Thomas G. Reichenauer

mRNA-Based Parallel Detection of Active Methanotroph Populations by Use of a Diagnostic Microarray

ABSTRACT A method was developed for the mRNA-based application of microbial diagnostic microarrays to detect active microbial populations. DNA- and mRNA-based analyses of environmental samples were compared and confirmed via quantitative PCR. Results indicated that mRNA-based microarray analyses may provide additional information on the composition and functioning of microbial communities.

Bacterial rhizosphere populations of black poplar and herbal plants to be used for phytoremediation of diesel fuel

Abstract Phytoremediation—an environmentally sound method to clean up moderate and superficial site contamination—is based on the stimulation of rhizosphere microorganisms by plant roots. Tolerance of plants to the contamination is a basic criterion for successful phytoremediation. Degradation may be accelerated by inoculation with appropriate microorganisms that are able to break down pollutants and to compete with indigenous microorganisms. An outdoor pot experiment, in which 16 black poplar clones and four herbal plant varieties were cultivated in soil contaminated with diesel fuel, was performed in order to identify plants with high tolerance to the pollution. The effect of plant variety and diesel contamination on rhizosphere bacterial communities was tested using a 16S rRNA-based cultivation-independent approach to characterize the rhizoflora. To obtain potential inoculant strains, diesel-degrading microbes were isolated from the rhizosphere and analyzed for their ability to utilize n-alkanes as carbon sources, the presence of genes encoding alkane hydroxylase (alkB) and their abundance in the rhizosphere. All plant varieties were strongly affected by the diesel contamination, however, black poplar clones exhibited higher sensitivities than herbal plants. One black poplar clone (Brandaris) showed significantly higher tolerance to diesel fuel contamination, whereas the growth of the remaining clones were severely inhibited. Analysis of rhizosphere communities indicated that the diesel contamination had a more pronounced effect than the plant variety. Fourteen diesel degrading strains were isolated from the rhizosphere and identified by 16S rRNA gene sequencing. Taking into account the presence of alk B genes, the ability to utilize alkanes as well as the abundance in the rhizosphere, three promising plant/microbe combinations were identified for future phytoremediation applications.

Soil type affects plant colonization, activity and catabolic gene expression of inoculated bacterial strains during phytoremediation of diesel.

The combined use of plants and associated microorganisms has great potential for cleaning up soils contaminated with petroleum hydrocarbons. Apart from environmental conditions the physicochemical properties of the soil are the main factors influencing the survival and activity of an inoculated strain as well as the growth of plants. This study examined the effect of different soil types (sandy, loamy sand and loam) on the survival, gene abundance and catabolic gene expression of two inoculated strains (Pseudomonas sp. strain ITRI53 and Pantoea sp. strain BTRH79) in the rhizosphere and shoot interior of Italian ryegrass vegetated in diesel contaminated soils. High colonization, gene abundance and expression in loamy soils were observed. By contrast, low colonization, gene abundance and absence of gene expression in sandy soil were found. The highest levels of genes expression and hydrocarbon degradation were seen in loamy soil that had been inoculated with BTRH79 and were significantly higher compared to those in other soils. A positive correlation was observed between gene expression and hydrocarbon degradation indicating that catabolic gene expression is necessary for contaminant degradation. These results suggest that soil type influences the bacterial colonization and microbial activities and subsequently the efficiency of contaminant degradation.

Hydrocarbon degradation, plant colonization and gene expression of alkane degradation genes by endophytic Enterobacter ludwigii strains

The genus Enterobacter comprises a range of beneficial plant-associated bacteria showing plant growth promotion. Enterobacter ludwigii belongs to the Enterobacter cloacae complex and has been reported to include human pathogens but also plant-associated strains with plant beneficial capacities. To assess the role of Enterobacter endophytes in hydrocarbon degradation, plant colonization, abundance and expression of CYP153 genes in different plant compartments, three plant species (Italian ryegrass, birdsfoot trefoil and alfalfa) were grown in sterile soil spiked with 1% diesel and inoculated with three endophytic E. ludwigii strains. Results showed that all strains were capable of hydrocarbon degradation and efficiently colonized the rhizosphere and plant interior. Two strains, ISI10-3 and BRI10-9, showed highest degradation rates of diesel fuel up to 68% and performed best in combination with Italian ryegrass and alfalfa. All strains expressed the CYP153 gene in all plant compartments, indicating an active role in degradation of diesel in association with plants.

Expression of alkane monooxygenase (alkB) genes by plant-associated bacteria in the rhizosphere and endosphere of Italian ryegrass (Lolium multiflorum L.) grown in diesel contaminated soil

For phytoremediation of organic contaminants, plants have to host an efficiently degrading microflora. To assess the role of endophytes in alkane degradation, Italian ryegrass was grown in sterile soil with 0, 1 or 2% diesel and inoculated either with an alkane degrading bacterial strain originally derived from the rhizosphere of Italian ryegrass or with an endophyte. We studied plant colonization of these strains as well as the abundance and expression of alkane monooxygenase (alkB) genes in the rhizosphere, shoot and root interior. Results showed that the endophyte strain better colonized the plant, particularly the plant interior, and also showed higher expression of alkB genes suggesting a more efficient degradation of the pollutant. Furthermore, plants inoculated with the endophyte were better able to grow in the presence of diesel. The rhizosphere strain colonized primarily the rhizosphere and showed low alkB gene expression in the plant interior.

Phylogenetic and functional diversity of alkane degrading bacteria associated with Italian ryegrass (Lolium multiflorum) and Birdsfoot trefoil (Lotus corniculatus) in a petroleum oil-contaminated environment

Twenty-six different plant species were analyzed regarding their performance in soil contaminated with petroleum oil. Two well-performing species, Italian ryegrass (Lolium multiflorum var. Taurus), Birdsfoot trefoil (Lotus corniculatus var. Leo) and the combination of these two plants were selected to study the ecology of plant-associated, culturable alkane-degrading bacteria. Hydrocarbon degrading bacteria were isolated from the rhizosphere, root interior and shoot interior and subjected to the analysis of 16S rRNA gene, the 16S and 23S rRNA intergenic spacer region and alkane hydroxylase genes. Furthermore, we investigated whether alkane hydroxylase genes are plasmid located. Higher numbers of culturable, alkane-degrading bacteria were associated with Italian ryegrass, which were also characterized by a higher diversity, particularly in the plant interior. Only half of the isolated bacteria hosted known alkane hydroxylase genes (alkB and cytochrome P153-like). Degradation genes were found both on plasmids as well as in the chromosome. In regard to application of plants for rhizodegradation, where support of numerous degrading bacteria is essential for efficient break-down of pollutants, Italian ryegrass seems to be more appropriate than Birdsfoot trefoil.

The Inoculation Method Affects Colonization and Performance of Bacterial Inoculant Strains in the Phytoremediation of Soil Contaminated with Diesel Oil

Plants in combination with microorganisms can remediate soils, which are contaminated with organic pollutants such as petroleum hydrocarbons. Inoculation of plants with degrading bacteria is one approach to improve remediation processes, but is often not successful due to the competition with resident microorganisms. It is therefore of high importance to address the persistence and colonization behavior of inoculant strains. The objective of this study was to determine whether the inoculation method (seed imbibement and soil inoculation) influences bacterial colonization, plant growth promotion and hydrocarbon degradation. Italian ryegrass was grown in non-sterilized soil polluted with diesel and inoculated with different alkane-degrading strains Pantoea sp. ITSI10, Pantoea sp. BTRH79 and Pseudomonas sp. MixRI75 individually as well as in combination. Inoculation generally had a beneficial effect on plant biomass production and hydrocarbon degradation, however, strains inoculated in soil performed better than applied by seed imbibement. Performance correlated with the colonization efficiency of the inoculated strains. The highest hydrocarbon degradation was observed in the treatment, in which all three strains were inoculated in combination into soil. Our study revealed that besides the degradation potential and competitive ability of inoculant strains the inoculation method plays an important role in determining the success of microbial inoculation.

Transcriptome analysis of nitrate assimilation in Aspergillus nidulans reveals connections to nitric oxide metabolism

Nitrate is a dominant form of inorganic nitrogen (N) in soils and can be efficiently assimilated by bacteria, fungi and plants. We studied here the transcriptome of the short‐term nitrate response using assimilating and non‐assimilating strains of the model ascomycete Aspergillus nidulans. Among the 72 genes positively responding to nitrate, only 18 genes carry binding sites for the pathway‐specific activator NirA. Forty‐five genes were repressed by nitrate metabolism. Because nirA‐ strains are N‐starved at nitrate induction conditions, we also compared the nitrate transcriptome with N‐deprived conditions and found a partial overlap of differentially regulated genes between these conditions. Nitric oxide (NO)‐metabolizing flavohaemoglobins were found to be co‐regulated with nitrate assimilatory genes. Subsequent molecular characterization revealed that the strongly inducible FhbA is required for full activity of nitrate and nitrite reductase enzymes. The co‐regulation of NO‐detoxifying and nitrate/nitrite assimilating systems may represent a conserved mechanism, which serves to neutralize nitrosative stress imposed by an external NO source in saprophytic and pathogenic fungi. Our analysis using membrane‐permeable NO donors suggests that signalling for NirA activation only indirectly depends on the nitrate transporters NrtA (CrnA) and NrtB (CrnB).

Free radicals generated during oxidation of green tea polyphenols: Electron paramagnetic resonance spectroscopy combined with density functional theory calculations

Electron paramagnetic resonance spectroscopy and density functional theory calculations have been used to investigate the redox properties of the green tea polyphenols (GTPs) (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), and (-)-epicatechin gallate (ECG). Aqueous extracts of green tea and these individual phenols were autoxidized at alkaline pH and oxidized by superoxide anion (O(2)(-)) radicals in dimethyl sulfoxide. Several new aspects of the free radical chemistry of GTPs were revealed. EGCG can be oxidized on both the B and the D ring. The B ring was the main oxidation site during autoxidation, but the D ring was the preferred site for O(2)(-) oxidation. Oxidation of the D ring was followed by structural degradation, leading to generation of a radical identical to that of oxidized gallic acid. Alkaline autoxidation of green tea extracts produced four radicals that were related to products of the oxidation of EGCG, EGC, ECG, and gallic acid, whereas the spectra from O(2)(-) oxidation could be explained solely by radicals generated from EGCG. Assignments of hyperfine coupling constants were made by DFT calculations, allowing the identities of the radicals observed to be confirmed.

Hydrocarbon degradation and plant colonization by selected bacterial strains isolated from Italian ryegrass and birdsfoot trefoil

Aims:  To assess the degradation potential and plant colonization capacity of four alkane‐degrading strains (ITSI10, ITRI15, ITRH76 and BTRH79) in combination with birdsfoot trefoil and Italian ryegrass and to evaluate the diversity of indigenous alkane‐degrading soil bacteria in the rhizo‐ and endosphere.