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Disinfection implies the elimination of pathogenic microorganisms in water treatment systems that facilitates the safe discharge or reuse of wastewater. Alkaline disinfection is the inactivation of pathogenic bacteria at high pH conditions, usually above 12. Wood fly ash has been known to increase the pH when introduced to water solutions. Fly ash is an industrial by-product of the combustion of different wood materials and is mostly disposed of as waste on landfills. Proposed applications of wood ash are in agriculture and forestry where it serves as a liming agent and increases the availability of nutrients in acid soils, as a component in concrete, cement, and mortar manufacturing, and as a catalyst for biodiesel synthesis. Usage of fly ash as a disinfectant has not been reported in the literature so we tested it by monitoring the removal of typical pathogen indicators (faecal coliforms and intestinal enterococci) in effluents of urban wastewater and landfill leachate treatment plants in order to simulate the disinfection by using wood fly ash as a potential tertiary treatment method. The addition of ash at a concentration of 10 g L-1 (1 %) caused an instant increase of pH in urban wastewater (Wastewater treatment plant of Velika Gorica, Croatia) and landfill leachate (Jakuševec landfill, Zagreb, Croatia). High pH (10.1–12.7) inactivated bacterial populations in both types of wastewater and the removal of faecal coliforms and intestinal enterococci after 6 h of contact was 100 % (below the detection limit

Technology of bacterial immobilization could be an alternative for improvement of biological phosphate removal from the wastewater. Immobilization of phosphate accumulating bacteria on suitable carriers offers higher density of bacterial cells and enhanced metabolic activity of desired bacteria in the bioreactors. In addition, immobilized bacteria are better adapted to survive periods of environmental stresses characteristic for wastewater treatment. The perlite, an expanded volcanic rock, and sepiolite, a naturally occurring clay mineral, were tested as carriers of phosphate-accumulating bacterium Acinetobacter junii. All three forms of tested perlite (non-expanded, semi-expanded and expanded) and both forms of tested sepiolite (original and purified) proved to be good carriers of A. junii. Highest number of immobilized bacteria was on the expanded perlite (12.65 × 109 CFU g-1) followed by semi expanded (5.57 × 109 CFU g-1) and non-expanded (1.68× 109 CFU g-1) perlite. The numbers of bacteria immobilized on sepiolite were 8.12 ×109 CFU g-1 on purified form and 5.57 × 109 CFU g-1 on original form of sepiolite. The immobilized bacteria were metabolically active and successfully removed phosphate from the simulative wastewater when incubated in pure culture or in bioaugmented activated sludge. Out of three perlite samples, the semi-expanded perlite proved to be the best carrier of A. junii in the activated sludge system due to good incorporation in the sludge. The expanded and raw perlite were, respectively, bulking and sinking in the bioreactors, thus diminishing the exposure and phosphate removal of immobilized bacteria in the wastewater. The addition of sepiolite with immobilized A. junii in activated sludge system resulted in significant improvement of phosphate removal from the wastewater, increase of biomass and better settling properties of sludge.

The bacteria in the form of biofilm are more adapted to survive environmental stresses. We prepared biofilm by immobilizing phosphate-accumulating bacterium Acinetobacter junii, a heterotrophic bacteria normally present in activated sludge, on the particles of natural zeolitized tuff. The obtained bioparticles had thick biofilm with number of immobilized bacteria 2.2±0.12×109 CFU g-1. To test the survival of such bacteria in harsh conditions of wastewater treatment plants, such as unfavourable pH, the bioparticles were introduced in reactors containing simulated wastewater of extreme pH values, namely, 2, 3, 4 and 5 during 24h. The bacteria immobilized on bioparticles showed great resistance to unfavourable pH. The bacteria were viable in the biofilm, but also detaching from the biofilm and continued its growth in the media as planktonic cells. At pH 4 the survival of bacteria was 82% of starting number as opposed to the control reactors where survival was 0%. At pH 3, there were 38% of bacterial cells able to survive, while at pH 2 no viable cells were present in the reactors after the incubation. The results proved that bacteria immobilized on natural zeolitized tuff are able to survive the extreme conditions of wastewater treatment plants such as 24h disturbances of pH. This way the survival of bacterial population is enabled while the system upsets are fixed and bioparticles could serve as seeds for regeneration of bacterial population in the treatment plant.

The hexadecyltrimethylammonium (HDTMA) bromide was used to modify the surface of natural zeolitized tuff (NZ) from partial monolayer to the bilayer coverage. The modification of NZ resulted in the change of zeta potential of the surface from negative to positive which enhanced the phosphate (P) adsorption capacity. Only in reactors containing the partial monolayer coverage of the surfactant-modified zeolites (SMZs), the P was efficiently removed from wastewater by combined adsorption onto the SMZs and bacterial uptake in the biomass. The SMZs with monolayer, partial bilayer or bilayer coverage showed the bactericidal effect.

Three types of natural zeolite tuffs of particle size  0.125 mm, originating from Croatia, Turkey and Serbia were examined. The tuff from Croatia consisted of approximately 50% clinoptilolite, subordinate plagioclase feldspars and quartz (10-15% of each) and traces of K-feldspars and celadonite (app. 5% of each). The tuff from Turkey contained approximately 70% clinoptilolite, subordinate opal-CT and quartz (10– 15% of each) and traces of K-feldspars and biotite (app. 5% of each). The tuff from Serbia contained approximately 75% clinoptilolite and several minor constituents were analcime, calcite, plagioclase feldspars, biotite and quartz (app. 5% of each). The tuff from Serbia had the largest cation exchange capacity of 1.71 meq/g, followed by tuff from Croatia with 1.60 meq/g and tuff from Turkey with 1.35 meq/g. The main exchangeable cation in samples of tuffs from Turkey and Serbia was Ca, whereas Na was the main exchangeable cation in the sample of tuff from Croatia. The particles of the tuff from Croatia had the most negative zeta potential of -25.14 mV, followed by tuff from Turkey (-23.12 mV) and tuff form Serbia (-17.60 mV). The phosphate adsorption capacity varied from 22.5 mg P/kg for Serbian tuff, to 30.2 mg P/kg for Croatian tuff and 77.5 mg P/kg for Turkish tuff. The interaction of zeolite tuffs and phosphate-accumulating bacterium Acinetobacter junii in simulated wastewater was investigated. After 24h of contact with minerals, most of the bacterial population was immobilized onto minerals by adsorptive growth, while the rest of the bacteria remained as planktonic cells in supernatant. The highest number of immobilized cells was obtained on the tuff from Turkey (74.91x108 CFU/g), followed by tuff form Croatia (48.39x108 CFU/g), while the tuff from Serbia had the lowest number of immobilized cells (6.14x108 CFU/g). The zeta potential of A. junii (-18.4 mV) was negative, as well as the zeta potential of mineral particles and no correlation with the number of immobilized cells and zeta potential of minerals was obtained. The best bacterial activity measured as phosphate uptake rate per CFU was obtained with tuff from Croatia, followed by tuff from Turkey and tuff from Serbia. As the result of bacterial activity, the final percent of phosphate removal from wastewater decreased in the order: 50.23%, 46.32% and 40.21% for tuff from Croatia, Turkey and Serbia, respectively. It can be concluded that zeolite tuff can be a good carrier of phosphate-accumulating bacteria. The zeta potential of zeolite tuff was not a crucial factor which determined the immobilization of bacteria. The clinoptilolite content in the zeolite tuff was not the prevailing factor for the immobilization of bacteria. Bacterial immobilization on the zeolite tuff was defined by the original structure of material. The extent of bacterial immobilization on single zeolite tuff cannot be predicted by mineralogical and chemical analysis of mineral, its cation exchange capacity or its zeta potential. The phosphate- accumulating bacteria immobilized onto zeolite tuff as a biocarrier can find application in the bioaugmentation of activated sludge in order to achieve better phosphate removal from wastewater.

The antibacterial effect of commercial zeolite A against the pure culture of phosphate- accumulating bacterium Acinetobacter junii was investigated. The estimated EC50 values of the zeolite for the inhibition of bacterial colony forming units was 0.33-0.37 g/L by investigated initial pH values of 6, 7 and 8. The toxic effect of zeolite is ascribed to the generation of Al (0.38-1.09 x 10-2 g/L) and Si (2.02-2.93 x 10-2 g/L) ionic species during the process of hydrolysis and dissolution of zeolite (leaching of Al, Si and Na). The toxic effect of Al was confirmed in separate experiments, where the aluminum toxicity (in the form of aluminum sulphate) was estimated as EC50 of 3.45-3.89 x 10-4 g/L. These results suggest the importance of controlling the amounts of synthetic zeolites which can be released to different compartments of environment and accumulate there as nondegradable material. Zeolite A may be the important source of Al in influent of the biological wastewater treatment systems, where they can cause unwanted antibacterial effects.

The phosphate (P)-accumulating bacteria are important for biological P removal from wastewater. Currently, attention is being drawn to the immobilisation of desired bacteria on different carriers in order to achieve a better efficiency of the wastewater treatment. In this study, two size fractions (0.1-1 and 0.1-2 mm) of different forms of expanded perlite (original, autoclaved and magnesium-exchanged) were investigated as possible carriers of P accumulating bacterium A. junii. After 24h of incubation, most of the cells in the reactors were immobilised onto the perlite particles by means of adsorptive growth, while the rest of the biomass remained as free cells in the supernatant. The highest immobilisation rate of 3.27 &plusmn ; ; 0.13x109 CFU/g along with the highest removal of P (49.05 &plusmn ; ; 0.97 mg/g of perlite) was in the reactors containing the magnesium-exchanged perlite of particle size 0.1-1 mm. Considering the very high bacterial biomass accompanied by high P removal and satisfactory reactor performance, the magnesium-exchanged perlite proved to be the most suitable carrier of bacteria among the materials tested in this study.

The aim of this study was to determine the potential of phosphate (P) removal from wastewater by P-accumulating bacteria and organic bentonite. Organic bentonite was prepared by treating bentonite clay with quaternary ammonium salt – cetyltrimetylammonium (CTA) bromide. Cation exchange capacity (CEC) of the bentonite was found to be 179.0 meq/100 g of the dry bentonite. The CTA occupied ca. 175% of the CEC. The modification of bentonite with CTA in amounts higher than 55% of the CEC resulted in the change of zeta potential of particles from negative to positive. Only in reactors containing organic bentonite samples occupied with 3.5 and 28% of the CEC the P was efficiently removed from wastewater by combined adsorption and bacterial uptake in the biomass. The organic bentonite samples with higher CTA loading (from 55 to 175% of the CEC) showed the bactericidal effect. To enhance the P removal from wastewater in the aerated biological system, the organic bentonite can be used, but the special attention should be given to the configuration and location of sorbed CTA molecules and its potential desorption.

Cilj ovog rada je bio istražiti i usporediti kako fosfat-akumulirajuća bakterija Acinetobacter junii preživljava nepovoljne vrijednosti pH u planktonskoj formi (slobodne stanice) i imobilizirana na mineralni nosač, u ovom slučaju prirodni zeolitni tuf, (biočestice), te kako to utječe na njenu sposobnost uklanjanja fosfata iz sintetske otpadne vode. Imobilizirane bakterije su bile otpornije na nepovoljne vrijednosti pH od planktonskih, a njihova stopa umnožavanja je bila značajno veća. Metaboličku aktivnost su zadržale i planktonske i imobilizirane bakterije, no postotak uklonjenog fosfata je bio veći u slučaju biočestica. Rezultati pokazuju da je prirodni zeolitni tuf obečavajući nosač fosfat-akumulirajućih bakterija u svrhu korištenja u postrojenjima za pročišćavanje otpadnih voda. The aim of this study was to determine and compare how phosphate-accumulating bacterium Acinetobacter junii survives unfavorable pH levels in planktonic form (free cells) and immobilised on a mineral carrier, in this case natural zeolitic tuff, (biosolids), and also how this affects the ability to remove phosphate from synthetic wastewater. Immobilised bacteria were better at withstanding unfavorable pH levels than planktonic, and their reproduction rates were signifcantly higher. Both planktonic and immobilised bacteria remaind metabolically active, but percentage of removed phosphate was higher for biosolids. Results show that natural zeolitic tuff is a promising carrier for phosphate accumulating bacteria to be used in wastewater treatment plants.

Teški metali zbog svoje toksičnosti, stabilnosti i sklonosti nakupljanja u živim organizmima, predstavljaju ozbiljni ekološki problem. Stoga ih je potrebno ukloniti iz otpadnih voda prije ispuštanja u prirodni prijemnik. Prirodni zeolitni tuf pokazao se kao pogodan materijal s velikim kapacitetom vezanja iona Cu2+ iz otpadnih voda. U radu je ispitana antibakterijska aktivnost zeolitnog tufa koji sadrži 2,60 % masenog udjela (eng. percentage by weight, wt.%) Cu2+ u odnosu na bakterije Escherichia coli i Staphylococcus aureus, nakon 1 h i 24 h kontakta s zeolitom u tri vodena medija: hranjivom mediju Luria Bertani (LB), sintetskoj otpadnoj vodi i realnoj otpadnoj vodi. Utvrđeno je da zeolitni tuf obogaćen bakrom pokazuje antibakterijsku aktivnost u odnosu na Gram - pozitivnu bakteriju Staphylococcus aureus i Gram - negativnu bakteriju Escherichia coli. Nakon 24 h kontakta dolazi do redukcije broja bakterija u sintetskoj (87,35 – 95,67%) i realnoj otpadnoj vodi (86,79 – 93,54%), dok u Luria Bertani (LB) mediju (5,54 – 10,69%) redukcija nije bila učinkovita. Ne dolazi do izlučivanja toksičnih Cu2+ iona u vodenu otopinu što zeolit s Cu2+ čini obećavajućim antibakterijskim materijalom za dezinfekciju otpadnih voda. Heavy metals pollution is one of the most important environmental problems, due of their toxicity, stability, and tendency to accumulate in living organisms. Therefore, heavy metals should be removed from the wastewater before it can be discharged into a natural recipient. Natural zeolitized tuff was proved to be a suitable material with high capacity binding of Cu 2+ ions from wastewater. The antibacterial activity of natural zeolitized tuff containing 2.60 percentage by weight (wt.%) Cu2+ were tested against bacteria Escherichia coli and Staphylococcus aureus after 1 h and 24 h of contact with zeolit in three different media, namely: Luria Bertani (LB) medium, synthetic wastewater and real wastewater. The copper – loaded zeolitized tuff showed antibacterial activity against Gram - positive bacteria Staphylococcus aureus and Gram - negative bacteria Escherichia coli, after 24 h of contact. The reduction of bacterial numbers was efficient in synthetic water (87,35 – 95,67%) and real effluent water (86,79 – 93,54%), while in the Luria Bertani (LB) medium the reduction was not efficient (5,54 – 10,69%). Since there was not leaching of toxic Cu2 + ions in the aqueous solution, Cu - loaded zeolite can be regarded as promising antibacterial material for disinfection of wastewater.