Extracellular Carbohydrates and Polysaccharides of the Alga Chlorella pyrenoidosa Chick S-39

In the growing culture of the thermophilic alga Chlorella pyrenoidosa Chick S-39, the amount of extracellular carbohydrates in the medium reached 5–17% of their content in the cells and 20–40% of the total content of extracellular organic matter. Experiments with the enrichment and synchronous algal cultures showed that the accumulation of extracellular carbohydrates and polysaccharides in the media occurred due to their release from the cells, rather than to cell lysis, and depended on cell photosynthetic activity and reproduction. Chromatographic determination of free sugars revealed the presence of saccharose, glucose, and fructose in the culture medium. Extracellular carbohydrates in C. pyrenoidosa cultures were represented mainly by water-soluble polysaccharides containing galactose, mannose, arabinose, xylose, ribose, fucose, and rhamnose.     

Terminal Oxidases of Chlorella pyrenoidosa

In studies of the kinetics of oxygen uptake by glucose-stimulated Chlorella pyrenoidosa, two terminal oxidases could be distinguished. The cytochrome oxidase of Chlorella has a Km (O₂) of 2.1 ± 0.3 μm, while the second oxidase has a Km (O₂) of 6.7 ± 0.5 μm, and a maximum capacity about one-quarter of that of the cytochrome system. The identity of the second oxidase is unknown, but it is not inhibited by carbon monoxide, 1 mm cyanide, 0.1 mm thiocyanate, or 1 mm 8-hydroxyquinoline. In fresh cultures, the second oxidase accounts for at most 35% of the total oxygen uptake.     

Protoplast induction in Chlorella pyrenoidosa

Chlorella pyrenoidosa (UTEX 1230) cells in late log phase of growth were induced to form viable protoplasts by enzymatic digestion only when incubated in 2-deoxy-d-glucose (2DG) for 24 h. The combination of hemicellulase (4% w/v), Cellulysin (4% w/v), and glucuronidase (5% v/v) with 0.8 M mannitol and 8 mM CaCl₂ in modified Bristol's solution, was most effective for obtaining viable protoplasts as determined by light and electron microscopy, and vital staining with primuline (0.01% w/v). Resistance of cell walls to extensive extraction (acetolysis), and infrared analysis indicated that sporopollenin is a component of the cell wall. Transmission electron miscroscopy of acetolysed cell walls also allowed visualization of the laminate nature of the wall. This is the first report of successful induction of protoplasts from algae which contain sporopollenin in their cell walls.     

Anaerobic lipoxygenase activity from Chlorella pyrenoidosa

The micro-alga Chlorella pyrenoidosa expresses an enzymatic activity that cleaves the 13-hydroperoxide derivatives of linoleic acid [13-hydroperoxy-9(Z),11(E)-octadecadienoic acid, 13-HPOD] and linolenic acid [13-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid, 13-HPOT] into volatile C(5) and non-volatile C(13) oxo-products. This enzymic activity initially was attributed to a hydroperoxide lyase enzyme; however, subsequent studies showed that this cleavage activity is the result of lipoxygenase activity under anaerobic conditions. Headspace analysis of the volatile products by GC/MS showed the formation of pentane when the substrate was 13-HPOD, whereas a more complex mixture of hydrocarbons was formed when 13-HPOT was the substrate. Analysis of the non-volatile cleavage products from 13-HPOD by liquid chromatography/MS indicated the formation of 13-oxo-9(Z),11(E)-tridecadienoic acid (13-OTA) along with the 13-keto-octadecadienoic acid derivative. When the substrate is 13-HPOT, liquid chromatography/MS analysis indicated the formation of 13-OTA as the major non-volatile product. Aldehyde dehydrogenase (AldDH) oxidizes 13-OTA to an omega-dicarboxylic acid, whereas alcohol dehydrogenase (ADH) reduces 13-OTA to an omega-hydroxy carboxylic acid. AldDH and ADH require the oxidized (NAD(+)) and reduced (NADH) forms of the cofactor NAD, respectively. By combining the action of AldDH and ADH into a continuous cofactor-recycling process, it is possible to simultaneously convert 13-OTA to the corresponding omega-dicarboxylic acid and omega-hydroxy carboxylic acid derivatives.     

The Growth of Chlorella pyrenoidosa on Glycollate

The utilization of glycollate as a substrate for photoheterotrophicgrowth by a strain of Chlorella pyrenoidosa has been demonstrated.Glycollate stimulated growth of this alga in basal medium overthe pH range 4.0 to 8.0 in the light, but did not support growthin the dark. Stimulation of growth in the light occurred overa wide range of glycollate concentrations and was optimal at30 mM. Enzyme and inhibitor experiments suggested that the synthesisof cell constituents during growth on glycollate is achievedby the same pathway which operates during the metabolism ofexogenous glycollate by autotrophically-grown cells. For algaeto metabolize and grow on exogenous glycollate the cells mustbe readily permeable to this compound. When the cells readilytake up exogenous glycollate, the level of activity of enzymesin the cell, in particular glycollate:DCPIP oxidoreductase,may regulate the over-all rate of glycollate metabolism.     

The Photometabolism of Acetate by Chlorella pyrenoidosa

Chlorella pyrenoidosa can utilize sodium acetate as a carbonsource for growth in the light. Growth proceeds under aerobicconditions both in the presence and in the absence of carbondioxide, but under anaerobic conditions only in its presence.The assimilation of acetate does not result from oxidation tocarbon dioxide followed by photosynthetic fixation because theproducts of ¹⁴C-acetate assimilation are different from theproducts of ¹⁴CO₂ fixation in the presence of unlabelled acetate. In aerobic conditions 10-⁶ M DCMU induces a pattern of acetateassimilation in the light similar to that in the dark. Thus,in the presence of DCMU in the light, less acetate carbon isincorporated into cells, particularly into lipids, polysaccharide,and protein, and more is released as carbon dioxide than inits absence. The effect of 4 x 10^-3 M MFA on acetate assimilationin the presence of 10^-6 M DCMU is the same in light and dark.Acetate assimilation is unaffected by desaspidine and sodiumbisulphite. The mean generation time of C. pyrenoidosa growing on acetatein the light under aerobic conditions is 20 hours. When 10^-5M DCMU is added the mean generation time is 60 hours, the sameas that for Chlorella growing on acetate in the dark. The activityof the enzymes of the glyoxylate cycle, isocitrate lyase (E.C.4.1.3.1.)and malate synthetase (E.C.4.1.3.2.) is repressed in the light,but activity of both enzymes increases markedly when DCMU isadded.     

Effects of Phenylethyl Alcohol on Chlorella pyrenoidosa

Asynchronous as well as synchronous cultures were used to study the effect of phenylethyl alcohol on Chlorella pyrenoidosa. The following observations were made:

• 1 Autotrophic growth and the synthesis of DNA and chlorophyll were inhibited more than 80% by 8.4 mM phenylethyl alcohol when added to synchronous suspensions containing autospores.
• 2 Autospore formation did not occur when fully grown cells capable of division were transferred to 4.2 mM phenylethyl alcohol.
• 3 Photosynthetic oxygen evolution of asynchronous cultures was inhibited more than 70 % by 21 mM phenylethyl alcohol.
• 4 Endogenous respiration in the dark was stimulated by 8.4 mM, while higher concentrations were inhibitory.
• 5 Glucose respiration in the dark was inhibited by all concentrations in the range from 8.4 mM to 21 mM. Assimilation of glucose in darkness was retarded by phenylethyl alcohol at concentrations which gave maximal stimulation of endogenous respiration.

It is concluded that phenylethyl alcohol is not a specific inhibitor of DNA synthesis. The prime effect appears to be on respiration and photosynthesis.     

Growth of Chlorella pyrenoidosa in Recycled Medium

Bench-scale studies with Chlorella pyrenoidosa 7-11-05 were conducted in a 4-liter culture vessel with a used and recycled medium. Algal cultures were maintained for periods of several weeks by supplementing the nutrient medium with minimal amounts of certain salts. An algal strain was maintained for a period of up to 72 days with a supplemented recycled medium. No inhibition was observed as the result of any autotoxic materials. Rather dense cultures were maintained in the presence of high bacterial populations.     

Experiments on Enzyme Induction in Chlorella pyrenoidosa

Tutorial computer assisted learning has been successfully introduced in two Open University biology courses. The purpose of the tutorials, how they were designed and written, and students' reaction to them are discussed in the expectation that other teachers will find the technique useful.     

Proline Uptake and Utilization by Chlorella pyrenoidosa

Conditions for proline uptake and utilization by Chlorella pyrenoidosa Chick are described. Proline is taken up by growing cultures during late log phase growth after depletion of glucose from the medium. However, proline uptake by stationary phase cultures requires the presence of glucose in the medium. The results are consistent with the interpretation that some carbohydrate is required for proline uptake but proline uptake is inhibited by the accumulation of intracellular carbohydrates.