Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by fed-batch cultures of Pseudomonas sp EL-2

Pseudomonas sp EL-2 was cultivated to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] from a structurally unrelated carbon source, glucose, by a fed-batch culture technique. Variation of the carbon to nitrogen (C/N) ratio of the medium produced optimal P(3HB-co-3HV) production at a C/N ratio of 95. Production of P(3HB-co-3HV) was favored by a dissolved oxygen tension of 40%. A maximum biomass concentration of 38 g L⁻¹ containing 53% P(3HB-co-3HV) was achieved after 45 h of cultivation. This corresponds to a volumetric productivity of 0.84 g L⁻¹ h⁻¹. The copolymer contained 7.5 mol% 3-hydroxyvalerate. Journal of Industrial Microbiology & Biotechnology (2000) 24, 36–40. Received 28 January 1999/ Accepted in revised form 11 September 1999     

Isolation and characterization of an extracellular thermoalkanophilic P(3HB-co-3HV) depolymerase from Streptomyces sp. IN1

Here, we report on the biodegradation of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] by a novel thermoalkanophilic extracellular esterase from the soil isolate Streptomyces sp. IN1. Preliminary screening and isolation of the bacterium was done using polyhydroxyalkanoate latex medium (PHALM). The isolate was cultured with P(3HB-co-3HV) as the only carbon source and by-products of degradation were derivatized with [N,O-bis(trimethylsilyl)trifluroacetamide] (BSTFA). These products were identified by gas chromatography/mass spectrometry (GC-MS) as silylated hydroxybutyric acid (3HB) and hydroxyvaleric acid, suggesting extracellular depolymerase activity by the isolate. The depolymerase was isolated by (NH₄)₂SO₄ fractionation, dialyzed and purified using fast protein liquid chromatography (FPLC), and confirmed using P(3HB-co-3HV) as a sole source of carbon. The molecular mass of the FPLC purified enzyme occurred between 45 and 66 kDa (SDS-PAGE), but was confirmed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to be 62 kDa. Enzyme activity was significantly inhibited by phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT), and Tween 80, but induced by azide (N³⁻). Sensitivity to PMSF, DTT, and Tween 80 suggests the involvement of serine as an active site amino acid with disulphide bonds contributing to the catalytic activity, as well as the presence of hydrophobic regions in the enzyme. Non-inhibition of activity by azide indicates that metal ions may not be required as cofactors for activity. This observation was further corroborated by the decrease in enzyme activity in the presence of metal ions such as Ca²⁺, Mg²⁺, Na⁺, and K⁺. The kinetic parameters, V_max and K_m, in the presence of p-nitrophenylbutyrate as substrate, were determined to be 5.06 × 10⁻¹ ??mol min⁻¹ and 6.73 × 10⁻¹ mM, respectively.     

Regulation of poly-(R)-(3-hydroxybutyrate-co-3-hydroxyvalerate) biosynthesis by the AtoSCDAEB regulon in phaCAB + Escherichia coli

AtoSC two-component system (TCS) upregulates the high-molecular weight poly-(R)-3-hydroxybutyrate (PHB) biosynthesis in recombinant phaCAB ⁺ Escherichia coli strains, with the Cupriavidus necator phaCAB operon. We report here that AtoSC upregulates also the copolymer P(3HB-co-3HV) biosynthesis in phaCAB ⁺ E. coli. Acetoacetate-induced AtoSC maximized P(3HB-co-3HV) to 1.27 g/l with a 3HV fraction of 25.5 % wt. and biopolymer content of 75 % w/w in a time-dependent process. The atoSC locus deletion in the ?atoSC strains resulted in 4.5-fold P(3HB-co-3HV) reduction, while the 3HV fraction of the copolymer was restricted to only 6.4 % wt. The ?atoSC phenotype was restored by extrachromosomal introduction of AtoSC. Deletion of the atoDAEB operon triggered a significant decrease in P(3HB-co-3HV) synthesis and 3HV content in ?atoDAEB strains. However, the acetoacetate-induced AtoSC in those strains increased P(3HB-co-3HV) to 0.8 g/l with 21 % 3HV, while AtoC or AtoS expression increased P(3HB-co-3HV) synthesis 3.6- or 2.4-fold, respectively, upon acetoacetate. Complementation of the ?atoDAEB phenotype was achieved by the extrachromosomal introduction of the atoSCDAEB regulon. Individual inhibition of β-oxidation and mainly fatty acid biosynthesis pathways by acrylic acid or cerulenin, respectively, reduced P(3HB-co-3HV) biosynthesis. Under those conditions, introduction of atoSC or atoSCDAEB regulon was capable of upregulating biopolymer accumulation. Concurrent inhibition of both the fatty acid metabolic pathways eliminated P(3HB-co-3HV) production. P(3HB-co-3HV) upregulation in phaCAB ⁺ E. coli by AtoSC signaling through atoDAEB operon and its participation in the fatty acids metabolism interplay provide additional perceptions of AtoSC critical involvement in E. coli regulatory processes towards biotechnologically improved polyhydroxyalkanoates biosynthesis.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer by <Emphasis Type="Italic">Azotobacter chroococcum</Emphasis> strain 7B

The ability of Azotobacter chroococcum strain 7B, producer of poly(3-hydroxybutyrate) (PHB), to synthesize its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) was studied. It was demonstrated, for the first time, that A. chroococcum strain 7B was able to synthesize P(3HB-co-3HV) with various molar rates of HV in the polymer chain when cultivated on medium with sucrose and carboxylic acids as precursors of HV elements in the PHB chain, namely, valeric (13.1–21.6 mol %), propanoic (3.1 mol %), and hexanoic (2.1 mol %) acids. Qualitative and functional differences between PHB and P(3HB-co-3HV) were demonstrated by example of the release kinetic of methyl red from films made of synthesized polymers. Maximal HV incorporation into the polymer chain (28.8mol %) was recorded when the nutrient medium was supplemented with 0.1% peptone on the background of 20 mM valerate. These results suggest that that the studied strain can be regarded as a potential producer of not only PHB but also P(3HB-co-3HV).     

High-Level Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) by Fed-Batch Culture of Recombinant Escherichia coli

Fermentation strategies for production of high concentrations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] with different 3-hydroxyvalerate (3HV) fractions by recombinant Escherichia coli harboring the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes were developed. Fed-batch cultures of recombinant E. coli with the pH-stat feeding strategy facilitated production of high concentrations and high contents of P(3HB-co-3HV) in a chemically defined medium. When a feeding solution was added in order to increase the glucose and propionic acid concentrations to 20 g/liter and 20 mM, respectively, after each feeding, a cell dry weight of 120.3 g/liter and a relatively low P(3HB-co-3HV) content, 42.5 wt%, were obtained. Accumulation of a high residual concentration of propionic acid in the medium was the reason for the low P(3HB-co-3HV) content. An acetic acid induction strategy was used to stimulate the uptake and utilization of propionic acid. When a fed-batch culture and this strategy were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 141.9 g/liter, 88.1 g/liter, 62.1 wt%, and 15.3 mol%, respectively. When an improved nutrient feeding strategy, acetic acid induction, and oleic acid supplementation were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 203.1 g/liter, 158.8 g/liter, 78.2 wt%, and 10.6 mol%, respectively; this resulted in a high level of productivity, 2.88 g of P(3HB-co-3HV)/liter-h.     

A novel <Emphasis Type="Italic">Bacillus</Emphasis> sp. accumulating poly (3-hydroxybutyrate-co-3-hydroxyvalerate) from a single carbon substrate

The objective of this paper was to report a bacterium designated as 88D, capable of producing poly (3-hydroxybutyrate-co-3-hydroxyvalerate) [P (3HB-co-3HV)] copolymer from a single carbon source, which was isolated from a municipal sewage treatment plant in Hyderabad, India. This microorganism, based on the phenotypical features and genotypic investigations, was identified as Bacillus sp. The optimal growth of Bacillus sp. 88D occurred between 28 and 30°C and at pH 7. The strain yielded a maximum of 64.62% dry cell weight (DCW) polymer in the medium containing glucose as carbon source, which was followed by 60.46% DCW polymer in glycerol containing medium. Bacillus sp. 88D produced P (3HB-co-3HV) from glucose or glycerol, when they were used as a single carbon substrate. This bacterium produced polyhydrxybutyrate (PHB) when sodium acetate was used as sole carbon substrate. The viscosity average molecular mass (Mv) of the copolymers ranged from 523 to 627 kDa. The physical, chemical and mechanical properties of the biopolymers were characterized.     

High-level production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by fed-batch culture of recombinant Escherichia coli.

Fermentation strategies for production of high concentrations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] with different 3-hydroxyvalerate (3HV) fractions by recombinant Escherichia coli harboring the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes were developed. Fed-batch cultures of recombinant E. coli with the pH-stat feeding strategy facilitated production of high concentrations and high contents of P(3HB-co-3HV) in a chemically defined medium. When a feeding solution was added in order to increase the glucose and propionic acid concentrations to 20 g/liter and 20 mM, respectively, after each feeding, a cell dry weight of 120.3 g/liter and a relatively low P(3HB-co-3HV) content, 42.5 wt%, were obtained. Accumulation of a high residual concentration of propionic acid in the medium was the reason for the low P(3HB-co-3HV) content. An acetic acid induction strategy was used to stimulate the uptake and utilization of propionic acid. When a fed-batch culture and this strategy were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 141.9 g/liter, 88.1 g/liter, 62.1 wt%, and 15.3 mol%, respectively. When an improved nutrient feeding strategy, acetic acid induction, and oleic acid supplementation were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 203.1 g/liter, 158.8 g/liter, 78.2 wt%, and 10.6 mol%, respectively; this resulted in a high level of productivity, 2.88 g of P(3HB-co-3HV)/liter-h.     

Regulation by Ammonium of Variation in Heterotrophic CO2 Fixation by Euglena gracilis During Growth Cycles*

SYNOPSIS Heterotrophic (dark) CO₂ fixation by Euglena gracilis strain Z varies with phase of batch culture growth and mode of nutrition. Increases in the fixation during growth cycles correlate closely with the depletion of exogenous NH₄* from the medium during growth. It is demonstrated that exogenous NH₄⁺ regulates a component of heterotrophic CO₂ fixation and that another component is independent of NH₄⁺. This is true for cells grown heterotrophically (glucose, dark), autotrophically (CO₂, light) and for a permanently bleached strain (E. gracilis SB3). Some kinetics of the NH₄⁺ regulation are presented.     

Estimation of the Number of Polyhydroxyalkanoate (PHA)-Degraders in Soil and Isolation of Degraders Based on the Method of Most Probable Number (MPN) Using PHA-Film

A new method to estimate the number of polyhydroxyalkanoates (PHA)-degraders in soil and to isolate degraders, called the film-MPN method, is proposed. The incubation time was measured by the first order reaction (FOR) model. This method was used to estimate numbers of poly (3-hydroxybutyrate-co-3-hydroxyvalerate)[P(3HB-co-3HV)]- and poly(3-hydroxyvalerate-co-4-hydroxybutyrate)[P(3HB-co-4HB)]-degraders in garden soil (4.30 × 10⁵ and 2.15 × 10⁵ aerobic degraders per gram of dry soil, respectively). The number of P(3HB-co-3HV)-degraders in paddy field soil was 5.06 × 10⁵ aerobic degraders per gram dry soil. Also, several P(3HB-co-3HV)-degraders were isolated directly from positive-growth tubes of high dilution.     

补料培养提高真养产碱杆菌产3-羟基丁酸和3-羟基戊酸共聚物生产强度的研究

对Alcaligenes eutrophus进行高密度培养,研究表明在发酵过程中进行有效控制,可以较大幅度地提高3－羟基丁酸和3－羟基戊酸共聚物[P(3HB-co-3HV)]的生产强度。实验中选择使用限氮的方法积累P(3HB-co-3HV),分别采用丙酸和戊酸为3HV前体,对摇瓶种子生长状态,停氮时机对菌体生产P(3HB-co-3HV)的影响以及补酸（3HV前体）策略进行了研究,在6．6L罐中,以葡萄糖为碳源,以丙酸为3HV前体培养50h,细胞干重,PHA产量,PHA含量分别达到149．9g/L,149.9g/L,83.3%(其中3HV组分占PHA的12．4mol%),生产强度达到2．50（g.h^-1.L^-1);以戊酸为3HV前体培养45h,细胞干重,PHA产量,PHA含量分别达到160．2g/L,119.0g/L,74.2%（其中3HV组分占PHA的17．7mol%)生产强度达到2．64（g.h^-1.L^-1)。     

Synthesis of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) by recombinant Escherichia coli

Several recombinant Escherichia coli strains, including XL1-Blue, JM109, HB101, and DH5alpha harboring a stable high-copynumber plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoate (PHA) biosynthesis genes were constructed. These recombinant strains were examined for their ability to synthesize and accumulate poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymer from glucose and either propionate or valerate. All recombinant E. coli strains could synthesize the P(3HB-co-3HV) copolymer in the medium containing glucose and propionate. However, only the homopolymer poly-(3-hydroxybutyrate) [P(3HB)] was synthesized from glucose and valerate. The PHA concentration and the 3HV fraction could be increased by inducing with acetate and/or oleate. When supplemented with oleate, the 3HV fraction increased by fourfold compared with that obtained without induction. Induction with propionate resulted in lower PHA concentration due to the inhibitory effect, but an 3HV fraction of as high as 33.0% could be obtained. These results suggest that P(3HB-co-3HV) can be efficiently produced from propionate by recombinant E. coli by inducing with acetate, propionate, or oleate. (c) 1996 John Wiley & Sons, Inc.     

Accumulation of PHA and its copolyesters by Methylobacterium sp. KCTC 0048

Summary Methylobacterium sp. KCTC 0048 isolated from soil, could synthesize a variety of copolyesters when secondary carbon substrates were added to nitrogen-limited cultures containing methanol as a major carbon and energy source. The copolyester of 3-hydroxy-butyrate and 3-hydroxyvalerate, P(3HB-co-3HV) accumulated when valeric acid, pentanol or heptanoic acid was added to the nitrogen-limited medium containing methanol. The copolyester of 3-hydroxybutyrate and 4-hydroxybutyrate, P(3HB-co-4HB) was synthesized from 4-hydroxybutyrate, 1,4-butanediol, or -butyrolactone, and the copolyester of 3-hydroxybutyrate and 3-hydroxypropionate (P(3HB-co-3HP)), from 3-hydroxypropionate as the secondary carbon substrates, respectively.     

Physico-chemical properties of polyhydroxyalkanoate produced by mixed-culture nitrogen-fixing bacteria

Ultra-high molecular weight polyhydroxyalkanoates (PHAs) with low polydispersity index (PDI = 1.3) were produced in a novel, pilot scale application of mixed cultures of nitrogen-fixing bacteria. The number average molecular weight (M _n) of the poly(3-hydroxybutyrate) (P(3HB)) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) was determined to be 2.4 × 10⁶ and 2.5 × 10⁶ g mol⁻¹, respectively. Using two types of carbon sources, biomass contents of the P(3HB) and P(3HB-co-3HV) were 18% and 30% (PHA in dry biomass), respectively. The extracted polymers were analysed for their physical properties using analytical techniques such as nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry (DSC) and gel permeation chromatography (GPC). NMR confirmed the formation of homopolymer and copolymer. DSC showed a single melting endotherm peak for both polymers, with enthalpies that indicated crystallinity indices of 44% and 37% for P(3HB) and P(3HB-co-3HV), respectively. GPC showed a sharp unimodal trace for both polymers, reflecting the homogeneity of the polymer chains. The work described here emphasises the potential of mixed colony nitrogen-fixing bacteria cultures for producing biodegradable polymers which have properties that are very similar to those from their pure-culture counterparts and therefore making a more economically viable route for obtaining biopolyesters.     

γ‐Aminobutyric acid addition alleviates ammonium toxicity by limiting ammonium accumulation in rice (Oryza sativa) seedlings

Excessive use of nitrogen (N) fertilizer has increased ammonium (NH₄⁺) accumulation in many paddy soils to levels that reduce rice vegetative biomass and yield. Based on studies of NH₄⁺ toxicity in rice (Oryza sativa, Nanjing 44) seedlings cultured in agar medium, we found that NH₄⁺ concentrations above 0.75 mM inhibited the growth of rice and caused NH₄⁺ accumulation in both shoots and roots. Use of excessive NH₄⁺ also induced rhizosphere acidification and inhibited the absorption of K, Ca, Mg, Fe and Zn in rice seedlings. Under excessive NH₄⁺ conditions, exogenous γ‐aminobutyric acid (GABA) treatment limited NH₄⁺ accumulation in rice seedlings, reduced NH₄⁺ toxicity symptoms and promoted plant growth. GABA addition also reduced rhizosphere acidification and alleviated the inhibition of Ca, Mg, Fe and Zn absorption caused by excessive NH₄⁺. Furthermore, we found that the activity of glutamine synthetase/NADH‐glutamate synthase (GS; EC 6.3.1.2/NADH‐GOGAT; EC1.4.1.14) in root increased gradually as the NH₄⁺ concentration increased. However, when the concentration of NH₄⁺ is more than 3 mM, GABA treatment inhibited NH₄⁺‐induced increases in GS/NADH‐GOGAT activity. The inhibition of ammonium assimilation may restore the elongation of seminal rice roots repressed by high NH₄⁺. These results suggest that mitigation of ammonium accumulation and assimilation is essential for GABA‐dependent alleviation of ammonium toxicity in rice seedlings.     

Comparative evaluation of physico-chemical characteristics of biopolyesters P(3HB) and P(3HB-co-3HV) produced by endophytic <Emphasis Type="Italic">Bacillus cereus</Emphasis> RCL 02

Background

Bacteria endogenously residing within the plant tissues have attracted significant attention for production of biopolyester, polyhydroxyalkanoates (PHAs). Bacillus cereus RCL 02 (MCC 3436), a leaf endophyte of oleaginous plant Ricinus communis L. accumulates 81% poly(3-hydroxybutyrate) [P(3HB)] of its cell dry biomass when grown in mineral salts (MS) medium.

Methods

The copolymer production efficiency of B. cereus RCL 02 was evaluated in valeric acid supplemented MS medium under biphasic cultivation condition. The copolymer so produced has been compared with the P(3HB) isolated from RCL 02 in terms of thermal, mechanical and chemical properties.

Results

Valeric acid supplementation as co-substrate in the medium has led to the production of copolymer of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) [P(3HB-co-3HV)] with 14.6 mol% 3HV. The identity of the polymers has been confirmed by X-ray diffraction (XRD) analysis, Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopic studies. Thermogravimetric analysis (TGA) revealed that P(3HB) and P(3HB-co-3HV) films degraded at 278.66°C and 273.49°C, respectively. The P(3HB-co-3HV) showed lower melting temperature (165.03°C) compared to P (3HB) (170.74°C) according to differential scanning calorimetry (DSC). Incorporation of 3HV monomers decreased the tensile strength (21.52 MPa), tensile modulus (0.93 GPa), storage modulus (E′) (0.99 GPa) and increased % elongation at break (12.2%) of the copolyester. However, P(3HB) showed better barrier properties with lower water vapor transmission rate (WVTR) of 0.55 g-mil/100 in²/24 h.

Conclusion

These findings emphasized exploration of endophytic bacterial strain (RCL 02) to produce biodegradable polyesters which might have significant potential for industrial application.

     

The modification of glucose levels and N source in the Hungate's medium to stimulate the production of fibrolytic enzymes of Anaeromyces sp. YQ3 grown on corn stalks

Hungate's method is a well-accepted protocol for the isolation or incubation of anaerobes with a roll tube technique. The aim of this study was to stimulate fungal enzyme production by optimizing the components of Hungate's medium for the growth of a rumen fungus Anaeromyces sp. YQ3. The organism was grown on corn stalks and incubated for 10 days in defined media with two glucose levels (G⁺, glucose in the Hungate's medium as a glucose control; G^?, glucose removed in a modified Hungate's medium) and four N sources (N1: yeast extract + tryptone + (NH₄)₂SO₄ in Hungate's medium (control); N2: yeast extract + (NH₄)₂SO₄; N3: tryptone + (NH₄)₂SO₄; and N4: tryptone + yeast extract). In the G^? media, the recovered activities of feruloyl esterase (FAE) (P<0.0001), acetyl esterase (AE) (P=0.0065) and xylanase (P<0.0001) were decreased, while the G⁺ media with N1 nitrogen stimulated the production of FAE and xylanase (P<0.0001). The G^? medium with N2 nitrogen increased the recovered activities of carboxymethyl cellulase (P=0.0001) and avicelase (P<0.0001), while the N3 and N4 media increased the recovered activity of AE (P=0.0015). The N4 medium was comparable to the N1 medium in stimulating the amount of recovered xylanase activity. The activities of FAE (P<0.0001), AE (P<0.0001), and xylanase (P<0.0001) showed a time-dependent increase and reached their peaks at day 10, while the avicelase activity peaked at day 8 (P=0.0071). The esterase activities (FAE and AE) were positively correlated with the enzyme activities of xylanase and carboxymethyl cellulase (r > 0.48, P<0.05). After a 10-day incubation, the glucose in the Hungate's media contributed to an increase in organic matter disappearance (P<0.0001) and volatile fatty acid (VFA) concentration (P<0.0001), except for molar acetate proportions. The N4 treatment increased organic matter disappearance and total VFA concentration (P=0.0002). The change in N source did not alter molar proportions of acetate, propionate and valerate, while the N2 treatment increased molar butyrate proportion (P<0.0035), and both N2 and N3 increased the molar proportion of branched chain VFAs (P<0.0041). In summary, the glucose in the Hungate's medium is beneficial for stimulating the production of esterases and xylanase, thereby promoting fungal growth. Amending the N source in Hungate's medium brings about different yields of rumen fungal esterases and polysaccharide hydrolases that have important nutritional impacts on fibre degradation in ruminant animals.     

Variation inRhizobium leguminosarum response to short term application of NH4NO3 to nodulatedPisum sativum L.

Summary This study was conducted to determine the effect of short term application of NH₄NO₃ on nodule function and to determine whether the rhizobial isolate used was a significant factor in this effect. Pea plants were inoculated with 10 differentRhizobium leguminosarum isolates and grown for 3 weeks in N-free medium before addition of 0, 1, 2 or 5 mM NH₄NO₃ for 2 to 7 days. Acetylene reduction and leghemoglobin content decreased with increasing exposure time to NH₄NO₃ and with increasing concentration of NH₄NO₃. NH ₄ ⁺ and NO ₃ ⁻ depletion from the nutrient medium were assayed in plants exposed to 5 mM NH₄NO₃ and mean uptake rates were similar for each ion. There were significant differences among isolates in the rate of decrease of C₂H₂ reduction with increasing NH₄NO₃ concentration (C₂H₂ reduction responsiveness to NH₄NO₃) 4 and 7 days after addition of NH₄NO₃ but no differences after 2 days of exposure to NH₄NO₃. There were significant differences among isolates in NH ₄ ⁺ depletion from the nutrient medium but these differences were not correlated with the differences observed in C₂H₂ reduction. Ranking of the isolates for C₂H₂ reduction responsiveness to NH₄NO₃ applied to plants with nodules was different from that obtained when NH₄NO₃ was applied at seeding. Isolates with varying sensitivity to NH₄NO₃ may be useful tools for determining the mechanisms responsible for inhibition of symbiotic N₂ fixation by combined nitrogen. NRCC paper no. 25863.     

The effect of the source of inorganic nitrogen on growth and enzymes of nitrogen assimilation in soybean and wheat cells in suspension cultures

Summary Soybean (Glycine max L. cv. Mandarin) and wheat (Triticum monococcum L.) cells were grown in media with NO₃ ^- plus NH₄ ⁺ (B₅) and NO₃ ^- without NH₄ ⁺ (B₅-NH₄) as nitrogen sources. Changes in pH, [NO₃ ^-] and [NH₄ ⁺] in media, and dry weight, protein content, nitrate reductase (NR) and glutamate dehydrogenase (GDH) in the cells were followed for about 170 h. With both NH₄ ⁺ and NO₃ ^- in the medium, NH₄ ⁺ was utilized very quickly. Soybean cells grew poorly in the absence of NH₄ ⁺ while wheat cells grew equally well on media with or without NH₄ ⁺. When soybean cells were grown in medium with NO₃ ^- plus NH₄ ⁺, dry weight and NR activity remained relatively low for several hours after which both increased rapidly. This coincided with the time NH₄ ⁺ was depleted from the medium. In the absence of NH₄ ⁺, soybean cell growth and NR activity remained low. NR activity in wheat cells, and GDH activity in soybean and wheat cells, did not vary significantly in the presence or absence of NH₄ ⁺.This work was supported by a grant in aid of research from the National Research Council of Canada to one of us (J. K.). NRCC No. 12521.     

Ammonia production from yeast extract and its effect on growth of the hyperthermophilic archaeonSulfolobus solfataricus

Utilization of yeast extract and formation of byproduct metabolite were investigated for hyperthermophilic archaeonSulfolobus solfataricus (DSM 1617). In both batch and fed-batch cultivations ofS. solfataricus, maximal cell density, NH₄ ⁺ ion production and pH change were highly dependent on the ratio of yeast extract to glucose in the medium. Variation of NH₄ ⁺ ion level was identified as a major cause of pH change during cultivation, and acidification of culture broth was attributed to consumption of NH₄ ⁺ ions rather than formation of acid byproducts. It was also observed that increase of NH₄ ⁺ ion concentrations in the medium resulted in greater degree of growth inhibition.     

Effects of N source on pH and nutrient exchange of extramatrical mycelium in a mycorrhizal Ri T-DNA transformed root system

 The influence of different N sources on medium pH variation and the effect of the external mycelium of arbuscular mycorrhizal fungi on nutrient dynamics were studied using a two-compartment, aseptic Petri plate system. VA mycorrhizal, transformed roots of carrot (Daucus carota L.) were cultured in the proximal compartment and external mycorrhizal mycelium in the distal compartment. The medium in the distal compartament contained N either as NO₃ ^– or as NH₄ ⁺. The pH and the anion and cation concentrations were measured every 15 days in filtrates prepared from the distal compartments. Thirteen weeks after colonization, there was a significant basification or a light acidification of the NO₃ ^– and NH₄ ⁺ medium, respectively. There was no change in NO₃ ^– concentration but a significant decrease in NH₄ ⁺ concentration. Treatments containing N as NO₃ ^– showed no variation in cations such asCa²⁺ and Mg²⁺ or anions such as PO₄ ^2–, and SO₄ ^2– but showed significant increases in the concentration of K⁺. Treatments containing N as NH₄ ⁺ showed no variation in cations or anions, except for increases in the concentrations of K⁺ and Cl^–. Accepted: 7 March 1996