Validation of a high-throughput fermentation system based on online monitoring of biomass and fluorescence in continuously shaken microtiter plates

Abstract

Background: An advanced version of a recently reported high-throughput fermentation system with online measurement, called BioLector, and its validation is presented. The technology combines high-throughput screening and high-information content by applying online monitoring of scattered light and fluorescence intensities in continuously shaken microtiter plates. Various examples in calibration of the optical measurements, clone and media screening and promoter characterization are given.

Results: Bacterial and yeast biomass concentrations of up to 50 g/L cell dry weight could be linearly correlated to scattered light intensities. In media screening, the BioLector could clearly demonstrate its potential for detecting different biomass and product yields and deducing specific growth rates for quantitatively evaluating media and nutrients. Growth inhibition due to inappropriate buffer conditions could be detected by reduced growth rates and a temporary increase in NADH fluorescence. GFP served very well as reporter protein for investigating the promoter regulation under different carbon sources in yeast strains. A clone screening of 90 different GFP-expressing Hansenula polymorpha clones depicted the broad distribution of growth behavior and an even stronger distribution in GFP expression. The importance of mass transfer conditions could be demonstrated by varying filling volumes of an E. coli culture in 96 well MTP. The different filling volumes cause a deviation in the culture growth and acidification both monitored via scattered light intensities and the fluorescence of a pH indicator, respectively.

Conclusion: The BioLector technology is a very useful tool to perform quantitative microfermentations under engineered reaction conditions. With this technique, specific yields and rates can be directly deduced from online biomass and product concentrations, which is superior to existing technologies such as microplate readers or optode-based cultivation systems. In particular, applications with strong demand on high-throughput such as clone and media screening and systems biology can benefit from its simple handling, the high quantitative information content and its capacity of automation.

Figure 1

Comparison between scattered light and optical density measurements (absorption). Hansenula polymorpha (wt) culture in YPG medium measured in 96 well MTP, with 200 μL filling volume, at 37°C temperature, 995 rpm shaking frequency and 3 mm shaking diameter, scattered light (ex: 620 nm/em: -, Gain: 40).

Figure 2

Limits of biomass monitoring with scattered light. Hansenula polymorpha (wt) in YPG medium; E. coli JM109 in TB medium, both were measured in 96 well MTP, with 200 μL filling volume, at 37°C temperature, 995 rpm shaking frequency and 3 mm shaking diameter, scattered light (ex: 620 nm/em: -, Gain: 5).

Figure 3

Comparison of different media – variation of carbon source concentration monitored with scattered light intensities. Hansenula polymorpha RB11-pC10-FMD-GFP culture in YNB-G medium with varying glycerol concentrations (5, 10, 15 and 20 g/L); measured in 96 well MTP, with 200 μL filling volume, at 30°C temperature, 995 rpm shaking frequency and 3 mm shaking diameter, scattered light (ex: 620 nm/em: -, Gain: 10).

Figure 4

Influence of pH conditions on growth – buffered/unbuffered medium monitored with scattered light intensities and NADH fluorescence intensities. Hansenula polymorpha RB11-pC10-FMD-GFP culture in buffered YNB medium with 10 g/L glycerol and 100 mM phosphate and unbuffered YNB medium with 10 g/L glycerol without phosphate; measured in 96 well MTP, with 200 μL filling volume, at 30°C temperature, 995 rpm shaking frequency and 3 mm shaking diameter, scattered light (ex: 620 nm/em: -, Gain: 20), NADH (ex: 340 nm/em: 460 nm, Gain: 20).

Figure 5

Comparison of different media – Hansenula polymorpha on complex and synthetic media with glucose and glycerol as carbon source monitored with scattered light intensities. Hansenula polymorpha RB11-pC10-Mox-GFP culture in YPG, YPD, buffered YNB-D and YNB-G media; measured in 96 well MTP, with 200 μL filling volume, at 37°C temperature, 995 rpm shaking frequency and 3 mm shaking diameter, scattered light (ex: 620 nm/em: -, Gain: 20).

Figure 6

Comparison of different media – growth and protein expression of a flavin mononucleotide (FMN)-based fluorescent protein (FbFP) in E. coli. E. coli BL21-Pet 28A ytvAC62A culture in LB, TB and WR media; measured in 96 well MTP, with 200 μL filling volume, at 30°C temperature, 995 rpm shaking frequency and 3 mm shaking diameter, induction at 7.3 h with 0.5 mM IPTG, scattered light (ex: 620 nm/em: -, Gain: 20), FbFP (ex: 460 nm/em: 520 nm, Gain: 10).

Figure 7

Clone Screening – comparison of growth and GFP protein expression of 90 different Hansenula polymorpha clones. (A) growth via scattered light intensities (B) protein expression via GFP fluorescence intensities; (C) volumetric productivity (P_V) – calculated as GFP formation rate without consideration of setup time of the equipment, the best clones are depicted by given the well number in the diagram; 45 clones of Hansenula polymorpha RB11-pC10-Mox-GFP and 45 clones of Hansenula polymorpha RB11-pC10-FMD-GFP in buffered YNB-G medium; measured in 96 well MTP, with 200 μL filling volume, at 37°C temperature, 995 rpm shaking frequency and 3 mm shaking diameter, scattered light (ex: 620 nm/em: -, Gain: 20), GFP (ex: 485 nm/em: 520 nm, Gain: 10).

Figure 8

Derivation of specific product yield (Y_P/X). Data taken from the clone screening of Fig. 7, specific product yield Y_P/X calculated as ratio of GFP intensities (protein concentration) to scattered light intensities (biomass concentration); data arranged to present the best clones from left to right given the well number on the abscissa, TOP10 represents the best ten clones in respect to Y_P/X.

Figure 9

Characterization of promoters. MOX and FMD promoter regulation in Hansenula polymorpha on glucose and glycerol growth medium monitored via GFP fluorescence intensities and parallel measurement of scattered light intensities; Hansenula polymorpha wt, RB11-pC10-Mox-GFP and RB11-pC10-FMD-GFP in YPD (10 g/L glucose) and YPG (20 g/L glycerol) medium; measured in 96 well MTP, with 200 μL filling volume, at 37°C temperature, 995 rpm shaking frequency and 3 mm shaking diameter, scattered light (ex: 620 nm/em: -, Gain: 20), GFP (ex: 485 nm/em: 520 nm, Gain: 10).

Figure 10

Influence of filling volume. Growth and pH monitoring of E. coli cultures with filling volumes from 200 μl to 290 μL; E. coli BL21 culture in TB medium, measured in 96 well MTP, with 200, 230, 260 and 290 μL filling volume, at 30°C temperature, 995 rpm shaking frequency and 3 mm shaking diameter, scattered light (ex: 620 nm/em: -, Gain: 20), HPTS (ex: 410 nm and 460 nm/em: 510 nm, Gain: 10), calibration parameters for the Boltzmann equation: I_{R, min} = 0.00, I_{R, max} = 3.00, dpH = 0.50, pH₀ = 7.20.