Rapid screening and cultivation of oleaginous microorganisms

Oleaginous microbial strains were cultivated to identify the best oil-producing strain amongst Yarrowia lipolytica (CGMCC 2.1398), Lipomyces starkeyi (CGMCC 2.1608), Rhodosporidium toruloides (CGMCC 2.1389), Mortierella isabellina (CGMCC 3.3410), Cunninghamella blakeleana (CGMCC 3.970), and Mycobacterium QJ311. A method for rapid determination of oil content and fatty acid composition was established to identify the optimum oil-producing strains. This method had a relative standard deviation of 4.09%, an average recovery ratio of 97.09% and a detection limit of 0.1-1.0 g. Mortierella isabellina CGMCC 3.3410 was identified as the best oil-producing strain amongst the six strains tested, with a total biomass of 75 g/10 L and a lipid content of 35%. A rapid screening method of oleaginous microorganisms is discussed for the first time.     

Colorimetric cell proliferation assay for microorganisms in microtiter plate using water-soluble tetrazolium salts

A colorimetric method to assay cell proliferation of microorganisms in 96-well microtiter plates using water-soluble tetrazolium salts and electron mediators was developed. Combinations of 6 kinds of water-soluble tetrazolium salts and 27 kinds of electron mediators that considered the metabolic efficiency of microorganisms and the influence with medium components were investigated. 2-Methyl-1,4-naphthoquinone (NQ) was reduced most effectively by various species of microorganisms, and a combination of WST-8 as a water-soluble tetrazolium salt with 2-methyl-1,4-NQ repressed the increase in background due to medium components. In the presence of 2-methyl-1,4-NQ, WST-8 was reduced by microbial cells to formazan, which exhibited maximum absorbance at 460 nm. The proposed tetrazolium method could be applied to measure proliferations of various microbial cells including 3 kinds of yeast, 9 kinds of Gram-positive bacteria, and 10 kinds of Gram-negative bacteria. Linear relationships between the absorbance and viable microbial cell density were obtained in all microorganisms, suggesting that the absorbance change reflected the microbial cell proliferation.     

Rapid and sensitive detection of D-hydantoinase producing microorganisms by using microtiter plate assay

Summary D-Hydantoinase activity of microorganisms was detected by a microtiter plate assay using dihydrouracil as substrate in 24 h. The detection gave a clear result compared to the spot-test in which the colour development of one colony interfered with colour development by adjacent colonies. The sensitivity of the detection was also higher than that of the spot-test.     

Respiration activity monitoring system for any individual well of a 48-well microtiter plate

Background

Small-scale micro-bioreactors have become the cultivation vessel of choice during the first steps of bioprocess development. They combine high cultivation throughput with enhanced cost efficiency per cultivation. To gain the most possible information in the early phases of process development, online monitoring of important process parameters is highly advantageous. One of these important process parameters is the oxygen transfer rate (OTR). Measurement of the OTR, however, is only available for small-scale fermentations in shake flasks via the established RAMOS technology until now. A microtiter plate-based (MTP) μRAMOS device would enable significantly increased cultivation throughput and reduced resource consumption. Still, the requirements of miniaturization for valve and sensor solutions have prevented this transfer so far. This study reports the successful transfer of the established RAMOS technology from shake flasks to 48-well microtiter plates. The introduced μRAMOS device was validated by means of one bacterial, one plant cell suspension culture and two yeast cultures.

Results

A technical solution for the required miniaturized valve and sensor implementation for an MTP-based μRAMOS device is presented. A microfluidic cover contains in total 96 pneumatic valves and 48 optical fibers, providing two valves and one optical fiber for each well. To reduce costs, an optical multiplexer for eight oxygen measuring instruments and 48 optical fibers is introduced. This configuration still provides a reasonable number of measurements per time and well. The well-to-well deviation is investigated by 48 identical Escherichia coli cultivations showing standard deviations comparable to those of the shake flask RAMOS system. The yeast Hansenula polymorpha and parsley suspension culture were also investigated.

Conclusions

The introduced MTP-based μRAMOS device enables a sound and well resolved OTR monitoring for fast- and slow-growing organisms. It offers a quality similar to standard RAMOS in OTR determination combined with an easier handling. The experimental throughput is increased 6-fold and the media consumption per cultivation is decreased roughly 12.5-fold compared to the established eight shake flask RAMOS device.

     

组学技术在产油微生物中的应用

微生物油脂是未来燃料和食品用油的重要潜在资源.近年来,随着系统生物学技术的快速发展,从全局角度理解产油微生物生理代谢及脂质积累的特征成为研究热点.组学技术作为系统生物学研究的重要工具被广泛用于揭示产油微生物脂质高效生产的机制研究中,这为产油微生物理性遗传改造和发酵过程控制提供了基础.文中对组学技术在产油微生物中的应用概...     

A microtiter plate assay for aspartate transcarbamylase

A discontinuous, colorimetric method for the assay of aspartate transcarbamylase has been adapted for use with 96-well microtiter plates. The method is based on that of L.M. Prescott and M.E. Jones (1969 Anal. Biochem. 32, 408-419) for the detection of ureido compounds, using monoxime and antipyrine. The enzymatic reaction is carried out in a volume of 150 microliters and is stopped by the addition of 100 microliters of a color mix. After development, the absorbance at 460 nm is directly proportional to the quantity of N-carbamyl-L-aspartate up to at least 0.125 mumol and to the quantity of Escherichia coli aspartate transcarbamylase up to about 7 ng. Kinetic parameters obtained from saturation curves for L-aspartate in 50 mM Tris-acetate, pH 8.0, are indistinguishable from those previously obtained: Vmax = 26,225 mumol h-1 mg-1; S0.5 = 14.7 mmol liter-1; hill constant = 2.5.     

A microtiter plate assay for N-acetyl-beta-D-glucosaminidase using a fluorogenic substrate

We describe a simple endpoint method for the determination of N-acetyl-beta-D-glucosaminidase (NAGase; EC 3.2.1.30). NAGase uses a fluorogenic substrate, 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide, at pH 4.6, liberating the fluorescent 4-methylumbelliferone. The method is reproducible and fast both at room temperature and at 37 degrees C. The procedure developed can be used, e.g., in the diagnosis of bovine subclinical mastitis, where elevated NAGase activities are found in raw milk samples. The assay procedure has a high capacity and high sensitivity and several hundred milk samples can be screened per hour using 96-well microtiter plates and an automated fluorescence reader. In addition to its use in mastitis diagnosis, the assay can be used in the diagnosis of some diseases of human origin.     

Thermal analysis for differentiating between oleaginous and non-oleaginous microorganisms

The potential of thermal analysis for differentiating between oleaginous and non-oleaginous microorganisms was investigated using thermogravimetry (TG) and differential thermal analysis (DTA). The model oleaginous microorganisms used in the present study were the fungi, Mortierella alpina IFO32281 and Mortierella alliacea YN-15, the unicellular alga, Aurantiochytrium sp. CB 15-5, and the yeast, Rhodosporidium toruloides DMKU3-TK 16. Escherichia coli JM109, Rhodococcus opacus B-4, and Saccharomyces cerevisiae were used as the control non-oleaginous microorganisms. In simultaneous TG and DTA, the furnace temperature was linearly increased from 30 to 280 °C, decreased to 30 °C, linearly increased from 30 to 360 °C, and then isothermally held at 360 °C for 30 min. This two-step linear temperature program was effective in resolving overlapping exothermic peaks in the DTA curves in the temperature range from 280 to 360 °C. Heat evolved from a microbial sample was estimated from the area under the exothermic peak between 280 and 360 °C using indium as a standard material. There was a linear relationship between the exothermic heat and total lipid content of the tested microorganisms. Exothermic heat per dry sample mass (kJ/g) in the temperature range from 280 to 360 °C is a promising measure for differentiating between oleaginous and non-oleaginous microorganisms.     

A microtiter plate assay for inorganic phosphate

A microtiter assay for the detection of picomolar quantities of inorganic phosphate has been described. The assay, linear between 50 and 1000 pmol of inorganic phosphate, is simple and rapid, with results obtainable in several minutes. Results from 5'-nucleotidase and (Ca2+ + Mg2+)ATPase assays using this method were compared with conventional phosphate assays and showed a high degree of correlation. The high sensitivity of this assay and the small sample size needed allows its widespread use in biochemical studies involving the generation of inorganic phosphate.     

Development and validation of a plate technique for screening of microorganisms that produce volatile sulfur compounds

Volatile sulfur compounds (VSCs) are of major importance for flavor development in foodstuffs such as cheeses. Such compounds originate from the amino acid l-methionine, which can be degraded to methanethiol (MTL), a common precursor to a variety of other VSCs. A plate assay based on double-layer petri dishes containing 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB), a chemical used for the estimation of free thiols, in the upper layer provides an easy and reliable detection method for thiol-producing, cheese-ripening microorganisms. MTL production was quantitated by measuring the yellow-orange color intensity resulting from reaction with DTNB. Using this method, 18 Geotrichum candidum strains isolated from cheeses were compared, and the color intensity was found to be correlated with the production of microbial VSCs as measured by gas chromatographic analysis.     

Periodic acid-Schiff's reagent assay for carbohydrates in a microtiter plate format

Microtiter plate colorimetric assays are widely used for analysis of carbohydrates and glycoconjugates. However, mucins are often not easily detected, as they have low neutral sugar content. We have adapted and optimised the periodic acid–Schiff’s reagent (PAS) staining for microtiter plate assay by examining five factors: concentration and volume of periodic acid, oxidation time, volume of Schiff’s reagent, and color development time. This assay requires just 25 μl of sample, utilises standardised Schiff’s reagent, and has decreased assay time (140 min to completion). Seventeen monosaccharides (acidic, neutral, basic, phosphorylated, and deoxy) and four disaccharides were assessed. PAS-positive carbohydrates (amino, N-acetylamino, deoxy, and certain neutral monosaccharides, and sialic acids) responded linearly within a 10–100 nmol range approximately, which varied for each carbohydrate. The assay response for fetuin and porcine gastric mucin (PGM) was linear up to 150 μg (highest concentration tested), with no response from nonglycosylated protein. A lower response for asialofetuin was observed, but desialylated PGM preparations were similar or higher in response than their sialylated counterparts. The simplicity and low sample consumption of this method make it an excellent choice for screening or quantitation of chromatographic fractions containing carbohydrates and glycoconjugates, especially in the case of mucins.     

A microtiter plate assay for protein kinase C

We report the development of a microtiter plate assay for protein kinase C. Reaction components and enzyme samples (protein kinase C purified by phosphatidylserine/cholesterol affinity or DEAE-Sephacel ion-exchange chromatography) were added to wells of a 96-well microtiter plate. The assay was started by the addition of [gamma-32P]ATP with a repeating pipet. After a 3-min incubation at 30 degrees C the wells were sampled six at a time with a 12-channel pipet and spotted onto phosphocellulose filter paper rectangles which were washed with tap water and acetone and counted for radioactivity. The microtiter plate method was more rapid than but gave results similar to those of a standard assay performed in plastic test tubes individually incubated in a 30 degrees C water bath. The microtiter plate procedure gave an intraassay (within one plate) variation of less than 9% and an interassay (between plates) variation of less than 5%. It was linear with time of incubation for 20 min and with amount of enzyme. This method can be used to expedite the assaying of column chromatography fractions for protein kinase C (and other kinase) activity.     

一种鉴定多糖水解酶类及其产生菌的新方法

利用曲里苯蓝能与多糖形成复合物的原理,建立起一种简便、快捷、灵敏的筛选多糖水解酶类及其产生菌的平板鉴定方法。曲里苯蓝对供试微生物无毒害作用,不影响酶的活性,并可高温灭菌。其最适浓度在0.005%~0.01%(W/V)之间。可用于纤维素水解酶类、淀粉水解酶类、甘露聚糖酶、普鲁兰酶等多糖水解酶,与传统方法相比,该方法不仅有着同样高的灵敏度,而且能够提高筛选效率,避免污染问题,同时适合于多种多糖底物。但不能用于木聚糖酶和菊粉酶的检测。     

Assessment of biofilm cell removal and killing and biocide efficacy using the microtiter plate test

Biofilm formation on surfaces has serious economic and environmental implications. Growth of biofilm within a water distribution system can lead to problems such as biocorrosion and biofouling accumulation. To prevent and control these occurrences, it is necessary to use suitable biocides to remove the biofilm and kill biofilm cells. In this study, the genera Actinobacillus, Branhamella, Bacillus, Micrococcus and Acinetobacter were isolated from biofilms formed on brass coupons exposed to a cooling water system. It was shown by the microtiter plate test that a mixed culture of the isolates and a single culture of Acinetobacter sp(2) produced high levels of biofilm formation. A microwell plate technique was applied for assessment of the ability of various biocides to remove and kill mixed-culture biofilm cells and Acinetobacter sp(2), the latter as a single-species biofilm with a high rate of biofilm production. The results showed that the mixed-culture biofilm cells had more resistance to removal and killing by some biocides, such as hydrogen peroxide and sulfathiazole, than the single-species biofilm cells (Acinetobacter sp(2)). Oxidising biocides, such as sodium hypochlorite and hydrogen peroxide, demonstrated a higher potential for biofilm removal and killing compared with non-oxidising biocides (sulfathiazole and glutaraldehyde).     

Development of a plate technique for screening of polysaccharide-degrading microorganisms by using a mixture of insoluble chromogenic substrates

A plate assay based on the visible solubilization of small substrate particles and the formation of haloes on Petri dishes, containing a mixture of different dye-labelled polysaccharides as substrates, provides a specific, reliable and rapid simultaneous detection of corresponding polysaccharide-degrading microorganisms. It has potential for increasing the efficacy of screening of microorganisms, utilizing different polysaccharides, in large numbers of natural samples. Diversely colored insoluble forms of amylose, xylan and hydroxyethyl-cellulose (HE-cellulose) were prepared as chromogenic substrates by using the cross-linking reagent 1,4-butanediol diglycidyl ether and the dyes Brilliant Red 3B-A, Cibacron Blue 3GA and Reactive Orange 14. Using the method, the bacteria with amylase or xylanase or cellulase or a combination of these activities were screened from soil and sludge samples, selected and identified according to 16S rDNA sequencing.     

A microtiter plate assay for polyglutamine aggregate extension

Polyglutamine (polyGln) aggregates are neuropathological markers of expanded CAG repeat disorders, and may also play a critical role in the development of these diseases. We have established a highly sensitive, fast, reproducible, and specific assay capable of monitoring aggregate-dependent deposition of polyglutamine peptides. This assay allows detailed studies on various aspects of aggregation kinetics, and also makes possible the detection and quantitation of low levels of "extension-competent" aggregates. In the simplest form of this assay, polyGln aggregates are made from chemically synthesized peptides and immobilized onto microplate wells. These wells are incubated for different times with low concentrations of a soluble biotinylated polyGln peptide. Europium-streptavidin complexation of the immobilized biotin, followed by time-resolved fluorescence detection of the deposited europium, allows us to calculate the rate (fmol/h) of incorporation of polyGln peptides into polyGln aggregates. This assay will make possible basic studies on the assembly mechanism of polyGln aggregates and on critical features of the reaction, such as polyGln length dependence. The assay also will be a valuable tool for screening and characterizing anti-aggregation inhibitors. It will also be useful for detection and quantitation of aggregation-competent polyGln aggregates in biological materials, which may prove to be of critical importance in understanding the disease mechanism.