Monitoring Oxygen and pH during Red Grape Fermentation

Advances in high-performance sensor materials and optoelectronics have enabled novel optical sensors for use in applications in life sciences, pharmaceuticals, biotechnology, food and beverage processing and more. Compared with traditional electrochemical sensing techniques and devices such as galvanic sensors, Ocean Optics optical sensors can be made in small and customizable form factors, allow non-intrusive measurements and do not consume the sample.

In this application note, we demonstrate the viability of optical O2 and pH sensors for real-time monitoring of biological parameters during red grape fermentation. Our optical sensors can be customized in probes and patches and allow non-intrusive measurements.

Principles of Sensor Operation

Ocean Optics optical sensors use light to measure the interaction between a sample and one of our proprietary sensing materials in order to quantify a chemical property of the sample.

The principle of operation is to trap an oxygen-sensitive fluorophore or pH indicator dye in a thin film host matrix that can be applied to the tip of a fiber, an adhesive membrane such as a patch, or a flat substrate such as a cuvette or microtiter plate. The indicator materials change optical properties in response to specific analytes in their immediate environment and electronics then measure the response. For oxygen, the NeoFox phase fluorometer measures the partial pressure of dissolved or gaseous oxygen; for pH, a miniature fiber optic spectrometer such as our Flame model measures the colorimetric (absorbance) response of the pH dye.

Experimental Conditions

To demonstrate the viability of optical oxygen and pH sensors for monitoring biological parameters in bioreactor environments, we placed oxygen- and pH-sensitive adhesive patches inside a bioflask in which red grape biofermentation occurred. Although we used table grapes and not the variety grown for wines and juices, the basic concepts of bioprocess monitoring still apply.

Bioreactors are closed-environment systems where the cells are cultured under specific conditions to synthesize the final product. Such systems require constant monitoring of DO and pH to optimize bioprocesses. Ocean Optics patches provide oxygen and pH measurements in both headspace and the liquid phase.

Figure 1. Optical oxygen and pH sensors are integrated into a biosystem in patch form for non-intrusive measurements.

Our RedEye® oxygen patches were attached to the container with adhesive backing to monitor the oxygen in headspace and in solution. The pH patches were placed in solution to monitor pH changes during the fermentation process (Figure 1).

- Fresh red table grapes were mashed and the must was left untouched for 48 hours.
- The juice was drained from the mixture and placed in a bioflask.
- Yeast cells and nutrients were added to begin fermentation.
- Non-intrusive oxygen and pH measurements during the aerobic and anaerobic processes were monitored over a 60-hour period.

Oxygen Measurements

To measure oxygen we used two NeoFox phase fluorometers equipped with bifurcated optical fibers for the excitation and detection of the RedEye patches. NeoFox measures the phase shift between a blue LED used to excite the oxygen indicator in the patch and the emission signal of the fluorescence. The fibers were situated normal to the outside surface of the flask pointing directly at the patches on the inside.

 
pH Measurements

To measure pH we used a Flame spectrometer with tungsten halogen light source and a bifurcated optical fiber. One leg of the fiber transmitted light to the patch inside the container and the other leg of the fiber read the response from the reflective patch inside the solution. Standard pH buffers were used for calibration. Absorbance curves were observed over time.

 
Results

In first two hours of fermentation, the RedEye oxygen sensor in the solution detected a quick drop from air saturation as soon as yeast cells and nutrients were added (Figure 2). The yeast cells had started consuming the oxygen through the liquid cell membrane interface by the diffusion process. The pH sensor in the solution measured a slight drop in absorbance as the oxygen decreased and CO2 was released. The same experiment could be extended to a single cell in a microfluidics well culture system.

Headspace remained at air saturation for approximately the first 2.5 hours of fermentation. Once the oxygen in solution was completely quenched, the yeast cells and nutrients started consuming the oxygen from the headspace.

Figure 2. With the addition of yeast cells and nutrients, air saturation in the solution dropped dramatically.

The limitations of electrochemical-based oxygen and pH sensing are overcome by Ocean Optics optical oxygen and pH patches. Such patches can be integrated easily within a small-scale biosystem such as a bioflask and provide continuous, non-intrusive monitoring of key system parameters (Figure 3). The ability to monitor DO and pH in real time without perturbing a sealed environment can lead to an improved understanding of the processes in the bioreactor and, ultimately, help to facilitate the development of new products, improved processes and higher quality output.

Figure Figure 3. Monitoring of red grape fermentation using optical sensors shows oxygen saturation in headspace and solution and pH changes in solution over 60 hours.