Bioreactors For Single Celled Bacterial Culture Biology Essay

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A 100 m3 sparged mechanically agitated bioreactor is operating and close to oxygen limitation due to high density single celled bacterial culture. However, a new scheme has been introduced that uses even higher cell densities with even higher oxygen demands. To undertake this issue, several different bio-reaction processes needs to be considered to be able to choose the most appropriate and compensating process for this new scheme. However, prior to this; parameters that are majorly important in bioreactor design needs to be highlighted and discussed.

Oxygen is needed by all cells for respiration, it must be available as dissolved oxygen (DO2), since oxygen solubility is quite small, about 6-7 mg per litre under normal cultivation conditions, the metabolic oxygen requirements is usually supplied by continuous aeration of the culture medium. Thus, a continuous supply of oxygen must be maintained for aerobic bioprocesses.

The need for oxygen is satisfied for most cells by keeping the dissolved oxygen concentration in the medium at about 1mg/L. If the oxygen decreases, with a concomitant decreases in cellular energy production and as a result, cell growth do not occurs.

Kinetic growth

Understanding cell growth kinetics is necessary for the correct design and operation of a bioreactor and bio production. Cell kinetics can be described as the consequential interaction of numerous complicated biochemical reactions and transport phenomena, involving multiple stages of multi component systems. During growth, a heterogeneous mixture of young and old cells is continually transforming and adapting to it changing environment.

Kinetics is virtually impossible. Therefore, to derive simple bioreactor operation and performance models can be expressed.


The fundamental problem in supplying sufficient oxygen is that this gas has a low solubility in aqueous systems. Only 0.3 mM O2 equivalent to 9mg/L, dissolves in 1 litre of water at 20oC in an air/water mixture at 1 atm(101 kPa). The solubility of oxygen decreases as the temperature and concentration of dissolved solutes increases for large-scale culture with high cell densities, the oxygen demand of the bacteria can be met only by forced aeration. In practice, this is achieved by blowing sterile air through the culture. The efficiency with which oxygen is transferred from air bubbles to the liquid phase principally depends on the surface area to volume ratio of the air bubbles and the residence time of the bubbles in the liquid. The smaller the bubbles, the greater the surface area to the volume ratio and the greater the oxygen transfer rate. The longer bubbles remains in the liquid, the greater will be the amount of oxygen that will diffuse into the liquid.

One way of decreasing bubbles size is to introduce the air through a sparger with multiple small arifices rather than through a single layer-bore tube. A second way for increasing the oxygen supply is to agitate the culture broth vigorously with a stirrer. In order to ensure most effective mixing by the impeller, baffles are installed vertically along the inside diameter of the bioreactor. Agitation and aeration of the culture medium can result in excessive foaming, therefore antifoam control system required.

The injected air at the base for mixing, drives the liquid around the reactor gently, generating negligible shear stress for nematodes (bacterial cells).

To resolve the conflict between high oxygen demand for the bacteria and low shear stress for the nematodes within a conventional bioreactor; low shear paddle impellers are used to churn the medium and a downward pointing air sparger is used.

The consequences of bio-reactor design and operation are significant. Generally, microbial cultures exhibit high oxygen demands and limited shear sensitivity. Therefore, CSTR type bioreactors are the preferred choice, offering high oxygen transfer rates and good mixing.

Shear sensitivity of cultured cells-shear stress can be described as cellular injury from excess torsion. In a bioreactor such forces may result from the movement of mechanical parts, such as the impeller or from the mechanical effects of fluid movement.

To combat forming, anti-foam agents (usually silicon based) may be used, but it is strongly recommended to establish dose response curves for these agents, to determine their effects on culture growth, cell viability and metabolic production prior to their use in fermentation.

Production of cellular products is divided into two types, based on when

they are produced within a biological cycle. Primary

metabolites are produced during growth and are essential

for continuing growth. Secondary metabolites are produced

after growth has ceased. Primary metabolites include amino

acids, nucleotides, nucleic acids, proteins, lipids and carbohydrates.

Examples of primary products for industrial use

include ethanol, citric acid, acetone, butanol, lysine,

polysaccharides and vitamins.

Secondary cellular products are formed from the intermediates

and products of primary metabolism, and tend to

be specific to a species or group of organisms. Not all microorganisms

produce secondary metabolites, but they are

widespread among the filamentous fungi and plants. Many

secondary products have toxic or antibiotic properties and

are, as such, the basis of much of the antibiotic industry.

Production of enzymes via bioreactions has displaced inefficient

extraction techniques with mutation and genetic manipulation.

Continuous Bioreactions

The defining characteristic of continuous bioreaction is

a perpetual feeding process. A culture medium that is either ther sterile or comprised of microorganisms is continuously

fed into the bioreactor to maintain the steady state. Of

course, the product is also drawn continuously from the reactor.

The reaction variables and control parameters remain

consistent, establishing a time-constant state within the reactor.

The result is continuous productivity and output.

These systems provide a number of advantages, including:

• Increased potential for automating the process.

• Reduced labor expense, due to automation.

• Less non-productive time expended in emptying, filling

and sterilizing the reactor.

• Consistent product quality due to invariable operating


• Decreased toxicity risks to staff, due to automation.

• Reduced stress on instruments due to sterilization.

The disadvantages of continuous bioreactors include:

• Minimal flexibility, since only slight variations in the

process are possible (throughput, medium composition,

oxygen concentration and temperature).

• Mandatory uniformity of raw material quality is necessary

to ensure that the process remains continuous.

• Higher investment costs in control and automation

equipment, and increased expenses for continuous sterilization

of the medium.

• Greater processing costs with continuous replenishment

of non-soluble, solid substrates such as straw.

• Higher risk of contamination and cell mutation, due to

the relatively brief cultivation period.

Continuous bioreaction is frequently used for processes

with high-volume production; for processes using gas, liquid

or soluble solid substrates; and for processes involving microorganisms

with high mutation-stability. Typical end products

include vinegar, baker's yeast and treated wastewater.

During this dynamic reaction period, cells, substrates (including

the nutrient salts and vitamins) and concentrations of the

products vary with time. Proper mixing keeps the differences

in composition and temperature at acceptable levels.

To promote aerobic cultivation, the medium is aerated to

provide a continuous flow of oxygen. Gaseous byproducts

formed, such as CO2, are removed, and aeration and gas-removal

processes take place semicontinuously.

Next, an acid or alkali is added if the pH needs to be

controlled. To keep foaming to acceptable levels, antifoaming

agents may be added when indicated by a foam


Continuous vs. batch

There are several major advantages to using continuous

bioreactions as opposed to the batch mode. First, continuous

reactions offer increased opportunities for system investigation

and analysis. Because the variables remain unchanged,

a benchmark can be determined for the process

results, and then the effects of even minor changes to physical

or chemical variables can be evaluated. Also, by

changing the growth-limiting nutrient, changes in cell

composition and metabolic activity can be tracked. The

constancy of continuous bioreaction also provides a more

accurate picture of kinetic constants, maintenance energy

and true growth yields.

Secondly, continuous bioreaction provides a higher degree

of control than does batch. Growth rates can be regulated

and maintained for extended periods. By varying the

dilution rate, biomass concentration can be controlled. Secondary

metabolite production can be sustained simultaneously

along with growth. In steady-state continuous bioreaction,

mixed cultures can be maintained using chemostat

cultures - unlike in batch bioreaction, where one organism

usually outgrows another. Chemostats are continuousflow

stirred-tank bioreactors (CFSTRs) in an idealized

steady-state, i.e., the feed- and outlet-stream compositions

and flows are constant, and perfect mixing occurs within

the CFSTR vessel. In chemostats, the outlet stream composition

is considered to be the same as within the bioreactor.

Bioreactors operated as chemostats can be used to enhance

the selectivity for thermophiles, osmotolerant strains, or

mutant organisms with high growth rates. Also, the medium

composition can be optimized for biomass and product

formation, using a pulse-and-shift method that injects nutrients

directly into the chemostat. As changes are observed,

the nutrient is added to the medium supply reservoir

and a new steady state is established.

A third advantage is the quality of the product. Because of

the steady-state of continuous bioreaction, the results are not

only more reliable, but also more easily reproducible. This

process also results in higher productivity per unit volume,

because time-consuming tasks, such as cleaning and sterilization,

are unnecessary. The ability to automate the process also

renders it less labour-intensive, and, therefore, more cost-efficient

and less sensitive to the impact of human error.

Along with the strengths of continuous bioreaction,

there are inherent disadvantages that may make this process

unsuitable for some types of bioreaction. For example,

one challenge lies in controlling the production of

some non-growth-related products. For this reason, the

continuous process often requires feed-batch culturing,

and a continuous nutrient supply. Wall growth and cell aggregation

can also cause wash-out or prevent optimum

steady-state growth.

Another problem is that the original product strain can

be lost over time, if it is overtaken by a faster-growing one.

The viscosity and heterogeneous nature of the mixture can

also make it difficult to maintain filamentous organisms.

Long growth periods not only increase the risk of contamination,

but also dictate that the bioreactor must be extremely

reliable and consistent, incurring a potentially larger initial

expenditure in higher-quality equipment.