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Glavni proizvod anaerobne digestije je bioplin, koji je obnovljivo gorivo, a sporedni proizvod ovog procesa je digestat, koji se koristi kao gnojivo bogato hranjivim tvarima. Dodatni pozitivni učinci anaerobne digestije su razgradnja organskog otpada te smanjenje neugodnih mirisa i koncentracije patogenih mikroorganizama. Bioplin se uglavnom koristi za proizvodnju električne energije i topline, a u nekim slučajevima se pročišćava da bi se dobio biometan koji se koristi u mreži prirodnog plina, kao gorivo za motore s unutarnjim sagorijevanjem ili kao polazna kemikalija za kemijsku industriju. Zbog svega navedenog, razvoj proizvodnje bioplina ima pozitivne društveno-ekonomske i ekološke učinke. Bioplin proizveden u Hrvatskoj većinom se koristi za proizvodnju električne i toplinske energije u kogeneracijskim postrojenjima. Iako su u Hrvatskoj dostupne različite obnovljive sirovine koje bi se mogle iskoristiti za proizvodnju bioplina, njihov je potencijal do sada bio nedovoljno iskorišten. Kao sirovine za proizvodnju bioplina u nas se pretežno koriste gnojovka i nusproizvodi poljoprivrede, klaonica i prehrambene industrije. Racionalnijim korištenjem zemljišta i razvojem prehrambene industrije mogla bi se povećati količina poljoprivrednih ostataka i nusproizvoda koji nastaju preradom hrane. Usmjeravanjem i poticanjem korištenja ovih nusproizvoda za anaerobnu digestiju može se stimulirati brži razvoj proizvodnje bioplina u Hrvatskoj. Pored mogućeg povećanja vlastite proizvodnje električne energije i goriva, radi se o ekološki povoljnoj tehnologiji koja ima pozitivan društveno-ekonomski učinak., The main product of anaerobic digestion is biogas, which is a renewable fuel, and the by-product of this process is digestate, which is used as a nutrient-rich fertilizer. Additional positive effects of anaerobic digestion are the decomposition of organic waste and the reduction of unpleasant odors and the concentration of pathogenic microorganisms. Biogas is mainly used for the production of electricity and heat, and in some cases it is purified to obtain biomethane which is used in the natural gas network, as a fuel for internal combustion engines or as a starting chemical for the chemical industry. Due to all of the above, the development of biogas production has positive socio-economic and ecological effects. Biogas produced in Croatia is mostly used for the production of electricity and thermal energy at cogeneration plants. Although various renewable raw materials are available in Croatia that could be used for biogas production, their potential has been underutilized until now. Manure and by-products of agriculture, slaughterhouses and the food industry are mainly used as raw materials for the production of biogas in our country. More rational use of land and development of the food industry could increase the amount of agricultural residues and by-products resulting from food processing. Directing and encouraging the use of these by-products for biogas production can stimulate a faster development of biogas production in our Croatia. In addition to the possible increase in own production of electricity and fuel, it is an environmentally friendly technology that has a positive socio-economic effect.

L-lactate and 2, 3-butanediol are important chemicals used in many industries. Lactate is industrially produced by lactic acid bacteria, while Klebsiella bacteria are considered for the production of 2, 3-butanediol. However, these bioprocesses have certain disadvantages such as reliance on raw materials that could be used for human consumption or the use of potentially pathogenic bacteria. These disadvantages can be avoided by using a suitable microorganism and therefore the possibility of using the yeast Kluyveromyces marxianus for the production of L- lactate and 2, 3-butanediol was investigated in this study. K. marxianus is a non-conventional yeast that has some industrially important characteristics such as rapid growth rates, thermotolerance, ability to grow on xylose, and due to its GRAS status, it is also suitable for use in the food industry. To examine the potential of K. marxianus yeast for L-lactate production, a strain containing optimized genes encoding L- lactate dehydrogenase (ldhL) isolated from Lactobacillus gasseri was constructed. To study the production of 2, 3-butanediol, optimized genes encoding α-acetolactate synthase (alsS) and α- acetolactate decarboxylase (alsD) isolated from Bacillus subtilis were expressed in K. marxianus, and the effect of overexpression of native gene encoding 2, 3-butanediol dehydrogenase (BDH1) was examined. Wildtype yeast K. marxianus produces neither L-lactate nor 2, 3-butanediol when grown on glucose, while genetically modified strains produce up to 2, 5 g/L of L-lactate and about 4 g/L of 2, 3-butanediol.

The term “non-conventional yeasts” refers to all yeast species other than Saccharomyces cerevisiae and Shizosaccharomyces pombe, the two species which are exceptionally well studied and widely utilized in biotechnological processes. In recent years the interest of researchers and industry steers towards non-conventional yeasts harbouring new and potentially exploitable metabolic pathways or other features favourable in biotechnological processes such as thermotolerance, halotolerance, resistance to various growth inhibitors etc. These yeasts are being investigated both as sources of interesting genes which can be transferred into conventional systems but also as the industrial work organisms themselves. In the latter case it is desirable that the work organism can be genetically transformed and even precisely genetically modified allowing construction of improved and more versatile industrial strains. The Schwanniomyces genus is comprised of seven species of budding yeasts which are fairly poorly explored but could have interesting biotechnological properties. Notably, most species form this genus can grow on D-xylose and cellobiose making them worth exploring in the context of production of valuable biochemicals on waste lignocellulose materials as a substrate. Furthermore, the most prominent member of the genus, S. occidentalis var. occidentalis, is potentially interesting producer of heterologous proteins. The aim of this work was to establish a set of basic genetic tools allowing genetic transformation and construction of replicative plasmids for yeasts belonging to the Schwanniomyces genus, thus facilitating further research on these yeasts as well as their industrial application. Using an electroporation protocol, we developed earlier for Brettamomyces bruxellensis several species form the Schwanniomyces genus were successfully transformed with fairly high transformation efficiency. Moreover, several plasmid replication origins belonging to various budding yeast species were tested and origin belonging to a closely related species Scheffersomyces stipitis was identified to be active in Schwanniomyces yeasts, allowing construction of completely stable replicative plasmid in certain Schwanniomyces species.

Značajne količine raznovrsnih ostataka (odnosno lignocelulozne biomase) nastaju u poljoprivredi, prehrambenoj industriji i šumarstvu. Stoga je važno podići svijest o mogućnostima primjene takvih materijala koji se u današnje vrijeme ne bi trebali tretirati kao otpad, već se mogu koristiti kao obnovljive biotehnološke sirovine za proizvodnju kemikalija, drugih visokovrijednih proizvoda i biogoriva. Lignocelulozni materijal uglavnom sadrži celulozu, hemicelulozu i lignin. Predmet interesa ovog rada je lignocelulozni otpad iz poljoprivrede i prehrambene industrije kao mogući sirovinski temelj za napredak održive biotehnološke proizvodnje u Republici Hrvatskoj. Razmotrene su dostupne količine ovih lignoceluloznih sirovina, tipovi bioprocesa u kojima se one mogu koristiti, postupci predobrade koje je neophodno provesti prije provedbe samog bioprocesa te vrste biotehnoloških proizvoda koje je moguće dobiti., Significant amounts of various residues (i.e. lignocellulosic biomass) are generated in agriculture, food industry and forestry. Therefore, it is important to raise awareness about the possibilities of using such materials, which nowadays should not be treated as waste, but can be used as renewable biotechnological raw materials for the production of chemicals, other high-value products and biofuels. Lignocellulosic material consists mainly of cellulose, hemicellulose and lignin. The subject of interest of this paper is lignocellulosic waste from agriculture and the food industry as a possible raw material basis for the progress of sustainable biotechnological production in the Republic of Croatia. The available amounts of these lignocellulosic raw materials, the types of bioprocesses in which they can be used, the pretreatment procedures that need to be carried out before the implementation of the bioprocess itself, and the types of biotechnological products that can be obtained have been considered.

Kefir drink represents a product which is obtained by fermentation of sugary medium, at room temperature, by different types of microorganisms contained within water kefir grains. It has been shown that water kefir has positive effects on human health including antihyperglycemic, antimutagenic, and antioxidative. In spite of these facts, the industrial production is still very limited because of varying fermentation conditions, and poorly explored and complex microorganism interaction present in water kefir grains. So far it was proven that water kefir drink can be produced using different initial sugar concentrations together with different types of fruit which are added as an additional carbon and nitrogen source for the working microorganisms. In this study, we tested the possibility of usage of non refined brown sugar from sugar cane as carbon source and its effect on the dynamics of water kefir production. In the first part of investigation the influence of two different initial sucrose concentrations ( 40 and 60 g L-1) on the composition of water kefir dring was tested, while in the second part the experiments were conducted with the addition of sweeteners (erythrol, xylitol and steviol). In this case, the initial sucrose concentration (cane sugar) was lower (20 g L-1) and the media contined the same concentration of added artificial sweetener. Obtined results show that water kefir production can be obtained with higher intitial sucrose concentrations and that it is possible to use cane sugar with no negative effects. Also, the addition of artificial sweeteners at the beginning of fermentation doesnt have any negative impacts on the fermentation process, respectively

Paenivacillus polymyxa is a gram-positive GRAS bacterium which produces wide range of useful biochemicals such as ethanol, lactate, 2, 3-butanediol, phytohormones, polysaccharides, antibiotics and enzymes. The most interesting product of P. polymyxa metabolism is 2, 3-butanediol due to its wide application in cosmetic industry, in food industry as aroma compound or in pharmaceutical industry as an antiseptic. The focus of this research was the cultivation of P. polymyxa DSM 742 strain and process optimization for enhanced production of 2, 3-butanediol. Carbon source for our cultivations were sugars stemming from pre-treated lignocellulosic biomass like glucose and xylose. The highest 2, 3-butanediol concentration of 18, 61gL-1 was achieved during cultivation in medium with 70 g L-1 of glucose during 40 h of fermentation. However, during bacterium cultivation in only xylose containing medium 2, 3-butanediol production was considerably diminished. In order to make our 2, 3-butanediol production economically and ecologically sustainable, the pre-treated brewers' spent grains (BSG) with diluted sulphuric acid were used as a substrate. Non-detoxified and detoxified liquid part of pre-treated BSG was used for bacterial cultivation as well as the enzymatically hydrolysed solid part of pre-treated BSG. Liquid part of pre-treated BSG was detoxified with activated carbon to remove inhibitory compounds. Our results showed very low concentrations of 2, 3-butanediol and ethanol after cultivation on non-detoxified liquid part of pre-treated BSG. During bacterial cultivation on detoxified liquid part of pre-treated BSG relatively high lactate concentration was observed (30 g L-1), while 2, 3-butanediol concentration was negligible. Cultivation on the enzymatically hydrolysed solid part of pre-treated BSG gave 9, 8 g L-1 of 2, 3-butanediol and 3, 93 g L-1 of ethanol. On the basis of obtained results it can be concluded that different experimental set-ups give the possibility of directing the metabolism of P. polymyxa DSM 742 towards the production of either 2, 3-butanediol and ethanol or lactate.

Gene targeting is a genetic technique that allows the modification of an endogenous gene by homologous recombination. It is achieved by the introduction of linear, non-replicative DNA fragments that should replace the homologous (targeted) sequence in the genome. It is generally assumed that gene targeting in the conventional yeast Saccharomyces cerevisiae almost exclusively occurs by homologous recombination and that it is usually higher than 90 %. Non-conventional yeast species are isolated from different niches and have interesting properties such as utilization of different carbon sources, fermentation potential, tolerance to different growth inhibitors and they are emerging as potential producers in different biotechnological processes. However, the efficiency of gene targeting in most of those species is still unknown. Using linear transformation cassette, a DNA that contains antibiotic resistance gene in the middle flanked by regions homologous to the target sequence in the genome, we have determined the efficiency of gene targeting in Brettanomyces/Dekkera bruxellensis, Kluyveromyces marxianus and Scheffersomyces stipitis. Depending on the yeast species gene targeting efficiency, obtained using transformation cassettes with flanking homologies longer than 1 kb, varied from less than 0, 1 % to 75 %. Our results suggest that in some species (e. g. K. marxianus) use of flanking homologies, longer than 1 kb, allows the introduction of targeted modifications in the genome, while in some other yeast species, advanced gene-editing techniques e.g. CRISPR/Cas system might be mandatory to allow precise modifications of the genome.

Fungi can degrade lignocellulolytic materials by secreting complex enzyme cocktails composed of cellulolytic and lignolytic enzymes. In this work, seven fungal species (Mucor indicus DSM 2185, Paecilomyces variotii CBS 372.70, Myceliophthora thermophila CBS 663.74, Thielavia terrestris CBS 456.75, Botryosphaeria dothidea JCM 2738, Fusarium oxysporum f.sp. langenariae JCM 8293 and Fusarium verticillioides JCM 23107) grown in four different media were screened for lignocellulose degrading activity. Enzymatic activity assays for the hydrolase’s xylanase, mannanase, arabinase, endo- and exo-glucanase, and pectinase, as well as for the oxidoreductase’s laccase, lignin peroxidase and cellobiose dehydrogenase were employed. From the seven tested fungi, Fusarium oxysporum f.sp. langenariae JCM 8293 and Fusarium verticillioides JCM 23107 were selected as the best producers of lignocellulolytic enzymes, and a more detailed investigation was performed. Sequential solid-state and submerged cultivation of F. oxysporum and F. verticilliodes was used to induce an increased production of lignocellulolytic enzymes. After six days of cultivation the fermentation broth was centrifuged, and the obtained supernatant was partially purified and concentrated. Enzymatic activities in both extracts were determined and the mold F. verticillioides showed higher xylanase activities than F. oxysporum, while the latter showed higher glucanase activities. It can be concluded that both strains can be used as producers of enzyme cocktails for the degradation of lignocellulose containing agro-waste.

The objectives of this study were to establish the impact of Fusarium culmorum infection and fungicide treatment on the occurrence of deoxynivalenol (DON), 3-acetyl deoxynivalenol, T-2 toxin, HT-2 toxin, nivalenol, fusarenon-X, diacetoxyscirpenol and zearalenone in wheat, wheat malt and wort (beer). The concentrations of these compounds were also measured in the germ/rootlets, spent grains and spent yeast because these are the most important by-products and are further used as food or feed additives. Two wheat genotypes were obtained from the Agricultural Institute in Osijek, Croatia. The Osk.110/09 genotype, the genotype more susceptible to Fusarium infections, and Lucija, the genotype less susceptible to Fusarium, were analysed in this research. Each genotype was treated in four different ways at the field: (A) control, (B) treated with fungicide Prosaro® 250, (C) inoculated with F. culmorum spores and treated with fungicide Prosaro® 250, and (D) inoculated with F. culmorum spores. All samples were malted and brewed according to standard procedures, products and by-products were analysed for the mycotoxins by using LC-MS/MS. Since the majority of trichothecenes are polar molecules, the water after steeping was also analysed with LC-MS/MS. Mycotoxin concentrations were lower in malt samples treated with the fungicide. Elevated mycotoxin concentrations were observed in samples of both genotypes exposed to F. culmorum. Fungicide treatment was observed to suppress mycotoxin production and accumulation. However, samples with notably high mycotoxin concentrations, especially DON, retained elevated mycotoxin concentrations throughout the entire beer production process, even after a six-month storage period. DON proved to be the most frequently occurring mycotoxin in all of the by-products. The highest concentration of this compound was found in the steeping water from sample D (Osk.110/09), at 20,326 μg/l, leaving the spent grains of this sample with no detectable levels of DON.

High flocculation efficiency of 97.46 % was obtained at chitosan concentrations above 25 mg L- 1 in the pH range 8.0-9.0. The optimal chitosan concentration for efficient flocculation of microalga was 25 mg L-1 at pH 8.0, with minimal volume of alkali used for the culture pH adjustment.

Mikroalge su raznovrsna skupina fototrofnih mikroorganizama koje proizvode niz različitih biotehnološki interesantnih tvari s biološkom aktivnošću. Biomasa mikroalgi je potencijalno održiv izvor sirovine za proizvodnju biogoriva, hrane, krmiva, kemikalija, finih kemikalija i za farmaceutsku industriju. U ovom istraživanju mikroalge će biti izolirane iz uzoraka vode uzorkovanih na nekoliko mjesta u Jadranskom moru i rijeci Gackoj. Kulture će biti obogaćene, pročišćene i identificirane upotrebom molekularnih biljega. Potencijal izoliranih mikroalgi bit će određen s obzirom na makromolekulski sastav (lipidi, proteini, ugljikohidrati), prinos biomase i produktivnost, udjel pigmenata, sastav masnih kiselina i antimikrobno, antimikotsko i antioksidativno djelovanje. Optimizacija uzgoja odabranih sojeva mikroalgi provest će se s obzirom na sastav hranjive podloge (ugljik, dušik, fosfor, silicij), način rasta (fototrofni, heterotrofni, miksotrofni) i način vođenja procesa (šaržni, šaržni s pritokom).

Cilj projekta Virtulab je poboljšati znanstveno- istraživačke i nastavne kapacitete pet sastavnica Sveučilišta u Zagrebu u sektoru primarnih i sekundarnih sirovina te dosegnuti europske i svjetske standarde praktične primjene znanja u gospodarstvu. Virtulab okuplja istraživačke kapacitete koji zadovoljavaju potrebe prospekcijskih istraživanja primarnih i sekundarnih sirovina, eksploatacije, oplemenjivanja, proizvodnih procesa, recikliranja i pronalaženja zamjenskih sirovina. Projekt doprinosi: 1) jačanju kvalitete znanstvenih istraživanja uvođenjem suvremene te povećanjem dostupnosti postojeće instrumentacije, 2) povećanju produktivnosti odnosno broja znanstveno- istraživačkih radova i broja prijava kompetitivnih znanstvenih i inovativnih projekata, 3) poboljšanju kvalitete nastave uz jačanje kompetencija studenata i time njihove konkurentnosti i zapošljivosti na tržištu rada i 4) transferu znanja i inovacija prema gospodarstvu. Implementacija projekta dugoročno će poboljšati dostupnost i iskorištenost primarnih i sekundarnih sirovina, pozitivno utjecati na okoliš i gospodarstvo i smanjniti uvoznu ovisnost Republike Hrvatske i Europske Unije u sektoru ne- energetskih mineralnih sirovina.

Lignocelulozna otpadna biomasa je široko dostupna i jeftina, a sadrži velike količine spojeva koji se potencijalno mogu koristiti u različitim biotehnološkim procesima. Intenzivno se istražuje potencijal nekonvencionalnih kvasaca, poput kvasaca iz roda Schwanniomyces, da efikasno prevode supstrate u vrijedne produkte. Međutim, biotehnološki potencijal nekog mikroorganizma se može u potpunosti iskoristiti samo ako se taj mikroorganizam može dodatno genetički modificirati. U ovom radu su, korištenjem protokola razvijenog za transformaciju nekonvencionalnog kvasca Brettanomyces bruxellensis, po prvi puta uspješno transformirani kvasci S. pseudopolymorphus i S. polymorphus var. africanus, kao i prethodno uspješno transformirani kvasac S. polymorphus var. polymorphus. Svi spomenuti kvasci transformirani su linearnim, nereplikativnim, heterolognim fragmentima s efikasnošću 2⋅10² - 8⋅10³ μg⁻¹. U radu su korišteni i replikativni plazmidi koji sadržavaju ishodišta replikacije izolirana iz drugih vrsta kvasaca, a upotrebom nekih od ovih plazmida postignuta je visoka efikasnost transformacije (do 5⋅10⁵ μg⁻¹). Ovaj rad financiran je od strane Hrvatske zaklade za znanost u sklopu projekta IP- 2018-01-9717.

Non-Saccharomyces yeasts represent a great potential of different yeasts that can be used in biotechnological processes. In comparison to Saccharomyces yeasts which are typically used in the industry, non-Saccharomyces yeasts show some unique characteristics that make them interesting for research. They can use different substrates for their growth, which makes them interesting for application in bioethanol production from lignocelulosic materials. Also, in addition, it has been shown that they have greater resistance to higher concentrations of inhibitors that are usually formed during the pre-treatment of such feedstock. Kluyveromyces marxianus NBRC 1777 is classified as thermotolerant yeasts, and some research has shown that it can grow at temperature of 52 °C. Also, it has been shown that it is a good ethanol producer. Therefore its application could lead to more sustainable and less energy demanding industrial bioethanol production. In this research, series of experiments were conducted using Kluyveromyces marxianus NBRC 1777 as a working microorganism at 4 different temperatures ( 20, 30, 40 and 50 °C) and specific growth rates and ethanol production parameters were determined (YP/S , YP).

In recent years microalgae have attracted considerable attention due to relatively fast growth coupled with high lipid, carbohydrate, protein and valuable nutrients contents. While freshwater microalgae are already used as food supplements, marine microalgae especially diatoms (Bacillariophyceae) have recently attracted considerable attention due to favourable fatty acids (FAs) profile abundant in long polyunsaturated omega-3 and omega-6 fatty acids (EPA, 20:5 n-3, DHA, 22:6 n-3, ARA, 20:4 n-6). They are considered as alternative to fish oil as a source of these essential omega-3 FAs but also as a source of numerous other bioactive molecules of interest such as carotenoids (β-carotene, fucoxanthin and astaxanthin) that also provide wide range of health benefits along with PUFAs. Omega-3 fatty acids are thought to have a role in reducing risk of coronary heart disease and improving cholesterol level. Several strains of microalgae isolated from Adriatic Sea were cultivated under phototrophic conditions and their fatty acid profile was determined and compered to the one obtained with green microalgae isolated from river Gacka. The analysis revealed the predominance of C14:0 (11%-21%) and C16:0 (30%-42%) and C16:1 cis 9 (13%-43%) fatty acids in all marine microalgae strains and polyunsaturated fatty acids 20:5n-3 (EPA) and 20:4n-3 (ARA) comprised a significant proportion of total fatty acid. In freshwater microalgae strains dominant fatty acids were C16:0 (8, 76-29, 44%), C17:0 (3, 57-36, 20%) and unsaturated fatty acids C18:2 cis 9, 12 (6, 48- 35, 27%) and C18:3 cis 9, 12, 15 (8, 82-33, 37%).

Xylitol is a sugar alcohol which can be found in vegetables and fruits in very small quantities. It is mostly used as a sweetener for diabetics and for other purposes (e.g. in toothpastes and chewing gums). Commercially it is manufactured through a chemical process that is costly, has high energy requirements, and needs extensive purification steps. However, xylitol can also be produced by biotechnological processes, which use agricultural and forestry by-products. The precursor xylose is produced by chemical and/or enzymatic hydrolysis of the lignocellulosic biomass. In this work, corn stover and corn cobs were subjected to dilute acid pre- treatment with 0.5 % w/w H2SO4 under different pre-treatment conditions. The resulting liquids were then separated from the solid parts and used for xylitol production using Mucor indicus as the working microorganism. The fermentations were carried out in baffled Erlenmeyer flasks under aerobic conditions. Xylitol production efficiencies ranged from 12.98-19.49% for corn stover, and 13.88-28.53% for corn cobs, calculated on the maximum theoretical xylitol yield (Yxylitol/xylose = 1.013 g g-1). Further improvement of bioprocess efficiency could be achieved by the lignocellulosic biomass hydrolysate detoxification before fermentation what could result in final higher xylitol concentration. Acknowledgments The research was financially supported by the Croatian Science Foundation under project No. HRZZ-IP-2018-01- 9717.

A large global industry of banana (Musa spp.) generates the high quantity of byproduct- banana peel, representing approximately 30 % of the fruit. Potential applications of banana peel depend on its chemical composition. Due to the valuable bioactive content of banana peel, the objective of this study was to develop alginate-pectin- and alginate/pectin- based edible films containing banana peel extracts and to characterize their physical (colour, solubility, thickness, dry matter) and mechanical (firmness and elasticity) properties, bioactive potential and release kinetics of bioactives from films in simulated gastrointestinal conditions. Among differently prepared banana peel extracts (conventional and ultrasound assisted extraction, acid hydrolysis, decoction and maceration), conventionally prepared extract (30 min, 80 ºC) was used in the formulation of edible films due to the highest total phenolic content (TPC) (19.08±0.35 mg GAE/g dw) and antioxidant capacity (AC) determined by ABTS assay (0.11±0.00 mmol TE/g dw). According to the values of physical- mechanical properties and determined prolonged release of TPC and AC in simulated gastrointestinal conditions, alginate- based edible film was the best evaluated. The obtained results indicate the promising application of banana peel extract in the formulation of bioactive enriched delivery systems.

In recent years marine microalgae have attracted considerable attention due to relatively fast growth coupled with high lipid, carbohydrate, protein and valuable nutrients contents. Among marine microalgae, diatoms (Bacillariophyceae) are the most abundant species in oceans and seas. Owing to favorable fatty acids (FAs) profile abundant in medium chain length, they are considered as suitable feedstock for biodiesel production, as well as alternative source of essential omega-3 FAs due to characteristically high levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Four strains of diatoms isolated from Adriatic Sea were cultivated under phototrophic conditions. Effect of nitrogen on lipid metabolism was studied by varying a sodium nitrate concentration in the culture medium (f/2 medium). The most abundant FAs in diatoms were palmitoleic (13%-43%) followed by palmitic (30%-42%) and myristic (11%-21%) acids, regardless of nitrogen concentration. Nitrogen concentration significantly affected the lipid and FAs content. The content of highly unsaturated FAs, EPA and ARA, in strain 5.4 grown in f/2 medium was 5.42% and 2.58%, respectively. Growth under nitrogen repletion stimulated accumulation of these FAs in diatom ; EPA and ARA content in FAs was 8.24% and 3.75%, respectively. On contrary, growth under the nitrogen limitation decreased the content of both FAs (EPA and ARA content was 2.45% and 0, 4%, respectively). The highest biomass and lipid productivity of 42.5 mg L-1 d-1 and 26.16 mg L-1 d-1, respectively, was obtained under nitrogen repletion conditions. In order to improve lipid and biomass productivity, optimization of culture medium was carried out using 2x, 4x and 10x concentrated f/2 growth medium. Furthermore, batch and fed-batch phototrophic cultivation of strain 5.4 was conducted in a photobioreactor using the optimized growth media.

In this study, the total lipid content and fatty acid composition of four diatom species isolated from Adriatic Sea (Šibenik, Croatia) were examined. Microalgae were isolated by serial dilution and the streaking plate method combined with use of antibiotics and physical separation methods. All four diatom species were screened for their capacity to produce lipids under phototrophic conditions. Moreover, the effect of nitrogen concentration on lipid production of isolated Navicula sp. was investigated under mixotrophic growth conditions with glucose as carbon source. Mixotrophic cultivations were performed at three NaNO3 concentrations: 8, 82 mM (original f/2 medium), 4, 42 mM (nitrogen limitation) and 17, 64 mM (nitrogen excess). In phototrophic cultivation lipid content varied between strains from 24% to 63% of dry weight. Lipid content was 53, 91% in presence of nitrogen excess and it was similar as in original medium where lipid content was 56, 57% of dry weight. The highest lipid content was obtained under nitrogen limitation. The analysis of the fatty acid composition revealed the predominance of C14:0 (11%-21%) and C16:0 (30%-42%) and C16:1 cis 9 (13%-43%) fatty acids in all strains for phototrophic conditions as well as in mixotrophic cultivation for all three nitrogen concentrations. In both experiments polyunsaturated fatty acids 20:5n-3 (EPA) and 20:4n-3 (ARA) comprised a significant proportion of total fatty acid. In phototrophic cultivation the highest content of EPA was obtained in strain S1 (7, 39%) and for ARA in strain S4 (8, 03%). Fatty acid analysis showed significant impact of nitrogen concentration on content of saturated and unsaturated fatty acids. In nitrogen excess conditions content of EPA and ARA was increased from 5, 42 % to 8, 24 % and from 2, 45% to 3, 75% respectively when compared with original medium. Nitrogen limitation resulted in significant reduction of these fatty acids content with obtained values of 2, 58 % for EPA and 0, 4% of ARA. Moreover, growth under nitrogen excess decreased content of saturated fatty acids from 41, 59 % to 34, 77 % comparing to original medium while nitrogen limitation resulted in increment up to 47, 56%. Consequently, content of mono- and poly- unsaturated fatty acid of 65, 54 % achieved in presence of nitrogen excess was higher than in original medium and under nitrogen limitation where their content was 58, 41% and 52, 44%, respectively.

Microbial lipids or single-cell oils (SCO) have been studied for decades and since the beginning of the 20th century they have been exploited as an alternative source of oils and fats for human consumption and animal feed. The number of "oleaginous" microorganisms belonging to the genera of microalgae, yeast, bacteria, and fungi have ability to accumulate substantial amounts of lipids, mostly in the form of triacylglycerols, under specific cultivation conditions. Lipids produced by oleaginous fungi and microalgae are source of long chain ω-3 and ω-6-polyunsaturated fatty acids. Microbial lipids could be also use as an alternative large-scale feedstock for biodiesel production instead of edible oils. However, the high expense of the carbon source for microorganism growth and lipid production, coupled with low microorganism productivity and the high costs of lipid extraction, are the main obstacles to the broader commercialization of the biodiesel production. Lignocellulose biomass is non-food, low cost and renewable alternative source of sugars that could be use as substrate for lipid production. Since highly productive oleaginous microorganisms lack cellulolytic activity, lignocellulosic biomass has to be hydrolysed to fermentable sugars using cellulolytic enzymes or thermochemical process at elevated temperature in the presence of acid catalyst. The enzymatic hydrolysis is preferred to the hydrolysis using acid catalyst since it produces fewer side-products and the reaction is carried out in non-corrosive environment. Unfortunately, lignocellulosic biomass is highly recalcitrant to enzymatic hydrolysis due to its structural features. Therefore, lignocellulosic biomass is subjected to pretreatment process that breaks down the physical and chemical barriers and makes cellulose amenable to the enzymatic hydrolysis. Microbial lipids production from lignocellulosic biomass is carried out by submerged and solid state cultivation. Different strategies are used for enzymatic hydrolysis and lipid production process, including separate hydrolysis and lipid production (SHLP), simultaneous saccharification and lipid production (SSLP) and consolidate bioprocessing (CBP).

materials for different biotechnological processes [1, 2]. Pre-treatment of such materials is necessary due to their complex structure. With desirable products formed during pretreatment as glucose and xylose for example [1] a variety of undesirable by products are formed as organic acids and aromatic compounds (eg. furfural, formic acid, hidroxymetil furfural, levulinic acid, acetic acid)[3]. Those compounds have inhibitory effect on selected microorganism and on bioprocess efficiency [1, 4]. Origin of inhibitors can be categorized in three notable groups. First group are inhibitors present in lignocellulosic structure. Compounds as aldehydes and phenols are created during depolymerization of lignin. Acids as acetic, formic, galacturonic are formed during degradation of hemicellulose [4]. Second group of inhibitors are compounds that are formed during degradation of lignocellulosic materials. In the structure of lignin, generally they are present as non-inhibitors (pentose and hexose sugars). Under high temperature and acidic conditions, pentose sugars are degraded to furfural (furan 2- carbaldehyde) and hexose sugars to 5- hydroxymethyl furfural. Also 5- hydroxymethyl furfural can be degraded to levulinic acid and formic acid [3, 4, 5]. Third group of inhibitors are compounds present on lignocellulosic materials and are not integral part of materials itself. Most common are nitrogen and sulphur chemical compounds, heavy metals and different pesticides or other chemicals used in agriculture [3, 4, 5]. Furfural and hydroxymethyl furfural are two most important inhibitors that are created during lignocellulosic pre-treatment. Influence of furans (furfural and hydroximethylfurfural) on the biomass growth and activity can be used as an inhibition indicator for other compounds present in hydrolysates too [6, 7]. Set of experiments were carried out to investigate the inhibitory effect of furfural on the growth and ethanol production with wild strain Saccharomyces cerevisiae. Selected microorganism is potential ethanol producer from hydrolysates obtained with weak acid pre- treatment of lignocellulosic materials as wheat straw.

Corn cob hydrolysate was used as carbon source for microbial lipids production by the oleaginous fungus Mortierella isabellina. In order to improve the enzymatic digestibility, corn cobs were subjected to alkaline pretreatment with NaOH and acid pretreatment with H2SO4. Pretreatment process was carried out at different NaOH and H2SO4 concentrations, temperature and residence time. Optimal pretreatment conditions were chosen based on glucose and xylose yield by enzymatic hydrolysis of pretreated biomass. The highest efficiency of enzymatic hydrolysis was obtained with corn cobs pretreated with 2 % w/w NaOH at 121oC and residence time of 1 hour, 2 % w/w NaOH at 50oC and residence time of 6 hours and 1 % w/w H2SO4 at 121oC and residence time of 20 min. Furthermore, the effect of enzyme and substrate loading rate on efficiency of enzymatic hydrolysis of pretreated corn cobs was also studied. The highest efficiency of glucan and xylan hydrolysis was achieved at 20 % (g/g) substrate loading rate and 30 FPU g-1 of enzyme loading rate. Oleaginous fungus Mortierella isabellina was cultivated on the enzymatic hydrolysate of corn cobs pretreated with 2 % w/w NaOH at 50 oC for 6 hours. The microbial lipid content in fungal biomass reached up to 45 % of biomass dry weight, while lipid productivity and lipid concentrations were 0, 051 gL-1h-1 and 24, 21 gL- 1, respectively.

Lignocellulose is a potential raw material for sustainable production of biofuels and biochemicals, but to use lignocellulose in bioprocesses it is necessary to convert cellulose and hemicellulose to fermentable sugars. In this work the results of wheat straw pre-treatment with 2% H3PO4, in high-pressure reactor at different temperatures (160°C - 200°C) and retention times (1 min – 10 min), are shown. During dilute acid pre-treatment hemicellulose is degraded to pentose sugars that cannot be used by industrial ethanol producing yeasts. Therefore, to improve overall bioprocess efficiency a genetically engineered Saccharomyces cerevisiae strain, that can utilize xylose, was used. Fermentations were performed on different hydrolysates in shake-flasks, and in horizontal rotating tubular bioreactor. Efficiencies for fermentations carried out in shake flasks were in the range from 19.6% to 74.5%. The maximum efficiency of 88.2% was achieved during fermentation in HRTB. The acquired results were also used for life cycle analysis of the ethanol production process. For the quantification of process sustainability, specific partial area atot was calculated using SPIonExcel program. Value of atot represents environmental impact, and economic and ecological sustainability. The partial specific area for this ethanol production process is 7782.06 m2 for 1 kg of ethanol produced, that is 2-3 times lower compared to, a simultaneous saccharification and fermentation process (SSF) described in Morikawa [1] atot=21401.5 m2, and a separate enzymatic hydrolysis and fermentation process (SHF) presented in Saha [2] atot=16701.9 m2.

Due to the constant increase in energy consumption, the production of biofuels from renewable sources, such as lignocellulosic waste, has become more frequent in the last several decades. Lignocellulosic waste consists of cellulose, hemicellulose and lignin and the yeast S. cerevisiae is not able to ferment it directly. Therefore, lignocellulosic waste is usually hydrolysed to release compounds that can be used for fermentation. However, the pretreatment also generates compounds that act as growth and fermentation inhibitors such as acetic and levulinic acid and 2-furaldehyde. In this study, newly constructed intraspecies hybrid diploids, obtained by crossing natural S. cerevisiae isolates, showed higher resistance to acetic and levulinic acid. Selected strains were also transformed with replicative vectors overexpressing yeast genes (e.g. ATR1, CTA1, FLR1, YAP1, ZWF1) encoding proteins involved in a stress response and the overexpression of YAP1 gene further increased the resistance to inhibitors. Therefore, our results suggest that the construction of intraspecies hybrids coupled with genetic engineering is a promising approach for construction of new biotechnologically relevant yeast strains.

Waste lignocellulosic raw materials are by- products of agriculture, forest and wood industry, and represent a valuable alternative for the production of biofuels and different biochemicals. Since lignocellulose is highly resistant to microbial degradation, the aim of this work was to conduct alkali pre-treatment of wheat straw and to determine the optimum conditions for wheat straw pre-treatment with sodium hydroxide solution. Pretreatment of wheat straw was performed in high-pressure reactor. The reactor was filled with 500 g of wheat straw (% w/w ; reduced in size to 5 mm pieces) and 10 L of 2 % (w/w) sodium hydroxide solution and homogenized. Pre-treatment was performed at 120, 160, 180 and 210°C and residence time of 1, 10 and 20 minutes. After pretreatment two phases were obtained, liquid and solid phase (pre-treated wheat straw). Composition of these phases was determined according to NREL method (Sluiter et al., 2011). Glucan content in the pretreated wheat straw ranged from 23.5 % to 86.1 %, while xylan and arabinan content ranged from 1.6 % to 24.8 % and 0.00 % to 11.4 %, respectively. Also, in these pretreatments 55.8 % to 97.1 % of lignin was removed. The highest glucan content (86.1 %) in solid phase was determined at 210°C and a residence time of 10 minutes while maximum lignin removal (97.1 %) in contrast to wheat straw composition was determined at 210°C and 20 minutes residence time.

Sugar and starch containing raw materials are widely used as food and feed thus, second generation raw materials like waste lignocellulosic biomass present sustainable alternative for production of different biochemicals, e.g. lactic acid. In this work, alkali pre-treatment of wheat straw with 2 % w/w sodium hydroxide solution in high-pressure reactor at different temperatures (from 120°C to 210°C) and residence times (from 1 to 20 minutes) was performed. After alkali pre-treatment of wheat straw, solid and liquid phase was obtained and composition of the two phases was determined. Solid phase (wheat straw cellulose) with high glucan content was used to study the fermentation capabilities of selected strain in two bioprocess types: Separate Hydrolysis and Fermentation (SHF) and Simultaneous Cellulose Hydrolysis and Fermentation (SCHF). Cellulose hydrolysis was performed with commercially obtained cellulases and β-glucosidases isolated from mold Aspergillus niger. In SHF bioprocess, after enzymatic hydrolysis of wheat straw cellulose, obtained hydrolysates were used for lactic acid production by lactic acid bacterium Lactobacillus amylovorus DSM 20531T. While, in SCHF bioprocess, enzymatic hydrolysis of wheat straw cellulose to simpler carbohydrates and fermentation by L. amylovorus DSM 20531T was performed simultaneously. Process efficiency parameters, like final lactic acid concentration, lactic acid yield and bioprocess productivity, in SCHF bioprocess were approximately 60 % higher than in SHF bioprocess.

The effect of dilute H2SO4 and NaOH in corn cob pretreatment was studied. As measure for pretreatment efficiency, enzymatic digestibility of the pretreated biomass was determined. The influence of acid and alkali concentration, temperature and time of pretreatment was studied. After enzymatic hydrolysis of pretreated biomass, the best conditions for alkali and acid pretreatment were selected. The effect of enzyme and substrate loading rate on efficiency of enzymatic hydrolysis of pretreated corn cobs was studied for selected pretreatment conditions. The highest efficiency was achieved at 20% substrate loading and 30 FPU g-1 glucan of enzyme loading. With corn cobs pretreated at 2 % NaOH at 50oC and residence time of 6 hour hydrolysis efficiency of glucan and xylan was 85, 71 % and 73, 53 %, respectively. Enzymatic hydrolysate of corn cobs pretreated on these conditions was used for cultivation of oleaginous fungus Mortierella isabellina, and microbial lipid concentration of 24, 21 g L-1 was achieved. Based on fatty acid composition it is proved that biodiesel produced from microbial lipids from Mortierella isabelina fulfill European biodiesel standard.

Biogas consists mainly of methane (55-70%) and carbon dioxide (most often 30-45%) and small amounts of other compounds. It is important to stress positive socio-economic and environmental impact of biogas production by anaerobic digestion (AD) from various renewable raw materials, e.g. manure, agricultural residues, organic wastes from food industry, organic fraction of municipal waste or dedicated energy crops. In Europe, biogas is mainly used for generating heat and electricity, and in some cases is upgraded to biomethane and utilized as vehicle fuel, instead of fossil fuels. This reduces the emissions of greenhouse gases and thus prevents the climate change. In most biogas-producing countries, anaerobic digestion is taking place in industrial and farming scale. Domestic biogas digesters are mostly abundant in developing countries and intended mainly for direct combustion of the produced biogas in household stoves and gas lamps. Various renewable raw materials for large scale biogas production are available in Croatia, and the opportunities for their use are insufficiently exploited. The biogas produced in Croatia is mostly used for the operation of electric power plants. Biogas production in Croatia is predominantly based on manure and by-products from slaughterhouses, agriculture and food industry. Aside from producing biogas as a renewable fuel, the additional positive effects of anaerobic digestion of animal manure and slurries are organic waste degradation, reducing odours and pathogens, as well as possibility of use digested residue (digestate) as a byproduct rich in nutrients that can be used to fertilize agricultural land. Concerning large unused agricultural areas, great potential lies in their rational exploitation for growing biomass, e.g. for energy crops or perennial grasses. Biogas production in Croatia can be negatively affected by lowering animal breeding capacity due to inability to cope with the EU standards and prices. Current state of biogas production in Croatia and its potential in industrial and farming sector will be discussed, as well as various obstacles preventing faster development of biogas production sector.

Sugar beet pulp (SBP) is a promising feedstock for the production of 2nd generation biofuels, but efficient enzymatic hydrolysis remains a key challenge ; therefore, new process designs and/or bioreactor designs are crucial to overcome this hurdle. In this regard, horizontal rotating tubular bioreactors (HRTB) offer the advantage of high substrate loadings while minimizing the space and energy demand compared with conventional stirred tank reactors. Here, a statistical approach is used to optimize the hydrolysis of sugar beet pulp in laboratory experiments, and it is shown that such a process can be implemented in a HRTB. Using the design of experiments (DOE) method, the reaction conditions of four commercial enzyme mixtures (Ultrazym AFP-L, Viscozyme L, Pectinase and Cellulase) was optimized for the degradation of SBP in small-scale experiments. Using Ultrazym AFP-L as the most efficient mixture, a 10 L scale conversion was performed in a HRTB. At a substrate loading of 135 g L−1 and optimized conversion parameters (enzyme load, pH and rotating speed of the reactor), 0.525 W dm−3 were needed to achieve solubilisation of 30% of the total mass of initial SBP after 24 h. DOE was found to be an easy-to-apply method that allowed optimizing the conditions for enzymatic hydrolysis of SBP, resulting in a higher sugar yield. The results could be transferred to an HRTB, which is a suitable system for enzymatic conversion and efficient saccharification of semi-solid or solid substrates with relatively low energy consumption.

Wheat straw hydrolysates (obtained by diluted sulphuric acid pre-treatment) were used as substrates for anaerobic cultivation of genetically modified strain of Saccharomyces cerevisiae. Wheat straw pre-treatment process was carried out in the high-pressure reactor under different temperatures (150 - 200 °C) and retention times (1 - 10 min) with 0, 5 % w/w sulphuric acid. The highest mass fraction of xylans (68, 64 % w/w) in the wheat straw hydrolysate was observed at 160 °C and residence time of 5 minutes and the highest mass fraction of glucans (30, 42 % w/w) at 200 °C and residence time of 10 minutes, respectively. Genetically modified strain of Saccharomyces cerevisiae has ability to use hexoses and pentoses as a carbon source. The initial kinetic study (e.g. growth rate and ethanol synthesis) of modified yeast strain was carried out in small scale. The most successful ethanol production was observed on the wheat straw hydrolysates obtained at 180 °C and residence time of 10 minutes. In these conditions, bioethanol production efficiency was 43, 25 % and bioprocess productivity of 0, 045 g L-1 h-1, respectively. During anaerobic cultivation of genetically modified S. cerevisiae in the horizontal rotating tubular bioreactor on the same hydrolysate, bioprocess productivity was 0, 143 g L-1 and efficiency of 64, 01 %, respectively.

BACKGROUND Biodiesel is an alternative to conventional fossil diesel fuel. Currently, it is mainly produced from vegetable oils and consequently it has an adverse impact on food price. Lipids from oleaginous microorganisms (e.g. microalgae, bacteria, fungi and yeasts) could be an alternative feedstock for biodiesel production and therefore the growth and lipid accumulation of Trichosporon oleaginosus was studied under different conditions. RESULTS The growth of T. oleaginosus on the xylose containing media enhanced the lipid accumulation and reduced cell growth, while on the glucose-containing media the opposite effect was observed. Trichosporon oleaginosus can accumulate above 50% of total lipids in dry cell mass during continuous and batch culture in the nitrogen-limited conditions. In the continuous culture relatively high productivity of total lipid accumulation (0.67 g dm−3 h−1) was observed on the glucose containing media. The type of substrate limitation has considerable impact on the fatty acid composition of yeast cell lipids. The predicted biodiesel properties (based on the fatty acids composition of lipids) were found to meet the CEN quality standards. CONCLUSION The results obtained show that T. oleaginosus has great potential for feasible biodiesel production, especially when sugars from industrial and agricultural waste are used as a substrate.

The depolymerisation of complex biomass such as lignocellulose by organisms depends on a network of enzymatic and chemical reactions. Fungi that can grow on lignocellulosic materials are equipped with a complex enzymatic degradation system, including a broad variety of hydrolases and oxidoreductases. In 2010, a new class of enzymes was discovered, which carry out oxidative cleavage of polysaccharides. These enzymes, today known as lytic polysaccharide monooxygenases (LPMOs, CAZy family AA9) are copper-dependent oxidoreductases that can hydroxylate the C1 or C4 positions of glucose and thereby cleave the glycosidic bonds in cellulose. These “cellulase activity boosters” provide hydrolytic enzymes access to crystalline cellulose by introducing chain breaks. LPMOs, despite their importance in biotechnology, are difficult to investigate because of their complex heterogeneous reaction mechanism. An open question is how LPMOs orchestrate the complex reaction between copper reduction, co-substrate activation and substrate binding. It is generally accepted that the first reaction step of LPMO is the reduction of the active-site copper by small molecule reductants or by other enzymes (CDH, GDH). In this work, we address the substrate binding of oxidized and reduced LPMO-02916 from Neurospora crassa to phosphoric acid swollen cellulose (PASC) and microcrystalline cellulose (MC) to investigate the role of the copper centre for substrate binding.

A biorefinery is a facility that integrates biomass conversion processes and equipment to produce bio-products from biomass. Biorefineries will have to employ the best possible processes (for biomass cultivation, harvesting, storage and transport, pre-treatment, fermentation and bio-chemical conversion) to ensure efficient bio-based production. Nevertheless, numerous technical, strategic and commercial challenges have to be overcome to reinforce importance of bio-based production. Mathematical models can be used to simulate all aspects of bio-based production systems including the process design, production economics, supply logistics and environmental impacts. Those models can provide powerful tools to optimize biorefinery processes and evaluate its technical feasibility as well as economics and environmental impacts. In the presentation, examples of different mathematical models will be discussed, from the physical base model including kinetic, transport and diffusion equations to the statistical base model and data analyses. In the end, life cycle assessment analysis of the ethanol production from the corn stover will be provided as the example of modelling application in better understanding of the bio-based production environmental impact.

Zadnjih desetljeća zabilježen je značajan porast potrošnje fosilnih izvora energije što će uzrokovati iscrpljivanje njihovih zaliha i povećanje potrebe za alternativnim izvorima energije. Stoga je pažnja usmjerena prema proizvodnji goriva iz obnovljivih izvora energije, najčešće otpada iz poljoprivrede i drvne industrije poznatih pod nazivom lignocelulozne sirovine. Najpoznatije alternativno gorivo je bioetanol kojeg proizvodi kvasac Saccharomyces cerevisiae procesom alkoholne fermentacije. Međutim, kvasac ne može fermentirati lignocelulozne sirovine pa ih je stoga neophodno prethodno obraditi kako bi nastali fermentabilni šećeri (npr. glukoza) iz kojih kvasac može proizvesti etanol. Dodatni je problem to što kemijskom obradom, odnosno hidrolizom lignoceluloznih sirovina nastaju takozvani inhibitori rasta, kemijski spojevi koji inhibiraju rast kvasca i alkoholnu fermentaciju, a nastaju i nefermentabilni ugljikohidrati. Stoga se metodama genetičkog inženjerstva nastoji konstruirati sojeve kvasca S. cerevisiae koji su rezistentni na inhibitore rasta i mogu fermentirati inače nefermentabilne ugljikohidrate.

Synthesis of lactic acid from petrochemicals has been cancelled and since 2011 the lactic acid has been exclusively produced by biotechnological processes - fermentation of glucose, sucrose or lactose by different microorganisms. Traditional use of the lactic acid and, nowadays, production of other chemicals and biodegradable polymers from the acid drive global lactic acid demand. Leading producers are investing in development of new bioprocesses for the lactic acid production from renewable raw materials by microorganisms which possess hydrolytic and fermentative activity. Main objective of investigation was development of new bioprocess that meets the European Union climate and energy goals - „20/20/20“ targets and the strategy „Innovating for Sustainable Growth: a Bioeconomy for Europe”. Application of novel highly efficient and sustainable bioprocess and construction of industrial plant for the lactic acid production depends on type of raw material to be used in market established manner in the European Union area. The new bioprocess of simultaneous saccharification of semi-solid substrate and fermentation to the lactic acid by amylolytic lactic acid bacterium was developed. The bacterium also possesses proteolytic activity and it can use proteins from the raw material as amino acid source. The developed bioprocess is suitable for scale up and it can be integrated in zero waste biorefineries in which other biochemical and biofuels can be produced from the renewable raw material.

Bioethanol production from raw lignocellulosic material requires a producer strain which tolerates high ethanol concentrations and presence of growth and fermentation inhibitors. Therefore we constructed hybrid diploid S. cerevisiae strains by mating two haploids having desirable traits. One haploid was isolated from wine thus tolerating high ethanol concentration while the other has been shown to be resistant to several growth and fermentation inhibitors. These haploids were mated and resulting homozygous (control) and heterozygous (hybrid) diploids were tested for their ability to grow in the presence of several inhibitors frequently found in different lignocellulosic hydrolysates (acetic and levulinic acid, 2- furaldehyde) and to produce ethanol. The results showed that some hybrid diploids were more resistant to inhibitors than parental haploid strains or homozygous diploids. Interestingly, constructed hybrids showed different fermentation ability in CO2 production test, suggesting genetic variability of constructed strains. All together, these results suggest that the approach used in this study may help to construct new strains as well as to improve traits of biotechnologically interesting strains.

Owing to the increased tendency of producing bioactives-enriched films and coatings for food applications, in this study the potential of formulating edible films from polymeric hydrocolloid materials (alginate, pectin, chitosan) in cocoa (Theobroma cacao L.) husk extract as the source of macroconstituent (polysaccharides) and bioactive compounds (polyphenols and methylxanthines) was investigated. Since cocoa husk extract represents the main cocoa processing by-product in the chocolate industry, valorization of this material by expoiting it as a source of added-value functional ingredients was examined. A comparative study of the physical, mechanical, bioactive and sensory properties of produced films, prior and after drying of films at 60°C, was undertaken, and the relation between specific properties (colour, sensory and mechanical properties) was deduced by correlation analysis. The effect of enrichment with polyphenolic compounds and theobromine derived from cocoa husk extract was determined by comparing the produced films with control films obtained only in distilled water. According to the obtained results, edible films were produced with thickness ranging from 30-100 μm, which was primarily in dependance of the solubilization media (water or cocoa husk extract), while the water and dry matter content were more affected by the employed biopolymer material. Control alginate film generally exhibited the best mechanical (highest tensile strenght - 6, 17 N and lowest deformation - 0, 73 %) and sensory properties, while the enriched chitosan film prepared in cocoa husk extract exhibited the poorest respective properties and the most potent thickness (104, 67 μm). By preparing the films in cocoa husk extract a significant increase of bioactive compounds content was achieved (even up to 3-fold higher) in relation to control films, while the bioactive properties remained preserved even after drying of films at 60°C. Enrichment of films in cocoa husk extract enabled to markedly enhance the bioactive content, but it negatively affected the physical, mechanical and sensory properties of produced films, which were better for control films (especially alginate one).

Lignocellulosic biomass have been studied extensively as renewable carbon source for production of second generation of biofuels such as bioethanol and biodiesel. In order to determine the content of unfermented carbon source as well as overall efficiency of the process it is necessary to determine the content of lignocellulosic carbohydrates. Therefore, a simple and reliable method for the analysis of major lignocellulosic components (glucan, xylan and lignin) was developed. Composition of three abundant lignocellulosic feedstocks in Croatia, corn cobs, corn husks and hay, was analysed. Firstly, lignocellulosic biomass was subjected to sulphuric acid hydrolysis prior to monosaccharide determination. The acid hydrolysis was optimized to give the maximal constituent monosaccharides. A high-performance liquid chromatography (HPLC)-based method using refractive (RI) was applied to determine concentration of monosaccharides in purified lignocellulosic hydrolysate. Monosaccharides were separated by HPLC using two different HPLC columns, i.e. Rezex RPM Monosaccharide Pb2+ (Phenomenex) and Supelcogel C610H (Sigma-Aldrich). Glucan and xylane content was calculated from the HPLC measured cellulose (glucose) and hemicellulose (xylose, galactose, mannose, arabinose) derived sugars, respectively. Furthermore, the content of acid soluble and acid insoluble lignin in lignocellulosic hydrolysate biomass was also determined.

Strains of species Lactobacillus amylovorus possess extracellular amylolytic activity that is essential for their accomodation in natural niches. In our comprehensive investigations strain Lactobacillus amylovorus DSM 20531T was employed for hydrolysis and fermentation of starch as sole carbon and energy source. HPLC analysis have shown that starch was degraded to maltose and maltodextrins, while glucose was not detected during the bioprocess. The aim of this work was to characterize the utilization of maltose for production of D-/L-lactic acid by L. amylovorus DSM 20531T at laboratory scale. Initial concentration of maltose was 20.0 g/L and fermentation was carried out at 40C. The maltose was not completely depleted from the medium over 24.0 h of the bioprocess and substrate to product conversion coefficient (YP/S) of only 0.87 g of D- and L-lactic acid (in equimolar amounts) per g of spent maltose was achieved. No other fermentation products were observed in explored experimental conditions, although this strain was reported to produce small amounts of acetate at low sugar concentration.

Corn cobs, which are available in large quantities in the continental Croatia, were subject of this work because they can be used as a readily available, renewable lignocellulosic feedstock for biotechnological production of biofuels, biochemicals and many other products. The effect of alkaline treatment on corn cobs composition was investigated, using gravimetric and spectrophotometric analytical methods. Portions of dry matter, ash, protein, extractive substances, lignin and cellulose were analysed in the starting raw material and compared with those obtained after treatment with sodium hydroxide. The results obtained for corn cobs were generally in line with the values reported in the literature, while minor deviations can be interpreted as the influence of genetic factors, growing conditions, modes of production and storage as well as the impact of the applied analytical methods. Portion of total lignin in the dry matter of corn cobs was 11.55 % (10.02 % and 1.54 % of insoluble and soluble lignin, respectively). The share of total extract in dry matter of corn cobs after alkaline treatment was 1.26 % (0.53 % of water extract and 0.73% of ethanol extract). This treatment has removed a large part of lignin. The treated corn cobs contained 5.19 % of the total lignin (2.46 % and 2.73 % of insoluble and soluble lignin, respectively) in the dry matter. At the same time there has been an increase of the portion of cellulose in the dry matter, with approximately 42.23 % in the raw material to 74.38 % in the alkaline treated feedstock. Portion of hemicellulose, calculated by subtracting other compounds from the total solids, were 39.00 and 18.52 % for corn cobs and alkaline treated feedstock, respectively. Numeric indicators, applicable for comparison of variations in raw materials, as well as for monitoring of lignocellulose pretreatments and solid-state fermentation, have also been introduced in this work.

Biodiesel is a renewable substitute fuel for traditional petroleum-based diesel. It consists of the methyl esters of the fatty acids produced by trans-esterification of triglycerides with an alcohol such as methanol under mild conditions in the presence of a base catalyst Main feedstocks used for commercial production of first generation biodiesel are plant oils(rapeseed, soybean. palm, sunflower), animal fats and waste oils. Compared to petroleum diesel, biodiesel is less toxic, biodegradable and does not contain sulphur or aromatics Despite these benefits, the major obstacle to widespread commercialization of biodiesel production is the high production cost relative to petroleum diesel. The high price of the feedstock is the major factor determining the biodiesel price. Intensive search for alternative feedstock brought back in focus the microbial lipids. An oleaginous microorganisms have ability to accumulate significant amounts of lipids (>20% of dry matter) under certain cultivation conditions. Production costs could be significantly reduced by replacing expensive carbon sources (e.g. glucose) with low-value substrates such as agricultural wastes, food waste streams and industrial by-products. In this work, oleaginous yeast Trichosporon o!eaginosus was used for the production of lipids from corn cobs hydrolysate Batch process of simultaneous saccharification and lipid production (SSLP) was conducted at different initial substrate loadings (5-20 % w/w) The highest final lipid yield of 92.4 mg lipid per g of dry matter of pretreated corn cobs (g/gDM_PCC) was obtained at substrate loading of 125% (w/w). A "fed-batch" strategy of sequential loading of substrate during SSLP process was investigated to achieve high cumulative substrate loadings, reduce viscosity of lignocellulosic slurry and obtain higher lipid yields. Batch additions of 2.5 and 5% (w/w) substrate resulted in cumulative substrate loading of 20% (w/w). The strategy of gradual substrate addition was related to the homogenous medium conditions during bioprocess due to the more efficient mixing process Fed batch process of SSLP with substrate additions of 2.5 % (w/w) significantly increased the lipid yield in comparison to the batch process Maximal lipid yield of 269.25 mg/ gDM_PCC was obtained after 312 hours of cultivation.

Lignocellulose consists of cellulose, hemicelluloses and lignin. During the acidic pre- treatment of lignocellulose, hemicellulose is degraded to pentose (xylose and arabinose) and hexose sugars (glucose, mannose, galactose), acids (acetic acid, formic acid and levulinic acid), and furans (furfural and 5-hydroxymethylfurfural). Obtained hydrolysates are usually detoxified by different expensive and long-lasting methods that can be used to detoxify obtained hydrolysates. In order to optimize the process of lactic acid production from lignocelluosic hydrolysates and possibly to avoid or minimize detoxification steps, it is necessary to determine the influence of inhibitors formed during acid pre-treatment of different lignocellulosic feedstocks. The effect of major toxic substances present in hydrolysates should be determined, but the concentration of furans can be used as a valuable indicator to predict the inhibition. Therefore, in this work the influence of different concentrations of furfural (from 100 to 5000 mg L-1) on growth and lactic acid production by lactic acid bacteria Lactobacillus amylovorus DSM 20531T was investigated. The results obtained show significant reduction in bioprocess efficiency parameters with increase of medium furfural concentration.

Surplus amounts of lignocellulosic waste materials, as corn stover, corn cobs and wheat straw have a good potential to be used as a substrate for microorganisms growth and production of biofuels and biochemicals. Pre- treatment of such lignocellulosic waste materials is necessary to obtain a substrate that is suitable for chosen microorganism. Pre- treatment was carried out in self-made high temperature, high pressure reactor with 1 % sulphuric acid as a medium for hydrolysis. Obtained liquid part was used for the growth of wild strain Saccharomyces cerevisiae. The selected wild strain yeast was isolated from Županja, Sugar factory. Obtained hydrolisate from pretreatment contains chemical compounds as furfural (among others), that has inhibitory effect on selected yeast. Set of experiments were conducted to investigate the inhibitory effect of furfural on growth and production of ethanol. Results have shown that the wild strain of selected yeast can grow in medium that has furfural concentration in amount that is present in obtained hydrolysates from pretreatment of lignocellulosic waste materials. Next step was cultivation of selected strain of yeast on liquid parts obtained from pretreatment of corn stover, corn cobs and wheat straw in high temperature, high pressure reactor. Experiments have shown that the growth and production of ethanol is possible in substrates obtained with that method of pre- treatment.

Zbog iscrpljenosti nalazišta nafte u bliskoj budućnosti se očekuje energetska kriza (kriza goriva) kao i kriza kemijskih sirovina koje se dobivaju iz nafte. Pretpostavlja se da će se u prvo vrijeme taj manjak kompenzirati kemikalijama i energentima proizvedenim iz obnovljivih šećernih, škrobnih i lignoceluloznih sirovina. S ciljem upotrebe lignoceluloznih sirovina za proizvodnju etanola potrebno je provesti proces njihove predobrade. Predobrada je važan korak u proizvodnji biogoriva i biokemikalija pri čemu se koriste različite biološke, kemijske i fizikalne metode s ciljem izdvajanja lignina i pripreme sirovine za enzimsku hidrolizu. Primjenom toplinske predobrade ostvarena je bolja razgradnja sirovine i veća koncentracija šećera koje kvasac, u daljnjim fazama procesa proizvodnje bioetanola, može metabolizirati. Uslijed djelovanja topline i mehaničke dekompresije dolazi do promjena u strukturi sirovine pri čemu polimerna vlakana lignocelulozih sirovina depolimeriziraju do jednostavnih šećera. Potpuna razgradnja lignocelulozne sirovine zahtjeva primjenu različitih vrsta pektinolitičkih, hemicelulolitičkih, lignolitičkih i celulolitičkih enzima. Stoga je nakon predobrade toplinom provedeno istraživanje enzimske hidrolize lignocelulozne sirovine (kukuruzni oklasci, pulpa šećerne repe, slama) s enzimima izdvojenim iz supernatanata fungi. Novine predloženog predtretmana temelje se na primjeni ekstracelularnog razradnog sustava dobivenog iz fungi sa ciljem poboljšanja razgradnje celulozne frakcije lignocelulozne sirovine, a nedavna istraživanja enzimske razgradnje lignoceluloznih sirovina otkrila su da je sinergističko djelovanje enzima ključno za uspješnu razgradnju. Za planiranje eksperimenata te za obradu i prikaz rezultata korišten je programski paket Statistica. Dobiveni rezultati prikazani su centralno kompozitnom metodom odzivnih površina (eng. Response Surface Methodology ; RSM) koja se koristi za modeliranje i analizu utjecaja nezavisnih varijabli (procesnih parametara) na promatrani odziv s ciljem poboljšanja, razvoja i optimiranja procesa. Potrebno je odabrati odgovarajući dizajn eksperimenta (eng. Design of experiments, DOE) ili plan pokusa kojim će se definirati eksperimenti koje je potrebno provesti, a provedbom pokusa u svim kombinacijama određenim dizajnom eksperimenta dobiva se odzivna ploha.

Spent brewer's yeast, a by-product of beer production, could be used as a raw-material for isolation of β-glucan. In spite of the fact that large quantities of brewer's yeast are used as feed, certain quantities are still treated as waste. β-Glucans are compounds that can achieve a greater commercial value than the brewer's yeast itself. Among various compounds that can be isolated from yeast, β-glucans are products that can be used as multipurpose ingredient and achieve a high price on the market. In addition of their immunostimulatory activity that makes them suitable for application in human and veterinary medicine, β-glucans can find application in pharmaceutical and chemical industries, food and feed production, as well as in production of cosmetics. Therefore, production of β-glucans, which have huge application potential, is suggested as an attempt to solve the problem of spent brewer's yeast.

Lactic acid bacteria are a ubiquitous group of Gram-positive nonsporulating cocci and bacilli that produce lactic acid as an end product of carbohydrate fermentation. They are closely associated with our everyday life as some ferment food or have health promoting effect while others spoil food or even cause diseases. Their relevance motivated whole genome sequencing of many isolates belonging to this clade. As a result, numerous sequencing reads are publicly available but it is unclear which bioinformatics tools can organize them into high-quality genome assemblies resulting in time-consuming trial-and- error based strategies. To ameliorate this problem, we tested 13 state- of-the-art assemblers using four specialized quality control tools. Assemblers were tasked with inferring genome sequence of a representative species of lactic acid bacteria, Lactobacillus amylovorus DSM 20531T, using Illumina-generated reads. Produced genome sequences varied considerably regarding total length, continuity and accuracy. Several scaffolding and assembly merging tools were tried to improve the quality of the assemblies further but had limited success. Visualization of final stage de Bruijn graph showed that many “bulges” limit continuity while graph’s shape indicated that the genome contains small plasmid. From these insights, we derive a set of guidelines which streamline the path to a high-quality genome assembly of lactic acid bacteria.

Agro-industrial wastes comprise different types of plant residues that are generated as by-products on the field or in the factory. Such biomass is characterised by high carbohydrate content and nowadays is becoming an increasingly studied raw material for their isolation and re-utilisation within the sustainable production concepts in food technology and biotechnology. Due to the complexity of plant cell wall matrix, recovery of polysaccharides is a demanding task, whether the aim is obtaining satisfactory yields, preserving native structures or, often, both. The present study aimed to define a suitable procedure for sequential extraction of polysaccharides from sugar beet pulp (SBP), walnut shell (WS), cocoa bean husk (CBH), and pea pods (PP), as well as to establish a reliable method for compositional evaluation of the obtained fractions. Alcohol insoluble residue (AIR) was prepared by repeated extractions with 70% ethanol. AIR was subsequently subjected to hot diluted acid (HCl) extraction, delignification with NaClO2 and CH3COOH and finally alkaline extraction with NaOH. Yields of the obtained fractions were measured gravimetrically, while the composition using developed HPLC-UV technique with 3-methyl-1-phenyl-5-pyrazoline-2-one pre-column labeling, after complete hydrolysis. Three fractions were obtained, namely, pectic (P), hemicellulosic (H) and cellulosic (C), with fraction mass ratios P:H:C dependent on the sample and the employed extraction protocol. SBP exhibited the highest yield of pectins (18.24% dwb), while WS and PP of hemicelluloses (28.65% and 22.32% dwb). Monomeric composition evaluation revealed typical galacturonic acid dominance for pectic fractions of SBP and CBH, while their hemicelluloses were of very heterogeneous composition. The obtained hemicelluloses of WS and PP were xylan-type, with relative xylose content of nearly 90% and 80%, respectively. The established procedure is regarded as suitable for pectin and hemicellulose extraction due to high total xylan (71.53% for WS, 71.43% for PP) and galacturonan recoveries (88.84% for SBP) from the starting material.

Amylolytic lactic acid bacterium Lactobacillus amylovorus DSM 20531T utilised glucose, sucrose and starch as a sole carbon and energy source. The three substrates were completely depleted from MRS medium during batch cultivations carried out in a laboratory scale stirred tank bioreactor at constant temperature (40 °C) and pH value (5.5). Under the tested conditions, the bacterium was capable of conducting simultaneously starch hydrolysis and fermentation. A mixture of two stereoisomers, D-(–)- and L-(+)-lactic acid, was produced in all cases by highly efficient homofermentative bioprocess with 0.93 to 1 g of lactate produced per g of total (consumed) substrate. The effect of temperature on the kinetics of cell growth and lactic acid production by the amylolytic strain in the starch-containing medium was also investigated. Efficient simultaneous saccharification and fermentation (SSF) was obtained at 35, 40 and 45 °C with completely degraded complex carbohydrate in 8 to 12 h and the product yield coefficient in the range from 0.91 to 0.93 g/g. Maximum values for substrate consumption rate (0.89 h^–1), maximum specific growth rate (0.87 h^–1), product formation rate (2.01 h^–1), and productivity of lactic acid (1.45 g/(L·h)) were obtained at 45 °C, while maximum biomass concentration (4.38 g/L) was attained at 40 °C. The ratio of the two stereoisomeric forms of produced lactic acid was strongly affected by the temperature. Unstructured kinetic model was used to describe the consumption of the three substrates, bacterial biomass formation and lactic acid production by L. amylovorus DSM 20531T. The dependence of biokinetic parameters on temperature was described by cardinal temperature model. The applied models successfully predicted all experimental data.

The subject of this presentation is possibility of using forest residues and wood waste as renewable lignocellulosic feedstock in biotechnology. Use of these materials is justified from the environmental but also from the economical standpoint. There is a large number of products that can be obtained from lignocellulosic raw materials, such as various chemicals, drugs, biofuels, enzymes and other high added value products. Forest residues and wood waste could therefore become the basis of the development of Croatian biotechnological production.