Microwave-Assisted Extraction of Multiple Trace Levels of Intermediate Metabolites for Camptothecin Biosynthesis in Camptotheca acuminata and Their Simultaneous Determination by HPLC-LTQ-Orbitrap-MS/MS and HPLC-TSQ-MS.

Camptothecin (CPT) has strong antitumor activity and is used as an anticancer therapeutic agent. To better understand and decipher the pathway of CPT biosynthesis in Camptotheca acuminata, the main purpose here was focused on creating an effective extraction strategy for a rich intermediate metabolite profile. In the present study, a 70% aqueous acetonitrile was verified as an optimal extraction solvent for microwave-assisted extraction (MAE) of metabolites by spiking experiments. Based on multi-objective optimization, the best extraction conditions of a solid-liquid ratio of 1:20, microwave power of 230 W, and a time of 4 min were achieved using a full factorial 34 experimental design. Crude extracts obtained from the shoot apex of C. acuminata using MAE have been qualitatively profiled by high-performance liquid chromatography coupled with linear ion trap quadrupole-orbitrap mass spectrometry (HPLC-LTQ-Orbitrap-MS/MS) and a HPLC triple quadrupole-MS (HPLC-TSQ-MS) analysis was conducted for their metabolite content in different tissues. CPT, and ten related metabolites and their isomers, including tryptamine, loganic acid, secologanic acid, strictosidinic acid, strictosamide, strictosamide epoxide, strictosamide diol, strictosamide ketolactam, pumiloside, and deoxypumiloside, were detected and tentatively identified. Scanning electron microscopy (SEM) imaging of the shoot apex demonstrated that severe cell disruption was evident after intensified extraction processes. The study showed the difference of metabolite profiles and the enhancement of metabolite content after microwave-pretreated techniques, and the established MAE procedure is an effective methodology to preserve valuable metabolite compounds for analysis.

[Tactic movement of microalgae and its application in targeted transport: a review].

Microalgae are a group of photosynthetic microorganisms, which have the general characteristics of plants such as photosynthesis, and some species have the ability of movement which resembles animals. Recently, it was reported that microalgae cells can be engineered to precisely deliver medicine-particles and other goods in microfluidic chips. These studies showed great application potential in biomedical treatment and pharmacodynamic analysis, which have become one of the current research hotspots. However, these developments have been rarely reviewed. Here, we summarized the advances in manageable movement exemplified by a model microalgae Chlamydomonas reinhardtii based on its characteristics of chemotaxis, phototaxis, and magnetotaxis. The bottlenecks and prospects in the application of microalgae-based tactic movement were also discussed. This review might be useful for rational design and modification of microalgal manageable movement to achieve targeted transport in medical and other fields.

[Improvement of inhibitors tolerance of Saccharomyces cerevisiae by overexpressing of long chain sphingoid kinases encoding gene LCB4].

By-products released from pretreatment process of lignocellulose seriously hinder the development of cellulosic fuel ethanol. Therefore, the great way to increase the efficiency of cellulosic ethanol production is improvement of Saccharomyces cerevisiae tolerance to these inhibitors. In this work, the effects of LCB4 gene overexpression on cell growth and ethanol fermentation in S. cerevisiae S288C under acetic acid, furfural and vanillin stresses were studied. Compared to the control strain S288C-HO, the recombinant strain S288C-LCB4 grew better on YPD solid medium containing 10 g/L acetic acid, 1.5 g/L furfural and 1 g/L vanillin. Ethanol yields of recombinant strain S288C-LCB4 were 0.85 g/(L·h), 0.76 g/(L·h) and 1.12 g/(L·h) when 10 g/L acetic acid, 3 g/L furfural and 2 g/L vanillin were supplemented into the fermentation medium respectively, which increased by 34.9%, 85.4% and 330.8% than the control strain S288C-HO. Meanwhile, ethanol fermentation time was reduced by 30 h and 44 h under furfural and vanillin stresses respectively. Further metabolites analysis in fermentation broth showed that the recombinant strain produced more protective compounds, such as glycerol, trehalose and succinic acid, than the control strain, which could be the reason for enhancing strain tolerance to these inhibitors from pretreatment process of lignocellulose. The results indicated that overexpression of LCB4 gene could significantly improve ethanol fermentation in S. cerevisiae S288C under acetic acid, furfural and vanillin stresses.

[Process development for continuous ethanol fermentation by the flocculating yeast under stillage backset conditions].

Propionic acid, a major inhibitor to yeast cells, was accumulated during continuous ethanol fermentation from corn meal hydrolysate by the flocculating yeast under stillage backset conditions. Based on its inhibition mechanism in yeast cells, strategies were developed for alleviating this effect. Firstly, high temperature processes such as medium sterilization generated more propionic acid, which should be avoided. Propionic acid was reduced significantly during ethanol fermentation without medium sterilization, and concentrations of biomass and ethanol increased by 59.3% and 7.4%, respectively. Secondly, the running time of stillage backset should be controlled so that propionic acid accumulated would be lower than its half inhibition concentration IC50 (40 mmol/L). Finally, because low pH augmented propionic acid inhibition in yeast cells, a higher pH of 5.5 was validated to be suitable for ethanol fermentation under the stillage backset condition.

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.

Bioethanol from lignocellulosic biomass.

China is suffering from a sustained shortage of crude oil supply, making fuel ethanol and other biofuels alternative solutions for this issue. However, taking into account the country's large population and dwindling arable land due to rapid urbanization, it is apparent that current fuel ethanol production from grain-based feedstocks is not sustainable, and lignocellulosic biomass, particularly agricultural residues that are abundantly available in China, is the only choice for China to further expand its fuel ethanol production, provided economically viable processes can be developed. In this chapter, cutting edge progress in bioethanol is reviewed, with a focus on the understanding of the molecular structure of the feedstock, leading pretreatment technologies, enzymatic hydrolysis of the cellulose component and strategies for the co-fermentation of the C5 and C6 sugars with engineered microorganisms. Finally, process integration and optimization is addressed with a case study on the COFCO Corporation's pilot plant, and challenges and perspectives for commercial production of bioethanol are highlighted.

[Impact of distillage recycling on the glycolysis key enzymes, stress response metabolites and intracelluler components of the self-flocculating yeast].

This research aimed to study the effect of distillage recycling on ethanol fermentation, the key glycolytic enzymes and cell composition of the self-flocculating yeast. With the self-flocculating yeast SPSC01 and medium composed of 220 g/L glucose, 8 g/L yeast extract and 6 g/L peptone, continuous ethanol fermentation was carried out at the dilution rate of 0.04 h(-1) with a 1.5 L tank bioreactor. Fermentation broth was collected every 3 days, and ethanol and other volatile byproducts were removed by distillation, but the stillage with high boiling byproducts was recycled to prepare the medium instead of fresh water. The system was run for 20 days, during which ethanol and biomass concentrations in the effluent decreased continuously, indicating the significant inhibition of the high boiling byproducts accumulated within the system. Thus, the activities of the key enzymes of the glycolytic pathway: hexokinase, 6-phosphofructose kinase, and pyruvate kinase were analyzed, and it was observed that all of them were inhibited. Furthermore, the biosynthesis of the stress response metabolites glycerol and trehalose was investigated, and it was found that glycerol production that can protect yeast cells against osmotic pressure stress was enhanced, but trehalose biosynthesis that can protect yeast cells against ethanol inhibition was not improved, correspondingly. And in the meantime, the biosynthesis of the major intracellular components proteins and hydrocarbons was adjusted, correspondingly.

[Continuous ethanol fermentation using self-flocculating yeast in multi-stage suspended bioreactors coupled with directly recycling of waste distillage].

A fermentation system composed of four airlift suspended-bed bioreactors in series and with a total working volume of 4800 mL was established. Continuous ethanol fermentation using self-flocculating yeast SPSC01, a fusant from Saccharomyces cerevisiae and Schizosaccharomyces pombe, and two-stage enzymatic hydrolyte of dry milling corn powder, was continuously run for 120 days. All of the backset distillage collected after distilling the final beer was used to mix the corn powder and no any other wastes except the solid residue of corn powder was discharged from the fermentation system, which guaranteed the distillage to be recycled at its maximum. The experimental results revealed that both ethanol and residual sugar in the final beer could be maintained relatively stable with their average levels of 93.6 and 7.9 g/L, respectively when the fermentation system was operated at the dilution rate of 0.05 h(-1). Parameter oscillations reported previously were also observed for the first and second bioreactors, but were effectively attenuated thereafter, which indicated that high yeast cell concentrations resulted from the self-immobilization of this special self-flocculating strain contributed to damp these oscillations. The monitoring of residual nitrogen and phosphor indicated that the accumulations of these nutritional elements occurred and the amount of these inorganic salts supplemented in the substrate should be decreased properly.