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UTILIZATION OF FOOD PROCESSING WASTE FOR LACTIC ACID AND ETHANOL PRODUCTION
Hudečková, Helena ; Kráčmar, Stanislav (referee) ; Buňka, František (referee) ; Márová, Ivana (advisor)
The doctoral thesis is focused on the microbial production of lactic acid and ethanol using food processing waste as substrate. Coffee processing waste (spent coffee grounds), wine production waste (grape pomace) and orange processing waste (orange peel) were chosen as substrates for experiments. The theoretical part is dedicated to summarizing current knowledge about waste from food production and possibilities of its processing. It also deals with selected metabolites (lactic acid, ethanol) to which these wastes can be used. Part of the experiments was focused on the characterization and optimization of hydrolysis to maximize the amount of fermentable saccharides. Different combinations of chemical, physical and enzymatic hydrolysis of selected substrates have been tested. Subsequently, a suitable strain for lactic acid and ethanol production was searched for. In the case of lactic acid production, 7 bacterial strains were selected (Lactobacillus casei CCM 4798, Bacillus coagulans CCM 2013, Bacillus coagulans CCM 2658, Lactobacillus rhamnosus CCM 1825T, Lactobacillus delbruckii subsp. bulgaricus CCM 7190, Lactobacillus plantarum CCM 7039T, Streptococcus thermophilus CCM 4757). These strains were first cultivated on the synthetic media containing different kind of saccharides. Afterward, the cultivation on the waste biomass hydrolysates were tested. In the case of ethanol production, 2 yeast strains kmeny (S. cerevisiae CNCTC 6646 a S. cerevisiae CNCTC 6651) were cultivated on hydrolysates of individual waste substrates. Subsequently, the experiments focused on the production of lactic acid and ethanol on hydrolysates of waste biomass in bioreactor were done. The last part of this doctoral thesis deals with the microaerobic pretreatment of lignocellulosic biomass to increase the production of organic acids during the acetogenic phase of anaerobic digestion.
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The Application of Separation Techniques Based on Gas and Liquid Chromatography with Different Types of Detectors for the Determination of Biologically Active Compounds and Selected Xenobiotics
Mravcová, Ludmila ; Hajšlová, Jana (referee) ; Kráčmar, Stanislav (referee) ; Chýlková,, Jaromíra (referee) ; Vávrová, Milada (advisor)
This work deals with the using and application of separation techniques for analysis of polymers degradation and polycyclic aromatic hydrocarbons. Thereby this work is separated to two special parts. In the first part, the degradation properties of synthetic biopolymers based on lactic acid, gylcolic acid and poly(ethyleneglycol) PLGA-PEG-PLGA and ITA-PLGA-PEG-PLGA-ITA (modified by itaconic acid) were studied. These copolymers (firstly their thermosensitive hydrogels) should be used for therapy of fractures in orthopedy (as adhesives). Therefore, the sol-gel and gel-sol phase diagrams were determinated for selected samples of copolymers. The samples forming gel at 37 C was used for other study. Polymer samples were depredated in phosphate buffer at 37°C. The degradation process of physical hydrogels was described by the decrease of molecular weight and the increase of concentration lactic acid and glycolic acid in phosphate buffer. The obtained results confirmed that the degradation of polymer modified by itaconic acid is faster process than no modified polymer and polymers with lower ratio PLGA/PEG degrade also faster than lower ration PLGA/PEG. The influence of pH it was also tested. The rate of degradation of polymers was follow pH 4,0
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Study of Factors Influencing Creation of Volatile Aroma Active Compounds in Natural Materials
Loupancová, Blanka ; Vávrová, Milada (referee) ; Kráčmar, Stanislav (referee) ; Fišera, Miroslav (advisor)
The production of high-quality foods requires the precise control of factors influencing their quality. The aim of this doctoral thesis was to monitor the influence of sterilization heating and storage time and temperature on selected parameters of model food matrix using suitable physical, chemical and sensory methods. Sterilized processed cheese was chosen as the model matrix. Processed cheeses are milk protein concentrates, produced by melting of mixture of natural cheeses, emulsifying salts, water and other dairy and/or non dairy ingredients. Desirable transformation of this mixture to homogenous, smooth, shiny mass with desired textural, structural, rheologic and sensory properties is caused by using high temperature (about 80-100 °C) during melting. Sterilized processed cheese is subjected to additional sterilization heating (above 100 °C) after production, significantly elongating its durability. The processed cheeses (dry matter 40 % w/w, fat in dry matter 45 % w/w) were analyzed in this work, the part of them was sterilized (117 °C 20 min). Cheeses were stored at various temperatures (cold 6 ± 2 °C, laboratory 23 ± 2 °C and elevated 40 ± 2 °C), sterilized cheeses for 2 years, non sterilized for 1 year. During storage, the samples for physical (instrumental measuring of colour and texture), chemical (assessment of fatty acids and aroma compounds) and sensory (paired preference test, ranking test and evaluation using scale) analyses were taken at regular intervals. The influence of sterilization heating and for another the influence of storage conditions on mentioned parameters were investigated. Non sterilized processed cheese kept their very good quality during all the declared durability (4 months), however, then the significant worsening of most sensory properties was observed. In consequence of sterilization heating the changes of cheese colour and texture were observed, confirmed by both sensory and instrumental assessment. The impairment of taste and aroma was also found. These changes are obvious immediately after production and differences were observed during all the durability. In the case of sterilized processed cheese, the significant impairment of single sensory properties was determined, the highest in the case of texture, taste and aroma. The results of instrumental techniques confirm conclusions from sensory evaluation. The measurements of colour and texture vindicate the formation of darker colour (increasing total difference of colour delta E*) and higher hardness (increasing Fmax) of sterilized processed cheeses, continuing during storage. The worsening of taste and aroma is in accordance with lower content of aroma active compounds and selected fatty acids.
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Metabolism of Bacterial Cells and the Effect of Stress on Biosynthesis of PHA
Kučera, Dan ; Kráčmar, Stanislav (referee) ; Ondrejovič,, Miroslav (referee) ; Obruča, Stanislav (advisor)
This thesis deals with the study of polyhydroxyalkanoate biosynthesis as a microbial product with the potential to replace current conventional plastics made from petroleum. The dissertation thesis is elaborated in the form of a discussed set of already published publications, which are then part of the thesis in the form of appendices. The work builds on relatively extensive knowledge in the field of polyhydroxyalkanoate production and brings new facts and possible strategies. Various possibilities of analysis of polyhydroxyalkanoates using modern methods were tested in this work, which brings especially speed, which can be crucial in real-time evaluation of production biotechnological process. Raman spectroscopy has proven to be a very promising technique for rapid quantification of PHA. Furthermore, the work deals with valorisation of waste of food and agricultural origin. Emphasis is placed on methods of detoxification of lignocellulose hydrolysates. In this context, adsorption of inhibitors to lignin was first used as an alternative to other detoxification techniques. Due to detoxification, selected production strains Burkholederia cepacia and B. sacchari were able to utilize softwood hydrolyzate for PHA production. In the next part of the work was also tested the possibility of using chicken feathers as a complex source of nitrogen. Evolutionary engineering was also used as a possible strategy to eliminate the inhibitory effect of levulic acid as a microbial inhibitor that results from the hydrolysis of lignocellulosic materials. Adaptation experiments were used to develop strains exhibiting higher resistance to levulic acid and the ability to accumulate a higher 3HV copolymer from the original wild-type C. necator strain. Another promising approach tested in the work was the use of extremophilic microbial strain, which leads to a reduction in the cost of biotechnological production. Selected Halomonas species have shown high potential as halophilic PHA producers. The final part of the thesis was devoted to the selection of the production strain with regard to the properties of the resulting PHA. The Cupriavidus malaysiensis strain was selected to produce a P(3HB-co-3HV-co-4HB) terpolymer which revealed significant differences in material properties over P3HB.
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Physicochemical Aspects of Ecological Heat-transfer Fluids
Skolil, Jan ; Kráčmar, Stanislav (referee) ; Vávrová, Milada (referee) ; Čorňák, Štefan (referee) ; Čáslavský, Josef (advisor)
This thesis develops composition of heat transfer fluids with antifreeze properties next to traditional one component glycols produced from propene and ethene. Based on assessment of physical properties it considers application of progressive antifreeze compounds of glycerine and propan-1,3-diole and their mixtures with classic glycols, which do not have negative influence on the environment and which are produced in last years from renewable sources. Due to the necessity of selection of certain compromise between their heat transfer properties, price, solubility and other factors physical properties of selected mixtures are determined together with synergy of each compound, including formulas with glycols: ethan 1,2 diol and propan 1,2 diol Another evaluated factor of studied formulations in addition to their heat transfer properties was anticorrosive protection of common corrosion inhibitors mixtures. The result of whole work is the proposal of several most perspective formulations based on mixtures of polyhydric alcohols in terms of their features enabling them heat transfer, reasonable costs, and the restrictions of impact of their use, particularly in heat transfer systems, to human health and the environment. The knowledge of physical properties of water solutions of polyhydric alcohols mixtures also helps to better using of recycled materials from planning recycling plant for glycols.
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Study of Exposure PBDE and Perfluorinated Compounds into Aquatic Ecosystem
Vondráčková, Ilona ; Chýlková, Jaromíra (referee) ; Hroch, Martin (referee) ; Kráčmar, Stanislav (referee) ; Vávrová, Milada (advisor)
In the presented PhD. thesis, problems covering occurrence of polybrominated diphenyl ethers (PBDE) and perfluorinated compounds in the environment were solved. The study was focused on identification and further verification of optimal methods in order to determine PBDEs and perfluorinated compounds sampled from the aquatic ecosystem. Polybrominated diphenyl ethers belong to persistent compounds classified as main pollutants; within the environmental constituents, they have been observed particularly in the last decade. For these studies, there were selected surface water matrices and sediments taken in various localities within the river Svratka basin. The analyses were to demonstrate whether they accumulate and remain in these specific matrices for longer time. There we assessed the congeners of polybrominated diphenyl ethers as follows: BDE-28, 47, 66, 85, 99, 100, 153, 154 and 183. In order to isolate them from matrices, various extraction techniques were used, i.e., ultrasonic extraction, microwave extraction, and pressure solvent extraction. Gas chromatography method with electron capture detection (GC/ECD) was selected for determination. The accomplished studies also assessed basic chemical, physical and environmental characteristics of diphenyl ethers in the environmental constituents. The attention was also paid to perfluorooctanoic compounds (PFOA), (PFOS) and (FOSA); physico-chemical properties of these organic pollutants were characterised and their toxicological and environmental aspects were evaluated. Usability of extraction techniques (ultrasonic extraction, pressure solvent extraction, solid phase extraction) applied to PFCs isolation from sediments samples was assessed. Identification and quantification of these analytes were performed using a high performance liquid chromatography/mass spectrometry method (HPLC/MS). After that, the optimized methods applied to real samples. Surface water and sediments were sampled within the river Svratka basin, in particular, from 19 sampling localities. PBDE occurrence was confirmed in sediments samples from the Svratka river (g.kg-1), PBDE were not detected in the surface water samples, their concentrations were below the detection limit, resp.; perfluorinated compounds were not detected in sediments and water samples at all.
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The Study of Distribution of Halogenated Diphenylethers to Parts of Environment
Hroch, Martin ; Hajšlová, Jana (referee) ; Kráčmar, Stanislav (referee) ; Šucman, Emanuel (referee) ; Vávrová, Milada (advisor)
In recent years considerable attention is paid to problems of brominated flame retardants (BFR's – Brominated Flame retardants), which are a diverse group of organic compounds. Even in the recent past have been the most often represented group particularly polybrominated diphenyl ethers (PBDEs), which have found wide use in many industrial sectors. The reason of aplication of these substances is the ability to slow down the combustion process and reduce the risk of ignition by the excessive heating of material. On the other hand, serious concerns about the use of PBDEs was added. The most serious trouble is particular their possible to release from consumer products during their normal use, toxicity and high persistence in parts of environment. Some of these contaminants are more characterized by a high degree of accumulation in biological systems. In this work by several sub-studies the issue of assessment the level of pollution of the aquatic ecosystem of selected localities of the Czech Republic just PBDEs was addressed. One of objectives was monitored the situation of breeding ponds near the village Záhlinice in Central Moravia. Here was assessed level of contamination in fish and birds as two consecutive segments of food chain. The obtained results largely confirmed the assumption that with increasing trophic level of organisms leads to bioaccumulation and thus also higher findings. For comparison, the capture of fish and birds in other localities of the CR was also implemented. In Bartošovice and Hustopeče nad Becvou levels at the buzzard and heron were detected as comparable. The cormorants of the order higher levels of PBDEs were found. Further were also evaluated levels of PBDEs detected in individuals of bioindicator kind of chub caught in two locations on the river Svratka in spring and autumn. Findings of PBDE in muscle, skin and intestines were compared. The obtained results show that between tissues and sites wasn’t significant variability respectively and the findings of controlled substances are practically comparable. With aim to determine the dependence of the concentration of PBDEs in organisms on the length of the river was conducted monitoring of Vír and Brno water reservoirs. In both locations was main monitored kind of bream, which was completed by the other species. The results of the analysis indicate a higher total concentration at the lower part of river Svratka. Differences were also the distribution of individual congeners in both sampling locations. For monitoring of terrestrial system contamination levels of PBDEs has been selected several types of plant bioindicators. These are mainly pine needles (Scots pine, Eastern white pine, Silver fir, Blue spruce) collected from different localities of the Czech Republic. Other matrices were chosen roughages (Common wheat, Naked barley, Alfalfa wheat, Red clover) and oilseeds (Rapeseed) from the Novy Jicin, where Agriculture plant school of the University of Veterinary and Pharmaceutical Sciences Brno. Detected values of PBDE concentrations were very low, generally ranged at the detection limits of the analytical method.
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Preconcentration Techniques for Determination of Uranium Using the Modified Sorbents.
Moos, Martin ; Řehůřková, Irena (referee) ; Kanický, Viktor (referee) ; Kráčmar, Stanislav (referee) ; Sommer, Lumír (advisor)
The work has been focused on the preconcentration techniques for determination of uranium on the modified sorbent, with the final use of ICP-OES and ICP-MS. First ICP-OES at a wavelength of 385.958 nm was used for determination of uranium. Parameters were optimized and the effect of mineral acids (HCl, HNO3), tensides (Septonex, Ajatin) and organic reagents like (4-(2-pyridylazo)resorcinol, ammonium pyrrolidinedithiocarbamate, 8-hydroxyquinoline-5-sulphonic acid and 1,2-dihydroxyanthraquinone-3-sulphonic acid) was observed. For ICP-MS an isotope 238U was chosen. As an internal standard 209Bi (200 ?g•l-1) was used. Modified silica gel was used (Silicagel tethered with C18, C8 alkyls and phenyl) and two types of Amberlite XAD 4 and XAD 16 for the preconcentration of uranium (VI). Commercially produced Amberlite was modified as follows: Drying at 100 °C (24 hours), milled, minced sorbent was selected and fractionated to particles size ranging from 0.32 to 0.63 ?m and finally activated in methanol. The deionised water and Septonex (5•10-3 mol•l-1) at pH 8 were used for Amberlite conditioning. The optimal sorption of uranium (VI) proceeded at pH 8 in the presence of 4-(2-pyridylazo)resorcinol or ammonium pyrrolidinedithiocarbamate with Amberlite XAD-16 moreover with 1,2-dihydroxyanthraquinone-3-sulphonic acid. All organic reagents have mass concentration five time larger than uranium . For the elution of uranium the mixture of 1 mol•l-1 HNO3 with acetone (ratio of 1:1) was used. Acetone was evaporated and the final analysis performed using ICP-OES. The preconcentration of uranium (VI) using Silicagel-C18 conditioning was performed with ethanol, deionised water and Zephyraminu (5•10-4 mol•l-1) at pH 8. For the Silica-C8 and Silicagel-Phenyl the same conditioning procedure without zephyramin was used. Silica-C18 had the highest sorption efficiency with 1,2-dihydroxyanthraquinone-3-sulphonic acid, Silicagel-C8 with ammonium pyrrolidinedithiocarbamate and Silicagel-Phenyl with 8-hydroxyquinoline-5-sulfonic acid, all organic reagents have mass concentration five time larger than uranium concentration. As an optimal elution mixture acetone and ethanol (ratio of 1:1) in the presence of 1 mol•l-1 HCl was evaluated. Acetone and ethanol was evaporated and the final analysis was performed using ICP-MS. Silicagel-C18 was evaluate as the most effective in the presence of zephyramin and 1,2-dihydroxyanthraquinone-3-sulphonic acid. Sorption was also tested in the presence of 20 ?g•l-1 microelements (Be, V, Co, Ni, Y, Pb, Th, Cd) and in the presence of a defined concentrations of K, Na, Ca, Mg, Al and Fe. Direct analysis of uranium was carried out on samples of water from the river Ploucnice and Turonian aquifers containing uranium, 3,5 and 19,3 ?g•l-1. Different type of water required preconcentration of uranium (VI) on Silica-C18 in the presence of 1,2-dihydroxyanthraquinone-3-sulphonic acid and zephyramin. The analysis results gave statistically satisfactory results, which were confirmed using standard addition of 20 ?g•l-1.
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Molecular Study of Intracellular Changes as Response of Microorganisms to Environment
Čarnecká, Martina ; Kráčmar, Stanislav (referee) ; Márová, Ivana (advisor)
Kvasinky žijú v neustále sa meniacom prostredí. Aby prežili tieto výkyvy ich okolitého prostredia, musia sa vedieť rýchlo a efektívne prispôsobiť novým podmienkam. Jedným z aspektov takejto bunkovej adaptácie je reorganizácia génovej expresie na program vyžadovaný pre rast v novom prostredí. Dôsledkom tejto reorganizácie genómu sú zmeny v metabolizme a fyziológií kvasiniek. Molekulárna odpoveď bunky určuje či sa organizmus adaptuje, prežije alebo zahynie. Predložená dizertačná práca sa zaoberá štúdiom vplyvu environmentálnych zmien na genóm a metabolóm vybraných karotenogénnych kvasiniek. Kvasinky boli kultivované jednak za optimálnych podmienok a jednak v oxidačnom a ozmotickom strese a na rôznych odpadových materiáloch (srvátke, zemiakovom odpade a pod.). V prítomnosti stresu bola pozorovaná zvýšená produkcia biologicky významných karotenoidov. Takáto obohatená biomasa môže nájsť svoje uplatnenie v biotechnologickom priemysle, napr. ako krmivo pre zvieratá. Možnosťou štúdia odozvy mikroorganizmov na environmentálny stres je aj príprava transformantov s deléciou vybraných génov a ich analýza. V ďalšej časti práce bola prevedená delécia vybraných génov kvasinky Schizosaccharomyces pombe. Zvolená technika je založená na knockoute konštruktov, ktoré obsahujú regióny homologické s deletovaným génom. Analýzou vytvorených transformantov boli identifikované proteíny potrebné pri meiotickej segregácií chromozómov.
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