R. R. Gokarn, M. A. Eiteman, and J. Sridhar

Department of Biological and Agricultural Engineering,
Driftmier Engineering Center, University of Georgia, Athens, GA 30602

Numerous anaerobic microorganisms synthesize succinic acid as a fermentation product. This chapter reviews the literature for succinate producing organisms and compares the growth and succinate production of two widely differing anaerobic bacteria, Fibrobacter succinogenes and Clostridium coccoides. F. succinogenes degrades simple sugars such as glucose as well as cellulosic materials such as pulped shredded office paper. The principal products after 90 hours from 10 g/L pulped paper are succinate (3.2 g/L) and acetate (0.58 g/L), with lower concentrations of formate (0.070 g/L). C. coccoides degrades simple sugars only, and after 24 hours the principal products from 5 g/L glucose are acetate (3.0 g/L), succinate (0.57 g/L) and lactate (0.58 g/L).

Succinic acid is a four-carbon aliphatic dicarboxylic acid having pKat = 4.2 and pKaj = 5.6. The depronated form succinate can be produced by many anaerobic microorganisms at their operating conditions, usually near neutral pH. Succinic acid can be used to manufacture specialty chemicals including tetrahydrofuran, 1,4- butanediol, maleic anhydride, adipic acid, and dimethyl succinate. Its derivatives are used in the food, pharmaceutical, cosmetics and polymer industries. Anaerobic processes from renewable resources are particularly appealing for the synthesis of succinate becuase of their high yields and straght-forward scale-up requirements. This chapter reviews the literature on anaerobic processes for succinate production as well as comparing the succinate production by two widely different microorganisms.

Rumen Bacteria

The rumen is a highly competitive microbial ecosystem of primarily anaerobic bacteria, fungi and protozoa. These microorganisms ferment cellulose, starch and various other carbohydrates into numerous low molecular weight products. The

© 1997 American Chemical Society

principal acid products from rumen fermentations are acetate, propionate and butyrate. The production of propionate in the rumen involves cross feeding between succinate — producing microorganisms and species that decarboxylate succinate to propionate and carbon dioxide (1,2). Therefore, even though succinate itself is not a product of the entire rumen ecosystem, numerous anaerobes have been isolated which synthesize succinate as a primary end product.

Fibrobacter succinogenes. Fibrobacter succinogenes (previously named Bacteroides succinogenes) is the predominant cellulolytic bacterial species found in the rumen. Hungate (3) isolated these organisms from bovine rumen and characterized them as obligately anaerobic, gram negative, cellulolytic, non-motile, non-sporeforming, rod­shaped bacteria whose morphology may change to lemon-shaped when cultivated in the laboratory. Strains of this organism have also been isolated from mice caeca (4), pig caeca (5), gut of horses (6), mice (7), langur monkeys (8) and several African ruminants (9). A more recent study (10) comparing the 16S ribosomal ribonucleic acid sequence demonstrated that this organism differs from other Bacteroides species, and hence the organism was renamed Fibrobacter.

Most F. succinogenes strains utilize glucose, cellobiose, maltose, dextrins, lactose, pectin or cellulose, while some strains also use starch as a carbon source (3). F. succinogenes requires one branched (i. e., isobutyrate or a-methyl butyrate) and one linear (i. e., valeric, caproic, heptaonic or caprylic) volatile fatty acid for the synthesis of long chain fatty acids and aldehydes incorporated into phospholipids (11,12). F. succinogenes has an absolute requirement for biotin (13) and for ions such as Na+, K+, Ca2+, Mg2+ and P043* (2). F. succinogenes synthesizes almost all of its cellular nitrogenous compounds from exogenous ammonia even when large amounts of amino acids and nucleotide precursors are present in the media. Although glutamine or asparagine may substitute for ammonia, ammonia is preferred when multiple nitrogen sources are present (14). Like all rumen bacteria which produce succinate as a major end product (15,16), F. succinogenes has an absolute requirement for carbon dioxide. Initiation of the growth of F. succinogenes is achieved at 0.02 to 0.05% of carbon dioxide, while optimal growth is observed when the carbon dioxide concentration is above 0.1% (15). F. succinogenes also fixes carbon dioxide during succinate production, with carbon dioxide incorporated in the carboxyl group of succinate (16).

F. succinogenes can degrade highly structured, crystalline cellulose such as cotton fibers (17). The cellulose degrading enzymes and mode of cellulose degradation by this organism have been extensively studied (18-20). In order to carry out cellulose degradation, cells must have intimate contact with cellulose fibers, since the cellulase enzyme endo-P-l,4-glucanase is membrane bound (18). This requirement for contact makes the available gross surface area of the substrate a major determinant factor of hydrolytic rate (21). Endoglucanase activity is about seven times greater when the organisms are grown on cellulose than when grown on cellobiose or glucose, suggesting that the enzyme system may be regulated by a catabolite repression mechanism (18). However, Hiltner and Dehority (22) found that the presence of glucose or cellobiose does affect cellulose digestion when pH is controlled, an observation which seems to contradict the catabolic repression hypothesis. In addition to cellulose degradation, F. succinogenes also degrades hemicellulose (18,23). However, the organism cannot utilize as a substrate for growth the pentoses which are released during hemicellulose degradation (24,25). The inability of F. succinogenes to utilize pentoses is attributed to the lack of key enzymes such as xylose permease, xylose isomerase and xylulokinase (26).

F. succinogenes has restricted ranges of redox potential and pH. The redox potential range for cell viability is -290 to +175 mV, and the most prevalent morphology at highest redox potential is greatly elongated cells (27). F. succinogenes has a pH range for growth of 6.1 to 6.9 (28), and cell wash out occurs at a pH of 6.0 when the organisms are grown on cellobiose in a chemostat (29). The highest cell yield on cellobiose and cellulose occurs at the lower pH limit (28,29). The inability of the organisms to grow at lower pH may be due to inhibition of the glucose transport system or due to low substrate affinity (28,30). When grown on microcrystalline cellulose, F. succinogenes has a maximum specific growth rate of 0.076 h’1 and a maintenance requirement of 0.04-0.06 g cellulose/g cells (28), while on cellobiose or cellodextrins F. succinogenes has a maximum specific growth rate of about 0.44 to 0.48 h*1 (31).

Figure 1 summaries the biochemical pathway for succinate production by F. succinogenes (34). The degradation products of cellulose, glucose and cellobiose, are transported into the cell by a highly specific active transport system. The glucose transport system is energized by a proton gradient, while the cellobiose transport system is energized by a sodium ion gradient (32). F. succinogenes possesses fructose 1,6-biphosphate aldolase (33) and glyceraldehyde-3-phosphate dehydrogenase (34). Oxaloacetate formation from phosphoenolpyruvate is accompanied by carbon dioxide fixation and is catalyzed by GDP-dependent phosphoenolpyruvate- carboxykinase. Reduction of oxaloacetate results in malate formation with NADPH or NADH acting as the electron donor. Even though fumarase activity has not been demonstrated, Miller (34) proposed that conversion of malate to fumarate is catalyzed by fumarase. Fumarate is then reduced to succinate by a flavin-dependent membrane — bound fumarate reductase (34). During the fumarate reduction, cytochrome b acts as an electron carrier, and the step may result in ATP generation via electron transport linked phosphorylation. This hypothesis is supported by observations of higher growth yields than can be explained solely by substrate level phosphorylation (27), and of decreased growth rates in the presence of electron uncouplers (35). Conversion of pyruvate to acetyl-CoA is accompanied by the reduction of FMN with carbon dioxide evolution. The formation of acetyl phosphate from acetyl-CoA is catalyzed by phosphotransacetylase, and acetate production from acetyl phosphate yields ATP. Studies with partially isolated phosphoenolpyruvate-carboxykinase indicate that this enzyme is active only in presence of bicarbonate, GDP and the Mn2+ ion (34).

Ruminococcus flavefaciens. Sijpesteijn described Ruminococcus flavefaciens as a gram positive, non-motile, anaerobic, cellulolytic, streptococci of 0.8-0.9 pm diameter (36). The cells can exist singly, in pairs or may form a chain. R. flavefaciens ferments xylans, cellobiose or cellulose, while the fermentations of glucose, xylose or other simple carbohydrates is restricted to only a few strains (36,37). R. flavefaciens is active on amorphous cellulose (17) and also breaks down hemicellulose, but most strains cannot utilize pentoses as an energy source (38). A distinct feature of R. flavefaciens is the production of a yellow pigment when grown on cellulose. The temperature range for growth is 30 to 45°C, with 39°C being the optimum. R.

Figure 1 Biochemical pathway for succinate production by F. succinogenes.

flavefaciens produces reducing sugars when grown in excess cellulose, although usual fermentation products include succinate, acetate and at least trace formate and lactate (36).

R. flavefaciens has a requirement for branched fatty acids, which are incorporated into lipids and amino acids (39). R. flavefaciens can utilize ammonia as a sole nitrogen source (14). Biotin is required by all strains, while vitamin B12 is required by some strains. p-Aminobenzoic acid has been shown to stimulate growth (13). R. flavefaciens has an absolute requirement for carbon dioxide: 0.05 to 0.1% carbon dioxide for growth initiation and above 0.1% for optimal growth (15).

R. flavefaciens possesses enzymes which are active on cellulose, hemicellulose and pectin. Most of these enzymes are believed to be cell wall associated, since cell attachment to cellulose fibers is necessary for plant wall degradation. R. flavefaciens has active exo-l,4-P-glycosidase enzymes which generate cellobiose and cellotriose from cellulose and xylobiose and xylotriose from xylan. R. flavefaciens also exhibits low levels of aryl p-glucosidase and aryl p-xylosidase activity (40). Presence of soluble sugars such as cellobiose seems not to affect cellulose digestion, although cellulose digestion may be influenced by a pH decrease when soluble sugars are rapidly fermented (22). R. flavefaciens has a poor affinity for cellobiose which may result in its poor utilization. With cellobiose in a continuous culture, cell wash out occurs at a pH of about 6.1, and cell yield decreases abruptly (29). An increase in growth rate results in a shift toward more acetate and formate with less succinate. On cellulose the organism has a low maintenance requirement of 0.07 g cellulose/g cell per hour (41).

For one R. flavefaciens strain isolated from sheep, succinate is the major product of glucose fermentation in the presence of carbon dioxide, but in the absence of carbon dioxide the fermentation shifts to a homolactic pattern (42). The pathway for succinate and acetate production by R. flavefaciens is believed to be identical to that of F. succinogenes shown in Figure 1 (42-44). Succinate formation is accompanied by fixation of carbon dioxide, which is incorporated in the carboxyl group. Formate can form from free carbon dioxide or from pyruvate (43). PEP — carboxykinase requires GDP and the bicarbonate ion as cosubstrates, and this enzyme is most effective in converting phosphoenolpyruvate to oxaloacetate in the presence of Mn2+ (44).

Ruminobacter amylophilus. Hamlin and Hungate (45) first isolated Ruminobacter amylophilus (Bacteroides amylophilus) from bovine rumen and characterized the species as an obligately anaerobic, gram negative, non-sporeforming, non-motile bacterium (45). A more recent study of the 16S ribosomal ribonucleic acid sequence showed that this organism differs from other Bacteroides species and hence was renamed Ruminobacter (46). R. amylophilus cells are rod-shaped, 0.9-1.6 pm, but may also exhibit larger irregular shapes. The organism has subsequently been isolated by Blackman and Hobson (47), Bryant and Hobson (48), Caldwell et al. (16), and Blackman (49). R. amylophilus uses only starch and maltose as substrates, and the organism’s population in the rumen is increased when the animal is fed a high starch diet (36). Fermentation products include acetate, succinate, formate and trace ethanol and lactate (45). The organisms grow at a pH of 6.5-7.8 and temperature range of 35 to 45°C (45). R. amylophilus growth occurs within a wide redox potential range of -320 mV to +250 mV, although the specific growth rate decreases above 0 mV (27). Above +200 mV more lactate is produced at the expense of succinate (27).

R. amylophilus has an absolute requirement for Na+, and this requirement cannot be replaced by K+, Li+, Cs+ or Rb+ (50). In addition to the Na+ ion, R. amylophilus requires K+, P043 and trace Mg2+ (50). The Na+ and K+ ions affect the growth rate and growth yield, while P043’ affects only the growth yield (50). R. amylophilus also has an absolute requirement of carbon dioxide for growth; however, bicarbonate is a suitable substitute (16). Growth is initiated at a carbon dioxide concentration between 4.5 x 10*3 M and 9 x 10’3 M, while optimal growth is achieved at 1.2 x 10‘3 M (16).

R. amylophilus possesses starch-degrading enzymes such as amylase and amylopectinase. The attachment between the cell and starch molecule is mediated by a protein or protein complex (51). R. amylophilus possesses enzymes of the Embden — Meyerhof-Pamas pathway (52). The presence of fumarate reductase enzyme suggests that succinate is produced by the reduction of fumarate (16). The production of succinate involves fixation of carbon dioxide which is incorporated as the carboxyl group (16). The pathway for succinate production is thought to be identical as the pathway for F. succinogenes shown in Figure 1.

Succinimonas amylolytica. Succinimonas amylolytica was isolated from bovine rumen (53). The organism is a gram negative, motile, anaerobic, non-sporeforming, short, rounded to coccoid bacterium 1.0 to 1.5 pm by 1.2 to 3 pm. S. amylolytica grows in a temperature range of 30 to 37°C and utilize glucose, maltose, starch or dextrin as a substrate. Fermentation products include succinate, acetate and trace propionate. The organisms grow well in media containing trypticase and yeast extract, and do not grow in the absence of either bicarbonate or carbon dioxide (53). The concentration of carbon dioxide required for the initiation of growth and optimal growth is at least 0.1% (15). S. amylolytica also requires acetate and other volatile fatty acids or casein hydrolysate (48). Even though S. amylolytica is normally associated with starch digestion in the rumen, the species possesses a wide range of glycoside hydrolases which aid in the utilization of plant cell wall degradation products (54).

Succinivibrio dextrinosolvens. Bryant and Small (55) first isolated Succinivibrio dextrinosolvens from a bovine rumen and described the species as an anaerobic, non­sporeforming, gram negative, mobile, helicoidal rod-shaped bacterium, 0.3 to 0.5 pm by 1 to 5 pm. The organisms can metabolize glucose, fructose, L-arabinose, D — xylose, galactose, maltose, sucrose, dextrins or pectins (55). S. dextrinosolvens cannot degrade cellulose, hemicellulose or starch, although the organism possesses a wide range of monosaccharide-generating glycoside hydrolases (54,55). Glucose fermentation yields principally acetate and succinate and is accompanied by significant carbon dioxide uptake (55). Formate is a minor product, while some strains also produce trace lactate (55). Similar organisms isolated from an ovine rumen by Wilson (56) were later also termed S. dextrinosolvens strains by Bryant (36).

S. dextrinosolvens has an absolute requirement for naphthoquinone, menadione or vitamin K5, with naphthoquinone resulting in best growth (57). S. dextrinosolvens possesses several nitrogen-assimilating enzymes such as urease, glutamate dehydrogenase and glutamine synthetase. Under ammonia limiting conditions the organism uses the ATP-driven glutamine synthetase system, while under excess ammonia the glutamate dehydrogenase enzyme is utilized (58). In addition to ammonia, S. dextrinosolvens requires an exogenous supply of amino acids to satisfy nitrogen requirements (48). In the absence of carbon dioxide, limited growth occurs after an extended lag phase (15). For optimal growth, a carbon dioxide concentration above 0.1% is required (55). S. dextrinosolvens requires volatile fatty acids and Na+ ion (which cannot be replaced by K+, Li+, Cs+ or Rb+). The Na+ concentration affects both the growth rate and the growth yield of S. dextrinosolvens (59).

The catabolic end products of glucose fermentation by S. dextrinosolvens are affected by the growth rate. Increased growth rate results in decreased succinate and acetate production and increased lactate formation (60). The pathway of succinate production appears to be identical to other rumen organisms already described (see Figure 1) with fixed carbon dioxide incorporated into the carboxyl group of succinate. S. dextrinosolvens has been shown to produce formate from free carbon dioxide present in the media (60).

Prevotella ruminocola. Bryant et al. (53) first isolated Prevotella ruminicola (previously named Bacteroides ruminicola) from bovine rumen and characterized the species as gram negative, non-motile, rod-shaped 0.8-1 pm by 0.8-3 pm, with slightly tapered, rounded ends (53). The organism recently was renamed Prevotella based on its genetic material (61). Several subspecies of P. ruminicola may be distinguished on the bases of morphology, substrates fermented and nutrient requirement. Most strains belonging to P. ruminicola subsp. ruminicola can utilize xylose, glucose and maltose. Some strains have the ability to hydrolyze starch and to utilize arabinose, sucrose and dextrins. Xylans and pectins are rapidly fermented by this particular subspecies (53). P. ruminicola subsp. ruminicola lacks the enzyme superoxide dismutase which is present in the subspecies brevis (62). Cells of P. ruminicola subsp. brevis are coccoid-to-oval shaped and do not require hemin (48,53). Most strains of this subspeices cannot utilize xylose and xylans, but can use pentoses as carbon sources. They also have the ability to hydrolyze starch and utilize maltose and sucrose (53).

P. ruminicola requires volatile fatty acids and acetate for growth. Use of casein hydrolysate can stimulate the growth of these organisms (48). As all other succinate producing rumen bacteria, P. ruminicola requires carbon dioxide for growth (63). The initiation of growth is achieved at 0.02-0.05% carbon dioxide, while above 0.1% optimal growth occurs (15). P. ruminicola exhibits proteolytic activity and possesses at least three different active proteinases (64).

P. ruminicola produces acetate, succinate and formate as products of sugar fermentation (53). In the presence of vitamin B12, strain 23 of P. ruminicola can also produce propionate (65). In this case the formation of propionate occurs via the direct reductive pathway (acrylate pathway) (66). P. ruminicola has low affinity for maltose, sucrose and cellobiose in comparison to glucose (67). The maintenance coefficient for P. ruminicola when grown on glucose is 0.135 g glucose /g cells • h (68). Glucose toxicity is observed with P. ruminicola strain B,4 (69). A pH below 5.7 halts the growth of P. ruminicola; however, growth is not significantly affected by pH changes above 5.7 (70).

P. ruminicola B{4 grows rapidly in a batch culture with a doubling time of 1.65 h (63). The conversion of glucose to phosphoenolpyruvate occurs via the Embden-Meyerhof pathway. The routes of synthesis for succinate and acetate are identical to previously described organisms (see Figure 1), with carbon dioxide again incorporated into the carboxyl group of succinate (63, 71).

Wolinella succinogenes. Wolinella succinogenes (previous named as Vibrio

succinogenes) was isolated by Wolin et al. (72) from bovine rumen and characterized as a curved rod-shaped, motile, anaerobic bacterium approximately 0.6 by 0.3 pm in size (72). A more recent study of the ribosomal ribonucleic acid content and evidence of the organism’s inability to ferment sugars resulted in their renaming to Wolinella succinogenes (73). W. succinogenes contains cytochromes which impart pink color to the cells. These organisms conserve energy by oxidation-reduction reaction in which hydrogen or formate acts as an electron donor and fumarate, malate, asparagine, nitrate, elemental sulfur or nitrous oxide acts as an electron acceptor (72,74,75). These oxidation-reduction reactions can be represented as (72, 75):

formate + H+ + fumarate —► C02 + succinate H2S + fumarate -► succinate + S formate + H+ + S -► C02 + H2S

Major fermentation products are carbon dioxide and succinate when these organism grow on fumarate and formate (72). Nitrous oxide can be reduced to nitrogen, nitrate to nitrite or ammonia, and elemental sulfur to hydrogen sulfide (74, 75).

W. succinogenes can use oxygen as an electron acceptor, but only at a low partial pressure of oxygen (72). At such low oxygen concentrations, hydrogen peroxide generated is degraded by peroxidase, but at high oxygen concentrations, hydrogen peroxide is hypothesized to accumulate and inhibit growth (76). W. succinogenes has a requirement for some succinate when grown on formate and nitrate. However, the presence of succinate does not appear to be necessary when the organisms are grown on fumarate, which is itself reduced to succinate (77).

The reduction of fumarate to succinate is associated with generation of ATP via electron transport phosphorylation (78). W. succinogenes has a doubling time of 3.2 h when grown on formate and fumarate, with growth yield of 4.8 g dry cell/mol formate (79). When grown on hydrogen sulfide and fumarate, the doubling time is 3.8 h and 6.0 g dry cell/mol fumarate is achieved. Use of formate and elemental sulfur results in a 1.2 h doubling time and a growth yield of 3.5 g dry cell/mol formate (75).

W. succinogenes can synthesize its cellular components from fumarate. Pyruvate acts as an intermediate for carbohydrates, nucleotides, phospholipids and for most of the amino acids. Glutamate is derived from a-ketoglutarate and further acts as a intermediate for the synthesis of amino acids belonging to its family (79).