Monitoring On-Line Desalted Lignocellulosic Hydrolysates by Microdialysis Sampling Micro-High Performance Anion Exchange Chromatography with Integrated Pulsed Electrochemical Detection/ Mass Spectrometry Karl Rumbold, 2 Harriet Okatch 1 , Nelson Torto 1 , Matti Siika-Aho, 3 1 Georg Gu Èbitz, 4 Karl-Heinz Robra, 4 Bernard Prior 2 1 Chemistry Department, University of Botswana, P/Bag UB 00704, Gaborone, Botswana; telephone: +267 3552502; fax: +267 3552836; e-mail:torton@mopipi.ub.bw 2 Department of Microbiology, University of Stellenbosch, P/Bag X1, 7602 Matieland, South Africa 3 VTT Biotechnology, Espoo, P.O. Box 1500, Finland 02044 4 Institute for Environmental Biotechnology, Graz University of Technology, 2 Petersgasse 12, 8010 Graz, Austria Received 27 August 2001; accepted 10 January 2002 DOI: 10.1002/bit.10264 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 ion- ization 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 de- gradation 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 lL) of at least 23 samples in a study of the sequential action of hydrolytic enzymes in an unmodi®ed environment where the enzymes and sub- strate 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 pro- duction of DP 1 after the addition of esterase, DP 2 and DP 3 after the addition of EG II and CBH I, from the dis- solving pulp substrate. Similar sequential enzyme addi- tion 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 limi- tation of the use of retention time alone for con®rmation 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 ef¯uent, provides a generic ap- proach to achieve real time monitoring of enzymatic hydrolysates when they are detected by a combination of IPED and MS. ã 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 822±828, 2002. Keywords: microdialysis sampling; 3 chromatography; enzymatic hydrolysates; sugar cane bagasse; Eucalyptus grandis; on-line desalting INTRODUCTION Lignocellulosic material is one of the most abundant sources of carbohydrates and thus has high industrial importance. Whenever there is shortage of resources, al- ternative raw materials as well as methods to access abundant natural sources are sought after extensively. Sincetheextractionofcellulosebymeansofpulpingisas old as paper, recently there have been eorts to ®nd new methodologiestomaketheprocesslessenergyconsuming and more environmentally friendly (Christov and Prior, 1998; Breen and Singleton, 1999). This has facilitated an intensive search for new enzymes to be employed in this new area that has become an industry of its own. Two examples of lignocellulosic materials that have attracted interest are dissolving pulp and sugar cane bagasse. Sugar cane bagasse is a waste product from the sugar cane industry that has been considered as a carbon Correspondence to: N. Torto ã 2002 Wiley Periodicals, Inc.