- Open Access
Application of 8-parallel micro-bioreactor system with non-invasive optical pH and DO biosensor in high-throughput screening of l-lactic acid producing strain
© The Author(s) 2018
- Received: 10 January 2018
- Accepted: 23 April 2018
- Published: 30 April 2018
Compared with shake flask, bioreactor offers a relatively stable and controllable extracellular environment for cell growth and metabolism. Meanwhile, parallel micro-bioreactor system could well meet the screening flux requirement in the process of high-throughput strain screening. In this study, a self-developed 8-parallel micro-bioreactor system with non-invasive optical biosensors was introduced to substitute the traditional shake flask.
Optical pH and DO biosensors could be well applied for the process monitoring and controlling in the cultures of commonly used microorganisms through maintaining constant temperature and bioreactor shading treatment. Subsequently, 8-parallel micro-bioreactor system was adopted in the rescreening procedure of high-throughput screening process, and it significantly increased the feasibility of scaling up a cultivating system without any sacrifice on the screening flux. As a result, a designated mutant strain Lactobacillus paracasei S4 was obtained, which presented an improvement of 18.9% on titer value of l-lactic acid. Moreover, the yield also increased from 0.903 ± 0.005 to 0.932 ± 0.013 g/g.
- High-throughput screening
- Lactic acid
- Lactobacillus paracasei
- Optical biosensor
Bioreactor, as a cultivator for microorganisms, can promote the efficiency of biochemical reactions with the advantages of offering a relatively stable and controllable extracellular environment. With the developments on manufacturing and information integrating technologies, industrial bioreactors present new trends towards large scale and informatization. Miniaturized, automated, and high-throughput bioreactors have been widely adopted for strain screening and bioprocess development (Chen et al. 2009; Rao 2007; Schäpper et al. 2009; Thomas et al. 2014).
As compared to a bioreactor commonly used in the laboratory, micro-bioreactor has smaller volume and structure, which generally includes micro-bioreactor with milliliter scale (Doig et al. 2005) and ultramicro-bioreactor with microliter or nanoliter scale (El-Ali et al. 2006). Nowadays, micro-bioreactors have been extensively applied in the bioprocess development and in the strain improvement to save labor and material cost through miniaturizing the cultivation process (Betts and Baganz 2006).
Traditionally, shake flask and microtiter plate (MTP) were adopted for high-throughput strain screening (Shi et al. 2015; Tan et al. 2014; Zhu et al. 2017). However, these facilities can only support simple input–output relationship, and more importantly, difficulties are normally encountered to obtain valuable process data. Particularly for pH and dissolved oxygen (DO), which play important roles in reflecting the cell physiological status and quality, the traditional probes working on the electrochemical principle have the problems of relatively large volume, high cost, and high contamination risk. Thus, achieving an on-line monitoring and controlling the pH and DO in a micro-bioreactor became a pivotal issue. Correspondingly, in the last three decades, many devoted studies have focused on the development of optical sensors for the determination of pH and DO (Harms et al. 2002; Zhang et al. 2005). However, it could be noted that due to the complex environment existing in the cell culture process, the detection of fluorescent intensity was always limited by various environmental factors, including lighting in the surrounding, the temperature of culture, and the heterogeneity of medium. Therefore, unlike shake flask and MTP, with the integration of optical biosensors as well as exhaust gas mass spectrum, micro-bioreactor could realize the whole-process dynamic analyses of cell growth and metabolism, which would be helpful to quickly understand the physiological characteristics of the mutants in high-throughput screening process.
In this study, optical pH and DO detection systems were optimized and were equipped subsequently on the self-developed 8-parallel micro-bioreactor for testing different microbial cultures. Finally, the micro-bioreactor system was introduced into high-throughput rescreening process to substitute the original shake flask for the screening of strain in the higher production of l-lactic acid.
Strains and media
Three microorganisms, Escherichia coli DH5α (Takara, China), Pichia pastoris GS115 (Ye et al. 2017), and Lactobacillus paracasei NCBIO01 (Tian et al. 2016), were used in this study. For high-throughput strain screening experiments, the parent strain of L. paracasei NCBIO01 was adopted.
Luria–Bertani (LB) medium was used for E. coli culture, which consisted of tryptone 10 g/l, yeast extract 5 g/l, NaCl 5 g/l. The medium for P. pastoris culture was composed of glucose 20 g/l, K2SO4 18.2 g/l, CaSO4 0.93 g/l, MgSO4·7H2O 14.9 g/l, KOH 4.13 g/l, H3PO4 26.8 ml/l, PTM1 12 ml/l, antifoam 1 ml/l. The medium components of L. paracasei culture contained: glucose 130 g/l, peptone 13.3 g/l, yeast extract 13.3 g/l, CH3COONa 0.67 g/l, MnSO4 0.0133 g/l, MgSO4 0.0133 g/l, FeSO4 0.0133 g/l, NaCl 0.0133 g/l, Tween-80 1 ml/l. 20 g/l agar, and 10 g/l CaCO3 were added in solid plate medium for L. paracasei culture.
High-throughput screening of L. paracasei with high performance of l-lactic acid production
Cell growth determination
A spectrophotometer was used at 600 nm to measure cell growth of E. coli, P. pastoris, and L. paracasei. The cell biomass was expressed as dry cell weight (DCW), which has a linearity with optical density (OD). One OD unit corresponds to 0.34 and 0.41 g/l DCW for P. pastoris and L. paracasei.
l-lactic acid and residual glucose concentrations determination
The pH indicator (bromocresol green) assay in 24-well MTPs was a qualitative method for organic acid concentration in preliminary screening process as described by Lv et al. (2016). The 24-well MTPs fermentation broth was centrifuged at 4000g for 10 min, and then 200 μl supernatant was transferred into a 96-well microplate (Corning, USA). The absorbance change was detected at 616 nm by Multiskan GO microplate reader (Thermo Fisher Scientific). The concentration of l-lactic acid was specifically detected by SBA-40E biosensor analyzer (Shandong Province Academy of Sciences, China). The glucose kit (Kexin Biotechnology Institute, China) was used for residual glucose concentration analysis.
Relative residual error calculation
Optimization of non-invasive optical pH and DO detecting systems
The volume limitation of a traditional electrochemical probe makes it difficult to be equipped within a micro-bioreactor, whereas the non-invasive optical pH and DO sensors which are made from fluorescent dye film can combine with dissolved H+ and oxygen, respectively, to change the molecular conformation. Thus, the fluorescence signals can be detected under the excitation wavelength and the signal intensities exhibit a positive relationship with the concentrations of H+ and oxygen.
On the other hand, a significant fluctuation could also be noted on the pH and DO fluorescent signals when the detection system was exposed to external light (Fig. 3b). It could be observed that the maximum deviation in the measurement of pH reached 0.3 unit in the presence of external light, while it decreased to 0.05 unit with bioreactor shading treatment. Similar for DO measurement, its maximum deviation reduced from more than 15% to about 2%. The relative standard deviations of pH and DO measurements decreased from 1.54 and 6.01% to 0.189 and 0.691%, respectively, which demonstrated the improvements in the accuracy and stability of pH and DO detection.
The broth culture is a mixed solution of water, oxygen, organic materials, inorganic ions etc. Environmental temperature will affect the ionization degree of the electrolytes as well as the solubility of oxygen, and thus will subsequently influence the concentrations of H+ and dissolved oxygen. Although many investigations have reported on the novel biosensors for pH and DO detection in terms of principle, material, and chromophore structure (Bambot et al. 1994; Shen et al. 2011), but their practical applications in the process of cell culture were seldom taken into consideration.
Microorganism cultures in the micro-bioreactor
Hot model experiments of the micro-bioreactors were conducted using three commonly used laboratory microbes (E. coli, P. pastoris, and L. paracasei) as the testing strains. Relatively a higher oxygen supply was required for E. coli and P. pastoris, while L. paracasei exhibited facultative anaerobic characteristics. Moreover, the pH required for an optimal growth and metabolism of the common microbes were in the range of 5.0–8.0. Thus, as compared to shake flask, micro-bioreactor presented significant advantages both in monitoring and controlling the pH and DO.
Escherichia coli culture in the micro-bioreactor with process pH detection and DO regulation
On the other hand, due to a minor change in the pH value, the broth pH was not controlled during the whole process. Through the comparison of on-line pH by an optical biosensor and the off-line pH by a pH meter, the former was always approximately 0.1 unit lower than the latter (Fig. 4b), which could be partly ascribed to the discrepancy between the buffer of normalized pH and real broth. Simultaneously, his deviation could be considered as a system error, and the non-invasive optical pH detecting system could effectively monitor the change of the process pH.
Pichia pastoris culture in the micro-bioreactor with pH control
To test the applicability and stability of non-invasive optical pH detection system on the change in pH during the P. pastoris culture, which would spontaneously decrease the broth pH to lower than 4.0, it was conducted by adding NH4OH solution as a neutralizing agent to maintain the pH at 5.5. Similar to the process in E. coli culture, a difference was also observed between on-line and off-line measurements also occurred, while it showed a little dependence on the fermentation process.
Lactobacillus paracasei culture in the micro-bioreactor with process pH control and exhausted gas analysis
Lactic acid bacteria which always need a small amount of oxygen for their cell growth and metabolism are defined as facultative anaerobic microorganisms. Thus, regular DO parameter detected by DO probe loses the role to represent cellular oxygen metabolism. Moreover, additional neutralizing agent needs to be added into the broth to maintain a suitable range of pH for the cell growth and metabolism, as lactic acid is the main product of glucose metabolism. In this experiment, apart from controlling the process pH at 6.0, an additional process mass spectrum was introduced for L. paracasei culture as described in 5- and 50-l bioreactors (Lu et al. 2016; Wang et al. 2010, 2016), in order to calculate on-line physiological parameters of OUR, CER, and respiratory quotient (RQ).
Overall, by comparing with a shake flask, the micro-bioreactor not only demonstrated the advantages on the applicability of subjecting to different culture conditions, but also could characterize the real cell metabolic status by introducing more physiological parameters.
Application and performance of micro-bioreactor system in high-throughput screening of high l-lactic acid production strain
Fermentation performances of parent strain, mutants S1 to S6 at 24 h with a 5-l bioreactor
Maximum DCW (g/l)
l-lactic acid (g/l)
9.76 ± 0.42
88 ± 2.45
0.903 ± 0.005
11.04 ± 0.32a
96 ± 2.45a
0.914 ± 0.009
10.61 ± 0.64
90 ± 1.63
0.904 ± 0.013
9.59 ± 0.28
100 ± 3.27a
0.914 ± 0.003a
10.97 ± 0.33a
104 ± 1.63a
0.932 ± 0.013a
9.93 ± 0.20
84 ± 1.63
0.918 ± 0.011
9.64 ± 0.35
88 ± 3.27
0.914 ± 0.005
Considering the optimized detection conditions of non-invasive optical pH and DO biosensors, the micro-bioreactor fully satisfied the requirements for the cultures of commonly used laboratory microorganisms with monitoring and regulating the process pH and DO. Additionally, in the rescreening process, 8-parallel micro-bioreactor system was introduced into high-throughput screening of high l-lactic acid production strain to substitute the traditional shake flasks. The results demonstrated that the micro-bioreactor presented comparable screening flux with shake flask, while it could significantly increase the scale-up possibility. Finally, a mutant of L. paracasei S4 was acquired which improved the l-lactic acid titer value by 18.9% as compared with the parent strain and enhanced the l-lactic acid yield to 0.932 ± 0.013 g/g. Overall, the outcomes from this investigation could be extended to other bacteria or yeasts in obtaining a high-performance strain by high-throughput screening strategy.
XT, WW, and ZG conducted the experiments and data analysis. XT wrote the manuscript. MZ provided technical support for 8-parallel bioreactor system. HH, CJ, YZ, SZ gave advices in the experiment design and data analysis. AM participated in manuscript writing and polished the English thoroughly. All authors read and approved the final manuscript.
This work was financially supported by the National Science Foundation for Young Scientists of China (31700038), the National Key Research and Development Program (2017YFB0309302) and the National Major Scientific Technological Special Project (2012YQ15008706).
The authors declare that they have no competing interests.
Availability of data and materials
The datasets supporting the conclusions of this article were included in the main manuscript.
Consent for publication
All authors have read and approved the manuscript before submitting it to bioresources and bioprocessing.
Ethic approval and consent to participate
This study did not contain any studies with human participants or animals performed by any of the authors.
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- Bambot SB, Holavanahali R, Lakowicz JR, Carter G, Rao G (1994) Phase fluorometric sterilizable optical oxygen sensor. Biotechnol Bioeng 43:1139–1145View ArticleGoogle Scholar
- Betts JI, Baganz F (2006) Miniature bioreactors: current practices and future opportunities. Microb Cell Fact 5:21–34View ArticleGoogle Scholar
- Bridle H, Millingen M, Jesorka A (2008) On-chip fabrication to add temperature control to a microfluidic solution exchange system. Lab Chip 8:480–483View ArticleGoogle Scholar
- Chen A, Chitta R, Chang D, Amanullah A (2009) Twenty-four well plate miniature bioreactor system as a scale-down model for cell culture process development. Biotechnol Bioeng 102:148–160View ArticleGoogle Scholar
- Doig SD, Diep A, Baganz F (2005) Characterisation of a novel miniaturised bubble column bioreactor for high throughput cell cultivation. Biochem Eng J 23:97–105View ArticleGoogle Scholar
- El-Ali J, Sorger PK, Jensen KF (2006) Cells on chips. Nature 442:403–411View ArticleGoogle Scholar
- Harms P, Kostov Y, Rao G (2002) Bioprocess monitoring. Curr Opin Biotechnol 13:124–127View ArticleGoogle Scholar
- Liu X, Tian XW, Hang HF, Zhao W, Wang YH, Chu J (2017) Influence of initial glucose concentration on seed culture of sodium gluconate production by Aspergillus niger. Bioresour Bioprocess 4:55–68View ArticleGoogle Scholar
- Lu F, Wang ZJ, Zhao W, Chu J, Zhuang YP (2016) A simple novel approach for real-time monitoring of sodium gluconate production by on-line physiological parameters in batch fermentation by Aspergillus niger. Bioresour Technol 202:133–141View ArticleGoogle Scholar
- Lv XY, Song JL, Yu B, Liu HL, Li C, Zhuang YP, Wang YH (2016) High-throughput system for screening of high l-lactic acid-productivity strains in deep-well microtiter plates. Bioprocess Biosyst Eng 39:1737–1747View ArticleGoogle Scholar
- Rao G (2007) Twenty-four-well plate miniature bioreactor high-throughput system: assessment for microbial cultivations. Biotechnol Bioeng 98:1017–1028View ArticleGoogle Scholar
- Schäpper D, Alam MN, Szita N, Eliasson Lantz A, Gernaey KV (2009) Application of microbioreactors in fermentation process development: a review. Anal Bioanal Chem 395:679–695View ArticleGoogle Scholar
- Shen LJ, Lu XY, Tian H, Zhu WH (2011) Along wavelength fluorescent hydrophilic copolymer based on naphthalene diimide as pH sensor with broad linear response range. Macromolecules 44:5612–5618View ArticleGoogle Scholar
- Shi F, Tan J, Chu J, Wang YH, Zhuang YP, Zhang SL (2015) A qualitative and quantitative high-throughput assay for screening of gluconate high-yield strains by Aspergillus niger. J Microbiol Methods 109:134–139View ArticleGoogle Scholar
- Tan J, Chu J, Wang YH, Zhuang YP, Zhang SL (2014) High-throughput system for screening of Monascus purpureus high-yield strain in pigment production. Bioresour Bioprocess 1:16–22View ArticleGoogle Scholar
- Thomas C, Scanlon TC, Dostal SM, Griswold KE (2014) A high-throughput screen for antibiotic drug discovery. Biotechnol Bioeng 111:232–243View ArticleGoogle Scholar
- Tian XW, Wang YH, Chu J, Zhuang YP, Zhang SL (2016) Improvement of l-lactic acid production with a two-step OUR control strategy. J Chem Technol Biotechnol 91:2496–2502View ArticleGoogle Scholar
- Venkatesh KV (1997) Metabolic flux analysis of lactic acid fermentation: effects of pH and lactate ion concentration. Process Biochem 32:651–655View ArticleGoogle Scholar
- Wang ZJ, Wang HY, Li YL, Chu J, Huang MZ, Zhuang YP, Zhang SL (2010) Improved vitamin B12 production by step-wise reduction of oxygen uptake rate under dissolved oxygen limiting level during fermentation process. Bioresour Technol 101:2845–2852View ArticleGoogle Scholar
- Wang ZJ, Shi HL, Wang P (2016) The online morphology control and dynamic studies on improving vitamin B12 production by Pseudomonas denitrificans with online capacitance and specific oxygen consumption rate. Appl Biochem Biotechnol 179:115–1127Google Scholar
- Ye R, Huang MZ, Lu HZ, Qian JC, Lin WL, Chu J, Zhuang YP, Zhang SL (2017) Comprehensive reconstruction and evaluation of Pichia pastoris genome-scale metabolic model that accounts for 1243 ORFs. Bioresour Bioprocess 4:22–33View ArticleGoogle Scholar
- Zhang Z, Szita N, Boccazzi P, Sinskey AJ, Jensen KF (2005) A well-mixed, polymer-based microbioreactor with integrated optical measurements. Biotechnol Bioeng 93:286–296View ArticleGoogle Scholar
- Zhu XD, Arman B, Chu J, Wang YH, Zhuang YP (2017) Development of a method for efficient cost-effective screening of Aspergillus niger mutants having increased production of glucoamylase. Biotechnol Lett 39:739–744View ArticleGoogle Scholar