Thorsten Bauersachs

An interlaboratory study of TEX86 and BIT analysis of sediments, extracts, and standard mixtures

Two commonly used proxies based on the distribution of glycerol dialkyl glycerol tetraethers (GDGTs) are the TEX86 (TetraEther indeX of 86 carbon atoms) paleothermometer for sea surface temperature reconstructions and the BIT (Branched Isoprenoid Tetraether) index for reconstructing soil organic matter input to the ocean. An initial round-robin study of two sediment extracts, in which 15 laboratories participated, showed relatively consistent TEX86 values (reproducibility +/- 3-4 degrees C when translated to temperature) but a large spread in BIT measurements (reproducibility +/- 0.41 on a scale of 0-1). Here we report results of a second round-robin study with 35 laboratories in which three sediments, one sediment extract, and two mixtures of pure, isolated GDGTs were analyzed. The results for TEX86 and BIT index showed improvement compared to the previous round-robin study. The reproducibility, indicating interlaboratory variation, of TEX86 values ranged from 1.3 to 3.0 degrees C when translated to temperature. These results are similar to those of other temperature proxies used in paleoceanography. Comparison of the results obtained from one of the three sediments showed that TEX86 and BIT indices are not significantly affected by interlaboratory differences in sediment extraction techniques. BIT values of the sediments and extracts were at the extremes of the index with values close to 0 or 1, and showed good reproducibility (ranging from 0.013 to 0.042). However, the measured BIT values for the two GDGT mixtures, with known molar ratios of crenarchaeol and branched GDGTs, had intermediate BIT values and showed poor reproducibility and a large overestimation of the true (i.e., molar-based) BIT index. The latter is likely due to, among other factors, the higher mass spectrometric response of branched GDGTs compared to crenarchaeol, which also varies among mass spectrometers. Correction for this different mass spectrometric response showed a considerable improvement in the reproducibility of BIT index measurements among laboratories, as well as a substantially improved estimation of molar-based BIT values. This suggests that standard mixtures should be used in order to obtain consistent, and molar-based, BIT values.

Fossilized glycolipids reveal past oceanic N2 fixation by heterocystous cyanobacteria

N2-fixing cyanobacteria play an essential role in sustaining primary productivity in contemporary oceans and freshwater systems. However, the significance of N2-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 N2-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 N2-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.

Distribution of heterocyst glycolipids in cyanobacteria

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.

Rapid analysis of long-chain glycolipids in heterocystous cyanobacteria using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry.

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.

Diazotrophic microbial community of coastal microbial mats of the southern North Sea

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.

Occurrence and abundance of soil‐specific bacterial membrane lipid markers in the Têt watershed (southern France): Soil‐specific BHPs and branched GDGTs

Recently, four bacteriohopanepolyols (BHPs), adenosylhopane, and structurally similar adenosylhopane-type 1, 2-methyl adenosylhopane, and 2-methyl adenosylhopane-type 1, have been suggested to be characteristic of soil microbial communities and therefore can serve as molecular markers for soil organic matter (OM) supply in river, lake, and marine sediments. In this study, we analyzed BHPs in peats and soils collected in the Tet watershed (southern France) and compared them with branched glycerol dialkyl glycerol tetraethers (GDGTs), a more established molecular tracer of soil OM. Adenosylhopane-type I is identified in all of the samples from the study area except one collected near the Tet River mouth with up to three of the related compounds also frequently present, particularly in the surface samples. The concentrations of soil-specific BHPs in peat environments have been shown to increase with lower delta(15)N values, providing evidence that N(2)-fixing bacteria are probably a major source of soil-specific BHPs in acidic environments. It seems likely that soil pH is a major factor controlling BHP occurrence based on statistical analysis of environmental parameters and BHP concentration data. The comparison of the soil-specific BHP concentrations with those of branched GDGTs shows no clear relationship in the Tet River system, supporting the concept that these two groups of soil-specific compounds are synthesized by different microbial organisms living in different niches in the soil profile (e.g., oxic top versus anoxic deep).

Biotechnological screening of microalgal and cyanobacterial strains for biogas production and antibacterial and antifungal effects.

Microalgae and cyanobacteria represent a valuable natural resource for the generation of a large variety of chemical substances that are of interest for medical research, can be used as additives in cosmetics and food production, or as an energy source in biogas plants. The variety of potential agents and the use of microalgae and cyanobacteria biomass for the production of these substances are little investigated and not exploited for the market. Due to the enormous biodiversity of microalgae and cyanobacteria, they hold great promise for novel products. In this study, we investigated a large number of microalgal and cyanobacterial strains from the Culture Collection of Algae at Göttingen University (SAG) with regard to their biomass and biogas production, as well antibacterial and antifungal effects. Our results demonstrated that microalgae and cyanobacteria are able to generate a large number of economically-interesting substances in different quantities dependent on strain type. The distribution and quantity of some of these components were found to reflect phylogenetic relationships at the level of classes. In addition, between closely related species and even among multiple isolates of the same species, the productivity may be rather variable.

Distribution of long chain heterocyst glycolipids in N2-fixing cyanobacteria of the order Stigonematales

N2-fixing heterocystous cyanobacteria have been shown to hold a suite of unique glycolipids, so-called heterocyst glycolipids (HGs), as part of the heterocyst cell envelope. It was also demonstrated that the distribution of these components bears a high level of chemotaxonomic information, which allows distinguishing heterocystous cyanobacteria of the order Nostocales on a family or even genus level. Here we report the heterocyst glycolipid composition of five representatives of the order Stigonematales (Fischerella muscicola, Fischerella sp., Nostochopsis lobatus, Westiellopsis prolifica and Westiellopsis sp.), which have largely escaped a detailed investigation of their HG content so far. All analyzed strains contained a similar qualitative mixture of HGs with 1-(O-hexose)-3,29,31-dotriacontanetriol (HG32 triol) dominating over minor quantities of 1-(O-hexose)-29-keto-3,31-dotriacontanediol (HG32 keto-diol). When viewed in conjunction with previous culture studies on the HG composition of heterocystous cyanobacteria, our results demonstrate that HG32 triols and their corresponding keto-diol varieties are characteristic biological markers for heterocystous cyanobacteria of the order Stigonematales. Given that these N2-fixers primarily occur in tropical to subtropical freshwater lakes and subaerial habitats, the presence of HG32 triols and keto-diols in sedimentary sequences may offer additional information on climatic conditions in palaeoenvironmental studies.

Influence of Different CO2 Concentrations on Microalgae Growth, α-Tocopherol Content and Fatty Acid Composition

A prerequisite for the growth of microalgae on an industrial scale is the optimization of cultivation conditions and the reduction of production costs. The nutrient elements, heat and carbon dioxide provided by flue gas can be used to increase biomass production. The aim of this study was to investigate the effects of different CO2 concentrations on algal strains with regard to biomass production, α-tocopherol content, fatty acid composition and fatty acid content. For this, we grew the three microalgae Coccomyxa sp., Desmodesmus sp. and Muriella terrestris that were isolated from two tufa-forming karstwater creeks characterized by high CO2 partial pressures. No significant differences in biomass production rate were observed when the cultures were gassed with air or 15% (v/v) CO2. However, cell growth increased substantially when 5% (v/v) CO2 was used. The content of α-tocopherol increased in the stationary phase compared to the logarithmic phase in all investigated strains. In contrast, it decreased significantly when the cultures were aerated with either 5% (v/v) CO2 or 15% (v/v) CO2. The saturation level of fatty acids was significantly higher in cultures aerated with CO2 in the stationary phase compared to the logarithmic phase. In contrast, it decreased in the stationary phase when the cultures were gassed with air. Fatty acid contents increased in the stationary phase compared to the logarithmic phase. It was also higher by gassing with 5% (v/v) CO2 and decreased when the cultures were aerated with air or 15% (v/v) CO2. When the biomass production rates were taken into consideration together with contents of α-tocopherol and total fatty acid, the productivity of both increased considerably when the cultures experienced gassing by a 5% (v/v) CO2.

Methanosarcina Spherical Virus, a Novel Archaeal Lytic Virus Targeting Methanosarcina Strains

ABSTRACT A novel archaeal lytic virus targeting species of the genus Methanosarcina was isolated using Methanosarcina mazei strain Gö1 as the host. Due to its spherical morphology, the virus was designated Methanosarcina spherical virus (MetSV). Molecular analysis demonstrated that MetSV contains double-stranded linear DNA with a genome size of 10,567 bp containing 22 open reading frames (ORFs), all oriented in the same direction. Functions were predicted for some of these ORFs, i.e., such as DNA polymerase, ATPase, and DNA-binding protein as well as envelope (structural) protein. MetSV-derived spacers in CRISPR loci were detected in several published Methanosarcina draft genomes using bioinformatic tools, revealing a potential protospacer-adjacent motif (PAM) motif (TTA/T). Transcription and expression of several predicted viral ORFs were validated by reverse transcription-PCR (RT-PCR), PAGE analysis, and liquid chromatography-mass spectrometry (LC-MS)-based proteomics. Analysis of core lipids by atmospheric pressure chemical ionization (APCI) mass spectrometry showed that MetSV and Methanosarcina mazei both contain archaeol and glycerol dialkyl glycerol tetraether without a cyclopentane moiety (GDGT-0). The MetSV host range is limited to Methanosarcina strains growing as single cells (M. mazei, Methanosarcina barkeri and Methanosarcina soligelidi). In contrast, strains growing as sarcina-like aggregates were apparently protected from infection. Heterogeneity related to morphology phases in M. mazei cultures allowed acquisition of resistance to MetSV after challenge by growing cultures as sarcina-like aggregates. CRISPR/Cas-mediated resistance was excluded since neither of the two CRISPR arrays showed MetSV-derived spacer acquisition. Based on these findings, we propose that changing the morphology from single cells to sarcina-like aggregates upon rearrangement of the envelope structure prevents infection and subsequent lysis by MetSV. IMPORTANCE Methanoarchaea are among the most abundant organisms on the planet since they are present in high numbers in major anaerobic environments. They convert various carbon sources, e.g., acetate, methylamines, or methanol, to methane and carbon dioxide; thus, they have a significant impact on the emission of major greenhouse gases. Today, very little is known about viruses specifically infecting methanoarchaea that most probably impact the abundance of methanoarchaea in microbial consortia. Here, we characterize the first identified Methanosarcina-infecting virus (MetSV) and show a mechanism for acquiring resistance against MetSV. Based on our results, we propose that growth as sarcina-like aggregates prevents infection and subsequent lysis. These findings allow new insights into the virus-host relationship in methanogenic community structures, their dynamics, and their phase heterogeneity. Moreover, the availability of a specific virus provides new possibilities to deepen our knowledge of the defense mechanisms of potential hosts and offers tools for genetic manipulation.