Solvents

Solvents are compounds that are essentially neutral in character and comprise organic alcohols and ketones. The major solvent products of microbial carbohydrate catabo-lism include ethanol, acetone, isopropanol, butanol, ace-toin, 1,3-propanediol, and butanediol. These are the most useful oxychemicals produced by fermentation. Solvents have various uses in the chemical industry, including the direct use as organic chemicals and indirect use as fuel additives, extenders, or feedstocks for further chemical synthesis. Butanol is primarily used as a feedstock chemical in the manufacture of lacquers, rayon, plasticizers, coatings, detergents, and brake fluids. It can also be used as a solvent for fats, waxes, resins, shellac, and varnish. In addition, butanol may also be used as an extractant and solvent in the food industry.

Butanol. The acetone-butanol fermentation currently has potential because butanol has many characteristics that makes it better than the currently used liquid-fuel extender, ethanol. Higher alcohols have several characteristics that are favorable for motor fuel use, either in gasoline blends or, in some cases, when used directly as fuel. They are miscible in gasoline; their heats of combustion per gallon are greater than those of methanol, and they have good octane-enhancing properties. Butanol, for example, has a heat of combustion 54% higher than methanol and 83% of that for gasoline (57,58). Although the octane numbers are less than that for methanol (RON 100 vs. 110), the research octane number (RON) value of 100 is still above that of gasoline (RON 92), and therefore useful for fuel blends. In addition, butanol has low miscibility with water and exhibits high miscibility with both diesel and gasoline. Owing to its high heat of combustion butanol solutions containing as much as 20% (v/v) water have the same combustion value as anhydrous ethanol.

In the early 1900s, butanol was used to produce butadiene, the most desirable raw material for synthetic rubber. The annual production of fermentation-derived bu-tanol was over 45 million pounds during 1945. But the fermentation-derived butanol process declined after World War II in the U.S. due to both changes in availability of renewable feedstocks (molasses, sugarcane) and the increase in availability of inexpensive petrochemical feedstocks. Butanol fermentation of beet molasses continued through the 1970s in the Soviet Union, and fermentation of sugarcane molasses continued through the 1980s in South Africa. In China, butanol was still produced fermen-tatively until recently, but the last viable industrial acetone-butanol-ethanol (ABE) fermentation in the Western world was carried out by National Chemical Products in Germiston, South Africa, using C. acetobutylicum.

Butanol is now synthesized chemically from petroleum-derived ethylene, propylene, and triethylaluminium, or carbon monoxide and hydrogen. The major domestic producers of butanol and its derivatives are BASF, Chem Service Inc., Dow Chemical, Eastman Chemical, Hoechst Ce-lanese, Shell, Union Carbide, and Vista. The current U.S. production of butanol is more than 1.2 billion pounds per year and is experiencing a growth of 3-4% annually.

Butanol pricing has been rising steadily in recent years due to an increase in demand. The bulk butanol pricing in the United States is $0.40-$0.50 per pound. The price of butanol in Europe and the Far East is higher than $0.50 per pound. If the production cost of fermentation-derived butanol could be in the range of $0.25-$0.30 per pound, the market for butanol will likely expand: The market penetration of fermentation-based butanol will drive the utilization of corn from 7.2 million bushels in 1995 to 153 million bushels in 2010. In addition, the supply of other alternative feedstock, such as biomass, is more available than corn. However, biomass requires effective pretreat-ment technology that has not yet been developed. Biomass-derived solvents produced by fermentation can enter into the current petrochemical synthetic pathways through a number of reactions, the most important of which is the dehydration of alkanols to alkenes to form ethylene, pro-pylene, butylene, and butadiene.

The ABE fermentation in batch culture is a sequential process characterized by a primary acid-producing or aci-dogenic phase that is coupled to growth, and a secondary solvent-producing or solventogenic phase. Thus, solvento-genesis appears as a secondary metabolic process caused by uncoupling of growth. Acetate and butyrate are produced as soluble fermentation products during the acido-genic phase. As the acids accumulate, the growth rate and the culture pH decreases, and solventogenesis begins. The acids and H2 are consumed and are reduced to solvents in the solventogenic phase. The "switch" from acidogenesis to solventogenesis is accompanied by a large number of physiological and biochemical changes, and is clearly a multi-factorial process. The exact mechanism that regulates this switch has not been clearly elucidated, but the concentration of undissociated butyric acid within the cytoplasm functions as a "bioregulator." The end products of the ABE fermentation alter and inhibit growth and metabolism. Acids are more toxic than solvents, and the more hydrophobic a product, the higher is its toxicity. It should be noted that toxicity of acids depends upon the pH. At neutral pH, high concentrations of organic anions (i.e., butyrate, acetate) are not toxic because they are dissociated. In normal batch-fermentation conditions, organic acids are produced, and then consumed during solventogenesis to prevent dissipation of the cellular proton-motive force. Butanol inhibits the fermentation at a relatively low concentration.

Butanol producers use a wide variety of carbohydrates, including hexoses, pentoses, oligosaccharides, and poly-saccharides as fermentation substrates, and including starch, molasses, whey, wood hydrolysates, pentosans, in-ulin, and sulfite liquor. Cellulosic materials are one of the most attractive substrates for chemicals, as they are the most abundant and least expensive raw material. Few at tempts have been made to use cellulosic substrates for the production of butanol. A simultaneous saccharification fermentation process was studied to produce butanol from alkali-pretreated wheat straw using Trichoderma reesicel-lulase and C. acetobutylicum (59). The final butanol concentration was 10.7 g/L from about 140 g of straw. Pen-toses did not accumulate during the fermentation, suggesting simultaneous use ofboth hemicellulose and cellulose components. Different substrates alter fermentation parameters of C. acetobutylicum, such as growth rate and the solvent production ratio. Higher growth and substrate consumption rates were obtained when glucose, cellobiose, or mannose were used than when xylose, arabinose, or galactose were used. The solvent production ratio with the first sugar group was 1:4:10 (ethanol:acetone:butanol), whereas ratios of 1:2:5 were obtained with the latter.

Starch is at present the most useful raw material for fermentation because it is abundant, has high density, and requires limited pretreatments to be made from biomass. Starch is produced from corn, cereals (wheat, oats, rice), and starchy roots (sweet potatoes, cassava). Typically, C. acetobutylicum produces 12-13 g/L butanol with about 2022 g/L total solvents. In recent years, the majority of investigators have focused their interests on the type culture strain C. acetobutylicum ATCC 824. Other cultures included DSM 792 strain and NCIMB 8052. The C. acetobutylicum ATCC 4259 strain and the equivalent DSM 1731, NCIMB 619, and NRRL B-530 strains are all derived from the original Weizmann industrial strain. An asporo-genous mutant ATCC 39236 derived from this strain has been patented by scientists at the Moffett Technical Center CPC International (60).

Recently, a limited sporulating C. acetobutylicum E604 mutant strain has been developed by treating the parent C. acetobutylicum strain (ATCC 4259) with ethane methane sulfonate (61). This mutant strain has been used to ferment a high-carbohydrate substrate concentration in a multistage, continuous-temperature programmed-fermen-tation process followed by batch fermentation (11), to yield an extraordinarily high concentration of butanol (>20 g/ L) and total solvent (>30 g/L) production (61). This mutant strain has higher butyrate uptake rate (0.33 g/L) in comparison to the parent strain (0.26 g/L) at a lower temperature of 30 °C (62). Butyrate up to an externally added concentration of 11.4 g/L did not inhibit butyrate uptake. Optimization studies for butyrate uptake by C. acetobutyl-icum suggested a direct correlation between minimum pH and butyrate concentration or temperature (63). These developments make it possible to commercially produce bu-tanol fermentatively with no residual butyrate.

The saccharolytic strain C. acetobutylicum P262, one of the NCP production strains, has been studied by groups working in South Africa and New Zealand. The various solvent-producing strains now tend to be regarded as varieties of C. acetobutylicum or C. beijerinckii. For batch ABE fermentation to be industrially viable, and to compete with the chemical process, concentrations of between 22 and 28 g/L must be obtained in 40-60 h (64). A large number of publications on butanol-producing microbial cultures and the process demonstrate the extensive research work that has been done in past years (33,60,64-68).

Simultaneous recovery of butanol from the broth during fermentation can eliminate product inhibition. This will allow complete utilization of sugar and continual fermentation of the substrate at relatively higher rates. In contrast to the traditional distillation processes, removal of butanol can be accomplished by integrating a pervapora-tion process with the multistage fermentation process. In this process, the solvents will pass through the membranes preferentially and the organic acids and cells will remain on the liquid side and return to the fermentor. The permeate stream that is enriched in solvent relative to the feed will separate into a low-butanol aqueous phase and a butanol-rich solvent phase. This aqueous phase could be recycled back to the feed tank. Pervaporation also provides for the retention of cells in the fermentor, which improves volumetric productivity.

Ethanol. In the United States, use of ethanol as a fuel is almost wholly confined to its use as an octane enhancer in the higher grade of gasoline. Currently, this fuel ethanol is obtained from both fermentation and chemical synthesis. All of the fermentation ethanol produced in the United States is made by yeast fermentation.

Interestingly, the substrate range for both yeasts and Zymomonas for ethanol production is similar. Traditionally, yeasts have been used to produce alcoholic beverages and, more recently, to produce a considerable portion of industrial ethanol all over the world. Among yeasts, Sac-charomyces is the most important and most commonly used organism. Saccharomyces cerevisiae converts glucose to ethanol and carbon dioxide. Other yeasts include Pach-ysolen tannophilus and Kluyveromyces marxianus. Many facultative and anaerobic bacteria produce varying amounts of ethanol as one of the metabolic end products. Bacterial species such as Zymomonas mobilis, Sarcina ventriculi, and Erwinia amylovorans utilize pyruvate de-carboxylase to convert pyruvate to CO2 and acetaldehyde, which is then reduced to ethanol. Zymomonas grows on glucose, fructose, and sucrose, but is unable to use starch, maltose, or pentose sugars. Z. mobilis is a promising bacterium with some potential to replace yeast as the major organism for production of industrial ethanol.

Z. mobilis was metabolically engineered to broaden its range of fermentable substrates to include the pentose sugar xylose (69). Two operons encoding xylose assimilation and pentose-phosphate-pathway enzymes were constructed and transformed into Z. mobilis in order to generate a strain that grew on xylose and efficiently fermented both glucose and xylose to ethanol. Subsequent metabolic-pathway engineering further expanded the fermentation substrate range of the ethanologenic bacterium Z. mobilis to include the pentose sugar L-arabinose (70). Direct fermentation of cassava starch to ethanol by Z. mobilis was obtained in the medium containing amylase-rich culture filtrate of Endomycopsis fibuligera (71). The ethanol concentration of 105 g/L could be increased to 132 g/L by further addition of glucoamylase enzyme at 0.01%.

A number of different groups of bacteria, including clos-tridia, produce ethanol via the reduction of acetyl CoA, which is generated by the cleavage of pyruvate produced during glycolysis. At least 30 species of Clostridium have been reported to produce ethanol in amounts varying from trace to close to the theoretical maximum of 2 mol ethanol/ mol glucose fermented. In most cases, the extent of ethanol production is dependent upon the nature and concentration of fermentation substrate and the fermentation conditions, such as pH and temperature. For a number of me-sophilic clostridial species, ethanol yields ranging from 1.7-1.9 mol/mol hexose fermented have been observed. In a single-step biomass to ethanol conversion process, C. thermocellum has been shown to produce 23.B g/L ethanol from an alkali-treated paddy straw (9). The bacterium C. ljungdahlii, a fast-growing bacteria, has been shown to produce ethanol from carbon monoxide in concentrations up to 48 g/L from synthesis gas in a CSTR with cell recycling (10).

In recent years, thermophilic bacteria have been shown to produce solvents and are considered potential candidates for use in process development because of certain advantages over mesophiles. Thermophiles have the ability to use complex plant polymers such as cellulose or starch, and offer high growth rates, fast fermentation, reduced contamination, increased process stability, and eliminate high energy demand in cooling the media and product recovery. The major disadvantage is production of end products in low concentrations and yield due to lower end-product tolerance. However, this can be partially overcome if the solvents are removed simultaneously.

There are increasing number of patented processes concerning the use of thermophilic microorganisms, including ethanol fermentation (72-75). The thermophilic anaerobic bacteria have provided a great deal of information on ethanol-fermentation pathways via acetyl-CoA (34). There are two different ethanol pathways in thermophiles (76). The type I pathway uses NADH-linked alcohol dehydrogenase and produces ethanol from acetaldehyde. The Type II pathway uses NADPH-linked alcohol dehydrogenase to produce ethanol from both acetyl CoA directly or acetal-dehyde. In Type II ethanologens, the NAD-linked alcohol dehydrogenase functions to consume ethanol. The Type I pathway is applicable in anaerobic bacteria such as C. ther-mocellum. The Type II pathway is present in Thermoan-aerobacter thermohydrosulfuricus, Thermoanaerobacter brockii, and Thermoanaerobacter ethanolicus. Ethanol yields of heterofermentations vary considerably with the specific growth conditions used. The biochemical basis for different reduced end-product ratios of thermophilic etha-nol producers that contain the same glycolytic pathways is related to subtle differences in the specific activities and regulatory properties of the enzymes that control electron flow during fermentation. Similarly, specific changes in culture conditions such as temperature and pH influence the rate and direction of the enzymatic machinery responsible for end-product formation.

T. ethanolicus 39E has a low tolerance for ethanol, with growth inhibition occurring at 2% (wt/vol) ethanol. An ethanol-tolerant strain (39EA) that was tolerant to 4% (wt/ vol) ethanol at 60 "C, produced ethanol under these conditions (77), and lacked the NAD-linked alcohol dehydrogenase was selected. Another strain (H8) can grow at 8% ethanol but produces lactic acid at high solvent concentra tions. The mechanism of high (i.e., 8%) ethanol tolerance in T. ethanolicus is related to its ability to produce unique transmembrane lipids (C30 to C34 fatty acids) that provide solvent tolerance (78). Alcohol increases membrane fluidity, and these transmembrane lipids may serve to reduce the fluidity and maintain membrane integrity.

1,3-Propanediol. 1,3-Propanediol (PD) is a versatile intermediate compound for the synthesis of heterocycles and as a monomer for the production of polymers such as poly-ethers, polyesters, and polyurethanes. PD can also be used as a solvent and an additive for lubricants. The microbial conversion of glycerol to PD is simple in comparison to the chemical conversion of acrolein. It has been shown that some Clostridium species are able to convert glycerol to PD with an appreciable yield (79). 1,3-Propanediol from glycerol was produced by C. butyricum DSM 5431 at 50-58 g/ L with productivities of 2.3-2.9 g/L per hour (13). The fermentations were conducted in 300- and 3000-L fermentors with almost similar results, indicating that the scale-up of this microbial fermentation process should not cause a major problem. The substrate glycerol is relatively cheap and its conversion to PD could help reduce glycerol surpluses in the market.

Organic Acids. Currently, organic acids such as acetic, propionic, butyric, fumaric, succinic, malic, and lactic acids can be produced by anaerobic fermentation technology. The key technical problems blocking the rapid advances in developing the bioprocess technology for organic-acid fermentations have been low product concentration, low specificity to desired product(s), low productivity or rate of fermentation, and inefficient or energy-intensive recovery processes. However, recently, significant technological advances have occurred in anaerobic fermentations for production of organic acids by fermentation of carbohydrates (80). Acidogenic fermentation of carbohydrates to volatile organic acids is well known (43). In general, anaerobic bacterial fermentations are of interest because of the wide range of products formed and substrate fermented, the high substrate-to-product conversion yields and rates, and the potential for enhanced process stability and product recovery due to physiological diversity of species (e.g., extreme thermophily, acidophily, halophily). For acetate fermentation, homacetogenic fermentation producing 3 mol of acetate from 1 mol of dextrose (e.g., by using C. ther-moaceticum) has been described by many authors. Currently, the available organisms produce salts of organic acids in low concentrations (400-800 mM for acetate, 200400 mM for propionate and 200-300 mM for butyrate) with low productivity of 0.2-2 g/L per hour (81). New fermentation processes with better yield and productivities have been developed, making it feasible to produce volatile as well as nonvolatile organic acids economically.

Lactic Acid. Lactic acid is a natural organic acid with a long history of use in the food industry. Lactic acid, because of its flavoring and preservation properties, is used as an acidulant, particularly in dairy products, confectionery, beverages, pickles, bread, and meat products. It is also used in the pharmaceutical and cosmetic industries. The cyclic dimers of lactic acid are used as raw materials in the synthesis of biodegradable polymers for a variety of uses, including absorbable surgical sutures, slow-release drugs, and prostheses. Ethyl lactate is the active ingredient in many anti-acne preparations. Crude grades of lactic acid are used for the deliming of hides in the leather industry, and it is used for fabric treatment in the textile and laundry industries. Its ability to form polymeric polylactic acids finds application in production of various resins. Because of its structure and its two functionally reactive groups, hydroxyl and carboxyl, lactic acid can be used to make numerous value-added functional chemicals (82). These include biodegradable polymers and copolymers, propylene glycol, propylene oxide, and acrylates. Lactic acid-based polymers and copolymers have potentially large-volume uses as biodegradable thermoplastics, pesticide formulation, and environmentally benign polymers, with a potential market exceeding several hundred million dollars per year.

The market for fermentation lactic acid is growing every year because it is now feasible to produce large-volume chemicals from lactic acid. Homofermentative lactic-acid bacteria produce none or only trace amounts of end products other than lactic acid, and are used for industrial processes. Numerous organisms are known to produce lactic acid with high (>90%) yield and productivity (>2 g/L per hour) from carbohydrate fermentation. Some of the common homolactic acid bacteria are: Lactobacillus casei, L. pentosus, L. leichmannii, L. acidophilus, L. delbrueckii, L. bulgaricus, Streptococcus cremoris, S. lactis, S. diacetylac-tis, Sporolactobacillus sp. and Pediococcus sp. Homolactic acid-producing bacteria ferment starch, hexoses, pen-toses, and cellobiose. Depending upon the cultural conditions, the yield of lactic acid by the homolactic acid bacterium L. casei and heterolactic acid bacterium T. brockii are nearly equivalent. In non-energy-limited batch culture, L. casei produces lactic acid as the sole end product, whereas in energy-limited, continuous culture, nearly equivalent amounts of lactic, acetic, and formic acids, and as well as ethanol, are produced (43). Lactate is the major fermentation product of T. brockii grown in high-yeast extract, batch fermentation. It should be noted that lactate yields in homolactic-acid bacteria depend upon specific growth conditions and that additional products (e.g., formic acid, acetic acid, and ethanol) can also be formed. Lactic acid yields are generally highest during glycolysis via the homolactic-acid-fermentation pathway. Theoretically, 2 mol of lactate and 2 mol of ATP are formed per 1 mol of glucose fermented.

Anaerobic fermentations for production of organic acids operate optimally at pHs where salts of organic acids, rather than free acids, are produced. The free acids and their derivatives are required for the manufacture of functional chemicals. The development of new technologies to recover and purify the fermentation acids from broth have reduced the costs associated with product recovery and purification, thus making the commodity production of organic acids such as lactic acid practical and economically attractive. One of the approaches that has been traditionally applied is simultaneous fermentation and neutralization with calcium salt to obtain calcium lactate precipi tate. The precipitate can be separated easily, and free lactic acid can be obtained after treating the salt with acids such as sulfuric acid. The disadvantage of such a system is in handling the solids and slurry. Another emerging technology for separation and recovery of lactic acid is electrodi-alysis (ED). ED is a rapid process for altering the composition and/or concentration of electrolytes in aqueous solutions by transferring ions across ion-exchange membranes under the influence of a direct electric current. The recovery and purification of lactic acid using ED has been successfully demonstrated (14). In this process, sodium lactate is first selectively separated from proteins, amino acids, and cells, then, using water-splitting membrane, the lactic acid salt is converted to free lactic acid and base. The traditional process using lime for neutralization followed by cell filtration, carbon adsorption, evaporation, acidula-tion, gypsum filtration, carbon adsorption, evaporation, filtration, esterification, and final purification is uneconomical (83). However, separation and recovery of lactic acid by ED seems a cost-effective and attractive approach.

Succinic Acid. Succinic acid is a relatively new nonhy-groscopic acidulant. Succinic acid is listed by the FDA as a GRAS additive for miscellaneous and/or general-purpose uses. It readily combines with proteins in modifying the plasticity of bread doughs. It is also an dibasic acid for producing edible synthetic fats with desirable thermal properties. Succinic acid is manufactured by the catalytic hydrogenation of maleic or fumaric acid. It has also been produced commercially by aqueous alkali or acid hydrolysis of succinonitrile, which is derived from ethylene bromide and potassium cyanide.

Succinic acid is a common intermediate in the metabolic pathway of several anaerobic microorganisms. For example, succinate is a key intermediate for anaerobic fermentations by propionate-producing bacteria, but it is only produced in low yields and in low concentrations. Succinate is also produced by anaerobic rumen bacteria. These bacteria include Ruminobacter amylophilus and Prevotella ruminicola. As such, accumulation of succinic acid in fermentation broth has been observed in a number of rumen microorganisms. Although the rumen bacteria give higher yields of succinate than do the propionate-producing bacteria, the reported fermentations were generally run in very dilute solutions and gave a variety of products in generally low yields. Moreover, the rumen organisms tend to lyse after a comparatively short fermentation time, thereby leading to unstable fermentations.

Anaerobiospirillum succiniciproducens, an anaerobic non-rumen bacterium, has been demonstrated to produce succinic acid in higher concentrations and yield (84,85). The pathway for succinic acid production in A. succinici-producens has been established (45). Two processes to produce succinic acid as sodium succinate (86) and calcium succinate (87) were developed using A. suciniciproducens, as this organism produced succinate in a relatively higher concentration and yield with fewer cofermentation products, had a faster growth rate, and could grow in an industrial grade medium.

In order to develop a commercially attractive process to produce succinic acid by fermentation, the product should be produced in a high concentration and in a high yield (wt%) using inexpensive raw materials and nutrients. A. succiniciproducens produces succinate and acetate in a ratio of about 4:1, thus a good amount of carbon is lost as acetate and the separation of succinate and acetate becomes very critical. Therefore, a fluoroacetate-resistant variant FA-10 of A. succiniciproducens was developed that produced very minute amounts, if any, of acetic acid (88). The organism produces enough succinate to allow simultaneous fermentation and calcium succinate precipitation under strictly controlled conditions and when the correct calcium salt is used for neutralization. However, the process seems to have a major disadvantage in handling gypsum at a commercial scale. The succinic acid fermentation in A. succiniciproducens is regulated by fermentation pH and the amount of carbon dioxide (45). For example, lac-tate is the major fermentation product at pH 6.5 and higher with limiting levels of CO2, whereas succinate is the major product at a pH of 6.0-6.2 with an abundance of CO2. Phosphoenolpyruvate carboxykinase has been purified and described for A. succiniciproducens (89), a non-ruminal anaerobic bacterium, as well as from a ruminal anaerobic bacterium, Ruminococcus flavefaciens (90). In E. coli, overexpression of PEP carboxikinase had no effect on succinic acid production, but succinic acid was produced as the major fermentation product by weight (3.5-fold increase in the concentration) by overexpression of PEP car-boxylase (91).

Recently, a rumen-facultative anaerobic bacteria, Acti-nobacillus sp. strain 130Z, has been shown to produce succinic acid in much higher concentration (60-80 g/L) than any of the previously known succinic acid-producing microorganisms (92). Acetate and small amounts of pyruvate and formate are produced as cofermentation products. The variants of Actinobacillus sp. strain 130Z have been developed to produce succinic acid in a concentration of >105 g/L and yield of 85-95 wt % (19) under neutralization with magnesium. The organism is reported to be robust with a wide pH range and tolerance to high substrate and product concentrations. The levels of key succinic acid-producing pathway enzymes, such as phosphoenolpyruvate (PEP) carboxykinase, a key CO2-fixing enzyme, malate dehydrogenase, and fumarase were significantly higher in Actinobacillus sp. strain 130Z than in E. coli K-12 (93). The key enzymes in end-product formation in Actinobacillus sp. 130Z were regulated by the energy substrates.

Butyric Acid. Butyric acid, butyrate esters, and other butyrate derivatives are important flavor ingredients in many natural and processed foods. For example, butyric acid is present in butter as an ester to the extent of 4-5%. Some of its esters serve as bases of artificial flavoring ingredients of certain liqueurs, soda water syrups, candies, and so on. Development of a bioprocess for production of natural butyric acid by anaerobic fermentation thus is likely to satisfy the potential market of natural fermentation products. In addition, ethyl and methyl esters ofbu-tyric acid can also be used as octane enhancers when added to gasoline (94). Some common anaerobic bacterial genera producing butyrate are Butyribacterium, Butyrivibrio,

Clostridium, Eubacterium, Fusobacterium, Haloanaero-bium, and Sarcina. Most butyric-acid producing bacteria ferment starch, hexose, pentose, and cellobiose, and form acetic acid, in addition to butyric acid, as the major (i.e., percentage of substrate weight converted to product) fermentation product. Butyric acid production is also related to cultural conditions of the specific species. For example, C. thermosaccharolyticum produces butyric acid as the fermentation product during exponential growth (95). B. methylotrophicum can produce either acetate or butyrate as the sole end product, or it can form a mixture ofbutyrate and acetate. B. methylotrophicum ferments glucose to acetate with small amounts of butyrate at near neutral pH; it produces high levels of butyrate at acidic pH (42,96). In addition, it will produce butyrate from methanol in the presence of carbon dioxide and acetate under the same pH conditions. Products of CO fermentation by this organism are acetate and butyrate in a ratio of approximately 30:1. However, the fermentation can be shifted towards buty-rate by decreasing the pH at the onset of the stationary phase (97). Theoretically, yields of 1 mol of butyrate and 2 mol of hydrogen and CO2 are obtainable from 1 mol of hex-ose via the butyrate fermentation path (43). B. methylotro-phicum or C. butyricum under cell-recycle can achieve this scenario.

In general, the most studied butyrate-producing microorganisms belonged to the genus Clostridium. A large number of fermentation substrates, including hydroly-sates of waste cellulosic material, lactose from whey, molasses, and cellulosic materials can be utilized to produce butyric acid. However, the low concentration in the fermentation broth did not make the process commercially attractive as a commodity product. In addition, a major characteristic of this fermentation is the concomitant production of acetate, which is observed in butyrate-producing Clostridium and other bacterial species. Recently, fed-batch fermentation of glucose with Clostridium tyrobutyr-icum have yielded butyrate in a concentration of 42.5 g/L with a selectivity of 0.90, a productivity of 0.82 g/L per hour and a weight yield of 36% (98). In glucose-limited, fed-batch cultures, initially produced acetate was reutilized, resulting in exclusive production of butyrate. Because the ratio of butyrate to total acids was strongly influenced by the growth rate of bacteria, acetate being produced along with butyrate at higher growth rates (16) achieved increases in butyrate concentrations, productivity, selectivity, and yield by controlling the substrate feeding by the rate of gas production. In a fermentation of wheat flour hydrolysate (380 g/L of glucose) with C. tyrobutyricum, a butyrate concentration of 62.8 g/L was obtained with a productivity of 1.25 g/L per hour, a selectivity of 91.5%, and a weight yield of 45% (16). Thus, production of butyrate for specialty uses by fermentation is feasible.

Propionic Acid. Propionic acid is a value-added specialty chemical used in various chemical and food-processing industries. It has wide-ranging applications, such as an antifungal agent in foods and feeds, and as an ingredient in thermoplastics, antiarthritic drugs, perfumes, flavors, and solvents. Currently, propionic acid is produced by chemical synthesis from petroleum feedstocks, but small amounts of natural propionic acid are produced by fermentation. Propionic acid is a major end product of fermentations carried out by a variety of anaerobic bacteria, many of which ferment glucose to propionate, acetate, and CO2. Lactate can also be fermented to propionate either via the acrylate pathway in a stepwise reduction to propionate, or via the succinate pathway, where lactate is converted to propionate via pyruvate and succinate.

Although several microorganisms can produce pro-pionic acid, fermentations using propionibacteria have been studied most extensively. The slow growth rate of pro-pionibacteria usually results in propionate production at a slow rate. Propionispira arboris ferments glucose to pro-pionate, acetate, and CO2 via the succinate pathway. The ratio of propionate to acetate is low, resulting in a carbon loss to cofermentation products. However, the ratio of pro-pionate to acetate can be changed from 2 to 16:1, approaching a homofermentation yield from glucose, by use of H2 as a cosubstrate because this species consumes H2 (44). Various fermentation systems have been examined to increase the propionate fermentation rate, including batch, fed-batch, continuous, continuous with cell recycle, extractive, and immobilized cell fermentations. Also, various substrates such as glucose, xylose, lactate, glycerol, food-processing waste, and whey have been examined to produce propionate not only with increased productivity, but also economically. Using Propionibacterium acidipropion-ici, propionate has been produced at 42 g/L from glycerol (18) and at 57 g/L from glucose (17) in batch and fed-batch fermentation systems, respectively. It seems fermentative production of propionate is feasible if an appropriate fermentation system and recovery technology can be integrated to obtain the acid in high concentration, yield, and productivity.

Was this article helpful?

0 0
Healthy Chemistry For Optimal Health

Healthy Chemistry For Optimal Health

Thousands Have Used Chemicals To Improve Their Medical Condition. This Book Is one Of The Most Valuable Resources In The World When It Comes To Chemicals. Not All Chemicals Are Harmful For Your Body – Find Out Those That Helps To Maintain Your Health.

Get My Free Ebook


Post a comment