Title: Improving a recombinant Zymomonas mobilis strain 8b through continuous adaptation on dilute acid pretreated corn stover hydrolysate

Abstract

Complete conversion of the major sugars of biomass including both the C₅ and C₆ sugars is critical for biofuel production processes. Several inhibitory compounds like acetate, hydroxymethylfurfural (HMF), and furfural are produced from the biomass pretreatment process leading to ‘hydrolysate toxicity,’ a major problem for microorganisms to achieve complete sugar utilization. Therefore, development of more robust microorganisms to utilize the sugars released from biomass under toxic environment is critical. In this study, we use continuous culture methodologies to evolve and adapt the ethanologenic bacterium Zymomonas mobilis to improve its ethanol productivity using corn stover hydrolysate. The results are the following: A turbidostat was used to adapt the Z. mobilis strain 8b in the pretreated corn stover liquor. The adaptation was initiated using pure sugar (glucose and xylose) followed by feeding neutralized liquor at different dilution rates. Once the turbidostat reached 60% liquor content, the cells began washing out and the adaptation was stopped. Several ‘sub-strains’ were isolated, and one of them, SS3 (sub-strain 3), had 59% higher xylose utilization than the parent strain 8b when evaluated on 55% neutralized PCS (pretreated corn stover) liquor. Using saccharified PCS slurry generated by enzymatic hydrolysis from 25% solids loading, SS3 generated an ethanolmore » yield of 75.5% compared to 64% for parent strain 8b. Furthermore, the total xylose utilization was 57.7% for SS3 versus 27.4% for strain 8b. To determine the underlying genotypes in these new sub-strains, we conducted genomic resequencing and identified numerous single-nucleotide mutations (SNPs) that had arisen in SS3. We further performed quantitative reverse transcription PCR (qRT-PCR) on genes potentially affected by these SNPs and identified significant down-regulation of two genes, ZMO0153 and ZMO0776, in SS3 suggesting potential genetic mechanisms behind SS3’s improved performance. In conclusion, we have adapted/evolved Z. mobilis strain 8b for enhanced tolerance to the toxic compounds present in corn stover hydrolysates. The adapted strain SS3 has higher xylose utilization rate and produce more ethanol than the parent strain. We have identified transcriptional changes which may be responsible for these phenotypes, providing foundations for future research directions in improving Z. mobilis as biocatalysts for the production of ethanol or other fuel precursors.« less

Authors:

Mohagheghi, Ali ^[1]; Linger, Jeffrey G. ^[1]; Yang, Shihui ^[1]; Smith, Holly ^[1]; Dowe, Nancy ^[1]; Zhang, Min ^[1]; Pienkos, Philip T. ^[1]

---

+ Show Author Affiliations

1. National Renewable Energy Lab. (NREL), Golden, CO (United States). National Bioenergy Center

Publication Date:

2015-03-31

Research Org.:

National Renewable Energy Lab. (NREL), Golden, CO (United States)

Sponsoring Org.:

USDOE Office of Energy Efficiency and Renewable Energy (EERE), Sustainable Transportation Office. Bioenergy Technologies Office

OSTI Identifier:

1220621

Report Number(s):

NREL/JA-5100-62769
Journal ID: ISSN 1754-6834

Grant/Contract Number:  

AC36-08GO28308

Resource Type:

Accepted Manuscript

Journal Name:

Biotechnology for Biofuels

Additional Journal Information:

Journal Volume: 8; Journal ID: ISSN 1754-6834

Publisher:

BioMed Central

Country of Publication:

United States

Language:

English

Subject:

09 BIOMASS FUELS; 59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Zymomonas mobilis; xylose; pretreated corn stover; adaptation; turbidostat; next generation sequencing (NGS)