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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