Microbial effects on transport processes (BioTran) : anaerobic flow-through experiments using crushed Diorite and Pseudomonas aeruginosa (April 2008-March 2009)

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 BioTran project has focussed on materials from the Aspo Underground Research Laboratory (URL) in the context of the geological containment of radioactive waste in hard rock (diorite) environments (Banwart et al., 1995). Early experimental work simulated the interactions of indigenous microbes with mineralogical surfaces associated with groundwater flow systems at Aspo (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). ). It has also been shown that minerals precipitated biogenically are chemically and physically very stable and persist in the system long after the original biofilm has decayed or been removed (Brydie et al., 2005). As a result of these studies, the BioTran project undertook a pilot study to examine the influences of biofilm growth by growing the bacterium Pseudomonas aeruginosa aerobically on groundwater flowing though crushed diorite taken from the Aspo Hard Rock Laboratory, Sweden (West et al., 2008). The results of this study demonstrated that biofilms could be grown in both flow cells and flow-through columns within a few days on a diorite substrate through which artificial groundwater containing minimal nutrients was passed. Also, biofilm development could be successfully imaged in several ways, including by visible and UV light microscopy and cryogenic scanning electron microscopy (cryoSEM). The flow-through column experiment has now been repeated in this current study but under anaerobic conditions over an extended experimental period of 147 days (3524 hours). The experiment showed that: • P. aeruginosa biofilms can be grown anaerobically using diorite and synthetic Aspo groundwater as a growth medium, although the rate of growth is significantly slower. The respiratory metabolism of P. aeruginosa is essentially aerobic; it is however known to grow in the absence of oxygen if nitrate is available as a respiratory electron acceptor. The Aspo groundwater used in this column experiment had negligible nitrate, the fact that growth was observed under anaerobic conditions demonstrates the metabolic versatility and minimal nutritional requirements of the P. aeruginosa strain; • Numbers of P. aeruginosa decreased over the duration of the experiment but were able to survive up to 2684 hours; • CryoSEM observations of the experimental column material showed that biofilm was formed during the course of the experiment; • Biofilm only developed in the first 0 cm to 4 cm of the column as demonstrated by direct observations and by petrographic analysis of the experimental column at cessation of the investigation; • Observations of bacteria-free control columns were vitally important as the experimental column material revealed many features that could easily have been superficially identified as being of biological origin; • The presence of porewater salt caused considerable ambiguity in the identification of biofilaments and biofilm because the salt structures often closely resembled amorphous features anticipated for biofilm development filaments; • No change in pH was observed during the course of the experiment; • The growth of biofilm in the anaerobic environment was insufficient to effect pressure and fluid flow through either column; • The fluid chemistry data shows a rapid reduction in both Mn and Ba concentrations despite no appreciable levels of Mn or Ba being detectable in the groundwater. It was therefore assumed that Mn and Ba ions were present in the inoculant or had originated from the column itself. Future investigations will move away from dioritic sand-packed columns and focus on biofilm growth within complete sandstone rock cores under representative in-situ conditions of pressure and groundwater chemistry.