Testing of on-line optical cell biomass probe linearity
and accuracy across changing process conditions
A long-standing challenge for on-line optical cell biomass probes has been to maintain biomass prediction accuracy across changing process conditions, particularly for organisms requiring highly variable rates of agitation and aeration in order to avoid oxygen-limited growth. For microbial organisms grown to high cell biomass densities, being able to accurately track the biomass across the entire growth cycle from inoculation to harvest has been difficult to achieve due to the limited linearity range of traditional on-line methods and variation in sensor readings with changing process conditions. A comparison study of the linear range and the effects of changing agitation and aeration conditions on biomass prediction error for a methanotrophic culture are compared for two commercially available on-line optical reflectance probes.
Online OD Measurement
Monitor biomass, non-invasively, in fermentors having a glass wall or glass viewing port. The instrument consists of an optical sensor that is mounted onto the exterior of the fermentation vessel, and a monitor that processes the sensor signals. By combining reflectance signals measured at multiple source-detector distances, linear response to biomass change is achieved over a wide range of biomass.
Development and Evaluation of Methods to Infer Biosynthesis and Substrate Consumption in Cultures of Cellulolytic Microorganisms
Near Infrared Optical density (OD850) data were obtained using the BE2100 “Bugeye” non-invasive biomass monitor by Buglab LLC Danville, CA. NIR optical density data from the BE2100 is shown as “OD850″. Four different on-line Optical Density systems were tested for measuring optical density and compared to off-line analysis data (elemental analysis/quantitative saccharification). The optical density signal was much better correlated with off-line data for the ex situ system reported herein than probes we tested that were immersed in the fermentation broth.
Extremum Seeking Control of Batch Cultures of Microalgae Nannochloropsis O culata in Pre-Industrial Scale Photobioreactors
Extremum seeking control is applied to batch cultures of microalgae Nannochloropsis Oculata with the objective of maximizing the growth rate through manipulation of the pH. A commercial biomass monitor (BugEye from Buglab) using on-line optical density (OD) measurements at 725 nm is used to provide signals relative to both biomass density and growth.
New miniature on-line optical cell biomass probe with wide linear range and bubble discrimination
Immersible on-line cell biomass probes have historically suffered from a limited range of linear response to biomass and sensitivity to process variables, such as agitation and gas sparge rate. A new optical probe has been developed that provides linear response over 4 orders of magnitude of biomass (e.g. 0.01-200 g/L yeast dry cell weight). The small probe diameter (e.g. 3 mm) and minimal optical penetration depth (e.g. <3 cm) make it suitable for a wide range of vessel types, including miniature bioreactors (e.g. 250 mL). Interference from bubbles is largely eliminated through a novel measurement technique, making the results nearly insensitive to agitation and aeration rate changes.
The Application of an On-Line Optical Sensor to Measure Biomass of a Filamentous Bioprocess
Monitoring of all critical process parameters in bioprocess engineering is essential. Sensors have been previously developed for specific parameters such as on-line temperature, pH or stirring control and data logging. However, biomass monitoring needs further development. All current non-invasive technology, such as Near Infra-Red, is limited on biomass measurement of animal and insect cells. Biomass monitoring of industrial bioprocesses of filamentous microorganisms still requires sample removal from the vessel, which could potentially compromise sterility. In this study we focused on the use of a new non-invasive optical sensor (BugEye® 100) for the real time monitoring of biomass of the filamentous microorganism Streptomyces coelicolor A3 (2). Raw output data from the biomass monitor were directly compared to data from the sensors measuring dissolved oxygen levels and off gas evolution and the results successfully demonstrate that the optical sensor is sensitive in identifying different levels of biomass. Therefore, it is possible to use the simple output data to provide real time information on biomass levels of filamentous microorganisms, a very powerful tool in bioprocess engineering.
Optimizing Sensor Linear Response Range Under Challenging Process Conditions
The recent trend in bioprocess development toward small bioreactors and high cell densities presents challenges for accurate on-line monitoring of liquid cell cultures. To accommodate the trend toward smaller vessels, a more recent approach (BE3000 probe), uses a small diameter (e.g. 3 mm) fiber optic probe measuring back-reflectance at 1310 nm.
In Situ Clostridium Dry Cell Weight Monitoring in Complex Cellulosic Feedstocks
The economically competitive bioproduction of specialty chemicals using microbial cultures often demands the use of complex feedstocks that have undergone minimal processing and purification. Such feedstocks may be high in cellulosic content, have dark color, contain a wide mixture of particle sizes, and exhibit substantial batch-to-batch variation. The real-time monitoring of cell density (or cell biomass) is essential to maintaining the health of the microbial culture and for optimizing the yield of bioproducts, but has been a long-standing challenge within such complex feedstocks. The traditional method of monitoring microbial cultures by optical density (“OD”) is hampered by strong absorbance and scattering from the feedstock itself, by the limited linear range of response to changes in cell biomass, and by run-to-run inconsistency. In this study, optical reflectance in the near infrared is demonstrated to accurately predict (9.7% standard error of prediction, r2=0.985) in situ clostridial dry cell weight grown in two different types of complex cellulosic feedstocks during exponential growth across multiple batch fermentations.