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Bioreactors

1.1. Introduction

1.1.1. What is a bioreactor?

A bioreactor is an enclosure containing a nutrient medium consisting of a cocktail of various molecules - referred to as "substrates" - upon which one or more populations of microorganisms grow, and as such the set of these microorganisms is called "biomass". Bioreactors are used to perform operations for transforming matter through biological pathways, most often accompanied, but not systematically, by the increase of biomass in the reaction medium. Microbiology teaches us that only soluble substrates - that have created chemical bonds with water molecules - are available for the growth of living cells. Within the context of this book, from a formal point of view, a biological reaction will therefore describe the transformation of elements existing in the medium in soluble form into a solid form, biomass and possibly into a certain number of metabolites and/or gas. However, it may happen that a number of resources are present in solid form. A so-called "hydrolysis" step is necessary to transform this solid substrate into a soluble form assimilable by microorganisms. The conception that claims that a microorganism "grabs" molecules passing nearby is somehow a figment of the imagination. For example, it is accepted in soils that most microorganisms, whether they be mobile or not, excrete enzymes around them and recover the nutrients that reach them by diffusion. It is therefore essential to properly distinguish between the different processes involved before initiating the modeling of phenomena as complex as the degradation of a set of substrates by microorganisms. This is the subject of this chapter that describes the most important processes involved in this type of matter transformation and the systematic approach widely adopted in process engineering to model them. In what follows, we will only cover microbial ecosystems that are implemented in most bioprocesses.

1.1.2. Classification of biological reactors

In process engineering, bioreactors are first classified according to their mode of operation, in other words the way in which they are supplied with matter, and depending on whether microorganisms are free in the medium (so-called "planktonic" organisms) or fixed on a support; the latter could itself be fixed or mobile.

As a result, it is possible to distinguish continuously-fed systems, systems whose supply is semi-continuous and those operating in closed mode. In continuous reactors, the reaction volume remains constant, in- and outflow rates being identical. It is the most commonly used operating mode in industries aiming to process a large amount of material arriving continuously, as it is the case, for example, in the treatment of water by biological means. We will often refer to this mode in this chapter insofar as this is one of the most significant industries in terms of quantities of processed materials. Semi-continuous (or fedbatch) reactors are systems whose inflow rate is not zero but whose outflow rate is zero. In such a system, the reaction volume is thus increasing over time from a minimal to a maximal value. This type of system is particularly suitable for the production of biomass as the amount of substrate can be supplied according to the specific needs of microorganisms. It is also used when the risk of inhibition due to the substrate accumulation or a metabolic intermediary in the medium is present. Depending on the physiological state of microorganisms, it is then possible to decrease, or on the contrary, to increase the amount of resource fed into the reactor. Finally, batch mode - or reactor - designates a closed system in the sense where there is neither supply nor withdrawal of the system: substrates (the different nutrients necessary for the growth of microorganisms) as well as the inoculum (biomass) are introduced at the initial time. Therefore, the reaction volume of the system is constant over time (if possible liquid-gas exchanges are neglected) and the reaction takes place up to the moment when it is measured (or considered) that it has completed. This operating mode is widely used in agri-food, pharmaceutical and chemical industries, notably for the production of molecules with high-added value, and more generally in cultures in which the risk of contamination through the feed is high.

Since biomass is the catalyst for reaction, the effectiveness of a biological system will be all the more significant when the substrate necessary for its growth is in an appropriate form (this is referred to as biodegradability) and accessible (so-called accessibility). The homogeneity of the medium as well as biomass and resource densities will consequently play essential roles in the operation of these systems. In order to maximize the concentration of the existing biomass - but mainly to facilitate the separation of the biomass from the residual reaction medium (in other words, to facilitate the separation of the liquid and solid phases of the medium) - it is possible to resort to using a support upon which biomass will tend to settle in the form of "biofilms"1. In laboratories, even today, many engineers are testing the effectiveness of all kinds of fixed or mobile supports and are studying the properties of associated processes. In fact, it is essentially based on these considerations - relating to the feeding modes of reactors and to the manner in which biomass is retained within the system - that different technologies of reactors have been proposed. Finally, the last major element for the classification of bioprocesses is linked to the same biological processes that condition bacterial growth. It designates the set of conditions that must prevail in the medium to enable the growth of microorganisms (this is often referred to as "environmental conditions"). They are essentially ecosystem-dependent. In the next section, we review a certain number of concepts that are necessary to understand the formalization of the model of the chemostat, which we will next present in several ways in the book.

1.1.3. A brief reminder of microbiology

To grow and multiply, a (micro-) organism needs a multitude of elements, including some at trace level. A natural manner to classify organisms is to refer to the mechanisms that they implement to capture the matter necessary for their growth and to produce their energy. When we concentrate on the major factors influencing growth, a source carbon and an energy source are in effect essentially needed. Carbon is found in two basic forms in nature: organic or inorganic. For simplicity, assume here that organic carbon is the carbon produced by living entities, inorganic carbon designating the CO2 in its different chemical forms (carbonic acid, bicarbonate, dissolved CO2; we will later on return to these elements to talk about the mutual influence of biological reactions and chemical balance of the medium, which are fundamental factors affecting the growth of microorganisms).

There are essentially two sources of energy: light and chemical energy. Organisms that extract their energy from light are called phototrophs, those who take it from chemistry being called chemotrophs. Regarding sources of carbon, organisms being able to utilize organic matter are called heterotrophs, those using CO2 are known as autotrophic. By combining the carbon and the energy source being utilized, four major classes of microorganisms can then be defined:

• - chemoautotrophs utilize CO2 as a carbon source and derive their energy from the consumption of inorganic substrates;
• - chemoheterotrophs utilize organic matter as a source of energy and carbon;
• - photoautotrophs utilize CO2 as a carbon source and derive their energy from light;
• - photoheterotrophs utilize organic material as source of carbon and light as energy source;

In environmental biotechnology, chemoheterotroph bacteria have been particularly studied because they degrade organic matter and are not constrained by light, which is difficult to evenly diffuse inside reactors of large volumes comprising high cell densities. The metabolism of these organisms enables energy to be produced and the final products of the degradation are CO2 and water. The other bacteria widely studied in the environmental field are chemoautotrophs because they are involved in the nitrogen cycle. They derive their energy from the presence in the medium of molecules such as ammoniacal nitrogen or nitrite and their source of carbon is CO2.

Why have we presented these various notions in a book dedicated to the chemostat? Based on the previous few examples, we will show in the next section that the apparent complexity of the functioning of microbial ecosystems can be formalized in a simple and natural manner at the populations level, regardless of the type of metabolism implemented by the microorganisms.

1.2. Modeling of biological reactions

1.2.1. Regarding the state variables of the model

As indicated at the beginning of this chapter, a chemostat consists of an enclosure in which resources are provided by means of a feed and from which the reaction medium is withdrawn at an outlet (Figure 1.1). Assuming that all the elements necessary to the growth of the microorganism that will develop inside the reactor are present in excess at all times, the velocity at which it could develop would be limited...