Chemical fixation methods for Raman spectroscopy-based analysis of bacteria

Preservation of biological samples for downstream analysis is important for analytical methods that measure the biochemical composition of a sample. One such method, Raman microspectroscopy, is commonly used as a rapid phenotypic technique to measure biomolecular composition for the purposes of identification and discrimination of species and strains of bacteria, as well as investigating physiological responses to external stressors and the uptake of stable isotope-labelled substrates in single cells. This study examines the influence of a number of common chemical fixation and inactivation methods on the Raman spectrum of six species of bacteria. Modifications to the Raman-phenotype caused by fixation were compared to unfixed control samples using difference spectra and Principal Components Analysis (PCA). Additionally, the effect of fixation on the ability to accurately classify bacterial species using their Raman phenotype was determined. The results showed that common fixatives such as glutaraldehyde and ethanol cause significant changes to the Raman spectra of bacteria, whereas formaldehyde and sodium azide were better at preserving spectral features

Assessment of the bimodality in the distribution of bacterial genome sizes

Bacterial genome sizes have previously been shown to exhibit a bimodal distribution. This phenomenon has prompted discussion regarding evolutionary forces driving genome size in bacteria and its ecological significance. We investigated the level of inherent redundancy in the public database and the effect it has on the shape of the apparent bimodal distribution. Our study reveals that there is a significant bias in the genome sequencing efforts towards a certain group of species, and that correcting the bias using species nomenclature and clustering of the 16S rRNA gene, results in a unimodal rather than the previously published bimodal distribution. The true genome size distribution and its wider ecological implications will soon emerge as we are currently witnessing rapid growth in the number of sequenced genomes from diverse environmental niches across a range of habitats at an unprecedented rate

Dark matter annihilation and decay in dwarf spheroidal galaxies: The classical and ultrafaint dSphs

Dwarf spheroidal (dSph) galaxies are prime targets for present and future gamma-ray telescopes hunting for indirect signals of particle dark matter. The interpretation of the data requires careful assessment of their dark matter content in order to derive robust constraints on candidate relic particles. Here, we use an optimised spherical Jeans analysis to reconstruct the `astrophysical factor' for both annihilating and decaying dark matter in 21 known dSphs. Improvements with respect to previous works are: (i) the use of more flexible luminosity and anisotropy profiles to minimise biases, (ii) the use of weak priors tailored on extensive sets of contamination-free mock data to improve the confidence intervals, (iii) systematic cross-checks of binned and unbinned analyses on mock and real data, and (iv) the use of mock data including stellar contamination to test the impact on reconstructed signals. Our analysis provides updated values for the dark matter content of 8 `classical' and 13 `ultrafaint' dSphs, with the quoted uncertainties directly linked to the sample size; the more flexible parametrisation we use results in changes compared to previous calculations. This translates into our ranking of potentially-brightest and most robust targets---viz., Ursa Minor, Draco, Sculptor---, and of the more promising, but uncertain targets---viz., Ursa Major 2, Coma---for annihilating dark matter. Our analysis of Segue 1 is extremely sensitive to whether we include or exclude a few marginal member stars, making this target one of the most uncertain. Our analysis illustrates challenges that will need to be addressed when inferring the dark matter content of new `ultrafaint' satellites that are beginning to be discovered in southern sky surveys.Comment: 19 pages, 14 figures, submitted to MNRAS. Supplementary material available on reques

Presence and abundance of microplastics in the Thames River Basin, UK

The global increase in plastic production has led to growing concern about the environmental impacts of plastics and their degradation products. Microplastics have been extensively observed and studied in the marine environment but little is known about their presence and abundance in freshwater environments. Although rivers are recognised as a significant source of microplastics to the oceans, they are seldom considered in studies of the environmental presence of microplastics and there are no data reported to date on microplastics in UK rivers (or indeed any freshwater bodies). This study aimed to identify and quantify the abundance and types of plastics in the Thames Basin where population densities and sewage inputs are well described. Ten sampling sites on the River Thames and its tributaries were selected, ranging from densely populated, urban areas to sparsely populated, rural areas. Sites are all downstream of sewage treatment works (STWs) serving known populations, allowing correlation between population density with plastic types and abundances found. In addition samples were collected from sites at known distances downstream of STW outfalls, as well as the effluent itself, to try and establish the proportion of plastics directly entering from STWs, and its fate and transport pathways. River sediment and water samples were collected at all sites. Sediment samples were initially searched by eye, followed by flotation and overflowing using ZnCl2 solution. Plastics collected from the sediments were subsequently identified by Raman spectroscopy. Initial observations indicate that coloured and manmade particles are obviously visible in sediments from sites with high population densities compared to few evident manmade particles in sediments from areas with low population densities. Further analysis will allow for correlation of the plastic types and abundance with population density and sewage inputs to understand the distribution of plastics in river systems

Large-scale survey of seasonal drinking water quality in Malawi using in situ tryptophan-like fluorescence and conventional water quality indicators

Faecally-contaminated drinking water is a risk to human health, with the greatest risks to those living in developing countries. UN Sustainable Development Goal 6 aims to address this issue. Tryptophan-like fluorescence (TLF) shows potential as a rapid method for detecting microbial contamination in drinking water, which could reduce the spread of waterborne diseases. This study is the first to investigate the effectiveness of TLF for a large-scale survey using a randomised, spot-sampling approach. The large-scale survey took place in Malawi, sub-Saharan Africa, in the dry season (n = 183). A subset of sources were revisited at the end of the following wet season (n = 41). The effectiveness of TLF was assessed by comparing TLF results to thermotolerant coliforms (TTC), humic-like fluorescence (HLF), inorganic hydrochemical data and sanitary risk scores. The most prominent differences in microbial water quality were observed between source types, with little variation between districts and seasons. TLF, TTCs, turbidity and sanitary risk scores were all elevated at alternative sources (shallow wells and tap stands) compared to hand-pumped boreholes. In the dry season, 18% of hand-pumped boreholes showed TTC contamination, which increase to 21% in the wet season. Groundwater recharge processes are likely responsible for seasonal variability of inorganic hydrochemistry at hand-pumped boreholes. TLF was able to distinguish no and low WHO risk classes (TTC 0–9 cfu/100 mL) from medium, high and very high risk classes (TTC 10 – >1000 cfu/100 mL). TLF failed to distinguish between no and low risk classes, which limits the use of TLF for assessing water quality to drinking water standards. This dataset indicates that HLF may raise baseline TLF for samples with low TLF values, increasing false positives. Therefore, TLF is better suited as a rapid high-level water quality screening tool to assess moderate and high levels of faecal contamination

PIPITS: an automated pipeline for analyses of fungal internal transcribed spacer sequences from the Illumina sequencing platform

1. Studying fungal biodiversit y using data generated from Illumina MiSeq sequencing platforms poses a number of bioinformatic challenges with the analysis typically involving a large number of tools for each analytical step from quality filtering to generating identified operational taxonomic unit (OTU) abundance tables. 2. Here, we introduce PIPITS, an o pen-source stand-alone suite of software for automated processing of Illumina MiSeq sequences for fungal community analysis. PIPITS e xploits a number of state of the art applications to process paired-end reads from quality filtering to producing OTU abundance tables. 3. We pro vide detailed descriptions of the pipeline and show its utility in the analysis of 9 396 092 sequences generated on the MiSeq platform from Illumina MiSeq. 4. PIPITS is the first automated bioinformatics pipeline dedicated for fungal ITS sequences which incorporates ITSx to extract subregions of ITS and exploits the latest RDP Classifier to classify sequences against the curatedUNITE fungal data set

Tryptophan-like fluorescence as a high-level screening tool for detecting microbial contamination in drinking water

Regular monitoring of drinking water quality is vital to identify contamination of potable water supplies. Testing for microbial contamination is important to prevent transmission of waterborne disease, but establishing and maintaining a water quality monitoring programme requires sustained labour, consumables and resources. In low resource settings such as developing countries, this can prove difficult, but measuring microbial contamination is listed as a requirement of reaching the UN's Sustainable Development Goal 6 for water and sanitation. A nine-month water quality monitoring programme was conducted in rural Malawi to assess the suitability of tryptophan-like fluorescence (TLF), an emerging method for rapidly detecting microbial contamination, as a drinking water quality monitoring tool. TLF data was compared with thermotolerant coliforms (TTCs, E. coli) and inorganic hydrochemical parameters. A large (n = 235) temporal dataset was collected from five groundwater drinking water sources, with samples collected once or twice weekly depending on the season. The results show that TLF can indicate a broader contamination risk but is not as sensitive to short term variability when compared to other faecal indicators. This is likely due to a broad association of TLF with elevated DOC concentrations from a range of different sources. Elevated TLF may indicate preferential conditions for the persistence of TTCs and/or E. coli, but not necessarily a public health risk from microbial contamination. TLF is therefore a more precautionary risk indicator than microbial culturing techniques and could prove useful as a high-level screening tool for initial risk assessment. For widespread use of TLF to be successful, standardisation of TLF values associated with different levels of risk is required, however, this study highlights the difficulties of equating TLF thresholds to TTCs or E. coli data because of the influence of DOC/HLF on the TLF signal

Catchment-scale biogeography of riverine bacterioplankton

Lotic ecosystems such as rivers and streams are unique in that they represent a continuum of both space and time during the transition from headwaters to the river mouth. As microbes have very different controls over their ecology, distribution and dispersion compared with macrobiota, we wished to explore biogeographical patterns within a river catchment and uncover the major drivers structuring bacterioplankton communities. Water samples collected across the River Thames Basin, UK, covering the transition from headwater tributaries to the lower reaches of the main river channel were characterised using 16S rRNA gene pyrosequencing. This approach revealed an ecological succession in the bacterial community composition along the river continuum, moving from a community dominated by Bacteroidetes in the headwaters to Actinobacteria-dominated downstream. Location of the sampling point in the river network (measured as the cumulative water channel distance upstream) was found to be the most predictive spatial feature; inferring that ecological processes pertaining to temporal community succession are of prime importance in driving the assemblages of riverine bacterioplankton communities. A decrease in bacterial activity rates and an increase in the abundance of low nucleic acid bacteria relative to high nucleic acid bacteria were found to correspond with these downstream changes in community structure, suggesting corresponding functional changes. Our findings show that bacterial communities across the Thames basin exhibit an ecological succession along the river continuum, and that this is primarily driven by water residence time rather than the physiochemical status of the river

Evaluating the use of lake sedimentary DNA in palaeolimnology:A comparison with long‐term microscopy‐based monitoring of the phytoplankton community

Palaeolimnological records provide valuable information about how phytoplankton respond to long-term drivers of environmental change. Traditional palaeolimnological tools such as microfossils and pigments are restricted to taxa that leave sub-fossil remains, and a method that can be applied to the wider community is required. Sedimentary DNA (sedDNA), extracted from lake sediment cores, shows promise in palaeolimnology, but validation against data from long-term monitoring of lake water is necessary to enable its development as a reliable record of past phytoplankton communities. To address this need, 18S rRNA gene amplicon sequencing was carried out on lake sediments from a core collected from Esthwaite Water (English Lake District) spanning ~105 years. This sedDNA record was compared with concurrent long-term microscopy-based monitoring of phytoplankton in the surface water. Broadly comparable trends were observed between the datasets, with respect to the diversity and relative abundance and occurrence of chlorophytes, dinoflagellates, ochrophytes and bacillariophytes. Up to 20% of genera were successfully captured using both methods, and sedDNA revealed a previously undetected community of phytoplankton. These results suggest that sedDNA can be used as an effective record of past phytoplankton communities, at least over timescales of <100 years. However, a substantial proportion of genera identified by microscopy were not detected using sedDNA, highlighting the current limitations of the technique that require further development such as reference database coverage. The taphonomic processes which may affect its reliability, such as the extent and rate of deposition and DNA degradation, also require further research