Cold plasma pretreatment reinforces the lignocellulose-derived aldehyde inhibitors tolerance and bioethanol fermentability for Zymomonas mobilis

Abstract

Background: Lignocellulose-derived aldehyde inhibitors seriously blocked the biorefinery of biofuels and biochemicals. To date, the economic production of lignocellulose-based products heavily relied on high productivities of fermenting strains. However, it was expensive and time-consuming for the achievable rational modification to strengthen stress tolerance robustness of aldehyde inhibitors. Here, it aimed to improve aldehyde inhibitors tolerance and cellulosic bioethanol fermentability for the chassis Zymomonas mobilis ZM4 pretreated using energy-efficient and eco-friendly cold plasma.

Results: It was found that bioethanol fermentability was weaker in CSH (corn stover hydrolysates) than that in synthetic medium for Z. mobilis, and thus was attributed to the inhibition of the lignocellulose-derived aldehyde inhibitors in CSH. Convincingly, it further confirmed that the mixed aldehydes severely decreased bioethanol accumulation through additional aldehydes supplementary assays in synthetic medium. After assayed under different processing time (10-30 s), discharge power (80-160 W), and working pressure (120-180 Pa) using cold atmosphere plasma (CAP), it achieved the increased bioethanol fermentability for Z. mobilis after pretreated at the optimized parameters (20 s, 140 W and 165 Pa). It showed that cold plasma brought about three mutation sites including ZMO0694 (E220V), ZMO0843 (L471L) and ZMO0843 (P505H) via Genome resequencing-based SNPs (single nucleotide polymorphisms). A serial of differentially expressed genes (DEGs) were further identified as the potential contributors for stress tolerance via RNA-Seq sequencing, including ZMO0253 and ZMO_RS09265 (type I secretion outer membrane protein), ZMO1941 (Type IV secretory pathway protease TraF-like protein), ZMOr003 and ZMOr006 (16S ribosomal RNA), ZMO0375 and ZMO0374 (levansucrase) and ZMO1705 (thioredoxins). It enriched cellular process, followed by metabolic process and single-organism process for biological process. For KEGG analysis, the mutant was also referred to starch and sucrose metabolism, galactose metabolism and two-component system. Finally, but interestingly, it simultaneously achieved the enhanced stress tolerance capacity of aldehyde inhibitors and bioethanol fermentability in CSH for the mutant Z. mobilis.

Conclusions: Of several candidate genetic changes, the mutant Z. mobilis treated with cold plasma was conferred upon the facilitated aldehyde inhibitors tolerance and bioethanol production. This work would provide a strain biocatalyst for the efficient production of lignocellulosic biofuels and biochemicals.

Keywords: Bioethanol; Cold atmosphere plasma (CAP); Genome resequencing; RNA-Seq sequencing; Zymomonas mobilis.

Fig. 1

Ethanol fermentability in RM synthetic medium and CSH for Z. mobilis ZM4. a Cell growth; b glucose consumption; c ethanol concentration; d aldehyde conversion in CSH

Fig. 2

The effect of aldehyde inhibitors on ethanol fermentability for Z. mobilis ZM4. a Cell growth; b glucose consumption; c ethanol concentration; d aldehyde inhibitors conversion; e mixed aldehyde conversion

Fig. 3

Ethanol fermentability of Z. mobilis ZM4 treated with cold plasma under the optimized parameters (20 s, 15 Pa, 140 W) in RM medium. a Cell growth; b glucose consumption; c ethanol concentration

Fig. 4

SNPs analysis for Z. mobilis ZM4 pretreated with cold plasma under the optimized parameters. a The whole genome mutation profile (WGMP). The outermost circle was the position coordinate axes of reference sequence. It showed InDel distribution, SNP numbers distribution, coverage depth of Reads, GC mol% content and GC skew value distribution of reference genome from the inside out. b Mutation of ZMO0694. c Mutation of ZMO0843

Fig. 4

SNPs analysis for Z. mobilis ZM4 pretreated with cold plasma under the optimized parameters. a The whole genome mutation profile (WGMP). The outermost circle was the position coordinate axes of reference sequence. It showed InDel distribution, SNP numbers distribution, coverage depth of Reads, GC mol% content and GC skew value distribution of reference genome from the inside out. b Mutation of ZMO0694. c Mutation of ZMO0843

Fig. 5

Transcriptomic analysis for Z. mobilis ZM4 pretreated with cold plasma using the optimized parameters. a The differentially expressed genes (DEGs); b GO analysis; c KEGG pathway analysis

Fig. 6

Ethanol fermentability in CSH for the pretreated Z. mobilis ZM4 using cold plasma. a The origin of glucose and aldehyde inhibitors in CSH; b speculation of ethanol fermentability and aldehyde inhibitor tolerance in CSH for the mutant; c validation of ethanol fermentability and aldehydes conversion tolerance in CSH for the mutant