| 1. |
- Andersson, Christian
(författare)
-
Biobased production of succinic acid by Escherichia coli fermentation
- 2009
-
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.
|
|
| 2. |
- Backlund, Emma, et al.
(författare)
-
Suppressing glucose uptake and acetic acid production increases membrane protein overexpression in Escherichia coli.
- 2011
-
Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 10:1, s. 35-
-
Tidskriftsartikel (refereegranskat)abstract
- The production of integral membrane spanning proteins (IMP's) constitutes a bottleneck in pharmaceutical development. It was long considered that the state-of-the-art was to produce the proteins as inclusion bodies using a powerful induction system. However, the quality of the protein was compromised and the production of a soluble protein that is incorporated into the membrane from which it is extracted is now considered to be a better method. Earlier research has indicated that a slower rate of protein synthesis might overcome the tendency to form inclusion bodies. We here suggest the use of a set of E. coli mutants characterized by a slower rate of growth and protein synthesis as a tool for increasing the amount of soluble protein in high- throughput protein production processes. RESULTS: A set of five IMP's was chosen which were expressed in three mutants and the corresponding WT cell (control). The mutations led to three different substrate uptake rates, two of which were considerably slower than that of the wild type. Using the mutants, we were able to express three out of the five membrane proteins. Most successful was the mutant growing at 50% of the wild type growth rate. A further effect of a low growth rate is a low acetic acid formation, and we believe that this is a possible reason for the better production. This hypothesis was further supported by expression from the BL21(DE3) strain, using the same plasmid. This strain grows at a high growth rate but nevertheless yields only small amounts of acetic acid. This strain was also able to express three out of the five IMP's, although at lower quantities. CONCLUSIONS: The use of mutants that reduce the specific substrate uptake rate seems to be a versatile tool for overcoming some of the difficulties in the production of integral membrane spanning proteins. A set of strains with mutations in the glucose uptake system and with a lower acetic acid formation were able to produce three out of five membrane proteins that it was not possible to produce with the corresponding wild type.
|
|
| 3. |
- Björlenius, Berndt, 1963-
(författare)
-
Pharmaceuticals – improved removal from municipal wastewater and their occurrence in the Baltic Sea
- 2018
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Pharmaceutical residues are found in the environment due to extensive use in human and veterinary medicine. The active pharmaceutical ingredients (APIs) have a potential impact in non-target organisms. Municipal wastewater treatment plants (WWTPs) are not designed to remove APIs.In this thesis, two related matters are addressed 1) evaluation of advanced treatment to remove APIs from municipal wastewater and 2) the prevalence and degradation of APIs in the Baltic Sea.A stationary pilot plant with nanofiltration (NF) and a mobile pilot plant with activated carbon and ozonation were designed to study the removal of APIs at four WWTPs. By NF, removal reached 90%, but the retentate needed further treatment. A predictive model of the rejection of APIs by NF was developed based on the variables: polarizability, globularity, ratio hydrophobic to polar water accessible surface and charge. The pilot plants with granular and powdered activated carbon (GAC) and (PAC) removed more than 95% of the APIs. Screening of activated carbon products was essential, because of a broad variation in adsorption capacity. Recirculation of PAC or longer contact time, increased the removal of APIs. Ozonation with 5-7 g/m3 ozone resulted in 87-95% removal of APIs. Elevated activity and transcription of biomarkers indicated presence of xenobiotics in regular effluent. Chemical analysis of APIs, together with analysis of biomarkers, were valuable and showed that GAC-filtration and ozonation can be implemented to remove APIs in WWTPs, with decreased biomarker responses.Sampling of the Baltic Sea showed presence of APIs in 41 out of 43 locations. A developed grey box model predicted concentration and half-life of carbamazepine in the Baltic Sea to be 1.8 ng/L and 1300 d respectively.In conclusion, APIs were removed to 95% by GAC or PAC treatment. The additional treatment resulted in lower biomarker responses than today and some APIs were shown to be widespread in the aquatic environment.
|
|
| 4. |
- Bostrom, M., et al.
(författare)
-
Process design for recombinant protein production based on the promoter, P-malK
- 2004
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 66:2, s. 200-208
-
Tidskriftsartikel (refereegranskat)abstract
- P-malK is induced through activation of MalT, by the formation of maltotriose and cyclic adenosine monophosphate ( cAMP). The possibility to influence endogenous inducer levels is used to vary the production rates in specifically designed production protocols. Induction based on a batch process protocol on maltose gives low production rates, as the result of a lack of cAMP, which is shown to be of major importance to fully induce this promoter. Two mechanisms are thus used to influence the levels of maltotriose and/or cAMP formation: ( 1) catabolite derepression achieved from low glucose concentration and ( 2) catabolite derepression/inducer exclusion from diauxic growth on glucose/maltose. Fed-batch processes based on limited amounts of glucose result in product accumulation of up to 10% of the total protein. Depending on the feed of limiting glucose, different production profiles are developed. The initial increase in the production rate is due to maltotriose formation from endogenous glycogen degradation while, later in the process, production can be further supported by elevated levels of cAMP, provided the feed rate is sufficiently low. The introduction of maltose after a preceding fed-batch process on glucose can be efficiently used to produce maltotriose in combination with cAMP formation in the event of catabolite derepression. This leads to higher production rates and a further increase in product accumulation of up to 30% of the total protein. The diauxic growth phase resulting from the shift in carbon source can be shortened and even avoided by the design of the preceding feed-rate of glucose. It is postulated that proper design of the inoculum and initial phases of production can reduce basal levels of product formation. With this promoter, the production rate can be as high as 65 units mg(-1) h(-1) and the time to reach a maximal production rate can be designed to take up to 8 h. Furthermore, the duration of the production rate can be as long as 7 h.
|
|
| 5. |
- Boström, Maria, et al.
(författare)
-
Effect of substrate feed rate on recombinant protein secretion, degradation and invlusion body formation in Escherichia coli
- 2005
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 68:1, s. 82-90
-
Tidskriftsartikel (refereegranskat)abstract
- The effect of changes in substrate feed rate during fedbatch cultivation was investigated with respect to soluble protein formation and transport of product to the periplasm in Escherichia coli. Production was transcribed from the P-malK promoter; and the cytoplasmic part of the production was compared with production from the P-lacUV5 promoter. The fusion protein product, Zb-MalE, was at all times accumulated in the soluble protein fraction except during high-feed-rate production in the cytoplasm. This was due to a substantial degree of proteolysis in all production systems, as shown by the degradation pattern of the product. The product was also further subjected to inclusion body fori-nation. Production in the periplasm resulted in accumulation of the full-length protein; and this production system led to a cellular physiology where the stringent response could be avoided. Furthermore, the secretion could be used to abort the diauxic growth phase resulting from use of the P-malK promoter. At high feed rate, the accumulation of acetic acid, due to overflow metabolism, could furthermore be completely avoided.
|
|
| 6. |
- Bäcklund, Emma, et al.
(författare)
-
Cell engineering of Escherichia coli allows high cell density accumulation without fed-batch process control
- 2008
-
Ingår i: Bioprocess and biosystems engineering (Print). - : Springer Science and Business Media LLC. - 1615-7591 .- 1615-7605. ; 31:1, s. 11-20
-
Tidskriftsartikel (refereegranskat)abstract
- A set of mutations in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was used to create Escherichia coli strains with a reduced uptake rate of glucose. This allows a growth restriction, which is controlled on cellular rather than reactor level, which is typical of the fed-batch cultivation concept. Batch growth of the engineered strains resulted in cell accumulation profiles corresponding to a growth rate of 0.78, 0.38 and 0.25 h(-1), respectively. The performance of the mutants in batch cultivation was compared to fed-batch cultivation of the wild type cell using restricted glucose feed to arrive at the corresponding growth profiles. Results show that the acetate production, oxygen consumption and product formation were similar, when a recombinant product was induced from the lacUV5 promoter. Ten times more cells could be produced in batch cultivation using the mutants without the growth detrimental production of acetic acid. This allows high cell density production without the establishment of elaborate fed-batch control equipment. The technique is suggested as a versatile tool in high throughput multiparallel protein production but also for increasing the number of experiments performed during process development while keeping conditions similar to the large-scale fed-batch performance.
|
|
| 7. |
- Bäcklund, Emma, et al.
(författare)
-
Fedbatch design for periplasmic product retention in Escherichia coli
- 2008
-
Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 135:4, s. 358-365
-
Tidskriftsartikel (refereegranskat)abstract
- The feed profile of glucose during fedbatch cultivation could be used to influence the retention of the periplasmic product ZZ-cutinase. An increased feed rate led to a higher production rate but also to an increased specific leakage, which reduced the periplasmic retention. Three growth rates: 0.3, 0.2 and 0.1 h-1 where studied and resulted in 20, 9 and 6%, respectively, of the total ZZ-cutinase accumulating in the medium. It was also shown that leakage during fedbatch production of a Fab fragment was also influenced by the feed rate in a similar manner to ZZ-cutinase. If intracellular product accumulation is desired the advantage of a high productivity, resulting from a high substrate feed rate, is diminished because of a reduced product retention. Biochemical analysis revealed that the growth rate, resulting from a glucose limited feed, influenced the outer membrane protein compositions with respect to OmpF and LamB, whilst OmpA was largely unaffected. As the feed rate increased the amount of total outer membrane protein decreased. When ZZ-cutinase was produced there were further reductions in outer membrane protein accumulation, by 82, 100 and 22% for OmpF, LamB and OmpA, respectively, and the total reduction was almost 60% with a high product formation rate. We suggest that the reduced titre of the outer membrane proteins, OmpF and LamB, may have contributed to a reduced ability for the cell to retain recombinant protein secreted to the periplasm.
|
|
| 8. |
- Bäcklund, Emma
(författare)
-
Growth rate control of periplasmic product retention in Escherichia coli
- 2008
-
Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The recombinant product is secreted to the periplasm in many processes where E. coli is used as host. One drawback with secretion is the undesired leakage of the periplasmic products to the medium. The aim of this work was to find strategies to influence the periplasmic retention of recombinant products. We have focused on the role of the specific growth rate, a parameter that is usually controlled in industrial bioprocesses. The hypothesis was that the stability of the outer membrane in E. coli is gained from a certain combination of specific phospholipids and fatty acids on one side and the amount and specificity of the outer membrane proteins on the other side, and that the specific growth rate influences this structure and therefore can be used to control the periplasmic retention. We found that is possible to control the periplasmic retention by the growth rate. The leakage of the product increased as the growth rate increased. It was however also found that a higher growth rate resulted in increased productivity. This resulted in equal amounts of product inside the cells regardless of growth rate. We also showed that the growth rate influenced the outer membrane composition with respect to OmpF and LamB while OmpA was largely unaffected. The total amount of outer membrane proteins decreased as the growth rate increased. There were further reductions in outer membrane protein accumulation when the recombinant product was secreted to the periplasm. The lowered amount of outer membrane proteins may have contributed to the reduced ability for the cell to retain the product in the periplasm. The traditional way to control the growth rate is through a feed of substrate in a fed-batch process. In this work we used strains with a set of mutations in the phosphotransferase system (PTS) with a reduced uptake rate of glucose to investigate if these strains could be used for growth rate control in batch cultivations without the use of fed-batch control equipment. The hypothesis was that the lowering of the growth rate on cell level would result in the establishment of fed-batch similar conditions. This study showed that it is possible to control the growth rate in batch cultivations by using mutant strains with a decreased level of substrate uptake rate. The mutants also produced equivalent amounts of acetic acid as the wild type did in fed-batch cultivation with the same growth rate. The oxygen consumption rates were also comparable. A higher cell density was reached with one of the mutants than with the wild type in batch cultivations. It is possible to control the growth rate by the use of the mutants in small-scale batch cultivations without fed-batch control equipment.
|
|
| 9. |
- Bäcklund, Emma
(författare)
-
Impact of glucose uptake rate on recombinant protein production in Escherichia coli
- 2011
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Escherichia coli (E. coli) is an attractive host for production of recombinant proteins, since it generally provides a rapid and economical means to achieve high product quantities. In this thesis, the impact of the glucose uptake rate on the production of recombinant proteins was studied, aiming at improving and optimising production of recombinant proteins in E. coli. E. coli can be cultivated to high cell densities in bioreactors by applying the fed-batch technique, which offers a means to control the glucose uptake rate. One objective of this study was to find a method for control of the glucose uptake rate in small-scale cultivation, such as microtitre plates and shake flasks. Strains with mutations in the phosphotransferase system (PTS) where used for this purpose. The mutants had lower uptake rates of glucose, resulting in lower growth rates and lower accumulation of acetic acid in comparison to the wild type. By using the mutants in batch cultivations, the formation of acetic acid to levels detrimental to cell growth could be avoided, and ten times higher cell density was reached. Thus, the use of the mutant strains represent a novel, simple alternative to fed-batch cultures. The PTS mutants were applied for production of integral membrane proteins in order to investigate if the reduced glucose uptake rate of the mutants was beneficial for their production. The mutants were able to produce three out of five integral membrane proteins that were not possible to produce by the wild-type strain. The expression level of one selected membrane protein was increased when using the mutants and the expression level appeared to be a function of strain, glucose uptake rate and acetic acid accumulation. For production purposes, it is not uncommon that the recombinant proteins are secreted to the E. coli periplasm. However, one drawback with secretion is the undesired leakage of periplasmic products to the medium. The leakage of the product to the medium was studied as a function of the feed rate of glucose in fed-batch cultivations and they were found to correlate. It was also shown that the amount of outer membrane proteins was affected by the feed rate of glucose and by secretion of a recombinant product to the periplasm. The cell surface is another compartment where recombinant proteins can be expressed. Surface display of proteins is a potentially attractive production strategy since it offers a simple purification scheme and possibilities for on-cell protein characterisation, and may in some cases also be the only viable option. The AIDA-autotransporter was applied for surface display of the Z domain of staphylococcal protein A under control of the aidA promoter. Z was expressed in an active form and was accessible to the medium. Expression was favoured by growth in minimal medium and it seemed likely that expression was higher at higher feed rates of glucose during fed-batch cultivation. A repetitive batch process was developed, where relatively high cell densities were achieved whilst maintaining a high expression level of Z.
|
|
| 10. |
|
|
