Production of extracellular 5-aminolevulinic acid byClostridium thermoaceticum grown in minimal medium

Summary A growth associated formation of extracellular 5-aminolevulinic acid (ALA) was found in the homoacetogenesis of glucose byClostridium thermoaceticum grown in minimal defined medium. The growth and ALA production was enhanced by L-cysteine HCl both in complex medium (UM) and minimal defined medium (MDM). The amount of ALA produced extracellularly in MDM wasca. 15 mg/L after 90-h anaerobic cultivation (cell-mass: 1.5 g/l; glucose consumed: 20 g/l).     

Living cell reaction process forl-isoleucine andl-valine production

Summary A new process (Living Cell Reaction Process) forl-isoleucine production using viable, non-growing cells ofBrevibacterium flavum AB-07 was optimised using ethanol as the energy source and -ketobutyric acid (-KB) as precursor.l-valine also could be produced from glucose at high yield by this process. This process differs from the usual fermentation method in that non-growing cells are used, and the production ofl-isoleucine andl-valine were carried out under conditions of repressed cell division and growth. Minimal medium missing the essential growth factor, biotin was employed as the reaction mixture for the production ofl-isoleucine andl-valine. The productivity ofl-isoleucine andl-valine were 200 mmol·l^–1 · day^–1 (molecular yield to -KB: 95%) and 300 mmol · l^–1 · day^–1 (molecular yield to glucose: 80%) respectively. The content ofl-isoleucine andl-valine in total amino acids produced in the each mixture were 97% and 96% respectively.     

Diacetyl production and growth of Lactobacillus rhamnosus on multiple substrates

Lactobacillus rhamnosus is a heterolactic acid bacterium, which can be used to produce flavour compounds like diacetyl and acetoin. Various startegies have been applied to improve the growth rate and diacetyl yield. The use of multiple substrates affected growth as well as the yield of diacetyl. Growth on a medium containing glucose demonstrated a diauxic growth profile, with the second phase of growth being on the product, lactic acid. L. rhamnosus also grew on a medium containing citrate. Growth on medium containing glucose+citrate demonstrated simultaneous utilization of carbon sources. L. rhamnosus did not grow in a medium containing acetate and also did not co-metabolize it with glucose. Maximum specific growth rate ( _max) was found to increase in the case of simultaneous utilization of glucose+citrate (0.38 h^–1) as compared to glucose as the sole carbon source (0.28 h^–1). The yields of diacetyl were also found to increase for glucose + pyruvate and glucose + citrate (0.10 and 0.05 g g^–1 of glucose, respectively) as compared to glucose alone (0.01 g g^–1 of glucose). The productivity of diacetyl on medium containing glucose and citrate was double that of a medium containing only citrate, although the yields were comparable.     

Light-oriented locomotion in certain myxobacter species

Summary Euglena gracilis, strain Z, was grown in synchronous culture. Carbon source used was either dl-lactic acid (L) or a mixture of l-glutamic and dl-malic acids (GM). Synchronisation was obtained by transfering the cells in the exponential growth phase, at regular intervals—each 3 days—to a fresh medium.Respiration (measured during a whole cell cycle, 12 h) was 20±6 l/H/10⁶ cells on (GM) and 46±7 l/H/10⁶ cells on (L) medium. At the same time as the increased rate of oxygen uptake on lactate medium, we observed the appearance of a giant chondriome in the cells. On glutamate—malate containing medium the size of mitochondria was normal.

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Les micrographies électroniques ont été réalisées par Mr. C. Mattei, Technicien CNRS.     

Effective extracellular trehalose production by<Emphasis Type="Italic"> Cellulosimicrobium cellulans</Emphasis>

A bacterium isolated from a petal of Casa Blanca Lily (ST26 strain) produced a marked amount of extracellular trehalose (-d-glucopyranosyl-[1,1]--d-glucopyranose) in culture medium containing glucose. 16S rDNA-based phylogeny showed that ST26 belongs to, or is related to, Cellulosimicrobium cellulans, a close relative of Cellulomonas spp. Various Cellulomonas strains obtained from culture collections also showed extracellular trehalose productivity, suggesting that trehalose production is a common property of this bacterial genus. ST26 accumulated trehalose in medium supplied with glucose but not with sucrose, glycerol or maltose. Effective extracellular trehalose production by ST26 was achieved by supplying 0.5–1% ammonium sulfate and 0.5–1% CaCO₃. The addition of CaCO₃ adjusted the pH of the culture to around 5.0. The optimized culture conditions yielded trehalose from glucose at a conversion rate of 61%. The addition of ammonium sulfate greatly reduced the dry cell weight of ST26 and intracellular content of trehalose, which suggests that the addition of ammonium sulfate makes ST26 cells leak trehalose into the medium. ST26 effectively propagated in minimal medium containing trehalose as a sole carbon source, which suggests that trehalose serves as a carbohydrate reserve of this organism.The nucleotide sequence of 16S rDNA of ST26 has been submitted to the DDBJ databank under accession number AB109293     

The appearance of exo-α-mannosidase in cultures of Penicillium charlessi

Summary Growth of the fungus Penicillium charlesii G. Smith on glucose, minimal salts medium results in the appearance of -d-mannopyranosidase activity capable of hydrolyzing p-nitrophenyl--d-mannopyranoside. No activity is found until depletion of the carbon source, after which the enzyme activity rapidly increases in the mycelium. Prolonged incubation of the culture results in the appearance of small amounts of -mannosidase activity in the growth medium. The initial release of a mannose-containing polysaccharide into the medium precedes the appearance of -mannosidase by several days.     

The use of various substrates and substrate concentrations by a Hyphomicrobium sp. isolated from soil: Effect on growth rate and growth yield

The content of Hyphomicrobium sp. was estimated from a clay loam soil using the most probable number technique with methanol as the sole carbon source. The method enumerated Hyphomicrobia as 0.2% of the total bacteria determined by acridine orange direct counts. Hyphomicrobium sp. was not able to use C-C compounds such as glucose or acetate for growth. Maximal growth yield and growth rate were obtained when the concentration of methanol was in the range of 0.5–5 mg C/liter. Substrate affinity measurements revealed K_s values of 0.8 m and 5.8 m when the methanol concentration was 0.5–2.5 m and 5–200 m, respectively. Hyphomicrobium sp. had the ability to assimilate volatile organic compounds from air for growth. A growth yield of 0.7 mg/liter cell carbon was obtained in a mineral medium that contained no additions of organic compounds but had been stored for 4 weeks in flasks, allowing volatile compounds from the air to dissolve in the medium. When air was pumped into the culture during cultivating, the growth yield was proportional to the flow rate of air into the culture. Correspondence to: Kari Aa     

How does l-glutamate transport relate to selection of mixed nitrogen sources in Rhizobium leguminosarum biovar trifolii MNF1000 and cowpea Rhizobium MNF2030?

When growing on a mixture of ammonia and l-glutamate as nitrogen sources, Rhizobium leguminosarum biovar trifolii MNF1000 utilizes ammonia exclusively, while cowpea Rhizobium MNF2030 utilizes both compounds at similar rates. l-Glutamate transport in both strain MNF1000 and MNF2030 is active, giving rise to a 60-fold concentration gradient across the membrane of cells of strain MNF2030. Both strains produce two kinetically distinguishable glutamate transport systems under all conditions of growth — a high affinity system with an apparent K _m of 0.06–0.17 M but of relatively low V _max, and a low affinity system with a K _m of 1.2–6.7\ M, but of higher overall capacity. l-Glutamate transport activity in cells of MNF2030 was relatively insensitive to the presence of ammonia in the growth medium. By contrast, ammonia in the growth medium resulted in low activities of glutamate transport in cells of MNF1000 which were provided with a carbon source, offering one explanation for the failure of this strain to use glutamate in the presence of ammonia. However, in cells of MNF1000 growing on glutamate as sole source of carbon and nitrogen, the glutamate transport system is synthesized, even in the presence of accumulated or added ammonia. This suggests that the regulation of the glutamate permease also depends on availability of carbon source.Abbreviations CCCP carbonyl cyanide m-chlorophenyl hydrazone - HEPES N-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Growth of the black yeast Exophiala jeanselmei on styrene and styrene-related compounds

The black yeast Exophiala jeanselmei can grow on styrene as the sole source of carbon and energy in concentrations up to 0.36 mm. No growth is observed at higher styrene concentrations. Styrene oxidation is induced by styrene or styrene-related compounds, whereas glucose represses this styrene oxidation. E. jeanselmei shows a broad substrate specificity: various aromatic compounds are used as the sole source of carbon and energy. Styrene-grown cells can oxidize styrene, styrene oxide, phenylacetaldehyde, phenylacetic acid and 2-phenylethanol at a rate of 1.3 to 3.2 g O₂·min^–1·mg^–1 protein. A pathway for the degradation of styrene in E. jeanselmei is suggested.     

Growth of Escherichia coli cells in the presence of cysteine on sulphate-deficient media

Summary The behaviour of E. coli B culture grown on SO₄ ^2--free minimal glucose-salt medium was examined in the presence of exogenous cysteine at various concentrations. This was done by means of using the following parameters: length of lag, growth rate and total population. Up to a concentration of cysteine at 0.2mm the growth sets in without a lag phase, the growth rate is optimal (identical with that of cultures grown on media containing Na₂SO₄ as source of sulphur), only the size of total population being decreased by cysteine. At concentrations of 0.2mm and upwards, after a concentration-dependent lag-period, the cultures were found to increase at various lower growth rates.The toxic effect of cysteine was reduced by leucine itself, as well as by a mixture of leucine, isoleucine, valine and threonine. The anti-cysteine action of these amino acids showed itself in the shortening of the lag period and in the recovery of the growth rate which, however, failed to reach the original level.Cysteamine failed to provide sulphur for cultures of E. coli B grown on the above medium. Neither was the utilization of cysteamine affected by the application of amino acids possessing an anti-cysteine action.We have postulated that beside the inhibition of the biosynthesis of amino acids having an anti-cysteine effect, toxic concentrations of cysteine posses additional sites of action.     

Glucose metabolism in Acetobacter aceti

Acetobacter aceti NCIB 8554 grows on a minimal medium with ethanol but not with glucose as carbon and energy source. Addition of glucose to a wild type culture on ethanol has no influence on growth of the organism. Growth of a glucose sensitive mutant A5 is inhibited by the addition of glucose until all glucose has disappeared from the medium. In order to determine the routes by which glucose is metabolised in wild type and mutant, radiorespirometric, enzymatic, and uptake experiments have been performed. For the radiorespirometric experiments of the continuous substrate feeding type an apparatus has been constructed.Of the glucose entering the cells about 30% is excreted as gluconate and 6% metabolised with liberation of C-1 as CO₂. The rest is accumulated intracellularly. No differences were found between wild type and mutant.Under different growth conditions and with different enzymatic assay methods no pyruvate kinase activity (EC 2.7.1.40) could be detected. This might explain the inability of A. aceti to grow on glucose.Abbreviations PPO 2,5-diphenyloxazole - DM-POPOP 2,2-p-phenylene bis(4-methyl-5-phenyloxazole) - TCA trichloroacetic acid     

Citric acid production from cellobiose by 2-deoxyglucose-resistant mutant strains of Aspergillus niger in semi-solid culture

Citric acid production from cellobiose by Aspergillus niger was studied by a semi-solid culture method using bagasse as a carrier. From the parental strain Yang no. 2, mutant strains showing resistance to 2-deoxy-d-glucose (DG) on minimal medium containing glucose as a carbon source were induced. The representative mutant strain M155 was selected and subjected to further mutation. The new series of mutant strains showing resistance to DG on minimal medium containing cellobiose as a carbon source was induced, and among them the best mutant strain C192 showed higher citric acid productivity than Yang no. 2 in semi-solid culture when glucose was used as a carbon source. Moreover, in semi-solid culture, the strain C192 produced 49.6 g/l of citric acid, 1.6 times as much citric acid as Yang no. 2 produced, from 100 g cellobiose/l and showed enhanced -glucosidase production. In shake culture, the extracellular -glucosidase activity of C192 was higher than that of Yang no. 2 when not only cellobiose but also glucose and glycerol, catabolite repressors, were used as a carbon source. These results indicate that mutant strains such as C192 are insensitive to catabolite repression. Correspondence to: S. Usami     

Effect of growth substrates on morphology of Nocardia corallina

Cells of Nocardia corallina ATCC 4273 form multiply branched coenocytic mycelia and subsequent fragment to spherical cells when grown on solidified complex media. In liquid shake cultures using complex media the organisms grow into pleomorphic but seldomly branched rods, divide as rods and then the rods fragment to spheres as the stationary phase is reached. In a defined liquid medium with glucose as carbon source, the organisms divide entively as spheres at a doubling time of 44 hrs. The addition of l-tyrosine, some fatty acids and tricarboxylic acid cycle intermediates or fructose to the glucose medium caused the cells to grow at considerably faster growth rates (2.8–8.5 hrs doubling times) and to undergo the sphere-rod-sphere growth cycle. Other amino acids, fatty acids or sugars added singly to the glucose medium did not produce the sphere to rod morphology change. Some amino acids when added to the medium in pairs effected sphere to rod morphopoiesis. None of these amino acids alone were effectors. Some of the culture grew as rods and the remainder as spheres when isoleucine and valine were added to the glucose medium. No other amino acid combination tested gave this result. The reason for the mixed growth response was traced to inhomogeneity of the parent culture. The life cycle of N. corallina is illustrated in a series of photomicrographs of two slide cultures.     

Continuous microbial production ofl-leucine with cell retention

Summary Continuous production ofl-leucine was carried out withCorynebacterium glutamicum, strain ATCC 13032 starting from-ketoisocaproic acid as the precursor, glucose as the carbon source and ammonium sulphate as the nitrogen source, with biotin in a mineral medium. By means of cross-flow microfiltration or centrifugal separation for cell retention in continuous fermentation an increase in cell density was achieved and the product solution was obtained cell-free. The cells were concentrated to over 70 g cell dry mass/1. In experiments of up to 42 days, conversion rates of 85%–99% andl-leucine yields of 85%–93% were achieved. With a substrate residence time of 3.6 h, 114 mmol/1l-leucine was produced with a space-time yield of 97 g/1 per day. A scale-up of the fermentation volume from 4 to 1001 provided comparable results.     

Symbiotically defective histidine auxotrophs of Bradyrhizobium japonicum

Four histidine auxotrophs of Bradyrhizobium japonicum strain USDA 122 were isolated by random transposon Tn5 mutagenesis. These mutants arose from different, single transposition events as shown by the comparison of EcoRI and XhoI-generated Tn5 flanking sequences of genomic DNA. The mutants grew on minimal medium supplemented with l-histidine or l-histidinol but failed to grow with l-histidinol phosphate. While two of the muants were symbiotically defective and did not form nodules on Glycine max cvs. Lee and Peking and on Glycine soja, the other two mutants were symbiotically competent. Reversion to prototrophy occurred at a frequency of about 10^-7 on growth medium without added antibiotics, but prototrophs could not be isolated from growth medium containing 200 g/ml kanamycin and streptomycin. The prototrophic revertants formed nodules on all the soybean cultivars examined. When histidine was supplied to the plant growth medium, both nodulation deficient mutants formed effective symbioses. On histidine unamended plants, nodules were observed infrequently. Three classes of bacterial colonies were isolated from such infrequent nodules: class 1 were kanamycin resistant-auxotrophs; class 2 were kanamycin sensitive-prototrophs; and class 3 were kanamycin-sensitive auxotrophs. Our results suggest that two Tn5 insertion mutations in B. japonicum leading to histidine auxotrophy, affect nodulation in some way. These mutations are in regions that show no homology to the Rhizobium meliloti common nodulation genes.     

Ethanol production during D-xylose,L-arabinose,and D-ribose fermentation by Bacteroides polypragmatus

Summary The specific growth rate () during cultivation of Bacteroides polypragmatus in 2.51 batch cultures in 4–5% (w/v) l-arabinose medium was 0.23 h^-1 while that in either d-xylose or d-ribose medium was lower (=0.19 h^-1). Whereas growth on arabinose or xylose occurred after about 6–8 h lag period, growth on ribose commenced after a 30 h lag phase. The maximum substrate utilization rate for arabinose, ribose and xylose in media with an initial substrate concentration of 4–5% (w/v) was 0.77, 0.76, and 0.60 g/l/h respectively. In medium containing a mixture of glucose, arabinose, and xylose, the utilization of all three substrates occurred concurrently. The maximum amount of ethanol produced after 72 h growth in 4–5% (w/v) of arabinose, xylose, and ribose was 9.4, 6.5, and 5.3 g/l, respectively. The matabolic end products (mol/mol substrate) of growth in 4.4% (w/v) xylose medium were 0.73 ethanol, 0.49 acetate, 1.39 CO₂, 1.05 H₂, and 0.09 butyrate.National Research Council of Canada No. 23406     

The substrate constant for the ammonium ion of growing

The relation between ammonium concentration and growth rate was studied in steady state continuous cultures of Saccharomyces cerevisiae in nitrogenlimited glucose ammonium medium. This relation could be described by the Monod equation. A maximum specific growth rate of 0.41 h^-1 and a substrate constant for ammonium of 5–11 M were calculated. Ammonium was determined by a modification of the phenol hypochlorite method. A discussion of the results in view of literature data on the substrate constants for other nutrients is given.     

Construction of an arginine-producing strain of Serratia marcescens

Summary In Serratia marcescens Sr41, l-canavanine was demonstrated to be a weak cell growth inhibitor in minimal medium containing glucose as the sole carbon source. The inhibition of cell growth was enhanced by changing the carbon source from glucose to l-glutamic acid. An arginine regulatory mutant (i.e., argR mutant) in which formation of l-arginine biosynthetic enzymes was genetically derepressed was isolated by selecting for l-canavanine resistance on the glutamate medium. Furthermore, an l-arginine-producing strain was constructed by introducing the mutation leading to feedback-resistant N-acetylglutamate synthase into the argR mutant. The resulting transductant produced about 40 g/l of l-arginine, whereas the wild strain produced no l-arginine and the argR mutant only 3 g/l.     

Growth and butyric acid production by Clostridium populeti

The growth of Clostridium populeti in 2% (w/v) glucose medium containing 0.2% (w/v) yeast extract was optimal with 10 mM NH₄Cl as the nitrogen source. Although the maximum specific growth rate (=0.32 h^-1) with 5 mM NH₄Cl was similar, the biomass yield was about 30% lower than that at the optimum. Either sodium sulphide or cysteine-HCl at an optimum concentration of 0.33 mM and 5.0 mM respectively, could serve as the sole sulphur source for growth. The growth rate was unaffected by initial glucose concentrations of up to 10% (w/v), but in the presence of 15% glucose it declined by about 35%. The molar yield of butyric acid (mol/mol glucose) declined from 0.70 in 1% (w/v) initial glucose medium to 0.39 in 10% glucose medium. In 5.7% initial glucose medium, butyric acid levels of 6.3 g/l were obtained (0.56 mol butyrate/mol glucose) after 72 h of incubation in 2.5 l batch cultures. A decrease of about 50% in the maximum specific growth rate of C. populeti was observed in the presence of an initial concentration of either 1.2 g/l of butyric acid or 18.9 g/l of acetic acid.This paper is issued as NRCC No. 29032     

A physiological study of Saksenaea vasiformis Saksena

Summary The physiological conditions governing growth and sporulation ofSaksenaea vasiformis Saksena, a fungus with outstanding morphological features quite peculiar for Mucorales, were determined. Earlier studies made byTiwari (1955) on a strain of the same species had shown that this fungus is incapable of sporulating on any synthetic medium normally employed for growing fungi.The fungus had been found to have a high tolerance for very low and high pH values. It showed maximum growth at two pH values, one near neutral point, at pH 6, and another at high alkaline pH value, i.e., pH 11. Reason for this behaviour of this fungus has already been discussed. The most suitable temperature for the growth of the fungus was found to be between 30–35° C.Nearly all the carbon, nitrogen and sulphur sources which generally favour growth of fungi were found to support vegetative growth of this fungus as well. However, sporulation in this fungus had peculiar nutritional needs. Only some of the carbon sources, viz., arabinose, rhamnose, sorbose, galactose, lactose and citric acid which supported poor growth, were found to support good or excellent sporulation. But it may be stated that not all carbon sources supporting poor growth could induce sporulation of the organism. Also none of the nitrogen or sulphur sources could induce the fungus to sporulate in presence of glucose as carbon source.