Microalga Chlorella Pyrenoidosa Research Articles

Improved high performance liquid chromatographic method for determination of carotenoids in the microalga Chlorella pyrenoidosa

Microalgae have become an important commercial source of carotenoids and microalgae-derived functional foods are consumed by people worldwide. Therefore, an HPLC method was developed to discern the variety and content of carotenoids in the microalga Chlorella pyrenoidosa. The microalga sample was powdered, extracted, saponified and subjected to HPLC analysis. A mobile phase of methanol–acetonitrile–water (84:14:2, v/v/v) (A) and methylene chloride (100%) (B) with the following gradient elution was developed: 100% A and 0% B in the beginning, maintained for 14 min, decreased to 95% A in 25 min, 75% A in 30 min, 74% A in 35 min, 45% A in 50 min and returned to 100% A in 55 min. A total of 32 carotenoids were resolved within 49 min by using a C30 column with flow rate at 1 mL/min and detection at 450 nm. An internal standard β- apo-8′-carotenal was used to quantify all the carotenoids. All- trans-lutein was present in exceptionally large amount (125034.4 μg/g), followed by cis isomers of lutein (27975.3 μg/g), all- trans-α-carotene (2465.8 μg/g), zeaxanthin (2170.3 μg/g), cis isomers of β-carotene (2159.3 μg/g), all- trans-β-carotene (2155.0 μg/g), cis isomers of α-carotene (1766.7 μg/g), β-cryptoxanthin (334.9 μg/g), neoxanthin and its cis isomers (199.7 μg/g), neochrome (65.2 μg/g), auroxanthin (38.5 μg/g) and violaxanthin and its cis isomers (38.1 μg/g).

Purification of lipoxygenase from Chlorella: production of 9- and 13-hydroperoxide derivatives of linoleic acid.

Oxygenation of linoleic acid by the enzyme lipoxygenase (LOX) that is present in the microalga Chlorella pyrenoidosa is known to produce the corresponding 9- and 13-hydroperoxide derivatives of linoleic acid (9- and 13-HPOD, respectively). Previous work with this microalga indicated that partially purified LOX, present in the 30-45 and 45-80% saturated (NH4)2SO4 precipitate fractions, produced both HPOD isomers but in different ratios. It was not clear, however, if the observed activity in the two isolates represented the presence of one or more isozymes. In the present work, LOX isolated from the intracellular fraction of Chlorella by (NH4)2SO4 precipitation (35-80% saturated) was purified by ion exchange and hydrophobic interaction chromatography to apparent homogeneity. Analysis of the purified protein by SDS-PAGE and subsequent native size exclusion chromatography demonstrated that LOX in Chlorella is a single monomeric protein with a molecular mass of approximately 47 kDa. The purified LOX produced both the 9-HPOD and 13-HPOD isomers from linoleic acid in equal amounts, and the isomer ratio was not altered over the pH range of 6 to 9. Optimal activity of LOX was at pH 7.5.

EFFECT AND MECHANISM OF INORGANIC CARBON ON THE BIODEGRADATION OF DIMETHYL PHTHALATE BY CHLORELLA PYRENOIDOSA

ABSTRACT The effect and mechanism of inorganic carbon (IC) on the biodegradation of dimethyl phthalate (DMP) by a green microalga Chlorella pyrenoidosa was investigated. It was indicated that DMP could be used as the sole carbon source to support the slow heterotrophic growth of C. pyrenoidosa, but both the growth of C. pyrenoidosa and the biodegradation rate of DMP were obviously increased when initial inorganic carbon concentration (IC) was increased from 0.6 to 23.7 mg/l. Phthalic acid (PA) was found to be an intermediate product of DMP biodegradation and accumulated in the culture solution, which caused a sharp decrease in pH of medium and inhibited both the growth of alga and the biodegradation of DMP. The role of IC for improving the biodegradation of DMP was both to supply a favorite carbon source to support the rapid growth of alga and to mitigate the decrease of pH because of the production of PA. A suggested second-order kinetic equation of organic pollutant biodegradation by microalgae (−dC/dt = K N r) fitted well with the experimental data and the correlation coefficients were all above 0.9. The second-order constant (K) apparently declined with the increase of initial IC because lower ratio between organic carbon from DMP and IC was used to support the growth of alga when initial IC increased.

Characterization of lipoxygenase oxidation products by high-performance liquid chromatography with electron impact-mass spectrometric detection.

Lipoxygenase (LOX) is an enzyme that oxygenates polyunsaturated fatty acids to their corresponding hydroperoxy derivatives. For example, LOX found in plants produce the corresponding 13- and 9-hydroperoxide derivatives of linoleic acid (13-HPOD and 9-HPOD). Identification of the HPOD products is usually accomplished by using gas chromatography with mass spectrometric (MS) detection, which requires extensive derivatization of the thermally unstable hydroperoxy group. Here we report a high-performance liquid chromatographic method in combination with electron impact (EI)-MS detection that separates and characterizes the HPOD isomers generated by soybean LOX type I oxygenation of linoleic (LA) and linolenic acids as well as HPOD products produced by photosensitized oxidation of LA. The method does not required derivatization of the hydroxyperoxide group, and location of its position can be determined by the EI-MS fragmentation pattern. The method has been used for the analysis of HPOD produced by action of partially purified LOX from the micro-alga Chlorella pyrenoidosa on LA. The study suggests the presence of two LOX isozymes in the micro-alga that oxygenate LA to its 13-HPOD and 9-HPOD derivatives. Moreover, the 9-LOX isozyme under anaerobic conditions cleaves 13-HPOD to 13-oxo-tridecadienoic acid and pentane but does not cleave 9-HPOD.

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.

Anaerobic lipoxygenase activity from Chlorella pyrenoidosa

The micro-alga Chlorella pyrenoidosa expresses an enzymatic activity that cleaves the 13hydroperoxide 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 C5 and non-volatile C13 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 ω-dicarboxylic acid, whereas alcohol dehydrogenase (ADH) reduces 13-OTA to an ω-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 ω-dicarboxylic acid and ω-hydroxy carboxylic acid derivatives.

Influence of light intensity on the kinetic and yield parameters of Chlorella pyrenoidosa mixotrophic growth

The influence of light intensity on growth of the freshwater microalga Chlorella pyrenoidosa has been studied. A mineral culture medium supplemented with glucose at concentrations of 0·1, 0·5 and 1 g litre −1 was used. The different growth phases observed have been described. In the exponential mixotrophic phase, the specific growth rate did not depend on light, since the algae were able to use glucose as an energy source, a fact demonstrated by the increase in the specific rate of glucose consumption on reducing the light intensity. When the organic substrate was completely consumed, and after an adaptation period, an autotrophic phase followed. The results show that it is advisable to add glucose only to light-limited cultures. The highest biomass/substrate yields are attained at 0·1 g litre −1.

96/03963 Energy consumption and greenhouse gas emissions from biomass production chains

This study presents experimental results that compare the use of hydrothermal liquefaction (HTL), alcoholysis (Al), pyrolysis (Py) and hydropyrolysis (HPy) for the production of bio-oil from a microalga (Chlorella pyrenoidosa) and the catalytic hydrothermal upgrading of crude bio-oils produced by these four conversion routes. The yields and compositions of bio-oil, solid residue, and gases were evaluated and compared. HTL resulted in a bio-oil that has a higher energy density and superior fuel properties, such as thermal and storage stabilities, compared with the other three conversion routes. The N in crude bio-oils produced from Py and HPy is more easily removed than that in the bio-oils produced from HTL and Al. The upgraded bio-oils contain reduced amounts of certain O-containing and N-containing compounds and significantly increased saturated hydrocarbon contents. All of the upgraded bio-oils have a larger fraction boiling below 350 °C than their corresponding crude bio-oils.