Ugotavljanje produktov razgradnje lignina z izbranimi sevi bakterij s tekočinsko kromatografijo, sklopljeno z masno spektrometrijo

Plešnik, Helena (author), Trontelj, Jurij (mentor), Kosjek, Tina (comentor)

Lignin je kompleksen heterogen aromatski biopolimer, ki se nahaja v stenah rastlinskih celic kot ena izmed komponent lignoceluloze. Velike količine lignina nastajajo kot stranski produkt v papirni industriji in industriji biogoriv. Ker predstavlja bogat naravni vir aromatskega ogljika, bi lahko potencialno predstavljal odličen vir za proizvodnjo goriv in kemikalij z dodano vrednostjo. Kljub temu pa še vedno velja za odpadni produkt in je v veliki meri porabljen kot nizkokakovostno gorivo. Predvsem zaradi energetske krize je v zadnjih letih zanimanje za lignin in njegovo valorizacijo precej zraslo, in sicer še posebej za bolj okolju prijazno razgradnjo lignina z mikroorganizmi. Raziskave so bile doslej večinoma osredotočene na glivno razgradnjo lignina, ki pa je zaradi kompleksnosti njihovih encimov manj primerna za uporabo v industriji. Ligninolitični encimi so v bakterijah sicer precej redkejši kot v glivah, a bi lahko predstavljali novo, boljšo možnost za valorizacijo lignina. Pri tej magistrski nalogi smo se osredotočili na bakterijsko razgradnjo lignina. Naš cilj je bil preučiti ligninolitično aktivnost 5 bakterijskih sevov, ki proizvajajo lakazo, pomemben encim z delovanjem na lignin. Tako smo si zastavili raziskovalno vprašanje: Ali je v katerem od vzorcev prišlo do biorazgradnje? Če je odgovor »da«, kateri produkti so pri tem nastali? Po pregledu literature smo izbrali 20 fenolnih spojin, ki bi se potencialno lahko pojavile kot produkt bakterijske razgradnje lignina. Za 13 izmed njih smo nato razvili dve analizni metodi za tarčno analizo razgradnih produktov lignina. Metodi temeljita na analizi vzorcev z reverznofazno tekočinsko kromatografijo, sklopljeno s tandemsko masno spektrometrijo. Pred LC-MS/MS analizo smo vzorce pripravili z ekstrakcijo na trdnem nosilcu, z uporabo ekstrakcijskih plošč na sistemu za nadtlačno ekstrakcijo. V naslednjem koraku smo analizno metodo testirali na realnih vzorcih. Ti so bili predhodno pripravljeni tako, da smo kot vir ogljika za posamezne bakterijske seve na gojiščih uporabili lignin. Pri tem smo predpostavili, da z določitvijo katerega od potencialnih produktov razgradnje lahko potrdimo, da je v posameznem vzorcu do biorazgradnje zares prišlo. Odsotnost potencialnih produktov razgradnje v vzorcih pa ne pomeni, da do biorazgradnje ni prišlo, pač pa samo, da se ni zgodila na načine, kot so opisani v literaturi. Ker v vzorcih nismo uspeli potrditi prisotnosti nobenega izmed iskanih analitov, smo izvedli še netarčno analizo, in sicer v načinu IDA (Information Dependent Acquisition, oziroma »od podatkov odvisna analiza«). Dobljeni kromatogrami nakazujejo očitno razliko med kontrolnimi in dejanskimi vzorci, kar nakazuje razgradnjo lignina. Nalogo smo predstavili tudi na 12. študentski konferenci Mednarodne podiplomske šole Jožefa Stefana.

• Language: Slovenian
• Keywords: lignin, bakterijska razgradnja, ekstrakcija na trdni fazi, tekočinska kromatografija, masna spektrometrija
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-121243
• Publication date in RUL: 02.10.2020

PLEŠNIK, Helena, 2020, Ugotavljanje produktov razgradnje lignina z izbranimi sevi bakterij s tekočinsko kromatografijo, sklopljeno z masno spektrometrijo [online]. Master’s thesis. [Accessed 25 March 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=121243

Secondary language

• Language: English
• Title: Determination of bacterial lignin degradation products by liquid chromatography coupled to mass spectrometry

Abstract

Lignin is a complex and heterogenous aromatic biopolymer that can be found in plant cell walls as one of the components of lignocellulose. Large quantities of lignin are daily generated as a by-product in paper and biofuel industries. To date, lignin is still treated as waste and is mostly burned as a low-quality fuel, even though it could potentially be a great source for producing value-added chemicals and fuels as it is an abundant source of renewable aromatic carbon. As a result of the energy crisis, the interest in lignin and its valorization has grown considerably in recent years, mainly focusing on more environmentally friendly lignin degradation by microorganisms. The majority of research has so far been concentrating on fungal lignin degradation. However, fungi unfortunately seem to be unsuitable for large-scale application because of the complexity of their enzymes. Ligninolytic enzymes are very rare in bacteria, but those that possess this physiological trait present a new, better option than using fungi to valorize lignin. Our research focuses on bacterial lignin degradation, with the objective to study the ligninolytic activity of 5 laccase-producing bacterial strains, laccase being an important lignin-modifying enzyme. Our research question was: ''Did biodegradation occur in any of the samples? In case the answer is affirmative, which products were generated by the degradation?'' Based on the existing literature we defined 20 phenolic compounds, which could potentially appear as products of bacterial lignin degradation. 13 of those compounds were then included in two analytical methods that we developed for targeted screening of lignin degradation products. The analytical methods were based on determination by reversed-phase liquid chromatography coupled to tandem mass spectrometry. Prior to the LC-MS/MS analysis, we purified the samples by solid-phase extraction using extraction plates on a positive pressure extraction system. The analytical method was then tested on real samples, containing lignin treated with either of the laccase-producing bacterial strains, presuming that detection of any of the potential degradation products in a sample would prove biodegradation. The absence of those potential degradation products does, however, not mean that biodegradation did not take place. It merely suggests the absence of the specific degradation pathways, found in the literature. Since the target analysis did not confirm the presence of the expected analytes, we also performed nontargeted analysis using information-dependent acquisition-mediated LC-MS/MS screening. Acquired chromatograms showed significant differences between lignin samples and blanks, confirming its degradation. We presented our research at the 12th Jožef Stefan international postgraduate school students' conference.

• Keywords: lignin, bacterial degradation, solid phase extraction, liquid chromatography, mass spectrometry