Agreement between amoA gene-specific quantitative PCR and fluorescence in situ hybridization in the measurement of ammonia-oxidizing bacteria in activated sludge.

Microbial abundance is central to most investigations in microbial ecology, and its accurate measurement is a challenging task that has been significantly facilitated by the advent of molecular techniques over the last 20 years. Fluorescence in situ hybridization (FISH) is considered the gold standard of quantification techniques; however, it is expensive and offers low sample throughput, both of which limit its wider application. Quantitative PCR (qPCR) is an alternative that offers significantly higher throughput, and it is used extensively in molecular biology. The accuracy of qPCR can be compromised by biases in the DNA extraction and amplification steps. In this study, we compared the accuracy of these two established quantification techniques to measure the abundance of a key functional group in biological wastewater treatment systems, the ammonia-oxidizing bacteria (AOB), in samples from a time-series experiment monitoring a set of laboratory-scale reactors and a full-scale plant. For the qPCR analysis, we tested two different sets of AOB-specific primers, one targeting the 16SrRNA gene and one targeting the ammonia monooxygenase (amoA) gene. We found that there was a positive linear logarithmic relationship between FISH and the amoA gene-specific qPCR, where the data obtained from both techniques was equivalent at the order of magnitude level. The 16S rRNA gene-specific qPCR assay consistently underestimated AOB numbers.

Nitrification-denitrification in waste stabilisation ponds: a mechanism for permanent nitrogen removal in maturation ponds.

A pilot-scale primary maturation pond was spiked with (15)N-labelled ammonia ((15)NH(4)Cl) and (15)N-labelled nitrite (Na(15)NO(2)), in order to improve current understanding of the dynamics of inorganic nitrogen transformations and removal in WSP systems. Stable isotope analysis of delta(15)N showed that nitrification could be considered as an intermediate step in WSP, which is masked by simultaneous denitrification, under conditions of low algal activity. Molecular microbiology analysis showed that denitrification can be considered a feasible mechanism for permanent nitrogen removal in WSP, which may be supported either by ammonia-oxidising bacteria (AOB) or by methanotrophs, in addition to nitrite-oxidising bacteria (NOB). However, the relative supremacy of the denitrification process over other nitrogen removal mechanisms (e.g., biological uptake) depends upon phytoplanktonic activity.

Single-molecule study of transcriptional pausing and arrest by E. coli RNA polymerase.

Using an optical-trap/flow-control video microscopy technique, we followed transcription by single molecules of Escherichia coli RNA polymerase in real time over long template distances. These studies reveal that RNA polymerase molecules possess different intrinsic transcription rates and different propensities to pause and stop. The data also show that reversible pausing is a kinetic intermediate between normal elongation and the arrested state. The conformational metastability of RNA polymerase revealed by this single-molecule study of transcription has direct implications for the mechanisms of gene regulation in both bacteria and eukaryotes.

Enhanced estrogen removal in activated sludge processes through the optimization of the hydraulic flow pattern.

The removal of ß-estradiol (E2) and α-ethinylestradiol (EE2) in biological wastewater treatment plants (WWTP) would need to be improved in order to comply with prospective Environmental Quality Standards (EQS) of 0.4 and 0.035 ng.L-1 respectively. The effluent concentration of a micropollutant in an activated sludge process is a function of the removal rate, the hydraulic retention time (HRT) and the flow pattern, which is usually overlooked. In order to better understand this aspect, we carried out tracer studies in eight WWTPs in the UK and found that relatively modest changes in aeration tanks would translate into tangible improvements in their flow pattern. We further evaluated the degradation rates for E1 (estrone), E2, E3 (estriol) and EE2 in each WWTP and we estimated that the modification of the flow pattern would be sufficient to place effluent concentrations of E2 (23.2 L∙gVSS-1∙d-1

Coupled virus - bacteria interactions and ecosystem function in an engineered microbial system.

Viruses are thought to control bacterial abundance, affect community composition and influence ecosystem function in natural environments. Yet their dynamics have seldom been studied in engineered systems, or indeed in any system, for long periods of time. We measured virus abundance in a full-scale activated sludge plant every week for two years. Total bacteria and ammonia oxidising bacteria (AOB) abundances, bacterial community profiles, and a suite of environmental and operational parameters were also monitored. Mixed liquor virus abundance fluctuated over an order of magnitude (3.18 × 108-3.41 × 109 virus's mL-1) and that variation was statistically significantly associated with total bacterial and AOB abundance, community composition, and effluent concentrations of COD and NH4+- N and thus system function. This suggests viruses play a far more important role in the dynamics of activated sludge systems than previously realised and could be one of the key factors controlling bacterial abundance, community structure and functional stability and may cause reactors to fail. These findings are based on statistical associations, not mechanistic models. Nevertheless, viral associations with abiotic factors, such as pH, make physical sense, giving credence to these findings and highlighting the role that physical factors play in virus ecology. Further work is needed to identify and quantify specific bacteriophage and their hosts to enable us to develop mechanistic models of the ecology of viruses in wastewater treatment systems. However, since we have shown that viruses can be related to effluent quality and virus quantification is simple and cheap, practitioners would probably benefit from quantifying viruses now.

A flow cytometry method for bacterial quantification and biomass estimates in activated sludge.

Absolute bacterial quantification receives little serious attention in the literature compared to sequencing, conceivably because it is considered unimportant and facile, or because existing methods are tedious, laborious and/or biased in nature. This is particularly true in engineered systems, including activated sludge, where such information underpins their design and operation. To overcome these limitations we built upon existing work and optimised and comprehensively validated, through comparison with epifluorescence microscopy (EFM), a rapid and precise flow cytometric protocol to enumerate total bacterial numbers in activated sludge. Insights into potential biases were evaluated using appropriate statistical analyses on this comparison, which spanned four orders of magnitude, as well as comparing volatile suspended solid (VSS) concentrations. The results suggest flow cytometry (FCM) is a rapid, reproducible and economical technique for quantifying total bacterial numbers and biomass concentrations in activated sludge, despite within order of magnitude discrepancies with EFM counts, which had inherent and evidently greater errors and biases than FCM. The use of FCM for routine monitoring over both EFM and VSS should help further understanding of the microbial ecology in, and the operation of, engineered systems.

Investigating glutamate receptor-like gene co-expression in Arabidopsis thaliana.

There is increasing evidence of the important roles of glutamate receptors (GLRs) in plant development and in adaptation to stresses. However, the studies of these putative ion channels, both in planta and in Xenopus oocytes, may have been limited by our lack of knowledge of possible GLR heteromer formation in plants. We have developed a modification of the single-cell sampling technique to investigate GLR co-expression, and thus potential heteromer formation, in single cells of Arabidopsis thaliana leaves. Micro-EXpression amplification (MEX) has allowed us to amplify gene transcripts from a single cell, enabling expression of up to 100 gene transcripts to be assayed. We measured, on average, the transcripts of five to six different AtGLRs in a single cell. However, no consistent patterns of co-expression or cell-type-specific expression were detected, except that cells sampled from the same plant showed similar expression profiles. The only discernible feature was the detection of AtGLR3.7 in every cell examined, an observation supported by GUS staining patterns in plants stably expressing promoter::uidA fusions. In addition, we found AtGLR3.7 expression in oocytes induces a Ba2+-, Ca2+- and Na+-permeable plasma membrane conductance.

Presence and activity of ammonia-oxidising bacteria detected amongst the overall bacterial diversity along a physico-chemical gradient of a nitrifying wastewater treatment plant.

We wished to discover if we could gain greater insights into how biological treatment plants function by contrasting the presence and activity of the most abundant Bacteria in plug flow and completely mixed activated sludge plants. Presence was assessed by amplifying 16S rRNA gene fragments (using PCR) and activity by amplifying native 16S rRNA, using reverse-transcriptase PCR (RT-PCR), using Bacteria-specific primers. The amplified sequences were compared using denaturing gradient gel electrophoresis (DGGE). The plug flow plant exhibited a strong physico-chemical gradient with an initial anoxic zone, whilst the two completely mixed reactors did not. Similarities were observed between the profile of the banding pattern for presence and activity. However, in the plug flow reactor one prominent band was detected in the active population (16S rRNA) but was absent from the corresponding profile of the 16S rRNA gene. Sequencing of this band revealed its identity as a Nitrosomonas-like sequence. The intensity of the 16S rRNA sequenced varied along the physico-chemical gradient of the plug-flow reactor in a manner that coincided with the growth of ammonia-oxidising bacteria (AOB) and the loss of ammonia. This band was also absent from the completely mixed reactors, although significant numbers of AOB were detected in all systems ( approximately 10(6)-10(8)cells ml(-1)) by fluorescence in situ hybridisation (FISH). An abundant and highly active AOB population was present in the anoxic zone of the plug-flow reactor where up to 60% of the total ammonia was removed. An examination of nitrogen removal/production rates, together with the above data, reveal that complex nitrogen removal processes occur in this system. These data also enabled the calculation of a specific in situ growth rate for the AOB as 0.12h(-1).

A universal threshold concept for hydrophobic mycolata in activated sludge foaming.

Recent studies using quantitative fluorescence in situ hybridization (FISH) have supported the principle that there are mycolata concentration thresholds, above which foaming is likely to occur. In this study, we surveyed 14 wastewater treatment plants (WWTPs) in the UK, using quantitative FISH, to establish that the principle of a mycolata threshold (2 x 10(6) mycolata cells ml(-1) mixed liquor suspended solids) is an empirical though widely held value. In addition, we designed, optimized and applied probes for members of the less hydrophobic mycolata genera Corynebacterium and Dietzia, to show that these organisms dominated the mycolata populations in two non-foaming WWTPs where the mycolata concentrations were above the threshold value. We propose that the mycolata threshold value is only applicable to hydrophobic members of the mycolata.

Epidemiology, clinical characteristics, and management of adults referred to a teaching hospital first seizure clinic.

BACKGROUND: There are scarce data describing the epidemiology, clinical characteristics, and management of adults who suffer a suspected first seizure. AIM: To describe the epidemiology, clinical characteristics, and management of adults with a suspected first seizure who are referred to a teaching hospital first seizure clinic over a one year period. DESIGN: Prospective descriptive study. METHODS: Data were collected on consecutive adults referred to the Royal Infirmary of Edinburgh between 4 February 2003 and 10 February 2004. RESULTS: 232 patients were referred to the first seizure clinic. Median age was 32 years; 53% of patients were male. Lower socioeconomic groups were more likely to present with a suspected first seizure. Nineteen per cent of patients were admitted to hospital after their suspected seizure episode. Appropriate driving advice was reported in 64% of cases. Seventy two per cent of patients were offered a first seizure clinic appointment within six weeks of referral. Nine per cent of patients had a subsequent seizure while awaiting review. Fifty two per cent of patients were confirmed as having a first seizure at the clinic, of which 56% were provoked by alcohol, recreational drugs, or sleep deprivation. Electroencephalography and computed tomography of the brain were the most common investigations ordered at the first seizure clinic (22% and 22% of patients respectively). CONCLUSION: Adults who suffer a suspected first seizure, and who make a full neurological recovery, can be safely managed as an outpatient. Around half of these patients will have a specialist diagnosis of first seizure and alcohol will be a common precipitating factor.

Early management of adults with an uncomplicated first generalised seizure.

A literature review of first seizures in adults was performed and a management algorithm was constructed. This review highlights the importance of a thorough history and examination, routine biochemistry and haematology, an electrocardiogram, selected neuroimaging, discharge planning with driving and lifestyle advice, and follow-up in a specialist clinic.

Flow cytometric quantification of viruses in activated sludge.

Viruses may play a critical role in the microbial dynamics of activated sludge systems; however the difficulty of their quantification makes long term and large scale studies costly, timely and challenging. Thus a flow cytometric protocol was optimised and employed to determine virus abundance in activated sludge samples. The best flow cytometry signature and highest virus count was obtained by separating the indigenous floc-associated viruses using Tween 80 and sodium pyrophosphate, diluting the sample with Tris-EDTA and staining with SYBR Green II. Using the optimised protocol viral concentrations from 25 activated sludge plants were determined, with average concentrations of 2.35 × 109 mL⁻¹ observed. Direct counts by transmission electron microscopy were highly correlated with flow cytometric counts (p = <0.05 and r² = 0.77), with concentrations from both quantification methods comparable at the order of magnitude level. The high counting efficiency, ease of preparation and rapidity and reproducibility of analysis makes flow cytometric quantification of viruses in activated sludge ideal for routine investigation and thus invaluable in unravelling the complexity of phage host interactions in such systems.