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Phospholipid-derived fatty acids (PLFAs) are commonly used to characterize microbial communities in situ and the phylogenetic positions of newly isolated microorganisms. PLFAs are obtained through separation of phospholipids from glycolipids and neutral lipids using silica column chromatography. We evaluated the performance of this separation method for the first time using direct detection of intact polar lipids (IPLs) with high-performance liquid chromatography-mass spectrometry (HPLC-MS). We show that under either standard or modified conditions, the phospholipid fraction contains not only phospholipids but also other lipid classes such as glycolipids, betaine lipids, and sulfoquinovosyldiacylglycerols. Thus, commonly reported PLFA compositions likely are not derived purely from phospholipids and perhaps may not be representative of fatty acids present in living microbes.

The heterocysts of limnetic nitrogen-fixing filamentous cyanobacteria contain unique glycolipids in their cell wall that create the distinctive gas impermeability of the heterocyst cell wall as well as serve as biomarker lipids for these microbes. It has been assumed that marine free-living and endosymbiotic cyanobacteria synthesize the same glycolipids although they have not been investigated in any detail. Here we report the glycolipid composition of several marine free-living heterocystous cyanobacteria as well as the heterocystous endosymbiont Richelia intracellularis found in the biogeochemically important diatoms Hemiaulus hauckii and Hemiaulus membranaceus. In the marine cyanobacteria Nostoc muscorum and Calothrix sp., we detected the same glycolipids as found in freshwater representatives of these genera. However, we did not detect these glycolipids in the Hemiaulus-Richelia association. Instead, we identified glycolipids which comprised a C₅ sugar, ribose, rather than the C₆ sugars normally encountered in glycolipids of free-living cyanobacteria. In addition, the glycolipids had slightly longer chain lengths (C₃₀ and C₃₂ versus C₂₆ and C₂₈) in the aglycone moiety. The different glycolipid composition of the marine endosymbotic heterocystous cyanobacteria compared to their free-living counterparts may be an adaptation to the high intracellular O₂ concentrations within their host. These glycolipids may provide unique tracers for the presence of these microbes in marine environments and permit exploration of the evolutionary origins of these symbioses., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The diazotrophic community in microbial mats growing along the shore of the North Sea barrier island Schiermonnikoog (The Netherlands) was studied using microscopy, lipid biomarkers, stable carbon (δ(13) C(TOC) ) and nitrogen (δ(15) N) isotopes as well as by constructing and analyzing 16S rRNA gene libraries. Depending on their position on the littoral gradient, two types of mats were identified, which showed distinct differences regarding the structure, development and composition of the microbial community. Intertidal microbial mats showed a low species diversity with filamentous non-heterocystous Cyanobacteria providing the main mat structure. In contrast, supratidal microbial mats showed a distinct vertical zonation and a high degree of species diversity. Morphotypes of non-heterocystous Cyanobacteria were recognized as the main structural component in these mats. In addition, unicellular Cyanobacteria were frequently observed, whereas filamentous heterocystous Cyanobacteria occurred only in low numbers. Besides the apparent visual dominance of cyanobacterial morphotpyes, 16S rRNA gene libraries indicated that both microbial mat types also included members of the Proteobacteria and the Cytophaga-Flavobacterium-Bacteroides group as well as diatoms. Bulk δ(15) N isotopes of the microbial mats ranged from +6.1‰ in the lower intertidal to -1.2‰ in the supratidal zone, indicating a shift from predominantly nitrate utilization to nitrogen fixation along the littoral gradient. This conclusion was supported by the presence of heterocyst glycolipids, representing lipid biomarkers for nitrogen-fixing heterocystous Cyanobacteria, in supratidal but not in intertidal microbial mats. The availability of combined nitrogen species might thus be a key factor in controlling and regulating the distribution of the diazotrophic microbial community of Schiermonnikoog., (© 2011 Blackwell Publishing Ltd.)

N(2)-fixing cyanobacteria play an essential role in sustaining primary productivity in contemporary oceans and freshwater systems. However, the significance of N(2)-fixing cyanobacteria in past nitrogen cycling is difficult to establish as their preservation potential is relatively poor and specific biological markers are presently lacking. Heterocystous N(2)-fixing cyanobacteria synthesize unique long-chain glycolipids in the cell envelope covering the heterocyst cell to protect the oxygen-sensitive nitrogenase enzyme. We found that these heterocyst glycolipids are remarkably well preserved in (ancient) lacustrine and marine sediments, unambiguously indicating the (past) presence of N(2)-fixing heterocystous cyanobacteria. Analysis of Pleistocene sediments of the eastern Mediterranean Sea showed that heterocystous cyanobacteria, likely as epiphytes in symbiosis with planktonic diatoms, were particularly abundant during deposition of sapropels. Eocene Arctic Ocean sediments deposited at a time of large Azolla blooms contained glycolipids typical for heterocystous cyanobacteria presently living in symbiosis with the freshwater fern Azolla, indicating that this symbiosis already existed in that time. Our study thus suggests that heterocystous cyanobacteria played a major role in adding "new" fixed nitrogen to surface waters in past stratified oceans.

Thirty-four axenic strains of cyanobacteria were analysed for their glycolipid content using high performance liquid chromatography coupled to electrospray ionisation tandem mass spectrometry (HPLC/ESI-MS(2)). Species of the families Nostocaceae and Rivulariaceae, capable of biosynthesising heterocysts, contained a suite of glycolipids consisting of sugar moieties glycosidically bound to long-chain diols, triols, keto-ols and keto-diols. The aglycone moiety consisted of C(26) or C(28) carbon-chains with hydroxyl groups at the C-3, omega-1 or omega-3 positions. Keto-ols and keto-diols contained their carbonyl functionalities likely at the C-3 position. These compounds were absent in all analysed unicellular and filamentous non-heterocystous cyanobacteria and in the heterocyst-forming cyanobacterium Anabaena CCY9922 grown in the presence of combined nitrogen, supporting the idea that the long-chain glycolipids are an important and unique structural component of the heterocyst cell envelope. The glycolipids 1-(O-hexose)-3,25-hexacosanediol and 1-(O-hexose)-3-keto-25-hexacosanol were ubiquitously distributed in species of the family Nostocaceae. 1-(O-hexose)-3,25,27-octacosanetriol and 1-(O-hexose)-3-keto-25,27-octacosanediol were dominant in members of the Calothrix genus, while traces of those compounds were detected only in one species of the Nostocaceae family. Their distribution in heterocystous cyanobacteria suggests a chemotaxonomic relevance that might allow distinguishing between species of different genera. Culture experiments indicate that the amount of keto-ols and keto-diols decreases relatively to their corresponding diols and triols counterparts with increasing temperature. Possibly, this is an adaptation to optimise the cell wall gas permeability, preventing inactivation of the oxygen-sensitive nitrogenase while allowing the highest diffusion of atmospheric dinitrogen into the heterocyst.

Under nitrogen-depleted conditions, N2-fixing cyanobacteria of the order Nostocales and Stigonematales differentiate vegetative cells into heterocysts. The cell envelope of these specialized cells contains unique glycolipids, consisting of a sugar moiety glycosidically bound to long-chain diols, triols and hydroxyketones. Only few reports have been published on these glycolipids in cultured cyanobacteria and none has reported them in natural environments. Here we show that heterocyst glycolipids can be rapidly and sensitively analyzed using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC/ESI-MS2). Positive ion mass spectra of the glycolipids consisted of protonated molecules and diagnostic product ions, indicating losses of sugar groups as well as hydroxyl and carbonyl functionalities from an alkyl chain. Using this method, heterocyst glycolipids were for the first time identified in a natural ecosystem, i.e., a microbial mat from the North Sea barrier island Schiermonnikoog, The Netherlands. This technique will facilitate the quick screening of cyanobacterial cultures and natural environments for the presence of heterocyst glycolipids, which may aid in assessing the role of heterocystous cyanobacteria in the global nitrogen cycle., (Copyright (c) 2009 John Wiley & Sons, Ltd.)