An investigation of the microbial hydrolysis of the lignin carbohydrate complex of grasses

Stevens, Gary Grant (2004-03)

Thesis (MSc)--University of Stellenbosch, 2004.

Thesis

ENGLISH ABSTRACT: The microbial degradation of the lignin carbohydrate complex of plant material is only partially understood. Lignin carbohydrate complex was extracted from wheat straw and subsequently analysed. An adjustment to the standard protocol was required to increase the amount of lignin carbohydrate complex extracted from wheat straw. Characterization of the lignin carbohydrate complex after trifluoacetic acid hydrolysis was done by capillary electrophoresis. HPLC proved ineffective, as baseline separation could not be achieved. Characterization of the lignin carbohydrate complex revealed that a large portion (68 %) consisted of carbohydrate and lignin (20 %). Capillary electrophoresis of the trifluoroacetic acid hydrolysates of the lignin carbohydrate complex revealed that the carbohydrates consisted of mannose, xylose, arabinose, galactose, glucose and rhamnose. The major monosaccharide present in the lignin carbohydrate complex was mannose which made up 34 % of the total carbohydrate composition. Ferulic and p-coumaric acid were present in the lignin carbohydrate complex, but in concentrations less than 1 % of the lignin carbohydrate complex. The lignin carbohydrate complex of wheat straw probably had a heterogenous structure consisting of a variety of molecules, as molecular weight determination could not be accurately determined. An estimated molecular weight of 5.9 kOa was determined. Ten fungal strains (Aspergillus niger, Aureobasidium pul/u/ans, Bjerkandera adusta, Corio/us versicolor, Lenzitus betu/ina, Phanerochaete chrysosporium, Pycnoporus coccineus, Pycnoporus sanguineus 294, Pycnoporus sanguineus K5-2-3 and Trichoderma reeseï; were evaluated for growth on the lignin carbohydrate complex. All strains except B. adusta showed growth after 5 days with A. niger, A. pul/u/ans, C. versicolor, P. chrysosoporium and T. reesei showing the best growth on the lignin carbohydrate complex. The culture fluid revealed a number of proteins secreted by these organisms. The protein determination was confirmed by SOS-PAGE which revealed an array of proteins ranging from 8 kOa to 180 kOA. Prominent bands between 26 kOa and 80 kOa could be observed in the culture fluid of A. niger, A. pul/ulans and T. reesei, but not in C. versicolor. Activity studies on the culture fluid of these four strains revealed activity for xylanase, xylosidase, arabinofuranosidase, ferulic acid esterase and laccase, with vast differences between the activities of the various fungi. The enzymes of these fungal strains were also evaluated for their ability to degrade xylan and sugar cane bagasse using capillary electrophoresis. It appeared that all the organisms produced enzymes to degrade birchwood xylan. However, the electropherograms revealed that the degradation patterns of birchwood xylan differed among these organisms over the same time interval, as xylotetraose, xylotriose, xylobiose and xylose were released in various concentrations. The electropherograms obtained from the enzyme hydrolysates of sugar cane bagasse, pointed to the substrate being inaccessible. Electropherograms of the culture fluid of A. niger, A. pul/ulans, C. versicolor and T. reesei, when incubated on the lignin carbohydrate complex indicated similar peaks to those obtained and identified in the trifluoroacetic acid hydrolysates. However, the electropherograms of the culture fluid of these organisms revealed additional smaller peaks which could not be identified. The electropherograms of the culture fluid of the various organisms also indicated a complete release of some sugars, using the trifluoacetic acid hydrolysate of the lignin carbohydrate complex as a control for the amount of sugars present. HPLC analyses revealed that after 72 h, no apparent degradation of the lignin carbohydrate complex took place as peak height and areas appeared to be similar. These peaks could however not be identified due to a lack of standards as well as baseline separation which could not be achieved.

AFRIKAANSE OPSOMMING: Tans word die mikrobiese afbraak van die lignienkoolhidraatkompleks van plant materiaal slegs gedeeltelik verstaan. Lignienkoolhidraatkompleks was vanaf koringstrooi geïsoleer en gevolglik geanaliseer. Daar moes van die standaard prosedure vir die ekstraksie van lignienkoolhidraatkompleks afgewyk word ten einde beter lignienkoolhidraatkompleks opbrengs te lewer. Karakterisering van die lignienkoolhidraatkompleks na trifluoroasynsuurvertering was deur kapillêre elektroforese bepaal. Dit wou voorkom asof kapillêre elektroforese "n beter opsie vir die analise van die verteerde monster van lignienkoolhidraatkompleks is, vergeleke met hoëdruk vloeistof chromatografie. Daar was gevind dat die lignienkoolhidraatkompleks uit 68 % koolhidraat en 20 % lignien bestaan. Kapillêre elektroforese het die teenwoordigheid van die volgende suikers bevestig naamlik, mannose, xilose, arabinose, glukose, galaktose en ramnose. Mannose was die dominerende suiker in die lignienkoolhidraatkompleks wat 34 % van die totale koolhidraat opbrengs uitgemaak het. Ferulien- en p-kumaarsuur kon ook identifiseer word, maar die twee sure het minder as 1 % van die totale inhoud van die lignienkoolhidraatkompleks uitgemaak. Vanuit resultate bekom wil dit voorkom dat die lignienkoolhidraatkompleks "n heterogene molekuul is omdat die molekulêre gewig daarvan nie akkuraat bepaal kon word nie. 'n Geskatte molekulêre grootte van ongeveer 5.9 kDa was bepaal met verwysing na die hoogste piek wat in die chromatogram waargeneem was. Tien fungus kulture was in die studie gebruik om hul vermoë te toets om op die lignienkoolhidraatkompleks te groei, naamlik Aspergillus niger, Aureobasidium pullulans, Bjerkandera adusfa, Goriolus versicolor, Lenziius betuline. Phanerochaefe chrysosporium, Pycnoporus coccineus, Pycnoporus sanguineus 294, Pycnoporus sanguineus K5-2-3 en Trichoderma reesei. B. eauste het nie groei na 5 dae getoon nie, en dit wou voorkom asof A. niger, A. pul/ulans, G. versicolor, P. chrysosoporium en T. reesei die beste kon groei op die substraat na 5 dae. Die kultuurvloeistof van die vier kulture het getoon dat proteïene deur hierdie organisms uitgeskei was. Hierdie proteinbepaling was ook bevestig deur SOS-PAGE, wat bande tussen 8 kDa en 180 kDa gelewer het. Prominente bande tussen 26 kDa en 80 kDa kon waargeneem word in die kultuurvloeistof van A. niger, A. pul/ulans, en T. reesei, maar nie in die kultuurvloeistof van C. versicolor nie. Aktiwiteitstudies op die kultuur vloeistowwe het getoon dat daar aktiwiteit was vir die volgende ensieme, naamlik xilanase, xilosidase, arabinofuranosidase en feruliensuur esterase. Hierdie aktiwiteit het aansienlik verskil tussen die verskillende organismes. Die ensieme van die vier organismes was ook gebruik om hul vermoë te toets om xilaan en suikerriet af te breek. Daar was gevind dat xilaanafbraak verskillend was vir die organisms oor dieselfde tydperk. Xilotetraose, xilotriose, xilobiose en xilose was in verskillende konsentrasies gevind vir die verskillende organismes. Die elektroferogramme van die kultuurvloeistof op suikerriet van die verskillende organismes het getoon dat die substraat nie toeganklik vir die ensieme was nie. Die elektroferogramme van die kultuurvloeistof op lignienkoolhidraatkompleks van die verskillende organismes het dieselfde pieke getoon soos geïdentifiseer in die elektroferogramme van die trifluoroasynsuur vertering. Die elektroferogramme met die ensiem vertering het egter addisionele pieke getoon wat nie sigbaar op die elektroferogramme van die trifluoroasynsuur vertering was nie. Hierdie pieke het min of meer dieselfde tyd ge-elueer as die monosakkariede. Kapillêre elektroforese het ook getoon dat die ensiematiese afbraak van die lignienkoolhidraatkompleks gelei het tot algehele vrystelling van sommige suikers, wanneer die trifluoroasynsuur vertering as maatstaaf dien vir die hoeveelheid suikers teenwoordig in die lignienkoolhidraatkompleks. Hoëdruk vloeistof chromatografie het getoon dat geen sigbare afbraak na 72 h van inkubasie met die ensieme op die lignienkoolhidraatkompleks plaasgevind het nie aangesien die piek hoogtes konstant gebly het. Hierdie pieke kon egter nie geïdentifiseer word nie as gevolg van lae resolusie van die pieke asook standaarde wat nie beskikbaar was nie.

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