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.

Over the last few decades, ethanol production from renewable resources has been of interest as an alternative fuel to the current fossil fuel, due to the unstable oil market and in order to decrease net emission of carbon dioxide which leads to global warming. According to analyses of DG Transport and Energy (TREN), it is not possible to reach the current biofuels directive promoting 5,75 % biofuel by the year 2010, due to the markets and technologies, but by the year 2020 achievement of 6.9% is expected. This new law will increase biofuel demand by 3,1 %.Lignocelluloses materials, which are relatively cheap and plentiful, are considered to be the main source of feedstock’s for low-cost bio-ethanol production. The general procedure to convert lignocelluloses material to bioethanol is hydrolysis of the hemicelluloses and the cellulose to its monomer sugars, fermentation and distillation.Bacteria, yeasts and filamentous fungi are able to ferment hydrolysates from different plants and convert it to bioethanol.Mucor indicus is a filamentous fungus; it is able to utilize a wide range of hexoses, phentoses and disaccharides (cellobiose) in order to produce ethanol. The Ethanol yield and productivity of this microorganism from hexoses are as same as Saccharomyces cerevisiae. But the reason that it is one of the candidates for ethanol production is the fungus ability to utilize xylose. The cell wall of M.indicus contains significant quantity of chitosan/chitin which can be easily extracted. Chitosan is the deacetylated products of chitin. They have many applications in chemistry, biotechnology, medicine, veterinary, dentistry, agriculture, food processing, environmental protection, water purification, cosmetic and textile industries.The results of the current work show that the glucose concentration in the medium had a great impact on the lag phase, glucose consumption and ethanol production in both aerobic and anaerobic conditions. The lag phase increased as the initial concentration of glucose increased. While the glucose concentration increased above 190 g/l in the medium the glucose consumption and ethanol production decreased in both aerobic and anaerobic conditions. The glucose tolerance of M.indicus in both aerobic and anaerobic condition is about 190 g/l and in the anaerobic condition the ethanol tolerance of this fungus is around 70 g/. / Uppsatsnivå: D

Syftet med detta examensarbete är att finna exoenzymer från Rhizopus sp, Mucor indicus och Rhizomucor pusillus som kan användas vid förbehandling av organiskt avfall. Syftet är även att finna kolkällor/energikällor som tidigare inte använts inom forskningen i ämnet resursåtervinning vid Högskolan i Borås.För att kunna undersöka vilka kolkällor mikroorganismerna bryter ner odlas dessa upp på agarplattor innehållande minimal-medium samt en specifik kolkälla. Efter fyra dagars inkubering i 30oC studerar man agarplattorna för att se om mikroorganismerna vuxit eller inte. Kan man urskilja tillväxt har de lyckats bryta ner kolkällan samt producera motsvarande exoenzym. Då vissa resultat är oklara odlas mikroorganismerna även i skakflaskor, detta för att se om det är själva agarn i agarplattorna som påverkar mikroorganismernas tillväxt.Resultatet visar att vissa mikroorganismer växer bättre än andra. Detta kan bero på kolkällornas struktur, det vill säga om de är komplicerade eller ej. Studerar man mikroorganismerna var för sig skiljer de sig lite åt. Rhizopus sp växer bäst på galaktan vilket indikerar att den lyckas producera exoenzymet galaktas. Mikroorganismen saknar produktion av exoenzym när den odlas på kolkällorna cellulosa och kitin.Studerar man mikroorganismen Mucor indicus har den bäst tillväxt på galaktan och potatismjöl, vilket indikerar att den producerar exoenzymerna galaktas samt α-amylas. Den kolkällan som ger sämst tillväxt är cellulosa.Rhizomucor pusillus har bäst tillväxt på galaktan samt triglycerider och producerar då exoenzymerna galaktas och lipas. Den lyckas inte bryta ner cellulosa eller kitin och saknar då produktion av exoenzymen cellulas samt kitinas.Både xylan och galaktan testas var för sig för att kunna dra slutsatser om någon produktion av exoenzymet hemicellulas finns. Detta görs eftersom det inte finns tillgång till något rent ämne med hemicellulosa. Xylan testas även endast för exoenzymet xylanas.En av de kolkällorna som gett minst tillväxt för alla de testade mikroorganismerna var cellulosa. För att styrka detta resultat odlas mikroorganismerna upp i skakflaskor, där ingen tillväxt skedde. Den lilla tillväxt som erhölls på agarplattorna tyder på att mikroorganismerna växer med den tillsatta agarn som kolkälla och inte utnyttjar själva kolkällan. Varför mikroorganismerna inte kan tillgodo se sig cellulosa kan bero på att cellulosa har en komplex struktur som gör den svår att bryta ner utan förbehandling.