Microbial Pretreatment of Cotton Stalks by Phanerochaete chrysosporium for Bioethanol Production

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Title: Microbial Pretreatment of Cotton Stalks by Phanerochaete chrysosporium for Bioethanol Production
Author: Shi, Jian
Advisors: Mari S. Chinn, Committee Member
Ralph A. Dean, Committee Member
Michael D. Boyette, Committee Co-Chair
Ratna R. Sharma-Shivappa, Committee Chair
Abstract: Lignocellulosic biomass has been recognized as a widespread, potentially low cost renewable source of mixed sugars for fermentation to fuel ethanol. Pretreatment, as the first step towards conversion of lignocellulose to ethanol, remains one of the main barriers to technical and commercial success of the processing technology. Existing pretreatment methods have largely been developed on the basis of physiochemical technologies which are considered relatively expensive and usually involve adverse environmental impacts. In this study, an environmentally benign alternative, microbial pretreatment using Phanerochaete chrysosporium, was explored to degrade lignin in cotton stalks and facilitate their conversion into ethanol. Two submerged liquid pretreatment techniques (SmC), shallow stationary and agitated cultivation, at three inorganic salt concentrations (no salts, modified salts without Mn2+, modified salts with Mn2+) were compared by evaluating their pretreatment efficiencies. Shallow stationary cultivation with no salt was superior to other pretreatment conditions and gave 20.7% lignin degradation along with 76.3% solids recovery and 29.0% carbohydrate availability over a 14 day period. The influence of substrate moisture content (65%, 75% and 80% M.C. wet-basis), inorganic salt concentration (no salts, modified salts without Mn2+, modified salts with Mn2+) and culture time (0-14 days) on pretreatment effectiveness in solid state (SSC) systems was also examined. It was shown that solid state cultivation at 75% M.C. without salts was the most preferable pretreatment resulting in 27.6% lignin degradation, 71.1% solids recovery and 41.6% carbohydrate availability over a period of 14 days. A study on hydrolysis and fermentation of cotton stalks treated microbially using the most promising SmC (shallow stationary, no salts) and SSC (75% moisture content, no salts) methods resulted in no increase in cellulose conversion with direct enzyme application (10.98% and 3.04% for SmC and SSC pretreated samples, respectively) compared with untreated cotton stalk samples (17.93%). Washing of pretreated cotton stalks alone caused no significant increase in cellulose conversion. However, a heat treatment (autoclaving) followed by washing remarkably improved (P<0.05) cellulose conversion to 14.94% and 17.81% for SmC and SSC pretreatment, respectively. Mathematical models describing holocellulose consumption, lignin degradation, cellulase and ligninolytic enzyme production, and oxygen uptake associated with the growth of P. chrysosporium during 14 days fungal pretreatment were developed. For SmC pretreatment, model parameters were estimated by nonlinear regression and validated using an independent set of experimental data. Models yielded sufficiently accurate predictions for holocellulose consumption (R2=0.9772 and 0.9837, 1d and 3d oxygen flushing, respectively), lignin degradation (R2=0.9879 and 0.8682) and ligninolytic enzyme production (R2=0. 8135 and 0.9693) under both 1 and 3 d oxygen flushing conditions. However, the prediction capabilities for fungal growth (1d and 3 d), cellulase production (3d) and oxygen uptake (3d) were limited. For SSC, the models were established in three phases (I: day 0-4, II: day 4-7, III: day 7-14). After validation it was shown that the developed models can yield sufficiently accurate predictions for fungal growth (R2 =0.9724), holocellulose consumption (R2 =0.9686), lignin degradation (R2=0.9309) and ligninolytic enzyme production (R2=0.9203); however predictions of cellulase production were fair (R2=0.6133). Although significant delignification occurred during fungal pretreatment indicating the presence of ligninolytic enzymes, common spectrophotometric enzyme assays failed to detect lignin peroxidase (LiP) and manganese peroxidase (MnP) activities in fungal pretreatment cultures. Efforts were made to overcome the drawbacks of standardized assays by performing protein gel electrophoresis and crude enzyme delignification studies. Results from this research are expected to be beneficial in the development of pretreatment technologies that are environment friendly and utilize naturally occurring microorganisms.
Date: 2007-07-31
Degree: PhD
Discipline: Biological and Agricultural Engineering
URI: http://www.lib.ncsu.edu/resolver/1840.16/3258

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