Instrumentation and control in bioreactors: control of pH. Arredondo Patiño Jonathan Israel Instituto Politécnico Nacional-Unidad Profesional Interdisciplinaria de Ingeniería Guanajuato October 8th, 2010 Abstract Maintaining the pH of the medium is an important factor in the control of bioreactors because every organism has a pH range within its growth is possible and usually has a well defined pH optimum range. A glass electrode was used to represent the way the control and measure of pH are controlled. It stood in contact with 600 mL of water to which the pH was varied with a solution of HCl 0.5 N and a solution of NaOH 0.5 N. The pH of this solution could be modified because of the reading of the meter-controller which was connected to the electrode. Introduction The growth of an organism in a bioreactor has to be controlled, so the operators have sufficient information about the state of the organism and the bioreactor conditions. As most bioreactors operate under sterile conditions, information can be obtained by taking samples or by in situ measurements (Najafpour, 2007) Bioreactors are equipped with specific instruments, which provide the record operating variables or parameters that keep the bioreactor operating conditions, all in order to obtain maximum productivity of any bioprocess. The objective of the instrumentation is kept to a minimum difference between the measured value and a desired value. The control of a particular parameter is carried to market through a system comprising a sensor or electrode and a controller (Standbury et al. 2003) The sensors can be connected directly to the bioreactor ("in line"), which are in direct contact with the liquid or solid mass of fermentation. They can also be "on line" which means that the value of the variable is known but can´t be controlled directly, and finally, when the sensors are not connected directly to the bioreactor, are "off line " (Najafpour, 2007). When the sensors are in line, are used to measure physical properties and variables of operation: temperature, pressure, agitation intensity or speed of the impellers, flow rates of liquids and gases. They are also used to measure certain chemical properties: pH, dissolved oxygen concentration and gas and dissolved CO2 concentration in the gas, the concentration of dissolved sugars, concentration of certain cellular products, etc. In order to know the status of an operation variable with sensors off-line, a sample must be taken aseptically for the measured of some property (Najafpour, 2007). The objective of this paper was to determine the use of a pH electrode for a pH measurement and its control in a bioreaction, because this factor affects largely the metabolism and growth of the microorganism (Madigan, 2004).
Key words: pH, bioreactor, pH electrode, sensor, control. Materials and methods Calibration of the electrode: For the measurement of pH, the electrodes were calibrated with 3 buffer solutions which had different values of pH (4, 7, and 12). Measurement and control of pH: A beaker was filled up to a 60% of its capacity. It was placed on the heating grate and a stirring speed of 300 rpm was set. A glass electrode was used to represent the way of how the pH is controlled and measured. It stood in direct contact with 600 mL of water, to which the pH was varied with a solution of 0.5 N HCl and NaOH 0.5. The samples were taken and the pH was measured off line. The pH was adjusted to a value of 7 with a solution of NaOH 0.5 N. An acidogenic reaction was simulated by the addition of 5 mL de HCl 0.5 N. Then NaOH 0.5 N was added until the pH reached to 7. Results Experimentally the following results were obtained by pH measurements (table 1): Table 1: Results of measurement of pH by the added of HCl 0.5 N and NaOH 0.5 N pH 8.556 7.052 5.5 7.04
Start Calibration Acidogenic reaction Alkaline reaction Comparison with theoretical results. Initial pH = 7.052 Concentration of
=
Concentration of 5 mL were added of HCl to acidify a pH of 5.5 Using the following formula.
pH= -log[ pH= 5.05
]
This result is very similar to the pH achieved in the practice, with a value of 5.5, between the theoretical and practical result there is only a variation of 0.45 which may be due to poor calibration of the electrode, interference during the measurements or the compositions of acid and basic solutions used during the practice. The graph of pH obtained vs. time is shown in the graphic 1: Graphic 1. - Concentration of pH during the experiment. 8 7 6 5 4 3 2 1 0
Concentration of pH vs time pH()
0
50
100
150
200
250
In the graphic any strong variation is observed, and although a simulation of an acid reaction was performed, there is not a maximum point in the reaction with HCl 0.5 N. Discussion Measurement of pH is based on the absolute standard of the electrochemical properties of the electrode. The basic part of the electrode is a thin glass membrane (0.2-0.5 mm). This layer exists on both sides of the membrane and is essential for proper operation and maintenance of the electrode. Hydrogen ions that exist within the gel layer are mobile and any difference between the ion activity on either side of the membrane will lead to the establishment of a pH-depended potential. This potential remains constant in the inner surface of the glass membrane to fill the catheter tube with a buffer solution, with precision and stable composition, and a specific activity of hydrogen ions (Najafpour, 2007). In any bioprocess is necessary to maintain a constant pH, as each microorganism has a pH range within its growth is possible and usually has a well defined pH optimum (Prescott, 2002). For example in fermentation, the pH of culture medium will change as microorganisms grow and produce metabolic products. Therefore, pH control is necessary for the course of fermentation, it has a great effect on microbial growth and production of metabolites, this influences the distribution of substrates and transport across the cell membrane . (Najafpour, 2007). Microorganisms are classified according to the pH necessary for their growth. Acidophilus grow at pH 0 and 0.5, neutrophils 5.5 to 8, and alkalophilic from 8.5 to 11.5. The organisms often have to adapt to changes in pH to survive. In bacteria antiport system potassium / proton and sodium / proton, probably correct slight variations in pH. Some synthesize new proteins in response to a change in pH. They frequently change the pH of the acidic or basic produce metabolites, which is necessary to use substances that absorb the change, because in high concentrations is toxic to the
cell. One of the substances used for this purpose are the buffers, which are included in the means of preventing the inhibition of pH in large changes in pH (Prescott, 2002). Conclusion: The optimal development of the organism or process intended to be done within a bioreactor, depends largely on good management of it. On the other hand, it also depends on the control of operating variables or specific parameters, to know exactly the extent of such variables that the microorganism need. Among these variables, the pH is one of the most important to control, because a drastic change in its concentration greatly affects the future of the bioprocess. References: 1. Najafpour, G.D. (2007). Biochemical engineering and biotechnology. Elsevier. Holanda. 421 pages. 2. Prescott, L. M. (2002). Microbiology. México: The McGraw - Hill. 3. Stanbury et al. (2003). Principles of Fermentation Technology. Great Britain: Butterworth Heinemann. 4.
Madigan, M.T. Martinko, J.M & Parker, J. (2004) Biology of microorganisms. Pearson.
