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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.

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).