Inhibition of Cyanide-Insensitive Respiration in <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> SYSU-011 by 8-Hydroxyquinolone

The inhibition of the cyanide (KCN)-insensitive respiration of Klebsiella oxytoca SYSU-011 by 8-hydroxyquinoline (8-HQ) was determined. Results showed that the profile of the rate of oxygen uptake of normal-grown and 8-HQ–grown K. oxytoca SYSU-011 was biphasic and similar, suggesting that 8-HQ did not inhibit the respiration of normal-grown K. oxytoca SYSU-011. A different biphasic KCN inhibition profile of respiration was observed for KCN-grown cells treated with and without 8-HQ. No decrease in respiration rate of KCN-grown cells and a 40% decrease in respiration rate of KCN-grown cells treated with 8-HQ were observed when KCN concentration was 10^–1 mM. Comparing differences of the profiles of oxygen uptake in KCN-grown cells with and without 8-HQ addition indicated that 8-HQ inhibited expression of the KCN-insensitive pathway carried out by nonheme oxidase. Greater inhibition of NADH oxidase activity by 2-n-heptyl-4-hydroxyquinoline-N-oxide from the cell membrane of the KCN-grown cells treated with 8-HQ, and more H₂O₂ production from these cells with than without 8-HQ, suggest that the function of the cyanide-insensitive pathway can stabilize the respiration of the cyanide-grown cells to prevent the production of H₂O₂.     

Proteomic Analysis of Low Nitrogen Stress-Responsive Proteins in Roots of Rice

In recent years, the application of proteomic approaches as a tool for global expression analysis and protein identification has been highly efficient in the field of plant research. A solution culture experiment involving two nitrogen treatments, 0.14 mM NH₄NO₃ (low nitrogen (N)) and 1.07 mM NH₄NO₃ (control), was conducted to investigate the response of rice root to low N stress. Root system architecture changed markedly under low N stress, with more lateral roots occurring on the lower part of adventitious roots and longer lateral roots on the upper part, compared to the control. A proteomic approach was employed to further study the rice responses to low N stress. Proteins extracted from roots were profiled by two-dimensional gel electrophoresis, and differentially expressed proteins were analyzed by mass spectrometry. Twelve protein spots were successfully identified by mass spectrometry, 11 of which had known functions. Of these, four were involved with the tricarboxylic acid cycle, two with adenylate metabolism, two with phenylpropanoid metabolism, and two with protein degradation. These differentially expressed proteins play an important role in the responsive mechanisms of rice root to low N stress, and uncovering how the rice proteins respond to low N stress could contribute to improving the nitrogen use efficiency.     

Enzymatic degradation of nitriles by Klebsiella oxytoca

Klebsiella oxytoca, isolated from cyanide-containing wastewater, was able to utilize many nitriles as sole source of nitrogen. The major objective of this study was to explore the ability of K. oxytoca to utilize some nitriles and then further evaluate the pathways of transformation of cyanide compounds by K. oxytoca. Results from this study indicate that succinonitrile and valeronitrile were the most optimal sources of nitrogen for the growth of K. oxytoca. The biodegradation of acetonitrile proceeded with the formation of acetamide followed by acetic acid. The production of ammonia was also detected in this biodegradation experiment. Similar results were observed in the propionitrile biodegradation experiments. Collectively, this study suggests that the breakdown of acetonitrile or propionitrile by this bacterium was via a two-step enzymatic hydrolysis with amides as the intermediates and organic acids plus with ammonia as the end products.     

Symbiotic effectiveness and response to mannitol-mediated osmotic stress of various chickpea–rhizobia associations

Thirty-six symbiotic associations involving six chickpea cultivars against six rhizobial strains were evaluated for symbiotic performance and responses to osmotic stress applied by mannitol (50 mM) in aerated hydroponic cultures. Analyses in different symbioses were focused on biomass production, nodulation, nitrogen fixation, and their modulation under osmotic stress conditions, as well as expression of nodular antioxidant enzymes. Mesorhizobium ciceri reference (835) and local (CMG6) strains, as well as the local (C₁₁) M. mediterraneum allowed the best symbiotic efficiency for all chickpea cultivars. The osmotic stress induces severe decrease ranging 30–50% in aerial biomass and 50–70% for nitrogen fixation. Nevertheless, plants inoculated with M. ciceri (835) and M. mediterraneum (C₁₁) preserve a relatively high growth (4 g plant⁻¹) with nitrogen-fixing activity (25 μmols h⁻¹ plant⁻¹). The bacterial partner was the most important factor of variance of the analysed parameters in osmotic stress or physiological conditions where it gets to 60–85%. The strains allowing the best competent symbioses were proposed for field assays. Under osmotic stress, nodular peroxidase (POX) and ascorbate peroxidase (APX) activities were significantly enhanced. The increase of POX and APX was inversely correlated with the inhibition of aerial biomass production (p = 0.05) and nitrogen-fixing capacity (p = 0.01), suggesting a protective role of these enzymes in nodules. Superoxide dismutase (SOD) was also activated in stressed nodules. However, the spectacular decrease in catalase (CAT) activity discounts its involvement in osmotic stress response.     

Production of Phenols and Alkyl Gallate Esters by Rhodobacter sphaeroides OU5

Rhodobacter sphaeroides OU5 grows phototrophically with generation doubling time of 18 h on l-phenylalanine when used as sole source of nitrogen. Phenols accumulated in the medium and gallate (0.5 mM) was identified as one of the major product. The others namely protocatechuate (0.2 mM) and caffeate (0.1 mM) and also three putative phenol alkyl esters were identified using Liquid chromatography–mass spectroscopy (LC–MS) analysis from the culture supernatant. Rhodobacter sphaeroides OU5 strain could explain its capability to produce the bioactive compounds during the growth. This study sheds production of bioactive and their biological exploring molecules.     

Reversibility of Soil Productivity Decline with Organic Matter of Differing Quality Along a Degradation Gradient

In the highlands of Western Kenya, we investigated the reversibility of soil productivity decline with increasing length of continuous maize cultivation over 100 years (corresponding to decreasing soil organic carbon (SOC) and nutrient contents) using organic matter additions of differing quality and stability as a function of soil texture and inorganic nitrogen (N) additions. The ability of additions of labile organic matter (green and animal manure) to improve productivity primarily by enhanced nutrient availability was contrasted with the ability of stable organic matter (biochar and sawdust) to improve productivity by enhancing SOC. Maize productivity declined by 66% during the first 35 years of continuous cropping after forest clearing. Productivity remained at a low level of 3.0 t grain ha^-1 across the chronosequence stretching up to 105 years of continuous cultivation despite full N–phosphorus (P)–potassium (K) fertilization (120–100–100 kg ha⁻¹). Application of organic resources reversed the productivity decline by increasing yields by 57–167%, whereby responses to nutrient-rich green manure were 110% greater than those from nutrient-poor sawdust. Productivity at the most degraded sites (80–105 years since forest clearing) increased in response to green manure to a greater extent than the yields at the least degraded sites (5 years since forest clearing), both with full N–P–K fertilization. Biochar additions at the most degraded sites doubled maize yield (equaling responses to green manure additions in some instances) that were not fully explained by nutrient availability, suggesting improvement of factors other than plant nutrition. There was no detectable influence of texture (soils with either 11–14 or 45–49% clay) when low quality organic matter was applied (sawdust, biochar), whereas productivity was 8, 15, and 39% greater (P < 0.05) on sandier than heavier textured soils with high quality organic matter (green and animal manure) or only inorganic nutrient additions, respectively. Across the entire degradation range, organic matter additions decreased the need for additional inorganic fertilizer N irrespective of the quality of the organic matter. For low quality organic resources (biochar and sawdust), crop yields were increasingly responsive to inorganic N fertilization with increasing soil degradation. On the other hand, fertilizer N additions did not improve soil productivity when high quality organic inputs were applied. Even with the tested full N–P–K fertilization, adding organic matter to soil was required for restoring soil productivity and most effective in the most degraded sites through both nutrient delivery (with green manure) and improvement of SOC (with biochar).     

Extracellular Enzyme Activities and Soil Organic Matter Dynamics for Northern Hardwood Forests receiving Simulated Nitrogen Deposition

Anthropogenic nitrogen enrichment alters decomposition processes that control the flux of carbon (C) and nitrogen (N) from soil organic matter (SOM) pools. To link N-driven changes in SOM to microbial responses, we measured the potential activity of several extracellular enzymes involved in SOM degradation at nine experimental sites located in northern Michigan. Each site has three treatment plots (ambient, +30 and +80 kg N ha⁻¹ y⁻¹). Litter and soil samples were collected on five dates over the third growing season of N treatment. Phenol oxidase, peroxidase and cellobiohydrolase activities showed significant responses to N additions. In the Acer saccharum–Tilia americana ecosystem, oxidative activity was 38% higher in the litter horizon of high N treatment plots, relative to ambient plots, while oxidative activity in mineral soil showed little change. In the A. saccharum–Quercus rubra and Q. velutina–Q. alba ecosystems, oxidative activities declined in both litter (15 and 23%, respectively) and soil (29 and 38%, respectively) in response to high N treatment while cellobiohydrolase activity increased (6 and 39% for litter, 29 and 18% for soil, respectively). Over 3 years, SOM content in the high N plots has decreased in the Acer–Tilia ecosystem and increased in the two Quercus ecosystems, relative to ambient plots. For all three ecosystems, differences in SOM content in relation to N treatment were directly related (r² = 0.92) to an enzyme activity factor that included both oxidative and hydrolytic enzyme responses.     

Degradation of methamidophos by <Emphasis Type="Italic">Hyphomicrobium</Emphasis> species MAP-1 and the biochemical degradation pathway

Methamidophos is one of the most widely used organophosphorus insecticides usually detectable in the environment. A facultative methylotroph, Hyphomicrobium sp. MAP-1, capable of high efficiently degrading methamidophos, was isolated from methamidophos-contaminated soil in China. It was found that the addition of methanol significantly promoted the growth of strain MAP-1 and enhanced its degradation of methamidophos. Further, this strain could utilize methamidophos as its sole carbon, nitrogen and phosphorus source for growth and could completely degrade 3,000 mg l⁻¹ methamidophos in 84 h under optimal conditions (pH 7.0, 30°C). The enzyme responsible for methamidophos degradation was mainly located on the cell inner membrane (90.4%). During methamidophos degradation, three metabolites were detected and identified based on tandem mass spectrometry (MS/MS) and gas chromatography-mass spectrometry (GC–MS) analysis. Using this information, a biochemical degradation pathway of methamidophos by Hyphomicrobium sp. MAP-1 was proposed for the first time. Methamidophos is first cleaved at the P–N bond to form O,S-dimethyl hydrogen thiophosphate and NH₃. Subsequently, O,S-dimethyl hydrogen thiophosphate is hydrolyzed at the P–O bond to release –OCH₃ and form S-methyl dihydrogen thiophosphate. O,S-dimethyl hydrogen thiophosphate can also be hydrolyzed at the P–S bond to release –SCH₃ and form methyl dihydrogen phosphate. Finally, S-methyl dihydrogen thiophosphate and methyl dihydrogen phosphate are likely transformed into phosphoric acid.     

Effect of nitrogen limitation on oleic acid biosynthesis in <Emphasis Type="Italic">Botryococcus braunii</Emphasis>

The influence of nitrogen (N) deficiency on the cell growth and intracellular lipid production of the alga Botryococcus braunii UTEX 572 was investigated. Biomass concentration and lipid content of B. braunii cultivated in modified Chu-13 medium containing 0.04, 0.37, and 3.66 mM nitrate were 0.23–0.38 g L⁻¹ and 36–63% of dry cell weight, respectively. The specific growth rate of B. braunii reached a constant of 0.185 day⁻¹ during cultivation with an initial nitrate feed of 3.66 mM. The maximum lipid content of B. braunii was 63% with 0.04 mM nitrate. However, the maximum lipid productivity of 0.019 g L⁻¹ day⁻¹ was achieved with 0.37 mM nitrate. The level of oleic acid, an important component of biodiesel, was higher at 86% of the total fatty acids under N-limited conditions (0.04 mM nitrate) compared to 69% under N-sufficient conditions (3.66 mM nitrate). Furthermore, expression of the stearoyl-ACP desaturase gene (sad) encoding a stearoyl-ACP desaturase involved in the synthesis of oleic acid was 2.6-fold higher under N-limited conditions than under N-sufficient conditions.     

Adventitious root suspension cultures of <Emphasis Type="BoldItalic">Hypericum perforatum</Emphasis>: effect of nitrogen source on production of biomass and secondary metabolites

The present study investigated the effect of nitrogen source (NH₄⁺; NO₃⁻) at different concentrations on the accumulation of biomass and secondary metabolites in adventitious root cultures of Hypericum perforatum L. Cultures were initiated in shake flasks by using half-strength Murashige and Skoog (MS) medium with B5 vitamins, 1.0 mg l⁻¹ indole-3-butyric acid, 0.1 mg l⁻¹ kinetin, 3% (w/v) sucrose, and different ratios of ammonium and nitrate (0:30, 5:25, 10:20, 15:15, 20:10, 25:5, and 30:0 mM, using NH₄Cl and KNO₃). The cultures were maintained in darkness. The medium supplemented with 5:25 (mM) NH₄⁺/NO₃⁻ resulted in the optimum accumulation of biomass and total phenols and flavonoids. The antioxidant potential of a methanolic extract, measured as the 1, 1-diphenyl-2-picrylhydrazyl and 2, 2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activities, of H. perforatum adventitious roots showed that antioxidant activity was high from root extracts that were grown on higher concentrations of NO₃⁻ nitrogen (15, 20, and 25 mM). Further, assessment of hydrogen peroxide (H₂O₂) and malondialdehyde content of the root extracts revealed that cultures supplemented with higher levels of NO₃⁻ nitrogen (15–30 mM) were under oxidative stress, which boosted the levels of secondary metabolites in the adventitious roots. These results suggest that optimal adventitious root biomass could be achieved with the supplementation of cultures with 5:25 ratios of MS nitrogen sources.     

Investigation of the Gracilaria gracilis (Gracilariales,Rhodophyta) proteome response to nitrogen limitation

Inorganic nitrogen has been identified as the major growth‐limiting nutritional factor affecting Gracilaria gracilis populations in South Africa. Although the physiological mechanisms implemented by G. gracilis for adaption to low nitrogen environments have been investigated, little is known about the molecular mechanisms of these adaptions. This study provides the first investigation of G. gracilis proteome changes in response to nitrogen limitation and subsequent recovery. A differential proteomics approach employing two‐dimensional gel electrophoresis and liquid chromatography–tandem mass spectrometry was used to investigate G. gracilis proteome changes in response to nitrogen limitation and recovery. The putative identity of 22 proteins that changed significantly (P < 0.05) in abundance in response to nitrogen limitation and recovery was determined. The identified proteins function in a range of biological processes including glycolysis, photosynthesis, ATP synthesis, galactose metabolism, protein‐refolding and biosynthesis, nitrogen metabolism and cytoskeleton remodeling. The identity of fructose 1,6 biphosphate (FBP) aldolase was confirmed by western blot analysis and the decreased abundance of FBP aldolase observed with two‐dimensional gel electrophoresis was validated by enzyme assays and western blots. The identification of key proteins and pathways involved in the G. gracilis nitrogen stress response provide a better understanding of G. gracilis proteome responses to varying degrees of nitrogen limitation and is the first step in the identification of biomarkers for monitoring the nitrogen status of cultivated G. gracilis populations.     

Degradation of cyanide in agroindustrial or industrial wastewater in an acidification reactor or in a single-step methane reactor by bacteria enriched from soil and peels of cassava

During cassava starch production, large amounts of cyanoglycosides were released and hydrolysed by plant-borne enzymes, leading to cyanide concentrations in the wastewater as high as 200 mg/l. For anaerobic degradation of the cyanide during pre-acidification or single-step methane fermentation, anaerobic cultures were enriched from soil residues of cassava roots and sewage sludge. In a pre-acidification reactor this culture was able to remove up to 4 g potassium cyanide/l of wastewater at a hydraulic retention time (t _HR) of 4 days, equivalent to a maximal cyanide space loading of 400 mg CN⁻ l⁻¹ day⁻¹. The residual cyanide concentration was 0.2–0.5 mg/l. Concentrated cell suspensions of the mixed culture formed ammonia and formate in almost equimolar amounts from cyanide. Little formamide was generated by chemical decay. A concentration of up to 100 mmol ammonia/l had no inhibitory effect on cyanide degradation. The optimal pH for cyanide degradation was 6–7.5, the optimal temperature 25–37 °C. At a pH of 5 or lower, cyanide accumulated in the reactor and pre-acidification failed. The minimal t _HR for continuous cyanide removal was 1.5 days. The enriched mixed culture was also able to degrade cyanide in purely mineralic wastewater from metal deburring, either in a pre-acidification reactor with a two-step process or in a one-step methanogenic reactor. It was necessary to supplement the wastewater with a carbon source (e.g. starch) to keep the population active enough to cope with any possible inhibiting effect of cyanide. Received: 29 April 1998 / Received revision: 8 June 1998 / Accepted: 14 June 1998     

Plant growth and responses of antioxidants of <Emphasis Type="Italic">Chenopodium album</Emphasis> to long-term NaCl and KCl stress

The effects of long-term NaCl and KCl treatment on plant growth and antioxidative responses were investigated in Chenopodium album, a salt-resistant species widely distributed in semi-arid and light-saline areas of Xinjiang, China. Growth parameters [plant height, branch number, leaf morphology and chlorophyll (Chl) content], the level of oxidative stress [superoxide anion radical (O₂ ⁻), hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) concentrations], activity of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), peroxidase (POX)], the contents of non-enzymatic antioxidants [carotenoids (Car) and ascorbic acid (AsA)] and expression of selected genes were investigated. Plants were grown in the presence of 0, 50, and 300 mM NaCl or KCl for 2 months. Growth was stimulated by 50 mM NaCl or KCl, maintained stable at 300 mM NaCl, but was inhibited by 300 mM KCl. Three hundred mM NaCl did not affect O₂ ⁻, H₂O₂, MDA, Car and AsA, but increased the activities of SOD, CAT and POX compared to the controls. RT-PCR analysis suggested that expression of some genes encoding antioxidant enzymes could be induced during long-term salt stress, which was consistent with the enzyme activities. Treatment with 300 mM KCl was associated with elevated oxidative stress, and significantly decreased Car and AsA contents. These results suggest that an efficient antioxidant machinery is important for overcoming oxidative stress induced by treatment with high NaCl concentrations in C. album. Other strategies of ion regulation may also contribute to the differential tolerance to Na and K at higher concentrations.     

The salt stress relief and growth promotion effect of Rs-5 on cotton

The effect of the Rs-5 bacteria strain, identified as Klebsiella oxytoca and isolated with ACC as the sole nitrogen source, on salt stressed cotton seedling growth was studied. It was demonstrated that Rs-5 could obviously relieve salt stress and promote cotton seedling growth. After treatment with Rs-5, the individual plant height and dry weight of cotton increased by 14.9 and 26.9%, respectively, compared to the control. Further analysis found that Rs-5 exhibited the ability to increase the cotton’s absorption of the N, P, K, and Ca elements and decrease the absorbability of the Na element under salt stress. In addition, Rs-5 itself could produce phytohormone-auxin, and was capable of dissolving phosphorus (P). The ratio of the dissolved P diameter to the colony diameter was 1.86. The dissolved P was 81.6 mg·l⁻¹ in media after four days of incubation. Responsible Editor: Petra Marschner.     

Cyanide inhibition and pyruvate-induced recovery of cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase

The mechanism of cyanide’s inhibitory effect on the mitochondrial cytochrome c oxidase (COX) as well as the conditions for its recovery have not yet been fully explained. We investigated three parameters of COX function, namely electron transport (oxygen consumption), proton transport (mitochondrial membrane potential Δψ _m) and the enzyme affinity to oxygen (p ₅₀ value) with regard to the inhibition by KCN and its reversal by pyruvate. 250 μM KCN completely inhibited both the electron and proton transport function of COX. The inhibition was reversible as demonstrated by washing of mitochondria. The addition of 60 mM pyruvate induced the maximal recovery of both parameters to 60–80% of the original values. When using low KCN concentrations of up to 5 μM, we observed a profound, 30-fold decrease of COX affinity for oxygen. Again, this decrease was completely reversed by washing mitochondria while pyruvate induced only a partial, yet significant recovery of oxygen affinity. Our results demonstrate that the inhibition of COX by cyanide is reversible and that the potential of pyruvate as a cyanide poisoning antidote is limited. Importantly, we also showed that the COX affinity for oxygen is the most sensitive indicator of cyanide toxic effects.     

Regulated alterations in redox and energetic status are the key mediators of salinity tolerance in the halophyte <Emphasis Type="Italic">Sesuvium portulacastrum</Emphasis> (L.) L

The present work addresses the importance of antioxidant, redox and energetic parameters in regulating salt-tolerance in Sesuvium portulacastrum. Experiments were conducted on 45 days old plants subjected to 250 and 1,000 mM NaCl stress for 2–8 days. Plants showed no significant change in growth parameters (shoot length, dry weight, and water content) at 250 mM NaCl as compared to control. However, growth of plants was significantly affected at 1,000 mM NaCl. The differential growth behaviour could be attributed to a greater decline in the energetic parameters (in terms of ratios of NADP/NADPH and ATP/ADP) at 1,000 mM NaCl than at 250 mM NaCl. The osmotic stress imposed to plants at 250 mM NaCl was presumably balanced by the accumulation of sodium ions (Na⁺), an energetically favorable process, and did not require an increased synthesis of proline. In contrast, to counter osmotic stress at 1,000 mM NaCl, plants accumulated Na⁺ as well as proline and were, therefore, energetically stressed. Further, the response of enzymatic and molecular antioxidants at 1,000 mM was either close to or even lower than that at 250 mM, which resulted in oxidative damage at 1,000 mM, particularly on longer durations. In conclusion, it is suggested that altered redox and energetic status of the plants could play a key role in mediating the tolerance of Sesuvium under salinity stress.     

Metabolomics demonstrates divergent responses of two <Emphasis Type="Italic">Eucalyptus</Emphasis> species to water stress

Past studies of water stress in Eucalyptus spp. generally highlighted the role of fewer than five “important” metabolites, whereas recent metabolomic studies on other genera have shown tens of compounds are affected. There are currently no metabolite profiling data for responses of stress-tolerant species to water stress. We used GC–MS metabolite profiling to examine the response of leaf metabolites to a long (2 month) and severe (Ψ_predawn < −2 MPa) water stress in two species of the perennial tree genus Eucalyptus (the mesic Eucalyptus pauciflora and the semi-arid Eucalyptus dumosa). Polar metabolites in leaves were analysed by GC–MS and inorganic ions by capillary electrophoresis. Pressure–volume curves and metabolite measurements showed that water stress led to more negative osmotic potential and increased total osmotically active solutes in leaves of both species. Water stress affected around 30–40% of measured metabolites in E. dumosa and 10–15% in E. pauciflora. There were many metabolites that were affected in E. dumosa but not E. pauciflora, and some that had opposite responses in the two species. For example, in E. dumosa there were increases in five acyclic sugar alcohols and four low-abundance carbohydrates that were unaffected by water stress in E. pauciflora. Re-watering increased osmotic potential and decreased total osmotically active solutes in E. pauciflora, whereas in E. dumosa re-watering led to further decreases in osmotic potential and increases in total osmotically active solutes. This experiment has added several extra dimensions to previous targeted analyses of water stress responses in Eucalyptus, and highlights that even species that are closely related (e.g. congeners) may respond differently to water stress and re-watering.     

Engineering <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for the production of pyruvate

A Corynebacterium glutamicum strain with inactivated pyruvate dehydrogenase complex and a deletion of the gene encoding the pyruvate:quinone oxidoreductase produces about 19 mM l-valine, 28 mM l-alanine and about 55 mM pyruvate from 150 mM glucose. Based on this double mutant C. glutamicum △aceE △pqo, we engineered C. glutamicum for efficient production of pyruvate from glucose by additional deletion of the ldhA gene encoding NAD⁺-dependent l-lactate dehydrogenase (LdhA) and introduction of a attenuated variant of the acetohydroxyacid synthase (△C–T IlvN). The latter modification abolished overflow metabolism towards l-valine and shifted the product spectrum to pyruvate production. In shake flasks, the resulting strain C. glutamicum △aceE △pqo △ldhA △C–T ilvN produced about 190 mM pyruvate with a Y _P/S of 1.36 mol per mol of glucose; however, it still secreted significant amounts of l-alanine. Additional deletion of genes encoding the transaminases AlaT and AvtA reduced l-alanine formation by about 50%. In fed-batch fermentations at high cell densities with adjusted oxygen supply during growth and production (0–5% dissolved oxygen), the newly constructed strain C. glutamicum △aceE △pqo △ldhA △C–T ilvN △alaT △avtA produced more than 500 mM pyruvate with a maximum yield of 0.97 mol per mole of glucose and a productivity of 0.92 mmol g_(CDW)⁻¹ h⁻¹ (i.e., 0.08 g g_(CDW) ⁻¹ h⁻¹) in the production phase.