| 1. |
- Munthe, Christian, 1962
(författare)
-
Precaution and Ethics: Handling risks, uncertainties and knowledge gaps in the regulation of new biotechnologies
- 2017
-
Bok (övrigt vetenskapligt/konstnärligt)abstract
- This volume outlines and analyses ethical issues actualized by applying a precautionary approach to the regulation of new biotechnologies. It presents a novel way of categorizing and comparing biotechnologies from a precautionary standpoint. Based on this, it addresses underlying philosophical problems regarding the ethical assessment of decision-making under uncertainty and ignorance, and discusses how risks and possible benefits of such technologies should be balanced from an ethical standpoint. It argues on conceptual and ethical grounds for a technology neutral regulation as well as for a regulation that not only checks new technologies but also requires old, inferior ones to be phased out. It demonstrates how difficult ethical issues regarding the extent and ambition of precautionary policies need to be handled by such a regulation, and presents an overarching framework for doing so.
|
|
| 2. |
- Mayers, Joshua, 1988, et al.
(författare)
-
Integrating Microalgal Production with Industrial Outputs - Reducing Process Inputs and Quantifying the Benefits
- 2016
-
Ingår i: Industrial Biotechnology. - : Mary Ann Liebert Inc. - 1550-9087 .- 1931-8421. ; 12:4, s. 219-234
-
Tidskriftsartikel (refereegranskat)abstract
- The cultivation and processing of microalgal biomass is resource- and energy-intensive, negatively affecting the sustainability and profitability of producing bulk commodities, limiting this platform to the manufacture of relatively small quantities of high-value compounds. A biorefinery approach where all fractions of the biomass are valorized might improve the case for producing lower-value products. However, these systems are still likely to operate very close to thresholds of profitability and energy balance, with wide-ranging environmental and societal impacts. It thus remains critically important to reduce the use of costly and impactful inputs and energy-intensive processes involved in these scenarios. Integration with industrial infrastructure can provide a number of residual streams that can be readily used during microalgal cultivation and downstream processing. This review critically considers some of the main inputs required for microalgal biorefineries - such as nutrients, water, carbon dioxide, and heat - and appraises the benefits and possibilities for industrial integration on a more quantitative basis. Recent literature and demonstration studies will also be considered to best illustrate these benefits to both producers and industrial operators. Additionally, this review will highlight some inconsistencies in the data used in assessments of microalgal production scenarios, allowing more accurate evaluation of potential future biorefineries.
|
|
| 3. |
- Skoog, Emma, 1983, et al.
(författare)
-
Biobased adipic acid – The challenge of developing the production host
- 2018
-
Ingår i: Biotechnology Advances. - : Elsevier BV. - 0734-9750. ; 36:8, s. 2248-2263
-
Forskningsöversikt (refereegranskat)abstract
- Adipic acid is a platform chemical, and is the most important commercial dicarboxylic acid. It has been targeted for biochemical conversion as an alternative to present chemical production routes. From the perspective of bioeconomy, several kinds of raw material are of interest including the sugar platform (derived from starch, cellulose or hemicellulose), the lignin platform (aromatics) and the fatty acid platform (lipid derived). Two main biochemical-based production schemes may be employed: (i) direct fermentation to adipic acid, or (ii) fermentation to muconic or glucaric acid, followed by chemical hydrogenation (indirect fermentation). This review presents a comprehensive description of the metabolic pathways that could be constructed and analyzes their respective theoretical yields and metabolic constraints. The experimental yields and titers obtained so far are low, with the exception of processes based on palm oil and glycerol, which have been reported to yield up to 50 g and 68 g adipic acid/L, respectively. The challenges that remain to be addressed in order to achieve industrially relevant production levels include solving redox constraints, and identifying and/or engineering enzymes for parts of the metabolic pathways that have yet to be metabolically demonstrated. This review provides new insights into ways in which metabolic pathways can be constructed to achieve efficient adipic acid production. The production host provides the chassis to be engineered via an appropriate metabolic pathway, and should also have properties suitable for the industrial production of adipic acid. An acidic process pH is attractive to reduce the cost of downstream processing. The production host should exhibit high tolerance to complex raw material streams and high adipic acid concentrations at acidic pH.
|
|
| 4. |
- Andersson, Viktor, 1983
(författare)
-
Excess heat utilisation in oil refineries - CCS and algae-based biofuels
- 2016
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The main objective of this thesis is to investigate two different concepts for CO2 mitigation, from a system perspective, in relation to the oil refining industry: CO2 capture and storage; and algae-based biofuels. For all these processes, process integration with an oil refinery is assumed. The oil refinery sector is a major emitter of CO2 and is responsible for 9% of the industrial emissions of CO2 worldwide. Oil refineries have large amounts of unused excess heat, which can be used to satisfy the heat demands of a CO2 capture plant, a land-based algal cultivation facility, or an algae-based biofuel process. The use of this excess heat significantly reduces the cost for CO2 capture, while an economic evaluation for algae-based biofuels has not been made.Since the amount of heat available from the oil refinery´s processes increase with decreasing temperature in the stripper reboiler, it was investigated how much heat was available at different temperatures. It was also investigated how the decreased temperature would affect the heat demand of CO2 capture processes that use MEA or ammonia as the absorbent. The findings show that it is possible to capture more CO2 using excess heat when the temperature in the stripper reboiler is decreased. For the MEA process, the lower limit of the temperature interval investigated showed the maximum CO2 capture rate, while the ammonia process benefitted from a lower temperature than the standard temperature but showed maximal CO2 capture rate above the lower limit. These results are valid only when using excess heat to satisfy the entire heat demand. At the case study refinery, the available excess heat could satisfy between 28% and 50% of the heat demand of the MEA process when treating the flue gases from all chimneys, depending on the temperature in the stripper reboiler. This utilisation of excess heat represents a way to reduce significantly the costs for CCS in an oil refinery. Land-based cultivation of algae proved to be unsuitable for the utilisation of excess heat. Since the cultivation pond is exposed to wind, rain, and cold, the heat demand fluctuates strongly over the year, making the pond an unstable recipient of the excess heat.Three types of biofuel processes based on microalgae and macroalgae were investigated with respect to integration with the oil refinery. For the algae-based biofuel processes, heat integration and material integration combined to increase the efficiency of the system. When two different build margin technologies (with different CO2 emission factors) are employed for electricity production, macroalgae-based biofuel production appears to be the more robust process from the perspective of CO2 due to the lower electricity demands of the algal cultivation and harvesting phases.
|
|
| 5. |
- Jansson, Ronnie, et al.
(författare)
-
Functionalized silk assembled from a recombinant spider silk fusion protein (Z-4RepCT) produced in the methylotrophic yeast Pichia pastoris
- 2016
-
Ingår i: Biotechnology Journal. - : Wiley-VCH Verlagsgesellschaft. - 1860-6768 .- 1860-7314. ; 11:5, s. 687-699
-
Tidskriftsartikel (refereegranskat)abstract
- Functional biological materials are a growing research area with potential applicability in medicine and biotechnology. Using genetic engineering, the possibility to introduce additional functions into spider silk-based materials has been realized. Recently, a recombinant spider silk fusion protein, Z-4RepCT, was produced intracellularly in Escherichia coli and could after purification self-assemble into silk-like fibers with ability to bind antibodies via the IgG-binding Z domain. In this study, the use of the methylotrophic yeast Pichia pastoris for production of Z-4RepCT has been investigated. Temperature, pH and production time were influencing the amount of soluble Z-4RepCT retrieved from the extracellular fraction. Purification of secreted Z-4RepCT resulted in a mixture of full-length and degraded silk proteins that failed to self-assemble into fibers. A position in the C-terminal domain of 4RepCT was identified as being subjected to proteolytic cleavage by proteases in the Pichia culture supernatant. Moreover, the C-terminal domain was subjected to glycosylation during production in P. pastoris. These observed alterations of the CT domain are suggested to contribute to the failure in fiber assembly. As alternative approach, Z-4RepCT retrieved from the intracellular fraction, which was less degraded, was used and shown to retain ability to assemble into silk-like fibers after enzymatic deglycosylation.
|
|
| 6. |
- Bergman, Alexandra Linda, 1985, et al.
(författare)
-
Heterologous phosphoketolase expression redirects flux towards acetate, perturbs sugar phosphate pools and increases respiratory demand in Saccharomyces cerevisiae
- 2019
-
Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 18:1
-
Tidskriftsartikel (refereegranskat)abstract
- Introduction: Phosphoketolases (Xfpk) are a non-native group of enzymes in yeast, which can be expressed in combination with other metabolic enzymes to positively influence the yield of acetyl-CoA derived products by reducing carbon losses in the form of CO2. In this study, a yeast strain expressing Xfpk from Bifidobacterium breve, which was previously found to have a growth defect and to increase acetate production, was characterized. Results: Xfpk-expression was found to increase respiration and reduce biomass yield during glucose consumption in batch and chemostat cultivations. By cultivating yeast with or without Xfpk in bioreactors at different pHs, we show that certain aspects of the negative growth effects coupled with Xfpk-expression are likely to be explained by proton decoupling. At low pH, this manifests as a reduction in biomass yield and growth rate in the ethanol phase. Secondly, we show that intracellular sugar phosphate pools are significantly altered in the Xfpk-expressing strain. In particular a decrease of the substrates xylulose-5-phosphate and fructose-6-phosphate was detected (26% and 74% of control levels) together with an increase of the products glyceraldehyde-3-phosphate and erythrose-4-phosphate (208% and 542% of control levels), clearly verifying in vivo Xfpk enzymatic activity. Lastly, RNAseq analysis shows that Xfpk expression increases transcription of genes related to the glyoxylate cycle, the TCA cycle and respiration, while expression of genes related to ethanol and acetate formation is reduced. The physiological and transcriptional changes clearly demonstrate that a heterologous phosphoketolase flux in combination with endogenous hydrolysis of acetyl-phosphate to acetate increases the cellular demand for acetate assimilation and respiratory ATP-generation, leading to carbon losses. Conclusion: Our study shows that expression of Xfpk in yeast diverts a relatively small part of its glycolytic flux towards acetate formation, which has a significant impact on intracellular sugar phosphate levels and on cell energetics. The elevated acetate flux increases the ATP-requirement for ion homeostasis and need for respiratory assimilation, which leads to an increased production of CO2. A majority of the negative growth effects coupled to Xfpk expression could likely be counteracted by preventing acetate accumulation via direct channeling of acetyl-phosphate towards acetyl-CoA.
|
|
| 7. |
- Westman, Johan, 1983, et al.
(författare)
-
Current progress in high cell density yeast bioprocesses for bioethanol production
- 2015
-
Ingår i: Biotechnology journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 10:8, s. 1185-1195
-
Forskningsöversikt (refereegranskat)abstract
- High capital costs and low reaction rates are major challenges for establishment of fermentation-based production systems in the bioeconomy. Using high cell density cultures is an efficient way to increase the volumetric productivity of fermentation processes, thereby enabling faster and more robust processes and use of smaller reactors. In this review, we summarize recent progress in the application of high cell density yeast bioprocesses for first and second generation bioethanol production. High biomass concentrations obtained by retention of yeast cells in the reactor enables easier cell reuse, simplified product recovery and higher dilution rates in continuous processes. High local cell density cultures, in the form of encapsulated or strongly flocculating yeast, furthermore obtain increased tolerance to convertible fermentation inhibitors and utilize glucose and other sugars simultaneously, thereby overcoming two additional hurdles for second generation bioethanol production. These effects are caused by local concentration gradients due to diffusion limitations and conversion of inhibitors and sugars by the cells, which lead to low local concentrations of inhibitors and glucose. Quorum sensing may also contribute to the increased stress tolerance. Recent developments indicate that high cell density methodology, with emphasis on high local cell density, offers significant advantages for sustainable second generation bioethanol production.
|
|
| 8. |
- Zambrano, Jesús, et al.
(författare)
-
Optimal steady-state design of zone volumes of bioreactors with Monod growth kinetics
- 2015
-
Ingår i: Biochemical Engineering Journal. - : Elsevier BV. - 1369-703X .- 1873-295X. ; 100, s. 59-66
-
Tidskriftsartikel (refereegranskat)abstract
- This paper deals with steady-state analysis and design of bioreactors consisting of a number of completely stirred tank reactors (CSTRs) in series. The study is confined to one consumed (substrate) and one consuming constituent (biomass). The specific microbial growth rate is assumed to be described by Monod kinetics. The death of biomass is assumed to be negligible. Two optimal design problems for a large number of CSTRs in series are studied: to minimize the effluent substrate concentration for a given total volume, and to minimize the total volume for a given effluent substrate concentration. As an appealing alternative to solve these problems numerically, it is proposed to consider the asymptotic case where the number of CSTRs tends to infinity. This is shown to correspond to one CSTR in series with a plug flow reactor (PFR). A CSTR with a sufficient large volume is needed to avoid wash-out of the biomass. The main result is that both design problems for the CSTR + PFR configuration have the same solution with respect to the optimal volume of the CSTR, which is given as an explicit function of the incoming substrate concentration, the volumetric flow rate and the coefficients of the Monod growth rate function. Numerical results indicate that the plug flow approach may be used as a feasible design procedure even for a reasonably low number of CSTRs in series.
|
|
| 9. |
- Franzén, Carl Johan, 1966, et al.
(författare)
-
Multifeed simultaneous saccharification and fermentation enables high gravity submerged fermentation of lignocellulose.
- 2015
-
Ingår i: Recent Advances in Fermentation Technology (RAFT 11), Clearwater Beach, Florida, USA, November 8-11, 2015. Oral presentation..
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Today, second generation bioethanol production is becoming established in production plants across the world. In addition to its intrinsic value, the process can be viewed as a model process for biotechnological conversion of recalcitrant lignocellulosic raw materials to a range of chemicals and other products. So called High Gravity operation, i.e. fermentation at high solids loadings, represents continued development of the process towards higher product concentrations and productivities, and improved energy and water economy. We have employed a systematic, model-driven approach to the design of feeding schemes of solid substrate, active yeast adapted to the actual substrate, and enzymes to fed-batch simultaneous saccharification and co-fermentation (Multifeed SSCF) of steam-pretreated lignocellulosic materials in stirred tank reactors. With this approach, mixing problems were avoided even at water insoluble solids contents of 22%, leading to ethanol concentrations of 56 g/L within 72 hours of SSCF on wheat straw. Similar fermentation performance was verified in 10 m3 demonstration scale using wheat straw, and in lab scale on birch and spruce, using several yeast strains. The yeast was propagated in the liquid fraction obtained by press filtration of the pretreated slurry. Yet, even with such preadaptation and repeated addition of fresh cells, the viability in the SSCF dropped due to interactions between lignocellulose-derived inhibitors, the produced ethanol and the temperature. Decreasing the temperature from 35 to 30°C when the ethanol concentration reached 40-50 g/L resulted in rapid initial hydrolysis, maintained fermentation capacity, lower residual glucose and xylose and ethanol concentrations above 60 g/L.
|
|
| 10. |
- Nickel, David, 1990, et al.
(författare)
-
Uncertainty analysis as a tool to consistently evaluate lignocellulosic bioethanol processes at different system scales
- 2018
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Lignocellulosic processes are highly prone to batch-to batch variability, e.g. of raw materials and enzyme activities. This variability can be propagated throughout system scales during process development and optimization, influencing the outputs of bioreaction models, techno-economic analyses and life cycle assessments. As these outputs are the main decision variables for designing and developing lignocellulose-based processes, tools are required to evaluate the influences of process variation at different system scales. Uncertainty analysis quantifies the effects of model input variations on model outputs. It is an effective tool to consistently propagate process variation throughout scales and analyse its influence on model outputs. As an example, we use a model describing multi-feed simultaneous saccharification and co-fermentation (SSCF) of wheat straw. During the process enzymes hydrolyse the lignocellulosic material to release glucose which can be converted by microorganisms into ethanol. To investigate the impact of batch-to-batch variability in enzyme cocktails, we collected literature data on the enzymatic activity of Cellic CTec2. Retrieved data were propagated in models at bioreactor, techno-economic analysis and life cycle assessment scale. We show how uncertainty analysis can be used to guide process development by comparing different modes of operation. The method can identify economically feasible process ranges with low environmental impact while increasing the robustness of bioprocesses with high variation in raw material inputs. Furthermore, uncertainty analysis could help to identify relevant parameters to choose as response variables in experimental designs.
|
|
| 11. |
- Wang, Ruifei, 1985, et al.
(författare)
-
Model-based optimization and scale-up of multi-feed simultaneous saccharification and co-fermentation of steam pre-treated lignocellulose enables high gravity ethanol production.
- 2016
-
Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 9:1, s. 88-
-
Tidskriftsartikel (refereegranskat)abstract
- High content of water-insoluble solids (WIS) is required for simultaneous saccharification and co-fermentation (SSCF) operations to reach the high ethanol concentrations that meet the techno-economic requirements of industrial-scale production. The fundamental challenges of such processes are related to the high viscosity and inhibitor contents of the medium. Poor mass transfer and inhibition of the yeast lead to decreased ethanol yield, titre and productivity. In the present work, high-solid SSCF of pre-treated wheat straw was carried out by multi-feed SSCF which is a fed-batch process with additions of substrate, enzymes and cells, integrated with yeast propagation and adaptation on the pre-treatment liquor. The combined feeding strategies were systematically compared and optimized using experiments and simulations.
|
|
| 12. |
- Nickel, David, 1990, et al.
(författare)
-
Multi-scale uncertainty analysis – A tool to systematically consider variability in lignocellulosic bioethanol processes
- 2018
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Bioethanol production processes from lignocellulosic raw materials are highly prone to batch-to-batch variations. For example, raw material compositions and enzymatic activities required to release fermentable sugars from lignocellulose vary significantly between batches. To develop lignocellulosic biofuel processes and evaluate their performance regarding economics and sustainability consistently, tools are required to cope with this variability. In this presentation we will propose a multi-scale uncertainty analysis strategy to propagate input variability throughout system scales. In a first step, we use meta-data obtained from literature to define uncertainties in the process inputs. Utilizing these meta-data, uncertainty analysis is performed on a macro-kinetic model by sampling from the defined uncertain input space. The results of this uncertainty analysis are transferred to process simulations to analyze the impact of input uncertainties on the process mass- and energy balances, and on the economics of building this type of bioprocess. The generated data from process simulations (mass flows, energy integration, and economic data) are in the next step extracted and used as input to an environmental impact assessment of the process. This is done whilst keeping the simulation and systems modeling parameters constant, thus the input variability is propagated throughout the different system scales. The data generated in this integrated approach will then be compared with the variations and uncertainties observed with relevance to the estimated parameters in the process simulation and environmental impact assessment. Based on this consistent strategy, we can analyze the impact of input variability from different system perspectives, identify important bottlenecks for development, and suggest robust and sustainable process designs for different conditions and under given uncertainties. In a case study we demonstrate how integrated kinetic modeling (in Matlab), process simulation (in SuperPro Designer), and environmental impact assessment together with statistical analysis can be used for assessing how variability in enzymatic activities in bioethanol production can be propagated throughout system scales. A macro-kinetic model is used to describe the enzymatic breakdown of lignocellulose-derived polysaccharides into fermentable sugars (saccharification) and the simultaneous fermentation to bioethanol. We discuss the integration of the simulation results of the macro-kinetic model into the flowsheeting software for mass and energy balance generation, and then further on to assess environmental impacts of the process. We will evaluate different process designs regarding their robustness towards input variability. Finally, we also show how propagated uncertainties at different system scales can be integrated to design experiments at laboratory scale so that these focus on the most important parameters for developing robust kinetic models, and include the parameters that are most important for sustainable design of processes and value chains.
|
|
| 13. |
- Skvaril, Jan, 1982-, et al.
(författare)
-
Applications of near-infrared spectroscopy (NIRS) in biomass energy conversion processes : A review
- 2017
-
Ingår i: Applied spectroscopy reviews (Softcover ed.). - : Informa UK Limited. - 0570-4928 .- 1520-569X. ; 52:8, s. 675-728
-
Forskningsöversikt (refereegranskat)abstract
- Biomass used in energy conversion processes is typically characterized by high variability, making its utilization challenging. Therefore, there is a need for a fast and non-destructive method to determine feedstock/product properties and directly monitor process reactors. The near-infrared spectroscopy (NIRS) technique together with advanced data analysis methods offers a possible solution. This review focuses on the introduction of the NIRS method and its recent applications to physical, thermochemical, biochemical and physiochemical biomass conversion processes represented mainly by pelleting, combustion, gasification, pyrolysis, as well as biogas, bioethanol, and biodiesel production. NIRS has been proven to be a reliable and inexpensive method with a great potential for use in process optimization, advanced control, or product quality assurance.
|
|
| 14. |
- Brechmann, Nils Arnold, et al.
(författare)
-
Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture
- 2019
-
Ingår i: Biotechnology progress (Print). - : AIChE. - 8756-7938 .- 1520-6033.
-
Tidskriftsartikel (refereegranskat)abstract
- High capacity magnetic protein A agarose beads, LOABeads PrtA, were used in the developmentof a new process for affinity purification of monoclonal antibodies (mAbs) from non-clarifiedCHO cell broth using a pilot-scale magnetic separator. The LOABeads had a maximum bindingcapacity of 65 mg/mL and an adsorption capacity of 25–42 mg IgG/mL bead in suspension for anIgG concentration of 1 to 8 g/L. Pilot-scale separation was initially tested in a mAb capture stepfrom 26 L clarified harvest. Small-scale experiments showed that similar mAb adsorptions wereobtained in cell broth containing 40 Å~ 106 cells/mL as in clarified supernatant. Two pilot-scalepurification runs were then performed on non-clarified cell broth from fed-batch runs of 16 L,where a rapid mAb adsorption ≥96.6% was observed after 1 h. This process using 1 L of magnetic beads had an overall mAb yield of 86% and 16 times concentration factor. After this single proteinA capture step, the mAb purity was similar to the one obtained by column chromatography, whilethe host cell protein content was very low, <10 ppm. Our results showed that this magnetic beadmAb purification process, using a dedicated pilot-scale separation device, was a highly efficientsingle step, which directly connected the culture to the downstream process without cell clarification.Purification of mAb directly from non-clarified cell broth without cell separation can providesignificant savings in terms of resources, operation time, and equipment, compared to legacy procedure of cell separation followed by column chromatography step.
|
|
| 15. |
- Martinez Avila, Hector, 1985
(författare)
-
Biofabrication, Biomechanics and Biocompatibility of Nanocellulose-based Scaffolds for Auricular Cartilage Regeneration
- 2015
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In about 2:10,000 births the external part of the ear, the auricle, is severely malformed or absent. Furthermore, tumors and trauma can cause defects to the auricle. For patients with dysplasia of the auricle, and especially for children, an inconspicuous outer appearance with life-like auricles is important for their psychological and emotional well being as well as their psycho-social development. Auricular reconstruction remains a great challenge due to the complexity of surgical reconstruction using rib cartilage. Despite the advances in stem cell technology and biomaterials, auricular cartilage tissue engineering (TE) is still in an early stage of development due to critical requirements demanding appropriate mechanical properties and shape stability of the tissue-engineered construct. This thesis has focused on developing patient-specific tissue-engineered auricles for one-step surgery using a novel biomaterial, bacterial nanocellulose (BNC), seeded with human nasoseptal chondrocytes (hNC) and bone marrow mononuclear cells (MNC).Biomechanical properties of human auricle cartilage were measured and used as a benchmark for tuning BNC properties. In order to meet the biomechanical requirements, a scaffold with bilayer architecture composed of a dense BNC support layer and a macroporous structure was designed. Firstly, the biocompatibility of the dense BNC layer was investigated, demonstrating a minimal foreign body response according to standards set forth in ISO 10993. Secondly, different methods to create macroporous BNC scaffolds were studied and the redifferentiation capacity of hNCs was evaluated in vitro; revealing that macroporous BNC scaffolds support cell ingrowth, proliferation and neocartilage formation. The bilayer BNC scaffold was biofabricated and tested for endotoxins and cytotoxicity before evaluating in long-term 3D culture, and subsequently in vivo for eight weeks—in an immunocompromised animal model. The results demonstrated that the non-pyrogenic and non- cytotoxic bilayer BNC scaffold offers a good mechanical stability and maintains a structural integrity, while providing a porous 3D environment that is suitable for hNCs and MNCs to produce neocartilage, in vitro and in vivo. Furthermore, patient-specific auricular BNC scaffolds with bilayer architecture were biofabricated and seeded with autologous rabbit auricular chondrocytes (rAC) for implantation in an immunocompetent rabbit model for six weeks. The results demonstrated the shape stability of the rAC-seeded scaffolds and neocartilage depositions in the immunocompetent autologous grafts. 3D bioprinting was also evaluated for biofabrication of patient-specific, chondrocyte-laden auricular constructs using a bioink composed of nanofibrillated cellulose and alginate. Bioprinted auricular constructs showed an excellent shape and size stability after in vitro culture. Moreover, this bioink supports redifferentiation of hNCs while offering excellent printability, making this a promising approach for auricular cartilage TE. Furthermore, the use of bioreactors is essential for the development of tissue-engineered cartilage in vitro. Thus, a compression bioreactor was utilized to apply dynamic mechanical stimulation to cell-seeded constructs as a means to enhance production of extracellular matrix in vitro.In this work, a potential clinical therapy for auricular reconstruction using tissue-engineered auricles is demonstrated; where BNC is proposed as a promising non-degradable biomaterial with good chemical and mechanical stability for auricular cartilage TE. Although the primary focus of this thesis is on auricular reconstruction, the methods developed are also applicable in the regeneration of other cartilage tissues such as those found in the nose, trachea, spine and articular joints.
|
|
| 16. |
- Huang, Mingtao, 1984, et al.
(författare)
-
Engineering the protein secretory pathway of Saccharomyces cerevisiae enables improved protein production
- 2018
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 115:47, s. E11025-E11032
-
Tidskriftsartikel (refereegranskat)abstract
- Baker’s yeast Saccharomyces cerevisiae is one of the most important and widely used cell factories for recombinant protein production. Many strategies have been applied to engineer this yeast for improving its protein production capacity, but productivity is still relatively low, and with increasing market demand, it is important to identify new gene targets, especially targets that have synergistic effects with previously identified targets. Despite improved protein production, previous studies rarely focused on processes associated with intracellular protein retention. Here we identified genetic modifications involved in the secretory and trafficking pathways, the histone deacetylase complex, and carbohydrate metabolic processes as targets for improving protein secretion in yeast. Especially modifications on the endosome-to-Golgi trafficking was found to effectively reduce protein retention besides increasing protein secretion. Through combinatorial genetic manipulations of several of the newly identified gene targets, we enhanced the protein production capacity of yeast by more than fivefold, and the best engineered strains could produce 2.5 g/L of a fungal α-amylase with less than 10% of the recombinant protein retained within the cells, using fed-batch cultivation.
|
|
| 17. |
- Janssen, Mathias, 1973, et al.
(författare)
-
Life cycle impacts of ethanol production from spruce wood chips under high gravity conditions
- 2016
-
Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 9:1, s. 53-
-
Tidskriftsartikel (refereegranskat)abstract
- BackgroundDevelopment of more sustainable biofuel production processes is ongoing, and technology to run these processes at a high dry matter content, also called high-gravity conditions, is one option. This paper presents the results of a life cycle assessment (LCA) of such a technology currently in development for the production of bio-ethanol from spruce wood chips.ResultsThe cradle-to-gate LCA used lab results from a set of 30 experiments (or process configurations) in which the main process variable was the detoxification strategy applied to the pretreated feedstock material. The results of the assessment show that a process configuration, in which washing of the pretreated slurry is the detoxification strategy, leads to the lowest environmental impact of the process. Enzyme production and use are the main contributors to the environmental impact in all process configurations, and strategies to significantly reduce this contribution are enzyme recycling and on-site enzyme production. Furthermore, a strong linear correlation between the ethanol yield of a configuration and its environmental impact is demonstrated, and the selected environmental impacts show a very strong cross-correlation (r^2 > 0.9 in all cases) which may be used to reduce the number of impact categories considered from four to one (in this case, global warming potential). Lastly, a comparison with results of an LCA of ethanol production under high-gravity conditions using wheat straw shows that the environmental performance does not significantly differ when using spruce wood chips. For this comparison, it is shown that eutrophication potential also needs to be considered due to the fertilizer use in wheat cultivation.ConclusionsThe LCA points out the environmental hotspots in the ethanol production process, and thus provides input to the further development of the high-gravity technology. Reducing the number of impact categories based only on cross-correlations should be done with caution. Knowledge of the analyzed system provides further input to the choice of impact categories.
|
|
| 18. |
- Jers, C., et al.
(författare)
-
Production of 3-hydroxypropanoic acid from glycerol by metabolically engineered bacteria
- 2019
-
Ingår i: Frontiers in Bioengineering and Biotechnology. - : Frontiers Media SA. - 2296-4185. ; 7:MAY
-
Forskningsöversikt (refereegranskat)abstract
- 3-hydroxypropanoic acid (3-HP) is a valuable platform chemical with a high demand in the global market. 3-HP can be produced from various renewable resources. It is used as a precursor in industrial production of a number of chemicals, such as acrylic acid and its many derivatives. In its polymerized form, 3-HP can be used in bioplastic production. Several microbes naturally possess the biosynthetic pathways for production of 3-HP, and a number of these pathways have been introduced in some widely used cell factories, such as Escherichia coli and Saccharomyces cerevisiae. Latest advances in the field of metabolic engineering and synthetic biology have led to more efficient methods for bio-production of 3-HP. These include new approaches for introducing heterologous pathways, precise control of gene expression, rational enzyme engineering, redirecting the carbon flux based on in silico predictions using genome scale metabolic models, as well as optimizing fermentation conditions. Despite the fact that the production of 3-HP has been extensively explored in established industrially relevant cell factories, the current production processes have not yet reached the levels required for industrial exploitation. In this review, we explore the state of the art in 3-HP bio-production, comparing the yields and titers achieved in different microbial cell factories and we discuss possible methodologies that could make the final step toward industrially relevant cell factories.
|
|
| 19. |
- Tang, Hongting, et al.
(författare)
-
Efficient yeast surface-display of novel complex synthetic cellulosomes
- 2018
-
Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 17:1
-
Tidskriftsartikel (refereegranskat)abstract
- Background: The self-assembly of cellulosomes on the surface of yeast is a promising strategy for consolidated bioprocessing to convert cellulose into ethanol in one step. Results: In this study, we developed a novel synthetic cellulosome that anchors to the endogenous yeast cell wall protein a-agglutinin through disulfide bonds. A synthetic scaffoldin ScafAGA3 was constructed using the repeated N-terminus of Aga1p and displayed on the yeast cell surface. Secreted cellulases were then fused with Aga2p to assemble the cellulosome. The display efficiency of the synthetic scaffoldin and the assembly efficiency of each enzyme were much higher than those of the most frequently constructed cellulosome using scaffoldin ScafCipA3 from Clostridium thermocellum. A complex cellulosome with two scaffoldins was also constructed using interactions between the displayed anchoring scaffoldin ScafAGA3 and scaffoldin I ScafCipA3 through disulfide bonds, and the assembly of secreted cellulases to ScafCipA3. The newly designed cellulosomes enabled yeast to directly ferment cellulose into ethanol. Conclusions: This is the first report on the development of complex multiple-component assembly system through disulfide bonds. This strategy could facilitate the construction of yeast cell factories to express synergistic enzymes for use in biotechnology.
|
|
| 20. |
- Wang, Ruifei, 1985
(författare)
-
Bioprocess development for biochemical conversion of lignocellulose
- 2017
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Due to its low environmental impact and high maturity of the fuel ethanol market, lignocellulosic ethanol is a promising option for reducing the carbon footprint in the transport sector. The characteristics of lignocellulosic feedstocks, such as varied sugar composition, low sugar density, low solubility, recalcitrance to enzymatic degradation, and inhibitors formed during thermochemical pretreatment, have so far limited the production process, and costs for conversion of lignocellulosic materials to ethanol are still high. In this thesis, I describe the development of a bioconversion process that pushes the limits of simultaneous saccharification and co-fermentation (SSCF) to achieve higher ethanol titre, yield and productivity on lignocellulosic feedstocks. I propose an integrated fed-batch strategy, Multi-Feed SSCF, including feeds of substrates, enzymes and adapted cells to tackle the technical challenges in operating a SSCF process at high substrate loadings. Using insights from experiments and a model-based feeding design, lignocellulose saccharification and fermentation at water insoluble solids (WIS) levels greater than 20% (w/w) was achieved. The multi-feed SSCF concept and model-aided substrate feeding design allowed rapid, reproducible, and scalable bioconversion of lignocellulose, as proven on several lignocellulosic feedstocks in both laboratory and demonstration scales. Ethanol production above 50 g/L in SSCF processes was found to be severely inhibited by the combined effects of ethanol, lignocellulose-derived inhibitors, and higher than standard cultivation temperature (35°C). Cell viability and fermentation improved significantly in a multi-feed SSCF process with a step change in temperature from 35 to 30°C, compared to operation at 35°C throughout. However, introducing the Erg3Tyr185 point mutation which has been reported to render thermotolerance in yeast, did not offer any significant improvement. Cell concentrations were determined by counting in a hemocytometer and colony forming unit assay. Their accuracy and reproducibility in lignocellulosic media, were verified by Design-of-Experiment-based calibration. Applic-ability of real time qPCR and dielectric spectroscopy as potential cell quantification methods was also investigated. With multi-feed of solid substrates, enzyme preparations, and adapted cells, the SSCF process produced > 60 g/L ethanol within 120 h, equivalent to 70% of the theoretical yield of the total sugar input, and 90% of the consumed sugar. The systematic optimisation reported in this work represents a robust and reproducible routine for developing lignocellulose-based processes. It could inspire continuous development of alternative strategies to current fossil-based chemical/fuel processes.
|
|
| 21. |
- Aulitto, Martina, 1991, et al.
(författare)
-
Seed culture pre-adaptation of Bacillus coagulans MA-13 improves lactic acid production in simultaneous saccharification and fermentation
- 2019
-
Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 12:1
-
Tidskriftsartikel (refereegranskat)abstract
- Background Lignocellulosic biomass is an abundant and sustainable feedstock, which represents a promising raw material for the production of lactic acid via microbial fermentation. However, toxic compounds that affect microbial growth and metabolism are released from the biomass upon thermochemical pre-treatment. So far, susceptibility of bacterial strains to biomass-derived inhibitors still represents a major barrier to lactic acid production from lignocellulose. Detoxification of the pre-treated lignocellulosic material by water washing is commonly performed to alleviate growth inhibition of the production microorganism and achieve higher production rates. Results In this study, we assessed the feasibility of replacing the washing step with integrated cellular adaptation during pre-culture of Bacillus coagulans MA-13 prior to simultaneous saccharification and lactic acid fermentation of steam exploded wheat straw. Using a seed culture pre-exposed to 30% hydrolysate led to 50% shorter process time, 50% higher average volumetric and 115% higher average specific productivity than when using cells from a hydrolysate-free seed culture. Conclusions Pre-exposure of B. coagulans MA-13 to hydrolysate supports adaptation to the actual production medium. This strategy leads to lower process water requirements and combines cost-effective seed cultivation with physiological pre-adaptation of the production strain, resulting in reduced lactic acid production costs.
|
|
| 22. |
|
|
| 23. |
- Antonopoulou, Io, 1989-
(författare)
-
Development of biocatalytic processes for selective antioxidant production
- 2018
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Feruloyl esterases (FAEs, EC 3.1.1.73) represent a subclass of carboxylic acid esterases that under normal conditions catalyze the hydrolysis of the ester bond between hydroxycinnamic acids (ferulic acid, sinapic acid, caffeic acid, p-coumaric acid) and sugar residues in plant cell walls. Based on their specificity towards monoferulates and diferulates, substitutions on the phenolic ring and on their amino acid sequence identity, they have been classified into four types (A-D) while phylogenetic analysis has resulted in classification into thirteen subfamilies (SF1-13). Under low water content, these enzymes are able to catalyze the esterification of hydroxycinnamic acids or the transesterification of their esters (donor) with alcohols or sugars (acceptor) resulting in compounds with modified lipophilicity, having a great potential for use in the tailor-made modification of natural antioxidants for cosmetic, cosmeceutical and pharmaceutical industries. The work described in this thesis focused on the selection,characterization and application of FAEs for the synthesis of bioactive esters with antioxidant activity in non-conventional media. The basis of the current classification systems was investigated in relation with the enzymes’ synthetic and hydrolytic abilities while the developed processes were evaluated for their efficiency and sustainability.Paper I was dedicated to the screening and evaluation of the synthetic abilities of 28 fungal FAEs using acceptors of different lipophilicity at fixed conditions in detergentless microemulsions. It was revealed that FAEs classified in phylogenetic subfamilies related to acetyl xylan esterases (SF5 and 6) showed increased transesterification rates and selectivity. In general, FAEs showed preference on more hydrophilic alcohol acceptors and in descending order to glycerol > 1-butanol > prenol. Homology modeling and small molecule docking simulations were employed as tools for the identification of a potential relationship between the predicted surface and active site properties of selected FAEs and the transesterification selectivity.Papers II- IV focused on the characterization of eight promising FAEs and the optimization of reaction conditions for the synthesis of two bioactive esters (prenyl ferulate and L-arabinose ferulate) in detergentless microemulsions. The effect of the medium composition, the donor and acceptor concentration, the enzyme load, the pH, the temperature and the agitation on the transesterification yield and selectivity were investigated. It was observed that the acceptor concentration and enzyme load were crucial parameters for selectivity. Fae125 (Type A, SF5) iiexhibited highest prenyl ferulate yield (81.1%) and selectivity (4.685) converting 98.5% of VFA to products after optimization at 60 mM VFA, 1.5 M prenol, 0.04 mg FAE mL-1, 40oC, 24 h, 53.4:43.4:3.2 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0. On the other hand, FaeA1 (Type A, SF5) showed highest L-arabinose ferulate yield (52.2 %) and selectivity (1.120) at 80 mM VFA, 55 mM L-arabinose, 0.02 mg FAE mL-1, 50oC, 8 h, 19.8: 74.7: 5.5 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0.In paper V, the effect of reaction media on the enzyme stability and transesterification yield and selectivity was studied in different solvents for the synthesis of two bioactive esters: prenyl ferulate and L-arabinose ferulate. The best performing enzyme (Fae125) was used in the optimization of reaction conditions in the best solvent (n-hexane) via response surface methodology. Both bioconversions were best described by a two-factor interaction model while optimal conditions were determined as the ones resulting in highest yield and selectivity.Highest prenyl ferulate yield (87.5%) and selectivity (7.616) were observed at 18.56 mM prenol mM-1VFA, 0.04 mg FAE mL-1, 24.5 oC, 24.5 h, 91.8: 8.2 v/v n-hexane: 100 mM sodium acetate pH 4.7. Highest L-arabinose ferulate yield (56.2%) and selectivity (1.284) were observed at 2.96 mM L-arabinose mM-1VFA, 0.02 mg FAE mL-1, 38.9 oC, 12 h, 90.5: 5.0: 4.5 v/v/v n-hexane: dimethyl sulfoxide: 100 mM sodium acetate pH 4.7. The enzyme could be reused for six consecutive reaction cycles maintaining 66.6% of its initial synthetic activity. The developed bioconversions showed exceptional biocatalyst productivities (> 300 g product g-1FAE) and the waste production was within the range of pharmaceutical processes.Paper VI focused on the investigation of the basis of the type A classification of a well-studied FAE from Aspergillus niger(AnFaeA) by comparing its activity towards methyl and arabinose hydroxycinnamic acid esters. For this purpose, L-arabinose ferulateand caffeate were synthesized enzymatically. kcat/Kmratios revealed that AnFaeA hydrolyzed arabinose ferulate 1600 times and arabinose caffeate 6.5 times more efficiently than methyl esters. This study demonstrated that short alkyl chain hydroxycinnamate esters which are used nowadays for FAE classification can lead to activity misclassification, while L-arabinose esters could potentially substitute synthetic esters in classification describing more adequately the enzyme specificitiesin the natural environment.
|
|
| 24. |
- Faisal, Abrar, et al.
(författare)
-
Recovery of l-Arginine from Model Solutions and Fermentation Broth Using Zeolite-Y Adsorbent
- 2019
-
Ingår i: ACS Sustainable Chemistry and Engineering. - : American Chemical Society (ACS). - 2168-0485. ; 7:9, s. 8900-8907
-
Tidskriftsartikel (refereegranskat)abstract
- Arginine was produced via fermentation of sugars using the engineered microorganism Escherichia coli. Zeolite-Y adsorbents in the form of powder and extrudates were used to recover arginine from both a real fermentation broth and aqueous model solutions. An adsorption isotherm was determined using model solutions and zeolite-Y powder. The saturation loading was determined to be 0.2 g/g using the Sips model. Arginine adsorbed from a real fermentation broth using either zeolite-Y powder or extrudates both showed a maximum loading of 0.15 g/g at pH 11. This adsorbed loading is very close to the corresponding value obtained from the model solution showing that under the experimental conditions the presence of additional components in the broth did not have a significant effect on the adsorption of arginine. Furthermore, a breakthrough curve was determined for extrudates using a 1 wt % arginine model solution. The selectivity for arginine over ammonia and alanine from the real fermentation broth at pH 11 was 1.9 and 8.3, respectively, for powder, and 1.0, and 4.1, respectively, for extrudates. To the best of our knowledge, this is the first time recovery of arginine from real fermentation broths using any type of adsorbent has been reported.
|
|
| 25. |
|
|
