Dissolved Oxygen and its measurement

What is dissolved oxygen? Without oxygen in the air that we breathe, we would not survive for a very long time. Imagine the animals that live underwater, well, they also need oxygen to survive. So, the question is how do they get oxygen inside the water? The answer is dissolved oxygen in the water.

If you ever mix salt/sugar in the water the crystals disappear, this is because the molecule of water is mixed with the salt/sugar molecules and is evenly distributed in the solution. Similarly, oxygen also evenly distributes in water to form a mixture. There are two ways for oxygen molecules to enter water bodies (ocean, lake). One is through the atmosphere, and another is through photosynthesis.

Oxygen makes up about a fifth of the atmosphere and its entry into water depends on the conditions like saltiness and temperature inside the water. If there is more salt present in the water, then no space will be there for oxygen molecules to stay. In the same way, if the water temperature is more then, the entropy will be more in water, thus, no space for oxygen. That is why there are some animals underwater who need more oxygen lives in colder water bodies and the ones who need less oxygen live in higher temperature water bodies.

We all know that plants use photosynthesis mechanisms to process their food but by doing so they also generate oxygen. Plants and other small living things like phytoplankton, cyanobacteria, dinoflagellates organisms who live underwater use photosynthesis and produce cum release oxygen which then further mix with water to form dissolved oxygen.

Effect of dissolved oxygen on culture/wastewater- Different people can relate DO differently based on their work. If we talk about a scientist, he/she will be interested in surface water as a direct indicator of the water body’s ability to support aquatic life. Students could use DO to help determine stable conditions for purging or to regulate the analysis of metals or volatile compounds. A plant operator would be concerned with DO levels for the microbes used to transform wastewater into a harmless product.

For an industry like Biotechnology or Bioprocesses, oxygen is necessary for cellular processes like respiration and cell division. However, DO impacts the pH of media and product quality. Increased level of DO is important for cell growth, controlling the upper limit of oxygen in cell culture because reactive oxygen species (ROS) can chemically degrade the protein of interest. Therefore, the operating range of DO typically falls 30-40% range. If these levels are too high or low, the cell culture process can be adversely affected by such issues as slow growth rates, poor nutrient uptake, cellular mutations, etc. DO can be measured through sensors or probes.

How does a sensor work? There is a total of three kinds of sensors that are generally used in bioreactors, vessels for bioprocesses, and water treatments. They are as follows:

• Optical DO sensor- the working of optical sensors uses fluorescence quenching measuring principle, in this method, the sensor is equipped with one LED, a photodiode, and a separating section covered by an oxygen-permeable membrane. This layer of the membrane has similar molecules of oxygen as are there in the medium, this means the partial oxygen layer is equal to the pressure in media. The membrane has macromolecules that are excited by an orange light and emit dark red fluorescent light. Meanwhile, oxygen molecules attach with these macromolecules and attenuate the fluorescent light. This attenuation of light is known as quenching, where fluorescent light is quenched by oxygen molecules. Photodiode will detect the light signal in the transmitter and display the required units on the board.

• In applications that involve higher temperatures like sterilization and CIP processes, sensors with a reference LED are used. Reference LED compensates for aging of measurement and delivers reliable results even in demanding applications. Advantages- faster availability, stable measured values, and extremely low maintenance. It can be used in quality assurance and process controls of various industries like wastewater, food and beverage, life sciences, chemical, and power.

• Galvanic DO sensor- generally, the Clark electrode is used as a sensor to measure dissolved oxygen in a solution. It consists of a glass tube, platinum electrode, silver chloride electrode, KCl electrolyte, Teflon membrane, and one o ring. Here platinum electrode acts as cathode and silver chloride electrode acts as an anode. Electrical connection between these two electrodes is completed by KCl solution. Electrode components are isolated from the solution by the Teflon membrane. This membrane has permeability to allow oxygen molecules through it. O-ring is a rubber ring that is used to hold the Teflon membrane on the glass tube, while measuring the dissolved oxygen in the solution, a Clark electrode probe is dipped into the solution. The anode and cathode probes are connected to an oxygen meter, the voltage is applied to electrodes, and oxygen molecules in the solution enter and slowly reach the electrodes through a permeable membrane by the process called diffusion. By a chemical reaction, it creates a small amount of electrical current to flow between electrodes. The amount of current flowing between electrodes is proportional to the oxygen concentration in the solution.

• This technique is highly dependent on the polarization concept, while taking measurement it consumes an amount of oxygen from solution equal to the diffusion in the sensor. So, the amount of oxygen nearby the probe is reduced. Hence to get an accurate measurement, the sensor must be stirred in the solution which is again a big task to perform.

• Polarographic DO sensors- Polarographic working principle is like the galvanic DO sensors as they both need cathode and anode electrodes and perform on the concept of polarization. The electron flow from the anode to the cathode represents the measuring signal which is proportional to the partial pressure of oxygen in the measured culture of media. The only difference between them is that the galvanic sensor requires a constant voltage for action and the polarographic sensor is self-polarizing due to the material properties of an anode (zinc or lead) and cathode (silver). Galvanic DO sensors can be used immediately after calibration, polarographic sensors need a warmup time of about 5-10 min. the applied voltage should be at least +401mv (magnitude of standard redox potential) at the cathode with reversed polarity. This negative voltage must be constant and stabilized against a reference electrode.

Calibration of the probe- DO sensor probes are usually calibrated through transmitters CM44x using zero solution (reference solution). Zero solution is made by adding 20gms of sodium sulfite in 1L of water and mixed properly. A single batch of this zero-water solution is good for repetition for 4-5 days. At first, the solution is stirred for 1 min continuously, the sensor is emerged in it for about 15 min and through the analyzer, the reference calibration is selected to acquire minimum data at parts per million and is saved for further use.

Probe maintenance- Old probes when start giving unusually low reading needs reconditioning at that particular time. For this, probes are taken out of the vessel and are first cleaned and then connected to a multimeter, check the voltage for 30sec max and if it is coming below 25mv then it’s confirmed that the probe needs maintenance. For reconditioning, a maintenance kit is required in which there is a syringe, a blunt tip needle, electrolyte solution, and a replacement membrane. From the old probe, the membrane cap is unscrewed, and the electrolyte is removed from the probe. Through syringe and blunt tip needle, the fresh electrolyte is injected into the probe and a new membrane cap is attached.

Next thing is to confirm whether it’ll give good readings in further batches or not. This is again tested by the connecting probe with a multimeter and if it shows data from 45mv to 55mv then it is ready to use.

Conclusion- Many liquids are essential in our daily life such as water, beverage, dairy product, acids, bases, and pharmaceutical products. The quality of the liquid is determined by its physical and chemical properties and to assess these properties, there are various principles of measurements used and one of them is DO in liquids.

For the dissolved oxygen measurement, many different types of sensors are available according to the operating environment of the culture in fermentation processes. Polarographic sensors use a permeable membrane to separate the sample from the electrochemical cell inside. They usually require monthly electrolyte and membrane replacement and even after service, the sensor cannot produce meaningful reading for several hours because of required polarization. Sensors are sensitive to sample flow rate and experience interference from other particles present.

Unlike traditional DO sensors, nowadays, sensors do not require polarization. Optical technology has provided fast, accurate, and low-maintenance analysis. Its measurement response is six times faster than polar sensors, not sensitive to sample flow rate, and not susceptible to damage from particles interference. There is no interior body replacement required and service only once a year is sufficient. Just one sensing element is replaced during the service and eventually provides reduced downtime for production plants.

Selection of probe- according to the above-listed pros and cons, the user should identify which probe will suit its process the most. Monitoring and controlling DO can mean the difference between high or low cell viability and can further play an important role in complex protein production. Each of the mentioned probes has a major role in scientific research. Users need to discover which type is right for the specific culture.

Reference: Mettler-Toledo., Sensorex