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Kvasci roda Spathaspora imaju sposobnost previranja heksoza i pentoza te se iz tog razloga istražuju za proizvodnju biokemikalija i biogoriva iz lignoceluloznih sirovina. U ovome radu istražen je rast kvasaca Spathaspora passalidarum CBS 10155 i Spathaspora arborarie CBS 11463 na arabinozi pri različitim brzinama okretaja tresilice (50, 100, 150 i 250 o minˉ¹). Također istražena je proizvodnja arabitola na hidrolizatu izluženih repinih rezanaca dobivenom kiselinskom hidrolizom. Uzgojem kvasca Spathaspora passalidarum CBS 10155 na podlozi s arabinozom utvrđeno je da povećanjem broja okretaja iznad 100 o minˉ¹ ne dolazi do značajnog povećanja koncentracije arabitola (2,7873 g Lˉ¹) dok kvascu Spathaspora arborarie CBS 11463 najviše pogoduje brzina od 250 o minˉ¹ za proizvodnju arabitola (4,1491 g Lˉ¹). Uzgojima pri svim brzinama okretaja tresilice utvrđene specifične brzine rasta kvasca Spathaspora arborarie CBS 11463 su manje od kvasca Spathaspora passalidarum CBS 10155. Uzgojem na kiselinskom hidrolizatu kvasac Spathaspora passalidarum CBS 10155 proizvedeno je 8,4810 g Lˉ¹ arabitola (YP/S=0,6038 g gˉ¹, Pr=0,1767 g Lˉ¹ hˉ¹). Na kraju uzgoja kvasca Spathaspora arborarie CBS 11463, koncentracija arabitola iznosila je 2,1759 g Lˉ¹(YP/S=0,5570 g gˉ¹, Pr=0,0453 g Lˉ¹ hˉ¹). Spathaspora yeasts have become of interest for biofuel and biochemicals production from lignocellulose because of their ability to co-ferment hexoses and pentoses. Arabinose fermentation by Spathaspora passalidarum CBS 10155 and Spathaspora arborarie CBS 11463 at different agitation speeds (50, 100, 150 and 250 rpm) was explored in this thesis. In addition, arabitol production from acid hydrolysate of exhausted sugar beet cossettes was investigated. By cultivating Spathaspora passalidarum CBS 10155 in arabinose-rich medium, it was determined that by increasing agitation speed above 100 rpm, there is not a significant increase in arabitol production (2,7873 g Lˉ¹) while the agitation speed of 250 rpm is favoured for arabitol production by Spathaspora arborarie CBS 11463 (4,1491 g Lˉ¹). During all cultivations, specific growth rate of Spathaspora arborarie CBS 11463 was lower than that of Spathaspora passalidarum CBS 10155 at the same agitation speed. When grown on the acid hydrolysate of exhausted sugar beet cossettes, yeast Spathaspora passalidarum CBS 10155 produced 8,4810 g Lˉ¹ of arabitol (YP/S=0,6038 g gˉ¹, Pr=0,1767 g Lˉ¹ hˉ¹) while Spathaspora arborarie CBS 11463 produced 2,1759 g Lˉ¹(YP/S=0,5570 g gˉ¹, Pr=0,0453 g Lˉ¹ hˉ¹).

Lignocelulozne sirovine dostupne u Hrvatskoj čini uglavnom otpad iz poljoprivrede, šumarstva, prehrambene i drvne industrije. Tri su osnovna strukturna polimera lignoceluloze: celuloza, hemiceluloza i lignin. Da bi se lignoceluloza mogla koristiti u biotehnološkim procesima, potrebno je provesti odgovarajuću predobradu sirovine kojom se razara njezina čvrsta struktura. Postoje različite metode predobrade, a dijele se na fizikalne, kemijske, fizikalno-kemijske i biološke. U ovom radu dan je pregled prednosti i nedostataka pojedinih metoda predobrade lignoceluloznih sirovina. Također je prikazan biotehnološki potencijal tvrdog drva s naglaskom na bukovinu. Lignocellulosic raw materials awailable in Croatia are mainly waste from agriculture, forestry, food and wood industry. Lignocellulose mainly consists of three structural polymers: cellulose, hemicellulose and lignin. In order to use lignocellulose in biotechnological processes, it is necessary to perform an appropriate pretreatment of the raw material which disrupts it's solid structure. There are various pretreatment methods which are classified into physical, chemical, physicochemical and biological treatment. This thesis points out some advantages and disadvantages of the most common methods for lignocellulosic biomass pretreatment. It also gives insight into the biotechnological potential of hardtwood with emphasis on beech wood.

Pojam lignocelulozne biomase podrazumijeva poljoprivredni i šumski otpad, drvne ostatke i trave te dio industrijskog otpada. Njezin osnovni sastav uključuje tri strukturna polimera: celulozu, hemicelulozu i lignin. Primjena lignoceluloznih materijala u biotehnologiji postaje sve popularnija jer su to obnovljive sirovine koje ne konkuriraju tržištu hrane. Lignocelulozna biomasa može se koristiti za dobivanje rezličitih proizvoda visoke dodane vrijednosti, ali njezina biotehnološka primjena zahtiejva odgovarajuću predobradu. Metoda predobrade se bira prema vrsti sirovine, proizvodu, radnom mikroorganizmu i vrsti procesa. Općenito, zbog visokog udjela lignina, meko drvo je teže obraditi od nekih drugih lignoceluloznih sirovina. U ovom radu prikazan je u biotehnološki potencijal mekog drva s naglaskom na jelovinu. The term lignocellulosic biomass implies agricultural and forest wastes, wood residues and grass, as well as a part of industrial waste. The primary composition of lignocellulose includes three structural polymers: cellulose, hemicellulose and lignin. The use of lignocellulosic materials in biotechnological processess is becoming more popular because they are renewable raw materials that do not compete on the food market. Lignocellulosic biomass can be used to produce various types of high value-added products, but its biotechnological application requires the appropriate pretreatment. The pretreatment method is selected according to the type of raw material, product, working microorganism and the type od the proces. Generally, because of it's high percentage of lignin, soft wood is harder to process than some other lignocellulosic raw materials. In this thesis, the biotechnological potential of softwood with emphasis on fir wood is presented.

Lignocelulozne sirovine obuhvaćaju poljoprivredne ostatke, šumski otpad i trave, a sastoje se od celuloze, hemiceluloze i lignina. Koriste se za biotehnološku proizvodnju bioetanola, enzima, organskih kiselina, mikrobne biomase i mnogih drugih proizvoda. Kako bi se mogle koristiti u biotehnološkim procesima, neophodna je predobrada kojom se razgrađuje čvrsta struktura sirovine. Metode predobrade dijele se na fizikalne, kemijske, fizikalno-kemijske i biološke. Odabir metode predobrade treba prilagoditi sastavu pojedine sirovine. Meko drvo u prosjeku sadrži veći udio lignina nego druge lignocelulozne sirovine. Ovaj rad daje pregled postupaka predobrade mekog drveta opisanih u literaturi, s posebnim naglaskom na predobradu jelovine. Ovi postupci predobrade provode se kao jednostupanjski ili višestupanjski postupci uz mogućnost kombinacije različitih metoda obrade. Lignocellulosic raw materials include agricultural residues, forest waste and grasses, and consist of cellulose, hemicellulose and lignin. They are used for biotechnological production of bioethanol, enzymes, organic acids, microbial biomass and many other products. In order to be used in biotechnological processes, pretreatment is necessary to decompose the solid structure of raw material. The pretreatment methods are divided into physical, chemical, physicochemical and biological methods. The choice of the pretreatment method should be made according to the composition of each raw material. Softwood on average contains a higher amount of lignin than the other lignocellulosic raw materials. This paper provides an overview of softwood pretreatment procedures described in the literature, with a special emphasis on fir pretreatment. These pretreatment processes are carried out as single-stage or multi-stage processes with the possibility of combining different processing methods.

Potencijalne lignocelulozne sirovine u Republici Hrvatskoj, koje bi se mogle koristiti kao temelj buduće biotehnološke proizvodnje, su otpad koji nastaje u poljoprivredi, šumarstvu, prehrambenoj i drvnoj industriji. Glavne komponente lignoceluloznih sirovina su celuloza, hemiceluloza i lignin. Kako bi se dobili fermentabilni šećeri, koje radni mikroorganizmi mogu metabolizirati, potrebno je podvrgnuti kompleksne lignocelulozne sirovine određenim postupcima predobrade i hidrolize. Cilj je postići što veći stupanj hidrolize hemiceluloze, ukloniti lignin te smanjiti kristaliničnost celuloze. Metode predobrade dijele se na fizikalne, kemijske, fizikalno-kemijske i biološke, a treba ih prilagoditi ovisno o sastavu sirovine koja se koristi. U radu su opisani primjeri predobrade tvrdog drveta s naglaskom na bukovinu te njihove prednosti i nedostatci. Potential lignocellulosic raw materials in Croatia, which could be used as the basis of future biotechnological production, are wastes derived from agriculture, forestry, food and wood industries. The main components of lignocellulose raw materials are cellulose, hemicellulose and lignin. In order to obtain fermentable sugars, which can be metabolized by working microorganisms, it is necessary to subject the complex lignocellulosic raw materials to certain pretreatment and hydrolysis procedures. The aim is to achieve the highest percentage of hemicellulose hydrolysis, to remove lignin and to reduce the crystallinity of cellulose. Pretreatment methods are divided into physical, chemical, physico-chemical and biological, and need to be adjusted depending on the composition of the used raw material. The paper describes examples of hardwood pretreatment with an emphasis on the beech wood as well as their advantages and disadvantages.

Poljoprivreda, šumarstvo i prehrambena industrija izvori su velike količine lignocelulozne biomase, koja može poslužiti kao lako dostupna i jeftina obnovljiva sirovina za dobivanje različitih bioproizvoda. Jedan od takvih proizvoda je i bioetanol. Ovaj rad daje pregled bakterija koje se koriste i/ili istražuju za proizvodnju bioetanola iz lignoceluloznih sirovina. U navedenim istraživanjima proizvodnje bioetanola pomoću bakterija primijenjuju se različiti pristupi kako bi se povećala ekološka i ekonomska efikasnost procesa. Pored uobičajenih bioprocesa, koji se provode u više faza i uz pomoć monokulture, razvijaju se i visokointegrirani (konsolidirani) bioprocesi uz primjenu mikrobnih kokultura., Agriculture, forestry and food industry are sources of large quantities of lignocellulosic biomass, which can be used as an easily accessible and cheap renewable raw material for production of different bioproducts. One of these bioproducts is also bioethanol. This paper provides an overview of bacteria used and/or investigated for bioethanol production from lignocellulose-containing feedstocks. In the research of bioethanol production using bacteria, various approaches are applied in order to increase ecological and economic efficiency of bioprocess. In addition to conventional multi-stage bioprocesses that are carried out using monoculture, highly integrated (consolidated) bioprocesses and applications of microbial cocultures are also in developing stage.

Pojam lignoceluloznih materijala odnosi se na biljnu biomasu koja uključuje poljoprivredne ostatke, šumski i drvni otpad, otpad koji nastaje u prehrambenoj industriji te komunalni otpad. Ove sirovine sastoje se od celuloze, hemiceluloze i lignina, a postoji sve veći interes za njihovu biotehnološku primjenu za dobivanje različitih proizvoda (npr. organskih kiselina, enzima, biogoriva, antibiotika, mikrobne biomase i biopesticida). Zbog složene strukture lignoceluloznih sirovina, prije početka biotehnološkog procesa potrebna je odgovarajuća predobrada. Veličina čestica sirovina može utjecati na ekonomičnost biotehnološkog procesa koji se temelji na lignoceluloznim sirovinama jer utječe na prinos, potrošnju energije i troškove cjelokupnog procesa. Potrebne dimenzije čestica ovise o odabranoj metodi predobrade i korištenoj sirovini, a usitnjavanje čestica provodi se mehaničkim postupcima, poput rezanja, sjeckanja i mljevenja, koji su energetski zahtjevni. Stoga je za svaki pojedini proces važno izabrati najefikasniji i najisplativiji proces usitnjavanja. The term lignocellulosic material refers to plant biomass which includes agricultural residues, forest and wood waste, waste generated in the food industry and communal waste. These raw materials consist of cellulose, hemicellulose and lignin, and there is an increasing interest for their biotechnological application to obtain various products (eg. organic acids, enzymes, biofuels, antibiotics, microbial biomass and biopesticides). Because of the complex structure of lignocellulosic raw materials, proper pretreatment is needed prior to the biotechnological process. The particle size of the raw material can affect the economics of a biotechnological process based on lignocellulosic raw materials because it affects yield, energy consumption and costs of the entire process. The required particle dimensions depend on the pretreatment method and raw material used, and the size reduction is carried out by mechanical procedures such as cutting, hacking and milling, which are energy-intensive. Therefore, it is important to choose the most efficient and most costeffective way of size reduction.

Lignocelulozne sirovine koriste se za proizvodnju biogoriva i biokemikalija. Zbog njihove složene strukture, prije samog bioprocesa, potrebno ih je podvrgnuti metodama predobrade i hidrolize. Tim postupcima nastaju, osim fermentabilnih šećera, brojni nusproizvodi od kojih neki mogu inhibirati radni mikroorganizam. Oleaginozna plijesan Mortierella isabellina ima sposobnost nakupljanja velikih količina intracelularnih lipida (do 80% suhe tvari biomase) što ju čini obećavajućim organizmom u proizvodnji biodizela. Cilj ovog rada bio je istražiti učinak fenolnih spojeva (vanilin i 4-hidroksibenzaldehid) nastalih tijekom predobrade lignocelulozne sirovine na rast M. isabellina i sintezu lipida. Rezultati su pokazali da vanilin i 4-hidroksibenzaldehid značajno inhibiraju rast plijesni i sintezu lipida. Pri koncentracijama vanilina i 4-hidroksibenzaldehida većim od 1,2 i 0,6 g L-1, rast i akumulacija lipida bili su u potpunosti inhibirani. Lignocellulosic raw materials are used in production of biofuels and biochemicals. Because of their complex structure, prior to the bioprocess itself, they have to be subjected to pre-treatment and hydrolysis. These processes generate, along with fermentable sugars, numerous byproducts, some of which may inhibit the working microorganism in the production process. The oleaginous fungus Mortierella isabellina has the ability to accumulate large quantities of intracellular lipids (up to 80% of dry cell weight) that makes it a promising organism for use in biodiesel production. The aim of this paper was to investigate effect of phenolic compounds (vanillin and 4-hydroxybenzaldehyde) produced during lignocellulose pretreatment on the growth of M. isabellina and lipid synthesis. The results showed that vanillin and 4-hydroxybenzaldehyde significantly inhibit the fungus growth and lipid synthesis. At concentrations of vanillin and 4-hydroxybenzaldehyde higher than 1,2 and 0,6 g L-1 respectively, the growth and lipid accumulation was completely inhibited.

Mikrobni lipidi, proizvedeni uz pomoć oleaginoznih mikroorganizama na lignoceluloznim supstratima, obećavajuća su alternativa nafti kao sirovina za proizvodnju dizela. Oleaginozni kvasac Trichosporon oleaginosus može nakupiti i do 80% lipida u biomasi prilikom rasta u uvjetima limitacije dušikom. Mikrobna proizvodnja lipida uključuje hidrolizu lignocelulozne sirovine do fermentabilnih šećera, koji se koriste za mikrobni rast i sintezu lipida. U ovom radu prikazan je novi proces odvojene hidrolize i proizvodnje lipida uz reciklaciju enzima, koji je proveden u četiri ponovljena ciklusa. Nehidrolizirani, teško razgradivi čvrsti ostatak s adsorbiranim celulazama, izdvojen je i nanovo korišten u narednim ciklusima enzimske hidrolize. Dobiveni rezultati pokazali su da se reciklacijom nehidrolizirane biomase u četiri ciklusa smanjuje dodana količina enzima do 30% njegove optimalne količine, bez značajnijeg utjecaja na učinkovitost hidrolize lignocelulozne sirovine i produktivnost nakupljanja lipida. Prosječna vrijednost koncentracije biomase iznosila je 23,23 gL-1, a udio lipida u biomasi 47,58 %. Microbial lipids produced from lignocellulosic biomass by oleaginous microorganisms are promising alternative feedstock to petroleum in biodiesel production. Oleaginous yeast Trichosporon oleaginosus has the ability to accumulate lipids up to 80% of the biomass under nitrogen-limited growth conditions. Microbial lipid production involves hydrolysis of lignocellulosic biomass into fermentable sugars, which are used for microbial growth and lipid synthesis. Here we report a novel process of the separate hydrolysis and lipid production with enzyme recycling conducted in four repeated cycles. Unhydrolysed recalcitrant solids with adsorbed cellulases were separated and recycled to the subsequent cycles of enzymatic hydrolysis. Obtained results indicated that enzyme recycling with the unhydrolysed solids reduced the enzyme loading up to 30% of the optimal enzyme loading without significant effect on efficiency of lignocellulose hydrolysis and lipid productivity. Average value of biomass concentration and lipid content was 23,23 gL-1 and 47,58 %, respectively.

Oleaginozni mikroorganizmi proizvode lipide koji se mogu koristiti kao zamjenska sirovina za proizvodnju biodizela umjesto biljnih ulja. Oleaginozna plijesan Mortierella isabellina nakuplja triacilglicerole kao stanične rezervne lipide čiji udjel u suhoj tvari biomase iznosi i do 80 %. Lignocelulozna biomasa izvor je jeftinih ugljikohidrata koji se mogu koristiti kao izvor ugljika za rast mikroorganizma i sintezu proizvoda (npr. bioetanola). Procesom predobrade poboljšava se enzimska razgradnja lignocelulozne sirovine s celulazama te se povećava prinos reducirajućih šećera nastalih hidrolizom. Međutim tijekom ovog procesa nastaje niz nusproizvoda iz lignocelulozne sirovine koji inhibiraju radni mikroorganizam i celulolitičke enzime. U ovom radu istražen je učinak furfurala i 5-hidroksimetil furfurala (HMF) na rast i proizvodnju lipida s plijesni M. isabellina. Rezultati pokazuju da furfural jače inhibira rast i sintezu lipida od HMF-a. Rast plijesni i sinteza lipida je potpuno inhibirana pri koncentraciji furfurala većoj od 0,6 g/L i HMF-a većoj od 2 g/L. Lipids produced by oleaginous microorganisms can be used as alternative feedstock for production of biodiesel instead of plant oils. Oleaginous fungus Mortierella isabellina has the ability to accumulate triacylglycerols, as cellular storage lipids, up to 80% of the biomass. Lignocellulosic biomass is source of cheap carbohydrates that can be used as carbon source for the microbial growth and product synthesis (e.g. bioethanol production). The pretreatment process improves enzymatic digestibility of lignocellulosic biomass by cellulases and enhanced the yield of reducing sugars by hydrolysis. However, during this process many lignocellulose-derived by-products are formed that inhibit microorganism and cellulolic enzymes. The influence of furfural and 5-hydroxymethyl furfural (HMF) on the growth and lipid production by fungus M. isabellina was investigated. The results indicate that furfural inhibits the growth and lipid synthesis markedly more than HMF. The growth and lipid accumulation of fungus was completely inhibited at concentration of furfural and HMF higher than 0,6 g/L and 2 g/L, respectively.

Velika količina lignoceluloznog otpada, kojeg čine uglavnom celuloza, hemiceluloza i lignin, nastaje u šumarstvu, poljoprivredi i prehrambenoj industriji. Obzirom na dostupne količine ovih jeftinih i obnovljivih sirovina u Republici Hrvatskoj, važno je razmotriti mogućnosti njihove primjene u proizvodnji bioetanola i raznih drugih biotehnoloških proizvoda. Lignocelulozni hidrolizati su složene smjese heksoza i pentoza te drugih spojeva od kojih neki mogu djelovati kao inhibitori fermentacije. U ovom radu dan je pregled kvasaca koji se koriste i/ili istražuju za proizvodnju etanola iz lignoceluloznih hidrolizata., Huge amounts of lignocellulosic waste, consisting mainly of cellulose, hemicellulose and lignin, are ge- nerated through forestry, agriculture and food industry. Given the available amount of these cheap and renewable raw materials in the Republic of Croatia, it is important to consider the possibility of their conversion into ethanol and other biotechnological products. Lignocellulosic hydrolysates are complex mixtures of hexoses, pentoses and other compounds, some of which may act as fermentation inhibitors. This paper reviews yeast strains that are used and/or studied for ethanol production by fermentation of lignocellulosic hydrolyzates.

Cilj ovog preglednog rada jest analiza biogoriva na osnovi lignoceluloze, te njihova usporedba s drugim biogorivima. Pregledom i proučavanjem potrebnih predtretmana kod obrade lingocelulozne biomase, može se zaključiti o vrijednosti, isplativosti i konkurentnosti biogoriva na osnovi lignoceluloze na tržištu. Kako bi razumijevanje predtretmana bilo potpuno, potrebno je imati uvid u samu strukturu lignocelulozne biomase, stoga, u ovom radu obrađeni su osnovni dijelovi od kojih se lignocelulozna biomasa sastoji; lignin, celuloza i hemiceluloza. Obzirom na to da je fermentacijski etanol najčešće proizvedeno biogorivo iz lignoceluloze, objašnjen je proces fermentacije i navedena su određena svojstva etanola. Usporedba sintetskog i fermentacijskog etanola, njihovih troškova proizvodnje i povrata investicije omogućuju bolju procjenu fermentacijskog etanola, a time i lignoceluloznog etanola u smislu konkurentnosti i isplativosti. Zaključak rada jest da lignocelulozni etanol ima budućnost na tržištu biogoriva samo ako se smanji broj potrebnih predtretmana lignocelulozne biomase, ili snize ukupni troškovi proizvodnje biogoriva na osnovi lignoceluloze. The aim of this study is to analyze lignocellulose based biofuels, their production and used biomass material. Comparing lignocellulosic biofuel with other biofuels is necessary for estimating its potential on the market. By reviewing and studying the required pre-treatment when processing lingocelullosic biomass, conclusions can be made about the economy and profitability of the process. In order to fully understand various pre-treatment processes, having an insight into the structure of the lignocellulosic biomass is unavoidable, therefore, this paper will cover the main components of which lignocellulosic biomass is composed; lignin, cellulose and hemicellulose. Given the fact that fermentation ethanol is usually the most commonly obtained biofuel from lignocellulosic biomass, an explanation of the fermentation process is provided as well as specifying certain properties of ethanol. A comparison of synthetic and fermentation ethanol, their production costs and return of investment allows a better understanding of lignocellulosic ethanol in terms of competitiveness on the market and profitability. Lignocellulosic ethanol will be more valued on the market once less pretreatments of lignocellulosic biomass are required resulting in a less expensive production process of lignocellulosic biomass.

Velika količina lignoceluloznog otpada, kojeg čine uglavnom celuloza, hemiceluloza i lignin, nastaju u šumarstvu, poljoprivredi i prehrambenoj industriji. Lignoceluloza je jeftina, ali i kompleksna obnovljiva sirovina koja se može prevesti u razne visokovrijedne biotehnološke proizvode. Da bi se iskoristio potencijal lignoceluloze potrebno je primijeniti prikladne metode predobrade. Lignocelulozni hidrolizati su složene smjese heksoza i pentoza te drugih spojeva od kojih neki mogu djelovati kao inhibitori fermentacije. Optimalni radni mikroorganizam trebao bi fermentirati pentoze i heksoze pri čemu se preferira da ih troši istodobno. Također bi trebao imati visoku toleranciju prema inhibitorima i produktima fermentacije, biti otporan na mikrobiološke kontaminacije te postizati visoku produktivnost i prinos proizvoda. Uz to, trebao bi tolerirati visoke temperature i niske pH-vrijednosti kako bi se smanjio rizik od kontaminacije. Poželjno je da radni mikroorganizam posjeduje i izlučuje celulolitičke i hemicelulolitičke enzime. Ova svojstva mogu biti prirodno prisutna u mikroorganizmu ili se mogu postići primjenom metoda genetičkog inženjerstva. U ovom radu dan je pregled prokariotskih i eukariotskih mikroorganizama koji su istraživani za dobivanje etanola, ali i nekih drugih ekonomski važnih proizvoda koji se mogu dobiti biotehnološkom preradom lignoceluloznih sirovina. Huge amounts of lignocellulosic waste, consisting mainly of cellulose, hemicellulose and lignin, are generated through forestry, agriculture and food industry. Lignocellulose is a cheap but complex renewable raw material that can potentially be converted into various value-added biotechnological products. In order to exploit lignocellulose potential, it is necessary to implement appropriate pretreatment methods. Lignocellulosic hydrolysates are complex mixtures of hexoses, pentoses and other compounds, some of which may act as inhibitors of fermentation. Optimal working microorganism should ferment pentoses and hexoses, which should be preferentially simultaneously consumed. It should also have a high tolerance to inhibitors and fermentation products, to be resistant to microbiological contamination as well as to achieve a high productivity and high product yields. In addition, it should tolerate high temperatures and low pH-values in order to reduce the risk of contamination. It is also desirable for working microorganism to produce and secrete cellulolytic and hemicellulolytic enzymes. These properties can be naturally present in the microorganism or can be achieved by using genetic engineering methods. This paper gives an overview of prokaryotic and eukaryotic microorganisms which have been investigated for production of ethanol as well as other economically important products which can be obtained by biotechnological processing of lignocellulosic raw materials.

Povećanje emisije stakleničkih plinova, energetska ovisnost i nestabilnost isporuke energenata posljedica su ubrzane potrošnje fosilnih goriva zbog ubrzanog rasta svjetske populacije i industrijalizacije. Bioetanol je postao atraktivno zamjensko biogorivo jer se proizvodi iz obnovljivih sirovina i ekološki je prihvatljiv. Upotrebljava se kao pogonsko gorivo i to kao hidrirani (96 %) ili bezvodni (u mješavinama s benzinom). Bioetanol se proizvodi fermentacijom s kvascem S. cerevisiae ili nekim drugim mikroorganizmom iz ugljikohidrata kao što su jednostavni šećeri, škrob i celuloza. Nakon fermentacije, bioetanol se izdvaja i pročišćava destilacijom i dehidracijom. Sastav je propisan specifikacijama za primjenu u motornim gorivima. Uobičajeni usjevi, kao što su kukuruz, šećerna repa i trska, zasad su osnovne sirovine za proizvodnju bioetanola. Međutim, proizvodnja bioetanola iz ovih sirovina ne može zadovoljiti globalne potrebe za bioetanolom, zbog njihove primarne uloge u prehrani ljudi i životinja. Lignoceluloza je pogodna sirovina za proizvodnju bioetanola jer je široko rasprostranjena, obnovljiva i ne upotrebljava se u prehrani. Proizvodnja bioetanola iz lignoceluloznih sirovina je složen proces, koji se u mnogim aspektima razlikuje od proizvodnje iz šećernih ili škrobnih sirovina. U ovom radu prikazane su dosadašnje i nove tehnologije u proizvodnji bioetanola uključujući primjenu različitih sirovina, postupaka proizvodnje i radnih mikroorganizama. Nadalje, navedene su mogućnosti integracije osnovnih koraka u bioprocesima proizvodnje s ciljem povećanja produktivnosti i smanjenja troškova proizvodnje., Rapidly growing fossil energy consumption, due to population and industrial growth, has caused increasing greenhouse-gas emissions, growing energy dependency and supply insecurity. Bioethanol has become an attractive alternative biofuel because of its environmental benefits and the fact that it is made from renewable resources. Ethanol is widely used as transport fuel, pure hydrous ethanol or anhydrous ethanol in mixtures with gasoline (Fig. 1). Bioethanol is produced from carbohydrates such as sugar, starch and cellulose by fermentation with yeast S. cerevisiae or other microorganisms. Thereupon, ethanol is separated and purifyed by distillation-rectification-dehydration to meet fuel specifications. Currently, conventional crops such as corn or sugarcane are the main feedstock for bioethanol production. Bioethanol production from the sucrose-containing feedstock is simpler compared to the starchy materials and the lignocellulosic biomass due to an additional step – feedstock hydrolysis. The process of ethanol production from starchy materials includes the hydrolysis of starch to glucose using α-amylase (1,4-α-D-glucan-4-glucanohydrolase) and glucoamylase (1,4-α-D-glucanglucohydrolase). Finally, the glucose is fermented to ethanol by yeast cells. Enzymatic hydrolysis of starch and fermentation of glucose can be carried out in different process configurations, such as separate hydrolysis and fermentation (SHF), and simultaneous saccharification and fermentation (SSF, Fig. 2). In consolidated bioprocessing (CBP), the conversion of starch into ethanol is performed in one step without added enzymes. This process configuration has potential to lower the cost of biomass processing due to elimination of operating and capital costs associated with dedicated enzyme production. Current bioethanol production from corn and sugarcane is unable to meet the global demand for bioethanol, due to their primary value as livestock feed and human food. The lignocellulosic biomass such as agricultural wastes (corn stover, crop straws, husks and bagasse), herbaceous crops (switchgrass), woody crops, forestry residue, waste paper and other wastes (municipal and industrial) is favourable feedstock for bioethanol production. The major advantages of lignocellulosic biomass are its renewable and ubiquitous nature and its noncompetitiveness with food crops. Ethanol production from lignocellulosic feedstock is complex and comprises two steps prior to fermentation: biomass pretreatment (breaking down the structure of the lignocellulosic matrix) and cellulose hydrolysis (enzymatic hydrolysis of cellulose to glucose). The lignocellulosic hydrolysate contains not only hexoses, but also pentoses that are not assimilated by yeast S. cerevisiae. Furthermore, the lignocellulosic hydrolysate contains a broad range of compounds that inhibit the yeast’s cells. The composition of the inhibitors depends on the type of the lignocellulosic material, and the chemistry and nature of the pretreatment process. The pretreated cellulose can be enzymatically hydrolysed either prior or simultaneously with glucose fermentation (Fig. 3). The four main steps involved in the process of lignocellulosic bioethanol production (pretreatment, cellulose hydrolysis, hexoses and pentoses fermentation) can be arranged in various process configurations, including separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), simultaneous saccharification and co-fermentation (SSCF) and consolidated bioprocessing (CBP, Fig. 3). Specific strains of bacteria and yeasts have been developed to ferment sugars released from lignocellulosic biomass and hydrolysed cellulose, through a selection of new strains and genetic engineering of traditional strains.