Growth of Synthrophomonas wolfei on unsaturated short chain fatty acids

The anaerobic fatty acid-degrading syntrophic bacterium, Syntrophomonas wolfei, was grown in pure culture with either trans-2-pentenoate, trans-2-hexenoate, trans-3-hexenoate, and trans, trans-2,4-hexadienoate as the substrate. Trans-2-pentenoate was fermented to acetate, propionate, butyrate, and valerate. Acetate, butyrate, and hexanoate were produced from the six-carbon mono- and di-unsaturated acids. Propionate was also product from the trans,trans-2,4-hexadienoate which suggested this compound was degraded by another pathway in addition to -oxidation. The transient production of trans-2-hexenoate from trans-3-hexenoate suggested that the position of the double bound shifted from carbon-3 to carbon-2 prior to -oxidation. The specific growth rate decreased with increasing carbon length and degree of unsaturation. Molar growth yields ranged from 8.4 to 17.5 mg (dry wt.) per mmol and suggested that energy was conserved not only from substrate-level phosphorylation, but also from the reduction of unsaturated substrate.     

Studies on the formation of long-chain dicarboxylic acids from puren-alkanes by a mutant ofCandida tropicalis

Summary Individualn-alkanes, from C₁₁–C₁₆, were metabolized by a mutant ofCandida tropicalis. This strain was selected for its inability to grow in the presence of dodecanedioic acid and dodecane as the sole carbon source. Transformations were studied in fed-batch cultures. Undecane was only poorly transformed, but from dodecane to hexadecane high transformation yields were achieved. Maximum yield of acid-precipitable long-chain dioic acids was obtained with tridecane as substrate. All the products were mixtures of different acids. Besides the ,-alkanedioic acids, the 3-hydroxy derivatives of long-chain ,-alkanedioic acids and dioic acids with a shortened carbon chain were found.     

Biosynthesis of the beta-diketones of barley spike epicuticular wax.

[1-¹⁴C]acetate and [2-¹⁴C]acetate were incorporated into the β-diketones of barley spike epicuticular wax via the peduncle. Utilizing column chromatography with dry copper acetate, the β-diketones were isolated and the labeling pattern in the hentriacontan-14, 16-dione determined after its degradation. A modified iodoform procedure was used to give myristic and palmitic acids. Radio-gas chromatography was then performed on the products of chemical α-oxidation of the separated fatty acids. This procedure, in effect, gave the specific activity of every carbon atom of hentriacontan-14,16-dione except carbon-1 to carbon-5 (from myristic acid) and carbon-27 to carbon-31 (from palmitic acid) for each labeled substrate. The specific activity of carbon-15 was determined by an indirect method. On the basis of these data it is suggested that the hentriacontan-14,16-dione is synthesized from the carbon-31 end of the molecule by elongation as follows. C₂ units are added, perhaps to a mixture of short chain precursors, to give a chain with 12 carbon atoms. This chain is then elongated to one with 16 carbon atoms so that the four added carbon atoms are uniformly labeled. Following this, the chain with 16 carbon atoms is elongated with C₂ units to give the complete molecule. Possibly some change in mechanism occurs in this last elongation process when the chain is 22 carbon atoms long. Barley spike wax β-diketones contain about 2% nonacosan-13, 15-dione which seems to be synthesized in an analogous manner.     

Biosynthesis of methyl branched hydrocarbons of the German cockroach Blattella germanica (L.) (orthoptera,blattellidae)

The precursors and directionality of synthesis of the methyl branched cuticular hydrocarbons and the female contact sex pheromone, 3,11-dimethyl-2-nonacosanone, of the German cockroach, Blattella germanica, were investigated by radiotracer and carbon-13 NMR techniques. The amino acids [G-³H]valine, [4,5-³H]isoleucine and [3,4-¹⁴C₂]methionine labeled the hydrocarbon fraction in a manner indicating that the carbon skeletons of all three amino acids serve as the methyl branch group donor. The incorporation of [1,4-¹⁴C₂]- and [2,3-¹⁴C₂]succinates into the hydrocarbon and acylglycerol/polar lipid fractions indicated that succinate also served as a precursor to methylmalonyl-CoA. Carbon-13 NMR analyses showed that [1-¹³C]propionate labeled the carbon adjacent to the tertiary carbon, and, for the 3,x-dimethylalkanes, that carbon-4 and not carbon-2 was enriched. [1-¹³C]Acetate labeled carbon-2 of these hydrocarbons. This indicates that the methyl branching groups of the 3,x-dimethylalkanes were inserted early in the chain elongation process. [3,4,5-¹³C₃]Valine labeled the methyl, tertiary and carbon adjacent to the tertiary carbon of the methyl branched alkanes. Thus, the methyl branched hydrocarbon was formed by the insertion of methylmalonyl units derived from propionate, isoleucine, valine, methionine and succinate early in chain elongation.     

Effect of culture conditions on mycelial growth and production of γ-linolenic acid by the fungus Mortierella ramanniana

Summary Effect of culture conditions on cell growth, lipid accumulation and -linolenic acid production is reported for four Mortierella species. The highest concentration as well as the highest productivity of -linolenic acid in lipid was determined in strains of M. ramanniana. M. ramanniana CBS 112.08 was used in the studies of the influence of medium composition, concentration of carbon- and nitrogen sources and growth temperature. Several carbon sources provided good growth and a high lipid content in biomass. The highest dry weights (11–12g/l) and lipid contents (24%, w/w), were observed if glucose or fructose was used as carbon source, whereas the highest amount of -linolenic acid (26%) was determined in starch-grown cells. The fatty acid composition in the lipid was influenced by the cultivation time, growth temperature and, to a minor extent, by the carbon source used. In fermentor cultures, both strains of Mortierella ramanniana showed relatively poor growth and incomplete consumption of glucose. M. vinacea, on the other hand, grew well in tower reactors. M. vinacea, which has a different morphology than M. ramanniana strains, also showed higher yields of biomass and lipid and higher yield coefficients than the latter.     

Uptake of free amino acids by the diatom,Melosira mediocris

Individual ¹⁴C-labelled amino acids are rapidly removed from dilute solution in artificial sea water (0.2 mol 1^–1) by suspensions of Meliosira medocris. The rate of disappearance of radioactivity corresponds closely to removal of primary amines as determined by measurement of the rate of decrease of fluorescamine-positive material. Net removal of naturally occurring free amino acids from the sea water habitat from which the alga was isolated is demonstrated using high performance liquid chromatography. Removal of amino acids from natural sources makes a significant contribution to the carbon requirements of the alga as well as supplying significant amounts of amino nitrogen.     

Controlling simultaneous production of endoglucanase and beta-glucosidase by Fusarium oxysporum in submerged culture

Summary The simultaneous production of endoglucanase and -glucosidase by Fusarium oxysporum was investigated in submerged culture. Consecutive optimization of growth conditions resulted in the correction of large activity differences, observed during production of enzymes, and substantially enhanced low enzyme yields. At optimum growth conditions yields as high as 1650 and 232 U per g of carbon source of endoglucanase and -glucosidase were obtained respectively competing favourably with those reported for microorganisms grown on the same carbon source. The most important kinetic characteristics of the enzymes were the high temperature optima of endoglucanase (60°C) and -glucosidase (65°C) and the exceptionally high thermostability of endoglucanase. The latter enzyme retained 50% of the activity at pH 5.0 after approximately 6.5 h at 70°C     

Synthesis of organic compounds from carbon monoxide and water by UV photolysis

The photolysis of water vapor with carbon monoxide at 1849 Å yields alcohols, aldehydes and organic acids, with an overall quantum yield of 3.3×10^–2. This rather high quantum yield could have led to a contribution of 10¹¹ organic molecules cm^–2 sec^–1 to the pool of organic material on the primitive Earth. The reactions are initiated by the photolysis of water molecules and the resulting hydrogen atoms reduce the carbon monoxide to a variety of one and two carbon compounds. The organic molecules are dissolved in water and thus escape destruction by photolysis. Photolysis of water vapor with carbon dioxide did not yield organic compounds under these conditions.     

The incorporation of acetate by the chemoautotroph Thiobacillus neapolitanus strain C

Summary Cultures of Thiobacillus neapolitanus strain C assimilate ¹⁴C-labelled acetate and aspartate. Both carbon atoms of acetate are incorporated, and 25% of the cell carbon can arise from acetate. Aspartate-¹⁴C contributes 4–5% of the cell carbon, and is found in pyrimidines and in protein as aspartate and its related amino acids. Acetate-¹⁴C contributes to lipid, glutamate, arginine, proline and leucine, but not to aspartate. Acetate assimilation by washed organisms requires carbon dioxide and energy from thiosulphate oxidation. Degradation of ¹⁴C-glutamic acid from acetate-¹⁴C-labelled bacteria; the accumulation of ¹⁴C-citrate in the presence of fluoroacetate and [¹⁴C] acetate; short-term kinetic experiments on acetate-¹⁴C turnover; and the demonstration of citrate synthesis by cell-free extracts all indicate glutamate synthesis from -ketoglutarate formed by reactions of the tricarboxylic acid cycle. The cycle is believed to be incomplete, probably not proceeding further than -ketoglutarate, and functions as a glutamate-synthesising system, using oxaloacetate derived solely from carbon dioxide fixation. Malate synthase (and the glyoxylate cycle) appear to be insignificant in the metabolism, but extracts did form citramalate from acetate and pyruvate.     

Autotrophic CO2 fixation in Chlorobium limicola. Evidence for the operation of a reductive tricarboxylic acid cycle in growing cells

Chlorobium limicola was grown on a mineral salts medium with CO₂ as the main carbon source supplemented with specifically labeled ¹⁴C propionate and the incorporation of ¹⁴C into alanine ( intracellular pyruvate), aspartate ( oxaloacetate), and glutamate ( -ketoglutarate) was studied in long term labeling experiments. During growth in presence of propionate 30% of the cell carbon were derived from propionate and 70% from CO₂. Propionate was not oxidized to CO₂.All three amino acids were found to be labeled. The labeling patterns indicate that propionate was assimilated via propionyl CoA, methylmalonyl CoA and succinyl CoA. When 1-¹⁴C propionate was the labeled precursor no radioactivity was found in the carboxyl group(s) of alanine, aspartate and glutamate, excluding the incorporation of propionate into the amino acids via succinate oxidation to fumarate. With 1-¹⁴C propionate preferentially aspartate (C-3) and glutamate (C-2) became labeled, with 2-¹⁴C propionate alanine (C-3) and glutamate (C-4). These findings indicate that propionate was incorporated into the amino acids via succinyl CoA, -ketoglutarate, isocitrate, and citrate, followed by a si-type cleavage of citrate to oxaloacetate and acetyl CoA (or acetate). Similar experiments with U-¹⁴C acetate confirm these conclusions. Thus, all reactions of the proposed reductive tricarboxylic acid cycle could be demonstrated in autotrophically growing cells.     

Studies on aspergilli xix. Role of carbon and nitrogen on citric acid production

Summary The effect of various carbon and nitrogen compounds on the production of citric acid byAspergillus luchuensis was studied under controlled conditions. Unhydrolysed sucrose was better than the hydrolysed one. The other carbon sources, viz., maltose, lactose, Gur, Ral and cane molasses were unsuitable for citric acid fermentation. With all these carbohydrates low yields were obtained. Sodium nitrate was found to be the best nitrogen source for the maximum yield of citric acid. It was closely followed by potassium nitrate. Ammonium compounds and amino acids, in general, were unsuitable for the production of citric acid.     

Substrate specificity of acyl-lipid Δ9-desaturase from <Emphasis Type="Italic">Prochlorothrix hollandica</Emphasis> cyanobacterium producing myristoleic acid

Chlorophyll b-containing cyanobacterium Prochlorothrix hollandica is characterized by a high content of esterified fatty acids (FA) with 14 and 16 carbon atoms in the membrane lipids. Depending on the conditions of cultivation, the relative amount of myristic (C_14:0) and myristoleic (C_14:1) acids can reach 35%, and palmitic (С_16:0) and palmitoleic (С_16:1) acids can reach 60% of the sum of all fatty acids in cells. Monounsaturated FAs are represented by C_14:1, and C_16:1 with an olefinic bond presumably located in the Δ⁹ position. We cloned the gene of acyl-lipid Δ9-desaturase, desC1, from Prochlorothrix hollandica and characterized its specificity to the length of the substrate using the heterologous expression in Escherichia coli cells adding C_14:0 or stearic (C_18:0) acids as exogenous substrates. The results show that DesC1 Δ⁹ desaturase generates olefinic bonds in the FAs with a length of 14 to 18 carbon atoms with an approximately equal efficiency. This indicates that the length of the FA chain in P. hollandica is determined by the activity of the FA synthase, and the chain is desaturated at the Δ⁹ position nonspecifically relatively to its length.     

Rapid and Efficient Synthesis of Peptides Containing α,α-dialkylamino acids employing KOBt

Several Fmoc-,-dialkylamino acids and their acid chlorides have been prepared, isolated and characterised. The synthesis of peptides containing sterically hindered dialkylamino acids has been accomplished using acid chloride/KOBt in dichloromethane. The yields as well as the purity of the peptides were satisfactory.     

The spark discharge synthesis of amino acids from various hydrocarbons

The spark discharge synthesis of amino acids using an atmosphere of CH₄ + N₂ + H₂O + NH₃ has been investigated with variable pNH₃. The amino acids produced using higher hydrocarbons (ethane, ethylene, acetylene, propane, butane, and isobutane) instead of CH₄ were also investigated. There was considerable range in the absolute yields of amino acids, but the yields relative to glycine (or -amino-n-butyric acid) were more uniform. The relative yields of the C₃ to C₆ aliphatic -amino acids are nearly the same (with a few exceptions) with all the hydrocarbons. The glycine yields are more variable. The precursors to the C₃-C₆ aliphatic amino acids seem to be produced in the same process, which is separate from the synthesis of glycine precursors. It may be possible to use these relative yields as a signature for a spark discharge synthesis provided corrections can be made for subsequent decomposition events (e.g. in the Murchison meteorite).     

Synthesis of Biologically Active Lipids by the Fungus Mucor lusitanicus 306D Grown on Media with Various Composition

Growth and lipogenesis of the fungusMucor lusitanicus306D, producing -linoleic acid, was studied under various conditions of nitrogen and carbon nutrition. Media containing food-industry wastes, such as maize extract, molasses, and protein hydrolysate, were used. The content of -linoleic acid was higher when carbohydrates (glucose and molasses) were used as carbon sources and urea was used as a nitrogen source. At a high glucose concentration (100 g/l), fed batch cultivation ensured high contents of -linoleic acid in lipids (1 g/l). After extraction of lipids, the fungus biomass was 42% protein, containing all essential amino acids. A defatted biomass was shown to be effectively assimilated by minks.     

Research for amino acids in lunar samples

Water extracts of lunar fines were analyzed for amino acids by a gas-liquid chromatographic technique whereby amino acids were converted to the N-trifluoroacetyln-butyl, esters prior to analysis. The lunar material studied included both Apollo 14 (14240 SESC and 14298) and Apollo 12 (12023) samples. The water extract of the special Apollo 14 sample (14240 SESC) was analyzed both for free and bound amino acids (hydrolysis with 6 N hydrochloric acid). In both the hydrolyzed and unhydrolyzed extracts, the amino acids were not observed above background levels.The analysis of Apollo 12 and 14 samples (12023 14298) yielded similar results. Detection limits were established at 300 pg to 1 ng for different amino acids. A large chromatographic peak with a retention temperature of 126°C was observed on analysis of sample, (12023); it was identified as oxalic acid by GC-MS. The concentration of amino acids in the Apollo 14 SESC samples processed and analyzed in the joint experiments at Ames by GLC and IEC were found to be extremely low (glycine at 3 to 4 ng g^–1). As the quantities were so minute, these identifications could not be confirmed by GLC-MS and therefore should still be considered as tentative. Other studies included the analysis of performance standards at the 2 to 6 ng level of each of 17 amino acids, and the analysis of 5 ml of H₂O containing 2 ppb of each amino acid. Recovery of amino acids added to lunar fines were conducted at the 10, 50, and 70 ng level of each amino acid with 50 to 70 mg of lunar material. The recoveries varied from as high as 80% for some of the aliphatics to complete loss of the amino acids ornithine and lysine.Contributed from Missouri Agricultural Experiment Station Journal Series No. 6255. Approved by the Director. Supported in part by grants from the National Aeronautics and Space Administration (NGR 26-004-011) and the Experiment Station Chemical Laboratories.     

Differential filtration studies of carbon flux from living algae to microheterotrophs,microplankton size distribution and respiration in Lake Kinneret

The flux of newly photosynthetically fixed, dissolved organic carbon (PDOC) from phototrophs to microheterotrophs in Lake Kinneret was examined by differential (3 and 0.4m) filtration after samples were incubated with¹⁴C-bicarbonate for 3 hours (in the light) and subsequently for 21 hours (in darkness). Only a small proportion (average about 10%) of the carbon fixed from¹⁴C-bicarbonate in the light was associated with particulate matter <3m. In 14 out of 16 experiments there was no significant decrease in the relative proportion of radioactivity associated with larger (>3m) organisms after the dark period, suggesting that the amount of PDOC taken up by unclumped, single bacteria (<3m) was not very great. Respiration rates, estimated by the decrease in¹⁴C particulate counts in the dark period, ranged from 3.4 to 21.2% of daylight net photosynthesis. In almost all cases, parallel experiments with additions of radioactive glucose or amino acids indicated that the majority of active heterotrophs passed through 3m filters. Apparent residence times for glucose and amino acids were 20 to 168 hours and 20 to 152 hours, respectively.     

Stereoconservative and stereoselective syntheses of rare and non-naturalα-amino acids from (S)-aspartic acid and (S)-malic acid

Summary A new strategy for stereoconservative and stereoselective syntheses of several types of amino acids starting from-functional carboxylic acids employing hexafluoroacetone as protecting and activating reagent is described. Outstanding features of this new method are the mild reaction conditions and the high yields for introduction and cleavage of the protective group allowing sensitive functional groups in the side chain to survive. Furthermore, the new concept results in saving of synthetic steps.     

Utilization of Amino Acids as a Sole Source of Nitrogen by Obligate Chemolithoautotroph Thiobacillus ferrooxidans

An obligate chemolithoautotroph, Thiobacillus ferrooxidans API 9–3, could utilize amino acids, other than glycine, methionine and phenylalanine, as a sole source of nitrogen. However, both the growth rate and growth yield were lower than those in Fe²⁺-NH4 -salts medium, suggesting that the ammonium ion was a superior nitrogen source for the strain compared to amino acids. Methionine and phenylalanine strongly inhibited the cell growth on Fe²⁺-NH4-salts medium at 10 mm. [¹⁴C]Glycine could not be taken up into the cells, and this meant the strain could not use glycine as a sole source of nitrogen. The uptake of [¹⁴C]leucine into the cells was dependent on the presence of Fe^{2 +}. When the strain was cultured on Fe^{2 +} - leucine (lOmm)-salts medium lacking an inorganic nitrogen source for 5 days at 30°C, 83.5% and 16.5% of the cellular carbon were derived from carbon dioxide and leucine, respectively, indicating that carbon dioxide was a superior carbon source for the bacterium compared to leucine. The ammonium ion did not inhibit the utilization of leucine for cellular carbon. Leucine uptake was markedly inhibited by inhibitors of protein synthesis, such as chloramphenicol (94.3% at 1 mm), streptomycin (57.2% at 5mm) and rifampin (77.2% at 0.1 mm), respectively. Carbon dioxide uptake was also completely inhibited by chloramphenicol at 4mm. These results suggest that the transport of both amino acids and carbon dioxide into the cells was dependent on protein synthesis.     

Intramitochondrial reductive carboxylation of 2-oxoglutarate in adipose tissue and its contribution to fatty acid synthesis

• 1.1. The reductive carboxylation of 2-oxoglutarate was found to proceed in mitochondria of rat epididymal fat pads and rabbit perirenal adipose tissue at a rate similar to that in liver mitochondria.
• 2.2. In rat fat pads the incorporation of ¹⁴C from [5-¹⁴C]2-oxoglutarate into fatty acids via the carboxylation was suppressed by butylmalonate by 30%.
• 3.3. 2-Oxoglutarate and glutamate stimulated the incorporation into fatty acids of ¹⁴C from [2-¹⁴C]acetate in rat fat pads with the simultaneous reduction of tissue NADP. These effects persisted after inhibition of succinate dehydrogenase by malonate.
• 4.4. It is concluded that in adipose tissue 2-oxoglutarate carboxylation proceeds in both the cytoplasm and mitochondria. Therefore, it can supply carbon atoms as well as NADPH for fatty acid synthesis.