| 1. |
- Chambi, Diego, et al.
(författare)
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Exopolysaccharides production by cultivating a bacterial isolate from the hypersaline environment of salar de uyuni (Bolivia) in pretreatment liquids of steam-exploded quinoa stalks and enzymatic hydrolysates of Curupaú sawdust
- 2021
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Ingår i: Fermentation. - : MDPI. - 2311-5637. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- The halotolerant bacterial strain BU-4, isolated from a hypersaline environment, was identified as an exopolysaccharide (EPS) producer. Pretreatment liquids of steam-exploded quinoa stalks and enzymatic hydrolysates of Curupaú sawdust were evaluated as carbon sources for EPS production with the BU-4 strain, and the produced EPS was characterized using FTIR, TGA, and SEM. Cultivation was performed at 30◦C for 48 h, and the cells were separated from the culture broth by centrifugation. EPS was isolated from the cell pellets by ethanol precipitation, and purified by trichloroacetic acid treatment, followed by centrifugation, dialysis, and freeze-drying. EPS production from quinoa stalks-and Curupaú sawdust-based substrates was 2.73 and 0.89 g L−1, respectively, while 2.34 g L−1 was produced when cultivation was performed on glucose. FTIR analysis of the EPS revealed signals typical for polysaccharides, as well as ester carbonyl groups and sulfate groups. High thermal stability, water retention capacity and gel-forming ability were inferred from SEM and TGA. The capability of the halotolerant isolate for producing EPS from pretreatment liquids and hydrolysates was demonstrated, and characterization of the EPS revealed their broad application potential. The study shows a way for producing value-added products from waste materials using a bacterium from a unique Bolivian ecosystem.
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| 2. |
- Daga-Quisbert, Jeanett, et al.
(författare)
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Analysis of the microbiome of the Bolivian high-altitude Lake Pastos Grandes
- 2023
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Ingår i: FEMS Microbiology Ecology. - : Oxford University Press (OUP). - 0168-6496 .- 1574-6941. ; 99:8
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Tidskriftsartikel (refereegranskat)abstract
- Lake Pastos Grandes in Bolivia is mainly composed of salt flats, which are sporadically and only partially submerged during the wet season. In the present study, the chemical composition of water samples of the lake and some influent rivers was determined. We found that it is likely that the lake was influenced by the dilution of metals from ancient evaporites. We performed the first metagenomic studies of this lake. Analyses of shotgun metagenomics revealed that the relative abundances of Burkholderiales and Pseudomonadales were noteworthy in the water samples, whereas the archaea belonging to the Halobacteriales and Cyanobacteria from subsection III had high abundances in the salt flat. The eukaryotes Crustacea and Diatomea exhibited the highest abundances in the water samples. We investigated further the potential effect of human activities on the nitrogen cycle mobilization in the lake and the propagation of antimicrobial resistance genes. This is the first report about the cycle in the lake. Additionally, rifamycin resistance genes and genes related to efflux pumps, which are not considered a hazard when identified in metagenomes, had the uppermost relative abundances in all sampling points. We found that Lake Pastos Grandes hitherto does not show an appreciable influence by anthropogenic actions. The microbiome of Lake Pastos Grandes, including microbial distribution, the nitrogen cycle and antibiotic resistance genes, was analyzed.
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| 3. |
- Daga-Quisbert, Jeanett, et al.
(författare)
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Assessing water quality of a hypereutrophic alkaline urban lake and its coagulation-treated water using metagenomic analysis
- 2024
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Ingår i: Water, Air and Soil Pollution. - : Springer Nature. - 0049-6979 .- 1573-2932. ; 235:6
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Tidskriftsartikel (refereegranskat)abstract
- The study on Lake Alalay, an urban alkaline lake facing increasing pollution, focused on the impact of coagulation treatment on its water quality and microbiome. The findings revealed higher nutrient concentrations, specifically phosphate and ammonium, compared to the 2019 benchmark. The lake was found to be dominated by Proteobacteria, followed by Cyanobacteria, with Desulfobacterota thriving in areas with low dissolved oxygen. Arthrospira and Roseobacter, halo-alkali-tolerant photosynthetic bacterial genera, were detected at all sampling points. Local phosphate and oxygen concentration variations led to distinct microbial communities on the lake's surface. Despite these differences, long-term ex-situ studies on water treatment with iron chloride and poly-aluminum chloride reduced the relative abundance of Cyanobacteria, promoting the presence of Proteobacteria and Bacteroidota. However, the coagulants required higher quantities than those typically used in small shallow lakes to precipitate phosphate and improve water quality effectively. Furthermore, the large-scale assay of lake water treatment with coagulants failed to eliminate Vibrio and Acinetobacter multidrug-resistant bacteria. In conclusion, the study underscores the need to prevent the inflow of polluted water into Lake Alalay and implement effective measures to deal with the existing chemical and biological contamination.
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| 4. |
- Delgado, Osvaldo, et al.
(författare)
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Nesterenkonia aethiopica sp nov., an alkaliphilic, moderate halophile isolated from an Ethiopian soda lake
- 2006
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Ingår i: International Journal of Systematic and Evolutionary Microbiology. - : Microbiology Society. - 1466-5026 .- 1466-5034. ; 56:6, s. 1229-1232
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Tidskriftsartikel (refereegranskat)abstract
- T Strain DSM 17733(T), isolated from the shore of Lake Abjata in Ethiopia, is a heterotrophic, alkaliphilic, moderately halophilic, Gram-positive, strictly aerobic, non-motile, non-endospore-forming bacterium. The organism grows optimally at 30-37 degrees C, pH 9 and 3 % (w/v) NaCl. Analysis of the cell wall showed the presence of murein of the type L-Lys-Gly-L-Glu, variation A4 alpha. The G + C content of the genomic DNA was 69(.)0 mol%. Sequence analysis of 16S rRNA gene sequence of strain DSM 17733(T) placed the isolate in the genus Nesterenkonia. DNA-DNA hybridization of DSM 17733(T) with those organisms with the closest phylogenetic affiliation, i.e. Nesterenkonia halobia, Nesterenkonia facusekhoensis and Nesterenkonia xinjiangensis, gave relatedness values of 48.5 %, 63(.)7 % (repetition, 57(.)2 %) and 35(.)7 % (repetition, 29(.)3 %), respectively. On the basis of both phenotypic and phylogenetic criteria and the low levels of DNA-DNA relatedness with the phylogenetically closest species N. xinjiangensis and N. halobia, it is proposed that the isolate be classified in a novel species, Nesterenkonia aethiopica sp. nov. The type strain is DSM 17733(T) (=CCUG 48939(T)).
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| 5. |
- Doan Van, Thuoc, et al.
(författare)
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High productivity of ectoines by Halomonas boliviensis using a combined two-step fed-batch culture and milking process.
- 2010
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 147:1, s. 46-51
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Tidskriftsartikel (refereegranskat)abstract
- A process comprising two-step fed-batch cultivation has been investigated for the production of ectoines using the halophilic bacterium Halomonas boliviensis DSM 15516(T). The first cultivation was performed under optimal conditions for cell growth and resulted in cell mass concentration of about 41gl(-1) after 24h of cultivation. During the second cultivation at higher salt concentration, accumulation of ectoines increased while cell mass decreased with increasing salt concentration. Maximum productivity of total ectoines reached was 10gl(-1)d(-1) with ectoine concentration of 6gl(-1) and hydroxyectoine concentration of 8gl(-1) after 9h of cultivation at 18.5% NaCl, which is among the highest reported so far. H. boliviensis cells were further recycled for the production process after releasing the ectoines. About 75% of the accumulated ectoines were released by subjecting the cells to hypoosmotic shock. On subsequent reincubation in a medium containing higher salt concentration the cells were able to re-synthesize the ectoines resulting in a global productivity of 11.1gl(-1)d(-1), and ectoine and hydroxyectoine productivities of 9.1gl(-1)d(-1) and 2.0gl(-1)d(-1), respectively.
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| 6. |
- Doan Van, Thuoc, et al.
(författare)
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Utilization of agricultural residues for poly(3-hydroxybutyrate) production by Halomonas boliviensis LC1
- 2008
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Ingår i: Journal of Applied Microbiology. - : Oxford University Press (OUP). - 1364-5072 .- 1365-2672. ; 104:2, s. 420-428
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Tidskriftsartikel (refereegranskat)abstract
- Aims: Utilization of cheap and readily available agricultural residues as cheap carbon sources for poly(3-hydroxybutyrate) (PHB) production by Halomonas boliviensis. Methods and results: Wheat bran was hydrolysed by a crude enzyme preparation from Aspergillus oryzae NM1 to provide a mixture of reducing sugars composed mainly of glucose, mannose, xylose and arabinose. Growth of H. boliviensis using a mixture of glucose (0.75% w/v) and xylose (0.25% w/v) in the medium led to a PHB content and concentration of 45 wt% and 1 g l(-1), respectively, after 30 h. A similar PHB concentration was attained when H. boliviensis was grown on wheat bran hydrolysate but with a lower PHB content, 34 wt%. In a batch cultivation mode in a fermentor, using 1.8% (w/v) reducing sugars, the maximum PHB accumulation by H. boliviensis was attained in 20 h, but was reduced to about 30 wt%. By adding butyric acid (0.8% v/v), sodium acetate (0.8% w/v) and decreasing the reducing sugars concentration to 1.0% w/v in the medium, PHB accumulation and concentration were increased to 50 wt% and 4 g l(-1), respectively, after 20 h. Butyric acid and sodium acetate for PHB production could also be provided by anaerobic digestion of solid potato waste. Conclusions: Cheap and readily available agricultural residues can be used as substrates to produce PHB. The production of PHB by H. boliviensis using wheat bran hydrolysate as source of carbon is expected to reduce the production cost and motivates further studies. Significance and Impact of the Study: Large-scale commercial utilization of PHB is mainly hampered by its high production cost. Carbon source for PHB production accounts up to 50% of the total production costs. Thus, the use of waste agricultural residues can substantially reduce the substrate cost (and in turn even provide value to the waste), and can downsize the production costs. This improves the market competitiveness. Studies on PHB production by moderate halophiles were recently initiated with H. boliviensis and findings show that it has potential for commercial exploitation. PHB production by H. boliviensis using wheat bran and potato waste is hence interesting.
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| 7. |
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| 8. |
- Guevara-Martínez, Mónica, 1989-, et al.
(författare)
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Regulating the production of (R)-3-hydroxybutyrate in Escherichia coli by N or P limitation
- 2015
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Ingår i: Frontiers in Microbiology. - : Frontiers Research Foundation. - 1664-302X. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- The chiral compound (R)-3-hydroxybutyrate (3HB) is naturally produced by many wild type organisms as the monomer for polyhydroxybutyrate (PHB). Both compounds are commercially valuable and co-polymeric polyhydroxyalkanoates have been used e.g., in medical applications for skin grafting and as components in pharmaceuticals. In this paper we investigate cultivation strategies for production of 3HB in the previously described E. coil strain AF1000 pJBGT3RX. This strain produces extracellular 3HB by expression of two genes from the PHB pathway of Halomonas boliviensis. H. boliviensis is a newly isolated halophile that forms PHB as a storage compound during carbon excess and simultaneous limitation of another nutrient like nitrogen and phosphorous. We hypothesize that a similar approach can be used to control the flux from acetylCoA to 3HB also in E coli; decreasing the flux to biomass and favoring the pathway to the product. We employed ammonium- or phosphate-limited fed-batch processes for comparison of the productivity at different nutrient limitation or starvation conditions. The feed rate was shown to affect the rate of glucose consumption, respiration, 3HB, and acetic acid production, although the proportions between them were more difficult to affect. The highest 3HB volumetric productivity, 1.5 g L-1 h(-1), was seen for phosphate-limitation.
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| 9. |
- Guevara-Martínez, Mónica, 1989-, et al.
(författare)
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The role of the acyl-CoA thioesterase YciA in the production of (R)-3-hydroxybutyrate by recombinant Escherichia coli
- 2019
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Ingår i: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; , s. 1-12
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Tidskriftsartikel (refereegranskat)abstract
- Biotechnologically produced (R)-3-hydroxybutyrate is an interesting pre-cursor for antibiotics, vitamins, and other molecules benefitting from enantioselective production. An often-employed pathway for (R)-3-hydroxybutyrate production in recombinant E. coli consists of three-steps: (1) condensation of two acetyl-CoA molecules to acetoacetyl-CoA, (2) reduction of acetoacetyl-CoA to (R)-3-hydroxybutyrate-CoA, and (3) hydrolysis of (R)-3-hydroxybutyrate-CoA to (R)-3-hydroxybutyrate by thioesterase. Whereas for the first two steps, many proven heterologous candidate genes exist, the role of either endogenous or heterologous thioesterases is less defined. This study investigates the contribution of four native thioesterases (TesA, TesB, YciA, and FadM) to (R)-3-hydroxybutyrate production by engineered E. coli AF1000 containing a thiolase and reductase from Halomonas boliviensis. Deletion of yciA decreased the (R)-3-hydroxybutyrate yield by 43%, whereas deletion of tesB and fadM resulted in only minor decreases. Overexpression of yciA resulted in doubling of (R)-3-hydroxybutyrate titer, productivity, and yield in batch cultures. Together with overexpression of glucose-6-phosphate dehydrogenase, this resulted in a 2.7-fold increase in the final (R)-3-hydroxybutyrate concentration in batch cultivations and in a final (R)-3-hydroxybutyrate titer of 14.3 g L-1 in fed-batch cultures. The positive impact of yciA overexpression in this study, which is opposite to previous results where thioesterase was preceded by enzymes originating from different hosts or where (S)-3-hydroxybutyryl-CoA was the substrate, shows the importance of evaluating thioesterases within a specific pathway and in strains and cultivation conditions able to achieve significant product titers. While directly relevant for (R)-3-hydroxybutyrate production, these findings also contribute to pathway improvement or decreased by-product formation for other acyl-CoA-derived products.
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| 10. |
- Guzmán, Daniel, et al.
(författare)
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Halomonas andesensis sp. nov., a moderate halophile isolated from the saline lake Laguna Colorada in Bolivia.
- 2010
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Ingår i: International Journal of Systematic and Evolutionary Microbiology. - : Microbiology Society. - 1466-5026 .- 1466-5034. ; 60, s. 749-753
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Tidskriftsartikel (refereegranskat)abstract
- Strain LC6T was isolated from a water sample of the lake Laguna Colorada in the Bolivian Andes. The bacterium was characterized as moderately halophilic, motile, Gram-negative rod. The major fatty acids present in the cell were C18:1omega7c, C16:1omega7c iso 2-OH, C16:0 and C12:0 3-OH. The respiratory ubiquinones found in strain LC6T were Q-9 (97%) and Q-8 (3%). The organism was found to be an aerobic, heterotroph, able to utilize various carbohydrates and other substrates as carbon source. The G+C content (mol %) of the genomic DNA was 52.5. The organism was able to grow in the pH range of 6.0-11.0 (optimum between 7.0-8.0), temperature of 4-45 degrees C (optimum at 30-35 degrees C) and sodium chloride concentration of 0.5-20 % (w/v) (optimum between 1-3% w/v). Based on 16S rRNA gene sequence analysis, LC6T was 98.8 % similar to Halomonas hydrothermalis DSM 15725T and Halomonas venusta DSM 4743T, and 98.4 % similar to Halomonas aquamarina DSM 30161T, Halomonas axialensis DSM 15723T and Halomonas meridiana DSM 5425T. However, its DNA-DNA relatedness with these type strains was considerably low. LC6T resembled other Halomonas species with respect to various physiological, biochemical, and nutritional characteristics. Combined phenotypic data and DNA-DNA hybridization data supported the conclusion that LC6T is a novel species of the genus Halomonas, for which the name Halomonas andesensis (LC6T=CCUG 54844T=LMG 24243T = DSM 19434T) is proposed.
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| 11. |
- Guzmán, Daniel, et al.
(författare)
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Preparation of hydrophilic poly(3-hydroxybutyrate) macroporous scaffolds through enzyme-mediated modifications
- 2011
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Ingår i: Journal of Bioactive and Compatible Polymers. - : SAGE Publications. - 0883-9115 .- 1530-8030. ; 26:5, s. 452-463
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Tidskriftsartikel (refereegranskat)abstract
- Scaffolds made from poly(3-hydroxybutyrate) were prepared by thermally induced phase separation from solutions in dioxane at temperatures above (23 degrees C) or below the freezing point of the solvent. At 23 degrees C, gelation occured with nano-fibrous network formation. The scaffolds prepared at -12 degrees C and -25 degrees C exhibited a highly porous morphology, with pores in the range of 3-25 mu m, caused by freezing of dioxane. The macroporous scaffolds, prepared at -12 degrees C, were treated with lipase to generate functional groups to which gelatin and glucosamine, respectively, were chemically coupled. The modified scaffolds had lower molecular weight, higher water content, lower melting temperature, and enthalpy. Cultivation of human embryonic fibroblasts on the macroporous scaffolds confirmed that the cells proliferated and adhered to the materials.
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| 12. |
- Guzmán, Hector, et al.
(författare)
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A process for the production of ectoine and poly(3-hydroxybutyrate) by Halomonas boliviensis.
- 2009
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 84, s. 1069-1077
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Tidskriftsartikel (refereegranskat)abstract
- The paper reports a study involving the use of Halomonas boliviensis, a moderate halophile, for co-production of compatible solute ectoine and biopolyester poly(3-hydroxybutyrate) (PHB) in a process comprising two fed-batch cultures. Initial investigations on the growth of the organism in a medium with varying NaCl concentrations showed the highest level of intracellular accumulation of ectoine (0.74 g L(-1)) at 10-15% (w/v) NaCl, while at 15% (w/v) NaCl, the presence of hydroxyectoine (50 mg L(-1)) was also noted. On the other hand, the maximum cell dry weight and PHB concentration of 10 and 5.8 g L(-1), respectively, were obtained at 5-7.5% (w/v) NaCl. A process comprising two fed-batch cultivations was developed-the first culture aimed at obtaining high cell mass and the second for achieving high yields of ectoine and PHB. In the first fed-batch culture, H. boliviensis was grown in a medium with 4.5% (w/v) NaCl and sufficient levels of monosodium glutamate, NH (4) (+) , and PO (4) (3-) . In the second fed-batch culture, the NaCl concentration was increased to 7.5% (w/v) to trigger ectoine synthesis, while nitrogen and phosphorus sources were fed only during the first 3 h and then stopped to favor PHB accumulation. The process resulted in PHB yield of 68.5 wt.% of cell dry weight and volumetric productivity of about 1 g L(-1) h(-1) and ectoine concentration, content, and volumetric productivity of 4.3 g L(-1), 7.2 wt.%, and 2.8 g L(-1) day(-1), respectively. At salt concentration of 12.5% (w/v) during the second cultivation, the ectoine content was increased to 17 wt.% and productivity to 3.4 g L(-1) day(-1).
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| 13. |
- Jarmander, Johan, et al.
(författare)
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Cultivation strategies for production of (R)-3-hydroxybutyric acid from simultaneous consumption of glucose, xylose and arabinose by Escherichia coli
- 2015
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Ingår i: Microbial Cell Factories. - : BioMed Central. - 1475-2859. ; 14:1, s. 51-
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundLignocellulosic waste is a desirable biomass for use in second generation biorefineries. Up to 40 % of its sugar content consist of pentoses, which organisms either take up sequentially after glucose depletion, or not at all. A previously described Escherichia coli strain, PPA652ara, capable of simultaneous consumption of glucose, xylose and arabinose was in the present work utilized for production of (R)-3-hydroxybutyric acid (3HB) from a mixture of glucose, xylose and arabinose.ResultsThe Halomonas boliviensis genes for 3HB production were for the first time cloned into E. coli PPA652ara leading to product secretion directly into the medium. Process design was based on comparisons of batch, fed-batch and continuous cultivation, where both excess and limitation of the carbon mixture was studied. Carbon limitation resulted in low specific productivity of 3HB (< 2 mg g-1 h-1) compared to carbon excess (25 mg g-1 h-1), but the yield of 3HB/cell dry weight (Y3HB/CDW) was very low (0.06 g g-1)during excess. Nitrogen-exhausted conditions could be used to sustain a high specific productivity (31 mg g-1 h-1) and to increase the yield of 3HB/cell dry weight to 1.38 g g-1. Nitrogen-limited fed-batch process design lead to further increased specific productivity (38 mg g-1 h-1) but also to additional cell growth (Y3HB/CDW = 0.16 g g-1). Strain PPA652ara did under all processing conditions simultaneously consume glucose, xylose and arabinose, which was not the case for a reference wild type E. coli, which also gave a higher carbon flux to acetic acid.ConclusionsIt was demonstrated that by using the strain E. coli PPA652ara it was possible to design a production process for 3HB from a mixture of glucose, xylose and arabinose where all sugars were consumed. An industrial 3HB production process is proposed to be divided into a growth and a production phase, and nitrogen depletion/limitation is a potential strategy to maximize the yield of 3HB/CDW in the latter. The specific productivity of 3HB by E. coli reported here from glucose, xylose and arabinose is further comparable to the current state of the art for production of 3HB from glucose sources.
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| 14. |
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| 15. |
- Lacayo, Martha, et al.
(författare)
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A toxaphene-degrading bacterium related to Enterobacter cloacae, strain D1 isolated from aged contaminated soil in Nicaragua
- 2005
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Ingår i: Systematic and Applied Microbiology. - : Elsevier BV. - 0723-2020 .- 1618-0984. ; 28:7, s. 632-639
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Tidskriftsartikel (refereegranskat)abstract
- Enterobacter sp. strain D1 is a facultative anaerobic, Gram-negative heterotrophic bacterium isolated from toxaphene-contaminated soil. This organism was identified and characterized through phylogenetic and taxonomic studies. Based on 16S rDNA analysis, the strain D1 was clustered closely with the species Enterobacter cloacae subsp. dissolvens (LMG 2683) and E. cloacae (ATCC 13047T). Strain D1 resembled these E. cloacae strains with respect to various biochemical and nutritional characteristics, but also exhibited differences. Moreover, strain D1 is able to grow and survive with toxaphene supplied in the medium in the range 3-96 mg/L. Amongst the chemical components of toxaphene, octachlorocamphenes, nonachlorobornanes and decachlorobornanes were seen to be rapidly metabolized, although levels of hexachlorocamphenes and heptachlorobornanes were found to be slowly degraded, and subsequently accumulated during the last stage of the cultivation.
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| 16. |
- Miranda, Diego A., et al.
(författare)
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Production and characterization of poly(3-hydroxybutyrate) from Halomonas boliviensis LC1 cultivated in hydrolysates of quinoa stalks
- 2023
-
Ingår i: Fermentation. - : MDPI. - 2311-5637. ; 9:6
-
Tidskriftsartikel (refereegranskat)abstract
- The global production of fossil-based plastics has reached critical levels, and their substitution with bio-based polymers is an urgent requirement. Poly(3-hydroxybutyrate) (PHB) is a biopolymer that can be produced via microbial cultivation, but efficient microorganisms and low-cost substrates are required. Halomonas boliviensis LC1, a moderately halophilic bacterium, is an effective PHB producer, and hydrolysates of the residual stalks of quinoa (Chenopodium quinoa Willd.) can be considered a cheap source of sugars for microbial fermentation processes in quinoa-producing countries. In this study, H. boliviensis LC1 was adapted to a cellulosic hydrolysate of quinoa stalks obtained via acid-catalyzed hydrothermal pretreatment and enzymatic saccharification. The adapted strain was cultivated in hydrolysates and synthetic media, each of them with two different initial concentrations of glucose. Cell growth, glucose consumption, and PHB formation during cultivation were assessed. The cultivation results showed an initial lag in microbial growth and glucose consumption in the quinoa hydrolysates compared to cultivation in synthetic medium, but after 33 h, the values were comparable for all media. Cultivation in hydrolysates with an initial glucose concentration of 15 g/L resulted in a higher glucose consumption rate (0.15 g/(L h) vs. 0.14 g/(L h)) and volumetric productivity of PHB (14.02 mg/(L h) vs. 10.89 mg/(L h)) than cultivation in hydrolysates with 20 g/L as the initial glucose concentration. During most of the cultivation time, the PHB yield on initial glucose was higher for cultivation in synthetic medium than in hydrolysates. The produced PHBs were characterized using advanced analytical techniques, such as high-performance size-exclusion chromatography (HPSEC), Fourier transform infrared (FTIR) spectroscopy, 1H nuclear magnetic resonance (NMR) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). HPSEC revealed that the molecular weight of PHB produced in the cellulosic hydrolysate was lower than that of PHB produced in synthetic medium. TGA showed higher thermal stability for PHB produced in synthetic medium than for that produced in the hydrolysate. The results of the other characterization techniques displayed comparable features for both PHB samples. The presented results show the feasibility of producing PHB from quinoa stalks with H. boliviensis.
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| 17. |
- Ortiz-Veizan, Nancy, et al.
(författare)
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Improvements of poly(3-hydroxybutyrate) production in an air-lift reactor using simple production media
- 2020
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Ingår i: Bioresources And Bioprocessing. - : Springer. - 2197-4365. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Halomonas boliviensis is a halophilic microorganism that accumulates poly(3-hydroxybutyrate) (PHB) using different carbons sources when nitrogen is depleted from the culture medium. This work presents an improved production of PHB using an air-lift reactor (ALR) that was fed with a concentrated solution of a carbon source, and was supplemented with an adequate airflow rate. Results: Simple production media were used to study PHB production by H. boliviensis in an ALR. Glucose was first used as the main carbon source and was fed during the exponential phase of cell growth. The maximum CDW and PHB content were 31.7 g/L and 51 wt%, respectively, when the airflow rate entering the reactor varied between 0.5 and 1.2 L/min. Changing the air inflow to 0.5-0.9 L/min resulted in an improvement in PHB accumulation (62 wt%). A cultivation was performed by using the latter range of airflow rate and feeding glucose only when nitrogen was depleted from the medium; a considerable enhancement in PHB content (72 wt%) and CDW (27 g/L) was achieved under these conditions. Moreover, PHB was also produced using molasses as the main carbon source. Residual cell mass was about the same to that achieved with glucose, however the PHB content (52 wt%) was lower. Conclusions: PHB production by H. boliviensis in an ALR using a simple medium is possible. CDW and PHB content in H. boliviensis can be improved with respect to batch cultivations previously reported when a carbon source is fed to the reactor. The best strategy for the production of PHB consisted of starting the cultivation in a batch mode while glutamate was present in the medium; glucose should be fed when glutamate is depleted from the medium to keep an excess of the carbon source during the synthesis of PHB.
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| 18. |
- Perez-Zabaleta, Mariel, 1987-, et al.
(författare)
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Comparison of engineered Escherichia coli AF1000 and BL21 strains for (R)-3-hydroxybutyrate production in fed-batch cultivation
- 2019
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Ingår i: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; 103:14, s. 5627-5636
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Tidskriftsartikel (refereegranskat)abstract
- Accumulation of acetate is a limiting factor in recombinant production of (R)-3-hydroxybutyrate (3HB) by E. coli in high-cell-density processes. To alleviate this limitation, this study investigated two approaches: (i) Deletion of phosphotransacetylase (pta), pyruvate oxidase (poxB) and/or the isocitrate-lyase regulator (iclR), known to decrease acetate formation, on bioreactor cultivations designed to achieve high 3HB concentrations. (ii) Screening of different E. coli strain backgrounds (B, BL21, W, BW25113, MG1655, W3110 and AF1000) for their potential as low acetate-forming, 3HB-producing platforms. Deletion of pta and pta-poxB in the AF1000 strain background was to some extent successful in decreasing acetate formation, but also dramatically increased excretion of pyruvate and did not result in increased 3HB production in high-cell-density fed-batch cultivations. Screening of the different E. coli strains confirmed BL21 as a low acetate forming background. Despite low 3HB titers in low-cell density screening, 3HB-producing BL21 produced 5 times less acetic acid per mol of 3HB, which translated into a 2.3-fold increase in the final 3HB titer and a 3-fold higher volumetric 3HB productivity over 3HB-producing AF1000 strains in nitrogen-limited fed-batch cultivations. Consequently, the BL21 strain achieved the hitherto highest described volumetric productivity of 3HB (1.52 g L-1 h-1) and the highest 3HB concentration (16.3 g L-1) achieved by recombinant E. coli. Screening solely for 3HB titers in low-cell-density batch cultivations would not have identified the potential of this strain, reaffirming the importance of screening with the final production conditions in mind.
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| 19. |
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| 20. |
- Perez-Zabaleta, Mariel, 1987-, et al.
(författare)
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Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply
- 2016
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Ingår i: Microbial Cell Factories. - : Springer. - 1475-2859. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: In a recently discovered microorganism, Halomonas boliviensis, polyhydroxybutyrate production was extensive and in contrast to other PHB producers, contained a set of alleles for the enzymes of this pathway. Also the monomer, (R)-3-hydroxybutyrate (3HB), possesses features that are interesting for commercial production, in particular the synthesis of fine chemicals with chiral specificity. Production with a halophilic organism is however not without serious drawbacks, wherefore it was desirable to introduce the 3HB pathway into Escherichia coli. Results: The production of 3HB is a two-step process where the acetoacetyl-CoA reductase was shown to accept both NADH and NADPH, but where the V-max for the latter was eight times higher. It was hypothesized that NADPH could be limiting production due to less abundance than NADH, and two strategies were employed to increase the availability; (1) glutamate was chosen as nitrogen source to minimize the NADPH consumption associated with ammonium salts and (2) glucose-6-phosphate dehydrogenase was overexpressed to improve NADPH production from the pentose phosphate pathway. Supplementation of glutamate during batch cultivation gave the highest specific productivity (q(3HB) = 0.12 g g(-1) h(-1)), while nitrogen depletion/zwf overexpression gave the highest yield (Y-3HB/CDW = 0.53 g g(-1)) and a 3HB concentration of 1 g L-1, which was 50 % higher than the reference. A nitrogen-limited fedbatch process gave a concentration of 12.7 g L-1 and a productivity of 0.42 g L-1 h(-1), which is comparable to maximum values found in recombinant E. coli. Conclusions: Increased NADPH supply is a valuable tool to increase recombinant 3HB production in E. coli, and the inherent hydrolysis of CoA leads to a natural export of the product to the medium. Acetic acid production is still the dominating by-product and this needs attention in the future to increase the volumetric productivity further.
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| 21. |
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| 22. |
- Quillaguaman, Jorge, et al.
(författare)
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Chromohalobacter sarecensis sp. nov., a psychrotolerant moderate halophile isolated from the saline Andean region of Bolivia
- 2004
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Ingår i: International Journal of Systematic and Evolutionary Microbiology. - : Microbiology Society. - 1466-5026 .- 1466-5034. ; 54:6, s. 1921-1926
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Tidskriftsartikel (refereegranskat)abstract
- A moderately halophilic, aerobic, motile, Gram-negative, rod-shaped bacterium (strain LV4T) was isolated from saline soil around the lake Laguna Verde in the Bolivian Andes. The organism is a heterotroph, able to utilize various carbohydrates as a carbon source. It showed tryptophan deaminase, oxidase and catalase activity, but was unable to produce indole or H2S; nitrate was not reduced. The G+C content of the genomic DNA was 56·1 mol%. The pH range for growth was 5–10, temperature range was 0–45 °C and the range of NaCl concentrations was 0–25 % (w/v). On the basis of 16S rRNA gene sequence analysis, strain LV4T was found to be closely related to Chromohalobacter canadensis DSM 6769T and Pseudomonas beijerinckii DSM 7218T; however, its DNA–DNA relatedness with these type strains was low. Strain LV4T resembled other Chromohalobacter species with respect to various physiological, biochemical and nutritional characteristics but also exhibited differences. Thus, a novel species, Chromohalobacter sarecensis sp. nov., is proposed, with LV4T (=CCUG 47987T=ATCC BAA-761T) as the type strain.
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| 23. |
- Quillaguaman, Jorge, et al.
(författare)
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Halomonas boliviensis sp nov., an alkalitolerant, moderate halophile isolated from soil around a Bolivian hypersaline lake
- 2004
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Ingår i: International Journal of Systematic and Evolutionary Microbiology. - : Microbiology Society. - 1466-5026 .- 1466-5034. ; 54:3, s. 721-725
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Tidskriftsartikel (refereegranskat)abstract
- Halomonas boliviensis sp. nov. is proposed for two moderately halophilic, psychrophilic, alkalitolerant bacteria, LC1(T) (=DSM 15516(T)=ATCC BAA-759(T)) and LC2 (=DSM 15517=ATCC BAA-760), both of which were isolated from a soil sample around the lake Laguna Colorada, located at 4300 m above sea level in the south-west region of Bolivia. The bacteria are aerobic, motile, Gram-negative rods that produce colonies with a cream pigment. Moreover, they are heterotrophs that are able to utilize various carbohydrates as carbon sources. The organisms reduce nitrate and show tryptophan deaminase activity. The genomic DNA G+C contents were 51.4 mol% for isolate LC1(T) and 52.6 mol% for isolate LC2. Based on 16S rDNA sequence analysis, isolates LC1(T) and LC2 were identified as members of the genus Halomonas and clustered closely with Halomonas variabilis DSM 3051(T) and Halomonas meridiana DSM 5425(T). However, DNA-DNA relatedness between the new isolates and the closest related Halomonas species was low.
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| 24. |
- Quillaguaman, Jorge
(författare)
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Novel Moderate Halophiles: Application of Halomonas boliviensis in Biopolyester Production
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The south-west Andean region of Bolivia has vast landscapes comprising salt deserts and dry lands interspersed with a number of saline lakes. Much of this region lies above 4000 m above sea level that allows the survival of limited and unique flora and fauna. This thesis involves studies on novel, moderately halophilic microorganisms isolated from this region. Strains LC1T and LC2 were isolated from a soil sample around the lake Laguna Colorada. Based on 16S rDNA analysis, the isolates were identified as members of the genus Halomonas. Due to the low DNA-DNA relatedness between the new isolates and related Halomonas spp. and also several taxonomic differences, strains LC1T and LC2 were classified as new bacterial species called Halomonas boliviensis. Strain LV4T was isolated from saline soil around the lake Laguna Verde. It showed close phylogenetic relationship with Chromohalobacter spp., but differed sufficiently to become a novel species, which was named Chromohalobacter sarecensis.Halomonas boliviensis is able to accumulate a biogegradable polymer, poly(b-hydroxybutyrate) (PHB), when grown under conditions of nutrient limitation and excess carbon source. Cultivation with butyric acid and sodium acetate as carbon sources and 4.5 % (w/v) sodium chloride under controlled conditions in a fermentor led to a maximum PHB yield of 88 wt% with respect to cell dry weight, but a low cell concentration of 2 g/L. The use of glucose and sucrose, respectively, as carbon source resulted in the production of PHB at an average level of 55 wt%. Starch hydrolysate, generated by the action of a recombinant maltooligosaccharide forming amylase from Bacillus halodurans LBK34, was also used as the carbon source for the production of PHB by H. boliviensis LC1. Optimization of the culture conditions containing sucrose led to an improvement in the cell concentration to 14 g/L with a PHB content of 54% in batch fermentations.
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| 25. |
- Quillaguaman, Jorge, et al.
(författare)
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Optimizing conditions for poly(beta-hydroxybutyrate) production by Halomonas boliviensis LC1 in batch culture with sucrose as carbon source
- 2007
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 74:5, s. 981-986
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Tidskriftsartikel (refereegranskat)abstract
- Halomonas boliviensis LC1 is able to accumulate poly(beta-hydroxybutyrate) (PHB) under conditions of excess carbon source and depletion of essential nutrients. This study was aimed at an efficient production of PHB by growing H. boliviensis to high cell concentrations in batch cultures. The effect of ammonium, phosphate, and yeast extract concentrations on cell concentration [cell dry weight (CDW)] and PHB content of H. boliviensis cultured in shake flasks was assayed using a factorial design. High concentrations of these nutrients led to increments in cell growth but reduced the PHB content to some extent. Cultivations of H. boliviensis under controlled conditions in a fermentor using 1.5% (w/v) yeast extract as N source, and intermittent addition of sucrose to provide excess C source, resulted in a polymer accumulation of 44 wt.% and 12 g l(-1) CDW after 24 h of cultivation. Batch cultures in a fermentor with initial concentrations of 2.5% (w/v) sucrose and 1.5% (w/v) yeast extract, and with induced oxygen limitation, resulted in an optimum PHB accumulation, PHB concentration and CDW of 54 wt.%, 7.7 g l(-1) and 14 g l(-1), respectively, after 19 h of cultivation. The addition of casaminoacids in the medium increased the CDW to 14.4 g l(-1) in 17 h but reduced the PHB content in the cells to 52 wt.%.
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| 26. |
- Quillaguaman, Jorge, et al.
(författare)
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Poly(3-hydroxybutyrate) production by Halomonas boliviensis in fed-batch culture.
- 2008
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 78:2, s. 227-232
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Tidskriftsartikel (refereegranskat)abstract
- High poly(3-hydroxybutyrate) (PHB) content and volumetric productivity were achieved by fed-batch culture of Halomonas boliviensis using a defined medium. Initial shake flask cultivations in a minimal medium revealed that the growth of H. boliviensis was supported only when the medium was supplemented with aspartic acid, glycine, or glutamine. Addition of 0.1% (w/v) glutamine in the medium resulted in the highest cell dry weight (CDW; 3.9 g l(-1)). Glutamine was replaced by the less expensive monosodium glutamate (MSG) in the medium without any notable change in the final cell density. Effect of initial concentrations of NH(4)Cl and K(2)HPO(4) on cell growth and PHB accumulation by H. boliviensis was then analyzed using a fed-batch fermentation system. The best conditions for PHB production by H. boliviensis were attained using 0.4% (w/v) NH(4)Cl and 0.22% (w/v) K(2)HPO(4) and adding MSG intermittently to the fermentor. Poly(3-hydroxybutyrate) content and CDW reached 90 wt.% and 23 g l(-1), respectively, after 18 h of cultivation. In order to increase CDW and PHB content, MSG, NH(4)Cl, and K(2)HPO(4) were initially fed to the fermentor to maintain their concentrations at 2%, 0.4%, and 0.22% (w/v), respectively, and subsequently their feed was suppressed. This resulted in a CDW of 44 g l(-1), PHB content of 81 wt.%, and PHB volumetric productivity of 1.1 g l(-1) h(-1).
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| 27. |
- Quillaguaman, Jorge, et al.
(författare)
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Poly(beta-hydroxybutyrate) production by a moderate halophile, Halomonas boliviensis LC1
- 2006
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Ingår i: Enzyme and Microbial Technology. - : Elsevier BV. - 0141-0229. ; 38:1-2, s. 148-154
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Tidskriftsartikel (refereegranskat)abstract
- The moderate halophile Halomonas boliviensis, isolated from a Bolivian saline soil sample, was able to accumulate poly(P-hydroxybutyrate) (PHB) when grown under conditions of nutrient limitation and excess carbon source. The concentration of sodium chloride in the medium influenced the cell-growth, -size, and rate of PHB accumulation. Cultivation in shake flasks led to a PHB accumulation of about 54 wt.% with respect to cell dry weight at 4.5% (w/v) NaCl in a medium with butyric acid and sodium acetate as carbon sources. The production of PHB was substantially improved to a maximum value of 88 wt.% during cultivation under controlled conditions of pH and oxygen concentration in a fermentor. The use of glucose and sucrose, respectively, as carbon source could also lead to the production of PHB at an average level of 55 wt.%. (c) 2005 Elsevier Inc. All rights reserved.
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| 28. |
- Quillaguaman, Jorge, et al.
(författare)
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Poly(beta-hydroxybutyrate) production by a moderate halophile, Halomonas boliviensis LC1 using starch hydrolysate as substrate
- 2005
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Ingår i: Journal of Applied Microbiology. - : Oxford University Press (OUP). - 1364-5072 .- 1365-2672. ; 99:1, s. 151-157
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Tidskriftsartikel (refereegranskat)abstract
- Aim: The objective of the present work was to enable the use of starch hydrolysate, generated by the action of a recombinant maltooligosaccharide forming amylase from Bacillus halodurans LBK 34, as the carbon source for the production of poly-beta- hydroxybutyrate ( PHB) by Halomonas boliviensis LC1. Methods and Results: In this work, different amounts of the alpha- amylase ( Amy 34) were utilized for starch hydrolysis, resulting in the production of mixtures of maltooligosaccharides ( G1 - G6) at varying ratios. The highest PHB accumulation ( 56 wt%) by H. boliviensis cultivated in shake flasks ( with agitation at 160 rev min(-1)) was obtained when 6.4 U ml(-1) of the amylase was used for starch hydrolysis. When H. boliviensis cells were grown in a fermentor with no oxygen limitation the accumulation of PHB was decreased to 35 wt%. Although some improvements in PHB accumulation and cell mass concentration were reached by the addition of peptone and phosphate, respectively, major enhancements were attained when oxygen limitation was induced in the fermentor. Conclusions: Halomonas boliviensis uses preferentially maltose for PHB formation from starch hydrolysate. It is also able to hydrolyse higher sugars if no other simpler carbon source is available but with a significantly lower polymer yield. Furthermore, H. boliviensis is able to adjust its metabolism to oxygen limitation, most probably by directing the excess NAD( P) H to PHB accumulation. Significance and Impact of the Study: There have been no reports related to PHB production amongst the members of the genus Halomonas. The use of a maltooligosaccharide forming alpha- amylase, which is active at a temperature and pH close to that required for growth of H. boliviensis, and the versatility of this bacterium in the selection of the carbon source may provide an attractive alternative for the utilization of starch- derived raw materials.
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| 29. |
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| 30. |
- Quillaguaman, Jorge, et al.
(författare)
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Synthesis and production of polyhydroxyalkanoates by halophiles: current potential and future prospects
- 2010
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 85:6, s. 1687-1696
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Forskningsöversikt (refereegranskat)abstract
- Biodegradable materials with plastic or elastomeric properties are in great demand for a variety of applications. Polyhydroxyalkanoates (PHAs), polyesters synthesized by microorganisms, possess such desired features. Industrial production of PHAs is currently achieved using recombinant Escherichia coli. Nevertheless, recent research on halophiles, salt requiring microorganisms, has shown a remarkable potential for biotechnological production of PHAs. The halophilic archaeon Haloferax mediterranei accumulates a co-polymer, i.e., poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in large amounts using glucose, starch, and hydrolyzed whey as carbon sources. Chemical composition and molecular weight of PHAs produced by H. mediterranei can be modified depending on the substrate utilized as precursor. Phylogenetic studies on haloarchaeal enzymes able to polymerize the components of PHAs (i.e., PHA synthases) reveal a novel cluster, with a close relationship with PHA polymerases of bacteria and archaea found in marine-related niches. On the other hand, sequences of PHA synthases of two halophilic bacteria are more closely affiliated to synthases of Proteobacteria. Several bacterial species of the family Halomonadaceae accumulate PHAs. Halomonas boliviensis reached PHA yields and volumetric productivities close to the highest reported so far. Furthermore, H. boliviensis and other Halomonas species are able to co-produce PHA and osmolytes, i.e., ectoines and hydroxyectoine, in one process.
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| 31. |
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