Chemotaxonomy of planktonic cyanobacteria based on non-polar and 3-hydroxy fatty acid composition

Twenty-eight axenio planktonic cyanobacterial strains (10 Microcystis, three Oscillatoria, one Spirulina, one Aphanizomenon, 13 Anabaena) were investigated for their fatty acid composition by measurement of non-polar and hydroxy fatty acids. No 2-hydroxy fatty acids were detected in any strain, but 3-hydroxy fatty acids were detected in minor quantities in 24 strains. The highest portion of total fatty acids were non-polar fatty acids. Qualitative and quantitative analyses of 3-hydroxy fatty acids showed no taxonomic value in these strains, while the type of non-polar fatty acid composition was shown to be consistent within Microcystis and Anabaena strains, distinguishing them as type 4, characterized by the presence of 18:4, and type 2, characterized by 18:3 (α) of the Kenyon-Murata system. Two Oscillatoria agardhii Gomont strains were also included in the type 2 group due to the presence of 18: 3 (α), but the difference in characteristics of 16:2 and 16:3 between O. agardhii and Anabaena further divided type 2 into two subgroups: type 2A for Anabaena and type 2B for O. agardhii. A simplified unweighted pair group method with arithmetic averages (UPGMA) dendrogram demonstrated that the classification of 28 strains (Microcystis spp., Anabaena spp., Aphanizomenon flos-aquae (Lemmermann) Ralfs f. gracile (Lemmermann) Elenkin, O. agardhii and Spirullnasubsalsa Oersted ex Gomont based on numerical analysis of non-polar fatty acids corresponded to morphological species criteria, suggesting that non-polar fatty acid composition is a valuable chemical marker in the taxonomy of planktonic cyanobacteria. However, the fatty acid composition in Oscillatoria raciborskii is similar to that of Microcystis and very different from that of O. agardhii, suggesting its special position in Oscillatoria and the chemical diversity in the genus Oscillatoria.     

Characterization of morphospecies and strains of the genus Microcystis (Cyanobacteria) for a reconsideration of species classification

For characterization of Microcystis species and strains, cell size, growth temperature optimum, salinity tolerance, dark chemoheterotrophy, photoheterotrophy, guanine + cytosine content in DNA, total fatty acid composition and restriction fragment length polymorphism of a polymerase chain reaction product (PCR-RFLP) of the cpcBA intergenic spacer and flanking region were examined using 24 strains of Microcystis isolated from various lakes and ponds in Japan. From the results obtained it was observed that Microcystis spp. displayed low phenotypic diversity. Cell diameters of these strains were overlapping and there was no clear correlation with morphospecies. Slight differences in growth temperature optimum and salinity tolerance were observed among all strains. No strains showed either chemoheterotrophy or photoheterotrophy. The fatty acids present were the same in different strains although the amounts were different. All the strains had a similar G + C content ranging from 39 to 43 mol%. The phonogram constructed from the PCR-RFLP analysis showed that the species assignment for Microcystis species by morphology did not correspond with the genetic background.     

Analysis of whole cellular fatty acids and anastomosis relationships of binucleate Rhizoctonia spp. associated with Ceratobasidium cornigerum

Previous research has demonstrated that whole cellular fatty acids analysis is a useful tool for identifying and establishing taxonomic relationships between anastomosis groups (AGs) and related Rhizoctonia isolates. In this experiment, the composition of fatty acid of 28 isolates of teleomorph genus Ceratobasidium cornigerum, consisting of binucleate Rhizoctonia, AG-A, AG-B(o), AG-C, AG-P, and AG-Q, was evaluated using gas chromatography. Eleven fatty acids identified, i.e., myristic, pentadecanoic, palmitic, 2-hydroxypalmitic, palmitoleic, heptadecanoic, 9-heptadecenoic, stearic, oleic, linoleic, and linolenic acids, were present in isolates of AG-A, AG-B(o), AG-C, AG-P, and AG-Q. The major fatty acids, palmitic, oleic, and linoleic acids, were common in all isolates, constituting 87.1% to 94.7% of the whole cellular fatty acids identified. Isolates within the same AG were closely clustered, whereas isolates from different AGs were clearly and distinctly clustered based on average linkage cluster analysis of whole cellular fatty acids. Principal-component analysis generated from all fatty acids also confirmed the divergent separation of the 5 AGs of binucleate Rhizoctonia.     

Individual Microcystis colonies harbour distinct bacterial communities that differ by Microcystis oligotype and with time

Interactions between bacteria and phytoplankton in the phycosphere have impacts at the scale of whole ecosystems, including the development of harmful algal blooms. The cyanobacterium Microcystis causes toxic blooms that threaten freshwater ecosystems and human health globally. Microcystis grows in colonies that harbour dense assemblages of other bacteria, yet the taxonomic composition of these phycosphere communities and the nature of their interactions with Microcystis are not well characterized. To identify the taxa and compositional variance within Microcystis phycosphere communities, we performed 16S rRNA V4 region amplicon sequencing on individual Microcystis colonies collected biweekly via high-throughput droplet encapsulation during a western Lake Erie cyanobacterial bloom. The Microcystis phycosphere communities were distinct from microbial communities in whole water and bulk phytoplankton seston in western Lake Erie but lacked ‘core’ taxa found across all colonies. However, dissimilarity in phycosphere community composition correlated with sampling date and the Microcystis 16S rRNA oligotype. Several taxa in the phycosphere were specific to and conserved with Microcystis of a single oligotype or sampling date. Together, this suggests that physiological differences between Microcystis strains, temporal changes in strain phenotypes, and the composition of seeding communities may impact community composition of the Microcystis phycosphere.     

Biosystematic studies on novel streptomycetes from soil

Members of three putatively novel Streptomyces species, designated Streptomyces groups A, B and C, were repeatedly isolated from environmental samples taken from four hay meadow plots at Cockle Park Experimental Farm, Northumberland (UK). Representative isolates were found to have properties consistent with their classification in the genus Streptomyces and were recovered in three taxa using different phenotypic criteria, namely morphological and pigmentation properties, rapid enzyme tests, and whole-organism fatty acid, protein electrophoretic and pyrolysis mass-spectrometric data. The isolates were rapidly characterised as three taxonomic groups using pyrolysis mass spectrometry. The three taxa were also distinguished from one another and from validly described species of Streptomyces using rapid enzyme tests based on the fluorophores 7-amino-methylcoumarin and 4-methylumbelliferone, and computer-assisted identification procedures. The results indicate that selective isolation and rapid characterisation of streptomycetes using pyrolysis mass spectrometry provide a practical way of determining the phenotypic species diversity of streptomycetes in natural habitats. The experimental data also indicate that representative sampling of cultivable streptomycetes from soil can best be achieved using a multi-step extraction procedure coupled with the use of selective isolation procedures.     

Fatty acid composition as an indicator of physiological condition of the cyanobacterium Microcystis aeruginosa

The present study examined the fatty acid composition of Microcystis aeruginosa grown in a batch culture and that of Microcystis-dominated plankton collected in an experimental enclosure in a shallow, eutrophic embayment of Lake Biwa (Akanoi Bay). In pure culture, we detected 16 : 0, 18 : 2ω6, 18 : 3ω3, 18 : 3ω6, and 18 : 4ω3 acids as major fatty acids of M. aeruginosa, with trace amounts of C₂₀ polyunsaturated fatty acids. In both pure culture and the field enclosure, the ratio of total fatty acid weight to dry weight decreased with decreasing availability of dissolved inorganic nitrogen. The ω3/ω6 ratios of C₁₈ polyunsaturated fatty acids [(18 : 3ω3 + 18 : 4ω3)/(18 : 2ω6 + 18 : 3ω6)] varied greatly (range, 2–5) in response to the changes in physical and chemical conditions for Microcystis growth. Most notably, the ω3/ω6 fatty acid ratios were significantly positively correlated with the growth rate of cells in a batch culture. We suggest that the fatty acid composition is a useful indicator of the physiological state of Microcystis in freshwater lakes. Received: March 2, 2001/ Accepted: December 19, 2001     

Comparative Study of Microbial Communities from Cultured and Natural Populations of the Mussel Mytilus trossulus in Peter the Great Bay

The 525 strains of heterotrophic bacteria isolated from natural and cultured populations of the mussel Mytilus trossulus and the surrounding seawater were identified to a genus level on the basis of phenotypic analysis and the fatty acid composition of cell lipids. Gram-negative isolates were dominated by six genera of the family Enterobacteriaceae and by the genera Pseudoalteromonas, Vibrio, Photobacterium, Cytophaga/Flavobacterium/Bacteroides, Pseudomonas, and Moraxella. Gram-positive isolates were mainly represented by the genus Streptomyces. The taxonomic compositions of natural and cultured populations of the mussel M. trossulus in Peter the Great Bay were similar.     

A Temperature Dependence of the Intra- and Extracellular Fatty-Acid Composition of Green Algae and Cyanobacterium

The effect of ambient temperature on the composition of intracellular fatty acids and the release of free fatty acids (FFA) into a medium by cyanobacterium Spirulina platensis and eukaryotic microalgae, Chlorella vulgaris and Botryococcus braunii, was studied using their batch cultures. It was found that all the species studied, regardless of their taxonomic status, responded to the temperature regime by similar changes in their intracellular fatty acid composition: the relative content of more unsaturated fatty acids decreased with the elevation of temperature. At the same time, in the prokaryote, this temperature shift blocked, first of all, the elongation of 16:0 to 18:0 and then their further desaturation. In eukaryotes, the change in the desaturation of dienoic to trienoic fatty acids was the most pronounced process. The ratio of dienoic to trienoic fatty acids remained almost unchanged in S. platensis. The relative content of extracellular unsaturated FFA increased in the prokaryotic organism S. platensis at a higher temperature. But no significant changes in the composition of extracellular unsaturated FFA were detected in eukaryotic algae upon temperature elevation.     

Agromonas oligotrophica gen. nov., sp. nov., a nitrogen-fixing oligotrophic bacterium

Phenotypic and chemotaxonomic characteristics of five isolates of acetylenereducing (nitrogen-fixing) oligotrophic bacteria from a paddy soil were investigated. They showed similar phenotypic characteristics: they were aerobic, asporogenous, gram-negative, motile by a polar flagellum, and irregular rods. On full strength nutrient broth (NB) growth was severely suppressed, but well supported on 10-to 10000-fold diluted NB. They consumed glucose but produced no acid, and also utilized phenolic acids such as ferulic acid or p-coumaric acid. The cellular fatty acid composition, quinone system and DNA base composition of the isolates were investigated. Cellular fatty acids mainly consisted of straightchain unsaturated C_{18 : 1} (62–81% of total fatty acids). Ubiquinone Q-10 and a high guanine-plus-cytosine content (65.1–66.0 mol%) were found. The taxonomic status of the isolates is discussed and a new genus, Agromonas, with a single species Agromonas oligotrophica sp. nov., is proposed for these isolates. The type strain of A. oligotrophica is JCM 1494.     

Contribution to the taxonomy of haemolytic corynebacteria

In an attempt to assess the taxonomic relationships among human (Corynebacterium haemolyticum), animal (Corynebacterium pyogenes bovis) haemolytio corynebacteria, typical corynebacteria (Corynebacterium diphteriae mitis, C. ovis, C. ulcerans) and group A and G streptococci, a number of biochemical parameters were established: the DNA content of G + C, the presence of the cytochrome system, composition of fatty acids in free lipids and production of carboxylic acids as end products of fermentation. It was found that according to the above criteria, streptococci differed significantly from the corynebacteria studied. In addition, it was possible to differentiate a subgroup of typically aerobic haemolytic corynebacteria (different from both human and animal corynebacteria), possessing a complete cytochrome system, producing propionic acid and having a different composition of fatty acids.     

Characterisation,differentiation and biochemical diversity of Fusarium solani and Fusarium proliferatum based on cellular fatty acid profiles

The biochemical relationships between Fusarium solani and Fusarium proliferatum isolates were investigated using fatty acid analysis. Cellular fatty acid composition showed that palmitic acid, stearic acid, oleic acid and linoleic acid were the most abundant fatty acids in these species and accounted for 93.88 and 94.02% of the fatty acid profiles in F. solani and F. proliferatum, respectively. The most predominant fatty acids were linoleic acid (37.44%) in F. solani and oleic acid (39.81%) in F. proliferatum. The fatty acid compositions of F. solani and F. proliferatum were significantly different (p?<?0.05) for most of the individual fatty acids. This study demonstrated that fatty acid profiles may be useful to characterise and differentiate F. solani and F. proliferatum isolates at the species level. Using fatty acid analysis, biochemical diversity was observed among isolates of these species. The dendrogramme revealed that F. solani and F. proliferatum formed two distinct clusters with a distance of 7.2. Isolates of each species were clustered with each other, having a Euclidean distance of 6 and 6.6 for F. solani and F. proliferatum, respectively.     

PHYSIOLOGICAL VARIABILITY WITHIN TEN STRAINS OF CHAETOCEROS MUELLERI (BACILLARIOPHYCEAE)1

The effects of temperature, ionic composition, and conductivity on growth rates of ten strains of Chaetoceros muelleri Lemmerman (mostly var. subsalsum Johan. & Rushf.) were studied. Lipid content of stressed and unstressed cells and fatty acid composition were also determined. Considerable physiological variability was observed in the ten strains, although principal components analysis of physiological data indicated that all strains fell into one of two major groups: C. muelleri (var. muelleri and var. subsalsum) and an undescribed Chaetoceros species morphologically close to C. muelleri var. subsalsum. A high degree of agreement was found among morphological, physiological, and biochemical data sets, indicating that physiological and biochemical data may be helpful in making taxonomic decisions in diatoms, particularly in taxa with few morphological characters. We also conclude that nonmorphological characters such as those employed in the present study can be used to test phylogenetic hypotheses formulated from traditional morphological data.     

Fatty Acid Composition of Planktonic Species of <Emphasis Type="Italic">Anabaena</Emphasis> (Cyanobacteria) with Coiled Trichomes Exhibited a Significant Taxonomic Value

Twenty-six axenic strains of planktonic Anabaena with coiled trichomes belonging to 13 species were investigated by analyzing the pattern and content of their fatty acid composition, and by comparing their fatty acid composition with their morphological properties. In general, the planktonic Anabaena with coiled trichomes contained 14:0, 16:0, 16:1(cis-), 18:0, 18:1, 18:2, and 18:3() as their major fatty acid component, and were classified as Type 2 according to the Kenyon-Murata System. The Type 2 was further divided into two subtypes: Type 2A with 16:2 and 16:3, and Type 2B without 16:2 and 16:3. Among these Anabaena strains with coiled form, A. oumiana (NIES-73 and Ana Kas1) and A. eucompacta (Ana Chiba) contained Type 2B fatty acid composition, and other strains contained Type 2A. Among the strains with the latter type, A. circinalis (Ana Da) and A. curva (Ana Ao) had low levels of 18:3(). Most Anabaena strains with coiled trichomes showed a strong correlation between morphological characteristics and fatty acid composition.     

QUANTITATIVE GENETICS OF FATTY ACID VARIATION IN ISOCHRYSIS GALBANA (PRYMNESIOPHYCEAE) AND PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Fatty acid variation among culture collection strains and 40 new isolates of Isochrysis galbana Parke was analyzed by quantitative genetic methods. Fatty acid variation among strains and among isolates was highly significant for major fatty acids showing the existence of a genetic component in the determination of differences in fatty acid content. The heritabilities for the major fatty acids ranged between 0.68 and 0.99 among collection strains and between 0.31 and 0.43 among isolates. Eicosapentaenoic acid (EPA) had the highest heritability in I. galbana, but the majority of remaining fatty acids also showed high heritability values. A similar experiment with five UTEX strains of Phaeodactylum tricornutum also showed the presence of a genetic component in four out of seven major fatty acids. Nevertheless, the UTEX strains did not differ significantly in EPA content, although they showed a heritability of 0.40 for this fatty acid. An additional experiment culturing the same isolates of I. galbana in larger volumes of media showed that there was a high significant positive linear relation between EPA content in different volumes. Therefore, EPA content in small volume cultures was an unbiased indicator of EPA content in larger volume cultures. Our results provide support for the genetic determination of fatty acid content in microalgae and suggest that selection, and mutation and selection, are likely to improve EPA content in I. galbana and probably in many other microalgae. Such a selection program can be carried out in small-volume cultures with high confidence.     

Survey of duckweed diversity in Lake Chao and total fatty acid,triacylglycerol, profiles of representative strains

Lemnaceae (duckweeds) are widely distributed aquatic flowering plants. Their high growth rate, starch content and suitability for bioremediation make them potential feedstock for biofuels. However, few natural duckweed resources have been investigated in China, and there is no information about total fatty acid (TFA) and triacylglycerol (TAG) composition of duckweeds from China. Here, the genetic diversity of a natural duckweed population collected from Lake Chao, China, was investigated using multilocus sequence typing (MLST). The 54 strains were categorised into four species in four genera, representing 12 distinct sequence types. Strains representing Lemna aequinoctialis and Spirodela polyrhiza were predominant. Interestingly, a surprisingly high degree of genetic diversification within L. aequinoctialis was observed. The four duckweed species revealed a uniform fatty acid composition, with three fatty acids, palmitic acid, linoleic acid and linolenic acid, accounting for more than 80% of the TFA. The TFA in biomass varied among species, ranging from 1.05% (of dry weight, DW) for L. punctata and S. polyrhiza to 1.62% for Wolffia globosa. The four duckweed species contained similar TAG contents, 0.02% mg·DW^?1. The fatty acid profiles of TAG were different from those of TFA, and also varied among the four species. The survey investigated the genetic diversity of duckweeds from Lake Chao, and provides an initial insight into TFA and TAG of four duckweed species, indicating that intraspecific and interspecific variations exist in the content and composition of both TFA and TAG in comparison with other studies.     

Specificity of Lipid Composition in Two Botryococcus Strains,the Producers of Liquid Hydrocarbons

The structure of liquid hydrocarbons and fatty acids produced by the green alga Botryococcus was identified. Two representatives of this alga, Botryococcus braunii Kütz, strain IPPAS H-252, introduced into culture earlier and an organism isolated for the first time from the Shira Lake, were used for this identification. Fatty acid composition of B. braunii, strain H-252, lipids was characterized by a high content of trienoic acids of C16–C18 series. The hydrocarbon composition of this strain was represented by straight-chain and branched-chain C14–C28 components; long-chain linear aliphatic C20–C27 hydrocarbons (54.4%) and 2,6,10,14-tetramethylhexadecane (20.5%) predominated among them. The strain H-252 differed in its fatty acid and hydrocarbon composition from the strains described earlier as Botryococcus braunii. The fatty acid composition of the Botryococcus isolate was represented mainly by C12–C32 saturated and monoenoic acids. The hydrocarbons formed by this isolate were represented by dienoic and trienoic components. C29 (48.9–56.3%) and C31 (11.1–16.3%) hydrocarbons predominated among the C23–C31 dienoic hydrocarbons, and C27, C29, and C31 trienoic hydrocarbons comprised 2.5–2.6% of total hydrocarbons. This type of hydrocarbons and the lipid fatty acid composition were characteristic for the race A of B. braunii.     

Cyanospira rippkae and Cyanospira capsulata (gen. nov. and spp. nov.): new filamentous heterocystous cyanobacteria from Magadi lake (Kenya)

New filamentous heterocystous cyanobacteria were isolated from the alkaline soda lake Magadi in Kenya. The characteristics of the isolates are summarized and their taxonomic position discussed.Uniform attributes of the strains, grouped in two types, Mag II 702 and Mag I 504 are the following: helical structure of the trichomes, immotility, gas vacuolation, obligate autotrophy, nitrogen fixation under aerobic conditions, and closely similar fatty acid composition, including the uncommon cis-vaccenic acid.For these organisms the assignment to a new genus named Cyanospira is proposed with the species C. rippkae and C. capsulata, separated on the basis of structural, chemical and mean DNA-base composition. Type strains 702 and 504 will be deposited at the ATCC and PCC.     

Variability of Xanthomonas Campestris pv. vasculorum From Sugarcane and Other Gramineae in Reunion Island. Characterization of a Different Xanthomonad

Stains presumed to be of Xanthomonas campestris pv. vasculorum (Cobb.) Dye, obtained from sugarcane and other gramineae in Réunion Island, were compared in terms of cultural aspects, pathogenic and physiological reactions, fatty acid profiles and restriction fragment length polymorphism (RFLP) of genomic DNA. The strains could be divided into two separate groups (G1 and G2). The G1 strains were identical to strains described as X. campestris pv. vasculorum; they showedan important variability in their cultural characteristics and in their aggressiveness. The G2 strains did not induce the usual symptoms of gumming disease on sugarcane cultivars infected under natural conditions or inoculated in the greenhouse. The G2 strains grew faster on agar medium, their colonies were more pigmented and less fluidal and had a different morphology on agar slant. Unlike the G1 strains, G2 strains hydrolyzed starch weakly and casein strongly; they utilized L-fucose and, to a lesser extent, melibioze. The fatty acid and genomic DNA profiles differed between the groups. Differences seemed large enough to support separation of G1 and G2 strains into distinct taxonomic entities, namely G1 as Xanthomonas campestris pv. vasculorum and G2 as a different pathovar of X. campestris. The taxonomic position of G2 strains is discussed.     

Mapping QTL controlling fatty acid composition in a doubled haploid rapeseed population segregating for oil content

Increasing oil content and improving the fatty acid composition in the seed oil are important breeding goals for rapeseed (Brassica napus L.). The objective of the study was to investigate a possible relationship between fatty acid composition and oil content in an oilseed rape doubled haploid (DH) population. The DH population was derived from a cross between the German cultivar Sollux and the Chinese cultivar Gaoyou, both having a high erucic acid and a very high oil content. In total, 282 DH lines were evaluated in replicated field experiments in four environments, two each in Germany and in China. Fatty acid composition of the seed oil was analyzed by gas liquid chromatography and oil content was determined by NIRS. Quantitative trait loci (QTL) for fatty acid contents were mapped and their additive main effects were determined by a mixed model approach using the program QTLMapper. For all fatty acids large and highly significant genetic variations among the genotypes were observed. High heritabilities were determined for oil content and for all fatty acids (h ² = 0.82 to 0.94), except for stearic acid content (h ²= 0.38). Significant correlations were found between the contents of all individual fatty acids and oil content. Closest genetic correlations were found between oil content and the sum of polyunsaturated fatty acids (18:2 + 18:3; r _G = −0.46), the sum of monounsaturated fatty acids (18:1 + 20:1 + 22:1; r _G = 0.46) and palmitic acid (16:0; r _G = −0.34), respectively. Between one and eight QTL for the contents of the different fatty acids were detected. Together, their additive main effects explained between 28% and 65% of the genetic variance for the individual fatty acids. Ten QTL for fatty acid contents mapped within a distance of 0 to 10 cM to QTL for oil content, which were previously identified in this DH population. QTL mapped within this distance to each other are likely to be identical. The results indicate a close interrelationship between fatty acid composition and oil content, which should be considered when breeding for increased oil content or improved oil composition in rapeseed.     

Comparative Characterization of the Microbial Community in Two Species of Sponges from Sea of Japan Using Fatty Acid Markers

The fatty acid composition of total lipids in two species of sponges (Halichondria panicea and Clathria pennata) inhabiting the same biotope in the Sea of Japan was examined. More than 80 fatty acids (FAs) were found for each species of sponges. Of them, 61 fatty acids were identified for H. panicea and 54 for C. pennata. The relative content of most FAs was less than 1%. The contribution of symbiotic microorganisms to the total fatty acids was higher in H. panicea than in C. pennata. Bacterial symbionts and microeukaryotes were found among the microorganisms associated with the sponges. The contribution of prokaryotic organisms was equal (7.8%) for both species of sponges, but the fatty acids were characteristic of different taxonomic groups of bacteria. The proportion of microeukaryotic fatty acids in the total lipids of H. panicea (19.6%) was two times that of C. pennata (10.1%).