To check the sterility

OICR-9429 datasheet of this medium, 1 ml aliquot was plated onto the sterile bacteriological agar purchased from Sigma Aldrich (Cape Town, South Africa) and incubated at 37°C for 24 h. Only flasks containing the sterile media were considered for the next step of the experimental study. Determination of the growth performance and heavy metal removal efficiency of test isolates in the industrial wastewater The laboratory batch reactors consisted of 500 ml Erlenmeyer containing 300 ml of the culture media. Separate flasks were aseptically inoculated with a fresh culture of bacterial isolates (~100 CFU/ml) or protozoan isolates (~100 Cells/ml). Nutrient broth and PPG (Sigma Aldrich, SA) were used to obtain the microbial inoculums for bacteria and protozoa, respectively. Two supplementary culture media were set up as negative and positive controls. The positive control flask contained the domestic wastewater mixed liquor free of heavy metals, but selleck screening library inoculated with the specific test isolate, while an LY2603618 concentration uninoculated industrial wastewater sample was used as the negative control. All the inoculated flasks as well as the controls were initially shaken in a shaking incubator (100 rpm) and exposed at 30°C ± 2°C. Aliquots of 40 ml were taken every day for five days to estimate the biomass and the quantity of

heavy metal removed. The microbial estimation for bacterial species was determined using the spread plate method after dilution [26]. Briefly, 100 μl of aliquot from each sample was transferred to Mannitol Thiamet G Egg Yolk Polymyxin (MYP) agar (Sigma Aldrich, SA), nutrient agar (NA)

(Merck, SA) and Pseudomonas isolation agar (PIA) (Sigma Aldrich, SA) for Bacillus licheniformis, Brevibacillus laterosporus and Pseudomonas putida, respectively. The plates were incubated at 50°C for Bacillus[25] and at 30°C for the two other bacterial isolates [28]. Protozoan density was determined by a visual count using an inverted microscope (Axiovert S100, Carl Zeiss) under × 100 to × 400 magnification. The first-order die-off rate (mortality rate) and specific growth rate of the bacterial and protozoan species were calculated using the formula as reported by Peng et al. [29] and Farrier-Pagès and Rassoulzadegan [30], respectively. The die-off rate coefficient was converted to a percentage by using the total inhibition/die-off of the colony/cell counts as the 100% die-off rate. The physico-chemical parameters such as pH, DO and COD were determined using standard methods [26]. To check the removal of heavy metals in the industrial wastewater by test organisms, an aliquot of 30 ml of the medium was taken on a daily basis, centrifuged (4000 ×g, 4°C, 15 min) and filtered using a 0.45 μm nylon filter. The remaining heavy metal concentrations were determined from the supernatants and compared with the initial heavy metal concentrations as described above.