Glutamate biosynthesis in Bacillus azotofixans. 15N NMR and enzymatic studies

Pathways of ammonia assimilation into glutamic acid in Bacillus azotofixans, a recently characterized nitrogen-fixing species of Bacillus, were investigated through observation by NMR spectroscopy of in vivo incorporation of 15N into glutamine and glutamic acid in the absence and presence of inhibitors of ammonia-assimilating enzymes, in combination with measurements of the specific activities of glutamate dehydrogenase, glutamine synthetase, glutamate synthase, and alanine dehydrogenase. In ammonia-grown cells, both the glutamine synthetase/glutamate synthase and the glutamate dehydrogenase pathways contribute to the assimilation of ammonia into glutamic acid. In nitrate-grown and nitrogen-fixing cells, the glutamine synthetase/glutamate synthase pathway was found to be predominant. NADPH-dependent glutamate dehydrogenase activity was detectable at low levels only in ammonia-grown and glutamate-grown cells. Thus, B. azotofixans differs from Bacillus polymyxa and Bacillus macerans, but resembles other N2-fixing prokaryotes studied previously, as to the pathway of ammonia assimilation during ammonia limitation. Implications of the results for an emerging pattern of ammonia assimilation by alternative pathways among nitrogen-fixing prokaryotes are discussed, as well as the utility of 15N NMR for measuring in vivo glutamate synthase activity in the cell.     

Ammonia assimilation in Bacillus polymyxa. 15N NMR and enzymatic studies

Pathways of ammonia assimilation into glutamic acid and alanine in Bacillus polymyxa were investigated by 15N NMR spectroscopy in combination with measurements of the specific activities of glutamate dehydrogenase, glutamine synthetase, glutamate synthetase, alanine dehydrogenase, and glutamic-alanine transaminase. Ammonia was found to be assimilated into glutamic acid predominantly by NADPH-dependent glutamate dehydrogenase with a Km of 2.9 mM for NH4+ not only in ammonia-grown cells but also in nitrate-grown and nitrogen-fixing cells in which the intracellular NH4+ concentrations were 11.2, 1.04, and 1.5 mM, respectively. In ammonia-grown cells, the specific activity of alanine dehydrogenase was higher than that of glutamic-alanine transaminase, but the glutamate dehydrogenase/glutamic-alanine transaminase pathway was found to be the major pathway of 15NH4+ assimilation into [15N]alanine. The in vitro specific activities of glutamate dehydrogenase and glutamine synthetase, which represent the rates of synthesis of glutamic acid and glutamine, respectively, in the presence of enzyme-saturating concentrations of substrates and coenzymes are compared with the in vivo rates of biosynthesis of [15N]glutamic acid and [alpha,gamma-15N]glutamine observed by NMR, and implications of the results for factors limiting the rates of their biosynthesis in ammonia- and nitrate-grown cells are discussed.     

A kinetic study of the assimilation of 15N-labelled ammonium in rice seedling roots

The pathway of ammonium nitrogen assimilation, its incorporationinto amino acids and synthesis of protein was studied with theaid of nitrogen-15. The analysis of ¹⁵N involves the use ofoptical emission spectrometry. Kinetic analysis of nitrogen assimilation by the roots indicatesthat glutamine and glutamic acid were the primary products ofammonium assimilation. Possibly some of the amino acids, suchas aspartic acid and alanine received their amino nitrogen directlyfrom free ammonia in the roots. Amino groups were transformedinto other amino acids from these primary products, especiallyfrom glutamic acid through transamination. (Received April 1, 1974; )     

Ammonium Assimilation and the Role of [gamma]-Aminobutyric Acid in pH Homeostasis in Carrot Cell Suspensions

In vivo 15N NMR spectroscopy was used to monitor the assimilation of ammonium by cell-suspension cultures of carrot (Daucus carota L. cv Chantenay). The cell suspensions were supplied with oxygen in the form of either pure oxygen ("oxygenated cells") or air ("aerated cells"). In contrast to oxygenated cells, in which ammonium assimilation had no effect on cytoplasmic pH, ammonium assimilation by aerated cells caused a decrease in cytoplasmic pH of almost 0.2 pH unit. This led to a change in nitrogen metabolism resulting in the accumulation of [gamma]-aminobutyric acid. The metabolic effect of the reduced oxygen supply under aerated conditions could be mimicked by artificially decreasing the cytoplasmic pH of oxygenated cells and was abolished by increasing the cytoplasmic pH of aerated cells. The activity of glutamate decarboxylase increased as the cytoplasmic pH declined and decreased as the pH recovered. These findings are consistent with a role for the decarboxylation of glutamate, a proton-consuming reaction, in the short-term regulation of cytoplasmic pH, and they demonstrate that cytoplasmic pH influences the pathways of intermediary nitrogen metabolism.     

Determination of the metabolic origins of the sulfur and 3'-nitrogen atoms in biotin of Escherichia coli by mass spectrometry

Two steps in the biosynthesis of biotin in Escherichia coli, incorporation of the nitrogen atom of methionine into 7-keto-8-aminopelargonic acid and of the sulfur atom into dethiobiotin, were examined. Sulfur and nitrogen metabolism were monitored by gas chromatography-mass spectrometry of volatile derivatives of internal (protein-bound) amino acids and excreted biotin. We were able to show that internal cysteine and excreted biotin were labeled to the same extent with 34S from either of two exogenous sulfur sources, 34SO4(2)-or L-[sulfane-34S]thiocystine. Internal methionine was eliminated from consideration, while cysteine, or possibly a closely related intermediate, was implicated as providing the sulfur atom for biotin biosynthesis. Also, in experiments designed to follow the metabolism of the nitrogen atom of methionine, it was found that biotin excreted into the culture medium by this organism grown with 95 atom % [15N]methionine contained greater than 70 atom % excess 15N in one of the nitrogens over that obtained from cultures grown with methionine of natural abundance 15N. These results provide evidence for the direct transfer of the methionine nitrogen as the role of S-adenosylmethionine in the conversion of 7-keto-8-aminopelargonic acid to 7,8-diaminopelargonic acid.     

Evidence for a role of glutamine synthetase in assimilation of amino acids as nitrogen source in the cyanobacterium Nostoc muscorum.

Methylammonium/ammonium ion, glutamine, glutamate, arginine and proline uptake, and their assimilation as nitrogen sources, was studied in Nostoc muscorum and its glutamine synthetase-deficient mutant. Glutamine served as nitrogen source independent of glutamine synthetase activity. Glutamate was not metabolised as a nitrogen source but still inhibited nitrogenase activity and diazotrophic growth. Glutamine synthetase activity was essential for the assimilation of N2, ammonia, arginine and proline as nitrogen sources but not for the control of their transport, heterocyst formation, and production of ammonia or aminoacid dependent repressor signal for N2-fixing heterocysts. These results also suggest that glutamine synthetase serves as the sole route of ammonia assimilation and glutamine synthesis, and ammonia per se as the repressor signal for N2-fixing heterocysts and methylammonium (ammonium) transport.     

Assimilation of Nitrogen in Mutants Lacking Enzymes of the Glutamate Synthase Cycle

¹⁵N labelling was used to investigate the pathway of nitrogenassimilation in photorespiratory mutants of barley (Hordeumvulgare cv. Maris Mink), in which the leaves have low levelsof glutamine synthetase (GS) or glutamate synthase, key enzymesof ammonia assimilation. These plants grew normally when maintainedin high CO₂, but the deletions were lethal when photorespirationwas initiated by transfer to air. Enzyme levels in roots weremuch less affected, compared to leaves, and assimilation oflabelled nitrate into amino acids of the root showed very littledifference between wild type and mutants. Organic nitrogen wasexported from roots in the xylem sap mainly as glutamine, levelsof which were somewhat reduced in the GS-deficient mutant andenhanced in the glutamate synthase deficient mutant. In theleaf, the major effect was seen in the glutamatesynthase mutant,which had an extremely limited capacity to utilize the importedglutamine and amino acid synthesis was greatlyrestricted. Thiswas confirmed by the supply of [¹⁵N]-glutamine directly to leaves.Leaves of the GS-deficient mutant assimilatedammonia at about75% the rate found for the wild type, and this was almost completelyeliminated by addition of the inhibitormethionine sulphoximine.Root enzymes, together with residual levels of the deleted enzymesin the leaves, have sufficient capacityfor ammonia assimilation,through the glutamate synthase cycle, to provide adequate inputof nitrogen for normal growth of themutants, if photorespiratoryammonia production is suppressed. Key words: Hordeum vulgare, ¹⁵N, glutamine synthetase, glutamate synthase, ammonia assimilation     

A kinetic study of the assimilation of 15N-labelled nitrate in rice seedlings

A ¹⁵N kinetic-analysis of the assimilation of nitrate nitrogenin the roots of rice seedlings indicated that (1) nitrate wasrapidly reduced to ammonia in the roots, where it was incorporatedinto glutamine and glutamic acids; (2) the pattern of nitrateassimilation into amino acids was very similar to that of ammoniumassimilation; and (3) the pattern of nitrogen incorporationinto protein was also similar to that of the incorporation withNH₄-feeding. In the shoots, alanine, serine, glutamic acid, -amino butyricacid and aspartic acid were relatively strongly labelled with¹⁵N as compared with the other amino acids. A different mechanismof nitrogen assimilation seems to operate in between the photosyntheticand non-photosynthetic organs of the plants. (Received August 19, 1974; )     

Induction of nutritional mutants ofMicrococcus glutamicus and their amino acid accumulation

Micrococcus glutamicus ATCC 13032, a glutamic acid-producing organism, was treated with 0.2M ethylmethane sulfonate, the auxotrophs isolated showing varied patterns of extracellular amino acids. Eighty auxotrophic strains were obtained, out of which 31 excreted 1.0-4.0 mg threonine per ml and all the auxotrophs required biotin for growth and production of the amino acid. Eleven auxotrophs produced 1.5 to 3.0 mg alanine per ml and these auxotrophs required amino acids for their growth. Other auxotrophs lost their excretion capacity in subsequent fermentation trials. Further mutation of the biotin-requiring auxotroph Micrococcus glutamicus EM with gamma rays resulted in the isolation of 89 auxotrophic strains, out of which 28 excreted threonine (up to 5.0 mg per ml) higher than the parent auxotroph. Exposure to X-rays yielded 97 auxotrophs, out of these 35 producing 1.0-3.0 mg methionine per ml and requiring biotin for growth and production of the amino acid. Other auxotrophs produced alanine (0.5 to 2.0 mg per ml) and threonine (2.0 to 3.3 mg per ml). Irradiation with gamma rays favoured the development of threonine producing auxotrophs while X-rays favoured methionine-producing auxotrophs.     

The ammonotelic African lungfish,<Emphasis Type="Italic"> Protopterus dolloi</Emphasis>, increases the rate of urea synthesis and becomes ureotelic after feeding

This study aimed to elucidate the role of urea synthesis in the slender African lungfish Protopterus dolloi in detoxifying ammonia after feeding. There were significant increases in the rate of ammonia excretion in P. dolloi between hours 6 and 15 after feeding. Simultaneously, there were significant increases in urea excretion rates between hours 3 and 18. Consequently, the percentage of total nitrogen (N) excreted as urea N increased to ~60% between hours 12 and 21 post-feeding. Hence, after feeding, the normally ammonotelic P. dolloi became ureotelic. Approximately 41% of the N intake from food was excreted within 24 h by P. dolloi, 55% of which was in the form of urea N. At hour 12 post-feeding, the accumulation of urea N was greater than the accumulation of ammonia N in various tissues, which indicates that feeding led to an increase in the rate of urea synthesis. This is contrary to results reported previously on the infusion of ammonia into the peritoneal cavity of the marine dogfish shark, in which a significant portion of the exogenous ammonia was excreted as ammonia. In contrast, feeding is more likely to induce urea synthesis, which is energy intensive, because feeding provides an ample supply of energy resources and leads to the production of ammonia intracellularly in the liver. The capacity of P. dolloi to synthesize urea effectively prevented a postprandial surge in the plasma ammonia level as reported elsewhere for other non-ureogenic teleosts. However, there was a significant increase in the glutamine content in the brain at hour 24, indicating that the brain had to defend against ammonia toxicity after feeding.Communicated by I.D. Hume     

Phytoplankton uptake and excretion of assimilated nitrate in a small Canadian shield lake

Nitrate uptake in the epilemnetic waters of a small eutrophic Canadian Shield lake was studied by using a 15N method during summer stratification. Concurrent with inhibition of primary production, 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibited NO3- assimilation. Nitrate up to 1 mg of N/liter did not affect the rate of primary production during 3 h of incubation. The NO3- fertilizer added to the lake weekly was consumed through algal assimilation in about 3 days. Excretion of the photoassimilated NO3- as dissolved organic nitrogen represented a significant portion of the nutrient incorporated by the cells. Only 40% of the NO3- -15N which disappeared could be accounted for in the particulate fraction. Although the rest was presumably excreted, only 15% of the 15N label was accounted for as cationic dissolved organic nitrogen by isotope assays. These excreted organic forms were predominantly serine and glycine in the dissolved free amino acid fraction. Bacteria as well as algae might be expected to contribute to and modify the extracellular nitrogen pool.     

Phytoplankton uptake and excretion of assimilated nitrate in a small Canadian shield lake.

Nitrate uptake in the epilemnetic waters of a small eutrophic Canadian Shield lake was studied by using a 15N method during summer stratification. Concurrent with inhibition of primary production, 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibited NO3- assimilation. Nitrate up to 1 mg of N/liter did not affect the rate of primary production during 3 h of incubation. The NO3- fertilizer added to the lake weekly was consumed through algal assimilation in about 3 days. Excretion of the photoassimilated NO3- as dissolved organic nitrogen represented a significant portion of the nutrient incorporated by the cells. Only 40% of the NO3- -15N which disappeared could be accounted for in the particulate fraction. Although the rest was presumably excreted, only 15% of the 15N label was accounted for as cationic dissolved organic nitrogen by isotope assays. These excreted organic forms were predominantly serine and glycine in the dissolved free amino acid fraction. Bacteria as well as algae might be expected to contribute to and modify the extracellular nitrogen pool.     

Growth yields,polysaccharide production and energy conservation in chemostat cultures of Rhizobium trifolii

Rhizobium trifolii was grown in a defined medium in chemostat cultures. Extracellular polysaccharide production was found in carbon-sufficient as well as in carbon-limited cultures. Extracellular polysaccharide production in limited cultures, asparagine was always totally depleted from the culture medium. Only when the asparagine supply was not sufficient to meet the nitrogen need of the culture, ammonia assimilation took place. Excess organic nitrogen was excreted as ammonia. Whether ammonia assimilation or ammonia excretion took place was also dependent on the growth rate. Respiration-coupled proton translocation measurements showed the presence of three energy conserving sites in an electron transport chain which is branched. Assuming a H⁺/P ratio of 4, a P/O ratio of 2.33 was found. Growth yield calculations indicated a P/O ratio of approximately 2. Sulphate limitation in the chemostat culture resulted in a decrease in the efficiency of oxidative phosphorylation and in a less stringent coupling between growth and energy yielding processes.The investigations were supported in part by the Foundation for Fundamental Biological Research (BION), which is subsidized by The Netherlands Organisation for the Advancement of Pure Research (ZWO).     

In vivo fluxes in the ammonium-assimilatory pathways in corynebacterium glutamicum studied by 15N nuclear magnetic resonance

Glutamate dehydrogenase (GDH) and glutamine synthetase (GS)-glutamine 2-oxoglutarate-aminotransferase (GOGAT) represent the two main pathways of ammonium assimilation in Corynebacterium glutamicum. In this study, the ammonium assimilating fluxes in vivo in the wild-type ATCC 13032 strain and its GDH mutant were quantitated in continuous cultures. To do this, the incorporation of 15N label from [15N]ammonium in glutamate and glutamine was monitored with a time resolution of about 10 min with in vivo 15N nuclear magnetic resonance (NMR) used in combination with a recently developed high-cell-density membrane-cyclone NMR bioreactor system. The data were used to tune a standard differential equation model of ammonium assimilation that comprised ammonia transmembrane diffusion, GDH, GS, GOGAT, and glutamine amidotransferases, as well as the anabolic incorporation of glutamate and glutamine into biomass. The results provided a detailed picture of the fluxes involved in ammonium assimilation in the two different C. glutamicum strains in vivo. In both strains, transmembrane equilibration of 100 mM [15N]ammonium took less than 2 min. In the wild type, an unexpectedly high fraction of 28% of the NH4+ was assimilated via the GS reaction in glutamine, while 72% were assimilated by the reversible GDH reaction via glutamate. GOGAT was inactive. The analysis identified glutamine as an important nitrogen donor in amidotransferase reactions. The experimentally determined amount of 28% of nitrogen assimilated via glutamine is close to a theoretical 21% calculated from the high peptidoglycan content of C. glutamicum. In the GDH mutant, glutamate was exclusively synthesized over the GS/GOGAT pathway. Its level was threefold reduced compared to the wild type.     

Ammonia assimilation pathways in nitrogen-fixing Clostridium kluyverii and Clostridium butyricum.

Pathways of ammonia assimilation into glutamic acid were investigated in ammonia-grown and N2-fixing Clostridium kluyverii and Clostridium butyricum by measuring the specific activities of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. C. kluyverii had NADPH-glutamate dehydrogenase with a Km of 12.0 mM for NH4+. The glutamate dehydrogenase pathway played an important role in ammonia assimilation in ammonia-grown cells but was found to play a minor role relative to that of the glutamine synthetase/NADPH-glutamate synthase pathway in nitrogen-fixing cells when the intracellular NH4+ concentration and the low affinity of the enzyme for NH4+ were taken into account. In C. butyricum grown on glucose-salt medium with ammonia or N2 as the nitrogen source, glutamate dehydrogenase activity was undetectable, and the glutamine synthetase/NADH-glutamate synthase pathway was the predominant pathway of ammonia assimilation. Under these growth conditions, C. butyricum also lacked the activity of glucose-6-phosphate dehydrogenase, which catalyzes the regeneration of NADPH from NADP+. However, high activities of glucose-6-phosphate dehydrogenase as well as of NADPH-glutamate dehydrogenase with a Km of 2.8 mM for NH4+ were present in C. butyricum after growth on complex nitrogen and carbon sources. The ammonia-assimilating pathway of N2-fixing C. butyricum, which differs from that of the previously studied Bacillus polymyxa and Bacillus macerans, is discussed in relation to possible effects of the availability of ATP and of NADPH on ammonia-assimilating pathways.     

Exploring symbiotic nitrogen fixation and assimilation in pea root nodules by in vivo 15N nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry

Nitrogen (N) fixation and assimilation in pea (Pisum sativum) root nodules were studied by in vivo (15)N nuclear magnetic resonance (NMR) by exposing detached nodules to (15)N(2) via a perfusion medium, while recording a time course of spectra. In vivo (31)P NMR spectroscopy was used to monitor the physiological state of the metabolically active nodules. The nodules were extracted after the NMR studies and analyzed for total soluble amino acid pools and (15)N labeling of individual amino acids by liquid chromatography-mass spectrometry. A substantial pool of free ammonium was observed by (15)N NMR to be present in metabolically active, intact nodules. The ammonium ions were located in an intracellular environment that caused a remarkable change in the in vivo (15)N chemical shift. Alkalinity of the ammonium-containing compartment may explain the unusual chemical shift; thus, the observations could indicate that ammonium is located in the bacteroids. The observed (15)N-labeled amino acids, glutamine/glutamate and asparagine (Asn), apparently reside in a different compartment, presumably the plant cytoplasm, because no changes in the expected in vivo (15)N chemical shifts were observed. Extensive (15)N labeling of Asn was observed by liquid chromatography-mass spectrometry, which is consistent with the generally accepted role of Asn as the end product of primary N assimilation in pea nodules. However, the Asn (15)N amino signal was absent in in vivo (15)N NMR spectra, which could be because of an unfavorable nuclear Overhauser effect. gamma-Aminobutyric acid accumulated in the nodules during incubation, but newly synthesized (15)N gamma-aminobutyric acid seemed to be immobilized in metabolically active pea nodules, which made it NMR invisible.     

Dilution rates influence ammonia-assimilating enzyme activities and cell parameters of Selenomonas ruminantium strain D in continuous (glucose-limited) culture

Aims: The objective of this study was to examine the effect of dilution rates (Ds, varying from 0·05 to 0·42 h^?1) in glucose‐limited continuous culture on cell yield, cell composition, fermentation pattern and ammonia assimilation enzymes of Selenomonas ruminantium strain D. Methods and Results: All glucose‐limited continuous culture experiments were conducted under anaerobic conditions. Except for protein, all cell constituents including carbohydrates, RNA and DNA yielded significant cubic responses to Ds with the highest values at Ds of either 0·10 or 0·20 h^?1. At Ds higher than 0·2 h^?1, fermentation acid pattern shifted primarily from propionate and acetate to lactate production. Succinate also accumulated at the higher Ds (0·30 and 0·42 h^?1). Glucose was most efficiently utilized by S. ruminantium D at 0·20 h^?1 after which decreases in glucose and ATP yields were observed. Under energy limiting conditions, glutamine synthetase (GS) and glutamate dehydrogenase (GDH) appeared to be the major enzymes involved in nitrogen assimilation suggesting that other potential ammonia incorporating enzymes were of little importance in ammonia assimilation in S. ruminantium D. GS exhibited lower activities than GDH at all Ds, which indicates that the bacterial growth rate is not a primary regulator of their activities. Conclusions: Studied dilution rates influenced cell composition, fermentation pattern and nitrogen assimilation of S. ruminantium strain D grown in glucose‐limited continuous culture. Significance and Impact of the Study: Selenomonas ruminantium D is an ecologically and evolutionary important bacterium in ruminants and is present under most rumen dietary conditions. Characterizing the growth physiology and ammonia assimilation enzymes of S. ruminantium D during glucose limitation at Ds, which simulate the liquid turnover rates in rumen, will provide a better understanding of how this micro‐organism responds to differing growth conditions.     

营养条件对光滑球拟酵母发酵生产丙酮酸的影响

丙酮酸是多种氨基酸、维生素及其它有用物质的重要前体,广泛应用于化工、制药及农用化学品工业。能够直接发酵生产丙酮酸的菌种主要有Ａｃｉｎｅｔｏｂａｃｔｅｒ[1],Ｅｎｔｅｒｏｂａｃｔｅｒ[2],Ｅｎｔｅｒｏｃｏｃｃｕｓ[3],Ｅｓｃｈｅｒｉｃｈｉａ[4],Ａｇａｒｉｃｕ?..     

Origin of the ammonia used for mitochondrial citrulline synthesis as revealed by 13C-15N spin coupling patterns observed by 13C NMR.

The sources of ammonia used by isolated, intact rat liver mitochondria in the production of citrulline have been investigated in situ using a novel methodology based on the analysis of 13C-15N heteronuclear couplings observed by 13C NMR. Isolated mitochondria from rat liver were incubated with ornithine, 13CO3H- and 15NH4Cl, using unlabeled glutamate or glutamine as alternative, intramitochondrial nitrogen donors. The production of (7-13C, 8-15N) or (7-13C, 8-14N) citrulline was determined in situ by 13C NMR and the relative proportions of 15N- and 14N-citrullines confirmed by high resolution 13C NMR analysis of the C-7 citrulline resonance observed in perchloric acid extracts prepared at the end of the incubations. The 15N fractional enrichment of the intramitochondrial NH3 pool was manipulated either by modifying the 15N enrichment of added 15NH4Cl, or by altering the concentration of the unlabeled nitrogen donors in the incubation medium. Fractional 15N enrichments measured in the N-8 nitrogen of the resulting (7-13C) citrulline closely paralleled those of the external 15NH4Cl with minor dilutions derived from the unlabeled nitrogen contribution from the alternative substrates. In the presence of 10 mM 15NH4Cl, 10 mM glutamate contributed 4% of the citrulline N-8 nitrogen. Under similar conditions, the contribution of nitrogen from 10 mM glutamine to N-8 citrulline was 6%. These results indicate that the primary source of ammonia used for citrulline synthesis by isolated, intact rat liver mitochondria is extramitochondrial, providing also an illustration of the use of 13C-15N spin coupling patterns observed by 13C NMR, as a new tool in the study of ammonia metabolism.     

Organic and inorganic nitrogen source effects on the metabolism of Clostridium acetobytulicum

Summary The effects of organic and inorganic nitrogen combinations on cell growth, solvent production and nitrogen utilization by Clostridium acetobutylicum ATCC 824 was studied in batch fermentations. Fermentations in media with 10 mM glutamic acid, as the organic nitrogen source, and 0 mM to 10 mM ammonium chloride, as the inorganic nitrogen source had a solvent yield of 0.8 to 1.08 mmol solvent/mmol glucose used, with a slow fermentation rate (2 mmol solvent/l h^-1). When media contained 20 mM or 30 mM glutamic acid as well as 2.5 to 7.5 mM ammonium chloride the fermentation rate increased (5.5 mmol/l h^-1) while the solvent yield remained constant (0.86 to 0.96 mmol solvent/mmol glucose used). Total solvent production was higher in media containing 20 mM or 30 mM glutamic acid than with 10 mM glutamic acid.