Biological phosphorus removal during high-rate, low-temperature, anaerobic digestion of wastewater.

We report, for the first time, extensive biologically-mediated phosphate removal from wastewater during high-rate anaerobic digestion (AD). A hybrid sludge bed/fixed-film (packed pumice stone) reactor was employed for low-temperature (12°C) anaerobic treatment of synthetic sewage wastewater. Successful phosphate removal from the wastewater (up to 78% of influent phosphate) was observed, mediated by biofilms in the reactor. Scanning electron microscopy and energy dispersive X-ray analysis revealed the accumulation of elemental phosphorus (~2%) within the sludge bed and fixed-film biofilms. 4’, 6-diamidino-2-phenylindole (DAPI) staining indicated phosphorus accumulation was biological in nature and mediated through the formation of intracellular inorganic polyphosphate (polyP) granules within these biofilms. DAPI staining further indicated that polyP accumulation was rarely associated with free cells. Efficient and consistent chemical oxygen demand (COD) removal was recorded, throughout the 732-day trial, at applied organic loading rates between 0.4-1.5 kg COD m-3 d-1 and hydraulic retention times of 8-24 hours, while phosphate removal efficiency ranged from 28-78% on average per phase. Analysis of protein hydrolysis kinetics and the methanogenic activity profiles of the biomass revealed the development, at 12˚C, of active hydrolytic and methanogenic populations. Temporal microbial changes were monitored using Illumina Miseq analysis of bacterial and archaeal 16S rRNA gene sequences. The dominant bacterial phyla present in the biomass at the conclusion of the trial were the Proteobacteria and Firmicutes and the dominant archaeal genus was Methanosaeta. Trichococcus and Flavobacterium populations, previously associated with low temperature protein degradation, developed in the reactor biomass. The presence of previously characterised polyphosphate accumulating organisms (PAOs) such as Rhodocyclus, Chromatiales, Actinobacter and Acinetobacter was recorded at low numbers. However, it is unknown as yet if these were responsible for the luxury polyP uptake observed in this system. The possibility of efficient phosphate removal and recovery from wastewater during AD would represent a major advance in the scope for widespread application of anaerobic wastewater treatment technologies.

Isotope dilution gas chromatography/mass spectrometry method for the determination of methionine sulfoxide in protein

We have developed a new technique for quantifying methionine sulfoxide (MetSO) in protein to assess levels of oxidative stress in physiological systems. In this procedure, samples are hydrolyzed with methanesulfonic acid (MSA) in order to avoid the conversion of MetSO to methionine (Met) that occurs during hydrolysis of protein in HCl. The hydrolysate is fractionated on a cation exchange column to remove the nonvolatile MSA from amino acids, and the amino acids are then derivatized as their trimethylsilyl esters for analysis by selected ion monitoring-gas chromatography/mass spectrometry. The limit of detection of the assay is 200 pmol of MetSO per analysis, and the interassay coefficient of variation is 5.8%. Compared to current methods, the SIM-GC/MS assay avoids the potential for conversion of Met to MetSO during sample preparation, requires less sample preparation time, has lower variability, and uses mass spectrometry for sensitive and specific analyte detection.

FoodCAP: acid-labile protein adducts of heterocyclic amines in human blood are not viable biomarkers of HCA dietary exposure

Intake of heterocyclic amines (HCAs, carcinogens produced during cooking of meat/fish, the most abundant being PhIP, DiMeIQx and MeIQx) is influenced by many factors including type/thickness of meat and cooking method/temperature/duration. Thus, assessment of HCA dietary exposure is difficult. Protein adducts of HCAs have been proposed as potential medium-term biomarkers of exposure, e.g. PhIP adducted to serum albumin or haemoglobin. However, evidence is still lacking that HCA adducts are viable biomarkers in humans consuming normal diets. The FoodCAP project, supported by World Cancer Research Fund, developed a highly sensitive mass spectrometric method for hydrolysis, extraction and detection of acid-labile HCAs in blood and assessed their validity as biomarkers of exposure. Multiple acid/alkaline hydrolysis conditions were assessed, followed by liquid-liquid extraction, clean-up by cation-exchange SPE and quantification by UPLC-ESI-MS/ MS. Blood was analysed from volunteers who completed food diaries to estimate HCA intake based on the US National Cancer Institute’s CHARRED database. Standard HCAs were recovered quantitatively from fortified blood. In addition, PhIP/MeIQx adducts bound to albumin and haemoglobin prepared in vitro using a human liver microsome system were also detectable in blood fortified at low ppt concentrations. However, except for one sample (5pg/ml PhIP), acid-labile PhIP, 7,8-DiMeIQx, 4,8-DiMeIQx and MeIQx were not observed above the 2pg/ml limit of detection in plasma (n=35), or in serum, whole blood or purified albumin, even in volunteers with high meat consumption (nominal HCA intake >2µg/day). It is concluded that HCA blood protein adducts are not viable biomarkers of exposure. Untargeted metabolomic analyses may facilitate discovery of suitable markers.

The structure of an unconventional HD-GYP protein from Bdellovibrio reveals the roles of conserved residues in this class of cyclic-di-GMP phosphodiesterases

UNLABELLED: Cyclic-di-GMP is a near-ubiquitous bacterial second messenger that is important in localized signal transmission during the control of various processes, including virulence and switching between planktonic and biofilm-based lifestyles. Cyclic-di-GMP is synthesized by GGDEF diguanylate cyclases and hydrolyzed by EAL or HD-GYP phosphodiesterases, with each functional domain often appended to distinct sensory modules. HD-GYP domain proteins have resisted structural analysis, but here we present the first structural representative of this family (1.28 Å), obtained using the unusual Bd1817 HD-GYP protein from the predatory bacterium Bdellovibrio bacteriovorus. Bd1817 lacks the active-site tyrosine present in most HD-GYP family members yet remains an excellent model of their features, sharing 48% sequence similarity with the archetype RpfG. The protein structure is highly modular and thus provides a basis for delineating domain boundaries in other stimulus-dependent homologues. Conserved residues in the HD-GYP family cluster around a binuclear metal center, which is observed complexed to a molecule of phosphate, providing information on the mode of hydroxide ion attack on substrate. The fold and active site of the HD-GYP domain are different from those of EAL proteins, and restricted access to the active-site cleft is indicative of a different mode of activity regulation. The region encompassing the GYP motif has a novel conformation and is surface exposed and available for complexation with binding partners, including GGDEF proteins.

IMPORTANCE: It is becoming apparent that many bacteria use the signaling molecule cyclic-di-GMP to regulate a variety of processes, most notably, transitions between motility and sessility. Importantly, this regulation is central to several traits implicated in chronic disease (adhesion, biofilm formation, and virulence gene expression). The mechanisms of cyclic-di-GMP synthesis via GGDEF enzymes and hydrolysis via EAL enzymes have been suggested by the analysis of several crystal structures, but no information has been available to date for the unrelated HD-GYP class of hydrolases. Here we present the multidomain structure of an unusual member of the HD-GYP family from the predatory bacterium Bdellovibrio bacteriovorus and detail the features that distinguish it from the wider structural family of general HD fold hydrolases. The structure reveals how a binuclear iron center is formed from several conserved residues and provides a basis for understanding HD-GYP family sequence requirements for c-di-GMP hydrolysis.

Acid-labile protein-adducted heterocyclic aromatic amines in human blood are not viable biomarkers of dietary exposure: A systematic study.

Heterocyclic aromatic amines (HCA) are carcinogenic mutagens formed during cooking of protein-rich foods. HCA residues adducted to blood proteins have been postulated as biomarkers of HCA exposure. However, the viability of quantifying HCAs following hydrolytic release from adducts in vivo and correlation with dietary intake are unproven. To definitively assess the potential of labile HCA-protein adducts as biomarkers, a highly sensitive UPLC-MS/MS method was validated for four major HCAs: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) and 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx). Limits of detection were 1e5 pg/ml plasma and recoveries 91e115%. Efficacy of hydrolysis was demonstrated by HCA-protein adducts synthesised in vitro. Plasma and 7-day food diaries were collected from 122 fasting adults consuming their habitual diets. Estimated HCA intakes ranged from 0 to 2.5 mg/day. An extensive range of hydrolysis conditions was examined for release of adducted HCAs in plasma. HCA was detected in only one sample (PhIP, 9.7 pg/ml), demonstrating conclusively for the first time that acid-labile HCA adducts do not reflect dietary HCA intake and are present at such low concentrations that they are not feasible biomarkers of exposure. Identification of biomarkers remains important. The search should concentrate on stabilised HCA peptide markers and use of untargeted proteomic and metabolomic approaches.