Metabolic varsality of bacteria isolated from surgace river sediment influenced by petrochemical effluentes: the case of Pseudomonassp. TN301 as polyhydroxyalkanoate producer

Mono- i poliaromatična jedinjenja predstavljaju široko rasprostranjene zagađivače životne sredine. Ova jedinjenja veoma dugo opstaju u datoj sredini, a mnoga od njih imaju i toksična, kancerogena i mutagena svojstva. Sredine zagađene aromatičnim jedinjenjima su najčešće kontaminirane i teškim metalima, a odlikuju se i neravnotežnim odnosom esencijalnih nutrijenata. Sa druge strane, ovakve sredine su dobar izvor bakterija sa sposobnošću preživljavanja u izuzetno teškim uslovima. Cilj ovog rada je bila izolacija robustnih bakterija sa potencijalnom biotehnološkom aplikacijom iz sedimenata koji su pod uticajem petrohemijskih efluenata. Upotrebom dve različite strategije izolovanja, toplotnog pretretmana i medijuma za rast i sporulaciju Actinobacteria, i predobogaćivanja uzoraka naftalenom u kombinaciji sa odabirom brzo rastućih bakterija sa sposobnošću rasta na naftalenu kao jedinom izvoru ugljenika i energije izolovane su 34 Gram-pozitivne i 4 Gram-negativne bakterije. Izolati sa sposobnošću degradacije od 9 do 15 najčešćih aromatičnih polutanata poput benzena, bifenila i naftalena pripadali su rodovima Bacillus, Arthrobacter, Rhodococcus, Gordonia, Streptomyces, Staphylococcus, Actinobacteria, Sinorhizobium i Pseudomonas. Rhodococcus sp. TN105, Gordonia sp. TN103 i Arthrobacter sp. TN221 su identifikovani kao novi sojevi. Od 11 izolata sa sposobnošću rasta u prisustvu bar jednog od testiranih teških metala (živa, kadmijum, nikl) u 100 mM koncentracijama, 8 je imalo sposobnost degradacije 15 različitih aromatičnih jedinjenja. Rezistenciju na bar jedan od testiranih antibiotika pokazalo je 60% izolata. Međutim, nije primećena visoka učestanost multirezistencije na antibiotike. Trećina izolata je imala sposobnost da akumulira bar jedan od biopolimera. Dvanaest izolata, uglavnom pripadnika rodova Bacillus i Arthrobacter su akumulirali polifosfate, 3 Bacillus sp. izolata su akumulirala polihidroksibutirat (PHB), Pseudomonas sp. TN301 je akumulirao polihidroksialkanoate srednje duzine niza (mcl-PHA), dok su 4 Grampozitivna izolata akumulirala egzopolisaharide. U ovom radu je po prvi put pokazana sposobnost pripadnika roda Bacillus da konvertuju toluen u PHB. Pseudomonas sp. TN301 se istakao zbog svoje sposobnosti da akumulira mcl-PHA do 22% suve ćelijske mase iz poliaromatičnog jedinjenja naftalena. Razvijen je metod za dodavanje izrazito hidrofobnih poliaromatičnih jedinjenja u kultivacioni medijum. Najveća masa i najviši nivo akumuliranog mcl-PHA postignuti su dodavanjem sintetičkog surfaktanta Tweena 80 (0.5 g l-1). Pokazano je da Pseudomonas sp. TN301 ima sposobnost akumulacije mcl-PHA iz širokog opsega poli- i monoaromatičnih jedinjenja, kao i njihovih smeša. Ovaj rad predstavlja prvu sveobuhvatnu studiju metabolički raznovrsnih, u prvom redu Gram-pozitivnih bakterija sa velikim aplikacionim potencijalom u procesu bioremedijacije sredina zagađenih mešovitim polutantima, ali i sa drugim biotehnološkim aplikacijama kao sto je konverzija otpadnog materijala u biopolimere. U ovom radu je po prvi put pokazana sposobnost bakterijskog soja da konvertuje različita poliaromatična jedinjenja i njihove smeše u biodegradabilni polimer (mcl- PHA). S obzirom na to da ima odlike biodegradabilnog termoelastomera, mcl-PHAsve više dobija na važnosti, pa je izolacija novih sojeva sa sposobnošću njegove produkcije od izuzetnog značaja.Mono- and polyaromatic compounds are among the most prevalent and persistent pollutants in the environment. They have been reported as toxic, carcinogenic and mutagenic. Contaminated sites usually contain complex mixture of aromatic compounds and heavy metals, and are characterized by essential nutrients imbalance. On the other hand, polluted sites proved to be valuable sources of bacteria capable of surviving in these harsh environments. The aim of this study was to obtain robust bacteria with potential biotechnological application from the river sediments affected by the proximity of a petrochemical industrial site. Using two different isolation strategies, heat pretreatment and media for growth and sporulation of Actinobacteria and naphthalene enrichment combined with the selection of fast growing bacteria capable of using naphthalene as the sole source of carbon and energy, 34 Gram-positive and 4 Gram-negative bacterial strains were isolated. Isolates that were able to use 9 or more common aromatic pollutants, such as benzene, biphenyl, naphthalene etc. as a sole source of carbon and energy included members of Bacillus, Arthrobacter, Rhodococcus, Gordonia, Streptomyces, Staphylococcus, Actinobacteria, Sinorhizobium, and Pseudomonas genus. Rhodococcus sp. TN105, Gordonia sp. TN103 and Arthrobacter sp. TN221 were identified as novel strains. Out of 11 isolates able to grow in the presence of one or more metals (mercury, cadmium, nickel) at high concentration (100 mM), eight could degrade 15 different aromatic compounds. Antibiotic resistance to at least one of the tested antibiotics was found among 60% of the isolates. However, high incidence of multiresistance was not observed. One third of the isolates could accumulate at least one biopolymer. Twelve isolates (mainly Bacillus sp. and Arthrobacter sp.) accumulated polyphosphate, three Bacillus sp. accumulated polyhydroxybutyrate (PHB), and Pseudomonas sp. TN301 accumulated medium chain length polyhydroxyalkanoate (mcl-PHA), while four Gram-positive isolates could accumulate exopolysaccharides. This study is the first report of a Bacillus strain capable of converting toluene to PHB. Pseudomonas sp. TN301 was distinguished by its ability to accumulate mcl-PHA up to 22% of cell dry weight from the naphthalene. A method of supplying highly hydrophobic polyaromatic hydrocarbons to a culture medium was developed. The best biomass and mcl-PHA yields were achieved with the addition of synthetic surfactant Tween 80 (0.5 g l-1). It was shown that Pseudomonas sp. TN301 can accumulate mcl-PHA from a wide range of poly- and monoaromatic hydrocarbons, and mixtures thereof. This is the first comprehensive study on the metabolically diverse, primarily Grampositive bacteria with great application potential for mixed pollution bioremediation studies, as well as in other biotechnological applications such as waste conversion to biopolymers. Also, this is the first report of the ability of a bacterial strain to convert a range of polyaromatic hydrocarbon compounds to the biodegradable polymer (mcl- PHA). Mcl-PHA is gaining importance as a promising biodegradable thermoelastomer and therefore isolation of new producing strains is highly significant