Limits...
High-throughput screening for a moderately halophilic phenol-degrading strain and its salt tolerance response.

Lu ZY, Guo XJ, Li H, Huang ZZ, Lin KF, Liu YD - Int J Mol Sci (2015)

Bottom Line: Bacterial enrichments were cultivated in 48 deep well microplates instead of shake flasks or tubes.Measurement of phenol concentrations was performed in 96-well microplates instead of using the conventional spectrophotometric method or high-performance liquid chromatography (HPLC).PCR detection of the functional genes suggested that the largest subunit of multicomponent phenol hydroxylase (LmPH) and catechol 1,2-dioxygenase (C12O) were active in the phenol degradation process.

View Article: PubMed Central - PubMed

Affiliation: State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, China. lzy1009a@163.com.

ABSTRACT
A high-throughput screening system for moderately halophilic phenol-degrading bacteria from various habitats was developed to replace the conventional strain screening owing to its high efficiency. Bacterial enrichments were cultivated in 48 deep well microplates instead of shake flasks or tubes. Measurement of phenol concentrations was performed in 96-well microplates instead of using the conventional spectrophotometric method or high-performance liquid chromatography (HPLC). The high-throughput screening system was used to cultivate forty-three bacterial enrichments and gained a halophilic bacterial community E3 with the best phenol-degrading capability. Halomonas sp. strain 4-5 was isolated from the E3 community. Strain 4-5 was able to degrade more than 94% of the phenol (500 mg · L(-1) starting concentration) over a range of 3%-10% NaCl. Additionally, the strain accumulated the compatible solute, ectoine, with increasing salt concentrations. PCR detection of the functional genes suggested that the largest subunit of multicomponent phenol hydroxylase (LmPH) and catechol 1,2-dioxygenase (C12O) were active in the phenol degradation process.

No MeSH data available.


Related in: MedlinePlus

The correlation between phenol concentration and the absorbance of the generated indoxyl antipyrine in a 96-well microplate screening system (upper panel); the color formation of indoxyl antipyrine at different phenol concentrations (lower panel).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4490417&req=5

ijms-16-11834-f002: The correlation between phenol concentration and the absorbance of the generated indoxyl antipyrine in a 96-well microplate screening system (upper panel); the color formation of indoxyl antipyrine at different phenol concentrations (lower panel).

Mentions: This study established a high-throughput assay for phenol measurement performed in 96-well microplates based on the 4-aminoantipyrine color reaction principle, which resolved the low throughput bottleneck in conventional measurement for phenol concentrations. This assay had a good linear detection range of phenol from 0.1–2 mg·L−1. The standard curve was y = 0.08808x + 0.00203 (R2 = 0.9988) (Figure 2), revealing that the assay was reliable. The relative standard deviation (RSD) between wells was less than 3%, indicating no significant difference between them. The data based on the high-throughput assay were in close agreement with those obtained from the conventional spectrophotometric method (ASTM Standard D1783-01,2012) and HPLC [17]; the high correlation coefficient (0.976 and 0.965) were obtained between them by statistical analysis, revealing the accuracy of the assay. Therefore, it could be used for phenol determination in a high-throughput screening system. The conventional spectrophotometric method was widely used for the measurement of phenol. However, it was time-consuming, with a large chemical reagent requirement, and inefficient, which was impractical for a high-throughput system. The coloring reaction was carried out in the colorimetric tubes. Each sample was sequentially detected, which resulted in detection errors. HPLC was a sensitive and fast method. However, the samples are required to flow through the column for separation in sequence, resulting in a low detection throughput. This study establishes a high-throughput phenol determination method based on conventional colorimetric principles. Colorimetric tubes were replaced with 96-well microplates, and the spectrophotometer was replaced with the microplate spectrophotometer, which contributed to the micro-volume quantification. The microplate spectrophotometer was able to read the information of the absorbance for the entire plate and to detect 96 samples simultaneously, achieving the purpose of high-throughput detection. The eight-channel pipettes transferred bacterial culture to 96-well microplates in 40 s; the absorbance values of all of the wells were obtained in 6 s using the microplate reader; data could be obtained well within 1 h. Each test had a 96-throughput. In this study, only three tests were required to obtain 258 phenol concentrations.


High-throughput screening for a moderately halophilic phenol-degrading strain and its salt tolerance response.

Lu ZY, Guo XJ, Li H, Huang ZZ, Lin KF, Liu YD - Int J Mol Sci (2015)

The correlation between phenol concentration and the absorbance of the generated indoxyl antipyrine in a 96-well microplate screening system (upper panel); the color formation of indoxyl antipyrine at different phenol concentrations (lower panel).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490417&req=5

ijms-16-11834-f002: The correlation between phenol concentration and the absorbance of the generated indoxyl antipyrine in a 96-well microplate screening system (upper panel); the color formation of indoxyl antipyrine at different phenol concentrations (lower panel).
Mentions: This study established a high-throughput assay for phenol measurement performed in 96-well microplates based on the 4-aminoantipyrine color reaction principle, which resolved the low throughput bottleneck in conventional measurement for phenol concentrations. This assay had a good linear detection range of phenol from 0.1–2 mg·L−1. The standard curve was y = 0.08808x + 0.00203 (R2 = 0.9988) (Figure 2), revealing that the assay was reliable. The relative standard deviation (RSD) between wells was less than 3%, indicating no significant difference between them. The data based on the high-throughput assay were in close agreement with those obtained from the conventional spectrophotometric method (ASTM Standard D1783-01,2012) and HPLC [17]; the high correlation coefficient (0.976 and 0.965) were obtained between them by statistical analysis, revealing the accuracy of the assay. Therefore, it could be used for phenol determination in a high-throughput screening system. The conventional spectrophotometric method was widely used for the measurement of phenol. However, it was time-consuming, with a large chemical reagent requirement, and inefficient, which was impractical for a high-throughput system. The coloring reaction was carried out in the colorimetric tubes. Each sample was sequentially detected, which resulted in detection errors. HPLC was a sensitive and fast method. However, the samples are required to flow through the column for separation in sequence, resulting in a low detection throughput. This study establishes a high-throughput phenol determination method based on conventional colorimetric principles. Colorimetric tubes were replaced with 96-well microplates, and the spectrophotometer was replaced with the microplate spectrophotometer, which contributed to the micro-volume quantification. The microplate spectrophotometer was able to read the information of the absorbance for the entire plate and to detect 96 samples simultaneously, achieving the purpose of high-throughput detection. The eight-channel pipettes transferred bacterial culture to 96-well microplates in 40 s; the absorbance values of all of the wells were obtained in 6 s using the microplate reader; data could be obtained well within 1 h. Each test had a 96-throughput. In this study, only three tests were required to obtain 258 phenol concentrations.

Bottom Line: Bacterial enrichments were cultivated in 48 deep well microplates instead of shake flasks or tubes.Measurement of phenol concentrations was performed in 96-well microplates instead of using the conventional spectrophotometric method or high-performance liquid chromatography (HPLC).PCR detection of the functional genes suggested that the largest subunit of multicomponent phenol hydroxylase (LmPH) and catechol 1,2-dioxygenase (C12O) were active in the phenol degradation process.

View Article: PubMed Central - PubMed

Affiliation: State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, China. lzy1009a@163.com.

ABSTRACT
A high-throughput screening system for moderately halophilic phenol-degrading bacteria from various habitats was developed to replace the conventional strain screening owing to its high efficiency. Bacterial enrichments were cultivated in 48 deep well microplates instead of shake flasks or tubes. Measurement of phenol concentrations was performed in 96-well microplates instead of using the conventional spectrophotometric method or high-performance liquid chromatography (HPLC). The high-throughput screening system was used to cultivate forty-three bacterial enrichments and gained a halophilic bacterial community E3 with the best phenol-degrading capability. Halomonas sp. strain 4-5 was isolated from the E3 community. Strain 4-5 was able to degrade more than 94% of the phenol (500 mg · L(-1) starting concentration) over a range of 3%-10% NaCl. Additionally, the strain accumulated the compatible solute, ectoine, with increasing salt concentrations. PCR detection of the functional genes suggested that the largest subunit of multicomponent phenol hydroxylase (LmPH) and catechol 1,2-dioxygenase (C12O) were active in the phenol degradation process.

No MeSH data available.


Related in: MedlinePlus