Screening of cellulases for biofuel production: Online monitoring of the enzymatic hydrolysis of insoluble cellulose using high‐throughput scattered light detection

Jäger, Gernot; Wulfhorst, Helene; Zeithammel, Erik U.; Elinidou, Efthimia; Spieß, Antje C.; Büchs, Jochen

doi:10.1002/biot.201000387

Cited by 24 publications

(20 citation statements)

References 55 publications

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“…Furthermore, Figure 9 illustrates the aforementioned differences in cellulose hydrolysis rates (Figure 7) for various substrates and pretreatments. In addition, it confirms the findings of other authors: (i) since smaller cellulose particle sizes lead to increased cellulase adsorption [25] (see previous section), hydrolysis rates increase with decreasing cellulose particle size [22-24]; (ii) since a reduction in CrI leads to increased cellulase adsorption and higher reactivity of adsorbed cellulases, hydrolysis rates correlate inversely with the CrI of the applied cellulose [24,26]. It should be noted that Figure 9 shows an empirical correlation for the conducted hydrolysis experiments.…”

Section: Resultssupporting

confidence: 90%

“…These properties, in particular, are the degree of polymerization, accessibility and crystallinity [15-18]. Cellulose accessibility, which is determined by cellulose particle size (external surface area) and porosity (internal surface area) [15,19], is the most important factor for hydrolysis [15,18,20-24]. This accessibility reflects the total surface area available for direct physical contact between cellulase and cellulose and, therefore, influences cellulase adsorption as well as the rate and extent of cellulose hydrolysis [21,25].…”

Section: Introductionmentioning

confidence: 99%

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How recombinant swollenin from Kluyveromyces lactisaffects cellulosicsubstrates and accelerates their hydrolysis

Jäger

Girfoglio

Dollo

et al. 2011

Biotechnol Biofuels

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BackgroundIn order to generate biofuels, insoluble cellulosic substrates are pretreated and subsequently hydrolyzed with cellulases. One way to pretreat cellulose in a safe and environmentally friendly manner is to apply, under mild conditions, non-hydrolyzing proteins such as swollenin - naturally produced in low yields by the fungus Trichoderma reesei. To yield sufficient swollenin for industrial applications, the first aim of this study is to present a new way of producing recombinant swollenin. The main objective is to show how swollenin quantitatively affects relevant physical properties of cellulosic substrates and how it affects subsequent hydrolysis.ResultsAfter expression in the yeast Kluyveromyces lactis, the resulting swollenin was purified. The adsorption parameters of the recombinant swollenin onto cellulose were quantified for the first time and were comparable to those of individual cellulases from T. reesei. Four different insoluble cellulosic substrates were then pretreated with swollenin. At first, it could be qualitatively shown by macroscopic evaluation and microscopy that swollenin caused deagglomeration of bigger cellulose agglomerates as well as dispersion of cellulose microfibrils (amorphogenesis). Afterwards, the effects of swollenin on cellulose particle size, maximum cellulase adsorption and cellulose crystallinity were quantified. The pretreatment with swollenin resulted in a significant decrease in particle size of the cellulosic substrates as well as in their crystallinity, thereby substantially increasing maximum cellulase adsorption onto these substrates. Subsequently, the pretreated cellulosic substrates were hydrolyzed with cellulases. Here, pretreatment of cellulosic substrates with swollenin, even in non-saturating concentrations, significantly accelerated the hydrolysis. By correlating particle size and crystallinity of the cellulosic substrates with initial hydrolysis rates, it could be shown that the swollenin-induced reduction in particle size and crystallinity resulted in high cellulose hydrolysis rates.ConclusionsRecombinant swollenin can be easily produced with the robust yeast K. lactis. Moreover, swollenin induces deagglomeration of cellulose agglomerates as well as amorphogenesis (decrystallization). For the first time, this study quantifies and elucidates in detail how swollenin affects different cellulosic substrates and their hydrolysis.

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Section: Resultssupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

How recombinant swollenin from Kluyveromyces lactisaffects cellulosicsubstrates and accelerates their hydrolysis

Jäger

Girfoglio

Dollo

et al. 2011

Biotechnol Biofuels

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“…Therefore, the fermentation conditions will have to match the pH-optima of cellulases around 4.8. This prerequisite applies more for U. maydis than for A. terreus as commonly available cellulases would not perform well under pH values below 3 [38]. In addition, the advantage of the yeast-like, single cell growth of U. maydis will facilitate a scale-up in larger stirred tank bioreactors.…”

Section: Resultsmentioning

confidence: 99%

Biomass pretreatment affects Ustilago maydis in producing itaconic acid

et al. 2012

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BackgroundIn the last years, the biotechnological production of platform chemicals for fuel components has become a major focus of interest. Although ligno-cellulosic material is considered as suitable feedstock, the almost inevitable pretreatment of this recalcitrant material may interfere with the subsequent fermentation steps. In this study, the fungus Ustilago maydis was used to produce itaconic acid as platform chemical for the synthesis of potential biofuels such as 3-methyltetrahydrofuran. No studies, however, have investigated how pretreatment of ligno-cellulosic biomass precisely influences the subsequent fermentation by U. maydis. Thus, this current study aims to first characterize U. maydis in shake flasks and then to evaluate the influence of three exemplary pretreatment methods on the cultivation and itaconic acid production of this fungus. Cellulose enzymatically hydrolysed in seawater and salt-assisted organic-acid catalysed cellulose were investigated as substrates. Lastly, hydrolysed hemicellulose from fractionated beech wood was applied as substrate.ResultsU. maydis was characterized on shake flask level regarding its itaconic acid production on glucose. Nitrogen limitation was shown to be a crucial condition for the production of itaconic acid. For itaconic acid concentrations above 25 g/L, a significant product inhibition was observed. Performing experiments that simulated influences of possible pretreatment methods, U. maydis was only slightly affected by high osmolarities up to 3.5 osmol/L as well as of 0.1 M oxalic acid. The production of itaconic acid was achieved on pretreated cellulose in seawater and on the hydrolysed hemicellulosic fraction of pretreated beech wood.ConclusionThe fungus U. maydis is a promising producer of itaconic acid, since it grows as single cells (yeast-like) in submerged cultivations and it is extremely robust in high osmotic media and real seawater. Moreover, U. maydis can grow on the hemicellulosic fraction of pretreated beech wood. Thereby, this fungus combines important advantages of yeasts and filamentous fungi. Nevertheless, the biomass pretreatment does indeed affect the subsequent itaconic acid production. Although U. maydis is insusceptible to most possible impurities from pretreatment, high amounts of salts or residues of organic acids can slow microbial growth and decrease the production. Consequently, the pretreatment step needs to fit the prerequisites defined by the actual microorganisms applied for fermentation.

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“…Fig. 1 Schematic representation of the BioLector [36] In contrast to other methods the shaking of the microtiter plate does not have to be stopped during the measurement. Taking samples during the reaction is not necessary and sources of error are avoided.…”

Section: Scattered Light Measurement Using Biolectormentioning

confidence: 99%

Enzymatic Degradation of Lignocellulose for Synthesis of Biofuels and Other Value-Added Products

Wulfhorst

Harwardt

Giese

et al. 2015

Notes on Numerical Fluid Mechanics and Multidisciplinary Design

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Wood is a renewable source for biofuels and chemicals. An efficient pretreatment is required to destroy the highly ordered and complex structure of wood fibres and to improve their enzymatic degradability. To understand the effectiveness of pretreatment on enzymatic degradability, high-throughput analysis of cellulose kinetics using insoluble cellulosic substrate is required. The BioLector technology enables online monitoring of scattered light intensity and fluorescence signals during the continuous shaking of cellulose samples in microtiter plates. It is used to monitor the hydrolysis of three different cellulosic substrates catalysed by a commercial cellulase preparation from Trichoderma reesei (Celluclast). Moreover, the reduction of crystallinity and particle size is a key determining factor for an efficient hydrolysis of cellulose particles in heterogeneous system. To increase the sugar release, crystallinity and particle size were decreased by the dissolution of spruce wood in the ionic liquid EMIM Ac resulting in high conversion and reaction rates. Additionally, the enzymatic action on lignin model substrates is characterised using an activity assay and cyclic voltammetry.

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Screening of cellulases for biofuel production: Online monitoring of the enzymatic hydrolysis of insoluble cellulose using high‐throughput scattered light detection

Cited by 24 publications

References 55 publications

How recombinant swollenin from Kluyveromyces lactisaffects cellulosicsubstrates and accelerates their hydrolysis

How recombinant swollenin from Kluyveromyces lactisaffects cellulosicsubstrates and accelerates their hydrolysis

Biomass pretreatment affects Ustilago maydis in producing itaconic acid

Enzymatic Degradation of Lignocellulose for Synthesis of Biofuels and Other Value-Added Products

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