Geological Survey of Denmark and Greenland Bulletin 7, 2004, p 37-40 37 Steam treatment of contaminated soil and aquifer sediment is a promising method of cleaning soil. The treatment is based on steam injection into a water saturated porous aqui- fer (Gudbjerg et al. 2004), by which the heat transfers the contaminants into the vapour phase, allowing entrapment in an active carbon filter connected to a large vacuum suction device. The treatment is effective against several important groundwater contaminants, including pentachlorophenole and perchloroethylene, typically found in association with industrial processes or dry cleaning facilities. Furthermore, as an example of removal of non-aqueous phase liquids (NAPLs) large amounts of creosote have been recovered after steam injection in a deep aquifer (Kuhlmann 2002; Tse & Lo 2002). Steam treatment is dependent on the complete heating of the soil volume under treatment. The steam has a strongly adverse impact on trees and other plants with deep root sy- stems within the soil, but no other visible effects have been reported. The aim of the activities undertaken during colla- borative projects carried out by the Geological Survey of Denmark and Greenland (GEUS) and the Danish Institute of Agricultural Sciences (DJF) for the Danish Environmental Protection Agency and the local authorities in Copenhagen (Københavns Amt) was to establish to what extent the micro- bial community was affected by the steam treatment of the soil. A few results from the literature indicate that the micro- bial activity increases in steam treated soil (Richardson et al. 2002), probably due to microbial degradation of the soil con- taminants in combination with microbial utilisation of heat- killed organisms. It is, however, not known whether this increased microbial activity is associated with the development of pathogenic micro-organisms; these are typically able to grow at higher temperatures than the general microbial community in soil. Geological Survey of Denmark and Greenland Bulletin 7, 37–40 (2005) © GEUS, 2005 Steam treatment of contaminated groundwater aquifers – development of pathogenic micro-organisms in soil Carsten Suhr Jacobsen, Susanne Elmholt, Carsten Bagge Jensen, Pia Bach Jakobsen and Mikkel Bender Denmark Hedehusene Copenhagen 100 km steam steam ground surface ground water level gases and polluted water removed and cleaned up steam steam collection well contaminated soil Fig. 1. Sketch of steam treatment facility at a strongly con- taminated industrial site at Hedehusene, west of Copen- hagen. Inset map shows location. 38 The steam treatment in Hedehusene Hedehusene is situated approximately 25 km to the west of Copenhagen, and the contaminated soil and groundwater aquifer here results from various industrial activities prima- rily carried out between 1920 and 1970. These activities include a dry cleaning facility and several small workshops. From both types of industry, trichloroethylene and tetra- chloroethylene are often found as groundwater contami- nants. The groundwater aquifer in Hedehusene was known to be contaminated with high concentrations of trichloroeth- ylene, which has been a constant hazard to an important drinking water production well downstream from the site. Pumping, treating and recycling water at the site over many years had controlled the distribution of the contamination, but the main contamination was still present at very high le- vels and has become a long-term threat to continued ground- water extraction. The steam treatment in Hedehusene was carried out dur- ing the winter 2001–2002. Wells delivering steam were buried nine metres below the land surface, allowing the trans- fer of steam below the contamination plume (Fig. 1). The steam was pumped continuously for a period of five months, until the temperature reached 90°C. Heating of the soil allowed the transfer of the contaminant to the vapour phase which was then trapped in an active carbon filter. Heat-tolerant micro-organisms found at the Hedehusene site The site was monitored by sampling surface soil and soil from approximately 50 cm depth on 11 September 2001 (before the steam treatment), and resampling during and after the steam treatment. Sampling was undertaken six times with the latest sampling on 26 October 2004. In general, it was found that the number of heat-tolerant micro-organisms increased after the heat treatment, and that some of the heat-tolerant micro-organisms could still be found three years after the 2001–2002 steam treatment. Heat-tolerant bacteria are defined as able to continue growing at temperatures of 42°C, and heat-tolerant fungi as those able to continue growing at 37°C. Such high tempera- tures do not occur naturally at the site, and soil micro-orga- nisms originating from this site are not expected to be able to grow at such high temperatures. Heat tolerance is one of the main characteristics that distinguish normal soil micro- organisms from pathogenic micro-organisms found in human patients. General microbial community adaptation to growth at high temperature The effect on the general microbial community was investi- gated by assessing its growth rate on 24 different microbial food sources. A small amount of soil was added to 24 diffe- rent microbial growth media and incubated at either 20°C or 42°C. This technique revealed that the microbial community in the control plot was very constant in its ability to utilise the different food sources during the sampling period. Further- more, the microbial community in the control plot showed little ability to utilise food sources at the elevated temperature (an area approximately 30 m away from the heating zone). In contrast, the heated soil showed a massive and long-lasting A B C D 5 cm Fig. 2. Changes of microbial metabolic fingerprints using comparisons of the ability of micro-organisms to grow on different carbon substrates. The appearance of coloration in each section indicates growth of micro- organisms. A high number of positive sections at 42°C indicate a high number of organisms able to grow at temperatures associated with path- ogenic micro-organisms. A: steam-treated soil with growth at 20°C. B: control soil with growth at 20°C. C: steam-treated soil with growth at 42°C. D: control soil with growth at 42°C. 39 increase in the ability of the microbial community to utilise the food sources at 42°C (Fig. 2). It is well known that micro-organisms differ in their abi- lity to survive in soil. Some are able to form spores that can stay inactive in the soil for years while others die out due to predation and competition with micro-organisms having a very low level of metabolic activity. We have chosen two dif- ferent representatives of heat-tolerant micro-organisms: a bacterium without the ability to form spores, and a fungus which forms conidia. Although these conidia are able to ger- minate and grow in the laboratory, they need not be active in the soil. Both species showed a clear response to soil heating as described below. Aspergillus fumigatus – an unusual pathogenic and allergenic micro-organism Aspergillus fumigatus is a remarkable and unusual pathogen because in addition to causing life-threatening invasive dis- ease of immuno-compromised human patients, it can also cause allergic reactions in persons with fully functional im- mune systems (Latgé 1999; Denning et al. 2002). A. fumigatus is easily identified and is distinguished by rapidly growing colonies in characteristic turquoise to dark green colours, by the phialides curving to be roughly parallel to each other and to the axis of the stipe, and the presence of small conidia in columns (Fig. 3; Klich & Pitt 1988). A. fumigatus is regularly reported as a dominant species in vari- ous types of compost, but never as a dominant species in soil (Domsch et al. 1993). A. fumigatus was only found in very low numbers in the untreated control plot, but in the heat-treated soil this fungal species was abundant. A. fumigatus was still present in ele- vated numbers at the last sampling in October 2004 in the heat-treated soil, but the numbers were slowly declining. It seems, however, likely that the elevated numbers of A. fumi- gatus will continue for some time due to the ability of the fungus to form conidia. Pseudomonas aeruginosa – an opportunistic pathogen Pseudomonas aeruginosa is an opportunistic pathogen, mean- ing that it exploits any defects in the human host defences to initiate an infection. It causes urinary tract infections, respi- ratory system infections and also bone and joint infections. Furthermore, it is associated with gastrointestinal infections and a variety of systemic infections, particularly in patients with severe burns and in immuno-compromised cancer and AIDS patients. P. aeruginosa infections are a serious problem for patients hospitalised with cancer, cystic fibrosis and burns. The case fatality rate for these patients is 50%. P. aeruginosa increased from non-detectable (less than 100 cells per gram of soil) in the non-treated soils to 105 cells per gram of soil in the heat-treated soil (Fig. 4). P. aeruginosa is a 1 cm Fig. 3. Colony of Aspergillus fumigatus isolated from steam-treated soil. Fig. 4. Pseudomonas aeruginosa isolated from steam-treated soil. The photograph was taken in ultraviolet light to show the characteristic flu- orescence of this bacteria genus. Diameter of view is 9 cm. representative of fast growing soil bacteria that are unable to form spores. In contrast to A. fumigatus, the population of P. aeruginosa decreased rapidly after the heat treatment, and after one year the numbers were again below the detection level. This reduction was probably due to predation and lack of competing abilities when the temperature decreased. Need for monitoring of microbial side- effects in relation to steam treatment Micro-organisms differ in their ability to develop resting forms. A. fumigatus develops conidia that can remain in soil for many years. These resting conidia may not be active in the soil, even if they can be detected on agar plates when analysed in the laboratory. In contrast, P. aeruginosa does not form resting spores, and detection on agar plates in the laboratory is connected to activity of the bacterium in the soil. The present project highlights the need for microbial risk assessments in connection with new steam treatment pro- jects. The high level of potentially pathogenic micro-orga- nisms expected after heat treatment of a soil points to the need for monitoring these organisms in connection with new steam treatment projects. References Denning, D.W., Anderson, M.J., Turner, G., Latgé, J.-P. & Bennett, J.W. 2002: Sequencing the Aspergillus fumigatus genome. The Lancet Infec- tious Diseases 2, 251–253. Domsch, K.H., Gams, W. & Anderson, T.-H. 1993: Compendium of soil fungi 1, 2, 2nd edition, 860 pp., 406 pp. Eching: IHW-Verlag. Gudbjerg, J., Trotschler, O., Farber, A., Sonnenborg, T.O. & Jensen, K.H. 2004: On spurious water flow during numerical simulation of steam injection into water-saturated soil. Journal of Contaminant Hydrology 75, 297–318. Klich, M.A. & Pitt, J.I. 1988: A laboratory guide to the common Asper- gillus species and their teleomorphs, 116 pp. North Ryde, Australia: CSIRO Division of Food Processing. Kuhlman, M.I. 2002: Analysis of the steam injection at the Visalia Superfund Project with fully compositional nonisothermal finite dif- ference simulations. Journal of Hazardous Materials 92, 1–19. Latgé, J.-P. 1999: Aspergillus fumigatus and Aspergillosis. Clinical Micro- biology Reviews 12, 310–350. Richardson, R.E., James, C.A., Bhupathiraju, V.K. & Alvarerez-Cohen, L. 2002: Microbial activity in soils following steam treatment. Biodegra- dation 13, 285–295. Tse, K.K.C. & Lo, S.-L. 2002: Desorption of PCP-contaminated soil: effect of temperature. Water Research 36, 284–290. Authors’ addresses C.S.J., P.J. & M.B., Geological Survey of Denmark and Greenland, Øster Voldgade 10, DK-1350 Copenhagen K, Denmark. E-mail: csj@geus.dk S.E., Danish Institute of Agricultural Sciences, Blichers Allé 20, DK- 8830 Tjele, Denmark. C.B.J., Copenhagen County, Soil and Groundwater Department, Stationsparken 27, DK-2600 Glostrup, Denmark. 40