CHARACTERISATION OF HEXANE-DEGRADING MICROORGANISMS FROM WASTE GAS BIOFILTERS DISSERTATION zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) Eingereicht am Fachbereich Biologie/Chemie der Universität Osnabrück 1. Gutachter: Prof. Dr. K. Altendorf 2. Gutachter: Priv.-Doz. Dr. A. Lipski vorgelegt von Michèle Martine Friedrich aus Osnabrück Osnabrück, November 2005 In loving memory of Inge Naismith Acknowledgements I am grateful that Prof. Dr. Karlheinz Altendorf gave me the opportunity to work and start my Ph.D. studies in his department and to work in such an interesting field of microbiological research. I thank Priv.-Doz. Dr. André Lipski for introducing me to bacterial taxonomy, biomarkers and among other things GC/MS. I really miss working with fatty acids. Maybe we will find means of future collaborations! Additionally, I would like to thank all colleagues in Osnabrück for having made my time in Osnabrück scientifically and privately a successful and content time. I would also like to thank my husband for his patience and support; and all my family and friends for their understanding. I am looking forward to share more spare time with all of you in the near future. Contents I. Introduction........................................................................................................................1 Biofiltration: Treatment of waste gas ..................................................................................1 Cultivation-dependent and -independent approaches ........................................................2 II. Materials and Methods.....................................................................................................5 Analyses of biofilter packing material .................................................................................5 Microbiological methods .....................................................................................................6 Molecular biological methods .............................................................................................9 Labelling of hexane-degrading microorganisms ...............................................................13 III. Results............................................................................................................................15 Characterisation of the biofilters under investigation ........................................................15 Differentiation of the isolated bacteria ..............................................................................16 Fluorescence in situ hybridisation (FISH) of biofilter samples and individual strains .......56 Isotope-based labelling of hexane degraders...................................................................63 IV. Discussion .....................................................................................................................69 Taxonomic affiliation and identification of the isolates......................................................69 Physiology of the isolated strains .....................................................................................80 Fluorescence in situ hybridisation and labelling of biofilter material .................................82 V. Summary.........................................................................................................................89 VI. Zusammenfassung .......................................................................................................93 References ..........................................................................................................................97 Appendix ...........................................................................................................................111 I. Introduction BIOFILTRATION: TREATMENT OF WASTE GAS The increased ecological awareness in the 80s and early 90s of the last century led to stricter environmental guidelines. Waste air was not merely regarded as a source of odour nuisance but also in terms of its health and environmental impacts. Thus, biological waste gas treatment was no longer used almost solely for odour control as in the 1960s and the application of biological waste gas treatment for the elimination of solvents in industrial waste gas followed. Waste gas emissions are a common problem in various branches of industry such as the chemical and pharmaceutical industry, the automotive, the furniture, the textile, the agricultural, and the food industry. Biological waste gas treatment is applicable when processes emit high volume loads containing degradable pollutants in relatively low concentrations. Only if pollutants are more concentrated than 5 g/m3, chemical or physical treatments are more suitable (Engesser et al., 1997). Because investment and operation costs of biological air remediation have proven to be considerably lower than those of chemical and physical techniques biological waste gas treatment has become very dominant (Ottengraf, 1986; Ottengraf and Diks, 1992; Menig et al., 1997). Apart from being cost- effective biological air remediation ideally leads to a complete degradation of the pollutants to carbon dioxide, water, salt and biomass (Engesser, 1992; Converse et al., 2003). Three forms of biological waste gas treatment have established on the market: biofilters, bioscrubbers and trickling filters, the biofilter being the most commonly applied version (Sabo et al., 1996). The simplest construction of a biofilter is the open biofilter. Generally, the pre-treated waste gas is pumped through the filterbed in an upward direction. The packing material usually consists of organic material such as heather-peat mixtures, wood chops, preferably crushed tree roots or tree bark compost. Apart from the advantage of being able to dispose off the organic packing material after usage as compost, the organic filter material also supports the microbiological population of the biofilter with additional nutrients and provides enough surface area for the microorganisms. Most organisms are thought to live in the water layer surrounding the packing material (Ottengraf, 1986). When air flows around the packing material a continuous mass transfer between the gas and water phase occurs. Biological degradation or consumption of the compounds in the water layer drives the continuous mass transfer by retaining the concentration gradient (Ottengraf and Diks, 1992). Thus, biofiltration is not a classical filtration process because the compounds are actually degraded and converted. Most studies on the degradation of hydrophobic substances such as hexane have focused on the technical biofiltration performance (Morgenroth et al., 1996; Budwill and Coleman, 1997; Kastner et al., 1999; Zhu et al., 1 Introduction 2004). This study rather focuses on the actual degrading microorganisms of the biofilter population. The ability to convert alkanes has been described for a variety of bacteria. The most commonly mentioned genera are Pseudomonas, Acinetobacter, Arthrobacter, Mycobacterium, and Nocardia (Britton, 1984; Witholt et al., 1990). More recently Alcanivorax, Rhodococcus, Nocardioides, and Gordonia have been shown to utilise alkanes including short-chained gaseous alkanes such as hexane (Kummer et al., 1999; Saadoun et al., 1999; Hamamura et al., 2001; Kasai et al., 2002; Saadoun, 2003). CULTIVATION-DEPENDENT AND -INDEPENDENT APPROACHES A number of combined culture-dependent and -independent studies have been conducted. Wagner and co-workers (1993) found the community structure obtained by the application of cultivation-independent rRNA-targeting probes to deviate substantially from that obtained by culturing. During an investigation of grassland soils the cultivation-independent cloning of environmental 16S rDNA led to a microbial community structure that did not correlate with the cultivation-dependent investigation (Felske et al., 1999). Usually, the enrichment seamed to have introduced the bias. Fatty acid analyses of cell pellets during enrichment phases showed strong shifts and pronounced deviations from the fatty acid profile of the original samples (Piehl, 2002). Studies have been conducted to isolate some bacteria whose rDNA is common in cultivation-independent analysis of ecosystems but even despite the wide spectrum of growth media employed the isolation of many of these strains failed (e.g. Rheims et al., 1999). Since current culture techniques do not always satisfy the need of providing a balanced picture of the microbial community, future developments in the study of bacterial diversity should include improvements in cultivation methods to approach as closely as possible the conditions of natural habitats (Palleroni, 1997). Thus, the isolation approach may not be designed to isolate only the fast-growing, easy-to-handle strains. In contrast, the scientist must also try and cultivate the fastidious strains as these may be of importance within the ecosystem to be analysed. Many properties that make these organisms important members of the living World are amenable to observation only through the study of living cultures. Additionally, analysis of phospholipid fatty acids (PLFAs) is not entirely isolation independent because interpretations are based on known fatty acid profiles (Spring et al., 2000). The same holds true for interpretations of data obtained by fluorescence in situ hybridisation (FISH) which obviously also depend on knowledge of known taxa. Concerning FISH the dependence on known taxa can be partly overcome