Graduate Thesis Or Dissertation

Hydrogen peroxide oxidation of annual ryegrass lignin and utilization of the products by Rhodotorula mucilaginosa

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  • Annual ryegrass (Lolium multiflorum) a major grass seed crop in the Willamette Valley, Oregon, is presently burned in the fields after harvest, causing considerable air pollution and general wastage of a material which could be used in the manufacture of economically important products. The cellulosic component of straw is relatively susceptible to biodegradation but the lignin fraction, approximately 15%, is extremely resistant. Modification of the lignin to relieve recalcitrance would be valuable in the complete biological degradation of the straw and under controlled conditions, the cellulosic and modified lignin components could be used as substrates for the propagation of microbial populations leading to the production of single cell protein. Klason lignin prepared from Lolium multiflorum was subjected as a 2.5% w/v suspension, to 1.0, 2. 5, 5.0, 10 and 15% H₂O₂ concentrations at pH 1 and 100 C until the peroxide was consumed. Rhodotorula mucilaginosa, which was unable to utilize the nonoxidized lignin as a sole source of carbon, was used to monitor the effect of lignin oxidation on growth. Lignin oxidation substrates obtained from all concentrations of H₂O₂ exhibited increased growth response to the Rhodotorula. The substrate from the 2.5% H₂O₂ treatment gave the maximum growth response of yeast in terms of cellular dry weight. A soluble polymer-like material which formed during the initial stages of oxidation was not utilized and reduced the overall efficiency of substrate conversion to cellular material. The maximum conversion efficiencies, 21 and 86%, measured as dissolved lignin solids and total carbon respectively occurred in the 10% H₂O₂ substrate. However, maximum growth, 5%, relative to total dissolved lignin was observed in the 1.0% H₂O₂ substrate. Gas-liquid chromatography-Rapid-Scan mass spectrometry and gas-liquid and thin-layer chromatography were used to determine lignin oxidation products. Succinic, lactic, maleic,malonic and oxalic acids were identified and methoxyhydroquinone, acetaldehyde, methyl alcohol, protocatechualdehyde, p-hydroxybenzaldehyde, vanillin, vanillic acid, and p-hydroxybenzoic acid tentatively identified. All of these compounds except methyl alcohol, oxalic and maleic acids and methoxyhydroquinone were utilized by the Rhodotorula strain. The appearance of protocatechualdehyde suggested that the degradation mechanism by H₂O₂ involved hydroxylation of the aromatic fragments. However, lack of muconic acids and the presence of succinic, oxalic, maleic, and malonic acids showed that a parahydroxylation mechanism was predominant. The occurrence of lactic acid was postulated to occur from the C₃-side chain of the phenylpropanoid building block. This was supported by the oxidation of guaiacylglycerol-β-coniferyl ether and p-hydroxybenzoic, vanillic, and syringic acids. Lactic acid and guaiacol were obtained from the ether whereas only guaiacol was obtained from vanillic acid while the other two acids gave neither guaiacol nor lactic acid. Formation of quinones and succinic, maleic, malonic and oxalic acids from p-hydroxybenzoic, vanillic, and syringic acids during H₂O₂ oxidation indicated that aromatic carboxylic acids were precursors to hydroquinones which were transformed to non-aromatic quinoid structures prior to cleavage to dicarboxylic acids. The mechanism was postulated to occur by a modified Dakin reaction. The ability of Rhodotorula mucilaginosa to utilize the oxidation products obtained during treatment of the lignin with H₂O₂ suggests that H₂O₂ oxidation of lignin can greatly improve biodegradation. A similar approach on other lignins utilizing different oxidizing agents and systems should be explored to obtain the maximum oxidative efficiency.
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