Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

In memoriam

Microcosm experiments were performed to identify the influence of bacterial cell surfaces on the morphology, mineralogy, size and solubility of CaCO₃ precipitated in response to the enzymatic hydrolysis of urea in an artificial groundwater (AGW) by the ureolytic bacteria, Bacillus pasteurii. In each microcosm, B. pasteurii were contained within a cellulose dialysis membrane (10 K Dalton MWCO), resulting in bacteria-inclusive and bacteria-free AGW solution. Urea hydrolysis by B. pasteurii resulted in the production of ammonium and an increase in pH in the whole AGW solution. This initiated predominantly rhombohedral calcite precipitation at the same critical saturation state ( S _critical = 12) in the B. pasteurii-inclusive and bacteria-free zone of the AGW, indicating the mineralogy and morphology of CaCO₃ precipitation is not controlled by B. pasteurii surfaces. However, the temporal evolution of distinctly different lognormal crystal-size-distributions in the B. pasteurii-inclusive and bacteria-free zone of the AGW resulted from identical changes in bulk solution chemistry. Specifically, B. pasteurii increased the size and size variance of crystals, and led to a greater crystal growth rate throughout the experiments, relative to bacteria-free AGW. Calculated crystal solubility (ln K _S0 ) was lower for crystals > 4000 nm in diameter, reflecting smaller molar surface areas. This suggests that the larger crystals generated in the presence of B. pasteurii have a lower affinity for re-dissolution than those generated in the bacteria-free AGW, which may act as a positive feedback to maintain larger crystal sizes in the presence of B. pasteurii. During ureolysis, higher bacterial concentrations may therefore generate larger and less soluble carbonate crystals. This has important implications for the adaptation of bacterial ureolysis as a method for precipitating calcium carbonate and co-precipitating metals and radionuclides in contaminated aquifers.     

Removal of Contaminating Metals from Soil by Sulfur-Based Bioleaching and Biogenic Sulfide-Based Precipitation

The potential of a hybrid process incorporating sulfur-based bioleaching and sulfide-based precipitation for treatment of metal-contaminated soil was examined in batch-type experiments. The sulfur-based soil bioleaching process with Acidithiobacillus sp. could be initiated at a wide range of initial pH from 4.0 to 6.3. After 15 days, 98% of Zn, 89% of Cu and 79% of Cd was bioleached. The gaseous sulfides recycling from Desulfovibrio sp.-mediated sulfate-reducing reactor via N₂ sparging efficiently treated metal-loaded soil leachate. With a sulfide/metal ratio of 3.0, 88% of Zn, 100% of Cu and 95% of Cd were precipitated, resulting in effluent metal concentrations of 3.5 mg Zn²⁺/L, 0.2 mg Cu²⁺/L and 0.03 mg Cd²⁺/L.

Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the supplemental file.     

Parameters Effects Evaluation of Microbial Strengthening of Sandy Soils in Mixing Experiments Using Taguchi Methodology

The effects of experimental parameters including soil type, curing duration, inoculum size, and biomass and nutrients concentration on soil strengthening due to calcite precipitation by Sporosarcina pasteurii PTCC 1645 were investigated. The laboratory-scale mixing experiments on remolded samples were designed by the Taguchi method. Soil type proved to be the most incorporating factor, followed by curing time and nutrient concentration. The main effect and the interactions of the parameters were presented and the optimal conditions were obtained. This suggests the importance of local conditions including soil type on any future large-scale, in situ application.     

Towards a metabolic engineering strain “commons”: An Escherichia coli platform strain for ethanol production

In the genome‐engineering era, it is increasingly important that researchers have access to a common set of platform strains that can serve as debugged production chassis and the basis for applying new metabolic engineering strategies for modeling and characterizing flux, engineering complex traits, and optimizing overall performance. Here, we describe such a platform strain of E. coli engineered for ethanol production. Starting with a fully characterized host strain (BW25113), we site‐specifically integrated the genes required for homoethanol production under the control of a strong inducible promoter into the genome and deleted the genes encoding four enzymes from competing pathways. This strain is capable of producing >30 g/L of ethanol in minimal media with <2 g/L produced of any fermentative byproduct. Using this platform strain, we tested previously identified ethanol tolerance genes and found that while tolerance was improved under certain conditions, any effect on ethanol production or tolerance was lost when grown under production conditions. Thus, our findings reinforce the need for a metabolic engineering “commons” that could provide a set of platform strains for use in more sophisticated genome‐engineering strategies. Towards this end, we have made this production strain available to the scientific community. Biotechnol. Bioeng. 2013; 110: 1520–1526. © 2013 Wiley Periodicals, Inc.     

油松天然群体的种实性状表型多样性研究

为了揭示油松天然种群在不同地理环境条件下表型变异的程度和规律,在油松整个天然分布范围内选择了12个具有代表性的居群作为研究对象,对其球果、种子、种翅等12个种实性状的变异程度及其与环境因子间关系进行了比较分析。结果显示:(1)各个性状在居群内和居群间均存在较大的变异(CV>12%)。其中千山(QS),曾家镇(ZJ)和互助(HZ)3个居群表现出了较高的变异(CV>20%),而球果干重(CDW)和种子长(CL)是所有表型性状中变异幅度最大的(CV分别为31%和21%),但种翅性状与其他性状相比具有较高的稳定性。(2)巢式设计方差分析表明,在居群内表型分化系数(Vst)变化在3.18%~89.86%之间,而群体间的Vst为38.97%;与其他针叶树种相比,油松拥有较高的表型分化系数,且居群内的变异程度远高于居群间的变异,尤其是千山(QS)、曾家镇(ZJ)和互助(HZ)3个居群,这说明油松具有较高的环境异质性适应能力或恶劣环境耐受能力。(3)相关性分析表明,该研究的各形态特征与潜在蒸发量均为负相关,且大部分形态指标间及它们与潜在蒸发量间存在显著相关性,表明潜在蒸发量是油松形态特征变化的最重要环境影响因子,预示油松最适宜生长于温暖潮湿的环境中;并表明因各形态特征间相互紧密关联,所以它们受环境条件影响而共变。     

Tetradecane 1,14-Dicarboxylic Acid Production from n-Hexadecane by Candida cloacae

The better condition of cultivation for tetradecane 1,14-dicarboxylic acid (DC-16) production from n-hexadecane (n-C₁₆) by Candida cloacae MR-12 was investigated by using acetic acid as carbon source for the growth. In general, the condition suitable for the growth was also favorable for the production of DC-16. The change of pH during cultivation, the use of NaOH solution as pH controlling agent after pH-change and the addition of antifoam stimulated the production of DC-16.

Under the optimum conditions where the culture medium contained 15% (v/v) n-C₁₆, 1.4% (w/v) acetic acid, inorganic salts and growth factors, and pH was changed from 6.5 to 7.75 at 16 hr after the inoculation, the highest level of DC-16 production was attained after about 72 hr cultivation and the amount of the product accumulated was 61.5 g per liter of the medium.

When a mixture of various n-alkanes was used as starting material, DCs corresponding to the respective n-alkanes were produced as mixture.     

Cleavage of Disulfide Bonds of Wheat Glutenin by Mercuric Chloride

The dissociation of wheat glutenin into subunits was observed by treatment with a small amount of mercuric chloride under moderate conditions, suggesting that the cleavage of inter-polypeptide chain disulfide bonds in the glutenin might occur. The dissociation into the subunits was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The electrophoretic patterns of the glutenin treated with mercuric chloride were essentially similar to those of the glutenin treated with 2-mercaptoethanol. Silver nitrate also had the same effects as mercuric chloride, and p-chloromercuribenzoate and N-ethylmaleimide showed no effect on the dissociation of the glutenin. Complete dissociation was achieved when the glutenin solution containing 0.5% SDS and 0.01 m phosphate buffer (pH 7.0) was incubated with 10^?3 m mercuric chloride (about four moles per mole of disulfide groups) at 30°C for 20 hr. Partial dissociation was also observed after 30 min incubation. Increasing temperature and SDS concentration promoted the rate of the dissociation of the glutenin by mercuric chloride.     

Promotion or suppression of glucose isomerization in subcritical aqueous straight- and branched-chain alcohols

The influence of water-miscible alcohols (methanol, 1-propanol, 2-propanol, and t-butyl alcohol) on the isomerization of glucose to fructose and mannose was investigated under subcritical aqueous conditions (180–200 °C). Primary and secondary alcohols promoted the conversion and isomerization of glucose to afford fructose and mannose with high and low selectivity, respectively. On the other hand, the decomposition (side-reaction) of glucose was suppressed in the presence of the primary and secondary alcohols compared with that in subcritical water. The yield of fructose increased with increasing concentration of the primary and secondary alcohols, and the species of the primary and secondary alcohols tested had little effect on the isomerization behavior of glucose. In contrast, the isomerization of glucose was suppressed in subcritical aqueous t-butyl alcohol. Both the conversion of glucose and the yield of fructose decreased with increasing concentration of t-butyl alcohol. In addition, mannose was not detected in reactions using subcritical aqueous t-butyl alcohol.