Development of a bioassay for maturity analysis of composts

Research project P 13953 Development of a bioassay for maturity analysis of composts Heribert INSAM 11.10.1999 Composting is used in one breath with environmentally sound and economic waste recycling, sustainability of agricultural production and saving of natural resources. Successful recycling of organic wastes by composting requires the establishment of realistic product goals, but reliable compost quality and maturity criteria beyond basic chemical parameters and simple plant growth tests are still lacking. Many of the classical tests give either ambiguous results, are not integrative, or are very time consuming. Albeit microbial activity is the major process for compost production and utilization, standard tests do not include any, let alone state-of-the-art microbiological aspects. New approaches in environmental microbiology do now offer new options. In this project, an approach based on substrate utilization profiles of the microbial community will be used. Such community level physiological profiles (CLPPs) are increasingly used to characterize catabolic patterns of microbial consortia of soils and sediments (e.g. Garland, 1997, FEMS Microbiol Ecol 24, 289-300) and have recently also been used to describe microbial communities during composting (e.g. Insam et al, 1996, Microb Ecol 3 1, 77-87). CLPPs will be related to dominant microorganisms throughout the composting stages by isolation and analysis of total-DNA extracts, PCR amplification, denaturing gradient gel electrophoresis (DGGE) and sequencing of isolates and DGGE bands. The aim of the project will be to deliver a database for developing a bioassay for maturity testing of composts based on CLPPs. Three composting sites (both medium and large scale) with well defined input material (organic wastes) and advanced process control will be selected and time sequences of a minimum of 8 composting runs will be analysed. A control of the composting process and compost maturity will be performed by standard chemical and biological methods. Carbon content, the C/N ratio, nitrate and ammonia contents and heat production in Dewar vessels, microbial biomass and basal respiration will be determined. Further, the F-400/E600 ratio will be measured as an index of humification. As process control parameters, temperature, C02 and 02 concentrations will be measured in situ. A culture based quantification of pathogens will also be performed. The community level physiological profile (CLPP) will be determined by using Biolog EcoPlates with 31 different C sources (Insam and Rangger, 1997, Microbial Communities; Springer Verlag). Extraction, inoculation and incubation conditions will be optimized, and appropriate biomathematical tools (curve parameter estimations and and multivariate statistics) will be employed to establish a CLPP data base for composts of different maturity. CLPPs of all maturation stages will be compared to the results of the classical tests, and will be related to isolates and DGGE patterns. At the end of the project, a CLPP data basis for establishing a standard procedure for testing compost maturity will be established. The obtained data basis form three composting sites studied in great detail will further be validated with samples from various composting processes from several composting plants in Europe.

Composting is the final process for most of the 109 tons of organic waste coming up annually in Europe. A successful merchandizing of the final product is based on various quality criteria indicating the end of the final degradation process. Traditional maturity tests are often time consuming and sometimes lacking significance and sufficient integrity. Although microorganisms are regarded as the driving factors of the degradation process, there is still no assay based on the measurement of microbial activity and physiology. The aim of this project was the compilation of data for the development of a bioassay based on microbial substrate use profiles. These so called `Community Level Physiological Profiles` (CLPPs) were applied for a qualitative evaluation of different composts. Process parameters like pH, temperature, water content, microbial biomass and respiration or CO 2 -content were measured. Additionally molecular methods were applied. Total microbial DNA was extracted and fragments of the 16S rDNA were separated by denaturing gradient gel electrophoresis (DGGE) after PCR amplification for further analysis. We sampled composting runs (one each from Italy and Austria) as well as different mature composts with well defined input materials like biowaste or sewage sludge from several composting plants in Austria. Based on the data gained in the CLPP experiments the composts could be well differentiated due to their maturity as well as to their input materials. By means of principal component analysis of the different substrate profiles it was possible to separate sewage sludge composts from biowaste composts. However, the data were insufficient for the setup of a databasis for the development of a bioassay as the great heterogeneity of feedstock was of significant importance for the substrate use profiles, superposing the effects of maturity. Results from the molecular approach indicated a succession of microbial communities during the composting process where more sequences at the beginning where affiliated with members of the Enterobacteriaceae and Lactobacilli, with low G+C Gram positives in the hot phase and to members of the Bacteroides-Cytophaga-Flexibacter-cluster at the end. Genetic fingerprints of the amplified microbial 16S rDNA fragments separated in gradient gels showed a highly diverse microbial community changing with the time of composting in all samples investigated. Also different input materials resulted in different banding patterns of microbial communities from mature composts. The kind of sample storage, however, showed no influence on the banding patterns.