Mechanisms of heavy metal tolerance in free living bacteria and bacterial biofilms

Teški metali, čija se koncentracija u sredini sve više povećava kao rezultat povećane industrijske aktivnosti i tehnološkog razvoja, ubrajaju se u najčešće zagađivače životne sredine. Kada su prisutni u većim koncentracijama toksični su i predstavljaju ozbiljnu pretnju kako za životnu sredinu tako i za zdravlje ljudi, jer se ne mogu razgraditi i jer se nagomilavaju u lancima ishrane. Sa druge strane, ovakve sredine predstavljaju dobar izvor mikroorganizama tolerantnih na teške metale, sa potencijalnom primenom u biološkom uklanjanju (bioremedijaciji) i oslobađanju (bioluženju) teških metala. Cilj ovog rada je bila izolacija, identifikacija, analiza diverziteta i aplikativnog potencijala novih bakterijskih sojeva sa sposobnošću tolerancije visokih koncentracija teških metala iz površinskog i podzemnog sedimenta iz rudnika bakra Bor. Metagenomskom analizom je detektovan veći mikrobiološki diverzitet u uzorku površinskog sedimenta u poređenju sa podzemnim sedimentom. Iz oba uzorka je izolovano 6 bakterija tolerantnih na visoke koncentracije teških metala, primenom direktne kultivacije. Ovi izolati su taksonomski identifikovani kao predstavnici rodova Arthrobacter i Staphylococcus. Pored ovih izolata, u ovu studiju je bilo uključeno još 8 sojeva za koje je tolerancija na metale prethodno opisana u literaturi, tako da je aplikativni potencijal slobodnoživećih bakterija i definisanih bakterijskih zajednica, kao i njihovih biofilmova, ispitan kod ukupno 14 sojeva. Među 14 testiranih sojeva, 6 sojeva je imalo sposobnost rasta u prisustvu nikla, 6 u prisustvu kadmijuma, 4 u prisustvu bakra, 3 u prisustvu hroma i 1 je imao sposobnost rasta u prisustvu žive, dok nijedan od sojeva nije imao sposobnost rasta u prisustvu gvožđa i cinka. Izdvojio se izolat Staphylococcus sp. MSI08, koji je jedini imao sposobnost rasta u prisustvu tri teška metala (nikl, kadmijum, hrom). Analizom genetičkih determinanti mehanizama tolerancije na teške metale, kod 2 soja je detektovano prisustvo gena za determinantu tolerancije na kadmijum, kod 2 soja prisustvo gena za determinantu tolerancije na hrom i kod 2 soja je detektovano prisustvo gena za determinantu tolerancije na bakar. Svi testirani sojevi su imali sposobnost formiranja biofilmova, s tim što su najveći potencijal za formiranje biofilmova pokazali Staphylococcus sp. MUI10, Arthrobacter sp. MSI31, Pseudomonas aeruginosa PAO1 i Cupriavidus metallidurans CH34, dok su Staphylococcus sp. MSI08, Rhodococcus sp. TN113, Pseudomonas putida KT2440 i Cupriavidus necator H16 pokazali nešto slabiji potencijal za formiranje biofilmova...Heavy metals are one of the most common environmental pollutants due to increased industrial activities and technological development. When present in excessive concentrations, heavy metals are highly toxic and they pose a serious threat both to environment and human health, due to their persistence in nature and accumulation in the food chain. On the other hand, polluted sites have proven to be valuable sources of bacteria capable to tolerate increased concentration of heavy metals that could be potentially applied in biological removal (bioremediation) and release (bioleaching) of heavy metals. The aim of this study was to obtain, identify, analyze diversity and application potential of novel bacterial strains with the ability to tolerate high heavy metal concentrations from surface and underground sediments from the copper mine Bor. Metagenomic analysis revealed a higher microbial diversity in surface sediment compared to underground sediment. From both sediments, 6 bacterial strains able to tolerate high heavy metal concentrations were isolated using direct cultivation approach. These isolates were taxonomically identified as members of Arthrobacter and Staphylococcus genus. In addition, this study involved 8 more strains with heavy metal tolerance previously described in the literature. Thus, 14 bacterial strains were included in analysis and evaluation of heavy metal tolerance and application potential of free living bacteria and defined bacterial consortia, as well as their biofilms. Among 14 tested strains, 6 strains had the ability to grow in the presence of nickel, 6 in the presence of cadmium, 4 in the presence of copper, 3 in the presence of chromium and 1 had the ability to grow in the presence of mercury, while none of the strains had the ability to grow in the presence of iron and zinc. Staphylococcus sp. MSI08 singled out as the only strain with the ability to grow in the presence of three heavy metals (nickel, cadmium, chromium). Analysis of genetic determinants conferring heavy metal tolerance revealed presence of cadmium tolerance genes in 2 strains, chromium tolerance genes in 2 strains, and copper tolerance genes were also detected in 2 strains. All strains showed ability to form biofilms, but Staphylococcus sp. MUI10, Arthrobacter sp. MSI31, Pseudomonas aeruginosa PAO1 and Cupriavidus metallidurans CH34 demonstrated the highest potential for the biofilm formation, while Staphylococcus sp. MSI08, Rhodococcus sp. TN113, Pseudomonas putida KT2440 and Cupriavidus necator H16 were less successful in biofilm formation..