Oxidation-reduction potential (ORP) as a tool for process monitoring of H2O2/LPMO assisted enzymatic hydrolysis of cellulose

Kadić, Adnan; Chylenski, Piotr; Hansen, Mads Anders Tengstedt; Bengtsson, Oskar; Eijsink, Vincent G. H.; Lidén, Gunnar

doi:10.1016/j.procbio.2019.08.015

Cited by 20 publications

(16 citation statements)

References 35 publications

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“…2 a). The signal in this case agrees qualitatively well with measurement made in a previous work [ 27 ] on enzymatic saccharification of sulfite pretreated spruce wood, in which the naturally present SSL liquid served as a reductant, but the absolute value was higher in the present study due to a different medium background. Feeding of H 2 O 2 increased the ORP, and higher feed rates led to an earlier and more rapid increase in the ORP (Fig.…”

Section: Resultssupporting

confidence: 91%

“…We have previously examined the suitability of an oxidation–reduction potential (ORP) redox electrode, which provides a “summarizing” signal about the redox state of the reaction, to monitor changes in LPMO activity during batch enzymatic saccharification of sulfite pretreated softwood pulp [ 27 ]. We found that under well-controlled laboratory-scale saccharification experiments with H 2 O 2 feeding, there was an increase in the ORP signal at the time point when the accumulation of LPMO products came to a halt.…”

Section: Introductionmentioning

confidence: 99%

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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

Kadić

Várnai

Eijsink

et al. 2021

Biotechnol Biofuels

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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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Section: Resultssupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

In situ measurements of oxidation–reduction potential and hydrogen peroxide concentration as tools for revealing LPMO inactivation during enzymatic saccharification of cellulose

Kadić

Várnai

Eijsink

et al. 2021

Biotechnol Biofuels

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“…Similarly to BGs, GH61s, today called LPMOs, have been incorporated in the Cellic CTec series [48,62,135,160,250]. Of note, while the contribution of LPMOs to the efficiency of today's cellulase cocktails is clear and important [49,65,146,167,[248][249][250], optimizing this impact is not easy and requires careful consideration of reaction conditions [60], as discussed below.…”

Section: Today's Cellulase Cocktails: What Are the Limitations And Homentioning

confidence: 99%

“…It has been shown for various reaction setups that too high feeding rates of externally added H 2 O 2 [200,248] or too high levels of in situ production of H 2 O 2 [185,269] lead to LPMO inactivation. Recent studies following the accumulation of LPMO products over the course of H 2 O 2 -assisted saccharification of industrial feedstocks [37,65,167,248] clearly indicate that LPMO inactivation occurs presumably due to the accumulation of H 2 O 2 in the reaction mixture, although the extent and rate of inactivation over time remain to be elucidated. Notably, there is a clear difference between LPMOs in terms of redox stability [66,272], partly due to the presence or absence of CBMs (discussed below).…”

Section: The Interplay Between Process Configuration and Enzyme Efficmentioning

confidence: 99%

“…Consequently, process robustness may be increased by screening for LPMOs with higher stability. Successful process optimization may further include control of the rate of addition or in situ generation of H 2 O 2 , control of dissolved oxygen levels, supplementation with catalase and/or superoxide dismutase to maintain low levels of H 2 O 2 and superoxide radicals [37] as well as online monitoring and control of the redox processes taking place during saccharification, e.g., through online monitoring of the oxidation-reduction potential [167]. Before the power of LPMOs can be leveraged to its fullest extent, however, further fundamental research is required to better understand the impact of reactive oxygen species generated in biotic and abiotic redox processes on LPMO activity and to unravel the mechanisms of LPMO inactivation in the presence of industrially relevant feedstocks.…”

Section: The Interplay Between Process Configuration and Enzyme Efficmentioning

confidence: 99%

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Enzymatic processing of lignocellulosic biomass: principles, recent advances and perspectives

Østby

Hansen

Horn

et al. 2020

Journal of Industrial Microbiology and Biotechnology

136

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Efficient saccharification of lignocellulosic biomass requires concerted development of a pretreatment method, an enzyme cocktail and an enzymatic process, all of which are adapted to the feedstock. Recent years have shown great progress in most aspects of the overall process. In particular, increased insights into the contributions of a wide variety of cellulolytic and hemicellulolytic enzymes have improved the enzymatic processing step and brought down costs. Here, we review major pretreatment technologies and different enzyme process setups and present an in-depth discussion of the various enzyme types that are currently in use. We pay ample attention to the role of the recently discovered lytic polysaccharide monooxygenases (LPMOs), which have led to renewed interest in the role of redox enzyme systems in lignocellulose processing. Better understanding of the interplay between the various enzyme types, as they may occur in a commercial enzyme cocktail, is likely key to further process improvements.

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Demonstration‐scale enzymatic saccharification of sulfite‐pulped spruce with addition of hydrogen peroxide for LPMO activation

de_Freitas_Costa

Kadić²,

Chylenski

et al. 2020

Biofuels Bioprod Bioref

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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.© 2020 The Authors. Biofuels, Bioproducts, and Biorefining published by Society of Chemical Industry and John Wiley & Sons, Ltd.

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Oxidation-reduction potential (ORP) as a tool for process monitoring of H2O2/LPMO assisted enzymatic hydrolysis of cellulose

Cited by 20 publications

References 35 publications

In situ measurements of oxidation–reduction potential and hydrogen peroxide concentration as tools for revealing LPMO inactivation during enzymatic saccharification of cellulose

In situ measurements of oxidation–reduction potential and hydrogen peroxide concentration as tools for revealing LPMO inactivation during enzymatic saccharification of cellulose

Enzymatic processing of lignocellulosic biomass: principles, recent advances and perspectives

Demonstration‐scale enzymatic saccharification of sulfite‐pulped spruce with addition of hydrogen peroxide for LPMO activation

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