Potential of the Red Alga Dixoniella grisea for the Production of Additives for Lubricants.

There is an increasing interest in algae-based raw materials for medical, cosmetic or nutraceutical applications. Additionally, the high diversity of physicochemical properties of the different algal metabolites proposes these substances from microalgae as possible additives in the chemical industry. Among the wide range of natural products from red microalgae, research has mainly focused on extracellular polymers for additive use, while this study also considers the cellular components. The aim of the present study is to analytically characterize the extra- and intracellular molecular composition from the red microalga Dixoniella grisea and to evaluate its potential for being used in the tribological industry. D. grisea samples, fractionated into extracellular polymers (EPS), cells and medium, were examined for their molecular composition. This alga produces a highly viscous polymer, mainly composed of polysaccharides and proteins, being secreted into the culture medium. The EPS and biomass significantly differed in their molecular composition, indicating that they might be used for different bio-additive products. We also show that polysaccharides and proteins were the major chemical compounds in EPS, whereas the content of lipids depended on the separation protocol and the resulting product. Still, they did not represent a major group and were thus classified as a potential valuable side-product. Lyophilized algal fractions obtained from D. grisea were found to be not toxic when EPS were not included. Upon implementation of EPS as a commercial product, further assessment on the environmental toxicity to enchytraeids and other soil organisms is required. Our results provide a possible direction for developing a process to gain an environmentally friendly bio-additive for application in the tribological industry based on a biorefinery approach.

The use of fluorescence microscopy and image analysis for rapid detection of non-producing revertant cells of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002.

BACKGROUND: Ethanol production via genetically engineered cyanobacteria is a promising solution for the production of biofuels. Through the introduction of a pyruvate decarboxylase and alcohol dehydrogenase direct ethanol production becomes possible within the cells. However, during cultivation genetic instability can lead to mutations and thus loss of ethanol production. Cells then revert back to the wild type phenotype. A method for a rapid and simple detection of these non-producing revertant cells in an ethanol producing cell population is an important quality control measure in order to predict genetic stability and the longevity of a producing culture. Several comparable cultivation experiments revealed a difference in the pigmentation for non-producing and producing cells: the accessory pigment phycocyanin (PC) is reduced in case of the ethanol producer, resulting in a yellowish appearance of the culture. Microarray and western blot studies of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002 confirmed this PC reduction on the level of RNA and protein. METHODS: Based on these findings we developed a method for fluorescence microscopy in order to distinguish producing and non-producing cells with respect to their pigmentation phenotype. By applying a specific filter set the emitted fluorescence of a producer cell with a reduced PC content appeared orange. The emitted fluorescence of a non-producing cell with a wt pigmentation phenotype was detected in red, and dead cells in green. In an automated process multiple images of each sample were taken and analyzed with a plugin for the image analysis software ImageJ to identify dead (green), non-producing (red) and producing (orange) cells. RESULTS: The results of the presented validation experiments revealed a good identification with 98 % red cells in the wt sample and 90 % orange cells in the producer sample. The detected wt pigmentation phenotype (red cells) in the producer sample were either not fully induced yet (in 48 h induced cultures) or already reverted to a non-producing cells (in long-term photobioreactor cultivations), emphasizing the sensitivity and resolution of the method. CONCLUSIONS: The fluorescence microscopy method displays a useful technique for a rapid detection of non-producing single cells in an ethanol producing cell population.

Fatty acid profiles and their distribution patterns in microalgae: a comprehensive analysis of more than 2000 strains from the SAG culture collection.

BACKGROUND: Among the various biochemical markers, fatty acids or lipid profiles represent a chemically relatively inert class of compounds that is easy to isolate from biological material. Fatty acid (FA) profiles are considered as chemotaxonomic markers to define groups of various taxonomic ranks in flowering plants, trees and other embryophytes. RESULTS: The fatty acid profiles of 2076 microalgal strains from the culture collection of algae of Göttingen University (SAG) were determined in the stationary phase. Overall 76 different fatty acids and 10 other lipophilic substances were identified and quantified. The obtained FA profiles were added into a database providing information about fatty acid composition. Using this database we tested whether FA profiles are suitable as chemotaxonomic markers. FA distribution patterns were found to reflect phylogenetic relationships at the level of phyla and classes. In contrast, at lower taxonomic levels, e.g. between closely related species and even among multiple isolates of the same species, FA contents may be rather variable. CONCLUSION: FA distribution patterns are suitable chemotaxonomic markers to define taxa of higher rank in algae. However, due to their extensive variation at the species level it is difficult to make predictions about the FA profile in a novel isolate.

A lipoxygenase with linoleate diol synthase activity from Nostoc sp. PCC 7120.

The dioxygenation of PUFAs (polyunsaturated fatty acids) in plants is mainly catalysed by members of the LOX (lipoxygenase) enzyme family. LOX products may be further metabolized, and are known as signalling substances in plant development and in responses to wounding and pathogen attack. In contrast with the situation in eukaryotes, information on the relevance of lipid peroxide metabolism in prokaryotic organisms is scarce. Therefore, we aimed to analyse LOXs and oxylipin patterns of cyanobacterial origin. A search of the genomic sequence of the cyanobacterium Nostoc sp. PCC 7120 suggested an open reading frame encoding a putative LOX named NspLOX that harboured an N-terminal extension. Individual analysis of recombinant C-terminal domain revealed enzymatic activity as a linoleate (9R)-LOX. Analysis of the full-length NspLOX protein, however, revealed linoleate diol synthase activity, generating (10E,12E)-9,14-dihydroxy-10,12-octadecadienoic acid as the main product from LA (linoleic acid) and (10E,12E,14E)-9,16-dihydroxy-10,12,14-octadecatrienoic acid as the main product from ALA (alpha-LA) substrates respectively, with ALA as preferred substrate. The enzyme exhibited a broad pH optimum between pH 7 and pH 10. Soluble extracts of Nostoc sp. contain more 9-LOX-derived hydroperoxides in sonified than in non-sonified cells, but products of full-length NspLOX were not detectable under the conditions used. As no other LOX-like sequence was identified in the genome of Nostoc sp. PCC 7120, the results presented suggest that (9R)-LOX-derived oxylipins may represent the endogenous products of NspLOX. Based on the biochemical results of NspLOX, we suggest that this bifunctional enzyme may represent a more ancient way to control the intracellular amount of oxylipins in this cyanobacterium.

Oxylipin formation in Nostoc punctiforme (PCC73102).

The dioxygenation of polyunsaturated fatty acids is mainly catalyzed by members of the lipoxygenase enzyme family in flowering plants and mosses. Lipoxygenase products can be metabolized further and are known as signalling substances that play a role in plant development as well as in plant responses to wounding and pathogen attack. Apart from accumulating data in mammals, flowering and non-flowering plants, information on the relevance of lipid peroxide metabolism in prokaryotic organisms is scarce. Thus we aimed to isolate and analyze lipoxygenases and oxylipin patterns from cyanobacterial origin. DNA isolated from Nostoc punctiforme strain PCC73102 yielded sequences for at least two different lipoxygenases. These have been cloned as cDNAs and named NpLOX1 and NpLOX2. Both proteins were identified as linoleate 13-lipoxygenases by expression in E. coli. NpLOX1 was characterized in more detail: It showed a broad pH optimum ranging from pH 4.5 to pH 8.5 with a maximum at pH 8.0 and alpha-linolenic acid was the preferred substrate. Bacterial extracts contain more 13-lipoxygenase-derived hydroperoxides in wounded than in non-wounded cells with a 30-fold excess of non-esterified over esterified oxylipins. 9-Lipoxygenase-derived derivatives were not detectable. 13-Lipoxygenase-derived hydroperoxides in esterified lipids were present at almost equal amounts compared to non-esterified hydroperoxides in non-wounded cells. These results suggest that 13-lipoxygenases acting on free fatty acids dominate in N. punctiforme strain PCC73102 upon wounding.

Lipid metabolism in arbuscular mycorrhizal roots of Medicago truncatula.

The peroxidation of polyunsaturated fatty acids, common to all eukaryotes, is mostly catalyzed by members of the lipoxygenase enzyme family of non-heme iron containing dioxygenases. Lipoxygenase products can be metabolized further in the oxylipin pathway by several groups of CYP74 enzymes. One prominent oxylipin is jasmonic acid (JA), a product of the 13-allene oxide synthase branch of the pathway and known as signaling substance that plays a role in vegetative and propagative plant development as well as in plant responses to wounding and pathogen attack. In barley roots, JA level increases upon colonization by arbuscular mycorrhizal fungi. Apart from this first result regarding JA, no information is available on the relevance of lipidperoxide metabolism in arbuscular mycorrhizal symbiosis. Thus we analyzed fatty acid and lipidperoxide patterns in roots of Medicago truncatula during mycorrhizal colonization. Levels of fungus-specific fatty acids as well as palmitic acid (16:0) and oleic acid (18:1 n - 9) were increased in mycorrhizal roots. Thus the degree of arbuscular mycorrhizal colonization of roots can be estimated via analysis of fungal specific esterified fatty acids. Otherwise, no significant changes were found in the profiles of esterified and free fatty acids. The 9- and 13-LOX products of linoleic and alpha-linolenic acid were present in all root samples, but did not show significant differences between mycorrhizal and non-mycorrhizal roots, except JA which showed elevated levels in mycorrhizal roots. In both types of roots levels of 13-LOX products were higher than those of 9-LOX products. In addition, three cDNAs encoding CYP74 enzymes, two 9/13-hydroperoxide lyases and a 13-allene oxide synthase, were isolated and characterized. The transcript accumulation of these three genes, however, was not increased in mycorrhizal roots of M. truncatula.