Biological Fermentation

Biological fermentation engineering is an important component of biotechnology, in which microorganisms utilise carbohydrates to produce various industrial solvents and chemical raw materials through fermentation. Ethanol, acetone–butanol, butanol–isopropanol, acetone–ethanol, 2,3-butanediol and glycerol fermentation are several forms of solvent fermentation carried out by microorganisms.

In any fermentation process, monitoring the growth status of microorganisms is crucial, as this status affects the conversion rate of nutrients, the production of by-products, and even the generation of toxic substances.

Fermentation is closely linked to human life; for example, in pharmaceutical production, biological fermentation technology is utilised to produce and develop medicines. Many foods, such as wine, soy sauce and bread, are also produced through microbial fermentation.

During fermentation, different types of microorganisms utilise raw materials such as carbohydrates and proteins, employing distinct metabolic pathways to produce various metabolites. Changes in the concentration of exhaust gas components reflect changes in the material balance throughout the fermentation process; in particular, variations in CO₂ and O₂ levels in the exhaust gas can indicate the fermentation status and the microscopic growth of the microbial cells.

Consequently, process mass spectrometers are being increasingly adopted by fermentation manufacturers. The ProC-TOF online mass spectrometer not only enables qualitative and quantitative analysis of gases produced during fermentation but also offers numerous advantages, including excellent stability, high accuracy and multi-channel capability.

Substances primarily monitored by the mass spectrometer: N₂, O₂, CO₂, methanol, ethanol, methane, ammonia, acetic acid and aromatic compounds

Advantages of online mass spectrometers in fermentation process analysis and control

Process mass spectrometers offer the advantages of strong quantitative and qualitative capabilities, high sensitivity, high analytical precision and rapid detection speeds.

(1) As a general-purpose gas analyser, in addition to detecting conventional O₂, CO₂, it can also perform high-precision, full-component detection of N₂, Ar, NH₃, ethanol and other volatile gases;

(2) Not only can it quantitatively detect known gases, but it can also perform full-scan analysis of unknown compounds to carry out preliminary qualitative analysis;

(3) Utilising multi-channel simultaneous detection technology, it can monitor more than a dozen fermentation tanks online at the same time, making it highly suitable for both research and industrial production needs;

(4) It offers high detection sensitivity, capable of detecting gases at concentrations in the ppb range;

(5) It exhibits excellent long-term stability, meeting the requirements for continuous, long-term monitoring in fermentation applications without the need for shutdowns.

Applications of Process Mass Spectrometers in Fermentation

The fermentation process involves a large number of complex biochemical reactions and transport phenomena. During fermentation, the concentrations of substrates, microbial cells and products vary over time; the properties of the fermentation broth—such as density, viscosity, rheological characteristics, surface tension, oxygen content and other properties including diffusion coefficients and oxygen saturation—also change over time.

As a result of a series of catabolic and anabolic processes, changes occur in temperature, pH, dissolved oxygen, redox potential, carbon dioxide emissions and oxygen emissions. In order to control fermentation effectively, it is necessary to obtain information on the changes in fermentation process variables and to monitor changes in fermentation process parameters.

Fermentation off-gas is an important indirect variable in the fermentation process; the oxygen consumption rate and CO₂ release rate (CER), obtained through online gas analysis, are currently the most effective indicators of biological metabolism.

The ProC-TOF online mass spectrometer can monitor the concentrations of O₂ and CO₂ in fermentation exhaust gas in real time, enabling the acquisition of key respiratory metabolic parameters such as the CO₂ release rate (CER), oxygen uptake rate (OUR) and respiratory quotient (RQ); it can also be combined with other online and offline data to provide a more comprehensive analysis of the entire fermentation process.

In recent years, research into the application of exhaust gas analysis in fermentation has become increasingly widespread and in-depth. This is of great significance for the in-depth study of fermentation mechanisms, the exploration of optimised fermentation processes, and the comprehensive control of fermentation processes; particularly in the biopharmaceutical sector, it can significantly accelerate the research and development of new products, facilitate stable industrial-scale production, and improve yield.

The use of the ProC-TOF online mass spectrometer to monitor O₂ and CO₂ concentrations in fermentation exhaust gases in real time offers advantages such as high resolution, excellent sensitivity, strong qualitative and quantitative capabilities, and speed; it has now become an essential tool and method for analysis in fermentation laboratories. The application of exhaust gas analysis technology in the field of fermentation has yielded significant results. It is believed that, with the widespread adoption of the ProC-TOF online mass spectrometer, research into fermentation applications will become more in-depth and further drive the development of the biopharmaceutical industry.

Explanation of some terms used in fermentation

Carbon dioxide release rate (CER)

In microbial metabolic processes, the carbon dioxide release rate (CER) is an important parameter that reflects the metabolic status of the microbial cells. The expression is as follows:

where QCO₂ is the rate of CO₂ release, expressed in mol CO₂/(g·h); X is the dry weight of the microbial biomass, expressed in g/l; Fin is the feed gas flow rate, expressed in mol; V is the volume of the fermentation broth, expressed in L; and in is the concentration of inert gas in the feed gas, expressed as a percentage by volume: CO₂out and CCO₂out denote the concentrations of oxygen and carbon dioxide in the exhaust gas, respectively, with units of % (volume fraction); Pin denotes the absolute pressure of the inlet gas, with units of Pa; T denotes the inlet gas temperature, with units of °C; and h denotes the relative humidity of the inlet gas, with units of %.

Oxygen Uptake Rate (OUR)

OUR reflects the metabolic activity of the microbial cells; the higher the microbial activity and the greater the microbial biomass, the higher the OUR value. It is calculated as follows:

Here, CO₂in represents the concentration of O₂ in the inlet gas, expressed as a percentage by volume (%). For further details on other parameters, please refer to the description in the CER.

Respiratory Quotient (RQ)

RQ is a key parameter in fermentation; it is defined as the ratio of CER to OUR. Its value provides insight into the utilisation of carbon sources by the microbial cells during metabolism, as well as their metabolic state.

Applications of process mass spectrometers in the online monitoring of biological fermentation

Online mass spectrometers are also utilised in other fermentation methods, playing a vital role in investigating the mechanisms of the fermentation process, exploring ways to optimise fermentation processes, and comprehensively controlling the fermentation process

Antibiotic production

Since Fleming’s discovery of penicillin in 1928, antibiotics have gained widespread recognition and undergone significant development. In recent years, antibiotics have consistently ranked first in terms of total sales across all major departments in Chinese hospitals, making an enormous contribution to human health. In addition to their use as human medicines, antibiotics are also extensively applied in fields such as veterinary medicine and pesticides. Antibiotics are primarily derived from microbial metabolism and produced through fermentation. The composition of the exhaust gases emitted throughout the fermentation process can be analysed using a process mass spectrometer. This includes conventional gases such as O₂, CO₂ and N₂, as well as volatile gases produced during certain metabolic processes—such as methanol and ethanol—which are expelled with the exhaust gases and can also be detected in real time using the ProC-TOF online mass spectrometer.

Fermentation Production for Human Medicines

Human antibiotics are classified into tetracyclines, β-lactams, aminoglycosides and macrolides; cephalosporin C is taken as an example for discussion.

In the fermentation of cephalosporin C, soya oil is fed in a continuous stream as a precursor to promote antibiotic production; the optimal rate and timing of oil addition are crucial. The continuous addition of soya oil causes fluctuations in the OUR and CER trends, generally for two reasons:

Firstly, the oil acts as a defoamer, breaking up foam in the fermentation broth and releasing CO₂, which causes an increase in CO₂ and a decrease in O₂ in the off-gas:

Secondly, prior to the oil addition, the carbon source is at a limiting concentration. By analysing the effluent gas using process mass spectrometry, and calculating the OUR, CER and RQ from the fermentation effluent data—combined with other online and offline data—it was further analysed that: when acting as a defoamer, parameters such as DO, KLa and RQ exhibit a relatively wide range of fluctuations; under limiting carbon source concentrations, the fluctuations in these parameters are not significant. The characteristic differences between the two situations are distinct. Generally, during the hydrogen production phase, maintaining the carbon source at a limiting concentration allows for the determination of the optimal timing and rate of oil supplementation, thereby ensuring high-yield fermentation.

Fermentation processes often encounter situations where dissolved oxygen (DO) levels drop too rapidly, become uncontrollable, or even fall to zero. Process mass spectrometry can precisely detect and calculate the OUR in fermentation off-gas. Given the synchronous relationship between OUR and dissolved oxygen (DO), in cases where DO levels are falling rapidly, combining online DO electrode readings with OUR data allows for timely metabolic adjustments to control DO levels. Furthermore, process mass spectrometry offers excellent data continuity, ensuring that even minute changes occurring over very short time intervals can be captured, thereby enabling early warning and the regulation of DO to an appropriate concentration. The ProC-TOF online mass spectrometer holds great potential for both research and development and production.

Veterinary Drug Fermentation Production

Biological veterinary medicines are environmentally friendly drugs characterised by safety, the absence of residues and pollution. They play a vital role in the development of the livestock industry and have yielded significant economic benefits in preventing coccidiosis and improving feed conversion rates in ruminants. They are already in widespread use in over 40 countries and regions worldwide, with usage volumes rising year on year, and they play an immeasurable role in human health. Salmystin is one of the more commonly used veterinary medicines; during the fermentation process, soya oil must be added as a precursor skeleton for the formation of the antibiotic. The appropriate dosage, rate and timing of addition are key to the success of process optimisation.

As soya oil is a relatively high-cost component of this product, excessive oil not only increases costs but also raises the viscosity of the fermentation broth, impairing oxygen transfer. Furthermore, excess oil adhering to the mycelium severely impairs respiration; therefore, the aim is to produce the maximum yield using the minimum amount of oil. Optimisation can be achieved by analysing online exhaust gas data (such as OUR and CER) detected by the ProC-TOF online mass spectrometer.

Fermentation Production of Pesticides and Antibiotics

Certain antibiotics are capable of killing insects and mites and are used as biological pesticides. Among these, avermectin is one of the most popular and competitive products on the current biological pesticide market, owing to its contact action against insects and mites and its low residue levels on fabric surfaces.

Process optimisation for avermectin primarily focuses on optimising the addition of carbon sources. The ProC-TOF online mass spectrometer analysed the fermentation exhaust gas; by restricting the air flow rate and limiting primary metabolism, the OUR decreased, causing more raw materials to be directed towards secondary metabolism, thereby increasing yield.

Production of amino acid-based food products

As essential nutrients for human growth, amino acids not only possess specific physiological functions but also fulfil unique roles in the food industry. The fermentation of glutamic acid serves as the most typical example. Monosodium glutamate, commonly known as MSG, is an important umami enhancer that intensifies flavour. It is widely used as a food flavouring agent, either on its own or in combination with other amino acids.

Given a specific strain of bacteria, the optimisation of the continuous sugar addition process during glutamic acid fermentation is of paramount importance. Factors such as the timing of the initial sugar addition, the rate of addition, and the residual sugar content during the addition period are crucial to the success of process optimisation. Excessive sugar can lead to the accumulation of catabolic by-products, whilst insufficient sugar content can impair metabolism, thereby limiting amino acid production.

The ProC-TOF online mass spectrometer integrates technical advantages from various fields, enabling accurate detection of OUR, CER and RQ during the fermentation process. Changes in RQ provide an accurate, real-time understanding of the fermentation status, ensuring the correct timing of sugar addition whilst maintaining sugar concentrations within limiting levels. This allows for the timely identification and resolution of issues arising during fermentation.

Production of Enzyme Preparations

Enzyme preparations offer advantages such as high efficiency, specificity and mild operating conditions, and are widely used in industries such as food processing. With the continuous emergence of new technologies, enzyme preparations are set to find even wider application in the food industry. Taking phytase as an example, the production of phytase via recombinant Pichia pastoris fermentation is one such method. The Pichia pastoris fermentation process requires the use of methanol to induce the expression of exogenous proteins; however, this methanol is carried off as a volatile component in the exhaust gas. Process mass spectrometry can detect the methanol content in the exhaust gas, thereby providing an indirect, real-time indication of the methanol concentration in the fermentation broth.

Not only has this mass spectrometer been specifically designed for biological fermentation applications, but its built-in sample preparation system effectively addresses interferences such as foam, particulate matter and water droplets present in the fermentation exhaust gas. This ensures that the exhaust gas is nearly equivalent to a standard gas before entering the mass spectrometer, thereby guaranteeing long-term stable operation of the instrument. Currently, the ProC-TOF online mass spectrometer has a significant number of representative clients in the biological fermentation industry.

Conclusion

The ProC-TOF online mass spectrometer holds broad prospects for application in the biofermentation industry; the online data provides crucial support for the control and optimisation of fermentation processes. Online measurements of OUR, CER and RQ serve as key physiological parameters, whilst online data on methanol, ethanol and other compounds can be used as references for metabolic flux calculations. These measurements hold significant value for both fermentation production and scale-up.