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Australasian Biotechnology, Vol. 11 No. 2, 2001, pp. 33-34 BIOPROCESSING PERSPECTIVES ON BIOPROCESSING Martin Playne Code Number: au01028 My view of bioprocessing is influenced by a career-long interest in gut fermentation. My Masters research and my PhD were on the biochemistry and microbiology of silage fermentation. Silage of course is the fermentation of plant materials usually by naturally-occurring bacteria, although inoculants of lactic acid bacteria, such as Lactobacillus plantarum are increasingly being used to make silage these days. Depending on the environmental conditions imposed on the plant material (e.g. anaerobic conditions, moisture content, sugar content), the mixed culture fermentation could be shifted to produce predominantly lactic acid, rather than acetic and butyric acids which tended to dominate if the silage was badly made. Silage is but one example of a number of products made by natural mixed culture processes, where the quality of the product is determined by the conditions imposed on the process, and the nature of the original substrate. Other examples include wine, cider and beer fermentations, cheese manufacture, and a variety of fermented milks. There are a large number of traditional food and beverage fermentations continuing to be used particularly in tropical countries, where acidification by lactic acid production is an important form of preservation of the food or beverage, in the absence of refrigeration. These processes, many very ancient, are all forms of bioprocessing. They appear relatively uncontrolled and irreproducible, but this is not the case, as they have been optimised over time, and the critical control points acknowledged by trial and error. I progressed from silage into rumen fermentation research. The cow and sheep are remarkably adept at conversion of ligno-cellulosic plant materials into volatile fatty acids (acetic, propionic and butyric), and then are able to efficiently use these acids as sources of energy for their body maintenance and for their production of muscle (meat), milk, and wool. Even the poorest grasses in the midst of the dry season of northern tropical Australia are able to be digested up to 40% by cattle. Such grasses may contain over 80% of heavily lignified cellulose and hemicelluloses. Around half of this is able to be converted to volatile fatty acids. The only pretreatment of this “straw” before it is digested, is chewing and regurgitation actions of the animal. A remarkable achievement. The gastro-intestine of the cow is a very complex fermentor, and recycling system (eg., an adult cow cycles somewhere over 100 litres per day of saliva). I progressed further in my career in bioprocessing by moving into research on the production of liquid fuels from cellulosic biomass. The concept was to develop an industrial process, which produced fuels for cars. This involved several steps which were:
This product development process had a number of limiting steps. Pretreatment could be expensive, and costs were involved in removal of alkali if used. Transport of cellulosic biomass to the plant could be costly, especially in the case of wheat straw. With sugar cane, the bagasse was already at a factory site. Without a pretreatment step, we found that we could not achieve an effective fermentation to volatile fatty acids - the raw material was too intractable. This made us appreciate the ability of a ruminant even more. It was also necessary to investigate how to manipulate the ratios of acetic, propionic and butyric acids in the final ferment to optimise the conversion. Finally, we also struggled with how to overcome product inhibition, and how to ferment under low pH conditions (pH4). We needed to get high concentrations of acids at a low pH to improve the efficiency of the membrane extraction process. It is difficult to achieve concentrations above 20g/L in the final ferment. Higher concentrations could be achieved by adaptation. Finally, we had to complete the fermentation within 3 days for the process to be economical. The membrane technology step also had problems. The major one was the emulsified interface (caused by proteins) between the fermentor and the membrane surface. The overall process costed out at about the same price as alcohol fermentations from carbohydrate wastes. Unfortunately, in 1980 and without subsidy, these processes could not compete with non-renewable petroleum fuel sources. Today, the story is gradually changing. From renewable fuels, I moved on into research in the production of various organic acids from carbohydrate wastes (propionic acid, citric acid) I will take the example of citric acid, because it was with this project that we used our accumulated expertise on product development. The concept was to make better use of the lactose in cheese whey, which was largely wasted both in Australia and globally. We believed we could add value by conversion of the lactose to citric acid. Traditional citric acid production is done by converting glucose or sucrose using Aspergillus species. Some Candida species of yeast can produce high concentrations (>60g/L) of citrate also, and have the advantage that they are much faster fermenters, and able to utilise galactose. Our task was to find Candida strains able to utilise lactose, galactose and glucose, and produce citrate in high yield. This time, we took the approach of developing a detailed techno-economic model of the process. This computerised model allowed us to assess the effect on costs of production of different scenarios (e.g. fermentation speed, yield effects, recovery of product at various points in the step process) Such a model also allows one to estimate where the most economic gain will be achieved in the process. Thus, enabling us to concentrate research effort in those particular parts of the process. Although the process development proceeded well, the price of citric acid continued to drop globally against expectations. This was partly due to suspected dumping by certain countries. In the end, we discontinued the project for those reasons. One conclusion could be that it is very difficult to compete in a commodity product, worth only a few dollars per kilogram, where there is already a mature technology. So there is still a whey waste disposal problem! A somewhat different product is microbial cells. Microbial cultures are, of course, sold to the bakery, brewing, wine, cheese and yoghurt industries. They are a market either satisfied in-house, or by specialist companies in Australia such as Rhodia, Chr Hansen Laboratories, DSM Food Specialties, and the Australian Starter Culture Research Centre. Here we see yet another emphasis in the process of growing cells - storage of product in a viable state without loss of viable numbers. Such storage is either as freeze-dried cultures (expensive and suffer 10-fold losses during freezing) or as frozen cultures (here we have the daily cost of refrigeration). Critical factors are the cheapness of the growth media, growing the cells rapidly to high concentrations in the fermentor, having the cells in a state where they are not only viable, but have good activity and “take off” rapidly when put into a vat of milk, for example. Major losses often occur when cells are removed from the fermentor, and when they are frozen. Such losses can often be 10-fold. This of course puts the cost of production up 10-fold also. So we see here yet another example of different bioprocessing problems. Leading on from this, is my final example. Probiotic bacteria are increasingly being used in food products. Examples are probiotic yoghurts, cheese, infant milk powders, fermented milk drinks and energy bars. The list expands every year. Yet, the cost of providing adequate numbers of the probiotic bacteria for human consumption is a problem for industry, and eventually the consumer. Probiotic products have to be designed so that the probiotic strain used is able to reach the bowel in sufficient numbers (e.g. 107 cells per gram of product). To do this, the cells have to survive the manufacturing steps described above, then survive shelf storage conditions for the period of the shelf life, then survive transit through the acid conditions of the stomach and the hydrolase and protease enzymes in the stomach and small intestine, and the surfactant effects of bile salts. On top of this, the bacteria also have to be able to compete in the bowel with a large number of other microorganisms already established in that gut region. Finally, they have to have been selected to impart some defined health effects on the host. So, here we have quite a different bioprocessing system - one where the product has to effect a “living fermentor” in a beneficial way. Designing fermentor systems which simulate the human gut ecosystem and all the physiological steps therein is difficult. Once again it is just another example where the skills of those versed in bioprocessing can be applied. I would like to express my great acknowledgment to all my many colleagues equally involved in all the above bioprocesses. I would particularly thank my former colleague, the late Dr Russell Smith, for the part he played in the renewable fuels project and his great expertise in membrane technology. Thanks are also due to several Divisions of CSIRO where this work took place.
Copyright 2001 - AusBiotech |
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