Influence of CO(2)-HCO(3) Levels and pH on Growth, Succinate Production, and Enzyme Activities of Anaerobiospirillum succiniciproducens

Growth and succinate versus lactate production from glucose by Anaerobiospirillum succiniciproducens was regulated by the level of available carbon dioxide and culture pH. At pH 7.2, the generation time was almost doubled and extensive amounts of lactate were formed in comparison with growth at pH 6.2. The succinate yield and the yield of ATP per mole of glucose were significantly enhanced under excess-CO(2)-HCO(3) growth conditions and suggest that there exists a threshold level of CO(2) for enhanced succinate production in A. succiniciproducens. Glucose was metabolized via the Embden-Meyerhof-Parnas route, and phosphoenolpyruvate carboxykinase levels increased while lactate dehydrogenase and alcohol dehydrogenase levels decreased under excess-CO(2)-HCO(3) growth conditions. Kinetic analysis of succinate and lactate formation in continuous culture indicated that the growth rate-linked production rate coefficient (K) cells was much higher for succinate (7.2 versus 1.0 g/g of cells per h) while the non-growth-rate-related formation rate coefficient (K') was higher for lactate (1.1 versus 0.3 g/g of cells per h). The data indicate that A. succiniciproducens, unlike other succinate-producing anaerobes which also form propionate, can grow rapidly and form high final yields of succinate at pH 6.2 and with excess CO(2)-HCO(3) as a consequence of regulating electron sink metabolism.     

Whey Fermentation by Anaerobiospirillum succiniciproducens for Production of a Succinate-Based Animal Feed Additive

Anaerobic fermentation processes for the production of a succinate-rich animal feed supplement from raw whey were investigated with batch, continuous, and variable-volume fed-batch cultures with Anaerobiospirillum succiniciproducens. The highest succinate yield, 90%, was obtained in a variable-volume fed-batch process in comparison to 80% yield in a batch cultivation mode. In continuous culture, succinate productivity was 3 g/liter/h, and the yield was 60%. Under conditions of excess CO₂, more than 90% of the whey-lactose was consumed, with an end product ratio of 4 succinate to 1 acetate. Under conditions of limited CO₂, lactose was only partially consumed and lactate was the major end product, with lower levels of ethanol, succinate, and acetate. When the succinic acid in this fermentation product was added to rumen fluid, it was completely consumed by a mixed rumen population and was 90% decarboxylated to propionate on a molar basis. The whey fermentation product formed under excess CO₂, which contained mainly organic acids and cells, could potentially be used as an animal feed supplement.     

Effects of Eliminating Pyruvate Node Pathways and of Coexpression of Heterogeneous Carboxylation Enzymes on Succinate Production by Enterobacter aerogenes

Lowering the pH in bacterium-based succinate fermentation is considered a feasible approach to reduce total production costs. Newly isolated Enterobacter aerogenes strain AJ110637, a rapid carbon source assimilator under weakly acidic (pH 5.0) conditions, was selected as a platform for succinate production. Our previous work showed that the ΔadhE/PCK strain, developed from AJ110637 with inactivated ethanol dehydrogenase and introduced Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PCK), generated succinate as a major product of anaerobic mixed-acid fermentation from glucose under weakly acidic conditions (pH <6.2). To further improve the production of succinate by the ΔadhE/PCK strain, metabolically engineered strains were designed based on the elimination of pathways that produced undesirable products and the introduction of two carboxylation pathways from phosphoenolpyruvate and pyruvate to oxaloacetate. The highest production of succinate was observed with strain ES04/PCK+PYC, which had inactivated ethanol, lactate, acetate, and 2,3-butanediol pathways and coexpressed PCK and Corynebacterium glutamicum pyruvate carboxylase (PYC). This strain produced succinate from glucose with over 70% yield (gram per gram) without any measurable formation of ethanol, lactate, or 2,3-butanediol under weakly acidic conditions. The impact of lowering the pH from 7.0 to 5.5 on succinate production in this strain was evaluated under pH-controlled batch culture conditions and showed that the lower pH decreased the succinate titer but increased its yield. These findings can be applied to identify additional engineering targets to increase succinate production.     

The Effect of pH, O(2), and Temperature on the CO(2) Compensation Point of Isolated Asparagus Mesophyll Cells

The effect of pH, O₂ concentration, and temperature on the CO₂ compensation point (Г[CO₂]) of isolated Asparagus sprengeri Regel mesophyll cells has been determined in a closed, aqueous environment by a sensitive gas-chromatographic technique. Measured values range between 10 and 100 microliters per liter CO₂ depending upon experimental conditions. The Г(CO₂) increases with increasing temperature. The rate of increase is dependent upon the O₂ concentration and is more rapid at high (250-300 micromolar), than at low (30-60 micromolar), O₂ concentrations. The differential effect of temperature on Г(CO₂) is more pronounced at pH 6.2 than at pH 8.0, but this pH-dependence is not attributable to a direct, differential effect of pH on the relative rates of photosynthesis and photorespiration, as the O₂-sensitive component of Г(CO₂) remains constant over this range. The Г(CO₂) of Asparagus cells at 25°C decreases by 50 microliters per liter when the pH is raised from 6.2 to 8.0, regardless of the prevailing O₂ concentration. It is suggested that the pH-dependence of Г(CO₂) is related to the ability of the cell to take up CO₂ from the aqueous environment. The correlation between high HCO₃⁻ concentrations and low Г(CO₂) at alkaline pH indicates that extracellular HCO₃⁻ facilitates the uptake of CO₂, possibly by increasing the flux of inorganic carbon from the bulk medium to the cell surface. The strong O₂− and temperature-dependence of Г(CO₂) indicates that isolated Asparagus mesophyll cells lack an efficient means for concentrating intracellular CO₂ to a level sufficient to reduce or suppress photorespiration.     

Homolactate Fermentation by Metabolically Engineered Escherichia coli Strains

We report the homofermentative production of lactate in Escherichia coli strains containing mutations in the aceEF, pfl, poxB, and pps genes, which encode the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, and phosphoenolpyruvate synthase, respectively. The process uses a defined medium and two distinct fermentation phases: aerobic growth to an optical density of about 30, followed by nongrowth, anaerobic production. Strain YYC202 (aceEF pfl poxB pps) generated 90 g/liter lactate in 16 h during the anaerobic phase (with a yield of 0.95 g/g and a productivity of 5.6 g/liter · h). Ca(OH)₂ was found to be superior to NaOH for pH control, and interestingly, significant succinate also accumulated (over 7 g/liter) despite the use of N₂ for maintaining anaerobic conditions. Strain ALS961 (YYC202 ppc) prevented succinate accumulation, but growth was very poor. Strain ALS974 (YYC202 frdABCD) reduced succinate formation by 70% to less than 3 g/liter. ¹³C nuclear magnetic resonance analysis using uniformly labeled acetate demonstrated that succinate formation by ALS974 was biochemically derived from acetate in the medium. The absence of uniformly labeled succinate, however, demonstrated that glyoxylate did not reenter the tricarboxylic acid cycle via oxaloacetate. By minimizing the residual acetate at the time that the production phase commenced, the process with ALS974 achieved 138 g/liter lactate (1.55 M, 97% of the carbon products), with a yield of 0.99 g/g and a productivity of 6.3 g/liter · h during the anaerobic phase.     

Increased Feeding and Nutrient Excretion of Adult Antarctic Krill,Euphausia superba,Exposed to Enhanced Carbon Dioxide (CO2)

Ocean acidification has a wide-ranging potential for impacting the physiology and metabolism of zooplankton. Sufficiently elevated CO₂ concentrations can alter internal acid-base balance, compromising homeostatic regulation and disrupting internal systems ranging from oxygen transport to ion balance. We assessed feeding and nutrient excretion rates in natural populations of the keystone species Euphausia superba (Antarctic krill) by conducting a CO₂ perturbation experiment at ambient and elevated atmospheric CO₂ levels in January 2011 along the West Antarctic Peninsula (WAP). Under elevated CO₂ conditions (∼672 ppm), ingestion rates of krill averaged 78 µg C individual⁻¹ d⁻¹ and were 3.5 times higher than krill ingestion rates at ambient, present day CO₂ concentrations. Additionally, rates of ammonium, phosphate, and dissolved organic carbon (DOC) excretion by krill were 1.5, 1.5, and 3.0 times higher, respectively, in the high CO₂ treatment than at ambient CO₂ concentrations. Excretion of urea, however, was ∼17% lower in the high CO₂ treatment, suggesting differences in catabolic processes of krill between treatments. Activities of key metabolic enzymes, malate dehydrogenase (MDH) and lactate dehydrogenase (LDH), were consistently higher in the high CO₂ treatment. The observed shifts in metabolism are consistent with increased physiological costs associated with regulating internal acid-base equilibria. This represents an additional stress that may hamper growth and reproduction, which would negatively impact an already declining krill population along the WAP.     

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.     

HCO3− Fixation by Naturally Occurring Tufts and Pure Cultures of Thiothrix nivea

Naturally occurring tufts of the mixotroph Thiothrix nivea blanketed the East Everglades (Dade County, Fla.) Chekika artesian well and runoff areas. The rate of HCO₃⁻ fixation by these Thiothrix tufts was determined to be 14.0 ± 5.4 nmol of HCO₃⁻ per min per mg of dry weight, which reflected a growth rate of 5.0%/h. The addition of 10 mM glucose, ribose, acetate, or pyruvate or 0.05% Casamino Acids (Difco Laboratories, Detroit, Mich.) did not appear to alter the HCO₃⁻ fixation rate. Whereas 1 mM acetate or 10 mM lactate, ethanol, glycerol, α-ketoglutarate, succinate, fumarate, or citrate slightly stimulated HCO₃⁻ fixation, 5 to 10 mM malate inhibited HCO₃⁻ fixation by 90%. Pure Thiothrix cultures isolated from Chekika fixed HCO₃⁻ at rates as high as 29.9 ± 2.8 nmol of HCO₃⁻ per min per mg of dry weight in the presence of growth medium. Malate did not have a suppressive effect but rather slightly stimulated in vivo HCO₃⁻ fixation.     

<Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> NAD-dependent, <Emphasis Type="SmallCaps">D</Emphasis>-fructose reducing,polyol dehydrogenases activity: screening,medium optimisation and application for enzymatic polyol production

Gluconobacter oxydans LMG 1489 was selected as the best strain for NAD(P)-dependent polyol dehydrogenase production. The highest enzyme activities were obtained when this strain was cultivated on a medium consisting of 30 g glycerol l^–1, 7.2 g peptone l–1 and 1.8 g yeast extract l^–1. Two D-fructose reducing, NAD-dependent intracellular enzymes were present in the G. oxydans cell-free extract: sorbitol dehydrogenase, and mannitol dehydrogenase. Substrate reduction occurred optimally at a low pH (pH 6), while the optimum for substrate oxidation was situated at alkaline pHs (pH 9.5–10.5). The mannitol dehydrogenase was more thermostable than the sorbitol dehydrogenase. The cell-free extract could be used to produce D-mannitol and D-sorbitol enzymatically from D-fructose. Efficient coenzyme regeneration was accomplished by formate dehydrogenase-mediated oxidation of formate into CO₂.     

Relationship between Succinate Transport and Production of Extracellular Poly(3-Hydroxybutyrate) Depolymerase in Pseudomonas lemoignei

The relationship between extracellular poly(3-hydroxybutyrate) (PHB) depolymerase synthesis and the unusual properties of a succinate uptake system was investigated in Pseudomonas lemoignei. Growth on and uptake of succinate were highly pH dependent, with optima at pH 5.6. Above pH 7, growth on and uptake of succinate were strongly reduced with concomitant derepression of PHB depolymerase synthesis. The specific succinate uptake rates were saturable by high concentrations of succinate, and maximal transport rates of 110 nmol/mg of cell protein per min were determined between pH 5.6 and 6.8. The apparent K_S0.5 values increased with increasing pH from 0.2 mM succinate at pH 5.6 to more than 10 mM succinate at pH 7.6. The uptake of [¹⁴C]succinate was strongly inhibited by several monocarboxylates. Dicarboxylates also inhibited the uptake of succinate but only at pH values near the dissociation constant of the second carboxylate function (pK_a2). We conclude that the succinate carrier is specific for the monocarboxylate forms of various carboxylic acids and is not able to utilize the dicarboxylic forms. The inability to take up succinate²⁻ accounts for the carbon starvation of P. lemoignei observed during growth on succinate at pH values above 7. As a consequence the bacteria produce high levels of extracellular PHB depolymerase activity in an effort to escape carbon starvation by utilization of PHB hydrolysis products.     

Genome-Based Metabolic Engineering of Mannheimia succiniciproducens for Succinic Acid Production

Succinic acid is a four-carbon dicarboxylic acid produced as one of the fermentation products of anaerobic metabolism. Based on the complete genome sequence of a capnophilic succinic acid-producing rumen bacterium, Mannheimia succiniciproducens, gene knockout studies were carried out to understand its anaerobic fermentative metabolism and consequently to develop a metabolically engineered strain capable of producing succinic acid without by-product formation. Among three different CO₂-fixing metabolic reactions catalyzed by phosphoenolpyruvate (PEP) carboxykinase, PEP carboxylase, and malic enzyme, PEP carboxykinase was the most important for the anaerobic growth of M. succiniciproducens and succinic acid production. Oxaloacetate formed by carboxylation of PEP was found to be converted to succinic acid by three sequential reactions catalyzed by malate dehydrogenase, fumarase, and fumarate reductase. Major metabolic pathways leading to by-product formation were successfully removed by disrupting the ldhA, pflB, pta, and ackA genes. This metabolically engineered LPK7 strain was able to produce 13.4 g/liter of succinic acid from 20 g/liter glucose with little or no formation of acetic, formic, and lactic acids, resulting in a succinic acid yield of 0.97 mol succinic acid per mol glucose. Fed-batch culture of M. succiniciproducens LPK7 with intermittent glucose feeding allowed the production of 52.4 g/liter of succinic acid, with a succinic acid yield of 1.16 mol succinic acid per mol glucose and a succinic acid productivity of 1.8 g/liter/h, which should be useful for industrial production of succinic acid.     

Chlorophyll a Fluorescence and Photosynthetic and Growth Responses of Pinus radiata to Phosphorus Deficiency, Drought Stress, and High CO(2)

Needles from phosphorus deficient seedlings of Pinus radiata D. Don grown for 8 weeks at either 330 or 660 microliters CO₂ per liter displayed chlorophyll a fluorescence induction kinetics characteristic of structural changes within the thylakoid chloroplast membrane, i.e. constant yield fluorescence (F_O) was increased and induced fluorescence ([F_P-F_I]/F_O) was reduced. The effect was greatest in the undroughted plants grown at 660 μl CO₂ L⁻¹. By week 22 at 330 μl CO₂ L⁻¹ acclimation to P deficiency had occurred as shown by the similarity in the fluorescence characteristics and maximum rates of photosynthesis of the needles from the two P treatments. However, acclimation did not occur in the plants grown at 660 μl CO₂ L⁻¹. The light saturated rate of photosynthesis of needles with adequate P was higher at 660 μl CO₂ L⁻¹ than at 330 μl CO₂ L⁻¹, whereas photosynthesis of P deficient plants showed no increase when grown at the higher CO₂ concentration. The average growth increase due to CO₂ enrichment was 14% in P deficient plants and 32% when P was adequate. In drought stressed plants grown at 330 μl CO₂ L⁻¹, there was a reduction in the maximal rate of quenching of fluorescence (R_Q) after the major peak. Constant yield fluorescence was unaffected but induced fluorescence was lower. These results indicate that electron flow subsequent to photosystem II was affected by drought stress. At 660 μl CO₂ L⁻¹ this response was eliminated showing that CO₂ enrichment improved the ability of the seedlings to acclimate to drought stress. The average growth increase with CO₂ enrichment was 37% in drought stressed plants and 19% in unstressed plants.     

Efficient Homolactic Fermentation by Kluyveromyces lactis Strains Defective in Pyruvate Utilization and Transformed with the Heterologous LDH Gene

A high yield of lactic acid per gram of glucose consumed and the absence of additional metabolites in the fermentation broth are two important goals of lactic acid production by microrganisms. Both purposes have been previously approached by using a Kluyveromyces lactis yeast strain lacking the single pyruvate decarboxylase gene (KlPDC1) and transformed with the heterologous lactate dehydrogenase gene (LDH). The LDH gene was placed under the control the KlPDC1 promoter, which has allowed very high levels of lactate dehydrogenase (LDH) activity, due to the absence of autoregulation by KlPdc1p. The maximal yield obtained was 0.58 g g⁻¹, suggesting that a large fraction of the glucose consumed was not converted into pyruvate. In a different attempt to redirect pyruvate flux toward homolactic fermentation, we used K. lactis LDH transformant strains deleted of the pyruvate dehydrogenase (PDH) E1α subunit gene. A great process improvement was obtained by the use of producing strains lacking both PDH and pyruvate decarboxylase activities, which showed yield levels of as high as 0.85 g g⁻¹ (maximum theoretical yield, 1 g g⁻¹), and with high LDH activity.     

Two Systems for Concentrating CO(2) and Bicarbonate during Photosynthesis by Scenedesmus

Scenedesmus cells grown on high CO₂, when adapted to air levels of CO₂ for 4 to 6 hours in the light, formed two concentrating processes for dissolved inorganic carbon: one for utilizing CO₂ from medium of pH 5 to 8 and one for bicarbonate accumulation from medium of pH 7 to 11. Similar results were obtained with assays by photosynthetic O₂ evolution or by accumulation of dissolved inorganic carbon inside the cells. The CO₂ pump with K_0.5 for O₂ evolution of less than 5 micromolar CO₂ was similar to that previously studied with other green algae such as Chlamydomonas and was accompanied by plasmalemma carbonic anhydrase formation. The HCO₃⁻ concentrating process between pH 8 to 10 lowered the K_0.5 (DIC) from 7300 micromolar HCO₃⁻ in high CO₂ grown Scenedesmus to 10 micromolar in air-adapted cells. The HCO₃⁻ pump was inhibited by vanadate (K_i of 150 micromolar), as if it involved an ATPase linked HCO₃⁻ transporter. The CO₂ pump was formed on low CO₂ by high-CO₂ grown cells in growth medium within 4 to 6 hours in the light. The alkaline HCO₃⁻ pump was partially activated on low CO₂ within 2 hours in the light or after 8 hours in the dark. Full activation of the HCO₃⁻ pump at pH 9 had requirements similar to the activation of the CO₂ pump. Air-grown or air-adapted cells at pH 7.2 or 9 accumulated in one minute 1 to 2 millimolar inorganic carbon in the light or 0.44 millimolar in the dark from 150 micromolar in the media, whereas CO₂-grown cells did not accumulate inorganic carbon. A general scheme for concentrating dissolved inorganic carbon by unicellular green algae utilizes a vanadate-sensitive transporter at the chloroplast envelope for the CO₂ pump and in some algae an additional vanadate-sensitive plasmalemma HCO₃⁻ transporter for a HCO₃⁻ pump.     

Effect of CO2 on succinate production in dual-phase Escherichia coli fermentations

Succinate production under different concentrations of carbon dioxide (CO₂) 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). A series of two-phase fermentations were conducted in which an aerobic cell growth phase was followed by an anaerobic succinate production phase using several constant concentrations of CO₂. As the concentration of CO₂ in the gas phase increased from 0% to 50%, the succinate specific productivity increased from 1.9 mg/g h to 225 mg/g h, and the succinate yield increased from 0.04 g/g to 0.75 g/g. Above 50% CO₂, succinate production did not increase further. Intracellular fluxes were determined at three different CO₂ concentrations (3%, 10%, and 50%) using ¹³C-label tracing coupled with LC–MS analysis. The fraction of carbon flux into the pentose phosphate pathway increased from 0.04 at 3% CO₂ to 0.17 at 50% CO₂. Also, the fractional flux through anaplerotic carboxylation at the phosphoenolpyruvate (PEP) node increased slightly from 0.53 at 3% CO₂ to 0.63 at 50% CO₂. The increased flux into the pentose phosphate pathway is attributed to an increased demand for reduced cofactors with elevated CO₂. A four-process explicit model to describe the CO₂ transfer and utilization was proposed. The model predicted that at CO₂ concentrations below about 30–40% the system becomes limited by gas phase CO₂, while at higher CO₂ concentrations the system is limited by PEP carboxylase enzyme kinetics.     

Change in Size of Chromatium minus Cells in Relation to Growth Rate, Sulfur Content, and Photosynthetic Activity: A Comparison of Pure Cultures and Field Populations

The size frequency distribution of planktonic cells of purple sulfur phototrophic bacteria was measured at several depths in a bacterial layer of Lake Cisó (Spain). The bacterioplankton was dominated by Chromatium minus (87 to 94% of the total biomass). The largest cells of C. minus were found in the top part of the bacterial layer. In addition, the in situ and potential specific photosynthetic activity (CO₂ fixation and acetate uptake) and specific pigment content were measured in relation to several key environmental parameters that determine the activity of cells. Potential growth rates were estimated from production rates and biomass. A maximal specific growth rate of 0.074 h⁻¹ was found for the top part of the bacterial layer. Photosynthesis versus light and versus sulfide curves among field samples indicated that light was the main limiting factor controlling the activity of C. minus in Lake Cisó. The specific bacteriochlorophyll a content was very high in all samples (0.27 to 0.36 μg μg of C⁻¹). Results of laboratory experiments performed with pure cultures indicated that the average cell volume changes from 5.9 to 20.0 μm³ and that differences in growth rate, breakdown, or synthesis of sulfur and glycogen and degradation of the photosynthetic apparatus are the main factors accounting for the observed changes in cell volume across the bacterial layer.     

Synergistic Interaction Between Anabaena and Zoogloea spp. in Carbon Dioxide-Limited Continuous Cultures

Flocs consisting of Anabaena and Zoogloea spp. were used as a model system for the study of planktonic phototroph-heterotroph interactions. In CO₂-limited continuous culture (3.2 μmol of NaHCO₃ liter⁻¹ h⁻¹, 1.5 μmol of glucose liter⁻¹ h⁻¹, pH 8.5, D = 0.026 h⁻¹), the biomass of the phototroph increased 8.6-fold due to association. However, direct CO₂ exchange accounted for only a 3.8-fold increase. When the glucose supply rate was increased to 7.5 μmol liter⁻¹ h⁻¹, there was a 26-fold increase in biomass. When CO₂ was supplied in excess, there was no difference due to association. In batch culture, using the same medium, the specific growth rate was 0.029 h⁻¹ for the phototroph alone and 0.047 h⁻¹ for the phototroph in association with the heterotroph. The stimulatory effect of the heterotroph was found only under CO₂-limiting conditions and was directly related to the concentration of organic matter supplied in the medium. Both the biomass and the growth rate of the Anabaena sp. were increased by association with the Zoogloea sp. Thus, dissolved organic matter may substitute for CO₂ to maximize both growth rate and biomass production by phototrophs when heterotrophic bacteria are present.     

Photosynthesis, growth, and the role of chloride

Previous studies with isolated chloroplasts have indicated that Cl⁻ is an essential cofactor for photosynthesis. Considerable support for the postulated Cl⁻ requirement in photosynthesis came from the observation that Cl⁻ is essential for growth. Data are presented which show that a 60% reduction in growth which occurred in Cl⁻ -deficient sugar beet (Beta vulgaris L.) was not due to an effect of Cl⁻ on the rate of photosynthesis in vivo (net CO₂ uptake per unit area of attached leaves). The principal effect of Cl⁻ deficiency was to lower cell multiplication rates in leaves, thus slowing down their growth and ultimately decreasing their area. The absence of an effect of Cl⁻ on photosynthesis in vivo was unlikely to have been due to Cl⁻ retention by the chloroplasts because their Cl⁻ concentration (measured after nonaqueous isolation) decreased progressively with decrease in leaf Cl⁻.     

Formation of formate and hydrogen, and flux of reducing equivalents and carbon in Ruminococcus flavefaciens Fd-1

A pathway for conversion of the metabolic intermediate phosphoenolpyruvate (PEP) and the formation of acetate, succinate, formate, and H₂ in the anaerobic cellulolytic bacterium Ruminococcus flavefaciens FD-1 was constructed on the basis of enzyme activities detected in extracts of cells grown in cellulose- or cellobiose-limited continuous culture. PEP was converted to acetate and CO₂ (via pyruvate kinase, pyruvate dehydrogenase, and acetate kinase) or carboxylated to form succinate (via PEP carboxykinase, malate dehydrogenase, fumarase, and fumarate reductase). Lactate was not formed even during rapid growth (batch culture, µ = 0.35/h). H₂ was formed by a hydrogenase rather than by cleavage of formate, and ¹³C-NMR and¹⁴ C-exchange reaction data indicated that formate was produced by CO₂ reduction, not by a cleavage of pyruvate. The distribution of PEP into the acetate and succinate pathways was not affected by changing extracellular pH and growth rates within the normal growth range. However, increasing growth rate from 0.017/h to 0.244/h resulted in a shift toward formate production, presumably at the presence of H₂. This shift suggested that reducing equivalents could be balanced through formate or H₂ production without affecting the yields of the major carbon-containing fermentation endproducts.     

The new facultatively chemolithoautotrophic, moderately halophilic, sulfate-reducing bacterium Desulfovermiculus halophilus gen. nov., sp. nov., isolated from an oil field

The new mesophilic, chemolithoautotrophic, moderately halophilic, sulfate-reducing bacterium strain 11-6, could grow at a NaCl concentration in the medium of 30–230 g/l, with an optimum at 80–100 g/l. Cells were vibrios motile at the early stages of growth. Lactate, pyruvate, malate, fumarate, succinate, propionate, butyrate, crotonate, ethanol, alanine, formate, and H₂/CO₂ were used in sulfate reduction. Butyrate was degraded completely, without acetate accumulation. In butyrate-grown cells, a high activity of CO dehydrogenase was detected. Additional growth factors were not required. Autotrophic growth occurred, in the presence of sulfate, on H₂/CO₂ or formate without other electron donors. Fermentation of pyruvate and fumarate was possible in the absence of sulfate. Apart from sulfate, sulfite, thiosulfate, and elemental sulfur were able to serve as electron acceptors. The optimal growth temperature was 37°C; the optimum pH was 7.2. Desulfoviridin was not detected. Menaquinone MK-7 was present. The DNA G+C content was 55.2 mol %. Phylogenetically, the bacterium represented a separate branch within the cluster formed by representatives of the family Desulfohalobiaceae in the class Deltaproteobacteria. The bacterium was assigned to a new genus and species, Desulfovermiculus halophilus gen. nov., sp. nov. The type strain is 11-6^T (= VKM B-2364), isolated from the highly mineralized formation water of an oil field.