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.     

Culture conditions for growth and solvent biosynthesis by a modified Clostridium acetobutylicum

Summary A modified strain of Clostridium acetobutylicum and the fermentation medium conditions for good growth of the culture and normal production of solvents are described. The pretreatment of the culture with butyric-acid-enriched medium increased the final solvent yield on sugar and lowered the residual butyric acid accumulation. In a complex medium, relatively high concentrations of yeast extract (7.5 g/l) and ammonium sulphate (3 g/l to 6 g/l) were required for normal solvent synthesis. The nitrogen requirements for cellular growth and solvent production were distinctively different. Production of solvents and growth of the culture were dependent on the concentration of para-aminobenzoic acid and relatively independent of the variations of the initial pH of the medium in the range of 4.6 to 6.3. Solvent production was obtained with initial glucose concentrations of 20.5 g/l to 70 g/l, resulting in a maximum solvent concentration of 22 g/l and a maximum yield on glucose of 32.7%.     

Influence of Ammonium Salts and Cane Molasses on Growth of Alcaligenes eutrophus and Production of Polyhydroxybutyrate

The production of polyhydroxybutyrate (PHB) by Alcaligenes eutrophus DSM 545 was studied in a synthetic medium with 3% glucose at pH 7.0 supplemented with several ammonium substrates and cane molasses. Growth was measured by dry cell weight, and the PHB content was measured by gas chromatography. The effects of ammonium sources such as sulfate, nitrate, phosphate, and chloride salts and those of different ammonium sulfate concentrations were evaluated. The best growth and PHB production were obtained with ammonium sulfate; however, NH(inf4)(sup+) concentrations between 0.5 and 1.5 g/liter showed no significant difference. Ammonium sulfate was therefore used as the sole source of NH(inf4)(sup+) for experiments with cane molasses as the growth activator. Optimal growth and PHB production were obtained with 0.3% molasses. However, the yields of biomass (39 to 48%) and PHB (17 to 26%) varied significantly among the different ammonium substrates and cane molasses concentrations.     

Effect of nitrogen source on biosynthesis of rapamycin by Streptomyces hygroscopicus

Six non-amino acid nitrogen compounds were examined as nitrogen source for growth of Streptomyces hygroscopicus and biosynthesis of rapamycin. Of the nitrogen sources studied, ammonium sulfate was the best with respect to formation of rapamycin, and supported cell growth comparable to the organic nitrogen sources used in the control chemically defined medium, ie, aspartate, arginine plus histidine. In the new chemically defined medium, which is buffered with 200 mM 2-(N-morpholino)ethanesulfonic acid to prevent decline of pH during fermentation, an ammonium sulfate concentration of 40 mM was optimal for biosynthesis of rapamycin. Rapamycin production increased by more than 30% on both volumetric and specific bases as compared to the previous medium containing the three amino acids as nitrogen source. Received 08 November 1996/ Accepted in revised form 07 April 1997     

Effects of glucose and glycerol on gamma-poly(glutamic acid) formation by Bacillus licheniformis ATCC 9945a

Bacillus licheniformis ATCC 9945a is one of the bacterial strains that produce gamma-poly(glutamic acid) (gamma-PGA). The use of carbohydrate medium components for gamma-PGA production was explored. Cells were grown in shake flasks or in controlled pH fermentors using medium formulations that contain different carbon sources. During the cultivations, aliquots were removed to monitor cell growth, carbon utilization, polymer production, and polymer molecular weight. Glucose was a better carbon source than glycerol for cell growth. Furthermore, glucose was utilized at a faster rate than glycerol, citrate, or glutamate. However, by using mixtures of glucose and glycerol in medium formulations, the efficiency of gamma-PGA production increased. For example, by increasing the glycerol in medium formulations from 0 to 40 g/L, the gamma-PGA broth concentration after 96 h increased from 5.7 to 20.5 g/L. Considering that glycerol utilization was low for the glucose/glycerol mixtures studied, it was unclear as to the mechanism by which glycerol leads to enhanced product formation. Cell growth and concomitant gamma-PGA production (12 g/L) at pH 6.5 was possible using glucose as a carbon source if trace amounts (0.5 g/L each) of citrate and glutamate were present in the medium. We suggested that citrate and glutamate were useful in preventing salt precipitation from the medium. In addition, glutamate may be preferred relative to ammonium chloride as a nitrogen source. The conversion of glucose to gamma-PGA by the strain ATCC 9945a was believed to occur by glycolysis of glucose to acetyl-CoA and tricarboxylic acid (TCA) cycle intermediates that were then metabolized via the TCA cycle to form alpha-ketoglutarate, which is a direct glutamate precursor.     

Fatty acid and carotenoid production by Sporobolomyces ruberrimus when using technical glycerol and ammonium sulfate

The production of carotenoids, lipid content, and fatty acid composition were all studied in a strain of Sporobolomyces ruberrimus when using different concentrations of technical glycerol as the carbon source and ammonium sulfate as the nitrogen source. The total lipids represented an average of 13% of the dry weight, and the maximum lipids were obtained when using 65.5 g/l technical glycerol (133.63 mg/ g). The optimal conditions for fatty acid production were at 27 degrees C using 20 g of ammonium sulfate and a pH range from 6 to 7, which produced a fatty acid yield of 32.5+/-1 mg/g, including 1.27+/- 0.15 mg of linolenic acid (LNA), 7.50+/-0.45 mg of linoleic acid (LLA), 5.50+/-0.35 mg of palmitic acid (PA), 0.60+/-0.03 mg of palmitoleic acid (PAL), 1.28+/-0.11 mg of stearic acid (SA), 9.09+/-0.22 mg of oleic acid, 2.50+/-0.10 mg of erucic acid (EA), and 4.25+/-0.20 mg of lignoceric acid (LCA), where the palmitic, oleic, and linoleic acids combined formed about 37% of the total fatty acids. The concentration of total carotenoids was 2.80 mg/g when using 20 g of ammonium sulfate, and consisted of torularhodin (2.70 mg/g) and beta-carotene (0.10 mg/ g), at 23 degrees C and pH 6. However, the highest amount with the maximum specific growth rate was obtained (micromax=0.096 h(-1)) with an ammonium sulfate concentration of 30 g/l.     

Effect of levulinic acid on pigment biosynthesis in Agmenellum quadruplicatum.

When levulinic acid was added to a growing culture of the cyanobacterium (blue-green alga) Agmenellum quadruplicatum PR-6, delta-aminoelevulinic acid accumulated in the medium and chlorophyll a synthesis and cell growth were inhibited, but there was a small amount of c-phycocyanin synthesis. The amount of delta-aminolevulinic acid produced in the treated culture did not fully account for the amount of pigment synthesized in the untreated control. Levulinic acid and either sodium nitrate or ammonium chloride were added to nitrogen-starved cultures of PR-6, and delta-aminolevulinic acid production and chlorophyll a and c-phycocyanin content were monitored. When ammonium chloride was added as a nitrogen source after nitrogen starvation, the cells recovered more rapidly than when sodium nitrate was added as a nitrogen source. In cultures recovering from nitrogen starvation, synthesis of c-phycocyanin occurred before synthesis of chlorophyll a.     

Influence of different vitamin-nitrogen sources on cell growth and propionic acid production from sucrose by Propionibacterium shermanii

Cell growth and organic acid production by Propionibacteria are dependent on the vitamin-nitrogen source in the culture medium. Final cell and propionic acid concentrations produced by Propionibacterium shermanii, using corn-steep liquor, were higher than those obtained utilizing yeast extracts. Since corn-steep liquor is much cheaper than yeast extract, the process becomes more attractive. By calculating the specific growth rates, it was observed that the critical propionic acid concentration, that prevents all growth (μ_X = 0), is different depending on the vitamin-nitrogen source used and its concentration. For example, for 5.0 and 15.0 g/l Oxoid yeast extract, those critical propionic acid concentrations were 16.0 and 27.0 g/l, respectively. Such propionic acid concentrations inhibit the cell growth, but not the formation of acid. The specific propionic acid production rate also indicates that the critical concentration for metabolic activity, when propionic acid is no longer produced (μ_P = 0), varies according to the vitamin-nitrogen source and its concentration in the medium. For 5.0 and 15.0 g/l Oxoid yeast extract, those concentrations were 22.1 and 30.1 g/l, respectively.     

戊糖乳杆菌发酵培养基氮源条件的优化

对戊糖乳杆菌发酵培养基的氮源条件进行了优化。通过单因素实验及响应面分析优化利用木糖高产乳酸的戊糖乳杆菌发酵培养基的不同氮源组合。优化得到的牛肉膏与柠檬酸氢二铵复合的最佳组成为牛肉膏17.72 g/L,柠檬酸氢二铵1.91 g/L,得到乳酸实际最大产量42.37 g/L。添加玉米浆与酵母粉和无机氮源复合的最佳组成为玉米浆46.54 g/L,酵母粉21.95 g/L,柠檬酸氢二铵9.95 g/L,可得到乳酸最大产量41.06 g/L。通过响应面优化减少了有机氮源的种类。牛肉膏与柠檬酸氢二铵的复合得到了更高的乳酸产量,且减少了有机氮源用量,节约了成本。玉米浆与酵母粉的复合解决了单一玉米浆造成的木糖利用速率过低的问题,同样得到较高浓度的乳酸。     

Induction,maintenance, and differentiation of rice callus cultures on ammonium as sole nitrogen source

Rice callus was initiated from cultured anthers and was maintained on medium containing ammonium as sole nitrogen source and supplemented with either a buffer or an organic acid. Plant regeneration also was obtained on this medium, but the morphogenetic capacity of the callus culture declined as rapidly as on a medium containing a mixture of ammonium and nitrate. The results of the studies suggest that organic acids provide an additional function beyond that of buffering the medium during growth on ammonium.     

Enhancement and Cultural Characteristics of Leucomycin Production by Streptomyces kitasatoensis in the Presence of Magnesium Phosphate

Stimulation of leucomycin production with Streptomyces kitasatoensis by magnesium phosphate (MgP), and a concomitant decrease of ammonium ion concentration in the culture supernatant have been reported from this laboratory. Cultural characteristics of the MgP-supplemented culture were further examined in relation to the stimulation of leucomycin production. When the leucomycin titer increased 4- to 5-fold in the presence of MgP, mycelial growth level and assimilation rate of a major carbon source (glycerol) increased about 2-fold. MgP-grown mycelia were not appreciably different in shape or in carbon and nitrogen contents from the control mycelia. Magnesium and phosphate ions were released from MgP into the medium, while the addition of magnesium or phosphate salts to the control medium was rather inhibitory for leucomycin production. The pH values of MgP-supplemented cultures were lower than those of the control culture. When modifications of the basal media and culture conditions were done so as to permit the pH-change almost identical with that of the control media, the leucomycin titer was still higher in the presence of MgP than in its absence. When uric acid, an insoluble compound, was used as the sole nitrogen source, the leucomycin production increased to a high level even in the absence of MgP, which was comparable to that seen when MgP was added to the ammonium lactate-containing medium. The ammonium ion concentration in the culture supernatant of the uric acid-containing medium was maintained at a considerably low level. While in the ammonium lactate-containing medium, leucomycin production was low and ammonium ion concentration remained at a high level. Thus, it was demonstrated that the stimulation of leucomycin production by MgP is related to the lowering of ammonium ion level, but not to the other factors examined such as growth level, carbon or phosphate regulation and pH-effect.     

耐铵型产琥珀酸放线杆菌的选育及铵离子对其生长代谢的影响

经硫酸二乙酯(DES)诱变,在含61～242mmol/LNH4+梯度平板中,筛选到一株耐铵型突变株YZ25,该菌株在含121mmol/LNH4+发酵培养基中,琥珀酸产量达32.68g/L,转化率为65.4%,比出发菌提高了180.5%。进一步考察了不同形态铵盐对YZ25生长的影响,结果表明添加少量铵盐能够提高突变菌的生长速率,但当超过一定量后菌株生长受到抑制,不同铵盐对菌株的抑制程度不同,硫酸铵、碳酸氢铵、氯化铵和硝酸铵对突变株YZ25的半抑制浓度分别为:215mmol/L、265mmol/L、235mmol/L、210mmol/L。为了考察铵离子对YZ25发酵产琥珀酸的影响过程,在3.0L发酵罐以氨水作为pH的调控剂发酵,结果表明在稳定期前菌株生长基本不受铵离子抑制,生物量能够达到正常水平,但是进入稳定期后铵离子抑制作用越来越明显,导致菌株生长提前结束,耗糖不完全,产酸受阻。最后结合产琥珀酸放线杆菌Actinobacillus succinogenes代谢途径分析了铵离子对菌株抑制作用的机理。     

Defined medium for Aquaspirillum serpens VHL effective in batch and continuous culture.

A defined medium for Aquaspirillum serpens VHL allows the replacement of the complex media now in use. It was developed by batch culture methods but supports growth in continuous culture. A basal salts medium supplemented with L-aspartic acid, L-alanine, and L-glutamic acid provided the best growth (turbidity), as long as ammonium chloride was omitted. Ammonium chloride caused either a lag or a reduction or a complete inhibition of the growth of A. serpens VHL on the above amino acids and other organic supplements depending on the combination used. Ammonium sulfate and ammonium hydroxide with L-glutamic acid allowed growth, but the lag period was increased in shake flask cultures. Vitamins, cysteine hydrochloride, and carbon dioxide had no effect on the growth rate. Viability (less than 50%) was inadequate to maintain continuous culture with L-glutamic acid as the sole source of carbon and nitrogen. Combinations of amino and carboxylic acids were then tested and, of these, L-glutamic acid (1 g/liter) and L-histidine (75 mg/liter) without ammonium chloride in the basal salts medium supported growth in batch and continuous culture. L-Glutamic acid was the limiting substrate for growth.     

SOME FACTORS AFFECTING GROWTH OF APHANOMYCES EUTEICHES IN SYNTHETIC MEDIA

Papavizas , G. C., and C. B. Davey . (USDA, ARS, Crops Research Division, Beltsville, Maryland.) Some factors affecting growth of Aphanomyces euteiches in synthetic media. Amer. Jour. Bot. 47(9) : 758–765. Illus. 1960.—Some nutritional requirements of 3 single-zoospore isolates of Aphanomyces euteiches Drechsler were studied in a synthetic medium (SM-1) consisting of mineral salts, D-glucose, DL-glutamic acid, and thioglycolic acid. Micronutrients were essential for growth, whereas vitamins were not. Controlled pH experiments showed that the fungus has a relatively wide pH range (5.2–7.7) for growth with an optimal range between 5.4 and 6.5. When the pH of the medium was maintained within the optimal range by periodic adjustments, ammonium nitrogen was utilized as the sole source of nitrogen. Nitrate nitrogen was unavailable to the fungus under all conditions. DL-glutamic acid was superior as a nitrogen source to either the D or the L isomer. Growth rate and final mycelial dry weight in a synthetic medium (SM-2) containing 14 amino acids in proportions found in 1.0% solution of yeast extract approximated those obtained in complex media. Mycelial dry weights in SM-2 were superior to those obtained in the glucose-glutamic acid-thioglycolic acid medium, but this superiority was due to DL-methionine, a sulfur-containing compound of the amino acid mixture. Sulfates were not utilized as sole source of sulfur, whereas cysteine, cystine, and thioglycolic acid supported fair amounts of growth. The optimal sulfur concentrations from thioglycolic acid for growth in SM-1 were between 128 and 192 mg/1. Mycelial yields equalling or exceeding those supported by complex media were obtained with high concentrations of D-glucose and DL-glutamic acid.     

Study of fermentation behavior and formulation of a semidefined nutrient medium based on acid-production measurements in Z. mobilis cultures

Batch fermentations of glucose to ethanol by Z. Mobilis.(ATCC 10988) were examined in several semidefined nutrient media. The measurement of acid produced by the microorganism was used to study its transient fermentation characteristics. Limitation of nitrogen source in the semidefined medium of Rogers and coworkers(2) was found to limit the growth of this microorganism in the late stages of batch fermentations, when the initial glucose concentration was 75 g/L and higher. The microorganism exhibits a preference for inorganic nitrogen over preformed organic nitrogen provided by yeast extract. The microbial growth occurs exponentially in the presence of ammonium sulfate and yeast extract. However, in the absence of ammonium sulfate, the growth occurs in a linear fashion. The "linear" growth phase is characterized by poor cell-mass yields, and during this phase, growth and ethanol production are decoupled. An improved semi-defined growth medium is established which supports better growth rate and cellular yield, without affecting the ethanol yield.     

Production of lactic acid and byproducts from waste potato starch by <Emphasis Type="Italic">Rhizopus arrhizus</Emphasis>: role of nitrogen sources

Summary Lactic acid was produced by Rhizopus arrhizus using waste potato starch as the substrate. The aim of this study was to identify the role of nitrogen sources and their impact on the formation of lactic acid and associated byproducts. Ammonium sulphate, ammonium nitrate, urea, yeast extract and peptone were assessed in conjunction with various ratios of carbon to nitrogen (C:N). Fermentation media with a low C:N ratio enhanced the production of lactic acid, biomass and ethanol, while a high C:N ratio favoured the production of fumaric acid. Ammonium nitrate appeared to be the most suitable nitrogen source for achieving a high and stable lactic acid yield, and minimizing the production of byproducts such as biomass and ethanol, while urea proved to be the least favourable nitrogen source. Yeast extract and peptone appeared to improve fungal cell growth. The kinetics data revealed that a high concentration of ammonium nitrate enhanced the lactic acid productivity. The maximum lactic acid concentration of 36.4 g/l, representing a yield of 91%, was obtained with addition of 0.909 g/l ammonium nitrate in 32 h.     

The effect of lactate addition on the growth of Penicillium camembertii on glutamate

The effect of an additional carbon source, lactate, on Penicillium camembertii growth on glutamate as both carbon and nitrogen sources was examined. Glutamate (and lactate) was present in excess in both media. Throughout the whole culture, similar growth time-courses were recorded on both media, indicating the absence of a lactate effect on growth. During the first part of growth, corresponding to an increasing amount of viable biomass, the rate of glutamate consumption remained high, as well as the related ammonium production, indicating its use as a carbon source in addition to being nitrogen source. The low growth rates recorded during the last part of growth resulted in low glutamate consumption, while lactate consumption continued mainly by a maintenance mechanism for the energy supply. A clear differentiation appeared therefore between the carbon source and the energy source: glutamate was mainly used as C source (and N source) for biosynthesis, while lactate was mainly assimilated for energy supply. Carbon and nitrogen yield examinations confirmed this result. Indeed, the C/N ratio found for P. camembertii cellular material (8.14) was about twice that of glutamate (4.29). From this, about half of the available nitrogen was used for biomass formation during growth on glutamate-lactate based medium, as experimentally confirmed (constant yield nitrogen from biomass on nitrogen from glutamate was found (0.49), while the excess nitrogen was released as ammonium). The constant and close to unit (0.99) yield carbon from CO2 on carbon from lactate, also recorded during growth on glutamate-lactate based medium, confirmed that lactate was mainly used as an energy source.     

Isolation and Characterization of NH+4-Excreting Mutants of Enterobacter gergoviae

NH4+-excreting mutants were isolated from Enterobacter gergoviae 57–7 wild type as methylamine resistant strains which were obtained by mutagenesis with a transposable element Tn5. The MG 61 mutants excreted 2 mmol/L of ammonium during a diazotrophic growth. The growth of MG 61 mutants were slower than the growth of wild types because of its excreting ammonium. MG 61 mutants expressed up to 86% of the fully depressed nitrogenase activity when grown in a medium containing 20 mmol/L ammonium. By contrast the ammonium grown cultures of wild type had no nitrogenase activity. In the presence of 5 mmol/L or 30 mmol/L of ammonium in the medium, the growth of MG 61 mutants was as same as CK and much slower than that of the wild types which means that the mutants could not utilize amonium very well in the medium. But MG 61 mutants could utilize glutamate as a sole nitrogen source. In the presence of nitrate (10 mmol/L) in the medium, MG 61 mutants grew slowly but excreted 7.8 mmol/L of ammonium.     

Effect of major nutrients on podophyllotoxin production in <Emphasis Type="Italic">Podophyllum hexandrum</Emphasis> suspension cultures

The effect of major medium ingredients (sugar, nitrogen source and phosphate) in Podophyllum hexandrum suspension cultures was investigated in order to increase the production of podophyllotoxin, the raw material in the synthesis of anticancer drugs. Amongst B5, Eriksson, MS, Nitsch, Street and White's medium, MS medium resulted in high growth and podophyllotoxin accumulation. The optimum level of nitrogen was found to be 60 mM, with a combination of ammonium salts and nitrate in the ratio of 1:2. The highest level of podophyllotoxin was obtained at 60 g glucose/l and at 1.25 mM phosphate after 30 days. Statistical design was adopted to determine the optimum levels of the parameters for cell growth and podophyllotoxin production.