Mengmeng Xu

A novel in situ gas stripping‐pervaporation process integrated with acetone‐butanol‐ethanol fermentation for hyper n‐butanol production

Butanol is considered as an advanced biofuel, the development of which is restricted by the intensive energy consumption of product recovery. A novel two‐stage gas stripping‐pervaporation process integrated with acetone‐butanol‐ethanol (ABE) fermentation was developed for butanol recovery, with gas stripping as the first‐stage and pervaporation as the second‐stage using the carbon nanotubes (CNTs) filled polydimethylsiloxane (PDMS) mixed matrix membrane (MMM). Compared to batch fermentation without butanol recovery, more ABE (27.5 g/L acetone, 75.5 g/L butanol, 7.0 g/L ethanol vs. 7.9 g/L acetone, 16.2 g/L butanol, 1.4 g/L ethanol) were produced in the fed‐batch fermentation, with a higher butanol productivity (0.34 g/L · h vs. 0.30 g/L · h) due to reduced butanol inhibition by butanol recovery. The first‐stage gas stripping produced a condensate containing 155.6 g/L butanol (199.9 g/L ABE), which after phase separation formed an organic phase containing 610.8 g/L butanol (656.1 g/L ABE) and an aqueous phase containing 85.6 g/L butanol (129.7 g/L ABE). Fed with the aqueous phase of the condensate from first‐stage gas stripping, the second‐stage pervaporation using the CNTs‐PDMS MMM produced a condensate containing 441.7 g/L butanol (593.2 g/L ABE), which after mixing with the organic phase from gas stripping gave a highly concentrated product containing 521.3 g/L butanol (622.9 g/L ABE). The outstanding performance of CNTs‐PDMS MMM can be attributed to the hydrophobic CNTs giving an alternative route for mass transport through the inner tubes or along the smooth surface of CNTs. This gas stripping‐pervaporation process with less contaminated risk is thus effective in increasing butanol production and reducing energy consumption. Biotechnol. Bioeng. 2016;113: 120–129.

Metabolic and process engineering of Clostridium cellulovorans for biofuel production from cellulose.

Production of cellulosic biofuels has drawn increasing attention. However, currently no microorganism can produce biofuels, particularly butanol, directly from cellulosic biomass efficiently. Here we engineered a cellulolytic bacterium, Clostridium cellulovorans, for n-butanol and ethanol production directly from cellulose by introducing an aldehyde/alcohol dehydrogenase (adhE2), which converts butyryl-CoA to n-butanol and acetyl-CoA to ethanol. The engineered strain was able to produce 1.42 g/L n-butanol and 1.60 g/L ethanol directly from cellulose. Moreover, the addition of methyl viologen as an artificial electron carrier shifted the metabolic flux from acid production to alcohol production, resulting in a high biofuel yield of 0.39 g/g from cellulose, comparable to ethanol yield from corn dextrose by yeast fermentation. This study is the first metabolic engineering of C. cellulovorans for n-butanol and ethanol production directly from cellulose with significant titers and yields, providing a promising consolidated bioprocessing (CBP) platform for biofuel production from cellulosic biomass.

Metabolic engineering of Clostridium tyrobutyricum for n‐butanol production through co‐utilization of glucose and xylose

The glucose‐mediated carbon catabolite repression (CCR) in Clostridium tyrobutyricum impedes efficient utilization of xylose present in lignocellulosic biomass hydrolysates. In order to relieve the CCR and enhance xylose utilization, three genes (xylT, xylA, and xylB) encoding a xylose proton‐symporter, a xylose isomerase and a xylulokinase, respectively, from Clostridium acetobutylicum ATCC 824 were co‐overexpressed with aldehyde/alcohol dehydrogenase (adhE2) in C. tyrobutyricum (Δack). Compared to the strain Ct(Δack)‐pM2 expressing only adhE2, the mutant Ct(Δack)‐pTBA had a higher xylose uptake rate and was able to simultaneously consume glucose and xylose at comparable rates for butanol production. Ct(Δack)‐pTBA produced more butanol (12.0 vs. 3.2 g/L) with a higher butanol yield (0.12 vs. 0.07 g/g) and productivity (0.17 vs. 0.07 g/L · h) from both glucose and xylose, while Ct(Δack)‐pM2 consumed little xylose in the fermentation. The results confirmed that the CCR in C. tyrobutyricum could be overcome through overexpressing xylT, xylA, and xylB. The mutant was also able to co‐utilize glucose and xylose present in soybean hull hydrolysate (SHH) for butanol production, achieving a high butanol titer of 15.7 g/L, butanol yield of 0.24 g/g, and productivity of 0.29 g/L · h. This study demonstrated the potential application of Ct(Δack)‐pTBA for industrial biobutanol production from lignocellulosic biomass. Biotechnol. Bioeng. 2015;112: 2134–2141.

Butanol production in acetone-butanol-ethanol fermentation with in situ product recovery by adsorption

Activated carbon Norit ROW 0.8, zeolite CBV901, and polymeric resins Dowex Optipore L-493 and SD-2 with high specific loadings and partition coefficients were studied for n-butanol adsorption. Adsorption isotherms were found to follow Langmuir model, which can be used to estimate the amount of butanol adsorbed in acetone-butanol-ethanol (ABE) fermentation. In serum-bottle fermentation with in situ adsorption, activated carbon showed the best performance with 21.9g/L of butanol production. When operated in a fermentor, free- and immobilized-cell fermentations with adsorption produced 31.6g/L and 54.6g/L butanol with productivities of 0.30g/L·h and 0.45g/L·h, respectively. Thermal desorption produced a condensate containing ∼167g/L butanol, which resulted in a highly concentrated butanol solution of ∼640g/L after spontaneous phase separation. This in situ product recovery process with activated carbon is energy efficient and can be easily integrated with ABE fermentation for n-butanol production.

Anaerobic Fermentation for Production of Carboxylic Acids as Bulk Chemicals from Renewable Biomass

Biomass represents an abundant carbon-neutral renewable resource which can be converted to bulk chemicals to replace petrochemicals. Carboxylic acids have wide applications in the chemical, food, and pharmaceutical industries. This chapter provides an overview of recent advances and challenges in the industrial production of various types of carboxylic acids, including short-chain fatty acids (acetic, propionic, butyric), hydroxy acids (lactic, 3-hydroxypropionic), dicarboxylic acids (succinic, malic, fumaric, itaconic, adipic, muconic, glucaric), and others (acrylic, citric, gluconic, pyruvic) by anaerobic fermentation. For economic production of these carboxylic acids as bulk chemicals, the fermentation process must have a sufficiently high product titer, productivity and yield, and low impurity acid byproducts to compete with their petrochemical counterparts. System metabolic engineering offers the tools needed to develop novel strains that can meet these process requirements for converting biomass feedstock to the desirable product.

Restriction modification system analysis and development of in vivo methylation for the transformation of Clostridium cellulovorans

Clostridium cellulovorans, a cellulolytic bacterium producing butyric and acetic acids as main fermentation products, is a promising host for biofuel production from cellulose. However, the transformation method of C. cellulovorans was not available, hindering its genetic engineering. To overcome this problem, its restriction modification (RM) systems were analyzed and a novel in vivo methylation was established for its successful transformation in the present study. Specifically, two RM systems, Cce743I and Cce743II, were determined. R. Cce743I has the same specificity as LlaJI, recognizing 5′-GACGC-3′ and 5′-GCGTC-3′, while M. Cce743I methylates the external cytosine in the strand (5′-GACGmC-3′). R. Cce743II, has the same specificity as LlaI, recognizing 5′-CCAGG-3′ and 5′-CCTGG-3′, while M. Cce743II methylates the external cytosine of both strands. An in vivo methylation system, expressing M. Cce743I and M. Cce743II from C. cellulovorans in Escherichia coli, was then established to protect plasmids used in electrotransformation. Transformants expressing an aldehyde/alcohol dehydrogenase (adhE2), which converted butyryl-CoA to n-butanol and acetyl-CoA to ethanol, were obtained. For the first time, an effective transformation method was developed for metabolic engineering of C. cellulovorans for biofuel production directly from cellulose.

Comparative genomic analysis of Clostridium acetobutylicum for understanding the mutations contributing to enhanced butanol tolerance and production

Clostridium acetobutylicum JB200 is a hyper butanol tolerant and producing strain obtained from asporogenic C. acetobutylicum ATCC 55025 through mutagenesis and adaptation in a fibrous bed bioreactor. The complete genomes of both strains were sequenced by the Illumina Hiseq2000 technology and assembled using SOAPdenovo approach. Compared to the genomic sequence of the type strain ATCC 824, 143 single nucleotide polymorphisms (SNPs) and 17 insertion/deletion variations (InDels) were identified in the genome of ATCC 55025. Twenty-nine mutations were in genes involved in sporulation, solventogenesis and stress response. Compared to ATCC 55025, there were seven additional point mutations in the chromosome of JB200. Among them, a single-base deletion in cac3319 encoding an orphan histidine kinase caused protein C-terminal truncation. Disruption of this gene in ATCC 55025 and ATCC 824 resulted in significantly elevated butanol tolerance and production. This study provides genome-level information for the better understanding of solventogenic C. acetobutylicum in several key aspects of cell physiology and metabolism, which could help further metabolic engineering of Clostridium for butanol production.