Hi my name's John Woodley from the Technical University of Denmark in Lyngby - that's just outside Copenhagen in Denmark. I've been working for 30 years in the area of biocatalysis and I'm the professor of biochemical engineering, so my specialism is much more in the engineering aspects and the process aspects of biocatalysis. And it's with that background that I've been working with Professor Nick Turner, here now at MIB in Manchester, for the past 30 years or so. In this MOOC, I'm going to talk about two aspects. One is concerned with biocatalysis and the implementation of biocatalytic and enzymatic processes and explain a little of the difference between that and fermentation processes. Then we'll also talk about the economics of processes and how to be able to implement them in industry and how we assess that. In this Module 3 of the Industrial Biotechnology MOOC, we're going to discuss biocatalysis and enzymatic processes. And the emphasis here is on the process, rather than on the reaction. But by way of background, perhaps it's worth saying that biocatalytic reactions, also known as enzymatic reactions, use one or more enzymes in order to convert a starting substrate molecule into a higher value chemical product. These types of enzymatic reactions have enormous value to synthetic and process chemists. In fact, biocatalysis today is a well established technique in the laboratory. But there are, of course, some special kinds of engineering considerations which are required when we try to implement this in industry. And that is what we're going to focus on in module three. Before I start on that, I'd like to explain a little bit some of the advantages of enzymatic catalysis, not just from a chemical perspective, but also from an engineering perspective. Enzymes of course have exquisite selectivity, both in terms of the reaction, but also stereo and regio selectivity as well. And that means that we can avoid many by-products, and the recovery of downstream processes are therefore much easier. This selectivity can also be achieved under very mild conditions. By that I mean, a pH around about neutral, temperature, around about ambient temperature and pressure around about atmospheric pressure. And that also avoids extra reactions which can take place under these extreme conditions, meaning also that this downstream process becomes significantly easier. Operating with high selectivity, and under mild conditions also of course, gives a very good environmental footprint to many of these reactions. And that's further enhanced by the fact that the catalyst is from a renewable resource. People don't often think about the fact that the enzyme itself is from a fermentation. And is growing on glucose or sugar source, and that of course is an enormous advantage for process like this. Perhaps even more important in the selectivity, in mild conditions and the catalyst operating from a renewable resource, is the ability to alter biocatalyst properties. And there's a technique known as protein engineering where we swap the amino acids around the protein. And we can do that inside the active site, which can alter the reactivity or the selectivity of the enzyme. Or we can do that sometimes far from the active site, and that may affect and improve the thermostability, the pH and the solvent tolerance of the enzyme. This ability to be able to alter the properties of the enzyme is very important from an engineering perspective. And enables us to design completely new types of processes operating under conditions far away from those found in nature. There are many opportunities for biocatalysis but important to point out that these opportunities are in all of the different value sectors. High-value molecules where we talk about multi-functional compounds which, of course, are sensitive, we find these a lot in the pharmaceutical industry and that's where we've seen the biggest application to date of biocatalysis - around about 150 processes are operating today in industry. Medium-value molecules where the selectivity is very important to improve the process and where the costs of the process starts to become important - also an opportunity for biocatalysis. And even low-value molecules, mostly the products of a previous fermentation from a renewable resource and also opportunities for biocatalysis down into $10 per kilogram a range. The basic concept of biocatalysis is worthwhile emphasizing because the primary engineering concept is that we exploit here the ability to be able to de-couple cell growth from reaction. Cell growth is what we normally refer to fermentation and the reaction we are are referring to here as biocatalysts. By separating these two unit operations, it means also that we can de-couple the rate of growth from the rate of product formation. And that means that we can achieve far higher values of what chemical engineers refer to as space time yield. Biologists frequently refer to this as the productivity of a process. But, in all cases, we talk about how much mass is made, per unit volume, per time. This ability to de-couple the fermentation from the biocatalysis also gives us the opportunity to recycle the biocatalyst, in much the same way we would as a conventional catalyst. And, that of course is provided that it has sufficient stability. Or in other words, that it can maintain its reaction activity over time. Many processes today operate with this recycleing in place. And worthwhile, perhaps, considering generic process flowsheets, which is what I show here on this next slide. We start at the left hand side with a reactor. The reactor is fed with substrate (given the symbol S here) by a catalyst (given the symbol B). Afterwards, we follow the reaction by a separation step where we separate the biocatalyst from substrate and product. That's called Separation 1 and the biocatalyst can be recycled back to the start of the reactor again, provided, as I said before, it has sufficient stability. The product and the substrates then go to separation 2 where they are then separated. It might be that it is useful in some cases to recycle the substrate back to the beginning of the process. But, in many cases, the conversion will be high enough that we make enough product at the end that we don't need to do this. This generic flow-sheet depends a little bit on the biocatalyst that we use. And in the next slide here I show the two formats we have for the biocatalyst. The top format refers to what we call a resting whole cell, or maybe a better term, microbial catalyst. We convert here a compound A to a compound B. And the first step of this is that the compound A needs to come into the cell, converted by an enzyme into product B and then B neede to come back out of the cell at the end. For natural compounds there is normally an uptake mechanism to assist A going into the cell, and there's also a similar secretion mechanism for B coming back out of the cell. For non-natural compounds, certainly the uptake of A can be a limitation. I indicate on this slide here also with the arrow on the right-hand side, the fact that we can recycle this cell. That of course is one of the ways that we're able to make sure that the cost contribution of the enzyme to the overall process is minimized. In the lower part of the slide, I show here an isolated enzyme. Well, we've now taken the enzyme out of the cell and this means that we don't then have the difficulty of having to get material into the cell and getting the product back out of the cell at the end. Again, we can recycle the enzyme. We frequently refer to such systems as cell free extracts because in an industrial context, we will not normally try to, we'll not normally try to, purify the enzyme, in order to reduce the cost. Such recycle can be achieved easily by immobilizing the enzyme on a solid support. This process biocatalyst format, helps to guide us a little bit for the types of process options that we have available as well. And I show here some different possibilities. At the top of the slide here, I show a fermentation. Fermentation is normally fed by a substrate but could also require other nutrients. For an aerobic fermentation we'll require oxygen as well and the cells from this then feed into a biocatalytic step, which of course need to be fed by a substrate. We purify the product at the end of this. An alternative to this could be the flow sheets which I show at the bottom of the slide here. Where I still have a fermentation and a biocatalysis step fed by substrate. But in between the two I now have the option of biocatalyst preparation. For whole cells that could mean that I simply re-suspend the cells in a different media. For example, in water, which means that the downstream purification will be very much easier. An alternative could be that I isolate the enzyme at this point, or even isolate the enzyme and put it onto a solid support. In any case, the option shown at the bottom gives us the possibility to be able to change the biocatalyst concentration and the media in which it operates. Reaction kinetics are of course paramount to this biocatalytic conversion and to be able to help it to operate effectively. And as shown in this next slide here, a simple plot of the reaction rate against the substrate concentration. For those used to chemical reactions, of course this is a somewhat unconventional plot. At the left hand size, we have a first order reaction where the reaction rate is essentially proportional to the substrate concentration. Towards the right-hand side of the plot, we have a zero order reaction, where the reaction rate is independent of the substrate concentration. This shape of the curve is very important for working out how to operate the reaction and the reactor in the best possible way. There are essentially three types of reactor which I show on this next slide here. On the left hand side we begin with what we refer to as a batch stirred tank reactor. This is a reactor, which is normally well mixed, meaning the concentrations at all points inside the reactor is the same. I feed my reactor in the beginning with a certain amount of substrate and biocatalyst and the reaction takes place over time. It's a very flexible system, meaning that I can operate for different lengths of time in order to get different levels of conversion. An alternative approach to that is to operate the reactor scheme I show in the middle of this slide, a continuous the tank reactor. In this scheme here I feed continuously sub strate to the reactor at a rate Q. And I remove substrates and product from the reactor, also at a rate Q, meaning that the reactor itself, operates at steady state. All concentrations are the same, both with respect to space, but also with respect to time, as well. In such a reactor, it's not possible to achieve 100% conversion, because now some of the substrate is required in order that the reaction can still take place. There could be advantages to operating in a continuous stirred tank reactor, especially at big scale. But one of the penalties to be paid very often we require a lot of enzyme in order to be able to make the conversion reasonable because it will operate at a low substrate concentration, the leaving substrate concentration. An alternative continuous reactor set up is shown in the third example on the right hand side of slide, the continuous plug flow reactor. Here, we feed the substrate at the same flow rate Q to a packed bed of enzyme, and remove at the end of it our product, and, if there's some unreacted substrate, also substrate, also at a rate Q. By being able to put all of the enzymes into the bed we're able to save a lot of volume here and make a much smaller reactorant. But it comes at the penalty of not being able to operate with a fast flow rate otherwise we have a high pressure drop through this column. And it is also necessary, of course, to immobilize or to keep the enzyme within the reactor configuration itself. A mathematical description of the reaction and indeed also the reactors has been carried out. I'm going back to the 1930s, the Michaelis-Menten kinetics describe such enzyme reactions. The equation shown on the screen here has on the left hand side the rate of change of product with respect to time, dP by dt, and on the right hand side we have a constant, that's the maximum rate of reaction, Vmax which is equal to a constant Kcat, specific to the particular enzyme, multiplied by the enzyme concentration. And this itself is multiplied then by the substrate concentration in the upper equation. That is divided by another constant, a Michaelis constant KM, added to the substrate concentration. There are different forms of this type of equation, but this equation describes, the parabolic that we have seen earlier. These reaction kinetics can also be integrated together with mass balances in order to provide reactor kinetics. And here I show the equations for the batch stirred tank reactor, the continuous plug flow reactor, and a continuous stirred tank reactor. This equation links the conversion, X, with the amount of enzyme, E, required in order to operate within a certain period of time for a given substrate concentration S0. These equations are very powerful because they help us to understand what happens as we change the enzyme and what happens as we change the amount of enzyme that we operate within the reactorant. The equations were developed in the 1960s and at that time the cost of the enzyme was prohibitive and therefore is very important to understand exactly how much enzyme was required. Today we have genetic engineering tools, the cost of the enzyme is much reduced. And that means that it is much easier for us to implement the process. Never-the-less, in many processes, especially of scale, the cost of the enzyme will be important. These equations help us to work out and to establish, which is the best way to operate the process. Some complications can never the less arise and I've listed here some typical modes of operation beyond these three reactive types. First we can have the situation that the substrates are inhibitory or toxic to the enzyme meaning that they lower the reaction rate reversibly or irreversibly respectively, and in such cases we need to feed the reactor. Fed-batch mode is a normal way of being able to overcome this. It can also be the case that the product is inhibitory or toxic to the enzyme. And here, we implement technologies such as in situ product removal, abbreviated here to ISPR. Where we're able to remove the product as we go along through the reaction itself. A final complication can arise where we have compounds which are interesting from a chemical or commercial perspective but have a low water solubility. And to deal with some substrates or products, techniques we developed in the 1980s, to be able to operate in a two liquid phase system, where we have an aqueous phase, and a water emissable, organic solvent phase, so solublize, the substrate, and the product. Beyond understanding the different types of reactors, it's also important to understand from an engineering prospective the limits to the biocatalyst concentration that we can use. At low biocatalyst concentrations it is of course the case that we can add more catalyst. And we get a return for the additional catalyst that we add and that's shown in this plot of limiting regimes on the left hand axis I have shown different metrics and these metrics are a way of measuring the process and the efficiency of the process. The blue line refers to the product concentration, given here in grams per liter. The red line refers to the space time yield, or productivity of the reaction in grams per liter per hour. The black line at the top refers to the biocatalyst yield, how many grams of product I get the gram of biocatalyst that I put in. As we change the biocatalyst concentration we move from a stability limited regime, at low biocatalyst concentrations, to a product limited regime, at medium biocatalyst concentrations, to a rate limiting regime at high biocatalyst concentrations. Understanding what limits the process helps us to design and to improve the reactor and its operation. In order to scale up these bioprocesses, it's important, not only to consider an increase in volume, but also to make sure that we have sufficient process intensity, meaning that the metrics need to be high enough that we can put in place a commercial process. In the schematic plot here, I've indicated, for example, that we need first to improve the intensity. That's the green arrow here. And secondly, to increase the volume. That's the red arrow that I've shown here. By making such an improvement in the process and by operating such a design paradigm, it's possible for us to be able to implement the process very effectively. Additionally it's worth saying that protein engineering has a very important role to play as well. In our task to move from nature into industrial application we need to move from a limited substrate scope to a broader substrate scope. And the green arrows on the schematic plot here indicate the types of improvements which are required and can be carried out by protein engineering. But it's also necessary that the process conditions are suitable to implement our process effectively, and that's shown by the blue arrows on the slide here as well. That's very much the job of the process engineer working together with protein engineers in order to move from natural conditions and substrate scope, to industrial application. Let us summarize what we have learnt in this module three. Biocatalytic processes offer a number of advantages from an engineering perspective over fermentations. The fact that we can decouple the fermentation from the reaction means that we have increased space time yield and the possibility to recycle the biocatalysts. There are a range of biocatalytic formats and reactors. They of course require selection. That's one of the primary jobs of the process engineer, since they determine the process flow sheet. We have kinetic equations already available to describe potential reactions. But also reactors so we can describe these mathematically, model, and simulate the process. Typical modes of operation also need to allow for some extra complexities and that depends a little bit on the characteristics of a given biocatalytic reaction. Issues such as poor water solubility or limited inhibition also need to be considered. And finally the scale-up needs to focus on process intensification.
