Comparison of CLPP and enzyme activity assay for functional characterization of bacterial soil communities

Abstract

Functional characterization of soil microbial communities by the use of BIOLOG GN microtiter plates was compared with an optimized assay of measuring the enzymatic activity of bacterial associated enzymes. Since the first use of BIOLOG GN microtiter plates for characterization of environmental microorganisms, this Community Level Physiological Profile (CLPP) has been widely used and is today routinely used in soil monitoring programmes in e.g. the Netherlands (Nielsen and Winding 2002). The CLPP method is based upon the metabolic capabilities of bacteria during growth in the wells of the microtiter plates at nutrient rich conditions compared to the conditions in soil. Inoculum preparation, incubation conditions, reading intervals and data treatment have all been the subject of many investigations. In spite of the various criticisms, the method remains to be widely used, to a large extent due to the ease and speed of performance. The Enzymatic Activity Assay (EAA) is based on enzymatic cleavage of model fluorogenic enzyme substrates (4-methylumbelliferyl, MUF), that upon cleavage forms a fluorescent product. The assay is independent of bacterial growth and can be used directly on bacteria extracted from soil, yielding a functional characterization of the bacterial communities based on the enzymatic activities. The substrates chosen here represent 14 different enzymes related to the degradation of different carbohydrates, including starch, chitin and cellulose. The microbial communities of 11 different agricultural and forest soils were collected around Sjaelland, Denmark, and characterized by CLPP and EAA. For CLPP the bacteria were extracted from the soils by dilution and shaking. For EAA the bacteria were extracted by Nycodenz buoyant density centrifugation to ensure that the enzymatic activities were associated to the bacteria and not to free soil enzymes. In addition, the culturable and total number of bacteria and soil respiration were used to describe the size and activity of the soil microbial communities. The data were subjected to principal component analysis followed by correlation analysis. Significant correlations were found between the principal components of CLPP and EAA. Hence, the functional characterization of the bacterial communities by the growth based CLPP correlated positively to the functional characterization by the EEA, while correlation to the abundance estimations of bacteria (CFU and total counts) correlated negatively. The overall assessment of soil activity, the soil respiration, did not correlate with either. Significant correlations were found between the principal components of pH, CLPP, EAA, and culturable and total number of bacteria, while no correlations were found to soil respiration, loss on ignition and soil moisture content. The pH in the test system might make differences between bacterial communities more detectable. The functional characterization of the bacterial communities by the growth based CLPP correlated positively to the bacterial community function assayed by the EAA, which is independent of growth and, hence, more closely reflects the in situ activities. Functional characterization of microbial communities aims to describe the in situ functions. However, most functional tests depends on manipulation, including incubation of the microbial communities, in the laboratory. Certainly, the CLPP assay is dependant on microbial growth in the wells, which has the potential to change the functional characteristis of the microbial communities. Our results show that the functional characterization of microbial communities by the growth based assay of CLPP correlated posistively to the EAA which is independent of growth and, hence, more closely should reflect the in situ activities. This observation is of importance for the current debate on assessment of the functions of soil microbial communities. The estimations of the abundance of bacteria (CFU and total counts), however, showed negative correlations to the functional characterizations. This highlights the questions on the identity and culturability versus dormancy of the functional bacterial players in soil. The two assays tested, CLPP and EAA, separated the soil bacterial communities similarly in vitro. Hence, depending on the objectives, either technique can be used to get a fingerprint of the soil bacterial community diversity.