Dialysis fermentation. I. Enhanced production of salicylic acid from naphthalene by Pseudomonas fluorescens

The conversion of naphthalene to salicylic acid by Pseudomonas fluorescens was studied as a model for dialysis fermentation. In a demonstration experiment, the continual removal of the product by dialysis and by intermittent replenishment of the dialysate reservoir caused cyclical changes in the concentrations of viable cells and product. The cumulative total amount of both cell mass and salicylate, however, continued to increase steadily until the experiment was terminated after 15 days. At this time the rate of product formation was highest and still increasing, although less than 10% of the cells were viable. The terminal amount of salicylate was about 20-fold greater than the maximum reached in the control fermentation, and was calculated to be 2.6-fold more productive even if the control were optimally recycled. Methods were projected to achieve still further improvements.     

Studies on the amino acid fermentation. Part 1. Production of L-glutamic acid by various microorganisms

Screening tests for glutamate producing strains were carried out, with the media containing carbohydrate and ammonia source as chief ingredients. Glutamate as well as certain other amino acids was detected by paper chromatography in culture broth of many microorganisms tested. Accumulation of L-glutamate in a significant amount (at least a few mg of glutamate per ml of broth) has been demonstrated by various strains of bacteria, streptomycetes, yeasts, and fungi. The highest level of glutamate production has been obtained by a new species of Micrococcus, yielding as much as 0.25 mole of it from one mole of glucose. The courses of fermentations mainly by known strains of microorganisms are shown. The importance of the cultural condition and strain specificity for the production of amino acids are briefly described.     

Degradation of naphthalene to salicylic acid by cultures ofPseudomonas denitrificans andAchromobacter sp. from the effluents of petroleum refinery

The microbial degradation of aromatic hydrocarbons from effluents of a petroleum refinery was investigated, with emphasis on the breakdown of naphthalene to salicylic acid. The microorganisms were grown in a synthetic medium with naphthalene as the sole carbon source. The effects of pH, temperature, aeration and naphthalene concentration were studied. The optimum conditions for degradation were found to be: pH 6.0, 28°C, 25 ml medium with 1% naphthalene in 500 ml flasks. Salicylic acid was estimated by colorimetric and chromatographic methods. Maximum of growth was found on the 5th day of cultivation.Pseudomonas denitrificans produced 9.0 μg salicylic acid per ml medium,Achromobacter sp. produced 7.1 μg/ml.     

Drosophila topoisomerase I: isolation, purification and characterization.

We have purified and characterized topoisomerase I from Drosophila melanogaster. The molecular weight of the enzyme is 135,000; 100,000, 90,000, and 65,000 molecular weight products result from degradation of the enzyme. The enzyme relaxes both positive and negative supercoiled DNA. Mg++ is not absolutely required, but stimulates the enzymatic activity considerably.     

Ammonia production by ruminal microorganisms and enumeration,isolation, and characterization of bacteria capable of growth on peptides and amino acids from the sheep rumen

Excessive NH(3) production in the rumen is a major nutritional inefficiency in ruminant animals. Experiments were undertaken to compare the rates of NH(3) production from different substrates in ruminal fluid in vitro and to assess the role of asaccharolytic bacteria in NH(3) production. Ruminal fluid was taken from four rumen-fistulated sheep receiving a mixed hay-concentrate diet. The calculated rate of NH(3) production from Trypticase varied from 1.8 to 19.7 nmol mg of protein(-1) min(-1) depending on the substrate, its concentration, and the method used. Monensin (5 micro M) inhibited NH(3) production from proteins, peptides, and amino acids by an average of 28% with substrate at 2 mg/ml, compared to 48% with substrate at 20 mg/ml (P = 0.011). Of the total bacterial population, 1.4% grew on Trypticase alone, of which 93% was eliminated by 5 micro M monensin. Many fewer bacteria (0.002% of the total) grew on amino acids alone. Nineteen isolates capable of growth on Trypticase were obtained from four sheep. 16S ribosomal DNA and traditional identification methods indicated the bacteria fell into six groups. All were sensitive to monensin, and all except one group (group III, similar to Atopobium minutum), produced NH(3) at >250 nmol min(-1) mg of protein(-1), depending on the medium, as determined by a batch culture method. All isolates had exopeptidase activity, but only group III had an apparent dipeptidyl peptidase I activity. Groups I, II, and IV were most closely related to asaccharolytic ruminal and oral Clostridium and Eubacterium spp. Group V comprised one isolate, similar to Desulfomonas piger (formerly Desulfovibrio pigra). Group VI was 95% similar to Acidaminococcus fermentans. Growth of the Atopobium- and Desulfomonas-like isolates was enhanced by sugars, while growth of groups I, II, and V was significantly depressed by sugars. This study therefore demonstrates that different methodologies and different substrate concentrations provide an explanation for different apparent rates of ruminal NH(3) production reported in different studies and identifies a diverse range of hyper-ammonia-producing bacteria in the rumen of sheep.     

Studies on isolated cell components: I. Nuclear isolation by differential centrifugation

A method is presented for isolating nuclei of rat and hamster liver in a high state of purity and in a condition optically similar to nuclei within living cells.The isolation procedure consists in the homogenization and differential centrifugation of fresh liver at 0–5 ° in a salt-sucrose solution buffered at pH 7.1. By layering the material to be centrifuged over a relatively large volume of a slightly denser solution the purification can be carried out in 4 or 5 centrifugations. The entire procedure can be completed in about 90 minutes. The yield as determined by measurements of desoxyribonucleic acid is about 5 per cent.The solutions contain KH₂PO₄, K₂HPO₄, NaHCO₃, and sucrose. The sucrose concentration is varied to give density differences required for layering. Salt-sucrose solutions maintain a large proportion of the isolated nuclei in a nongranular condition for 6 hours at 0 °. Pure sucrose is less satisfactory for maintenance.The protein-desoxyribonucleic acid ratio for the isolated nuclei averages 5.1 with a range of 2.7 to 8.9.     

The basal bodies of Chlamydomonas reinhardtii. Formation from probasal bodies, isolation, and partial characterization

The assembly and composition of basal bodies was investigated in the single-celled, biflagellate green alga, Chlamydomonas reinhardtii, using the cell wall-less strain, cw15. In the presence of EDTA, both flagellar axonemes remained attached to their basal bodies while the entire basal body-axoneme complex was separated from the cell body, without cell lysis, by treatment with polyethylene glycol-400. The axonemes were then removed from the basal bodies in the absence of EDTA, leaving intact basal body pairs, free from particulate contamination from other regions of the cell. The isolated organelles produced several bands on sodium dodecyl sulfate-urea polyacrylamide gels, including two tubilin bands which co-electrophoresed with flagellar tubulin. The formation of probasal bodies was observed by electron microscopy of whole mount preparations. Synchronous cells were lysed, centrifuged onto carbon-coated grids, and either negatively stained or shadowed with platinum. The two probasal bodies of each cell appeared shortly after mitosis as thin "annuli," not visible in thin sections, each consisting of nine rudimentary triplet microtubules. Each annulus remained attached to one of the mature basal bodies by several filaments about 60 in diameter, and persisted throughout interphase until just before the next cell division. It then elongated into a mature organelle. The results revive the possibility of the nucleated assembly of basal bodies.     

Occurrence and characterization of oils rich in gamma-linolenic acid Part I: Echium seeds from Macaronesia

Nineteen species of the genus Echium (Fam. Boraginaceae) collected in Macaronesia were surveyed in a search for new sources of gamma-linolenic acid (GLA, 18:3omega6). High amounts of this acid were found in all of them, ranging from 9.15% (E. plantagineum) to 26.31% (E. callithyrsum) of total seed fatty acids. The amounts of GLA related to total seed weight were also significant, ranging from 1.77% (E. sventenii) to 5.02% (E. nervosum). In addition, considerable amounts of stearidonic acid (SA, 18:4omega3) were detected, ranging from 3.03% (E. auberianum) to 12.94% (E. plantagineum) of total fatty acids. These data allow us to consider tile members of the genus Echium from Macaronesia as one of the richest sources of gamma-linolenic acid found so far in nature. The results obtained from multivariable data analysis and the taxonomic relationships among the species is discussed.