Optimization of polyhydroxyalkanoates fermentations with on-line capacitance measurement
Introduction
In a bioprocess, the amount of viable biomass is a crucial physiological parameter, which is highly correlated to the cell growth, metabolism and productivity (Carvell and Dowd, 2006). Dry cell weight (DCW), optical density (OD) and packed mycelial volume (PMV) are the conventional off-line indicators of biomass concentration. Nevertheless, these measurements are time-consuming and cannot reflect the microbial viability. Although the number of colony forming units (CFU) measures merely viable cells, the measurement is also time-consuming and difficult to reproduce (Xiong et al., 2008).
Currently, the employment of advanced instruments to monitor and control the microbial physiological state has become popular in the industrial fermentations. On-line measurements of physiological parameters are beneficial to comprehend the variation of cell status and culture condition in time. The constant physiological parameters such as oxygen uptake rate (OUR) have been a scale-up criterion to promote the erythromycin production (Zou et al., 2009). Redox electrode can measure redox potential to optimize the ethanol fermentation (Yu and Lin, 2012). Respiratory quotient (RQ) is controlled in micro-aerobic fermentation of ethanol production to improve the yield (Franzen, 2003).
In the latest studies, a few of instruments for biomass determinations have been developed, such as in situ near-infrared measurement (Arnold et al., 2002), impedance spectroscopy measurement (Sarro et al., 2012), fluorescence measurement (Zhao et al., 2011) and capacitance measurement (Markx et al., 1991). Among them, Biomass Monitor (Aber Instruments Ltd., Aberystwyth, UK) has become an effective tool to evaluate the concentration of viable biomass. Cells with intact plasma membranes are considered as tiny capacitors after polarized under the influence of a radio frequency electric field (Kiviharju et al., 2008). After choosing an optimal frequency with β-dispersion, the capacitance values are proportional to concentrations of viable cells. Dead cells and other particles without intact cell membrane do not contribute to the capacitance signals (Carvell and Dowd, 2006). This technology satisfies Process Analytical Technology (PAT) requirements initiated by FDA (2004). It has been applied successfully to monitor concentrations of various cells, such as bacteria (Kedia et al., 2013), yeasts (Xiong et al., 2008), plant cells (Markx et al., 1991), insect cells (Zeiser et al., 1999) and mammalian cells (Neves et al., 2000).
In this study, Biomass Monitor is adopted during the polyhydroxyalkanoates (PHAs) fermentation by Ralstonia eutropha (reclassified from Alcaligenes eutrophus). PHAs are a class of biological polyesters, which can be biosynthesized by microbe utilizing starch as carbon resource (Pan et al., 2012). In previous study, it has been found that the R. eutropha is capable of producing the PHAs containing approximately 10 mol% 3-hydroxybutyrate (3HB) and 90 mol% 3-hydrocyvalerate (3HV). Because PHAs are nontoxic and biodegradable, they turn out to be the potential substitutes of fossil-fuel plastics. Typically, in this fermentation process when the bacteria encounter an excess supply of carbon but limitation of nitrogen, phosphorus or oxygen, bacterial sizes begin to expand with the PHAs accumulation (Madison and Huisman, 1999). As the morphological changes are closely related to the PHAs production, an automated method is developed for the quantification of microbial morphology. Meanwhile, the capacitance measurement can also display the dynamic changes regarding the visibility of biomass and the morphology, and is utilized as a key physiological parameter for the fed-batch control strategy in this work.
So far, few investigations focus on analyzing capacitance combined with the microscope image of bacteria and other parameters. In addition, a fed-batch control strategy judging by capacitance is first proposed, which is a convenient and feasible method for improving the industrial PHAs production.
Section snippets
Strain, media and cultivation
The strain used in this study is R. eutropha TA-032, which is kindly provided by Tianan Pharmacy Corporation (Zhejiang, China).
1 L pre-cultured R. eutropha are inoculated in 29 L of fermentation medium in a 50 L bioreactor (NCBIO) using Biostar database at 34 °C. The medium consists of, in (g/L): yeast extract (8.0), glucose (20.0), Na2HPO4 (6.78), KH2PO4 (3.0), NH4Cl (1.0), NaCl (0.5), CaCl2 (4.0) and MgSO4 (0.24). During the fed-batch culture, the concentration of residual sugar maintains at 15.0 ±
Comparison of on-line and off-line biomass measurements
A typical time course of the PHAs fermentation by R. eutropha is shown in Fig. 1. On-line capacitance measurement is compared with three conventional off-line biomass measurements: the number of colony forming units (CFU), optical density at 540 nm (OD540) and dry cell weight (DCW). The number of CFU represents viable biomass, while OD540 and DCW are used to estimate total biomass concentration. After the inoculation, the four biomass measurements increase synchronously during 8–32 h.
Conclusions
Our data demonstrate that on-line capacitance measurement at one single frequency can accurately estimate the viable biomass concentration during the PHAs fermentations. First, the mean projected cell area acquired by automatically quantification of microbial morphology enables a deeper understanding of the capacitance measurements. Second, capacitance-based and μ are verified as key parameters related to the PHAs productions. Third, the controlling strategies of phosphate concentration
Acknowledgements
This work was financially supported by a Grant from the Major State Basic Research Development Program of China (973 Program), No. 2012CB721006, National High Technology Research and Development Program, No. 2012AA021201, and National Key Technology Support Program, No. 2011BAF02B04. We also thank Tianan Pharmacy Corporation (Zhejiang, China) for donating the industrial strain Ralstonia eutropha TA-032.
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