FOOD AND INDUSTRIAL MICROBIOLOGY

Module 5. Bioprocessing

Lesson 21
GROWTH CULTIVATION- BATCH AND CONTINUOUS TYPE

21.1 Introduction

The fermentation process involves actual growth of the microorganism and formation of the product under agitation and aeration, to provide uniform environment and adequate oxygen to the cell for growth, survival, and product formation.

A fermentation system is usually operated in one of the following modes: batch, fed batch, or continuous fermentation. The choice of the fermentation mode is dependent on the relation of consumption of substrate to biomass and products.

The fermentation unit in industrial microbiology is analogous to a chemical plant in the chemical industry. A fermentation process is a biological process and, therefore, has requirements of sterility and use of cellular enzymatic reactions instead of chemical reactions aided by inanimate catalysts, sometimes operating at elevated temperature and pressure. Industrial fermentation processes may be divided into two main types, with various combinations and modifications. These are batch fermentations and continuous fermentations.

21.2 Batch Fermentations

A tank of fermenter (Figure 21.3) is filled with the prepared mash of raw mate­rials to be fermented. The temperature and pH for microbial fermen­tation is properly adjusted, and occassionally nutritive supplements are added to the prepared mash. The mash is steam-sterilized in a pure culture process. The inoculum of a pure culture is added to the fermenter, from a separate pure culture vessel. Fermentation proceeds, and after the proper time the contents of the fermenter, are taken out for further processing. The fermenter is cleaned and the process is repeated. Thus each fermentation is a discontinuous process divided into batches.

21.3 Continuous Fermentation

Growth of microorganisms during batch fermentation confirms to the characteristic growth curve, with a lag phase followed by a loga­rithmic phase. This, in turn, is terminated by progressive decrements in the rate of growth until the stationary phase is reached. This is because of limitation of one or more of the essential nutrients. In continuous fermentation, the substrate is added to the fermenter continuously at a fixed rate. This maintains the organisms in the logari­thmic growth phase. The fermentation products are taken out conti­nuously. The design and arrangements for continuous fermentation are somewhat complex.

21.4 Batch Growth

It involves a closed system where all nutrients are present at the start of the fermentation within a fixed volume.

Batch cultivations are generally employed to grow cells for desired metabolite production as it is simple and involves least requirement of labor and equipments as opposed to other modes of cultivation (Fed-batch/ Continuous cultivations). However these cultivations work as closed system and thereby feature highly dynamic growth conditions and have less yield and productivity of desired product. This is particularly because of high non production time (Cleaning/Sterilization/Cooling etc) & due to significantly long Lag/Stationery phases in this mode of cultivation. Generally the higher activity featuring exponential growth (Balanced growth wherein all components of the cell grow by same proportion) of the culture is observed for only 25-40 % of total cultivation time of batch cultivation wherein the culture produces the metabolites of interest in growth associated fermentation processes. Growth rate in batch fermentation is normally uncontrolled and is highest at the start. Productivity of batch fermentation: The productivity of batch fermentation is calculated by the final concentration of biomass or product being produced divided by the complete time of batch, which includes fermentation time and turnaround time (time for emptying, cleaning, sterilizing, and refilling) (Figure 21.2).

Fig. 21.1 Batch fermentation system (Crueger and A. Cruegar, 2000)

Fig. 21.2 Productivity in batch fermentation (Crueger and A. Cruegar, 2000)

If however, during the exponential phase of growth, a constant volume is maintained by ensuring an arrangement for a rate of broth outflow which equals the rate of inflow of fresh medium, then the microbial density (i.e., cells per unit volume) remains constant. This is the principle of continuous culture.

21.5 Continuous Culture

The concept of the continuous cultures dates from the 19th Century when a continuous process for the conversion of waste beers and wines to vinegar was developed. In this reactor, natural acetic acid bacterial populations were immobilized on wood shavings. Beer or wine was added through the top of the reactor and allowed to trickle through the shavings. Vinegar was collected at an outlet located at the base of the reactor.

It is an approach that provides a spectrum of exciting possibilities for studying bacteria under conditions that more closely resemble the way they grow naturally is continuous culture. In continuous culture, microorganisms are placed in an environment where the feed rate to the system and from the system is fixed. Thus, microorganisms experience a constant, and steady supply of limiting substrate and nutrients. Consequently, they can (over time) adjust their enzyme levels, pH and osmotic gradients, macromolecular composition etc. to achieve an “optimal growth”. This situation is generally referred to as “steady state” and may take up to 10-21 generations to achieve.

Continuous culture systems can be operated as chemostats or as turbidostats. In a chemostat the flow rate is set at a particular value with the help of a flow rate regulator and the rate of growth of the culture adjusts to this flow rate. That is, the sterile medium is fed-into the vessel at the same rate as the media containing microorganisms is removed.

21.5.1 Chemostat

Continuous fermentation is an open system to maintain cells in a state of balanced growth by continuously adding fresh medium and removing the culture medium at the same rate. Essentially, the two modes of operation for continuous fermentation are chemostats and auxostats. The commonly used auxostats include turbidostats, the pHauxostat, and the nutristat. One type of system that is widely used for continuous cultivation is the chemostat. This system depends on the fact that the concentration of an essential nutrient within the culture vessel controls the growth rate of the cells. In general, one nutrient is limited to an amount that restricts growth, and the culture is removed at the same rate as nutrients are added (Figure 21.3).

The chemostat invented in the early 1940's marked the advent of serious continuous fermentation. Here, planktonic cultures in a fermentor are fed with a nutrient solution to maintain a bacterial population in the exponential or log phase of growth.The continuous culture reaches "balanced growth" in which the levels of bacteria, bacterial products, media components, and waste products are constant. This condition is referred to as "steady-state" growth. The culture volume and the cell concentration are both kept constant by allowing fresh, sterile medium to enter the culture vessel at the same rate that "spent" medium, containing cells, is removed from the growing culture. Under these conditions, the rate at which new cells are produced in the culture vessel is exactly balanced by the rate at which cells are being lost through the overflow from the culture vessel.

Presently, the chemostat is the most widely used apparatus for studying microorganisms under constant environmental conditions. It is a continuous fermentation

process performed in a Continuous Stirred Tank Reactor (CSTR). A CSTR operates by maintaining a growth rate through continuously feeding a growth limiting nutrient and withdrawing part of medium at the same rate, thereby achieving steady state growth. The growth limiting nutrient may be carbon, nitrogen, phosphorus, or any other essential nutrient, which influences the specific growth rate. A significant advantage of chemostat mode over batch mode is that by changing the feed rate of growth limiting nutrient, the growth rate can be varied .

21.5.1.1 Auxostat

An auxostat is a continuous culture technique wherein the dilution rate is regulated based on an indication of the metabolic activity of the culture. A chemostat is essentially used for operation at moderate to low dilution rates, but an auxostat is used at

high dilution rates.

21.5.1.2 pHauxostat

In pH auxostat the feed rate is regulated by measurement and control of the pH of the fermentation medium. This can be applied only if there is a change in pH consequent to the growth of microorganism. The pHauxostat has been used for continuous mass cultivation of bacteria for isolation of intracellular products.

Fig. 21.3 Schematic representation of a chemostat (Najafpour, 2007)

21.5.2 Turbidostat

A turbidostat is a continuous culturing method where the turbidity of the culture is held constant by manipulating the rate at which medium is fed. If the turbidity tends to increase, the feed rate is increased to dilute the turbidity back to its set point. When the turbidity tends to fall, the feed rate is lowered so that growth can restore the turbidity to its setpoint. The problem of growth or other materials fouling the optical surfaces of whatever method is used to measure turbidity has not been solved. While a turbidostat may operate well for a brief time, the control signal for turbidity soon becomes unreliable. In a turbidostat the system includes an optical sensing device (photoelectric device) which continuously monitors the culture density in the growth vessel and controls the dilution rate to maintain the culture density at a constant rate (Figure 21.4). If the culture density becomes too high the dilution rate is increased, and if it becomes too low the dilution rate is decreased. The turbidostat differs from the chemostat in many ways (Table 21.1). The dilution rate in a turbidostat varies rather then remaining constant, and its culture medium lacks a limiting nutrient. The turbidostat operates best at high dilution rates; the chemostat is most stable and effective at low dilution rates.

Fig. 21.4 Schematic representation of a turbidostat (Najafpour, 2007)

Table 21.1 Comparison between turbidostat and chemostat continuos flow cultures

As discussed above, the stationary phase sets in partly because of the exhaustion of various nutrients and partly because of the introduction of an unfavorable environment produced by metabolites such as acid. Either of these two groups of factors can be used to maintain the culture at a constant density. In fed-batch systems fresh medium or medium components are fed continuously, intermittently or are added as a single supplement and the volume of the batch increases with time.

21.6 Fed Batch Fermentation

Fed batch fermentation, which is a technique in between batch and continuous fermentation, is a more recent development in industrial fermentation systems. Neither batch nor continuous fermentation is suitable for non growth associated products. In order to produce such products, it is first necessary to build up a high concentration of cells in the growth or batch phase, and then switch the metabolism of the

cell to arrest cell growth by feeding product precursors, carbon, and oxygen at a rate sufficient to meet the maintenance and product synthesis requirements. Essentially, fed batch fermentation involves two phases: growth phase and production phase. After the initial growth phase, one or more of the nutrients are supplied to the fermentor while cells and product remain in the fermentor.

Fed batch fermentation is well suited for production of compounds during very slow growth where there is no possibility of cell washout. Fed batch fermentation is well suited for producing product or cells when: (1) Substrate is inhibitory and there is a need to maintain low substrate concentration to avoid the cells being inhibited (e.g., citric acid, amylase), and (2) Product or biomass yields at low substrate concentrations are high (e.g.,baker’s yeast, antibiotic production).

There are two methodologies in the fed batch approach, namely fixed volume fed batch and variable volume fed batch. In fixed volume fed batch fermentation, a very concentrated feed nutrient is fed to the fermentor so that there is no appreciable increase in volume. The specific growth rate decreases with time and the biomass increases directly with time. In variable volume fed batch fermentation, there is an increase in volume due to nutrient inflow and no outflow. The specific growth rate is solely dependent on the concentration of the limiting nutrient.

Fig. 21.5 Holding vessels for baych type culture cultivation (Crueger and A. Cruegar, 2000)

Screw-neck borosilicate glass vessel with medium/inoculum addition assembly. (a) Stainless steel rod; (b) Silicon tubing; (c) Silicon disc; (d) Hypodermic needle; (e) Air vent; (f) Screw cap; (g) Magnetic bar. B. Aspirator-type vessel for introducing an inoculum of filamentous fungi into the fermentor. (a) Cotton-wool plug; (b) Magnetic stirrer bar.