Nanoscale Imaging of Microbial-Mineral Interactions (NIMMI)

Lead Research Organisation: University of Manchester


Microbial processes mediate the redox state of many metals and radionuclides, which in turn controls their mobility in the environment and the stability of a wide range of mineral phases. Organics are also influenced by these processes, acting either as electron donors for metal/mineral reduction, or as competing electron acceptors, for example in the case of chlorinated solvents. Advances in molecular ecology, genomics and post-genomic technologies have given us significant insight into the diversity of the organisms responsible for these important processes, and the underpinning physiology, often at a genetic level. In parallel, developments in nano-scale imaging and spectroscopy now offer the potential to reveal how these biological processes impact on the geosphere at an atomic-scale. The aim of this project is to gain a deeper understanding of key microbial-mineral interactions at the nano-scale using a combination of new state of the art synchrotron imaging techniques, including STXM, alongside microbiological, microscopy, geochemical and modelling approaches. Three complementary and interlinked systems of major environmental importance are the foci of this investigation. First, we will address the microbial reduction of insoluble Fe(III) oxyhydroxides, which plays a major role in many coupled biogeochemical processes in subsurface environments, to identify the nano-scale processes controlling this ubiquitous form of anaerobic respiration. Next, we will focus on the reductive mobilisation of As(V) sorbed onto Fe(III) oxyhydroxides, to identify the mechanism of this bioprocess thought to be catalysed by Fe(III)-reducing bacteria, and threatening the lives of tens of millions worldwide. Finally, the bioreduction of U(VI) in Fe(III)-reducing systems, which potentially limits uranium solubility, will also be studied as it has considerable relevance to the management of our existing soil contamination and our legacy nuclear waste, but is poorly understood at a mechanistic level.
Through this programme, the applicants aim to obtain definitive evidence to help us understand the precise mechanisms of these important processes, which have been studied for more than a decade by many groups world-wide, but remain elusive. Impact will be across a broad scientific community, underpinning more robust conceptual and numerical models in very high profile areas including contaminant bioremediation, trace metal/metalloid biogeochemistry and the safe long-term stewardship of our legacy nuclear waste. Stakeholder engagement will be through strong links that already exist between Manchester and key centres involved in land and water quality and nuclear waste disposal, in the UK and worldwide. We will also enable the transfer of soft x-ray and STXM expertise from synchrotron facilities in North America and Europe to the UK Diamond synchrotron (beamline under construction), while training of key personal in the UK to make maximal use of these exciting and powerful new world-class facilities.

Planned Impact

The work described in this proposal will provide underpinning science of the highest quality that will support international efforts in areas including those working on the bioremediation of organics and metals, predicting and mitigating arsenic pollution issues worldwide (especially in SE Asia), bioenergy production, the synthesis of Fe-based functional nanomaterials and in underpinning the safety case(s) for geological disposal of radioactive wastes. The following will benefit:
The Synchrotron Community especially the STXM community. The extensive research community that is investigating the causes and mitigation of arsenic pollution in groundwaters. The global contaminated land clean up market. The Nuclear Decommissioning Authority and NDA-Radioactive Waste Management Directorate as well as other nuclear stakeholders such as Sellafield Ltd. and National Nuclear Labs. In its broadest sense, society will benefit from this independent, underpinning research.

The communities listed will benefit as follows:
The arsenic-groundwater community will be able to use important new information on As-mobilisation processes to predict areas that will be at risk and also effective mitigation processes. The developments of techniques to monitor interfacial bioremediation processes will help generate robust data sets on the mechanisms of bioremediation of metals and organics, and the fate of the contaminants.
Sellafield Ltd. & other nuclear stakeholders will also benefit from the work, which will contribute to the remediation options for uranium-contaminated land. NDA RWMD is the organisation tasked with implementing geological disposal of UK radioactive wastes. This research will contribute significantly to underpinning the safety case for geological disposal. NNL is responsible for supporting the UK's strategic nuclear R&D and for preserving and developing specialised high level skills. The high profile publications that we hope to generate from this cutting edge work, will also raise public awareness of these significant environmental challenges. Strong, independent research will be crucial to inspiring public confidence in management of the nuclear legacy and management of the arsenic legacy in SE Asia.

Methods for disseminating data/knowledge/skills will include;
VC is on the UK DIAMOND Synchrotron STXM working group and will be involved in key decisions on the capabilities of the UK STXM. In 2014, we will hold a workshop for the UK environmental communities and request £7000 to cover accommodation, travel and food, logistics. JRL is Royal Society Industrial Fellow, between 2010-2014, working closely with NNL and nuclear stakeholders. This will deliver knowledge transfer in areas linked to and including the proposed work programme. RADP is head of Nuclear Education at the University of Manchester and will develop CPD programmes to inform industry of the latest research results from Manchester. Outreach activities through the extant SE Asia Links will be actively sustained by meetings with key stakeholders, web-based communications, including podcasts, media presentations and via publication in magazines. Through the unique Fe nanobiomineralogy research programme and we will transfer knowledge, data and expertise to collaborators and stakeholders in the area. Networking via VC's L'Oreal-UNESCO Fellowship will occur. Web-based Communication will use existing WRC and RCRD websites for detailing the grant activities and linking to wider members interests will be set up. The cost of the website will be £2000 per annum, £6000 total. The team will attend radioactive and media communications training courses to facilitate outreach to wider audiences. We will develop new materials for Gifted and Talented (G&T) activities, identify suitable students for work experience secondments over summer vacations and work with MOSI. Outreach cost is estimated at £2200.
Total requested impact costings are £15,500.


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Description Development of new method for imaging metal reducing bacteria:
We have developed a new set of experimental procedures for culturing Fe(III)-reducing bacteria in a synthetic system which mimics environmental subsurface conditions and allows the bacteria and mineral phases to be imaged in situ. This involves making a thin film of ferrihydrite on the appropriate sample holder (e.g. glass for brightfield/fluorescence microscopy or gold grids for electron or x-ray microscopy.) by spray coating or spin coating. These samples can then be incubated anaerobically with Fe(III)-reducing bacteria and later removed for imaging. This system preserves the spatial arrangement of bacteria and so will give a more accurate impression of the bacteria mineral interface compared with mineral / bacteria slurries previously studied.

Image analysis method for automated counting of bacteria from micrographs:
We developed a new method for quickly estimating the number of bacteria in micrographs. Previously this has been very difficult, especially in mineral, soil, sediment and water samples due to the turbidity. However we have combined fluorescent labelling of cells with epifluorescent imaging and subsequent analysis using the open source software imageJ to allow monitoring of cell populations over time in previously opaque samples.

Cryo-electron tomography study of metal-reducing bacteria:
High resolution 3D images of Geobacter sulfurreducens cells and associated Fe minerals were acquired using this technique, however there were some difficulties with reproducibility. The samples had a mixture of Fe bearing minerals and bacteria, but in many cases the mineral layer was too thick to allow penetration of the electrons through the sample to the detector and so it was not possible to record images. This is a difficult constraint of applying the technique to mineral samples such that it may not be tenable to pursue this further. Cryo-electron tomography is suitable for imaging bacteria in liquid culture and the results from these samples are very promising and may be useful for another study where it would be beneficial to characterise bacterial nanostructures.

Cryo soft X-ray tomography of bacteria
We have collaborated with a team led by Liz Duke at Diamond Light Source where they are developing a new microscopy which uses soft x-rays, can produce 3D tomography images and is useful for imaging biological samples. We imaged some metal reducing bacteria using and found some interesting patterns of x-ray absorption in the cells, opening up new research avenues. There has been very limited research on bacteria using this technique and it may have useful applications in imaging environmental microbial samples. We are also working closely with RAL on imaging U(VI)-reducing bacteria as part of a new collaboration with Manchester radiochemists.

STEM-EDX (Scanning transmission electron microscopy - energy dispersive X-ray) spectrum imaging
STEM-EDX was, for the first time, applied to bacteria enclosed in a hydrated liquid chamber. It was used to analyse the microbe-metal interface of bacteria decorated with bimetallic nanocrystals. The ability to image and measure the chemical composition of bacteria and metals in liquid is an important advancement which negates the need for normal intrusive sample preparation which can alter the cell morphology. An article describing this work is currently in submission.

Functional imaging of the bacteria mineral interface during Fe(III) reduction
We have employed the use of fluorescent probes to detect changes in the Fe(III) bearing minerals caused by Fe(III)-reducing bacteria. Specifically, using confocal microscopy, we have been able to map regions of bio-reduced Fe(II) on mineral films in relation to cell position alongside monitoring biochemical activity such as localised (sub-micron) changes in redox potential.

NanoSIMS of arsenic mobilisation during Fe(III) reduction
We have used NanoSIMS to measure the changes in arsenic distribution on a Fe-As mineral coated surface cause by the proliferation of Fe(III)-reducing bacteria. It was also possible to measure the metabolic activity of the bacteria by their uptake of 13C.
Exploitation Route The system for imaging minerals and bacteria could be used to research a wide variety of iron biogeochemistry questions. Amorphous minerals or sediments could be converted to thin films that would support bacterial activity and then studied using a suite of imaging techniques, combined with chemical analysis to get a fuller understanding of these obscure processes. Likewise, the method for automatically counting bacterial cells from images will have broad application in many areas of microbiology where bacteria are growing on surfaces of minerals, plants, tissue or in biofilms and need to be counted.
Our work applying liquid STEM-EDX for bacteria metal systems is potentially useful to study the formation of nanocatalysts. Additionally, as the bacteria can remain active during imaging, the technique could be taken further to study the formation of nanocrystals over time, which may help to further refine green production of metal catalysts and have widespread applications in the biotechnology sectors.
In the longer term when this research is extended to studying the fate of toxic elements in the subsurface, there will be broader implications in environmental planning, the nuclear industry and in science policy.
Sectors Agriculture, Food and Drink,Chemicals,Energy,Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

Description The findings have been used during a workshop with researchers and NGOs at the Royal University of Phnom Penh, Cambodia, to communicate the role of bacteria in the mobilisation of arsenic in aquifers. This involved communicating our findings with people who work in areas affected by arsenic contamination of ground water and as a consequence this transfer of knowledge may influence the development of water treatment technologies. Additionally, this research contributed to the development of a workshop for Cambodian Environmental Science undergraduate students. The workshop provided students with background information on scientific approaches to understand water quality issues affecting their country, including a session on microbiology and arsenic.
First Year Of Impact 2014
Sector Environment
Impact Types Cultural,Societal

Description JRL gave Pint of Science talk, 17th MAY 2017 Bluu Smithfield Market Hall 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Approx 50 general public attended Pint of Science talk, 17th MAY 2017 Bluu Smithfield Market Hall on microbes in the environment (one of three speakers)
Year(s) Of Engagement Activity 2017
Description School visit, Winchester 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact JRL gave talk to 6th form students at Peter Symonds College, Winchester. Feedback positive ... better engagement with environmental science/geomicrobiology
Year(s) Of Engagement Activity 2017