Isolation of microorganisms involved in reduction of crystalline iron(III) oxides in natural environments.

Reduction of crystalline Fe(III) oxides is one of the most important electron sinks for organic compound oxidation in natural environments. Yet the limited number of isolates makes it difficult to understand the physiology and ecological impact of the microorganisms involved. Here, two-stage cultivation was implemented to selectively enrich and isolate crystalline iron(III) oxide reducing microorganisms in soils and sediments. Firstly, iron reducers were enriched and other untargeted eutrophs were depleted by 2-years successive culture on a crystalline ferric iron oxide (i.e., goethite, lepidocrocite, hematite, or magnetite) as electron acceptor. Fifty-eight out of 136 incubation conditions allowed the continued existence of microorganisms as confirmed by PCR amplification. High-throughput Illumina sequencing and clone library analysis based on 16S rRNA genes revealed that the enrichment cultures on each of the ferric iron oxides contained bacteria belonging to the Deltaproteobacteria (mainly Geobacteraceae), followed by Firmicutes and Chloroflexi, which also comprised most of the operational taxonomic units (OTUs) identified. Venn diagrams indicated that the core OTUs enriched with all of the iron oxides were dominant in the Geobacteraceae while each type of iron oxides supplemented selectively enriched specific OTUs in the other phylogenetic groups. Secondly, 38 enrichment cultures including novel microorganisms were transferred to soluble-iron(III) containing media in order to stimulate the proliferation of the enriched iron reducers. Through extinction dilution-culture and single colony isolation, six strains within the Deltaproteobacteria were finally obtained; five strains belonged to the genus Geobacter and one strain to Pelobacter. The 16S rRNA genes of these isolates were 94.8-98.1% identical in sequence to cultured relatives. All the isolates were able to grow on acetate and ferric iron but their physiological characteristics differed considerably in terms of growth rate. Thus, the novel strategy allowed to enrich and isolate novel iron(III) reducers that were able to thrive by reducing crystalline ferric iron oxides.

Determinants of Acidobacteria activity inferred from the relative abundances of 16S rRNA transcripts in German grassland and forest soils.

16S rRNA genes and transcripts of Acidobacteria were investigated in 57 grassland and forest soils of three different geographic regions. Acidobacteria contributed 9-31% of bacterial 16S rRNA genes whereas the relative abundances of the respective transcripts were 4-16%. The specific cellular 16S rRNA content (determined as molar ratio of rRNA : rRNA genes) ranged between 3 and 80, indicating a low in situ growth rate. Correlations with flagellate numbers, vascular plant diversity and soil respiration suggest that biotic interactions are important determinants of Acidobacteria 16S rRNA transcript abundances in soils. While the phylogenetic composition of Acidobacteria differed significantly between grassland and forest soils, high throughput denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism fingerprinting detected 16S rRNA transcripts of most phylotypes in situ. Partial least squares regression suggested that chemical soil conditions such as pH, total nitrogen, C : N ratio, ammonia concentrations and total phosphorus affect the composition of this active fraction of Acidobacteria. Transcript abundance for individual Acidobacteria phylotypes was found to correlate with particular physicochemical (pH, temperature, nitrogen or phosphorus) and, most notably, biological parameters (respiration rates, abundances of ciliates or amoebae, vascular plant diversity), providing culture-independent evidence for a distinct niche specialization of different Acidobacteria even from the same subdivision.

Environmental factors affect Acidobacterial communities below the subgroup level in grassland and forest soils.

In soil, Acidobacteria constitute on average 20% of all bacteria, are highly diverse, and are physiologically active in situ. However, their individual functions and interactions with higher taxa in soil are still unknown. Here, potential effects of land use, soil properties, plant diversity, and soil nanofauna on acidobacterial community composition were studied by cultivation-independent methods in grassland and forest soils from three different regions in Germany. The analysis of 16S rRNA gene clone libraries representing all studied soils revealed that grassland soils were dominated by subgroup Gp6 and forest soils by subgroup Gp1 Acidobacteria. The analysis of a large number of sites (n = 57) by 16S rRNA gene fingerprinting methods (terminal restriction fragment length polymorphism [T-RFLP] and denaturing gradient gel electrophoresis [DGGE]) showed that Acidobacteria diversities differed between grassland and forest soils but also among the three different regions. Edaphic properties, such as pH, organic carbon, total nitrogen, C/N ratio, phosphorus, nitrate, ammonium, soil moisture, soil temperature, and soil respiration, had an impact on community composition as assessed by fingerprinting. However, interrelations with environmental parameters among subgroup terminal restriction fragments (T-RFs) differed significantly, e.g., different Gp1 T-RFs correlated positively or negatively with nitrogen content. Novel significant correlations of Acidobacteria subpopulations (i.e., individual populations within subgroups) with soil nanofauna and vascular plant diversity were revealed only by analysis of clone sequences. Thus, for detecting novel interrelations of environmental parameters with Acidobacteria, individual populations within subgroups have to be considered.