Proceedings of the Ninth Annual Biochemical Engineering Symposium

This report presents the proceedings of the Biochemical Engineering Symposium held at Kansas State University, April 28, 1979. Since a number of the contributions will be published in detail elsewhere, only brief reports of each contribution are included here. Requests for further information on work at Iowa State University should be directed to Dr. Peter J. Reilly; at Colorado State University to Drs. V. G. Murphy and A. R. Moreira, and at Kansas State University to Drs. L. T. Fan and L. E. Erickson. ContentProperties of a Homogeneous Xylobiohydrolase from Aspergillus niger, Mary M. Frederick, Iowa State University Kinetic Studies on the Enzymatic Hydrolysis of Cellulose–Absorption and Desorption of Cellulase onto Cellulose and the Behavior of Absorbed Cellulase, Yong-Hyun Lee and L. T. Fan, Kansas State University Properties of a Homogeneous Endo-Xylanase from Aspergillus niger, Ricardo Fournier A., Iowa State University Solid State Fermentation of Manure Fibers, D. C. Ulmer, Colorado State University Analysis and Consistency of Experimental Data for Microbial Growth on Renewable Resources, B. 0. Solomon, Kansas State University Biochemical Mechanisms of Enzyme Regulation, Frederick A. Blum, Colorado State University An Evaluation of Cellulose Pretreatments for Enzymatic Hydrolysis, David H. Beardmore, Kansas State University Use of Immobilized 8-Amylase/Glucoamylase Mixtures to Produce High Maltose Syrups, Carol G. Bohnenkamp, Iowa State University Effect of Viscosity on Bubble Behavior in an Airlift Fermentor, Vasanti Deshpande, Kansas State University

Proceedings of the 10th Annual Biochemical Engineering Symposium

This the tenth in a series of symposia devoted to talks by students on their biochemical engineering research. The first, third, fifth, and ninth were at Kansas State University in Manhattan, the second and fourth were at the University of Nebraska–Lincoln, the sixth was in Kansas City in conjunction with the 81st American Institute of Chemical Engineers National Meeting, the seventh was at Iowa State University in Ames, and the eighth was held at the University of Missouri–Columbia. Contents"Combined Autohydrolysis-Organosolv Pretreatment of Lignocellulosic Materials," Robert A. Lewis, Colorado State University "An Investigation of Cellulase Activity Assays," Minhhuong Nguyen, University of Missouri–Columbia "Action Pattern of a Xylobiohydrolase from Aspergillus niger," Mary M. Frederick, Iowa State University "Estimation of Heats of Combustion of Biomass from Elemental Analysis Using Available Electron Concepts," Snehal A. Patel, Kansas State University "Design of a Wheat Straw to Ethanol Conversion Facility," Michael M. Meagher, Colorado State University "Effects of Salt, Heat, and Physical Form on the Fermentation of Bananas," Carl Drewel, University of Missouri–Columbia "Gas Hold-up in the Downflow Section of a Split Cylinder Airlift Column," Vasanti Deshpande, Kansas State University "Measurement of Michaelis Constants for Soluble and Immobilized Glucoamylase," Robert A. Lesch, Iowa State University "Kinetics of Alkaline Oxidation and Degradation of Sugars," Alfred R. Fratzke, Iowa State University "Stability of Cereal Protein During Microbial Growth on Grain Dust," Bamidele O. Solomon, Kansas State University

Proceedings of the 13th Annual Biochemical Engineering Symposium

The symposium reported here was the thirteenth of a series devoted to talks by students on their biochemical engineering research. The first, third, fifth, ninth, and twelfth were at Kansas State University, the second and fourth were at the University of Nebraska–Lincoln, the sixth was in Kansas City and was hosted by Iowa State University, the seventh and tenth were at Iowa State, and the eighth and eleventh were at the University of Missouri–Columbia and Colorado State University, respectively. All symposia have been followed by proceedings edited by faculty of the host institution. Because final publication usually takes place elsewhere, papers here are brief, and often cover research in progress. ContentSequential Utilization of Mixed Sugars by Clostridium acetobutylicum, B. Hong, N. H. Choi, and L. T. Fan, Kansas State University The Effects of Dilution Rate on the Kinetics. of Anaerobic Acidogenesis, C. J. Huang, Colorado State University Ethanol Production by Zymomonas mobilis in Anaerobic Glucose-Limited Culture: A Yield Study, Mehmet D. Oner, Kansas State University Hydrolysis of Cellulosics by Enterobacteria, Michael R. Sierks, Iowa State University The Cellulase System of Chaetomium cellulolyticum, Nikhil Mehta, Colorado State University DNA Measurement as a Tool for Estimating Biomass Concentration in the Presence of Interfering Solids, Bamidele 0. Solomon, Kansas State University The Effect of Cellulose Crystallinity on Enzymatic Hydrolysis, Maria S. Bertran, Colorado State University High Performance Liquid Chromatography of Di- and Trisaccharides, Michael M. Meagher, Iowa State University Dynamics of Bubble Size .Distributions in Air-Lift Fermentors, c. H. Lee and Snehal A. Patel, Kansas State University A Thermal Coagulation Study of Alfalfa Leaf Proteins by Differential Scanning Calorimeter, Khalif Ahmed and Bruce Dale, Colorado State University Thermodynamic Efficiency of Photoautotrophic Growth, Hyeon Y. Lee, Kansas State University

Proceedings of the 14th Annual Biochemical Engineering Symposium

The Annual Biochemical Engineering Symposium series is devoted to presentations by students on their research topics. The fourteenth event, held in 1984, was organized at the University of Missouri–Columbia. It was attended by the biochemical engineering faculty and the students from Colorado State University, Iowa State University, Kansas State University, University of Missouri–Columbia, University of Missouri–Rolla and Washington University, St. Louis. Contents"Estimation of Product Formation Kinetics and Microbial Yield Parameters for Anaerobic Organic Acid and Solvent Production," M.D. Oner, Kansas State University "Characterization of Soy Protein Texturization in a Complex Bioreactor," J.L. Ibave, Colorado State University "Acid and Solvent Fermentations Using Mixed Cultures," D. Stevens, University of Missouri–Columbia "Preliminary Process Design for Ethanol from Sweet Sorghum Ensilage Feedstock," Keith D. Lange, Colorado State University "Lamella Settlers in Ethanol Fermentation," Yong Jayanata, University of Missouri–Columbia "Bubble Size Distribution in the Down Flow Section of an Air-Lift Column," Snehal A. Patel and C.H. Lee, Kansas State University "The Sensitivity of Plant Cells to Shear Stress," Morris Z. Resenberg and Eric H. Dunlap, Washington University, St. Louis "Estimation of Growth Yield Parameters Associated with Microbial Growth," Hyeon Y. Lee, Kansas State University "Capillary Gas Chromatography of Trimethylsilylated Trisaccharides," Etienne J.M. Selosse, Iowa State University "Subsite Mapping of an Endo-Xylanase Labeled Xylooligo-saccharides," Bernard Y. Tao, Iowa State University "Cellulase Enzyme Recycle," Kate M.V. Baptie, Colorado State University "Non-Homogeneous Poisson Renewal Reward Process for Modelling Enzymatic Hydrolysis of Cellulose," M.M. Gharpuray and L.T. Fan, Kansas State University

Effect of compost and slow-release fertilizers addition on soil biochemistry and yield of maize (Zea mays L.) in Oaxaca, Mexico

The effect of Bokashi (B, a fermented compost), slow-release fertilizers (SRFs) and their combined application on mycorrhizal colonization (MC), soil invertase, cellulase, acid (AcP) and alkaline (AlP) phosphatases activities and maize (Zea mays L.) yield was investigated in terrace (TS) and valley (VS) soils in Oaxaca, Mexico. A complete randomized design, seven fertilizer treatments and four replications were used: unamended control (C); conventional fertilization (90-46-00 NPK) (CF); B; SRF1 (Multigro 6®, 21-14-10 NPK); SRF2 (Multigro 3®, 24-05-14 NPK); B+SRF1; B+SRF2. Highest root colonization percentage: CF in VS, and SRF2 in TS. Highest extraradical mycelium length: B, B+SRF1, CF in VS, and B+SRF1 in TS. In both soils, B increased the spore number. Highest AcP activity: B, SRF2 in VS, and B+SRF1, B+SRF2 in TS. Highest AlP activity: B+SRF1, CF in VS, and C in TS. Highest invertase activity: B+SRF1, SRF2, CF in VS, and B in TS. Grain yield only increased with B in VS. The significant interaction soil type × fertilizer treatment for the majority of the biological soil properties analyzed suggests that MC and soil enzyme activity response to fertilization was influenced by soil type. Bokashi, alone or combined with SRFs improves biological soil fertility in maize fields.

Treatment of tropical forages with exogenous fibrolytiic enzymes: effects on chemical composition and in vitro rumen fermentation

The effects of three treatments of fibrolytic enzymes (cellulase from Trichoderma longibrachiatum (CEL), xylanase from rumen micro-organisms (XYL) and a 1:1 mixture of CEL and XYL (MIX) on the in vitro fermentation of two samples of Pennisetum clandestinum (P1 and P2), two samples of Dichanthium aristatum (D1 and D2) and one sample of each Acacia decurrens and Acacia mangium (A1 and A2) were investigated. The first experiment compared the effects of two methods of applying the enzymes to forages, either at the time of incubation or 24 h before, on the in vitro gas production. In general, the 24 h pre-treatment resulted in higher values of gas production rate, and this application method was chosen for a second study investigating the effects of enzymes on chemical composition and in vitro fermentation of forages. The pre-treatment with CEL for 24 h reduced (p < 0.05) the content of neutral detergent fibre (NDF) of P1, P2, D1 and D2, and that of MIX reduced the NDF content of P1 and D1, but XYL had no effect on any forage. The CEL treatment increased (p < 0.05) total volatile fatty acid (VFA) production for all forages (ranging from 8.6% to 22.7%), but in general, no effects of MIX and XYL were observed. For both P. clandestinum samples, CEL treatment reduced (p < 0.05) the molar proportion of acetate and increased (p < 0.05) that of butyrate, but only subtle changes in VFA profile were observed for the rest of forages. Under the conditions of the present experiment, the treatment of tropical forages with CEL stimulated their in vitro ruminal fermentation, but XYL did not produce any positive effect. These results showed clearly that effectiveness of enzymes varied with the incubated forage and further study is warranted to investigate specific, optimal enzyme-substrate combinations.

Occurrence of Cello-Oligosaccharides in the Apoplast of Auxin-Treated Pea Stems

Treatment of pea (Pisum sativum L.) hypocotyl segments with indole-3-butyric acid, which promotes segment elongation, increased the solubilization of both xyloglucan and cello-oligosaccharides in the apoplast of auxin-treated pea stems. The cello-oligosaccharides were isolated from the apoplastic solution with a charcoal/Celite column and were identified as cellobiose, cellotriose, and cellotetraose after subsequent thin-layer chromatography and paper electrophoresis. Cello-oligosaccharides in the apoplastic fraction were monitored using cellobiose dehydrogenase. Both xyloglucan and cello-oligosaccharides appeared to be formed concurrently within 30 min after treatment with the auxin, and the cello-oligosaccharides increased with stem elongation even after 2 h. The total activity of cellulase did not increase for up to 4 h.

Two Pine Endo-β-1,4-Glucanases Are Associated with Rapidly Growing Reproductive Structures

Two cDNA clones encoding endo-β-1,4-glucanases (EGases) were isolated from a radiata pine (Pinus radiata) cDNA library prepared from immature female strobili. The cDNAs PrCel1 (Pinus radiata cellulase 1) and PrCel2 encode proteins 509 and 515 amino acids in length, respectively, including putative signal peptides. Both proteins contain domains conserved in plant and bacterial EGases. The proteins PRCEL1 and PRCEL2 showed strong similarity to each other (76% amino acid identity), and higher similarity to TPP18 (73 and 67%, respectively), an EGase cloned from tomato (Lycopersicon esculentum) pistils, than to any other reported EGases. Northern-blot analyses indicated that both genes displayed a similar pattern of expression. The only significant difference was in the level of expression. In situ hybridizations were used to demonstrate that, within differentiating pine reproductive structures, PrCel1 expression was greatest in microsporangia in pollen strobili and near the developing ovule in the seed strobili. Expression was also found in vegetative tissues, especially in regions experiencing cell elongation, such as the elongating region of root tips. Both proteins have an ability to degrade carboxymethylcellulose in vitro. Genomic-blot analysis indicated the presence of a family of EGase genes in the radiata pine genome, and that PrCel1 and PrCel2 are transcribed from distinct one-copy genes.

Molecular evidence that the asexual industrial fungus Trichoderma reesei is a clonal derivative of the ascomycete Hypocrea jecorina.

The relationship of the important cellulase producing asexual fungus Trichoderma reesei to its putative teleomorphic (sexual) ancestor Hypocrea jecorina and other species of the Trichoderma sect. Longibrachiatum was studied by PCR-fingerprinting and sequence analyses of the nuclear ribosomal DNA region containing the internal transcribed spacers (ITS-1 and ITS-2) and the 5.8S rRNA gene. The differences in the corresponding ITS sequences allowed a grouping of anamorphic (asexual) species of Trichoderma sect. Longibrachiatum into Trichoderma longibrachiatum, Trichoderma pseudokoningii, and Trichoderma reesei. The sexual species Hypocrea schweinitzii and H. jecorina were also clearly separated from each other. H. jecorina and T. reesei exhibited identical sequences, suggesting close relatedness or even species identity. Intraspecific and interspecific variation in the PCR-fingerprinting patterns supported the differentiation of species based on ITS sequences, the grouping of the strains, and the assignment of these strains to individual species. The variations between T. reesei and H. jecorina were at the same order of magnitude as found between all strains of H. jecorina, but much lower than the observed interspecific variations. Identical ITS sequences and the high similarity of PCR-fingerprinting patterns indicate a very close relationship between T. reesei and H. jecorina, whereas differences of the ITS sequences and the PCR-fingerprinting patterns show a clear phylogenetic distance between T. reesei/H. jecorina and T. longibrachiatum. T. reesei is considered to be an asexual, clonal line derived from a population of the tropical ascomycete H. jecorina.

Induction of a Cryphonectria parasitica cellobiohydrolase I gene is suppressed by hypovirus infection and regulated by a GTP-binding-protein-linked signaling pathway involved in fungal pathogenesis.

Extracellular cellulase activity is readily induced when the chestnut blight fungus Cryphonectria parasitica is grown on cellulose substrate as the sole carbon source. However, an isogenic C. parasitica strain rendered hypovirulent due to hypovirus infection failed to secrete detectable cellulase activity when grown under parallel conditions. Efforts to identify C. parasitica cellulase-encoding genes resulted in the cloning of a cellobiohydrolase (exoglucanase, EC 3.2.1.91) gene designated chb-1. Northern blot analysis revealed an increase in cbh-1 transcript accumulation in a virus-free virulent C. parasitica strain concomitant with the induction of extracellular cellulase activity. In contrast, induction of cbh-1 transcript accumulation was suppressed in an isogenic hypovirus-infected strain. Significantly, virus-free C. parasitica strains rendered hypovirulent by transgenic cosuppression of a GTP-binding protein alpha subunit were also found to be deficient in the induction of cbh-1 transcript accumulation.

The anchorage function of CipA (CelL), a scaffolding protein of the Clostridium thermocellum cellulosome.

Enzymatic cellulose degradation is a heterogeneous reaction requiring binding of soluble cellulase molecules to the solid substrate. Based on our studies of the cellulase complex of Clostridium thermocellum (the cellulosome), we have previously proposed that such binding can be brought about by a special "anchorage subunit." In this "anchor-enzyme" model, CipA (a major subunit of the cellulosome) enhances the activity of CelS (the most abundant catalytic subunit of the cellulosome) by anchoring it to the cellulose surface. We have subsequently reported that CelS contains a conserved duplicated sequence at its C terminus and that CipA contains nine repeated sequences with a cellulose binding domain (CBD) in between the second and third repeats. In this work, we reexamined the anchor-enzyme mechanism by using recombinant CelS (rCelS) and various CipA domains, CBD, R3 (the repeat next to CBD), and CBD/R3, expressed in Escherichia coli. As analyzed by non-denaturing gel electrophoresis, rCelS, through its conserved duplicated sequence, formed a stable complex with R3 or CBD/R3 but not with CBD. Although R3 or CBD alone did not affect the binding of rCelS to cellulose, such binding was dependent on CBD/R3, indicating the anchorage role of CBD/R3. Such anchorage apparently increased the rCelS activity toward crystalline cellulose. These results substantiate the proposed anchor-enzyme model and the expected roles of individual CipA domains and the conserved duplicated sequence of CelS.

Relation between cell disruption conditions, cell debris particle size, and inclusion body release

The efficiency of physical separation of inclusion bodies from cell debris is related to cell debris size and inclusion body release and both factors should be taken into account when designing a process. In this work, cell disruption by enzymatic treatment with lysozyme and cellulase, by homogenization, and by homogenization with ammonia pretreatment is discussed. These disruption methods are compared on the basis of inclusion body release, operating costs, and cell debris particle size. The latter was measured with cumulative sedimentation analysis in combination with membrane-associated protein quantification by SDS-PAGE and a spectrophotometric pepticloglycan quantification method. Comparison of the results obtained with these two cell debris quantification methods shows that enzymatic treatment yields cell debris particles with varying chemical composition, while this is not the case with the other disruption methods that were investigated. Furthermore, the experiments show that ammonia pretreatment with homogenization increases inclusion body release compared to homogenization without pretreatment and that this pretreatment may be used to control the cell debris size to some extent. The enzymatic disruption process gives a higher product release than homogenization with or without ammonia pretreatment at lower operating costs, but it also yields a much smaller cell debris size than the other disruption process. This is unfavorable for centrifugal inclusion body purification in this case, where cell debris is the component going to the sediment and the inclusion body is the floating component. Nevertheless, calculations show that centrifugal separation of inclusion bodies from the enzymatically treated cells gives a high inclusion body yield and purity. (C) 2004 Wiley Periodicals, Inc.

Isolation and selection of plant growth-promoting bacteria associated with sugarcane

v. 46, n. 2, p. 149-158, apr./jun. 2016.

Alternativas de uso da borra de café através de hidrólise ácida, enzimática, ou como substrato para a produção das enzimas: lacase e celulase

In the industrial production of soluble coffee, huge amounts of extracted coffee residues are generated; onaverage, for eachtonne of green coffee extracted, 480 kg of coffee ground waste is produced. This is a solid residue currently used to generate energy at the steam boilers from the soluble coffee industry. Some is also used or as fertilizer on agriculture fields. Seeking a better end use, the work reported here aimed to study the viability of hydrolyzing the coffee ground residue for the production of carbohydrates. Hydrolysis was undertaken with hydrochloric acid at different temperatures and pressures, using a water bath or autoclave.An enzymatic hydrolysis with Viscozyme Lwas developed using Whatman filter paper No1 and the optimal conditions were determined using a rotational central composite experimental design (DCCR).The best conditions to hydrolyze filter paper cellulose were 50 FBG (Fungal β-glucanase) of Viscozyme L at pH 4.0 for 1.0 h and 45 ºC. The ground coffee was hydrolyzed under the same conditions as described above for filter paper, however this enzymatic hydrolysis was not efficient. A combination of enzymatic hydrolysis as a pre-treatment for the ground coffee followed by acid hydrolysis using HCl conducted in an autoclave (120 C for 2.0 h) resulted in higher production of glucose as analyzed by HPLC. Another end use of the ground coffee evaluated was as source of substrate in the culture medium to grow Botryosphaeria rhodina MAMB-05 to produce the enzymes laccase and cellulase. Highest enzyme titres obtained were with 8% (w/v) coffee grounds to which was added a minimum salts medium(Vogel), under agitation conditions (180 rpm) at 28ºC. The phenolic compounds present in the coffee grounds appear to have induced laccase by Botryosphaeria rhodina.