FERMENT
A
TION TECHNOLOGIES
aeration has been emphasized on by many scientists. However, it should always be remembered that inoculum development and sterilization difficulties may be the reason for a decrease in yie1d when a process is scaled up and that achieving the correct aeration & agitation system is not the only problem to be addressed.
•
Fine, then how do scale up the aeration and agitation operations?As we have seen, the laboratory scale experimentations usually start with erlenmeyer flasks which are the
conventional vessels for fermentation. However, these flasks, even when shaken on a reciprocating or rotary mechanical shaker, provide a poor estimate of the fermentation potential for a microorganism and its medium because of the relatively poor aeration characteristics associated with these vessels. Similar flasks with small glass baffles mounted in the bottom provide better aeration but do not provide the favorable aeration conditions obtainable in it
fermentation tank. Small laboratory fermentation tanks of 1 to 10 or 12 liter sizes are more ideal for these studies, since their aeration and agitation conditions can be varied, and since the overall fermentation conditions of these tanks more closely resemble those of the larger production tanks. Thus, the most important criteria for a particular
fermentation must be established and the scale-up based on reproducing those characteristics
•
Do the scales up operations depend on different types of fermentors?Yes. Bubble column and air-lift vessels ten to be scaled-up on the basis of geometric similarity and constant gas velocity. The major difference will be the height of the vessel resulting in increased pressure at the base of larger vessel. This would result in higher oxygen and carbon dioxide solubility. The other problem in the scale-up of air-lift systems is that the organism is exposed to extremes of oxygen levels in the riser and downcomer and the effects of these conditions should be investigated on the laboratory scale.
•
Is scale up needed where solid substrates are used? Of course. Though at present, most industrial fermentation use the submerged fermentation process (SMF) where the microorganisms are grown in liquid media that are stirred or aerated within a large vessel, another fermentation process, solid state fermentation (SSF), has been shown to be a promising alternative for some biotransformation processes. Solid-state fermentation is the growth of microorganisms on solid materials without the presence of free water. Interest in SSF has intensified for the past decade because it may be used to produce a wide variety of substances, the most promising of which enzymes, organic acids, biopesticides, aroma compounds, and other bioactive compounds.•
That was scale up. What do we mean by scale down? Scale-down is the situation where laboratory- or pilot-scale experiments are conducted under conditions which mimic the industrial-scale conditions. This approach is important inboth the development of a new product and the improvement of an existing full-scale fermentation. The procedure has been reviewed by Jem (1989).
Frequently, conditions achievable on a laboratory scale are impractical on an industrial scale which means that if inappropriate conditions have been used in the laboratory unrealistic yield objectives may be set for the scaled-up process. The aspects to consider in the design of laboratory- or pilot-plant experiments in the context of scale-down may be summarized as follows:
1. Medium design: Media relevant to the industrial situation should be used in development experiments.
2. Medium sterilization: lf the medium is to be batch sterilized on the large scale its exposure time at a high temperature will be much greater than that experienced in the laboratory or pilot plant. Thus, the sterilization times on the smaller scales should be increased to mimic the industrial situation. Alliteratively, medium sterilized in the production fermentor may be used in the laboratory and pilot plant. This highlights the advantage of continuous sterilization where little loss of medium quality occurs. Furthermore, the same continuous sterilizer may be used for both full-scale and pilot scale vessels.
3. lnoculation procedures: Due to a range of circumstances, it may not always be possible to inoculate every production fermentation with inoculum in optimum condition. The scale down approach can be used to predict the consequences of such events by mimicking these situations in the
laboratory, for example by storing inoculum or using inocula of different ages.
4. Number of generations:Industrial scale fermentation requires a greater number of generations than does a laboratory one; this may place more severe stability criteria on the process strain than may have been appreciated on the small scale. The industrial situation may be modeled in the laboratory by using serial sub-culture to ensure that the strain is sufficiently stable. This approach is particularly pertinent in the development of recombinant fermentations.
5. Mixing: As indicated in the previous section it is almost inevitable that the degree of mixing will decrease with an increase in scale. Thus. it is possible to model inadequate mixing in the laboratory by subjecting the organism to pulse medium feeds or fluctuating process conditions such as oxygen concentration , pH and temperature. Such scaled- down experiments then allow predictions to be made about the suitability of new strains for industrial exploitation. 6. Oxygen transfer rate Far higher oxygen transfer rates can
be achieved in laboratory Fermentors than in industrial-scale ones. Thus, unrealistic demands may be made of a fermentation plant if the development work has been done at very high oxygen-transfer rates. Therefore, the laboratory and pilot Fermentors should reflect the oxygen transfer rates achievable in the full-scale Fermentors.
FERMENT
A
TION TECHNOLOGIES
•
How do we carry out the scale up operations in solid state fermentations?We all know what solid state fermentations are. The need for solid state fermentations was felt primarily due to the need to ferment agricultural raw materials. Modern Society is critically dependent on a wide variety of products derived from the processing of raw materials from nature. During the 20th century, with the great advances in chemistry and chemical engineering, these products were commercially produced using chemical manufacturing methods. However, with the recent progress in biotechnology and biochemical engineering, the production of chemicals and bioactive compounds (e.g. antibiotics) using biological agents such as microorganisms, cells, or enzymes is being considered as a superior alternative to the traditional chemical methods. Biotransformation processes that involve enzymatic or microbial biocatalysts, when compared to their chemical counterparts, offer the advantages of high selectivity, mild operating conditions, and the use of a wide variety of inexpensive materials such as agricultural wastes as substrate. At present, most industrial fermentation use the submerged fermentation process (SMF) where the microorganisms are grown in liquid media that are stirred or aerated within a large vessel. However, another fermentation process, solid state fermentation (SSF), has been shown to be a promising alternative for some biotransformation processes.
Solid-state fermentation is the growth of microorganisms on solid materials without the presence of free water. Interest in SSF has intensified for the past decade because it may be used to produce a wide variety of substances, the most promising of which enzymes, organic acids, biopesticides, aroma compounds, and other bioactive compounds.
SSF offers several advantages over conventional stirred and aerated liquid fermentation. These are:
The absence of large amounts of water. The requirement of no free water in SSF greatly reduces the volume of
fermentation media per mass of substrate as compared to liquid fermentation. This smaller volume gives the following benefits:
a. Since the yield in SSF per mass of substrate is comparable to those typically achieved in liquid fermentations, the fact that there is less volume of media per mass of substrate means that a much smaller fermentor is needed to synthesize a given amount of product.
b. After fermentation there is no large quantities of liquid waste to dispose off.
c. Since we get the same amount of product per mass of substrate, the absence of large amounts of water means that after fermentation the product concentration is greater. The higher final concentration makes it easier to purify the product, thus lowering downstream costs.
Minimal risk of contamination. The absence of free water in SSF makes it less prone to contamination by microorganisms other than molds. Most bacteria require a liquid environment and do not thrive in SSF. This translates to less stringent
conditions for SSF and may altogether require no sterilization or aseptic procedures during operation.
The use of simple, naturally occurring media. These are usually agricultural materials, in contrast to the synthetic media for liquid fermentation.
These advantages make SSF less capital intensive than SMF. The absence of large amounts of wastewater would preclude the use of expensive waste treatment facilities while the minimal risk of contamination means that costly facilities for providing sterile conditions are not needed. Solid-state fermentation is therefore an attractive technology for developing countries like the Philippines where capital is scarce. Also, the use of agricultural wastes as substrate will increase the income of local farmers and decrease disposal costs.
Several studies have shown that the primary limiting factor for large-scale SSF is the poor removal of the large metabolic heat generated by the microorganism. This leads to high temperature gradients within the bioreactor that limit the growth of
microorganisms.
One of the methods used to increase heat transfer in SSF is forced aeration in a packed bed bioreactor but this has produced only limited success. It is hypothesized that this is because the particles of the solid substrates used are very small (<2 mm) and this greatly decreases the spaces between them. This forces the air to pass through the column only along curtain paths (channeling). The main heat transfer mechanism within the fungi-substrate mass will still be conduction and since organic matter are poor conductors, the heat transfer rate will be low, and the temperatures may rise to as high as 50°C. A way of increasing the porosity of the packed bed is by using larger particles but this is not possible for wheat bran and rice bran, materials that have been proven to be superior SSF substrates, since they are by nature in powder form. However, forming pellets from them may produce aggregates of wheat bran and rice bran of any desired size. The pelletized media will now be large enough so that the forced air can pass between them and therefore increase convection heat transfer within the bed. Scale- up of SSF may become possible because it is expected that the performance of a fermentor using the pelletized media will be the same whether a small or a large fermentor is used since the air spaces between the pellets will remain the same. The use of pelletized media for SSF in an aerated packed bed bioreactor has not been reported in the literature.
The study was conducted to compare the performance of an aerated packed bed bioreactor for SSF using pelletized and unpelletized media. The system used was the production of a- amylase by Aspergillus oryzae in SSF based on the method developed by Dr. Teresita M. Espino (1999) of Biotech for static trays. This process was chosen because SSF is particularly suited for enzyme production by filamentous fungi since they naturally secrete these during growth. The media used was a mixture of 90 mass % rice bran and 10%
cassava starch. The pelletized medium had an effective diameter of 5 mm compared to less than 0.833 mm for the unpelletized one. The column bioreactor used had a diameter of 100 mm and a total bed volume of 1.3 dm³. The schematic diagram of the experimental set-up is shown in Fig. 1.
FERMENT
A
TION TECHNOLOGIES
All fermentation runs showed that the pelletized medium gave amylase activities that were very much higher than the
unpelletized. At an aeration rate of 1.20 vessel volume per minute (vvm) and 84 hours fermentation time, the pelletized medium gave a yield of 589 dextrinixing unit (DUN)/g dry medium compared to 179 (DUN)/g dry medium for the unpelletized medium at the same conditions. Compared to that reported for SSF in static trays, the pelletized medium gave 5.25 greater value. At 3.40 vvm aeration rate, the yield (pelletized medium) was 611 DUN/g dry medium, which was 1.55 times that for the unpelletized medium and 5.44 times that for trays (Fig. 2). The higher amylase production was attributed to more fungal growth in the pelletized medium because of better heat transfer.
The effects of aeration rate and length of fermentation were further investigated for the pelletized medium. The results show that the yield of a-amylase did not vary significantly (a = 5%) for aeration rates of 1.20, 2.06, 2.81 and 3.40 vvm. This lack of effect may be explained by the contributions of the conduction and convection heat transfer in the bioreactor. It was possible that the convection heat transfer in the pelletized media had been greatly increased so that the gas phase resistance was negligible compared to the conduction resistance. As such, variations in the aeration rate would no longer affect the total rate of heat transfer. This implies that SSF using pelletized media does not require high aeration rates. A fermentation time of 120 h produced a-amylase yields which were 60% greater than that for 84 h. This was attributed to secondary growth from the spores produced by the initial inoculum.
The study showed that the use of pelletized media increases a- amylase production by as much as nine times (120 hrs.) than that obtained using static tray SSF. The higher productivity will significantly decrease the cost of products from SSF. The higher enzyme concentration in the crude extract should make purification easier and less wastewater will be generated. The media requirements for a given amount of enzyme production will be decreased considerably. This will give substantial savings in raw material costs, sterilization costs and capital outlay (lower bioreactor volume) The cost of palletizing is small and since only low airflows rates are required for pelletized media, the air pumping costs will be minimal. Since the higher productivity is attributed to the greater interparticle space in pelletized medium, it is expected that the same results will be obtained for large- scale SSF since the size of the air spaces will remain the same in a small or large bioreactor. The use of pelletized medium will therefore allow the economical scale-up of SSF.
FERMENT
A
TION TECHNOLOGIES
Learning Objectives
In this lecture, you will learn•
IP methodologies•
IP in bacteria, yeasts and moulds•
IP & morphology of organism Good morning students;We have seen what is needed to formulate a correct
fermentation medium. We have also seen how the appropriate microbial culture offering maximum productivity can be isolated through screening. Let us now see how to develop a seed culture required for the fermentation. This process is called