Andrew J. Schuler

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.

Parameters characterization and optimization of activated carbon (AC) cathodes for microbial fuel cell application.

Activated carbon (AC) is employed as a cost-effective catalyst for cathodic oxygen reduction in microbial fuel cells (MFC). The fabrication protocols of AC-based cathodes are conducted at different applied pressures (175-3500 psi) and treatment temperatures (25-343°C). The effects of those parameters along with changes in the surface morphology and chemistry on the cathode performances are comprehensively examined. The cathodes are tested in a three-electrode setup and explored in single chamber membraneless MFCs (SCMFCs). The results show that the best performance of the AC-based cathode is achieved when a pressure of 1400 psi is applied followed by heat treatment of 150-200°C for 1h. The influence of the applied pressure and the temperature of the heat treatment on the electrodes and SCMFCs is demonstrated as the result of the variation in the transfer resistance, the surface morphology and surface chemistry of the AC-based cathodes tested.

Effects of integrated fixed film activated sludge media on activated sludge settling in biological nutrient removal systems

Integrated fixed film activated sludge (IFAS) is an increasingly popular modification of conventional activated sludge, consisting of the addition of solid media to bioreactors to create hybrid attached/suspended growth systems. While the benefits of this technology for improvement of nitrification and other functions are well-demonstrated, little is known about its effects on biomass settleability. These effects were evaluated in parallel, independent wastewater treatment trains, with and without IFAS media, both at the pilot (at two solids residence times) and full scales. While all samples demonstrated good settleability, the Control (non-IFAS) systems consistently demonstrated small but significant (p<0.05) improvements in settleability relative to the IFAS trains. Differences in biomass densities were identified as likely contributing factors, with Control suspended phase density>IFAS suspended phase density>IFAS attached phase (biofilm) density. Polyphosphate content (as non-soluble phosphorus) was well-correlated with density. This suggested that the attached phases had relatively low densities because of their lack of anaerobic/aerobic cycling and consequent low content of polyphosphate-accumulating organisms, and that differences in enhanced biological phosphorus removal performance between the IFAS and non-IFAS systems were likely related to the observed differences in density and settleability for the suspended phases. Decreases in solids retention times from 8 to 4 days resulted in improved settleability and increased density in all suspended phases, which was related to increased phosphorus content in the biomass, while no significant changes in density and phosphorus content were observed in attached phases.

Double‐Chamber Microbial Fuel Cell with a Non‐Platinum‐Group Metal Fe–N–C Cathode Catalyst

Non-Pt-group metal (non-PGM) materials based on transition metal-nitrogen-carbon (M-N-C) and derived from iron salt and aminoantipyrine (Fe-AAPyr) of mebendazole (Fe-MBZ) were studied for the first time as cathode catalysts in double-chamber microbial fuel cells (DCMFCs). The pH value of the cathode chamber was varied from 6 to 11 to elucidate the activity of those catalysts in acidic to basic conditions. The Fe-AAPyr- and Fe-MBZ-based cathodes were compared to a Pt-based cathode used as a baseline. Pt cathodes performed better at pH 6-7.5 and had similar performances at pH 9 and a substantially lower performance at pH 11 at which Fe-AAPyr and Fe-MBZ demonstrated their best electrocatalytic activity. The power density achieved with Pt constantly decreased from 94-99 μW cm(-2) at pH 6 to 55-57 μW cm(-2) at pH 11. In contrast, the power densities of DCMFs using Fe-AAPyr and Fe-MBZ were 61-68 μW cm(-2) at pH 6, decreased to 51-58 μW cm(-2) at pH 7.5, increased to 65-75 μW cm(-2) at pH 9, and the highest power density was achieved at pH 11 (68-80 μW cm(-2) ). Non-PGM cathode catalysts can be manufactured at the fraction of the cost of the Pt-based ones. The higher performance and lower cost indicates that non-PGM catalysts may be a viable materials choice in large-scale microbial fuel cells.

High catalytic activity and pollutants resistivity using Fe-AAPyr cathode catalyst for microbial fuel cell application.

For the first time, a new generation of innovative non-platinum group metal catalysts based on iron and aminoantipyrine as precursor (Fe-AAPyr) has been utilized in a membraneless single-chamber microbial fuel cell (SCMFC) running on wastewater. Fe-AAPyr was used as an oxygen reduction catalyst in a passive gas-diffusion cathode and implemented in SCMFC design. This catalyst demonstrated better performance than platinum (Pt) during screening in “clean” conditions (PBS), and no degradation in performance during the operation in wastewater. The maximum power density generated by the SCMFC with Fe-AAPyr was 167 ± 6 μW cm−2 and remained stable over 16 days, while SCMFC with Pt decreased to 113 ± 4 μW cm−2 by day 13, achieving similar values of an activated carbon based cathode. The presence of S2− and showed insignificant decrease of ORR activity for the Fe-AAPyr. The reported results clearly demonstrate that Fe-AAPyr can be utilized in MFCs under the harsh conditions of wastewater.

Development and validation of a flux-based stoichiometric model for enhanced biological phosphorus removal metabolism

Abstract Enhanced biological phosphorus removal (EBPR) is a wastewater treatment process involving metabolic cycling through several biopolymers (polyphosphate, polyhydroxyalkanoates, and glycogen). This metabolic cycling is induced in microorganisms by treatment systems that alternate between initial carbon-rich, anaerobic environments followed by carbon-poor, aerobic environments. While the appearance and disappearance of these biopolymers has been documented, the intracellular pressures that lead to their synthesis and degradation are not well understood. To understand how carbon, energy, and redox potential are channeled through the metabolic pathways in each treatment process stage, a metabolic flux model that contained a complete set of the pathways involved in biomass synthesis and energy production in bacteria was developed. The model accounts for the energy requirements of macromolecule synthesis and of metabolite transport across the cell membrane. The equations for the 163 reversible and 166 irreversible reactions were solved using linear optimization. Data from a laboratory scale sequencing batch reactor performing EBPR were used as model inputs. Given polyhydroxyalkanoate synthesis and glycogen degradation rates in the anaerobic phase, the model predicted reasonable anaerobic acetate uptake and polyphosphate consumption rates. In the aerobic phase, the polyphosphate and glycogen synthesis rates were used to predict the polyhydroxyalkanoate consumption rate. In addition, the model predicted the ratio of acetate uptake to phosphate release observed experimentally, as well as an inverse relationship between polyhydroxyalkanoate and polyphosphate consumption. The model provides information on the pathways by which the energy-rich molecules ATP, NADH, and NADPH are produced and consumed during the EBPR processes. In doing so, it supports the hypothesis that biopolymer metabolism provides a means for organisms to balance intracellular energy supplies. Moreover, the model suggests pathways at which metabolic regulation should occur and provides a comprehensive account of EBPR metabolism.

Influence of anode surface chemistry on microbial fuel cell operation

Self-assembled monolayers (SAMs) modified gold anodes are used in single chamber microbial fuel cells for organic removal and electricity generation. Hydrophilic (N(CH3)3(+), OH, COOH) and hydrophobic (CH3) SAMs are examined for their effect on bacterial attachment, current and power output. The different substratum chemistry affects the community composition of the electrochemically active biofilm formed and thus the current and power output. Of the four SAM-modified anodes tested, N(CH3)3(+) results in the shortest start up time (15 days), highest current achieved (225 μA cm(-2)) and highest MFC power density (40 μW cm(-2)), followed by COOH (150 μA cm(-2) and 37 μW cm(-2)) and OH (83 μA cm(-2) and 27 μW cm(-2)) SAMs. Hydrophobic SAM decreases electrochemically active bacteria attachment and anode performance in comparison to hydrophilic SAMs (CH3 modified anodes 7 μA cm(-2) anodic current and 1.2 μW cm(-2) MFCs power density). A consortium of Clostridia and δ-Proteobacteria is found on all the anode surfaces, suggesting a synergistic cooperation under anodic conditions.

Mighty small: Observing and modeling individual microbes becomes big science

Progress in microbiology has always been driven by technological advances, ever since Antonie van Leeuwenhoek discovered bacteria by making an improved compound microscope. However, until very recently we have not been able to identify microbes and record their mostly invisible activities, such as nutrient consumption or toxin production on the level of the single cell, not even in the laboratory. This is now changing with the rapid rise of exciting new technologies for single-cell microbiology (1, 2), which enable microbiologists to do what plant and animal ecologists have been doing for a long time: observe who does what, when, where, and next to whom. Single cells taken from the environment can be identified and even their genomes sequenced. Ex situ, their size, elemental, and biochemical composition, as well as other characteristics can be measured with high-throughput and cells sorted accordingly. Even better, individual microbes can be observed in situ with a range of novel microscopic and spectroscopic methods, enabling localization, identification, or functional characterization of cells in a natural sample, combined with detecting uptake of labeled compounds. Alternatively, they can be placed into fabricated microfluidic environments, where they can be positioned, exposed to stimuli, monitored, and their interactions controlled “in microfluido.” By introducing genetically engineered reporter cells into a fabricated landscape or a microcosm taken from nature, their reproductive success or activity can be followed, or their sensing of their local environment recorded.

Low Acetate Concentrations Favor Polyphosphate-Accumulating Organisms over Glycogen-Accumulating Organisms in Enhanced Biological Phosphorus Removal from Wastewater

Glycogen-accumulating organisms (GAOs) are thought to compete with polyphosphate-accumulating organisms (PAOs) in enhanced biological phosphorus removal (EBPR) wastewater treatment systems. A laboratory sequencing batch reactor (SBR) was operated for one year to test the hypothesis that PAOs have a competitive advantage at low acetate concentrations, with a focus on low pH conditions previously shown to favor GAOs. PAOs dominated the system under conventional SBR operation with rapid acetate addition (producing high in-reactor concentrations) and pH values of 7.4-8.4. GAOs dominated when the pH was decreased (6.4-7.0). Decreasing the acetate addition rate led to very low reactor acetate concentrations, and PAOs recovered, supporting the study hypothesis. When the acetate feed rate was increased, EBPR failed again. Dominant PAOs and GAOs were Candidatus Accumulibacter phosphatis and Defluviicoccus Cluster 2, respectively, according to fluorescent in situ hybridization and 454 pyrosequencing. Surprisingly, GAOs were not the immediate causes of PAO failures, based on functional and population measurements. Pyrosequencing results suggested Dechloromonas and Tetrasphaera spp. may have also been PAOs, and additional potential GAOs were also identified. Full-scale systems typically have lower in-reactor acetate concentrations than laboratory SBRs, and so, previous laboratory studies may have overestimated the practical importance of GAOs as causes of EBPR failure.

Experimental and theoretical examination of surface energy and adhesion of nitrifying and heterotrophic bacteria using self-assembled monolayers.

Biofilm-based systems, including integrated fixed-film activated sludge and moving bed bioreactors, are becoming increasingly popular for wastewater treatment, often with the goal of improving nitrification through the enrichment of ammonia and nitrite oxidizing bacteria. We have previously demonstrated the utility of self-assembled monolayers (SAMs) as tools for studying the initial attachment of bacteria to substrata systematically varying in physicochemical properties. In this work, we expanded these studies to bacteria of importance in wastewater treatment systems and we demonstrated attachment rates were better correlated with surface energy than with wettability (water contact angle). Toward the long-term goal of improving wastewater treatment performance through the strategic design of attachment substrata, the attachment rates of two autotrophic ammonia-oxidizing bacteria (Nitrosomonas europaea and Nitrosospira multiformis) and a heterotroph (Escherichia coli) were evaluated using SAMs with a range of wettabilities, surface energies, and functional properties (methyl, hydroxyl, carboxyl, trimethylamine, and amine terminated). Cell attachment rates were somewhat correlated with the water contact angles of the SAMs with polar terminal groups (hydroxyl, carboxyl, trimethylamine, and amine). Including all SAM surfaces, a better correlation was found for all bacteria between attachment rates and surface free energy, as determined using the Lewis Acid-Base approach. The ammonia-oxidizers had higher adhesion rates on the SAMs with higher surface energies than did the heterotroph. This work demonstrated the successful application of SAMs to determine the attachment surface preferences of bacteria important to wastewater treatment, and it provides guidance for a new area of research aimed at improving treatment performance through rational attachment surface design.