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
Effects of BT Maize (MON810) crop and its residues on selected soil biological properties and N and P release in a sandy loam soil from Alice, Eastern Cape, South Africa
- Authors: Landzela, Besule
- Date: 2013
- Subjects: Bacillus (Bacteria) , Bacillus thuringiensis , Corn -- Planting , Biomass , Plant proteins , Enzymes , Soil fertility , Crop residue management
- Language: English
- Type: Thesis , Masters , MSc Agric (Crop Science)
- Identifier: vital:11870 , http://hdl.handle.net/10353/d1007542 , Bacillus (Bacteria) , Bacillus thuringiensis , Corn -- Planting , Biomass , Plant proteins , Enzymes , Soil fertility , Crop residue management
- Description: There are apprehensions that genetic modification of maize with Bacillus thuringiensis (Bt) may have negative effects on soil biodiversity, ecosystem processes and functions. This study aimed at determining the effect of Bt maize crop, Bt maize residues and its genetic modification on microbial biomass carbon (MBC), selected enzyme activities, vesicular arbuscular mycorrhizal (VAM) fungi and N and P release patterns. The study was conducted under field, glasshouse and laboratory conditions. In 2010/2011 season, four maize cultivars; DKC 61-25B (Bt), DKC 61-24 (non-Bt), PAN 6Q-321B (Bt) and PAN6777 (non-Bt) were planted. Determination of MBC, enzyme activities and fungal spore count was done at 42, 70, and 105 days after planting (DAP). A loam soil amended with Bt or non-Bt maize leaf residues from a study of 2009/2010 season was incubated to investigate effects of Bt maize residues on MBC and soil enzyme activities. Leaf residues of Bt and non-Bt maize cultivars (DKC 61-25B, DKC 61-24, PAN 6Q-321B and PAN6777) were used and soil without residues was used as a control. Samples were collected at 7, 28 and 56 days of incubation (DOI). An incubation study was also carried out in the laboratory to determine the effect of Bt maize residues (i.e. leaf, stem and root) and its genetic modification on N and P release patterns. Residues of DKC 61-25B, DKC 61-24, PAN 6Q-321B and PAN6777and soil without residues as a control were incubated in the laboratory. After destructive sampling at 0, 7, 14, 28, and 56 DOI, N in the form of NH4-N and NO3-N and P mineralisation were determined. Amendment of soil with residues enhanced MBC (p < 0.05) at all the sampling dates. For example MBC increased from 95 in the control to 146.3 mg/kg in the DKC 61-25B treatment at the end of the glasshouse trial. In the field DKC 61-25B had 9.1 mg/kg greater MBC than DKC 61-24, while PAN 6Q-321B had 23.9 mg/kg more MBC than PAN6777 at the end of the trial. However, no differences (p < 0.05) were observed in enzyme activities under field and glasshouse conditions except for dehydrogenase that had greater activity where DKC 61-25B and PAN 6777 were grown. There were no differences between the type of residues (Bt and non-Bt) on enzyme activities tested. However, differences were observed among the sampling dates. No effects of Bt maize crop on fungal spore count were observed. Similarly no differences were observed in leaf, stem and root tissues composition between Bt and non-Bt maize cultivars. Net N and P mineralisation from Bt maize cultivars did not differ from that of non-Bt maize cultivars. However, differences were observed among the cultivars. The results of this study suggested that Bt maize with Bt MON810 event can be grown in the central region of the Eastern Cape (EC), South Africa without affecting MBC, soil enzyme activities, VAM, and release of N and P nutrients from its residues.
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- Date Issued: 2013
Effect of BT maize on earthworm activity, silage quality and residue decomposition in the central Eastern Cape
- Authors: Kamota, Agathar
- Date: 2011
- Subjects: Corn -- South Africa -- Eastern Cape , Corn -- Silage , Bacillus thuringiensis , Earthworm culture , Biomass , Plant proteins
- Language: English
- Type: Thesis , Masters , MSc Agric (Crop Science)
- Identifier: vital:11863 , http://hdl.handle.net/10353/413 , Corn -- South Africa -- Eastern Cape , Corn -- Silage , Bacillus thuringiensis , Earthworm culture , Biomass , Plant proteins
- Description: There are concerns that genetic modification of maize with Bacillus thuringiensis may influence its structural and chemical composition which, together with the Cry1Ab proteins, may affect agro-ecosystem processes and feed quality. This study was aimed at investigating the expression of Cry1Ab protein in leaves, stems and roots of Bt maize and the effect of genetic modification (MON810) on activity of earthworms, silage quality and decomposition of residues in the field. In 2009/10 four maize cultivars; DKC61-25B, DKC61-24, PAN6Q-321B and PAN6777 were planted. Expression of Cry1Ab in leaves, stems and roots was analyzed at 6, 8, 10 and 12 weeks after planting (WAP). Earthworms were also sampled from the same treatments at 6, 9 and 18 WAP. Two silage experiments were conducted using maize cultivars, DKC80-12B and DKC80-10 produced in the 2008/09 season and DKC61-25B, DKC61-24, PAN6Q-321B and PAN6777, produced in 2009/10. The silage was sampled at 0, 2, 4, 8, 15 and 42 days in 2008/09 and 0, 8 and 42 days in 2009/10 and analyzed for Ash Free Dry Matter, Crude Fiber, Neutral Detergent Fiber, Acid Detergent Fiber, Acid Detergent Lignin, Crude Protein and Total Digestible Nutrients. Two litter-bag decomposition studies were also carried out (i) in 2008 (surface applied) using maize cultivars DKC80-12B, DKC80-10 and DKC6-125 residues and (ii) in 2009 (soil incorporated) using DKC75-15B, CRN3505, PAN6Q-321B v and PAN6Q-121. Ash-free dry matter and Cry1Ab protein were measured throughout the incubation time. There were no differences between DKC61-25B and PAN6Q-321B in terms of expression of Cry1Ab in leaves, stems and roots over time. The Cry1Ab expression levels were in decreasing order: leaves > stems > roots. No effects of Bt maize on earthworm numbers and biomass were observed. There were no differences in all silages parameters except NDF and ADF, which were higher in the Bt maize silage than that of the non-Bt maize from the 2008/09 season. The Cry1Ab levels were essentially not reduced during ensiling. The maize residues (both Bt and non-Bt maize) degraded to similar levels, either when surface-applied or incorporated into soil but soil-incorporated residues decomposed faster than surface-applied ones. Cry1Ab degraded as the plant matrix decomposed. The findings suggested that maize genetically modified with the Bt MON810 event can be grown in the Central Eastern Cape without affecting earthworm numbers and biomass, silage quality and decomposition of maize residues.
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- Date Issued: 2011