Priest Pot Research Articles

Some Personal Memories of Bland J. Finlay and Work on Protists in “Priest Pot“

With his scientific curiosity and deep understanding of unicellular organisms, Bland J. Finlay could demonstrate that water bodies as small and innocuous as "Priest Pot" in the English Lake District represent a very broad range of protists and their interactions. From this, not only the field of protistan ecology benefitted greatly, but also my personal career. Enclosed are some scientific and personal memories to say "thank you" to Bland.

Some Personal Memories of Bland J. Finlay and Work on Protists in “Priest Pot“

Some Personal Memories of Bland J. Finlay and Work on Protists in “Priest Pot“

Priest Pot in the English Lake District: a showcase of microbial diversity

Summary 1. The distinctive characteristic of the water column of Priest Pot in summer is its intense physical and chemical stratification. The changing dissolved oxygen tension with depth is the main factor determining which microbes live where; distinct microbial communities exploit the different microbial niches at different depths and times. 2. Most biological activity in Priest Pot, whether in sediment or water column, is driven by microorganisms. The number of protist species recorded thus far is 883. Much remains to be discovered, particularly concerning scale-bearing protists. 3. Transmission electron microscopy has been used to study the microscales from, and on, the surfaces of microbial eukaryotes living in Priest Pot. The biological material was collected from fresh samples of the water column, from the sediment surface and from the pond’s immediate surrounding habitats. ‘Subfossils’ from the superficial sediment of the pond at the end of summer were also examined. 4. A total of 220 species of scale-bearing protists have been documented in the pond, belonging to the three functional groups or ‘body plans’ of protozoa: the amoeboid protozoa, the flagellates and the ciliates. The following genera are reported and illustrated: Acanthocystis, Chrysochromulina, Chrysolepidomonas, Chrysosphaerella, Cochliopodium, Cyathobodo, Gyromitus, Korotnevella, Lepidotrachelophyllum, Luffisphaera, Mallomonas, Paraluffisphaera, Paraphysomonas, Pompholysophrys, Pseudodendromonas, Raphidiophrys, Spiniferomonas, Synura, Thuamatomastix and Trachelophyllum/Bylamellophrya.

Turbulence and stratification in Priest Pot, a productive pond in a sheltered environment

Priest Pot is an example of the abundant ponds that, collectively, contribute crucially to species diversity. Despite extensive biological study, little has been reported about the physical framework that supports its ecological richness. This article elucidates the physical character of Priest Pot’s water column and thus that of similar water bodies. Vertical thermal microstructure profiles were recorded during summer 2003 and analyzed alongside concurrent meteorological data. During summer stratification, the thermal structure appeared to be dominated by surface heat fluxes. Surface wind stress, limited by sheltering vegetation, caused turbulent overturns once a surface mixed layer was present but appeared to contribute little to setting up the thermal structure. Variations in full-depth mean stratification occurred predominantly over seasonal and ∼5-day time scales, the passage of atmospheric pressure systems being posited as the cause of the latter. In the uppermost ∼0.5 m, where the stratification varied at subdaily time scales, turbulence was active (sensu Ivey and Imberger 1991) when this layer was mixed, with dissipation values e ∼ 10−8 m2 s−3 and vertical diffusivity KZ = 10−4 — 10−6 m2 s−1. Where the water column was stratified, turbulence was strongly damped by both buoyancy and viscosity, and KZ was an order of magnitude smaller. Vertical transport in the mixed layer occurred via many small overturns (Thorpe scale r.m.s. and maximum values were typically 0.02 m and 0.10 m, respectively), and seston were fully mixed through the water column.

Development of Temporal Temperature Gradient Electrophoresis for Characterising Methanogen Diversity

Temporal temperature gradient electrophoretic (TTGE) analysis of 16S rDNA sequences was optimized to monitor the methanogen population present in water and sediments of a small eutrophic lake, Priest Pot, in the English Lake district. The production of nonrepresentative TTGE profiles due to the generation of polymerase chain reaction (PCR) artifacts initially proved problematical. The use of a proofreading polymerase in the PCR was found to be essential and fully optimized protocols were established and tested to ensure confidence that the TTGE profiles truly reflected sequence diversity. TTGE analysis revealed the methanogen population to be less diverse in water than in sediment. The most genetic diversity was observed in TTGE profiles of sediment DNA isolated in winter and the least was in sediment DNA isolated in summer. DNA sequencing analysis of bands recovered from TTGE gels revealed the presence of two methanogen communities. One clustered with Methanosaeta species and the other with the Methanomicrobiales. Many sequences showed low DNA sequence similarity to known methanogens, suggesting that Priest Pot harbors previously undescribed methanogen species.

Temporal distribution of viruses, bacteria and phytoplankton throughout the water column in a freshwater hypereutrophic lake

Analytical flow cytometry (AFC) and denaturing gradient gel electrophoresis (DGGE) were used to investigate the diversity and dynamics of viruses, bacteria and phytoplankton commu- nities in a hypereutrophic freshwater lake. Samples were taken from different depths throughout the water column over an annual cycle. Priest Pot is a small lake in the Lake District in NW England (UK), and has been well characterised in previous studies; however, little is known of the diversity and dynamics of the virus community. Virus abundance was shown to change over both spatial and tem- poral scales, and appeared to be closely linked to other biotic and abiotic parameters. The highest virus concentrations occurred in the deepest part of the lake at a depth of 3.2 m, in the anoxic layer, at the same time as a peak of abundance of green sulphur bacteria. Sequence analysis of a DGGE band that occurred at the same time suggested a bacterium similar to Chlorobium spp., a green sulphur bacterium, comprised part of this bacterial bloom.

Hydropyrolysis of algae, bacteria, archaea and lake sediments; insights into the origin of nitrogen compounds in petroleum

Nitrogen compounds are ubiquitous in fossil fuels and yet our understanding of their origins in the geosphere is limited. In this study, high hydrogen pressure pyrolysis was performed on sample material representing potential contributors to sedimentary organic matter (algae, bacteria and archaea) and sediments representing early diagenetic accumulations from Lake Pollen (Norway) and Priest Pot (UK). Previous workers demonstrated the structurally conservative nature of high hydrogen pressure pyrolysis in that the technique maximizes the yields of covalently bound hydrocarbon biomarkers from organic matter without adversely affecting their stereochemistry ( Love et al., 1995). Release of covalently bound biomarkers in high yields from kerogen via catalytic hydropyrolysis. In this study, the types and distributions of organic nitrogen compounds in the hydropyrolysates were characterised under similar conditions to such experiments where biomarker hydrocarbons undergo minimal rearrangement. Compounds identified by gas chromatography–mass spectrometry included alkyl-substituted indoles, carbazoles, benzocarbazoles, quinolines and benzoquinolines. Indoles are present in all hydropyrolysates, suggesting a common origin. A potential source of indoles is represented by tryptophan which was shown to degrade through a series of alkylated intermediates to indole. Carbazole, quinoline and benzoquinoline were also found in the hydropyrolysates of algae, bacteria and archaea. The presence of these petroleum-related nitrogen compounds in hydropyrolysates generated from biomass suggests an early origin for petroleum nitrogen compounds. A potential source of naturally occurring nitrogen compounds such as that in the alkaloids has yet to be realised. Benzocarbazoles were absent from hydropyrolysates of algae, bacteria and archeae, but present in those from recent sediments, suggesting their presence may be related to processes occurring during early diagenesis at, or immediately below, the sediment–water interface. In sediments from Lake Pollen, changes in the benzocarbazole ratio [ a]/([ a] + [ c]) ratio coincides with the interval described as a transition from fjord to lake environment, suggesting that benzocarbazoles are sensitive to changes in depositional environment and may have potential to act as a marker for environmental conditions.

Analysis of polyacrylamide gels for trace metals using diffusive gradients in thin films and laser ablation inductively coupled plasma mass spectrometry.

A simple method for the analysis of polyacrylamide diffusive gradients in thin film (DGT) gels by laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS), employing a novel use of (115)In internal standardization, has been developed. This method allows the determination of Co, Ni, Cu, Zn, Cd, and Pb concentrations (at the DGT filter face) or fluxes in sediments at a spatial resolution of 100 microm. Single-layered gels, using an optimized laser defocus of 4000 microm at 400 mJ power, showed high precision (generally approximately 10%) and a linear response during solution deployment. Of the elements Sc, In, Ba, La, Ce, and Tb, Ba most closely tracked variations in laser energy and showed the highest analytical precision but could not be used as an internal standard due to its elevated presence in natural sediments. Therefore, internal standardization, necessary to normalize data collected on different days, was carried out using (115)In contained within a second layer of backing gel and dried along with the analyte layer as a dual-gel disk. This multilayered gel standard required a laser defocus setting of 1000 microm and a laser power of approximately 800 mJ. Analytical precision for a 64-spot ablation grid at 100-microm spacing was approximately 10%. Verification of this method was carried out on DGT sediment probes deployed in Priest Pot (English Lake District). Results obtained by conventional slicing techniques and aqueous elution agreed with laser ablation results when the different sampling areas were considered. The elution results varied by a factor of <2, whereas the laser ablation technique showed a variability of approximately 4, indicating localized elevated concentrations of Co. This higher resolution LA-ICPMS method could ultimately lead to an improved understanding of the geochemical processes responsible for metal uptake and release in sediments.

Ultrastructure and geographic distribution of the genus Paradermamoeba (Gymnamoebia, Thecamoebidae)

The genus Paradermamoeba includes two species with a distinct and unusually thick cell coat. This coat consists of densely packed helical glycostyles, up to 520 nm in length. Perhaps due to the impenetrable properties of the cell coat, we were unable to achieve satisfactory quality of EM fixation in these species until recently. In this study the treatment of cells with dilute Triton X-100 detergent prior to fixation has improved preservation of the ultrastructure of both Paradermamoeba species. Paradermamoeba valamo has an unusual central nucleolus consisting of densely packed fibrils of nucleolar material. A newly isolated strain of P. levis from a UK pond, Priest Pot, forms rounded, single-walled cysts. Both species have enigmatic trichocyst-like bodies in the cytoplasm. Despite the presence of a thick and highly differentiated cell coat, dictyosomes in both species are few and are not more developed than in many other species of gymnamoebae. Neither ultrastructure nor LM morphology indicates the phylogenetic position of Paradermamoeba. It seems that the family Thecamoebidae is a heterogeneous assemblage of unrelated genera. Recent isolation of both P. valamo and P. levis in Switzerland and in the UK indicates the wide distribution of both species in Europe.

Bacterioviridins: novel sedimentary chlorins providing evidence for oxidative processes affecting palaeobacterial communities

A series of novel sedimentary transformation products of bacteriochlorophyll a, 7,8-didehydro derivatives (bacterioviridins), has been identified using high performance liquid chromatography with photodiode array detection and atmospheric pressure chemical ionisation liquid chromatography-multistage mass spectrometry. The derivatives occur in a range of sediments including Priest Pot (UK), les Salines de la Trinital, Ebre delta (Spain), Sutton-on-the-Forest (UK) and Certes (France). The bacterioviridins are products of oxidative transformation of bacteriochlorophyll a, and their presence in sediments indicates exposure of the bacterial community to oxygen.

Analysis of methanogen diversity in a hypereutrophic lake using PCR-RFLP analysis of mcr sequences.

The incidence and diversity of methanogens in Priest Pot, a dynamic and active lake, were monitored by analysing mcrA gene sequences generated from total DNA samples obtained at different times of the year and amplified using the polymerase chain reaction. A number of mcrA clones were analysed by developing an RFLP-based protocol to generate a number of restriction patterns that were assigned to a number of classes. The RFLP patterns for each class were compared with published sequence information for mcrA from cultured methanogens as well as with those from other experimental studies. They could be used to assign tentative identification for some of the Priest Pot clones and also revealed the presence of a number of clones that could not be affiliated to any known methanogens. The limitations of using RFLP profiles of mcrA gene sequences for studying methanogen ecology are discussed.

Evidence for the rapid incorporation of hopanoids into kerogen

Hopanoids bound into the insoluble organic matter (kerogen) of Recent sediments from a freshwater lake (Priest Pot) and an anoxic sulphidic fjord (Framvaren) were released by hydropyrolysis and examined by gas chromatography-mass spectrometry. Bound hopanoids are present in high concentration (190–1400 μg/g TOC) and represent 22 to 86% of the total analysable hopanoids (i.e., bound and solvent-soluble), this proportion increasing with depth in Framvaren Fjord. The hopanes generated by hydropyrolysis contain higher amounts of the C 35, C 32, and C 30 homologues, reflecting the carbon number distribution of the bound hopanoids and indicating that both biohopanoids (C 30 and C 35) and their diagenetic products (dominated by C 32) are incorporated into the kerogen on a timescale of only 0 to 350 years. Sequential (multiple temperature) hydropyrolysis experiments gave an indication of the relative strengths of bonds being cleaved in association with hopane generation: The hopanoids of a sediment from Priest Pot are almost entirely bound by strong covalent bonds, interpreted to be mainly ether linkages, whilst a Framvaren sediment contains hopanoids that are bound by a mixture of weak di-/polysulphide linkages and stronger ether bonds. Labelling with deuterium indicated that the strong covalent linkages dominate, even for the Framvaren sediment.

Development and application of a high resolution liquid chromatographic method for the analysis of complex pigment distributions

Ternary and binary gradient systems have been developed for the high-performance liquid chromatographic analysis of complex pigment distributions typical of natural samples. Improved chromatographic resolution reveals significantly more pigment components in extracts from a sediment (Priest Pot, Cumbria, UK), a microbial mat (les Salines de la Trinital, South Catalonia, Spain) and a culture ( C. phaeobacteroides) including novel bacteriochlorophyll derivatives. The methods developed are directly suited to LC–MS analysis and the automated acquisition of MS/MS data for pigments.

Characterisation of the culturable heterotrophic bacterial community in a small eutrophic lake (Priest Pot)

The community composition and structure of planktonic heterotrophic bacteria (903 isolates) sampled from a small eutrophic lake in northern England (Priest Pot) was studied with respect to season (four samples) and depth (to 3.1 m). Bacteria (887) were isolated on tryptic soy broth agar and identified to 48 genera using fatty acid methyl ester analysis. The two most abundant genera isolated were Aeromonas and Pseudomonas which, respectively, dominated the middle to bottom depths in August and all depths in February. The structure of the sampled community was described using: species richness, Simpson's index and the Shannon-Wiener index. All three indices detected a number of significant differences with depth demonstrating stratification. The greatest stratification of the bacterial community was observed in August when bacterial counts correlated strongly and negatively with diversity. Using structural measures was found to be preferable to the use of species frequencies in the analysis of perturbation and succession in community structure. Insensitivity to one or more of eight antibiotics was observed in 71% (61/86) of the isolates tested particularly in Gram-negative genera. Bacteriocinogeny and lysogeny was observed in 36% (32/90) of isolates. Using sensitive indicator strains, two of 10 producing strains produced virus, while the others produced bacteriocins.

Characterisation of the culturable heterotrophic bacterial community in a small eutrophic lake (Priest Pot)

The community composition and structure of planktonic heterotrophic bacteria (903 isolates) sampled from a small eutrophic lake in northern England (Priest Pot) was studied with respect to season (four samples) and depth (to 3.1 m). Bacteria (887) were isolated on tryptic soy broth agar and identified to 48 genera using fatty acid methyl ester analysis. The two most abundant genera isolated were Aeromonas and Pseudomonas which, respectively, dominated the middle to bottom depths in August and all depths in February. The structure of the sampled community was described using: species richness, Simpson’s index and the Shannon–Wiener index. All three indices detected a number of significant differences with depth demonstrating stratification. The greatest stratification of the bacterial community was observed in August when bacterial counts correlated strongly and negatively with diversity. Using structural measures was found to be preferable to the use of species frequencies in the analysis of perturbation and succession in community structure. Insensitivity to one or more of eight antibiotics was observed in 71% (61/86) of the isolates tested particularly in Gram-negative genera. Bacteriocinogeny and lysogeny was observed in 36% (32/90) of isolates. Using sensitive indicator strains, two of 10 producing strains produced virus, while the others produced bacteriocins.

A novel sedimentary chlorin: structural evidence for a chlorophyll origin for aetioporphyrins

A novel sedimentary transformation product of chlorophyll, purpurin-7 phytyl ester, has been identified by atmospheric pressure chemical ionisation liquid chromatography mass-spectrometry in sediments from Lake Baikal, Russia, Loch Ness and Priest Pot, UK. This product of oxidative transformation is an intermediate on the transformation pathway leading to sedimentary aetioporphyrins and its occurrence lends strong support to the view that aetioporphyrins derive mainly from precursor chlorophylls.

Novel polyfunctionalised geohopanoids in a recent lacustrine sediment (Priest Pot, UK)

The sediment of a highly productive lake (Priest Pot, UK) contains the biosynthesised hopanoid bacteriohopanetetrol (BHT) and a series of hopanediols and triols. These polyols represent intermediate stages in the conversion of biologically produced hopanoids to the diagenetic products, hopanes. Two of these geohopanoids, assigned as trishomohopane-32,33-diol and bishomohopane-30,31,32-triol have not been previously reported in recent sediments, and their identification, in addition to that of tetrakishomohopane-32,33,34-triol and a bishomohopanediol gives valuable information on the original composition and subsequent diagenesis of bacteriohopanepolyols within recent sediments.

Fluxes of methane and carbon dioxide from a small productive lake to the atmosphere

The fluxes of CH4 and CO2 to the atmosphere, and the relative contributions of ebullition and molecular diffusion, were determined for a small hypertrophic freshwater lake (Priest Pot, UK) over the period May to October 1997. The average total flux of CH4 and CO2 (estimated from 7 sites on the lake) was approximately 52 mmol m−2 d−1 and was apportioned 12 and 40 mmol m−2 d−1 toCH4 and CO2 respectively. Diffusion across the air-water interface accounted for the loss of 0.4and 40 mmol m−2 d−1 of CH4 and CO2 respectively whilst the corresponding figures for ebullition losses were 12.0 (CH4) and 0.23 (CO2) mmol m−2 d−1. Most CH4 (96%) was lost by ebullition, and most CO2 (99%) by diffusive processes. The ebullition of gas, measured at weekly intervals along a transect of the lake, showed high spatial and temporal variation. The CH4 content of the trapped gas varied between 44 and 88% (by volume) and was highest at the deepest points. Pulses of gas ebullition were detected during periods of rapidly falling barometric pressure. Therelevance of the measurements to global estimates ofcarbon emission from freshwaters are discussed.

Identification of novel bacterial lineages as active members of microbial populations in a freshwater sediment using a rapid RNA extraction procedure and RT-PCR.

A rapid method for the extraction of RNA from the indigenous bacterial communities in environmental samples was developed. The method was tested using anoxic sediment samples from a productive freshwater lake (Priest Pot, Cumbria, UK). The simple protocol yielded rRNA and mRNA of a purity suitable for amplification by reverse transcriptase PCR (RT-PCR). The integrity of the RT-PCR was demonstrated by amplifying 16S rRNA and mRNA for the mercury resistance regulatory gene merR. The diversity of 16S rRNA sequences recovered from RNA and DNA extracted from anoxic Priest Pot sediments was analysed. The 5' end of extracted 16S rRNA was amplified by RT-PCR and the 16S rRNA PCR products were cloned and sequenced to identify active constituents of the sediment bacterial community. Corresponding analyses were performed upon DNA templates from the same sediment samples. Partial 16S rRNA sequences were obtained from a total of 147 clones (71 rRNA-derived and 76 rDNA-derived). The clone libraries included sequences related to Pirellula staleyi, an aerobic planktonic member of the Planctomycetales, and the recently described candidate bacterial division OP5. Sequences from these groups were recovered in libraries generated from a DNA template but were also present in RNA-derived libraries. Previous studies of anoxic environments have identified sequences most closely related to Pirellula spp. This study, which utilized RT-PCR of 16S rRNA, has provided the first evidence that Pirellula-like bacteria are active in situ in an anoxic environment. Furthermore, members of the recently described candidate division, OP5, were also identified as active constituents of the bacterial community of anoxic Priest Pot sediments. This not only supports the widespread occurrence of OP5 members in diverse environments but suggests that they are active under anoxic conditions.

Planktonic Ciliate Species Diversity as an Integral Component of Ecosystem Function in a Freshwater Pond

A diverse and dynamic community of ciliated protozoa lives in the stratified water column of the productive freshwater pond known as 'Priest Pot'. As part of a long-term continuous monitoring programme, this community was examined with 10 cm-scale vertical sampling in August 1995 and June 1997, and found to be dominated by species with endosymbiotic algae (1995), or by a quite different set of species, feeding on the dinoflagellate Peridinium (1997). On both occasions, the community structure was comprehensible in terms of the preceding sequence of reciprocal interactions involving microbiological, physical and chemical factors (e.g. oxygen depletion, thermal gradient, essential nutrient concentrations). In this one pond, very different ciliate communities appear at different times, yet each community may be nothing more than a transient bi-product of dynamic ecosystem functions. The facility with which the ciliate community (or any other microbial community) transforms in a continuously changing environment probably depends on a large local diversity of rare and encysted species and the rapidity with which these species fill vacant niches.