Microbial effects on transport processes (BioTran) : experimental methologies and results (April 2007 - March 2008)

Risk assessments for landfills and geological repositories for radioactive waste are primarily based on the precepts of contaminant transport; and are concerned with understanding the movement of gas, wastes and solutes through engineered barriers and natural groundwater systems, within the concept of ‘Source’, ‘Pathway’, and ‘Receptor’. The emphasis on solute migration for landfill investigations is reflected in the theoretical development used during numerical simulation. However, microbes living in such environments can have an impact on transport processes (Bateman et al., 2006; Chapelle 2000; Cunningham et al.,1997; Fredrickson et al., 1989; Keith-Loach and Livens 2002; West and Chilton 1997). Microbial activity in any environment is generally located on chemical or physical interfaces, usually within biofilms, and the impacts can be both physical (e.g. altering porosity) and/or chemical (e.g. changing pH, redox conditions) and may result in intracellular or extracellular mineral formation or degradation (Beveridge et al., 1997; Ehrlich 1999; Konhauser et al., 1998; Milodowski et al., 1990; Tuck et al., 2006). These processes could all impact on fluid flow through fractures and porous media by, for example, blocking of constrictions in fracture flow pathways and pore throats. The BioTran project was initiated to examine the effects of microbes on transport processes, especially in the context of contaminant properties of host rocks. An understanding of these microbial processes will also be relevant to other areas such as bioremediation of contaminated land, borehole and reservoir clogging and enhanced oil recovery. More broadly, these processes impinge on aquifer recharge, pathogen survival, and ultimately on groundwater protection. To date, the project has comprehensively reviewed the available literature and developed methodologies for experimental studies to provide information and data for existing transport models (Bateman et al., 2006; Coombs et al., 2008; Wagner et al., 2007). The experimental work in BioTran project has focussed on materials from the Äspö Underground Research Laboratory (URL) in the context of the geological containment of radioactive waste in hard rock (diorite) environments. The significance of microbiological processes in the containment of radioactive waste has long been recognised (West and McKinley, 2002) and, consequently, detailed evaluations of the biofilms present on the walls of the URL and on the significance of indigenous microbial populations has been a key area of work (Pedersen 1999). An in-situ study at the URL examined the redox buffering of groundwater in vertical fracture-zones penetrated by recent, oxidising, meteoric water and showed that indigenous bacteria were capable of maintaining reducing conditions in the deep groundwaters (Banwart, 1995). Experimental work has also simulated the interactions of indigenous microbes with mineralogical surfaces associated with groundwater flow systems at Äspö (Hama et al., 2001); and ascertained that these microbes can either concentrate relevant chemical species for mineral formation in localised microenvironments or accelerate clay formation, the implications of this being that local hydrological conditions can be changed by microbial activity (Tuck et al., 2006). Also, biogenic mineral precipitates and trapped mineral matter are much more chemically and physically stable than a biofilm, persisting in the system long after the biofilm has decayed or been removed (Brydie et al., 2005). As a result of these studies, the BioTran project has undertaken pilot studies to examine the influences of biofilm growth, from the bacteria Pseudomonas aeruginosa, on groundwater flow though crushed diorite from the Äspö Hard Rock Laboratory, Sweden. This report details these experimental methodologies and results from pilot studies. The aims were to: 1. Develop methodologies to grow and observe biofilms in flow-through cells and columns: 2. Undertake a pilot study to quantitatively evaluate the effects of biofilms on fluid flow; 3. Evaluate the effect of a pH change and introduction of bacteriophage on these biofilms and on fluid flow. The work described in this report has shown that: • P. aeruginosa biofilms can be grown reliably using Äspö diorite and synthetic groundwater as a growth medium; • P. aeruginosa biofilms can be grown in a variety of laboratory flowing systems; • Biofilm growth and development can be imaged using a variety of techniques. The pilot quantitative study evaluated the effects of biofilms on fluid flow through crushed Äspö diorite over a total period of 2141 hours. It showed that: • Numbers of P. aeruginosa gradually increased suggesting that the bacteria were not only surviving, but were also growing in the flow systems; • Biofilms developed in the two columns studied as evidenced by direct observations and by petrographic analysis of both columns at the end of the experiment; • Quantitative results could be obtained using the methodologies described in this report, allowing calculation of hydraulic conductivity. The fluctuations in transport properties during the experiments demonstrate the complexity of systems which include a biological component. The results also show, indirectly, the effect of biofilm on transport properties which is particularly evident after 500 hours when flow in both columns dropped to very low rates. However, use of these data in existing and microbial transport and clogging models is limited because few of them provide the option of coupling flow to microbial growth; • Changes in pH do not appear to influence transport properties in the described flowing systems containing P. aeruginosa biofilms over the experimental exposure period. It is possible that column material and the biofilms may not have been exposed to the pH change because of the low flow rates which will need to be further investigated in future experiments; • Introduction of phage E79 does not appear to influence transport properties in these flowing systems containing P. aeruginosa biofilms. However, it is possible that the phage were not active in these experiments; • No observable fluid/solid interactions took place in either column. Future work will concentrate on developing the quantitative methodologies further so that studies can be undertaken under anaerobic conditions. Further development work is also required to directly observe biofilm growth in columns, particularly for petrographic analysis. Consideration is also needed on how best to utilise the quantitative transport measurements as existing transport models are very limited in their ability to couple biological processes with flow calculations. Development of the BGS in-house model PRECIP model may be the most appropriate.