enzymes for the feed industry

Virtually all chemical reactions in biological systems are catalyzed by macromolecules called enzymes. Chemical reactions in vivo rarely proceed at perceptible rates in the absence of enzymes while reaction rates increase as much as a million times when enzymes are present. These proteins are among the most remarkable biomolecules known.

The name ‘enzyme’, meaning ‘in yeast’, was not used until 1877; however much earlier it was suspected that biological catalysts were involved in the fermentation of sugar to form alcohol. These catalysts were termed ferments (Lehninger, 1975). Eduard Buchner extracted the enzymes catalyzing alcoholic fermentation in 1897. This demonstrated that enzymes could function independently of cell structure. In 1860, Louis Pasteur postulated that enzymes are linked with the structure of the yeast cell. The ability of enzymes to function outside a cell has greatly increased their use in a large variety of commercial products and reactions.

A wide range of industries use commercial enzymes. The world annual sales of industrial enzymes was recently valued at $1 billion (Bron et al., 1999). Three quarters of the market is for enzymes involved in the hydrolysis of natural polymers, of which about two-thirds are proteolytic enzymes used in the detergent, dairy and leather industries; and one third are carbohydrases used in the animal feed, baking, brewing, distilling, starch and textile industries. Detergent manufacturers use 45% of all industrial enzymes produced in spot remover and detergent products containing proteases and lipases. This industry is expected to have a 10% annual growth rate for the next five years. Food processing enzymes including "-amylases, glucose isomerase and pectinases account for about 45% of enzyme usage. The starch processing industry uses half of the enzymes in the food industry, approximately 25% are used by the dairy industry and 10% by the brewers, fruit juice and wine producers. The textile and paper industry (6%) uses primarily amylases and hemicellulases and the leather industry (2%) uses proteases. Enzyme supplements for animal feeds account for about 1% (Amado, 1993). Table 1 lists some industrially important enzymes and their applications.



Table 1. Commercial applications of enzymes.*


To enlarge the image, click here
^{*Modified from Stanbury et al., 1995.}



The acceptance of enzymes by the animal feed industry has become widespread in the last decade. As the understanding of enzymes and their properties has grown, so have both their use and their effectiveness as feed supplements. The purpose of using enzymes in monogastric animals is to improve availability of nutrients in feedstuffs. The result is improved feed utilization and reduced impact of anti-nutritional components.


Methods for production of commercial enzymes

Industrial enzymes are produced by plants, animals, and microbes. By far the most exploited for the use of industrial enzymes has been the microbial population. Short generation times and high yields, together with the fact that microorganisms produce extracellular enzymes, which are easy to harvest, make microbes the enzyme source of choice. Production of enzymes by microorganisms has also expanded because of the vast amounts of genetic information now available. Several industrially important microbial genomes have been sequenced; and the understanding of gene expression systems in microorganisms is much more advanced when compared to other gene expression systems. This knowledge has made it possible to select a variety of microorganisms suitable for enzyme production with traditional submerged liquid fermentation (Bailey and Ollis, 1986). An alternative fermentation method for enzyme production is solid substrate fermentation (Mitchell and Lonsane, 1992).


SUBMERGED LIQUID FERMENTATION

Submerged liquid fermentations are traditionally used in the United States for the production of microbially derived enzymes. Submerged fermentation involves submersion of the microorganism in an aqueous solution containing all the nutrients needed for growth. A research team led by Chaim Weizmann in Great Britian developed a process for production of acetone by submerged liquid fermentation using Clostridium acetobutylicum, which eventually led to the first large-scale aseptic fermentation vessel (Stanbury et al., 1995). The first large-scale aerobic fermenters were used in central Europe in the 1930s for production of compressed yeast (de Becze and Liebmann, 1944). In 1943, the British government decided that solid substrate fermentation was inadequate for the production of penicillin. This decision forced the development of liquid fermenters that are aseptic and contain adequate aeration and agitation. Construction of the first large-scale plant to produce penicillin by liquid fermentation began in 1943 (Callahan, 1944).


Organisms used in submerged fermentations

Fermentation using bacillus species accounts for about half of the world’s production of industrial enzymes. The main classes of bacillus enzymes and the strains used to produce them are listed in Table 2. Two enzymes dominate the industrial market: alkaline protease and "-amylase. Alkaline protease used in detergents represents the single largest enzyme market. Although bacillus species are the primary enzyme-producing organisms, other microbes are also used. Through genetic modifications the bacterium Escherichia coli is able to produce insulin and human growth hormones. Penicillum chrysogenum is used to produce penicillin. Other microorganisms used on an industrial scale include Saccharomyces cerevisiae for ethanol production and the fungi Aspergillus and Trichoderma for carbohydrase production.



Table 2. Industrial enzymes produced by Bacillus species.*


^{Bron et al., 1999.}



Selection of microorganisms for the fermentation industries in the past involved a hit-and-miss screening approach. With the development of genetic engineering techniques, organisms can be engineered to produce high yields of a great variety of products. Most of the information has been developed in bacterial systems and has resulted in very efficient enzyme production.

Genetic manipulation of organisms can increase yield 100 fold or more over wild type strains (Stanbury et al., 1995). The genetic manipulation of genomes is common for organisms used in submerged liquid fermentations.


Fermenter design

The main function of a fermenter is to provide a controlled environment for growth of microorganisms in order to obtain a desired product. Two important criteria for a submerged liquid fermenter include the ability to operate aseptically for a number of days and provide adequate aeration and agitation to meet the metabolic requirements of the microorganism. Many different types of fermenters have been described in the literature, but very few proved satisfactory for industrial aerobic fermentations. The most common designs are based on a stirred upright cylinder with sparger aeration (Stanbury et al., 1995). Fermenter sizes can range from flasks used in the laboratory to production fermenters of 8,000 liters or more (Figure 1).


Fermenter operation

Many biochemical processes involve batch growth of cell populations. After seeding a liquid medium with an inoculum of living cells, only gas is added or removed from the culture as growth proceeds. Typically in such a batch reactor, the concentrations of nutrients, cells and products vary with time as growth proceeds. In addition, it is often desirable to add liquid streams to a batch bioreactor as the reaction process occurs. This can be done to add precursors for desired products, to add regulating compounds such as inducers at a point in the batch operation, to maintain low nutrient levels to minimize catabolite repression or to extend the stationary phase by nutrient addition (Baily and Ollis, 1986). When the fermenter is used in this manner it is known as a ‘fed-batch’ fermentation.





Figure 1. Deep tank liquid fermenter (250 liter).



The success of a fermentation depends upon the existence of defined environmental conditions for biomass and product formation. The temperature, pH, degree of agitation, oxygen concentration in the medium and other factors may need to be kept constant during the process. Careful monitoring of the fermentation is performed to regulate these parameters. Table 3 lists the variety of process sensors included in a submerged liquid fermentation.


SOLID SUBSTRATE CULTIVATION

In addition to submerged liquid fermentation, an ancient fermentation technology known as solid substrate fermentation is also used to produce enzymes. Solid substrate fermentations are generally characterized by growth of microorganisms on water-insoluble substrates in the presence of varying amounts of free water (Mitchell and Lonsane, 1992). This process is also referred to as solid state fermentation (SSF). Table 4 shows differences between the SSF and submerged liquid fermentation.



Table 3.Process sensors and their possible control functions.*


^{*Stanbury et al., 1995.}



Table 4.Differences between solid-substrate fermentation and submerged liquid cultures.*


^{*Mitchell and Lonsane, 1992.}



In 1896, Takamine produced a digestive enzyme, Takadiastatse, by SSF employing Aspergillus niger on wheat bran (Takamine, 1914). This led to the application of SSF in other food and beverage industries. The most profitable applications of SSF are in Asian and African countries where SSF processes have been perfected over long periods. In western countries, traditional applications of SSF are scarce. Solid substrate fermentation has been largely neglected since the 1940s; and negligible research and development efforts have been made. The selection of submerged liquid instead of SSF in western countries was not based on economic comparisons of submerged liquid and SSF techniques; the choice was linked to slow growth of the microbial cultivation industries across the world (Ralph, 1976; Hesseltine, 1976).

The origin of SSF can be traced back to bread-making in ancient Egypt. Solid substrate fermentations also include a number of well known microbial processes such as soil growth, composting, silage production, wood rotting and mushroom cultivation. In addition, many familiar western foods such as mold-ripened cheese, bread, sausage and many foods of Asian origin including miso, tempeh and soy sauce are produced using SSF. Beverages derived from SSF processes include ontjom in Indonesia, shao-hsing wine and kaoliang (sorghum) liquor in China and sake in Japan (Mudgett, 1986). Table 5 gives examples of foods that involve an SSF process at some point in production.



Table 5.Examples of foods produced by solid substrate fermentation.


^{Mitchell and Lonsane, 1992.}



While commercial use of SSF is not widespread in North America, industrial enzyme production by SSF has occurred for a number of years (Takamine, 1914; Underkofler et al, 1958). After World War II, Underkofler et al. (1947) and Terui et al. (1957) used heaped bed cultures with forced aeration to produce enzymes and citric acid. Tempeh production has been established on a small scale in the US (Hesseltine, 1987) because it has been accepted as a meat substitute by vegetarians. Mushrooms are cultivated in western countries; and soy sauce production has become highly industrialized and is widely used across the world. Kikkoman Foods has built a state-of-the-art facility completed in 1998 for soy sauce production in Folsom, California.


General features of solid substrate fermentation

The single most important feature of SSF is the low moisture content of the medium, which makes SSF very different from submerged liquid cultures. Water is essential for microbial growth; and the limited water in SSF has several consequences. It is adsorbed and to some extent held tightly; and there may even be some free water in the interior and on the surface. Water activity can be below 0.99 in SSF, where free water is virtually absent. These conditions favor filamentous fungi, many of which grow well between water activities of 0.93 and 0.98 (Corry, 1973). Bacteria and yeast grow above a water activity of 0.99.

Heat transfer is restricted in SSF, which can lead to overheating problems in large scale fermentations (Laukevics et al., 1984). Evaporative cooling is the most effective cooling method, although this will reduce water availability (Trevelyan, 1974). Proper temperature conditions during the fermentation are a balance between the need for heat removal and the necessity of keeping the substrate sufficiently moist to support growth. The insoluble substrates used in SSF are composite and heterogeneous products from agriculture or by-products of agro-industries. For many processes, substrates are chosen because they are readily available and therefore inexpensive. Most substrates have a common macromolecular structure. The macromolecular portion often provides a structural matrix for the substrate as well as serve as the carbon and energy source (e.g. cellulose) for the microorganism. If the macromolecule serves as a structural matrix only, the carbon and energy source is provided by a non-structural macromolecule such as starch or a smaller, soluble compound (e.g. soluble sugar).


Differences in enzymes produced by SSF and submerged liquid fermentation

Evidence has been accumulating to support the view that SSF processes are qualitatively different than submerged liquid fermentations. The data suggest that microbial physiology and regulation within the cell are influenced by the fermentation environment (Viniegra-Gonzalez, 1997). Ayers et al. (1952) reported that pectinases produced by SSF had noticeable biochemical differences from those produced by submerged fermentation. A glucosidase produced by Aspergillus phoenicis in SSF was more thermotolerant than when produced in submerged liquid fermentation (Deschamps and Huet, 1984). Alazard and Raimbault (1981) showed that amylases produced by A. niger using SSF were more resistant to heat denaturation than those produced in submerged liquid fermentation by the same strain. Other differences have also been reported (Romero et al., 1993; Villegas et al., 1993) and reinforced by the observation that the induction and repression patterns of pectinase production by A. niger are different for each fermentation technique (Solis-Pereira et al., 1993).

Exoenzyme production in SSF systems results in increased amounts of some enzymatic activities not produced by cultures in liquid fermentation. A phytase produced by SSF (Allzyme Phytase, Alltech Inc.) also contained a mixture of activities not found in enzyme preparations from submerged culture systems (Table 6). The complex nature of feedstuffs makes these side activities beneficial to the animal industry (Classen, 1996). In vitro comparisons have shown increased rates of reducing sugar and amino nitrogen, and an associated increase in phosphate release by an SSF phytase product (Allzyme Phytase) (Figures 2-4, Filer et al., 1999).



Table 6.Comparison of enzyme activities of two commercially available phytases.





Microorganisms for solid state fermentation

Bacteria, yeast and fungi can all grow on solid substrates and have applications in SSF processes. However, filamentous fungi are the best adapted species for SSF and dominate in the research and practical applications around the world. Bacterial SSF fermentations are rarely used for large scale enzyme production, but are very important in nature and in the fermented food industry. In composting, moist solid organic wastes are decomposed by a succession of naturally-occurring microorganisms. Ensiling processes are dominated by lactobacilli producing lactic acid. Natto is a fermented food involving Bacillus subtilis. The fermentative yeast Endomycopsis burtonii is involved in the production of a traditional Indonesian fermented food, tape (Steinkraus, 1983).

Filamentous fungi are the most important group of microorganisms for enzyme production in SSF. The hyphal mode of growth gives a major advantage to filamentous fungi over unicellular microorganisms in the colonization of solid substrates and the utilization of available nutrients. The filamentous fungi have the power to penetrate solid substrates. Hydrolytic enzymes are excreted at the hyphal tip, without large dilution. This makes the action of hydrolytic enzymes very efficient and allows penetration into most solid substrates. This is critical for the growth of the fungi. Fungi cannot transport macromolecular substrates across the cell wall, so the macromolecule must be hydrolyzed externally into soluble units that can be transported into the cell (Knapp and Howell, 1980).





Figure 2. Effect of phytase enzyme source on reducing sugar release from four substrates at recommended use rates (abMeans differ, P<0.05).





Figure 3. Effect of phytase enzyme source on amino nitrogen release from four substrates at recommended enzyme use rates (abMeans differ, P<0.05).





Figure 4.Effect of phytase enzyme source on phosphate release from four substrates at recommended enzyme use rates (abMeans differ, P<0.05).



Many submerged liquid fermentations are performed using pure cultures.The substrate is sterilized and then inoculated with a single culture. In the case of SSF a range of culture types are commonly used:

Most applications involve one of these inoculation schemes using fungi. Among the filamentous fungi, three classes have gained importance: phycomycetes such as the genera mucor and rhizopus, the ascomycetes with the genera aspergillus and penicillium, and basidiomycetes (Moo-Young et al., 1983).


Advantages and disadvantages of using solid substrate fermentations

Solid state fermentation systems have a number of advantages (Cannel and Moo-Young, 1980; Mudgett, 1986):

a) The medium is often simple, consisting of unrefined agricultural product, which may contain all the nutrients for microbial growth. Examples of substrates are cereal grains, wheat bran, and wheat straw.

b) Substrates require less pretreatment compared to liquid fermentation. Pretreatment for SSF must increase the accessibility of nutrients, while pretreatment for liquid fermentation must achieve extraction of the nutrients into the bulk liquid phase.

c) The restricted availability of water helps to select against undesirable contaminants, especially bacteria and yeast.

d) Forced aeration is often easier in solid state cultures than in liquid cultures because the interparticle spaces allow transfer of fresh air to thin films of water at the substrate surfaces.

e) Downstream processing and waste disposal is simplified or minimized. If drying is required, less water is present to be removed.

Solid state fermentation as an enzyme production technique is not without difficulties that must be overcome. A number of disadvantages must be addressed to make a successful product (Cannel and Moo-Young, 1980; Mudgett, 1986):

a) Restricted to microorganisms that grow at reduced moisture levels. The majority of commercially profitable processes involve fungi, however.

b) Removal of metabolic heat can be a problem in large scale fermentations. Depending on the organism, heat can drastically influence end product production. This problem can be lessened by using organisms that are heat tolerant.

c) The solid nature of the substrate presents problems in monitoring process parameters. Changes in pH are not easily identified and controlled in SSF; and the control of moisture content and substrate concentrations is extremely difficult. Heat production, oxygen consumption and carbon dioxide are parameters that can be measured.

d) Many important basic scientific and engineering aspects are poorly understood. Little is known about the mode of growth of fungi within substrate masses composed of irregularly shaped solid particles.

e) Cultivation times are often longer.

Bioreactor design

Reactor design is important in developing an efficient SSF process. The design of solid state reactors has to date been mostly empirical. Three basic types of reactors can be distinguished based on the mixing regime and the aeration mode. These include tray bioreactors, packed bed reactors, and agitated bioreactors.

The simplest SSF reactor is the tray. In a tray bioreactor a relatively thin layer of substrate is spread over a large horizontal area. There is no forced aeration, although the base of the tray may be perforated and air forced around the tray. Mixing, if any, is by simple automatic devices or manual. Internal temperature may vary with ambient temperature; or the tray may be placed in a temperature-controlled room. Tray bioreactors have been used successfully at laboratory, pilot, semi-commercial and commercial scale (Ahmed et al, 1987; Hesseltine, 1987). Although the design of the reactor is simple, extensive mechanization and automation have been reported in Japan (Lonsane et al., 1985).

Packed bed bioreactors are characterized by having a static substrate supported on a perforated base plate through which forced aeration is applied. Many variations of this basic design are possible (Lonsane et al., 1985). A tall, thin cylindrical column is the typical design. Most commonly the forced air is applied to the bottom. The humidity of the incoming air is kept high to minimize water loss from the substrate. The temperature of the incoming air can be changed to aid in temperature regulation of the substrate (Narahara et al., 1984). The advantage of packed bed reactors is that they remain simple while allowing better process control than trays.

Two general types of agitated fermenters have been designed. The first is a rotating drum reactor consisting of a horizontal or inclined cylinder that rotates around a central axis and causes a tumbling motion of the substrate. Aeration is supplied in the headspace. Mixing is gentle, although problems can arise if microorganisms are sensitive to the agitation. Temperature control is difficult because the reactor is difficult to water jacket (Lonsane et al., 1985). The second type of agitated fermenter, a stirred reactor, has the reactor placed either on a horizontal or a vertical axis. Horizontal reactors are similar to rotating drums except the mixing is provided by an internal scraper or paddles, rather than rotation of the reactor. Vertical stirred reactors are subjected to forced aeration and are agitated continuously or intermittently.


Process methodology

The steps involved in solid state fermentation process consist of (Lonsane et al, 1985):

1. The preparation of a solid substrate, often with pretreatment to decrease the particle size or increase the availability of nutrients in the substrate.
   
   
2. A cooking step which sterilizes or at least pasteurizes the substrate and causes the absorption of water into the substrate particles.
   
   
3. Growth of a suitable inoculum.
   
   
4. Inoculation of the moist solids.
   
   
5. Incubation in appropriate culture vessels.
   
   
6. Maintenance of optimal conditions to the extent possible.
   
   
7. Harvest of the solids.
   
   
8. Drying or extraction of the product from the solids
   
   
9. Downstream processing.

The Alltech solid state fermentation program

The many advantages of enzyme production by SSF have convinced Alltech that it is a valuable technology for the production of enzymes. As a result of this commitment an entire SSF program has been developed. The program includes small-scale lab fermentations up to production size facilities. The development from idea to commercial production employed the following steps:

1. Culture isolation, screening and selection.
   
   
2. Standardization of the process at small scale.
   
   
3. Scale-up studies.
   
   
4. Design and construction of the plant.
   
   
5. Operation of the plant.

The development of a SSF fermentation program at Alltech is intended to produce a more useful enzyme employing a procedure with economics that make it practical for the animal feed and fuel ethanol industries. With the completion of a production facility in Serdan, Puebla, Mexico the idea became a reality.


CULTURE ISOLATION, SCREENING AND SELECTION: NON-GMO ORGANISMS

The SSF program starts with the cultures. Two isolates have gone through a rigorous screening and selection process and are currently used extensively in the program. An Aspergillus niger has been naturally selected for overproduction of phytase. Of all the organisms surveyed, A. niger produces the most active extracellular phytase reported (Wodzinski and Ullah, 1996).

Through numerous rounds of screening and selection, overproduction by about 400 fold has been achieved. A point that is becoming more and more critical for many consumers is that the organism is a non-genetically modified organism (non-GMO) able to produce phytase at a significant level for commercial production. Use of non-GMO organisms in SSF processes is common. The SSF growth environment is conducive to overproduction of a number of different enzymes; and genetic engineering is not required for production of large amounts. In contrast, submerged liquid systems generally use GMOs designed for overproduction of a particular enzyme.

The second organism currently being studied extensively is Rhizopus oryzae, which produces glucoamylase. Glucoamylase sequentially cleaves glucose molecules from the nonreducing end of a starch molecule and is used extensively in the ethanol industry. The organism is not genetically modified and has been naturally selected for overproduction of glucoamylase. Rhizopus sp. have been reported to produce amylases (Raimbault, 1998). This organism is currently being used in lab scale and pilot scale tray fermentations and deep bed lab scale fermentations. Completion of these studies will allow the organism to be used at a commercial level.


STANDARDIZATION OF THE PROCESS AT SMALL SCALE

The selection of strains involves SSF on a small scale. Small scale fermentations occur in 500 ml flasks. The substrate used for the majority of fermentations is wheat bran. In these flasks the typical moisture, temperature and extraction conditions as well as the length of fermentation and inoculation rate have been determined and maximized. The information from these systems is important, although certain limitations do exist. In addition to thin layer tray systems, deep bed systems of 30–50 cm will be used in production. In order to determine if organisms are suitable for growth in deep layer fermentations, lab scale deep layer fermenters have been designed. Conditioned air is blown in through the bottom with an exit port on the top. The substrate sits on top of a perforated screen. The vessel has ports for thermocouples to monitor temperature. The airflow, relative humidity of the air, and oxygen content within the chamber can all be measured. These parameters are monitored and the data collected and sent to a computer. The entire fermenter is placed in a temperature and humidity controlled room. With this system we are able to generate data that reflect the heat produced during the fermentation and determine the amount of air required to maintain temperature in the target range. The substrate cannot be agitated during the fermentation and water is not easily added. These systems can produce data on the success of the fermentation in deep layers and the amount of heat produced during the fermentation.


SCALE-UP STUDIES

The scale-up step is a crucial linkage in the process since it determines whether the process will operate at a commercial scale. The scale-up should theoretically result in the same overall performance that can be achieved in the laboratory. This is rarely the case since a number of additional parameters influence the fermentation. Heat removal must be addressed; and there are no simple solutions since thermodynamic and kinetic properties become more complex.

Once the fermentation conditions have been determined in the flasks, the organism can be grown in lab scale tray systems. These systems are designed to mimic the conditions in a production tray system. The initial system designed was a plexiglass chamber able to hold 12 trays that hold up to 200 g of substrate. The chamber is incubated in a temperature-controlled room. Water saturated air is added from the top. In order to gain more information from tray fermentations, a second-generation tray fermenter was designed and built by the Departmento de Biotechnologia, Universidad Autonoma Metroplitana Iztapalapa. Figure 5 depicts a tray fermentation system. Four trays fit inside the reactor and conditioned air is forced in at the lower left portion of the reactor. Deflectors direct airflow over the tray or away from it. The reactor is equipped with thermocouples, flow meter, oxygen sensor and relative humidity sensor. Data acquisition equipment allows better understanding of heat and carbon dioxide production. This information is then used in designing production systems.





Figure 5.Pilot scale tray fermentation system.



Alltech is interested in production of enzymes in deep layers as well as thin layer tray systems. A pilot scale deep bed fermentation system was constructed (Figure 6). The reactor is a vertical stirred reactor based on a modified lauter tun design. The system is designed to be used at a maximum depth of about 50 cm. The entire unit sits on load cells to measure weight loss during the fermentation. The substrate is sterilized by a rotary mixer that can be pressurized and heated. During the fermentation, temperature, oxygen, carbon dioxide, airflow and relative humidity can be monitored. The reactor will enable studies to determine if organisms can perform in deep layers at pilot scale levels and develop an understanding of heat production.





Figure 6.A pilot scale deep bed fermenter.



DESIGN AND CONSTRUCTION OF THE SSF PLANT

There is no information available in the literature on SSF with respect to the theoretical and experimental comparisons of different kinds of bioreactors, methods for controlling cultivation parameters, automation, design and scale up criteria or downstream processing options. The design process for large scale production of enzymes has been based on information obtained in lab scale and pilot scale studies as well as experience gained in the areas of fermentation and downstream processing.

From the data generated and the scant literature available it became apparent that for initial production a tray fermentation system would reach optimum production levels in the shortest amount of time. The data generated in the lab and pilot tray systems were used in the development of a production tray fermentation system. The facility has been built in Serdan, Pueblo, Mexico. This facility houses pretreatment, inoculation facilities, fermentation facilities, and downstream processing facilities. A separate building at the site contains a laboratory for initial strain manipulation. The initial phase of the design was intended for construction of a facility that will contain about 10,000 trays.


Future potential

Most profitable applications of SSF are confined to Asian and African countries and are scarce elsewhere in the world. A resurgence of interest has occurred in western and European countries in response to the everincreasing demand for economy in enzyme production. The facility in Serdan is believed to be the first commercial enzyme production facility in North America that uses SSF technology. Its success will lead to expansion. The future of the SSF program at Alltech includes the development of new strains for enzyme production, to enhance current enzyme systems and development of new enzyme applications. Solid substrate fermentations will also lead us in the direction of new substrates. The use of a variety of waste products will be investigated as well as the potential for using inert supports for fermentation. The SSF technology also has the potential to be used for purposes other than enzyme production. Other metabolites such as ethanol, flavors, and other microbial by-products can be produced. SSF can be used for upgrading agro-industrial by-products that can be used in animal feed applications. Knowledge gained about the SSF process will allow construction of systems that better monitor and control fermentation parameters and utilize a wider range of substrates, as well as microorganisms.



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