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The research focuses on developing a bioleaching procedure for red mud samples. Bacteria were isolated using red mud from three different locations, after that, grown bacteria were applied to the red mud samples from Mostar, Bosnia and Herzegovina. Samples were heated to 100°C and filtered with MiliQ water. Geochemical analysis of red mud samples with bacteria, eluates and red mud on filter papers was carried out.

Hydroxyapatite (HAp) is the most widely used calcium phosphate for hard tissue applications due to its compositional similarity to biological apatite, biocompatibility and bioactivity. As bone apatite is a multi-substituted carbonated HAp containing trace elements, one of the used approaches to improve the biological and physicochemical properties of HAp is ionic substitution. To achieve a good balance between cell biomaterial interactions and anti-bacterial properties, a Sr-substituted HAp with potential regenerative properties was co-substituted with Ag+ ions to offset the cytotoxicity of higher substitution levels of Ag+ ions and stimulate osteogenic growth. A series of single-substituted HAp with Sr2+ and Ag+ ions, and Ag/Sr-co-substituted HAp with different substitution degrees (0, 1, 2.5 and 5 mol%) were obtained by wet precipitation method from cuttlefish bone in order to obtain biomimetic multi-substituted HAp. Rietveld refinement studies revealed an increase of cell volume (Å3), a-axis (Å), b-axis (Å) and c-axis (Å) for all obtained HAp powders, due to larger ionic radius of Sr2+ (1.12 Å) and Ag+ (1.15 Å) in comparison to substituted Ca2+ (0.99 Å) ion. The element mapping reflected the uniform distribution of the Ca, P, Ag and Sr elements in the precipitated powders. The zeta-potential values of the Ag/Sr-co-substituted HAp samples revealed that all the samples were negatively charged and the obtained zeta-potential values expressed a downward trend with increasing buffer pH. The antibacterial effect was evaluated by inhibition zone and spread plate analysis, while the surface of samples was observed after inhibition zone analysis by scanning electron microscopy. The antibacterial effect was confirmed for all HAp powders substituted with Ag+ ions against Gram-positive S. aureus and Gram-negative E. coli, representative bacteria in clinical bone infections. Further, the non-cytotoxicity was confirmed by using human embryonic kidney 293 and human mesenchymal stem cells.

Bacterium Acinetobacter baumannii is nowadays an emerging hospital pathogen. From colonised or infected patients, A. baumannii is disseminated into the natural water environments. Due to the resistance of this bacterium to the conventional methods of water disinfection, alternative methods of A. baumannii removal from water are needed. There is no literature data how the hydrophobicity level of A. baumannii isolates influences the immobilization onto hydrophilic surface of natural zeolitized tuff (NZ). The immobilization of hydrophilic and hydrophobic isolate of A. baumannii onto NZ was examined. Both hydrophilic and hydrophobic isolate was successfully immobilized onto NZ, whereby hydrophobic isolate in a higher abundance than hydrophilic one. The NZ is a promising material for the capture of isolates of pathogenic bacteria A. baumannii from water, and could find application in water treatment technology.

Intestinal enterococci (Ie) are well known bacterial indicators of faecal contamination in food as well as in environmental samples. In total 19 soils under the anthropogenic influence in Croatia were analysed: 15 influenced by illegal dumpsites of human solid waste and 4 agricultural soils fertilized with animal manure. Three samples were control soils without the visible anthropogenic influence. The number of Ie was measured after suspension of soil in peptone water and subsequent confirmation on Slanetz Bartley and Bile esculin azide agar. The pH was measured in soil suspension in distilled water. The pH values of soils varied from 4.9 to 9.7. Ie were not detected (˂1 CFU/g) in control soils saved from visible human activities. Ie also were not detected in agricultural soils fertilized with swine or poultry manure. All soils influenced by illegal dumpsites contained the Ie at concentration from 1.3 to 6.2 log CFU per g of wet soil. Ie could be used as indicators of illegal dumps, since they could be leached from the human solid waste by storm water and infiltrated into the underling soil. The traditional use of animal manure as a soil fertilizer has no appreciable influence on the occurrence of Ie. More stringent monitoring and prevention of illegal dumps is needed to avoid the negative human influence on the soil.

In order to improve hydrophilicity, dyeing and antimicrobial properties of wool, the potentialapplication of low-pressure oxygen plasma pretreatment was explored. After optimization of process parameters, samples of wool fabrics were dyed with natural dye extracted from Punica granatum L. The effect of plasma pretreatment and mordants on colorimetric and antibacterial properties against Staphylococcus aureus and Klebsiella pneumoniae was investigated. The results revealed a significant improvement in wicking ability and tensile strength of all tested samples. Plasma pretreatment proved to be suitable for improving color yield and even replacing the application of certain mordants in the process of dyeing wool with pomegranate dye. All samples exhibited good antibacterial activity against tested bacteria. Only a slight decrease in color strength and antibacterial activity is visible on plasma pretreated samples after 28 days of aging.

Carbapenems are considered as last-resort antibiotics, and the carbapenem-resistant bacteria (CRB) are currently deemed as the biggest microbial threat to human welfare. Since soil is considered as one of the most important environments for emergence of antibiotic-resistant bacteria, we searched for cultivable CRB in soil samples from 14 locations, mainly those influenced by illegal dumps, across Croatia. We proposed cultivation of CRB from environmental samples at two temperatures to differentiate between ones that are intrinsically resistant (37°C) and ones with acquired resistance, which are presumably human-associated and clinically relevant (42°C). The Stenotrophomonas sp. were predominant among CRB37. Among CRB42 we found mostly bacteria from the family Enterobacteriaceae, Acinetobacter and Burkholderia species. The CRB37 were found in every soil, excluding one sample where pH was extremely low, and another where water content was extremely low. The CRB42 were found in most soil samples, but were absent from samples of non-contaminated pasture and forest, indicating that human-associated CRB are unlikely to be found in soils deprived of anthropogenic influence. The appropriate deposition of human solid waste is thus one of the means to prevent the spreading of CRB of clinical relevance to the soil.

Acinetobacter baumannii is an emerging human opportunistic pathogen that causes nosocomial as well as community-acquired infections. A. baumannii is assigned to ESKAPE group of pathogens due to its ability to „escape“ the biocidal activity of antibiotics. The aim of this study was to test the behaviour of A. baumannii isolates in water media under different temperature and pH conditions in order to predict its behaviour in the environment. Survival of 5 A. baumannii isolates was monitored during 7 weeks in commercially available spring water and diluted Nutrient broth. Tested temperatures at neutral pH were: -20, 4, 22, 35, 44, 50 and 63 °C. Tested pH values were 2, 7 and 12 at 22 °C. Bacterial numbers were determined on Nutrient agar and expressed as log CFU mL-1 from which survival rates were calculated. A. baumannii successfully survived at temperatures ranging from -20 to 44 °C during 7 weeks. It was able to survive classic pasteurization procedure (63 °C/30 min) and was destroyed after 2h of contact. Prolonged survival of A. baumannii at extreme temperatures and pH values indicates that this pathogen is resilient and persistent under different conditions that are unfavourable for the majority of other mesophilic non-sporogenic bacteria. Optimal conditions for the growth and survival of this pathogen are room temperature and neutral pH, which is a prerequisite for the existence of isolates in hospital environment. Multidrug-resistant isolates survive better in harsh environmental conditions than sensitive isolates, which represents a major healthcare concern.

An emerging hospital pathogen, bacterium Acinetobacter baumannii occurs in the natural environment influenced by human liquid or solid waste. Bacterial biofilm formation on abiotic or biotic solid surfaces is considered as an important virulence factor of this pathogen. There are no data on the quantification of viable bacterial cells of A. baumannii (either of clinical or environmental origin) that are immobilized onto the solid surfaces. In this study, the environmental isolate of A. baumannii was immobilized onto the particles of natural zeolitizied tuff (NZ) at 20°C in the water media of different nutrient concentrations. Numbers of immobilized viable bacterial cells ranged from 6.7-9.1 log CFU/g. The intensity of bacterial immobilization onto the NZ was a function of total bacterial concentration which was statistically significantly positively correlated with the COD of surrounding water media. Immobilization of bacteria onto the NZ occurred via the bacterial extracellular substances that enable longer survival of bacteria in the environment. The presence of A. baumannii in the natural environment will possess a serious public health concern about the spread of this emerging human pathogen.

Multi-drug resistant (MDR) Acinetobacter baumannii were found in untreated as well as in biologically or chemically treated hospital and municipal wastewaters. However, there is no evidence about the fate of A. baumannii in the wastewater treatment process. The aim of this study was to screen the sewage sludge after its stabilization by anaerobic mesophilic digestion for the presence of A. baumannii. On 9 sampling occasions at the municipal wastewater treatment plant of the City of Zagreb, 17 isolates of A. baumannii were recovered form digested sludge with MALDI-TOF MS score values ranging from 2.026-2.288. 3/17 isolates were sensitive to all antibiotics, while 14 MDR isolates shared the resistance to carbapenems and fluoroquinolones but sensitivity to colistin. Technology of anaerobic sludge digestion was performed at 36°C, neutral pH and digestion time of 21-36 days. In laboratory controlled anaerobic conditions isolates were able to survive on Nutrient agar in anaerocultA system during 30 days, after which multiplied normally in aerobic conditions. However, isolates were not able to multiply directly in anaerocultA. The finding confirms the need of proper management and disposal of sewage sludge generated at wastewater treatment plants in order to prevent the spread of MDR A. baumannii in nature.

Acinetobacter baumannii is an emerging opportunistic pathogen associated mainly with hospital infections. Since there is insufficient data regarding the successfulness of A. baumannii in the natural environment, the aim of this investigation was to examine the survival rates of A. baumannii in different types of natural water media. Multi-drug resistant (MDR) environmental isolates were cultivated on CHROMagar Acinetobacter at 42°C/24h. Bacterial suspension was inoculated into bottles containing the 100 mL of sterile spring water, seawater and effluent water from the secondary type of municipal wastewater treatment plant, and incubated at 20°C with stirring (150 rpm) during 28 days. Number of bacteria was followed by cultivation on Nutrient agar plates at 42°C/24h, from which the survival rates were calculated. In effluent water the successful multiplication of A. baumannii was evident (124% survival rate after 28 days), while in natural spring water and seawater no multiplication occurred. The survival rate in spring water was constant (100%) with a slight decrease (94%) after 21 days and 89% after 28 days, while in seawater the survival rate was constant for the first 2 days (100%), and decreased to 64% after 28 days. The chemical oxygen demand of effluent was considerably higher (24 mgO2/L) than the spring (3 mgO2/L) and seawater (4 mgO2/L). These findings indicate that MDR isolates of A. baumannii successfully persist in the natural water media and that the survival rates are dependent on the availability of nutrients.

Acinetobacter baumannii is well known emerging hospital pathogen of the 21st century. Multi- drug resistant (MDR) A. baumannii are found in untreated as well as in chemically treated hospital wastewaters. However, there is no clear evidence about their origin. The aim of this study was to screen the hospital wastewater for the presence of viable A. baumannii and to find the correlation between recovered wastewater and clinical isolates. The sampling of the hospital wastewater was done at the central manhole of the Special Hospital for Pulmonary Diseases in Zagreb, Croatia. The clinical isolate was recovered on the exact date from bronchial aspirate of patient hospital. The isolation of A. baumannii was performed on CHROMagar Acinetobacter supplemented after incubation at 42°C/48h. One clinical and six isolates from hospital wastewater gave reliable MALDI-TOF MS score values ranging from 2.021-2.271 confirming the identity of A. baumannii colonies. All isolates were MDR and shared the resistance to carbapenems and fluoroquinolones, but sensitivity to colistin. MLST analysis following Oxford scheme revealed that all isolates belong to the ST-195 clustering into the CC92 within the IC2. This study confirmed the occurrence of viable MDR A. baumannii in hospital wastewater in Zagreb. Close relatedness of isolates from hospital wastewater with the clinical isolates from the same hospital in the period of monitoring suggests the possible origin of recovered wastewater A. baumannii. This finding confirms the need for proper treatment and disposal of untreated hospital wastewaters in order to prevent the spread of MDR A. baumannii in nature.

The carbapenem resistance in Gram-negative bacteria, which is usually associated with resistance to several other classes of antibiotics, has become a global problem of this century in hospital settings. Reports regarding the occurrence of viable carbapenem- resistant bacterial population (CRBP) outside medical institutions are globally scarce. The aim of this study was to quantify the numbers of viable CRBP through the secondary type of municipal wastewater treatment process (WWTP). The study was performed at the WWTP of the Croatian capital city of Zagreb, on 20 sampling occasions from September 2015 to June 2016. The CRBP was enumerated on CHROMagar Acinetobacter supplemented with CR102, which allows the growth of carbapenem-resistant isolates after incubation at 37C/72 h. The CRBP were found in influent, effluent, fresh activated sludge, anaerobically digested sludge, but not in lime- treated stabilized sludge of pH 12. Similar abundance (p>0.05) of CRBP was detected in influent and fresh activated sludge (median value 4.0 log CFU/mL) and significant decrease (p

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

Any natural or artificial substrate that is immersed in seawater is rapidly covered by unavoidable biofouling. Growth of such fouling also known as biofilm formation is complex and not yet thoroughly investigated. Roughly known stages of biofilm generation are: adsorption of organic and inorganic macromolecules ; migration of microbial cells to the surface ; attachment of bacteria onto surface ; production of extra-cellular polymers ; and development of a more complex 3D community with the presence of micro- and macroalgae, leading to attachment of larger macro-invertebrates. Succession of diatoms and bacteria was investigated during a one month period incubation on artificial substrate mounted above the seafloor at five meters depth. Samples were collected after 1h, 12h, 24h, 48h, 4 days, 7 days and 30 days of contact. For bacteriological analysis the biofilm was detached from plexiglass plates (polymer of methyl methacrylate) using an ultrasonic probe and samples were inoculated onto selective agar plates. Biofilm for algological analysis was scraped with adopted toothbrush and resuspended in subsamples for epiphluorescent, light and electronic microscopy, as well as for HPLC pigment analysis. During monitoring of biofilm development, abundance of all biofilm components increased and reached maximum values after one month incubation. In the bacterial community heterotrophic marine bacteria were dominant and reached 1.94x104 CFU cm-2. Faecal coliforms and faecal enterococci were not detected in the biofilm. Phototrophic component of the biofilm was dominated by diatoms and reached 6.10x105 cells cm-2, which was supported by pigment analysis with fucoxantin as dominant pigment in concentration up to 110 ng cm-2. Dominant diatoms were Cylindrotheca closterium and unidentified penate benthic diatoms. More detailed taxonomic analysis of those diatoms by electronic microscopy revealed up to 20 different taxa of diatoms. The study confirmed that the plexiglass surface in a marine environment is susceptible to biofouling within 30 days of contact.

Quercetin, a protective phenolic compound isolated from plants, exerts antibacterial activity against many strains of bacteria. It is discovered that some positive biological effects of free phenolics can be improved by their chemical derivatization with metals. Thus, we synthesized a complex of the phenolic compound quercetin (Q) with the rare earth metal europium (Eu3+) in a biological buffer, and compared its activity against Staphylococcus aureus cells with that of free Q. While free Q did not show antibacterial activity against S. aureus, its complex with Eu3+ did. Using confocal laser scanning microscope we screened for the ability of Q/Eu3+ complex, free Q and Eu3+ to penetrate into S. aureus cells. Images revealed all the three chemical forms penetrated into S. aureus cells. However, while free Q did not disrupt cell morphology, Q/Eu3+ complex and free Eu3+ caused degradation of cell structure – most of the cells were lysed, their structures were amorphous, and only few cells showed minor fluorescence signal corresponding to the Q/Eu3+ or free Eu3+. Thus, the antibacterial activity of Q/Eu3+ was attributed to degradation of complex and antibacterial effect of released free Eu3+. Confocal laser scanning microscopy was successfully employed for visualization of anti-S. aureus effect of Q/Eu3+ complex.

Two commercially available materials, MiraCarbon® carbon fibers (CF) and CultiSpher-S® microcarrier (CS), were used as support materials for immobilization of phosphate (P)-accumulating bacterium Acinetobacter junii. The CF are filamentous microscopic fibers intended to be used for mass adhesion of activated sludge thus preventing excess of waste sludge. The CS are macroporous gelatin spheres intended to be used as support for growth of animal cells. The immobilization of A. junii was performed by aerobically incubating the pure bacterial culture and selected materials in simulative wastewater with low P concentrations (2 mg L-1) for 24 h. After the incubation the numbers of immobilized bacteria were 1.97±0.95×109 CFU g-1 of CF and 4.96±1.0×109 CFU g-1 of CS. The materials with immobilized bacteria were now transferred to simulative wastewater with high P concentrations (20 mg L-1) to test the P-removing capacity of such prepared substrate. During 24 h of aerobic incubation a part of the cells were detaching from the surface of the CF thus bioaugmenting the surrounding media while majority of the bacteria remained firmly in the biofilm ; the bacteria were metabolically active and removed 50% of starting P from the wastewater. On the other hand, the CS dissolved after 24 h of incubation resulting in blur and gelatinous solution which excluded CS for potential usage in the treatment systems, although the bacteria were still viable and metabolically active. The CF were rigid throughout the experiment, showed great potential for implementation and should further be tested in activated sludge systems.

To test new methods of activated sludge bioaugmentation we immobilised Acinetobacter junii and Pseudomonas aeruginosa onto natural zeolitic tuff and tested their ability to remove phosphates and heavy metals (copper, zinc, nickel) from synthetic and real wastewater. A. junii strain used is a known phosphate (P)-accumulating bacterium and P. aeruginosa strain was isolated from metalworking fluids. We immobilized the bacteria (bioparticle formation) by aerobically incubating pure bacterial cultures and zeolitic tuff in nutrient medium, obtaining A. junii bioparticles (AJBP) and P. aeruginosa bioparticles (PABP). We conducted four sets of experiments in order to: i) compare heavy metal tolerance of planktonic and immobilized bacteria, ii) compare their ability to remove phosphates and heavy metals from synthetic wastewater, iii) investigate if combined system with AJBP and PABP can be as efficient as systems with only AJBP or PABP, iv) test the ability of combined AJBP and PABP to remove phosphates and metals from real wastewater. Immobilised bacteria were significantly more tolerant to heavy metal stress than planktonic bacteria and removed significantly more metals. The two bacteria in form of bioparticles can survive in the same system, multiply and remove phosphates and heavy metals more successfully than when used separately. By simultaneous use of both bioparticles in real wastewater achieved values of phosphate and metal removal were from 15.2 to 17.0 mg/L P-PO4, 1.1 to 11.4 mg/L Cu2+, 1.2 to 32.8 mg/L Zn2+ and 1.0 to 39.6 mg/L Ni2+. This high efficiency gives the opportunity to use these bioparticles in wastewater treatment plants.

E. coli DSM 498 was precultivated on the Tryptic glucose yeast agar (Biolife, Italy) at 36±0.1°C/2d. Biomass was subcultured and incubated at 36±0.1°C/2d on/in 8 different commercial media suitable for the cultivation of E. coli (Biolife, Italy): Nutrient agar, EC X-GLUC agar, Endo agar, Mac Conkey agar, Mac Conkey broth, m-Faecal Coliform agar, m-LES Endo agar, TTC Tergitol 7 agar base. The culture of E. coli from each medium was stained by Gram method and examined by light microscopy with magnification of 7520x. The length and width of bacteria were determined using DinoCapture 2.0 software by measuring at least 100 cells per medium. The cells of E. coli cultivated on the media with proteins as the carbon source (Nutrient agar and EC X-GLUC agar) had an average length of 1.592±0.338 m, width 0.651±0.106 m and width/length ratio 0.422±0.086. The cells of E. coli cultivated on/in the media with proteins and lactose as the carbon sources (Endo agar, Mac Conkey agar, Mac Conkey broth, m-Faecal Coliform agar, m-LES Endo agar, TTC Tergitol 7 agar base) were statistically significant longer with an average length of 3.595±1.193 m, wider with 0.680±0.131 m and with lower width/length ratio of 0.202±0.050. In the literature the cells belonging to the genus Ecsherichia are generally described as straight cylindrical rods 2.0-6.0 x 1.1-1.5 m. The size of E. coli varies when cultivated on/in different commercial media. Although the size of E. coli is not a determinative characteristic, the variation of cell size when cultured on/in different commercial media can be of great help during routine laboratory work.

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.

This research aimed to determine the microbial colonization and potential biodegradation of classical high density polyethylene (HDPE) garbage bags in conditions which refl ects its real disposal without preliminary abiotic pretreatment. The HDPE fi lms were buried during 14 weeks in containers with humifi ed soil, activated sludge and its mixture. The colonization of heterotrophic mesophilic aerobic and anaerobic bacteria on the HDPE was monitored. The bacteria and fungi formed biofi lms on the surface of HDPE, but did not have the potential to degrade it since no changes in FTIR spectra of HDPE fi lms were observed. The classical HDPE is primary nontoxic inert material, which is not biodegradable in environmental conditions, what represents a serious ecological problem.

Natural zeolitized tuffs (NZ) rich in clinoptilolite are good adsorbents of heavy metals due to their high cation exchange capacity. The NZ containing 70 wt. % of clinoptilolite was obtained from the sedimentary deposit Zlatokop, Serbia. The particle size of the sample was in the range 0.063-0.1 mm. The NZ was firstly converted into the Na-form by treating the NZ with 2M of NaCl solution, in order to improve the clinoptilolite exchange capacity without affecting the crystallinity of NZ. The experiments on Zn2+ removal from water were performed at 30C during 24h using the batch method and ZnCl2 solution in concentration of 400 mg Zn/L with mass concentration of NZ of 1g/100mL. On this way NZ containing Zn2+ ions (named ClZn2+) was obtained. The ClZn2+ was than completely dehydrated at 550ºC under air. The dehydration led to a generation of ZnO nano-particles (about 5 nm) widespread over the surface of NZ. This material was assigned as ClZnO. The leaching test of ClZn2+ and ClZnO was performed using the 0.05M NaCl solution of pH 7.0 at 37C during 24h. The resulting leached concentration of Zn2+ in water was 0.67 mg/L for ClZn2+ whereas no leached Zn2+ was found for ClZnO. After the use of clinoptilolite for zinc removal from wastewater, we looked for future application of the used material, one of which can be removal of pathogenic bacteria in the tertiary stage of wastewater treatment. The antibacterial activity of ClZn2+ and ClZnO was tested against representative pathogenic Gram-negative (Escherichia coli, DSM 498) and Gram-positive (Staphylococcus aureus, clinical isolate at Institute of Public Health, Zagreb) bacteria. The antibacterial assay was carried out whit mass concentration of material of 1g/100mL in three different water media: Luria Bertani or LB medium, synthetic wastewater and real effluent water from the secondary stage of the biological wastewater treatment. The initial pH of used media was 7.0 and the chemical composition was as follows (in mg/L): LB medium (bacto-tryptone 10, 000 ; bacto-yeast extract 5, 000 ; NaCl 10, 000 ; COD 14, 000), synthetic wastewater (Na-propionate 1, 000 ; peptone 100 ; MgSO4 10 ; CaCl2 6 ; KCl 30 ; yeast extract 10 ; KH2PO4 20 ; COD 1, 206), effluent water (T-N 28.3 ; T-P 2.27 ; COD 31.4). All media and materials were autoclaved before the experiments were to commence. The experiments were carried out at 37C. The CFUs of bacteria were measured on LB agar at the start and after 24h of experiment. The antibacterial activity of material was expressed as percentage of log CFU reduction when compared to control where no material was added (Table 1). The percentage of inhibition of bacteria in the presence of ClZn2+ and ClZnO showed significantly negative correlation with the COD of water media used in assay (R = -0.987 and -0.949, respectively). The ClZn2+ showed better antibacterial activity than ClZnO, which is ascribed to the difference in leaching of Zn2+ ions from materials. The E. coli seems to be more resistant to Zn2+ ions than S. aureus. The difference in final pH among control and experimental reactors was not higher than 0.59 units. No significant antibacterial activity of materials was observed after 1h of contact with bacteria. Table 1. Antibacterial activity of ClZn2+ and ClZnO against E. coli and S. aureus after 24h of incubation in different water media when compared to control. c0 E. coli (106 CFU/mL) = 3.85±0.89 ; c0 S. aureus (107 CFU/mL) = 1.42±0.24 ; significantly different when compared to A-LB medium, B-synthetic wastewater, C-ClZn2+. The antibacterial activity of zinc containing clinoptilolite (mass concentration 1g/100mL, 24h of contact) was depended on the following factors in decreasing order: COD of the water medium, type of material and species of bacteria. The antibacterial activity of certain compound tested in the synthetic medium can be significantly different from those in real water medium.

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 applicable methods for technical stabilization of waterfall are conditioned by natural, architectural, historical values of the landscape. That is a reason why concrete or reinforced concrete structures should be avoided and why binders for natural tuff detritus that do not change the shape or color of naturally hardened tuff are preferred. In this paper the possibilities of technical interventions in waterfall area that are formed in heterogeneous tuff sediments are described. The rivers Pliva waterfall is a unique natural phenomenon, placed in the middle of an old town Jajce, and therefore is an ideal and unique test field. This landscape uniqueness, conditions the innovative approach to the waterfall erosion problem. As a possibility of waterfall erosion protection injection suspensions were examined. Laboratory experiments have shown good results, on a large number of samples of tuff beds, when injected with micro-cement. Also the usability of other injection suspensions was examined within the research region. The injection suspensions were adjusted according to the tuff lithological composition. Comparison of the control samples have shown acceptable results of the applied methods, both in the laboratory and on the site.

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 possibilities of technical interventions in waterfall area formed in heterogeneous tuff sediments are described in this paper. The river Pliva's waterfall was chosen for the test site, as it is a unique natural phenomenon placed in the middle of the old town of Jajce. The applicable methods for technical stabilization of waterfall are conditioned by natural, urban, architectural, historical and specific values of the town landscape. That is why that concrete or reinforced concrete structure should be avoided and priority given to binders for natural tuff detritus that do not change the shape or color of naturally hardened tuff are preferred. Laboratory experiments on a large number of samples have shown good results when micro-cement was injected into tuff beds, but the usability of other injection suspensions was also examined on the research region. The injection suspensions were adjusted to the tuff lithological composition. Comparisons of the control samples in laboratory and on site have shown acceptable results of the applied methods., U članku se opisuju mogućnosti prihvatljivih tehničkih intervencija u prostoru slapova formiranih u heterogenim naslagama sedre. Kao istražni poligon odabran je slap rijeke Plive, izuzetan prirodni fenomen smješten u samom središtu povijesnog grada Jajca. Metode istraživanja primjenjivosti tehničkih rješenja stabilnosti slapa uvjetovane prirodnim urbanističkim, arhitektonskim, povijesnim, te specifičnim pejzažnim vrijednostima gradskog prostora zbog čega betonske i armirano betonske konstrukcije treba izbjegavati, a prioritet dati stabiliziranju prirodnog sedrenog detritusa vezivima koja ne mijenjaju izgled i boju prirodno očvrsle sedre. Laboratorijski eksperimenti na velikom uzorku pokazali su dobru injektibilnost mikrocementa u sedreni detritus, a primjena ostalih suspenzija ispitivana je na istražnom poligonu gdje su injekcijske suspenzije prilagođavane litološkom sastavu sedrenih naslaga. Kontrolna ispitivanja u laboratoriju i na istražnom poligonu potvrdila su uspješnost primijenjenih metoda.

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.

Objectives: Autism is a neurodevelopmental syndrome, with onset in early childhood, characterized by social and communication dysfunction and stereotypic behaviors. Recent findings indicate that disturbances in serotonergic (5HT) neurotransmission represent one of the biological substrates of this disorder. Alterations in the expression of one or more 5HT-related genes might lead to the dysregulation of 5HT transmission in the brain, affecting so its early development and resulting in autistic behavioral symptoms. We have studied the association of the following 5HT-related genes: tryptophan hydroxylase 1 and 2 (Tph1, Tph2), monoamine oxidase A and B (MAOA, MAOB), 5HT transporter (5HTT) and 5HT2A receptor (5HT2Ar) with autism in the Croatian population. Materials and Methods: DNA was isolated from blood samples of 103 autistic and 368 control subjects and genotyped, using polymerase chain reaction and restriction analysis, for A218C Tph1, G703T Tph2, uVNTR MAOA, A/G MAOB, 5HTT-LPR and -1438AG 5HT2Ar polymorphisms. Distributions of alleles and genotypes were compared between the autistic and control groups. Results: There were no significant differences in the distribution of alleles and genotypes of the Tph, MAO and 5HTT polymorphisms between autistic and control subjects. However, a significant difference in the distribution of genotypes (p = 0.014) adn alleles (p = 0.0054) of the 5HT2Ar polymorphism has been observed, with an increased incidence of G allele and GG genotype in the autistic group. In addition, an increase in the relative autistic to control subject ratio followed the increase in number of G alleles. The observed trend was significant (p = 0.0035). Conclusion: The results provide evidence for the association of the 5HT2Ar gene with autism in the Croatian population and indicate a possible role of this 5HT-related gene in susceptibility to autism spectrum disorders.

Depending on the type of zeolite, different interactions between material and examined PAB A. junii were observed. Natural clinoptilolite tuff is excellent bacterial carrier and the main mechanism of interaction is immobilization of bacteria onto the surface of material and increase of the total bacterial biomass concentration. In spite, the synthetic zeolites display the toxic effect against the same PAB. Future investigations should be done in order to investigate the influence of different types of zeolite on other bacteria.

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 effects of the pH (3-10) of synthetic wastewater on the pure culture of phosphate (P) – accumulating bacterium Acinetobacter junii were studied. The pH 7 was set as an optimal value for growth, aerobic P-uptake and anaerobic P-release of A. junii. The wastewater having pH 3, 4 and 5 showed negative influence to the growth and metabolism of A. junii. The addition of clay mineral in the reactors having unfavourable pH (4 and 5) resulted in the shift of pH toward the neutral pH 7 due to its buffering ability. The addition of clay enabled the survival of A. junii at low pH 4 and 5. The majority of the cultured bacterial population was immobilized onto the clay particles, showing that clay can be good carrier of bacteria. It can be concluded that the addition of clay could impair the influence of unfavourable pH of the wastewater on the A. junii due to its buffering capacity and immobilization of bacteria.

The harmful effects of surfactants to the environment are well known. The antibacterial effect of cationic surfactants against the pure culture of phosphate (P)-accumulating bacterium Acinetobacter junii was investigated. The estimated EC50 values of the N-dodecylpyridinium chloride (DPC) for growth inhibition was 1.4&plusmn ; ; ; 0.5 x 10-6 mol L-1 and for the inhibition of the P-uptake rates 7.3&plusmn ; ; ; 2.6 x 10-5 mol L-1. The estimated EC50 values of the N-cetylpyridinium chloride (CPC) for growth inhibition was 4.9&plusmn ; ; ; 1.3 x 10-7 mol L-1 and for the inhibition of the P-uptake rates 7.7&plusmn ; ; ; 2.9 x 10-6 mol L-1. The estimated EC50 values of the hexadecyltrimethylammonium bromide (HDTMA) for the inhibition of CFUs in the pure culture of A. junii was 3.27&plusmn ; ; ; 1.12 x 10-7 mol L-1 and for the inhibition of the P-uptake rates 2.47&plusmn ; ; ; 0.51 x 10-6 mol L-1. The results proved a high acute toxicity of the three investigated cationic surfactants against A. junii. This suggests the importance of controlling the amounts of cationic surfactants in influent of the wastewater treatment systems in order to avoid the possible failure of the biological P removal from wastewaters.

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.