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The massive production of urban and industrial wastes has created a clear need for alternative waste management processes. One of the more promising strategies is to use waste as raw material for the production of biopolymers such as polyhydroxyalkanoates (PHAs). In this work, a lactate-enriched stream obtained by anaerobic digestion (AD) of wastewater (WW) from a candy production plant was used as a feedstock for PHA production in wild-type Cupriavidus necator H16. Unexpectedly, we observed the accumulation of poly(3-hydroxybutyrate)/poly(lactic acid) (P(3HB)/PLA), suggesting that the non-engineered strain already possesses the metabolic potential to produce these polymers of interest. The systematic study of factors, such as incubation time, nitrogen and lactate concentration, influencing the synthesis of P(3HB)/PLA allowed the production of a panel of polymers in a resting cell system with tailored lactic acid (LA) content according to the GC–MS of the biomass. Further biomass extraction suggested the presence of methanol soluble low molecular weight molecules containing LA, while 1 % LA could be detected in the purified polymer fraction. These results suggested that the cells are producing a blend of polymers. A proteomic analysis of C. necator resting cells under P(3HB)/PLA production conditions provides new insights into the latent pathways involved in this process. This study is a proof of concept demonstrating that LA can polymerize in a non-modified organism and paves the way for new metabolic engineering approaches for lactic acid polymer production in the model bacterium C. necator H16.

[Abstract] The environmental concerns regarding fossil plastics call for alternative biopolymers such as polyhydroxyalkanoates (PHAs) whose manufacturing costs are however still too elevated. Autotrophic microbes like Cupriavidus necator, able to convert CO2 and H2 into PHAs, offer an additional strategy. Typically, the preferred source for CO2 and H2 are expensive pure gases or syngas, which has toxic compounds for most PHAs-accumulating strains. In this work, for the first time, H2 and CO2 originating from an acidogenic reactor were converted autotrophically into poly(3-hydroxybutyrate) P(3HB). During the first stage, a mixed microbial community continuously catabolized melon waste into H2 (26.7 %) and CO2 (49.2 %) that were then used in a second bioreactor by C. necator DSM 545 to accumulate 1.7 g/L P(3HB). Additionally, the VFAs (13 gCOD/L) produced during acidogenesis were processed into 2.7 g/L of P(3HB-co-3HV). This is the first proof-of-concept of using acidogenic-derived H2 and CO2 from fruit waste to produce PHAs.

BackgroundIntracellular biomacromolecules, such as industrial enzymes and biopolymers, represent an important class of bio-derived products obtained from bacterial hosts. A common key step in the downstream separation of these biomolecules is lysis of the bacterial cell wall to effect release of cytoplasmic contents. Cell lysis is typically achieved either through mechanical disruption or reagent-based methods, which introduce issues of energy demand, material needs, high costs, and scaling problems. Osmolysis, a cell lysis method that relies on hypoosmotic downshock upon resuspension of cells in distilled water, has been applied for bioseparation of intracellular products from extreme halophiles and mammalian cells. However, most industrial bacterial strains are non-halotolerant and relatively resistant to hypoosmotic cell lysis.ResultsTo overcome this limitation, we developed two strategies to increase the susceptibility of non-halotolerant hosts to osmolysis using Cupriavidus necator, a strain often used in electromicrobial production, as a prototypical strain. In one strategy, C. necator was evolved to increase its halotolerance from 1.5% to 3.25% (w/v) NaCl through adaptive laboratory evolution, and genes potentially responsible for this phenotypic change were identified by whole genome sequencing. The evolved halotolerant strain experienced an osmolytic efficiency of 47% in distilled water following growth in 3% (w/v) NaCl. In a second strategy, the cells were made susceptible to osmolysis by knocking out the large-conductance mechanosensitive channel (mscL) gene in C. necator. When these strategies were combined by knocking out the mscL gene from the evolved halotolerant strain, greater than 90% osmolytic efficiency was observed upon osmotic downshock. A modified version of this strategy was applied to E. coli BL21 by deleting the mscL and mscS (small-conductance mechanosensitive channel) genes. When grown in medium with 4% NaCl and subsequently resuspended i

Burgeoning concerns on the adverse environmental impacts of fossil-derived products are propelling the pursuit of material production from sustainable resources. Resource recovery from waste is a key component of meeting the environmental sustainability agendas set by the United Nations. Municipal organic wastes present a significant opportunity for resource recovery due to their inherent organic content. Volatile fatty acids (VFAs), the intermediary products of anaerobic digestion of waste streams, can serve as building block chemicals with a wide range of applications. Meanwhile, microbiologically produced biopolymers called polyhydroxyalkanoates (PHAs) hold an enormous potential as an alternative to petrochemical-based plastics, given their comparable physiochemical properties, biodegradability and biocompatibility. In view of this, the focus of this thesis was on bio-based VFA production from food waste (FW) and PHA production from municipal organic waste by exploring process optimization and microbial community dynamics of mono and co-cultures as well as mixed microbial cultures (MMCs). The link between different inocula, retention time and pH on VFA production from FW was elucidated. This part of the study employed three distinct inocula under initial acidic (pH 5) and alkaline (pH 10) conditions for a period of 30 days. Waste-derived VFAs were employed for mono and co-culture PHA biosynthesis with bacteria, Cupriavidus necator, Burkholderia cepacia and Bacillus megaterium. The highest PHA yields of 78 ± 5.7% of cell dried weight (CDW) was obtained with C. necator and a PHA yield of 55 ± 3.7% of CDW was achieved with B. cepacia. In the next part of the study, activated sludge MMC was enriched over short (3 and 5 days) periods in combination with bioaugmentation of C. necator and B. cepacia in both mono and co-culture modes. While bioaugmentation did not increase the total PHA accumulation capacity, the microbial composition of the different bioreactors was m, En växande oro för de negativa miljöeffekterna av fossilbaserade produkter driver strävan efter materialproduktion från hållbara resurser. Resursåtervinning från avfall är en nyckelkomponent för att uppfylla FN:s miljöpolitiska hållbarhetsagendor. Kommunalt organiskt avfall utgör en betydande möjlighet till resursåtervinning på grund av sitt inneboende organiska innehåll. Flyktiga fettsyror (VFA), mellanprodukter från anaerob rötning av avfallsströmmar, kan fungera som byggstenskemikalier med ett brett spektrum av tillämpningar. Samtidigt har mikrobiologiskt framställda biopolymerer som kallas polyhydroxialkanoater (PHA) en enorm potential som ett alternativ till petrokemiskt baserad plast, med tanke på deras jämförbara fysiokemiska egenskaper, biologiska nedbrytbarhet och biokompatibilitet. Mot bakgrund av detta låg fokus för denna avhandling på biobaserad VFA-produktion från matavfall (FW) och PHA-produktion från kommunalt organiskt avfall genom att utforska processoptimering och mikrobiell gemenskapsdynamik hos mono- och samkulturer samt blandade mikrobiella kulturer (MMC).Kopplingen mellan olika inokula, retentionstid och pH på VFA-produktion från FW klargjordes. Denna del av studien använde tre distinkta inokula under initiala sura (pH 5) och alkaliska (pH 10) betingelser under en period av 30 dagar.Avfallshärledda VFAs användes för mono- och samodling av PHA-biosyntes med bakterier, Cupriavidus necator, Burkholderia cepacia och Bacillus megaterium. De högsta PHA-utbytena på 78  5,7 % av celltorkad vikt (CDW) erhölls med C. necator och ett PHA-utbyte på 55  3,7 % av CDW uppnåddes med B. cepacia.I nästa del av studien berikades aktivt slam MMC under korta (3 och 5 dagar) perioder i kombination med bioaugmentation av C. necator och B. cepacia i både mono- och samodling. Även om bioaugmentering inte ökade den totala PHA-ackumuleringskapaciteten, modifierades den mikrobiella sammansättningen av de olika bioreaktorerna.Denna Ph.D. projektet gav insikter om återvin, QC 20230912

Burgeoning concerns on the adverse environmental impacts of fossil-derived products are propelling the pursuit of material production from sustainable resources. Resource recovery from waste is a key component of meeting the environmental sustainability agendas set by the United Nations. Municipal organic wastes present a significant opportunity for resource recovery due to their inherent organic content. Volatile fatty acids (VFAs), the intermediary products of anaerobic digestion of waste streams, can serve as building block chemicals with a wide range of applications. Meanwhile, microbiologically produced biopolymers called polyhydroxyalkanoates (PHAs) hold an enormous potential as an alternative to petrochemical-based plastics, given their comparable physiochemical properties, biodegradability and biocompatibility. In view of this, the focus of this thesis was on bio-based VFA production from food waste (FW) and PHA production from municipal organic waste by exploring process optimization and microbial community dynamics of mono and co-cultures as well as mixed microbial cultures (MMCs). The link between different inocula, retention time and pH on VFA production from FW was elucidated. This part of the study employed three distinct inocula under initial acidic (pH 5) and alkaline (pH 10) conditions for a period of 30 days. Waste-derived VFAs were employed for mono and co-culture PHA biosynthesis with bacteria, Cupriavidus necator, Burkholderia cepacia and Bacillus megaterium. The highest PHA yields of 78 ± 5.7% of cell dried weight (CDW) was obtained with C. necator and a PHA yield of 55 ± 3.7% of CDW was achieved with B. cepacia. In the next part of the study, activated sludge MMC was enriched over short (3 and 5 days) periods in combination with bioaugmentation of C. necator and B. cepacia in both mono and co-culture modes. While bioaugmentation did not increase the total PHA accumulation capacity, the microbial composition of the different bioreactors was m, En växande oro för de negativa miljöeffekterna av fossilbaserade produkter driver strävan efter materialproduktion från hållbara resurser. Resursåtervinning från avfall är en nyckelkomponent för att uppfylla FN:s miljöpolitiska hållbarhetsagendor. Kommunalt organiskt avfall utgör en betydande möjlighet till resursåtervinning på grund av sitt inneboende organiska innehåll. Flyktiga fettsyror (VFA), mellanprodukter från anaerob rötning av avfallsströmmar, kan fungera som byggstenskemikalier med ett brett spektrum av tillämpningar. Samtidigt har mikrobiologiskt framställda biopolymerer som kallas polyhydroxialkanoater (PHA) en enorm potential som ett alternativ till petrokemiskt baserad plast, med tanke på deras jämförbara fysiokemiska egenskaper, biologiska nedbrytbarhet och biokompatibilitet. Mot bakgrund av detta låg fokus för denna avhandling på biobaserad VFA-produktion från matavfall (FW) och PHA-produktion från kommunalt organiskt avfall genom att utforska processoptimering och mikrobiell gemenskapsdynamik hos mono- och samkulturer samt blandade mikrobiella kulturer (MMC).Kopplingen mellan olika inokula, retentionstid och pH på VFA-produktion från FW klargjordes. Denna del av studien använde tre distinkta inokula under initiala sura (pH 5) och alkaliska (pH 10) betingelser under en period av 30 dagar.Avfallshärledda VFAs användes för mono- och samodling av PHA-biosyntes med bakterier, Cupriavidus necator, Burkholderia cepacia och Bacillus megaterium. De högsta PHA-utbytena på 78  5,7 % av celltorkad vikt (CDW) erhölls med C. necator och ett PHA-utbyte på 55  3,7 % av CDW uppnåddes med B. cepacia.I nästa del av studien berikades aktivt slam MMC under korta (3 och 5 dagar) perioder i kombination med bioaugmentation av C. necator och B. cepacia i både mono- och samodling. Även om bioaugmentering inte ökade den totala PHA-ackumuleringskapaciteten, modifierades den mikrobiella sammansättningen av de olika bioreaktorerna.Denna Ph.D. projektet gav insikter om återvin, QC 20230912

The nature of air-inactivation of the formate dehydrogenase FdsDABG from Cupriavidus necator has been investigated. It is found that superoxide, generated in the reaction of reduced enzyme with oxygen, is responsible for the loss of activity and that superoxide dismutase protects the enzyme from air-inactivation. Inhibition appears to be due to the reaction of superoxide with the catalytically essential MoS group of the enzyme's molybdenum center in such a way that generates sulfite. SYNOPSIS: Superoxide generated in the reaction of reduced formate dehydrogenase FdsDABG from Cupriavidus necator with O2 is found to be responsible for the loss of activity. Catalytic amounts of superoxide dismutase are found to protect FdsDABG just as well as more generally used stabilizing inhibitors such as nitrate.

The nature of air-inactivation of the formate dehydrogenase FdsDABG from Cupriavidus necator has been investigated. It is found that superoxide, generated in the reaction of reduced enzyme with oxygen, is responsible for the loss of activity and that superoxide dismutase protects the enzyme from air-inactivation. Inhibition appears to be due to the reaction of superoxide with the catalytically essential MoS group of the enzyme's molybdenum center in such a way that generates sulfite. SYNOPSIS: Superoxide generated in the reaction of reduced formate dehydrogenase FdsDABG from Cupriavidus necator with O2 is found to be responsible for the loss of activity. Catalytic amounts of superoxide dismutase are found to protect FdsDABG just as well as more generally used stabilizing inhibitors such as nitrate.

The chemolithotroph Cupriavidus necator H16 is known as a natural producer of the bioplastic-polymer PHB, as well as for its metabolic versatility to utilize different substrates, including formate as the sole carbon and energy source. Depending on the entry point of the substrate, this versatility requires adjustment of the thermodynamic landscape to maintain sufficiently high driving forces for biological processes. Here we employed a model of the core metabolism of C. necator H16 to analyze the thermodynamic driving forces and PHB yields from formate for different metabolic engineering strategies. For this, we enumerated elementary flux modes (EFMs) of the network and evaluated their PHB yields as well as thermodynamics via Max-min driving force (MDF) analysis and random sampling of driving forces. A heterologous ATP:citrate lyase reaction was predicted to increase driving force for producing acetyl-CoA. A heterologous phosphoketolase reaction was predicted to increase maximal PHB yields as well as driving forces. These enzymes were then verified experimentally to enhance PHB titers between 60 and 300% in select conditions. The EFM analysis also revealed that PHB production from formate may be limited by low driving forces through citrate lyase and aconitase, as well as cofactor balancing, and identified additional reactions associated with low and high PHB yield. Proteomics analysis of the engineered strains confirmed an increased abundance of aconitase and cofactor balancing. The findings of this study aid in understanding metabolic adaptation. Furthermore, the outlined approach will be useful in designing metabolic engineering strategies in other non-model bacteria., QC 20221024

The chemical industry is transitioning to more sustainable and biobased processes. One key element of this transition is coupling energy fluxes and feedstock utilization for optimizing processes, routes and efficiencies. Here, we show for the first time the coupling of the Kolbe electrolysis at the anode with a subsequent microbial conversion of the cathodically produced co-product hydrogen. Kolbe electrolysis of valeric acid yields the liquid drop-in fuel additive n-octane. Subsequently, the solvent isopropanol is produced by resting Cupriavidus necator cells using gaseous electrolysis products (esp. CO2 and H2). The resting microbial cells show carbon efficiencies of up to 41% and coulombic/faradaic efficiencies of 60% and 80% for anodic and cathodic reactions, respectively. The implementation of a paired electrolyzer resulted in superior process performances with overall efficiencies of up to 64.4%.

The increasing threat of climate change combined with the prospected growth in the world population puts an enormous pressure on the future demand for sustainable protein sources for human consumption. In this review, hydrogen oxidizing bacteria (HOB) are presented as a novel protein source that could play a role in fulfilling this future demand. HOB are species of bacteria that merely require an inflow of the gasses hydrogen, oxygen, carbon dioxide, and a nitrogen source to grow in a conventional bioreactor. Cupriavidus necator is proposed as HOB for industrial cultivation due to its remarkably high protein content (up to 70% of mass), suitability for cultivation in a bioreactor, and the vast amount of available background information. A broad overview of the unique aspects of the bacteria will be provided, from the production process, amino acid composition, and source of the required gasses to the future acceptance of HOB into the market.

The “list of microorganisms with QPS status” first established in 2007, has been revised and updated annually until 2014 via QPS Opinions; since 2014 the updates are carried out and published every 3 years. If new information is retrieved from extended literature searches (ELS) that would change the QPS status of a TU or its qualifications, this is also published in the Panel Statement covering the previous 6-months period. The ELS protocol can be found at https://doi.org/10.5281/zenodo.3607190 and the Search strategies are available at: https://doi.org/10.5281/zenodo.3607193. The QPS Panel Statement also includes the evaluation of microbiological agents notified to EFSA within the 6-month period for an assessment for feed additives, food enzymes, food additives and flavourings, and novel foods or plant protection products for a possible QPS status. The new QPS status recommendations are incorporated into the 2019 updated “list of microorganisms with QPS status” is available in this upload. The list of “Microbiological agents as notified to EFSA” from 2007, in the context of technical dossiers to EFSA Units, for intentional use in feed and/or food or as sources of food and feed additives, enzymes and plant protection products (PPPs) for safety assessment can be found at https://doi.org/10.5281/zenodo.3607184.

An astonishing variety of functions has been attributed to polyphosphate (polyP) in prokaryotes. Besides being a reservoir of phosphorus, functions in exopolysaccharide formation, motility, virulence and in surviving various forms of stresses such as exposure to heat, extreme pH, oxidative agents, high osmolarity, heavy metals and others have been ascribed to polyP. In this contribution, we will provide a historical overview on polyP, will then describe the key proteins of polyP synthesis, the polyP kinases, before we will critically assess of the underlying data on the multiple functions of polyP and provide evidence that - with the exception of a P-storage-function - most other functions of polyP are not relevant for survival of Ralstonia eutropha, a biotechnologically important beta-proteobacterial species.

Decelerating global warming is one of the predominant challenges of our time and will require conversion of CO2 to usable products and commodity chemicals. Of particular interest is the production of fuels, because the transportation sector is a major source of CO2 emissions. Here, we review recent technological advances in metabolic engineering of the hydrogen-oxidizing bacterium Cupriavidus necator H16, a chemolithotroph that naturally consumes CO2 to generate biomass. We discuss recent successes in biofuel production using this organism, and the implementation of electrolysis/artificial photosynthesis approaches that enable growth of C. necator using renewable electricity and CO2. Last, we discuss prospects of improving the nonoptimal growth of C. necator in ambient concentrations of CO2.

Bacteria must balance the different needs for substrate assimilation, growth functions, and resilience in order to thrive in their environment. Of all cellular macromolecules, the bacterial proteome is by far the most important resource and its size is limited. Here, we investigated how the highly versatile 'knallgas' bacterium Cupriavidus necator reallocates protein resources when grown on different limiting substrates and with different growth rates. We determined protein quantity by mass spectrometry and estimated enzyme utilization by resource balance analysis modeling. We found that C. necator invests a large fraction of its proteome in functions that are hardly utilized. Of the enzymes that are utilized, many are present in excess abundance. One prominent example is the strong expression of CBB cycle genes such as Rubisco during growth on fructose. Modeling and mutant competition experiments suggest that CO2-reassimilation through Rubisco does not provide a fitness benefit for heterotrophic growth, but is rather an investment in readiness for autotrophy., See also peer review documents at DOI 10.7554/eLife.69019.sa0 10.7554/eLife.69019.sa1 10.7554/eLife.69019.sa2QC 20220111

Decelerating global warming is one of the predominant challenges of our time and will require conversion of CO2 to usable products and commodity chemicals. Of particular interest is the production of fuels, because the transportation sector is a major source of CO2 emissions. Here, we review recent technological advances in metabolic engineering of the hydrogen-oxidizing bacterium Cupriavidus necator H16, a chemolithotroph that naturally consumes CO2 to generate biomass. We discuss recent successes in biofuel production using this organism, and the implementation of electrolysis/artificial photosynthesis approaches that enable growth of C. necator using renewable electricity and CO2. Last, we discuss prospects of improving the nonoptimal growth of C. necator in ambient concentrations of CO2.

An astonishing variety of functions has been attributed to polyphosphate (polyP) in prokaryotes. Besides being a reservoir of phosphorus, functions in exopolysaccharide formation, motility, virulence and in surviving various forms of stresses such as exposure to heat, extreme pH, oxidative agents, high osmolarity, heavy metals and others have been ascribed to polyP. In this contribution, we will provide a historical overview on polyP, will then describe the key proteins of polyP synthesis, the polyP kinases, before we will critically assess of the underlying data on the multiple functions of polyP and provide evidence that - with the exception of a P-storage-function - most other functions of polyP are not relevant for survival of Ralstonia eutropha, a biotechnologically important beta-proteobacterial species.

An astonishing variety of functions has been attributed to polyphosphate (polyP) in prokaryotes. Besides being a reservoir of phosphorus, functions in exopolysaccharide formation, motility, virulence and in surviving various forms of stresses such as exposure to heat, extreme pH, oxidative agents, high osmolarity, heavy metals and others have been ascribed to polyP. In this contribution, we will provide a historical overview on polyP, will then describe the key proteins of polyP synthesis, the polyP kinases, before we will critically assess of the underlying data on the multiple functions of polyP and provide evidence that - with the exception of a P-storage-function - most other functions of polyP are not relevant for survival of Ralstonia eutropha, a biotechnologically important beta-proteobacterial species.

The Calvin – Benson cycle is the most widespread metabolic pathway capable of fixing CO2 in nature and a target of very high interest to metabolic engineers worldwide. In this study, 12 metabolites (ATP, AMP, NADP, NADPH, 2PG, 3PGA, FBP, RuBP, PEP, AKG, Ac-CoA and phenylalanine) were tested for protein – metabolite interactions against the proteome of Cupriavidus necator (previously Ralstonia eutropha) in the hopes of finding potential examples of allosteric regulation of the Calvin – Benson cycle. This is accomplished through the use of the LiP-SMap method, a recently developed shotgun proteomics method described by Piazza et al. capable of testing a metabolite of interest for interactions with the entire proteome of an organism at once. A functional protocol was developed and 234 protein – metabolite interactions between ATP and the proteome of C. necator are identified, 103 of which are potentially novel. Due to time constraints and setbacks in the lab, significant results were not produced for the other 11 metabolites tested. C. necator is an industrially relevant chemolithoautotroph that can be engineered to produce many valuable products and is capable of growth on CO2 and hydrogen gas. The bacteria were grown in continuous cultures after which the proteome was extracted while retaining its native state. Subsequently, the proteome was incubated with a metabolite of interest and subjected to limited, non-specific proteolysis. The resulting peptide mix was analyzed by liquid chromatography coupled tandem mass spectrometry (LC – MS/MS)., Calvin-Benson-cykeln är den mest utbredda metaboliska processen i naturen med vilken det är möjligt att fixera CO2 och en måltavla av högsta intresse för bioteknologer världen över. I den här studien testades 12 metaboliter (ATP, AMP, NADP, NADPH, 2PG, 3PGA, FBP, RuBP, PEP, AKG, Ac-CoA and phenylalanine) för interaktioner mot proteomet från Cupriavidus necator (tidigare Ralstonia eutropha) i hopp om att hitta potentiella exempel på allosterisk reglering av Calvin-Benson-cykeln. Detta uppnåddes genom användning av LiP-SMap-metoden, en nyligen utvecklad proteomikmetod beskriven av Piazza et al. kapabel av att testa en metabolit av intresse mot en organisms hela proteom simultant. Ett funktionellt protokoll utvecklades och 234 interaktioner mellan ATP och proteomet av C. necator identifierades, varav 103 potentiellt är nyupptäckta. På grund av tidsbrist och motgångar i labbet producerades inga signifikanta resultat för de resterande 11 metaboliterna som testades. C. necator är en industriellt relevant kemolitoautotrof som kan växa på CO2 och vätgas, samt manipuleras till att producera många värdefulla produkter. Bakterierna odlades i kemostater varefter proteomet extraherades i sitt naturliga tillstånd. Sedan inkuberades proteomet med en metabolit av intresse och utsattes för begränsad, icke-specifik proteolys. Den resulterande peptidblandningen analyserades via tandem masspektrometri kopplad till vätskekromatografi (LC – MS/MS).

Formate oxidation to carbon dioxide is a key reaction in one-carbon compound metabolism, and its reverse reaction represents the first step in carbon assimilation in the acetogenic and methanogenic branches of many anaerobic organisms. The molybdenum-containing dehydrogenase FdsABG is a soluble NAD+-dependent formate dehydrogenase and a member of the NADH dehydrogenase superfamily. Here, we present the first structure of the FdsBG subcomplex of the cytosolic FdsABG formate dehydrogenase from the hydrogen-oxidizing bacterium Cupriavidus necator H16 both with and without bound NADH. The structures revealed that the two iron-sulfur clusters, Fe4S4 in FdsB and Fe2S2 in FdsG, are closer to the FMN than they are in other NADH dehydrogenases. Rapid kinetic studies and EPR measurements of rapid freeze-quenched samples of the NADH reduction of FdsBG identified a neutral flavin semiquinone, FMNH•, not previously observed to participate in NADH-mediated reduction of the FdsABG holoenzyme. We found that this semiquinone forms through the transfer of one electron from the fully reduced FMNH-, initially formed via NADH-mediated reduction, to the Fe2S2 cluster. This Fe2S2 cluster is not part of the on-path chain of iron-sulfur clusters connecting the FMN of FdsB with the active-site molybdenum center of FdsA. According to the NADH-bound structure, the nicotinamide ring stacks onto the re-face of the FMN. However, NADH binding significantly reduced the electron density for the isoalloxazine ring of FMN and induced a conformational change in residues of the FMN-binding pocket that display peptide-bond flipping upon NAD+ binding in proper NADH dehydrogenases.

The Calvin – Benson cycle is the most widespread metabolic pathway capable of fixing CO2 in nature and a target of very high interest to metabolic engineers worldwide. In this study, 12 metabolites (ATP, AMP, NADP, NADPH, 2PG, 3PGA, FBP, RuBP, PEP, AKG, Ac-CoA and phenylalanine) were tested for protein – metabolite interactions against the proteome of Cupriavidus necator (previously Ralstonia eutropha) in the hopes of finding potential examples of allosteric regulation of the Calvin – Benson cycle. This is accomplished through the use of the LiP-SMap method, a recently developed shotgun proteomics method described by Piazza et al. capable of testing a metabolite of interest for interactions with the entire proteome of an organism at once. A functional protocol was developed and 234 protein – metabolite interactions between ATP and the proteome of C. necator are identified, 103 of which are potentially novel. Due to time constraints and setbacks in the lab, significant results were not produced for the other 11 metabolites tested. C. necator is an industrially relevant chemolithoautotroph that can be engineered to produce many valuable products and is capable of growth on CO2 and hydrogen gas. The bacteria were grown in continuous cultures after which the proteome was extracted while retaining its native state. Subsequently, the proteome was incubated with a metabolite of interest and subjected to limited, non-specific proteolysis. The resulting peptide mix was analyzed by liquid chromatography coupled tandem mass spectrometry (LC – MS/MS)., Calvin-Benson-cykeln är den mest utbredda metaboliska processen i naturen med vilken det är möjligt att fixera CO2 och en måltavla av högsta intresse för bioteknologer världen över. I den här studien testades 12 metaboliter (ATP, AMP, NADP, NADPH, 2PG, 3PGA, FBP, RuBP, PEP, AKG, Ac-CoA and phenylalanine) för interaktioner mot proteomet från Cupriavidus necator (tidigare Ralstonia eutropha) i hopp om att hitta potentiella exempel på allosterisk reglering av Calvin-Benson-cykeln. Detta uppnåddes genom användning av LiP-SMap-metoden, en nyligen utvecklad proteomikmetod beskriven av Piazza et al. kapabel av att testa en metabolit av intresse mot en organisms hela proteom simultant. Ett funktionellt protokoll utvecklades och 234 interaktioner mellan ATP och proteomet av C. necator identifierades, varav 103 potentiellt är nyupptäckta. På grund av tidsbrist och motgångar i labbet producerades inga signifikanta resultat för de resterande 11 metaboliterna som testades. C. necator är en industriellt relevant kemolitoautotrof som kan växa på CO2 och vätgas, samt manipuleras till att producera många värdefulla produkter. Bakterierna odlades i kemostater varefter proteomet extraherades i sitt naturliga tillstånd. Sedan inkuberades proteomet med en metabolit av intresse och utsattes för begränsad, icke-specifik proteolys. Den resulterande peptidblandningen analyserades via tandem masspektrometri kopplad till vätskekromatografi (LC – MS/MS).

These data belong to a metabolic engineering project that introduces the reductive glycine pathway for formate assimilation in Cupriavidus necator. As part of this project we performed short-term evolution of the bacterium Cupriavidus necator H16 to grow on glycine as sole carbon and energy source. Some mutations in a putiative glycine transporting systems facilitated growth, and we performed transcriptomics on the evolved strain growing on glycine. Analysis of these transcriptomic data lead us to the discovery of a glycine oxidase (DadA6), which we experimentally demonstrated to play a key role in the glycine assimilation pathay in C. necator. Overall design: Transcriptomes are performed for the evolved strain growing on glyine (in triplicate)., These data belong to a metabolic engineering project that introduces the reductive glycine pathway for formate assimilation in Cupriavidus necator. As part of this project we performed short-term evolution of the bacterium Cupriavidus necator H16 to grow on glycine as sole carbon and energy source. Some mutations in a putiative glycine transporting systems facilitated growth, and we performed transcriptomics on the evolved strain growing on glycine. Analysis of these transcriptomic data lead us to the discovery of a glycine oxidase (DadA6), which we experimentally demonstrated to play a key role in the glycine assimilation pathay in C. necator. Overall design: Transcriptomes are performed for the evolved strain growing on glyine (in triplicate).

Direct biocatalytic conversion of CO2 to formic acid is an attractive means of reversibly storing energy in chemical bonds. Formate dehydrogenases (FDHs) are a heterogeneous group of enzymes that catalyze the oxidation of formic acid to carbon dioxide, generating two protons and two electrons. Several FDHs have recently been reported to catalyze the reverse reaction, i.e., the reduction of carbon dioxide to formic acid, under appropriate conditions. The main challenges with these enzymes are relatively low rates of CO2 reduction and high oxygen sensitivity. Our earlier studies (Yu et al. (2017) J. Biol. Chem. 292, 16872-16879) have shown that the FdsABG formate dehydrogenase from Cupriavidus necator is able to effectively catalyze the reduction of CO2, using NADH as a source of reducing equivalents, with a good oxygen tolerance. On the basis of this result, we have developed a highly thermodynamically efficient and cost-effective biocatalytic process for the transformation of CO2 to formic acid using FdsABG. We have  cloned the full-length soluble formate dehydrogenase (FdsABG) from C. necator and expressed it in Escherichia coli with a His-tag fused to the N terminus of the FdsG subunit; this overexpression system has greatly simplified the FdsABG purification process. Importantly, we have also combined this recombinant C. necator FdsABG with another enzyme, glucose dehydrogenase, for continuous regeneration of NADH for CO2 reduction and demonstrated that the combined system is highly effective in reducing CO2 to formate. The results indicate that this system shows significant promise for the future development of an enzyme-based system for the industrial reduction of CO2.

Direct biocatalytic conversion of CO2 to formic acid is an attractive means of reversibly storing energy in chemical bonds. Formate dehydrogenases (FDHs) are a heterogeneous group of enzymes that catalyze the oxidation of formic acid to carbon dioxide, generating two protons and two electrons. Several FDHs have recently been reported to catalyze the reverse reaction, i.e., the reduction of carbon dioxide to formic acid, under appropriate conditions. The main challenges with these enzymes are relatively low rates of CO2 reduction and high oxygen sensitivity. Our earlier studies (Yu et al. (2017) J. Biol. Chem. 292, 16872-16879) have shown that the FdsABG formate dehydrogenase from Cupriavidus necator is able to effectively catalyze the reduction of CO2, using NADH as a source of reducing equivalents, with a good oxygen tolerance. On the basis of this result, we have developed a highly thermodynamically efficient and cost-effective biocatalytic process for the transformation of CO2 to formic acid using FdsABG. We have  cloned the full-length soluble formate dehydrogenase (FdsABG) from C. necator and expressed it in Escherichia coli with a His-tag fused to the N terminus of the FdsG subunit; this overexpression system has greatly simplified the FdsABG purification process. Importantly, we have also combined this recombinant C. necator FdsABG with another enzyme, glucose dehydrogenase, for continuous regeneration of NADH for CO2 reduction and demonstrated that the combined system is highly effective in reducing CO2 to formate. The results indicate that this system shows significant promise for the future development of an enzyme-based system for the industrial reduction of CO2.

Qualified presumption of safety (QPS) was developed to provide a generic safety evaluation for biological agents to support EFSA's Scientific Panels. The taxonomic identity, body of knowledge, safety concerns and antimicrobial resistance are assessed. Safety concerns identified for a taxonomic unit (TU) are where possible to be confirmed at strain or product level, reflected by ‘qualifications’. No new information was found that would change the previously recommended QPS TUs and their qualifications. The list of microorganisms notified to EFSA was updated with 54 biological agents, received between April and September 2019; 23 already had QPS status, 14 were excluded from the QPS exercise (7 filamentous fungi, 6 Escherichia coli, Sphingomonas paucimobilis which was already evaluated). Seventeen, corresponding to 16 TUs, were evaluated for possible QPS status, fourteen of these for the first time, and Protaminobacter rubrum, evaluated previously, was excluded because it is not a valid species. Eight TUs are recommended for QPS status. Lactobacillus parafarraginis and Zygosaccharomyces rouxii are recommended to be included in the QPS list. Parageobacillus thermoglucosidasius and Paenibacillus illinoisensis can be recommended for the QPS list with the qualification ‘for production purposes only’ and absence of toxigenic potential. Bacillus velezensis can be recommended for the QPS list with the qualifications; the absence of toxigenic potential and the absence of aminoglycoside production, including the genes encoding this. Cupriavidus necator, Aurantiochytrium limacinum and Tetraselmis chuii can be recommended for the QPS list with the qualification; for production purposes only. Pantoea ananatis is not recommended for the QPS list due to lack of body of knowledge in relation to its pathogenicity potential for plants. Corynebacterium stationis, Hamamotoa singularis, Rhodococcus aetherivorans and Rhodococcus ruber cannot be recommended for the QPS list due to lack

We investigate photorespiration pathways in the chemoautotroph Cupriavidus necator H16 when growing autotropically on hydrogen and carbon dioxide via the Calvin Cycle. We demonstrate an upregulation of the glycerate pathway under photorespiration (ambient carbon dioxide) conditions Overall design: Transcriptomes are compared for growth under photorespiration conditions (H2, ambient CO2) versus growth at H2 and 10% CO2, as versus heterotrophic growth on pyruvate, biological triplicates are used (9 samples total), We investigate photorespiration pathways in the chemoautotroph Cupriavidus necator H16 when growing autotropically on hydrogen and carbon dioxide via the Calvin Cycle. We demonstrate an upregulation of the glycerate pathway under photorespiration (ambient carbon dioxide) conditions Overall design: Transcriptomes are compared for growth under photorespiration conditions (H2, ambient CO2) versus growth at H2 and 10% CO2, as versus heterotrophic growth on pyruvate, biological triplicates are used (9 samples total)

The ability of the FdsABG formate dehydrogenase from Cupriavidus necator (formerly known as Ralstonia eutropha) to catalyze the reverse of the physiological reaction, the reduction of CO2 to formate utilizing NADH as electron donor, has been investigated. Contrary to previous studies of this enzyme, we demonstrate that it is in fact effective in catalyzing the reverse reaction with a kcat of 11 ± 0.4 s-1 We also quantify the stoichiometric accumulation of formic acid as the product of the reaction and demonstrate that the observed kinetic parameters for catalysis in the forward and reverse reactions are thermodynamically consistent, complying with the expected Haldane relationships. Finally, we demonstrate the reaction conditions necessary for gauging the ability of a given formate dehydrogenase or other CO2-utilizing enzyme to catalyze the reverse direction to avoid false negative results. In conjunction with our earlier studies on the reaction mechanism of this enzyme and on the basis of the present work, we conclude that all molybdenum- and tungsten-containing formate dehydrogenases and related enzymes likely operate via a simple hydride transfer mechanism and are effective in catalyzing the reversible interconversion of CO2 and formate under the appropriate experimental conditions.

Medium-chain-length polyhydroxyalkanoates (mcl-PHA) are biotechnologically useful natural products found in many bacteria. This biopolymer functions as a carbon and energy storage reservoir in cells but has physical and mechanical properties that make it a promising bioplastic with applications ranging from adhesives to medical implants. Therefore, there is much interest in understanding the biology of mcl-PHA synthesis and metabolism. Increased knowledge of PHA biology serves as a foundation for the bioengineering of PHA and its eventual use as a biologically derived product. This chapter covers the state of knowledge on mcl-PHA, including its synthesis and its central role in cellular metabolism. Moreover, this chapter discusses methods for bioengineering mcl-PHA production in bacteria as well as synthetic biology methods for its study and production in the natural mcl-PHA producer, Pseudomonas putida.

Methanol utilization by methylotrophic or non-methylotrophic organisms is the first step toward methanol bioconversion to higher carbon-chain chemicals. Methanol oxidation using NAD-dependent methanol dehydrogenase (Mdh) is of particular interest because it uses NAD(+) as the electron carrier. To our knowledge, only a limited number of NAD-dependent Mdhs have been reported. The most studied is the Bacillus methanolicus Mdh, which exhibits low enzyme specificity to methanol and is dependent on an endogenous activator protein (ACT). In this work, we characterized and engineered a group III NAD-dependent alcohol dehydrogenase (Mdh2) from Cupriavidus necator N-1 (previously designated as Ralstonia eutropha). This enzyme is the first NAD-dependent Mdh characterized from a Gram-negative, mesophilic, non-methylotrophic organism with a significant activity towards methanol. Interestingly, unlike previously reported Mdhs, Mdh2 does not require activation by known activators such as B. methanolicus ACT and Escherichia coli Nudix hydrolase NudF, or putative native C. necator activators in the Nudix family under mesophilic conditions. This enzyme exhibited higher or comparable activity and affinity toward methanol relative to the B. methanolicus Mdh with or without ACT in a wide range of temperatures. Furthermore, using directed molecular evolution, we engineered a variant (CT4-1) of Mdh2 that showed a 6-fold higher K cat/K m for methanol and 10-fold lower K cat/K m for n-butanol. Thus, CT4-1 represents an NAD-dependent Mdh with much improved catalytic efficiency and specificity toward methanol compared with the existing NAD-dependent Mdhs with or without ACT activation.

Methanol utilization by methylotrophic or non-methylotrophic organisms is the first step toward methanol bioconversion to higher carbon-chain chemicals. Methanol oxidation using NAD-dependent methanol dehydrogenase (Mdh) is of particular interest because it uses NAD(+) as the electron carrier. To our knowledge, only a limited number of NAD-dependent Mdhs have been reported. The most studied is the Bacillus methanolicus Mdh, which exhibits low enzyme specificity to methanol and is dependent on an endogenous activator protein (ACT). In this work, we characterized and engineered a group III NAD-dependent alcohol dehydrogenase (Mdh2) from Cupriavidus necator N-1 (previously designated as Ralstonia eutropha). This enzyme is the first NAD-dependent Mdh characterized from a Gram-negative, mesophilic, non-methylotrophic organism with a significant activity towards methanol. Interestingly, unlike previously reported Mdhs, Mdh2 does not require activation by known activators such as B. methanolicus ACT and Escherichia coli Nudix hydrolase NudF, or putative native C. necator activators in the Nudix family under mesophilic conditions. This enzyme exhibited higher or comparable activity and affinity toward methanol relative to the B. methanolicus Mdh with or without ACT in a wide range of temperatures. Furthermore, using directed molecular evolution, we engineered a variant (CT4-1) of Mdh2 that showed a 6-fold higher K cat/K m for methanol and 10-fold lower K cat/K m for n-butanol. Thus, CT4-1 represents an NAD-dependent Mdh with much improved catalytic efficiency and specificity toward methanol compared with the existing NAD-dependent Mdhs with or without ACT activation.

Photovoltaic cells have considerable potential to satisfy future renewable-energy needs, but efficient and scalable methods of storing the intermittent electricity they produce are required for the large-scale implementation of solar energy. Current solar-to-fuels storage cycles based on water splitting produce hydrogen and oxygen, which are attractive fuels in principle but confront practical limitations from the current energy infrastructure that is based on liquid fuels. In this work, we report the development of a scalable, integrated bioelectrochemical system in which the bacterium Ralstonia eutropha is used to efficiently convert CO2, along with H2 and O2 produced from water splitting, into biomass and fusel alcohols. Water-splitting catalysis was performed using catalysts that are made of earth-abundant metals and enable low overpotential water splitting. In this integrated setup, equivalent solar-to-biomass yields of up to 3.2% of the thermodynamic maximum exceed that of most terrestrial plants. Moreover, engineering of R. eutropha enabled production of the fusel alcohol isopropanol at up to 216 mg/L, the highest bioelectrochemical fuel yield yet reported by >300%. This work demonstrates that catalysts of biotic and abiotic origin can be interfaced to achieve challenging chemical energy-to-fuels transformations.

Glycerol is a co-product of many industrial processes and is generated in large quantities from different origins. In this study, glycerol is used as a cheap carbon source for the production of poly(3- hydroxybutyrate) (PHB) with two different collection strains, Cupriavidus necator and Burkholderia sacchari, in order to provide an alternative outlet for glycerol and produce value-added bioproducts. The objective of this work was to study the influence of this carbon source on their growth kinetics, on their polymer production, and on the molecular mass of the produced biopolymer. Therefore, fermentations in bioreactors were carried out with these strains. Different results for both strains were obtained showing, for the first time, a high cell dry mass and growth rate, when glycerol was used together with glucose in the fermentation with C. necator. In the first fermentation with B. sacchari using glycerol as a sole carbon source, the strain properly developed synthesizing PHB. The biopolymers obtained from both fermentations with glycerol showed low molecular masses about 300 kDa with a polydispersity of 4.72 with C. necator, and 200 kDa with polydispersity of 2.50 with B. sacchari., Postprint (author's final draft)

2-Hydroxyisobutyryl-coenzyme A mutase, originally discovered in the context of methyl tert-butyl ether degradation in Aquincola tertiaricarbonis L108, catalyzes the isomerization of 3-hydroxybutyryl-coenzyme A (3-HB-CoA) to 2-hydroxyisobutyryl-CoA. It thus constitutes the basis for a biotechnological route from practically any renewable carbon to 2-hydroxyisobutyrate (2-HIB) via the common metabolite 3-hydroxybutyrate. At first sight, recombinant Cupriavidus necator H16 expressing the mutase seems to be well suited for such a synthesis process, as a strong overflow metabolism via (R)-3-HB-CoA is easily induced in this bacterium possessing the poly-3-hydroxybutyrate metabolism. However, the recently established stereospecificity of the mutase, dominantly preferring the (S)-enantiomer of 3-HB-CoA, calls for a closer investigation of C. necator as potential 2-HIB production strain and raised the question about the strain’s potential to yield 2-HIB from substrates directly providing (S)-3-HB-CoA. We compared two mutase-expressing C. necator H16 strains for their capability to synthesize 2-HIB from fructose and butyrate, delivering either (R)- or (S)-3-HB-CoA. Our results indicate that due to the enantiospecificity of the mutase, fructose is a weaker substrate for 2-HIB synthesis than butyrate. Production rates achieved with the PHB-negative strain H16 PHB−4 on butyrate were higher than on fructose. Using the wild-type did not significantly improve the production rates as the latter showed a 34-fold and a 5-fold lower 2-HIB synthesis rate compared to H16 PHB−4 on fructose and butyrate, respectively. Moreover, both strains showed concomitant excretion of undesired side products, such as pyruvate and 3-hydroxybutyrate, significantly decreasing the 2-HIB yield.

Cupriavidus necator JMP134 has been extensively studied because of its ability to degrade chloroaromatic compounds, including the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 3-chlorobenzoic acid (3-CB), which is achieved through the pJP4-encoded chlorocatechol degradation gene clusters: tfdCIDIEIFI and tfdDIICIIEIIFII. The present work describes a different tfd-genes expression profile depending on whether C. necator cells were induced with 2,4-D or 3-CB. By contrast, in vitro binding assays of the purified transcriptional activator TfdR showed similar binding to both tfd intergenic regions; these results were confirmed by in vivo studies of the expression of transcriptional lacZ fusions for these intergenic regions. Experiments aimed at investigating whether other pJP4 plasmid or chromosomal regulatory proteins could contribute to the differences in the response of both tfd promoters to induction by 2,4-D and 3-CB showed that the transcriptional regulators from the benzoate degradation pathway, CatR1 and CatR2, affected 3-CB- and 2,4-D-related growth capabilities. It was also determined that the ISJP4-interrupted protein TfdT decreased growth on 3-CB. In addition, an ORF with 34% amino acid identity to IclR-type transcriptional regulator members and located near the tfdII gene cluster module was shown to modulate the 2,4-D growth capability. Taken together, these results suggest that tfd transcriptional regulation in C. necator JMP134 is far more complex than previously thought and that it involves proteins from different transcriptional regulator families. [Int Microbiol 2009; 12(2):97-106]

Cupriavidus necator JMP134 has been extensively studied because of its ability to degrade chloroaromatic compounds, including the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 3-chlorobenzoic acid (3-CB), which is achieved through the pJP4-encoded chlorocatechol degradation gene clusters: tfdCIDIEIFI and tfdDIICIIEIIFII. The present work describes a different tfd-genes expression profile depending on whether C. necator cells were induced with 2,4-D or 3-CB. By contrast, in vitro binding assays of the purified transcriptional activator TfdR showed similar binding to both tfd intergenic regions; these results were confirmed by in vivo studies of the expression of transcriptional lacZ fusions for these intergenic regions. Experiments aimed at investigating whether other pJP4 plasmid or chromosomal regulatory proteins could contribute to the differences in the response of both tfd promoters to induction by 2,4-D and 3-CB showed that the transcriptional regulators from the benzoate degradation pathway, CatR1 and CatR2, affected 3-CB- and 2,4-D-related growth capabilities. It was also determined that the ISJP4-interrupted protein TfdT decreased growth on 3-CB. In addition, an ORF with 34% amino acid identity to IclR-type transcriptional regulator members and located near the tfdII gene cluster module was shown to modulate the 2,4-D growth capability. Taken together, these results suggest that tfd transcriptional regulation in C. necator JMP134 is far more complex than previously thought and that it involves proteins from different transcriptional regulator families. [Int Microbiol 2009; 12(2):97-106]

The nucleotide sequence of the biphenyl catabolic transposon Tn4371 has been completed and analyzed. It confirmed that the element has a mosaic structure made of several building blocks. In addition to previously identified genes coding for a tyrosine recombinase related to phage integrases and for biphenyl degradation enzymes very similar to those of Achromobacter georgiopolitanum KKS102, Tn4371 carries many plasmid-related genes involved in replication, partition, and other, as-yet-unknown, plasmid functions. One gene cluster contains most of the genes required to express a type IV secretion-mating pair formation apparatus coupled with a TraG ATPase, all of which are related to those found on IncP and Ti plasmids. Orthologues of all Tn4371 plasmid-related genes and of the tyrosine recombinase gene were found, with a very similar organization, in the chromosome of Ralstonia solanacearum and on the yet-to-be-determined genomic sequences of Erwinia chrysanthemi and Azotobacter vinelandii. In each of these chromosomal segments, conserved segments were separated by different groups of genes, which also differed from the Tn4371 bph genes. The conserved blocks of genes were also identified, in at least two copies, in the chromosome of Ralstonia metallidurans CH34. Tn4371 thus appears to represent a new family of potentially mobile genomic islands with a broad host range since they reside in a wide range of soil proteobacteria, including plant pathogens., SCOPUS: ar.j, info:eu-repo/semantics/published

The nucleotide sequence of the biphenyl catabolic transposon Tn4371 has been completed and analyzed. It confirmed that the element has a mosaic structure made of several building blocks. In addition to previously identified genes coding for a tyrosine recombinase related to phage integrases and for biphenyl degradation enzymes very similar to those of Achromobacter georgiopolitanum KKS102, Tn4371 carries many plasmid-related genes involved in replication, partition, and other, as-yet-unknown, plasmid functions. One gene cluster contains most of the genes required to express a type IV secretion-mating pair formation apparatus coupled with a TraG ATPase, all of which are related to those found on IncP and Ti plasmids. Orthologues of all Tn4371 plasmid-related genes and of the tyrosine recombinase gene were found, with a very similar organization, in the chromosome of Ralstonia solanacearum and on the yet-to-be-determined genomic sequences of Erwinia chrysanthemi and Azotobacter vinelandii. In each of these chromosomal segments, conserved segments were separated by different groups of genes, which also differed from the Tn4371 bph genes. The conserved blocks of genes were also identified, in at least two copies, in the chromosome of Ralstonia metallidurans CH34. Tn4371 thus appears to represent a new family of potentially mobile genomic islands with a broad host range since they reside in a wide range of soil proteobacteria, including plant pathogens., SCOPUS: ar.j, info:eu-repo/semantics/published

Bacterial predation of bacteria in soil, or in other habitats, is a relatively new field of study. To data, only a few bacterial predators of bacteria have been found. Some of them are obligate predators, others are not. I shall only briefly mention obligate bacterial predation of bacteria, for example, as with Bdellovibrio bacteriovorus, since this is not the subject of the present review. The subject is included because, as noted below, nonobligate strains of this bacterium have been described. Nonobligate bacterial predators of bacteria are present and active in soil. Each predator can attack a few, or in some cases, many prey species. Some predator species can attack other predator species, and the arrays of sensitive prey species for the various predatory species are not necessarily the same, some of the sensitive prey species for a given predator may survive relatively well if the predator is, in turn, attacked by another predator. Ecological predictions based on this predator-on-predator attack are further complicated by the attack-counterattack phenomenon. Thus, in certain cases, it is possible for the prey species (actually a predator in its own right) to counterattack the predator species. Certain gram-negative predator bacteria (e.g., strain N-1) are at or near the top of the bacterial predation hierarchy in soil. They attack both gram-positive and gram-negative prey species, including other predator bacteria, and they can operate as predators in both nutritionally poor and nutritionally rich soil, although less well in the latter., Pub. in Microbiol Ecology v15 p1-8 1988.

Tato bakalářská práce se zabývá vlivem akumulace PHA na stresovou odolnost bakterií vůči dezinfekčním prostředkům. Jako modelové mikroorganismy, využité pro analýzy v experimentální části práce, byly zvoleny dva kmeny: PHA akumulující Cupriavidus necator H16 a její mutantní kmen neschopný PHA syntézy Cupriavidus necator PHB-4. Tyto bakterie byly exponovány třem vybraným dezinfekčním prostředkům (ethanol, savo a ajatin) o třech různých koncentracích. Byla stanovena viabilita bakterií při působení daných detergentů, a to plotnovou metodou a průtokovým cytometrem. Vliv dezinfekčních prostředků na bakterie byl dále zkoumán bujónovou diluční metodou, která spočívala v měření absorbance v čase. Poslední využitou technikou byla agarová difúzní metoda. Z výsledků je patrné, že produkce PHA pozitivně napomáhá obranyschopnosti buněk vůči dezinfekčním prostředkům, jelikož bakteriální kmen Cupriavidus necator H16 je více odolný než jeho mutantní kmen Cupriavidus necator PHB-4. Ze tří vybraných dezinfekčních prostředků je neúčinnější ajatin., This bachelor thesis deals with the influence of acumulation of PHA balancing on the resistence of bacteria against desinfecting means. Two bacterial strains, PHA accumulation Cupriavidus necator H16 and its mutant strain insoluble PHA synthase Cupriavidus necator PHB-4 were used as model microorganisms. These bacteria were exposed to three different concentration of three desinfecting means (etanol, savo and ajatin). The viability of bacteria by the action of disinfectants was determined by the plate method and by the flow cytometry. The influence of desinfecting means against bacteria was examined by the broth dilution method which rested in measurement of absorbance over time. Agar diffusion method was the last one technique use. The results show that PHA production positively aids cell defence against desinfectants because bacterial strain Cupriavidus necator H16 is more resistant than its mutant strain Cupriavidus necator PHB-4. Of the free selected disinfectants, ajatin is the most effective.

Předmětem bakalářské práce bylo studium Gram-negativních bakterií se schopností akumulovat polyhydroxyalkanoáty pomocí mikroskopie atomárních sil. V rámci experimentální části práce byly zkoumány bakteriální kmeny Cupriavidus necator H16 a jeho mutantní kmen Cupriavidus necator PHB-4, Rhodospirillum rubrum a Halomonas halophila. Konkrétním cílem bylo optimalizovat tuto mikroskopickou metodu pro výzkum jak bakterií na vzduchu, tak živých bakterií v jejich přirozeném prostředí. Optimalizace byla provedena s ohledem na jednoduchost, časovou náročnost a reprodukovatelnost provedení měření. Pro měření na vzduchu byla zvolena imobilizace 10× zředěné bakteriální suspenze vysušením na skleněném substrátu. Skenování bylo provedeno pomocí hrotu TESPA-V2 se sníženou konstantou tuhosti v QITM módu. V rámci měření bakteriálních vzorků v kapalině byly zkoumány různé postupy imobilizace bakterií na skleněném substrátu pomocí Poly-L-lysinu a Poly(ethyleniminu). Jako nejvhodnější fixace byla zvolena metoda s využitím Poly-L-lysinu. Pro měření v QITM módu v kapalném prostředí byly voleny hroty MLCT – A nebo SNL – B s konstantou tuhosti okolo 0,1 N/m. Nakonec byl proveden pokus o zisk informací o adhezi a Youngově modulu vysušených i živých bakterií s ohledem na předpokládané ovlivnění mechanických vlastností buněk s rozdílným obsahem polyhydroxybutyrátu u bakteriálních kmenů Cupriavidus necator H16 a Cupriavidus necator PHB-4., The purpose of this bachelor thesis was to study Gram-negative bacteria with the ability to accumulate polyhydroxyalkanoates using atomic force microscopy. During the experimental part were studied bacterial strains Cupriavidus necator H16 and its mutant strain Cupriavidus necator PHB-4, Rhodospirillum rubrum, and Halomonas halophila. The main aim was to optimise this microscopy method to study either bacteria in the air or bacteria in liquid medium under physiological conditions. The optimisation was done considering the procedure's simplicity, time demands, and reproducibility. 10× diluted bacterial suspense was dried on a glass surface for measurements in the air. Scanning was performed using TESPA-V2 tip with lowered Spring constant in QITM mode. Various procedures for immobilising bacteria on a glass substrate using Poly-L-lysine and Poly(ethyleneimine) were tested for measurements in liquid. The method using Poly-L-lysine was chosen as the most sufficient way of immobilisation. The tips MLCT – A or SNL – B with the spring constant around 0,1 N/m were applied for measurements in liquid using QITM mode. Finally, an attempt was made to obtain information about the adhesion and Young's modulus of dried and live bacteria concerning the high difference of polyhydroxybutyrate content between the bacterial strains Cupriavidus necator H16 and Cupriavidus necator PHB-4.

Tato bakalářská práce se zabývá stresovou odolností bakterií produkující PHA vůči nepříznivým podmínkám v gastrointestinálním traktu. Za tímto účelem byly zvoleny dva bakteriální kmeny jako modelové mikroorganismy. Cupriavidus necator H16 produkující PHA a její mutantní kmen Cupriavidus necator PHB-4 bez schopnosti akumulace PHA. Pro experimentální část byly nejprve vybrány tři šťávy o různých koncentracích, konkrétně žaludeční, žlučová a pankreatická. Dále byla stanovována viabilita stresovaného bakteriálního kmene pomocí plotnové metody a průtokového cytometru. Mezi další využité metody patří antimikrobiální testy. První z těchto testů byla použita agarová difúzní metoda, díky které byly stanoveny a porovnány velikosti inhibičních zón. Poslední metoda, která byla provedena, je bujónová diluční metoda, sloužící k porovnání absorbancí čistých bakteriálních suspenzí a stresovaných suspenzích s danou šťávou o určité koncentraci. Z výsledků je patrné, že produkce PHA představuje v kontextu expozice stresovým podmínkám gastrointestinálního traktu především nevýhodu pro akumulující bakteriální kmen., This bachelor thesis is focused on stress resistance of PHA accumulating bacteria against unfavourable conditions associated with gastrointestinal tract. Two bacterial strains were chosen as model microorganisms for this purpose. Cupriavidus necator H16 producing PHA and its mutant strain Cupriavidus necator PHB-4 without the ability of PHA accumulation. Firstly, three gastrointestinal juices of different concentrations were chosen for the experimental part, namely gastric, bile and pancreatic juices. The stressed bacterial strain was then determined using a spread plate method and a flow cytometry. Other methods used include antimicrobial tests. The agar diffusion method was first tested to determine the size of the inhibition zones. The last method that has been performed is the broth dilution method, which serves to compare the absorbance of pure bacterial suspensions and stressed suspensions with the given juice of a certain concentration. The results show that the production of PHA represents a disadvantage in context of conditions associated with gastrointestinal tract of accumulating bacterial strain.

Cílem práce je studium a návrh fyzikálně-chemické metody na ověření fotoprotektivních vlastností PHB granulí pomocí bakterie Cupriavidus necator H16 a její mutantní kmen Cupriavidus necator PHB-4 lišíci se schopností produkce polyhydroxyalkanoátů. V teoretické části jsou zpracované základní informace ku polymerním agregátů a metodám využitých při zkoumaní fotoprotektivity. Byli optimalizovány metody jako nefelometrie a kontinuální fluorescenční mikrofotolýza (CFM) sloužíci k charakterizaci mechanizmu způsobu interakce světla s granulemi. V experimentální časti byly stanovovány rozptylové vlastnosti bakterií a získány charakteristické křivky. Dále byla porovnávána fotostabilita fluorescenční sondy specifická pro nukleové kyseliny v bakteriích. Tato metoda měla dokázat větší odolnost vůči UV světlu kmenu H16. Zadefinovala také možnou interakci nukleových kyselin a zkoumaných granulí., The objective of this work is study and design of physical-chemical methods for verification of photoprotective properties PHB inclusions using bacteria Cupriavidus necator H16 and its mutated bacteria strain Cupriavidus necator PHB-4 which differs in production capability of polyhydroxyalkanoates. Basic information about of polymer inslusions and methods used for proving photoprotectivity are discussed in theoretical part. Methods such as nephelometry and continous fluorescence microphotolysis (CMF), which is used to characterize the mechanism of interaction of light with inclusions, were optimized. In experimental part properties of light scattering in bacteria were determined and characteristic curves were obtained. Further the photostability of fluorescence probe specific for nucleic acids were compared in bacteria strains. This method should prove bigger tolerance of bacteria H16 for UV light. It should also define possible interaction of nucleic acids with PHB inclusions.

Táto bakalárska práca sa zaoberá vplyvom oxidačného stresu na bakteriálny kmeň Cupriavidus necator v dvoch podobách: Cupriavidus necator H16 tvoriaci PHB granule a Cupriavidus necator PHB-4 ako mutant, ktorý nedokáže granule syntetizovať. Práca porovnáva vplyvy vyvolávajúce oxidačný stres, ktorý sa prejavuje výskytom ROS v bunke. Bunky boli ďalej analyzované na prietokovom cytometre a fluorescenčnom mikroskope. V analýze bolo potrebné pracovať s fluorescenčnými sondami rôzneho typu. Na vytvorenie oxidačného stresu boli použité viaceré koncentrácie peroxidu vodíka, ďalej bola vyskúšaná Fentonova reakcia s použitím síranu železnato–amonného a nakoniec hexahydrát chloridu kobaltnatého. Na zistenie viability bakteriálnych buniek sa používala fluorescenčná sonda propidium jodid, ako lipofilné farbivo bolo testované BODIPY. Na záver to boli fluorescenčné sondy na detekciu ROS v bunke H2DCFDA a CM–H2DCFDA, tieto sondy museli byť optimalizované pre danú techniku. Záverom práce je potvrdenie hypotézy, že schopnosť produkcie PHA granúl zvyšuje odolnosť C. necator proti oxidačnému stresu., This bachelor thesis deals with the impact of oxidative pressure on the bacterial strain Cupriavidus necator in its two forms: Cupriavidus necator H16 producing PHB granules and Cupriavidus necator PHB-4 as a mutant that is not capable of producing granules. The thesis compares different influences causing oxidative stress that is demonstrated by the occurrence of ROS in the cell. Furthermore, the cells were analysed with a flow cytometer and a fluorescence microscope. During the analysis it was necessary to use different types of fluorescence probes. The oxidative stress was created by means of hydrogen peroxide at various concentrations. Further tests focused on Fenton’s reaction including ammonium iron sulfate and cobalt chloride hexahydrate. Propidium iodide as a fluorescence probe was used to determine the viability of the bacterial cells, and BODIPY was tested as a lipophilic dye. Finally, the ROS in the cell was detected by H2DCFDA and CM–H2DCFDA, fluorescence probes that had to be optimised for the respective technique. The result of this bachelor thesis is the confirmation of the hypothesis that PHA granules production increase the resistance of Cupriavidus necator against oxidative stress.

Bakalářská práce se zabývá srovnáním a optimalizací metod studia biologických membrán pomocí fluorescenční spektroskopie a mikroskopie s využitím bakterie Cupriavidus necator H16 (obsahující PHB granule) a PHB-4 (neobsahující PHB granule) jako modelového organismu. Experimentálně byla určena vhodná koncentrace sondy laurdan pro barvení biologických membrán a čas potřebný k jejímu průniku do buňky. Takto obarvené organismy byly dále studovány pomocí steady-state spektrofluorimetrie, při které byl zkoumán vliv teploty na tvar emisního spektra a také možnost promývání jako jednoho z kroků při přípravě vzorků. Obě tato měření naznačují výhodnost promývání, ale neposkytují nezpochybnitelné výsledky. Rozdíl mezi dvěma kmeny bakterie byl ale pozorován. Dále byly bakterie podrobeny fluorescenční mikroskopii. Tato metoda se projevila jako potenciálně užitečná pro studium výraznějších variací ve stupni uspořádání membrán buněčné membrány a pro zobrazování dalších větších útvarů. Distribuce laurdanu v rámci Cupriavidu necator H16 působí nerovnoměrněji ve srovnání s PHB-4. Získané poznatky tak naznačují vliv přítomnosti granulí při barvení fluorescenčními sondami, nelze je ale interpretovat jednoznačně., This bachelor's thesis deals with the comparison and optimization of methods for studying biological membranes using fluorescence spectroscopy and microscopy, utilizing the bacteria Cupriavidus necator H16 (containing PHB granules) and PHB-4 (not containing PHB granules) as model organisms. The appropriate concentration of the membrane probe laurdan for staining biological membranes and the time required for its penetration into the cell were determined. Organisms stained in this way were further studied using steady-state spectroscopy, which helped examine the influence of temperature on the shape of the emission spectrum and the possibility of washing as one of the steps in sample preparation. Both of these measurements suggest the advantages of washing but do not provide definite results. However, a clear difference between the two bacterial strains was observed. Furthermore, the bacteria were studied by fluorescence microscopy. This method proved to be potentially useful for studying significant variations in the membrane order of the cell membrane and for imaging other larger structures. The distribution of laurdan within Cupriavidus necator H16 appears more uneven compared to PHB-4. The findings thus suggest the influence of granule presence when staining with fluorescent probes, but they cannot be interpreted definitively.

Techniky analýzy jednotlivých buněk neboli single-cell analýzy, jsou velmi důležité v kontextu studia důležitých biotechnologických mikroorganismů. V biotechnologických procesech je optimální mít rychlé, efektivní a real-time analýzy, které lze provést s minimální přípravou vzorku. Proto jsme v rámci předložené disertační práce využili autofluorescenci jako marker fyziologického stavu mikrobiální kultury, který je možné analyzovat pomocí fluorescenční mikroskopie a/nebo průtokové cytometrie. Nejdříve bylo potvrzeno, na základně emisních spekter, že zelená autofluorescence emitovaná bakteriemi Cupriavidus necator H16 a jejím polyhydroxyalkanoáty (PHA) neprodukujícím mutantem Cupriavidus necator PHB-4, pochází skutečně z flavinů. Emisní maximum bylo naměřeno v rozmezí 510–550 nm pro FAD, FMN a riboflavin – bohužel jednotlivá spektra od sebe nebylo možné rozeznat, a 515 nm pro oba bakteriální kmeny Cupriavidus necator. Byly nalezeny dvě maxima excitačních spekter, jedno v rozmezí 360–370 nm a druhé okolo 440 nm. Z těchto poznatků byl sestaven nový protokol pro fluorescenční mikroskopii – laser byl nastaven na hodnotu 467 nm a emisní filtr pro detekci byl vybrán 520/35 nm. Flaviny a jejich autofluorescence může být take použita v kombinaci s jinými fluorofory, když jsou použity multiparametrické analýzy, ale je důrazně doporučeno, aby vybrané fluorescenční sondy buď emitovaly v jiném rozmezí, než je 500–550 nm anebo byla jejich doba života excitovaného stavu jiná, než je interval 3,2–3,7 a 4,2 – 4,9 ns. Tyto doby života navíc zůstaly konstantní, i když byly buňky vystaveny kultivačním a stresovým podmínkám. To z nich dělá velmi stabilní marker v porovnání s dynamickým charakterem intenzit fluorescence. Dále byl optimalizován protokol pro značení bakterie C. necator s využitím PHA specifické sondy BODIPY 493/503. Optimální koncentrace této lipofilní sondy byla stanovena na 2,5 µg ml-1 a sondu je možné dle našich dat aplikovat společně s viabilitní sondou propidium iodide, Single-cell analysis techniques are very important when studying relevant biotechnological microorganisms. In biotechnological processes, the optimum is to have fast, efficient and real-time analyses that can be done with minimal preparations. Therefore, we looked at autofluorescence as a possible and natural marker when employing fluorescence microscopy and flow cytometry. First, it has been established, based on the emission spectra, that the green autofluorescence of the bacteria used – Cupriavidus necator H16 and its non polyhydroxyalkanoates (PHA) producing mutant Cupriavidus necator PHB-4, is caused predominantly by flavins. The maxima of emission spectra were 510 – 550 nm for FAD, FMN and riboflavin whose spectra could not be distinguished, and 515 nm for both strains of Cupriavidus necator. Two maxima of excitation spectra of the bacteria were found – one in the range 360 – 370 nm and another around 440 nm, and so a new protocol for fluorescence microscopy has been established - the laser used was 467 nm and the emission filter chosen for the detection was 520/35 nm. Flavins and their autofluorescence can also be used in combination with other fluorophores when the need for multi-parametrical analyses arises, but it’s highly recommended the other dyes emit other than green fluorescence in the band of 500 – 550 nm or/and have an average fluorescence lifetime other than 3.2 – 3.7 and 4.2 - 4.9 ns. These values stayed constant even when the cells were exposed to various cultivation and even stress conditions. That makes them a very stable marker compared to the dynamic marker of fluorescence intensity that changes immediately after a change in the environment. A new staining protocol for C. necator using PHA-specific hydrophobic probe BODIPY 493/503 was optimised. The optimal staining concentration of the lipophilic dye BODIPY 493/503 was determined to be 2.5 µg ml-1 and it can be used in combination with propidium iodide. The BODIPY 493/503 staining protocol h

Techniky analýzy jednotlivých buněk neboli single-cell analýzy, jsou velmi důležité v kontextu studia důležitých biotechnologických mikroorganismů. V biotechnologických procesech je optimální mít rychlé, efektivní a real-time analýzy, které lze provést s minimální přípravou vzorku. Proto jsme v rámci předložené disertační práce využili autofluorescenci jako marker fyziologického stavu mikrobiální kultury, který je možné analyzovat pomocí fluorescenční mikroskopie a/nebo průtokové cytometrie. Nejdříve bylo potvrzeno, na základně emisních spekter, že zelená autofluorescence emitovaná bakteriemi Cupriavidus necator H16 a jejím polyhydroxyalkanoáty (PHA) neprodukujícím mutantem Cupriavidus necator PHB-4, pochází skutečně z flavinů. Emisní maximum bylo naměřeno v rozmezí 510–550 nm pro FAD, FMN a riboflavin – bohužel jednotlivá spektra od sebe nebylo možné rozeznat, a 515 nm pro oba bakteriální kmeny Cupriavidus necator. Byly nalezeny dvě maxima excitačních spekter, jedno v rozmezí 360–370 nm a druhé okolo 440 nm. Z těchto poznatků byl sestaven nový protokol pro fluorescenční mikroskopii – laser byl nastaven na hodnotu 467 nm a emisní filtr pro detekci byl vybrán 520/35 nm. Flaviny a jejich autofluorescence může být take použita v kombinaci s jinými fluorofory, když jsou použity multiparametrické analýzy, ale je důrazně doporučeno, aby vybrané fluorescenční sondy buď emitovaly v jiném rozmezí, než je 500–550 nm anebo byla jejich doba života excitovaného stavu jiná, než je interval 3,2–3,7 a 4,2 – 4,9 ns. Tyto doby života navíc zůstaly konstantní, i když byly buňky vystaveny kultivačním a stresovým podmínkám. To z nich dělá velmi stabilní marker v porovnání s dynamickým charakterem intenzit fluorescence. Dále byl optimalizován protokol pro značení bakterie C. necator s využitím PHA specifické sondy BODIPY 493/503. Optimální koncentrace této lipofilní sondy byla stanovena na 2,5 µg ml-1 a sondu je možné dle našich dat aplikovat společně s viabilitní sondou propidium iodide, Single-cell analysis techniques are very important when studying relevant biotechnological microorganisms. In biotechnological processes, the optimum is to have fast, efficient and real-time analyses that can be done with minimal preparations. Therefore, we looked at autofluorescence as a possible and natural marker when employing fluorescence microscopy and flow cytometry. First, it has been established, based on the emission spectra, that the green autofluorescence of the bacteria used – Cupriavidus necator H16 and its non polyhydroxyalkanoates (PHA) producing mutant Cupriavidus necator PHB-4, is caused predominantly by flavins. The maxima of emission spectra were 510 – 550 nm for FAD, FMN and riboflavin whose spectra could not be distinguished, and 515 nm for both strains of Cupriavidus necator. Two maxima of excitation spectra of the bacteria were found – one in the range 360 – 370 nm and another around 440 nm, and so a new protocol for fluorescence microscopy has been established - the laser used was 467 nm and the emission filter chosen for the detection was 520/35 nm. Flavins and their autofluorescence can also be used in combination with other fluorophores when the need for multi-parametrical analyses arises, but it’s highly recommended the other dyes emit other than green fluorescence in the band of 500 – 550 nm or/and have an average fluorescence lifetime other than 3.2 – 3.7 and 4.2 - 4.9 ns. These values stayed constant even when the cells were exposed to various cultivation and even stress conditions. That makes them a very stable marker compared to the dynamic marker of fluorescence intensity that changes immediately after a change in the environment. A new staining protocol for C. necator using PHA-specific hydrophobic probe BODIPY 493/503 was optimised. The optimal staining concentration of the lipophilic dye BODIPY 493/503 was determined to be 2.5 µg ml-1 and it can be used in combination with propidium iodide. The BODIPY 493/503 staining protocol h

Bakalářská práce byla zaměřena na studium možností využití odpadních produktů růže šípkové získaných po výrobě likérů jako sekundárních uhlíkových zdrojů pro produkci poly(3- hydroxybutyrátu) (PHB). Pro biosyntézu PHB byla použita bakterie Cupriavidus necator. Veškeré kultivace byly vedeny jako submerzní batch kultivace v minerálním médiu s obsahem mikroelementů v erlenmeyerových baňkách po dobu 72 hodin. Jako sekundární zdroj uhlíku se použil šípkový olej získaný extrakcí hexanem ze zrníček plodu šípku a enzymatický hydrolyzát připravený z odpadní dužiny plodu šípku. Výtěžky po 72-hodinové fermentaci ukázaly, že šípkový olej je porovnatelným uhlíkovým zdrojem s fruktózou pro kultivaci bakterie C. necator, na rozdíl od enzymatického hydrolyzátu. Fermentací za použití 20 g/l šípkového oleje bylo vyprodukováno 10,82 g/l biomasy a 6,20 g/l PHB, což bylo o 21,6 % víc biomasy a o 13,1 % víc PHB v porovnání s fermentací za použití 20 g/l fruktózy. Fermentací C. necator za použití enzymatického hydrolyzátu o koncentraci redukujících cukrů 20 g/l se získalo jenom 2,92 g/l biomasy a 0,12 g/l PHB. Ani kombinace enzymatického hydrolyzátu s fruktózou se neukázala vhodná pro produkci PHB. V rámci této bakalářské práce byl dále v likéru, šípkovém oleji a enzymatickém hydrolyzátu stanoven obsah celkových redukujících cukrů, fenolických látek, a celková antioxidační aktivita. V šípkovém oleji byl také stanoven profil mastných kyselin a obsah barviv. Výsledky ukázali, že všechny stanovované vzorky vykazovali významný obsah celkových polyfenolů (2,04 – 12,4 g GAE/l vzorku) a antioxidační aktivitu (1,3 – 2,0 mmol TE /l vzorku)., The bachelor thesis was focused on studying the possibilities of using waste rosehip byproducts obtained after the production of liqueurs as secondary carbon sources for the production of poly (3-hydroxybutyrate) (PHB). Cupriavidus necator was used for PHB biosynthesis. All cultures were run as submerged batch cultures in mineral media containing microelements in Erlenmeyer flasks for 72 hours. Rosehip oil obtained by hexane extraction from rosehip seeds and an enzymatic hydrolysate prepared from rosehip waste pulp were used as secondary carbon sources. After 72 hours of fermentation, yields showed that rosehip oil is a comparable carbon source to fructose for culturing of C. necator, in contrast to the enzymatic hydrolysate. Fermentation using 20 g / l rosehip oil produced 10.82 g / l biomass and 6.20 g / l PHB. This is about 21.6% more biomass and 13.1% more PHB than was received by fermentation using 20 g / l fructose. Fermentation of C. necator using an enzymatic hydrolysate with a reducing sugar concentration of 20 g / l gave only 2.92 g / l biomass and 0.12 g / l PHB. Even the combination of enzymatic hydrolysate with fructose did not prove satisfying yields for the production of PHB. Next, in liqueur, rosehip oil and enzymatic hydrolysate was also determined the content of total reducing sugars, phenolics, and total antioxidant activity. Moreover, the fatty acid profile and the content of dyes was determined in rosehip oil. The results showed that all selected rosehip products showed a significant range of total polyphenols (2.04 - 12.4 g GAE /l sample) and antioxidant activity (1.3 - 2.0 mmol TE /l sample).

Bakalářská práce je zaměřena na sledování a uplatnění metod obrazové analýzy na bakteriích druhu Bacillus megaterium a Cupriavidus necator. V úvodu práce jsou popsány obecné znalosti o bakteriích, mikroskopických metodách a metodách obrazové analýzy. K pozorování buněk bakterií, bylo nutné nejprve připravit vhodné mikroskopické preparáty pomocí metody Gramova barvení. Obrazy jednotlivých snímků bakterií byly zaznamenány pomocí CCD kamery a mikroskopu a potom analyzovány. Nejprve byla metodou Box–Counting zjištěna optimální hodnota prahu a jí odpovídající fraktální míry (resp. plochy pokryté bakteriemi) a fraktální dimenze (resp. entropie jejich uspořádání na ploše). Prahované obrázky byly dále zpracovány pomocí metody Mass–Radius, která umožnila zjistit distribuci bakterií vzhledem k těžišti jejich shluku., This bachelor thesis is focused on monitoring and application of image analysis methods on Bacillus megaterium and Cupriavidus necator bacteria. The introduction describes a general knowledge about bacteria, microscopic and image analysis methods. At first it was necessary to prepare suitable microscopic specimens using Gram staining for the observation of a bacteria cell. Every picture of bacteria was taken using a CCD camera and a microscope. Box–Counting method was used to identify the optimal threshold value, corresponding to their fractal measure (or surface areas covered with bacteria) and fractal dimension (or entropy of their arrangement on the surface area). After thresholding, the images were further processed using the Mass–Radius method, which defines a distribution of bacteria relative to the centroid of a cluster.

V rámci této práce byly nejprve připraveny artificiální PHB granule. Pomocí dynamického a elektroforetického rozptylu světla byl studován vliv použitého rozpouštědla PHB, vliv teploty a délky působení ultrazvukové lázně, vliv teploty odpaření rozpouštědla a vliv povahy stabilizačních látek. Nejvhodnější parametry byly pozorovány u granulí připravených rozpuštěním PHB v chloroformu, stabilizací CTAB nebo lecitinem, ultrasonifikací při 35 °C a následným odpařováním chloroformu při 60 °C. Na základě výsledků ATR-FTIR a Ramanovy spektroskopie bylo zjištěno, že PHB v artificiálních granulích byl krystalický. Nativní PHB granule byly izolovány z bakterií Cupriavidus necator pomocí lysozymu, deoxyribonukleázy a rozrušením buněk ultrazvukem nebo digescí buněk s alkalázou, SDS a EDTA. Granule získané oběma izolačními postupy byly charakterizovány pomocí ATR-FTIR, Ramanovy spektroskopie, technikami rozptylu světla a DSC. Výsledky analýz ukázaly, že druhý zmíněný postup je pro získání polymeru v amorfním stavu mnohem efektivnější. Polymer uvnitř granulí izolovaných tímto způsobem byl navíc i tepelně stabilnější. Nakonec byly vzorky nativních PHB granulí vystaveny vlivu acetonu, lipázy a chlornanu sodného s tím, že by tyto látky měly v různé míře zvýšit krystalinitu polymeru. Nejvyšší krystalinity PHB bylo dosaženo po jejich ošetření lipázou., First artificial PHB granules were prepared under the terms of this thesis. The effect of used PHB solvent, ultrasonic bath temperature and time, solvent evaporation temperature and stabilizing agent nature was investigated using dynamic and electrophoretic light scattering. The most proper parameters were demonstrated at samples which were prepared by dissolving of PHB in chloroform, stabilizing with CTAB or lecithin, ultrasonifying at 35 °C followed by chloroform evaporating at 60 °C. Based on ATR-FTIR and Raman spectroscopy results, it was found out that PHB within the artificial granules were in crystalline form. The native PHB granules were isolated from Cupriavidus necator using either lysosyme, deoxyribonuclease and cell disruption by ultrasonification or digestion with alcalase, SDS and EDTA. Granules obtained by both isolation procedures were characterized by ATR-FTIR, Raman spectroscopy, light scattering techniques and DSC. According to the analyses results, the second mentioned procedure turned out to be more effective for obtaining the polymer in amorphous state. Furthermore, the polymer within granules recovered by using this procedure was thermally more stable. Last but not least, the native PHB granules samples were exposed to effect of acetone, lipase and sodium hypochlorite, assuming that polymer crystallinity should be increased by these chemicals in varying degrees. The highest degree of crystallinity was achieved after their treatment with lipase.

Táto diplomová práca sa zaoberá možnosťami analýzy stresovej odolnosti bakteriálnych buniek Cupriavidus necator H16 a PHB-4 pomocou spektroskopických metód a otestovaním vhodnosti akridinovej oranži ako viabilitného farbiva. Na základe literárnej rešerše boli navrhnuté vhodné analytické metódy, konkrétne prietokový cytometer a fluorescenčný mikroskop. Prvá časť experimentálnej práce bola zameraná na fluorescenčný mikroskop, ktorým bol potvrdený základný charakter akridinovej oranži. Na sledovanie viability boli vybrané tri stresové faktory, 50% a 70% etanol a kyslé pH (pH = 1). Baktérie po expozícii etanolom fluoreskovali zelenou farbou a vedľa buniek sa nachádzali červené škvrny, čo naznačuje ich stratu integrity. V kyslom prostredí baktérie fluoreskovali červene, pretože došlo k čiastočnému porušeniu DNA. Výsledky boli overené kombináciou propídium jodidu so SYTO9 a pri tejto metóde sa osvedčila vhodnosť akridinovej oranži. Obrazové záznamy boli spracované pomocou obrazovej analýzy. V druhej časti bola akridinová oranž použitá pri sledovaní fluorescencie pomocou prietokového cytometra. Výsledkom merania bola fluorescencia vyjadrená vo forme histogramov pre jednotlivé kanály, kde fluorescencia bola charakterizovaná mediánom a priemernou intenzitou. Porovnaním použitých metód sa akridinová oranž javí ako vhodnejšie fluorescnečné farbivo pre mikroskop ako pre prietokový cytometer, v ktorom bolo zložitejšie získať informácie o životaschopnosti buniek. V poslednej časti experimentálne práce boli skúmané zaujímavé fotofyzikálne vlastnosti akridinovej oranži., This diploma thesis deals with the possibilities of stress resistance analysis of the Cupriavidus necator H16 and PHB-4 bacterial cells by spectroscopic methods and by testing the suitability of acridine orange as a viable dye. Based on research in literature, suitable analytical methods have been proposed, namely flow cytometer and fluorescence microscope. The first part of the experimental work was focused on the fluorescence microscope, which confirmed the basic character of acridine orange. Three stress factors, 50% and 70% ethanol, and acidic pH (pH = 1) were selected for viability monitoring. The bacteria fluoresced with green color after exposure to ethanol and red spots were found next to the cells, indicating their loss of integrity. In an acidic environment, the bacteria fluoresced red because of a partial DNA breakdown. The results were verified by the combination of propidium iodide with SYTO9 and the acridine orange suitability proved to be useful in this method. Image records were processed using image analysis. In the second part, acridine orange was used to monitor fluorescence using a flow cytometer. The result of the measurement was fluorescence expressed as histograms for individual channels, where fluorescence was characterized by median and mean intensity. By comparing the methods used, the acridine orange appears to be a more suitable fluorescent dye for the microscope than for a flow cytometer in which it was more difficult to obtain cell viability information. In the last part of the experimental work interesting photophysical properties of acridine orange were investigated.

Bakalářská práce byla zaměřena na studium možností využití odpadních produktů růže šípkové získaných po výrobě likérů jako sekundárních uhlíkových zdrojů pro produkci poly(3- hydroxybutyrátu) (PHB). Pro biosyntézu PHB byla použita bakterie Cupriavidus necator. Veškeré kultivace byly vedeny jako submerzní batch kultivace v minerálním médiu s obsahem mikroelementů v erlenmeyerových baňkách po dobu 72 hodin. Jako sekundární zdroj uhlíku se použil šípkový olej získaný extrakcí hexanem ze zrníček plodu šípku a enzymatický hydrolyzát připravený z odpadní dužiny plodu šípku. Výtěžky po 72-hodinové fermentaci ukázaly, že šípkový olej je porovnatelným uhlíkovým zdrojem s fruktózou pro kultivaci bakterie C. necator, na rozdíl od enzymatického hydrolyzátu. Fermentací za použití 20 g/l šípkového oleje bylo vyprodukováno 10,82 g/l biomasy a 6,20 g/l PHB, což bylo o 21,6 % víc biomasy a o 13,1 % víc PHB v porovnání s fermentací za použití 20 g/l fruktózy. Fermentací C. necator za použití enzymatického hydrolyzátu o koncentraci redukujících cukrů 20 g/l se získalo jenom 2,92 g/l biomasy a 0,12 g/l PHB. Ani kombinace enzymatického hydrolyzátu s fruktózou se neukázala vhodná pro produkci PHB. V rámci této bakalářské práce byl dále v likéru, šípkovém oleji a enzymatickém hydrolyzátu stanoven obsah celkových redukujících cukrů, fenolických látek, a celková antioxidační aktivita. V šípkovém oleji byl také stanoven profil mastných kyselin a obsah barviv. Výsledky ukázali, že všechny stanovované vzorky vykazovali významný obsah celkových polyfenolů (2,04 – 12,4 g GAE/l vzorku) a antioxidační aktivitu (1,3 – 2,0 mmol TE /l vzorku)., The bachelor thesis was focused on studying the possibilities of using waste rosehip byproducts obtained after the production of liqueurs as secondary carbon sources for the production of poly (3-hydroxybutyrate) (PHB). Cupriavidus necator was used for PHB biosynthesis. All cultures were run as submerged batch cultures in mineral media containing microelements in Erlenmeyer flasks for 72 hours. Rosehip oil obtained by hexane extraction from rosehip seeds and an enzymatic hydrolysate prepared from rosehip waste pulp were used as secondary carbon sources. After 72 hours of fermentation, yields showed that rosehip oil is a comparable carbon source to fructose for culturing of C. necator, in contrast to the enzymatic hydrolysate. Fermentation using 20 g / l rosehip oil produced 10.82 g / l biomass and 6.20 g / l PHB. This is about 21.6% more biomass and 13.1% more PHB than was received by fermentation using 20 g / l fructose. Fermentation of C. necator using an enzymatic hydrolysate with a reducing sugar concentration of 20 g / l gave only 2.92 g / l biomass and 0.12 g / l PHB. Even the combination of enzymatic hydrolysate with fructose did not prove satisfying yields for the production of PHB. Next, in liqueur, rosehip oil and enzymatic hydrolysate was also determined the content of total reducing sugars, phenolics, and total antioxidant activity. Moreover, the fatty acid profile and the content of dyes was determined in rosehip oil. The results showed that all selected rosehip products showed a significant range of total polyphenols (2.04 - 12.4 g GAE /l sample) and antioxidant activity (1.3 - 2.0 mmol TE /l sample).