Application of COMPOCHIP Microarray to Investigate the Bacterial Communities of Different Composts

A microarray spotted with 369 different 16S rRNA gene probes specific to microorganisms involved in the degradation process of organic waste during composting was developed. The microarray was tested with pure cultures, and of the 30,258 individual probe-target hybridization reactions performed, there were only 188 false positive (0.62%) and 22 false negative signals (0.07%). Labeled target DNA was prepared by polymerase chain reaction amplification of 16S rRNA genes using a Cy5-labeled universal bacterial forward primer and a universal reverse primer. The COMPOCHIP microarray was applied to three different compost types (green compost, manure mix compost, and anaerobic digestate compost) of different maturity (2, 8, and 16 weeks), and differences in the microorganisms in the three compost types and maturity stages were observed. Multivariate analysis showed that the bacterial composition of the three composts was different at the beginning of the composting process and became more similar upon maturation. Certain probes (targeting Sphingobacterium, Actinomyces, Xylella/Xanthomonas/ Stenotrophomonas, Microbacterium, Verrucomicrobia, Planctomycetes, Low G + C and Alphaproteobacteria) were more influential in discriminating between different composts. Results from denaturing gradient gel electrophoresis supported those of microarray analysis. This study showed that the COMPOCHIP array is a suitable tool to study bacterial communities in composts

Enumeration and detection of anaerobic ferrous iron-oxidizing, nitrate-reducing bacteria from diverse European sediments

Anaerobic, nitrate-dependent microbial oxidation of ferrous iron was recently recognized as a new type of metabolism, In order to study the occurrence of three novel groups of ferrous iron-oxidizing, nitrate-reducing bacteria (represented by strains BrG1, BrG2, and BrG3), 16S rRNA-targeted oligonucleotide probes mere developed, In pure-culture experiments, these probes were shown to be suitable for fluorescent in situ hybridization, as well as for hybridization analysis of denaturing gradient gel electrophoresis (DGGE) patterns. However, neither enumeration by in situ hybridization nor detection by the DGGE-hybridization approach aas feasible with sediment samples, Therefore, the DGGE-hybridization approach was combined with microbiological methods. Freshwater sediment samples from different European locations were used for enrichment cultures and most-probable-number (MPN) determinations, Bacteria with the ability to oxidize ferrous iron under nitrate-reducing conditions were detected in all of the sediment samples investigated, At least one of the previously described types of bacteria was detected in each enrichment culture. MPN studies showed that sediments contained from 1 x 10(5) to 5 x 10(8) ferrous iron-oxidizing, nitrate-reducing bacteria per g (dry weight) of sediment, which accounted for at most 0.8% of the nitrate-reducing bacteria growing with acetate, Type BrG1, BrG2, and BrG3 bacteria accounted for an even smaller fraction (0.2% or less) of the ferrous iron-oxidizing, nitrate-reducing community, The DGGE patterns of MPN cultures suggested that more organisms than those isolated thus far are able to oxidize ferrous iron with nitrate, A comparison showed that among the anoxygenic phototrophic bacteria, organisms that have the ability to oxidize ferrous iron also account for only a minor fraction of the population

Screening for genetic diversity of isolates of anaerobic Fe(II)-oxidizing bacteria using DGGE and whole-cell hybridization

Nitrate-reducing bacteria, which grow anaerobically with Fe(II) as electron donor, were isolated from freshwater mud samples. Since extensive phylogenetic and physiological characterization of multiple strains is very time-consuming and labour-intensive, the isolates were first screened for genetic diversity by denaturing gradient gel electrophoresis (DGGE) and whole-cell hybridization. DGGE analysis of 16S rDNA fragments amplified from 12 strains indicated that three different types of bacteria had been independently isolated (type A-C). Whole-cell hybridization with domain- and group-specific oligonucleotide probes suggested that the type-A and -C isolates were members of the beta-subdivision of the Proteobacteria. The type-B isolates hybridized only with the bacterial probe but not with any of the probes specific for the alpha-, beta- or gamma-Proteobacteria. Based on these results representative strains of each type were chosen for further phylogenetic characterization using 16S rDNA sequencing. This analysis confirmed that the type-A and -C isolates were members of the beta-Proteobacteria. The type-B isolate was shown to be a member of the Xanthomonas group of the gamma-Proteobacteria. Our results demonstrate that probe GAM42a (specific for gamma-Proteobacteria; MANZ et al., 1992) does not hybridize to the 23S rRNA target sequence of this group of deep branching gamma-Proteobacteria: this was confirmed by hybridization experiments with Xanthomonas fragariae, which also failed to hybridize to this probe. 23S rDNA sequence analysis revealed that probe GAM42a has one mismatch with the target sequence of the type-B isolate and two mismatches with the target sequence of X. fragariae; Furthermore it was shown that double stranded DNA fragments of 626 bp length, which differed by as many as two to three nucleotides were not separated by DGGE. This suggested that rDNA fragments of closely related bacteria (99.8% sequence similarity or more) are not resolved by DGGE. We propose that the combined use of DGGE and whole-cell hybridization provides a rapid way to distinguish distantly related microbial isolates

The use of biologically produced ferrihydrite for the isolation of novel iron-reducing bacteria

Ferric iron was produced anaerobically from ferrous iron through the metabolic activity of recently described ferrous iron-oxidizing, nitrate-reducing bacteria. It was identified as poorly crystallized 2-line ferrihydrite with a particle size of 1-2 nm. This biologically produced ferrihydrite was shown to be a suitable electron acceptor for dissimilatory ferric iron-reducing bacteria in freshwater enrichment cultures, and was completely reduced to the ferrous state; no magnetite formation occurred. Geobacter metallireducens was also able to completely reduce the biologically produced ferrihydrite. These results indicate the possibility of an anaerobic, microbial cycling of iron. Using the biologically produced ferric iron, two isolates of obligately anaerobic, dissimilatory ferric iron-reducing bacteria, strains Dfr1 and Dfr2, were obtained from freshwater enrichment cultures. Analysis of 16S rRNA gene sequences revealed an affiliation with the Geobacter cluster within the family Geobacteraceae. The sequence similarity between strains Dfr1 and Dfr2 is 92.5%. The closest relative of strain Dfr1 is Geobacter sulfurreducens with 92.9%, and of strain Dfr2 Geobacter chapelleii with 93.7% sequence similarity In addition, strains Dfr1 and Dfr2 are both able to grow by dissimilatory reduction of Mn(IV), S degrees, and fumarate. Furthermore, strain Dfr2 is able to reduce akaganeite (beta-FeOOH), a more crystallized type of ferric iron oxide

Anoxic aggregates - An ephemeral phenomenon in the pelagic environment?

Radial microscale distributions of oxygen and pH were studied in ca 1.5 mm large laboratory-made aggregates composed of phytoplankton detritus and fecal pellets. Microsensor measurements were done at spatial increments down to 0.05 mm in a vertical flow system in which the individual aggregates stabilized their position in the water phase according to the upward flow velocity. The aggregates were surrounded by a diffusive boundary layer with steep gradients of oxygen and pH. They were highly heterotrophic communities both under natural light conditions and in darkness. pH was lowered from 8.2 in the surrounding water to 7.4 in the center of an anoxic aggregate. Sulfide was not detectable by use of sulfide microelectrodes in anoxic aggregates, and methanogenic bacteria could not be detected after PCR (polymerase chain reaction) amplification using archaebacterial-specific primers. The oxygen respiration rate decreased exponentially over time with a T1/2 of 2.3 d. Theoretical calculations of the volumetric oxygen respiration rate needed to deplete oxygen inside aggregates was compared to the density of organic matter in natural marine aggregates. These calculations showed that carbon limitation of heterotrophic processes would limit anoxic conditions to occurring only over a few hours, depending on the size of the aggregates. Therefore slow-growing obligate anaerobic microorganisms such as sulfate reducing bacteria and methanogenic bacteria may be limited by the relatively short persistence of anoxia in marine aggregates

Diversity of ferrous iron-oxidizing, nitrate-reducing bacteria and their involvement in oxygen-independent iron cycling

In previous studies, three different strains (BrG1, BrG2, and BrG3) of ferrous iron-oxidizing, nitrate-reducing bacteria were obtained fromfreshwater sediments. All three strains were facultative anaerobes and utilized a variety of organic substrates and molecular hydrogen with nitrate as electron acceptor. In this study, analyses of 16S rDNA sequences showed that strain BrG1 was affiliated with the genus Acidovorax, strain BrG2 with the genus Aquabacterium, and strain BrG3 with the genus Thermomonas. Previously, bacteria similar to these three strains were detected with molecular techniques in MPN dilution series for ferrous iron-oxidizing, nitrate-reducing bacteria inoculated with different freshwater sediment samples. In the present study, further molecular analyses of theseMPNcultures indicated that the ability to oxidize ferrous iron with nitrate is widespread amongst the Proteobacteria and may also be found among the Gram-positive bacteria with high GC content of DNA. Nitrate-reducing bacteria oxidized ferrous iron to poorly crystallized ferrihydrite that was suitable as an electron acceptor for ferric iron-reducing bacteria. Biologically produced ferrihydrite and synthetically produced ferrihydrite were both well suited as electron acceptors inMPNdilution cultures. Repeated anaerobic cycling of iron was shown in a coculture of ferrous iron-oxidizing bacteria and the ferric iron-reducing bacterium Geobacter bremensis. The results indicate that iron can be cycled between its oxidation states +II and +III by microbial activities in anoxic sediments