Accelerostat study in conventional and microfluidic bioreactors to assess the key role of residual glucose in the dimorphic transition of Yarrowia lipolytica in response to environmental stimuli.

Yarrowia lipolytica, with a diverse array of biotechnological applications, is able to grow as ovoid yeasts or filamentous hyphae depending on environmental conditions. This study has explored the relationship between residual glucose levels and dimorphism in Y. lipolytica. Under pH stress conditions, the morphological and physiological characteristics of the yeast were examined during well-controlled accelerostat cultures using both a 1 L-laboratory scale and a 1 mL-microfluidic bioreactor. The accelerostat mode, via a smooth increase of dilution rate (D), enabled the cell growth rate to increase gradually up to the cell wash-out (D ≥µmax of the strain), which was accompanied by a progressive increase in residual glucose concentration. The results showed that Y. lipolytica maintained an ovoid morphology when residual glucose concentration was below a threshold value of around 0.35-0.37 mg L-1. Transitions towards more elongated forms were triggered at this threshold and progressively intensified with the increase in residual glucose levels. The effect of cAMP on the dimorphic transition was assessed by the exogenous addition of cAMP and the quantification of its intracellular levels during the accelerostat. cAMP has been reported to be an important mediator of environmental stimuli that inhibit filamentous growth in Y. lipolytica by activating the cAMP-PKA regulatory pathway. It was confirmed that the exogenous addition of cAMP inhibited the mycelial morphology of Y. lipolytica, even with glucose concentrations exceeding the threshold level. The results suggest that dimorphic responses in Y. lipolytica are regulated by sugar signaling pathways, most likely via the cAMP-PKA dependent pathway.

A perfusion-capable microfluidic bioreactor for assessing microbial heterologous protein production.

We present an integrated microfluidic bioreactor for fully continuous perfusion cultivation of suspended microbial cell cultures. This system allowed continuous and stable heterologous protein expression by sustaining the cultivation of Pichia pastoris over 11 days. This technical capability also allowed testing the impact of perfusion conditions on protein expression. This advance should enable small-scale models for process optimization in continuous biomanufacturing.

Polymer waveguide backplanes for optical sensor interfaces in microfluidics.

A polymer optical backplane capable of generic luminescence detection within microfluidic chips is demonstrated using large core polymer waveguides and vertical couplers. The waveguides are fabricated through a new process combining mechanical machining and vapor polishing with elastomer microtransfer molding. A backplane approach enables general optical integration with planar array microfluidics since optical backplanes can be independently designed but still integrated with planar fluidic circuits. Fabricated large core waveguides exhibit a loss of 0.1 dB cm(-1) at 626 nm, a measured numerical aperture of 0.50, and a collection efficiency of 2.86% in an n = 1.459 medium, comparable to a 0.50 NA microscope objective. In addition to vertical couplers for out-of-plane collection and excitation, polymer waveguides are doped with organic dyes to provide wavelength selective filtering within waveguides, further improving optical device integration. With large core low loss waveguides, luminescence collection is improved and measurements can be performed with simple LEDs and photodetectors. Fluorescein detection via fluorescence intensity with a limit of detection (3sigma) of 200 nM in a 1 microL volume is demonstrated. Phosphorescence lifetime based oxygen detection in water in an oxygen controllable microbial cell culture chip with a limit of detection (3sigma) of 0.08% or 35 ppb is also demonstrated utilizing the waveguide backplane. Single waveguide luminescence collection performance is equivalent to a back collection geometry fiber bundle consisting of nine 500 microm diameter collection fibers.

Microbioreactor arrays with integrated mixers and fluid injectors for high-throughput experimentation with pH and dissolved oxygen control.

We have developed an integrated array of microbioreactors, with 100 microL working volume, comprising a peristaltic oxygenating mixer and microfluidic injectors. These integrated devices were fabricated in a single chip and can provide a high oxygen transfer rate (k(L)a approximately 0.1 s(-1)) without introducing bubbles, and closed loop control over dissolved oxygen and pH (+/-0.1). The system was capable of supporting eight simultaneous Escherichia coli fermentations to cell densities greater than 13 g-dcw L(-1) (1 cm OD(650 nm) > 40). This cell density was comparable to that achieved in a 4 litre reference fermentation, conducted with the same strain, in a bench scale stirred tank bioreactor and is more than four times higher than cell densities previously achieved in microbioreactors. Bubble free oxygenation permitted near real time optical density measurements which could be used to observe subtle changes in the growth rate and infer changes in the state of microbial genetic networks. Our system provides a platform for the study of the interaction of microbial populations with different environmental conditions, which has applications in basic science and industrial bioprocess development. We leverage the advantages of microfluidic integration to deliver a disposable, parallel bioreactor in a single chip, rather than robotically multiplexing independent bioreactors, which opens a new avenue for scaling small scale bioreactor arrays with the capabilities of bench scale stirred tank reactors.