Jürgen Köster

Microbial diversity of a heavily polluted microbial mat and its community changes following degradation of petroleum compounds

ABSTRACT We studied the microbial diversity of benthic cyanobacterial mats inhabiting a heavily polluted site in a coastal stream (Wadi Gaza) and monitored the microbial community response induced by exposure to and degradation of four model petroleum compounds in the laboratory. Phormidium- and Oscillatoria-like cyanobacterial morphotypes were dominant in the field. Bacteria belonging to different groups, mainly the Cytophaga-Flavobacterium-Bacteriodes group, the γ and β subclasses of the class Proteobacteria, and the green nonsulfur bacteria, were also detected. In slurry experiments, these communities efficiently degraded phenanthrene and dibenzothiophene completely in 7 days both in the light and in the dark. n-Octadecane and pristane were degraded to 25 and 34% of their original levels, respectively, within 7 days, but there was no further degradation until 40 days. Both cyanobacterial and bacterial communities exhibited noticeable changes concomitant with degradation of the compounds. The populations enriched by exposure to petroleum compounds included a cyanobacterium affiliated phylogenetically with Halomicronema. Bacteria enriched both in the light and in the dark, but not bacteria enriched in any of the controls, belonged to the newly described Holophaga-Geothrix-Acidobacterium phylum. In addition, another bacterial population, found to be a member of green nonsulfur bacteria, was detected only in the bacteria treated in the light. All or some of the populations may play a significant role in metabolizing the petroleum compounds. We concluded that the microbial mats from Wadi Gaza are rich in microorganisms with high biodegradative potential.

Specific Bacterial, Archaeal, and Eukaryotic Communities in Tidal-Flat Sediments along a Vertical Profile of Several Meters

ABSTRACT The subsurface of a tidal-flat sediment was analyzed down to 360 cm in depth by molecular and geochemical methods. A community structure analysis of all three domains of life was performed using domain-specific PCR followed by denaturing gradient gel electrophoresis analysis and sequencing of characteristic bands. The sediment column comprised horizons easily distinguishable by lithology that were deposited in intertidal and salt marsh environments. The pore water profile was characterized by a subsurface sulfate peak at a depth of about 250 cm. Methane and sulfate profiles were opposed, showing increased methane concentrations in the sulfate-free layers. The availability of organic carbon appeared to have the most pronounced effect on the bacterial community composition in deeper sediment layers. In general, the bacterial community was dominated by fermenters and syntrophic bacteria. The depth distribution of methanogenic archaea correlated with the sulfate profile and could be explained by electron donor competition with sulfate-reducing bacteria. Sequences affiliated with the typically hydrogenotrophic Methanomicrobiales were present in sulfate-free layers. Archaea belonging to the Methanosarcinales that utilize noncompetitive substrates were found along the entire anoxic-sediment column. Primers targeting the eukaryotic 18S rRNA gene revealed the presence of a subset of archaeal sequences in the deeper part of the sediment cores. The phylogenetic distance to other archaeal sequences indicates that these organisms represent a new phylogenetic group, proposed as “tidal-flat cluster 1.” Eukarya were still detectable at 360 cm, even though their diversity decreased with depth. Most of the eukaryotic sequences were distantly related to those of grazers and deposit feeders.

Mono-, di- and trimethyl-branched alkanes in cultures of the filamentous cyanobacterium Calothrix scopulorum

The extractable lipids of the cyanobacterium Calothrix scopulorum contain a complex suite of mono-, di- and trimethylheptadecanes. We identified 21 isomers; most of them have not been reported previously. The major compounds are 4,13-, 5,13-, and 4,5-dimethyl-, and 4,5,13- and 4,5,14-trimethylheptadecane. Methyl substitution occurs preferentially, but not exclusively, at positions 4 and 5 (or ω-4 and ω-5) at either end of the carbon chain. In mass spectra, the location of methyl groups at opposite ends of the carbon chain can be deduced from the presence of a characteristic secondary CnH2n−1+ fragment ion. The interpretation of mass spectra was confirmed by calculated retention index values which closely match the measured data. Our data provide evidence that branched alkanes with 17 to 20 carbon atoms can be used as biomarkers for cyanobacterial contributions to sedimentary organic matter.

Degradation of petroleum model compounds immobilized on clay by a hypersaline microbial mat

In this study the degradation of hydrophobic petroleum model compounds (phenanthrene, pristane, octadecane and dibenzothiophene) added to a submersed hypersaline microbial mat was investigated. Montmorillonite with an artificially altered, hydrophobic surface was used as carrier material, forming an organo-clay complex (OCC) with the attached mixture of petroleum model compounds. 6 mg/cm2 OCC were applied to cyanobacterialmat pieces, containing ∼33.3 μg/mg OCC of each compound. The degradationexperiment was performed under controlled laboratory conditions and accompanied bychemical analyses by GC/GC-MS, molecular analyses by PCR and DGGE as well asfunctional analyses by microsensor measurements of oxygen, photosynthesis, sulfide,pH and light. All applied model compounds were degraded, but residues were stillpresent after 18 weeks. The aromatic compounds phenanthrene (5.1 μg/mg OCC)and dibenzothiophene (4.3 μg/mg OCC) were preferentially degraded compared to the alkanes pristane (12.4 μg/mg OCC) and n-octadecane (13.4 μg/mg OCC). Metabolic changes during the degradation process could not be detected by microsensor measurements. The molecular population analyses did not reveal any significant community changes concomitant with the decrease of the petroleum model compounds. We conclude, that the pristine mats represent an intact, robust ecosystem in which the enzymatic requirements for the degradation of the applied pollutants exist. The slow degradation process did not affect the usual high internal turnover rates and did not favor a certain population in the community of the mats.

Natural, anthropogenic and fossil organic matter in river sediments and suspended particulate matter: a multi-molecular marker approach.

Different classes of organic matter (OM) have been systematically investigated in sediments and suspended particulate matter (SPM) along the Danube River in order to understand causes of compositional changes. Analytical pyrolysis revealed the dominance of natural organic matter (NOM) in most of the samples. The predominance of aquatic biomass is evident mainly from the abundance of organonitrogen compounds and phenol distributions. As the river enters a forested gorge, the terrestrial component of the NOM in sediments is more significant. This is reflected in abundant methoxyphenols and a very high carbon preference index. SPM sample from a tributary shows a unique geochemical signature. It contains abundant carboxylic acids, amines, isoprenoids in the pyrolyzate, and is dominated by phytol and 24-methyl-cholesta-5,24(28)-dien-3β-ol in the extract, produced by a diatom bloom. Wax esters with a relatively high proportion of short, methyl-branched alkyl-chains appear together with abundant phytadienes and n-C(17) alkane in some samples, suggesting a microbial origin. Anthropogenic OM from runoff and atmospheric deposition was evident from a minor input of polycyclic aromatic hydrocarbons (PAHs) originating from mixed combustion sources. Multivariate analysis using PAH data led us to define simple molecular ratios to distinguish the PAH composition in sand and silty sediments. The newly defined ratios are the alkylated phenanthrenes and anthracenes ratio (APA; C(1)-C(3)/C(0)-C(3) phenanthrenes and anthracenes) and the PAH ring number ratio (RN; 5-6 ring parent PAHs/all parent PAHs). This demonstrates that alkylated, as well as 5-6 ring PAHs are better preserved in the finer than in coarser grained sediments. A ubiquitous, but minor input of petroleum-related contamination with a uniform composition was evident in all samples as revealed by the analysis of petroleum biomarkers. This study demonstrates that the investigation of different classes of riverine OM requires a detailed molecular analysis, applying a series of analytical techniques and adequate statistical data treatment.

Gammaproteobacteria as a possible source of eicosapentaenoic acid in anoxic intertidal sediments.

Eicosapentaenoic acid (EPA; n-20:5ω3) was found to be a constituent of phospholipids in three mesophilic strains of Gammaproteobacteria, which were isolated from anoxic most probable number series prepared with sediments from an intertidal flat of the German North Sea coast. Their partial 16S rRNA gene sequences identified the isolates as close relatives of Shewanella colwelliana, Vibrio splendidus, and Photobacterium lipolyticum. So far, eicosapentaenoic acid has mainly been reported to occur in eukaryotes and some piezophilic or psychrophilic bacteria. With decreasing temperature, relative contents of EPA (up to 14% of total fatty acids) increased in all strains. Additionally, Shewanella and Vibrio spp. showed a significant increase in monounsaturated fatty acids with lower growth temperature. Analysis of the phospholipid compositions revealed that EPA was present in all three major phospholipid types, namely, phosphatidyl glycerol (PG), cardiolipin and phosphatidyl ethanolamine (PE). However, EPA was enriched in PG and cardiolipin relative to PE. In the tidal flat sediments from which the isolates were obtained, substantial amounts of EPA-containing PG were detected, whereas other typical microeukaryotic phospholipids—being also a possible source of EPA—were abundant at the sediment surface but were present in clearly lower amounts in the anoxic layers beneath 5xa0cm depth. Therefore, the EPA-containing PG species in the deeper layers in these sediments may indicate the presence of Gammaproteobacteria closely related to the isolates. These bacteria appear to be an important source of EPA in buried, anoxic sediments beneath the layers harboring significant populations of benthic eukaryotes.

Occurrence of two novel benzothiophene hopanoid families in sediments

Abstract Two novel families of C34–C35 benzothiophene hopanoids have been identified in laminated marlstones and limestones of Hauptdolomit (Germany, Upper Triassic), Calcaires en Plaquettes (France, Upper Jurassic) and Ghareb (Jordan, Upper Cretaceous) Formations. Structures were assigned on the basis of Raney nickel desulphurization, deuteriated nickel boride desulphurization, mass spectral data and GC behaviour. Based on quantitative analysis, a precursor-product relationship between thiophene hopanoids and benzothiophene hopanoids seems likely. This means that the novel hopanoids are not formed by sulphur incorporation into benzohopanes, but instead cyclization and aromatization of the hopanoid side chain occur after sulphur incorporation.

High Variations in Endospore Numbers within Tidal Flat Sediments Revealed by Quantification of Dipicolinic Acid

Bacterial endospores are resting stages without detectable metabolism that can remain viable for extended periods and hence might accumulate in sediments during burial. They can be stained by fluorescence dyes and are suggested to contribute to total cell counts, but cannot be distinguished from vegetative cells by epifluorescence microscopy. Viable counts obtained after pasteurization of sediment samples have suggested a significant contribution of spores to sediment microbiota. However, the actual contribution of endospores to total cell counts in marine sediments is largely unknown, because of the lack of methods for reliable quantification. In the present study, dipicolinic acid (DPA), which is accumulated in the endospore core, was used to quantify endospores in up to 5.5 m long sediment cores collected from tidal flats off the German North Sea coast (Wadden Sea). Dipicolinic acid contents were determined fluorimetrically using a highly sensitive post-column complexation HPLC approach and ranged from 0.02 to 4.4 nmol DPA g −1 sediment dry weight. Dipicolinic acid contents of spores of pure cultures, that were isolated from the sampling area, were used for the conversion into endospore numbers. Estimated numbers ranged from 1× 10 5 to 2× 10 7 spores g −1 sediment dry weight. In the uppermost 50 cm of the sediment section endospore numbers represented less than 1% of the total cell counts. However, in the layers beneath their contribution to total cell counts apparently increased with depth reaching up to 10% of total cell counts. The endospore depth profile was irregular, but reflected the vertical changes in lithology. The highest endospore numbers were found in thin black mud layers, significantly lower numbers in sandy sediments.

Degradation of 2,4-Dichlorophenoxyacetic Acid (2,4-D) by a Hypersaline Microbial Mat and Related Functional Changes in the Mat Community

Microbial mats possibly possess degradation capacities for haloorganic pollutants because of their wide range of different functional groups of microorganisms combined with extreme diurnal changes in pH, oxygen, and sulfide gradients. In this study, 20xa0mg/l of the chlorinated herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was applied to a pristine hypersaline cyanobacterial mat from Guerrero Negro, Mexico, under a light regime of 12xa0h dark/12xa0h light (600xa0μmol photons/m2s). The loss of 2,4-D was followed by chemical GC analysis; functional changes within the mat were determined with microelectrodes for oxygen, photosynthesis, pH, and sulfide. The depletion of 2,4-D due to photooxidation or sorption processes was checked in control experiments. Within 13 days, the light/dark incubated mats degraded 97% of the herbicide, while in permanent darkness only 35% were degraded. Adsorption of 2,4-D to the mat material, agar, or glass walls was negligible (4.6%), whereas 21% of the herbicide was degraded photochemically. The 2,4-D removal rate in the light/dark incubations was comparable to values reported for soils. The phototrophic community of the mat was permanently inhibited by the 2,4-D addition by 17% on average. The sulfate reduction in the entire mat and the respiration in the photic zone were inhibited more strongly but returned to original levels. Since at the end of the experiment the photosynthetic and respiratory activity of the mats were almost as high as in the beginning and 2,4-D almost completely disappeared, we conclude that the examined mats represent a robust and effective system for the degradation of the herbicide where probably the aerobic heterotrophic population is a major player in the degradation process.

Characterization of Microbial Mats from a Desert Wadi Ecosystem in the Sultanate of Oman

ABSTRACT Desert wadis are widespread in the Arabian Peninsula and play a vital role in the ecology of the region; nevertheless, these ecosystems are among the least studied. Various types of microbial mats are predominant in wadis, but information on their bacterial diversity and spatial distribution is very scarce. We investigated bacterial diversity, pigments and lipid composition of ten mats located at the down-, mid- and upstream of a desert wadi in Oman. Direct microscopy revealed the existence of different unicellular and filamentous cyanobacteria, with the dominance of the heterocystous genera Calothrix and Scytonema. The majority of MiSeq 16S rRNA sequences (44-76%) were affiliated to Cyanobacteria and Proteobacteria. While Alphaproteobacteria was the most dominant proteobacterial class (10 to 48% of total sequences), Gamma- and Deltaproteobacteria were subdominant. Cluster analysis showed that the mats’ bacterial communities at the different locations along the wadi were different and shared less than 60% of their operational taxonomic units (OTUs). Chlorophyll a and scytonemin were the most predominant pigments in all mats. Different saturated, branched and mono- and poly-unsaturated fatty acids were detected in all mats, with C16 and C18 compounds as most dominant. The detected pigments and fatty acids indicate a major role of cyanobacteria in the wadi mats and the adaptation of microorganisms therein to the harsh wadi environment. Detection of diadinoxanthin and fucoxanthin confirmed the presence of diatoms. We conclude that microbial mats are important elements in wadi ecosystems and exist in a great variety of structure and community composition.