The Application of Continuous Gas Monitoring in Biotechnology
Nigel Bridger, Anglo Scientific Instruments
Introduction (Back to Top)
Biotechnology is the commercial application of biological processes to provide more efficient routes to such important end products as drugs, antibiotics, chemicals, food and drink. The importance of biotechnology and the effect that its developments will have on our future lifestyle has been compared to that of the changes that have stemmed from the advent of electronics.
Our hopes for finding cures to killer diseases, safer ways to control crop infestations, improve yields, and even take short cuts to producing healthy foodstuffs rest with the researchers in a wide range of commercial and academic centers which are now put under the umbrella of biotechnology.
The Process (Back to Top)
Many different types of biological process can be harnessed for commercial exploitation and some of them are certainly not new. The earliest form of fermentation for wine production was being used by the Egyptians in 5000 BC, and today yeasts are still important precursors in the production of alcohol and other arguably more useful products.
The common denominator is that the biotechnological process involves a living organism and this organism needs some basic requirements before it can flourish and replicate. Commonly encountered organisms of commercial importance would include yeasts, bacteria, mammalian cells and plant cells.
In order to optimize the process it is necessary to control the environment in which the organism is being held so that temperature, light and mixing are to the liking of the particular microbe and do not become growth-limiting factors. Two important requirements from both the organisms point of view and the cost efficiency of the process control is the provision of nutrient and the gas or gases needed to sustain life. In order to provide enough of these two commodities to keep the organism growing at the maximum rate but not allow excesses which waste money, it is necessary to form an idea of the population of the organism present at any given point in the process.
This estimation of the biomass is in fact quite difficult and indirect methods such as pH, pO2, weight and optical density measurements have been and are being used with differing degrees of success. Many industrial microorganisms have respiratory cycles like ourselves, i.e. they take in oxygen and give out carbon dioxide. It is therefore necessary to pump air into the vessels (bioreactors) to provide oxygen, and to provide an exit from the vessel for the exhaust gases. This is where gas phase mass spectrometry comes to the assistance of the process engineer.
Quadrupole Mass Spectrometry (Back to Top)
Mass spectrometry has been used for the analysis of gases for many years, but it was not until the advent of the quadrupole analyser that it was possible to produce compact, fast scanning, easy-to-use mass spectrometers for non-specialist users.
There are two basic modes of operation for any mass spectrometer, scanning and peak selection. In the scanning mode, the operator selects a start mass and a scan range and the mass spectrometer will scan through and "look" for any gas that produces molecular or fragment ions within this mass range. A scan of normal air from say mass 10 to 50 will include spectral peaks of nitrogen (28 and 14), oxygen (32 and 16), argon (40 and 20), water (18 and 17) and carbon dioxide (44, 32, and 28). Characteristic mass spectra are produced by each gas and can be used to identify the components of gas mixtures.
Where the gases of interest are already known, time can be saved by avoiding scanning over a range and jumping to the masses where you expect to find your target gases. This is called peak selection. For a normal aerobic fermentation, a number of useful control parameters can be identified by monitoring the levels of oxygen and carbon dioxide in the gas streams entering and leaving the bioreactor. This is achieved by selecting mass 32 for oxygen and mass 44 for carbon dioxide.
Information Obtained by Continuous Gas Monitoring (Back to Top)
There are two important derivations obtained from continuous monitoring of the oxygen and carbon dioxide levels on inlet and outlet streams, the first is the respiratory quotient (Rq) and the other is the Oxygen Uptake Rate (OUR). Both give good status reports on the amount of biomass present and if growth is positive or negative. Rq is the ratio between the increase in carbon dioxide level against the reduction in oxygen, while OUR is the reduction in oxygen level with respect to time.
During some plant cell cultivations, the mass spectrometer can be used to monitor the nitrogen levels on the inlet and exhaust lines so as to identify the amount of nitrogen fixation taking place. In all applications it is possible to include measurement of the four main air gases—nitrogen, oxygen, argon and carbon dioxide—on both sides of the bioreactor so as to achieve a so called mass balance and check the accuracy of results.
The A.S.I. Bioquad System (Back to Top)
Anglo Scientific Instruments (A.S.I.; Devon, UK) has developed a complete multicomponent, multichannel gas analysis system specifically designed for the gas analysis of processes such as fermentation and cell culture. The latest DQC 2000 computer integrated mass spectrometer is used and this instrument features a color VDU, easy to use mouse operated software, and a valve manifold which permits up to 32 gas streams to be connected to the central vacuum system for analysis.
The user can select up to 16 gases of interest in peak select mode, although oxygen and carbon dioxide are frequently the only two needed, and the switching from one channel to another is controlled by sequencing software incorporated into the standard computer control package. Users may select changeover delays to allow for reading stabilization after switching channels, then the number of readings required before moving on to the next channel can be programmed.
Data logging is independently selectable and allows gas concentration information to be logged onto the computers floppy or hard disc under specific batch numbers, time, date and channel identification.
Membrane Probe for the Analysis of Dissolved Species (Back to Top)
The BIOQUAD not only offers tremendous flexibility in the monitoring of multiple off gas streams but can also be supplied with a membrane probe to allow the analysis of gases and volatiles extracted directly from the fermentation broth.
The membrane probe used for this work consists of a stainless steel holder that fits into standard DN 25 ports on bioreactors. The probe has a replaceable stainless steel membrane holder and the gases that permeate through the membrane are taken to the mass spectrometer analyzer by a carrier gas. If gases or volatiles other than those present in air that are of interest then air itself can be used as a carrier gas, otherwise helium or some other gas that does not interfere with the analysis may be chosen.
Because the probe does not involve high vacuum operation, it is easy to incorporate multiple probes to the same multichannel inlet system as for off gas analysis. Data logged from inlet and exhaust gas streams, together with membrane probe results from the same bioreactor, provides the most useful process control information yet available.
For more information: Nigel Bridger, Anglo Scientific Instruments, 7 Devon Units Torrington, Devon EX38 7HL, UK. Fax: 44-1-805-625-112. From the UK: 01805-625-112. Email sales@a-s-i.demon.co.uk