Application of Supercritical Fluid Extraction in Biotechnology

ABSTRACT

In the present paper recent investigations on the applications of supercritical fluid extraction (SCE) from post fermentation biomass or in situ extraction of inhibitory fermentation products as a promising method for increasing the yield of extraction have been reviewed. Although supercritical CO₂ (SC-CO₂) is unfriendly, or even toxic, for some living cells and precludes direct fermentation in dense CO₂, it does not rule out other useful applications for in situ extraction of inhibitory fermentation products and fractional extraction of biomass constituents. This technique is a highly desirable method for fractional extraction of biomass constituents, and intracellular metabolites due to the potential of system modification by physical parameters and addition of co-solvents to selectively extract compounds of different polarity, volatility and hydrophilicity without any contamination.     

用GC-MS分析不同方法提取的茶油脂肪酸

用超临界CO2、超声波、微波和机榨4种方法提取茶油,利用GC-MS分析茶油成分。结果表明：超临界CO2萃取的茶油与超声波和微波提取的主要化学成分几乎无差异,机榨茶油的化学成分与前3种的有一些差异,未测出维生素E和角鲨烯。超临界CO2萃取的茶油已达到国家成品茶油的一级标准,超临界CO2萃取法为提取茶油的较理想方法。     

益智的抗氧化作用

本文探讨了益智(Alpinia oxyphylla Miquel)超临界CO2提取物及其渣的水提物、正丙醇提取物和乙酸乙酯提取物的抗氧化作用,测定了总酚含量、黄酮含量、抗氧化力、还原能力、DPPH清除率.结果表明,益智超临界CO2提取物和正丙醇提取物的总酚含量最高,均为5.53%,乙酸乙酯提取物的总酚含量为4.04%,水提物总酚含量最低,为O.89%.抗氧化力与酚含量相关(R2=0.703).四种提取物中黄酮含量顺序为:乙酸乙酯提取物(6.29%)＞丙醇提取物(5.81%)＞水提取物(4.85%)＞超临界CO2提取物(4.70%).在还原能力、清除DPPH自由基和羟自由基方面,乙酸乙酯提取物表现出了很强的抗氧化能力,呈现剂量依赖关系.     

Escherichia coli inactivation mechanism by pressurized CO2

The effects of pressurized CO2 on the survival of Escherichia coli and the mechanism of cell inactivation were studied. Bacterial cultures were inoculated in nutrient broth and incubated at 30 degrees C for 18 h. Exposure of the cells to CO2 under pressures ranging from 2.5 to 25 MPa and at temperatures between 8 and 40 degrees C was performed in a double-walled reactor with a 1 L capacity. The effect of the treatment on the cells was evaluated by plating and by transmission and scanning electron microscopy observation. Vapour CO2 generated a bacteriostatic effect. In liquid or supercritical state, CO2 provided a bactericidal effect. The bactericidal effect increased with pressure and temperature. The mechanism of cell inactivation by liquid CO2 involved two stages. First, cell stress caused by the CO2 penetration provoked cell wall collapse and cellular content precipitation. Second, the cell death caused by supercritical extraction of intracellular substances and cell envelope perforation resulted in leaking of intracellular constituents. In supercritical conditions, the cell inactivation process had one single phase: cellular death.     

鱼腥草挥发油的化学成分

The volatile oils from Houttuynia cordata Thunb. were obtained with supereritical CO2, steam distillation and petroleum ether extraction and analyzed qualitatively and quantitatively by GC-MS. The results showed that the chemical constituents of the volatile oils by three different methods are very different. The extraction rates of volatile oil by above-mentioned three extraction methods are 1.764%, 0.040% and 0.082%, respectively. The volatile oil extracted by supercritical CO2 extraction, which the content of houttuyninum reached 14.393 %, is better than those with traditional methods.     

迷迭香植物精油对白纹伊蚊的驱避作用及其化学成分

应用水蒸气蒸溜法和超临界二氧化碳萃取法提取生长于云南的迷迭香茎叶植物精油,测试2种精油对白纹伊蚊Aedes albopictus Skuse的驱避作用,并利用GC/MS联用仪分析2种精油的化学成分。结果表明:超临界二氧化碳提取的迷迭香精油对白纹伊蚊的驱避时间是(5.82±1.13)h,达到国标驱避剂评价的B级标准。2种精油化学成分中都含有驱纹活性物质:柠檬烯、樟脑、Eucalyptol、(+)-4-Carene、1-methyl-4-(1-methyle thylidene)-cyclohexene、Beta-pinene等,且驱蚊活性成分在挥发油中占有较高比例,显示该植物精油在驱蚊方面有一定开发价值。     

Bioproduction of butanol from biomass: from genes to bioreactors

Butanol is produced chemically using either the oxo process starting from propylene (with H2 and CO over a rhodium catalyst) or the aldol process starting from acetaldehyde. The key problems associated with the bioproduction of butanol are the cost of substrate and butanol toxicity/inhibition of the fermenting microorganisms, resulting in a low butanol titer in the fermentation broth. Recent interest in the production of biobutanol from biomass has led to the re-examination of acetone-butanol-ethanol (ABE) fermentation, including strategies for reducing or eliminating butanol toxicity to the culture and for manipulating the culture to achieve better product specificity and yield. Advances in integrated fermentation and in situ product removal processes have resulted in a dramatic reduction of process streams, reduced butanol toxicity to the fermenting microorganisms, improved substrate utilization, and overall improved bioreactor performance.     

Supercritical fluid extraction of algae enhances levels of biologically active compounds promoting plant growth

The aim of this research was to screen plant growth biostimulant properties of supercritical CO₂ macroalgal extracts. To this end secondary metabolites were isolated from the biomass of marine macroalgae from the Baltic Sea (species of Polysiphonia, Ulva and Cladophora). Chemical characteristics of the algal extracts were determined by inductively coupled plasma atomic emission spectroscopy for inorganic constituents and high-performance liquid chromatography and spectrophotometry for organic constituents. Inorganic (macro- and microelements) and organic (plant hormones: auxins and cytokinins; polyphenols) compounds were detected in the extract. Algal extracts were tested primarily on garden cress (Lepidium sativum L.; Brassicaceae) and wheat (Triticum aestivum L.; Poaceae). The extracts enhanced chlorophyll and carotenoid content in plant shoots, as well as root thickness and above-ground biomass. The most effective method of application of the extract was by foliar feed on cress and seed maceration for wheat. Algal extracts obtained by supercritical fluid extraction (SFE) were found to be a novel natural source of compounds, stimulating growth of cultivated plants. However, field trials will be required to show that the extracts can act as plant biopesticides and growth biostimulants.     

Simultaneous measurement of fluoroquinolones in eggs by a combination of supercritical fluid extraction and high pressure liquid chromatography

Simultaneous detection of the fluoroquinolone antibiotics ciprofloxacin, enrofloxacin, ofloxacin, and norfloxacin in eggs by a combination of supercritical fluid extraction (SFE) and high pressure liquid chromatography (HPLC) was studied. Lipid matrices that have been considered to result in poor extraction and isolation of fluoroquinolones in eggs were removed first by SFE with supercritical CO(2) alone, and then the fluoroquinolones were extracted by SFE with supercritical CO(2) containing 20% (v/v) methanol for HPLC analysis. A time-course study of the extraction of lipid matrices of eggs suggested that the SFE method successfully removed the matrices within 20 min. When the fluoroquinolones added to control eggs were extracted by SFE, the extraction efficiency was similar to that by the solvent extraction method, giving the recovery percentages from 83 to 96% in a 40 min-extraction time. The fluoroquinolones extracted from eggs by SFE were analyzed simultaneously by HPLC equipped with a fluorescence detector with detection sensitivity at about 10 ppb for the detection limit. The standard calibration profiles of fluoroquinolones showed linear responses to HPLC, showing more than 0.995 for the mean r(2) value. This is the first report of the simultaneous measurement of fluoroquinolones in eggs by a combination of SFE and HPLC. Using the SFE method allowed us to avoid extensive sample preparation such as solvent extraction and chromatographic cleanup that are basically required in extraction of fluoroquinolones.     

超临界CO2萃取红景天中红景天苷、苷元酪醇的研究

采用超临界CO₂ 萃取法和乙醇常温浸提法相比较, 研究从红景天中提取红景天苷、苷元酪醇的工艺条件, 结论是:采用超临界CO₂ 萃取法能萃取出红景天生药中红景天苷的1.2%, 提取率不高, 但该方法能萃取出80%的苷元酪醇, 萃取液中苷元酪醇的相对含量可达45.68%;乙醇常温浸提法能将红景天苷、苷元酪醇同时有效萃取, 且得率较高, 但是萃取液中两物质相对含量较低, 进一步分离纯化将有难度。本研究结果表明, 将超临界CO₂ 萃取法和乙醇常温浸提法有效结合, 可实现两物质的有效分离, 推进红景天有效成分的产业化进程。     

Supercritical fluid extraction of a lignocellulosic hydrolysate of spruce for detoxification and to facilitate analysis of inhibitors

This work describes a novel approach to detoxify lignocellulosic hydrolysates and facilitate the analysis of inhibitory compounds, namely supercritical fluid extraction (SFE). The efficiency of the fermentation of lignocellulosic dilute-acid hydrolysates depends upon the composition of the hydrolysate and the organism used. Furthermore, it has been shown that inhibitors in the hydrolysate reduce the fermentation yield. This knowledge has given rise to the need to identify and remove the inhibiting compounds. Sample clean-up or work-up steps, to provide a clean and concentrated sample for the analytical system, facilitate the characterization of inhibitors, or indeed any compound in the hydrolysates. Removal of inhibitors was performed with countercurrent flow supercritical fluid extraction of liquid hydrolysates. Three different groups of inhibitors (furan derivatives, phenolic compounds, and aliphatic acids) and sugars were subsequently analyzed in the hydrolysate, extracted hydrolysate, and extract. The effect of the SFE treatment was examined with respect to fermentability with Saccharomyces cerevisiae. Not only did the extraction provide a clean and concentrated sample (extract) for analysis, but also a hydrolysate with increased fermentability as well as lower concentrations of inhibitors such as phenolics and furan derivatives.     

皂荚皂甙提取工艺的研究

研究了以皂荚为原料,对传统有机溶剂提取与水提取和超临界CO2萃取三萜皂甙进行了比较,得出水最适合提取皂荚皂甙;水提工艺部分,在单因素实验的基础上采用正交设计实验优化工艺,得出提取皂荚皂甙的最佳工艺。     

Selective separation of cis-trans geometrical isomers of beta-carotene via CO2 supercritical fluid extraction

We investigated a novel method for the selective separation of beta-carotene isomers from a freeze-dried powder of the algae Dunaliella bardawil using supercritical fluid extraction. The separation method relies on the different dissolution rate of the 9Z and all-E isomers of beta-carotene in SC-CO(2). At first, the equilibrium solubility of the two isomers in SC-CO(2) was determined at the extraction conditions of 44.8 MPa and 40 degrees C. The solubility of the 9Z isomer was found to be nearly 4 times higher than that of the all-E isomer (1.92 x 10(-5) g all-E isomer/g CO(2) compared to 7.64 x 10(-5) g 9Z isomer/g CO(2)). When supercritical fluid extraction was applied to a carotenoid concentrate from the algae (29 wt% beta-carotene) or a freeze-dried powder of the algae (3.1% beta-carotene), a selective separation of the 9Z/all-E isomers of beta-carotene was obtained. Thirty-nine percent recovery of beta-carotene with 80% purity of 9Z isomer was achieved at the initial stages of extraction (40 mL CO(2)). The extraction rate of beta-carotene from the freeze-dried algae powder was slower than that from the carotenoid concentrate, resulting in a reduction in the recovery and purity of the 9Z isomer. This indicates that even at the initial stage of the extraction the internal mass resistance is significant. Isomer purity and recovery could be enhanced upon grinding of the algae powder.     

Formation of bioerodible polymeric microspheres and microparticles by rapid expansion of supercritical solutions.

Polyhydroxy acids [poly(L-lactic acid) (L-PLA), poly(D,L-lactic acid) (DL-PLA), and poly-(glycolic acid) (PGA)], biocompatible and bioerodible polymers that are being investigated for controlled delivery of pharmaceuticals and are approved by the Food and Drug Administration for in vivo sutures and bone repair implants, have been dissolved in supercritical CO2 and precipitated by rapid expansion of the resulting supercritical solutions (RESS). The formation of these microparticles and microspheres is a first step toward the goal of producing, in a single processing step, drug-loaded polymeric microspheres for use in controlled release applications. Nucleation of poly(L-lactic acid) from CO2 and CO2-acetone mixtures produced microparticles and microspheres ranging from 4 to 25 microns. Microspheres (2-20 microns) were also obtained with chlorotrifluoromethane as solvent. Commercial L-PLA precipitated after extraction of low molecular weight oligomers showed degradation kinetics similar to that of the starting material. The precipitation of DL-PLA from CO2 produced irregular-sized particles (10-20 microns). PGA, a polymer insoluble in most organic solvents, was found to be soluble in supercritical CO2. Nucleation of PGA from CO2 produced both regular-sized particles and needles of 10-40-microns length. The total solubility of commercial L-PLA in supercritical CO2 at 250 bar and 55 degrees C decreased from 0.14 wt % to less than 0.05 wt % and then leveled off as the cumulative flow of CO2 per unit mass of L-PLA loaded in the extractor increased beyond 20 standard L of CO2/g of L-PLA. Use of acetone (1 wt %) as a cosolvent increased L-PLA solubility by approximately 500%.     

Improvement of ethanol fermentation under hyperbaric conditions

Recently more and more interest is manifested in the utilization of high-pressure extraction using supercritical gases for the purification of products in biochemical processes. Some researchers have examined the possibility of circulating continuously a supercritical gas through the fermentor, under hyperbaric pressure, to recover the desired product while the fermentation is taking place. However, an earlier study has demonstrated that fermentation with baker's yeast was inhibited by a long exposure under hyperbaric pressure. This article is concerned with the improvement of ethanol production under hyperbaric pressure in view of the development of an integrated fermentation-extraction process where supercritical carbon dioxide would be used for the in situ recovery of ethanol. The selection of the best yeast strain and operation under cyclic pressures are considered.     

Molecular mechanisms of yeast tolerance and in situ detoxification of lignocellulose hydrolysates

Pretreatment of lignocellulose biomass for biofuel production generates inhibitory compounds that interfere with microbial growth and subsequent fermentation. Remediation of the inhibitors by current physical, chemical, and biological abatement means is economically impractical, and overcoming the inhibitory effects of lignocellulose hydrolysate poses a significant technical challenge for lower-cost cellulosic ethanol production. Development of tolerant ethanologenic yeast strains has demonstrated the potential of in situ detoxification for numerous aldehyde inhibitors derived from lignocellulose biomass pretreatment and conversion. In the last decade, significant progress has been made in understanding mechanisms of yeast tolerance for tolerant strain development. Enriched genetic backgrounds, enhanced expression, interplays, and global integration of many key genes enable yeast tolerance. Reprogrammed pathways support yeast functions to withstand the inhibitor stress, detoxify the toxic compounds, maintain energy and redox balance, and complete active metabolism for ethanol fermentation. Complex gene interactions and regulatory networks as well as co-regulation are well recognized as involved in yeast adaptation and tolerance. This review presents our current knowledge on mechanisms of the inhibitor detoxification based on molecular studies and genomic-based approaches. Our improved understanding of yeast tolerance and in situ detoxification provide insight into phenotype-genotype relationships, dissection of tolerance mechanisms, and strategies for more tolerant strain development for biofuels applications.     

不同方法提取的艾叶挥发油指纹图谱分析

采取超临界CO2、微波和水蒸气蒸馏三种方法提取艾叶挥发油,利用GC-MS分析了不同方法提取的 艾叶挥发油指纹图谱。超临界CO2和微波萃取的艾叶挥发油的化学成分相似,水蒸气蒸馏与前两种方法提 取的挥发油的化学成分有些差异。超临界CO2萃取的艾叶挥发油更具天然性,超临界CO2萃取法为提取艾 叶挥发油的较理想方法;微波辅助萃取也不失为一种可行的方法。     

沙蚕毒素的等温加湿超临界CO_2-萃取法研究

本文研究了用等温加湿超临界ＣＯ２从海洋蠕虫状动物沙蚕体腔细胞中,提取沙蚕毒素的萃取条件（温度、压力、时间）对萃取过程的影响。探讨了等温加湿超临界ＣＯ２萃取法与传统提取方法比较的最佳选择分离方法。     

Effect of partial pressure of CO2 on the production of thermostable alpha-amylase and neutral protease by Bacillus caldolyticus

Controlling the concentration of dissolved oxygen is a standard feature in aerobic fermentation processes but the measurement of dissolved CO2 concentrations is often neglected in spite of its influence on the cellular metabolism. In this work room air and room air supplemented with 5% and 10% carbon dioxide were used for aeration during the cultivation of the thermophilic microorganism Bacillus caldolyticus (DSM 405) on starch to produce alpha-amylase (E.C. 3.2.1.1) and neutral protease (E.C. 3.4.24.27/28). The increased CO2 concentrations resulted in a 22% raise in activity of secreted alpha-amylase and a 43% raise in protease activity when compared with aeration with un-supplemented room air. There was no effect on the final biomass concentration. Furthermore, the lag-phase of fermentation was reduced by 30%, further increasing the productivity of alpha-amylase production. Determinations of dissolved CO2 in the culture broth were conducted both in situ with a probe as well as using exhaust gas analysis and both the methods of quantification showed good qualitative congruence.     

Online monitoring and characterization of flocculating yeast cell flocs during continuous ethanol fermentation

Both intrinsic and observed kinetic investigations for those ethanol fermentations using self-flocculated yeast strains have been hindered by the lack of real online monitoring techniques and proper characterization methods for the flocs. An optical detecting technique, the focused beam reflectance measurement probe developed by Lasentec (Redmond, WA) was inserted into a fermentor to monitor the floc chord length distributions. Using a simulating system composed of the floc-buffer suspensions, the total floc chord length counts per second were directly correlated with the floc biomass concentrations so that the floc biomass concentrations can be in situ detected. Furthermore, a characterization method of the flocs was established by properly weighted treatments of the detected floc chord length distributions. When a real yeast floc ethanol fermentation system was detected during its intrinsic kinetic investigations in which the floc size needed to be controlled at a level of micrometer scale to eliminate inner mass transfer limitations, it was found and validated that CO(2) produced during fermentation exerted significant disturbances. By applying 1/length-weighted treatment, these disturbances were effectively overcome.