Phthalate esters (PEs) are endocrine-disrupting pollutants that are ubiquitous in the environment and can be degraded by microorganisms. In this study, we investigated the kinetics and pathway of biodegradation of di-n-butyl phthalate (DBP), diethyl phthalate (DEP), and dimethyl phthalate (DMP) by cyanobacteria Anabaena flos-aquae G. S. West (strain 4054) and two strains of Microcystis aeruginosa (Kütz.) Kütz. (strain 2396 and strain SM). Gas chromatography/mass spectroscopy (GC/MS) and a deuterium-labeled compound were used to analyze the degrading intermediates. The findings revealed that all three organisms were capable of metabolizing PE, and that among these organisms, A. flos-aquae achieved the highest degradation. Additionally, the biodegradation of DBP, DEP, and DMP followed first-order kinetics. Moreover, the results of the enzymatic study suggested that PE was degraded through transesterification on the side chains rather than deesterification. Finally, experiments using deuterium-labeled DBP showed that there were two degradation pathways: C16→ C14→ C12→ C10→ C8 and C16→ C15→ C13→ C11→ C9. Based on our results, the biodegradation pathway of PE for cyanobacteria was suggested.
Saturday, October 23, 2010
BIODEGRADATION OF PHTHALATE ESTERS BY CYANOBACTERIA1
ENDOGENOUS CYTOKININS, AUXINS, AND ABSCISIC ACID IN RED ALGAE FROM BRAZIL1
Endogenous cytokinins, auxins, and abscisic acid (ABA) were identified and quantified in 11 red algae collected from the Brazilian coast. Field materials and two isolates cultured in the laboratory were extracted with various solvents and buffers containing a mixture of appropriate internal standards, purified by solid-phase extraction followed by immunoaffinity chromatography, and analyzed by liquid chromatography–tandem mass spectrometry. Isoprenoid cytokinins (free and conjugated forms of isopentenyladenine [iP], cis-zeatin [cZ], and trans-zeatin [tZ]) were detected in all species with concentrations of cZ and iP forms being higher than tZ forms. Dihydrozeatin (DHZ) and its metabolites were only detected at very low levels in nine of the studied species. Aromatic cytokinins (6-benzylaminopurine [BA], ortho- and meta-topolin [oT and mT]) were not detected in any of the samples. The cytokinin profile of Chondracanthus teedei (Mert. ex Roth) Kütz. was distinct in comparison to other species with para-topolin (pT) derivatives detected in low concentrations. The main auxins present in all species were free indole-3-acetic acid (IAA) and indole-3-acetamide (IAM). Indole-3-ethanol (IEt), indole-3-acetyl glutamic acid (IAGlu), and indole-3-acetyl leucine (IALeu) were detected in a few species at low concentrations. ABA was present in all species analyzed except for Hypnea nigrescens Grev. ex J. Agardh. No ABA conjugates were detected in any species. These results confirm that cytokinins, auxins, and ABA were common constituents in red seaweeds, with this being the first report of the occurrence of ABA in Rhodophyta. The complexity of the hormone profiles suggests that plant hormones play a role in regulating physiological processes in Rhodophyta.
MICROSENSOR MEASUREMENTS OF THE EXTERNAL AND INTERNAL MICROENVIRONMENT OF FUCUS VESICULOSUS (PHAEOPHYCEAE)1
We investigated the O2, pH, and irradiance microenvironment in and around the tissue of the brown alga Fucus vesiculosus L. using microsensors. Microsensors are ideal tools for gaining new insights into what limits and controls macroalgal activity and growth at very fine spatial (<100 μm) and temporal (seconds) scales. This first microsensor investigation of a fucoid macroalga revealed differences in the microenvironment and metabolic activities at the level of different cell layers and thallus structures. F. vesiculosus responded quickly to rapid shifts in irradiance resulting in a highly dynamic microenvironment around and within its thallus. In combination with detailed morphological studies and molecular approaches, microsensors offer a promising toolbox to quantitatively describe structural and functional adaptations of macroalgae to environmental conditions, such as flow and light climate, as well as their physiological responses to environmental stressors.
ALLOPOLYPLOIDY IN NATURAL AND CULTIVATED POPULATIONS OF PORPHYRA (BANGIALES, RHODOPHYTA)1
To confirm whether allopolyploidy occurs in samples of previously identified Porphyra yezoensis Ueda, P. tenera Kjellm., and P. yezoensis × P. tenera from natural and cultivated populations, we examined these samples by using PCR-RFLP and microsatellite analyses of multiple nuclear and chloroplast regions [nuclear regions: type II DNA topoisomerase gene (TOP2), actin-related protein 4 gene (ARP4), internal transcribed spacer (ITS) rDNA and three microsatellite loci; chloroplast region: RUBISCO spacer]. Except for the ITS region, these multiple nuclear markers indicated that the wild strain MT-1 and the cultivated strain 90-02 (previously identified as P. yezoensis × P. tenera and cultivated P. tenera, respectively) are heterozygous and possess both genotypes of P. tenera and P. yezoensis in the conchocelis phase. Furthermore, gametophytic blades of two pure lines, HG-TY1 and HG-TY2 (F1 strains of MT-1 and 90-02, respectively), were also heterozygous, and six chromosomes per single cell could be observed in each blade of the two pure lines. These results demonstrate that allopolyploidy occurs in Porphyra strains derived from both natural and cultivated populations, even though ITS genotypes of these strains showed homogenization toward one parental ITS.
EFFECT OF TEMPERATURE ON THE SENSITIVITY OF NITROGENASE TO OXYGEN IN TWO HETEROCYSTOUS CYANOBACTERIA1
The effect of temperature and oxygen on nitrogenase activity in two heterocystous cyanobacteria, Anabaena variabilis Kütz. ATCC29413 and Nostoc sp. PCC7120, was investigated. The cyanobacteria were grown under a 12:12 light:dark (L:D) cycle at 27°C and were subsequently exposed to different temperatures (27, 36, 39, and 42°C) at different steady-state O2 concentrations (20, 10, 5, 0%). Light response curves of nitrogenase activity were recorded under each of these conditions using an online acetylene reduction assay combined with a sensitive laser photoacoustic ethylene detection method. The light response curves were fitted with the rectangular hyperbola model from which the model parameters Nm, Nd, and α were derived. In both strains, nitrogenase activity (Ntot = Nm + Nd) was the highest at 39°C–42°C and at 0% O2. The ratio Ntot/Nd was 4.1 and 3.1 for Anabaena and Nostoc, respectively, indicating that respectively 25% and 33% of nitrogenase activity was supported by respiration (Nd). Ntot/Nd increased with decreasing O2 concentration and with increasing temperature. Hence, each of these factors caused a relative increase in the light-driven nitrogenase activity (Nm). These results demonstrate that photosynthesis and respiration both contribute to nitrogenase activity in Anabaena and Nostoc and that their individual contributions depend on both O2 concentration and temperature as the latter may dynamically alter the flux of O2 into the heterocyst.
CONTRIBUTION OF THE CLASS CRYPTOPHYCEAE TO PHYTOPLANKTON STRUCTURE IN THE GERMAN BIGHT1
The class Cryptophyceae (Division Cryptophyta) contains ecologically relevant species, which are widespread in aquatic environments. However, classification, identification, and enumeration of cryptophytes are challenged by a morphology that must be usually examined with EM to permit species identification. The quantitative importance of this group has been revealed by HPLC data. But ecological information assessing the occurrence or seasonality of cryptophytes in the marine environment is still scarce. Molecular techniques allow for a refined assessment of taxonomically challenging taxa, such as the cryptophytes. In our laboratory, a Phylochip was developed to facilitate and refine the assessment of cryptophyte microalgae. Here, we present the results of an environmental study that took advantage of the Phylochip. The study was designed to elucidate the seasonality and contribution of cryptophytes to phytoplankton structure in the German Bight. The occurrence of cryptophytes in total plankton versus the picoplankton fraction was assessed with the Phylochip between the years 2004 and 2006. Our data indicate that cryptophytes are an important and constant contributor to phytoplankton structure of the German Bight, especially in the picoplankton fraction.
A ONE-SHOT SOLUTION TO BACTERIAL AND FUNGAL CONTAMINATION IN THE GREEN ALGA CHLAMYDOMONAS REINHARDTII CULTURE BY USING AN ANTIBIOTIC COCKTAIL1
Keeping sterile stocks or cultures of microalgae is fundamental to microalgae biotechnology as well as basic scientific research. However, contamination by bacteria and/or fungi in microalgae cultures or stocks is often a problem. Here, we have developed a strategy for reducing or eliminating bacterial and fungal contamination by using a cocktail of antibiotics. Chlamydomonas reinhardtii P. A. Dang., a widely used unicellular green alga, has been used as a testing organism. A combination of ampicillin, cefotaxime, and carbendazim removed or reduced contamination by three different bacteria and two different fungi tested. A step-by-step procedure is provided, which is simple, economical, and effective.
MOLECULAR PHYLOGENY OF GRACILARIA SPECIES INFERRED FROM MOLECULAR MARKERS BELONGING TO THREE DIFFERENT GENOMES1
The nucleotide sequence data of molecular markers 18S rRNA, RUBISCO spacer, and cox2-3 intergenic spacer were integrated to infer the phylogeny of Gracilaria species, collected from the western coast of India, reducing the possibility of misidentification and providing greater phylogenetic resolution. A phylogenetic tree was constructed using cox2-3 and RUBISCO spacer sequences, exhibiting the same clustering but differing slightly from that of the rRNA-based phylogenetic tree. The phylogeny inferred from the combined data set confers an analogous pattern of clustering, compared with those of trees constructed from individual data sets. The combined data set resulted in a phylogeny with better resolution, which supported the clade with higher consistency index, retention index, and bootstrap values. It was observed that Gracilaria foliifera (Forssk.) Børgesen is closer to G. corticata (J. Agardh) J. Agardh varieties, while G. salicornia (C. Agardh) E. Y. Dawson and G. fergusonii J. Agardh both originated from the same clade. The position of G. textorii (Suringar) De Toni faltered and toppled between G. salicornia and G. dura (C. Agardh) J. Agardh; however, G. gracilis (Stackh.) M. Steentoft, L. M. Irvine et W. F. Farnham was evidently distant from the rest of the species.
ISOLATION AND CHARACTERIZATION OF A LYCOPENE β-CYCLASE GENE FROM THE ASTAXANTHIN-PRODUCING GREEN ALGA CHLORELLA ZOFINGIENSIS (CHLOROPHYTA)1
The isolation, characterization, and regulation by light and nitrogen of the lycopene β-cyclase gene from Chlorella zofingiensis Dönz (CzlcyB), involved in the biosynthesis of astaxanthin and lutein, have been performed in this work. These carotenoids are of high commercial value as dyes in food and as nutraceuticals. The open reading frame (ORF) of CzlcyB encoded a polypeptide of 546 amino acids. A single copy of CzlcyB has been found in C. zofingiensis. The chararacteristic Rossmann or dinucleotide binding fold, present in most lycopene cyclases, has been also identified in the LCYb of C. zofingiensis (CzLCYb). Heterologous genetic complementation in Escherichia coli showed the ability of the predicted protein to cycle both lycopene and δ-carotene. Phylogenetic analysis has shown that the deduced protein forms a cluster with the rest of the lycopene β-cyclases (LCYb) of the chlorophycean microalgae studied, being very closely related to LCYb of plants. Transcript levels of CzlcyB were increased under nitrogen deprivation, but no increase was observed under high-light conditions. However, high irradiance triggered astaxanthin synthesis, while nitrogen deprivation by itself could not induce it. The combination of high irradiance and nitrogen deprivation led to a significant enhancement of the astaxathin accumulation.
