Biolog EcoPlates

Functional diversity of microbial communities in the 25 compost samples was determined using Biolog EcoPlates (Biolog, Inc., CA, United States), which have been used widely in soil sciences (Campbell et al., 1997; Preston-Mafham et al., 2002). Biolog EcoPlates allow determining a metabolic fingerprint of microbial communities by analyzing the characteristic reaction pattern of carbon metabolism at defined time intervals. They contain 31 of the most useful carbon sources for soil community analysis, including carboxylic acids, carbohydrates, amino acids, polymers, and amines/amides (Supplementary Table 2). A control well containing water is also included.

About 3 g of fresh material was added to 27,0 mL of Ringer Solution (Merck) and shaken for 30 minutes. Starting from that dilution (10^–1), a ten-fold serial dilution was prepared until 10^–7. Subsequently, each dilution was plated on Nutrient Agar (Merck) plates via spread plate method. Dilutions that resulted in 10 to 100 colonies were inoculated on Biolog EcoPlates. For each sample, three technical replicates were inoculated. Thereafter, the plates were incubated for 7 days at 25 °C. The optical density (OD590 nm) was measured directly after inoculation, after 1, 2, 3, 5 and 7 days with a microplate reader (VersaMax). To correct for background, readings of the initial well at time zero were subtracted from subsequent readings. Wells with a resulting optical density higher than 0.5 were considered as positive. Relative well optical density values that were negative or under 0.06 were set to zero (Classen et al., 2003).

The average well color development (AWCD) was calculated using formula 1. The AWCD is an indicator of total activity and can give an overall trend of metabolic activity of the microbial communities in time (Tanase et al., 2017).

With C_i the optical density value of each reaction well at 590 nm, R the optical density value of the control well and n the number of wells.

For further analysis, data from day 7 were used because of the largest difference in optical density values. The optical densities of each well after 7 days were used to calculate the Shannon diversity index (H), substrate evenness (E) and Simpson diversity index (D):

where P_i is the proportional optical density value of the ith well over total optical density of all wells of a plate,

where S represents the total number of utilized carbon sources (i.e., substrate utilization richness),

These indices reflect the metabolic functional diversity of the microbial communities of the composts.

Relative optical density values after 7 days were divided by the AWCD to minimize the influence of inoculum density differences between plates (Garland and Mills, 1991; Graham and Haynes, 2005).