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Formulation of an enzyme cocktail, HoloMix, using cellulolytic and xylanolytic enzyme core-sets for effective degradation of various pre-treated hardwoods

  • Authors: Malgas, Samkelo
  • Date: 2018
  • Subjects: Biomass , Cellulase , Hardwoods , Xylanases
  • Language: English
  • Type: Doctoral theses , text
  • Identifier: http://hdl.handle.net/10962/62827 , vital:28297 , DOI https://doi.org/10.21504/10962/62827
  • Description: Currently, there is a growing interest in utilising hardwoods as feedstocks for bioethanol production due to the vast advantages they have over other feedstocks for fermentable sugar production. In this study, two selected hardwoods, Acacia and Populus spp., were subjected to two pre-treatment processes (Sodium chlorite delignification and Steam explosion) and compared with respect to how these pre-treatments affect their enzymatic saccharification. Hardwoods were selected for this study, because hardwoods are easier to delignify when compared to softwoods, and therefore their polysaccharides are more easily accessible by enzymes for the purpose of producing fermentable sugars. Currently available commercial enzyme mixtures have been developed for optimal hydrolysis of acid-pre-treated corn stover and are therefore not optimal for saccharification of pre-treated hardwoods. In this work, we attempted the empirical design of a hardwood specific enzyme cocktail, HoloMix. Firstly, a cellulolytic core-set, CelMix (in a ratio of Egl 68%: Cel7A 17%: Cel6A 6%: Bgl1 9%), for the optimal release of glucose, and a xylanolytic core-set, XynMix (in a ratio of Xyn2A 60%: XT6 20%: AguA 11%: SXA 9%), for the optimal release of xylose, were formulated using an empirical enzyme ratio approach after biochemically characterising these enzymes. As it is well ̶ known that biomass pre-treatment may result in the generation of compounds that hamper enzymatic hydrolysis and microbial fermentation, the effects of these compounds on CelMix and XynMix were evaluated. Using the optimised CelMix and XynMix cocktails, a HoloMix cocktail was established for optimal reducing sugar, glucose and xylose release from the various pre-treated hardwoods. For delignified biomass, the optimized HoloMix consisted of CelMix to XynMix at 75% to 25% protein loading, while for the untreated and steam exploded biomass the HoloMix consisted of CelMix to XynMix at 93.75% to 6.25% protein loading. Sugar release by the HoloMix at a loading of 27.5 mg protein/g of biomass (or 55 mg protein/g of glucan) after 24 h gave 70-100% sugar yield. Treatment of the hardwoods with a laccase from Agaricus bisporus, especially wood biomass with a higher proportion of lignin, significantly improved saccharification by the formulated HoloMix enzyme cocktails. This study provided insights into the enzymatic hydrolysis of various pre-treated hardwood substrates and assessed whether the same lignocellulolytic cocktail can be used to efficiently hydrolyse different hardwood species. The present study also demonstrated that the hydrolysis efficiency of the optimised HoloMix was comparable to (if not better) than commercial enzyme preparations during hardwood biomass saccharification. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
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  • Date Issued: 2018

An investigation into the synergistic action of cellulose-degrading enzymes on complex substrates

Nanofiber immobilized cellulases and hemicellulases for fruit waste beneficiation

An investigation into the synergistic association between the major Clostridium cellulovorans cellulosomal endoglucanase and two hemicellulases on plant cell wall degradation

  • Authors: Beukes, Natasha
  • Date: 2008
  • Subjects: Clostridium , Cellulose , Hemicellulose , Cellulase , Biomass conversion , Biomass energy -- South Africa , Energy crops -- South Africa , Bagasse -- Biodegradation , Pineapple -- Biodegradation
  • Language: English
  • Type: Thesis , Masters , MSc
  • Identifier: vital:3968 , http://hdl.handle.net/10962/d1004027 , Clostridium , Cellulose , Hemicellulose , Cellulase , Biomass conversion , Biomass energy -- South Africa , Energy crops -- South Africa , Bagasse -- Biodegradation , Pineapple -- Biodegradation
  • Description: The cellulosome is a multimeric enzyme complex that has the ability to metabolise a wide variety of carbonaceous compounds. Cellulosomal composition may vary according to the microbe’s nutritional requirement and allows for the anaerobic degradation of complex substrates. The complex substrates of interest in this research study were sugarcane bagasse and pineapple fibre waste, as they represent two important lignocellulosic, South African agricultural crops. The effective degradation of complex plant biomass wastes may present a valuable source of renewable compounds for the production of a variety of biofuels, for example bioethanol, and a variety of biocomposites of industrial importance. The identification of renewable energy sources for the production of biofuels is becoming increasingly important, as a result of the rapid depletion of the fossil fuels that are traditionally used as energy sources. An effective means of completely degrading lignocellulose biomass still remains elusive due to the complex heterogeneity of the substrate structure, and the fact that the effective degradation of the substrate requires a consortium of enzymes. The cellulosome not only provides a variety of enzymes with varying specificities, but also promote a close proximity between the catalytic components (enzymes). The close proximity between the enzymes promotes the synergistic degradation of complex plant biomass for the production of valuable energy products. Previous synergy studies have focused predominantly on the synergistic associations between cellulases; however, the synergy between hemicellulases has occasionally been documented. This research project established the synergistic associations between two Clostridium cellulovorans hemicellulases that may be incorporated into the cellulosome and a cellulosomal endoglucanase that is conserved in all cellulosomes. This research study indicated that there was indeed a synergistic degradation of the complex plant biomass (sugarcane bagasse and pineapple fibre). The degrees of synergy and the ratio of the enzymes varied between the two complex substrates. The initial degradation of the bagasse required the presence of all the enzymes and proceeded at an enhanced rate under sulphidogenic conditions; however, there was a low production of fermentable sugars. The low quantity of fermentable sugars produced by the degradation of the bagasse may be related to the chemical composition of the substrate. The sugarcane contains a high percentage of lignin forming a protective layer around the holocellulose, thus the glycosidic bonds are shielded extensively from enzymatic attack. In comparison, the initial degradation of the pineapple fibre required the action of hemicellulases, and proceeded at an enhanced rate under sulphidogenic conditions. The initial degradation of the pineapple fibre produced a substantially larger quantity of fermentable sugars in comparison to the bagasse. The higher production of fermentable sugars from the degradation of the pineapple fibre may be explained by the fact that this substrate may have a lower percentage of lignin than the bagasse, thus allowing a larger percentage of the glycosidic bonds to be exposed to enzymatic attack. The data obtained also indicated that the glycosidic bonds from the hemicellulosic components of the pineapple fibre shielded the glycosidic bonds of the cellulose component. The identification of the chemical components of the different substrates may allow for the initial development of an ideal enzyme complex (designer cellulosome) with enzymes in an ideal ratio with optimal synergy that will effectively degrade the complex plant biomass substrate.
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  • Date Issued: 2008

Isolation of a Clostridium Beijerinckii sLM01 cellulosome and the effect of sulphide on anaerobic digestion

  • Authors: Mayende, Lungisa
  • Date: 2007
  • Subjects: Cellulose , Clostridium , Cellulase , Sulfides
  • Language: English
  • Type: Thesis , Masters , MSc
  • Identifier: vital:3973 , http://hdl.handle.net/10962/d1004032 , Cellulose , Clostridium , Cellulase , Sulfides
  • Description: Cellulose is the most abundant and the most resistant and stable natural organic compound on earth. Enzyme hydrolysis is difficult because of its insolubility and heterogeneity. Some (anaerobic) microorganisms have overcome this by having a multienzyme system called the cellulosome. The aims of the study were to isolate a mesophilic Clostridium sp. from a biosulphidogenic bioreactor, to purify the cellulosome from this culture, to determine the cellulase and endoglucanase activities using Avicel and carboxymethylcellulose (CMC) as substrates and the dinitrosalicyclic (DNS) method. The organism was identified using 16S rDNA sequence analysis. The sequence obtained indicated that a strain of Clostridium beijerinckii was isolated. The cellulosome was purified from the putative C. beijerinckii sLM01 host culture using affinity chromatography purification and affinity digestion purification procedures. The cellulosomal and non-cellulosomal fractions of C. beijerinckii sLM01 were separated successfully, but the majority of the endoglucanase activity was lost during the Sepharose 4B chromatography step. These cellulosomal and non-cellulosomal fractions were characterised with regards to their pH and temperature optima and effector sensitivity. Increased additions of sulphide activated the cellulase activity of the cellulosomal and non-cellulosomal fractions up to 700 %, while increased additions of sulphate either increased the activity slightly or inhibited it dramatically, depending on the cellulosomal and non-cellulosomal fractions. Increased additions of cellobiose, glucose and acetate inhibited the cellulase and endoglucanase activities. pH optima of 5.0 and 7.5 were observed for cellulases and 5.0 for endoglucanases of the cellulosomal fraction. The noncellulosomal fraction exhibited a pH optimum of 7.5 for both cellulase and endoglucanase activities. Both fractions and enzymes exhibited a temperature optimum of 30 °C. The fundamental knowledge gained from the characterisation was applied to anaerobic digestion, where the effect of sulphide on the rate-limiting step was determined. Sulphide activated cellulase and endoglucanase activities and increased the % chemical oxygen demand (COD) removal rate. Levels of volatile fatty acids (VFAs) were higher in the bioreactor containing sulphide, substrate and C. beijerinckii. Sulphide therefore accelerated the rate-limiting step of anaerobic digestion.
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  • Date Issued: 2007

The role of cellulases and glucohydrolases in the solubilisation of primary sewage sludge

  • Authors: Ngesi, Nosisa
  • Date: 2002 , 2013-05-09
  • Subjects: Sewage sludge , Sewage sludge digestion , Cellulase , Glucosidase inhibitors , Hydrolases , Sulfates
  • Language: English
  • Type: Thesis , Masters , MSc
  • Identifier: vital:4080 , http://hdl.handle.net/10962/d1007454 , Sewage sludge , Sewage sludge digestion , Cellulase , Glucosidase inhibitors , Hydrolases , Sulfates
  • Description: Biological sulph ate reduction has been identi fied as a potentially valuable process for removing sulphate and heavy metals from indllstrial effluents. The role of sulphate reducing bacteria (SRB) in this process has attracted the attention of biotechnologists and recently of enzymologists due to its fundamental properties and possible role in AMD bioremediation. These obligatory anaerobic sulphate-reducing bacteria are commonly known to dissimilate sulphate for energy. Under anaerobic conditions SRB oxidize simple organic compounds such as lactic acid with the sulphate and thereby generate hydrogen sulphide (a stTong reducing agent) and bicarbonate ions. The hydrogen sulphide in turn reacts with contaminant metals contained in AMD and precipitates them out of solution as metal sulphides. Bicarbonate ions neutralize AMD by reaction with protons to form carbon dioxide and water. Organic matter in the municipal sewage sludge has been identified as a potential source of electron donors for su lphate reduction. However, this organic matter is in the polymeric form that cannot be util ised by SRB. The latter depend on the activities of other hydrolytic bacteria for the degradation of complex polymers. Hence the availability of these monomeric substrates is a major factor, which may constrain further process development and is considered a rate-limiting step. Thi s study is therefore undertaken to investigate the bacterial glucohydrolase enzymes involved in the digestion of the polysaccharides present in the sewage sludge with specific interest in cellulases and/or p-glucosidase enzymes. The goals of the research are to: isolate, identify, purify and quantify these enzymes; study their distribution with respect to time, pH, and temperature; maximize and quantify the hydrol ys is products; study whether sulphide and sulphate have an enhancing or an inhibitory effect on the activity of enzymes; optimize the enzyme activity against substrate and/or product inhibition and soluble heavy metal salts. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
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  • Date Issued: 2002
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