Monitoring on-line desalted lignocellulosic hydrolysates by microdialysis sampling micro-high performance anion exchange chromatography with integrated pulsed electrochemical detection/mass spectrometry

Karl Rumbold, Harriet Okatch, Nelson Torto (Corresponding Author), Matti Siika-aho, Georg Gubitz, Karl-Heinz Robra, Bernard Prior

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

An on‐line system based on microdialysis sampling (MD), micro‐high performance anion exchange chromatography (micro‐HPAEC), integrated pulsed electrochemical detection (IPED), and electrospray ionization mass spectrometry (MS) for the monitoring of on‐line desalted enzymatic hydrolysates is presented.
Continuous monitoring of the enzymatic degradation of dissolving pulp from Eucalyptus grandis as well as degradation of sugar cane bagasse in a 5‐mL reaction vessel was achieved up to 24 h without any additional sample handling steps. Combining MD with micro‐HPAEC‐IPED/MS and on‐line desalting of hydrolysates enabled injection (5 μL) of at least 23 samples in a study of the sequential action of hydrolytic enzymes in an unmodified environment where the enzymes and substrate were not depleted due to the perm‐selectivity of the MD membrane (30 kDa cut‐off).
Xylanase, phenolic acid esterase and a combination of endoglucanase (EG II) with cellobiohydrolase (CBH I) resulted in the production of DP 1 after the addition of esterase, DP 2 and DP 3 after the addition of EG II and CBH I, from the dissolving pulp substrate. Similar sequential enzyme addition to sugar cane bagasse resulted in DP 1 production after the addition of esterase and DP 1, DP 2 and DP 3 production after the addition of the EG II and CBH I mixture.
Combining MS on‐line with micro‐HPAEC‐IPED proved to be a versatile and necessary tool for such a study compared to conventional methods. The mass selectivity of MS revealed complementary information, including the co‐elution of saccharides as well as the presence of more than one type of DP 2 in the case of dissolving pulp and several types of DP 2 and DP 3 for sugar cane bagasse.
This study demonstrates the limitation of the use of retention time alone for confirmation of the identity of saccharides especially when dealing with complex enzymatic hydrolysates. In situ sampling and sample clean‐up combined with on‐line desalting of the chromatographic effluent, provides a generic approach to achieve real time monitoring of enzymatic hydrolysates when they are detected by a combination of IPED and MS.
Original languageEnglish
Pages (from-to)822-828
JournalBiotechnology and Bioengineering
Volume78
Issue number7
DOIs
Publication statusPublished - 2002
MoE publication typeA1 Journal article-refereed

Fingerprint

Microdialysis
Chromatography
Canes
Anions
Mass spectrometry
Mass Spectrometry
Ion exchange
Negative ions
Sugar cane
Bagasse
Sampling
Monitoring
Pulp
Salt removal
Enzymes
Esterases
Cellulose 1,4-beta-Cellobiosidase
Acetylesterase
Online Systems
Eucalyptus

Keywords

  • microdialysis sampling
  • chromatography
  • enzymatic hydrolysates
  • sugar cane bagasse
  • Eucalyptus grandis
  • on-line desalting

Cite this

@article{4ce513d161f14f13b8f79b1f18b0cc92,
title = "Monitoring on-line desalted lignocellulosic hydrolysates by microdialysis sampling micro-high performance anion exchange chromatography with integrated pulsed electrochemical detection/mass spectrometry",
abstract = "An on‐line system based on microdialysis sampling (MD), micro‐high performance anion exchange chromatography (micro‐HPAEC), integrated pulsed electrochemical detection (IPED), and electrospray ionization mass spectrometry (MS) for the monitoring of on‐line desalted enzymatic hydrolysates is presented. Continuous monitoring of the enzymatic degradation of dissolving pulp from Eucalyptus grandis as well as degradation of sugar cane bagasse in a 5‐mL reaction vessel was achieved up to 24 h without any additional sample handling steps. Combining MD with micro‐HPAEC‐IPED/MS and on‐line desalting of hydrolysates enabled injection (5 μL) of at least 23 samples in a study of the sequential action of hydrolytic enzymes in an unmodified environment where the enzymes and substrate were not depleted due to the perm‐selectivity of the MD membrane (30 kDa cut‐off). Xylanase, phenolic acid esterase and a combination of endoglucanase (EG II) with cellobiohydrolase (CBH I) resulted in the production of DP 1 after the addition of esterase, DP 2 and DP 3 after the addition of EG II and CBH I, from the dissolving pulp substrate. Similar sequential enzyme addition to sugar cane bagasse resulted in DP 1 production after the addition of esterase and DP 1, DP 2 and DP 3 production after the addition of the EG II and CBH I mixture. Combining MS on‐line with micro‐HPAEC‐IPED proved to be a versatile and necessary tool for such a study compared to conventional methods. The mass selectivity of MS revealed complementary information, including the co‐elution of saccharides as well as the presence of more than one type of DP 2 in the case of dissolving pulp and several types of DP 2 and DP 3 for sugar cane bagasse. This study demonstrates the limitation of the use of retention time alone for confirmation of the identity of saccharides especially when dealing with complex enzymatic hydrolysates. In situ sampling and sample clean‐up combined with on‐line desalting of the chromatographic effluent, provides a generic approach to achieve real time monitoring of enzymatic hydrolysates when they are detected by a combination of IPED and MS.",
keywords = "microdialysis sampling, chromatography, enzymatic hydrolysates, sugar cane bagasse, Eucalyptus grandis, on-line desalting",
author = "Karl Rumbold and Harriet Okatch and Nelson Torto and Matti Siika-aho and Georg Gubitz and Karl-Heinz Robra and Bernard Prior",
year = "2002",
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Monitoring on-line desalted lignocellulosic hydrolysates by microdialysis sampling micro-high performance anion exchange chromatography with integrated pulsed electrochemical detection/mass spectrometry. / Rumbold, Karl; Okatch, Harriet; Torto, Nelson (Corresponding Author); Siika-aho, Matti; Gubitz, Georg; Robra, Karl-Heinz; Prior, Bernard.

In: Biotechnology and Bioengineering, Vol. 78, No. 7, 2002, p. 822-828.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Monitoring on-line desalted lignocellulosic hydrolysates by microdialysis sampling micro-high performance anion exchange chromatography with integrated pulsed electrochemical detection/mass spectrometry

AU - Rumbold, Karl

AU - Okatch, Harriet

AU - Torto, Nelson

AU - Siika-aho, Matti

AU - Gubitz, Georg

AU - Robra, Karl-Heinz

AU - Prior, Bernard

PY - 2002

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N2 - An on‐line system based on microdialysis sampling (MD), micro‐high performance anion exchange chromatography (micro‐HPAEC), integrated pulsed electrochemical detection (IPED), and electrospray ionization mass spectrometry (MS) for the monitoring of on‐line desalted enzymatic hydrolysates is presented. Continuous monitoring of the enzymatic degradation of dissolving pulp from Eucalyptus grandis as well as degradation of sugar cane bagasse in a 5‐mL reaction vessel was achieved up to 24 h without any additional sample handling steps. Combining MD with micro‐HPAEC‐IPED/MS and on‐line desalting of hydrolysates enabled injection (5 μL) of at least 23 samples in a study of the sequential action of hydrolytic enzymes in an unmodified environment where the enzymes and substrate were not depleted due to the perm‐selectivity of the MD membrane (30 kDa cut‐off). Xylanase, phenolic acid esterase and a combination of endoglucanase (EG II) with cellobiohydrolase (CBH I) resulted in the production of DP 1 after the addition of esterase, DP 2 and DP 3 after the addition of EG II and CBH I, from the dissolving pulp substrate. Similar sequential enzyme addition to sugar cane bagasse resulted in DP 1 production after the addition of esterase and DP 1, DP 2 and DP 3 production after the addition of the EG II and CBH I mixture. Combining MS on‐line with micro‐HPAEC‐IPED proved to be a versatile and necessary tool for such a study compared to conventional methods. The mass selectivity of MS revealed complementary information, including the co‐elution of saccharides as well as the presence of more than one type of DP 2 in the case of dissolving pulp and several types of DP 2 and DP 3 for sugar cane bagasse. This study demonstrates the limitation of the use of retention time alone for confirmation of the identity of saccharides especially when dealing with complex enzymatic hydrolysates. In situ sampling and sample clean‐up combined with on‐line desalting of the chromatographic effluent, provides a generic approach to achieve real time monitoring of enzymatic hydrolysates when they are detected by a combination of IPED and MS.

AB - An on‐line system based on microdialysis sampling (MD), micro‐high performance anion exchange chromatography (micro‐HPAEC), integrated pulsed electrochemical detection (IPED), and electrospray ionization mass spectrometry (MS) for the monitoring of on‐line desalted enzymatic hydrolysates is presented. Continuous monitoring of the enzymatic degradation of dissolving pulp from Eucalyptus grandis as well as degradation of sugar cane bagasse in a 5‐mL reaction vessel was achieved up to 24 h without any additional sample handling steps. Combining MD with micro‐HPAEC‐IPED/MS and on‐line desalting of hydrolysates enabled injection (5 μL) of at least 23 samples in a study of the sequential action of hydrolytic enzymes in an unmodified environment where the enzymes and substrate were not depleted due to the perm‐selectivity of the MD membrane (30 kDa cut‐off). Xylanase, phenolic acid esterase and a combination of endoglucanase (EG II) with cellobiohydrolase (CBH I) resulted in the production of DP 1 after the addition of esterase, DP 2 and DP 3 after the addition of EG II and CBH I, from the dissolving pulp substrate. Similar sequential enzyme addition to sugar cane bagasse resulted in DP 1 production after the addition of esterase and DP 1, DP 2 and DP 3 production after the addition of the EG II and CBH I mixture. Combining MS on‐line with micro‐HPAEC‐IPED proved to be a versatile and necessary tool for such a study compared to conventional methods. The mass selectivity of MS revealed complementary information, including the co‐elution of saccharides as well as the presence of more than one type of DP 2 in the case of dissolving pulp and several types of DP 2 and DP 3 for sugar cane bagasse. This study demonstrates the limitation of the use of retention time alone for confirmation of the identity of saccharides especially when dealing with complex enzymatic hydrolysates. In situ sampling and sample clean‐up combined with on‐line desalting of the chromatographic effluent, provides a generic approach to achieve real time monitoring of enzymatic hydrolysates when they are detected by a combination of IPED and MS.

KW - microdialysis sampling

KW - chromatography

KW - enzymatic hydrolysates

KW - sugar cane bagasse

KW - Eucalyptus grandis

KW - on-line desalting

U2 - 10.1002/bit.10264;

DO - 10.1002/bit.10264;

M3 - Article

VL - 78

SP - 822

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JO - Biotechnology and Bioengineering

JF - Biotechnology and Bioengineering

SN - 0006-3592

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