| Author: | Rehman, Shazia |
| Title: | Sustainable 2,3-butanediol production from lignocellulosic biorefinery : characterization, optimization, and omics-based studies on newly isolated fermentation strains |
| Advisors: | Leu, Shao-yuan Ben (CEE) |
| Degree: | Ph.D. |
| Year: | 2022 |
| Subject: | Biomass energy Biomass -- Refining |
| Department: | Department of Civil and Environmental Engineering |
| Pages: | xxiv, 168 pages : color illustrations |
| Language: | English |
| Abstract: | The gradual substitution of petroleum compounds with biomass-derived products is a critical research aim toward sustainability. Biorefinery plays a prime role in the bioconversion of lignocellulosic biomass into 2,3-butanediol (2,3-BDO), a bulk chemical and fuel, and applied in aviation, polymer, food, pharmaceutical, and cosmetic industries. However, microbial production of 2,3-BDO via fermentation technology has encountered a few limitations, such as incomplete valorization of lignocellulosic biomass; ineffective utilization of hemicelluloses by the fermentation bacteria; and effects on sustainability and commercial viability. In this dissertation, two bacterial strains, Klebsiella pneumoniae strain PM2 and Enterococcus faecalis strain OPS1, were newly isolated and characterized for their tremendous ability to produce 2,3-BDO by valorizing the hemicellulosic (xylose) sugars. Sustainability challenges of 2,3-BDO biorefinery and genomic insights of xylose assimilation were also evaluated in the bioproduction processes for 2,3-BDO. The first bacterial strain, K. pneumoniae PM2 isolated from palm oil mill effluent (POME), was introduced to convert the biomass "whole sugars" into high value 2,3-BDO in biorefinery. The fermentation conditions were optimized (30 °C, pH 7, and 150 rpm agitation) using glucose for maximum 2,3-BDO production in batch systems. A sulfite pretreated oil palm empty fruit bunches (EFB) whole slurry (substrate hydrolysate of 119.5 g/L total glucose mixed with pretreatment spent liquor of 80 g/L xylose) was fed to strain PM2 for fermentation. The optimized biorefinery process resulted in 75.03 ± 3.17 g/L of 2,3-BDO with 0.78 ± 0.33 g/L/h productivity and 0.434 g/g yield (87% of theoretical value) via a modified staged separate hydrolysis and fermentation process. This result is equivalent to approximately 135 kg 2,3-BDO and 14.5 kg acetoin precursors from 1 ton of EFB biomass without any wastage of both C6 and C5 sugars. The progress of the development of 2,3-BDO biorefinery has also been affected by many factors, i.e., prices of crude oil, food, and carbon. To quantify the environmental and social impacts of the technologies, the second study constructed a sustainability index for calculating two new bio-BDO production processes with oil palm empty fruit bunches as an example feedstock. The performance of organosolv pretreatment using butanediol was compared with the whole slurry conversion process using sulfite pretreated biomass (from the first study), over the petroleum refinery, and first-generation biorefinery with food crop feedstock. The organosolv biorefinery process successfully converted the biomass into 77.3 ± 1.63 g/L of bio-BDO (0.45 g/g yield), which is slightly higher (5.5%) than that of the sulfite-based process. The integration of biorefinery techniques, with oil palm farming, shall result in 6.8 kg-CO2 and 0.5 kg-food benefits per kg 2,3-BDO produced, yielding a sustainability index of 7.30. The food index for the first-generation biorefinery is -1.04 kg food per kg 2,3-BDO produced. Using empty fruit bunches for 2,3-BDO production could save 1.54 kg of food crop consumption, which turns the "food vs. fuel competition" into a "food plus fuel nexus". Sustainable bioconversion of plant biomass sugars for 2,3-BDO production is a carbon-neutral practice, however, its economic feasibility has been hindered by inefficient utilization of xylose. Our third study evaluated the second novel strain Enterococcus faecalis OPS1, isolated from palm oil-polluted soil, for 2,3-BDO production through xylose assimilation. While this wild-type strain OPS1 was suppressed with an increase in xylose concentration (60 g/L), strain OPS2 constructed using an adaptive laboratory evolution and achieved a desirable 2,3-BDO production as a result of increased xylose (>100 g/L) tolerance. Whole-genome sequencing analysis reconstructed the metabolic pathways facilitating xylose assimilation and 2,3-BDO production in these two novel strains that evolved within from a lactic acid-producing genus Enterococcus. A rpiB gene encoded for ribose-5-phosphate isomerase B enzyme of xylose metabolism in these two strains was identified to be horizontally acquired from a different family Enterobacteriaceae. The detection of a bacteriocin gene UviB and an antitoxin gene hicB in strains OPS1 and OPS2 which are absent in the E. faecalis type strain indicates their beneficial probiotic and antitoxin features. A mutation in uge (UDP-xylose 4-epimerase) was identified between strain OPS1 and OPS2 which may cater to the high-xylose stress. The evolved E. faecalis OPS2 showed a 3-fold increase in xylose consumption rate (2.19 g/L/h) and 0.67 g/L/h of 2,3-BDO production rate than OPS1. For the first time, a maximum of 65.3 g/L 2,3-BDO was successfully produced in fed-batch xylose fermentation by OPS2 strain. These results provide genomic insights into Enterococci xylose regulatory metabolism and pave the way for industrial implication as a microbial biocatalyst in a 2,3-BDO biorefinery. |
| Rights: | All rights reserved |
| Access: | open access |
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