Gregory R. Crocetti

Identification of Polyphosphate-Accumulating Organisms and Design of 16S rRNA-Directed Probes for Their Detection and Quantitation

ABSTRACT Laboratory-scale sequencing batch reactors (SBRs) as models for activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Enrichment for polyphosphate-accumulating organisms (PAOs) was achieved essentially by increasing the phosphorus concentration in the influent to the SBRs. Fluorescence in situ hybridization (FISH) using domain-, division-, and subdivision-level probes was used to assess the proportions of microorganisms in the sludges. The A sludge, a high-performance P-removing sludge containing 15.1% P in the biomass, was comprised of large clusters of polyphosphate-containing coccobacilli. By FISH, >80% of the A sludge bacteria were β-2 Proteobacteria arranged in clusters of coccobacilli, strongly suggesting that this group contains a PAO responsible for EBPR. The second dominant group in the A sludge was the Actinobacteria. Clone libraries of PCR-amplified bacterial 16S rRNA genes from three high-performance P-removing sludges were prepared, and clones belonging to the β-2 Proteobacteria were fully sequenced. A distinctive group of clones (sharing ≥98% sequence identity) related to Rhodocyclus spp. (94 to 97% identity) andPropionibacter pelophilus (95 to 96% identity) was identified as the most likely candidate PAOs. Three probes specific for the highly related candidate PAO group were designed from the sequence data. All three probes specifically bound to the morphologically distinctive clusters of PAOs in the A sludge, exactly coinciding with the β-2 Proteobacteria probe. Sequential FISH and polyphosphate staining of EBPR sludges clearly demonstrated that PAO probe-binding cells contained polyphosphate. Subsequent PAO probe analyses of a number of sludges with various P removal capacities indicated a strong positive correlation between P removal from the wastewater as determined by sludge P content and number of PAO probe-binding cells. We conclude therefore that an important group of PAOs in EBPR sludges are bacteria closely related toRhodocyclus and Propionibacter.

A review and update of the microbiology of enhanced biological phosphorus removal in wastewater treatment plants

Enhanced biological phosphorus removal (EBPR) from wastewater can be more-or-less practically achieved but the microbiological and biochemical components are not completely understood. EBPR involves cycling microbial biomass and influent wastewater through anaerobic and aerobic zones to achieve a selection of microorganisms with high capacity to accumulate polyphosphate intracellularly in the aerobic period. Biochemical or metabolic modelling of the process has been used to explain the types of carbon and phosphorus transformations in sludge biomass. There are essentially two broad-groupings of microorganisms involved in EBPR. They are polyphosphate accumulating organisms (PAOs) and their supposed carbon-competitors called glycogen accumulating organisms (GAOs). The morphological appearance of microorganisms in EBPR sludges has attracted attention. For example, GAOs as tetrad-arranged cocci and clusters of coccobacillus-shaped PAOs have been much commented upon and the use of simple cellular staining methods has contributed to EBPR knowledge. Acinetobacter and other bacteria were regularly isolated in pure culture from EBPR sludges and were initially thought to be PAOs. However, when contemporary molecular microbial ecology methods in concert with detailed process performance data and simple intracellular polymer staining methods were used, a betaproteobacteria called ‘Candidatus Accumulibacter phosphatis’ was confirmed as a PAO and organisms from a novel gammaproteobacteria lineage were GAOs. To preclude making the mistakes of previous researchers, it is recommended that the sludge ‘biography’ be well understood – i.e. details of phenotype (process performance and biochemistry) and microbial community structure should be linked.

Variability and abundance of the epiphytic bacterial community associated with a green marine Ulvacean alga.

Marine Ulvacean algae are colonized by dense microbial communities predicted to have an important role in the development, defense and metabolic activities of the plant. Here we assess the diversity and seasonal dynamics of the bacterial community of the model alga Ulva australis to identify key groups within this epiphytic community. A total of 48 algal samples of U. australis that were collected as 12 individuals at 3 monthly intervals, were processed by applying denaturing gradient gel electrophoresis (DGGE), and three samples from each season were subjected to catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). CARD-FISH revealed that the epiphytic microbial community was comprised mainly of bacterial cells (90%) and was dominated by the groups Alphaproteobacteria (70%) and Bacteroidetes (13%). A large portion (47%) of sequences from the Alphaproteobacteria fall within the Roseobacter clade throughout the different seasons, and an average relative proportion of 19% was observed using CARD-FISH. DGGE based spatial (between tidal pools) and temporal (between season) comparisons of bacterial community composition demonstrated that variation occurs. Between individuals from both the same and different tidal pools, the variation was highest during winter (30%) and between seasons a 40% variation was observed. The community also includes a sub-population of bacteria that is consistently present. Sequences from excised DGGE bands indicate that members of the Alphaproteobacteria and the Bacteroidetes are part of this stable sub-population, and are likely to have an important role in the function of this marine epiphytic microbial community.

Structure analysis and performance of a microbial community from a contaminated aquifer involved in the complete reductive dechlorination of 1,1,2,2-tetrachloroethane to ethene.

An anaerobic microcosm set up with aquifer material from a 1,1,2,2‐tetrachloroethane (TeCA) contaminated site and amended with butyrate showed a complete TeCA dechlorination to ethene. A structure analysis of the microbial community was performed by fluorescence in situ hybridization (FISH) with already available and on purpose designed probes from sequences retrieved through 16S rDNA clone library construction. FISH was chosen as identification tool to evaluate in situ whether the retrieved sequences belong to primary bacteria responsible for the biodegradative reactions. FISH probes identified up to 80% of total bacteria and revealed the absence or the marginal presence of known TeCA degraders and the abundance of two well‐known H2‐utilizing halorespiring bacteria, Sulfurospirillum (32.4 ± 8.6% of total bacteria) and Dehalococcoides spp. (14.8 ± 2.8), thereby providing a strong indication of their involvement in the dechlorination processes. These results were supported by the kinetic and thermodynamic analysis which provided indications that hydrogen was the actual electron donor for TeCA dechlorination. The specific probes, developed in this study, for known dechlorinators (i.e., Geobacter, Dehalobacter, and Sulfurospirillum species) represent a valuable tool for any future in situ bioremediation study as well as a quick and specific investigation tool for tracking their distribution in the field. Biotechnol. Bioeng. 2008;99: 240–249.

Visualization of Helicobacter Species Within the Murine Cecal Mucosa Using Specific Fluorescence In Situ Hybridization

Background.  Members of the genus Helicobacter have been associated with colitis development in a number of immunodeficient animal models. While it is known that these organisms can initiate colitis development, the location and spatial distribution of these bacteria within the intestinal tract is currently unknown. In this study, we developed and optimized fluorescence in situ hybridization (FISH) probes specifically for Helicobacter species.