What are Biomass Sensors?
Biomass sensors are designed to deliver real-time data on the density of biomass in solution without the need to take samples and analyze them offline. Biomass sensors utilize optical techniques to determine the cell density or biomass. NIR light passes through robust sapphire widows into the media and depending upon the technique used, measures either absorbance or backscatter of that light. The amount of absorbance or scatter detected can be correlated to known values in units such as g/l or to A600 / A660 values to generate a measurement of cell density.
Biomass sensors can deliver accurate and repeatable measurements at the cell concentrations in the media rather than diluted samples offline, removing both the risk of contamination through sampling and error through dilution. Absorbance sensors are well suited for lower-level concentrations while backscatter sensors are ideal for high level (200 OD or more) concentrations.
Typical applications include:
- Measurement of cell growth
- Concentration measurement of microorganisms
- End point determination
- Feed rate control
- Determination of dilution rates
- Monitoring/trending of fermentation processes
BIOMASS SENSORS DELIVER ACCURATE INLINE MEASUREMENTS IN REALTIME.
Do you have any of these concerns?:
MEASUREMENT DRIFT
“Our cell culture process lasts for several weeks and the cells are adherent. We’ve found that they stick to optical windows and cause the measurements to drift.”
This is a problem with many optical sensors. However, the Exner EXcell sensor can be installed in an Exner EXtract retractable housing (the same type as used for pH and DO2 probes) making it possible to withdraw the biomass probe aseptically from time to time to remove any coating that may be on the window surfaces. The probe can then be re-sterilized in the retractable housing before being reinserted into service.
PRECISION
“We have a process that requires gas sparge at a high rate. This causes biomass probes to be erratic and the results aren’t very useful. Why would the Kemtrak unit be any different?”
The Kemtrak unit has a flat smooth surface that doesn’t allow air to become trapped on its surface. Even in the more energetic agitation systems, probes that have windows that are set into the end of the probe, however slightly, will cause a single large bubble to form over the surface. That bubble just hangs out there and rolls around. The Kemtrak probe design doesn’t allow this to happen. The window surface is flush with the probe tip and angled at 45°, ensuring that the probe stays in full contact with the media. User adjustable bubble filter and damping settings means that the instrument can be tuned to provide a fairly smooth and more importantly, repeatable trend of biomass up to very high OD values.
NURTURING BIOMASS
"How do we streamline the monitoring of biomass growth and provision of adequate nutrition?"
In both the food and pharmaceutical industries, processes that grow biomass to feed into manufacturing lines downstream need to be carefully monitored. Biomass needs careful nurturing with the correct amount of nutrients in liquid and gas feeds. As the biomass in a process increases, the nutrient quantities needed also increase. What better way to control this than by directly measuring the biomass concentration with an NIR biomass probe?
These probes can monitor cell growth in a wide range of biomass types. Biomass probes are particularly useful in fermentation process monitoring, as they read cell mass in real time, as compared to traditional off-line sample testing techniques. NIR biomass probes are cost effective when compared to most traditional offline methods that require more equipment, and effort, and time.
In addition, NIR biomass probes aren’t restricted to mass manufacturing processes. Scientists have long used NIR biomass probes for research purposes. For example, the USDA Agricultural Research Service used NIR analysis in a study of switchgrasses. Their results are now being used by other scientists in their research of various warm-season perennial grasses. NIR biomass probes can be calibrated to monitor cell growth processes in any needed field.
STERILIZATION
“We are concerned about autoclaving biomass sensors with onboard electronics as they are expensive and we do not want to have failures.”
Biomass sensors are designed to be sterilized in an autoclave at high temperature. The most temperature sensitive part is often the light source. Exner probes have built in sensors that will power down the light source during events such as insitu steam sterilization to ensure damage does not occur. During autoclaving, the probes are not powered up, so this is not an issue.
Exner EXcell NIR Biomass Probe for Biopharmaceutical Process Monitoring
EXcell sensors are high precision digital NIR-absorbance devices designed specifically for monitoring biomass measurement from small scale laboratory work to full scale production process in the biotech, pharmaceutical, food and beverage industries. Sensors consist of an optical head and built-in amplifier/electronic unit designed around a standard 12mm probe footprint, similar to pH and dissolved oxygen (DO) probes. Connectivity is through RS485 and USB, with configuration through a PC-based EXpert software package. Accessories are available to integrate EXcell sensors into supervisory control systems and data collection devices.
KEMTRAK NBP007 FOR BIOMASS MEASUREMENT
The Kemtrak NBP007 is ideal for high biomass concentration measurement. With a huge dynamic range, the Kemtrak NBP007 measures real time biomass at levels unreachable by other probes. Each unit consists of an advanced analyzer electronic unit and fiber optic coupled probe. Built in bubble reject filtering allows use in highly aerated applications. Probes are available in Triclamp or 12mm diameter (similar to standard pH probe footprint) format and are CIP/SIP. The electronics has a multiline display and four operator buttons for local use. Units have ethernet connectivity for HTML/Java Web access or Modbus over TCP/IP.
Biomass Applications
Cell Culture Production
Drugs such as injectable biologics and vaccines produced by biotech companies are often produced using mammalian cell culture techniques. To produce these products, cells are cultivated in a rich nutrient media and infected with a virus. As the cells grow and multiply, they produce more of the virus. One of the key metrics required in cell culture is cell mass or biomass as this relates to cell count or density. Knowing the density of cells within the media allows for the proper control of nutrient and gas addition to the vessel to keep the cells in a healthy state and maximizes yield.
Traditionally, cell mass has been determined by manually extracting a sample from the bioreactor and performing a cell count in an offline laboratory (often referred to as OD600 or OD660). This method is prone to error due to sample handling and counting variations between technicians. Furthermore, with manual sampling comes the risk of contamination to the culture and this can have undesirable and costly consequences.
Cell mass measurement can be made online using an NIR absorbance technique. A probe with a known optical path is used to measure the optical density (OD) of the culture in situ. Probes can be inserted through vessel top plates or installed in side wall ports and sterilized with the rest of the apparatus. Once in process, the media circulating within the vessel continuously moves through the optical path of the probe and OD is measured through absorbance. The higher the cell count, the more absorbance of the NIR light occurs.
Cell density can be used as an OD trending measurement that follows the growth of cell mass within the media. Its value can be used to determine processing stages and end points or correlated to optical density by the user over the course of several batches to provide an actual assay of the cell density (i.e., cell count per ml).
Cell mass probes provide highly repeatable results and are a valuable tool in cell culture. As a measured variable, they can be used to control feed rates of nutrients, etc. while providing real time trending of growth rates. Using cell mass probes increases batch efficiency and yield while dramatically reducing the need for manual sampling. A reduction in manual sampling reduces both risk of contamination and lab time overhead.
Bacterial Fermentation
Bacterial fermentation is a far more challenging process than cell culture in which to measure biomass. Bacterial cultures typically have far more aggressive agitation and much greater rates of air and/or gas purge, creating a stirred media full of gas bubbles. Because of the bubbles within the media, many optical instruments provide noisy, erratic measurements and are quickly overwhelmed at higher cell densities, so in order to maintain a record of cell density during the process, frequent manual samples are necessary and each must be prepared and counted. Manual sampling is undesirable as it carries with it the risk of infection plus results are necessarily delayed from real time.
The standard technique for measuring cell density is absorbance of light at 600 or 660nm. Correlating cell density of samples against insitu OD readings provides a way of automatically recording biomass throughout the fermentation process. Fermentation processes often run over days, even weeks so are left unattended at night or over weekends. By using a biomass probe, continuous data is captured from the fermentation, filling the gaps that would otherwise be there in the overall record from the lack of sample testing.
For fermentation processes that have low biomass, a standard absorbance/transmission probe such as the Exner EXcell series is an excellent choice to allow monitoring of biomass growth. With electronics built in to the actual probe head, it has a very small footprint, making it ideal for crowded head plate installation on small bioreactors and in laboratory where space is at a premium. Several probes can be connected to a common PC on a desk running the intuitive Exner EXpert software package and log data simultaneously. Modbus communications provide easy connection to a variety of data systems, including popular programs like LabView®.
For dense and very dense biomass processes, reliable biomass measurement is critical and the use of a Kemtrak NBP007 insitu backscatter probe radically changes the measurement dynamic. With an angled window surface facing the direction of agitation, build up and fouling is greatly minimized. Software enhanced bubble rejection and damping settings within the analyzer effectively eliminate the bubble noise on the trends generated by many optical probes in bacterial applications and in doing so, provides a huge reliable dynamic measurement range, in some cases from 0.5 to 200 OD or more. The connected analyzer has an intuitive menu system and is available as standard with ethernet communications.
Both Exner and Kemtrak biomass solutions use NIR light sources to exclude influence from media color and smart filtering and bubble rejection to provide measurement where other sensors become unreliable or fail. As a result, insitu detection of process variations causing reduced cell growth rates is made possible, allowing operators can make adjustments to maintain the continued viability of the process or to determine growth end point and begin harvesting and other activities.
Microalgae Cultivation
Microalgae represents a renewable raw material and contains an array of valuable proteins, unsaturated fatty acids, oils, vitamins and minerals. They are already used in a large number of applications and are seen as being very promising for future development in a range of industries. Microalgae are already used in the production of products in the pharmaceutic, cosmetic, chemical and bioenergy sectors. Use has grown in the food industry where microalgae such as Arthrospira (Spirulina) and Chlorella are used to produce products such as food supplements.
Microalgae are cultivated in photobioreactors where they are provided with light, nutrients, and CO2. Light is needed for functioning photosynthesis and it is therefore critical that light level be controlled perfectly for efficient algae production. The process itself takes place as individual batches or a fed batch operation, depending on the scale of the production plant. Accordingly, there are various designs and sizes of photobioreactors.
Currently, there are many research projects being undertaken around the world to optimize the cultivation of algae biomass to improve efficiency and product yield. Insitu Biomass measurement is of particular importance in these projects as process cycles last over many days, even months, and the trend of biomass provides valuable data regarding growth patterns and speed. The purpose of biomass measurements are the monitoring of growth phases, reaction to bioreactor condition changes and determination of optimal harvesting time. Sensors such as the Exner EXcell biomass probe are ideal for these measurements. As they use only NIR as a light source, they do not promote the growth of algae on their optical surfaces and therefore maintain a reliable measurement at all times.
Yeast Cell Cultivation
Research performed with the yeast Saccharomyces cerevisiae has significantly improved our understanding of important cellular processes such as regulation, aging, and cell death. S. cerevisiae is widely commercially available and is favored by many laboratories for testing and teaching purposes. However, proper maintenance of this organism is critical for the success of the many experiments it is used in.
Yeast is cultured in a rich media that contains all of the nutrients the cells need to grow and multiply. When producing yeast, the culture goes through three main stages – lag growth, log growth and stationary phase. Once media is inoculated with a small sample of the yeast cells, the lag phase beings. During this phase, the cells become acclimatized to the media and secrete metabolites that condition the media for further growth. The end of this phase is indicated by a rapid logarithmic growth of cells within the media. As the biomass increases, so the nutrients in the media are depleted, causing a slowing of growth. At this point the culture transitions to a stationary phase where biomass remains fairly constant. The media is further depleted throughout this phase, eventually leading to a reduction in the number of cells as the culture runs out of “food”.
The use of a biomass sensor within the culture allows the phases of growth to be followed closely. By monitoring the amount of biomass within the culture, producers and researchers alike can add additional material to the media or choose the optimum moment to harvest the culture, obtaining the maximum yield possible.
Biomass measurement using both Exner and Kemtrak biomass solutions provides real time insight to the growth phases of yeast cultures. Biomass probes use NIR light sources to exclude influence from media color and smart filtering and bubble rejection to provide reliable measurement when high sparge rates of CO2 are being used. By following the growth trend in real time, operators have the information available observe the effects of formulation changes made, make decisions on gas and nutrient feed rates for continued viability of the process, or to determine growth end point and begin harvesting and other end-of-culture activities.
No matter the application or industry, South Fork Instruments can develop a superior optical sensor-based measurement solution.
We have provided NIR optical sensor solutions to clients in such industries as food and beverage, chemical processing, pharmaceutical manufacturing, and biotechnology, and can assist you in developing and effective and efficient means of fulfilling your measurement needs.
SOUTH FORK INSTRUMENTS
3845 Buffalo Road
Auburn, CA 95602
Tel: (+1) 925-461-5059
