Sewage Sludge Microbial Carbon Utilization

The capability of sewage sludge microbial communities to utilize a variety of carbon sources was assessed by using Biolog EcoPlate [22 ]. Every plate had 96 wells containing 31 different carbon sources plus a blank well, in three replications. The rate of utilization of the carbon sources was pointed by the reduction of tetrazolium violet redox dye, which changed from colorless to purple if added microorganisms utilize the substrate [23 ]. EcoPlate was prepared in the following way: 1 g of sewage sludge was suspended in 99-ml sterile peptone water and shaken for 20 min at 20 °C and then was incubated at 4 °C for 30 min [24 (link)]. Next, each well of the Biolog EcoPlate was inoculated by 120 μl of the prepared suspension and incubated at 25 °C. Absorbance at 590 nm was measured on Biolog Microstation after 24, 48, 72, 96, 120, and 144 of incubation hours. Optical density (OD_i) value from each well was corrected by subtracting the control (blank well) values from each plate well. Optical density values obtained at 120 h of incubation represented the optima range of optical density readings, so 120 h of incubation results was used for the assessment of microbial functional diversity and statistical analyses. In addition, substrates were subdivided into five group substrates, carbohydrates, carboxylic and ketonic acids, amines and amides, amino acids, and polymers, according to Weber and Legge [25 (link)].
Microbial activity in each microplate was expressed as average well color development (AWCD). Substrate richness values (R) were calculated as the number of utilized substrates and evenness were calculated according to Zak et al. [26 (link)] (Table 2).Table 2

Formulae for calculations

Index	Definition	Formulae	Definitions
Average well color development		AWCD = Σ ODi/31	pi = proportional color development of the well over total color development of all wells of a plate H = Shannon index of diversity S = number of wells with color development (substrate utilization richness)
Shannon diversity	Measure of richness	H = −Σpi(lnpi)
Shannon evenness	Evenness calculated from Shannon index	E = H/lnS