dHPLC, a system to investigate anaerobic populations

For economic and ecological reasons anaerobic digestion has turned out as an interesting alternative to handle biowaste. Digesters are closed systems thus preventing negative effect like bad odours and pests and the CH4- production can contribute in appreciable amount to the energy demand. However, despite the considerable amount of investigations dealing with various aspects of this technique several aspects of the basic process and especially possible reasons for its disturbances remain a "black box". Thus an increase in the knowledge about both, the engaged micro-organisms per se and their functional role within the digester is urgently needed. To meet these requirements we want to develop and establish a denaturing High Performance Liquid Chromatography (dHPLC) system. dHPLC is a well established method in a couple of research areas, ranging from mutation analysis to clone mapping. The method is quick and cheap, allows quantification and also a subsequent collection and investigation (e.g. sequencing) of isolated types of nucleic acid. Moreover dHPLC facilitates the application of other approaches, which so far are dependent on expensive, lab-intense, and time-consuming cloning (e.g. microarray). However to our knowledge no attempts have yet been made to use this powerful tool for investigating ecological topics in such complex environments as digester sludges. Irrespective of the respective molecular biological method used to investigate microbial populations it is quite easy to accumulate a huge amount of data. However, it is another thing to link these data to functionality of the respective organisms. We will be able to relate molecular analysis to classical culture based and physiological microbial properties (e.g., quantification of total cell counts, number of methanogens, O-demetylating acetogens, profiles of C-source-utilization) and to physical, chemical and biochemical parameters (contents of total fat, VFA, Ctot, Ntot, organic acids etc.) of the fermenter sludge and the input material. It is this combination which will allow a causal investigation of the anaerobic microbial populations and should avoid hoarding up data without further increase in knowledge. A further upgrading of the data material will occur due to a linkage of our data to process parameters of an anaerobic, thermophilic 750,000 litre digester, as we have full and unlimited access to all (online) data of one of the four great anaerobic digesters in Austria. We do the data analysis of this plant in a present investigation right now, and this collaboration will continue. Finally knowledge from this linkage might enable operators of digesters to intervene at an early stage, before problems emerge. Although our knowledge about microbiology of anaerobic digesters is, at best, superficially it is sure that engaged microbial communities can drastically change. By establishing and applying a dHPLC method which allows a rapid investigation of unculturable microorganisms we want to study how the communities change. Finally, through the linkage to physiological data, process parameters and especially to properties of the input material we want to go a step further and discuss why they change.

Within the project "dHPLC, a system to investigate anaerobic populations" a denaturing high performance liquid chromatography (dHPLC) method for the investigation of microbial populations was established using a conventional, in many (micro)biological laboratories available HPLC system. The biocompatible HPLC consists of a gradient pumping system with a flow path of PEEK tubings, fittings, and filters, a UV-detector (254 or 260 nm) and a column oven capable of temperatures between 50 and 80C with an accuracy of 0.1C. Two columns turned out to be suited for the separation (DNA-Sep, Transgenomic, and Helix DVB, Varian). After extended PCR optimisation work a method could be established for the separation and quantification of DNA-amplicons of same size varying only in base sequence and composition. This method was successfully applied for the investigation of the microbial community of soil, compost, and digester sludge. Moreover, the anaerobic population of a biogas plant was evaluated with this system in combination with various physico-chemical as well as micro- and molecular biological parameters. Although the described dHPLC system can be successfully used for different applications, there still remain questions concerning sample preparation and especially PCR problems have to be solved in the future.