295 research outputs found
Abcg2 overexpression represents a novel mechanism for acquired resistance to the multi-kinase inhibitor Danusertib in BCR-ABL-positive cells in vitro.
No full textThe success of Imatinib (IM) therapy in chronic myeloid leukemia (CML) is compromised by the development of IM resistance and by a limited IM effect on hematopoietic stem cells. Danusertib (formerly PHA-739358) is a potent pan-aurora and ABL kinase inhibitor with activity against known BCR-ABL mutations, including T315I. Here, the individual contribution of both signaling pathways to the therapeutic effect of Danusertib as well as mechanisms underlying the development of resistance and, as a consequence, strategies to overcome resistance to Danusertib were investigated. Starting at low concentrations, a dose-dependent inhibition of BCR-ABL activity was observed, whereas inhibition of aurora kinase activity required higher concentrations, pointing to a therapeutic window between the two effects. Interestingly, the emergence of resistant clones during Danusertib exposure in vitro occurred considerably less frequently than with comparable concentrations of IM. In addition, Danusertib-resistant clones had no mutations in BCR-ABL or aurora kinase domains and remained IM-sensitive. Overexpression of Abcg2 efflux transporter was identified and functionally validated as the predominant mechanism of acquired Danusertib resistance <i>in vitro</i>. Finally, the combined treatment with IM and Danusertib significantly reduced the emergence of drug resistance <i>in vitro</i>, raising hope that this drug combination may also achieve more durable disease control <i>in vivo</i>
Engineering NADH/NAD<sup>+</sup> ratio in Halomonas bluephagenesis for enhanced production of polyhydroxyalkanoates (PHA)
No full textHalomonas bluephagenesis has been developed as a platform strain for the next generation industrial biotechnology (NGIB) with advantages of resistances to microbial contamination and high cell density growth (HCD), especially for production of polyhydroxyalkanoates (PHA) including poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). However, little is known about the mechanism behind PHA accumulation under oxygen limitation. This study for the first time found that H. bluephagenesis utilizes NADH instead of NADPH as a cofactor for PHB production, thus revealing the rare situation of enhanced PHA accumulation under oxygen limitation. To increase NADH/NAD+ ratio for enhanced PHA accumulation under oxygen limitation, an electron transport pathway containing electron transfer flavoprotein subunits α and β encoded by etf operon was blocked to increase NADH supply, leading to 90% PHB accumulation in the cell dry weight (CDW) of H. bluephagenesis compared with 84% by the wild type. Acetic acid, a cost-effective carbon source, was used together with glucose to balance the redox state and reduce inhibition on pyruvate metabolism, resulting in 22% more CDW and 94% PHB accumulation. The cellular redox state changes induced by the addition of acetic acid increased 3HV ratio in its copolymer PHBV from 4% to 8%, 4HB in its copolymer P34HB from 8% to 12%, respectively, by engineered H. bluephagenesis. The strategy of systematically modulation on the redox potential of H. bluephagenesis led to enhanced PHA accumulation and controllable monomer ratios in PHA copolymers under oxygen limitation, reducing energy consumption and scale-up complexity.Accepted Author ManuscriptOLD BT/Cell Systems Engineerin
Production of a newly discovered PHA family member with an isobutyrate-fed enrichment culture
No full textAbstract: Using microbial enrichment cultures for the production of waste-derived polyhydroxyalkanoates (PHAs) is a promising technology to recover secondary resources. Volatile fatty acids (VFAs) form the preferred substrate for PHA production. Isobutyrate is a VFA appearing in multiple waste valorization routes, such as anaerobic fermentation, chain elongation, and microbial electrosynthesis, but has never been assessed individually on its PHA production potential. This research investigates isobutyrate as sole carbon source for a microbial enrichment culture in comparison to its structural isomer butyrate. The results reveal that the enrichment of isobutyrate has a very distinct character regarding microbial community development, PHA productivity, and even PHA composition. Although butyrate is a superior substrate in almost every aspect, this research shows that isobutyrate-rich waste streams have a noteworthy PHA-producing potential. The main finding is that the dominant microorganism, a Comamonas sp., is linked to the production of a unique PHA family member, poly(3-hydroxyisobutyrate) (PHiB), up to 37% of the cell dry weight. This is the first scientific report identifying microbial PHiB production, demonstrating that mixed microbial communities can be a powerful tool for discovery of new metabolic pathways and new types of polymers. Key points: • PHiB production is a successful storage strategy in an isobutyrate-fed SBR • Isomers isobutyrate and butyrate reveal a very distinct PHA production behavior • Enrichments can be a tool for discovery of new metabolic pathways and polymers Graphical abstract: [Figure not available: see fulltext.].BT/Environmental Biotechnolog
A Collection of Notes on Pha Beuk
No full textCollection of notes from A.E. Davidson on the region of Pha Beuk, specifically regarding fishing and three areas: Savannakhet, Pakse, and Khong Islan
The influence of essential growth nutrients on PHA production from waste
No full textVolatile fatty acids (VFA) may serve as building blocks for Polyhydroxyalkanoates (PHA) production and can be derived from waste streams. Ideal streams for PHA production contain a high Chemical Oxygen Demand (COD) to nutrient ratio, such as (waste)water from a paper-mill factory or candy-bar factory. The (waste)water generated by these companies are usually treated anaerobically with the final product being methane containing biogas. Usually, the methane is burned to produce either heat or electricity. Potentially, more value can be added to these streams by producing VFA and/or PHA. PHA can be produced using microbial enrichment cultures that can be established by cultivation in a selective environment that favours the growth of PHA producing microorganisms. Some advantages of using open cultures are that no sterilization and expensive equipment is required compared to pure culture biotechnology. Open culture biotechnology can be effectively applied when the right selection pressure for a specific microbial trait is identified. The microorganism that is most effective in the given conditions will win the competition, i.e. the strongest will survive. A selection criteria for PHA productis is consuming substrate very fast by first making a storage polymer (in this case PHA) from the supplied substrate. The PHA producers prefer VFA as substrate, hence it is important to maximize the VFA content in the substrate stream. For the production of VFA in the product chain towards PHA it is important to minimize the solid content in the feedstock for PHA production. The objective of the research described in this thesis was to gain more insight in the two-step upstream process for PHA production from agricultural waste streams. The first step concerns the maximization of the VFA concentration in the feedstock. Optimization of VFA production was investigated using the granular sludge process in order to maximize the volumetric VFA production capacity and to minimize the solids concentration in the effluent. Two process variables were investigated regarding the PHA production process. Firstly, the influence of the presence of nutrients on PHA production was investigated using PHA producing enrichment cultures. Secondly, the production of PHA was investigated using the leachate of the organic fraction of municipal solid waste (OFMSW) at pilot scale.BT/Environmental Biotechnolog
Process optimization for polyhydroxyalkanoate (PHA) production from waste via microbial enrichment cultures
No full textPolyhydroxyalkanoates (PHA) are compounds naturally produced by microorganisms, with many industrial applications, either as bioplastics or as precursors for production of chemicals. Until now, industrial PHA production was conducted with pure strains of bacteria fed with well-defined feedstocks, making the overall process non-economically feasible. The last decades research on PHA was devoted on producing them in open enrichment cultures using wastewater as substrate, and making the process continuous, decreasing the production costs. Laboratory research with well-defined VFA-based substrates enables high accumulation of PHA, up to 90wt% of the total biomass. After demonstrating the potential for PHA production via enrichment cultures, research was devoted on applying this research. Several wastestreams and operational conditions were used to test PHA production on pilot scale and maybe on short notice on industrial scale. Until now, it was shown that also when using fermented industrial wastewater (e.g. paper mill, food) a high cellular PHA-content could be achieved.The object of this thesis was to tackle problems associated with PHA production when operating the process using wastewater and to make it feasible to apply the strategy universally.In the first chapter general information about PHA (process- and material- based) are given.In the second chapter leachate from the source separated organic fraction of municipal solid waste (OFMSW) was evaluated as a substrate for polyhydroxyalkanoates (PHA) production. Initially, biomass enrichment was conducted directly on leachate in a feast-famine regime. Maximization of the cellular PHA content of the enriched biomass yielded to a low PHA content (29 wt%), suggesting that the selection for PHA-producers was unsuccessful. When the substrate for the enrichment was switched to a synthetic volatile fatty acid (VFA) mixture -resembling the VFA carbon composition of the leachate- the PHA-producers gained the competitive advantage and dominated. Subsequent accumulation with leachate in nutrient excess conditions resulted in a maximum PHA content of 78wt%. Based on the experimental results, enriching a PHA-producing community in a “clean” VFA stream, and then accumulating PHA from a stream that does not allow for enrichment but does enable a high cellular PHA content, enables a high cellular PHA content, contributing to the economic feasibility of the process. The estimated overall PHA yield on substrate can be increased four-fold, in comparison to direct use of the complex matrix for both enrichment and accumulation.The success of enriching PHA-producers in a feast-famine regime strongly depends on the substrate utilized. A distinction can be made between substrates that select for PHA-producers (e.g. volatile fatty acids) and substrates that select for growing organisms (e.g. methanol). In the third chapter the feasibility of using such a mixed substrate for PHA-production was evaluated. A sedimentation step was introduced in the cycle after acetate depletion and the supernatant containing methanol was discharged. This process configuration resulted in an increased maximum PHA storage capacity of the biomass from 48wt% to 70wt%. A model based on the experimental results indicated that the length of the pre-settling period and the supernatant volume that is discharged play a significant role for the elimination of the side population. The difference of the kinetic properties of the two different populations determines the success of the proposed strategy.Double-limitation systems have shown to induce polyhydroxyalkanoates (PHA) production in chemostat studies limited in e.g. carbon and phosphate. In the fourth chapter the impact of double limitation on the enrichment of a PHA producing community was studied in a sequencing batch process. Enrichments at different C/P concentration ratios in the influent were established and the effect on the PHA production capacity and the enrichment community structure was investigated. Experimental results demonstrated that when a double limitation is imposed at a C/P ratio in the influent in a range of 150 (C-mol/mol), the P-content of the biomass and the specific substrate uptake rates decreased. Nonetheless, the PHA storage capacity remained high (with a maximum of 84wt%). At a C/P ratio of 300, competition in the microbial community is based on phosphate uptake, and the PHA production capacity is lost. Biomass specific substrate uptake rates are a linear function of the cellular P-content, offering advantages for scaling-up the PHA production process due to lower oxygen requirements.In the fifth chapter, PHA accumulating microbial enrichment cultures were established in an anaerobic/aerobic sequencing batch reactor (SBR) with glucose as sole substrate. The effect of different solid retention times (SRT; 2 and 4 days) on PHA accumulation were investigated. The experimental results revealed that at both SRT conditions, glucose was first stored anaerobically as glycogen with energy generation from lactate fermentation. Subsequently lactate and glycogen were fermented to acetate and propionate in the anaerobic phase. At 2 d SRT operation, during the aerobic phase the fermentation products where rapidly sequestered by aerobic PHA accumulating microorganisms. When (limiting) nutrients were applied under aerobic conditions PHA formation occurred under anaerobic conditions. At a longer SRT of 4 days the fermentation products where already sequestered in the anaerobic phase into PHA by glycogen accumulating organisms (GAO). In all systems the glucose uptake rate was very fast (-2.7 C-mol/C-mol/h), making it the primary competition factor. Under the conditions tested direct conversion of glucose to PHA was not possible.In the sixth chapter some recommendations and questions that remain unanswered are addressed. As suggested, process could be improved by using a continuous system which would include a settling tank for the removal of carbon that is slowly consumed and leads to growth of “inert” biomass. The possibility of operating the process at lower oxygen concentrations, or completely anoxically, is also discussed.<br/
Production of medium-chain-length PHA in octanoate-fed enrichments dominated by Sphaerotilus sp.
No full textMedium-chain-length polyhydroxyalkanoate (mcl-PHA) production by using microbial enrichments is a promising but largely unexplored approach to obtain elastomeric biomaterials from secondary resources. In this study, several enrichment strategies were tested to select a community with a high mcl-PHA storage capacity when feeding octanoate. On the basis of analysis of the metabolic pathways, the hypothesis was formulated that mcl-PHA production is more favorable under oxygen-limited conditions than short-chain-length PHA (scl-PHA). This hypothesis was confirmed by bioreactor experiments showing that oxygen limitation during the PHA accumulation experiments resulted in a higher fraction of mcl-PHA over scl-PHA (i.e., a PHA content of 76 wt% with an mcl fraction of 0.79 with oxygen limitation, compared to a PHA content of 72 wt% with an mcl-fraction of 0.62 without oxygen limitation). Physicochemical analysis revealed that the extracted PHA could be separated efficiently into a hydroxybutyrate-rich fraction with a higher Mw and a hydroxyhexanoate/hydroxyoctanoate-rich fraction with a lower Mw. The ratio between the two fractions could be adjusted by changing the environmental conditions, such as oxygen availability and pH. Almost all enrichments were dominated by Sphaerotilus sp. This is the first scientific report that links this genus to mcl-PHA production, demonstrating that microbial enrichments can be a powerful tool to explore mcl-PHA biodiversity and to discover novel industrially relevant strains.</p
Optimize the measurement of Poly-hydroxy-alkanoates (PHA) in biomass
No full textPoly-hydroxy-alkanoate (PHA) is an intracellular polymer that can be used as an energy and carbon source by microorganisms. Measuring PHA is important for understanding the microbial metabolism of enhanced biological phosphorus removal (EBPR) and aerobic granular sludge (AGS) systems. There is a commonly used method to measure PHA, which is based on organic solvent extraction and gas chromatography (GC). However, there are different versions of the same method with different parameters, but the role of some of these parameters is unclear. When different types of biomass are analyzed, there is a requirement to understand the parameters and obtain an optimal protocol. In this study, the effect of various digestion times, different alcohols and organic solvents, and acid concentrations were tested to obtain the optimal protocol. The results showed that a minimum digestion time was required to get the maximum yield of PHA, and the time might differ when using different types of biomass. Methanol was shown to be better for GC separation than propanol. Using different organic solvents didn’t affect the final concentration, and an optimal acid concentration was required to determine by comparison. The GC temperature program optimization showed that lower oven temperature in GC is more beneficial for peak separation. From the analysis, it would be suggested to use methanol and chloroform for digestion and keep the digestion time for 24 hours.CIE5050-09Civil Engineering | Environmental Engineerin
Process for selecting polyhydroxyalkanoate (PHA) producing micro-organisms
No full textThe invention relates to a process for selecting a polyhydroxyalkanoate (PHA) producing micro-organism from a natural source comprising a variety of micro-organisms, comprising steps of preparing a fermentation broth comprising the natural source and nutrients in water; creating and maintaining aerobic conditions in the fermentation broth; applying a feast- famine cycle comprising a feast phase and a famine phase, wherein the feast-famine cycle is started with feeding a carbon source to the fermentation broth, nutrients are fed in such amounts to the fermentation broth that the carbon source becomes limiting for the growth of the micro-organisms during the famine phase; the feast-famine cycle has a cycle time of at least 8 hours, and the feeding of the carbon source is performed during a small initial fraction of the feast famine cycle time; withdrawing a part of the micro-organisms during and/or at the end of the feast-famine cycle (referred to as solids withdrawal step); and repeating the feast-famine- cycle and the solids withdrawal step, such that an average solids retention time (SRT) of less than 4 days results. The invention also relates to a mixed culture or isolated microorganism which has an activity of producing PHA, obtainable by the said selection process, use thereof in a process for producing a PHA, and the resulting PHA containing microorganism.BiotechnologyApplied Science
Scale-Up Aspects of PHA Production by Microbial Enrichment Cultures
No full textPolyhydroxyalkanoates (PHAs) are microbial storage polymers accumulated by many different prokaryotes as an intracellular carbon and energy reserve. The polymer properties make PHA interesting as bioplastic. Moreover, the monomers could serve as chiral building blocks or be used as a biofuel.Currently, PHA is commercially produced using pure cultures and well-defined substrates. To reduce the cost of PHA production and allow broad application, microbial enrichment cultures could be used. This eliminates the need for axenic conditions and allows the use agro-industrial waste streams as substrate, contributing to the development of a circular economy. The aim of this thesis was to investigate scale-up aspects of the PHA production by microbial enrichment cultures. A translation of the previously developed laboratory process to industrial application raises new questions concerning the impact of (variable) wastewater composition and process design, for example. Chapter 2 and 3, therefore, focus on the fate of different constituents of an acidified waste stream, and the second part (Chapter 4-6) of the thesis focusses on alternative process configurations. Chapter 1 provides a general introduction to the topic and describes the research preceding this thesis. Chapter 7 summarizes and integrates the main findings
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