1. Removal of heavy metals and phosphates from wastewater by bioparticles. The 2 nd International Symposium “Vera Johanides” BIOTECHNOLOGY IN CROATIA BY 2020 Zagreb, 10 – 11 May, 2013 Lucija Nuskern, mag. oecol. Jasna Hrenović, PhD

2. 1. Introduction Need for wastewater treatment prior to release into receiving environment ◦ Heavy metals – toxic activity, bioacumulation ◦ Phosphates – eutrophication BIOPARTICLE – particle composed of inorganic carrier and biofilm of bacteria ◦ higher density of bacterial population, bacteria protected from biotic and abiotic stresses, no bacterial wash-off  better treatment efficiency

3. Aim of the study Design bioparticles able to: 1)tolerate exposure to high concentrations of Cu 2+, Zn 2+ and Ni 2+ 2)Simultaneously remove those heavy metals and phosphates (PO 4 3- ) from wastewater

4. 2. Materials and methods Natural zeolitic tuff (Vranjska Banja, Srbija) – carrier material Bacteria: Acinetobacter junii DSM 1531 ◦ P - accumulation Pseudomonas aeruginosa DSM 15980 ◦ Heavy metal tolerance Mineral composition (wt %): clinoptilolite (72.6), plagioclase (14.6), quartz (12.8) Particle size: 0.063 – 0.121 mm

5. Bacterial immobilisation Incubation of zeolitic tuff with pure bacterial culture of A. junii or P. aeruginosa in liquid medium, 24 h/30 °C with aeration Wet bioparticles with immobilised A. junii (AJBP) Wet bioparticles with immobilised P. aeruginosa (PABP)

6. Experimental procedure 1. Planctonic bacteria 2. Immobilised bacteria (bioparticles - BP) 1 mL A. junii or P. aeruginosa C1 – C5: 1, 5, 10, 25 and 50 mg Cu 2+ /L 1, 5, 10, 35 and 70 mg Zn 2+ /L 1, 5, 10, 25 and 50 mg Ni 2+ /L 100 mL P - medium + 1 g wet AJBP or PABP 100 mL P - medium + C1, C3 and C5: 1, 10 and 50 mg Cu 2+ /L 1, 10 and 70 mg Zn 2+ /L 1, 10 and 50 mg Ni 2+ /L

7. Experimental procedure 3. Mixed AJBP and PABP in synthetic wastewater 4. Mixed AJBP and PABP in real wastewater 0.5 g wet AJBP and 0.5 g wet PABP 100 mL P - medium + 0.5 g wet AJBP and 0.5 g wet PABP 100 mL efluent + C1, C3 and C5: 1, 10 and 50 mg Cu 2+ /L 1, 10 and 70 mg Zn 2+ /L 1, 10 and 50 mg Ni 2+ /L C1, C3 and C5: 1, 10 and 50 mg Cu 2+ /L 1, 10 and 70 mg Zn 2+ /L 1, 10 and 50 mg Ni 2+ /L

8. 3. RESULTS - Heavy metal tolerance PLANCTONIC vs. IMMOBILISED bacteria A. juniiP. aeruginosa

9. 3. RESULTS - Heavy metal removal PLANCTONIC vs. IMMOBILISED bacteria Highest heavy metal removal was achieved with PABP: 84,1 % Cu 2+, 100 % Zn 2+ and 83,7 % Ni 2+

10. 3. RESULTS Mixed AJBP and PABP in real wastewater AJBP and PABP survived, multiplied and removed heavy metals and phosphates when in the same system Mixed AJBP and PABP removed heavy metals and phosphates from real wastewater more efficiently than from synthetic wastewater

11. 4. CONCLUSION Bioparticles composed of immobilised bacteria A. junii and P. aeruginosa on natural zeolitic tuff could be used to simultaneously remove heavy metals and phosphates from wastewater

12. THANK YOU FOR YOUR ATTENTION!