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Molecular Microbial Ecology
Abstract Book
Molecular Microbial Ecology
Meeting
2011
Abstract Book
Molecular Microbial Ecology
2
Welcome note
3
MMEG 2011 Programme
23 speakers in 5 sessions talks will be 15 minutes with 5 minutes for questions.
Tuesday 13th
December
12.00-1.45
Registration (lunch can be bought in refectory/cafes nearby)
Session 1 - Chair - Graeme Nicol
2.00-2.20 Jason Stephenson Methanotrophy in Movile Cave
2.20-2.40 Caitlin Burns Characterising the functional role of mycorrhizal fungi in
Miscanthus bioenergy cropping systems
2.40-3.00 Stephen Summers Microbial diversity and the potential influences on silicate
weathering.
3.00-3.20 James Houghton Cellulose degradation in anaerobic environments: molecular
biological approaches.
3.20-3.40 Joanne Preston Of grave conservation concern: molecular characterization of
microorganisms associated with the Mary Rose warship
reveals a diverse community of bacteria contributing to acid
production in her timbers.
3.40- 4.10
Coffee/Tea
Session 2 - Chair - Christopher van der Gast
4.10-4.30 Siobhan Watkins Beyond Compliance Monitoring: a Hierarchy of Investigative
Techniques, Including Molecular Microbiological Methods,
Provide Benefits for Operators of Biological Nutrient Removal
Package Sewage Treatment Systems
4.30-4.50 David Rooks Molecular methods for the study of Free viruses in the
Environment
4.50-5.10 Benjamin Folwell Novel approaches towards the bioremediation of recalcitrant
naphthenic acids and high molecular weight polycyclic
aromatic hydrocarbons in aquatic environments.
5.10-5.30 Agnieszka Kowalczyk The effect of inoculum quantity and quality on the
biodegradation of para-nitrophenol
5.30-5.50 Lawrence Davies The effect of light on soil microbial community structure and
the degradation of crop protection products
5.50-6.10 Lindsay Newbold Marine Picoplankton Responses to Ocean Acidification
6.10-6.30
Business meeting (location of MMEG 2012)
Followed by Wine reception at King Henry Building
20.00 Dinner at ‘The Old Customs House’ (map at back of abstract booklet)
4
Wednesday 14th
December
Session 3 - Chair - Joy Watts
9.10-9.30 Ian Lidbury Response of microbial biofilm communities to ocean
acidification in a natural carbon dioxide vent ecosystem
9.30-9.50 Paola Gomez-Pereira
Similar light-enhanced transport of organic molecules by
oceanic Prochlorococcus and SAR11 bacterioplankton
9.50-10.10 Adam Hamilton The role of microzooplankton grazing in regulating
phytoplankton and bacterioplankton biomass in the sub-
Arctic Atlantic Ocean
10.10-10.30 Will Gilbertson Effects of bioturbation on ammonia oxidising microbial
communities in marine sediments.
10.30-11.00
Coffee/Tea
Session 4 - Chair - Alan McCarthy
11.00-11.20 Jessica Poole Toxicity and impact of engineered nanoparticles on aquatic
microbial communities and their processes.
11.20-11.40 Daniela Wischer Methylated amines: nitrogen and carbon source for microbes
of the Movile Cave food web
11.40-12.00 Phillip James Contrasting Microbial Diversity over Soil Gradients with
Pyrosequencing
12.00-12.20 Matthew Wade and
Elizabeth Heidrich
Reliable Next Generation Sequencing for microbial
applications
12.20-1.20
Lunch (provided)
Session 5 – Chair - Kevin Purdy
1.20-1.40 Rachel Walton The role of extracellular DNA in microbial attachment to
surfaces.
1.40-2.00 Christopher Barnes
Mycorrhizal fungi community structures within bioenergy
crops.
2.00-2.20 Leah Cuthbertson Chronic respiratory infections: From ecological insights to
clinical benefit
2.20-2.40 Gregory Amos Analyses of the sediment metagenome and resistome
associated with waste water treatment plant effluent
2.40-2.50 Close of meeting
2.50-3.30
Coffee/ Tea
Session 1 5
Methanotrophy in Movile Cave
Jason Stephenson, Andrew Whiteley Colin Murrell School of life Sciences, University of Warwick, Coventry, West Midlands, CV4
7AL Movile Cave was discovered in 1986 after a 25 meter shaft was created in order to carry out
geological investigations (1,2). The cave is a sulfidic karst system situated in the south-
eastern corner of Romania along a geological fault about two kilometres from the Black Sea.
Movile Cave is unique in that it is an entirely sealed ecosystem that receives no input of
carbon from the upper photosynthetically driven environment. In spite of this there is a rich,
diverse flora and fauna that is supported by chemolithoautotrophy and methanotrophy,
most of which is thought to take place in floating biofilms found in discrete airbell
structures. One of the primary carbon sources is methane, of geological origin, that bubbles
up through groundwater flooding the lower region of the cave. Methane is oxidised by
methanotrophic bacteria to produce biomass and energy. The methanotrophs will release
carbon into their surrounding environment either by excretion or lysis, providing carbon for
organisms at subsequent trophic levels. As the methanotrophic bacteria are a buttress
supporting the Movile Cave biome, it is essential to understand the composition of the
microbial community and to determine those that are contributing significantly to the
microbial food web. A DNA Stable Isotope Probing approach was used with 13C methane in
order to determine the active methanotroph community and to follow the flow of carbon
into non-methanotrophs. 16S rRNA gene sequences from DGGE profiles of 13C enriched
samples suggest cross-feeding of the labelled carbon. Functional gene analysis of a clone
library of the pmoA gene encoding the alpha subunit of the particulate methane
monooxygenase indicated that Methylocystis, Methylomonas and Methylococcus species
were among the most dominant methanotrophs. These data are also in agreement with the
results of pmoA microarray analyses carried out using unlabeled DNA.
[1] Sarbu SM & Kane TC, 1995. NSS Bullitin. 57: 91–98, [2] Sarbu SM et al., 1996. Science. 272: 1953–1955.
Characterising the functional role of mycorrhizal fungi in Miscanthus
bioenergy cropping systems
Caitlin Burns1 Gary Bending1, Niall McNamara2
1 University of Warwick, 2 Centre of Ecology and Hydrology, Lancaster Arbuscular mycorrhizal fungi (AMF) live in symbiosis with around 80% of plant species, and
broadly increase plant yield, biomass, disease resistance and shoot P. Plants exchange
carbon sugars for nutrients scavenged by AMF, including phosphate. There is little known
about AMF in association with Miscanthus, a productive bioenergy crop grown across the
UK. My hypotheses are, 1) AMF are beneficial for plant growth and nutrition, 2) AMF species
vary across area and season, and 3) the most important pathway of P uptake to Miscanthus
is through AMF. Molecular techniques will then be used to identify which AMF species are
most important in P uptake in Miscanthus. Field samples from Lincolnshire were analysed
Session 1 6
using genetic and staining techniques. AMF were found to be present in Miscanthus roots,
and colonisation decreased significantly between June, August and October. These roots
host around ten species of AMF, many of which are uncultured glomus strains, and rare in
Britain. Further molecular work will determine if AMF community varies between seasons.
Microbial diversity and the potential influences on silicate
weathering.
Stephen Summers1,2
, Charles S. Cockell3
, Bruce Thomson2 and Andrew
Whiteley2
1Geomicrobiology Research Group, PSSRI, the Open University, Walton Hall,
MK7 6AA, UK , 2Molecular Microbial Ecology Laboratory, Centre for Ecology &
Hydrology, Benson Lane, Crowmarsh Gifford, OX10 8BB and 3 School of Physics
and Astronomy, James Clerk Maxwell Building, The Kings Building, University
of Edinburgh, Edinburgh, EH9 2JZ, UK. The rock-soil interface (critical zone) is where a variety of important earth system processes
occur, such as the sequestration of CO2 and pedogenesis from silicate weathering. This zone
is an important subsurface region of microbial activity in extreme environments because
bedrock dissolves and provides nutrients that are otherwise unobtainable to flora and
fauna. Yet the diversity and role of microorganisms at the critical zone is not well
understood. We examined microbial communities in vegetated and unvegetated critical
zones near Skorradalur Lake, Iceland, as well as conducted soil geo-chemical analysis at
these sites. Cultivation of microorganisms using organics-limited media produced isolates
associated from soils within cold environments (Polaromonas and Psychrobacter spp).
However, measures of isolate growth rates show most isolates have optimal growth at
temperature above 15°C suggesting that organisms are not optimally adapted to the critical
zone environment. Molecular analysis of the 16S rRNA gene clone libraries shows that the
communities are dominated by Rhizobiales, Actinobacteriales and Pseudomonadales;
indicating that culturing techniques have identified a fraction of the total diversity
The bacterial community at the critical zone is diverse in structure although whether this
diverse community is having any effect on the rate of weathering is still being investigated.
Initial experiments show that the bacteria increase the rate of rock weathering at the critical
zone and that many are capable of using the necromass from other organisms as a source of
carbon for growth in organic poor environments. These experiments show that microbial
processes are an important driver of weathering processes in the critical zone.
Session 1 7
Cellulose degradation in anaerobic environments: molecular
biological approaches.
James Houghton Microbiology Research Group, Institute of Integrative Biology, University of
Liverpool, james.houghton@liverpool.ac.uk. The microbial communities responsible for cellulose degradation in anaerobic environments
are not well understood. The use of molecular ecological methods allows the diversity of
these communities to be examined whilst sidestepping the issue of cultivating strictly
anaerobic, fastidious species. Focussing on landfill leachate, we have detected the presence
of species associated with cellulose degradation including relatives of the genus Fibrobacter
that were previously thought to exist only in the digestive tract of animals with a cellulose-
rich diet. Quantitative PCR data have provided evidence that these fibrobacters, along with
members of the genus Clostridium, have an important role in cellulose degradation in the
environment. These novel organisms could contain undescribed cellulase genes that may be
exploited in the production of second-generation biofuels. . We are therefore investigating
the metatranscriptomes of two anaerobic environments, freshwater lake sediment and
landfill leachate, using high-throughput pyrosequencing.
Of grave conservation concern: molecular characterization of
microorganisms associated with the Mary Rose warship reveals a
diverse community of bacteria contributing to acid production in her
timbers.
Joanne Preston1, Julian Mitchell1, Mark Jones2.
1School of Biological Sciences, University of Portsmouth, 2The Mary Rose Trust,
Portsmouth
The Mary Rose Tudor warship provides a unique environment for research. Submersion and
burial in anoxic marine sediment for almost 500yrs favoured reductive cycling of iron and
sulfur compounds in the Mary Rose hull and associated artefacts. Consequently, timbers of
the Mary Rose hull contain approximately 2 mass % reduced sulfur and iron compounds.
The microbial community associated with archaeological timbers under different states of
preservation have been characterized using both molecular and microbiological culturing
techniques. Acidophilic, iron and sulfur oxidizing cultures have been enriched using a range
of Mary Rose samples. Initial inocula included PEG preserved Mary Rose hull timber, biofilm
samples associated with the ship, unpreserved timber previously containing an iron bolt,
sulfur infested wood, and yellow deposits associated with degraded timber. Experimental
data using XANES (X-ray absorption near edge spectroscopy) analysis of pyrite (FeS 2)
impregnated oak demonstrates the biologically driven oxidation of reduced sulfur and iron
compounds by halotolerant bacteria enriched from the Mary Rose warship. Molecular
characterisation of the enriched acidophilic microbial communities and Mary Rose samples
has been achieved using 16S ribosomal DNA sequences derived from clone libraries. These
reveal that a diverse group of bacteria are contributing to acid production in timbers of the
Mary Rose Warship utilising a range of metabolic pathways.
Session 2 8
Beyond Compliance Monitoring: a Hierarchy of Investigative
Techniques, Including Molecular Microbiological Methods, Provide
Benefits for Operators of Biological Nutrient Removal Package Sewage
Treatment Systems
Siobhan C. Watkins1, John B. Williams2, Joy E.M. Watts1, Eric May1 1School of Biological Sciences, University of Portsmouth, 2School of Civil
Engineering and Surveying, University of Portsmouth
Wastewater treatment performed by package activated sludge systems is ideal for isolated
sources of commercial, industrial and agricultural waste. As overloading of sewage
treatment plants is becoming a frequent problem due to population increases, package
systems are emerging as a financially viable supporting technology. A package system
capable of treating a population equivalent of 100 was installed to perform biological
nutrient removal, and physical, chemical and microbiological parameters were assessed.
Presented here is an overview of the assessment of the system, using a range of
methodology including compliance testing, physico-chemical monitoring and culture-based
and non-culture based microbiological methods, including denaturing gradient gel
electrophoresis (DGGE). Using molecular microbiological methods, an indication of the level
of diversity of the microbial community composition and stability of this package system
was investigated and linked to overall treatment performance. This multidisciplinary
approach is presented as a monitoring hierarchy, results from which may be used by
operatives maintaining package systems in the field, the practical application of which was
an example of a knowledge transfer exercise. The success of the hierarchy of investigative
techniques was found to be dependent on the implementation of all types of methodology.
Molecular methods for the study of Free viruses in the Environment
David J. Rooks The University of Liverpool, Institute of Integrative Biology, Microbiology
Research Group, Biosciences Building, L69 7ZB, Liverpool, UK
david.rooks@liv.ac.uk
Conversion of a member of the enteropathogenic E. coli (EPEC) group to a hypervirulent
enterohaemorrhagic E. coli (EHEC) was primarily due to the acquisition of Shiga toxin
encoding temperate bacteriophages that are diverse but share a distinct genome
organization with bacteriophage lambda. Ruminants are the primary reservoir of Shiga
toxin producing E. coli (STEC) which are a recognized public health concern worldwide
causing diarrhoea, hemorrhagic colitis (HC) haemolytic uremic syndrome (HUS) and
thrombotic thrombocytopenic purpura (TTP) in humans. Although there are data on the
distribution of STEC, the occurrence and characterization of Stx phage as free entities has
received little attention, and consequently the epidemiological significance of Stx phages in
the environment is unknown. Furthermore, it is likely that the phage population in any
environment is underestimated in any given environment because of the limitations of
traditional identification and propagation techniques. The stx gene is located downstream
Session 2 9
of the anti-terminator gene Q, and exhibits a degree of sequence conservation which
enabled the design of qPCR primers. Application of these primer sets to water samples
possessing E. coli infecting phages detectable by plaque assay, demonstrated that one in 103
free lambdoid bacteriophages carried a Shiga toxin operon (stx). We have also developed a
novel method for harvesting viral DNA, which does not contain any detectable levels of
cellular DNA by end point PCR, or by homology searches of a 454 pyrosequence derived
metagenome library. The pyrosequenced Virome generated from a freshwater sample
contained a total of 41, 916 sequence fragments (~224 bp) of which 24% were assigned an
identity by BLASTX using the SEED database. We used qPCR to quantify the number of
lambdoid and Stx phages in the sample, and the ratios were similar to the abundance ratios
calculated from the 454 virome data. However the absolute numbers of lambdoid and Stx
phages obtained by qPCR suggest that the depth of sequencing performed had enabled us
to examine only c.a. 5% of the total Virome. These methods represent a novel approach to
epidemiological/ecological studies of free viruses in the environment.
Novel approaches towards the bioremediation of recalcitrant
naphthenic acids and high molecular weight polycyclic aromatic
hydrocarbons in aquatic environments.
Benjamin D Folwell1, Andrew Price2, Terry J McGenity1 and Corinne
Whitby1 1Department of Biological Sciences, University of Essex, Wivenhoe Park,
Colchester, CO4 3SQ, UK.
2Oil Plus Ltd., Dominion House, Kennet Side, Newbury, RG14 5PX, UK.
Email : bdfolw@essex.ac .
Currently >50% of global oil reserves are found as biodegraded heavy super-heavy oils in oil-
sand deposits which are not yet fully exploited. During oil-sand refining, vast quantities of
oil-sands tailings pond waters (TPW) are generated that contain complex mixtures of
carboxylic acids known as naphthenic acids (NAs). NAs are highly toxic and thus the removal
of TPW is of great environmental concern. High molecular weight polycyclic aromatic
hydrocarbons (HMW-PAHs) are also natural components of fossil fuels and are classified as
priority pollutants of aquatic environments.
The aim of this study is to investigate the biodegradation of recalcitrant NAs and HMW-
PAHs. TPW samples were incubated in minimal media containing hydrophobic filters with
HMW-PAHs (pyrene, benzo[a]pyrene and benzo[b]fluroanthene at 200 mg L-1) sorbed onto
the filters. DGGE analysis revealed that the biofilm communities enriched on the filters were
80% - 95% similar to planktonic communities for each PAH with 70% similarity in community
composition between the different HMW-PAHs. Further, benzo[a]pyrene and
benzo[b]fluroanthene enriched for more similar communities, while communities enriched
on pyrene were more distinct. Significantly, all the communities enriched on HMW-PAHs
shared only 55% similarity with the original TPW community. In all cases PAH-specific DGGE
bands were also obtained; suggesting that different microbial communities are required to
metabolise recalcitrant HMW-PAHs.
Session 2 10
The potential of artificial bacterial algal co-cultures of NA-degrading Pseudomonas putida,
Pseudomonas fluorescens and Chlorella vulgaris (a NA-tolerant alga), to degrade NAs was
also assessed. C. vulgaris had a high tolerance to NAs, and grew well (OD600 0.126) after 10
days incubation compared to Dunaliella minuta (OD600 0.068) and Cyclotella
pseudostelligera (OD600 0.008). Stable artificial co-cultures were established for use in NA
biodegradation experiments.
These findings will enable more efficient bioremediation strategies to be developed and
facilitate the removal of these important recalcitrant pollutants from aquatic environments.
The effect of inoculum quantity and quality on the biodegradation of
para-nitrophenol
Kowalczyk A1, van Egmond RA2, Finnegan CJ2, Price OR2, Schäfer H1,
Bending GD1 1 School of Life Sciences, Wellesbourne Campus, The University of Warwick,
Wellesbourne, Warwick CV35 9EF, 2 Safety & Environmental Assurance Centre, Unilever, Colworth Science Park,
Sharnbrook, Bedfordshire, MK44 1LQ, UK
OECD chemical biodegradation tests lack environmental realism and do not consider test
inoculum quantity and quality. In the present study we investigated the effect of inoculum
source and size on the biodegradation of para-nitrophenol (PNP), as well as associated
effects of Sewage Treatment Plant (STP) effluent on the quality of river water and sediment
inoculum. River water and sediment samples were collected from the River Dene, upstream
and downstream of Wellesbourne STP along with STP effluent. A culture-dependent
approach was used to determine the size of collected inoculum and to isolate PNP
degraders. Molecular microbial ecology methods (16S rRNA TRFLP analysis and qPCR
analysis of PNP functional markers (pnpA and mar)), were applied to study the community
structure and biodegradation potential of tested inoculum. HPLC analysis revealed that PNP
biodegradation was not affected significantly by inoculum source. Dominant bacterial
strains isolated after PNP degradation were identified as Pseudomonas syringae,
Pseudomonas putida and Pseudomonas fluorescens. TRFLP profiles of bacterial 16S rRNA
genes were significantly different only in effluent before incubation, and a shift in
community structure due to proliferation of PNP-degrading bacteria was observed at the
end of PNP degradation for all types of inoculum. Effluent from the Wellesbourne STP did
not affect the biodegradability potential of river water and sediment samples which were
collected downstream of STP. However, the copy number of both PNP functional markers
pnpA and mar was much higher in effluent samples before PNP degradation in comparison
to freshly collected river water and sediment samples.
Keywords: inoculum source, para-nitrophenol, biodegradability potential, functional marker
Session 2 11
The effect of light on soil microbial community structure and the
degradation of crop protection products
Lawrence O Davies1,2
, Hendrik Schäfer1, Sam Marshall
2, Irene Bramke
2,
Robin Oliver2, and Gary D Bending
1
1School of Life Sciences, University of Warwick, Wellesbourne, Warwick, CV35
9EF, UK.
2Syngenta, Product Safety, Jealott’s Hill International Research Centre,
Bracknell, Berkshire, RG42 6EY, UK
The biological soil crust (BSC), or the top millimeter of soil under the influence of light, has
functional importance in arid lands; it has roles in reducing soil erosion, improving water
infiltration, and nitrogen (N2) cycling. The importance of BSCs on agricultural land has not
been investigated, however, phototrophs in the BSC represent the first point of contact for
crop protection products (CPPs). In this study we investigated the effect of non-UV light on
the rate of degradation of a wide range of CPPs by comparing degradation under light and
dark conditions.
The DT50 (time it takes for 50% degradation) of fungicide A was approximately halved from
373d to 183d under light conditions. The DT90 (90% degradation) of chlorotoluron was
similarly halved from 79d to 35d under light conditions. There was also a significant increase
in the rate of degradation for prometryn (4%), imidacloprid (8%), and fludioxonil (24%)
under light. In contrast, there was a significant reduction in the rate of cinosulfuron (14%)
degradation under light conditions, and no difference for propiconazole and lufenuron.
This study also investigated the diversity of phototrophs in the BSC using molecular tools by
analysis of 23S rRNA genes in the top 3mm of soil under light. Phototrophs with close
homology to N2-fixing cyanobacteria Nostoc and Anabaena accounted for >50% of
sequences and terminal restriction fragment length polymorphism (TRFLP) found their
abundance to increase over time. A range of algae, cyanobacteria, diatoms, mosses, and
xanthophyta were also detected. TRFLP analysis of bacteria (16S rRNA) showed light to have
a significant time-dependent effect on bacterial community structure.
This is the first work to show that non-UV light has an effect on: (i) The rate of CPP degradation; (ii) Bacterial community structure.
Session 2 12
Marine Picoplankton Responses to Ocean Acidification
Lindsay Newbold1,2
, Anna Oliver1, Tim Booth
1, Bela Tiwari
1, Todd DeSantis
3,
Michael Maguire2, Gary Andersen
3 , Christopher van der Gast
1 and Andrew
S. Whiteley1
1Centre for Ecology and Hydrology, Wallingford, Benson lane, Crowmarsh
Gifford, Wallingford, OX10 8BB, U.K 2
Civil Engineering and Geosciences, University of Newcastle upon Tyne,
Newcastle Upon Tyne, NE1 7RU, U.K 3 Earth Sciences Division, Lawrence Berkeley National Laboratory, Cyclotron
Lane, Berkley, CA 94720, USA.
Since industrialisation global CO2 emissions have increased, and as a consequence oceanic
pH is predicted to drop by 0.3-0.4 units before the end of the century - a process coined
‘ocean acidification’ (OA). Consequently, there is significant interest in how pH changes will
affect the oceans’ biota and integral processes. Here, we investigated marine picoplankton
(0.2-2µm diameter) community composition and abundance in response to predicted end of
century CO2 concentrations via a 1‘high CO2’ (~750ppm) large volume (11,000 L) contained
seawater mesocosm approach. We found little evidence of changes occurring in bacterial
abundance or community composition due to elevated CO2 treatment under both
phytoplankton pre-bloom/bloom and post-bloom conditions. In contrast, significant
differences were observed between treatments for a number of key picoeukaryote
community members. Further, a classical predator-prey relationship was noted between
bacteria and picoeukaryotes suggesting the two populations were inherently linked by
trophic interaction.
These data indicated that a key outcome of ocean acidification is a more rapid exploitation
of elevated CO2 levels by photosynthetic picoeukaryotes, in tandem with probable
mixotrophic utilisation of bacterial biomass. Our study indicates the need for a more
thorough understanding of picoeukaryote mediated carbon flow within ocean acidification
experiments, both in relation to picoplankton carbon sources and sinks, and the ultimate
effect of carbon transfer to higher trophic levels.
Session 3 13
Response of microbial biofilm communities to ocean acidification in a
natural carbon dioxide vent ecosystem
Ian Lidbury1,2,3
, Colin Munn2
and Michael Cunliffe3
1University of Warwick, UK
2The University of Plymouth, Plymouth, UK
3The Marine Biological Association of the United Kingdom, Plymouth, UK
Due to anthropogenic fossil fuel consumption, the oceans are set to experience a significant
drop in seawater pH coupled with an increase in dissolved CO2, a process commonly refe
rred to as ocean acidification. A number of studies have shown that the structure and
function of both phytoplankton and bacterioplankton assemblages are affected by ocean
acidification, however no work has been conducted on marine biofilm communities. Marine
biofilms are integral cogs of marine ecosystems because they present an important food
source for marine herbivores. Natural CO2
vents sites are ideal systems to study the
potential effects of ocean acidification on marine microbial communities. Here we report,
for the first time, that biofilm structure and function are altered along a naturally-occurring
pH gradient. Biofilm extracellular polysaccharide substance (EPS) production increases in
areas of high CO2
partial pressure (pCO2) along with overall biofilm biomass. DGGE profiles
suggest the structure of both the bacterial and eukaryotic communities in high pCO2
areas is
different. Furthermore, sequencing a number of the 18S rRNA gene DGGE bands revealed
that areas of high pCO2
stimulated the photoautotrophic community. These results provide
direct evidence that elevated seawater pCO2
has the potential to increase primary
production in marine ecosystems, which is in agreement with previous short term CO2
perturbation experiments. This study opens the door to further research aiming to predict
microbial community responses to ocean acidification.
This work was carried out at the The Marine Biological Association of the United Kingdom in conjunction with the University of Plymouth prior to joining the University of Warwick.
Similar light-enhanced transport of organic molecules by oceanic
Prochlorococcus and SAR11 bacterioplankton
Paola R. Gomez-Pereira1, Manuela Hartmann
1, Carolina Grob
2, Adrian P.
Martin1, David J. Scanlan
2 and Mikhail V. Zubkov
1
1National Oceanography Centre, Ocean Biogeochemistry & Ecosystems
Research Group, European Way, Southampton, SO14 3ZH, United Kingdom 2School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4
7AL, United Kingdom
Bacterioplankton are the most abundant organisms in the surface ocean, where light is a
plentiful and reliable resource. The hypothesis that bacterioplankton harness light for
Session 3 14
transporting organic molecules was tested in the oligotrophic North Atlantic subtropical
Gyre, using α-33
P-ATP and 35
S-methionine as tracers for uptake of nucleotides and amino
acids, respectively. Total bacterioplankton depicted a consistent light enhanced uptake of
both substrates in all experiments performed. When exposed to light, the uptake rates of
ATP and methionine increased by 27% and 23%, respectively. In order to identify which
groups were responsible for the higher uptake in the light, we evaluated the uptake rate of
the major populations by flow cytometric sorting of labelled cells. The two dominant
bacterioplankton groups were the cyanobacteria Prochlorococcus and the non pigmented
bacteria with low nucleic acid (LNA) content. For both bacteria the uptake rate of ATP was
lower than the uptake rate of methionine. However , the light-induced increase in the
uptake of ATP and methionine was remarkably similar for both groups. When exposed to
light, Prochlorococcus cells took 31% more ATP and 36% more methionine, whereas the
SAR11 cells took 35% more ATP and 32% more methionine. Thus, we showed that
bacterioplankton groups with potentially different light harvesting mechanisms increased
the transport of organic molecules in comparable proportions. The above data provides
experimental evidence that two major oceanic bacterioplankton groups could ecologically
benefit from a photoheterotrophic lifestyle.
The role of microzooplankton grazing in regulating phytoplankton
and bacterioplankton biomass in the sub-Arctic Atlantic Ocean
A.S.Hamilton1, M.S.Hale
1, G.R.Fones
1 and R.B.Rivkin
2
1 School of Earth & Environmental Sciences, University of Portsmouth, Burnaby
Building, Burnaby Road, Portsmouth PO1 3QL, UK 2 Ocean Sciences Centre, Memorial University of Newfoundland, St. John’s, NL A1C
5S7, Canada
Given the indications of residual nitrate concentrations and low phytoplankton biomass, an
essential question is whether the sub-Arctic North Atlantic is a HNLC region due periodic Fe
limitation, or whether low phytoplankton biomass is due, in part, to top down grazing
pressure from microzooplankton. Fieldwork was carried out during May and July 2010
aboard the RRS Discovery in the sub-Arctic Atlantic. Water was collected at 20 m, using a
Titanium rosette CTD set and modified trace metal clean dilution assays were conducted
with and without Fe additions to determine growth and loss of phytoplankton and bacteria,
and to investigate grazer responses to Fe-stimulated phytoplankton and bacterioplankton
growth. Microzooplankton bacterivory and herbivory were determined from changes in
bacteria abundances and chlorophyll a concentrations, during 48 hr incubations, assuming
exponential growth. Initial results show a difference in phytoplankton growth and mortality
rates between control and Fe-enriched conditions and suggest that grazing plays an
important role in maintaining phytoplankton biomass. Microzooplankton responded rapidly
to increases in phytoplankton biomass in Fe-enriched experiments and maintained strong
top-down control, suggesting that microzooplankton are important in regulating
phytoplankton and bacterioplankton biomass in the hypothesised seasonally Fe-limited sub-
Arctic North Atlantic.
Session 3 15
Effects of bioturbation on ammonia oxidising microbial communities
in marine sediments.
Will Gilbertson1,2, Martin Solan1,3, James I Prosser2 1 Oceanlab, Institute of Biological and Environmental Sciences, University of
Aberdeen, Newburgh, Aberdeenshire, Scotland, AB41 6AA 2 Institute of Biological and Environmental Sciences, University of Aberdeen,
Cruickshank Building, St Machar Drive, Aberdeen, Scotland, AB24 3UU 3 Ocean and Earth Science, National Oceanography Centre, Southampton,
University of Southampton, Waterfront Campus, European Way, Southampton,
SO14 3ZH.
Mixing caused by invertebrate activity (bioturbation) has a well established role in a number
of key microbe-mediated processes in marine sediments, including nutrient and organic
matter cycling; however the microbial mechanisms underlying rate changes are largely
unknown. To investigate these mechanisms, microbial community structure and function
was examined in large mesocosms containing one of three functionally differing
invertebrates (a polychaete, Hediste diversicolor; amphipod, Corophium volutator; and mud
snail, Hydrobia ulvae) and across the burrow profile of cross-sectional microcosms
containing a large polychaete, Allita virens. In large mesocosms, nitrogen transformation
rates increased under all three invertebrate species treatments; nitrification was highest
under C. volutator whilst denitrification and mineralisation were highest under H.
diversicolor. However, overall abundance (determined by quantitative PCR of amoA gene) of
ammonia oxidising bacteria (AOB) and ammonia oxidising archaea (AOA) and DGGE
community profiles did not change significantly in surface sediment. AOB consistently
outnumbered AOA, though the AOB:AOA ratio did increase significantly under C. volutator
from ~ 5:1 to 10:1. In Allita virens cross-sectional microcosms, burrow wall sediment
contained higher abundance of AOA than ambient sediment, both at the surface and at
depth. In contrast, AOB abundance was fairly uniform throughout the sediment profile, but
AOB amoA transcriptional activity was higher in burrow wall sediment at depth, resembling
the transcriptional activity in surface sediment. The clear differences in microbial function
observed here with invertebrate burrowing, appear to be linked with community changes
restricted to within burrow structures, but with differing responses in AOB and AOA.
Session 4 16
Toxicity and impact of engineered nanoparticles on aquatic microbial
communities and their processes.
Jessica Poole*1, Bjorn Stolpe
2, Paula Cole
2, Jamie Lead
2, Melanie Sapp
3, Ian
Colbeck1 & Corinne Whitby
1.
1Dept. of Biological Sciences, University of Essex, Colchester, UK, CO4 3SQ;
2School of Geography, Earth and Environmental Sciences, University of
Birmingham, Edgbaston, UK, B15 2TT; 3CEFAS, Lowestoft Laboratory,
Lowestoft, UK, NR33 OHT. *Email jpoolea@essex.ac.uk
Engineered nanoparticles (ENP) often exhibit novel or improved properties over larger
particles and are increasingly used in consumer products ranging from cosmetics, to
clothing, electronic goods and household appliances. At present, very little is known about
the fate or behaviour of ENP in the environment, which has led to concern over the
potential risks ENP pose to living organisms and biological systems. Some ENP (e.g. silver)
are powerful antimicrobial agents with the potential to disrupt environmental microbial
communities and their processes . However there is currently little information on the
effect of ENP on in situ microbial communities. The aims of this study were to characterise
different nanoparticle species including silver (AgNP), titanium dioxide (TiO2NP) and carbon
nanotubes (CNT), measure their toxicity to pure bacterial cultures, and investigate their
effects on key microbial processes such as nitrification and hydrocarbon biodegradation.
Nanoparticle toxicity as measured by Microtox and bacterial growth assays revealed that
AgNP were more toxic than TiO2NP or CNT; where AgNP completely inhibited Escherichia
coli and Bacilus subtilis at 50 mg L-1, CNT and TiO2NP had no significant effect on the
growth of five bacterial species tested at concentrations up to 50 mg L-1. In freshwater
sediments, AgNP (at 50 mg L-1) caused a 28% reduction in microbial enzyme activity after 7
days. In addition there were significant changes in microbial community structure after 7
days in the presence of crude oil and AgNP (50 mg L-1) compared to controls. Despite this,
AgNP did not affect total cell numbers which remained between 2.8-16.6 x106 cells g dry
weight sediment-1 throughout a 14 day exposure period, and hydrocarbon degradation was
unaffected by the presence of AgNP.
TEM image s of NP-bacterial cell interactions indicate that AgNP and Ag+ (at 50 mg L-1) have
the potential to cause cell membrane damage and may even enter cells. Our results suggest
that although CNT and TiO2NP may not pose a risk to environmental microbial communities,
AgNP and Ag+ ions released from such particles may have a detrimental effect on aquatic
microbial communities and their processes.
Session 4 17
Methylated amines: nitrogen and carbon source for microbes of the
Movile Cave food web
Daniela Wischer, Yin Chen and J. Colin Murrell School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK
Methylated amines, produced during putrefaction, are used as carbon and energy source by
some methylotrophic bacteria. They are also nitrogen sources for a wide range of non-
methylotrophic bacteria. The role of methylated amines in the food web of Movile Cave, an
unusual underground ecosystem sustained exclusively by chemolithoautotrophy and
methanotrophy, is being studied. Carbon fixation by non-phototrophic microorganisms in
the cave is the ultimate driver of a complex food web involving bacteria, fungi and diverse
macrofauna. Extensive microbial mats grow at the redox interface between reduced, sulfidic
waters and the oxygenated atmosphere. Recycling of organic matter in Movile Cave
produces high amounts of degradation products, including methylated amines. Stable
isotope probing enrichments set up with 13C-labelled methylamine confirmed the presence
of active methylotrophs in the waters of the cave, including Movile Cave isolates obtained
with methylated amines as sole carbon and nitrogen source. Both known and novel
methylotrophs were identified as dominant methylamine utilisers. Isolates were
characterised with regard to the range of methylated amines used and their methylamine
metabolic pathways. The recently characterised indirect methylamine oxidation pathway
involving gamma- glutamylmethylamide [GMA] and N-methylglutamate [NMG](1,2,3) was
the dominant pathway amongst both methylotrophs and non-methylotrophs, as revealed by
PCR using a newly developed set of primers targeting the GMA synthetase (gmaS) gene. The
gene for methylamine dehydrogenase (mauA), involved in the conventional, direct
methylamine oxidation pathway, was detected in a number of methylotrophic isolates in
addition to gmaS. Out of all the characterised isolates, only one of the Paracoccus spp.
contained mauA but not gmaS, indicating horizontal transfer of this gene. The new gmaS
primers are currently being tested on environmental samples and have great potential as a
biomarker for identifying a wide range of methylamine-utilising bacteria not detected by
current primer sets that target only methylotrophs possessing the mauA gene.
(1) Latypova, E., S. Yang, Y.-S. Wang, T. Wang, T. A. Chavkin, M. Hackett, H. Schäfer, and M. G. Kalyuzhnaya.2010. Genetics of the glutamate-mediated methylamine utilization pathway in the facultative methylotrophic beta-proteobacterium Methyloversatilis universalis FAM5. Mol Microbiol 75:426-439. (2) Chen, Y., Scanlan, J., Song, L., Crombie, A., Rahman, M.T., Schäfer, H., and Murrell, J.C. 2010. γ-Glutamylmethylamide is an essential intermediate in the metabolism of methylamine by Methylocella silvestris. Appl Environ Microbiol 76:4530–4537. (3) Chen, Y., McAleer, K.L., and Murrell, J.C. 2010. Monomethylamine as a nitrogen source for a nonmethylotrophic bacterium, Agrobacterium tumefaciens. Appl Environ Microbiol 76:4102–4104.
Session 4 18
Contrasting Microbial Diversity over Soil Gradients with
Pyrosequencing
James, P.L.,1,2 Thomson, B.C.,1 Whiteley A.S.,1 Bailey, M.J.,1 Griffiths, R.I.,1
1Centre for Ecology & Hydrology, Maclean Building, Benson Lane, Crowmarsh
Gifford, Wallingford, Oxfordshire, OX10 8BB 2 School of Biology, Ridley Building, University of Newcastle, Newcastle upon
Tyne NE1 7RU Using 16S rRNA targeted Terminal Restriction Fragment Length Polymorphism (T-RFLP)
analysis of over 1100 soils from across the United Kingdom, we show that soil pH was the
most important determinant of bacterial community structure and diversity. Conversely,
when examining the Internally Transcribed Spacer region, no environmental variable was
significantly correlated with fungal diversity or community structure. To gain a more
taxonomically meaningful insight into community changes brought about by variance in soil
pH, nucleic acid extracts from 15 geographically independent samples representing low,
medium, and high pH soils underwent pyrosequencing analysis. Multiple gene regions were
targeted as candidates for optimal taxonomic inference. In the case of soil bacteria the V1 –
V3 and V6 – V9 regions of the 16S rRNA gene were targeted, and the 18S rRNA gene was
targeted for fungal community analysis. Analysis of both kingdoms via pyrosequencing
suggests a restriction of diversity within acidic soils. Principle coordinate analysis (PCoA) of
fungal and bacterial Unifrac based dissimilarity matrices showed a clear separation of
samples based upon pH group. The reported taxonomic composition of the bacterial
communities was highly dependent upon the region of the 16S rRNA gene targeted.
However, in summation, the large differences in community structure seen between the low
and high pH soils in bacterial communities was predominantly attributed to the ratio of
group 1 to group 6 Acidobacteria. Within fungal populations, the proportional abundance of
the Leotiomycetes (Ascomycota) decreased dramatically with increasing soil pH, and the
Chytridiomycetes (Chytridiomycota) exhibited the opposite trend. The Tremellomycetes
(Basidiomycota) showed a preference for more neutral soils. The impact of this research
aids in the construction of a basic ecological framework for two dominant soil microbial
kingdoms, and provides a foundation for further studies to generate a more detailed insight
into how ecosystems operate.
Session 4 19
Reliable Next Generation Sequencing for microbial applications
Dr. Matthew Wade & Dr. Liz Heidrich School of Civil Engineering & Geosciences
Newcastle University
Next generation sequencing is fast becoming the primary tool for microbial applications.
Decreasing material and hardware costs have facilitated the emergence of robust and
powerful software tools necessary for processing and analysing the large datasets
generated.
Many challenges still exist that scientists must address in order to rapidly and effectively
produce outputs from raw sequences. A major factor in microbial ecology is the role of
noise in the misclassification of OTU identification. It has been shown that PCR amplification
errors (e.g. single base substitutions), sequencing errors and PCR chimeras play a major role
in inflating the estimate of microbial diversity in 16s rRNA samples generated from high-
throughput sequencers.
The performance of an amplicon noise removal algorithm (Quince et al., 2011) coupled with
high performance computing techniques (parallelisation with multiple core processing) was
examined on 21 environmental sample datasets.
The samples were taken from various bioelectrochemical reactors with an abundance of
exoelectrogenic organisms. Samples of wastewater and soil used to seed these reactors
were also sequenced. The massive sequencing effort of this study has divulged information
about the selection process within reactors and diversity of the biofilms at different
temperatures and with different substrates.
The results showed that the noise removal step produced greatly reduced numbers of OTUs
in each sample and a corresponding improvement in accuracy of the predicted microbial
diversity. Gaining a true measure of diversity and abundance allows for a depth of
understanding in the microbial ecology of engineered systems not previously possible.
Session 5 20
The role of extracellular DNA in microbial attachment to surfaces.
Rachel C Walton, Stephen A Rolfe, Julie Scholes, Colin L Freeman, John H
Harding, Wei E Huang, Steven Banwart. Department of Animal and Plant Sciences, University of Sheffield, Western
Bank, Sheffield, S10 2TN Understanding the mechanisms that control cell-substrate and cell-cell attachments is
necessary for biofilm formation and has applications in areas as diverse as bioremediation
through to biomedical devices. The aim of this project is to combine an experimental
approach with theoretical molecular modelling to explore the role that extracellular DNA
(eDNA) plays in cell-surface interactions. We have selected eDNA as a model system as it
known that macromolecules on the bacterial cell surface involved in surface attachments
vary between genera [1]
but in some cases extracellular eDNA is crucial [2]
. DNA is easily
manipulated through molecular biology techniques and also has a simpler structure than
macromolecules such as proteins, thus facilitating molecular simulations. For these reasons
we have chosen to study an environmental bacteria (Pseudomonas sp. Pse1) known to form
biofilms in an eDNA-dependent manner[1]
.
We have developed an assay to quantify cell attachment to fused silica slides in liquid
media. Image analysis of the substratum enables us to quantify how many, and how firmly,
cells are attached and to distinguish these from unattached cells moving freely in the
medium. Results show that removal of eDNA from the cell surface by DNAse treatment
causes a significant decrease in cell adherence to the substratum and that this can be
restored with either Pse1 genomic DNA or salmon sperm DNA. Cell adherence can also be
restored with DNA of different lengths from 200 bp to 20 kbp. We now intend to quantify
how much DNA is bound to the each cell (using radiolabelled DNA), the role of inorganic
ions in attachment and the biophysical properties of DNA-cell and DNA surface interactions
(using Atomic Force Microscopy). These data are key data for numerical modelling studies.
[1] J.S. Andrews et al. Environ. Microbio. 12 (2010) 2496 [2] C.B. Whitechurch et al. Science 295 (2002) 1487
Mycorrhizal fungi community structures within bioenergy crops.
Christopher Barnes1, Dr. Gary Bending1, Dr. Niall McNamara2 1University of Warwick, 2CEH Lancaster c.j.barnes@warwick.ac.uk
Bioenergy crops occupy a growing proportion of farmland within the UK but little is known
on their long-term environmental impacts. Mycorrhizal fungi can influence plant diversity
and vitality, assisting their host in gathering nutrients. Again, very little is known about the
ecological role of mycorrhizal fungi within a bioenergy crop context. The aim of this project
is to compare and contrast mycorrhizal fungi community structures within Willow and
Miscanthus plantations. Mycorrhizal fungi’s role in sequestering carbon as soil organic
matter will also be analysed. Stable isotope probing using 13
C will be used to determine the
fate and longevity of recently derived photosynthate. The use of root and hyphal exclusion
Session 5 21
cores should alter soil communities so that individual community member’s contribution to
carbon cycling can be assessed using the label. So far this research has demonstrated that
ectomycorrhizal fungi dominate a Willow plantation in Brattleby, Lincolnshire, and that the
community structure of these vary spatially. Soil nutrients such as pH and phosphorus seem
to be the key determinants on soil community. It was also shown that there are high levels
of diversity, with 30 different species being found across a 180 m line transect.
Chronic respiratory infections: From ecological insights to clinical
benefit
Leah Cuthbertson1,2, Anna Oliver1, Geraint Rogers2, Alan Walker3, Ken
Bruce2, Christopher van der Gast1 1 NERC Centre for Ecology and Hydrology, Wallingford, UK; 2 Pharmaceutical Science Division, Molecular Microbiology Research
Laboratory, King’s College London, London, UK; 3 Wellcome Trust Sanger Institute, Hinxton,Cambridge, UK. Cystic fibrosis (CF) patients suffer from chronic bacterial lung infections that lead to death in
the majority of cases. The need to maintain lung function in these patients means that
characterising these infections is vital. Analysis of clinical specimens has traditionally relied
upon culture dependent methods for identification, and research has focused upon only a
few targeted pathogenic bacterial species. Molecular based studies however, are showing
that the level of bacterial diversity in CF sputum is much higher than previously accepted.
Although known pathogens clearly are important, it is now being recognised that chronically
colonized CF airways represent a surprisingly complex and diverse ecosystem.
Understanding the CF lung in terms of its community ecology could benefit our
understanding of disease progression and influence treatment regimens.
DNA derived signals can originate from both viable and non-viable cells. In order to draw
ecological conclusions from community-based analyses of samples from the CF lung it is
important that only the viable microorganisms are investigated. To ensure only DNA from
viable cells are analysed, samples can be treated with propidium monoazide (PMA), a
membrane impermeable dye, which covalently binds to DNA within non-viable cells or
extracellular DNA upon exposure to light thus inhibiting PCR amplification.
Here, the effect of PMA treatment on the microbial communities within the CF lung was
investigated. Individual sputum samples collected from different patients were split; half
were PMA treated, while the other half was not. DNA extracted from treated and untreated
samples were subsequently analysed by directed 454, high-throughput, pyrosequencing and
statistically compared.
PMA treatment may prove useful for the observation of active CF microbial communities
with implications for future treatment regimens. It may also have wider implications for the
investigation of microbial communities from other ecosystems.
Session 5 22
Analyses of the sediment metagenome and resistome associated with
waste water treatment plant effluent
Amos G.C.A., Gaze W.H., Zhang L., Wellington E.M.H. School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK
Waste water treatment plants (WWTPs) receive complex mixtures of chemicals and bacteria
from multiple origins, creating hotspots for selection and transfer of adaptive genes. Little is
known about the impacts of liquid effluent released into rivers.
In this study sediment samples were taken from points upstream and downstream of a
WWTP outfall to analyse the impact on the microbial resistome. Isolation was performed
using selective plates amended with various antibiotics and isolates were screened by PCR
for class 1 integrons. A significantly higher proportion of coliform isolates resistant to
clinically important antibiotics, was found downstream compared to upstream. A
significantly higher carriage of class 1 integrons and complex class 1 integrons was seen
downstream compared to upstream. PCR walking found several resistance genes in
association with these elements.
Real time PCR on metagenomic DNA revealed a significantly higher class 1 integron
prevalence downstream compared to upstream
Expression clone libraries were constructed and functionally screened for antibiotic
resistance phenotypes with results showing a significantly greater number of resistance
genes prevalent downstream compared to upstream.
In conclusion WWTP effluent produced significant impacts on the sediment resistome with
data suggesting liquid effluent disseminates and / or selects for resistance determinants in
the wider environment.
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