgaris was evaluated for its capacity to support high-density cultures on the basis of elemental stoichiometric composition of C. vulgaris. This analysis showed that the N-8 medium is deficient in iron, magnesium, sulfur, and nitrogen at high cell densities. N-8 medium was redesigned to contain stoichiometrically balanced quantities of the four deficient elements to support a biomass concentration of 2% (v/v). The redesigned medium, called M-8 medium, resulted in up to three- to fivefold increase in total chlorophyll content per volume of culture as compared to N-8 medium. Further experiments showed that addition of each of the four elements separately to N-8 medium did not improve culture performance and that balanced supplementation of all four deficient elements was required to yield the improved performance. Long-term (24 d) C. vulgaris culture in M-8 medium showed continuous increase in chlorophyll content and biomass throughout the period of cultivation. In contrast, the increase in chlorophyll content and biomass ceased after 7 and 12 d, respectively in N-8 medium, demonstrating the higher capacity of M-8 medium to produce biomass. Thus, the performance of high cell density photobioreactors can be significantly enhanced by proper medium design. The elemental composition of the biomass generated is an appropriate basis for medium design.
Light-dependent photosynthesis of Chlorella vulgaris was investigated by using a novel photosynthesis measurement system that could cover wide ranges of incident light and cell density and reproduce accurate readings. Various photosynthesis models, which have been reported elsewhere, were classified and/or reformulated based upon the underlying hypotheses of the light dependence of the algal photosynthesis. Four types of models were derived, which contained distinct light-related variables such as the average or local photon flux density (APFD or LPFD) and the average or local photon absorption rate (APAR or LPAR). According to our experimental results, the LPFD and LPAR models could predict the experimental data more accurately although the APFD and APAR models have been widely used for the kinetic study of microalgal photosynthesis.
The characteristics of the flow of culture medium significantly affects the photosynthetic productivity of bioreactors incorporating microalgae. Therefore, in order to optimize the performance of a conical helical tubular photobioreactor (CHTP) designed to be useful in practical applications, we characterized the flow pattern of the culture medium through the reactor. The effects of medium flow conditions on the photosynthetic productivity of Chlorella sp. were investigated using several different CHTP units with 0.50-m2 installation areas which were designed to vary the direction and rate of flow driven by airlift. In addition, the performance of two- and four-unit systems constructed by combining individual CHTP units was evaluated. We found that when medium flowed from the bottom to the top of the photostage, it exhibited smoother flow of culture medium than when flowing from top to bottom, which led to higher photosynthetic productivity by the former. Consistent with theoretical calculations, varying the lengths of vertical flow passages caused flow rates to vary, and higher flow rates meant smoother circulation of medium and better photosynthetic performance. Flow of medium through a four-unit CHTP system was similar to that in single units, enabling a photosynthetic productivity of 31.0 g-dry biomass per m2-installation area per day to be achieved, which corresponded to a photosynthetic efficiency of 7.50% (photosynthetically active radiation (PAR; 400-700 nm)). This high photosynthetic performance was possible because smoother medium flow attained in single units was also attained in the four-unit system.
The influence of the chlorides of K, Na, Ca, Zn, Cd, Pb, and Ce on the membrane potential of Chlorella fusca (Shihira et Krauss) 211-8b, Göttingen, has been studied. The potential depended on the concentration of Na und K much less than predicted by the Goldman equation. The dependence on the concentrations of Ca, Zn, and Cd was qualitatively similar to that in the case of the alkali ions. With Pb and Ce, the potential was entirely suppressed already at moderate concentrations, i.e., in the millimolar range. The contrast between the effects of the inessential elements Cd and Pb on the potential corresponds to the difference between their paths of uptake. The dependence of the membrane potential on time has also been studied. With K, Na, and Ca stationary states were reached within about 1 hour. The time course of the depression of the potential by Pb depended on the concentration. At moderate concentration, suppression could be partly reversed by subsequent treatment of the cells with diluted KCl.
Certain microalgae are considered to be a potential source of canthaxanthin, which possesses strong antioxidant and anticancer activities. A high-speed counter-current chromatography (HSCCC) method was developed for the separation and purification of canthaxanthin from the microalga Chlorella zofingiensis. The crude canthaxanthin was obtained by extraction with organic solvents after the microalgal sample had been saponified. Preparative HSCCC, with a two-phase solvent system composed of n-hexane-ethanol-water (10:9:1 v/v), was successfully performed yielding canthaxanthin at 98.7% purity from 150 mg of the crude extract (2.1% canthaxanthin) in a one-step separation. The recovery of canthaxanthin was 92.3%. This was the first report that canthaxanthin was successfully separated and purified from microalgae.
Kinetics of lutein production by heterotrophic Chlorella protothecoides was investigated with respect to pH and temperature. Flask cultures with initial pH 5.0-8.0 were carried out, and it was found that pH 6.0 was optimal for the algal growth. Further tests in fermentors showed that the highest biomass concentration, maximum cellular lutein content and lutein yield were achieved at pH 6. 6. In addition, it was shown that optimal biomass concentration and lutein yield were obtained at 28 degrees C, while application of 35 degrees C resulted in the highest cellular lutein content. A mathematical model was developed for the description of the processes under these cultivation conditions and the kinetic model fitted well to the experimental data. The obtained results may contribute to the commercial production of lutein by C. protothecoides.
Crude polysaccharide extracts were obtained from aqueous extracts of the microalgae Chlorella stigmatophora and Phaeodactylum tricornutum. The crude extracts were fractionated by ion-exchange chromatography on DEAE-cellulose columns. The molecular weights of the polysaccharides in each fraction were estimated by gel filtration on Sephacryl columns. The crude polysaccharide extracts of both microalgae showed anti-inflammatory activity in the carrageenan-induced paw edema test. In assays of effects on the delayed hyper-sensitivity response, and on phagocytic activity assayed in vivo and in vitro, the C. stigmatophora extract showed immunosuppressant effects, while the P. tricornutum extract showed immunostimulatory effects.
A pharmacological study of hydrosoluble and liposoluble extracts of the marine microalgae Chlorella stigmatophora and Phaeodactylum tricornutum indicated that hydrosoluble components of both species show significant antiinflammatory, analgesic and free radical scavenging activity. These activities were not detected in the liposoluble fractions.
The continuous culture of fresh green algae for use as food for Daphnia magna in toxicity tests is labor intensive. The possibility of using freeze-dried Chlorella vulgaris as an off-the-shelf food was investigated. The performance of D. magna provided with freeze-dried food was investigated over several generations and was found to satisfy the current EC validity criteria. However, a number of life-history parameters, for example, fecundity and neonate size, were significantly reduced when compared to animals fed fresh algae. The effect of freeze drying on cell size and availability are investigated and discussed in relation to daphnid performance.
A new biosensor is constructed for the detection of some herbicides based on kinetic measurements of chlorophyll-a fluorescence in Chlorella vulgaris cells. The microalgae are immobilized on removable membranes placed in front of the tip of an optical fiber bundle inside a homemade microcell. C. vulgaris was easily cultivated in laboratory and very sensitive to herbicides that effect the photosynthesis process. The response of the algal biosensor is studied in terms of detection limits, reversibility, and long-term activity. The effects of temperature and pH are also reported. The biosensor can be used to measure the concentration of a toxic chemical in the form of a single drop or dissolved in a continuous flow. The detection of 0.1 microg small middle dotL(-1) of a single herbicide as is required by European Community legislation for drinking water is possible with this algal biosensor especially for atrazine, simazine, and diuron.
The effects on the green alga Chlorella vulgaris of 40 herbicides in 19 different chemical structure classes and with 11 dissimilar modes of action were studied through 96-h acute toxicity tests. Experimental results indicated that the average acute toxicity of acetolactate synthase (ALS)-inhibiting herbicides to C. vulgaris was close to those of the acetyl-CoA carboxylase (ACCase)-inhibiting herbicides and the lipid synthesis-inhibiting protoporphyrinogen oxidase (Protox) inhibitor. Their acute toxicities were higher than those of the microtubule process inhibitor, mitotic process inhibitor, the glutamine synthase inhibitors, and 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSP synthase) inhibitors. The acute toxicities of auxin herbicides to C. vulgaris were the lowest among all herbicides tested and that of the photosynthesis-inhibiting herbicides was the highest.
Growth-inhibiting tests were carried out for 12 pesticides (including 11 fungicides: fosetyl-aluminum, benomyl, metalaxyl, iprodione, dimetachlone, carbendazim, thiophanate-methyl, bismerthiazol, procymidone, zineb, chlorothalonil, and the acaricide abamectin) in the green algae Chlorella pyrenoidosa and Scenedesmus obliqnus and the differential sensitivities of the two green algae to those pesticides were compared. The results indicate that the acute toxicity of benomyl to C. pyrenoidosa and S. obliqnus is the highest among all of the pesticides tested and is close to that of the photosynthesis-inhibiting herbicides atrazine, simazine, and chlorotoluron. Meanwhile, algal species vary widely in their response to the pesticides. The results demonstrated that there was a differential response to various pesticides by the two species of algae and that the sensitivity of various species of algae exposed to chlorothalonil varied by nearly two orders of magnitude; sensitivity to thiophanate-methyl varied by more than one order. Investigations using different algal species as test organisms have demonstrated that algae vary greatly in their response to chemicals. Differential sensitivity of green species to the compounds could induce species shifts within communities.
The termini of the chlorella virus PBCV-1 330-kb dsDNA genome consist of 35-nucleotide-long, incompletely base-paired, covalently closed hairpin loops that exist in one of two forms. The two forms are complementary when the 35 nucleotide sequences are inverted with respect to one another (flip and flop). Each hairpin loop structure is followed by an identical 2221-bp inverted repeat sequence after which the DNA sequence diverges. The strategy for cloning the PBCV-1 DNA hairpin ends may be useful for cloning other hairpin termini.
Forty-three kb of DNA, located at the left end (45 to 88 kb) of the 330-kb Chlorella virus PBCV-1 genome, was sequenced and analyzed. Eighty-six open reading frames (ORFs) 65 codons or longer were identified; 47 were classified as major ORFs. These 47 major ORFs are densely packed on both strands of PBCV-1 DNA. Seventeen of these major ORFs resemble genes in the sequence databases, including three putative gene products involved in manipulating sugars (glucosamine synthetase, GDP-D-mannose dehydratase, and N-acetylglucosaminyltransferase), two transcription factors, beta-1,3-glucanase, aspartate transcarbamylase, ubiquitin carboxy terminal hydrolase, RNA guanyl transferase, an exonuclease, and a helicase. This is the first time some of these putative PBCV-1 genes have been found in a virus genome. One of the transcription factor-like genes contains a type IB self-splicing intron. Since a spliceosomal processed intron was reported previously in the PBCV-1 DNA polymerase gene, PBCV-1 is the first virus known to contain two different types of introns.