| 1. |
- Costa, Thales H.F., et al.
(author)
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Demonstration-scale enzymatic saccharification of sulfite-pulped spruce with addition of hydrogen peroxide for LPMO activation
- 2020
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In: Biofuels, Bioproducts and Biorefining. - : Wiley. - 1932-104X .- 1932-1031. ; 14:4, s. 734-745
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Journal article (peer-reviewed)abstract
- The saccharification of lignocellulosic materials like Norway spruce is challenging due to the recalcitrant nature of the biomass, and it requires optimized and efficient pretreatment and enzymatic hydrolysis processes to make it industrially feasible. In this study, we report successful enzymatic saccharification of sulfite-pulped spruce (Borregaard's BALI™ process) at demonstration scale, achieved through the controlled delivery of hydrogen peroxide (H2O2) for the activation of lytic polysaccharide monooxygenases (LPMOs) present in the cellulolytic enzyme preparation. We achieved 85% saccharification yield in 4 days using industrially relevant conditions – that is, an enzyme dose of 4% (w/w dry matter of substrate) of the commercial cellulase cocktail Cellic CTec3 and a substrate loading of 12% (w/w). Addition of H2O2 and the resulting controlled and high LPMO activity had a positive effect on the rate of saccharification and the final sugar titer. Clearly, the high LPMO activity was dependent on feeding the reactors with the LPMO co-substrate H2O2, as in situ generation of H2O2 from molecular oxygen was limited. These demonstration-scale experiments provide a solid basis for the use of H2O2 to improve enzymatic saccharification of lignocellulosic biomass at large industrial scale.
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| 2. |
- Kadic, Adnan, et al.
(author)
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Does sugar inhibition explain mixing effects in enzymatic hydrolysis of lignocellulose?
- 2017
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In: Journal of Chemical Technology and Biotechnology. - : Wiley. - 0268-2575. ; 92:4, s. 868-873
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Journal article (peer-reviewed)abstract
- BACKGROUND: Enzymatic hydrolysis of lignocellulose is associated with mixing issues, which are likely to affect process performance. The aim of this study was to investigate how viscosity and sugar inhibition influence the mixing-dependence in hydrolysis of steam-pretreated spruce. RESULTS: The effect of agitation on low-viscosity, low-solid hydrolysis (5% water insoluble solid (WIS)) was marginal, as the conversion after 72 h decreased by 9% when decreasing the agitation rate from 600 to 100 rpm. However, when the viscosity at 5% WIS was increased by Xanthan addition, the effect of agitation was greater, and conversion decreased by 21% when decreasing agitation from 600 to 100 rpm. For high-viscosity, high-solid hydrolysis (16% WIS), the conversion decreased by 54% when decreasing the agitation from 600 to 100 rpm. However, when the product concentration was kept low by simultaneous saccharification and fermentation (SSF), the effect of agitation was weaker, and conversion decreased by only 14%. CONCLUSION: The results of this study strongly suggest that poor mixing in viscous lignocellulose hydrolysis causes local product accumulation, leading to increased inhibition and decreased hydrolysis rates. Decreasing the glucose and cellobiose concentration removes the mixing-dependence, highlighting SSF as an attractive option for large-scale hydrolysis and fermentation of steam-pretreated softwood.
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| 3. |
- Kadic, Adnan, et al.
(author)
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Effects of agitation on particle-size distribution and enzymatic hydrolysis of pretreated spruce and giant reed.
- 2014
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In: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 7
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Journal article (peer-reviewed)abstract
- Mixing is an energy demanding process which has been previously shown to affect enzymatic hydrolysis. Concentrated biomass slurries are associated with high and non-Newtonian viscosities and mixing in these systems is a complex task. Poor mixing can lead to mass and/or heat transfer problems as well as inhomogeneous enzyme distribution, both of which can cause possible yield reduction. Furthermore the stirring energy dissipation may impact the particle size which in turn may affect the enzymatic hydrolysis. The objective of the current work was to specifically quantify the effects of mixing on particle-size distribution (PSD) and relate this to changes in the enzymatic hydrolysis. Two rather different materials were investigated, namely pretreated Norway spruce and giant reed.
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| 4. |
- Kadić, Adnan, et al.
(author)
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In situ measurements of oxidation–reduction potential and hydrogen peroxide concentration as tools for revealing LPMO inactivation during enzymatic saccharification of cellulose
- 2021
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In: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 14:1
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Journal article (peer-reviewed)abstract
- Background: Biochemical conversion of lignocellulosic biomass to simple sugars at commercial scale is hampered by the high cost of saccharifying enzymes. Lytic polysaccharide monooxygenases (LPMOs) may hold the key to overcome economic barriers. Recent studies have shown that controlled activation of LPMOs by a continuous H2O2 supply can boost saccharification yields, while overdosing H2O2 may lead to enzyme inactivation and reduce overall sugar yields. While following LPMO action by ex situ analysis of LPMO products confirms enzyme inactivation, currently no preventive measures are available to intervene before complete inactivation. Results: Here, we carried out enzymatic saccharification of the model cellulose Avicel with an LPMO-containing enzyme preparation (Cellic CTec3) and H2O2 feed at 1 L bioreactor scale and followed the oxidation–reduction potential and H2O2 concentration in situ with corresponding electrode probes. The rate of oxidation of the reductant as well as the estimation of the amount of H2O2 consumed by LPMOs indicate that, in addition to oxidative depolymerization of cellulose, LPMOs consume H2O2 in a futile non-catalytic cycle, and that inactivation of LPMOs happens gradually and starts long before the accumulation of LPMO-generated oxidative products comes to a halt. Conclusion: Our results indicate that, in this model system, the collapse of the LPMO-catalyzed reaction may be predicted by the rate of oxidation of the reductant, the accumulation of H2O2 in the reactor or, indirectly, by a clear increase in the oxidation–reduction potential. Being able to monitor the state of the LPMO activity in situ may help maximizing the benefit of LPMO action during saccharification. Overcoming enzyme inactivation could allow improving overall saccharification yields beyond the state of the art while lowering LPMO and, potentially, cellulase loads, both of which would have beneficial consequences on process economics.
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| 5. |
- Kadic, Adnan
(author)
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The effects of mixing on the enzymatic hydrolysis of lignocellulosic biomass
- 2017. - 1
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Doctoral thesis (other academic/artistic)abstract
- Biorefining of lignocellulosic biomass into biofuels and chemicals can help replace fossil resources and decrease anthropogenic greenhouse gas emissions. This thesis is focused on the effects of mixing on the enzymatic hydrolysis of pretreated biomass. Two different types of biomass were studied: softwood (Norway spruce and Scots pine), and the energy grass giant reed. Before enzymatic hydrolysis, the biomass was pretreated by either steam or sulfite pretreatment. The first part of the work concerns the connection between particle morphology and rheology of pretreated biomass, how such properties change during the course of enzymatic hydrolysis, and how the changes are influenced by reactor mixing. The second part examines the effects of mixing in stirred tank reactors on the enzymatic hydrolysis of different pretreated materials, and also attempts to explain the mechanisms behind the observed phenomena.The particle size reduction during enzymatic hydrolysis of steam pretreated spruce was primarily driven by reactor agitation. In the case of steam pretreated giant reed the particle size was mainly reduced by enzymatic hydrolysis. The rapid reduction in particle size of giant reed coincided with a rapid liquefaction. For steam pretreated softwood, the viscosity in fact increased at the beginning of enzymatic hydrolysis, followed by a gradual decrease during the remainder of the hydrolysis. This interesting phenomenon was in part linked to the type of pretreatment used on the softwood biomass. In contrast to steam pretreated softwood, the viscosity of sulfite pretreated spruce decreased rapidly during enzymatic hydrolysis. Efficient viscosity reduction in sulfite pretreated spruce was also achieved with very low doses of pure endoglucanase enzymes (0.1 mg protein per g glucan) without significant glucose release.The effect of mixing on the enzymatic hydrolysis was in part determined by the viscosity of the pretreated biomass. For steam pretreated spruce at low solid loading, decreasing the agitation rate had little effect on the the enzymatic hydrolysis. However, if the viscosity was increased by the addition of a thickening agent, the effect of agitation was much larger. For a substrate that underwent rapid initial viscosity reduction, such as steam pretreated giant reed, the enzymatic hydrolysis was almost independent of agitation rate. Another important factor determining the effect of mixing on the enzymatic hydrolysis was the level of product inhibition. If the glucose and cellobiose concentrations were high, as during high solid hydrolysis of steam pretreated spruce, low agitation rate had a large negative effect on the enzymatic hydrolysis. However, if the product concentration was kept low, as during SSF, the effect of agitation was much weaker. Overall, the results indicate that the decrease in hydrolysis rate occurred due to increased local product inhibition, caused by mass transfer limitations in the stagnant zones, formed in the reactor volume when under low intensity mixing. The rate of enzymatic hydrolysis appeared to be determined by flow regime, i.e. Reynolds number, rather than specific mixing power input. This implies that the negative effects of low agitation rate will be less of a problem in larger reactors.
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| 6. |
- Kadić, Adnan, et al.
(author)
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Viscosity reduction of pretreated softwood by endoglucanases
- 2018
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In: Journal of Chemical Technology and Biotechnology. - : Wiley. - 0268-2575. ; 93:8, s. 2440-2446
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Journal article (peer-reviewed)abstract
- BACKGROUND: Cost-effective processing of lignocellulosic biomass into sugar derived products, such as biofuels or biochemicals, needs to be performed at high water insoluble solid (WIS) loading. However, the difficult rheology of such materials presents significant challenges. The aim of this study was to investigate if a Cel5A endoglucanase can be used to reduce the viscosity of two types of pretreated softwood: steam pretreated Scots pine and sulfite pretreated Norway spruce. RESULTS: The viscosity of steam pretreated pine increased (by more than 60%) during the first 20min of enzymatic hydrolysis, followed by a gradual decrease. A slightly lower viscosity during prolonged hydrolysis could be obtained by replacing 25% of the protein in Cellic CTec3 with the Cel5A endoglucanase. Very different results were obtained with sulfite pretreated spruce. The viscosity of this material was rapidly reduced by either CTec3 or the Cel5A endoglucanase, without a transient initial increase in viscosity. Even very low doses of Cel5A (0.1mg protein per g glucan) decreased the viscosity of sulfite pretreated spruce 30-fold within 6h. CONCLUSION: Low endoglucanase doses can be used to reduce the viscosity of sulfite pretreated softwood, whereas the viscosity of steam pretreated softwood is less affected by endoglucanase activity.
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| 7. |
- Palmqvist, Benny, et al.
(author)
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Scale-up of high-solid enzymatic hydrolysis of steam-pretreated softwood : the effects of reactor flow conditions
- 2016
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In: Biomass Conversion and Biorefinery. - : Springer Science and Business Media LLC. - 2190-6815 .- 2190-6823. ; 6:2, s. 173-180
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Journal article (peer-reviewed)abstract
- The importance of flow conditions during scale-up of high-solid enzymatic hydrolysis of steam-pretreated spruce was demonstrated by comparing hydrolysis rates between laboratory (2 L) and demonstration (4 m3) scale. A positive effect of increased agitation speed on the rate of enzymatic hydrolysis was found regardless of scale. Importantly, the hydrolysis rate was higher at the larger scale when compared at similar specific power inputs. Changes in the rheological properties of the pretreated material during the hydrolysis were followed by off-line measurements of apparent viscosity. This information was used to estimate the flow conditions in the reactors, i.e., average Reynolds numbers, which together with measured mixing power consumptions enabled a more detailed comparison between the scales. The hydrolysis yields correlated better with average Reynolds numbers than specific power input over the different scales. This indicates that mass transport limitations, caused by insufficient bulk flow, likely play a decisive role in determining the rate of enzymatic hydrolysis.
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