Experimental Systems for Studying Kinetics

In order to establish the kinetic profile, a small-scale experimental system should be used so that heat transfer and inter-particle mass transfer will not be limiting. The idea is that the conditions within the substrate bed are those that you wish the organism to experience; therefore heat and mass transfer limitations should not cause significant deviations from these conditions. In other words, the aim is to characterize the growth kinetics of the organism without interference from bulk transport phenomena, to the extent that is possible. Of course, when empirical equations are used to describe the kinetics, the intra-particle transport phenomena are subsumed in the overall kinetic equation. This is impossible to avoid, since in-tra-particle transport limitations are an intrinsic characteristic of SSF (Chap. 2).

As mentioned in Chap. 14, you will undertake these kinetic studies once you have identified a substrate composition and environmental conditions that allow reasonably good growth of the organism. The most important conditions to control are the gas phase composition and the temperature and the water activity of the substrate bed. The two basic experimental strategies available are: (1) The use of multiple erlenmeyer flasks (or similar vessels) within an incubator and (2) the use of multiple columns within a waterbath.

Kinetic studies are typically done in these systems, rather than in laboratory-scale bioreactors, because such bioreactors are commonly not well-mixed, and therefore it is difficult to remove representative samples from them (Fig. 15.1). The problem is most evident in the case where it is desirable to leave the bed totally static, in which case it is impossible to avoid heterogeneity in beds containing even as little as a few hundred grams of substrate. There will be differences between inner and outer regions of the bed, and samples cannot be removed from anywhere other than the exposed surface without disrupting the bed. This disruption will affect growth of the microorganism in the part of the bed left behind after the sample is removed. In systems that involve multiple flasks or columns, individual units can be sacrificed at each sampling time. Even though within individual flasks or columns with less than 100 g of substrate there might still be some heterogeneity in the substrate bed (for example, from top to bottom of a column or from the inner to the outer regions within a flask), each flask or column should be identically heterogeneous, and therefore representative of all the other flasks.

Even though each flask or column should be identical with the others, there will always be some variation. Therefore it is important to establish, before the fermentation, the order in which the flasks or columns will be removed. If the decision were made at the time of sampling, then it would be possible to be influenced by the relative appearance of the different flasks or columns. Also, given the possibility that the conditions in a waterbath or incubator might vary with position due to imperfect circulation patterns, the pattern of removal should be random (Fig. 15.1(b)).

there may be heterogeneity within each flask, but each flask is identical with the others a larger unagitated bioreactor (such as a tray) will have temperature and gas concentration gradients, resulting in heterogeneous growth i individual flasks can be removed without disturbing the other flasks

samples cannot be removed from the interior without disturbing other parts of the bed flasks or columns should not be removed in a sequential order the removal order should be random

Fig. 15.1. Basic considerations about kinetic studies. (a) It is better to use multiple small containers in which individual containers are sacrificed at each sampling time rather than to remove subsequent samples from a larger mass; (b) The individual containers should be removed in random order

15.1.1. Flasks in an Incubator

This system is very commonly used. Its basic features are shown in Fig. 15.2. Ideally the substrate layer should not be thicker than 1 to 2 cm, although even with this thickness growth at the bottom of the layer may be limited by poor O2 supply.

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Fig. 15.2. Considerations in the use of flasks for kinetic studies. (a) The manner in which the flask is "closed" must be considered carefully; (b) It may be appropriate to bubble air through a tray in the bottom of the incubator in order to maintain a high humidity and reduce evaporative losses from the substrate

Fig. 15.2. Considerations in the use of flasks for kinetic studies. (a) The manner in which the flask is "closed" must be considered carefully; (b) It may be appropriate to bubble air through a tray in the bottom of the incubator in order to maintain a high humidity and reduce evaporative losses from the substrate

It is important to consider the control of water content of the substrate and the O2 concentration in the gas phase. In order to provide a well-oxygenated gas phase, it would be preferable to leave the flasks open, allowing the headspace within the flask to communicate directly with the airspace in the incubator. However, if the relative humidity of the incubator atmosphere is not controlled, then it is likely that this will promote evaporation and drying out of the substrate. Also, open flasks provide no barrier to the entry of contaminants.

If necessary, water can be added to flasks at various intervals during the fermentation. In this case it would be desirable to mix the substrate bed in order to distribute the water evenly. If mixing is undesirable (due to adverse effects on the microorganism) then it is more challenging to add the water in a uniform manner. This might be achieved by adding the water as a fine spray over a thin layer of substrate.

In order to minimize water losses and therefore minimize the need to add water, it may be desirable to close the flask. However, in this case the O2 concentration in the headspace will fall quickly. Note that cotton wool plugs can provide a significant barrier to O2 transfer, so it would be wrong to assume that the headspace gas in plugged flasks has the composition of air. It would be necessary to take gas samples and analyze them.

15.1.2. Columns in a Waterbath

This system, involving small columns submerged in a waterbath (Fig. 15.3), has come to be known as "Raimbault columns", after their use by Maurice Raimbault in the 1980s. This system allows for forced aeration of the substrate bed, and therefore provides better control of the composition of the gas phase within the bed than can be obtained with fermentations carried out in flasks.

The columns need to be relatively thin, possibly only of 1-2 cm width, in order to minimize radial temperature gradients. Note that Saucedo-Castaneda et al. (1990) found significant radial temperature gradients in a 6-cm-diameter column. The height of the column could vary between 10-20 cm or even more, although the higher the column, the more likely that a special waterbath will have to be constructed.

Fig. 15.3. Basic features of the Raimbault column system. Only four columns are shown, but the number is in fact only limited by the size of the waterbath. The diagram at the right shows detail of an individual column

It is important to saturate the incoming air to minimize the drying out of the bed in the column, because it is not practical to add water to the bed during the fermentation. Typically this will require at least two humidification steps. Note that temperature control is also important in minimizing evaporation, since if the bed temperature were allowed to rise above the inlet air temperature, the air would heat up and evaporate water from the bed, even if it were saturated at the air inlet.

It is also important to regulate the airflow to each column independently, with a separate rotameter on each line. If a single manifold were used then, in the absence of individual controls, any differences in the resistance to flow through the various columns would lead to different columns receiving different flow rates.

Whole columns are removed from the bed and sacrificed as samples for analysis. Of course, when the column is removed its air line must be closed, and it may be necessary to regulate the airflow through the remaining columns. In the analysis of the column that is removed as a sample, it may be interesting to check how homogeneous the growth is with height, dividing the sample into various bands that are analyzed separately, instead of mixing the whole bed contents together.

The outlet gas from each column can be analyzed separately, allowing the growth process to be monitored on the basis of O2 consumption or CO2 evolution. This will be most simple if an automatic switching system is available to cycle each of the outlet air flows through the analyzer in turn.

15.1.3. Comparison of the Two Systems

Most laboratories will already have an incubator, so the flask/incubator system is typically the cheaper to apply. However, the control of the air phase in the bed is obviously better in the column/waterbath system.

If sufficient incubator space is available, it is easier to increase the number of flasks than the number of columns, which may require the construction or purchase of more waterbaths and, even if waterbaths are available, will require the construction of more columns. Due to these considerations, the flask system typically allows more replicates in the same fermentation than does the column system.

The on-line monitoring of growth through gas metabolism that the column system allows is advantageous, since gas metabolism, especially O2 consumption, is intimately linked with heat production, and data on the heat production rate will be needed within the energy balance in the bioreactor model.

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