Growth and pigment production byArthrobacter pyridinolis n. sp.

A new bacterium capable of growing on 2-hydroxypyridine as sole source of carbon and nitrogen was isolated from soil. During its growth on solid medium, approximately 50% of this substrate was converted to a brilliant blue crystalline pigment which was deposited extracellularly in the colony mass. The pigment was identical to that produced byArthrobacter crystallopoietes during its growth on 2-hydroxypyridine. The new isolate exhibited the typical cycle of morphogenesis characteristic of the genusArthrobacter. The organism is different from all other reported species ofArthrobacter. It is proposed that the organism be namedArthrobacter pyridinolis n. sp.List of Abbreviations MSP mineral salts phosphate basal culture medium containing 2-hydroxypyridine, yeast extract and trace salts - 2-HP 2-hydroxypyridine - PFU plaque forming units - G+C guanine+cytosine - T _m midpoint of thermal denaturation     

Characteristics of the Vegetative Growth of Bacillus popilliae

Growth characteristics of the insect pathogen, Bacillus popilliae Dutky, were studied by propagation in shaken flasks and in 2-liter fermentors. Maximal populations between 5 × 10⁸ and 2 × 10⁹ viable cells per milliliter of culture medium routinely were obtained in incubation periods of 18 to 24 hr at 30 C in a medium composed of 1.5% yeast extract, 0.6% K₂HPO₄, and 0.2% glucose or trehalose. The carbohydrate required for growth in liquid media was fermented with the formation of 2 meq of acid per mmole of carbohydrate utilized; acid products ordinarily were not subsequently metabolized. B. popilliae is an aerobe, and the amount of growth obtained varied with aeration to an optimum at oxygen absorption rates of about 0.5. Maximal populations persist in a culture for periods of only 1 to 4 hr; cessation of growth was followed immediately by rapid death of cultures, so that less than 1% of the cells remained viable after 48 hr, and viability often was lost entirely by the end of 72 hr of incubation. No cytological evidence for spore formation was observed under any growth condition. Death was not associated with lysis of the cells, although extensive granulation ultimately occurred. Continuous neutralizaiton, augmented buffering, various techniques of dialysis, or slow feeding of the carbohydrate did not markedly alleviate the characteristic death of the cultures.     

Synthesis of Glycols by Microbial Transformation of Some Monocyclic Terpenes

The transformation of three monocyclic terpenes by three soil microorganisms have been studied. The organisms were isolated on, and grew rapidly in, mineral salts medium containing the appropriate terpene substrates as sole carbon sources. These organisms belong to the class Fungi Imperfecti, and two of them have been tentatively identified as Cladosporium species. A Cladosporium species designated T₁ was isolated from terpene-soaked soil, using 1-menthene as the sole source of carbon. The major catabolic product isolated from the growth medium of this organism was found to be a cyclic 1,2-diol identified as trans-p-methane-1,2-diol. A similar but biochemically distinct Cladosporium sp. designated T₇ was isolated on D-limonene. After growth, the medium of this organism contained 1.5 g/liter of the analogous product, trans-limonene-1,2-diol. Minor quantities of the corresponding cis-1,2-diol were also isolated. The third organism, designated as laboratory culture T₈, was isolated on 3-menthene and yielded a diol identified as trans-p-menthane-3,4-diol. From these results it is concluded that the formation of diols is a common intermediate in the fungal metabolism of monocyclic terpenes.     

Growth of Pediococcus soyae nov. sp. in Highly Concentrated Solutions of Inorganic Salts and Sugars

Pediococcus soyae nov. sp., which has an inherited salt tolerant nature, is grown in solutions of high osmotic pressure. When this strain is transferred from 0.5% salted medium to a new medium containing 18% sodium chloride, the viable counts of this organism firstly decrease from about one half to one-third of the inoculated cells, and then normal growth occurs. This indicates the occurrence of physiological adaptation at an early stage of growth.

The growth of this lactic acid bacterium is observed in concentrated solutions of various inorganic salts. The solutions containing Na⁺, K⁺, Cl^?, NO₃^? and SO₄^{– –} ions are not toxic for the organism, and the organism can grow in solutions of 133 atm. osmotic pressure, generally. However, Li⁺, Ca⁺⁺, Mg⁺⁺ and Br⁺ are, toxic for growth.

In concentrated sugar solutions, this organism also propagates well, and growth is observed in the media containing 50% glucose or 60% sucrose, osmotic pressure being 105 and 84 atm., respectively. Therefore, Pediococcus soyae nov. sp. is osmotolerant.     

Isolation and characterization of a marine Anabaena sp. capable of rapid growth on molecular nitrogen

A marine filamentous cyanobacterium capable of rapid growth under N₂-fixing conditions has been isolated from the Texas Gulf Coast. This organism appears to be an Anabaena sp. and has been given the strain designation CA. Cultures grown on mineral salts medium bubbled with 1% CO₂-enriched air at 42°C show a growth rate of 5.6±0.1 generations per day with molecular nitrogen as the sole nitrogen source. This growth rate is higher than any other reported in the literature to date for heterocystous cyanobacteria growing on N₂. Under similar growth conditions, 7.5 mM NH₄Cl yields a growth rate of 6.6±0.1 generations per day while 7.5 mM KNO₃ allows for a growth rate of 5.8±0.4 generations-day. Nitrogen-fixation rates, as measured by acetylene reduction, show maximum activity values in the range of 50–100 nmoles ethylene produced/minxmg protein. These values compare favorably with those obtained from heterotrophic bacteria and are much higher than values reported for other cyanobacteria. Growth experiments indicate that the organism requires relatively high levels of sodium and grows maximally at 42°C. Because of its high growth rate on N₂, this newly isolated organism appears ideal for studying nitrogen metabolism and heterocyst development among the cyanobacteria.     

Thermal enzymes. IV. Partial separation of an adenosinetriphosphatase from an apyrase fraction

Adenosinetriphosphatase with a Q_P from 450 to 1400 was obtained from a cell granule fraction isolated from cells of a thermophilic bacterium. The enzyme withstands inactivation for 2 hr. at 65 °C., the growing temperature of the organism. At 75 °C. the enzyme is slowly inactivated. Magnesium plays a small part in providing protection against inactivation at 75 °C.     

Blood-Free Medium for the Rapid Growth of Pasteurella tularensis

A medium composed of (in g/100 ml) Tryptose broth with thiamine (Difco), 2.6; cysteine-HCl, 0.12; glucose, 1; FeSO₄, 7H₂O, 0.005; KCl, 0.02; histidine, 0.1; tris(hydroxymethyl)aminomethane (tris) buffer, 0.3; and agar, 1; will support rapid growth of the fully virulent SCHU-S4 strain of Pasteurella tularensis. Although the test organism grew rapidly on medium from which KCl and tris buffer were omitted, these two components increased the stability of the medium upon storage at 4 C. It was necessary to (i) control carefully the relative concentration of the ferrous iron and cysteine-HCl, (ii) incubate the prepared medium overnight prior to use, and (iii) incubate the inoculated plates in an atmosphere of high relative humidity. Rapid growth of the organism was obtained also from very small inocula in the liquid form of the medium. Biochemical studies designed to elucidate the mechanisms involved in the enhancement of growth of P. tularensis in this relatively simple blood-free medium were initiated.     

Heat-labile Enzymes in a Psychrophilic Bacterium

The oxidative and fermentative activities of a psychrophilic bacterium (strain 82), whose maximal growth temperature is 35 C, were completely destroyed by exposure of the cells to 46 C for about 1 hr, whereas those of mesophilic Escherichia coli were unaffected. Similar results were obtained with cell-free extracts. In attempts to determine some of the specific enzymes inactivated in strain 82 by exposure to 46 C, it was found that reduced nicotinamide adenine dinucleotide oxidase was completely inactivated at 46 C in 2 hr. Also, cytochrome c reductase was completely destroyed at 46 C in 1 hr and was 70% destroyed at 40 C in 2 hr. The heat lability of the latter may determine the maximal growth temperature of the organism. In addition, the results indicated that the enzymes of strain 82 involved in the clastic split of pyruvate to formate and acetate are inactivated by exposure to 46 C and that the lactic and glycerol dehydrogenases are more heat-labile than those in E. coli. Succinic, nicotinamide adenine dinucleotide phosphate-alcohol, and glucose-6-phosphate dehydrogenases, however, in both strain 82 and E. coli, were essentially unaffected by exposure to 46 C for 2 hr.     

Isolation and description ofHaloanaerobium praevalens gen. nov. and sp. nov., an obligately anaerobic halophile common to Great Salt Lake sediments

A new halophilic species is described that was isolated from the hypersaline (>20%) surface sediments of Great Salt Lake, Utah, via transfer from MPN end-dilution tubes that contained a complex organic medium. The organism was an obligate anaerobe that proliferated optimally at approximately 13% salt, but did not grow significantly at <2% or ≥30% salt. It stained Gram-negative, was nonmotile, nonsporing, and contained an outer-wall membranous layer. The complex lipids of the organism were fatty acid esters that did not change dramatically during growth at 5% or 25% NaCl. The DNA base composition was 27.0±1 mol% guanosine plus cytosine. The temperature range for growth was >5°C and <60°C, the pH range was between 6.0 and 9.0. The doubling time for growth in complex medium with 25% NaCl was 7 h. The organism utilized carbohydrates, peptides, and amino acids. Butyrate, acetate, propionate. H₂, and CO₂ were the major fermentation end products formed. Glucose, mannose, fructose,n-acetyl glucosamine, and pectin were used as energy sources for growth. Methylmercaptan was produced from methionine degradation. The nameHaloanaerobium praevalens gen. nov. sp. nov. is proposed for the type strain GSL which has been deposited as DSM 2228. The taxonomic relationships ofH. praevalens to other obligate halophiles and anaerobes, as well as its biological role in the Great Salt Lake microbial ecosystem, are discussed.     

Isolation and characterization of a new thermophilic and autotrophic methane producing bacterium:Methanobacterium thermoaggregans spec. nov.

Methanobacterium thermoaggregans is a new thermophilic autotrophic rod-shaped methane producing bacterium. The organism likes to form aggregates during growth and utilizes only H₂ and CO₂ as substrates. Growth optimum is at 65°C with a doubling time of 3.5 h. Optimal growth occurs at pH-values between 7 and 7.5. The addition of yeast extract to the mineral salt medium stimulates growth. The DNA base composition is 42 mol% G+C. The organism was isolated from mud taken from a cattle pasture. Because of its optimal growth temperature and its tendency to form aggregates the nameMethanobacterium thermoaggregans is suggested.Abbreviations G+C Guanine+cytosine     

Continuous real-time monitoring of metabolic parameters in growing bacterial cultures

Continuous monitoring of a bacterial culture for pH, growth, CO₂, and NH₃ has been accomplished by means of in situ probes. Changes in the metabolic parameters of Proteus cultures generally occurred about an hour before changes in growth were observed. The time of maximum CO₂ production preceded that of NH₃ elaboration by this organism; however, the sequence was reversed when urea was added to the medium. This type of in situ monitoring system has great potential for the study of the metabolism of growing organisms as well as for the early detection of growth in liquid culture.     

Growth and respiratory activity of mold fungus (Trichoderma lignorum)

The growth ofTrichoderma lignorum was studied in relation to the carbon balance of the culture system and the respiratory activity at different growth phases. Conidia, after inoculation into the medium, swelled and germinated rapidly. The growth rate of the hyphae at the exponential phase was 0.46 hr⁻¹ (2.2 hr for mass doubling) at 25 C. The yield efficiency of hyphal biomass-C at the cost of glucose-C was 67%, while those of the waste products-C excreted and of CO₂-C evolved were, 6.5% and 26.5%, respectively. The yield efficiency of conidia-C to the decrease of hyphae-C was 34%. The germination, growth rate and carbon balance were not affected by different concentrations of glucose from 10 to 2×10³ mg glucose-C/l. Carbon dioxide was needed as the growth factor for the initiation of the germination of conidia, but there was no increase in yield efficiency as a result of CO₂ fixation. The respiratory rate of the fungus changed drastically as the growth proceeded. The rate of endogenous respiration of conidia was less than 0.2 mg CO₂-C/g conidia-C/hr which increased immediately after inoculation into the medium. The highest respiratory rate of hyphae (100–110 mg CO₂-C/g hyphae-C/hr) was obtained throughout the exponential phase. Thereafter, decreasing rapidly, the respiratory rate of submerged hyphae of 1-week-old showed only 1.8 mg CO₂-C/g biomass-C/hr, whereas the rate of aerial hyphae forming conidia increased again, but did not exceed 10 mg CO₂-C/g biomass-C/hr.     

Spirochaeta halophila sp. n., a facultative anaerobe from a high-salinity pond

A facultatively anaerobic spirochete isolated from a high-salinity pond grew optimally when 0.75 M NaCl, 0.2 M MgSO₄, and 0.01 M CaCl₂ were present in media containing yeast extract, peptone, and a carbohydrate. The organism failed to grow when any one of these three salts was omitted from the medium. Aerobically-grown colonies of the spirochete were red, whereas anaerobically-grown colonies showed no pigmentation. Non-pigmented mutants of the spirochete were isolated.The spirochete used carbohydrates, but not amino acids, as energy sources. Glucose was fermented to CO₂, H₂, ethanol, acetate, and a small amount of lactate. Determinations of radioactivity in products formed from glucose-1-¹⁴C and enzymatic assays indicated that glucose was dissimilated to pyruvate mainly via the Embden-Meyerhof pathway. Pyruvate was metabolized through a clostridial-type clastic reaction.Cells growing acrobically performed an incomplete oxidation of glucose mainly to CO₂ and acetate. Comparison of aerobic and anaerobic growth yields indicated that oxidative phosphorylation occurred in cells growing aerobically. The guanine + cytosine content of the DNA of the spirochete was 62 moles%. It is proposed that the spirochete described herein be considered a new species and that it be namedSpirochaeta halophila.     

Effect of Sulfur-Containing Compounds on Growth of Methanosarcina barkeri in Defined Medium

Methanosarcina barkeri Fusaro (DSM 804) could grow on methanol in a mineral medium containing cysteine or thiosulfate as the sole sulfur source. Optimum growth occurred at cysteine concentrations of 1 to 2.8 mM and at thiosulfate concentrations of 2.5 to 5 mM. No inhibition of growth was observed even when these concentrations were doubled in the culture medium. Under the optimum cysteine and thiosulfate concentrations, the generation times of the organism were about 8 to 10 and 10 to 12 h, respectively, giving a cell yield of about 0.14 to 0.17 and 0.08 to 0.11 g (dry weight)/g of methanol consumed. The organism metabolized cysteine and thiosulfate during growth, giving rise to sulfide in the culture medium. H₂S evolution from cysteine and thiosulfate was catalyzed by two enzymes, namely cysteine desulfhydrase and thiosulfate reductase, respectively, as revealed by enzyme assay in the crude cell-free extract of the organism.     

Glucose Utilization by Euglena gracilis var. bacillaris at Higher pH*

SYNOPSIS. Euglena gracilis var. bacillaris is able to grow luxuriantly on glucose in a mineral salts medium at pH 6.8–7.1 following an adaptation period of about 200 hr. If adapted cells are used as an inoculum or if 0.1% glycine is included in the medium, the lag is shortened to 70–100 hr. Inclusion of 0.1% acetate in the medium produces a diphasic growth pattern, with acetate being metabolized first, followed by the later (about 400 hr) utilization of the glucose. Glucose utilization was found to be sensitive to pH as compared to growth on ethyl alcohol. However, glycine partially overcame this sensitivity. Glycine is maximally stimulatory with regard to growth on glucose at pH 7.0 at a concentration of 0.03%, thus suggesting that it functions as a sparking substance. Glycine markedly stimulates the assimilation of ¹⁴C-glucose. A number of Krebs cycle acids and amino acids were also found to stimulate ¹⁴C-glucose assimilation at neutral pH. Adaptation to glucose utilization at neutral pH was due to the appearance of mutants able to grow more rapidly under these conditions. The nature of this mutation was not determined.     

Rhizobium trifolii 0403 Is Capable of Growth in the Absence of Combined Nitrogen

Rhizobium trifolii 0403 was treated with 16.6 mM succinate and other nutrients and thereby induced to grow in nitrogen-free medium. The organism grew microaerophilically on either semisolid or liquid medium, fixing atmospheric nitrogen to meet metabolic needs. Nitrogen fixation was measured via ¹⁵N incorporation (18% ¹⁵N enrichment in 1.5 doublings) and acetylene reduction. Nitrogen-fixing cells had a K_m for acetylene of 0.07 atm (ca. 7.09 kPa), required about 3% oxygen for optimum growth in liquid medium, and showed a maximal specific activity of 5 nmol of acetylene reduced per min per mg of protein at 0.04 atm (ca. 4.05 kPa) of acetylene. The doubling time on N-free liquid medium ranged from 1 to 5 days, depending on oxygen tension, with an optimum temperature for growth of about 30°C. Nodulation of white clover by the cultures showing in vitro nitrogenase activity indicates that at least part of the population maintained identity with wild-type strain 0403.     

Influence of medium components on growth kinetics of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Starting with the standard complex medium HL-5C, the influence of different medium components on the growth behavior of Dictyostelium discoideum was investigated. For this purpose, each component was individually deleted from the complex medium HL-5C, and the overall concentration of all components as well as the kinds and concentrations of carbon source were varied. The effects of the supplementation with major components of synthetic medium like vitamins, trace metals, inorganic salts were also investigated. When glucose being the carbon source was deleted from the standard HL-5C medium, the maximal cell density then had a drastic drop. Deletion of yeast extract also caused lower maximal cell density. Of all the carbohydrates investigated only maltose and glucose led to higher final cell concentration. Adding inorganic salts such as CaCl₂ and MgCl₂ to HL-5C medium significantly improved the cell growth, whereas addition of vitamins or trace metals had little effect on cell growth. A semi-empirical model is employed to simulate the cell growth.     

CHARACTERIZATION OF BLEACHING IN CHLORELLA INDUCED BY SUBOPTIMAL GROWTH TEMPERATURES1

Incubation of cultures of a high-temperature strain of chlorella at 10 C stopped growth and bleached all chlorophyll in the cells in 24 hr. Optimal conditions of light (3.0 mw/cm²), gas (1% CO₂-in-air), and inorganic medium for maximal growth at 39 C were maintained in the transfer from 39 to 12, 10, or 5 C. The bleaching process at 10 C is characterized by a lag period for the first few hours followed by a linear decrease in chlorophyll content of cultures. The amounts of time required to bleach half of the chlorophyll initially present (effective half-times) at 10 C were 14 hr for chlorophyll a and 17 hr for chlorophyll b. Effective half-times of bleaching for total chlorophyll were 47 hr at 12 C and 6 hr at 5 C. Addition of glucose to inorganic medium delayed but did not prevent bleaching. Use of argon gas instead, of 1% CO₂-in-air prevented cells from bleaching in both inorganic and glucose media, and indicated an oxygen requirement for bleaching. Incubation of 6 additional strains of Chlorella at 10 C resulted in responses ranging from bleaching to no growth to growth.     

Chemically defined media for growth and sporulation of Entomophthora virulenta

Amino acids, salts, and vitamins were combined with dextrose to test their effect on growth and sporulation of Entomophthora virulenta in liquid shake culture. The addition of a vitamin solution to the tested media did not enhance growth or sporulation. MgSO₄·7H₂O was the only salt individually tested that allowed for good growth and sporulation. MgSO₄·7H₂O concentrations exceeding 250 mg/liter in media lacking other salts inhibited sporulation. A simple medium of l-arginine, l-leucine, glycine, and mineral salts allowed high growth and sporulation.     

Production of high-quality edible protein from Candida yeast grown in continuous culture

Candida utilis NRRL Y-900 was grown in aerobic continuous culture with cane molasses as the source of the growth-limiting carbon. At 1% reducing sugar in the chemostal (10 liter working volume) feed medium, addition of Zn (25μM) to a minimal salts medium resulted in an increase in the biomass productivity of the chemostat from 1.7 to 2.6 g/liter/hr with a growth yield of 0.55 g dry biomass/g reducing sugar utilized at D_max. On the average, the yeast biomass was 50–55% protein. At S_R > 2% sugar, the biomass productivity was limited by the oxygen supply. With O₂-supplemented aeration (at S_R = 4.2%)the maximum biomass productivity Was 7.25 g/liter/hr. Aerobic ethanol production was not observed. A highquality undenatured protein fraction was isolate from the yeast homogenate by isoelectric precipitation at pH 4.5. Contaminating nucleic acid was removed as an insoluble complex by chelation with an organic cation (cetavlon). The final protein product contained about 3% RNA (DWB) and was suitable for use as a food additive.