In light of rising costs in fossil fuels and petroleum, as well as the strain on this largely non-renewable resource, the conversion of biomass, in this case waste biomass, to value-added products is becoming more attractive. In this study, municipal sewage sludge and biosolids were used to determine their potential for glucose recovery. This research focused on three pretreatment processes including drying/grinding, as well as acid and alkaline pretreatments, followed by acid hydrolysis on primary sludge, activated sludge and biosolids. After each pretreatment under specified conditions, the residues remaining from the sludges and biosolids underwent a 2 % H2SO4 acid hydrolysis at 120oC for 1 hr. Compared with activated sludge and biosolids, primary sludge was found to demonstrate the highest potential for glucose recovery in this study. Primary sludge with 1.0 N HCl pretreatment over a 24 hour contact period yielded the highest glucose conversion result as 5.67±0.24%. The best KOH pretreatment condition for primary sludge was a 0.5 N KOH concentration for a 0.5 hour contact period. However, no consistent glucose recovery trend as a function of reagent concentration or contact time was identified for any of the sludges or biosolids in this study. Drying and grinding were also found to efficiently improve the acid hydrolysis results. Fibre content analysis was also performed on the sludge and biomass feedstocks and their residues following pretreatment and acid hydrolysis during this study, to better understand the conversion of these waste biomass feedstock. The Van Soest methods for neutral-detergent, acid-detergent and acid insoluble lignin analysis and the Weende crude fibre analysis were applied to the sewage sludge and biosolids samples prior to and after acid hydrolysis to determine the fibre content including cellulose, hemicellulose and lignin. A modification to the Weende crude fibre analysis was introduced, where a centrifuge step was added prior to the second filtration after the alkaline digestion of the procedure to reduce filter clogging problems. The centrifuge modification effectively reduced the filtering time from one day to 30 minutes; however, there was an average loss of 46% in crude fibre with the addition of this centrifugation step. It was found that most of cellulose content in the feedstock samples was hydrolyzed to glucose after the acid hydrolysis process and most hemicellulose content was likely to have been solubilized and washed away during acid and alkaline pretreatments and acid hydrolysis. The lignin content did not appear to be affected by the pretreatments applied nor the acid hydrolysis. / Thesis (Master, Civil Engineering) -- Queen's University, 2008-07-25 16:10:42.518

Dentre os materiais de fontes naturais e renováveis, a celulose se destaca pela sua abundância, podendo ser encontrada em diversos organismos vivos, como plantas, amebas, bactérias, fungos e alguns animais marinhos. Suas dimensões podem ser reduzidas por quebra das cadeias amorfas, com possibilidade de atingir escalas nanométricas, obtendo-se assim as chamadas nanopartículas de celulose ou nanocelulose. Devido à alta cristalinidade, a nanocelulose possui altos valores de módulo elástico, proporcionando alta capacidade de reforço em matrizes poliméricas, combinados com baixo peso, área superficial elevada e biodegradabilidade. A borracha natural é uma matéria-prima de fonte natural, sendo extraída das seringueiras na forma de látex - dispersão coloidal de partículas de borracha e substâncias não-borrachas em um meio aquoso, com aspecto leitoso. No presente trabalho foram estudados compósitos de borracha natural e nanocelulose. Inicialmente, foi realizada uma análise do látex de nacionalidade brasileira, centrifugado, contendo 60% em massa de sólidos. Os resultados de caracterização do látex centrifugado comercial, a qual consistiu na análise de concentração de sólidos totais e na medida do pH, estavam de acordo com os dados apresentados pelo fornecedor. Além disso, a análise de distribuição de tamanho de partícula indicou que o material apresenta uma população, com tamanho médio de 1,0 ?m. A borracha coagulada com ácido acético apresentou, após mastigação em cilindro aberto, viscosidade Mooney e extrato acetônico igual a, respectivamente, 52,8 e 2,57%. As nanoceluloses foram obtidas por hidrólise com ácido ortofosfórico (NC P) e sulfúrico (NC S), sendo classificadas como nanocristal de celulose (NC). NC P apresentaram comprimento médio, razão de aspecto e cristalinidade igual a, respectivamente, 270 ± 89 nm, 50 ± 24 e 78%; e as NC S apresentaram 209 ± 51 nm, 29 ± 10 e 75%.. Os compósitos de borracha natural com nanocristais de celulose apresentaram, nos ensaios de tração, aumentos nos valores de todas as propriedades analisadas, quando comparados à borracha natural pura. Ao adicionar-se 10 phr de nanocelulose preparada com ácido fosfórico na borracha natural, os valores de resistência à tração na ruptura, alongamento na ruptura e módulo a 300% aumentaram, respectivamente, em 90%, 16% e 52%. Já com a adição de 10 phr de NC S, essas propriedades aumentaram, respectivamente, em 68%, 5% e 109%. O mesmo foi observado para a dureza Shore A. Com a adição de 10 phr de nanocelulose obtida por ácido fosfórico à composição da borracha natural, a dureza Shore A aumentou em cerca de 22%; já com a adição de 10 phr de NCs S, a dureza da borracha natural aumentou em 36%. / Among the natural and renewable sources\' materials, cellulose stands out for its abundance, it can be found in many living organisms, such as plants, amoebas, bacteria, fungi and some marine animals. Its dimensions can be reduced by breaking the amorphous chains, with the possibility of reaching nanometric scales, obtaining the nanocellulose or cellulose nanoparticles. Due to the high crystallinity, the nanocellulose has high elastic modulus value, providing high reinforcement capacity combined with low weight, high surface area and biodegradability. Natural rubber is a raw material from a natural source, extracted from the latex - colloidal dispersion of rubber particles and non-rubbers in a milkylooking aqueous solution. At this work, composites of natural rubber and nanocellulose were studied. Initially, a Brazilian centrifuged latex with 60% of its weight in solids was characterized, by analyzing if the total solids concentration and the pH measurement is in agreement with the data presented by the supplier. In addition, particle size distribution analysis demonstrated that the material had an average size of 1.0 ?m. Then, the mastication in the open cylinder and the Mooney viscosity and acetone extract was measured and them were equal to, respectively, 52.8 and 2.57%. The nanocelluloses obtained by hydrolysis with phosphoric and sulfuric acids are classified as cellulose nanocrystal. NC P present average length, aspect ratio and crystallinity equal to 270 ± 89 nm, 50 ± 24 and 78%; and the NC S had 209 ± 51 nm, 29 ± 10 and 75%. In the tensile test, it was observed that there was an increase in all the mechanical properties analyzed for natural rubber when adding the nanocellulose in its composition. By adding 10 phr of prepared nanocellulose with phosphoric acid in the natural rubber the values of tensile strength at rupture, strain at rupture and modulus at 300% increased, respectively, by 90%, 16% and 52%. When added 10 phr of NC S, these properties increased, respectively, by 68%, 5% and 109%. The same was observed for Shore A hardness. When adding 10 phr of nanocellulose obtained by phosphoric acid in its composition, the Shore A hardness increased by about 22%; When adding 10 phr of NCs S, the hardness increased by 36%.

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.

De forma sustentável o setor sucroalcooleiro nacional apresenta grande potencial para o desenvolvimento tecnológico de fontes alternativas de energia. A partir das novas concepções de emprego de energias alternativas, o resíduo industrial do processamento da cana-de-açúcar passa a ser uma matéria-prima com grande potencial e valor. De forma ilustrativa da potencialidade deste material, os dados de produção de bagaço de cana e suas aplicações estão destacados na parte introdutória, compreendendo a possível integração das usinas com as biorrefinarias. A utilização do bagaço de cana-de-açúcar como combustível para co-geração de energia atende à necessidade de mercado, sendo que a rentabilidade de energia elétrica é altamente viável. Porém, considerando-se que esta matéria-prima apresenta características peculiares para a biorrefinaria, objetivou-se o fracionamento da biomassa. As metodologias são investigadas por abranger a conversão da biomassa(bagaço e palha de cana-de- açúcar) para uma variedade de produtos de maior valor agregado (energia, biomateriais e produtos químicos). As etapas de fracionamento, ou seja, de pré-tratamentos foram: préhidrólise, deslignificação e branqueamento. A pré-hidrólise apresentou-se seletiva para a remoção das hemiceluloses, já para a etapa de deslignificação, mostrou-se mais seletiva para a remoção de ligninas, embora houvesse grande remoção também das hemiceluloses. Para a etapa de branqueamento, a remoção residual de lignina foi atingida, porém também foi removido celulose. A avaliação das etapas de pré-tratamentos foi realizada pela determinação dos rendimentos de processos, da seletividade reacional, bem como pela quantificação dos produtos e resíduos obtidos. Os materiais obtidos nas etapas de pré-tratamentos foram caracterizados e posteriormente submetidos ao processo de hidrólise ácida. São apresentados os resultados de rendimento de obtenção de glicose a partir de celulose, em base celulose inicial e em base celulose reagida. Na primeira parte, discutiram-se os rendimentos gravimétricos das reações de hidrólise ácida que para o material in natura resultaram em maiores rendimentos de hidrólise. Os maiores rendimentos obtidos no estudo de hidrólise de celulose foram obtidos para substratos derivados do bagaço integral na concentração de ácido de 0,14%. Os resultados da quarta, relativa a obtenção de coprodutos, HMF e furfural revelou a geração de baixas concentrações desses inibidores da fermentação etanólica. De forma conclusiva, a associação dos resultados dos processos de pré-tratamentos com os resultados obtidos nas reações de hidrólise poderá ser empregada para compreender as vantagens e desvantagens de cada etapa, mostrando caminhos para um melhor aproveitamento da biomassa vegetal, em especial o bagaço de cana de açúcar. Considerando-se a produção de etanol de segunda geração, determinaram-se também as quantidades de inibidores de fermentação etanólica. / Sustainably the Brazilian sugarcane sector presents great potential for technological development of alternative sources of energy. From the new concepts of employment of alternative energy sources, industrial waste from processing of sugarcane becomes a raw material with great potential and value. Illustratively of the potential of this material, the production data of sugarcane bagasse and their applications are highlighted in the introductory section, including the possible integration of the sugarcane mills to biorefineries. The utilization of crushed sugarcane as fuel for co-generation of energy meets the market need, and the profitability of electricity is highly feasible. However, considering that this raw material has peculiar characteristics to the biorefinery, this work aimed at the fractionation of biomass. The methodologies are investigated for covering the conversion of biomass (bagasse and straw from sugarcane). For a variety of products with higher added value (energy, biomaterials and chemicals). The steps of fractionation, or pre-treatments were pre-hydrolysis, delignification and bleaching. The pre-hydrolysis showed up selective removal of hemicelluloses. On the other hand, the delignification step was more selective for the removal of lignin, although there was also large removal of hemicelluloses. For the bleaching stage, the removal of residual lignin was achieved, but some cellulose was also removed from the pulps. The evaluation of the pre-treatments steps were performed by determined the yields of the processes, the selectivity and the quantification of the reaction products and wastes obtained. The materials obtained by the pre-treatment steps were characterized and further submitted to acid hydrolysis. It were presented the results of glucose yields of production base on initial cellulose and reacted cellulose. The first part of the work discusses the gravimetric yield of the acid hydrolysis reaction that produced the highest yield for the in natura material. The highest yields obtained in this study of cellulose hydrolysis were obtained for substrates derivates from integral bagasse at acid concentration of 0.14%. The results of the fourth step, concerning the production of HMF and furfural, revealed the production of low concentrations of these inhibitors of ethanol fermentation. Conclusively, the association of the results obtaining in the pre-treatment processes with that ones obtained by the hydrolysis processes can be used to understand the advantages and disadvantages of the individual steps, showing trends towards a better utilization of vegetal biomass, especially crushed sugarcane. In terms of second generation ethanol, it was presented the results of the quantification of potential inhibitors of ethanolic fermentation.

Os métodos analíticos empregados atualmente para a caracterização química de materiais lignocelulósicos foram desenvolvidos para a madeira e são empregados com pequenas modificações ao bagaço e à palha da cana-de-açúcar. A não existência de metodologia específica para estes materiais leva a obtenção de resultados inadequados e dificultam tanto o planejamento quanto a interpretação de resultados. Sendo assim, o objetivo principal deste trabalho é desenvolver metodologias analíticas específicas para a caracterização química da palha e do bagaço de cana-de-açúcar. A determinação de lignina foi estudada a partir da hidrólise e solubilização da celulose e hemiceluloses em solução de ácido sulfúrico. A fração insolúvel foi analisada por análise elementar, espectroscopia no infravermelho, espectrometria de ressonância magnética nuclear de C13 no estado sólido e análise termogravimétrica (TGA). Os açúcares hidrolisados e produtos derivados destes foram analisados por meio de cromatografia líquida de alta eficiência (CLAE). Foi realizada uma análise de holocelulose que é complementar à determinação de lignina. O procedimento consiste no tratamento do material lignocelulósico (pré-extraído com cicloexano/etanol) com solução de clorito de sódio em meio ácido. Os resultados obtidos não apresentaram similaridade nas correlações entre os métodos analíticos, entretanto mostraram que o bagaço integral e suas frações separadamente apresentam comportamentos diferentes, frente ao tratamento ácido, o que ressalta a necessidade de métodos analíticos específicos. / The analytical methods currently used for chemical characterization of lignocellulosic materials were developed for wood and are applied with minor modifications for sugar cane bagasse and straw analysis. The lack of specific methodology for these materials leads to inadequate results and hamper both the planning and the interpretation of results. Thus, the main aim of this work is to develop specific analytical methodologies to the chemical characterization of sugar cane bagasse and straw. The determination of lignin was studied by the hydrolysis and dissolution of the polysaccharide fraction in sulfuric acid solution. The insoluble fraction was analyzed by elemental analysis, Fourier Transform Infrared (FT-IR), Carbon-13 nuclear magnetic resonance spectroscopy (13C NMR), Gel permeation chromatography (GPC) and Thermogravimetric analysis (TGA). The sugars and derivatives of these hydrolysates were analyzed by High performance liquid chromatography (HPLC). It was also performed a complementary analysis from the holocellulose content determinations in order to check the values obtained by the klason procedure. The results showed the dependence of sulfuric acid concentration on lignin content determinations and the role of condensation reactions in the lignin characteristics. Despite the similarities in chemical composition, klason lignins obtained from straw exhibited very low molar masses. Preliminary results obtained from holocellulose determinations showed also the need for optimized oxidation procedures in order to be successful applied to sugar cane bagasse analysis.

Gas chromatographic acquisition of representative 'Total' cellular fatty acid profiles from bacteria or bacteria-containing samples (e.g., environmental or clinical materials) tends to be dependent on the method used to released the fatty acids and convert them to derivatives suitable for analysis. Alkaline saponification or interesterification methods, while preserving acid-sensitive components such as cyclopropane fatty acids, are often insufficient to release amide-linked components, such as hydroxylated fatty acids. Acid-catalyzed hydrolyses or interesterifications, on the other hand, while more efficiently releasing the predominantly amide-linked hydroxylated components, have been shown to cause severe and unpredictable degradation of cyclopropane fatty acids. We report studies of a single-tube method involving sequential alkaline/acid release of fatty acids in which fatty acids released by the alkaline step are partitioned into an organic epiphase during the aqueous acid hydrolysis step. After hydrolysis, the epiphase and the released fatty acids are extracted into an hypophasic solvend and esterified at moderate temperature under relatively low acid concentrations. Under these conditions, cyclopropane as well as hydroxylated fatty acids are recovered in high yield.

Previous studies have shown that pullulan films are able to stabilize enzymes and other labile molecules from thermal and oxidative degradation. Solutions made with commercially available pullulan are extremely viscous and difficult to process limiting the ability to use low-cost printing systems, such as inkjet printers, to format pullulan-containing. In this work, we show that pullulan can be made printable by decreasing its chain length by acid hydrolysis. The acid hydrolysis reaction was modelled using statistical software; the molecular weight of pullulan decreased with increasing reaction time, temperature and acid concentration. Interactions between time and temperature, and temperature and acid concentration were determined to be significant to the reaction as well. The mechanical properties and oxygen permeability of films made from pullulan with different molecular weights were also measured. The films were found to have similar tensile properties and oxygen permeabilities to each other and to those obtained using native pullulan. Using a thermally unstable enzyme (acetylcholinesterase) and an easily oxidizable small molecule (indoxyl acetate) as test materials, it was found that these films have the same ability to stabilize the enzyme and to serve as an oxygen barrier, as the films made with native pullulan. It was also found that pullulan is inkjet printable as long as the molecular weight is 56 kDa. Poor jetting and clogging of the printhead was observed when pullulan with a molecular weight higher than this threshold was used. Microarray printing was also demonstrated by a printing acetylcholinesterase/pullulan in nano-sized volumes using a Dimatix inkjet printer and showing activity of the enzyme after printing and storage at ambient conditions. Proof of concept of microarray printing opens up the potential for future applications of pullulan in other high throughput applications. / Thesis / Master of Applied Science (MASc)

<p> The c^13 isotope effect in acid and alkaline hydrolysis of both ethyl and tertiary butyl benzoate-carboxyl-c^14 have been measured. Significant isotope effects were found in all the hydrolyses except acid hydrolysis of the tertiary butyl oster. These results are additional evidence for the suggestion of Choen and Schneider that alkyl oxygen rather than normal acyl oxygen bond cleavage occurs when tertiary butyl benzoate is hydrolyzed in acid solution. No appreciable c^13 isotope effect was found to occur in thermal, aqueous, or acid-catalysed decarboxylation of anthranilic acid. From this observation and other experimental evidence, it is concluded that anthranilic acid decarboxylates by a bimolecular process in which the attack of the hydrogen ion is rate controlling. It is further suggested that the hydrogen ion attack is on the zwitterion at the c carbon position. Both the c^13 and the c^14 isotope effect in the decarboxylation of mesitoic acid were measured using a mass spectrometer. The c^14 isotope effect was found to be two and one half times the magnitude of the c^13 isotope effect instead of twice as present theory predicts. </p> / Thesis / Doctor of Philosophy (PhD)

Nos últimos anos, pesquisas sobre a produção de nanocristais de amido (NCA) receberam interesse crescente devido a suas diversas aplicações, principalmente como material de reforço de matrizes poliméricas. Nesse contexto, o amido de quinoa (AQ) apresenta características desejáveis na produção de NCA tais como tamanho de grânulo pequeno e conteúdo de amilose relativamente baixo. Assim, o objetivo desta pesquisa foi produzir NCA de quinoa (NCAQ) por hidrólise ácida em diferentes temperaturas (30, 35 e 40) °C. Além disso, foi estudado o efeito da adição dos NCAQ nas propriedades estruturais e físicas de filmes de amido de mandioca. O AQ apresentou diferentes percentagens de hidrólise, no quinto dia, 63%, 73% e 91% para (30, 35 e 40) °C, respectivamente. O AQ (k = 0,59 dias-1) foi hidrolisado mais rápido que o amido de milho ceroso (k = 0,39 dias-1) a 40 °C. O rendimento dos NCAQ diminuiu com o incremento da temperatura de 30 a 40 °C; enquanto que a cristalinidade relativa dos NCAQ não foi alterada (~35%). A morfologia dos NCAQ produzidos a 30 °C foi irregular com tamanho micrométrico, enquanto que os produzidos a 35 e 40 °C apresentaram forma de paralelepípedo com tamanhos entre (50 e 100) nm e (400 e 900) nm (agregados). O diâmetro hidrodinâmico e as propriedades térmicas dos NCAQ diminuíram com o aumento da temperatura da hidrólise; enquanto que a intensidade das bandas FTIR e o potencial zeta aumentaram. As propriedades indicaram que NCAQ foram produzidos somente a (35 e 40) °C com rendimentos de 22,7% e 6,8%, respectivamente. Dessa forma, considerando o rendimento e a temperatura de transição, os NCAQ produzidos a 35 °C foram selecionados para aplicação em filmes de amido de mandioca. Os filmes foram preparados pela técnica do casting, com 4 g de amido de mandioca/100 g de dispersão filmogênica; 25 g glicerol/ 100 g de amido; e 0; 2,5; 5,0 e 7,5 g de NCAQ/ 100 g de amido. Os difratogramas de raios X confirmaram a presença dos NCAQ nos filmes. A adição de NCAQ nos filmes aumentou a rugosidade e o ângulo de contato em concentrações de 5% e 7,5%, a resistência à tração e o módulo elástico, os parâmetros de cor L* e a* em concentrações 7,5%, e a opacidade; enquanto que diminuiu a deformação na ruptura, a permeabilidade ao vapor de água na concentração de 5%, e o brilho. Outras propriedades dos filmes como espessura, umidade, solubilidade, propriedades térmicas não foram alteradas pela adição de NCAQ. Os resultados indicaram que os NCAQ produzidos a 35 °C podem ser usados como reforço em filmes nanocompósitos para melhorar suas propriedades mecânicas. / Recently researches on starch nanocrystals (SNC) production have become of interest due to their many applications, especially as reinforcement in polymeric matrices. Quinoa starch (QS) has desirable characteristics for SNC production such as small granule size and relatively low amylose content. Thus, the objective of this research was to produce quinoa SNC (QSNC) by acid hydrolysis at different temperatures (30, 35 and 40) °C. Furthermore, the effect of QSNC addition on the structural and physical properties of cassava starch films was studied. QS presented different percentages of hydrolysis on the fifth day, 63%, 73% and 91% for (30, 35 and 40) °C, respectively. QS (0.59 days-1) was hydrolyzed more rapidly than waxy maize starch (0.39 days-1) at 40 °C. QSNC yields decreased with temperature increase from (30 to 40) °C, while the relative crystallinity was not altered (~35%). The morphology of QSNC produced at 30 °C was irregular with micrometric size while those produced at 35 °C and 40 °C presented parallelepiped shapes with sizes between 50 nm and 100 nm and 400 nm to 900 nm (aggregates). The hydrodynamic diameter and the thermal properties of QSNC decreased with temperature increase, while the FTIR band intensities and the zeta potential increased. The properties indicated that quinoa QSNC were only obtained at (35 and 40) °C with yields of 22.8% and 6.8%, respectively. QSNC produced at 40 °C presented lower yield and crystallinity than waxy maize SNC, but a lower hydrodynamic diameter. Thus, based on the yield and transition temperature, QSNC produced at 35 °C was selected for application in cassava starch films. The films were prepared by casting technique, with 4 g of cassava starch / 100 g of film forming dispersion; 25 g glycerol / 100 g starch; and 0; 2.5; 5.0 and 7.5 g of QSNC / 100 g of starch. X-ray diffractograms confirmed the presence of QSNC in the films. Addition of QSNC to films increased the roughness and the contact angle at 5.0% and 7.5% concentrations, the tensile strength and elastic modulus, the color parameters L* and a* at 7.5% concentration, and the opacity; while decreasing deformation at break, water vapor permeability at 5.0% concentration, and gloss. Other film properties such as thickness, moisture content, solubility, thermal properties were not affected by QSNC addition. The results indicated that the QSNC produced at 35 ° C can be used as reinforcement in nanocomposite films to improve their mechanical properties.

For the citrus processing industry the disposal of fresh peels has become a major concern for manyfactories. Orange peels are the major solid by-product. Dried orange peels have a high content ofpectin, cellulose and hemicellulose, which make it suitable as fermentation substrate when hydrolyzed.The present work aims at utilizing orange peels for the production of ethanol by using the fungusMucor indicus. Hence, producing a valuable product from the orange peel waste. The biomass growthwas also examined, since the biomass of the fungus can be processed into chitosan, which also is avaluable material.The work was first focused on examining the fungus ability to assimilate galacturonic acid and severalother sugars present in orange peel hydrolyzate (fructose, glucose, galactose, arabionose, and xylose).Fructose and glucose are the sugars which are consumed the fastest whereas arabinose, xylose andgalacturonic acid are assimilated much slower.One problem when using orange peels as raw material is its content of peel oils (mainly D-limonene),which has an immense antimicrobial effect on many microorganism even at low concentrations. Inorder to study M. indicus sensitivity to peel oil the fungus was grown in medium containing differentconcentrations of D-limonene.At very low limonene concentrations the fungal growth was delayed only modestly, hence a couple ofhours when starting from spores and almost nothing when starting with biomass. Increasing theconcentration to 0.25% (v/v) and above halted the growth to a large extent. However, the fungus wasable to grow even at a limonene concentration of 1.0%, although, at very reduced rate. Cultivationsstarted from spore-solution were more sensitive than those started with biomass.Orange peels were hydrolyzed by two different methods to fermentable sugars, namely by dilute acidhydrolysis (0.5% (v/v) H2SO4) at 150 °C and by enzymatic hydrolysis by cellulase, pectinase and β-glucosidase. The fungus was able to produce ethanol with a maximum yield of about 0.36 g/g after 24h when grown on acid hydrolyzed orange peels both by aerobic and anaerobic cultivation. Apreliminary aerobic cultivation on enzymatic hydrolyzed orange peels gave a maximum ethanol yieldof 0.33 g/g after 26 h.The major metabolite produced during the cultivations was ethanol. Apart from ethanol, glycerol wasthe only component produced in significant amounts. In cultivations performed aerobically on acidandenzymatic hydrolyzed orange peels the glycerol yields were 0.048 g/g after 24 h.Two different techniques were also examined in order to evaluate if the methods could be use asbiomass determining methods when solid particles are present in the culture medium. The problemwith solid particles is that they will be buried inside the fungal biomass matrix. Hence makingseparation impossible prior to dry weight determination in the ordinary way. However, none of themethods involving chitin extraction or chitosan extraction did show any good results.The results from the present work are rather clear, M. indicus was able to grow and produce bothethanol and biomass even when limonene was present in the culture medium. The maximum ethanolyield was achieved after about 24 h in cultivations performed on both acid hydrolyzed and enzymatichydrolyzed orange peels. However, in order to say if the method can be applicable at industrial scaleand made economically feasible the subject has to be investigated further.