Hansen, Gustav Hammerich

Hovedformålet med denne afhandling var at bestemme måder til at forbedre lignocellulose omdannelses potentialet af Aspergillus carbonarius. Tilgangene til at forbedre nedbrydningen af lignocellulose inkluderede; screening efter svampe der kan producere cellulaser, medie og vækst optimering, genetisk modificering og supplering med enkelte enzymer. Svampe stammer blev screenet for at afgøre hvilke rå enzym ekstrakter der kunne blive suppleret, som forstærker, af A. carbonarius egne rå enzym ekstrakt når det bliver anvendt i lignocellulose hydrolyse. Svampene stammede fra forskellige miljømæssige nicher, der alle havde det til fælles at de indeholdte tilgængeligt lignocellulose som substrat. Femten forskellige arter blev udvalgt og sammenlagt blev 18 isolater screenet. Monokultur dyrkninger af disse isolater blev udført i både et fast og et flydende medie, til produktion af rå enzymer. Enzymerne blev analyseret for deres potentiale i hydrolyse af hvede strå ved både at anvende monokulture og ved at supplere dem til rå enzymer fra A. carbonarius. For de rå enzymer fra faste dyrkninger var der otte isolater der viste synergistisk interaktion, resulterende i fordobling og tredobling af glukose frigørelsen i hvede strå hydrolyse. En helt anderledes profil af synergi blev observeret for rå enzymer fra flydende kulturer, da der kun var tre isolater der forøgede glukose frigørelsen. Kun et af de tre isolater havde vist synergi effekt ved dyrkning i et fast medie. Screeningen efter synergistiske aktiviteter bør derfor gøre brug af mere end et medie til produktion af enzymerne der skal screenes.En mere målbevidst screening efter cellulase producerende svampe og potentielle forstærkere af A. carbonarius blev også lavet for udvalgte indendørs isolater. Indendørs miljøet indeholder lignocellulose i form af bygge materialer. Svampe isolaterne der blev screenet var 21 almindelige indendørs svampe tilhørende flere slægter. De vigtigste resultater fra denne screening var at både Cladosporium sphaerospermum og Penicillium chrysogenum havde en overordnet stærk enzym profil. Disse to isolater havde de højeste endo-cellulase, β-glucosidase, mannase, β-galactanase og arabinanase aktiviteter, hvilket gør dem til gode kandidater for rå-enzymer til at supplere A. carbonarius eller til co-kultivering med denne.Baseret på litteratur studier af de opnåede cellulase aktiviteter for adskillige ascomyceter i fast medie fermenteringer (FMF) og flydende fermenteringer (FF) blev det fundet at FMF er en process der kan yde højere enzym koncentration. Derfor blev forskellige FMF medier og vækstbetingelser analyseret for at bestemme den optimale strategi for A. carbonarius produktion af rå-enzymer til anvendelse i lignocellulose hydrolyse. Seks forskellige medier blev sammensat, alle baseret på lignocellulose affald som substrat. Med hensyn til den højeste glukose frigivelse opnået ved anvendelsen af rå enzymer i hvede strå hydrolyse, så var det mest optimale medie have og park affald (HPA) suppleret med to nitrogen kilder (HPA/N). De supplerede nitrogen kilder var urea og (NH4)2SO4, og det blev observeret at de var essentielle for den øgede enzym produktion. En alternativ nitrogen kilde i form af husholdnings affald blev tilsat til HPA mediet, men det viste sig ikke at være et godt alternativ. HPA/N mediet blev observeret til at give det optimale enzym udbytte efter kultivering i 5 dage ved 30 °C for både Trichoderma reesei Rut-C30 og for A. carbonarius. Dette var tydeligt, selvom en højere xylanase aktivitet blev observeret for 5 dages vækst i et hvedeklid/lactose medie og højere β-glucosidase blev opnået efter 14 dages vækst i et hvedeklid sphagnum tørv (H/ST) medie. Resultaterne af dette studie understreger at en bæredygtig metode kan blive etableret til produktionen af enzymer og potentielt organiske syre ved brug af A. carbonarius groet på HPA.Det blev fastlagt at en metode til at følge biomasse produktionen og dermed væksten af A. carbonarius i FMF var nødvendig, da dette kunne lede til en bedre forståelse af potentielle vækstrater på lignocellulose substrater. To indirekte målinger af biomasse blev evalueret, beroende på det membran associeret enzym, β-N-acetylhexosaminidase, og en membran sterol, ergosterol. Biomasse målingerne blev udført for totalt fire svampe, fra slægterne Aspergillus, Trichoderma og Talaromyces efter kultivering i det faste H/ST medie. Der var uheldigvis en stor forskel i reproducerbarheden af målingerne, med NAHA enzymet havende den højeste standard afvigelse på 11 – 23 % i forhold til 2 – 8 % standard afvigelse for ergosterol målingerne. Desuden var det primært ergosterol målingerne der viste en forøget biomasse når man sammenlignede 7 og 14 dages kultiveringerne. I følge ergosterol målingerne havde Aspergillus sachharolyticus og A. carbonarius produceret den laveste mængde biomasse relativt til T. reesei Rut-C30 og Talaromyces pinophilus. Fra dette studium kunne det konkluderes at ergosterol rettere end β-N-acetylhexosaminidase bør blive brugt til bestemmelse af svampe biomasse i FMF. Desuden blev det bestemt at enzym aktivitet ikke kan korreleres linært til mængden af Erg (biomasse) målt.I et andet studie udført undervejs i Ph.D. projektet blev A. carbonarius rå enzym ekstrakt blandet med enten lytisk polysaccharide monooxygenase (LPMO) fra Podospora anserina eller Thermoascus aurantiacus i kombination med en cellobiose dehydrogenase (CDH) fra Neurospora crassa. Formålet var at evaluere hvorvidt den oxidative nedbrydning af cellulose i hvede strå kunne forbedre glukose frigørelsen, når der samtidigt anvendes rå enzymer fra A. carbonarius i hydrolysen. Det blev observeret at tilsætningen af oprenset P. anserina LPMO og enten CDH eller ascorbate ikke kunne forøge glukose frigørelsen i hvede strå hydrolysen. Imidlertig kunne en lille, men signifikant forøgelse, i glukose frigivelse observeres når en høj koncentration af P. anserina LPMO, 4,21 mg/g cellulose (mg/g), blev suppleret til blandingen af de kommercielle enzym præparater Novozym 188 og Celluclast 1.5 L. En stor forøgelse af glukose frigørelsen blev iagttaget for suppleringen af T. aurantiacus LPMO, både ved den høje koncentration af 4,21 mg/g og ved den lave koncentration af 0,21 mg/g. Glukose frigørelsen steg fra 5,92 g/L til 7,2 eller 7,3 g/L. Dette svarer til en forbedring af cellulose omdannelsen på 6,8 %. Aktiviteten af denne LPMO blev ikke forbedret ved tilføjelse af CDH, men ved tilsætning af ascorbat til den høje koncentration af LPMO observeredes en signifikant forøgelse resulterende i den højeste glukose frigivelse på 7,63 g/L. Netop dette var den mest optimale forbedring af A. carbonarius rå enzym hydrolyse potentiale der blev opnået, tilsvarende en total cellulose omdannelses effektivitet på 32 %. Derved blev det vist at en stærk forøgelse af rå enzymer kunne opnås ved tilsætning af et enkelt enzym, T. aurantiacus LPMO’en, og at der er en stor forskel i effekten afhængigt af oprindelsen og typen af LPMO.Endeligt blev det analyseret hvorvidt den samme P. anserina LPMO og CDH’en fra N. crassa ville vise en forbedring af de rå enzymer når de blev heterologt udtrykt i A. carbonarius. De resulterende mutanter udtrykte LPMO og CDH konstitutivt, men der var ikke nogen forbedring af glukose frigørelsen opnået fra hvede strå. Selvom der var en manglende effekt på glukose frigivelsen, var der dog en signifikant effekt observeret for xylose frigivelsen i hydrolyse af hvede strå. Forbedringen af xylose frigivelsen vekslede mellem 0,83 g/L til 1,2 g/L. Derfor kan tilsætningen af LPMO fra P. anserina og CDH fra N. crassa stadig forstærke hydrolysen af hvedestrå med henblik på hemicellulose omdannelse, som kan gøre cellulosen mere tilgængelig.Resultaterne opnået igennem dette Ph.D. projekt repræsenterer succesfulde strategier til forbedring af A. carbonarius lignocellulose hydrolyse. Strategierne som blev anvendt successfuldt var blanding af monokulture og optimering af kultiverings betingelser. Ved optimering af kultiveringsbetingelserne blev det endvidere vist at denne svamp kan blive brugt til udnyttelse af lignocellulose affald i form af HPA til effektiv produktion af enzymer. Den mest virksomme forbedring af A. carbonarius rå enzymer blev opnået ved tilsætning af det enkelte LPMO enzym fra T. aurantiacus. Til vækst på eller omdannelse af lignocellulose kan A. carbonarius derfor forstærkes ved supplering med LPMO.The primary aim of this thesis was to determine ways for enhancing the lignocellulose conversion potential of Aspergillus carbonarius. Approaches for enhancing the degradation of lignocellulose included: screening for fungal cellulase producers, media and growth optimization, genetic engineering and single enzyme supplementation. Fungal strains were screened in order to determine crude enzyme extracts that could be supplemented as boosters of A. carbonarius own crude enzyme extract, when applied in lignocellulose hydrolysis. The fungi originated from different environmental niches, which all had in common that they contained available lignocellulose as substrate. Fifteen different species were selected and in total 18 isolates were screened. Monoculture cultivations of these isolates were performed in both a solid and a liquid medium, for production of crude enzymes. The enzymes were analyzed for their potential in hydrolysis of wheat straw both by application of monocultures and by supplementing to crude enzymes of A. carbonarius. For the crude enzymes from solid cultivations there were eight isolates that showed synergistic interaction resulting in doubling and tripling of the glucose release in wheat straw hydrolysis. A completely different profile of synergy was observed for crude enzymes from liquid cultivations, as there were only three isolates that enhanced glucose release. Only one of these three isolates had shown synergistic effects when cultivated in a solid medium. The screening for synergistic activities should therefore utilize more than one medium for production of the enzymes to be investigated.A more dedicated screening for cellulase producing fungi and potential boosters of A. carbonarius was also performed on selected indoor isolates. The indoor environment contains lignocellulose in the form of building materials. The fungal isolates screened were 21 common indoor fungi belonging to several genera. The most important finding of this screening was the overall strong enzyme profiles of both Cladosporium sphaerospermum and Penicillium chrysogenum. These two isolates had the highest endo-cellulase, β-glucosidase, mannase, β-galactanase and arabinanase activities, which makes them good candidates for crude enzymes to supplement A. carbonarius or for co-cultivation with it.Based on a literature study of cellulase activities obtained for several ascomycetes in solid state fermentation (SSF) and submerged fermentation (SmF) it was found that SSF is a process that may yield higher enzyme titers. Therefore different SSF media and growth parameters were analyzed to determine the optimal strategy for A. carbonarius production of crude enzymes to be used in lignocellulose hydrolysis. Six different media were composed, all based on lignocellulosic waste as substrate. With regards to highest glucose release achieved in wheat straw hydrolysis by crude enzyme application, the most optimal medium was garden and park waste (GPW) supplemented by two nitrogen sources (GPW/N). The nitrogen sources supplemented were urea and (NH4)2SO4, and they were found to be essential for the enhanced enzyme production. An alternative nitrogen source in the form of municipal waste was added to the GPW medium, but it did not prove a good alternative. The GPW/N medium was found to yield optimal enzymes after cultivation for 5 days at 30 °C for both Trichoderma reesei Rut-C30 and for A. carbonarius. This was apparent although higher xylanase activity was observed after 5 days growth in a wheat bran/lactose medium and higher β-glucosidase activities were obtained after 14 days growth in a wheat bran sphagnum peat (WB/SP) medium. The results of this study emphasize that a sustainable method can be established for production of enzymes and potentially organic acids by using A. carbonarius grown on GPW.A means to track the biomass production and thereby the growth of A. carbonarius in SSF was needed, as this could lead to better understanding of the potential growth rates on lignocellulosic substrates. Two indirect measurements of biomass were evaluated relying on the membrane associated enzyme, β-N-acetylhexosaminidase, and a membrane sterol, ergosterol. The biomass measurements were performed for a total of four fungi from the genera Aspergillus, Trichoderma and Talaromyces when cultivated in a solid WB/SP medium. There was unfortunately a large difference in the reproducibility of the measurements, with the enzyme having highest standard deviation of 11 - 23 % compared to the 2 - 8 % standard deviations of ergosterol measurements. Also it was primarily the ergosterol measurements that showed an increased biomass when comparing 7 and 14 days cultivations. According to the ergosterol measurements Aspergillus saccharolyticus and A. carbonarius produced the least amount of biomass relative to T. reesei Rut-C30 and Talaromyces pinophilus. From this study it was concluded that ergosterol rather than β-N-acetylhexosaminidase should be used for determining fungal biomass in SSF. It was also found that enzyme activity was not linearly correlated to the amount of Erg (biomass) measured.In another study conducted during this Ph.D. project, the A. carbonarius crude enzyme extract was mixed with either lytic polysaccharide monooxygenase (LPMO) from Podospora anserina or Thermoascus aurantiacus in combination with a cellobiose dehydrogenase (CDH) from Neurospora crassa. The aim was to evaluate whether the oxidative degradation of cellulose in wheat straw could enhance the glucose release when at the same time applying crude enzymes of A. carbonarius in the hydrolysis. It was found that the addition of purified P. anserina LPMO and either CDH or ascorbate could not increase the glucose release in wheat straw hydrolysis. However, when a high concentration of the P. anserina LPMO, 4.21 mg/g cellulose (mg/g), was supplemented to the mixture of commercial enzyme preparations Novozym 188 and Celluclast 1.5L a slight but significant increase in glucose was observed. A large increase in glucose release was observed for the supplementation of T. aurantiacus LPMO, both at the high concentration of 4.21 mg/g and at the low concentration of 0.21 mg/g. The glucose release rose from 5.92 to 7.2 or 7.3 g/L. This corresponds to an improvement of the cellulose conversion by 6.8 %. The activity of the LPMO was not enhanced by addition of CDH, but when adding ascorbate to the high concentration of LPMO a significant boosting effect was observed resulting in the highest glucose release of 7.63 g/L. Indeed this was the most optimal improvement of A. carbonarius crude enzyme hydrolysis potential that was achieved corresponding to a total cellulose conversion efficiency of 32 %. Thereby it was shown that a strong boosting of the crude enzymes could be obtained by a single enzyme, the T. aurantiacus LPMO, and that there is a large difference in the effect depending on the origin and type of the LPMO. The effect of LPMO depends on the amount of cellulases applied as well, as it was found that supplementing Ta_LPMO to Celluclast 1.5 L and Novozym 188 gave an increased efficiency of 25.9 %.Finally it was analyzed whether the same P. anserina LPMO and the CDH from N. crassa would show an improvement of the crude enzymes when heterologously expressed in A. carbonarius. The resulting mutants constitutively expressed LPMO and CDH, however, there was no improvement of the glucose release obtained from wheat straw. Despite the lack of effect on glucose release, a significant effect was though observed for xylose release in hydrolysis of wheat straw. The improvement in xylose release ranged from 0.83 g/L to 1.2 g/L. Therefore the addition of LPMO from P. anserina and CDH from N. crassa may still boost the hydrolysis of wheat straw with regards to hemicellulose conversion, which may make the cellulose more accessible.The results obtained during this Ph.D. project successfully represent strategies by which A. carbonarius hydrolysis of lignocellulose may be improved. The successfully applied strategies were blending of monocultures and optimization of cultivation conditions. By optimization of cultivation conditions it was further shown that this fungus can be employed to utilize lignocellulosic waste in the form of GPW for effective production of enzymes. The most potent improvement of A. carbonarius crude enzymes was achieved by the supplementation of a single LPMO enzyme from T. aurantiacus. For A. carbonarius growth on or conversion of lignocellulose a boosting can therefore be achieved by supplementing with LPMO

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Improving <i>Aspergillus carbonarius</i> crude enzymes for lignocellulose hydrolysis

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Improving <i>Aspergillus carbonarius</i> crude enzymes for lignocellulose hydrolysis

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