Investigation of the fumarate metabolism of the syntrophic propionate-oxidizing bacterium strain MPOB

The growth of the syntrophic propionate-oxidizing bacterium strain MPOB in pure culture by fumarate disproportionation into carbon dioxide and succinate and by fumarate reduction with propionate, formate or hydrogen as electron donor was studied. The highest growth yield, 12.2 g dry cells/mol fumarate, was observed for growth by fumarate disproportionation. In the presence of hydrogen, formate or propionate, the growth yield was more than twice as low: 4.8, 4.6, and 5.2 g dry cells/mol fumarate, respectively. The location of enzymes that are involved in the electron transport chain during fumarate reduction in strain MPOB was analyzed. Fumarate reductase, succinate dehydrogenase, and ATPase were membrane-bound, while formate dehydrogenase and hydrogenase were loosely attached to the periplasmic side of the membrane. The cells contained cytochrome c, cytochrome b, menaquinone-6 and menaquinone-7 as possible electron carriers. Fumarate reduction with hydrogen in membranes of strain MPOB was inhibited by 2-(heptyl)-4-hydroxyquinoline-N-oxide (HOQNO). This inhibition, together with the activity of fumarate reductase with reduced 2,3-dimethyl-1,4-naphtoquinone (DMNH₂) and the observation that cytochrome b of strain MPOB was oxidized by fumarate, suggested that menequinone and cytochrome b are involved in the electron transport during fumarate reduction in strain MPOB. The growth yields of fumarate reduction with hydrogen or formate as electron donor were similar to the growth yield of Wolinella succinogenes. Therefore, it can be assumed that strain MPOB gains the same amount of ATP from fumarate reduction as W. succinogenes, i.e. 0.7 mol ATP/mol fumarate. This value supports the hypothesis that syntrophic propionate-oxidizing bacteria have to invest two-thirds of an ATP via reversed electron transport in the succinate oxidation step during the oxidation of propionate. The same electron transport chain that is involved in fumarate reduction may operate in the reversed direction to drive the energetically unfavourable oxidation of succinate during syntrophic propionate oxidation since (1) cytochrome b was reduced by succinate and (2) succinate oxidation was similarly inhibited by HOQNO as fumarate reduction. Received: 18 March 1997 / Accepted: 10 November 1997     

Glucose fermentation by Propionibacterium microaerophilum: effect of pH on metabolism and bioenergetic

pH affected significantly the growth and the glucose fermentation pattern of Propionibacterium microaerophilum. In neutral conditions (pH 6.5-7.5), growth and glucose fermentation rate (qs) were optimum producing propionate, acetate, CO(2), and formate [which together represented 90% (wt/wt) of the end products], and lactate representing only 10% (wt/wt) of the end products. In acidic conditions, propionate, acetate, and CO(2) represented nearly 100% (wt/wt) of the fermentation end products, whereas in alkaline conditions, a shift of glucose catabolism toward formate and lactate was observed, lactate representing 50% (wt/wt) of the fermentation end products. The energy cellular yields ( Y(X/ATP)), calculated (i) by taking into account extra ATP synthesized through the reduction of fumarate into succinate, was 6.1-7.2 g mol(-1). When this extra ATP was omitted, it was 11.9-13.1 g mol(-1). The comparison of these values with those of Y(X/ATP) in P. acidipropionici and other anaerobic bacteria suggested that P. microaerophilum could not synthesize ATP through the reduction of fumarate into succinate and therefore differed metabolically from P. acidipropionici.     

Succinic acid production by <Emphasis Type="Italic">Actinobacillus succinogenes</Emphasis> from batch fermentation of mixed sugars

Succinic acid production from the monosaccharides xylose, arabinose, glucose, mannose and galactose was studied using the bacterium Actinobacillus succinogenes. In Duran bottle cultures, containing 10 g/L of each of sugar, succinic acid was produced from all sugars except for galactose. The highest succinate yield, 0.56 g/g, was obtained with glucose, whereas the succinate yield was 0.42, 0.38 and 0.44 g/g for xylose, mannose and arabinose, respectively. The specific succinate productivity was 0.7 g/g h for glucose, but below 0.2 g/g h for the other sugars. Batch bioreactor fermentations were carried out using a sugar mixture of the five sugars giving a total concentration of 50 g/L, mimicking the distribution of sugars in spent sulfite liquor (SSL) from Eucalyptus which is rich in xylose. In this mixture, an almost complete conversion of all sugars (except galactose) was achieved resulting in a final succinate concentration of 21.8–26.8 g/L and a total yield of 0.59–0.68 g/g. There was evidence of co-consumption of glucose and xylose, whereas mannose was consumed after glucose. The main by-products were acetate 0.14–0.20 g/g and formate 0.08–0.13 g/g. NADH balance calculations suggested that NADH required for succinate production was not met solely from formate and acetate production, but other means of NADH production was necessary. Results from mixed sugar fermentations were verified using SSL as substrate resulting in a succinate yield of 0.60 g/g. In addition, it was found that CO₂ sparging could replace carbonate supply in the form of MgCO₃ without affecting the succinate yield.     

Glucose Fermentation by <Emphasis Type="Italic">Propionibacterium microaerophilum</Emphasis>: Effect of pH on Metabolism and Bioenergetic

pH affected significantly the growth and the glucose fermentation pattern of Propionibacterium microaerophilum. In neutral conditions (pH 6.5–7.5), growth and glucose fermentation rate (qs) were optimum producing propionate, acetate, CO₂, and formate [which together represented 90% (wt/wt) of the end products], and lactate representing only 10% (wt/wt) of the end products. In acidic conditions, propionate, acetate, and CO₂ represented nearly 100% (wt/wt) of the fermentation end products, whereas in alkaline conditions, a shift of glucose catabolism toward formate and lactate was observed, lactate representing 50% (wt/wt) of the fermentation end products. The energy cellular yields (Y _X/ATP), calculated (i) by taking into account extra ATP synthesized through the reduction of fumarate into succinate, was 6.1–7.2 g mol⁻¹. When this extra ATP was omitted, it was 11.9–13.1 g mol⁻¹. The comparison of these values with those of Y _X/ATP in P. acidipropionici and other anaerobic bacteria suggested that P. microaerophilum could not synthesize ATP through the reduction of fumarate into succinate and therefore differed metabolically from P. acidipropionici. Received: 8 April 2002 / Accepted: 8 May 2002     

pH and base counterion affect succinate production in dual-phase <Emphasis Type="Italic">Escherichia coli</Emphasis> fermentations

Succinate production was studied in Escherichia coli AFP111, which contains mutations in pyruvate formate lyase (pfl), lactate dehydrogenase (ldhA) and the phosphotransferase system glucosephosphotransferase enzyme II (ptsG). Two-phase fermentations using a defined medium at several controlled levels of pH were conducted in which an aerobic cell growth phase was followed by an anaerobic succinate production phase using 100% (v/v) CO₂. A pH of 6.4 yielded the highest specific succinate productivity. A metabolic flux analysis at a pH of 6.4 using ¹³C-labeled glucose showed that 61% of the PEP partitioned to oxaloacetate and 39% partitioned to pyruvate, while 93% of the succinate was formed via the reductive arm of the TCA cycle. The flux distribution at a pH of 6.8 was also analyzed and was not significantly different compared to that at a pH of 6.4. Ca(OH)₂ was superior to NaOH or KOH as the base for controlling the pH. By maintaining the pH at 6.4 using 25% (w/v) Ca(OH)₂, the process achieved an average succinate productivity of 1.42 g/l h with a yield of 0.61 g/g.     

Production of formate,acetate, and succinate by anaerobic fermentation of Lactobacillus pentosus in the presence of citrate

Summary The formation of acetate, formate and succinate was studied in Lactobacillus pentosus. These compounds were produced in addition to lactic acid when cells were exposed to anaerobic growth conditions with limited carbohydrates and in the presence of citrate. Citrate was metabolised via oxalacetate serving as an H-acceptor in a joint process together with lactate. The metabolism of citrate resulted in stoichiometric amounts of succinate and acetate. Lactate was degraded to formate and acetate in a reaction catalysed by pyruvate formate lyase. These fermentation products can potentially affect the flavour of fermented food but ecological factors in fermenting meat, e.g. the presence of glucose, nitrate or nitrite prevent this reaction. Offprint requests to: G. Wolf     

Kinetic evaluation of products inhibition to succinic acid producers <Emphasis Type="Italic">Escherichia coli</Emphasis> NZN111, AFP111, BL21, and <Emphasis Type="Italic">Actinobacillus succinogenes</Emphasis> 130Z<Superscript>T</Superscript>

Succinic acid is one of the platform compounds and its production via natural feedstocks has drawn worldwide concerns. To evaluate the inhibitory effects of fermentation products on the growth of Actinobacillus succinogenes 130Z^T and Escherichia coli NZN111, AFP111, BL21, fermentations with addition of individual products in medium were carried out. The cell growth was inhibited when the concentrations of formate, acetate, lactate, and succinate were at range of 8.8–17.6 g/L, 10–40 g/L, 9–18 g/L, and 10–80 g/L, respectively. For these two species of bacteria, E. coli was more resistant to acid products than A. succinogenes, while both endured succinate rather than by-products. As a result of end product inhibition, succinate production yield by A. succinogenes decreased from 1.11 to 0.49 g/g glucose. Logistic and Monod mathematical models were presented to simulate the inhibition kinetics. The Logistic model was found more suitable for describing the overall synergistic inhibitory effects.     

Impact of carbon sources on growth and oxalate synthesis by the phytopathogenic fungus <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

The impact of various supplemental carbon sources (oxalate, glyoxylate, glycolate, pyruvate, formate, malate, acetate, and succinate) on growth and oxalate formation (i.e., oxalogenesis) by Sclerotinia sclerotiorum was studied. With isolates D-E7, 105, W-B10, and Arg-L of S. sclerotiorum, growth in an undefined broth medium (0.1% soytone; pH 5) with 25 mM glucose and 25 mM supplemental carbon source was increased by the addition of malate and succinate. Oxalate accumulation occurred in the presence of glucose and a supplemental carbon source, with malate, acetate, and succinate supporting the most oxalate synthesis. With S. sclerotiorum Arg-L, oxalate-to-biomass ratios, an indicator of oxalogenic potential, were dissimilar when the organism was grown in the presence of different carbon sources. The highest oxalate-to-biomass ratios were observed with pyruvate, formate, malate, acetate, and succinate. Time-course studies with acetate-supplemented cultures revealed that acetate and glucose consumption by S. sclerotiorum D-E7 coincided with oxalogenesis and culture acidification. By day 5 of incubation, oxalogenesis was halted when cultures reached a pH of 3 and were devoid of acetate. In succinate-supplemented cultures, oxalogenesis essentially paralleled glucose and succinate utilization over the 9-day incubation period; during this time period, culture pH declined but never fell below 4. Overall, these results indicate that carbon sources can regulate the accumulation of oxalate, a key pathogenicity determinant for S. sclerotiorum.     

The influence of acetic acid on penicillin production

Summary Fed-batch fermentations with Penicillum chrysogenum, strain S 3723, were fed with glucose as carbon source or with a mixture of glucose and acetic acid. When 20% of the carbon source was acetic acid, yields of penicillin-V were 25% higher than in fermentations where glucose was the only carbon source in the feed. The increased yield was due to higher specific productivity and/or cell mass. The effect was seen in fermentations where the carbon source was fed at a constant rate and the pH kept automatically at 6.5 by addition of inorganic acid or base, as well as in fermentations where pH controlled the addition of feed.     

Substrate and product inhibition kinetics in succinic acid production by Actinobacillus succinogenes

The inhibition of substrate and products on the growth of Actinobacillus succinogenes in fermentation using glucose as the major carbon source was studied. A. succinogenes tolerated up to 143 g/L glucose and cell growth was completely inhibited with glucose concentration over 158 g/L. Significant decrease in succinic acid yield and prolonged lag phase were observed with glucose concentration above 100 g/L. Among the end-products investigated, formate was found to have the most inhibitory effect on succinic acid fermentation. The critical concentrations of acetate, ethanol, formate, pyruvate and succinate were 46, 42, 16, 74, 104 g/L, respectively. A growth kinetic model considering both substrate and product inhibition is proposed, which adequately simulates batch fermentation kinetics using both semi-defined and wheat-derived media. The model accurately describes the inhibitory kinetics caused by both externally added chemicals and the same chemicals produced during fermentation. This paper provides key insights into the improvement of succinic acid production and the modelling of inhibition kinetics.     

Quantitative assessment of anaplerosis from propionate in pig heart in vivo

Normal cardiac metabolism requires continuous replenishment (anaplerosis) of catalytic intermediates of the citric acid cycle. Little is known about the quantitative aspects of propionate as a substrate of in vivo anaplerosis; therefore, we measured the rate of propionate entry into the citric acid cycle in hearts of anesthetized pigs. [U-(13)C(3)]propionate (0.25 mM) was infused in a coronary artery branch for 1 h via an extracorporeal perfusion circuit, and cardiac biopsies were analyzed for the mass isotopomer distribution of citric acid cycle intermediates. Infusion of propionate did not affect myocardial oxygen consumption, heart rate, or contractile function. In the infused territory, propionate infusion did not affect uptake of glucose and lactate but decreased free fatty acid uptake by one-half (P < 0.05). Propionate extraction and uptake were 57.4 +/- 3.3% and 0.078 +/- 0.009 micromol x min(-1) x g(-1). Anaplerosis from propionate, calculated from the mass isotopomer distribution of succinate, accounted for 8.9 +/- 1.3% of the citric acid cycle flux. Propioylcarnitine release accounted for only 0.033 +/- 0.002% of propionate uptake. Methylcitrate did not accumulate. Thus administration of a low concentration of propionate appears to be a convenient and safe way to boost anaplerosis in the heart.     

Engineering Escherichia coli for propionic acid production through the Wood–Werkman cycle

Native to propionibacteria, the Wood–Werkman cycle enables propionate production via succinate decarboxylation. Current limitations in engineering propionibacteria strains have redirected attention toward the heterologous production in model organisms. Here, we report the functional expression of the Wood–Werkman cycle in Escherichia coli to enable propionate and 1-propanol production. The initial proof-of-concept attempt showed that the cycle can be used for production. However, production levels were low (0.17 mM). In silico optimization of the expression system by operon rearrangement and ribosomal-binding site tuning improved performance by fivefold. Adaptive laboratory evolution further improved performance redirecting almost 30% of total carbon through the Wood–Werkman cycle, achieving propionate and propanol titers of 9 and 5 mM, respectively. Rational engineering to reduce the generation of byproducts showed that lactate (∆ldhA) and formate (∆pflB) knockout strains exhibit an improved propionate and 1-propanol production, while the ethanol (∆adhE) knockout strain only showed improved propionate production.     

Glucose overflow metabolism and mixed-acid fermentation in aerobic large-scale fed-batch processes with Escherichia coli

Industrial 20-m³-scale and laboratory-scale aerobic fed-batch processes with Escherichia coli were compared. In the large-scale process the observed overall biomass yield was reduced by 12% at a cell density of 33 g/l and formate accumulated to 50 mg/l during the later constant-feeding stage of the process. Though the dissolved oxygen signal did not show any oxygen limitation, it is proposed that the lowered yield and the formate accumulation are caused by mixed-acid fermentation in local zones where a high glucose concentration induced oxygen limitation. The hypothesis was further investigated in a scale-down reactor with a controlled oxygen-limitation compartment. In this scale-down reactor similar results were obtained: i.e. an observed yield lowered by 12% and formate accumulation to 238 mg/l. The dynamics of glucose uptake and mixed-acid product formation (acetate, formate, d-lactate, succinate and ethanol) were investigated within the 54 s of passage time through the oxygen-limited compartment. Of these, all except succinate and ethanol were formed; however, the products were re-assimilated in the oxygen-sufficient reactor compartment. Formate was less readily assimilated, which accounts for its accumulation. The total volume of the induced-oxygen-limited zones was estimated to be 10% of the whole liquid volume in the large bioreactor. It is also suggested that repeated excretion and re-assimilation of mixed-acid products contribute to the reduced yield during scale-up and that formate analysis is useful for detecting local oxygen deficiency in large-scale E. coli processes. Received: 7 November 1998 / Received revision: 4 February 1999 / Accepted: 5 February 1999     

Effects of DL-Malate on In Vitro Forage Fiber Digestion by Mixed Ruminal Microorganisms

The objective of this study was to evaluate the effects of 0, 4, 8, and 12 mM DL-malate on the in vitro mixed ruminal microorganism fermentation of alfalfa hay and Coastal bermudagrass hay. When alfalfa hay was the substrate, 4 and 8 mM DL-malate numerically increased propionate concentration, and 12 mM DL-malate increased (P < 0.10) propionate. All three concentrations of DL-malate decreased (P < 0.05) the acetate:propionate ratio. In Coastal bermudagrass hay fermentations, all three DL-malate concentrations increased (P < 0.05) propionate and decreased (P < 0.05) the acetate:propionate ratio, while 4 and 12 mM DL-malate numerically increased in vitro dry matter disappearance. When mixed ruminal microorganisms were incubated with 6.25 mM DL-lactic acid and alfalfa hay, 8 and 12 mM DL-malate increased (P < 0.05) final pH, and 12 mM DL-malate increased (P < 0.10) propionate and decreased (P < 0.10) the acetate:propionate ratio. DL-Malate treatment had little effect on in vitro dry matter disappearance. Addition of 8 and 12 mM DL-malate to Coastal bermudagrass hay plus DL-lactic acid fermentations increased (P < 0.05) final pH, and 8 mM DL-malate increased (P < 0.10) in vitro dry matter disappearance. Even though DL-malate treatment consistently increased final pH values in fermentations that included DL-lactic acid, there was not a corresponding increase in in vitro dry matter disappearance of either alfalfa hay or Coastal bermudagrass hay in the 48-h batch culture incubations.     

Efficient succinic acid production from glucose through overexpression of pyruvate carboxylase in an Escherichia coli alcohol dehydrogenase and lactate dehydrogenase mutant

An adhE, ldhA double mutant Escherichia coli strain, SBS110MG, has been constructed to produce succinic acid in the presence of heterologous pyruvate carboxylase (PYC). The strategic design aims at diverting maximum quantities of NADH for succinate synthesis by inactivation of NADH competing pathways to increase succinate yield and productivity. Additionally an operational PFL enzyme allows formation of acetyl-CoA for biosynthesis and formate as a potential source of reducing equivalents. Furthermore, PYC diverts pyruvate toward OAA to favor succinate generation. SBS110MG harboring plasmid pHL413, which encodes the heterologous pyruvate carboxylase from Lactococcus lactis, produced 15.6 g/L (132 mM) of succinate from 18.7 g/L (104 mM) of glucose after 24 h of culture in an atmosphere of CO(2) yielding 1.3 mol of succinate per mole of glucose. This molar yield exceeded the maximum theoretical yield of succinate that can be achieved from glucose (1 mol/mol) under anaerobic conditions in terms of NADH balance. The current work further explores the importance of the presence of formate as a source of reducing equivalents in SBS110MG(pHL413). Inactivation of the native formate dehydrogenase pathway (FDH) in this strain significantly reduced succinate yield, suggesting that reducing power was lost in the form of formate. Additionally we investigated the effect of ptsG inactivation in SBS110MG(pHL413) to evaluate the possibility of a further increase in succinate yield. Elimination of the ptsG system increased the succinate yield to 1.4 mol/mol at the expense of a reduction in glucose consumption of 33%. In the presence of PYC and an efficient conversion of glucose to products, the ptsG mutation is not indispensable since PEP converted to pyruvate as a result of glucose phosphorylation by the glucose specific PTS permease EIICB(glu) can be rediverted toward OAA favoring succinate production.     

Measurement and analysis of intracellular ATP levels in metabolically engineered Zymomonas mobilis fermenting glucose and xylose mixtures

Intracellular adenosine-5'-triphosphate (ATP) levels were measured in a metabolically engineered Zymomonas mobilis over the course of batch fermentations of glucose and xylose mixtures. Fermentations were conducted over a range of pH (5-6) in the presence of varying initial amounts of acetic acid (0-8 g/L) using a 10% (w/v) total sugar concentration (glucose only, xylose only, or 5% glucose/5% xylose mixture). Over the design space investigated, ethanol process yields varied between 56.6% and 92.3% +/- 1.3% of theoretical, depending upon the test conditions. The large variation in process yields reflects the strong effect pH plays in modulating the inhibitory effect of acetic acid on fermentation performance. A corresponding effect was observed on maximum cellular specific growth rates, with the rates varying between a low of 0.15 h(-1) observed at pH 5 in the presence of 8 g/L acetic acid to a high of 0.32 +/- 0.02 h(-1) obtained at pH 5 or 6 when no acetic acid was initially present. While substantial differences were observed in intracellular specific ATP concentration profiles depending upon fermentation conditions, maximum intracellular ATP accumulation levels varied within a relatively narrow range (1.5-3.8 mg ATP/g dry cell mass). Xylose fermentations produced and accumulated ATP at much slower rates than mixed sugar fermentations (5% glucose, 5% xylose), and the ATP production and accumulation rates in the mixed sugar fermentations were slightly slower than in glucose fermentations. Results demonstrate that higher levels of acetic acid delay the onset and influence the extent of intracellular ATP accumulation. ATP production and accumulation rates were most sensitive to acetic acid at lower values of pH.     

Effect of specific oxygen uptake rate on Enterobacter aerogenes energetics: carbon and reduction degree balances in batch cultivations

The effect of oxygen availability on the metabolism of Enterobacter aerogenes NCIMB 10102 was studied through batch fermentations of glucose performed increasing the specific oxygen uptake rate up to 72.7 mmol(O2) C-mol(DW) (-1) x h(-1). The final concentrations of fermentation products of this biosystem (2,3-butanediol, hydrogen, acetoin, formate, acetate, carbon dioxide, ethanol, lactate, succinate, and biomass) were utilized to check the use of simple carbon mass and reduction degree balances for the study of microbial energetics even in batch cultivations.     

The role of carbon dioxide in glucose metabolism of Bacteroides fragilis

The effect of CO₂ concentration on growth and glucose fermentation of Bacteroides fragilis was studied in a defined mineral medium. Batch culture experiments were done in closed tubes containing CO₂ concentrations ranging from 10% to 100% (with appropriate amounts of bicarbonate added to maintain the pH at 6.7). These experiments revealed that CO₂ had no influence on growth rate or cell yield when the CO₂ concentration was above 30% CO₂ (minimum available CO₂–HCO ₃ ^- , 25.5 mM), whereas a slight decrease in these parameters was observed at 20% and 10% CO₂ (available CO₂–HCO ₃ ^- , 17 and 8.5 mM, respectively). If CO₂–HCO ₃ ^- concentrations were below 10 mM, the lag phase lengthened and a decrease in maximal growth rate and cell yield were observed. The amount of acetate made decreased, while d-lactate concentration increased. A net production of CO₂ allowed growth under conditions of extremely low concentrations of added CO₂.When B. fragilis was grown in continuous culture with 100% CO₂ or 100% N₂, the dilution rate influenced the concentrations of acetate, succinate, propionate, d-lactate, l-malate and formate formed. Decreasing the dilution rate favored propionate and acetate production under both conditions. When the organism was grown with 100% N₂, the amount of propionate formed was greater than the amount of succinate formed at all dilution rates. Except at slow dilution rates the reverse was true when 100% CO₂ was used. B. fragilis was unable to grow at dilution rates faster than 0.154 h^-1 when grown with 100% N₂; the Y _glc ^max was 67.9 g DW cells/mol glucose and m _s was 0.064 mmol glucose/g DW·h. If the gas atmosphere was 100% CO₂ the organism was washed out of the culture when the dilution rate exceeded 0.38 h^-1; the Y _glc ^max was 59.4 g DW cells/mol glucose and m _s was 0.094 mmol glucose/g DW·h.Measurement of the phosphoenolpyruvate (PEP) carboxykinase (E.C. 4.1.1.49) with whole, permeabilized cells of B. fragilis showed an increase of specific enzyme activity with decreasing CO₂ concentrations. The mechanisms used by B. fragilis to adjust to low levels of CO₂ are discussed.     

Clostridium grantii sp. nov., a new obligately anaerobic,alginolytic bacterium isolated from mullet gut

A gram-positive, motile, rod-shaped, strictly anaerobic, sporulating bacterium was isolated from an enrichment initiated with mullet gut contents. The organism grew optimally at 30°C and pH6.5, and at a salinity of 1–10³. Out of a variety of polysaccharides tested as growth substrates, only alginate supported growth in either semidefined or complex culture medium. The organism also grew on a variety of mono- and disaccharides. Moles product per 100mol of alginate monomer degraded were: acetate, 186; ethanol, 19; formate, 54; and CO₂, 0.19. Moles product per 100mol of hexose in cellobiose or glucose degraded were: acetate, 135; ethanol,61; formate, 63: and CO₂, 61. Hydrogen was not detectable during the incubations (detection limit, <10^-5atm) and propionate, butyrate, lactate, or succinate were not produced as fermentation end products (<2 mol per 100 mol of monomer). The G+C content of DNA from the bacterium was 30.2±0.3 mol%, and the cell walls contained the peptidoglycan component meso-diaminopimelic acid. A phylogenetic analysis of the 16S rDNA sequence indicated that the organism grouped closely with members of the RNA-DNA homology group 1 of the genus Clostridium. However, it differed from other species of the genus with regard to morphology, growth temperature optimum, substrate range, and fermentation pattern and is therefore designated as a new species of Clostridium; the type strain is A-1 (DSM 8605).     

Evaluation of succinic acid continuous and repeat-batch biofilm fermentation by <Emphasis Type="Italic">Actinobacillus succinogenes</Emphasis> using plastic composite support bioreactors

Continuous and repeat-batch biofilm fermentations using Actinobacillus succinogenes were performed with immobilized and suspended-cell systems. For the immobilized continuous system, plastic composite supports (PCS) containing 50% (w/w) polypropylene (PP), 35% (w/w) ground soybean hulls, 5% (w/w) dried bovine albumin, 2.5% (w/w) soybean flour, 2.5% (w/w) yeast extract, 2.5% (w/w) dried red blood cells, and 2.5% (w/w) peptone, or PP tubes (8.5 cm in length) were arranged around the agitator shaft in a grid formation. Agitation was controlled at 125 rpm and 150 rpm. Samples were taken at dilution rates of 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 h^–1 and analyzed for succinic acid production and glucose consumption (g l^–1). For PCS bioreactors, the highest final succinic acid concentrations (10.1 g ^–1, 10.4 g l^–1) and percentage yields (62.6%, 71.6%) occurred at the dilution rate of 0.2 h^–1. PCS disks were evaluated in a repeat-batch biofilm reactor. Suspended-cell batch fermentations were performed in flasks and a repeat-batch bioreactor. The maximum concentration of succinic acid produced was 40 g l^–1. Peak succinic acid percentage yields in continuous and repeat-batch fermentations of A. succinogenes were observed in suspended-cell continuous fermentations at a dilution rate of 1.0 h^–1 (76.2%) and in PCS repeat-batch fermentations with an initial glucose concentration of 40 g l^–1 (86.7%).