| 1. |
- de Maré, Lena, et al.
(författare)
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A cultivation technique for E. coli fed-batch cultivations operating close to the maximum oxygen transfer capacity of the reactor
- 2005
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Ingår i: Biotechnology Letters. - : Springer Science and Business Media LLC. - 0141-5492 .- 1573-6776. ; 27:14, s. 983-990
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Tidskriftsartikel (refereegranskat)abstract
- A cultivation strategy combining the advantages of temperature-limited fed-batch and probing feeding control is presented. The technique was evaluated in fed-batch cultivations with E. coli BL21(DE3) producing xylanase in a 3 liter bioreactor. A 20% increase in cell mass was achieved and the usual decrease in specific enzyme activity normally observed during the late production phase was diminished with the new technique. The method was further tested by growing E. coli W3110 in a larger bioreactor (50 l). It is a suitable cultivation technique when the O2 transfer capacity of the reactor is reached and it is desired to continue to produce the recombinant protein.
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| 2. |
- Andersson, Christian
(författare)
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Biobased production of succinic acid by Escherichia coli fermentation
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The prospects of peak oil, climate change and the dependency of fossil carbon have urged research and development of production methods for the manufacture of fuels and chemicals from renewable resources (biomass). The present thesis illustrates different aspects of biobased succinic acid production by a metabolically engineered E. coli strain. The main areas of the thesis are sugar utilisation and feedstock flexibility, and fermentation inhibition, both due to toxic compound derived from the raw material and the fermentation products themselves.The first part of this thesis aimed to investigate the fermentation characteristics of AFP184 in a medium consisting of corn steep liquor, inorganic salts and different sugar sources without supplementation with high-cost nutrients such as yeast extract and peptone. The effects of different sugars, sucrose, glucose, fructose, xylose, equal mixtures of glucose-fructose and glucose-xylose, on succinic acid production kinetics and yields in an industrially relevant medium were investigated. AFP184 was able to utilise all sugars and sugar combinations except sucrose for biomass generation and succinate production. Using glucose resulted in the highest yield, 0.83 (g succinic acid per g sugar consumed anaerobically). Using a high initial sugar concentration resulted in volumetric productivities of almost 3 g L-1 h-1, which is above estimated values for economically feasible production. However, succinic acid production ceased at final concentrations greater than 40 g L-1. To further increase succinic acid concentrations, fermentations using NH4OH, NaOH, KOH, K2CO3, and Na2CO3 as neutralising agents were performed and compared. It was shown that substantial improvements could be made by using alkali bases to neutralise the fermentations. The highest concentrations and productivities were achieved when Na2CO3 was used, 77 g L-1 and 3 g L-1 h-1 respectively. A gradual decrease in succinate productivity was observed during the fermentations, which was shown to be due to succinate accumulation in the broth and not as a result of the addition of neutralising agent or the subsequent increase in osmolarity.To maintain high succinate productivity by keeping a low extracellular succinic acid concentration fermentations were interrupted and cells recovered and resuspended in fresh media. By removing the succinate it was possible to maintain high succinic acid productivity for a prolonged time. Cells subjected to high concentrations of succinate were also able to regain high productivity once transferred into a succinate-free medium.In the last part of the thesis succinic acid production from softwood dilute acid hydrolysates was demonstrated. This study involved establishing the degree of detoxification necessary for growth and fermentation using industrial hydrolysates. Detoxification by treatment with lime and/or activated carbon was investigated and the results show that it was possible to produce succinate from softwood hydrolysates in yields comparable to those for synthetic sugars.The work done in this thesis increases the understanding of succinic acid production with AFP184, illustrate its limitations, and suggests improvements in the current technology with the long term aim of increasing the economical feasibility of biochemical succinic acid production.
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| 3. |
- Andersson, Christian, et al.
(författare)
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Effect of different carbon sources on the production of succinic acid using metabolically engineered Escherichia coli
- 2007
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Ingår i: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 23:2, s. 381-388
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Tidskriftsartikel (refereegranskat)abstract
- Succinic acid (SA) is an important platform molecule in the synthesis of a number of commodity and specialty chemicals. In the present work, dual-phase batch fermentations with the E. coli strain AFP184 were performed using a medium suited for large-scale industrial production of SA. The ability of the strain to ferment different sugars was investigated. The sugars studied were sucrose, glucose, fructose, xylose, and equal mixtures of glucose and fructose and glucose and xylose at a total initial sugar concentration of 100 g L-1. AFP184 was able to utilize all sugars and sugar combinations except sucrose for biomass generation and succinate production. For sucrose as a substrate no succinic acid was produced and none of the sucrose was metabolized. The succinic acid yield from glucose (0.83 g succinic acid per gram glucose consumed anaerobically) was higher than the yield from fructose (0.66 g g-1). When using xylose as a carbon source, a yield of 0.50 g g-1 was obtained. In the mixed-sugar fermentations no catabolite repression was detected. Mixtures of glucose and xylose resulted in higher yields (0.60 g g-1) than use of xylose alone. Fermenting glucose mixed with fructose gave a lower yield (0.58 g g-1) than fructose used as the sole carbon source. The reason is an increased pyruvate production. The pyruvate concentration decreased later in the fermentation. Final succinic acid concentrations were in the range of 25-40 g L-1. Acetic and pyruvic acid were the only other products detected and accumulated to concentrations of 2.7-6.7 and 0-2.7 g L-1. Production of succinic acid decreased when organic acid concentrations reached approximately 30 g L-1. This study demonstrates that E. coli strain AFP184 is able to produce succinic acid in a low cost medium from a variety of sugars with only small amounts of byproducts formed.
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| 4. |
- Andersson, Christian, et al.
(författare)
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Effects of neutralising agent, organic acids, and osmolarity on succinic acid production by Escherichia coli AFP184
- 2008
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Using a low-cost medium Escherichia coli AFP184 has previously been reported to produce succinic acid with volumetric productivities close to 3 g L-1 h-1. At a total organic acid concentration of 30 g L-1 the productivity decreased drastically resulting in final succinate concentrations of 40 g L-1. The economical viability of biochemical succinic acid production would benefit from higher final succinic acid concentrations and volumetric productivities maintained at >2.5 g L-1 h-1 for an extended period of time. In the present work the effects of osmolarity and neutralising agent (NH4OH, KOH, NaOH, K2CO3, and Na2CO3) on succinic acid production by AFP184 were investigated. Highest concentration of succinic acid was obtained with Na2CO3, 75 g L-1. It was also found that the osmolarity resulting from succinate production and subsequent base addition, only marginally affected the productivity per viable cell. Organic acid inhibition due to the produced succinic acid on the other hand significantly reduced succinic acid productivity per viable cell. When using NH4OH productivity completely ceased at approximately 40 g L-1. Volumetric productivities remained at 2.5 g L-1 h-1 for 5 to 10 hours longer when using K- or Na-bases than when using NH4OH. However, loss of cell viability occurred, and together with the acid inhibition decreased the volumetric productivities. In this study it was demonstrated that by altering the neutralising agent it was possible to increase the period of high volumetric productivity in the anaerobic phase and improve the final succinic acid concentration by almost 100 %
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| 5. |
- Andersson, Christian, et al.
(författare)
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Inhibition of succinic acid production in metabolically engineered Escherichia Coli by neutralizing agent, organic acids, and osmolarity
- 2009
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Ingår i: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 25:1, s. 116-123
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Tidskriftsartikel (refereegranskat)abstract
- The economical viability of biochemical succinic acid production is a result of many processing parameters including final succinic acid concentration, recovery of succinate, and the volumetric productivity. Maintaining volumetric productivities >2.5 g L-1 h(-1) is important if production of succinic acid from. renewable resources should be competitive. In this work, the effects of organic acids, osmolarity, and neutralizing agent (NH4OH, KOH, NaOH, K2CO3, and Na2CO3) on the fermentative succinic acid production by Escherichia coli AFP184 were investigated. The highest concentration of succinic acid, 77 g L-1. was obtained with Na2O3. In general, irrespective of the base used, succinic acid productivity per viable cell was significantly reduced as the concentration of the produced acid increased. Increased osmolarity resulting from base addition during succinate production only marginally affected the productivity per viable cell. Addition of the osmoprotectant glycine betaine to cultures resulted in an increased aerobic growth rate and anaerobic glucose consumption rate, but decreased succinic acid yield. When using NH4OH productivity completely ceased at a succinic acid concentration of similar to 40 g L-1. Volumetric productivities remained at 2.5 g L-1 h(-1) for tip to 10 h longer when K- or Na-bases where used instead of NH4OH. The decrease in cellular succinic acid productivity observed during the anaerobic phase was found to be due to increased organic acid concentrations rather than medium osmolarity.
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| 6. |
- Andersson, Christian, et al.
(författare)
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Process for producing succinic acid from sucrose
- 2005
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Patent (populärvet., debatt m.m.)abstract
- A process for hydrolyzing sucrose to glucose and fructose using succinic acid is described. The hydrolysate can be used to produce purified glucose and/or fructose or can be used as a carbon source for fermentations to produce various chemicals including succinic acid.
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| 7. |
- Andersson, Christian
(författare)
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Succinic acid production using metabolically engineered Escherichia coli
- 2007
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The prospects of peak oil, climate change and the dependency of fossil carbon have urged research and development of production methods for the manufacture of fuels and chemicals from renewable resources (biomass). To date, the primary emphasis has been placed on the replacement of oil for transportation fuels. A highly significant subset of petroleum usage is the production of chemicals, which represents 10-15% of the petroleum usage. White biotechnology, also called industrial biotechnology, is a fast evolving technology with a large potential to have a substantial impact on the industrial production of fuels and chemicals from biomass. This work addresses the issue of chemical production by investigating the production of bio-based succinic acid, which can be used in a wide range of applications to replace petroleum based chemicals. Succinic acid can be produced by fermentation of sugar by a number of organisms; one is Escherichia coli (E. coli). It is known that E. coli under anaerobic conditions produces a mixture of organic acids. In order to obtain a cost-effective production it is necessary to metabolically engineer the organism to produce succinic acid in greater yield than the other acids. In the current work, E. coli mutant AFP184 was used. AFP184 originates from a near wild type strain, the C600 (ATCC 23724), which can ferment both five and six carbon sugars and has mutations in the glucose specific phosphotransferase system (ptsG), the pyruvate formate lyase system (pfl) and in the fermentative lactate dehydrogenase system (ldh). The previous studies using different organisms have all used cultivation mediums supplemented to some degree with different nutrients like biotin, thiamine and yeast extract. In order to apply the technology to large scale, production must be cost-effective and it is important to minimise the use of additional supplements. The first part of this work aimed to investigate the fermentation characteristics of AFP184 in a medium consisting of corn steep liquor, inorganic salts and different sugar sources without supplementation of other additional nutrients. It addresses questions regarding the effect of different sugars on succinic acid kinetics and yields in an industrially relevant medium. In order to gain a sustainable production of succinic acid from biomass feedstocks (sugar from biomass) it is important to investigate how well the organism can utilise different sugars in the biomass. The sugars studied were sucrose, glucose, fructose, xylose and equal mixtures of glucose-fructose and glucose-xylose at a total initial sugar concentration of 100 g L-1. AFP184 was able to utilise all sugars and sugar combinations except sucrose for biomass generation and succinate production. Using glucose resulted in the highest yield, 0.83 (g succinic acid per g sugar consumed anaerobically). Fructose resulted in a yield of 0.66 and xylose of 0.5. Using a high initial sugar concentration made it possible to obtain volumetric productivities of almost 3 g L-1h-1, which is above estimated values for feasible economic production. Succinic acid production ceased at final concentrations greater than 40 g L-1. In order to further increase succinic acid concentrations, this inhibitory effect was studied in the second part of the present work. The inhibitory effects can be two-fold including pH-based inhibition and an anion specific effect on metabolism. It has been reported that high concentrations of ammonia inhibit E. coli growth and damage cell membranes. In order to limit toxic and inhibitory effects different neutralising agents were tested. First the use of NH4OH was optimised with respect to fermentation pH and it was found that the best results were obtained at pH 6.5-6.7. Optimal pH was then used with NaOH, KOH, and Na2CO3 as neutralising agents and it was shown that NaOH, KOH, and Na2CO3 neutralised fermentations could reach succinic acid concentrations of 69 and 61 and 78 g L-1 respectively without any significant decrease in succinic acid productivity. It was observed that cells lost viability during the cause anaerobic phase. It resulted in decreasing succinic acid productivities. It is believed that the viability decrease is a combined effect of organic acids concentration and the osmolarity of the medium. The work done in this thesis is aimed towards increasing the economical feasibility of a biochemical succinic acid production.
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| 8. |
- Berglund, Kris, et al.
(författare)
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Fermentative Upgrading of Xylose
- 2009
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Ingår i: NWBC-2009. - Helsinki : KCL Re-inventing paper.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Chemical pulp mills such as Kraft, soda, or sulfite mills are current examples of biorefineries that can convert lignocellulosic biomass into energy, pulp or cellulose derivatives, and tall oil. While existing viscose pulps use a hemicellulose extraction to generate soluble sugars for ethanol production, in general there still exists a large potential for other more profitable applications of the biomass (Fig. 1), i.e. the mill needs to present a widespread product portfolio. The biofuels under development from fermentation that will be discussed are the diesel fuel oxygenates dibutyl succinate and diethyl succinate to be used for reduced particulate emissions and fossil fuel replacement for diesel engines and butanol for Otto engines. It's important to stress that succinic acid, butanol and ethanol, needed for the production of the diesel additives and gasoline replacement will be produced from renewable resources and hence replacing products currently produced from non-renewable fossil sources. Since wood will be used, there will be no issue of competing with raw material used for food production. Besides biofuel production, succinic acid and butanol, can be used directly or further refined into numerous different products classified as green chemicals.
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| 9. |
- Berglund, Kris, et al.
(författare)
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Process for the production of succinic acid
- 2006
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Patent (populärvet., debatt m.m.)abstract
- A process for the production of succinic acid can comprise supplying a media with E. coli AFP 184 and a high sugar concentration under aerobic conditions, then converting the media to aerobic conditions. Such a process can be useful when performed in conjunction with the production of ethanol in a biorefmery .
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| 10. |
- Berglund, Kris
(författare)
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Succinic acid from xylan : fermentations using mixed sugar feedstocks
- 2008
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Ingår i: NWBC 2008. - Stockholm : STFI-Packforsk. - 9789186018139 ; , s. 121-124
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Konferensbidrag (refereegranskat)abstract
- The next generation of cost effective, economically viable biorefinery scenarios requires the development and deployment of higher valued added products whose production can be integrated into a forest biorefinery. We present preliminary results for the production of one such product, succinic acid, from mixed sugars streams including glucose and xylose, as would be present from the hydrolysis of xylan. Examples are presented for the base laboratory case, softwood hydrolysis streams, and hardwood extracts
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