Bioaugmentation with Pseudomonas sp. strain MHP41 promotes simazine attenuation and bacterial community changes in agricultural soils

Bioremediation is an important technology for the removal of persistent organic pollutants from the environment. Bioaugmentation with the encapsulated Pseudomonas sp. strain MHP41 of agricultural soils contaminated with the herbicide simazine was studied. The experiments were performed in microcosm trials using two soils: soil that had never been previously exposed to s -triazines (NS) and soil that had >20 years of s -triazine application (AS). The efficiency of the bioremediation process was assessed by monitoring simazine removal by HPLC. The simazine-degrading microbiota was estimated using an indicator for respiration combined with most-probable-number enumeration. The soil bacterial community structures and the effect of bioaugmentation on these communities were determined using 16S RNA gene clone libraries and FISH analysis. Bioaugmentation with MHP41 cells enhanced simazine degradation and increased the number of simazine-degrading microorganisms in the two soils. In highly contaminated NS soil, bioaugmentation with strain MHP41 was essential for simazine removal. Comparative analysis of 16S rRNA gene clone libraries from NS and AS soils revealed high bacterial diversity. Bioaugmentation with strain MHP41 promoted soil bacterial community shifts. FISH analysis revealed that bioaugmentation increased the relative abundances of two phylogenetic groups ( Acidobacteria and Planctomycetes ) in both soils. Although members of the Archaea were metabolically active in these soils, their relative abundance was not altered by bioaugmentation.     

Determination of the Diversity of Rhodopirellula Isolates from European Seas by Multilocus Sequence Analysis

In the biogeography of microorganisms, the habitat size of an attached-living bacterium has never been investigated. We approached this theme with a multilocus sequence analysis (MLSA) study of new strains of Rhodopirellula sp., an attached-living planctomycete. The development of an MLSA for Rhodopirellula baltica enabled the characterization of the genetic diversity at the species level, beyond the resolution of the 16S rRNA gene. The alleles of the nine housekeeping genes acsA, guaA, trpE, purH, glpF, fumC, icd, glyA, and mdh indicated the presence of 13 genetically defined operational taxonomic units (OTUs) in our culture collection. The MLSA-based OTUs coincided with the taxonomic units defined by DNA-DNA hybridization experiments. BOX-PCR supported the MLSA-based differentiation of two closely related OTUs. This study established a taxon-area relationship of cultivable Rhodopirellula species. In European seas, three closely related species covered the Baltic Sea and the eastern North Sea, the North Atlantic region, and the southern North Sea to the Mediterranean. The last had regional genotypes, as revealed by BOX-PCR. This suggests a limited habitat size of attached-living Rhodopirellula species.The biogeography of microorganisms describes the habitat size of the species and the distribution of microorganisms on Earth. The experimental approaches depend on the focus of the studies. Habitats are often analyzed by environmental microbiologists with genetic-fingerprinting techniques, with up to 200 bands or fragments representing the whole community. Although the taxonomic resolution of these operational taxonomic units (OTUs) is limited, the studies revealed a community biogeography (22). Medical microbiologists analyze the alleles of housekeeping genes of microorganisms to gain insight into the epidemiology of pathogens, the population biogeography (2). This strain-specific, fine-scale taxonomic resolution within a species is well suited to observance of recent dispersal events. At the species level, multilocus sequence typing (MLST) and analysis (MLSA), which were developed for intraspecies and intragenus specific studies, respectively, consist of the sequences of several (at least seven) housekeeping gene fragments concatenated to an approximately 5-kilobase alignment (17). Recent MLSA studies revealed its applicability to marine isolates and the analysis of biogeographic patterns: Alteromonas macleodii isolates could be grouped in an epipelagic and an abyssal clade (6), and strains of Pseudomonas aeruginosa were genetically well separated into groups of coastal and oceanic origin (8). However, for Salinibacter ruber strains, biogeographical distinctness was not resolved in an MLSA study but showed allopatry in a metabolic analysis (31). Several studies used MLSA together with DNA-DNA hybridization (DDH) for the delineation of new species, e.g., for Vibrio and Ensifer spp. (20, 36).In the biogeography of microorganisms, the experimental proof of a local genetic evolution was first revealed at sample sites that were physically separated by over 18,000 km (39). Large populations and the small size of microbes have been considered as facilitators for dispersal over long distances, eventually establishing cosmopolitan microbial populations. On the other hand, the smallest spatial scale of a microbial species in an open system has not been investigated. Attached-living bacteria disperse only during a distinct, short time span in their lives. This limitation of the dispersal time stimulated this study of the biogeography of Rhodopirellula baltica in European seas.R. baltica is a planctomycete with typical morphological features. The peptidoglycanless bacteria have an intracellular compartmentation: the riboplasm with the nucleoid is separated by a membrane from the surrounding paryphoplasm. Cells attach with a holdfast substance to surfaces or, in culture, to themselves, forming typical rosettes. Proliferation occurs by budding, and offspring cells live free in the water column: they are motile with a flagellum until they settle on the sediment (4).Seventy recently isolated strains affiliated according to the 16S rRNA gene analysis with R. baltica SH1^T as the closest validly described species (40). The 16S rRNA gene sequences do not offer sufficient information at the species level. A dissimilarity of the 16S rRNA genes of more than 3%, recently reduced to 1.3% (34, 35), indicates that the strains under consideration belong to two species. These thresholds yielded in our strain collection, according to an ARB-based calculation, five or eight operational taxonomic units besides the species R. baltica (40). For strains with highly identical sequences, whole-genome DDH experiments have to be performed to identify the affiliation to established species. Recently, multilocus sequence analyses have emerged as a possible alternative method. Our strain collection comprised many strains with a 16S rRNA gene sequence very closely related to that of R. baltica SH1^T. To gain insight into the genetic identity of the isolates on the species level and the habitat sizes of the species, we developed a multilocus sequence analysis and applied it to the strain collection. The MLSA results were calibrated with a DDH study. The closely related strains were additionally characterized by BOX-PCR, a fingerprinting method (15). Transmission electron microscopy (EM) was performed on some isolates to support the identification as Planctomycetes and to visualize morphological differences between strains.     

Phylogenetic position of Salinibacter ruber based on concatenated protein alignments

A total of 22 genes from the genome of Salinibacter ruber strain M31 were selected in order to study the phylogenetic position of this species based on protein alignments. The selection of the genes was based on their essential function for the organism, dispersion within the genome, and sufficient informative length of the final alignment. For each gene, an individual phylogenetic analysis was performed and compared with the resulting tree based on the concatenation of the 22 genes, which rendered a single alignment of 10,757 homologous positions. In addition to the manually chosen genes, an automatically selected data set of 74 orthologous genes was used to reconstruct a tree based on 17,149 homologous positions. Although single genes supported different topologies, the tree topology of both concatenated data sets was shown to be identical to that previously observed based on small subunit (SSU) rRNA gene analysis, in which S. ruber was placed together with Bacteroidetes. In both concatenated data sets the bootstrap was very high, but an analysis with a gradually lower number of genes indicated that the bootstrap was greatly reduced with less than 12 genes. The results indicate that tree reconstructions based on concatenating large numbers of protein coding genes seem to produce tree topologies with similar resolution to that of the single 16S rRNA gene trees. For classification purposes, 16S rRNA gene analysis may remain as the most pragmatic approach to infer genealogic relationships.     

Occurrence of rhizobia in the gut of the higher termite Nasutitermes nigriceps

Wood-eating termites feed on a diet highly deficient in nitrogen. They must complement their diet with the aid of nitrogen-fixing bacteria. Nitrogen fixation in the gut has been demonstrated, but information about nitrogen-fixing bacteria in pure culture is scarce. From the higher termite Nasutitermes nigriceps the symbiotic bacterial strain M3A was isolated, which thrives in the hindgut contents. The Gram-negative strain exhibited similarities to the species of the genus Ensifer (including Sinorhizobium) on the basis of morphological and physiological/biochemical features. The 16S rRNA gene analysis showed the highest sequence similarity of the isolate M3A to Ensifer adhaerens (>99%; ATCC 33499). The DNA-DNA hybridization revealed a similarity of 66% with E. adhaerens (NCIMB12342(T)). In contrast to the type strain the isolate M3A possesses the capacity to nodulate plant roots. This is the first report on the detailed identification of a rhizobia-related strain from the intestinal tract of animals. Strain M3A has been deposited with two culture collections (DSM10169; ATCC BAA-396).     

Microbial Diversity in Maras Salterns, a Hypersaline Environment in the Peruvian Andes

Maras salterns are located 3,380 m above sea level in the Peruvian Andes. These salterns consist of more than 3,000 little ponds which are not interconnected and act as crystallizers where salt precipitates. These ponds are fed by hypersaline spring water rich in sodium and chloride. The microbiota inhabiting these salterns was examined by fluorescence in situ hybridization (FISH), 16S rRNA gene clone library analysis, and cultivation techniques. The total counts per milliliter in the ponds were around 2 × 10⁶ to 3 × 10⁶ cells/ml, while the spring water contained less than 100 cells/ml and did not yield any detectable FISH signal. The microbiota inhabiting the ponds was dominated (80 to 86% of the total counts) by Archaea, while Bacteria accounted for 10 to 13% of the 4′,6′-diamidino-2-phenylindole (DAPI) counts. A total of 239 16S rRNA gene clones were analyzed (132 Archaea clones and 107 Bacteria clones). According to the clone libraries, the archaeal assemblage was dominated by microorganisms related to the cosmopolitan square archaeon “Haloquadra walsbyi,” although a substantial number of the sequences in the libraries (31% of the 16S rRNA gene archaeal clones) were related to Halobacterium sp., which is not normally found in clone libraries from solar salterns. All the bacterial clones were closely related to each other and to the γ-proteobacterium “Pseudomonas halophila” DSM 3050. FISH analysis with a probe specific for this bacterial assemblage revealed that it accounted for 69 to 76% of the total bacterial counts detected with a Bacteria-specific probe. When pond water was used to inoculate solid media containing 25% total salts, both extremely halophilic Archaea and Bacteria were isolated. Archaeal isolates were not related to the isolates in clone libraries, although several bacterial isolates were very closely related to the “P. halophila” cluster found in the libraries. As observed for other hypersaline environments, extremely halophilic bacteria that had ecological relevance seemed to be easier to culture than their archaeal counterparts.     

The response of the microbial community of marine sediments to organic carbon input under anaerobic conditions.

Cyanobacterial biomass was added to anaerobic sediment to simulate the natural input of complex organic substrate that occurs in nature after algae blooms. Sediments were incubated at 0 degree C, 8 degrees C and 24 degrees C for 13 days. Community dynamics were measured by fluorescence in situ hybridisation (FISH), denaturing gradient gel electrophoresis (DGGE), and sequencing of 16S rDNA PCR products. Metabolic changes were followed by the analysis of total carbon mineralisation, sulfate reduction, and ammonium production rates. The addition of organic material resulted in significant changes in the composition of the microbial community at all temperatures tested. Sulfate reduction was the main mineralisation process detected. However, not sulfate-reducers but rather members of the Cytophaga-Flavobacterium phylogenetic cluster showed the highest increase in the bacterial cells as detected by FISH. We conclude that these organisms play an important role in the anaerobic decomposition of complex organic material perhaps because they are the main catalysts of macromolecule hydrolysis and fermentation. The molecular methods also indicated a stimulation of ribosome synthesis. The detection of a large number of rRNA-rich cells belonging to the Cytophaga-Flavobacterium phylogenetic cluster further supports the importance of their role in the degradation of complex organic material in anaerobic marine sediments. Their detection in high numbers in the field may indicate recent deposition events.     

Coexistence of Two Distinct Copies of Naphthalene Degradation Genes in Pseudomonas Strains Isolated from the Western Mediterranean Region

We analyzed the occurrence of the naphthalene degradation upper-pathway (nah) genes in the western Mediterranean region. The amplification, restriction, and sequence analysis of internal fragments for several nah genes (nahAc, nahB, nahC, and nahE) from naphthalene-degrading strains isolated from this geographical area proved the coexistence of two distinct sets of nah genes.     

Phototrophic utilization of toluene under anoxic conditions by a new strain of Blastochloris sulfoviridis

The capacity of anoxygenic phototrophic bacteria to utilize aromatic hydrocarbons was investigated in enrichment cultures with toluene. When mineral medium with toluene (provided in an inert carrier phase) was inoculated with activated sludge and incubated under infrared illumination (> 750 nm), a red-to-brownish culture developed. Agar dilution series indicated the dominance of two types of phototrophic bacteria. One type formed red colonies, had rod-shaped cells with budding division, and grew on benzoate but not on toluene. The other type formed yellow-to-brown colonies, had oval cells, and utilized toluene and benzoate. One strain of the latter type, ToP1, was studied in detail. Sequence analysis of the 16S rRNA gene and DNA-DNA hybridization indicated an affiliation of strain ToP1 with the species Blastochloris sulfoviridis, a member of the α-subclass of Proteobacteria. However, the type strain (DSM 729) of Blc. sulfoviridis grew neither on toluene nor on benzoate. Light-dependent consumption of toluene in the presence of carbon dioxide and formation of cell mass by strain ToP1 were demonstrated in quantitative growth experiments. Strain ToP1 is the first phototrophic bacterium shown to utilize an aromatic hydrocarbon. In the supernatant of toluene-grown cultures and in cell-free extracts incubated with toluene and fumarate, the formation of benzylsuccinate was detected. These findings indicate that the phototrophic bacterium activates toluene anaerobically by the same mechanism that has been reported for denitrifying and sulfate-reducing bacteria. The natural abundance of phototrophic bacteria with the capacity for toluene utilization was examined in freshwater habitats. Counting series revealed that up to around 1% (1.8 × 10⁵ cells per gram dry mass of sample) of the photoheterotrophic population cultivable with acetate grew on toluene. Received: 29 March 1999 / Accepted: 1 July 1999     

Microbial colonization in the seagrass Posidonia spp. roots

The pattern of colonization by microorganisms on root surfaces from three species of seagrass belonging to the genus Posidonia was assessed. Microbial abundance on roots was measured by two electronic microscope techniques. Trends in microbial colonization between species and root order were defined. In addition, eutrophication status of the sampling sites and physiological status of Posidonia oceanica (L.) Delile roots have been taken into account. Our results show high microbial abundance in the Mediterranean species P. oceanica, in comparison with the low rates of colonization found in the Australian species P. australis Hook f. and P. sinuosa Cambridge et Kuo. Microbial density tended to decrease as root order increased, and living roots always showed higher microbial abundance than dead ones. Colonization of P. oceanica roots at the three sites with different environmental status follows different trends according to root order. It is suggested that root age influences the rate of microbial colonization of seagrass roots and that colonization of root surface by microorganisms is associated with organic exudates from the roots rather than with decaying root tissues.