Naslov (srp)

Metagenomski pristup analizi mikrobiološkog diverziteta rizosfera endemoreliktnih biljaka i izolacija bakterija koje razgrađuju fenol

Autor

Đokić, Lidija, 1980-

Doprinosi

Morić, Ivana, 1971-
Vasiljević, Branka, 1955-
Radović, Svetlana, 1958-
Stevanović, Branka, 1944-

Opis (srp)

Fenol i njegovi derivati, kao najčešči zagađivači prirodne sredine, javljaju se kao prirodni proizvodi i proizvodi ljudskih aktivnosti. Glavni proizvođači fenolnih jedinjenja su rafinerije nafte i fenolna industrija. Oni se javlja i pri razgradnji organskog materijala. Takođe, više biljke sintetišu fenolna jedinjenja, koja potom izlučuju u zemljište. Fenolna jeinjenja se iz prirodne sredine mogu ukloniti fizičkim, hemijskim i biološkim tehnikama (biološki oporavak). Najefikasniji, sa ekonomskog i ekološkog aspekta, je biološki oporavak.Rizosfere endemoreliktnih biljaka Ramonda serbica i Ramonda nathaliae se odlikuju povećanom koncentracijom fenola. Fenol selekcijom mikroorganizama koji mogu da ga tolerišu ili razgrađuju oblikuje mikrobiološke zajednice. U ovom radu analizirane su mikrobiološke zajednice rizosfera, a izolovane su i bakterije koje razgrađuju fenol. Mikrobiološke zajednice su proučavane metagenomskim pristupom i FISH-om. Geni za 16S rRNK umnoženi su iz metagenomskih DNK izolovanih iz zemljišta upotrebom univerzalnih bakterijskih prejmera. Dve konstruisane sredinske biblioteke 16S rRNK genskih klonova, analizirane su restrikcionom analizom (RFLP). Od ukupno 192 klona, 35 različitih RFLP tipova definisano je iz rizosfere R. nathaliae, a 13 iz rizosfere R. serbica od ukupno 80 klonova. Reprezentativni klonovi su sekvencirani. Najveći broj sekvenci je pokazao veoma malu sličnost sa kultivisanim bakterijama. FISH analiza je pokazala da je samo 5% bakterija u uzorku aktivno. Takođe, iz rizosfera su izolovane 4 bakterije koje imaju sposobnost razgradnje fenola. Filogenetska analiza sekvenci 16S rRNK gena, pokazala je da su sojevi PSl i PS12 najsrodniji sojevima roda Bacillus, a sojevi PS12 i PNl najsrodniji sojevima roda Streptonn/ces. Analizirani su i enzimi (fenol hidroksilaza, katehol 1,2-dioksigenaza i katehol 2,3-dioksigenaza) uključeni u razgradnju fenola. Testirani enzimi su pokazali izuzetno visoku kako ekstraćelijsku tako i intraćelijsku aktivnost. Sva četiri analizirana soja razgrađuju fenol u orto putu, dok sojevi PSll i PNl fenol razgrađuju i u meta putu. Fenol hidroksilaza iz svih analiziranih sojeva je pokazala veliku supstratnu specifičnost, sa Michaelis-ovom konstantom od 51 do 56 nM, i maksimalnom brzinom reakcije od 0,018 do 0,022 U/min. Kako postoji mali broj podataka o bacilusima i streptomicetama koje razgrađuju fenol, ovo istraživanje pruža podatke za bolje razumevanje rasprostranjenosti puteva razgradnje fenola među bakterijskim vrstama, a predstavlja i osnovu za primenu ovih bakterija u biološkom oporavku.

Opis (srp)

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Opis (eng)

Among many environmental pollutants, phenol and its derivatives emerge as a result of human activities as well as they naturally occur. Oil refineries and phenolic resin industries are major producers of phenolics. On the other hand, organic matter decomposition products contain phenol. Furthermore, higher plants svnthesize phenolics and release them into the soil. Removal of phenolic compounds from the environment employs different methods, such as physical, chemical and biological techniques (bioremediation). Bioremediation approach appears to be the most efficient regarding economical and environmental aspects.Rhizospheres of endemorelict plants Ramonda serbica and Ramonda nathaliae are characterised by unusually elevated phenol concentration, which acts as selective pressure, shaping indigenous microbial communities by selecting those capable to tolerate or degrade it. In this work microbial commimities were analysed in these rhizospheres and also bacteria capable to degrade phenol were isolated. Microbial communities were analysed using metagenomic approach and FISH analysis. The 16S rRNA genes were amplified directly from metagenomic DNAs isolated from soils by universal Bacteria-specific rRNA gene primers. Two constructed environmental 16S rRNA gene libraries were screened by RFLP. Among 192 clones, 35 imique RFLP types were determined from rhizosphere of R. nathaiiae, and 13 from library of R. serbica out of total 80 clones. Representative clones were sequenced. Majority of sequences from soil showed very low similarity to any cultured Bacteria. FISH experiments showed that the active bacteria represent only a small fraction, approximately 5% of total soil bacteria obtained by DAPI staining. Also, four bacterial strains able to degrade phenol, were isolated. Phylogenetic analyses based on 16S rRNA gene sequencing, placed strains PSl and PSll in the genus Bacillus, while PS12 and PNl belong to the genus Streptomyces. Key enzymes involved in pathways of phenol catabolism, phenol hydroxylase, catechol l,2-dioxygenase and catechol 2,3- dioxygenase, were analysed. Tested enzymes showed extremely high intracellular as well as extracellular activities. All four strains used ortho degradation pathway, while PSll and PNl strains used meta pathway in addition. Phenol hydroxylases from all analysed strains showed high substrate affinity with Michaelis constant values in the range from 51 to 56 nM and maximal velocity between 0.018 and 0.022 U/min. Since data on bacillus and streptomycetes phenol degrading strains are scant present study provides better understanding of distribution of phenol degradation pathways across the bacterial species and supplies the basis for possible application of these common soil strains in phenol bioremediation.

Jezik

srpski

Datum

2010

Licenca

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