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A thermophilic Geobacillus bacterial strain, WSUCF1 contains different carbohydrate-active enzymes (CAZymes) capable of hydrolyzing hemicellulose in lignocellulosic biomass. We used proteomic, genomic, and bioinformatic tools, and genomic data to analyze the relative abundance of cellulolytic, hemicellulolytic, and lignin modifying enzymes present in the secretomes. Results showed that CAZyme profiles of secretomes varied based on the substrate type and complexity, composition, and pretreatment conditions. The enzyme activity of secretomes also changed depending on the substrate used. The secretomes were used in combination with commercial and purified enzymes to carry out saccharification of ammonia fiber expansion (AFEX)-pretreated corn stover and extractive ammonia (EA)-pretreated corn stover. When WSUCF1 bacterial secretome produced at different conditions was combined with a small percentage of commercial enzymes, we observed efficient saccharification of EA-CS, and the results were comparable to using a commercial enzyme cocktail (87% glucan and 70% xylan conversion). It also opens the possibility of producing CAZymes in a biorefinery using inexpensive substrates, such as AFEX-pretreated corn stover and Avicel, and eliminates expensive enzyme processing steps that are used in enzyme manufacturing. Implementing in-house enzyme production is expected to significantly reduce the cost of enzymes and biofuel processing cost.
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Adaptive Enrichment of a Thermophilic Bacterial Isolate for Enhanced Enzymatic Activity
The mimicking of evolution on a laboratory timescale to enhance biocatalyst specificity, substrate utilization activity, and/or product formation, is an effective and well-established approach that does not involve genetic engineering or regulatory details of the microorganism. The present work employed an evolutionary adaptive approach to improve the lignocellulose deconstruction capabilities of the strain by inducing the expression of laccase, a multicopper oxidase, in Geobacillus sp. strain WSUCF1. This bacterium is highly efficient in depolymerizing unprocessed lignocellulose, needing no preprocessing/pretreatment of the biomasses. However, it natively produces low levels of laccase. After 15 rounds of serially adapting this thermophilic strain in the presence of unprocessed corn stover as the selective pressure, we recorded a 20-fold increase in catalytic laccase activity, at 9.23 ± 0.6 U/mL, in an adapted yet stable strain of Geobacillus sp. WSUCF1, compared with the initial laccase production (0.46 ± 0.04 U/mL) obtained with the unadapted strain grown on unprocessed corn stover before optimization. Chemical composition analysis demonstrated that lignin removal by the adapted strain was 22 wt.% compared with 6 wt.% removal by the unadapted strain. These results signify a favorable prospect for fast, cost competitive bulk production of this thermostable enzyme. Also, this work has practical importance, as this fast adaptation of the Geobacillus sp. strain WSUCF1 suggests the possibility of growing industrial quantities of Geobacillus sp. strain WSUCF1 cells as biocatalysts on reasonably inexpensive carbon sources for commercial use. This work is the first application of the adaptive laboratory evolution approach for developing the desired phenotype of enhanced ligninolytic capability in any microbial strain.
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- Award ID(s):
- 1849206
- PAR ID:
- 10251443
- Date Published:
- Journal Name:
- Microorganisms
- Volume:
- 8
- Issue:
- 6
- ISSN:
- 2076-2607
- Page Range / eLocation ID:
- 871
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/microorganisms8060871
