Microbiome and photoperiod interactively determine thermal sensitivity of polar and temperate diatoms

The effect of temperature on ectothermic organisms in the context of climate change has long been considered in isolation (i.e. as a single driver). This is challenged by observations demonstrating that temperature-dependent growth is correlated to further factors. However, little is known how the chronobiological history of an organism reflected in its adaptation to re-occurring cyclic patterns in its environment (e.g. annual range of photoperiods in its habitat) and biotic interactions with its microbiome, contribute to shaping its realized niche. To address this, we conducted a full-factorial microcosm multi-stressor experiment with the marine diatoms Thalassiosira gravida (polar) and Thalassiosira rotula (temperate) across multiple levels of temperature (4°C; 9°C; 13.5°C) and photoperiod (4 h; 16 h; 24 h), both in the presence or absence of their microbiomes. While temperature-dependent growth of the temperate diatom was constrained by short and long photoperiods, the polar diatom coped with a 24 h photoperiod up to its thermal optimum (9°C). The algal microbiomes particularly supported host growth at the margins of their respective fundamental niches except for the combination of the warmest temperature tested at 24 h photoperiod. Overall, this study demonstrates that temperature tolerances may have evolved interactively and that the mutualistic effect of the microbiome can only be determined once the multifactorial abiotic niche is defined

In Silico Modeling of Spirolides and Gymnodimines: Determination of S Configuration at Butenolide Ring Carbon C-4

Only few naturally occurring cyclic imines have been fully structurally elucidated or synthesized to date. The configuration at the C-4 carbon plays a pivotal role in the neurotoxicity of many of these metabolites, for example, gymnodomines (GYMs) and spirolides (SPXs). However, the stereochemistry at this position is not accessible by nuclear Overhauser effect—nuclear magnetic resonance spectroscopy (NOE-NMR) due to unconstrained rotation of the single carbon bond between C-4 and C-5. Consequently, the relative configuration of GYMs and SPXs at C-4 and its role in protein binding remains elusive. Here, we determined the stereochemical configuration at carbon C-4 in the butenolide ring of spirolide- and gymnodimine-phycotoxins by comparison of measured 13C NMR shifts with values obtained in silico using force field, semiempirical and density functional theory methods. This comparison demonstrated that modeled data support S configuration at C-4 for all studied SPXs and GYMs, suggesting a biosynthetically conserved relative configuration at carbon C-4 among these toxins

Allelochemical defense against epibiosis in the macroalga Caulerpa racemosa var. turbinata

The abundance and diversity of microorganisms on the surface of the tropical green macroalga Caulerpa racemosa var. turbinata and the effect of algal surface and waterborne compounds on fouling organisms were investigated both in laboratory and field experiments. As shown via electron microscopic enumeration, the abundance of epibiotic bacteria and diatoms on algal frond surfaces was not significantly different from the reference biofilms harvested from stones in the C. racemosa habitat. The analysis of Terminal Restriction Fragment Length Polymorphism of DNA from algal surface-associated bacterial communities revealed that despite a similar abundance of these bacteria, the community profile on algal frond surfaces differed significantly from that of inanimate, undefended substrates. These results suggest that the alga regulate the occurrence of certain bacterial ribotypes. This result was in accordance with the fact that different bacterial communities formed on the artificial substrata (i.e. Petri dishes) placed in the C. racemosa habitat and alga-free control sites. Neither C. racemosa conditioned seawater (CCW) nor hexane surface extracts affected the growth of bacterial isolates from biofilms. However, only CCW exhibited a toxic effect on the larvae of the fouling polychaete Hydroides elegans, and evoked abnormal larval development in a concentration-dependent fashion. At sublethal concentrations, the <1 kD fraction of CCW inhibited the larval settlement of H. elegans and the bryozoan Bugula neritina. Caulerpenyne, the prominent bioactive metabolite in the genus Caulerpa, was not detected in CCW by chromatographic procedures. Our data suggest that waterborne compounds other than caulerpenyne are involved in the chemical defense of the alga C. racemosa

Siderophore purification with titanium dioxide nanoparticle solid phase extraction

Siderophores are metal chelators produced by microorganisms to facilitate binding and uptake of iron. The isolation and characterization of siderophores are impeded by typically low siderophore yields and the complexity of siderophore-containing extracts generated with traditional purification methods. We investigated titanium dioxide nanoparticle solid-phase extraction (TiO2 NP SPE) as a technique to selectively concentrate and purify siderophores from complex matrices for subsequent LC-MS detection and identification. TiO2 NP SPE showed a high binding capacity (15.7 ± 0.2 μmol mg−1 TiO2) for the model siderophore desferrioxamine B (DFOB) and proved robust to pH changes and the presence of EDTA. These are significant advances in comparison to immobilized metal affinity chromatography (IMAC). The TiO2 NP SPE was highly selective and recovered 77.6 ± 6.2% of DFOB spiked to a compositionally complex bacterial culture supernatant. The simple clean-up procedure removed the majority of contaminants and allowed direct detection of siderophores from the LC-MS base peak chromatogram. The ‘untargeted’ purification and analysis of an untreated supernatant of iron-deprived bacterial culture allowed for the direct identification of two known and three novel ferrioxamines. Thus, TiO2 NP SPE in combination with LC-MS offers great potential as a discovery platform for the purification and subsequent quantification or identification of novel siderophores of microbial origin

Niche-based assembly of bacterial consortia on the diatom Thalassiosira rotula is stable and reproducible

With each cell division, phytoplankton create new space for primary colonization by marine bacteria. Although this surface microenvironment is available to all planktonic bacterial colonizers, we show the assembly of bacterial consortia on a cosmopolitan marine diatom to be highly specific and reproducible. While phytoplankton–bacteria interactions play fundamental roles in marine ecosystems, namely primary production and the carbon cycle, the ecological paradigm behind epiphytic microbiome assembly remains poorly understood. In a replicated and repeated primary colonization experiment, we exposed the axenic diatom Thalassiosira rotula to several complex and compositionally different bacterial inocula derived from phytoplankton species of varying degrees of relatedness to the axenic Thalassiosira host or natural seawater. This revealed a convergent assembly of diverse and compositionally different bacterial inocula, containing up to 2071 operational taxonomic units (OTUs), towards a stable and reproducible core community. Four of these OTUs already accounted for a cumulative abundance of 60%. This core community was dominated by Rhodobacteraceae (30.5%), Alteromonadaceae (27.7%), and Oceanospirillales (18.5%) which was qualitatively and quantitatively most similar to its conspecific original. These findings reject a lottery assembly model of bacterial colonization and suggest selective microhabitat filtering. This is likely due to diatom host traits such as surface properties and different levels of specialization resulting in reciprocal stable-state associations

Selective purification of catecholate, hydroxamate and α-hydroxycarboxylate siderophores with titanium dioxide affinity chromatography

Siderophores, high affinity iron chelators, play a key role in the uptake of iron by microorganisms and regulate many biological functions. Siderophores are categorized by their chelating group, e.g., catecholates, hydroxamates, α-hydroxycarboxylates. Natural concentrations of siderophores are often either too low or sample matrices are too complex for direct analysis by, e.g., liquid chromatography – mass spectrometry. Therefore, both concentration and purification are prerequisite for reliable analyses. However, a chromatographic technique that is selective for all siderophore classes and affords high levels of purification is lacking. We developed a titanium dioxide affinity chromatography (TDAC) solid-phase extraction (SPE) that affords the selective purification of these siderophore classes from complex sample matrices with recoveries up to 82%. The one-step purification removed most non-ligand sample ‘contaminants’, therefore, affording the straightforward identification of siderophore peaks in base peak chromatograms. As a proof of concept, the bioinformatic processing, dereplication of known features and selection of significant features in the TDAC eluates afforded a fast identification of six novel siderophores (woodybactines) from bacterial supernatants. We propose TDAC SPE as a fast and cost-effective methodology to screen for known or discover novel siderophores in natural samples in combination with untargeted bioinformatic processing by, e.g., XCMS. The method is scalable and yielded large amounts of highly purified siderophores from bacterial culture supernatants, providing an effective quantitative sample clean-up for, e.g., NMR structure elucidation

Probabilistic modeling of LCF failure times using a epidemiological crack percolation model

The analysis of standardized low cycle fatigue (LCF) experiments shows that the failure times widely scatter. Furthermore, mechanical components often fail before the deterministic failure time is reached. A possibility to overcome these problems is to consider probabilistic failure times. Our approach for probabilistic life prediction is based on the microstructure of the metal. Since we focus on nickel-base alloys we consider a coarse grained microstructure, with random oriented FCC grains. This leads to random distributed Schmid factors and different anisotropic stress in each grain. To gain crack initiation times, we use Coffin-MansonBasquin and Ramberg-Osgood equation on stresses corrected with probabilistic Schmid factors. Using these single grain crack initiation times, we have developed an epidemiological crack growth model over multiple grains. In this mesoscopic crack percolation model, cracked grains induce a stress increase in neighboring grains. This stress increase is realized using a machine learning model trained on data generated from finite element simulations. The resulting crack clusters are evaluated with a failure criterion based on a multimodal stress intensity factor. From the generated failure times, we calculate surface dependent hazard rates using a Monte Carlo framework. We compare the obtained failure time distributions to data from LCF experiments and find good coincidence of predicted and measured scatter bands

Evaluation of a Commercial Enzyme Linked Immunosorbent Assay (ELISA) for the Determination of the Neurotoxin BMAA in Surface Waters

The neurotoxin ß-N-methylamino-L-alanine (BMAA) is suspected to play a role in Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Because BMAA seems to be produced by cyanobacteria, surface waters are screened for BMAA. However, reliable analysis of BMAA requires specialized and expensive equipment. In 2012, a commercial enzyme-linked immunosorbent assay (ELISA) for determination of BMAA in surface waters was released. This kit could enable fast and relatively cheap screening of surface waters for BMAA. The objective of this study was to determine whether the BMAA ELISA kit was suitable for the determination of BMAA concentrations in surface waters. We hypothesised that the recovery of spiked samples was close to 100% and that the results of unspiked sample analysis were comparable between ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. However, we found that recovery was higher than 100% in most spiked samples, highest determined recovery was over 400%. Furthermore, the ELISA gave a positive signal for nearly each tested sample while no BMAA could be detected by LC-MS/MS. We therefore conclude that in its current state, the kit is not suitable for screening surface waters for BMAA

Crystalline iron oxides stimulate methanogenic benzoate degradation in marine sediment- derived enrichment cultures

Elevated dissolved iron concentrations in the methanic zone are typical geochemical signatures of rapidly accumulating marine sediments. These sediments are often characterized by co-burial of iron oxides with recalcitrant aromatic organic matter of terrigenous origin. Thus far, iron oxides are predicted to either impede organic matter degradation, aiding its preservation, or identified to enhance organic carbon oxidation via direct electron transfer. Here, we investigated the effect of various iron oxide phases with differing crystallinity (magnetite, hematite, and lepidocrocite) during microbial degradation of the aromatic model compound benzoate in methanic sediments. In slurry incubations with magnetite or hematite, concurrent iron reduction, and methanogenesis were stimulated during accelerated benzoate degradation with methanogenesis as the dominant electron sink. In contrast, with lepidocrocite, benzoate degradation, and methanogenesis were inhibited. These observations were reproducible in sediment-free enrichments, even after five successive transfers. Genes involved in the complete degradation of benzoate were identified in multiple metagenome assembled genomes. Four previously unknown benzoate degraders of the genera Thermincola (Peptococcaceae, Firmicutes), Dethiobacter (Syntrophomonadaceae, Firmicutes), Deltaproteobacteria bacteria SG8_13 (Desulfosarcinaceae, Deltaproteobacteria), and Melioribacter (Melioribacteraceae, Chlorobi) were identified from the marine sediment-derived enrichments. Scanning electron microscopy (SEM) and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) images showed the ability of microorganisms to colonize and concurrently reduce magnetite likely stimulated by the observed methanogenic benzoate degradation. These findings explain the possible contribution of organoclastic reduction of iron oxides to the elevated dissolved Fe2+ pool typically observed in methanic zones of rapidly accumulating coastal and continental margin sediments