| 1. |
- Bjornsson, L., et al.
(författare)
-
Utilization of a palladium-metal oxide semiconductor (Pd-MOS) sensor for on-line monitoring of dissolved hydrogen in anaerobic digestion
- 2001
-
Ingår i: Biotechnology and Bioengineering. - 0006-3592 .- 1097-0290. ; 73:1, s. 35-43
-
Tidskriftsartikel (refereegranskat)abstract
- The use of a hydrogen-sensitive palladium-metal oxide semiconductor (Pd-MOS) sensor in combination with a membrane for liquid-to-gas transfer for the detection of dissolved hydrogen was investigated. The system was evaluated with known concentrations of dissolved hydrogen in water. The lowest concentration detected with this set-up was 160 nM. The method was applied to monitoring of a laboratory-scale anaerobic digestion process employing mixed sludge containing mainly food/industrial waste. Pulse loads of glucose were added to the system at different levels of microbial activity, and the microbial status of the culture was reflected in the dissolved hydrogen response. Simultaneous headspace hydrogen measurements were performed, and at the lower levels of dissolved hydrogen no corresponding headspace hydrogen could be detected. When glucose was added to a resting culture the dissolved hydrogen response was rapid and the first response could be detected 9 min after addition of glucose, whereas headspace hydrogen concentrations increased only after 80 to 110 min. This indicates limitations in the liquid-to-gas hydrogen transfer and illustrates the importance of hydrogen monitoring in the liquid. The sensor system developed is flexible, the membrane is easily replaceable, and the probe for liquid-to-gas hydrogen transfer can be adjusted easily to large-scale applications. © 2001 John Wiley & Sons, Inc.
|
|
| 2. |
- Boer, H., et al.
(författare)
-
Characterization of Trichoderma reesei cellobiohydrolase CeI7A secreted from Pichia pastoris using two different promoters
- 2000
-
Ingår i: Biotechnology and Bioengineering. - 0006-3592 .- 1097-0290. ; 69:5, s. 486-494
-
Tidskriftsartikel (refereegranskat)abstract
- Heterologous expression of T. reesei cellobiohydrolase Cel7A in a methylotrophic yeast Pichia pastoris was tested both under the P. pastoris alcohol oxidase (AOX1) pro meter and the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter in a fermenter. Production of Ce17A with the AOX1 promoter gave a better yield, although part of the enzyme expressed was apparently not correctly folded. Cel7A expressed in P. pastoris is overglycosylated at its N-glycosylation sites as compared to the native T. reesei protein, but less extensive than Cel7A expressed in Saccharomyces cerevisiae. The k(cat) and K-m values for the purified protein on soluble substrates are similar to the values found for the native Trichoderma Cel7A, whereas the degradation rate on crystalline substrate (BMCC) is somewhat reduced. The measured pH optimum also closely resembles that of purified T. reesei Cel7A. Furthermore, the hyperglycosylation does not affect the thermostability of the enzyme monitored with tryptophane fluorescence and activity measurements. On the other hand, CD measurements indicate that the formation of disulfide bridges is an important step in the correct folding of Cel7A and might explain the difficulties encountered in heterologous expression of T. reesei Cel7A. The constitutive GAP promoter expression system of P. pastoris is nevertheless well suited for activity screening of cellulase activities in microtiter plates. With this type of screening method a faster selection of site-directed and random mutants with, for instance, an altered optimum pH is possible, in contrast to the homologous T. reesei expression system.
|
|
| 3. |
- Taherzadeh, Mohammad J, 1965-, et al.
(författare)
-
On-line control of fed-batch fermentation of dilute-acid hydrolyzates
- 2000
-
Ingår i: Biotechnology and Bioengineering. - New York, NY, United States : John Wiley & Sons Inc. - 0006-3592 .- 1097-0290. ; 69:3, s. 330-338
-
Tidskriftsartikel (refereegranskat)abstract
- Dilute-acid hydrolyzates from lignocellulose are, to a varying degree, inhibitory to yeast. In the present work, dilute-acid hydrolyzates from spruce, birch, and forest residue, as well as synthetic model media, were fermented by Saccharomyces cerevisiae in fed-batch cultures. A control strategy based on on-line measurement of carbon dioxide evolution (CER) was used to control the substrate feed rate in a lab scale bioreactor. The control strategy was based solely on the ratio between the relative increase in CER and the relative increase in feed rate. Severely inhibiting hydrolyzates could be fermented without detoxification and the time required for fermentation of moderately inhibiting hydrolyzates was also reduced. The feed rate approached a limiting value for inhibiting media, with a corresponding pseudo steady-state value for CER. However, a slow decrease of CER with time was found for media containing high amounts of 5-hydroxymethyl furfural (HMF). The success of the control strategy is explained by the conversion of furfural and HMF by the yeast during fed-batch operation. The hydrolyzates contained between 1.4 and 5 g/l of furfural and between 2.4 and 6.5 g/l of HMF. A high conversion of furfural was obtained (between 65-95%) at the end of the feeding phase, but the conversion of HMF was considerably lower (between 12-40%). (C) 2000 John Wiley and Sons, Inc.Dilute-acid hydrolyzates from lignocellulose are, to a varying degree, inhibitory to yeast. In the present work, dilute-acid hydrolyzates from spruce, birch, and forest residue, as well as synthetic model media, were fermented by Saccharomyces cerevisiae in fed-batch cultures. A control strategy based on on-line measurement of carbon dioxide evolution (CER) was used to control the substrate feed rate in a lab scale bioreactor. The control strategy was based solely on the ratio between the relative increase in CER and the relative increase in feed rate. Severely inhibiting hydrolyzates could be fermented without detoxification and the time required for fermentation of moderately inhibiting hydrolyzates was also reduced. The feed rate approached a limiting value for inhibiting media, with a corresponding pseudo steady-state value for CER. However, a slow decrease of CER with time was found for media containing high amounts of 5-hydroxymethyl furfural (HMF). The success of the control strategy is explained by the conversion of furfural and HMF by the yeast during fed-batch operation. The hydrolyzates contained between 1.4 and 5 g/l of furfural and between 2.4 and 6.5 g/l of HMF. A high conversion of furfural was obtained (between 65-95%) at the end of the feeding phase, but the conversion of HMF was considerably lower (between 12-40%).
|
|
| 4. |
- Bylund, F., et al.
(författare)
-
Influence of scale-up on the quality of recombinant human growth hormone
- 2000
-
Ingår i: Biotechnology and Bioengineering. - 0006-3592 .- 1097-0290. ; 69:2, s. 119-128
-
Tidskriftsartikel (refereegranskat)abstract
- The aerobic fed-batch production of recombinant human growth hormone (rhGH) by Escherichia coli was studied. The goal was to determine the production and protein degradation pattern of this product during fed-batch cultivation and to what extent scale differences depend on the presence of a fed-batch glucose feed zone. Results of laboratory bench-scale, scale-down (SDR), and industrial pilot-scale (3-m(3)) reactor production were compared. In addition to the parameters of product yield and quality, also cell yield, respiration, overflow, mixed acid fermentation, glucose concentration, and cell lysis were studied and compared. The results show that oxygen limitation following glucose overflow was the critical parameter and not the glucose overflow itself. This was verified by the pattern of byproduct formation where formate was the dominating factor and not acetic acid. A correlation between the accumulation of formate, the degree of heterogeneity, and cell lysis was also visualized when recombinant protein was expressed. The production pattern could be mimicked in the SDR reactor for all parameters, except for product quantity and quality, where 30% fewer rhGH-degraded forms were present and where about 80% higher total yield was achieved, resulting in 10% greater accumulation of properly formed rhGH monomer.
|
|
| 5. |
- Adlercreutz, Dietlind, et al.
(författare)
-
Synthesis of phosphatidylcholine with defined fatty acid in the sn-1 position by lipase-catalyzed esterification and transesterification reaction.
- 2002
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 78:4, s. 403-411
-
Tidskriftsartikel (refereegranskat)abstract
- The incorporation of caproic acid in the sn-1 position of phosphatidylcholine (PC) catalyzed by lipase from Rhizopus oryzae was investigated in a water activity-controlled organic medium. The reaction was carried out either as esterification or transesterification. A comparison between these two reaction modes was made with regard to product yield, product purity, reaction time, and byproduct formation as a consequence of acyl migration. The yield in the esterification and transesterification reaction was the same under identical conditions. The highest yield (78%) was obtained at a water activity (a(w)) of 0.11 and a caproic acid concentration of 0.8 M. The reaction time was shorter in the esterification reaction than in the transesterification reaction. The difference in reaction time was especially pronounced at low water activities and high fatty acid concentrations. The loss in yield due to acyl migration and consequent enzymatic side reactions was around 16% under a wide range of conditions. The incorporation of a fatty acid in the sn-1 position of PC proved to be thermodynamically much more favorable than the incorporation of a fatty acid in the sn-2 position.
|
|
| 6. |
- Andersson, Mats, et al.
(författare)
-
Toward an enzyme-based oxygen scavenging laminate. Influence of industrial lamination conditions on the performance of glucose oxidase
- 2002
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 79:1, s. 37-42
-
Tidskriftsartikel (refereegranskat)abstract
- The laminate consisted of several polymer layers, aluminium, and one cellulose-based layer containing the active enzymatic system (e.g., glucose oxidase, catalase, glucose, and CaCO3). During the industrial lamination process, the enzyme layer was exposed to three temperature spikes up to 325degreesC without significant enzyme inactivation. Ninety-seven percent of the glucose oxidase activity still remained after the lamination process. The best laminate had an oxygen absorbing capacity of 7.6 +/- 1.0 L/m(2). A reference that was not laminated expressed a corresponding oxygen absorbing capacity of 7.1 +/- 0.8 L/m(2).
|
|
| 7. |
- Batstone, Damien, et al.
(författare)
-
Kinetics of thermophilic, anaerobic oxidation of straight and branched chain butyrate and valerate
- 2003
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 84:2, s. 195-204
-
Tidskriftsartikel (refereegranskat)abstract
- The degradation kinetics of normal and branched chain butyrate and valerate are important in protein-fed anaerobic systems, as a number of amino acids degrade to these organic acids. Including activated and primary wastewater sludge digesters, the majority of full-scale systems digest feeds with a significant or major fraction of COD as protein. This study assesses the validity of using a common kinetic parameter set and biological catalyst to represent butyrate, n-valerate, and i-valerate degradation in dynamic models. The i-valerate degradation stoichiometry in a continuous, mixed population system is also addressed, extending previous pure-culture and batch studies. A previously published mathematical model was modified to allow competitive uptake of i-valerate, and used to model a thermophilic manure digester operated over 180 days. The digester was periodically pulsed with straight and branched chain butyrate and valerate. Parameters were separately optimized to describe butyrate, i-valerate, and n-valerate degradation, as well as a lumped set optimized for all three substrates, and nonlinear, correlated parameter spaces estimated using an F distribution in the objective function (A Each parameter set occupied mutually exclusive parameter spaces, indicating that all were statistically different from each other. However, qualitatively, the influence on model outputs was similar, and the lumped set would be reasonable for mixed acid digestion. The main characteristic not represented by Monod kinetics was a delay in i-valerate uptake, and was compensated for by a decreased maximum uptake rate (k(m)). Therefore, the kinetics need modification if fed predominantly i-valerate. Butyrate (i- and n-) and n-valerate could be modeled using stoichiometry consistent with beta-oxidation degradation pathways. However, i-valerate produced acetate only, supporting the stoichiometry of a reaction determined by other researchers in pure culture. Therefore, lumping i-valerate stoichiometry with that of n-valerate will not allow good system representation, especially when the feed consists of proteins high in leucine (which produces i-valerate), and the modified model structure and stoichiometry as proposed here should be used. This requires no additional kinetic parameters and one additional dynamic concentration state variable (i-valerate) in addition to the variables in the base model.
|
|
| 8. |
- Castan, A., et al.
(författare)
-
Formate accumulation due to DNA release in aerobic cultivations of Escherichia coli
- 2002
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 77:3, s. 324-328
-
Tidskriftsartikel (refereegranskat)abstract
- Three different aerobic fed-batch processes of Escherichia coli were studied, two for the production of a recombinant protein and one process with a wild-type E. coli strain. In all three processes, an accumulation of formate could be observed in the latter part of the process. Analysis of the concentration of DNA in the medium revealed that the release of DNA coincided with the accumulation of formate. It was found that increasing concentrations of DNA correlated in almost linearly increasing concentrations of formate. Formate accumulation is caused by mixed acid fermentation, although no oxygen limitation was measured with the DO electrode. It is proposed that extracellular DNA restrained mass transfer between the bulk medium and the cell. To investigate if the DNA accumulation caused formate production, DNA was removed by continuous feeding of a DNA binding polymer to the medium. The addition of the polymer decreased the content of free DNA in the broth and the formate was reassimilated. Furthermore, additional DNA early in the process resulted in early formate accumulation.
|
|
| 9. |
- Collén, Anna, et al.
(författare)
-
Primary recovery of a genetically engineered Trichoderma reesei endoglucanase I (Cel 7B) fusion protein in cloud point extraction systems.
- 2002
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 78:4, s. 385-394
-
Tidskriftsartikel (refereegranskat)abstract
- Here we present data to demonstrate how partitioning of a hydrophilic enzyme can be directed to the hydrophobic detergent-enriched phase of an aqueous two-phase system by addition of short stretches of amino acid residues to the protein molecule. The target enzyme was the industrially important endoglucanase I, EGI (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei. We investigated the partitioning of three EGI variants containing various C-terminal peptide extensions including Trp-Pro motifs of different lengths and localizations. Additionally, a recently developed system composed of the thermoseparating copolymer HM-EOPO was utilized to study the effects of fusion tags. The addition of peptides containing tryptohan residues enhanced the partitioning of EGI to the HM-EOPO-rich phase. The system composed of a nonionic detergent (Agrimul NRE1205) resulted in the highest partition coefficient (K = 31) and yield (90%) with the construct EGI(core-P5)(WP)(4) containing (Trp-Pro)(4) after a short linker stretch. A recombinant strain of T. reesei Rut-C30 for large-scale production was constructed in which the fusion protein EGI(core-P5)(WP)(4) was expressed from the strong promoter of the cellulase gene cbh1. The fusion protein was successfully expressed and secreted from the fungus during shake-flask cultivations. Cultivation in a 28-L bioreactor however, revealed that the fusion protein is sensitive to proteases. Consequently, only low production levels were obtained in large-scale production trials.
|
|
| 10. |
|
|
