Benzene is Monomer for p-Polyphenyl Under very mild conditions, benzene can be polymerized to give a homopolymer, p-polyphenyl 142ND
ACS NATIONAL
MEETING
Petroleum Chemistry
Benzene can be polymerized to p-polyphenyl under very mild conditions, according to Dr. Peter Kovacic and Alexander Kyriakis of Case Institute of Technology, Cleveland, Ohio. This seems to be the first use of benzene as a monomer in a well defined polymerization leading to a homopolymer. The Case chemists carry out the polymerization in a system containing Lewis acid catalyst, cocatalyst, and oxidizing agent. In addition to the novel polymerization method, the properties of the polymer are of special interest. The p-polyphenyl is extremely insoluble in solvents, and has remarkable thermal stability, according to Dr. Kovacic. Since low molecular weight polyphenyls have been considered as moderators in fission reactors, the polymeric material may prove of interest in this connection. The Case scientists are now making a further evaluation of the polymer's properties, he notes.
Polymers containing polyphenyl structures have been previously prepared in a number of ways. For instance, Dr. Carl S. Marvel and Dr. G. E. Hartzell, while at the University of Illinois, synthesized impure p-polyphenyl by dehydrogenating poly-1,3cyclohexadiene. In general, other investigators have used the Fittig, the Ullmann, or Grignard routes, which gave ill-defined polymers. The Case chemists find that the polymerization of benzene proceeds smoothly in the system containing aluminum chloride, water, and cupric chloride in about 15 minutes to give the p-polyphenyl, a brown solid. Aluminum chloride is the catalyst, water the cocatalyst, and cupric chloride the oxidizing agent. In control experiments, no polymerization occurs in the absence of catalyst or oxidizing agent. The structure of crystalline p-polyphenyl was determined from elemental analyses, infrared and x-ray data, pyrolysis products, and oxidative degradation. The evidence indicates essentially an all-para configuration for p-polyphenyl, according to the Case workers. The polymer has good thermal sta-
Benzene Goes Through a Wei I-Defined Polymerization
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bility up to about 525° C , but breaks down at 750° to 800° C. to yield volatile species. These include biphenyl and lower molecular weight p-polyphenyls. The benzene polymerization produces almost no low molecular weight organic species, according to Dr. Kovacic. This is characteristic of addition polymerizations. The extreme insolubility of p-polyphenyl precludes its molecular weight determination by the usual methods, he adds. Dr. Kovacic and Mr. Kyriakis propose that the reaction proceeds by oxidative cationic polymerization of the aromatic nucleii. According to their hypothesis, initiation of the polymerization entails formation of a sigma complex, the benzenonium ion. This undergoes propagation as a growing carbonium ion. Dehydrogenation by the oxidizing agent prevents reversibility of the propagation step by restoring aromaticity. Except for the oxidative aspects, this proposed mechanism is closely analogous to the mechanism for cationic polymerization of olefins. The role of catalyst and oxidizing agent can be performed by a single reagent such as ferric chloride, Dr. Kovacic and Dr. Chisung Wu showed earlier [/. Polymer Sci., 47, 45 ( I 9 6 0 ) ] . Water and other Bronsted acids were shown to play vital roles as cocatalysts. However, in this case, the picture is complicated by the apparent presence of polynuclear structures in the polymeric product.
RECORDING. University of Arizona's Dr. Cornelius Steelink (foreground) and Dr. Gordon Tollin are recording the electron paramagnetic resonance spectrum of soil humic acid. The work shows the presence of free radicals in humic acid
Humic Acid Contains Stable Free Radicals Quinone and related species may be present; evidence may help explain how humic acid is formed 142ND
ACS NATIONAL
MEETING
Cellulose, Wood, and Fiber Chemistry
Three chemists at the University of Arizona have come up with some new evidence that may help explain how humic acid is formed. Dr. Cornelius Steelink, Dr. Gordon Tollin, and Ted Reid find that humic acid contains stable organic free radicals. The nature of the radical species is still uncertain. But electron paramagnetic resonance measurements strongly suggest the presence of a number of radicals based on quinone, semiquinone, and quinhydrone species. No one knows exactly how soil humic acid is formed. But the work of the Arizona chemists supports the speculation that the acid is formed by the free radical polymerization of plant and microbiological polyphenols and quinones related to lignin and resorcinol.
Scientists have been interested in soil humic acid for more than 200 years. Yet its origin and structure as well as its formation are still uncertain. The acid is generally defined as a chocolate-brown, polymeric substance found in humus. More specifically, humic acid refers to the soil fraction that is soluble in bases and insoluble in acids and alcohol. Humic acid plays an important role in soil fertility, plant respiration, soil stabilization, root growth stimulation, and the transport of metal ions. It's used occasionally in commercial fertilizers and is sold as a water conditioner for industrial boilers. It also goes into inks, dyes, and drilling mud. What's in It. Earlier degradation studies show that humic acid contains phenolic structural elements related to lignin breakdown products and resorcinol. Infrared spectrophotometry analyses proved the presence of carboxy 1 and hydroxyl bands. Some investigators inferred the presence of aromatic and quinoid moieties on the
basis of infrared and potentiometric measurements. "Our chief interest in humic acid is the elucidation of its chemical structure as a basis for understanding its biosynthesis and function in the soil/' Dr. Steelink says. "A report by Dr. R. W. Rex, California Research Corp., on the presence of stable free radicals in coals, lignin, and humic substances prompted us to investigate electron paramagnetic resonance as a possible probe into the structure of humic acid," Dr. Steelink explains. The probe was successful. Late last year the group confirmed that soil humic acid contains stable, organic free radicals. Fractionation studies reveal that the radicals are not caused by impurities or by unstable species trapped in the soil humic acid polymer, but rather are an integral part of the molecule. The radical species is present in concentrations of about 10 18 spins per gram, thus indicating that it's an important structural feature of the humic acid molecule, Dr. Steelink says. Humic acid, along with melandn and possibly lignin, is one of the few biological materials that contains known stable free radicals, he adds. Electron paramagnetic resonance measurements led the three chemists to believe that humic acid contains two radical species. One of these, they felt, could be a semiquinone radical, the other a quinhy drone-type radical. To find out more about the free radicals, they observed the effects of reduction, oxidation, and hydrolysis on the nature of the electron paramagnetic signals. Reduction. Alkaline reduction of soil humic acid causes marked increase in radical concentration, Dr. Steelink says. One explanation for this, he feels, is on the basis of a quinone/semiquinone model for humic acid. For example, it is well known that chemical reduction of quinones in basic media leads to stable semiquinone radical ions. The most significant increase in spins per gram was registered by a sodium-alcohol-treated sample which was isolated as the sodium salt. However, when the sodium salt was dissolved in water and acidified, the product had a considerably reduced free radical content. This, Dr. Steelink explains, is consistent with the known instability of semiquinone radical ions in acid solution. It also indicates that reduction is destroying a porSEPT.
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tion of the original radical species, he says. The large increase in radical content that's obtained merely by forming the sodium salt indicates that the increase in the free radical concentration of chemically reduced humic acid is due, in part, to the formation of the anion, and in part to the reduction of a quinone moiety. The presence of a quinhydrone moiety in humic acid is further indicated by the fact that the EPR spectrum for sodium humate is very similar to that for known quinhydrone salts, the Arizona workers say. The radical species in humic acid is fairly stable toward alkaline oxidation, Dr. Steelink says. Some of the oxidized samples did exhibit a slight increase in spin concentration, but this may be due to a reduction in molecular weight by alkaline hydrolysis, he points out. Two or More. "It is evident that two or more radical species exist in humic acid" from high resolution electron paramagnetic resonance studies of aqueous alkaline solutions of humic acid, Dr. Steelink says. The studies reveal a hyperfine structure in the spectra which is not evident in solid samples. "These spectra undoubtedly represent the superposition of absorptions by two or more radical species," he maintains. Acid hydrolysis causes a number of chemical changes in humic acid, many of which need further study. For example, the treatment has previously been shown to remove carbohydrate and protein material, materials usually not considered a part of the humic acid molecule. It also causes rather fundamental changes in the structure of the humic acid "core" or macromolecule. Dr. Steelink says that their preliminary studies of hydrolyzates tend to show that stepwise hydrolysis puts carbohydrate and protein fractions into the aqueous solution. Then, the remaining humic "core," having an increasing time of hydrolysis, would show a regular increase in spins per gram. Also, a fundamental change in chemical structure, perhaps caused by splitting off of polyphenol fragments and polymerization, tends to create new radical species as well as increase the total spin concentration. Change due to acid hydrolysis is an area of humic acid chemistry that the three Arizona chemists are now studying. 54
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N2F4 Forms Difluoramino Radical Compound's dissociation into NF2 explains its reactions with Cl2, NO, alkyl groups 142ND
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Inorganic Chemistry The discovery of tetrafluorohydrazine, N 2 F 4 , and of its dissociation into the difluoramino free radical, NF 2 , has opened a new area of both organic and inorganic chemistry, says Dr. Jeremiah P. Freeman of Rohm & Haas' Redstone Arsenal research division, Hunts ville, Ala. The compound takes part in a variety of free radical reactions with chlorine, nitric oxide, and alkyl radicals. Tetrafluorohydrazine's dissociation has been examined by a variety of spectral techniques, Dr. Frederic A. Johnson, also of Rohm & Haas, points out. Mass spectroscopy, infrared, electron paramagnetic resonance (EPR), and ultraviolet spectroscopy have all contributed to the study. Rohm & Haas scientists have been studying free radical reactions of tetrafluorohydrazine and the difluoramino radical since the company's Dr. C. B. Colburn and A. Kennedy first prepared tetrafluorohydrazine from nitrogen trifluoride under hot tube conditions in 1958. Dr. Colburn and Mr. Kennedy found that N 2 F 4 decomposes in moist air to produce various nitrogen oxides. Coupling of difluoramino radicals with oxygen undoubtedly causes the reaction, since oxygen should not affect undissociated N2F4. When passed over hot arsenic, "wet" nitrogen trifluoride converts to difluoramine, Dr. Colburn and Mr. Kennedy also found. In another study, the same two scientists and Dr. Freeman found that arsine is the hydrogen transfer agent. Thus, they reasoned that a radical abstraction reaction was taking place in the hot tube. The free radical hypothesis was confirmed when the group found a dependable method for making difluoramine by reacting N 2 F 4 with mercaptans—known to be good hydrogen atom sources. Dissociation. Many studies of the dissociation of N 2 F 4 into N F 2 have been made. The radical is present at room temperature and 1 atmosphere
only to the extent of 0.05%, Dr. Johnson points out. The radical concentration reaches 90% at 300° C. and 1 atmosphere, or at 150° C. and 1 mm., or at 25° C , and 10 - 1 0 atmosphere. Thus, usual methods of characterization are not practical; modern spectral methods, however, have been more fruitful. Mass spectra show that the main species present under ionizing chamber conditions (10 tS mm. and 175° C.) is the N F 2 radical. Mass spectral work gives 52 as the radical molecular weight. The parent ion of N 2 F 4 is observed only in molecular beams or with an unheated ionization section. Infrared studies by Dr. M. D. Harmony and co-workers at the National Bureau of Standards and the University of California, Berkeley, show a bent structure for NF 2 , with an F — N — F angle of 104° and F—N bond length of 1.37 A. Dr. L. Piette of Varian Associates, and Rohm & Haas' Dr. Johnson, Dr. K. A. Booman, and Dr. Colburn have found that the single unpaired electron of N F 2 has broad EPR absorption at 104 gauss. Lack of fine structure is due to the relatively high temperature and pressure of their study (40 mm. and above 80° C ) , Dr. Johnson says. He has little hope of more detailed EPR data on the molecule except possibly in matrix studies, since a sufficient concentration of radicals requires either high pressure or high temperature; both are incompatible with maximum resolution. Reactions. Tetrafluorohydrazine takes part in two inorganic reactions, both involving the difluoramino radical. It reacts with chlorine in the presence of light to form C1NF 2 . It also reacts with NO to produce NFoNO. Chlorine and N 2 F 4 react under the influence of UV to produce chlorodifluoramine, C1NF 2 . High chlorine concentration at 80° C. favors the reaction. In the NO reaction, NF 2 NO is present only to the extent of about 0.1%, even at 15 atmospheres total pressure of nitric oxide and tetrafluorohydrazine. N F 2 N O is identified under these circumstances by its ab-
sorption at 550 n y , imparting a blueviolet color to the mixture. Difluoramino radicals take part in a number of organic free radical reactions, Dr. Freeman points out. For instance, he and Dr. R. C. Petry find that the radicals abstract hydrogen from aliphatic aldehydes to produce difluoramine. In this reaction, the acyl radical that's generated couples with an N F 2 radical to produce a new class of organic compounds, the N,Ndifluoramides. The reactions are:
J. W. Frazer of University of California's Lawrence radiation lab has found that alkyl radicals also react with N 2 F 4 . He has prepared methyl and ethyl difluoramine by irradiating the corresponding iodides with UV in the presence of N 2 F 4 . Dr. Petry and Dr. Freeman made difluoraminoisobutyronitrile ( C H 3 ) 3 C ( C N ) N F 2 , and tert-butyl difluoramine ( C H 3 ) 3 C N F 2 , in similar alkyl radical reactions, heating the proper azo compound with N2F4. Dr. Frazer and co-workers have recently found that N 2 F 4 reacts with trifluoronitrosomethane. A new class of compounds—the N-fluoroazoxy compounds—stems from this reaction:
New Lube Oil Detergents Developed 142ND
ACS NATIONAL
MEETING
Petroleum Chemistry
A new class of high molecular weight monomers, polyethylene oxide methacrylates, is the key ingredient in ashless detergents-viscosity index improvers developed at California Research Corp., the research arm of Standard Oil (Calif.). They go into high performance lubricating oils tailored for passenger car engines in stop-and-go service. These monomers make copolymers that differ from conventional polyalkyl methacrylates by having long-chain polyethylene glycols as polar groups substituted periodically down the polymer "backbone."
The materials were developed by Dr. W. T. Stewart, Dr. F. A. Stuart, and J. A. Miller of Calresearch to control sludge and varnish in automobile engines operated at low temperatures and to act as viscosity index improvers needed for multigrade oils which have come into wide use. Polar substituents evaluated by the group include both those that are proton donors and acceptors—such as amino, carboxyl, and hydroxyl—and those that are only proton acceptors, such as polyalkylene glycols. Materials with these substituents form hydrogen bonds with acidic and other carbonyl compounds in engine deposits and with low molecular weight precursors of these compounds. As organic compounds, these types of detergents received research emphasis because they leave no ash, compared to metal salt detergents. Some ash left by metal salt detergents may cause an increase in octane requirement, preignition, and valve burning of engines. The polyethylene oxide methacrylate monomers have molecular weights as high as several thousand, Dr. Stewart told the Symposium on Polymers in Lubricating Oils. They consist of a polyglycol chain capped with an ether group at one end and a methacrylate or methyl methacrylate group at the other. Being water soluble, they provide a high degree of surface activity when incorporated into a largely hydrocarbon methacrylate polymer. Two Routes. The Calresearch chemists make the polar substituted polymers by two routes; these are either versatile or specific. The versatile method leads to a variety of derivatives for exploratory screening. First, a copolymer of mixed alkyl methacrylate and methacrylic acid is made. Saponification with potassium hydroxide adjusts the alkyl-to-acid ratio in the polymer. The copolymers produced in the saponification are esterified with a material such as polyethylene glycol to give the desired substituent. Adjustment of the quantities of materials and reaction conditions gives varying levels of esterification. In the specific method, alkyl methacrylates and the appropriate substituted methacrylate monomers are copolymerized, according to Dr. Stewart. Standard engine tests of oils containing detergents with either small or nonpolymeric substituents, such as
amino, carboxylic, or hydroxyl groups, show that detergency performance achieved with these various polar groups is relatively constant, Dr. Stewart says. At a level of 1.5% in base oil, these polymers provide engine cleanliness (FL-2 Chevrolet Test) significantly superior to metal salt detergents, the Calresearch workers say. More complex small substituents (but not polymers) and mixtures of small substituents in polyalkyl methacrylates fail to improve detergency performance significantly. Superior performance isn't achieved until polarity is moved out from the polymethacrylate backbone in the form of a polyglycol chain, or freed of the "shroud" of the hydrocarbon side chain. However, the amount of polarity must be limited to prevent formation of "lead paint" deposits or gels of resinous materials and lead halides. Lead paint deposits cause engine failures due to blocking oil lines and plugging oil ring slots, Dr. Stewart explains. Detergency Mechanism. Detergents in lubricating oils have a basic objective of keeping resinous materials that result from blow-by of unburned fuel components from agglomerating and depositing on engine surfaces. The detergents are designed to form films on resin particles to keep them apart. Since the resinous material· contains free carboxyl and carbonyl groups, the way to adsorb materials on the resin particles is to take advantage of the hydrogen bonding of these groups, Dr. Stewart points out. The detergent, therefore, should have substituent groups that participate in hydrogen bonding as proton donors or acceptors —for example, —COOH, —OH, and (-CH2CH2-0-)n. Several other chemists have observed and reported hydrogen bonding of ether oxygens with acidic hydrogens, Dr. Stewart says, including polyethylene glycols with polyacrylic acids. Others have studied thicknesses of adsorbed films of methacrylates and have found that substituted types have substantially thicker films than unsubstituted polyalkyl methacrylates. With all this evidence supporting hydrogen bonding and their own work, Dr. Stewart points out that the superiority of polymeric detergents over conventional detergents is due to their ability to form thicker protective films. SEPT.
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Protein Is Key Factor in Photosynthesis Chloroplast ferredoxin is electron carrier in triphosphopyridine nucleotide reduction Studies of an iron-containing protein at the University of California, Berkeley, provide strong support for the electron flow theory of the basic photochemical act, according to the California scientists. The protein is referred to as chloroplast ferredoxin by Dr. Kunio Tagawa and Dr. Daniel I. Anion. According to these chemists, chloroplast ferredoxin is the same substance that Dr. R. Hill, Dr. H. E. Davenport, and Dr. F. R. Whatley isolated from chloroplasts in 1952 at Cambridge, England [Proc. Roy. Soc., B, 139, 346 (1952)]. In 1956, Dr. A. San Pietro of the Charles F. Kettering Foundation, Yellow Springs, Ohio, while working at Johns Hopkins University, identified a factor from spinach that catalyzed the reaction of triphosphopyridine nucleotide in light [Science, 124, 118 (1956)]. Dr. Arnon, Dr. Whatley, and Dr. Allen identified this in 1957 as the triphosphopyridine nucleotide reducing factor of chloroplasts [Nature, 180, 182 (1957)]. And in 1958, Dr. A. San Pietro extensively purified and investigated it further. Dr. San Pietro refers to it as photosynthetic pyridine nucleotide reductase [/. Biol. Chem., 231, 211 (1958)]. Dr. Tagawa and Dr. Arnon now find that chloroplast ferredoxin is not an enzyme but an electron carrier that reduces triphosphopyridine nucleotide, with the aid of a flavin enzyme of chloroplasts. The reduction takes place either in light or in the dark at the expense of hydrogen gas. Recently [Nature, 195, 543 (1962)], they have presented evidence that ferredoxin is the most electronegative electron carrier in cellular physiology. And it is the most reducing constituent of the photosynthetic system yet isolated. It is reversibly oxidized and reduced, and can stimulate chloroplasts to both utilize or give off hydrogen gas. Mechanism. Research into the role of light in photosynthesis has been increasingly directed toward the mechanism of photosynthetic phosphorylation. In 1954, Dr. Arnon and his associates at Berkeley reconstructed the photosynthetic sequence from 56
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carbon dioxide to starch outside the living cell in isolated chloroplasts. This allowed study of light utilization without interference from other biochemical processes within the cell. At the same time, the Berkeley scientists discovered the process of photosynthetic phosphorylation in chloroplasts—their capacity to form adenosine triphosphate (ATP) at the expense of radiant energy without a net consumption of chemical substrate or molecular oxygen—referred to as cyclic photophosphorylation. A variant of this process—noncyclic photophosphorylation—in which ATP formation is linked with the photoreduction of triphosphopyridine nucleotide (TPN) was discovered in 1957. Dr. Arnon and his co-workers later devised a method of separating the chloroplast itself into two parts, a green and a nongreen portion. ATP and TPN buildup takes place in the green portion; carbon dioxide fixation occurs in the nongreen portion. Thus, energy buildup requires light, but conversion of carbon dioxide to carbohydrate takes place in the dark, they conclude. Once these two processes were recognized, the search began for the mechanism by which absorption of light produces ATP and TPN. Dr. Arnon's electron flow theory is based on experimental evidence from chloroplasts and from parallel experiments with cell-free photosynthesis in photosynthetic bacteria. The theory states that the chloroplast uses light energy to transfer a high energy electron from chlorophyll to a suitable electron acceptor which is thereby reduced. The electronic energy is stored as ATP and reduced TPN. Hydrogen or Light. Certain photosynthetic bacteria are able to use and produce hydrogen gas. The electron flow theory should be applicable to these processes also, the Berkeley scientists reasoned. And it should be possible to induce (experimentally) photoproduction of hydrogen gas from chloroplasts in place of the familiar evolution of oxygen in light. Last year, Dr. Antonio Paneque and Dr. Akira Mitsui at Dr. Arnon's laboratory did this with isolated chloro-
plasts. They used a blocking agent to prevent oxygen production, then added bacterial hydrogenase. Hydrogen formed only when they added a viologen dye to act as an electron carrier, they found. This was the case for hydrogenase from several bacterial species. Thus the nature of the physiological electron carrier was still in doubt, according to the Berkeley workers. Then early this year, Dr. L. E. Mortenson, Dr. R. C. Valentine, and Dr. J. E. Carnahan at Du Pont isolated a natural electron-transferring cofactor from the nitrogen fixing bacterium Clostridium pasteurianum [Biochem. Biophijs. Res. Commun., 7, 448 (1962)]. They called this ferredoxin. It links the bacterium's hydrogenase enzyme with several electron donors and acceptors. Dr. Paneque and Dr. Arnon find that ferredoxin from C. pasteurianum can replace viologen as an electron carrier in the chloroplast reaction. Dr. Tagawa and Dr. Arnon have crystallized the ferredoxin protein and examined its properties. It is oxidized and reduced reversibly with a redox potential of —417 m v. at p H 7.55. This closely resembles hydrogens electronegativity of —420 at p H 7; it is much more reducing than TPN, which has a redox potential value of - 3 2 4 mv. at p H 7. This led to a search for a counterpart in spinach chloroplasts. They isolated this protein—which they call "chloroplast ferredoxin"—by a procedure similar to that used in the isolation of Clostridium ferredoxin. Like Clostridium ferredoxin, the protein may be reversibly oxidized and reduced. It is somewhat more electronegative, —432 mv. compared to —417 mv. at p H 7.55. Its absorption spectra are similar. Iron content is about 1%. The molecular weight of Clostridium ferredoxin may be about 12,000. This is the tentative value resulting from the initial work of Dr. Phil G. Squire at Berkeley's hormone research laboratory, using the analytical ultracentrifuge. Dr. Arnon's iron analysis gives a preliminary value of 10 iron atoms per molecule. Dr. Arnon and Dr. Tagawa find that chloroplasts with ferredoxin added can utilize hydrogen gas in the dark and reduce TPN. This means that as in bacteria, hvdrogen reduces ferredoxin without an input of radiant energy as is the case
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with C. pasteurianum. The chloroplast catalyzes electron transfer from ferredoxin to TPN. It does this through an enzyme, probably a flavoprotein, they believe. However, under physiological conditions, the chloroplast reduces ferredoxin at the expense of radiant energy, then the flavoprotein aids the electron transfer from ferredoxin to the pyridine nucleotide, they add. Dr. Arnon believes that these findings point to an important basic biochemical unity between the electron transfer processes in photosynthesis and the corresponding chemical reactions in bacteria. They also indicate that the key redox reactions in the light phase of photosynthesis take place around the potential of the hydrogen electrode, that is about —420 mv.
Microorganisms Transform Hydrocarbons 142ND
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vapor pressure and the effects of lower alkanes on cell lipids are the reasons for poor growth, or even toxicity. But both these ideas need full study. The literature suggests that branched-chain alkanes are more easily attacked by microorganisms than normal alkanes. But Dr. Kallio finds that this is not always true. Studies with Micrococcus cerificans and Nocardia corallina and their utilization of 14 different hexadecane monomers show that a single methyl group on the chain may prevent its assimilation by some strains of micrococcus tested, he says. Although single methyl and single ethyl branches permit assimilation of hexadecane isomers by certain strains of Micrococcus, propyl derivatives do not. Propyl branches do not prevent assimilation by Nocardia or Mycobacterium species. But addition of more than one propyl branch appears to prevent far-reaching oxidation. A great many kinds of reaction sequences seem to operate in microbial hydrocarbon oxidations. Many of these may be related, but all will have to await a more detailed analysis.
Microbial Chemistry and Technology
"It is no more proper to consider hydrocarbon assimilating microorganisms in a special category, or to speak of 'hydrocarbon bugs' than it is to speak of glucose organisms or amino acid organisms," according to Dr. R. E. Kallio and his co-workers, Dr. A. Markovetz and Eva J. McKenna of the University of Iowa. Contrary to the general impression that hydrocarbon utilizing organisms are few, and must be found by classical enrichment techniques, Dr. Kallio and others find that the ability to assimilate hydrocarbons is a widespread characteristic among microorganisms. Utilization of hydrocarbons by microorganisms is getting a lot of attention today. Contamination of jet fuels is one of the main reasons; microbial degradation (or the lack of it) of detergents in streams is another. Although they will say little about it, petroleum companies are also very interested in the possibilities of using microorganisms to carry out various chemical transformations. Generally, lower alkanes—with fewer than eight or 10 carbon atoms—do not support growth of organisms well. One explanation may be that the high 58
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Amino Acids Change Bacterial Growth Pattern 142ND
ACS
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MEETING
Biological Chemistry
When either threonine or valine, both of which are essential nutrients, is limiting in the culture medium of Streptococcus faecalis, the dry weight of the culture increases markedly after the limiting amino acid has been used up. But if lysine is limiting and used up, bacterial lysis occurs and runs to completion, according to Dr. Gerrit Toennies, Dr. Gerald D. Shockman, and Joseph J. Kolb of the Institute for Cancer Research and the department of microbiology of Temple University's school of medicine, Philadelphia, Pa. When valine is limiting, there is a postexponential gain of about 50% in dry weight in about 20 hours, largely accounted for by a 100% gain in wall material. Under similar conditions, when threonine is limiting, the dry weight gain at the end of 40 hours is about 100%, mostly because of a
200% gain in cell wall substance. Further, postvaline depletion growth shows little gain in non wall nitrogen, whereas there is a 20% gain in nonwall nitrogen—mostly nucleic acid formation—following threonine depletion. Phenomena intermediate between complete lysis and increased dry weight occur when other amino acids are limiting, the Philadelphia group says. Studies of these phenomena led the Temple workers to look at the role of the bacterial membrane in the postexponential growth phase. They chose both lipid phosphorus and the petroleum-ether-soluble fraction of the methanol-soluble lipid in the cell as quantitative indexes for the bacterial membrane. Lipid phosphorus, a major component of the membrane, is not found in either cell wall or cytoplasm. The relative occurrence of both membrane lipid and lipid phosphorus in a cell sample, as well as in a sample of membrane substance, is determined. Then, using either membrane lipid or lipid phosphorus data, the membrane content of the whole cell substance is calculated. DNA synthesis is absent in valinedeficient cells. However, in threoninedeficient cells, DNA synthesis takes place. And RNA synthesis in threonine-deficient cells is greater than in valine-deficient ones. In valine-deficient cells, membrane gain is larger than in threonine-deficient cells, while cell wall gain is larger in threoninedeficient cells. Assuming that the average amount of DNA per cellular unit remains constant during growth on the deficient media, and also that the cellular unit of S. faecalis is spherical, Dr. Toennies says that DNA values suggest that the relationship of the volumes of cells in the exponential phase, valine-deficient cells, and threonine-deficient cells, is 1.00 to 1.28 to 0.63. The data further indicate that threonine depletion results in a tripling of cell numbers, but valine depletion is followed by little if any cell division. When numbers of cells, their volumes, and membrane and wall contents are considered together and compared with controls, Dr. Toennies concludes that wall thickness doubles in both valine- and threonine-deficient cells. Meanwhile, membrane thickness doubles in the case of the valine-deficient cells, but remains unchanged in threonine-deficient cells.
