Le remplacement des solvants organiques classiques par une nouvelle génération de solvants moins toxiques et moins polluants est un défi majeur pour l'industrie chimique. Les liquides ioniques (LIs) ont été largement identifiés comme substituts intéressants aux solvants traditionnels. Le but de ce travail est d'étudier la solubilité des sucres ou des constituants issus de la biomasse dans les liquides ioniques afin de pallier au manque de données expérimentales sur les équilibres de phases de systèmes {sucres + LIs} ou {biomasse + LIs}. Les données de solubilité ont été corrélées avec succès en utilisant les modèles thermodynamiques NRTL et UNIQUAC. Cette étude démontre que la méthode de l'antisolvant est une bonne technique pour l'extraction des sucres des LIs. Par conséquent, les liquides ioniques peuvent être facilement recyclés pour être réutilisés. Les natures fondamentales des interactions entre les sucres et les liquides ioniques ont été définies en utilisant le calcul ab initio. Les résultats obtenus par simulation sont en accord avec les données expérimentales et indiquent que les liquides ioniques interagissent avec les sucres par liaisons hydrogène. La seconde partie de ce travail met en évidence que le prétraitement du miscanthus avec les liquides ioniques permet d'obtenir une bonne production d'éthanol (jusqu'à 150 g d'éthanol par kg de miscanthus). Les résultats montrent que les liquides ioniques sont des solvants performants dans le domaine de la conversion des matières premières issues de la biomasse en biocarburant. Ainsi, l'application à l'échelle industrielle de ces procédés d'extraction de la cellulose pourrait être d'un grand intérêt / The replacement of conventional organic solvents by a new generation of solvents less toxic, less flammable and less polluting is a major challenge for the chemical industry. Ionic liquids have been widely promoted as interesting substitutes for traditional solvents. The purpose of this work is to study the solubility of carbohydrates or biomass based materials in ionic liquids in order to overcome the lack of experimental data on phase equilibria of {biomass or carbohydrate-ILs} mixtures. Solubility data were successfully correlated using NRTL and UNIQUAC thermodynamic models. It was found that the antisolvent method is a good technique for the extraction of carbohydrates from ILs. Ionic liquids could be then recycled successfully for reuse. The fundamental natures of the interaction between carbohydrates and ionic liquids were investigated using ab initio calculations. The theoretical results are in good agreement with experimental data. It was concluded that ionic liquids mainly interact with carbohydrates via hydrogen bonding formation. This confirms that the process of dissolution and regeneration of cellulose in ionic liquids is accompanied only with a physical change. The preatreatment of miscanthus with ionic liquids resulted in the regeneration of amorphous, porous cellulose almost free of lignin, which is suitable for enzymatic hydrolysis and fermentation processes. A successful ethanol production was obtained with an overall ethanol yield reached up to 150 g ethanol kg-1 miscanthus. This indicates the high performance of ionic liquids in converting biomass feedstocks into biofuel. Indeed, applying the cellulose extraction processes on the industrial scale could be of great interest

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The need to mitigate the twin crises of peak oil and climate change has driven a headlong rush to biofuels. This study was aimed at the development of a process to efficiently convert steam explosion pretreated (STEX) sugarcane bagasse into ethanol by using combinations of commercial enzyme cocktails and recombinant Saccharomyces cerevisiae strains. Though enzymatic saccharification is promising in obtaining sugars from lignocellulosics, the low enzymatic accessibility of the cellulose and hemicellulose is a key impediment thus necessitating development of an effective pretreatment scheme and optimized enzyme mixtures with essential accessory activities. In this context, the effect of uncatalysed and SO2 catalysed STEX pretreatment of sugarcane bagasse on the composition of pretreated material, digestibility of the water insoluble solids (WIS) fraction and overall sugar recovery was investigated. STEX pretreatment with water impregnation was found to result in a higher glucose recovery (28.1 g/ 100 bagasse) and produced WIS with a higher enzymatic digestibility, thus was used in the optimization of saccharification and fermentation. Response surface methodology (RSM) based on the 33 factorial design was used to optimize the composition of the saccharolytic enzyme mixture so as to maximize glucose and xylose production from steam exploded bagasse. It was established that a combination of 20 FPU cellulase/ g WIS and 30 IU -glucosidases/ g WIS produced the highest desirability for glucose yield. Subsequently the optimal enzyme mixture was used to supplement enzyme activities of recombinant yeast strains co-expressing several cellulases and xylanases in simultaneous saccharification and fermentations SSFs. In the SSFs, ethanol yield was found to be inversely proportional to substrate concentration with the lowest ethanol yield of 70% being achieved in the SSF at a WIS concentration of 10% (w/v). The ultimate process would however be a one-step “consolidated” bio-processing (CBP) of lignocellulose to ethanol, where hydrolysis and fermentation of polysaccharides would be mediated by a single microorganism or microbial consortium without added saccharolytic enzymes. The cellulolytic yeast strains were able to autonomously multiply on sugarcane bagasse and concomitantly produce ethanol, though at very low titres (0.4 g/L). This study therefore confirms that saccharolytic enzymes exhibit synergism and that bagasse is a potential substrate for bioethanol production. Furthermore the concept of CBP was proven to be feasible. / AFRIKAANSE OPSOMMING: Die behoefte om die twee krisisse van piek-olie en klimaatsverandering te versag, het veroorsaak dat mense na biobrandstof as alternatiewe energiebron begin kyk het. Hierdie studie is gemik op die ontwikkeling van 'n proses om stoomontplofde voorafbehandelde (STEX) suikerriet bagasse doeltreffend te omskep in etanol deur die gebruik van kombinasies van kommersiële ensiem mengsels en rekombinante Saccharomyces cerevisiae stamme. Alhoewel ensiematiese versuikering belowend is vir die verkryging van suikers vanaf lignosellulose, skep die lae ensiematiese toeganklikheid van die sellulose en hemisellulose 'n hindernis en dus is die ontwikkeling van' n effektiewe behandelingskema en optimiseerde ensiemmengsels met essensiële bykomstige aktiwiteite noodsaaklik. In hierdie konteks, was die effek van ongekataliseerde en SO2 gekataliseerde stoomontploffing voorafbehandeling van suikerriet bagasse op die samestelling van voorafbehandelde materiaal, die verteerbaarheid van die (WIS) breuk van onoplosbare vastestowwe in water (WIS), en die algehele suikerherstel ondersoek. Daar was bevind dat stoomontploffing behandeling (STEX) met water versadiging lei tot 'n hoër suikerherstel (21.8 g/ 100g bagasse) en dit het WIS met ‘n hoër ensimatiese verteerbaarheid vervaardig en was dus gebruik in die optimalisering van versuikering en fermentasie. Reaksie oppervlak metodologie (RSM), gebasseer op die 33 faktoriële ontwerp, was gebruik om die samestelling van die ‘saccharolytic’ ensiemmengsel te optimaliseer om sodoende die maksimering van glukose en ‘xylose’ produksie van stoomontplofde bagasse te optimaliseer. Daar was bevestig dat ‘n kombinasie van 20 FPU sellulase/ g WIS en 30 IU ‘ -glucosidases/ g’ WIS die hoogste wenslikheid vir glukose-opbrengs produseer het. Daarna was die optimale ensiemmengsel gebruik om ensiemaktiwiteit van rekombinante gisstamme aan te vul, wat gelei het tot die medeuitdrukking van verskillende ‘cellulases’ en ‘xylanases’ in gelyktydige versuikering en fermentasie SSFs. In die SSFs was daar bevind dat die etanol-produksie omgekeerd proporsioneel is tot substraat konsentrasie, met die laagste etanolopbrengs van 70% wat bereik was in die SSF by ‘n WIS konsentrasie van 10% (w/v). Die uiteindelike proses sal egter 'n eenmalige "gekonsolideerde" bioprosessering (CBP) van lignosellulose na etanol behels, waar die hidrolise en fermentasie van polisakkariede deur' n enkele mikroorganisme of mikrobiese konsortium sonder bygevoegde ‘saccharolytic’ ensieme bemiddel sal word. Die ‘cellulolytic’ gisstamme was in staat om vanself te vermeerder op suikerriet bagasse en gelyktydig alkohol te produseer, al was dit by baie lae titres (0.4 g/L). Hierdie studie bevestig dus dat ‘saccharolytic’ ensieme sinergisme vertoon en dat bagasse 'n potensiële substraat is vir bio-etanol produksie. Daar was ook onder meer bewys dat die konsep van CBP uitvoerbaar is. / The National Research Foundation (NRF) for financial support

It is widely known that fossil fuels are limited; consequently, the generation of new sources of energy in a clean and environmentally friendly manner is a research priority. Bioethanol appears to be one potential solution, especially second-generation production from renewable biomass. In order to use lignocellulosic feedstock to produce bioethanol, its polysaccharide components, cellulose and hemicellulose, must be hydrolysed into soluble sugars, which can then be converted into ethanol by fermentative microorganisms such as Geobacillus thermoglucosidasius TM242 used by the company ReBio Technologies Ltd. To date, the cost of commercial enzymes used during the hydrolysis process remains a major economic consideration in the production of second-generation bioethanol as an alternative fuel. The research project presented in this thesis aims to improve this rate-limiting step of microbial bioethanol production through an investigation of the different enzymes associated with hemicellulose hydrolysis. Firstly, the TM242 genome sequence revealed a number of genes encoding glycoside-hydrolases. Six of these genes were cloned and expressed in E. coli and the recombinant enzymes characterised; three of them, two β-xylosidases and an α arabinofuranosidase, are relevant to xylan hydrolysis, and were found to be highly active and thermostable. Crystallisation of one of the β-xylosidases permitted the determination of a high-resolution (1.7 Å) structure of the apo-enzyme along with a lower resolution (2.6 Å) structure of the enzyme-substrate complex, resulting in the first reported structure of a GH52 family member (Espina et al., 2014). Secondly, as the TM242 microorganism lacks xylanase enzymes, four genes encoding xylanases from closely-related Geobacillus strains were cloned and expressed in E. coli, with one of them being also successfully cloned and expressed in G. thermoglucosidasius TM242. This heterologous xylanase was secreted in active form representing an enhanced biomass utilisation by TM242. In conclusion, it is felt that the findings presented here have the potential to make a valuable contribution towards second-generation bioethanol production.

A celulose é o biopolímero de maior abundância no mundo e tem potencial para se tornar fonte de energia renovável através de sua transformação em açúcares fermentáveis, que por sua vez serão transformados em etanol. A recalcitrância da celulose, principal dificuldade encontrada no processo, pode ser superada com o auxílio de enzimas (celulases). Ao menos três enzimas celulolíticas são necessárias para a degradação total da celulose, incluindo as celobioidrolases, que hidrolisam as ligações glicosídicas das extremidades redutoras e não redutoras da cadeia, as endoglucanases, que clivam a cadeia de celulose amorfa randomicamente, e as beta-glicosidases, que produzem glicose através dos celo-oligômeros. Mas para que esse processo se torne financeiramente viável é necessário conhecer o funcionamento, otimizar a atividade e aumentar a produção dessas celulases. Com o intuito de avançar na compreensão da função e estrutura dessas enzimas, o presente trabalho teve como objetivo o estudo estrutural de beta-glicosidases da família GH1 e endoglucanases da família GH5. Na primeira parte do trabalho, a expressão da endoglucanase II de Trichoderma reesei não foi alcançada, mesmo utilizando diferentes organismos e condições de expressão. Porém, na segunda etapa, foi obtida a expressão, purificação e os primeiros ensaios de cristalização de 11 beta-glicosidases bacterianas da família GH1 e 8 endoglucanases bacterianas da família GH5. Dentre elas, três beta-glicosidases e uma endoglucanase de Bacillus licheniformis foram cristalizadas e tiveram sua estrutura resolvida. As beta-glicosidases, apesar de possuírem o enovelamente similar, apresentaram variações no tamanho e posição das alças formadoras da fenda catalítica e divergem em relação a um dos aminoácidos importantes para a estabilização do substrato. Essas diferenças podem ajudar a explicar o mecanismo dessas enzimas para reconhecer substratos distintos. A endoglucanase da família GH5, possuindo dois módulos acessórios, foi cristalizada tanto na forma apo quanto complexada ao substrato celotetraose. O segundo módulo acessório possivelmente é um domínio de ligação à celulose (CBM) e seus resíduos aromáticos, que são responsáveis pela interação com o substrato, parecem complementar o sítio catalítico, sendo assim um novo mecanismo de auxílio enzimático de um CBM. O primeiro módulo acessório não possui um aparente sítio de interação com carboidratos e provavelmente funciona como um conector entre domínio catalítico e o CBM. O posicionamento do substrato no sítio de ligação é parecido com outras estruturas já determinadas, porém, suscita algumas dúvidas sobre a função dos resíduos catalíticos que é conservada na família. O carbono anomérico do substrato possui uma densidade eletrônica contínua com o glutamato da fita β4 (que deveria ser o ácido/base) e está mais próximo dele que do glutamato da fita β7 (que deveria ser o nucleófilo). / Cellulose is the most abundant biopolymer in the world and can become a renewable energy source through its transformation in fermentable sugars, which will be converted in bioethanol. The cellulose recalcitrance, main difficulty in the process, can be overcome with the aid of enzymes (cellulases). At least three cellulolytic enzymes are required for complete hydrolysis of cellulose, including cellobiohydrolases for hydrolyzing the glycosidic linkages from the reducing and non-reducing chain ends, endoglucanases for randomly cleaving cellulose chains in the amorphous regions, and beta-glucosidases for producing glucose from the solubilized cello-oligomers. But, to become a financially viable process it is necessary to know the mechanism, optimize the activity and improve the production of these cellulases. In order to advance the understanding of the structure and function of these enzymes, the present work intended to study the structure of beta-glucosidases from family GH1 and endoglucanases from family GH5. In the first part of the work, the expression of endoglucanase II from Trichoderma reesei was not achieved, even using different organisms and expression conditions. However, in the second part, the expression, purification and the crystallization first trials of eleven bacterial beta-glucosidases and eight bacterial endoglucanases were achieved. Among them, three beta-glucosidases and one endoglucanase from Bacillus licheniformis were crystallized and had their structures solved. Beta-glucosidases, although having a similar folding, showed variations in the length and position of the loops that form the catalytic cleft and diverge in relation to one of the amino acids that are important in substrate stabilization. These differences may help explain the mechanism of these enzymes to recognize distinct substrates. The endoglucanase, which has two accessory modules, was crystallized in the apo form and complexed with the substrate celotetraose. The second accessory module probably is a cellulose binding domain (CBM) and its aromatic residues, which are responsible for the substrate interaction, seem to complement the catalytic site. Therefore it can be a new mechanism of CBM assistance in the enzymatic activity. The first accessory module has no apparent interaction site with carbohydrates and probably works as a connector between the catalytic domain and CBM. The positioning of the substrate in the binding site is similar to other structures already solved but raises some questions about the role of the catalytic residues, that are conserved in the family. The anomeric carbon of the substrate has a continuous electron density with glutamate from sheet-β4 (which should be the acid/base) and is closer to it than to glutamate from sheet-β7 (which should be the nucleophile).

O esgotamento das fontes de combustíveis fósseis e questões ambientais têm gerado grandes preocupações para a sociedade, e alternativas sustentáveis e eficientes para solucionar e trazer inovações na produção de biocombustíveis estão cada vez mais próximas. O uso de biomassa pode ser uma vantajosa opção, mas para isso, é necessária a degradação das moléculas constituintes de sua parede celular a açúcares fermentáveis. A biotecnologia tem melhorado e aumentado as opções para suprir fontes sustentáveis e preservação do meio ambiente. A transformação de biomassa na escala industrial para produção de bioetanol depende da capacidade de otimizar o processo de hidrólise enzimática da celulose utilizando enzimas de complexo celulolítico, principalmente de fontes bacterianas e fungos filamentosos. Dessa forma, estudamos a principal endoglucanase de Xanthomonas campestris pv. Campestris ATCC33913, que foi inicialmente identificada através de zimogramas e espectrometria de massas que mostrou boa cobertura de sequência e purificada utilizando passos de precipitação com sulfato de amônio seguida do uso de uma coluna de exclusão molecular. Esta enzima teve seus parâmetros cinéticos determinados, mostrando-se ativa para diversos substratos testados e também grande estabilidade para variações de pH e temperatura, com condições ótimas de pHótimo = 7.0 e Tótima = 45°C. Além da caracterização enzimática, a posição relativa entre o domínio catalítico e de ligação da celulose (CBM) da Endoglucanase por SAXS também foram determinadas, e ainda, para finalizar os estudos estruturais, a estrutura cristalográfica do domínio catalítico da enzima foi determinada com resolução de 2.8Å, contendo quatro moléculas por unidade assimétrica. / The exhaustion of fossil fuels and the environment issues related have been generated many concerns for the society and searching for sustainable and effective alternative are being done in order to solve and bring innovations for biofuel production. Biomass can be a good alternative, but, for the success, it will be necessary degrading the cellular wall molecules into fermentable sugar. Biotechnology has improved and increased options in order to supply sustainable sources and environment preservation. The biomass transformation for bioethanol production, at the industrial scale, depends on the capacity of optimize the enzymatic hydrolysis of cellulose using enzymes of the cellulolytic complex, mainly produced by bacteria and filamentous fungi. We have studied the main endoglucanase of Xanthomonas campestris pv. Campestris ATCC33913 which has been initially identified by zymograms and mass spectrometry with high coverage sequence, and purified using precipitation with ammonium sulfate followed by size exclusion column. This enzyme had its kinetic parameters determined showing activity for the substrates tested and also has showed stability for pH and temperature changes, with optimal conditions of pHopt = 7.0 and Topt = 45°C. Following this enzymatic characterization, the relative position of the catalytic domain and cellulose binding domain (CBM) was determined by SAXS. In order to complete the structural studies, the crystallographic structure of the catalytic domain has been determined with 2.8Å of resolution, showing four molecules by asymmetric unity.

<p>To ensure the sustainability of bioethanol production, major attention has been directed to develop feedstocks which provide an alternative to food-crop biomass. Lignocellulosic (LC) biomass, which is chiefly composed of industrial plant residues, is a carbon-rich reservoir that is presently attracting much attention. However LC material is highly recalcitrant to bioprocessing and requires a mixture of physical and enzymatic pretreatment in order to liberate fermentable sugars. Thermostable enzymes are extremely desirable for use in thermophilic fermentations due to their inherent stability. Hemicellulose, a core constituent of LC, requires a cascade of hemicellulases to stimulate the depolymerisation of its xylan backbone. &alpha / -L-arabinofuranosidase (AFase) increases the rate of lignocellulose biodegradation by cleaving arabinofuranosyl residues from xylan thereby increasing the accessibility of other hemicellulases. Twenty thermophilic Actinomycete isolates were screened for AFase activity using pnp-arabinofuranoside as the substrate. Three strains (ORS #1, NDS #4 and WBDS #9) displayed significant AFase activity and were identified as Streptomyces species with 16S rRNA gene sequence analysis. Genomic DNA was isolated from these strains and a cosmid library constructed in the shuttle vector pDF666. Subsequent functional and PCR-based screening revealed no positive clones.</p>

Liquid Biofuels mainly Bioethanol and biodiesel are the main replacement for fossil fuels in the current world. But there are questions and concerns about the present biofuels production, mainly when it comes to matter of sustainability. In this thesis paper,Strategic Life Cycle Management data along with Life Cycle Analysis data has been used to analyze the sustainable biofuels condition in Sweden and the Netherlands. Data also has been collected through the interview from different stakeholders in Sweden and the Netherlands.

Sweet sorghum (Sorghum bicolor) has potential as a bioenergy feedstock due to its high yield potential and the production of simple sugars for fermentation. Sweet sorghum cultivars are typically tall, high biomass types with juicy stalks and high sugar concentration. These sorghums can be harvested, milled, and fermented to ethanol using technology similar to that used to process sugarcane. Sweet sorghum has advantages in that it can be planted by seed with traditional planters, is an annual plant that quickly produces a crop and fits well in crop rotations, and it is a very water-use efficient crop. Processing sweet sorghum is capital intensive, but it could fit into areas where sugarcane is already produced. Sweet sorghum could be timed to harvest and supply the sugar mill during the off season when sugarcane is not being processed, be fit into crop rotations, or used in water limiting environments. In these ways, sweet sorghum could be used to produce ethanol in the Southern U.S and other tropical and subtropical environments. Traditionally, sweet sorghum has been grown as a pureline cultivar. However, these cultivars produce low quantities of seed and are often too tall for efficient mechanical harvest. Sweet sorghum hybrids that use grain-type seed parents with high sugar concentrations are one way to overcome limitation to seed supply and to capture the benefits of heterosis. There are four objectives of this research. First to evaluate the importance of genotype, environment, and genotype-by-environment interaction effects on the sweet sorghum yield and composition. The second objective is to determine the presence and magnitude of heterosis effects for traits related to sugar production in sweet sorghum. Next: to study the ability of sweet sorghum hybrids and cultivars to produce a ratoon crop and determine the contribution of ratoon crops to total sugar yield. The final objective is to evaluate variation in composition of sweet sorghum juice and biomass. Sweet sorghum hybrids, grain-type sweet seed parents, and traditional cultivars that served as male parents were evaluated in multi-environment trials in Weslaco, College Station, and Halfway, Texas in 2007 and 2008. Both genotype and environment influenced performance, but environment had a greater effect than genotype on the composition of sweet sorghum juice and biomass yield. In comparing performance, elite hybrids produced fresh biomass and sugar yields similar to the traditional cultivars while overcoming the seed production limitations. High parent heterosis was expressed among the experimental hybrids for biomass yield, sugar yield and sugar concentration. Additional selection for combining ability would further enhance yields and heterosis in the same hybrid. Little variation was observed among hybrids for juice and biomass composition suggesting that breeding efforts should focus on yield before altering plant composition.

The cost of pretreatment process for saccharification from biomass and the cost of dilute ethanol purification are significant components of the overall cost for fuel grade ethanol production through fermentation or other biological routes. This work focuses on developing optimal designs of dilute ethanol purification process and the new acid hydrolysis technology for the production of fermentable sugars from biomass where the overarching goal is to reduce the cost of ethanol production from biomass. In this thesis, the ethanol separation process with the reverse osmosis membrane pretreatment is developed to reduce separation cost and energy consumption especially when the feed is dilute. In addition, the new solid phase reactive separation system for biomass saccharification via acid hydrolysis is proposed. This new process is applied for both dilute and concentrated acid hydrolysis where the goal is to increase sugar yield and to reduce byproduct formation. The reaction kinetics of the concentrated acid hydrolysis is investigated through batch experiment. All of these use optimization approaches for seeking the best process designs and for parameter estimations.

Η βιοαιθανόλη παράγεται από την αλκοολική ζύμωση σακχαρούχων, αμυλούχων και κυτταρινούχων πρώτων υλών. Η χρήση της ως εναλλακτικό καύσιμο (βιοκαύσιμο), μπορεί να συμβάλει σημαντικά στην μείωση της ατμοσφαιρικής ρύπανσης. Στην βιοτεχνολογική παραγωγή αιθανόλης έχει μελετηθεί εκτεταμένα η χρήση ακινητοποιημένων κυττάρων, ως μέσο για τη αύξηση της παραγωγικότητας των ζυμώσεων. Στην παρούσα εργασία μελετήθηκε η προοπτική αξιοποίησης δύο νέων φυσικών υλικών, ως φορέων ακινητοποίησης των κυττάρων της ζύμης για την παραγωγή βιοαιθανόλης. Ο φορείς αυτοί είναι η ελίφη που αποτελεί το εσωτερικό του αποξηραμένου καρπού του φυτού Luffa cylindrica και τεμάχια του βλαστού του φυτού Arundo donax (καλάμι). Σε όλα τα πειράματα με βιοαντιδραστήρες διαλείποντος έργου, παρουσία των φορέων η παραγωγικότητα σε αιθανόλη αυξήθηκε σημαντικά και μάλιστα σε ορισμένες περιπτώσεις διπλασιάστηκε σε σχέση με τις αντίστοιχες ζυμώσεις απουσία του φορέα. Η βελτίωση είναι ιδιαίτερα σημαντική σε διαλύματα υψηλής αρχικής πυκνότητας σακχάρων (> 18 oBe), στα οποία τα κύτταρα της Saccharomyces cerevisiae, απουσία του φορέα αδυνατούν να αξιοποιήσουν το μεγαλύτερο ποσοστό των διαθέσιμων σακχάρων. Η θετική επίδραση των φορέων, παρουσίασε σταθερότητα κατά την χρήση των ίδιων φορέων σε διαδοχικές ζυμώσεις, γεγονός που αποτελεί ένα επιπλέον θετικό στοιχείο που αφορά στη διαχρονική χρήση τους. Η ακινητοποίηση των κυττάρων στους φορείς, μελετήθηκε με παρατή¬ρηση σε ηλεκτρονικό μικροσκόπιο σάρωσης (SEM). Επιβεβαιώθηκε πώς η επιφάνεια των φορέων καλύφθηκε από ένα εκτεταμένο βιολογικό υμένιο βιοκαταλύτη, το οποίο αυξάνει σημαντικά και με μικρό κόστος την παραγωγικότητα του βιοαντιδραστήρα. Συμπερασματικά, τόσο η ελίφη, όσο και το καλάμι, φαίνεται ότι συγκεντρώνουν σημαντικά πλεονεκτήματα για να αποτελέσει η χρήση τους μια ουσιαστική πρόταση για βελτίωση της τεχνολογίας παραγωγής βιοαιθανόλης. / Bioethanol is produced by the alcoholic fermentation process of sugar-containing, starchy and cellulosic raw materials. Its use as an alternative fuel (biofuel) could significantly contribute in air pollution reduction. Considerable amount of research has been carried out to develop new, highly efficient fermentation processes. One such area that has received great attention has been the use of immobilized cell systems. In the present study, we investigated the perspective to use two new natural materials as carriers for yeast cell immobilization. These materials are loofa sponge, obtained from the matured dried fruit of Luffa cylindrica and fragments of the stalk of Arundo donax (cane). All batch fermentations that carried out in the presence of carriers, had enhanced productivities (even 100% increase related to the fermentations without the carrier). This was more intense at high sugar densities (18 oBe). The positive effect of the carriers showed significant viability, when the same carriers were used in repeated batch fermentations. Cell immobilization on the carriers, was confirmed after observation in Scanning Electron Microscope. Carrier’s surface was covered by layers of biocatalyst (biofilm). Consequently, it was demonstrated that both loofa and cane, assemble enough advantages in order to constitute an effective suggestion, to improve the alcohol production technology.