Analysis of the Diversity of Substrate Utilisation of Soil Bacteria Exposed to Cd and Earthworm Activity Using Generalised Additive Models

Abstract

Biolog EcoPlates™ can be used to measure the carbon substrate utilisation patterns of microbial communities. This method results in a community-level physiological profile (CLPP), which yields a very large amount of data that may be difficult to interpret. In this work, we explore a combination of statistical techniques (particularly the use of generalised additive models [GAMs]) to improve the exploitation of CLPP data. The strength of GAMs lies in their ability to address highly non-linear relationships between the response and the set of explanatory variables. We studied the impact of earthworms (Aporrectodea caliginosa Savigny 1826) and cadmium (Cd) on the CLPP of soil bacteria. The results indicated that both Cd and earthworms modified the CLPP. GAMs were used to assess time-course changes in the diversity of substrate utilisation (DSU) using the Shannon-Wiener index. GAMs revealed significant differences for all treatments (compared to control -S-). The Cd exposed microbial community presented very high metabolic capacities on a few substrata, resulting in an initial acute decrease of DSU (i.e. intense utilization of a few carbon substrata). After 54 h, and over the next 43 h the increase of the DSU suggest that other taxa, less dominant, reached high numbers in the wells containing sources that are less suitable for the Cd-tolerant taxa. Earthworms were a much more determining factor in explaining time course changes in DSU than Cd. Accordingly, Ew and EwCd soils presented similar trends, regardless the presence of Cd. Moreover, both treatments presented similar number of bacteria and higher than Cd-treated soils. This experimental approach, based on the use of DSU and GAMs allowed for a global and statistically relevant interpretation of the changes in carbon source utilisation, highlighting the key role of earthworms on the protection of microbial communities against the Cd.

Influence of red mud on soil microbial communities: Application and comprehensive evaluation of the Biolog EcoPlate approach as a tool in soil microbiological studies

“…The absorbance was measured with DIALAB EL800 Microplate Reader at every 24 hours for 120 hours at 490 nm wavelength. The Biolog EcoPlate method usually measures optical density (OD) at 590 nm because the peak absorbance of the tetrazolium dye occurs at 590 nm (Garland, 1997, Muñiz et al, 2014, Garau et al, 2007. Nevertheless we used absorbance values at 490 nm because our microplate reader was equipped with 405, 450, 490 and 630 nm filters, but the optimal OD values were provided at 490 nm (Nagy et al, 2013).…”

“…The Shannon index (H) resulted from H= -ΣPi ln(Pi), where Pi=ODi/ ΣODi, which is the proportional color development of the well over total color development of all wells of a plate (Garland and Mills, 1991, Tam et al, 2001, Muñiz et al, 2014. The number of substrates oxidized (substrate richness, SR) was calculated as the sum of the number of cells where ODi value reached 0.15 after 120 h (Garau et al, 2007).…”

“…Biolog-derived data for evaluation generally include: the Average Well Colour Development (AWCD) and the Shannon diversity index (H) (Stefanowicz, 2006, Frąc et al, 2012, Kenarova et al, 2014, Muñiz et al, 2014. AWCD is an indicator of the general potential metabolic activity of the microbial community, thus it is an index of the total bioactivity for the Biolog plates (Frąc et al, 2012, Kenarova et al, 2014.…”

“…The Shannon diversity index (H) is used to calculate the physiological diversity of bacterial communities (Kenarova et al, 2014). Microbial communities that are able to degrade more substrates or/and to degrade them with similar efficiency would have higher values of H compared to that part of the community which is not metabolically active and is not capable of growing in plate conditions (Muñiz et al, 2014). Some authors calculated also several additional Biolog EcoPlate derived parameters, such as substrate richness (SR) and Shannon evenness (E) (Gryta et al, 2014, Garau et al, 2007.…”

Farming and soil urban occupation in the water quality of Jaboticabal and Cerradinho streams

“…EcoPlate (Biolog) is a rapid and convenient method to mainly investigate and estimate the impact of stressors on microbial communities from several environmental sources: soil (Muniz et al, 2014;FloresRentería et al, 2016), water (Patel et al, 2014;Xiao;Tinglin;Haihan, 2015), wastewater from different technologies (Jałowiecki et al, 2016) and many other sources.…”