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What Is Dissolved Oxygen (DO)?

The term 'dissolved oxygen' refers to the level of free, non-compound oxygen ions present in an aqueous solution. In bioprocessing, the presence of dissolved oxygen is vital for the growth and metabolism of aerobic microorganisms, as it serves as a crucial substrate for their energy production. DO is typically measured and reported as milligrams per liter [mg-DO/L of water (mg/L)], percent saturation (%), or partial pressure (pO2).

The DO concentration in a bioprocess is influenced by various factors, including temperature, pressure, and the rate of oxygen consumption by the organisms. As with pH, which is the measure of acidity or alkalinity in a solution, dissolved oxygen is an essential parameter that needs to be carefully monitored and controlled to optimize bioprocess outcomes.

Classification of Microorganisms

Types-of-Aerobes-9-26

While there are many variables that are important to cellular growth, oxygen is by far one of the most Critical Process Parameters (CPPs) in bioprocessing. Microorganisms can be divided into three main groups based on their oxygen requirements:

1. Obligate aerobes can produce energy only via aerobic respiration and require molecular oxygen to live.

2. Obligate anaerobes cannot tolerate any molecular oxygen and they require an oxygen-free environment to grow.

3. Facultative anaerobes on the other hand, can grow in both environments – in the presence of molecular oxygen and in its absence. Within the group of facultative anaerobes, two subgroups can be defined.

4. Aerotolerant organisms can grow in the presence of molecular oxygen, but they cannot use it. They would always produce their energy via anaerobic fermentation. Lactobacilli are prominent representatives of aerotolerant organisms.

5. The second subgroup of facultative anaerobic organisms, including many yeast species, would use oxygen for energy production if present and switch to the anaerobic fermentation when oxygen is depleted.

In addition, microaerobic organisms are organisms that need molecular oxygen to preserve energy (making them obligate aerobes), but they cannot tolerate the partial pressure of oxygen of the atmosphere. These organisms require lower oxygen partial pressures to survive. 


Check out our blog Microorganism Spotlight - Anaerobic Organism to learn more.

 

The Basics of DO in Bioprocessing

Efficient oxygen transfer is essential for a successful aerobic cultivation. Regardless of vessel type or application, determining the volumetric oxygen transfer coefficient (kLa) and the oxygen transfer rate (OTR) can offer some important benefits, including:

  • Ensuring healthy microbial colonies
  • Increasing product yields
  • Reducing waste from suboptimal oxygen concentrations

What is kLa?

The volumetric mass transfer coefficient (kLa) refers to the pace at which oxygen can move between the gas phase to the liquid phase. Consider a gas bubble containing oxygen in liquid. The kLa, can be represented by an equation.

kLa = kL × a

kL = mass transfer coefficient, describes the rate of molecular diffusion through the gas-liquid interface

a = the surface area available for diffusion

In most bioprocesses the most important step is the OTR, hence engineers and scientists try to affect it by changing their kLa.

gas-liquid-barrier
oxygen-transfer-in-shake-flasks

Oxygen Transfer

There are several key terms used to describe the points at which oxygen transfer occurs in bioprocessing. 

  1. Oxygen Transfer Rate (OTR) refers to how fast oxygen molecules can move from a gas bubble to the liquid media. To do so, O2 molecules must diffuse through a stagnant region called the gas-liquid interface. The OTR is influenced by the volumetric mass transfer coefficient (kLa).
  2. The amount of oxygen currently present in the liquid medium is represented by DO. 
  3. Oxygen Uptake Rate (OUR) is used to describe how fast oxygen, now present in the media, can overcome the liquid-cell interface to be available for use by the cell or aggregate.

When working with bioreactors, engineers use many different means to influence the kLa for a better OTR. This most often includes increasing small bubbles as well as pressure. Increasing OTR in shake flasks can be influenced by several factors.

Factors Affecting OTR in Shake Flasks

In shake flasks, oxygen transfer from the headspace to the cells is not driven by bubbles but by the thickness of the liquid film.

shake flask shaking speed graphic
Shaking
Speed

OTR increases with shaking speed.

8
Filling
Volume

Lower filling volumes result in higher OTR.

shake flask media viscosity
Media
Viscosity

Higher viscosities will have lower OTR.

7
Baffled vs
Non-Baffled

Baffles can be used to increase aeration.

10
Closure
Types

Some closure types allow for more oxygen in the vessel than others.

shake flask shaking diameter graphic
Shaking Diameter

A larger shaking diameter can increase the mixing and aeration of the liquid.

Why Monitor DO?

Ph-1-svg

Changes in Cellular Morphology and Function

All cells have an optimal pH level that supports their growth. While this can vary some, most cells prefer to be around neutral. When pH levels fall outside of the acceptable range, cell culture growth can become inhibited or altered and the titer of their products reduced. pH levels that are too acidic or alkaline can actually destabilize the genetic material of cells, which can cause mutations and ultimately cell death if not corrected.

Indication of Contamination

It is not possible to visualize contamination in clear media, however, sudden changes in pH are a good indicator of bacterial or fungal contamination. Most cases of bacterial contamination in the cell culture laboratory are caused by aerobes.

When working with indicator dyes, the media will become acidic and appear yellow if contaminated with aerobic bacteria. However, if the bacteria are anaerobic, the contamination will cause the medium to become basic and will appear pink. A fungal contamination will cause the media to turn hazy.

Contamination is a concern because bacteria often produce toxins that disrupt cell function and can ultimately destroy cell cultures. Cell cultures are especially susceptible to contaminations because they grow much slower than most bacteria and fungi, so the latter easily overgrow mammalian cells in nutrient rich media.

pH-Flasks-Yellow-and-Pink-Medium (1)
pH-6-Flask-with-K

Availability of Nutrients

Culture media, also known as growth media, is any solution comprised of amino acids, vitamins, inorganic salts, glucose, and other substrates that support the health and growth of cells in culture. Many also contain serums as a source of growth factors, hormones, and attachment factors.

Depending on the cell type and preferred outcome, nutrient supplementation in cell culture can be used to increase cell viability and genomic stability. The surrounding pH can influence how cells take in these nutrients. Macronutrients such as nitrogen, potassium, calcium, magnesium, and sulfur are more readily available at pH 6.0–6.5, while micronutrients become less available at higher, alkaline levels of greater that 7.0.

Want to learn more about monitoring DO? Our experts have the answers.

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The Pitfalls of Oxygen Limitations

Oxygen limitations occur when the amount of available molecular oxygen falls below a certain threshold. Without close monitoring, shake flasks can often fall victim to oxygen limitations. While you cannot visually identify oxygen limitations, you can see the effects they have on your fermentation. The best options to identify oxygen limitations in cultivations include biomass and dissolved oxygen monitoring.

biomass monitoring OTR

Biomass Monitoring

Lack of oxygen results in slowed growth of microorganisms, often associated with a lack of an exponential phase. While a linear curve does not correlate solely to oxygen limitation, secondary parameters can assist in determining the root cause.

oxygen limitation graph

DO Montoring

The organism utilizes oxygen to metabolize key nutrients. Without the addition of new resources, the levels of DO fall below the organism's threshold resulting in a limitation. 

How Is DO Measured​?

  • Chemosensors
  • Polarographic Sensors
  • Galvanic Sensors
dissolved-oxygen-chemosensor

Principle of Measurement: Spectroscopy - Luminescence

A chemosensor containing DO luminescent dye indicators is embedded in a matrix. A sensor emits light at one wavelength, exciting the chemosensors which show luminescence in another wavelength. Depending on the concentration of oxygen present in the solution, the amount of luminescence changes. The sensor measures this phase shift which is then calculated to represent a DO value.

Technologies:

Available in different form factors (patches, integrated in flow cells, pills) for continuous measurements in small vessels.

Learn More About DO Sensor Pills
  • Advantages
      • Does not consume oxygen
      • Little to no maintenance
      • Can be stored dry
      • On-line & in-line options
      • Can be automated (measurement and data handling)
  • Disadvantages
      • Sufficient flow is needed for accurate measurements
      • Running costs for consumables (OPEX)
      • Not compatible with all setups
polarographic-probe-dissolved-oxygen

Principle of Measurement: Electrochemical

Also commonly referred to as aerometric or Clark electrode, these sensors are common in bioprocessing. The sensor consists of two electrodes, a noble metal anode and silver cathode, contained in an electrolyte-filled chamber divided by a gas permeable membrane. A voltage is applied between the electrodes (cathode and anode). As oxygen diffuses across the membrane, it undergoes a reduction reaction at the cathode, where it is given an electron, while the anode undergoes an oxidation reaction, releasing electrons into the solution. The electron flow creates a current that is used to determine the dissolved oxygen concentration.

Technologies:

Used in invasive probes to spot check small vessels, or for continuous measurements in bioreactors.
  • Advantages
      • Well-known technology
      • Less frequent maintenance (vs galvanic)
      • More affordable (than optical)
      • On-line & in-line options
      • Can be automated (measurement and data handling)
  • Disadvantages
      • Requires a warm-up period
      • Consumes oxygen
      • Media must be in motion for accurate measurements
      • Requires frequent calibration
galvanic-probe-dissolved-oxygen

Principle of Measurement: Electrochemical

Very similar to the working principle of polarographic probes, galvanic probes also consist of a cathode and anode, present in an electrolyte-containing chamber with a gas permeable membrane. The main difference is that galvanic sensors use different materials. The anode is lead or zinc, and the cathode is gold or silver. The use of dissimilar metals creates an internal electric potential, so no outside voltage is required to start measuring. 

Technologies:

Used in invasive probes to spot check small vessels, or for continuous measurements in bioreactors.
  • Advantages
      • No warm-up period needed
      • Very stable at low DO levels
      • Fast response time
      • On-line & in-line options
      • Can be automated (measurement and data handling)
  • Disadvantages
      • Media must be in motion for readings
      • Requires regular maintenance to ensure accurate readings
      • Requires frequent calibration
Chemosensors
dissolved-oxygen-chemosensor

Principle of Measurement: Spectroscopy - Luminescence

A chemosensor containing DO luminescent dye indicators is embedded in a matrix. A sensor emits light at one wavelength, exciting the chemosensors which show luminescence in another wavelength. Depending on the concentration of oxygen present in the solution, the amount of luminescence changes. The sensor measures this phase shift which is then calculated to represent a DO value.

Technologies:

Available in different form factors (patches, integrated in flow cells, pills) for continuous measurements in small vessels.

Learn More About DO Sensor Pills
  • Advantages
      • Does not consume oxygen
      • Little to no maintenance
      • Can be stored dry
      • On-line & in-line options
      • Can be automated (measurement and data handling)
  • Disadvantages
      • Sufficient flow is needed for accurate measurements
      • Running costs for consumables (OPEX)
      • Not compatible with all setups
Polarographic Sensors
polarographic-probe-dissolved-oxygen

Principle of Measurement: Electrochemical

Also commonly referred to as aerometric or Clark electrode, these sensors are common in bioprocessing. The sensor consists of two electrodes, a noble metal anode and silver cathode, contained in an electrolyte-filled chamber divided by a gas permeable membrane. A voltage is applied between the electrodes (cathode and anode). As oxygen diffuses across the membrane, it undergoes a reduction reaction at the cathode, where it is given an electron, while the anode undergoes an oxidation reaction, releasing electrons into the solution. The electron flow creates a current that is used to determine the dissolved oxygen concentration.

Technologies:

Used in invasive probes to spot check small vessels, or for continuous measurements in bioreactors.
  • Advantages
      • Well-known technology
      • Less frequent maintenance (vs galvanic)
      • More affordable (than optical)
      • On-line & in-line options
      • Can be automated (measurement and data handling)
  • Disadvantages
      • Requires a warm-up period
      • Consumes oxygen
      • Media must be in motion for accurate measurements
      • Requires frequent calibration
Galvanic Sensors
galvanic-probe-dissolved-oxygen

Principle of Measurement: Electrochemical

Very similar to the working principle of polarographic probes, galvanic probes also consist of a cathode and anode, present in an electrolyte-containing chamber with a gas permeable membrane. The main difference is that galvanic sensors use different materials. The anode is lead or zinc, and the cathode is gold or silver. The use of dissimilar metals creates an internal electric potential, so no outside voltage is required to start measuring. 

Technologies:

Used in invasive probes to spot check small vessels, or for continuous measurements in bioreactors.
  • Advantages
      • No warm-up period needed
      • Very stable at low DO levels
      • Fast response time
      • On-line & in-line options
      • Can be automated (measurement and data handling)
  • Disadvantages
      • Media must be in motion for readings
      • Requires regular maintenance to ensure accurate readings
      • Requires frequent calibration

sbi's DO Monitoring Solutions

For Shake Flasks

multiparameter-sensor-shake-flask-DO-pill-motion-4

DO Sensor Pills for Shake Flasks

  • Novel, patented pill technology

  • Single-use pill: Factory-calibrated and pre-sterilized for immediate use

  • Drop & Go: Easy handling and fast experiment setup

  • Combine with LIS for DO-based feeding in shake flasks

For Bioreactors

ph-flow-cells

DO Flow Cells For Flow Loops

  • Continuously monitor DO in flow loops using fiber optic sensors.

  • DO range: 0-50% O2 (gas), 0-100% O2 (liquid) 

  • Factory-calibrated and pre-sterilized

  • Single-use design reduces risk of contamination

View The Success Stories

dissolved oxygen graph kitana
"Incorporating sbi’s flow cells into our system removed the need for manual sampling, saving us time, reducing the risk of contamination, and providing information on how the cells are growing even when we are not in the lab. With availability of this more detailed view of our culture, we can make informed improvements to our cell expansion process."

-- Kitana Manivone Kaiphanliam (Washington State University)
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