Chuang Xue

High-titer n-butanol production by clostridium acetobutylicum JB200 in fed-batch fermentation with intermittent gas stripping

Acetone–butanol–ethanol (ABE) fermentation with a hyper‐butanol producing Clostridium acetobutylicum JB200 was studied for its potential to produce a high titer of butanol that can be readily recovered with gas stripping. In batch fermentation without gas stripping, a final butanol concentration of 19.1u2009g/L was produced from 86.4u2009g/L glucose consumed in 78u2009h, and butanol productivity and yield were 0.24u2009g/Lu2009h and 0.21u2009g/g, respectively. In contrast, when gas stripping was applied intermittently in fed‐batch fermentation, 172u2009g/L ABE (113.3u2009g/L butanol, 49.2u2009g/L acetone, 9.7u2009g/L ethanol) were produced from 474.9u2009g/L glucose in six feeding cycles over 326u2009h. The overall productivity and yield were 0.53u2009g/Lu2009h and 0.36u2009g/g for ABE and 0.35u2009g/Lu2009h and 0.24u2009g/g for butanol, respectively. The higher productivity was attributed to the reduced butanol concentration in the fermentation broth by gas stripping that alleviated butanol inhibition, whereas the increased butanol yield could be attributed to the reduced acids accumulation as most acids produced in acidogenesis were reassimilated by cells for ABE production. The intermittent gas stripping produced a highly concentrated condensate containing 195.9u2009g/L ABE or 150.5u2009g/L butanol that far exceeded butanol solubility in water. After liquid–liquid demixing or phase separation, a final product containing ∼610u2009g/L butanol, ∼40u2009g/L acetone, ∼10u2009g/L ethanol, and no acids was obtained. Compared to conventional ABE fermentation, the fed‐batch fermentation with intermittent gas stripping has the potential to reduce at least 90% of energy consumption and water usage in n‐butanol production from glucose. Biotechnol. Bioeng. 2012; 109: 2746–2756.

Two-stage in situ gas stripping for enhanced butanol fermentation and energy-saving product recovery.

Two-stage gas stripping for butanol recovery from acetone-butanol-ethanol (ABE) fermentation with Clostridium acetobutylicum JB200 in a fibrous bed bioreactor was studied. Compared to fermentation without in situ gas stripping, more ABE (10.0 g/L acetone, 19.2 g/L butanol, 1.7 g/L ethanol vs. 7.9 g/L acetone, 16.2 g/L butanol, 1.4 g/L ethanol) were produced, with a higher butanol yield (0.25 g/g vs. 0.20 g/g) and productivity (0.40 g/L·h vs. 0.30 g/L·h) due to reduced butanol inhibition. The first-stage gas stripping produced a condensate containing 175.6 g/L butanol (227.0 g/L ABE), which after phase separation formed an organic phase containing 612.3g/L butanol (660.7 g/L ABE) and an aqueous phase containing 101.3 g/L butanol (153.2 g/L ABE). After second-stage gas stripping, a highly concentrated product containing 420.3 g/L butanol (532.3 g/L ABE) was obtained. The process is thus effective in producing high-titer butanol that can be purified with much less energy.

Integrated butanol recovery for an advanced biofuel: current state and prospects

Butanol has recently gained increasing interest due to escalating prices in petroleum fuels and concerns on the energy crisis. However, the butanol production cost with conventional acetone–butanol–ethanol fermentation by Clostridium spp. was higher than that of petrochemical processes due to the low butanol titer, yield, and productivity in bioprocesses. In particular, a low butanol titer usually leads to an extremely high recovery cost. Conventional biobutanol recovery by distillation is an energy-intensive process, which has largely restricted the economic production of biobutanol. This article thus reviews the latest studies on butanol recovery techniques including gas stripping, liquid–liquid extraction, adsorption, and membrane-based techniques, which can be used for in situ recovery of inhibitory products to enhance butanol production. The productivity of the fermentation system is improved efficiently using the in situ recovery technology; however, the recovered butanol titer remains low due to the limitations from each one of these recovery technologies, especially when the feed butanol concentration is lower than 1xa0% (w/v). Therefore, several innovative multi-stage hybrid processes have been proposed and are discussed in this review. These hybrid processes including two-stage gas stripping and multi-stage pervaporation have high butanol selectivity, considerably higher energy and production efficiency, and should outperform the conventional processes using single separation step or method. The development of these new integrated processes will give a momentum for the sustainable production of industrial biobutanol.

Redox potential control and applications in microaerobic and anaerobic fermentations

Many fermentation products are produced under microaerobic or anaerobic conditions, in which oxygen is undetectable by dissolved oxygen probe, presenting a challenge for process monitoring and control. Extracellular redox potentials that can be detected conveniently affect intracellular redox homeostasis and metabolism, and consequently control profiles of fermentation products, which provide an alternative for monitoring and control of these fermentation processes. This article reviews updated progress in the impact of redox potentials on gene expression, protein biosynthesis and metabolism as well as redox potential control strategies for more efficient production of fermentation products, taking ethanol fermentation by the yeast Saccharomyces under microaerobic conditions and butanol production by the bacterium Clostridium under anaerobic conditions as examples.

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 (27.5u2009g/L acetone, 75.5u2009g/L butanol, 7.0u2009g/L ethanol vs. 7.9u2009g/L acetone, 16.2u2009g/L butanol, 1.4u2009g/L ethanol) were produced in the fed‐batch fermentation, with a higher butanol productivity (0.34u2009g/Lu2009·u2009h vs. 0.30u2009g/Lu2009·u2009h) due to reduced butanol inhibition by butanol recovery. The first‐stage gas stripping produced a condensate containing 155.6u2009g/L butanol (199.9u2009g/L ABE), which after phase separation formed an organic phase containing 610.8u2009g/L butanol (656.1u2009g/L ABE) and an aqueous phase containing 85.6u2009g/L butanol (129.7u2009g/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 441.7u2009g/L butanol (593.2u2009g/L ABE), which after mixing with the organic phase from gas stripping gave a highly concentrated product containing 521.3u2009g/L butanol (622.9u2009g/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.

Engineering Clostridium acetobutylicum with a histidine kinase knockout for enhanced n-butanol tolerance and production

Clostridium acetobutylicum JB200, a mutant strain of C. acetobutylicum ATCC 55025 obtained through strain evolution in a fibrous bed bioreactor, had high butanol tolerance and produced up to ~21xa0g/L butanol from glucose in batch fermentation, an improvement of ~67xa0% over the parental strain (~12.6xa0g/L). Comparative genomic analysis revealed a single-base deletion in the cac3319 gene leading to C-terminal truncation in its encoding histidine kinase (HK) in JB200. To study the effects of cac3319 mutation on cell growth and fermentation, the cac3319 gene in ATCC 55025 was disrupted using the ClosTron group II intron-based gene inactivation system. Compared to ATCC 55025, the cac3319 HK knockout mutant, HKKO, produced 44.4xa0% more butanol (18.2u2009±u20091.3 vs. 12.6u2009±u20090.2xa0g/L) with a 90xa0% higher productivity (0.38u2009±u20090.03 vs. 0.20u2009±u20090.02xa0g/Lxa0h) due to increased butanol tolerance, confirming, for the first time, that cac3319 plays an important role in regulating solvent production and tolerance in C. acetobutylicum. This work also provides a novel metabolic engineering strategy for generating high-butanol-tolerant and high-butanol-producing strains for industrial applications.

Recent advances and state-of-the-art strategies in strain and process engineering for biobutanol production by Clostridium acetobutylicum

Butanol as an advanced biofuel has gained great attention due to its environmental benefits and superior properties compared to ethanol. However, the cost of biobutanol production via conventional acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum is not economically competitive, which has hampered its industrial application. The strain performance and downstream process greatly impact the economics of biobutanol production. Although various engineered strains with carefully orchestrated metabolic and sporulation-specific pathways have been developed, none of them is ideal for industrial biobutanol production. For further strain improvement, it is necessary to develop advanced genome editing tools and a deep understanding of cellular functioning of genes in metabolic and regulatory pathways. Processes with integrated product recovery can increase fermentation productivity by continuously removing inhibitory products while generating butanol (ABE) in a concentrated solution. In this review, we provide an overview of recent advances in C. acetobutylicum strain engineering and process development focusing on in situ product recovery. With deep understanding of systematic cellular bioinformatics, the exploration of state-of-the-art genome editing tools such as CRISPR-Cas for targeted gene knock-out and knock-in would play a vital role in Clostridium cell engineering for biobutanol production. Developing advanced hybrid separation processes for in situ butanol recovery, which will be discussed with a detailed comparison of advantages and disadvantages of various recovery techniques, is also imperative to the economical development of biobutanol.

A carbon nanotube filled polydimethylsiloxane hybrid membrane for enhanced butanol recovery

The carbon nanotubes (CNTs) filled polydimethylsiloxane (PDMS) hybrid membrane was fabricated to evaluate its potential for butanol recovery from acetone-butanol-ethanol (ABE) fermentation broth. Compared with the homogeneous PDMS membrane, the CNTs filled into the PDMS membrane were beneficial for the improvement of butanol recovery in butanol flux and separation factor. The CNTs acting as sorption-active sites with super hydrophobicity could give an alternative route for mass transport through the inner tubes or along the smooth surface. The maximum total flux and butanol separation factor reached up to 244.3u2005g/m2·h and 32.9, respectively, when the PDMS membrane filled with 10u2005wt% CNTs was used to separate butanol from the butanol/water solution at 80°C. In addition, the butanol flux and separation factor increased dramatically as temperature increased from 30°C to 80°C in feed solution since the higher temperature produced more free volumes in polymer chains to facilitate butanol permeation. A similar increase was also observed when butanol titer in solution increased from 10u2005g/L to 25u2005g/L. Overall, the CNTs/PDMS hybrid membrane with higher butanol flux and selectivity should have good potential for pervaporation separation of butanol from ABE fermentation broth.

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.

Effect of the size of yeast flocs and zinc supplementation on continuous ethanol fermentation performance and metabolic flux distribution under very high concentration conditions

Taking continuous ethanol fermentation with the self‐flocculating yeast SPSC01 under very high concentration conditions as an example, the fermentation performance of the yeast flocs and their metabolic flux distribution were investigated by controlling their average sizes at 100, 200, and 300u2009µm using the focused beam reflectance online measurement system. In addition, the impact of zinc supplementation was evaluated for the yeast flocs at the size of 300u2009µm grown in presence or absence of 0.05u2009gu2009L−1 zinc sulfate. Among the yeast flocs with different sizes, the group with the average size of 300u2009µm exhibited highest ethanol production (110.0u2009gu2009L−1) and glucose uptake rate (286.69u2009Cu2009mmolu2009L−1u2009h−1), which are in accordance with the increased flux from pyruvate to ethanol and decreased flux to glycerol. And in the meantime, zinc supplementation further increased ethanol production and cell viability comparing with the control. Zinc addition enhanced the carbon fluxes to the biosynthesis of ergosterol (28.6%) and trehalose (43.3%), whereas the fluxes towards glycerol, protein biosynthesis, and tricarboxylic acid cycle significantly decreased by 37.7%, 19.5%, and 27.8%, respectively. This work presents the first report on the regulation of metabolic flux by the size of yeast flocs and zinc supplementation, which provides the potential for developing engineering strategy to optimize the fermentation system. Biotechnol. Bioeng. 2010;105: 935–944.