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Assessment of wood degradation by Pycnoporus sanguineus when co-cultured with selected fungiVan Heerden, Andrea 03 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: It is commonly known that a diversity of fungi, including yeasts, may occur on
plant surfaces. Similarly, on fallen trees an ecological succession of different
fungal species is known to occur during wood degradation. Some of these fungi
may be pioneer fungi contributing to the initial degradation process, while others
may be yeasts associated with the fruiting bodies of macro-fungi which in turn
are able to utilize the more recalcitrant polymers in wood. Previously, it was
revealed that an increase occurs in the wood degradation rate of certain white-rot
fungi when co-cultured with selected yeast species.
A well known inhabitant of decomposing trees is the white rot fungus Pycnoporus
sanguineus. It was found by some that this fungus is capable of selective
delignification while growing on the wood of poplar trees, while other authors
found a simultaneous delignification pattern on Eucalyptus grandis trees. In the
latter case cellulose and lignin are degraded simultaneously.
We were interested in how yeasts occurring on the surface of P. sanguineus
fruiting bodies, and the pioneer fungus Aspergillus flavipes, impact on wood
degradation by this white-rot fungus. Restriction Fragment Length
Polymorphisms (RFLP) analyses were used to obtain an indication of the species
composition of the culturable yeast community associated with fruiting bodies of
P. sanguineus. The impact of the most dominant of these yeasts species, i.e.
Pichia guilliermondii and Rhodotorula glutinis, as well as A. flavipes, on wood
degradation by P. sanguineus was then determined by analyzing the major wood
components after growth of co-cultures on hot water washed E. grandis wood
chips. Co-cultures of P. sanguineus with the other fungi were prepared by
inoculating the wood chips, contained in solid state bioreactors and
supplemented with molasses and urea, with the an appropriate volume of fungal
inoculum, resulting in an initial moisture content of 60%. After two weeks of
incubation at 30°C with constant aeration, the chips were harvested. Standard protocol (TAPPI Standard Methods), commonly used by the paper and pulp
industry, were then employed to determine the percentage cellulose, Klason
Lignin, as well as polar and solvent-borne extractives in the chips. The resulting
data were analyzed using box plots, as well as biplots. No degradation of Klason
lignin was observed, while the percentage cellulose did decrease during fungal
degradation. Taking into account the inherent shortcomings of the Klason Lignin
determination, the results supported the findings of others that P. sanguineus
shows a simultaneous delignification pattern while growing on E. grandis wood.
In addition, it was found that the yeasts played no significant role in the
degradation ability of P. sanguineus, while A. flavipes showed an antagonistic
effect on P. sanguineus with respect to cellulose degradation. However, it was
clear that the analytical methods used in this study were inadequate to accurately
determine fungal degradation of wood. In addition, it was obvious that the
methods used did not distinguish between fungal biomass and wood
components. Nevertheless, the methods provided us with a fingerprint of each
culture growing on E. grandis wood, allowing us to compare the chemical
composition of the different cultures and the un-inoculated hot water washed
wood chips. The question, therefore, arose whether the effect of a particular coculture,
on the chemical composition of wood, differs between tree species.
Consequently, chemical alterations in different tree species, induced by a P.
sanguineus / A. flavipes co-culture, were investigated in the next part of the
study. Wood chips originating from four tree species, i.e. Acacia mearnsii,
Eucalyptus dunnii, E. grandis, and Eucalyptus macarthurii, were inoculated with
this co-culture. The culture conditions and subsequent analyses of the wood
components were the same as in the first part of the study. From the box- and
biplots constructed from the resulting data, it was clear that the chemical
composition of each tree species were altered in a different manner by the coculture.
Lignin content showed an apparent increase in A. mearnsii, while E.
dunnii showed a decrease in cellulose content. The results indicate that wood of
different tree species are degraded in a different manner and this phenomenon
should be taken into account in selecting fungi for biopulping. / AFRIKAANSE OPSOMMING: Dit is algemeen bekend dat 'n verskeidenheid fungi, insluitend giste, op
plantoppervlaktes mag voorkom. Dit is ook bekend dat 'n ekologiese
opeenvolging van verskillende fungusspesies tydens hout-afbraak op omgevalle
bome voorkom. Van hierdie fungi mag pionierfungi wees wat bydra tot die
aanvanklike afbraakproses, terwyl ander giste mag wees wat geassosieer word
met die vrugliggame van makro-fungi, wat op hul beurt weer in staat is om die
meer weerstandbiedende polimere in hout te benut. Dit is voorheen
bekendgemaak dat daar 'n toename plaasvind in die tempo van houtafbraak deur
sekere witvrot-fungi wanneer dit in ko-kulture met geselekteerde gisspesies
voorkom.
'n Bekende bewoner van verrottende bome is die wit-vrotfungus Pycnoporus
sanguineus. Dit is gevind dat hierdie fungus tot selektiewe delignifikasie in staat
is terwyl dit op die hout van populierbome groei, terwyl ander outeurs 'n
gelyktydige patroon van delignifisering op Eucalyptus grandis bome gevind het.
In laasgenoemde geval is sellulose en lignien gelyktydig afgebreek.
Ons was geïnteresseerd in die effek van giste op die oppervlak van vrugliggame
van P. sanguineus, en die pionierfungus Aspergillus flavipes, op die houtafbraak
deur hierdie wit-vrotfungus. Restriction Fragment Length Polymorphisms (RFLP)
analises is gevolglik gebruik om 'n aanduiding te kry van die spesiesamestelling
van die kweekbare gisgemeenskap wat met die vrugliggame van P. sanguineus
geassosieer word. Die impak van die mees dominante van hierdie gisspesies,
naamlik Pichia guilliermondii en Rhodotorula glutinis, asook A. flavipes, op
houtafbraak deur P. sanguineus is voorts bepaal deur die analise van die
belangrikste houtkomponente na die kweek van ko-kulture op E. grandis
houtskyfies wat met warm water gewas is. Ko-kulture van P. sanguineus met die
ander fungi is voorberei deur die houtskyfies in vaste fase bioreaktore, aangevul
met melasse en ureum, te inokuleer met 'n toepaslike volume van die fungus inokulum om 'n aanvanklike voginhoud van 60% te verkry. Na twee weke se
inkubasie by 30°C met konstante belugting is die skyfies ge-oes. Standaard
protokol (TAPPI Standard Methods), algemeen deur die papier en pulpindustrie
gebruik, is ingespan om die persentasie sellulose, Klason Lignien, asook polêre
en oplosmiddel-gedraagde ekstrakte in die skyfies te bepaal. Die gevolglike data
is geanaliseer deur gebruik te maak van box plots en biplots. Daar is geen
afbraak van Klason Lignien bespeur nie, terwyl die persentasie sellulose wel
toegeneem het tydens fungus degradasie. Met die inherente tekortkominge van
die Klason Lignien bepaling inaggenome, het die resultate die bevindings
ondersteun van andere wat getoon het dat P. sanguineus 'n gelyktydige
delignifikasiepatroon openbaar terwyl dit op E. grandis hout groei. Daarby is dit
gevind dat die giste geen beduidende rol in die afbraakvermoeë van P.
sanguineus gespeel het nie, terwyl A. flavipes 'n antagonisiese effek ten opsigte
van die sellulose degradering van P. sanguineus getoon het. Dit was egter
duidelik dat die analitiese metodes wat in hierdie studie gebruik is, onvoldoende
was om die degradering van hout akkuraat te bepaal. Daarby was dit duidelik
dat die metodes nie tussen fungus biomassa en houtkomponente kon onderskei
nie. Nogtans het die metodes 'n vingerafdruk verskaf van elke kultuur wat op E.
grandis hout groei, wat ons toegelaat het om die chemiese samestelling van die
verskillende kulture en die ongeïnokuleerde, met warm water gewasde
houtskyfies te vergelyk. Die vraag het gevolglik ontstaan of die effek van 'n
bepaalde ko-kultuur op die chemiese samestelling van hout van boomspesie tot
boomspesie verskil. Gevolglik is die chemiese wisselinge in verskillende
boomspesies, geïnduseer deur 'n P. sanguineus / A. flavipes ko-kultuur, in die
volgende gedeelte van die studie ondersoek. Houtskyfies van vier boomspesies,
naamlik Acacia mearnsii, Eucalyptus dunnii, E. grandis, en Eucalyptus
macarthurii, is met hierdie ko-kultuur geïnokuleer. Die kultuurkondisies en
daaropvolgende analises van die houtkomponente was dieselfde as in die eerste
deel van die studie. Van die box- en biplots wat van die resultate getrek is, is dit
duidelik dat die chemiese samestelling van elke boomspesie op 'n verskillende
manier deur die ko-kulture verander is. Lignien-inhoud het ’n waarskynlike toename getoon in A. mearnsii, terwyl E. dunnii 'n afname in sellulose-inhoud
getoon het. Die resultate toon dat hout van verskillende boomspesies op
verskillende maniere afgebreek word en dat hierdie fenomeen in aanmerking
geneem moet word wanneer fungi vir bioverpulping geselekteer word.
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Genetic and enzymatic characterisation of wood degrading strains of Phanerochaete speciesDe Koker, T. H. (Theodorus Hermanus), 1965- 03 1900 (has links)
Thesis (PhD)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: White rot fungi are of interest in the paper and pulp industry because of their
removal of lignin from wood. In this study over 600 Basidiomycete fungi were
isolated from indigenous forests as well as from commercial Eucalyptus spp.
and Pinus spp. plantations in South Africa. One hundred isolates were
identified to genus level. Biochemical tests were done to screen the fungal
cultures for characteristics that are favourable for biopulping, e.g. low
cellulase activity with concomitant high activity of ligninolytic enzymes.
Various Phanerochaete isolates with potentially high ligninolytic activity were
identified.
Although Phanerochaete chrysosporium Burds. has previously been isolated
from the indigenous forest at Knysna in South Africa, this study showed that
P. chrysosporium was a natural coloniser of wood chip piles in South Africa,
indicating potential for application in industry. A possible new species of
Phanerochaete, viz. Phanerochaete pseudomagnoliae nom. provo (strain
PP25) from decayed wood collected in Stellenbosch, South Africa, was
described and illustrated. It differs from previously described Phanerochaete
species in having smaller basidiospares, and in the formation of few
chlamydospores on malt extract agar but more on xylose containing media.
The potential of using internal transcribed spacer DNA sequences (ITS) to
infer phylogenetic relationships among species of the genus Phanerochaete
was investigated. Consensus phylogenetic trees could be presented, but the
presence of ambiguous aligning sequences within the ITS made inferring of
phylogenetic relationships within the whole genus difficult.
Fifty-five South African strains of P. chrysosporium were screened for lignin
peroxidase (liP), manganese peroxidase (MnP) and glyoxal oxidase (GLOX)
enzymes. Manganese peroxidase activity was quantified on agar media. The
liP and GLOX activities of 13 selected strains, including control strains and
P. pseudomagnoliae (PP25), were also quantified on agar media. Differences
in MnP and GLOX activities existed among the strains. Preliminary
biochemical characterisation of strain PP25 indicated that the most important
difference was the apparent unique regulation of ligninolytic enzymes. Under
low nitrogen, liP activity of the selected strains showed no significant variation, whereas strain PP25 had significantly increased liP levels under
high nitrogen conditions. Restriction fragment length polymorph isms of the
lignin and manganese peroxidase gene DNA fragments showed variability
among strains, whereas there was probably only allelic variation for the glox
gene DNA fragments.
Previous research has indicated xylose oxidation activity within
P. chrysosporium. To investigate whether GLOX can oxidise xylose, a purified
recombinant GLOX (rGLOX) from P. chrysosporium BKM-F-1767 Burds. was
used in this study. This rGLOX oxidised D-xylose and D-glucose (D-xylose >
D-glucose) to produce H202. Xylose was oxidised to xylono-1 ,4-lactone with a
1:1 stoichiometric relationship between H202 produced and xylose used.
Xylono-1,4-lactone was converted non-enzymatically to xylonate. This
suggested that the furanose form of xylose, rather than the pyranose form, is
a substrate of GLOX. The production of H202 and the removal of inhibitory
compounds by GLOX could enhance ligninolytic activity. .
To conclude, unique strains of P. chrysosporium have been isolated from
South Africa with potential biotechnological use in paper manufacturing. The
relationship of P. pseudomagnoliae nom. provo to other Phanerochaete
species was evaluated and light was shed on the possible role of GLOX in
lignin degradation. / AFRIKAANSE OPSOMMING: Witvrot swamme is van belang vir die papier en pulp industrie omdat hulle
lignin vanaf hout kan verwyder. Meer as 600 Sasidiomiseet fungi, afkomstig
vanaf inheemse woude asook kommersiële Eucalyptus spp. en Pinus spp.
plantasies, IS geïsoleer. Een honderd isolate is tot op genusvlak
geïdentifiseer. Die isolate is biochemies vir eienskappe wat voordelig vir
"bioverpulping" kon wees, bv. die gelyktydige produksie van lae sellulosemaar
hoë ligninolitiese ensiemaktiwiteit, getoets. Verskeie isolate met
potensieel hoë vlakke van ligninolitiese aktiwiteit is verkry.
Alhoewel Phanerochaete chrysosporium Surds. vantevore in die Knysna
inheemse woud in Suid-Afrika geïsoleer is, het hierdie studie gewys dat
P. chrysosporium natuurlik op hope houtblokkies voorgekom, met moontlike
toepasing in die industrie. Isolaat PP25, geisoleer vannaf verrottende hout te
Stellenbosch, Suid Afrika, is as 'n potensieel nuwe spesie van die genus
Phanerochaete beskryf en as Phanerochaete pseudomagnoliae nom. provo
benoem. Hierdie isolaat verskil van ander Phanerochaete-spesies daarin dat
dit kleiner basidiospore vorm en nie klamydospore op moutekstrakagar
produseer nie, maar wel op media wat xilose bevat. Die potensiaal van intern
getranskribeerde spasieerder ONS opeenvolging vir die aflei van filogenetiese
verhoudings tussen spesies van die genus Phanerochaete is ondersoek.
Konsensus filogenetiese bome kon bepaal word, maar die teenwoordigheid
van varieerbare areas het die afleiding van filogenetiese verwantskappe vir
die hele genus bemoeilik.
Vyf-en-vyftig Suid-Afrikaanse isolate van P. chrysosporium is vir die
teenwoordigheid van lignienperoksidase- (liP), mangaanperoksidase- (MnP)
en glioksaaloksidase (GLOX)-aktiwiteit getoets. Vlakke van MnP-aktiwiteit is
op agarplate gekwantifiseer. Vlakke van LiP- en GLOX-ensieme op agarplate
is vir 13 geselekteerde isolate, insluitend kontroles en ras PP25,
gekwantifiseer. Aktiwiteit van MnP en GLOX het statisties betekenisvol tussen
isolate verskil. Lignienperoksidase-aktiwiteit onder lae stikstof toestande het
nie statisties betekenisvol van mekaar verskil nie. Onder hoë stikstof
toestande het isolaat PP25 wel verhoogde liP-aktiwiteit getoon. Restriksie
fragment polimorfismes van die lignien- en mangaanperoxidase-gene het variasie getoon, terwyl waarskynlik slegs alleliese variasie vir die glox geen
waargeneem IS.
Rekombinante GLOX (GLOX vanaf P. chrysosporium BKM-F-1767) het xilose
en glukose (D-xilose > D-glukose) geoksideer met meegaande produksie van
H202. Xilose is na xilono-1,4-laktoon geoksideer met 'n 1:1 stoigiometrie
tussen H202-produksie en xilose verbruik. Xilono-1,4-laktoon is nieensiematies
na xilonaat omgeskakel. Bogenoemde resultaat dui aan dat die
furanose vorm van xilose die werklike substraat vir GLOX is. Deur die
meegaande produksie van H202 en die verwydering van inhiberende produkte
word lignoliese aangehelp.
Ten slote, unieke P. chrysosporium rasse met potensiële gebruik in
papiervervaardiging is in Suid-Afrika geisoleer. Die genetiese diversiteit van 'n
nuwe spesie, P. pseudomagnoliae, is bepaal en nuwe lig is op die potensiële
rol van GLOX in lignienafbraak gewerp.
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