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1、Rapid monitoring of glycerol in fermentation growth media: Facilitating crude glycerol bioprocess developmentSergi Abad, Xavier Prez, Antoni Planas, Xavier TuronnBioengineering Department, Institut Qumic de Sarri (IQS), Universitat Ramon Llull. Via Augusta 390, 08017 Barcelona, Spaina r t i c l e i
2、n f oArticle history: Received 16 July 2013 Received in revised form 5 December 2013 Accepted 11 December 2013 Available online 9 January 2014Keywords: Monitoring Fermentation Glycerol HPLC TLC Bioprocessa b s t r a c tRecently, the need for crude glycerol valorisation from the biodiesel industry ha
3、s generated many studies for practical and economic applications. Amongst them, fermentations based on glycerol media for the production of high value metabolites are prominent applications. This has generated a need to develop analytical techniques which allow fast and simple glycerol monitoring du
4、ring fermentation. The methodology should be fast and inexpensive to be adopted in research, as well as in industrial appli- cations. In this study three different methods were analysed and compared: two common methodologies based on liquid chromatography and enzymatic kits, and the new method based
5、 on a DotBlot assay coupled with image analysis. The new methodology is faster and cheaper than the other conventional methods, with comparable performance. Good linearity, precision and accuracy were achieved in the lower range (10 or 15 g/L to depletion), the most common range of glycerol concentr
6、ations to monitor fermentations in terms of growth kinetics.and fatty acids as AA, EPA and DHA 29,30. All the efforts in bioprocess development based on crude glycerol require analytical methods to quantify glycerol. Cell growth kinetics are substrate-dependent, so the calculation of growth related
7、parameters essential to the fermentation designrequire carbon source quantification. Nowadays, many labs andcompanies determine glycerol concentrations using techniques based on separation (liquid chromatography), enzymatic techni- ques and potentiometric methods. When developing fermenta- tions, th
8、e carbon source monitoring requires reliable, inexpensive and particularly fast methodologies to quantify the residual glycerol. A large number of samples is typically generated, and rapid monitoring is crucial to the decision-making process during batch, fed-batch or continuous fermentation. High p
9、erformance liquid chromatography (HPLC) is widely used to analyse glycerol content in fermentation media. HPLC methods offer high accuracy. Enzymatic based kits are also popular in glycerol based fermentations 2932. Enzymatic kits might offer less accuracy compared to HPLC, and post a direct cost pe
10、r sample analysed. Both methods can reach high accuracy but they are time consuming with a response delay often critical for fermentation monitoring close to real time. Current HPLC meth- odologies spent around 30 min per sample considering sample preparation injection and peak analysis. Depending o
11、n the column, the time can be reduced from 30 down to 15 min per sample. Enzymatic kits are faster than HPLC methods, but the cost per each assay is relatively high. Therefore it discourages exhaustive monitoring of the fermentation, due to the amount of samples. Potentiometric methodologies are sim
12、ple and cheaper than HPLC and enzymatic kits. However, this methodology needs larger volumes of samples and could not be suitable for monitoring different fermentations the at same time. The nature of the samples to analyse can be crucial in the methodology selection. Medium composition (e.g. high s
13、alinity and organic compoundsContents lists available at ScienceDirectjournal homepage: front matter fax: 34 93 205 6266.E-mail address: xavier.turoniqs.edu (X. Turon).Talanta 121 (2014) 210214interfering with the methods) or metabolites generated mayintroduce errors or invalidate the methodology.
14、For the specific case of enzymatic kits, the interference of the metals present in the medium in some of the enzymatic reactions could also mislead the determination. Potentiometric methods could not be applic- able in presence of organic compounds (e.g. coming from Yeast extract or metabolites prod
15、uced) containing more than two hydroxyl groups on adjacent carbon atoms 33. The current work presents a method developed to monitor either pure or crude glycerol residual concentration from fermen- tation samples. The method relies on a DotBlot assay (DB) coupled with image processing algorithms. A
16、method presenting several analogies but using Thin Layer Chromatography (TLC) was reported to quantify residual glycerol in biodiesel samples 34. Here, thesimple DB developed assay has specific staining adapted and validated for bioprocess development in aqueous medium with high salinity. Subsequent image processing and analysis has the advantage of rapid output with enough accuracy for bioprocess monitoring with a short respon
