Juerg Keller

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.

Microbial ecology meets electrochemistry: electricity-driven and driving communities

Bio-electrochemical systems (BESs) have recently emerged as an exciting technology. In a BES, bacteria interact with electrodes using electrons, which are either removed or supplied through an electrical circuit. The most-described type of BES is microbial fuel cells (MFCs), in which useful power is generated from electron donors as, for example, present in wastewater. This form of charge transport, known as extracellular electron transfer, was previously extensively described with respect to metals such as iron and manganese. The importance of these interactions in global biogeochemical cycles is essentially undisputed. A wide variety of bacteria can participate in extracellular electron transfer, and this phenomenon is far more widespread than previously thought. The use of BESs in diverse research projects is helping elucidate the mechanism by which bacteria shuttle electrons externally. New forms of interactions between bacteria have been discovered demonstrating how multiple populations within microbial communities can co-operate to achieve energy generation. New environmental processes that were difficult to observe or study previously can now be simulated and improved via BESs. Whereas pure culture studies make up the majority of the studies performed thus far, even greater contributions of BESs are expected to occur in natural environments and with mixed microbial communities. Owing to their versatility, unmatched level of control and capacity to sustain novel processes, BESs might well serve as the foundation of a new environmental biotechnology. While highlighting some of the major breakthroughs and addressing only recently obtained data, this review points out that despite rapid progress, many questions remain unanswered.

Use of Stable-Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescence In Situ Hybridization-Microautoradiography To Study a Methanol-Fed Denitrifying Microbial Community

ABSTRACT A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO3−-N mg of mixed-liquor volatile suspended solids (MLVSS)−1 h−1 to a steady-state value of 0.06 mg of NO3−-N mg of MLVSS−1 h−1 over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [13C]methanol to biomark the DNA of the denitrifiers. The extracted [13C]DNA and [12C]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [13C]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [12C]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [14C]methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges.

Cathodic oxygen reduction catalyzed by bacteria in microbial fuel cells.

Microbial fuel cells (MFCs) have the potential to combine wastewater treatment efficiency with energetic efficiency. One of the major impediments to MFC implementation is the operation of the cathode compartment, as it employs environmentally unfriendly catalysts such as platinum. As recently shown, bacteria can facilitate sustainable and cost-effective cathode catalysis for nitrate and also oxygen. Here we describe a carbon cathode open to the air, on which attached bacteria catalyzed oxygen reduction. The bacteria present were able to reduce oxygen as the ultimate electron acceptor using electrons provided by the solid-phase cathode. Current densities of up to 2.2 A m−2 cathode projected surface were obtained (0.303±0.017 W m−2, 15 W m−3 total reactor volume). The cathodic microbial community was dominated by Sphingobacterium, Acinetobacter and Acidovorax sp., according to 16S rRNA gene clone library analysis. Isolates of Sphingobacterium sp. and Acinetobacter sp. were obtained using H2/O2 mixtures. Some of the pure culture isolates obtained from the cathode showed an increase in the power output of up to three-fold compared to a non-inoculated control, that is, from 0.015±0.001 to 0.049±0.025 W m−2 cathode projected surface. The strong decrease in activation losses indicates that bacteria function as true catalysts for oxygen reduction. Owing to the high overpotential for non-catalyzed reduction, oxygen is only to a limited extent competitive toward the electron donor, that is, the cathode. Further research to refine the operational parameters and increase the current density by modifying the electrode surface and elucidating the bacterial metabolism is warranted.

Biological nutrient removal efficiency in treatment of saline wastewater

There is very little known about the effectiveness of wastewater treatment systems for saline wastewater generated by seafood processing industries, aquaculture and tourism activities. In particular, the effect of salinity on nitrogen and phosphorus removal in wastewater treatment processes is not well understood. Therefore we devised experiments to examine the treatment of highly saline wastewater, by using artificial seafood processing wastewater, for removal of nitrogen and phosphorus. Lab scale sequencing batch reactors (SBR) were initially operated at low, and then at increasing salt levels, to determine the overall effects of salinity on the nutrient removal performance. The microbial populations during these experiments were monitored to determine the specific effect of salinity on the various bacterial groups responsible for nutrient removal. The methods used were whole cell probing with fluorescently labelled RNA-directed oligonucleotide probes. Experimental data showed that the SBRs achieved good biological nutrient removal (BNR) when salinity levels in the influent were low (0.03% to 0.2% NaCl) but showed difficulties with biological phosphorus removal at salinity levels of 0.5%. It was found that there was a dominance of Gram-positive bacteria with a high mol% G+C in their DNA in the SBR treating wastewater with NaCl at 0.03% to 0.2%. The addition of acetate to improve BNR performance increased the proportion of bacteria from the beta Proteobacterial subclass.

Computational fluid dynamic modelling of wastewater ponds to improve design

Wastewater ponds are a popular treatment alternative in Australia, especially in the meat industry. However, increasingly stringent Australian environmental legislation is raising questions about the continued viability of ponds. Traditional design methods do not address the hydrodynamic problems (i.e. short-circuiting) nor can they predict the effects of measures like baffles or repositioning inlets or outlets to improve performance. This is because the microscale interactions between the fluid and solids, and the biological reactions are ignored. This paper presents a tool — computational fluid dynamic (CFD) modelling and explores its potential as a new design tool for wastewater ponds. FIDAP, a finite element CFD program, is one of the new generation of commercial CFD packages available. This program has been used to qualitatively investigate the hydrodynamics of four pond systems. These models are limited to 2-dimensional (D), steady-state simulations in a laminar flow regime. They form the first step in the process to address the microscale fluid flow, mixing and biology in wastewater ponds. Considerably more modelling and validation work is yet to be done.

Bio-P and non-bio-P bacteria identification by a novel microbial approach

Culturing bacteria from activated sludge with enhanced biological phosphorus removal (EBPR) has strongly implicated Acinetobacter with the process. However, using fluorescent in-situ hybridisation (FISH) probing to analyse microbial populations, we have shown evidence opposing this widespread belief. We describe the phosphorus (P) removing performance and microbial population analyses of sludges obtained in a laboratory scale EBPR reactor. Two sludges with extremely high P removing capabilities were examined, the P content of these sludges was 8.6% (P sludge) and 12.3% (S sludge) of the MLSS. Identification of bacteria using FISH probing indicated both sludges were dominated by microbes from the beta proteobacteria and high mol% G+C Gram positive bacteria. Acinetobacter could make up only a small proportion of the cells in these sludges. Sludge with extremely poor P removal (P content of 1.5%, referred to as T sludge) was then generated by reducing the P in the influent. Bacteria resembling the G-bacteria became abundant in this sludge and these were identified using FISH probing. The anaerobic transformations of the T and P sludges correlated well with that of the non-EBPR and EBPR biological models respectively, indicating that bacteria in the T sludge have the potential to inhibit P removal in EBPR systems.

Modelling anaerobic degradation of complex wastewater. II: parameter estimation and validation using slaughterhouse effluent

This is the second part of a two-part paper developing and validating a structural model to describe the high rate anaerobic digestion of complex wastewater. A full-scale, two-stage hybrid upflow anaerobic reactor treating pig slaughterhouse effluent is assessed. Parameters and hydraulics are proposed for this system and validated. Characterisation of the system indicated that influent was a combination of equal parts (in terms of COD) particulate fat, particulate protein, and soluble protein. Reactor hydraulics were close to completely stirred tank reactor (CSTR) hydraulics, as indicated by a residence time distribution (RTD) study. The Monday morning abattoir start-up was identified as a key disturbance suitable for parameter estimation. A stepwise parameter estimation procedure was used as proposed in the previous paper. An experimental data set without recycle was used for parameter estimation. The model with these parameters performed well on independent data sets with and without recycle. The model identified several anomalies in the experimental data that may be unique to reactors treating complex wastewater.

Characterisation of the bacterial consortium involved in nitrite oxidation in activated sludge

A sequencing batch reactor (SBR) was operated to selectively grow a nitrite oxidising microbial community and was called the nitrite oxidising SBR (NOSBR). The nitrite oxidising characteristics of the reactor biomass were studied as well as the microbial composition. Molecular biological methods of clone libraries were used to evaluate the microorganisms in both the seed sludge and in the NOSBR sludge. We have found that the nitrite oxidation in the NOSBR was due the presence of bacteria from the Nitrospira phylum and not because of the presence of Nitrobacter which were in very low numbers in the NOSBR and not detected in the seed sludge. We hypothesize that the unknown nitrite oxidising bacteria in wastewater treatment plants are a range of species related to Nitrospira moscoviensis. A suite of primers were developed from the clone sequence data and used in a diagnostic polymerase chain reaction to prove the presence of these novel nitrite oxidisers in a range of full scale and laboratory scale activated sludge plants.

Characterisation of enhanced biological phosphorus removal activated sludges with dissimilar phosphorus removal performances

A sequencing batch reactor (SBR) was operated for enhanced biological phosphorus removal (EBPR) and dramatic differences to the P removing capabilities were obtained in different stages of the operation. At one stage extremely poor P removal occurred and it appeared that bacteria inhibiting P removal overwhelmed the reactor performance. Changes were made to the reactor operation and these led to the development of a sludge with high P removing capability. This latter sludge was analysed by fluorescent in situ hybridisation (FISH) using a probe specific for Acinetobacter. Very few cells were detected with this probe indicating that Acinetobacter played an insignificant role in the P removal occurring here. Analysis of the chemical transformations of three sludges supported the biochemical pathways proposed for EBPR and non-EBPR systems in biological models. A change in operation that led to the improved P removal performance included permitting the pH to rise in the anaerobic periods of the SBR cycle.