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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.

Lactic acid bacterium Lactobacillus coryniformis subsp. torquens is classified to unique phylogenetic group of genus Lactobacillus and, according to key characteristics of glucose fermentation, to facultatively heterofermentative-type group. The strain rank is based on very limited amount of genotypic, biochemical, and physiological data. However, this lactic acid bacterium attracted interest as a potential D-lactic acid producer to be employed in sustainable production of exclusively one isomeric form of lactic acid. Only few papers report on use of L. coryniformis subsp. torquens in production of lactic acid with D-configuration which can be largely utilized in poly(lactic acid) synthesis. Further improvement of the bioprocess includes utilization of renewable resources as carbon and nitrogen sources and scale-up procedure. Before application of the strain in a large scale bioprocess better understanding of carbohydrate metabolism and its regulation is required. Our lab-scale experiments indicated that basic rules of energy-generating metabolism in the bacterial cells, based mainly on conversion of glucose presumably via glycolysis and phosphogluconate pathway, are not absolutely valid. Configuration of produced lactic acid and fermentation under carbohydrate limitation to (un)desired end-products were characterized. Further investigation was related to the impact of oxygen on D-lactic acid production and biomass yield. Flexibility of the metabolism under chosen conditions was probed in batch cultures at relatively low temperature. Our results can provide novel clues for the optimization of growth conditions of L. coryniformis subsp. torquens as well as other lactic acid bacteria as industrially relevant microorganisms.

Biomasa mikroalgi predstavlja vrijednu sirovinu za proizvodnju farmaceutskih, prehrambenih i kozmetičkih proizvoda (proteina, masnih kiselina, vitamina, anti-oksidansa, pigmenata, lijekova i imunostimulansa), te proizvodnju biogoriva (biodizela). Temelj učinkovite proizvodnje biomase mikroalgi za dobivanje biogoriva primjena je bioprocesno inženjerskih znanja povezanih s izborom mikroalge, hranjive podloge, uvjeta i postupka vođenja bioprocesa u različitim bioreaktorskim sustavima, te metode izdvajanja i pročišćavanja biomase mikroalgi. Kriteriji izbora mikroalge određeni su fiziološkim potencijalom i mogućnošću mikroalge da uspješno nakuplja lipide tijekom bioprocesa. Izbor hranjive podloge, uvjeta i postupka vođenja bioprocesa ključni su za proizvodnju biomase mikroalgi s velikim udjelom lipida. Za dobivanje što veće količine biomase mikroalgi s ciljem proizvodnje biogoriva koriste se različiti bioreaktorski sustavi s jednom ili više podjedinica koji su opremljeni različitim sustavima za nadzor bioprocesa. Osim uvjeta i postupka vođenja bioprocesa u bioreaktoru za uspješnu proizvodnju biogoriva potrebno je izabrati i adekvatne metode za izdvajanje i pročišćavanje biomase odnosno lipida iz biomase mikroalgi. Stoga je u ovom radu dan i pregled metoda za izdvajanje i pročišćavanje biomase odnosno lipida iz biomase mikroalgi nakon uzgoja u bioreaktoru.

Fossil fuels can be replaced by renewable raw materials and the raw materials can be used in production of several thousands of platform compounds and final products. Availability of particular renewable as well as climate, geo-political and economical environment defines boundaries of innovative technology to be implemented in such production. In addition, need for novel products and affiliated market dictate investments in plants for industrial-scale production of defined range of the platform compounds and other biochemicals.Biotechnological production of optically pure lactic acid, very important C3 platform compound, opens up numerous routes for its catalytic transformation and manufacturing commodity and high-value chemicals. Nowadays, the production of lactates depends on two main renewables - corn and sugarcane. The bioprocess sustainability has to be improved and moved to second and third generation of the raw materials. The sustainable technologies includes employment of efficient biocatalyst, mild bioprocess conditions, near 100% yields and relatively simple down-stream processes, which bring the system to hybrid integrated bioprocesses and, further, to factories of future.

Gushing is uncontrolled, eruptive foaming of beer after a bottle has been opened, without previous shaking. This is a phenomenon that affects breweries worldwide and all brands and types of beer. Gushing can be divided into primary (secretion of hydrophobins class II and other fungal metabolites from Fusarium fungi) and secondary gushing (calcium oxalate precipitations and metal ions). One of the methods to reduce the risk of gushing is the early detection of gushing potential of raw materials such as wheat or barley malt. The aim of this study was to determine the impact of wheat infection by Fusarium culmorum and the fungicide treatment on the gushing potential of wheat malt. Standard micro-malting procedure was performed using wheat variety „Lucija" (less susceptible to infections by Fusarium spp.), the sample „A" was the control sample, the sample „B" was treated with fungicide, sample „C" was infected with F. culmorum and treated with fungicide and sample „D" was infected with F. culmorum and was not treated with fungicide. Analyses were performed on dried and stabilized wheat malt samples in order to determine the correlation between the F. culmorum infection and the malt quality parameters (the share of soluble N in malt, Kolbach index, total proteins, soluble proteins and the gushing potential of malt). All analyses were done according to the methods from Analytica EBC. In conclusion, results obtained from this research indicate that the infection of wheat by F. culmorum significantly affects and increases the gushing potential of wheat malt, and this is particularly evident in samples „C“ and „D“ (high gushing potential). By overlooking the selected malt quality parameters (the share of soluble nitrogen in malt which ranges from 0.92 % in sample “A” to 0.98 % in sample “C” ; Kolbach index which ranges from 49 in sample “A” to 51 in sample “D” ; total proteins range from sample “A” - 11.8 % to sample “C” - 12.5 % ; soluble proteins range from 5.75 % in sample “A” to 6.12 % in sample “C”) it is evident that they are correlated with Fusarium infection, as that they are increasing in samples infected with Fusarium

A crisis of energy storing molecules (fuels) which are currently produced from crude mineral oil is expected in the future due to limited resources. One strategy to compensate a part of the oil deficiency is the production of biofules from lignocelluloses waste materials from agriculture and wood waste biomass. This work investigates and efficient and economical approach for the integrated enzymatic hydrolisation and fermentation of lignocellulosess (sugar beet pulp, corn cob and beech bark) to bioethanol through a new aspect of bioethanol production processes by investigating sustainability of the design of experiment to optimize the effectiveness of different enzymes (cellulase, pectinase, Ultrazym AFP.L and Viscozym L).

Ethanol production (as a biofuel) from renewable raw materials is still more expensive than gasoline production from fossil oil. Therefore, integration of different technological stages into one single stage could result in a more cost-effective and energy saving bioprocess. In this research, intermediates of sugar beet processing were studied as a substrate for ethanol production in order to reduce ethanol price as a biofuel. For ethanol production from the raw sugar beet juice in the stirred tank bioreactor batch and fed batch cultivation techniques were used. In horizontal rotating tubular bioreactor (HRTB) batch ethanol production from raw sugar beet cossettes was studied by different initial inoculum quantity (9.1 - 23.1 v/m). In both cases, Saccharomyces cerevisiae was used as a production microorganism. During batch ethanol production from the raw sugar beet juice, bioprocess efficiency was 59.48 % and in the fed batch process 72.49 %, respectively. At the same time, ethanol production in the HRTB from the raw sugar beet cossettes with inoculum of 16.7 % v/m (raw sugar beet cossettes) resulted in the highest bioprocess efficiency of 79.92 %. On the basis of experimental data it is clear that both intermediates of sugar beet processing can be successfully used for ethanol production.

The aim of this research was to show the applicability of the design of experiment (DOE) in the chemical, biotechnological and food industry. As a model experiment we present an optimization of the corrosion effects occurring in the stainless-steel coatings in the microbrewery tanks. For this purpose different concentrations of heavy metal ions (Al, Cu, Fe, Mo and Sn) were monitored in the water after rinsing of laboratory brewery vessel. Rinsing process was optimized considering the time of rinsing and the concentration of HNO3 acid in the solution prepared for the rinsing of brewery vessel. Experiment design, rinsing optimization and system prediction was made by using computer program Design Expert (version 7, State-Ease Company, USA). Optimization conditions were determined (optimal rinsing time and concentration of HNO3 acid in the solution prepared for vessel rinsing) using two criteria: minimum concentration of HNO3 allowed for satisfying vessel rinsing (according to the brewery sanitation protocol) and maximum concentration of HNO3 allowed (according to the Water Act NN 107/95, 150/05). The results of model prediction were tested by additional experiments, and very good agreement was found between modeled and experimental results. In addition, calculated optimized values satisfied the criteria of brewery sanitation so therefore they can be used in the brewery sanitation. Based on our results, we can recommend the DOE approach as a cost reductive and effective tool for optimization of complex systems such are occurring in different chemical, biotechnological and food industries.

Market for lactic acid and its biopolymers and consequently capacities for their production continue their steady growth since 2006. Versatility of chemical and pharmaceutical applications of (poly)lactic acid substantially support research activities and innovations focused on sustainable production of the acid from biological resources including waste. Submerged and semi-solid state simultaneous saccharification and fermentation of starch to lactic acid by amylolytic lactic acid bacterium were investigated and novel bioprocess was developed. Defined ratios of two stereoisomeric forms, D- and L-lactic acid, were produced in highly efficient direct conversion of starch and starch-containing raw material. Suitable analytical framework for monitoring key values in liquid and semi-solid phase was formed. Limited availability of amino acids in raw materials was identified as one of major parameter which constrained bacterial amylolytic and fermentative activity as well as efficiency of the semi-solid state bioprocess. Implementation of two different types of bioreactors, stirred tank bioreactor and horizontal rotating tubular bioreactor, was tested on the lab-scale and obtained results represent strong basis for efficient upscaling of the bioprocess and development of integrated bioprocess for lactic acid production.

U ovom istraživanju proveden je uzgoj alge Euglena gracilis s ciljem proizvodnje vitamina A i vitamina E. U svrhu određivanja najpovoljnijih parametara za proizvodnju vitamina u bioreaktoru s mješalom provedena je optimizacija sastava hranjive podloge i uvjeta uzgoja E. gracilis, u Erlenmeyer tikvicama na laboratorijskoj tresilici. Tijekom uzgoja alge E. gracilis u bioreaktoru s mješalom primijenjen je dvostupanjski uzgoj. U prvom stupnju proveden je fotoheterotrofni uzgoj na Hutner-ovoj podlozi, a u drugom stupnju miksotrofni uzgoj na podlozi s ograničenim izvorom ugljika. Primjena dvostupanjskog procesa osigurala je relativno visoku produktivnost proizvodnje vitamina A (PrA = 0, 07 mg/Lh) i vitamina E (PrE= 0, 27 mg/Lh), a prinosi su iznosili vitamina A (YA = 6, 53 mg/L) odnosno vitamina E (YE = 26, 26 mg/L). Visoki prinosivitamina E ukazuju na mogućnost primjene dvostupanjskog procesa njegove proizvodnje u industrijskom mjerilu.

Implementation of novel whole-cell catalyzed bioprocesses is considered as a key technology for conversion of renewable raw materials into biochemical(s). The microbial cells which synthesize so called critical enzymes (e.g. amylase, protease) have attracted special attention because they can be employed in direct conversion of the renewables within sustainable industrial-scale production. By their application conventional metabolic frame, intracellular metabolism of simpler substrates, can be connected to balanced extracellular degradation of polymeric substrates and considerably expanded. In the case of lactic acid bacteria (LAB) which produce at least one of the critical enzymes - amylase, it means that traditional fermentative industrial production of lactic acid (LA) can be expanded into amylolytic-fermentative bioprocesses. Amylolytic LAB (ALAB) Lactobacillus amylovorus DSM 20531T was used as a potent biocatalyst which possesses interdependent amylolytic-fermentative activity and it carried out highly efficient direct conversion of starch-containing renewable raw material to LA. Raw-starch binding alpha-amylase was produced by the ALAB in submerged fermentations and semi-solid substrate saccharification and fermentation [1]. The enzyme produced by L. amylovorus strains was only partially characterized [1-4]. Basic characteristics (reaction type, amino acid sequence, stability, etc.) of the enzyme have not yet been determined (BRaunschweig ENzyme DAtabase, http://www.brenda-enzymes.org). Amino acid sequence of forty alpha-amylases (EC 3.2.1.1) from genus Lactobacillus was accessed (2013-06-13) via The National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov) and compared by using Vector NTI® Express Software (http://www.invitrogen.com). Mining the alpha-amylases from Lactobacillus sp. represents key step in routing further investigation and application of novel biocatalysts in direct production of the LA from the renewables.

The aim of this research was to optimize and investigate effects of corrosion process during cleaning of stainless-steel coatings in microbrewery tanks and to predict their resistance to nitric acid in washing solution. Experimental conditions were limited with minimal concentration of acid that satisfied requirements of sanitation protocol, and maximal concentration of acid that did not cause corrosion and release of metals higher then prescribed law limits. Process was monitored by release of metal ions and optimized by design of experiment (DOE) using software Design-Expert. This approach proved to be very beneficial, cost reductive and effective for optimization of complex systems. The results of experiments prediction were compared to additional testing, and their matching proved the effectiveness of modeling. Therefore DOE is an effective tool for estimation of steel coating corrosion processes. In addition, optimized values satisfied required criteria, so the proposed protocol can be used in the effective sanitation of industrial tanks and pipelines in brewing industry.

U radu je opisana proizvodnja etanola na svježom repinim rezancima

Starch containing raw materials represent preferable ingredients of more or less complex media to be used in sustainable biotechnological production of lactic acid. After upstream processing, prepared starch-based medium can adopt structure of suspension, gel, or semi-solid material with relatively low content of free liquid (water), as well as a combination thereof. Utilization of such heterogeneous media in bioprocess for lactic acid production requires additional efforts related to: adaptation of bioreactor construction and the medium mixing, definition of mass and energy transfer, selection of applicable analytical methods, their adaptation for bioprocess monitoring, etc. Several analytical methods were selected, adapted and tested for monitoring of Simultaneous Semi- Solid State Saccharification and Fermentation (S5F) of starch to lactic acid. Suspension of corn grits in demineralized water that consisted of two easy-separable phases (liquid and semi-solid phase) was used as a minimal medium for direct production of D-/L-lactic acid by Lactobacillus amylovorus DSM 20531T. Settling rate of corn grits particles, absorbance maxima and initial composition of heterogeneous medium were determined. During the S5F the reduction of semi- solid phase portion and concentration of polymeric substrates in the two phases were followed by using customized analytical methods.

Biotechnological research and manufacture are currently focused on the production of fine chemicals, biofuels, biopolymers, different functional food products as well as on the environment protection. In order to achieve sustainability of these bioprocesses life cycle analysis (LCA) has to be performed and it should resulted in the improvement of bioprocess performance. In this work overview of previously mentioned bioprocesses will be done. Industrial production of bioethanol (as a biofuel) is currently based on the sugar and polysaccharides containing raw materials, but intensive research is performed on the lignocellulosic raw materials. Many companies and research institutions are working on the production of fine chemicals and one example of these products is β-glucan that could be produced by algae Euglena gracilis. Biopolymers production and their sustainability also attract a lot interest of companies and institutions and good examples are production of polylactic acid (PLA) and polyhydroxyalkanoates (PHA). Food companies and institutions continuously work on the improvement of the quality of different functional food products. Bioprocess engineers are working on new bioreactor constructions (especially for bioprocesses on the semi-solid and solid substrates) for different bioprocesses in order to improve the bioprocess performance. Heavy metals removal process is a good example of research in the field of environment protection.

The aim of this research was to apply statistical design of experiment for investigation of cleaning effects in microbrewery. The stainless steel resistance to nitric acid was monitored by determination of the release of metal ions (Al, Cu, Fe, Mo and Sn) in cleaning solution. Cleaning process was optimized by 25 preliminary experiments considering the time of rinsing and the concentration of HNO3 acid in the solution prepared for rinsing the laboratory vessel. Experimental design, rinsing optimization and system point prediction was made using computer program Design Expert (version 7, State-Ease Company, USA). Optimization conditions were determined (optimal rinsing time and concentration of HNO3 acid in the solution prepared for vessel rinsing) using two criteria: minimum concentration HNO3 allowed for satisfying vessel rinsing (according to brewery sanitation protocol) and maximum concentration of HNO3 allowed which will prevent corrosion and metal release into wastewaters (according to Water Act NN 107/95, 150/05). Calculated optimal values were afterwards proved by additional experiments, and very good agreement was found for majority of parameters. The obtained optimal results satisfied all necessary criteria, so the proposed protocol can be used in the effective sanitation of industrial tanks and pipelines in brewing industry. The design of experiment can be in addition recommended to other food industries for optimization of cleaning processes.

U ovom istraživanju proučavana je proizvodnja bioetanola na svježem soku šećerne repe s pomoću kvasca Saccharomyces cerevisiae. Za vođenje procesa proizvodnje bioetanola primjenjene su ove tehnike: šaržni proces, šaržni proces s pritokom supstrata i polukontinuirani proces. Tijekom šaržnog procesa proizvodnje bioetanola stupanj konverzije supstrata u produkt (YP/S) je iznosio 0, 375 g/g, učinkovitost bioprocesa (E) 69.70 %, te produktivnost (Pr) procesa 0, 53 g/Lh. U šaržnom procesu s pritokom supstrata dobivene su ove vrijednosti pokazatelja uspješnosti bioprocesa: YP/S = 0, 502 g/g, E = 93, 40 % i Pr = 0, 50 g/Lh. Nadalje, tijekom polukontinuirane proizvodnje bioetanola pokazatelji uspješnosti bioprocesa su imali ove vrijednosti: YP/S = 0, 360 g/g, E = 66, 91 % i Pr = 0, 66 g/Lh. Na osnovi prethodno prikazanih rezultata jasno je da šaržni proces s pritokom supstrata i polukontinuirani proces imaju značajno veći potencijal za primjenu u industrijskoj proizvodnji bioetanola iako je potrebna daljnja optimizacija polukontinuirane tehnike vođenja bioprocesa.

Utilization of different substrates (glucose, sucrose, maltose, celobiose, starch, microcristalline cellulose ; combination of the two substrates), as well as complex raw material (corn grits) by three lactic acid bacteria (LAB), Lactobacillus rhamnosus DSM 20021T, L. amylovorus DSM 20531T (1) and L. coryniformis subsp. torquens 20004T, was investigated in order to develop new bioprocess for lactic acid (LA) production. The bioprocesses were carried out in lab-scale stirred tank bioreactors and horizontal rotating tubular bioreactor (HRTB) (V= 5, 6, 10 i 12 L) in batch, fed-batch, semicontinuous and continuous mode. Procedures for preparation of (heterogenous) samples and their analysis by array of the methods, especially for the samples whithdrawn during semi-solid substrate simultaneous saccharification and fermentation (S5F), was adapted and optimized. Concentration of different carbon sources (glucose, fructose, sucrose, celobiose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, starch) and products (LA, acetic acid, ethanol) was determined by HPLC method (1, 2), while concentration of stereoisomers of D- and L-LA was estimated by enzymatic method (1, 2). Derivatization of free aminoacids in water solution, in different media and in corn grits suspension, as well as extraction of the derivates to organic phase were optimized. Fourteen out of twenty amino acid esters were detected and their concentration was determined by using optimized GC method (3). Beside analysis of liquid phase (supernatant), content of (semi-)solid phase of heterogenous samples was estimated (portion of solid particles, concentration of reducing sugars, concentration of nitrogen, thickness of biofilm formed on the inner surface of HRTB). Amylolytic LAB L. amylovorus DSM 20531T catalyzes S5F and different ratios of D- and L-LA were produced, depending on carbon source and temperature of cultivation (1, 2, 4). Different substrates were fermented by L. rhamnosus DSM 20021T to L-LA while L. coryniformis subsp. torquens 20004T produces only D-LA. Amylolytic enzymes produced by L. amylovorus DSM 20531T were partially purified by affinity chromatography. Starch was hydrolyzed by purified amylases in similar manner as it was case with α- and β-amylase isolated from Bacillus sp. and barley, respectively. The tree LAB strains can be distinguished by spectrum of used substrates, consumption of metabolic energy in synthesis of bacterial biomass and/or in synthesys of amylolytic and/or proteolytic enzymes (5) and, thus, employed in different bioprocesses for LA production.

U ovom istraživanju provedeno je ispitivanje utjecaja ukupne koncentracije kisika u staklenoj boci na kakvoću piva donjeg vrenja tijekom skladištenja. Pivo je bilo skladišteno na temperaturi od 22 - 25°C u tamnoj prostoriji, a u pivu je bio prisutan kisik u tri različite ukupne početne koncentracije: 0, 09 mg/L, 0, 29 mg/L i 0, 63 mg/L. Tijekom skladištenja (98 dana) u pivu je praćena promjena koncentracije trans-2-nonenala kao indikatora procesa oksidacije (starenja) piva, a dobiveni rezultati pokazuju da koncentracija trans-2-nonenala nije direktno ovisna o ukupnoj koncentraciji kisika u boci piva. U ovom istraživanju provedena je i senzorska analiza uzoraka piva koja je pokazala da povećane ukupne koncentracije kisika u boci izravno utječu na ocjenu kakvoće piva.

Sugar beet pulp (SBP) was hydrolyzed with combinations of commercially available fungal enzymes (Ultrazym AFP-L, Viscozyme L, Pectinase and Cellulase) and wood-rotting fungi (Trametes multicolor, Sclerotium rolfsii and Neurospora crassa) lignocellulolytic enzymes. Among the tested enzymes, the plant pathogen Sclerotium rolfsii was found to be the best-performing fungus and it was chosen for further research. Current research activities were focused on the optimization of the hydrolytic process using a factorial design approach and response surface methodology (RSM). The effect of various experimental parameters and their interactions with each other was evaluated using those methods and consequently the optimum conditions were accomplished. The set-up parameters were the enzyme loads (1, 2, 3, 4 μL total enzymes / mg SBP), pH (3, 4, 5, 6, 7) and temperature (20, 30, 40, 50 oC). Using statistical design methodology, it was observed dramatically reduce of the required enzyme loads to only 2 μL total enzymes / g SBP, at the optimal pH and temperature value. Optimal parameters of hydrolytic process would apply in the further investigation of SBP hydrolysis to obtain more economical process for biofuels, biopolymers or biochemicals production.

Enzimska hidroliza pulpe šećerne repe (PŠR) provedena je u Erlenmeyer tikvicama na laboratorijskoj tresilici, korištenjem enzimskih otopina pripravljenih tijekom uzgoja Trametes multicolor, Sclerotium rolfsii i Neurospora crassa na različitim supstratima. Najbolji rezultati ostvareni su korištenjem enzimskih otopina Sclerotium rolfsi uzgojene na PŠR i celulozi. Optimalni uvjeti hidrolize u tikvicama na laboratoriskoj tresilici ostvareni su u vodenim otopinama s 66 g/L PŠR pri 40 °C, 150 okr/min i koncentraciji enzima od 5 ml/L. Povećanje koncentracije enzima nije pogodovalo povećanju koncentraciji reducirajućih šećera, ali je kombinacijom enzimskih otopina S1c i S1sb te dodatkom pulpe u obrocima tri puta po 22 g/L odnosno 44 g/L PŠR ostvareno povećanje koncentracije reducirajućih šećera na razinu pogodnu za proizvodnju bioetanola (52, 46 g/L).

The objective of this study was to compare the ethanol separation from aqueous solutions by extraction and gas stripping process. Liquid–liquid extraction (LLE) of ethanol was performed using 1-octanol as the extractant. The distribution coefficient of ethanol in water /1-octanol system was determined (KDE = 0, 6). Equilibrium distribution coefficients for ternary system (ethanol + 1-octanol + water) were determined at 25 °C under atmospheric pressure and related separation factor for extractive ethanol removal from ethanol-water mixture have been calculated (α = 6). Ethanol elimination from aqueous solutions by gas stripping (GS) in the stripping column was investigated. A stream of air, nitrogen and carbon dioxide was sparged through a stripping column with the porous sintered glass (porosity 1) as gas sparger in order to remove the ethanol from the same aqueous solution as by LLE. Mass transfer coefficients of ethanol for this system were determined. LLE and GS were integrated in biotechnological process of bioethanol production. Mathematical model of simultaneous extraction-fermentation-product-recovery system (SEFPR) for ethanol production from sugar beet cossettes was used for the mathematical description of integrated bioprocess and for the calculation of its benefits (in order to compare new process toward classical ethanol production).

The production of sustainable liquid fuels such as bioethanol from renewable resources has become a primary worldwide concern. To date, the major problem of biorefinery processes is the high production cost of enzymes used for the degradation of lignocelluloses into fermentable sugars. Here we report the production of lignocellulolytic, hemicellulolytic and pectinolytic enzymes by three fungi using sugar beet pulp as relatively cheap and readily available substrate. The fungi under investigation were the white-rot basidiomycete Trametes multicolor and the plant pathogens Sclerotium rolfsii and Neurospora crassa. In cultivation experiments the activities of extracellular enzymes, namely cellulases, pectinases, xylanases, arabinases, mannanases, manganese peroxidase, laccase and cellobiose dehydrogenase were differently induced and monitored. By optimization of the cultivation conditions highly efficient enzyme preparations were obtained, which have hydrolytic activity towards sugar beet pulp. The highest arabinase (1.3 U mL−1), pectinase (0.78 U mL−1) and carboxymethyl cellulase (0.49 U mL−1) activities were observed in cultures of Sclerotium rolfsii grown on sugar beet pulp. The highest xylanase (0.88 U mL−1), manganese peroxidase (0.3 U mL−1) and laccase activities were detected in Trametes multicolor cultures. Neurospora crassa does not perform equally well as the other two fungi. The clearified culture supernatants were used to degrade sugar beet pulp enzymatically and found to hydrolyze all polymers efficiently and fast.

Ethanol as biofuel can significantly reduce the negative environmental impacts of fossil fuels. However, bioethanol production from renewable resources is still more expensive than gasoline production from fossil oil. Integrated bioprocess combines different steps in bioethanol production (fermentation, separation) in one single unit thus making it more cost-effective and energy saving. Different cultivation techniques, weather using liquid or solid substrate, can significantly increase ethanol yield and consequently improve the performance of the process. Production of ethanol on the raw sugar beet juice was studied. For ethanol production on the raw sugar beet juice batch and fed batch cultivation techniques were used during research in the stirred tank bioreactor. Saccharomyces cerevisiae was used as a working microorganism. All concentrations (supstrate and products) were measured using HPLC. Experimental data served for the development of mathematical models describing supstrate utilization and product and biomass formation via computational program Madonna. During batch ethanol production on the raw sugar beet juice ethanol yield was 59.89 g/L and efficiency 78.8 %. In fed batch process ethanol yield was 92.78 g/L and efficiency 93.4 %, respectively. Unstructured kinetic models correspond well with obtained experimental data. Experimental results suggest that sugar beet juice can be successfully used for ethanol production.

Sugar beet pulp (SBP) is a valuable lignocellulose’s waste raw material for bioethanol production. Bioethanol production process consists of following steps: lignocellulose pretreatment, fermentation and bioethanol separation and purification. Lignocellulose pretreatment is the main step for successful bioethanol production and wide investigation efforts were performed in order to develop the new efficient and cheap pretreatment method. Different physical, chemical and biological pretreatment methods could be used depending on the lignocellulose material source and composition. In this study, combination of physical and biological pretreatment was utilized in two consecutive steps. In the first step, thermal treatment or ultrasound disruption and in the second step enzymatic hydrolysis was used, respectively. For the enzyme hydrolysis commercial cellulases as well as combination of commercial cellulases and pectinases were utilized. Ultrasound disruption of SBP was carried out by ultrasound homogenisator SONOPLUS HD 2200 in continuous mode for 15 min with 50% of maximum power (max. 0.2 kW). Thermal treatment was performed at 121 OC for 15 min. Commercial cellulases (CN) and pectinases (PN) were used for the hydrolysis of SBP. Determination of hydrolyzed substrates and fermentation products was performed by high-pressure liquid chromatography (HPLC). It is obvious from obtained results that both pretreatment methods (thermal and ultrasound) of SBP have great potential for successful degradation of lignocellulose’s raw material. This observation can be most easily proved by the increase of mono– and disaccharide concentrations in cultivation medium. The extraction of remain sucrose (Suc) from SBP was considerably supported by the ultrasound treatment of SBP. Degradation of the lignocelluloses components of SBP to glucose (Glc) and galactose (Gal) was significantly increased by the thermal treatment. The treatment of SBP with celluloses and pectinases was related to the increase of glucose (Glc), galactose (Gal), arabinose (Ara) and galacturonic acid (GalA) concentrations. On the basis of obtained results it is clear that the combination of thermal treatment and enzyme hydrolysis is the most efficient SBP pretreatment method for bioethanol production.

INTRODUCTION The application of ultrasound to biological systems offers a range of possibilities in food technology and biotechnology. The biological parameters which influence the intensity of the ultrasound effects are: microbial strain tested, the suspending medium, the size of the cell. The objective of this research was to study the possible lethality of the combined effect of ultrasound and mild heat treatment on brewer’s yeast Saccharomyces uvarum viable cells suspended in wort. The effect of the amplitude of the sonication waves was also evaluated. METHODOLOGY Ultrasound at 20 kHz and 108 m (25 %), 173 m (40 %) and 260 m (60 %) of wave amplitude was applied to the brewer’s yeast S. uvarum cells wort suspension (pH 5.6 ; temperature 45 and 55 oC) with an immersed 13-mm diameter probe (1-10 min). Decimal reduction time (D) value was calculated at a given temperature and sonic wave amplitude. Determinations of cell size and percent of dead cells (methylene blue staining) were measured by light microscopy with digital camera and recorded onto a PC. RESULTS AND DISCUSSION The resistance of S. uvarum cells suspended in wort at pH 5.6 to the action of ultrasound (20 kHz, wave amplitude in the range 108-260 m) was analyzed at 45 and 55 oC. The inactivation rate where a first-order kinetic was observed exhibited D values between 1 and 25 min. At 45 oC, D values were decreased by the simultaneous effect of ultrasound from 19.6 to 14.5, but at 55 oC no advantages were obtained by adding sonication. Physiological studies of cell size and percent of dead cells proved that the ultrasonic waves do not destroy the brewer’s yeast cells ; they damage them, thus increasing their activity to heat. However, when ultrasound was applied at 55 oC, no structural differences were appreciated between sonicated cells and only heat-treated cells. CONCLUSIONS 1. Studies of survival of S. uvarum suspended in wort at 45 and 55 oC at different ultrasonic wave amplitude show that application of ultrasound at non-lethal temperature (45 oC) did not display a deactivation action while synergy between ultrasound and heat was confirmed at the 55 oC. 2. Determinations of cell size and percent of dead cells proved that the ultrasonic waves do not destroy the brewer’s yeast cells ; they damage them, thus increasing their activity to heat.

In the future, due to limited resources a crisis of energy storing molecules (fuels), which are currently produced from crude mineral oil, is expected. One strategy to compensate a part of the oil deficiency is the production of biodiesel from microalgae lipid. In this research, algae Euglena gracilis was chosen as model microorganism for lipid production. Photomixotrophic cultivation was done in self-constructed photobioreactor. During cultivation, chlorophyl, vitamine (E, A), biomass, substarte and cultivation conditions (temperature, pH, rpm, pO_2, CO_2, hν) were monitored. Due to the optimization of medium composition and cultivation conditions, lipid production was increased from 0.4 % to 60 % of biomass dry weight. Promising results obtained in this reserch encouraged us for futher investigation.

The bioethanol production on sugar beet pulp (SBP) can be divided into three main steps: SBP pretreatment, fermentation and ethanol recovery. In this research, different pretreatment methods were used to hydrolyze celluloses and pectin fractions of dried sugar beet pulp into fermentable sugars. Comparison between enzymes and acid pretreatment methods pointed out that higher sugars production efficiency was achieved by enzymes pretreatment method (pH 3.5-5 and temperature 30-50oC). Different combinations (ratio and concentration) of proteases, cellulases and pectinases were examined in order to define the most suitable enzymes combination for hydrolysis of SPB. In this research, for fermentation of sugar beet pulp Saccharomyces cerevisiae was used. During fermentation of untreated SBP ethanol yield was only 0.3 g/L. In fermentation process of enzymes pretreated SBP at 40oC and pH 4.8 with the most suitable enzymes combination considerable increase of ethanol yield (8 g/L) was observed. At the same time, the conversion rate of fermentable sugars into ethanol was 0.13 g ethanol/g dry weight SBP.

Pollution by heavy metals is a major environmental problem because they are not subject to natural degradation and therefore it comes to their bioaccumulation in tissues, what can cause serious health problems. Generally used bioreactors for metal removal from waste water include rotating biodisc reactors, trickle filters, fixed bed and fluidized beds, air- lift reactors and other biofilm reactors [1]. In this research, removal of metal (Co, Cr and Mn) by biosorption process in horizontal rotating tubular bioreactor (HRTB), was monitored. HRTB is designed as a combination of thin-layer and biodisc reactor and therefore has characteristics of both reactors. Discs are used as carriers for microbial biomass, which removes metals present in water. Biosorption can be defined as the removal of metal or metalloid species, compounds and particles from solution by biological material [2] and it was monitored by thin-layer chromatography. Before analysis of waste water samples, it was necessary to determine the optimal chromatographic system for separating metal ions. In optimization procedure, binary, ternary and quaternary solvent systems with different volume shares were used. Total mixture volume was 10 mL. Therefore, standard solutions of metal salts in distilled water were prepared in range of 0.1-1.0 g/L. These solutions were used for optimization and metal ion identification during real-sample analysis. Based on the obtained chromatograms, calculated retention factors (RF values) and plate development time, the adequate chromatographic system for separation and identification of analysed metal ions was chosen.

In this investigation, the production of ethanol on the raw sugar beet juice as well as on the raw sugar beet chips was studied. For ethanol production on the raw sugar beet juice batch and fed batch cultivation techniques were used during research in the stirred tank bioreactor. However, batch ethanol production on the raw sugar beet chips was carried out in horizontal rotating tubular bioreactor. In both cases, Saccharomyces cerevisiae was used as a working microorganism. During batch ethanol production on the raw sugar beet juice ethanol yield was 59.89 g/L and efficiency 78.8 %, respectively. In fed batch process ethanol yield was 92.78 g/L and efficiency 93.4 %, respectively. At the same time, ethanol production in HRTB on the raw sugar beet chips with 16.7 % v/m (raw sugar beet chips) inoculum resulted in the highest ethanol yield of 54.53 g/L and efficiency 79.5 %, respectively. Obtained results also clearly show that both intermediates of sugar beet processing can be successfully used for ethanol production.

The main goal of this research was to define the most favorable cultivation procedure for production of β-1, 3-glucan (paramylon) by heterotrophic cultivation of algae Euglena gracilis in stirred tank bioreactor. Paramylon is biologicaly active polysaccharide possessing imunostimulating and an antitumor effect, lowers the blood cholesterol level and his sulphur derivates inhibit the HIV-virus activity. In this investigation algae E. gracilis was cultivated on synthetic (Hutner) medium with different carbon sources (glucose, ethanol and Na-acetate) as well as on complex medium with different industrial by-products (e.g. molasses and corn steep liquor). Batch and continuous cultivation techniques were used for E. gracilis cultivation. The highest bioprocess efficiency parameters were observed during batch cultivation on complex medium with 20 g/L glucose and 35g/L corn steep liquor.

Nitrogen is one of the most commonly occurring elements in nature and forms many inorganic ionic species, such are ammonium, nitrate and nitrite ions, being most important. The simultaneous determination of ammonium, nitrate and nitrite ions in water, food, biological and environmental matrices is of vital significance due to their toxicity to humans (infants) and water systems (eutrophication). Ion–selective electrodes (ISE) are a promising approach because of their small size, rapid response, and ability to directly measure the analyte. In this study the simultaneous determination of ammonium, nitrate and nitrite ions was performed in modelled and real systems (wastewater) using commercial ISE (ELIT, NICO2000 Ltd.).

Nitrogen is one of the most commonly occurring elements in nature and forms many inorganic ionic species, such are ammonium, nitrate and nitrite ions, being most important. The simultaneous determination of ammonium, nitrate and nitrite ions in water, food, biological and environmental matrices is of vital significance due to their toxicity to humans (infants) and water systems (eutrophication). Ion-selective electrodes (ISE) are a promising approach because of their small size, rapid response, and ability to directly measure the analyte. In this study the simultaneous determination of ammonium, nitrate and nitrite ions was performed in modelled and real systems (wastewater) using commercial ISE (ELIT, NICO2000 Ltd.). The used ISE respond to ammonium, nitrate and nitrite ion according to the Nernst equation. The accuracy of determination (95, 8% - 101, 0%) can be considered satisfactory for the ammonium, nitrate and nitrite determination in complex matrices. A satisfactory fit of the theoretical models to the experimental values was obtained in all cases for the tested sensors and calibration parameters.

β -glukani se u prirodi mogu naći u različitim prirodnim izvorima i velik broj istraživanja pokazao je njihove jake imunostimulacijske učinke u različitim organizmima. β -glukani mogu naći primjenu u medicini i farmaciji, prehrambenoj, kozmetičkoj i kemijskoj industriji, kao i u veterinarskoj medicini i proizvodnji krmiva. Iskorišteni pivski kvasac, dobiven pri proizvodnji piva kao sekundarni proizvod, može se koristiti kao sirovina za izdvajanje β -glukana. Unatoč činjenici da se velike količine pivskog kvasca koriste kao krmivo, određene količine se još uvijek tretiraju kao tekući otpad. β -Glukan je jedna od komponenata koja može postići veću komercijalnu vrijednost nego sam pivski kvasac te tako može maksimalizirati ukupnu profitabilnost procesa proizvodnje piva. Stoga proizvodnja β -glukana, kao i drugih visokovrijednih proizvoda baziranih na β -glukanu, iz iskorištenog pivskog kvasca predstavlja isplativ tehnološki i ekonomski izbor za pivovare.

In this work, the bioadsorption process was studied in a pilot scale horizontal rotating tubular bioreactor (HRTB). On the basis of the results obtained via inductively coupled plasma sector field mass spectrometry an integrated mathematical model of heavy metal removal (Co2+, Cr6+ and Mn2+) in HRTB was established. It combines hydrodynamics (mixing) and kinetics in order to define bioprocess conduction in the bioreactor. Mixing in HRTB was described by a structured cascade model and metal ion removal by a combined diffusion-bioadsorption model, respectively. Differences between experimental and simulated metal ion concentrations were in the range of the experimental uncertainty, demonstrating that the established bioprocess model successfully predicts metal ion concentrations in HRTB over a wide range of process parameters [rotation speed (n = 5 - 30 min-1) and inflow rate (F = 0.5 - 2.0 L h-1)]. Furthermore, the obtained results also pointed out that the established model is accurate and robust and has therefore a high potential for use in a scale-up procedure.

Fermentation of food and beverages is one of the oldest ways of food processing. Popular products such as pickle, beer, bread, wine, cheese, sour milk and sausages have been around for millenia. Strictly speaking, the term "fermentation" relates to anaerobic energy metabolism only. Presently, fermentation of foods is defined more broadly as bioprocessing, using microorganisms and their enzymes to achieve desirable quality characteristics. Among the microorganisms all groups, namely bacteria, yeast and moulds are encountered as functional microorganisms in food processing. This lecture deals with: 1. the use of molecular techniques to monitor the fate of functional as well as contaminating microflora during processing and storage of the fermented products, 2. the contribution of fermented food (components) to human and animal health, 3. the legal aspects of the use of functional food (ingredients) and international legislation regarding labeling and the tailor-made design of fermented products, 4. the composition of fermented food products and the role that “ omics” can play in the future development of safe and healthy food and 5. the short review of our (Department of Biochemical Engineering) results in the area of biotechnological production of food and drinks.

Azotobacter chroococcum has ability to fix nitrogen from the atmosphere and therefore it could be used as an ingredient of biofertilizers. In order to use bacterial biomass for above mentioned purpose cultivation of A. chroococcum was done in different experimental conditions. During this research, optimization of chemically defined and complex media content were performed to define the most appropriate media content for bacterium cultivation in large scale. In this investigation, different cultivation techniques (batch, fed batch and repeated batch) were used for cultivation of bacterium A. chroococcum. The growth of bacterium A. chroococcum was monitored by determination of substrate and biomass concentration as well as other process parameters (e.g. pH, pO2 etc.). Comparison between different cultivation techniques was done on the basis of bioprocess efficiency parameters and it was observed that repeated batch technique is the most efficient and appropriate for use in industrial scale.