Solid-state fermentation involves the growth of microorganisms on solid, normally organic, material in the absence or near absence of free water. The substrates used are often cereal grains, bran, legumes and lignocellulosic materials, such as straw, wood chippings, etc.
On the left a technician is sampling the liquid phase of a solid-state 4 L column fermenter. In this situation the solid phase is finely chopped bagasse (sugar cane waste) seeded with the fungus Fusarium solani. The liquid phase is being recycled from bottom to top to assist aeration and mixing. Temperature control is by means of a water jacket. Some additional aeration is accomplished by means of pumping air in through the bottom and its removal at the top of the column. The fungus is producing cellulases to break down the cellulose into xylose, glucose, galactose, mannose, arabinose, glucuronic acid, glacturonic acid, 4-O-methyl-D-glucuronic acid, rhamnose, and fucose. The microorganism for additional growth immediately uses some of the products formed. The majority of the products are the sugars xylose and glucose which are later fermented to ethanol using an appropriate yeast (in a separate stirred fermenter).
Solid-state fermentations lack the sophisticated control mechanisms that are usually associated with submerged fermentations. Their use is often hampered by lack of knowledge of the intrinsic kinetics of microbial growth, under these operating conditions. Control of the environment within the bioreactors is also difficult to achieve, particularly the simultaneous maintenance of optimal temperature, pH, and appropriate moisture content (water activity) (23, 111, 115, 141, 150, 151, 152, 228, 247).
Xylose or wood sugar is the second most abundant sugar in nature after glucose, and is widely distributed in plant materials, especially in wood (maple, cherry), in straw, in cottonseed hulls, peanut shells, bagasse, etc. It is not found in free state, but in the form of xylan, a polysaccaride built from D-xylose units and occurring in association with cellulose. Xylose has been used in tanning, dying, and as a diabetic food.
Upon H2SO4 acid hydrolysis, bagasse yields a mixture of sugars, including glucose, but with the pentose D-xylose as the major product. Saccharomyces cerevisiae (baker's yeast) and Schizosaccharomyces pombe can ferment the glucose portion of the hydrolysate directly, but the D-xylose portion must first be isomerized to its ketoisomer, D-xylulose, before ethanol fermentation is possible. The D-xylose can be isomerized to D-xylulose by bacterial glucose isomerase (which is in fact a xylose isomerase) as shown in the following reaction :
xylose ___>[xylose isomerase]___> xylulose ___>[xylulokinase]___> xylulose 5-phosphate ___>(glycolysis)___> ethanol + CO2
On the right is a view of a 40 Liter water jacketed glass column packed with commercially available immobilized glucose isomerase. The column is brown-black in color, 2 meters in height, with wire mesh around it as a safety precaution. The inverted white 80 Liter tank is a feed reservoir. The blue boxes are pH controllers.
Thirty-five liters of acid hydrolyzed bagasse, neutralized by lime (calcium oxide), was continually pumped up through the base of the column, flowing out the top, and into a 50 liter fermenter. Previous experiments had shown that optimal temperature for isomerization of xylose to xylulose was 50 oC. The water jacketed column was maintained at this temperature throughout the run. Experiments had also shown that optimal pH was between 6.5 and 7.5. This range of pH was controlled with addition of gaseous ammonia by the use of an external side-stream bubble column. It had been shown previously that if borate was incorporated into the system, a shift in the equilibrium constant to enhance xylulose production could occur. Thus, borate was also added to the hydrolysate.
The above treated hydrolysate, containing the equilibrium mixture of glucose, xylose, and xylulose, was continuously sent to the 50 liter fermenter inoculated with the yeast Schizosaccharomyces pombe. With a relatively short residence time, the fermenter broth (hydrolysate + yeast) was sent through a continuous centrifuge with yeast being sent back to the fermenter and hydrolysate back through the glucose isomerase column. This procedure was continued for several passes.
Initially all of the glucose was completely exhausted before significant quantities of xylulose or xylose were used. The yeast used this initial glucose for growth of additional cell mass as was shown by an increase of optical density of the fermentation broth and plate counts. Then ethanol was continually produced at a rate proportional to the available xylulose converted by the glucose isomerase column (6, 111, 115, 141, 150, 151, 152, 228, 247).
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