Li-Jie Chen

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.

Effect of zinc supplementation on acetone–butanol–ethanol fermentation by Clostridium acetobutylicum

In this article, effect of zinc supplementation on acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum was studied. It was found that when 0.001 g/L ZnSO4·7H2O was supplemented into the medium, solventogenesis was initiated earlier, with 21.0 g/L ABE (12.6 g/L butanol, 6.7 g/L acetone and 1.7 g/L ethanol) produced with a fermentation time of 40 h, compared to 19.4 g/L ABE (11.7 g/L butanol, 6.4 g/L acetone and 1.3g/L ethanol) produced with a fermentation time of 64 h in the control without zinc supplementation, and correspondingly ABE and butanol productivities were increased to 0.53 and 0.32 g/L/h from 0.30 and 0.18 g/L/h, increases of 76.7% and 77.8%, respectively, but their yields were not compromised. The reason for this phenomenon was attributed to rapid acids re-assimilation for more efficient ABE production, which was in accordance with relatively high pH and ORP levels maintained during the fermentation process. The maximum cell density increased by 23.8%, indicating that zinc supplementation stimulated cell growth, and consequently facilitated glucose utilization. However, more zinc supplementation exhibited an inhibitory effect, indicating that zinc supplementation at very low levels such as 0.001 g/L ZnSO4·7H2O will be an economically competitive strategy for improving butanol production.

Observed quasi-steady kinetics of yeast cell growth and ethanol formation under very high gravity fermentation condition

Using a generalSaccharomyces cerevisiae as a model strain, continuous ethanol fermentation was carried out in a stirred tank bioreactor with a working volume of 1,500 mL. Three different gravity media containing glucose of 120, 200 and 280 g/L, respectively, supplemented with 5 g/L yeast extract and 3 g/L peptone, were fed into the fermentor at different dilution rates. Although complete steady states developed for low gravity medium containing 120 g/L glucose, quasi-steady states and oscillations of the fermented parameters, including residual glucose, ethanol and biomass were observed when high gravity medium containing 200 g/L glucose and very high gravity medium containing 280 g/L glucose were fed at the designated dilution rate of 0.027 h−1. The observed quasi-steady states that incorporated these steady states, quasi-steady states and oscillations were proposed as these oscillations were of relatively short periods of time and their averages fluctuated up and down almost symmetrically. The continuous kinetic models that combined both the substrate and product inhibitions were developed and correlated for these observed quasi-steady states.

Evaluation of asymmetric polydimethylsiloxane-polyvinylidene fluoride composite membrane and incorporated with acetone-butanol-ethanol fermentation for butanol recovery.

The polydimethylsiloxane-polyvinylidene fluoride (PDMS-PVDF) composite membrane was studied for its pervaporation performance to removal of butanol from butanol/ABE solution, fermentation broth as well as incorporated with acetone-butanol-ethanol (ABE) fermentation. The total flux and butanol titer in permeate through the PDMS-PVDF membrane were up to 769.6 g/m(2)h and 323.5 g/L at 80 °C, respectively. The butanol flux and total flux increased with increasing the feed temperature as well as the feed butanol titer. The butanol separation factor and butanol titer in permeate decreased slightly in the presence of acetone and ethanol in the feed due to their preferential dissolution and competitive permeation through the membrane. In fed-batch fermentation incorporated with pervaporation, butanol titer and flux in permeate maintained at a steady level with the range of 139.9-154.0 g/L and 13.3-16.3 g/m(2)h, respectively, which was attributed to the stable butanol titer in fermentation broth as well as the excellent hydrophobic nature of the PDMS-PVDF matrix. Therefore, the PDMS-PVDF composite membrane had a great potential in the in situ product recovery with ABE fermentation, enabling the economic production of biobutanol.

Enhanced removal of Zn2+ or Cd2+ by the flocculating Chlorella vulgaris JSC-7

Microalgae are attracting attention due to their potentials in mitigating CO2 emissions and removing environmental pollutants. However, harvesting microalgal biomass from diluted cultures is one of the bottlenecks for developing economically viable processes for this purpose. Microalgal cells can be harvested by cost-effective sedimentation when flocculating strains are used. In this study, the removal of Zn(2+) and Cd(2+) by the flocculating Chlorella vulgaris JSC-7 was studied. The experimental results indicated that more than 80% Zn(2+) and 60% Cd(2+) were removed by the microalgal culture within 3 days in the presence up to 20.0mg/L Zn(2+) and 4.0mg/L Cd(2+), respectively, which were much higher than that observed with the culture of the non-flocculating C. vulgaris CNW11. Furthermore, the mechanism underlying this phenomenon was explored by investigating the effect of Zn(2+) and Cd(2+) on the growth and metabolic activities of the microalgal strains. It was found that the flocculation of the microalga improved its growth, synthesis of photosynthetic pigments and antioxidation activity under the stressful conditions, indicating a better tolerance to the heavy metal ions for a potential in removing them more efficiently from contaminated wastewaters, together with a bioremediation of other nutritional components contributed to the eutrophication of aquatic ecosystems.

Improved ethanol production in Jerusalem artichoke tubers by overexpression of inulinase gene in Kluyveromyces marxianus

Ethanol production from Jerusalem artichoke tubers through a consolidated bioprocessing (CBP) strategy using the inulinase-producing yeast Kluyveromyces marxianus is an economical and competitive than that from a grainbased feedstock. However, poor inulinase production under ethanol fermentation conditions significantly prolongs the fermentation time and compromises ethanol productivity. Improvement of inulinase activity appears to be promising for increasing ethanol production from Jerusalem artichoke tubers by CBP. In the present study, expression of the inulinase gene INU with its own promoter in K. marxianus (K/INU2) was explored using the integrative cassette. Overexpression of INU was explored using chromosome integration via the HO locus of the yeast. Inulinase activity and ethanol were determined from inulin and Jerusalem artichoke tubers under fed-batch operation. Inulinase activity was 114.9 U/mL under aerobic conditions for K/INU2, compared with 52.3 U/mL produced by the wild type strain. Importantly, inulinase production was enhanced in K/INU2 under ethanol fermentation conditions. When using 230 g/L inulin and 220 g/L Jerusalem artichoke tubers as substrates, inulinase activities of 3.7 and 6.8 U/mL, respectively, were measured using K/INU2, comparing favorably with 2.4 and 3.1 U/mL, respectively, using the wide type strain. Ethanol concentration and productivity for inulin were improved by the recombinant yeast to 96.2 g/L and 1.34 g/L/h, respectively, vs 93.7 g/L and 1.12 g/L/h, respectively, by the wild type strain. Ethanol concentration and productivity improvements for Jerusalem artichoke tubers were 69 g/L and 1.44 g/L/h, respectively, from the recombinant strain vs 62 g/L and 1.29 g/L/h, respectively, from the wild type strain.

Engineering an industrial Saccharomyces cerevisiae strain with the inulinase gene for more efficient ethanol production from Jerusalem artichoke tubers

Engineering industrial microbial strains with inulinase production for developing a consolidated bioprocessing (CBP) strategy is a desirable solution to biofuel production from Jerusalem artichoke tubers. In this study, the integrative vector pFA6a‐rDNA‐PGK‐INU was generated by fusing the 3.3‐kb ribosomal DNA fragment with the phosphoglycerate kinase promoter to regulate the expression of the inulinase gene isolated from Kluyveromyces marxianus, which was then integrated into the chromosome of an industrial Saccharomyces cerevisiae strain for ethanol production from Jerusalem artichoke tubers. Compared to the host strain, no significant difference was observed in the growth of the recombinant yeast, but its inulinase production was enhanced: 22.9 versus 10.6 U/mL for aerobic seed cultures and 14.5 versus 10.0 U/mL for ethanol fermentation, which consequently facilitated the CBP process for ethanol production: 72.5 g/L ethanol produced in a fermentation time of 48 h, while only 67.0 g/L ethanol was produced by the host strain in a fermentation time of 60 h. Thus, the ethanol productivity was increased from 1.12 to 1.51 g/L/h, presenting an increase of 34.8%.

Improvements of metabolites tolerance in Clostridium acetobutylicum by micronutrient zinc supplementation

Micronutrient zinc is of great importance for acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum. The effect of zinc supplementation on toxic metabolites (formic, acetic, butyric acid and butanol) tolerance during ABE fermentation was investigated under various stress-shock conditions without pH control. Great improvements on cell growth, glucose utilization and butanol production were achieved. In the presence of 0.45 g/L formic acid, zinc contributed to 11.28 g/L butanol produced from 55.24 g/L glucose compared to only 5.27 g/L butanol from 29.49 g/L glucose in the control without zinc supplementation. More importantly, relatively higher levels of 7.5 g/L acetic acid, 5.5 g/L butyric acid and 18 g/L butanol could be tolerated by C. acetobutylicum with zinc supplementation while no fermentation was observed under the same stress-shock condition respectively, suggesting that the acids and butanol tolerance in C. acetobutylicum could be significantly facilitated by pleiotropic regulation of micronutrient zinc. Thus, this paper provides an efficient bioprocess engineering strategy for improving stress tolerance in Clostridium species.

Synergistic effect of calcium and zinc on glucose/xylose utilization and butanol tolerance of Clostridium acetobutylicum

Biobutanol outperforms bioethanol as an advanced biofuel, but is not economically competitive in terms of its titer, yield and productivity associated with feedstocks and energy cost. In this work, the synergistic effect of calcium and zinc was investigated in the acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum using glucose, xylose and glucose/xylose mixtures as carbon source(s). Significant improvements associated with enhanced glucose/xylose utilization, cell growth, acids re-assimilation and butanol biosynthesis were achieved. Especially, the maximum butanol and ABE production of 16.1 and 25.9 g L(-1) were achieved from 69.3 g L(-1) glucose with butanol/ABE productivities of 0.40 and 0.65 g L(-1) h(-1) compared to those of 11.7 and 19.4 g/L with 0.18 and 0.30 g L(-1) h(-1) obtained in the control respectively without any supplement. More importantly, zinc was significantly involved in the butanol tolerance based on the improved xylose utilization under various butanol-shock conditions (2, 4, 6, 8 and 10 g L(-1) butanol). Under the same conditions, calcium and zinc co-supplementation led to the best xylose utilization and butanol production. These results suggested that calcium and zinc could play synergistic roles improving ABE fermentation by C. acetobutylicum.

Improving Fructose Utilization and Butanol Production by Clostridium acetobutylicum via Extracellular Redox Potential Regulation and Intracellular Metabolite Analysis

Jerusalem artichoke (JA) can grow well in marginal lands with high biomass yield, and thus is a potential energy crop for biorefinery. The major biomass of JA is from tubers, which contain inulin that can be easily hydrolyzed into a mixture of fructose and glucose, but fructose utilization for producing butanol as an advanced biofuel is poor compared to glucose‐based ABE fermentation by Clostridium acetobutylicum. In this article, the impact of extracellular redox potential (ORP) on the process is studied using a mixture of fructose and glucose to simulate the hydrolysate of JA tubers. When the extracellular ORP is controlled above −460 mV, 13.2 g L−1 butanol is produced from 51.0 g L−1 total sugars (40.1 g L−1 fructose and 10.9 g L−1 glucose), leading to dramatically increased butanol yield and butanol/ABE ratio of 0.26 g g−1 and 0.67, respectively. Intracellular metabolite and q‐PCR analysis further indicate that intracellular ATP and NADH availabilities are significantly improved together with the fructose‐specific PTS expression at the lag phase, which consequently facilitate fructose transport, metabolic shift toward solventogenesis and carbon flux redistribution for butanol biosynthesis. Therefore, the extracellular ORP control can be an effective strategy to improve butanol production from fructose‐based feedstock.