scholarly journals The characteristics of insoluble softwood substrates affect fungal morphology, secretome composition, and hydrolytic efficiency of enzymes produced by Trichoderma reesei

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Vera Novy ◽  
Fredrik Nielsen ◽  
Daniel Cullen ◽  
Grzegorz Sabat ◽  
Carl J. Houtman ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Laser Scanning ◽  
State Of The Art ◽  
Steam Pretreatment ◽  
Confocal Laser ◽  
Cellulolytic Enzyme ◽  
Specific Substrate ◽  
Fungal Morphology ◽  
Adaptation Mechanism ◽  
Cellulose Accessibility

Abstract Background On-site enzyme production using Trichoderma reesei can improve yields and lower the overall cost of lignocellulose saccharification by exploiting the fungal gene regulatory mechanism that enables it to continuously adapt enzyme secretion to the substrate used for cultivation. To harness this, the interrelation between substrate characteristics and fungal response must be understood. However, fungal morphology or gene expression studies often lack structural and chemical substrate characterization. Here, T. reesei QM6a was cultivated on three softwood substrates: northern bleached softwood Kraft pulp (NBSK) and lodgepole pine pretreated either by dilute-acid-catalyzed steam pretreatment (LP-STEX) or mild alkaline oxidation (LP-ALKOX). With different pretreatments of similar starting materials, we presented the fungus with systematically modified substrates. This allowed the elucidation of substrate-induced changes in the fungal response and the testing of the secreted enzymes’ hydrolytic strength towards the same substrates. Results Enzyme activity time courses correlated with hemicellulose content and cellulose accessibility. Specifically, increased amounts of side-chain-cleaving hemicellulolytic enzymes in the protein produced on the complex substrates (LP-STEX; LP-ALKOX) was observed by secretome analysis. Confocal laser scanning micrographs showed that fungal micromorphology responded to changes in cellulose accessibility and initial culture viscosity. The latter was caused by surface charge and fiber dimensions, and likely restricted mass transfer, resulting in morphologies of fungi in stress. Supplementing a basic cellulolytic enzyme mixture with concentrated T. reesei supernatant improved saccharification efficiencies of the three substrates, where cellulose, xylan, and mannan conversion was increased by up to 27, 45, and 2800%, respectively. The improvement was most pronounced for proteins produced on LP-STEX and LP-ALKOX on those same substrates, and in the best case, efficiencies reached those of a state-of-the-art commercial enzyme preparation. Conclusion Cultivation of T. reesei on LP-STEX and LP-ALKOX produced a protein mixture that increased the hydrolytic strength of a basic cellulase mixture to state-of-the-art performance on softwood substrates. This suggests that the fungal adaptation mechanism can be exploited to achieve enhanced performance in enzymatic hydrolysis without a priori knowledge of specific substrate requirements.

scholarly journals The Characteristics of Insoluble Softwood Substrates Affect Fungal Morphology, Secretome Composition, and Hydrolytic Efficiency of Enzymes Produced by Trichoderma Reesei

2021 ◽  
Author(s):  
Vera Novy ◽  
Fredrik Nielsen ◽  
Daniel Cullen ◽  
Grzegorz Sabat ◽  
Carl J. Houtman ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Laser Scanning ◽  
State Of The Art ◽  
Steam Pretreatment ◽  
Confocal Laser ◽  
Cellulolytic Enzyme ◽  
Specific Substrate ◽  
Fungal Morphology ◽  
Adaptation Mechanism ◽  
Cellulose Accessibility

Abstract BackgroundOn-site enzyme production using Trichoderma reesei can improve yields and lower the overall cost of lignocellulose saccharification by exploiting the fungal gene regulatory mechanism that enables it to continuously adapt enzyme secretion to the substrate used for cultivation. To harness this, the interrelation between substrate characteristics and fungal response must be understood. However, fungal morphology or gene expression studies often lack structural and chemical substrate characterization. Here, T. reesei QM6a was cultivated on three softwood substrates: northern bleached softwood Kraft pulp (NBSK) and lodgepole pine pretreated by dilute-acid-catalyzed steam pretreatment (LP-STEX) and mild alkaline oxidation (LP-ALKOX). With different pretreatments of similar starting materials, we presented the fungus with systematically modified substrates. This allowed the elucidation of substrate-induced changes in the fungal response and the testing of the secreted enzymes’ hydrolytic strength towards the same substrates.ResultsEnzyme activity time courses correlated with hemicellulose content and cellulose accessibility. Specifically, increased amounts of side chain-cleaving hemicellulolytic enzymes in the protein produced on the complex substrates (LP-STEX; LP-ALKOX) was observed by secretome analysis. Confocal laser scanning micrographs showed that fungal micromorphology responded to changes in cellulose accessibility and initial culture viscosity. The latter was caused by surface charge and fiber dimensions, and likely restricted mass transfer, resulting in morphologies of fungi in stress. Supplementing a basic cellulolytic enzyme mixture with concentrated T. reesei supernatant improved saccharification efficiencies of the three substrates, where cellulose, xylan, and mannan conversion was increased by up to 27%, 45%, and 2800%, respectively. The improvement was most pronounced for proteins produced on LP-STEX and LP-ALKOX on those same substrates, and in the best case, efficiencies reached those of a state-of-the-art enzyme preparation.ConclusionCultivation of T. reesei on LP-STEX and LP-ALKOX produced a protein mixture that increased the hydrolytic strength of a basic cellulase mixture to state-of-the-art performance on softwood substrates. This suggests that the fungal adaptation mechanism can be exploited to achieve enhanced performance in enzymatic hydrolysis without a priori knowledge of specific substrate requirements.

scholarly journals Quantifying cellulose accessibility during enzyme-mediated deconstruction using 2 fluorescence-tagged carbohydrate-binding modules

2019 ◽  
Vol 116 (45) ◽  
pp. 22545-22551 ◽  
Author(s):  
Vera Novy ◽  
Kevin Aïssa ◽  
Fredrik Nielsen ◽  
Suzana K. Straus ◽  
Peter Ciesielski ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Degradation Mechanism ◽  
Length Distribution ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Distribution Analysis ◽  
Cellulose Accessibility ◽  
Carbohydrate Binding Modules

Two fluorescence-tagged carbohydrate-binding modules (CBMs), which specifically bind to crystalline (CBM2a-RRedX) and paracrystalline (CBM17-FITC) cellulose, were used to differentiate the supramolecular cellulose structures in bleached softwood Kraft fibers during enzyme-mediated hydrolysis. Differences in CBM adsorption were elucidated using confocal laser scanning microscopy (CLSM), and the structural changes occurring during enzyme-mediated deconstruction were quantified via the relative fluorescence intensities of the respective probes. It was apparent that a high degree of order (i.e., crystalline cellulose) occurred at the cellulose fiber surface, which was interspersed by zones of lower structural organization and increased cellulose accessibility. Quantitative image analysis, supported by 13C NMR, scanning electron microscopy (SEM) imaging, and fiber length distribution analysis, showed that enzymatic degradation predominates at these zones during the initial phase of the reaction, resulting in rapid fiber fragmentation and an increase in cellulose surface crystallinity. By applying this method to elucidate the differences in the enzyme-mediated deconstruction mechanisms, this work further demonstrated that drying decreased the accessibility of enzymes to these disorganized zones, resulting in a delayed onset of degradation and fragmentation. The use of fluorescence-tagged CBMs with specific recognition sites provided a quantitative way to elucidate supramolecular substructures of cellulose and their impact on enzyme accessibility. By designing a quantitative method to analyze the cellulose ultrastructure and accessibility, this study gives insights into the degradation mechanism of cellulosic substrates.

3-D imaging of cells using a confocal laser scanning microscope and digital image processing

1988 ◽  
Vol 46 ◽  
pp. 96-97
Author(s):  
Thomas M. Jovin ◽  
Michel Robert-Nicoud ◽  
Donna J. Arndt-Jovin ◽  
Thorsten Schormann
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
Biochemical Processes ◽  
Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

3-Dimensional immunolabeling and in situ hybridization detection using fluorescence photooxidation and intermediate-voltage Electron Microscopy

1994 ◽  
Vol 52 ◽  
pp. 164-165
Author(s):  
Thomas J. Deerinck ◽  
Maryann E. Martone ◽  
Varda Lev-Ram ◽  
David P. L. Green ◽  
Roger Y. Tsien ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Reactive Oxygen ◽  
Confocal Laser Scanning Microscope ◽  
Fluorescent Dye ◽  
Confocal Laser ◽  
3 Dimensional ◽  
Voltage Electron ◽  
Dimensional Distribution ◽  
Electron Microscopes ◽  
New Generation

The confocal laser scanning microscope has become a powerful tool in the study of the 3-dimensional distribution of proteins and specific nucleic acid sequences in cells and tissues. This is also proving to be true for a new generation of high contrast intermediate voltage electron microscopes (IVEM). Until recently, the number of labeling techniques that could be employed to allow examination of the same sample with both confocal and IVEM was rather limited. One method that can be used to take full advantage of these two technologies is fluorescence photooxidation. Specimens are labeled by a fluorescent dye and viewed with confocal microscopy followed by fluorescence photooxidation of diaminobenzidine (DAB). In this technique, a fluorescent dye is used to photooxidize DAB into an osmiophilic reaction product that can be subsequently visualized with the electron microscope. The precise reaction mechanism by which the photooxidation occurs is not known but evidence suggests that the radiationless transfer of energy from the excited-state dye molecule undergoing the phenomenon of intersystem crossing leads to the formation of reactive oxygen species such as singlet oxygen. It is this reactive oxygen that is likely crucial in the photooxidation of DAB.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

A confocal laser scanning microscope for fluorescence and reflection at video rates

1989 ◽  
Vol 47 ◽  
pp. 134-135
Author(s):  
P.M. Houpt ◽  
A. Draaijer
Keyword(s):  
Light Source ◽  
Laser Scanning ◽  
Focal Point ◽  
Confocal Laser Scanning Microscope ◽  
Confocal Laser ◽  
Point Light Source ◽  
Volume Of Interest ◽  
Ion Laser ◽  
Point Light ◽  
Point Detector

In confocal microscopy, the object is scanned by the coinciding focal points (confocal) of a point light source and a point detector both focused on a certain plane in the object. Only light coming from the focal point is detected and, even more important, out-of-focus light is rejected.This makes it possible to slice up optically the ‘volume of interest’ in the object by moving it axially while scanning the focused point light source (X-Y) laterally. The successive confocal sections can be stored in a computer and used to reconstruct the object in a 3D image display.The instrument described is able to scan the object laterally with an Ar ion laser (488 nm) at video rates. The image of one confocal section of an object can be displayed within 40 milliseconds (1000 х 1000 pixels). The time to record the total information within the ‘volume of interest’ normally depends on the number of slices needed to cover it, but rarely exceeds a few seconds.

Use of confocal laser scanning microscopy and a computer model to understand ink cavitation and filamentation

TAPPI Journal ◽  
2010 ◽  
Vol 9 (10) ◽  
pp. 7-15
Author(s):  
HANNA KOIVULA ◽  
DOUGLAS BOUSFIELD ◽  
MARTTI TOIVAKKA
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Print Quality ◽  
Length Scale ◽  
Offset Printing ◽  
Significant Difference ◽  
Printing Speed

In the offset printing process, ink film splitting has an important impact on formation of ink filaments. The filament size and its distribution influence the leveling of ink and hence affect ink setting and the print quality. However, ink filaments are difficult to image due to their short lifetime and fine length scale. Due to this difficulty, limited work has been reported on the parameters that influence filament size and methods to characterize it. We imaged ink filament remains and quantified some of their characteristics by changing printing speed, ink amount, and fountain solution type. Printed samples were prepared using a laboratory printability tester with varying ink levels and operating settings. Rhodamine B dye was incorporated into fountain solutions to aid in the detection of the filaments. The prints were then imaged with a confocal laser scanning microscope (CLSM) and images were further analyzed for their surface topography. Modeling of the pressure pulses in the printing nip was included to better understand the mechanism of filament formation and the origin of filament length scale. Printing speed and ink amount changed the size distribution of the observed filament remains. There was no significant difference between fountain solutions with or without isopropyl alcohol on the observed patterns of the filament remains.

ACTIVATED SLUDGE FLOC CHARACTERIZATION BY CONFOCAL LASER SCANNING MICROSCOPY

2012 ◽  
Vol 11 (3) ◽  
pp. 669-674 ◽  
Author(s):  
Szabolcs Szilveszter ◽  
Botond Raduly ◽  
Szilard Bucs ◽  
Beata Abraham ◽  
Szabolcs Lanyi ◽  
...  
Keyword(s):  
Activated Sludge ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser
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