Chlorella Vulgaris Beijerinck Research Articles

Cyanide formation in preparations from Chlorella vulgaris Beijerinck: Effect of sonication and amygdalin addition

A critical evaluation of a method for recovering HCN from cell extracts is presented. Since crude extracts often bind or metabolize HCN extensively, the HCN recovered by distillation at room temperature represents only the difference between production and consumption. Sonication leads to HCN release from the alga, Chlorella vulgaris Beijerinck. Illumination of extracts at high light intensity in oxygen, with added Mn(2+) and peroxidase, also stimulates HCN production. In both processes, the HCN is probably formed by oxidation of nitrogenous precursors. Chlorella extracts cause formation of HCN from added amygdalin. No evidence was found, however, for the presence of cyanogenic glycosides in the algae.

Cyanide formation in preparations from Chlorella and New Zealand spinach leaves: Effect of added amino acids

As part of an effort to identify the natural precursor(s) of HCN in the alga Chlorella vulgaris Beijerinck, and in leaves of New Zealand spinach (Tetragonia expansa, Murr.), HCN release was measured after addition of various amino acids to illuminated algal extracts and grana preparations. Histidine is particularly effective as an HCN precursor, both with Chlorella extracts and leaf grana. With the algal extracts, D-histidine is about ten times more effective than L-histidine and histamine, whereas the two isomers (and histamine) are about equally effective with leaf grana. In the presence of leaf grana plus added Mn(2+) and peroxidase, L-tyrosine and L-cysteine like-wise cause HCN formation; but these amino acids cause little or no HCN formation in the presence of Chlorella extracts. A stimulation of HCN production by L-histidine was observed with intact Chlorella cells. Because of the limitations of the assay method, the possibility can not be excluded that other substances than histidine may also lead to HCN generation in Chlorella vulgaris, but the results show that histidine has an important role in HCN generation by this species.

The effects of water-soluble petroleum components on the growth of Chlorella vulgaris Beijerinck

The effects of the water-soluble components of petroleum on a selected algal test species, Chlorella vulgaris, have been investigated under controlled laboratory conditions. Aqueous extracts of seven different crude oils and one refined product exhibited marked differences in toxicity to Chlorella, resulting in a 5% to 41% reduction of cell numbers during the first 48 h. However, this toxicity was only of a short-term nature, apparently resulting in a lag phase prior to the onset of growth. Work with oil extracts allowed to evaporate for 0, 24, 48 or 120 h prior to their inoculation with algae, suggests that the observed toxicity of oil extracts is due to highly volatile compounds which are largely lost from the culture flasks during the first 25 h. Lower concentrations of benzene, toluene, o-xylene and napthalene resulted in a growth inhibition similar to that caused by crude oil. Higher concentrations caused total inhibition of growth. The growth rate of cells was normal once the lag phase had ended. A significant stimulation of growth was observed with three of the oil extracts after their toxic compounds had evaporated. It is not known at present if this stimulation is a reflection of an actual ability to utilise certain hydrocarbons heterotrophically from the extract. Other possible interpretations are discussed.

Nitrate Reductase and Chlorate Toxicity in Chlorella vulgaris Beijerinck

A study of the growth-inhibiting effect of chlorate on the Berlin strain of Chlorella vulgaris Beijerinck provided complete confirmation of the theory of chlorate toxicity first proposed by Aberg in 1947. Chlorate was toxic to the cells growing on nitrate, and relatively nontoxic to the cells growing on ammonium. The latter cells contained only 0.01 as much NADH-nitrate reductase as the nitrate-grown cells. Chlorate could substitute for nitrate as a substrate of the purified nitrate reductase with Km = 1.2 mm, and V(max) = 0.9V(max) for nitrate. Bromate, and to a much smaller extent, iodate, also served as alternate substrates. Nitrate is a reversible competitive inhibitor of chlorate reduction, which accounts for the partial reversal, by high nitrate concentrations, of the observed inhibition of cell growth by chlorate. During the reduction of chlorate by NADH in the presence of purified nitrate reductase, there was a progressive, irreversible inhibition of the enzyme activity, presumably brought about by the reduction product, chlorite. Both the NADH-nitrate reductase activity and the associated NADH-cytochrome c reductase activity were inactivated to the same extent by added chlorite. The spectral properties of the cytochrome b(557) associated with the purified enzyme were not affected by chlorite. The inactivation of the nitrate reductase by chlorite could account for the toxicity of chlorate to cells grown on nitrate, though the destruction of other cell components by chlorite or its decomposition products cannot be excluded.

Metabolism of urea by Chlorella vulgaris.

Urea metabolism was studied with nitrogen-starved cells of Chlorella vulgaris Beijerinck var. viridis (Chodat), a green alga which apparently lacks urease. Incorporation of radioactivity from urea-(14)C into the alcohol-soluble fraction was virtually eliminated in cell suspensions flushed with 10% CO(2) in air. This same result was obtained when expected acceptors of urea carbon were replenished by adding ornithine and glucose with the urea. Several carbamyl compounds, which might be early products of urea metabolism and a source of the (14)CO(2), were not appreciably labeled. If cells were treated with cyanide at a concentration which inhibited ammonia uptake completely and urea uptake only slightly, more than half of the urea nitrogen taken up was found in the medium as ammonia. Cells under nitrogen gas in the dark were unable to take up urea or ammonia, but the normal rate of uptake was resumed in light. Since 3-(3,4-dichlorophenyl)-1,1-dimethylurea did not selectively inhibit this uptake, an active respiration supported by light-dependent oxygen evolution in these cells was ruled out. A tentative scheme for urea metabolism is proposed to consist of an initial energy-dependent splitting of urea into carbon dioxide and ammonia. This reaction in Chlorella is thought to differ from a typical urease-catalyzed reaction by the apparent requirement of a high energy compound, possibly adenosine triphosphate.

Chorella vulgaris Assay of the Activity of Soil Herbicides

An assay technique utilizing Chlorella vulgaris Beijerinck gave highly significant responses to low concentrations of aqueous 3-(p-chlorophenyl)-l,l dimethylurea (monuron), 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), and 2,4-bis(isopropylamino)-6-methylmercapto-s-triazine (prometryne) following 6 days of incubation. Water extracts of diuron from soil assayed with this organism were highly correlated with diuron added to the soil. Similar correlations for oats (Avena sativa L.) grown on the soil and for diuron in the water extracts as dichloroaniline serve to substantiate the relative effectiveness of the method. Assay of soil samples from field residue plots taken 150 days after herbicidal application showed that active residues of diuron and prometryne were detectable by the organism. Although the chemical method was faster, it has the disadvantage of measuring any dichloroaniline residues rather than only the active residues. The method developed insures a considerable saving in time over herbicide assays involving higher plants and gives reasonably good quantitative data on low concentrations of toxic monuron, diuron, and prometryne.

Chlorophyllase of Chlorella vulgaris Beijerinck

Some properties of chlorophyllase (E.C.3.1.1.14 = chlorophyll chlorophyllido-hydrolase) in homogenates from Chlorella vulgaris Beijerinck 211-11h are described. Chlorophyllase is localized in the chloroplast; the enzyme is easily solubilized and can be strongly activated after a short incubation with trypsin. Detergents do not influence activity. The possible function of the enzyme is discussed with relation to both the chlorophyll-bearing chloroplast protein (structural protein) and the formation and destruction of chlorophyll.

THE MEASUREMENT OF AVAILABLE LIGHT BY THE USE OF CHLORELLA

During a study of seasonal variations in tropical high forest and savanna woodland measurements of various microclimatic factors including light intensity were required. The measurement of light intensity for ecological purposes has always been unsatisfactory. This is partly due to the lack of a suitable instrument for measuring the 'photosynthetic efficiency' (or some similar value) of light and partly to the constantly changing light intensity found in natural conditions. It was decided to try to use the unicellular alga Chlorella vulgaris Beijerinck as a phytometer. There were three reasons for this choice. First, it can be grown in pure culture under controlled and repeatable conditions so far as the chemical composition of the medium is concerned. Secondly, it can be grown in flasks which protect it from predators and parasites. Thirdly, intensive laboratory studies have been carried out on the effect of external factors on the growth rate of Chlorella; in particuar Myers (I946) has shown that the growth-rate graph is a 'typical light saturated curve'. (Temperature also influences the growth rate of Chlorella but its effects are relatively small under conditions of light limitation.) The investigation was successful and as the method has been used satisfactorily for almost 2 years it was felt that it may be of use to other workers. The Chlorella was grown in 250 ml conical flasks each containing ioo ml of a standard liquid medium. The choice of both strains of Chlorella and medium are relatively unimportant provided that the same ones are used for a complete series of observations. For these observations a high temperature strain (designated Chlorella Tx7II5) was grown in a modified Knops solution, a medium in which it has been found to grow successfully (Myers, personal communication). A stock of Chlorella growing on agar slopes and in liquid medium in flasks was always kept over a cold light in the laboratory. These flasks were shaken twice daily to prevent the clumping of divided daughter cells. The number of cells per unit volume in a flask from which the inoculum was to be taken was determined by haemocytometer count. From this was calculated the volume of inoculum to be added to each flask in order to give these flasks an initial population of about 8oo,ooo cells - a suitable value found from preliminary experiments. (High cell densities are to be avoided as they limit the supply of both light and carbon dioxide to much of the culture.) Inoculation was done by pipette in a sterile chamber and the inoculated flasks were closed by a sterile rubber bung. The flasks were then transported in the dark in specially made boxes to the experimental site where the rubber bungs were removed and replaced by cotton-wool plugs and beaker covers. During this process a spirit lamp was used for sterilization. At a specified time the flasks were removed from the dark and placed in the positions

Biochemical Contributions to the Taxonomy of the Genus Chlorella

SINCE Warburg's1 work, in 1919, green algae of the genus Chlorella have been among the experimental organisms most frequently used for plant physiological and biochemical research. However, due to the little morphological differentiation of their more or less spherical cells, the taxonomy of these algae has been rather confused (cf. ref. 2). This holds especially true for the members of the section Euchlorella comprising the most common species, that is, Chlorella vulgaris Beijerinck, Chl. pyrenoidosa Chick, and Chl. ellipsoidea Gerneck. The criteria given by Pascher3 for their identification (size and shape of cells and chloroplasts, visibility of the pyrenoid, etc.) have been shown to be highly variable and ambiguous, and to depend to a large extent on culture conditions and developmental stage of the cells4. Therefore, in most cases it is very difficult, if not impossible, to identify these species by morphological criteria. Since the characteristics traditionally used for taxonomy are obviously insufficient in this case, we propose to use mainly physiological and biochemical rather than morphological criteria for the identification of these Chlorella species.