| 1. |
- Sassner, Per, et al.
(författare)
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Integration Options for High Energy Efficiency and Improved Economics in a Wood-to-Ethanol Process
- 2008
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 1:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: There is currently a steady increase in the use of wood-based fuels for heat and power production in Sweden. A major proportion of these fuels could serve as feedstock for ethanol production. In this study various options for the utilization of the solid residue formed during ethanol production from spruce, such as the production of pellets, electricity and heat for district heating, were compared in terms of overall energy efficiency and production cost. The effects of changes in the process performance, such as variations in the ethanol yield and/or the energy demand, were also studied. The process was based on SO2-catalysed steam pretreatment, which was followed by simultaneous saccharification and fermentation. A model including all the major process steps was implemented in the commercial flow-sheeting program Aspen Plus, the model input was based on data recently obtained on lab scale or in a process development unit. Results: For the five base case scenarios presented in the paper the overall energy efficiency ranged from 53 to 92%, based on the lower heating values, and a minimum ethanol selling price from 3.87 to 4.73 Swedish kronor per litre (0.41–0.50 EUR/L); however, ethanol production was performed in essentially the same way in each base case scenario. (Highly realistic) improvements in the ethanol yield and reductions in the energy demand resulted in significantly lower production costs for all scenarios. Conclusion: Although ethanol was shown to be the main product, i.e. yielding the major part of the income, the co-product revenue had a considerable effect on the process economics and the importance of good utilization of the entire feedstock was clearly shown. With the assumed prices of the co-products, utilization of the excess solid residue for heat and power production was highly economically favourable. The study also showed that improvements in the ethanol yield and reductions in the energy demand resulted in significant production cost reductions almost independently of each other.
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| 2. |
- Almeida, Joao, et al.
(författare)
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Pichia stipitis xylose reductase helps detoxifying lignocellulosic hydrolysate by reducing 5-hydroxymethyl-furfural (HMF)
- 2008
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Pichia stipitis xylose reductase (Ps-XR) has been used to design Saccharomyces cerevisiae strains that are able to ferment xylose. One example is the industrial S. cerevisiae xyloseconsuming strain TMB3400, which was constructed by expression of P. stipitis xylose reductase and xylitol dehydrogenase and overexpression of endogenous xylulose kinase in the industrial S. cerevisiae strain USM21. Results: In this study, we demonstrate that strain TMB3400 not only converts xylose, but also displays higher tolerance to lignocellulosic hydrolysate during anaerobic batch fermentation as well as 3 times higher in vitro HMF and furfural reduction activity than the control strain USM21. Using laboratory strains producing various levels of Ps-XR, we confirm that Ps-XR is able to reduce HMF both in vitro and in vivo. Ps-XR overexpression increases the in vivo HMF conversion rate by approximately 20%, thereby improving yeast tolerance towards HMF. Further purification of Ps-XR shows that HMF is a substrate inhibitor of the enzyme. Conclusion: We demonstrate for the first time that xylose reductase is also able to reduce the furaldehyde compounds that are present in undetoxified lignocellulosic hydrolysates. Possible implications of this newly characterized activity of Ps-XR on lignocellulosic hydrolysate fermentation are discussed.
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| 3. |
- Bengtsson, Oskar, et al.
(författare)
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Xylose reductase from Pichia stipitis with altered coenzyme preference improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae
- 2009
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 2
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Tidskriftsartikel (refereegranskat)abstract
- Background: Xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis are the two enzymes most commonly used in recombinant Saccharomyces cerevisiae strains engineered for xylose utilization. The availability of NAD+ for XDH is limited during anaerobic xylose fermentation because of the preference of XR for NADPH. This in turn results in xylitol formation and reduced ethanol yield. The coenzyme preference of P. stipitis XR was changed by site-directed mutagenesis with the aim to engineer it towards NADH-preference. Results: XR variants were evaluated in S. cerevisiae strains with the following genetic modifications: overexpressed native P. stipitis XDH, overexpressed xylulokinase, overexpressed non-oxidative pentose phosphate pathway and deleted GRE3 gene encoding an NADPH dependent aldose reductase. All overexpressed genes were chromosomally integrated to ensure stable expression. Crude extracts of four different strains overexpressing genes encoding native P. stipitis XR, K270M and K270R mutants, as well as Candida parapsilosis XR, were enzymatically characterized. The physiological effects of the mutations were investigated in anaerobic xylose fermentation. The strain overexpressing P. stipitis XR with the K270R mutation gave an ethanol yield of 0.39 g (g consumed sugars)(-1), a xylitol yield of 0.05 g (g consumed xylose)(-1) and a xylose consumption rate of 0.28 g (g biomass)(-1) h(-1) in continuous fermentation at a dilution rate of 0.12 h(-1), with 10 g l(-1) glucose and 10 g l(-1) xylose as carbon sources. Conclusion: The cofactor preference of P. stipitis XR was altered by site-directed mutagenesis. When the K270R XR was combined with a metabolic engineering strategy that ensures high xylose utilization capabilities, a recombinant S. cerevisiae strain was created that provides a unique combination of high xylose consumption rate, high ethanol yield and low xylitol yield during ethanolic xylose fermentation.
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| 4. |
- Bettiga, Maurizio, et al.
(författare)
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Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains
- 2008
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose. Results: The specific aerobic arabinose growth rate was identical, 0.03 h-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells)-1 h-1 than the xylose isomerase strain, which only reached 0.03 h-1 and 0.02 g (g cells)-1h-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g-1 compared with 0.18 g g-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g-1 compared with 0.32 g g-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l-1 for the xylose reductase/ xylitol dehydrogenase strain compared with 11.8 g l-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells)-1 h-1 compared with 0.01 g (g cells)-1 h-1 for the xylose reductase/ xylitol dehydrogenase strain and the xylose isomerase strain, respectively. Conclusion: The combination of the xylose reductase/xylitol dehydrogenase pathway and the bacterial arabinose isomerase pathway resulted in both higher pentose sugar uptake and higher overall ethanol production than the combination of the xylose isomerase pathway and the bacterial arabinose isomerase pathway. Moreover, the flux through the bacterial arabinose pathway did not increase when combined with the xylose isomerase pathway. This suggests that the low activity of the bacterial arabinose pathway cannot be ascribed to arabitol formation via the xylose reductase enzyme.
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| 5. |
- Hahn-Hägerdal, Bärbel, et al.
(författare)
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Welcome to biotechnology for biofuels.
- 2008
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 1:1
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Tidskriftsartikel (refereegranskat)
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| 6. |
- Kaper, Thijs, et al.
(författare)
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Fluorescence resonance energy transfer sensors for quantitative monitoring of pentose and disaccharide accumulation in bacteria
- 2008
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Engineering microorganisms to improve metabolite flux requires detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo. Fluorescence resonance energy transfer sensors represent a promising technology for measuring metabolite levels and corresponding rate changes in live cells. These sensors have been applied successfully in mammalian and plant cells but potentially could also be used to monitor steady-state levels of metabolites in microorganisms using fluorimetric assays. Sensors for hexose and pentose carbohydrates could help in the development of fermentative microorganisms, for example, for biofuels applications. Arabinose is one of the carbohydrates to be monitored during biofuels production from lignocellulose, while maltose is an important degradation product of starch that is relevant for starch-derived biofuels production. Results: An Escherichia coli expression vector compatible with phage. recombination technology was constructed to facilitate sensor construction and was used to generate a novel fluorescence resonance energy transfer sensor for arabinose. In parallel, a strategy for improving the sensor signal was applied to construct an improved maltose sensor. Both sensors were expressed in the cytosol of E. coli and sugar accumulation was monitored using a simple fluorimetric assay of E. coli cultures in microtiter plates. In the case of both nanosensors, the addition of the respective ligand led to concentration-dependent fluorescence resonance energy transfer responses allowing quantitative analysis of the intracellular sugar levels at given extracellular supply levels as well as accumulation rates. Conclusion: The nanosensor destination vector combined with the optimization strategy for sensor responses should help to accelerate the development of metabolite sensors. The new carbohydrate fluorescence resonance energy transfer sensors can be used for in vivo monitoring of sugar levels in prokaryotes, demonstrating the potential of such sensors as reporter tools in the development of metabolically engineered microbial strains or for real-time monitoring of intracellular metabolite during fermentation.
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| 7. |
- Kovacs, Krisztina, et al.
(författare)
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Enzymatic hydrolysis of steam-pretreated lignocellulosic materials with Trichoderma atroviride enzymes produced in-house
- 2009
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 2
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Tidskriftsartikel (refereegranskat)abstract
- Background: Improvement of the process of cellulase production and development of more efficient lignocellulose-degrading enzymes are necessary in order to reduce the cost of enzymes required in the biomass-to-bioethanol process. Results: Lignocellulolytic enzyme complexes were produced by the mutant Trichoderma atroviride TUB F-1663 on three different steam-pretreated lignocellulosic substrates, namely spruce, wheat straw and sugarcane bagasse. Filter paper activities of the enzymes produced on the three materials were very similar, while beta-glucosidase and hemicellulase activities were more dependent on the nature of the substrate. Hydrolysis of the enzyme preparations investigated produced similar glucose yields. However, the enzymes produced in-house proved to degrade the xylan and the xylose oligomers less efficiently than a commercial mixture of cellulase and beta-glucosidase. Furthermore, accumulation of xylose oligomers was observed when the TUB F-1663 supernatants were applied to xylan-containing substrates, probably due to the low beta-xylosidase activity of the enzymes. The efficiency of the enzymes produced in-house was enhanced by supplementation with extra commercial beta-glucosidase and beta-xylosidase. When the hydrolytic capacities of various mixtures of a commercial cellulase and a T. atroviride supernatant produced in the lab were investigated at the same enzyme loading, the glucose yield appeared to be correlated with the beta-glucosidase activity, while the xylose yield seemed to be correlated with the beta-xylosidase level in the mixtures. Conclusion: Enzyme supernatants produced by the mutant T. atroviride TUB F-1663 on various pretreated lignocellulosic substrates have good filter paper activity values combined with high levels of beta-glucosidase activities, leading to cellulose conversion in the enzymatic hydrolysis that is as efficient as with a commercial cellulase mixture. On the other hand, in order to achieve good xylan conversion, the supernatants produced by the mutant have to be supplemented with additional beta-xylosidase activity.
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| 8. |
- Monavari, Sanam, et al.
(författare)
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The influence of solid/liquid separation techniques on the sugar yield in two-step dilute acid hydrolysis of softwood followed by enzymatic hydrolysis.
- 2009
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 2:1
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Tidskriftsartikel (refereegranskat)abstract
- ABSTRACT: BACKGROUND: Two-step dilute acid hydrolysis of softwood, either as a stand-alone process or as pretreatment before enzymatic hydrolysis, is considered to result in higher sugar yields than one-step acid hydrolysis. However, this requires removal of the liquid between the two steps. In an industrial process, filtration and washing of the material between the two steps is difficult, as it should be performed at high pressure to reduce energy demand. Moreover, the application of pressure leads to more compact solids, which may affect subsequent processing steps. This study was carried out to investigate the influence of pressing the biomass, in combination with the effects of not washing the material, on the sugar yield obtained from two-step dilute acid hydrolysis, with and without subsequent enzymatic digestion of the solids. RESULTS: Washing the material between the two acid hydrolysis steps, followed by enzymatic digestion, resulted in recovery of 96% of the mannose and 81% of the glucose (% of the theoretical) in the liquid fraction, regardless of the choice of dewatering method (pressing or vacuum filtration). Not washing the solids between the two acid hydrolysis steps led to elevated acidity of the remaining solids during the second hydrolysis step, which resulted in lower yields of mannose, 85% and 74% of the theoretical, for the pressed and vacuum-filtered slurry, respectively, due to sugar degradation. However, this increase in acidity resulted in a higher glucose yield (94.2%) from pressed slurry than from filtered slurry (77.6%). CONCLUSION: Pressing the washed material between the two acid hydrolysis steps had no significant negative effect on the sugar yields of the second acid hydrolysis step or on enzymatic hydrolysis. Not washing the material resulted in a harsher second acid hydrolysis step, which caused greater degradation of the sugars during subsequent acid hydrolysis of the solids, particularly in case of the vacuum-filtered solids. However, pressing in combination with not washing the material between the two steps enhanced the sugar yield of the enzymatic digestion step. Hence, it is suggested that the unwashed slurry be pressed to as high a dry matter content as possible between the two acid hydrolysis stages in order to achieve high final sugar yields.
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| 9. |
- Olofsson, Kim, et al.
(författare)
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A short review on SSF - an interesting process option for ethanol production from lignocellulosic feedstocks.
- 2008
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 1:1
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Tidskriftsartikel (refereegranskat)abstract
- ABSTRACT: Simultaneous saccharification and fermentation (SSF) is one process option for production of ethanol from lignocellulose. The principal benefits of performing the enzymatic hydrolysis together with the fermentation, instead of in a separate step after the hydrolysis, are the reduced end-product inhibition of the enzymatic hydrolysis, and the reduced investment costs. The principal drawbacks, on the other hand, are the need to find favorable conditions (e.g. temperature and pH) for both the enzymatic hydrolysis and the fermentation and the difficulty to recycle the fermenting organism and the enzymes. To satisfy the first requirement, the temperature is normally kept below 37 degrees C, whereas the difficulty to recycle the yeast makes it beneficial to operate with a low yeast concentration and at a high solid loading. In this review, we make a brief overview of recent experimental work and development of SSF using lignocellulosic feedstocks. Significant progress has been made with respect to increasing the substrate loading, decreasing the yeast concentration and co-fermentation of both hexoses and pentoses during SSF. Presently, an SSF process for e.g. wheat straw hydrolyzate can be expected to give final ethanol concentrations close to 40 g L-1 with a yield based on total hexoses and pentoses higher than 70%.
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| 10. |
- Wiman, Magnus, et al.
(författare)
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Prefermentation improves xylose utilization in simultaneous saccharification and co-fermentation of pretreated spruce.
- 2009
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 2:1
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Tidskriftsartikel (refereegranskat)abstract
- ABSTRACT: BACKGROUND: Simultaneous saccharification and fermentation (SSF) is a promising process option for ethanol production from lignocellulosic materials. However, both the overall ethanol yield and the final ethanol concentration in the fermentation broth must be high. Hence, almost complete conversion of both hexoses and pentoses must be achieved in SSF at a high solid content. A principal difficulty is to obtain an efficient pentose uptake in the presence of high glucose and inhibitor concentrations. Initial glucose present in pretreated spruce decreases the xylose utilization by yeast, due to competitive inhibition of sugar transport. In the current work, prefermentation was studied as a possible means to overcome the problem of competitive inhibition. The free hexoses, initially present in the slurry, were in these experiments fermented before adding the enzymes, thereby lowering the glucose concentration. RESULTS: This work shows that a high degree of xylose conversion and high ethanol yields can be achieved in SSF of pretreated spruce with a xylose fermenting strain of Saccharomyces cerevisiae (TMB3400) at 7% and 10% water insoluble solids (WIS). Prefermentation and fed-batch operation, both separately and in combination, improved xylose utilization. Up to 77% xylose utilization and 85% of theoretical ethanol yield (based on total sugars), giving a final ethanol concentration of 45 g L-1, were obtained in fed-batch SSF at 10% WIS when prefermentation was applied. CONCLUSION: Clearly, the mode of fermentation has a high impact on the xylose conversion by yeast in SSF. Prefermentation enhances xylose uptake most likely because of the reduced transport inhibition, in both batch and fed-batch operation. The process significance of this will be even greater for xylose-rich feedstocks.
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