Cultivation Systems

A microbiological culture is a laboratory method that allows the multiplication of microorganisms, both prokaryotic and eukaryotic, in a growth environment called medium.

Cultivation of microorganisms represents one of the main diagnostic methods in microbiology, thanks to its capability of determining the type and amount of microorganism present in the analyzed sample. In Biotechnology, the cultivation of microorganisms plays a fundamental role for the final success of the bioprocess, which aims at obtaining specific products.

The subject of microbial cultivation may appear simple on the surface, but there are various factors that make this topic considerably complex (Prakash et al., 2013). As a matter of fact, microorganisms require proper environmental conditions and precise nutrients to grow and to make replicas of themselves. In particular, the chemical composition of microorganisms reveals which are the elements that must be provided with nutrients and that, additionally, should be in a metabolically accessible form. The nutrients present in the growth media can be divided in different categories, according to the elements that they supply:

carbon, nitrogen, sulfur, phosphorus and mineral sources are essential for living organisms.

Another requirement for microbial growth is the metabolic energy, fundamental for the synthesis of macromolecules and for the maintaining of essential chemical gradients across microorganisms‘ membranes. Metabolic energy can be generated by three main mechanisms:

fermentation, respiration and photosynthesis. It is clear that every microorganism has its specific demand of nutrients and metabolic pathway to grow. To satisfy all these necessities, the accurate control of the following environmental factors during the growth is determining:

nutrients, pH, temperature, aeration, salt concentration and ionic strength of the medium (Brooks et al., 2013).

The cultivation method and the choice of the medium depend on the investigation type, which may aim at obtaining one among the following objectives: growing cells of a given species, isolation of a microorganism species from a natural source, microbiological analysis of the sample. Moreover, microbiological cultures can be categorized by the physical state of the medium culture, that can be solid or liquid, such as agar and broth, respectively.

Agar is a gel used for plating, a technique that consists in growing a pure culture of microorganisms in the so-called Petri dish, see Fig. 2-4 (A). This device, thanks to its transparent lid, allows to observe the colonies and to limit contamination from other species.

The physical properties of agar, which is an acidic polysaccharide extracted from certain red

Microorganisms and Biotechnology

algae, allow to immobilize the cells in this solid medium and to let them grow in different colonies, originated by single cells. The streak-plate technique is made up of few steps, starting with the preparation of the Petri dish: agar, once dissolved in water at 100 °C, is enriched with nutrients and stored at 50 °C, then it is poured in the plate and its gel formation occurs at 45 °C. With a sterile wire loop some cells are caught from the inoculum and they are streaked on a limited area of the gel. Afterwards the wire loop is sterilised again, rubbed on the previous area to catch some cells and then streaked on another area, see Fig. 2-4 (B). By repeating this procedure, the cells deposited on the gel by the wire loop are more and more isolated, so that it is possible to identify the different colonies after the incubation time that for most routine laboratories is 5 days circa, although most of the pathogens grow after 24--28 hours (Brooks et al., 2013; Lagier et al., 2015).

(B)

Fig. 2-4 The streak-plate technique: (A) Petri dish with agar and a microbial culture. (B) Streaking patterns made with a wire loop showing the corresponding procedural steps (Brooks et al., 2013).

A broth culture consists of liquid growth medium and microorganisms: the sterile growth medium must contain all the necessary nutrients for the microorganisms that will be here inoculated.

An example of liquid growth medium is Luria Broth (LB), which is widely used for bacterial culture. It is a very rich medium that contains tryptone, yeast extract and NaCl (MacWilliams and Laio, 2006). The potential of a broth culture is revealed by the chance of growing a large number of microorganisms in a quite small amount of time; additionally it is chosen as a method of storage in sealed shaking flasks.

Broth culture is generally used in bioreactors to grow and use microorganisms; in this case it is called submerged fermentation. However, there is also a fermentation process called solid-state fermentation (SSF) that works with a solid matrix in absence, or nearly absence, of water, hence it is more appropriate when dealing with filamentous fungi, because they are microorganisms that are naturally adapted to this condition (Spier et al., 2012).

Bioreactors are the main unit operations for industrial biochemical transformation, in which the treated materials promote the biotransformation by the action of the living cells or by the cellular components, such as enzymes (Pandey et al., 2008). The purposes that can be achieved through these reactors are various: biomass production, metabolite formation such as organic acids and antibiotics, transformation of substrates, and production of enzymes. In general, bioreactors are cylindrical vessels or tanks, with a volume that goes from a liter to some cubic meters, depending on the design and on the operation mode (Spier et al., 2012).

Due to their great potentials and to the various operations that can be carried out, there exists a very wide variety of bioreactors, that can be considered the heart of the bioprocesses (Cinar et al., 2003).

(A)

Microorganisms and Biotechnology

10 | p a g e

The operation mode of a bioreactor can be classified in batch, fed-batch and continuous processes, although batch systems are definitely prevalent and extensively used (Spier et al., 2012). Generally, in a batch bioprocess the system (fermenter) is partially closed and the required materials are supplied before the starting of the operation in sterile conditions, and removed together with the products at the end.

In fed-batch processes, instead, sterile substrate and nutrients are continuously added inside the bioreactor, in which the volume increases due to the lack of an outflow. Hereafter, the main types of submerged bioreactors, that are the ones dealing with broth culture, are briefly presented, according to Spier et al. (2012).

Fig. 2-5 Important types of bioreactors: (A) stirred tank reactor, (B) bubble column reactor, (C) airlift column with external loop and with internal loop.

The mainly used bioreactor is the stirred tank reactor (STR), see Fig. 2-5 (A), where one or more impellers, according to the design specifications, promote the mechanical agitation of the broth, improving mass and heat transfer. There are aerated and non-aerated STRs; when aeration is required, its rate must be over-stoichiometric to promote higher contact between the broth and the air bubbles (Doran, 1995). The broth generally occupies 70–80% of the total volume of the fermenter, in order to have a headspace used for gas exhaust and foam formation.

Pneumatic mixing is instead performed in a bubble column bioreactor (BCR), where a sparger is equipped at the bottom of the cylindrical tank for the formation of air bubbles, see Fig. 2-5 (B). For this type of reactor, the choice of the sparger is determinant on the size and on the number of the bubbles, hence on the mass transfer.

A variation of the BCR is the airlift bioreactor (ALR), where the pneumatic mixing is promoted by the presence of components that help to circulate the fluid, see Fig. 2-5 (C). The different configurations of ALRs can be classified in two main types: external loop and internal loop.

Bioreactors own an additional complexity as compared to other reactors, due to the presence of the biological phase, which requires the establishment of favorable environmental conditions and the supply of proper nutrients for the growth of the living organisms. Hence, it is fundamental, for a good performance of the bioreactor, to pay attention and to control all the factors that can threaten the health of the microorganisms, i.e. in terms of cell stress. To achieve this, it is indispensable to translate the necessities of the biotic phase in physical quantities that can be measured and controlled in the reactor.

In the Sections 2.2–2.4, some important quantites of fluid dynamics are defined, with the intention of enhancing the clearness of the topic discussed in Section 2.5, concerning the stress on microorganisms.

Fluid Rheology