Chen-Guang Liu

Prospective and development of butanol as an advanced biofuel.

Butanol has been acknowledged as an advanced biofuel, but its production through acetone-butanol-ethanol (ABE) fermentation by clostridia is still not economically competitive, due to low butanol yield and titer. In this article, update progress in butanol production is reviewed. Low price and sustainable feedstocks such as lignocellulosic residues and dedicated energy crops are needed for butanol production at large scale to save feedstock cost, but processes are more complicated, compared to those established for ABE fermentation from sugar- and starch-based feedstocks. While rational designs targeting individual genes, enzymes or pathways are effective for improving butanol yield, global and systems strategies are more reasonable for engineering strains with stress tolerance controlled by multigenes. Compared to solvent-producing clostridia, engineering heterologous species such as Escherichia coli and Saccharomyces cerevisiae with butanol pathway might be a solution for eliminating the formation of major byproducts acetone and ethanol so that butanol yield can be improved significantly. Although batch fermentation has been practiced for butanol production in industry, continuous operation is more productive for large scale production of butanol as a biofuel, but a single chemostat bioreactor cannot achieve this goal for the biphasic ABE fermentation, and tanks-in-series systems should be optimized for alternative feedstocks and new strains. Moreover, energy saving is limited for the distillation system, even total solvents in the fermentation broth are increased significantly, since solvents are distilled to ~40% by the beer stripper, and more than 95% water is removed with the stillage without phase change, even with conventional distillation systems, needless to say that advanced chemical engineering technologies can distil solvents up to ~90% with the beer stripper, and the multistage pressure columns can well balance energy consumption for solvent fraction. Indeed, an increase in butanol titer with ABE fermentation can significantly save energy consumption for medium sterilization and stillage treatment, since concentrated medium can be used, and consequently total mass flow with production systems can be reduced. As for various in situ butanol removal technologies, their energy efficiency, capital investment and contamination risk to the fermentation process need to be evaluated carefully.

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.

Development of redox potential-controlled schemes for very-high-gravity ethanol fermentation.

Fermentation redox potential reflects the momentary physiological status of organisms. Controlling redox potential can modulate the redistribution of intracellular metabolic flux to favor the formation of the desired metabolite. Accordingly, we have developed three redox potential-controlled schemes to maximize their effects on the very-high-gravity (VHG) ethanol fermentation. They are aeration-controlled scheme (ACS), glucose-controlled feeding scheme (GCFS), and combined chemostat and aeration-controlled scheme (CCACS). These schemes can maintain fermentation redox potential at a prescribed level (i.e., -50, -100, and -150 mV) by supplementing sterile air, fresh glucose media, or a combination of sterile air and fresh glucose media into a fermenter to counteract the decline of redox potential due to yeast growth. When ACS was employed, the fermentation efficiency at -150 mV is superior to the other two redox potential levels especially when the initial glucose concentration is higher than 250 g/l. The redox potential-controlled period for ACS, GCFS, and CCACS at -150 mV under the same 200 g glucose/l condition was 2.5, 21.7 and 64.6h and the corresponding fermentation efficiency was 85.9,89.3 and 92.7%, respectively.

Very high gravity ethanol fermentation by flocculating yeast under redox potential-controlled conditions

BackgroundVery high gravity (VHG) fermentation using medium in excess of 250 g/L sugars for more than 15% (v) ethanol can save energy consumption, not only for ethanol distillation, but also for distillage treatment; however, stuck fermentation with prolonged fermentation time and more sugars unfermented is the biggest challenge. Controlling redox potential (ORP) during VHG fermentation benefits biomass accumulation and improvement of yeast cell viability that is affected by osmotic pressure and ethanol inhibition, enhancing ethanol productivity and yield, the most important techno-economic aspect of fuel ethanol production.ResultsBatch fermentation was performed under different ORP conditions using the flocculating yeast and media containing glucose of 201u2009±u20093.1, 252u2009±u20092.9 and 298u2009±u20093.8 g/L. Compared with ethanol fermentation by non-flocculating yeast, different ORP profiles were observed with the flocculating yeast due to the morphological change associated with the flocculation of yeast cells. When ORP was controlled at −100 mV, ethanol fermentation with the high gravity (HG) media containing glucose of 201u2009±u20093.1 and 252u2009±u20092.9 g/L was completed at 32 and 56 h, respectively, producing 93.0u2009±u20091.3 and 120.0u2009±u20091.8 g/L ethanol, correspondingly. In contrast, there were 24.0u2009±u20090.4 and 17.0u2009±u20090.3 g/L glucose remained unfermented without ORP control. As high as 131.0u2009±u20091.8 g/L ethanol was produced at 72 h when ORP was controlled at −150 mV for the VHG fermentation with medium containing 298u2009±u20093.8 g/L glucose, since yeast cell viability was improved more significantly.ConclusionsNo lag phase was observed during ethanol fermentation with the flocculating yeast, and the implementation of ORP control improved ethanol productivity and yield. When ORP was controlled at −150 mV, more reducing power was available for yeast cells to survive, which in turn improved their viability and VHG ethanol fermentation performance. On the other hand, controlling ORP at −100 mV stimulated yeast growth and enhanced ethanol production under the HG conditions. Moreover, the ORP profile detected during ethanol fermentation with the flocculating yeast was less fluctuated, indicating that yeast flocculation could attenuate the ORP fluctuation observed during ethanol fermentation with non-flocculating yeast.

Assessment and regression analysis on instant catapult steam explosion pretreatment of corn stover

Instant catapult steam explosion (ICSE) offers enormous physical force on lignocellulosic biomass due to its extremely short depressure duration. In this article, the response surface methodology was applied to optimize the effect of working parameters including pressure, maintaining time and mass loading on the crystallinity index and glucose yield of the pretreated corn stover. It was found that the pressure was of essential importance, which determined the physical force that led to the morphological changes without significant chemical reactions, and on the other hand the maintaining time mainly contributed to the thermo-chemical reactions. Furthermore, the pretreated biomass was assessed by scanning electron microscope, X-ray diffraction and Fourier transform infrared spectra to understand mechanisms underlying the ICSE pretreatment.

Ageing vessel configuration for continuous redox potential-controlled very-high-gravity fermentation

The development of continuous very-high-gravity (VHG) fermentation is hindered by ineffective glucose uptake in order to result in zero discharge in the effluent stream. To overcome the problem, we proposed a continuous redox potential-controlled fermentation configuration, consisting of a Chemostat vessel connected with two ageing vessels installed in parallel, and the relevant design criteria are also specified. The Chemostat vessel is subjected to redox potential control to maintain yeast viability, and the ageing vessels are used to completely utilize glucose before discharging to next process unit. Two ageing vessels are scheduled alternatively, resulting in continuously-like operation. The size of ageing vessel is governed by the Chemostat size, dilution rate and filling time. The guideline to choose proper dilution rate is provided and the selection criterion of the proposed continuous configuration over batch fermentation is derived. The excess ethanol produced by the proposed continuous configuration over batch fermenter is quantified. As an illustration, a bio-ethanol plant is typically operated 8000 h per annum and the downtime between batches is 6h. Given that the fermenter size of 100 m(3) for both batch fermenter and Chemostat vessel, and glucose fed at 300 g/l, if the proposed continuous redox potential-controlled fermentation configuration (operated at 0.028 h(-1) and controlled at -50 mV) is selected, it will take 191 h for this configuration to outperform the batch counterpart, and the excess amount of ethanol being produced will be 1142 t.

Flocculating Zymomonas mobilis is a promising host to be engineered for fuel ethanol production from lignocellulosic biomass

Whereas Saccharomyces cerevisiae uses the Embden-Meyerhof-Parnas pathway to metabolize glucose, Zymomonas mobilis uses the Entner-Doudoroff (ED) pathway. Employing the ED pathway, 50% less ATP is produced, which could lead to less biomass being accumulated during fermentation and an improved yield of ethanol. Moreover, Z. mobilis cells, which have a high specific surface area, consume glucose faster than S. cerevisiae, which could improve ethanol productivity. We performed ethanol fermentations using these two species under comparable conditions to validate these speculations. Increases of 3.5 and 3.3% in ethanol yield, and 58.1 and 77.8% in ethanol productivity, were observed in ethanol fermentations using Z. mobilis ZM4 in media containing ∼100 and 200 g/L glucose, respectively. Furthermore, ethanol fermentation bythe flocculating Z. mobilis ZM401 was explored. Although no significant difference was observed in ethanol yield and productivity, the flocculation of the bacterial species enabled biomass recovery by cost-effective sedimentation, instead of centrifugation with intensive capital investment and energy consumption. In addition, tolerance to inhibitory byproducts released during biomass pretreatment, particularly acetic acid and vanillin, was improved. These experimental results indicate that Z. mobilis, particularly its flocculating strain, is superior to S. cerevisiae as a host to be engineered for fuel ethanol production from lignocellulosic biomass.

Global gene expression analysis of Saccharomyces cerevisiae grown under redox potential‐controlled very‐high‐gravity conditions

Redox potential (ORP) plays a pivotal role in yeast viability and ethanol production during very-high-gravity (VHG) ethanol fermentation. In order to identify the correlation between redox potential profiles and gene expression patterns, global gene expression of Saccharomyces cerevisiae was investigated. Results indicated that significant changes in gene expression occurred at the periods of 0 - 6 h and 30 - 36 h, respectively. Changes noted in the period of 0 - 6 h were mainly related to carbohydrate metabolism. In contrast, gene expression variation at 30 - 36 h could be attributed primarily to stress response. Although CDC19 was down-regulated, expression of PYK2, PDC6 and ADH2 correlated inversely with ORP. Meanwhile, expression of GPD1 decreased due to the depletion of dissolved oxygen in the fermentation broth, but expression of GPD2 correlated with ORP. Transcription of genes encoding heat shock proteins was characterized by uphill, downhill, valley and plateau expression profiles, accordingly to specific function in stress response. These results highlight the role of ORP in modulating yeast physiology and metabolism under VHG conditions.

Redox potential driven aeration during very-high-gravity ethanol fermentation by using flocculating yeast

Ethanol fermentation requires oxygen to maintain high biomass and cell viability, especially under very-high-gravity (VHG) condition. In this work, fermentation redox potential (ORP) was applied to drive the aeration process at low dissolved oxygen (DO) levels, which is infeasible to be regulated by a DO sensor. The performance and characteristics of flocculating yeast grown under 300 and 260u2009g glucose/L conditions were subjected to various aeration strategies including: no aeration; controlled aeration at −150, −100 and −50u2009mV levels; and constant aeration at 0.05 and 0.2u2009vvm. The results showed that anaerobic fermentation produced the least ethanol and had the highest residual glucose after 72u2009h of fermentation. Controlled aerations, depending on the real-time oxygen demand, led to higher cell viability than the no-aeration counterpart. Constant aeration triggered a quick biomass formation, and fast glucose utilization. However, over aeration at 0.2u2009vvm caused a reduction of final ethanol concentration. The controlled aeration driven by ORP under VHG conditions resulted in the best fermentation performance. Moreover, the controlled aeration could enhance yeast flocculating activity, promote an increase of flocs size, and accelerate yeast separation near the end of fermentation.

Fermentation and Redox Potential

Redox potential, known as oxidation–reduction or oxidoreduction potential (ORP), not only indicates the reduction and oxidation capacity of the environment but also reflects the metabolic activity of microorganisms. Redox potential can be monitored online and controlled in time for more efficient fermentation operation. This chapter reviews the enzymes that modulate intracellular redox potential, the genetically engineered strains that harbor specific redox potential–regulated genes, the approaches that were used to manipulate and control redox potential toward the production of desired metabolites, the role of redox potential in metabolic pathway, and the impact of redox potential on microbial physiology and metabolism. The application of redox potential–controlled ethanol fermentation and the development of three redox potential–controlled fermentation processes are illustrated. In the end, the future perspective of redox potential control is provided.