Patterns of carbon flow from glucose to methane in a thermophilic anaerobic bioreactor

[U-¹⁴C]Glucose, added carrier-free to sludge from a thermophilic anaerobic bioreactor being fed a lignocellulose waste, was rapidly turned over with less than one-third of the original radiolabel remaining as glucose after 5 s of incubation. The primary labeled products found were [¹⁴C]acetate and ¹⁴CO₂, which were in a ratio near 2:1. Further incubation resulted in the disappearance of [¹⁴C]acetate and the appearance of an equivalent amount of label as ¹⁴CH₄ and ¹⁴CO₂. No significant production of [¹⁴C]propionate, butyrate, lactate, or ethanol was detected from [¹⁴C]glucose, even if these potential intermediates (unlabeled) were added to the sludge at a concentration of 1 mM to trap any label entering their pools. Addition of 0.8 atm (80 kPa) of H₂ to the headspace over sludge resulted in some accumulation of [¹⁴C]lactate and a corresponding decrease in [¹⁴C]acetate produced from [¹⁴C]glucose. Production of [¹⁴C]propionate, butyrate and ethanol were still not significant in the presence of H₂. Incubation of sludge for 1 h in the presence of hydrogen resulted in increases in the lactate and formate concentrations, but not those of propionate, butyrate, or ethanol. These results demonstrate that glucose was metabolized directly to acetate, CO₂, and H₂ by the microbial populations in the bioreactor with little carbon from glucose flowing through other intermediates, indicating a high degree of coupling between glucose fermentation and hydrogen uptake. The short-term response of these microbial populations to elevated H₂ partial pressures was to increase lactate production.     

Deamination of amino acids by Bacillus pasteurii

Resting cells of Bacillus pasteurii as employed in the treatment of distillery waste showed deamination of amino acids. The deamination of l-glutamic acid and dl-aspartic acid was found to be oxidative while that of dl-serine, dl-threonine and l-asparagine was non-oxidative. dl-Alanine and glycine were not deaminated when present individually but when incubated together showed oxidative deamination. NAD stimulated the oxidation of l-glutamic acid and α,α′-dipyridyl completely inhibited it.     

Fermentative degradation of putrescine by new strictly anaerobic bacteria

Three strains of new strictly anaerobic, Grampositive, non-sporeforming bacteria were isolated from various anoxic sediment samples with putrescine as sole carbon and energy source. Optimal growth in carbonate-buffered defined medium occurred at 37°C at pH 7.2–7.6. The DNA base ratio of strain NorPut1 was 29.6±1 mol% guanine plus cytosine. In addition to a surface layer and the peptidoglycan layer, the cell wall contained a second innermost layer with a periodic arrangement of subunits. All strains fermented putrescine to acetate, butyrate, and molecular hydrogen; the latter originated from both oxidative putrescine deamination and 4-aminobutyraldehyde oxidation. In defined mixed cultures with methanogens or homoacetogenic bacteria, methane or additional acetate were formed due to interspecies hydrogen transfer. Also 4-aminobutyrate and 4-hydroxybutyrate were fermented to acetate and butyrate, but no hydrogen was released from these substrates. No sugars, organic acids, other primary amines or amino acids were used as substrates. Neither sulfate, thiosulfate, sulfur, nitrate nor fumarate was reduced. Most of the enzymes involved in putrescine degradation could be demonstrated in cell-free extracts. A pathway of putrescine fermentation via 4-aminobutyrate and crotonyl-CoA with subsequent dismutation to acetate and butyrate is suggested.     

Differences in amino acids composition and coupling patterns between mesophilic and thermophilic proteins

Summary. Thermophilic proteins show substantially higher intrinsic thermal stability than their mesophilic counterparts. Amino acid composition is believed to alter the intrinsic stability of proteins. Several investigations and mutagenesis experiment have been carried out to understand the amino acid composition for the thermostability of proteins. This review presents some generalized features of amino acid composition found in thermophilic proteins, including an increase in residue hydrophobicity, a decrease in uncharged polar residues, an increase in charged residues, an increase in aromatic residues, certain amino acid coupling patterns and amino acid preferences for thermophilic proteins. The differences of amino acids composition between thermophilic and mesophilic proteins are related to some properties of amino acids. These features provide guidelines for engineering mesophilic protein to thermophilic protein. Authors’ addresses: Yuan-Jiang Pan, Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Zhejiang University Road 38, Hangzhou 310027, China; Wei-Fen Li, Microbiology Division, College of Animal Science, Zhejiang University, Hangzhou 310029, China     

Mesophilic syntrophic acetate oxidation during methane formation by a triculture at high ammonium concentration

In a mesophilic (37°C) triculture at a high ammonium concentration and pH8, methanogenesis from acetate occurred via syntrophic acetate oxidation. Studies with ¹⁴C-labelled substrates showed that the amount of labelled methane formed from 1-¹⁴C-labelled acetate was equal to that formed from 2-¹⁴C-labelled acetate. Labelled methane was also formed from H¹⁴CO₃ ^-. These results clearly showed that both the methyl and carboxyl groups of acetate were oxidized to CO₂ and that CO₂ was reduced to CH₄ through hydrogenotrophic methanogenesis. During growth of the triculture, a significant isotopic exchange between the carboxyl group of acetate and bicarbonate occurred. As a result, there was a decrease in the specific activity of 1-¹⁴C-acetate, and the production of ¹⁴CO₂ was slightly higher from 1-¹⁴C- than from 2-¹⁴C-acetate. For each mole acetate degraded, 0.94 mol methane was formed; 9.2 mmol acetate was metabolized during the 294 days of incubation.     

End products of metabolism in the anaerobic ciliate Trimyema compressum

Abstract The products of anaerobic and micro-aerobic (0.8% O₂) metabolism of the sapropelic ciliate Trimyema compressum strain N were studied. Under anaerobic conditions ethanol was formed in large amounts representing 44% of the total carbon excreted. Acetate, lactate, formate, CO₂ and H₂ were minor products, while succinate was formed in hardly detectable amounts. Under micro-aerobic conditions O₂ was consumed, CO₂ and formate were produced as major end products and no H₂, ethanol and succinate were formed.     

Effects of amino acid analogues on protein degradation in isolated rat hepatocytes

Analogues and derivatives of six of the amino acids which most effectively inhibit protein degradation in isolated rat hepatocytes (leucine, asparagine, glutamine, histidine, phenylalanine and tryptophan) were investigated to see if they could antagonize or mimic the effect of the parent compound. No antagonists were found. Amino alcohols and amino acid amides tended to inhibit protein degradation strongly, apparently by a direct lysosomotropic effect as indicated by their ability to cause lysosomal vacuolation. Amino acid alkyl esters and dipeptides inhibited degradation to approximately the same extent as did their parent amino acids, possibly by being converted to free amino acids intracellularly. Of several leucine analogues tested, four (L-norleucine, L-norvaline, D-norleucine and L-allo-isoleucine) were found to be as effective as leucine in inhibiting protein degradation. None of the analogues had any effect on protein synthesis. Since leucine appears to play a unique role as a regulator of bulk autophagy in hepatocytes, the availability of active leucine agonists may help tj elucidate the biochemical mechanism for control of this important process.     

Glucocorticoids inhibit precursor incorporation into protein in splenic lymphocytes by stimulating protein degradation and expanding intracellular amino acid pools

In the presence of tracer concentrations of extracellular leucine (5 μM), treatment of rat splenic lymphocyte suspensions in vitro with 1 μM dexamethasone for 2.5–4 h caused a 30–35% inhibition of [³H]leucine incorporation into protein. As the extracellular leucine concentration was raised to 5 mM, this inhibition was progressively reduced to 0–12%. This phenomenon correlated with a marked dependence on extracellular leucine concentration of the dexamethasone-dependent enlargement of free intracellular leucine pools in splenic lymphocytes: a 123% increase in pool size with tracer extracellular leucine; a 10% increase with 5 mM leucine. Varying extracellular leucine had no effect on: (1) nuclear [³H]dexamethasone binding by the cells; (2) the concentration of dexamethasone needed for half-maximal inhibition of [³H]leucine incorporation; (3) the time course of onset and maximal expression of the hormonal inhibition of [³H]leucine incorporation; or (4) the magnitude of dexamethasone-dependent inhibition of [³H]uridine incorporation into RNA by these cells. There was no detectable effect of dexamethasone on uptake and retention of [³H]leucine by the cells, regardless of the extracellular leucine concentration. Treatment of splenic lymphocytes for 4 h in vitro with 1 μM dexamethasone caused a small shift of ribosomes from larger aggregate polysomes to smaller forms. Thus, glucocorticoid-induced inhibition of amino acid incorporation in splenic lymphocytes is a multicomponent response, of which an actual decrease in protein synthesis is only a small part. Enlargement of free intracellular amino acid pools, probably resulting from increased protein degradation, is the major contributing factor to the hormonal inhibition of amino acid incorporation.     

Permeability of membranes to amino acids and modified amino acids: Mechanisms involved in translocation

Summary The amino acid permeability of membranes is of interest because they are one of the key solutes involved in cell function. Membrane permeability coefficients (P) for amino acid classes, including neutral, polar, hydrophobic, and charged species, have been measured and compared using a variety of techniques. Decreasing lipid chain length increased permeability slightly (5-fold), while variations in pH had only minor effects on the permeability coefficients of the amino acids tested in liposomes. Increasing the membrane surface charge increased the permeability of amino acids of the opposite charge, while increasing the cholesterol content decreased membrane permeability. The permeability coefficients for most amino acids tested were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium (approximately 10^–12–10^–13 cm · s^–1). This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. Hydrophobic amino acids were 10² more permeable than the hydrophilic forms, reflecting their increased partition coefficient values.External pH had dramatic effects on the permeation rates for the modified amino acid lysine methyl ester in response to transmembrane pH gradients. It was established that lysine methyl ester and other modified short peptides permeate rapidly (P = 10^–2 cm · s^–1) as neutral (deprotonated) molecules. It was also shown that charge distributions dramatically alter permeation rates for modified di-peptides. These results may relate to the movement of peptides through membranes during protein translocation and to the origin of cellular membrane transport on the early Earth.Abbreviations DCP dicetylphosphate - DMPC dimyristoyl phosphatidylcholine - EPC egg phosphatidylcholine - LUV large unilamellar vesicle - MLV multilamellar vesicle - PLM planar lipid membrane - SUV small unilamellar vesicle - pH transmembrane pH gradient     

Impact of separating amino acids between plasma, extracellular and intracellular compartments on estimating protein synthesis in rodents

Summary. Three models representing different separations of amino acid sources were used to simulate experimental specific radioactivity data and to predict protein fractional synthesis rate (FSR). Data were from a pulse dose of ¹⁴C-U Leu given to a non-growing 20 g mouse and a flooding dose of ³H Phe given to a non-growing 200 g rat. Protein synthesis rates estimated using the combined extracellular and intracellular (Ec + Ic) source pool and extracellular and plasma (Ec + Pls) source pool mouse models were 78 and 120% d⁻¹ in liver, 14 and 16% d⁻¹ in brain and 15 and 14% d⁻¹ in muscle. Predicted protein synthesis rates using the Ec + Ic, Ec + Ic + Tr (combined extracellular, intracellular and aminoacyl tRNA source pool) and Ec + Pls rat models were 57, 3.4 and 57% d⁻¹ in gastrocnemius, 58, 71 and 62% d⁻¹ in gut, 8.3, 8.4 and 7.9% d⁻¹ in heart, 32, 23 and 25% d⁻¹ in kidney, 160, 90 and 80% d⁻¹ in liver, 57, 5.5 and 57% d⁻¹ in soleus and 56, 3.4 and 57% d⁻¹ in tibialis. The Ec + Ic + Tr model underestimated protein synthesis rates in mouse tissues (5.0, 27 and 2.5% d⁻¹ for brain, liver and muscle) and rat muscles (3.4, 5.5 and 3.4% d⁻¹ for gastrocnemius, soleus and tibialis). The Ec + Pls model predicted the mouse pulse dose data best and the Ec + Ic model predicted the rat flooding dose data best. Model predictions of FSR imply that identification and separation of the source specific radioactivity is critical to accurately estimate FSR. Received June 11, 2000 Accepted September 26, 2000     

Methanogenic degradation of acetone by an enrichment culture

An anaerobic enrichment culture degraded 1 mol of acetone to 2 mol of methane and 1 mol of carbon dioxide. Two microorganisms were involved in this process, a filament-forming rod similar to Methanotrix sp. and an unknown rod with round to slightly pointed ends. Both organisms formed aggregates up to 300 m in diameter. No fluorescing bacteria were observed indicating that hydrogen or formate-utilizing methanogens are not involved in this process. Acetate was utilized in this culture by the Methanothrix sp. Inhibition of methanogenesis by bromoethanesulfonic acid or acetylene decreased the acetone degradation rate drastically and led to the formation of 2 mol acetate per mol of acetone. Streptomycin completely inhibited acetone degradation, and neither acetate nor methane was formed. ¹⁴CO₂ was incorporated exclusively into the C-1 atom of acetate indicating that acetone is degraded via carboxylation to an acetoacetate residue. It is concluded that acetone is degraded by a coculture of an eubacterium and an acetate-utilizing methanogen and that acetate is the only intermediate transferred between both. The energetical problems of the eubacterium converting acetone to acetate are discussed.     

Fermentation of isoleucine and arginine by pure and syntrophic cultures of Clostridium sporogenes

Abstract The fermentation of isoleucine, arginine and isoleucine + arginine by pure and syntrophic cultures of Clostridium sporogenes was investigated. Growth of C. sporogenes on isoleucine, if any, was poor, but some isoleucine was fermented to 2-methylbutyrate and hydrogen. In syntrophic cultures with Methanobacterium formicicum or Methanosarcina barkeri growth was better, and isoleucine was completely fermented, the hydrogen being used for methane production. Pure cultures of C. sporogenes grew on arginine and produced 5-aminovalerate, ornithine and acetate. The reducing equivalents for 5-aminovalerate production from intermediarily formed proline were provided by oxidative conversion of arginine to acetate and by oxidative metabolism of some amino acids present in the yeast extract. However, when isoleucine was available together with arginine in syntrophic cultures of C. sporogenes and M. formicicum , the reducing equivalents for arginine fermentation came mainly from the oxidation of isoleucine (Stickland reaction), and the hydrogen produced in excess served for the reduction of CO₂ to methane.     

High-rate thermophilic methane fermentation on short-chain fatty acids in a down-flow anaerobic packed-bed reactor

In order to maximize the efficiency of methane fermentation on short-chain fatty acids, growth media containing acetic acid and butyric acid as major carbon sources were supplied to a thermophilic down-flow anaerobic packed-bed reactor. The organic loading rate (OLR) to the reactor ranged from 0.2 to 169 kg-dichromate chemical oxygen demand(CODcr)/m³-reactor/day, corresponding to a hydraulic retention time (HRT) of between 1.4 h and 20 days. Stable methane production was maintained at HRTs as short as 2 h (OLR=120 kg-CODcr/m³/day), with the short-chain fatty acids in the feed almost completely removed during the process. The apparent substrate removal efficiency, determined from the total CODcr values in the influent and effluent, was 75% at short HRTs. However, the actual substrate removal efficiency must have been greater than 75%, since a fraction of substrate was also utilized in microbial cell synthesis, and these cells were part of the measured total CODcr.     

Synthesis of symmetric and asymmetric diamides of citric acid and amino acids

Summary A convenient method for the synthesis of symmetric and asymmetric diamides of amino acids including DOPA and citric acid from 2-tert-butyl-1,3-di(N-hydroxysuccinimidyl)citrate and 1-tert-butyl-2,3-di(N-hydroxysuccinimidyl)citrate is described.Abbreviations AcOtBu tert-butyl acetate - i-Bu iso-butyl - tBu tert-butyl - Bzl benzyl - p-OH-Bzl p-hydroxybenzyl - m,p-(OH)₂-Bzl m,p-dihydroxybenzyl - DCCI dicyclohexylcarbodiimide - Et ethyl - Me methyl - Su succinimidyl - SuOH N-hydroxysuccinimide - Ph phenyl     

A survey of hydrogen bond geometries in the crystal structures of amino acids

An analysis of the geometries of the hydrogen bonds observed by neutron diffraction in thirt-two crystal structures of amino acids shows the following results. Of the 168 hydrogen bonds in the data set, 64 involve the zwitterion groups 

and $\text{C}$O₂. Another 18 are from

to sulphate or carbonyl oxygens. The majority, 46, of these

H … O bonds are three-centered (bifurcated). Nine are four-centered (trifurcated). The geometry in which the three-centered hydrogen bond involves both oxygens of the same carboxylate group is not especially favoured. When it does occur, one hydrogen bond is generally shorter and the other longer, than when the bonding involves oxygens on different carboxylate groups. The shortest hydrogen bonds are the OH … O $\text{C}$, from a carboxylic acid hydroxyl to a carboxylate oxygen, and NH … O$\text{C}$ when the nitrogen is the ring atom in histidine or proline. Carboxylate groups, on average, accept six hydrogen bonds, with no examples of less than four bonds. The reason for the large number of three-centered

H … O$\text{C}$ bonds is therefore a proton deficiency arising from the disparity between the tripled donor property of the

groups and the sextuple, on average, acceptor property of the carboxylate groups. There is good geometrical evidence for the existence of H … O and H … Cl^? hydrogen bonds, especially involving the hydrogen atoms on α-atoms.     

Free amino acids are important for the retention of protein and non-protein meals by the midgut of Aedes aegypti females

There is a relationship between the normal progress of digestion and the retention or elimination of the proteins ingested with the meal by Aedes aegyti females. The addition of soybean trypsin inhibitor (STI) to a protein meal prevented digestion and resulted in a rapid elimination of the undigested proteins. The addition of a mix of free amino acids to a protein meal together with STI resulted in a significant increase in the retention of the undigested proteins during the first 10-15 hrs after feeding. The effect of the free amino acids on the retention of the proteins was concentration-dependent between 250 microg/ml and 5 mg/ml. Free amino acids were also important for the retention of non-protein meals. When females were fed a meal containing FITC-dextran (20 kD), most of this compound was eliminated into the feces by 10 hrs; the addition of free amino acid resulted in a significant increase in the retention of the FITC-dextran by the midgut during the first 15 hrs after feeding. The presence of free amino acids in the midgut lumen seems to be an important signal used by the mosquito to regulate the retention of the meal.     

Influence of triiodothyronine and dexamethasone on renal amino acid handling in rats loaded with various amino acid mixtures

Summary In adult female rats, the influence of dexamethasone or triiodothyronine on renal amino acid handling was investigated in amino acid loaded animals. Amino acids were administered intravenously as two mixtures, each containing four amino acids to overload amino acid reabsorption capacity. Bolus injections of both mixtures were followed by temporary increase in fractional excretion of the administered amino acids as well of the amino acids which were not covered in the mixtures. The administration of the two mixtures was followed by different interactions between various amino acid carriers.After dexamethasone pretreatment (60µg/100g b.wt. for 3 days, once daily) a stimulation of the renal amino acid handling could be shown. Triiodothyronine (20µg/100g b.wt. for 3 days, once daily) did not increase tubular reabsorption capacity for amino acids. It even increased fractional amino acid excretion in amino acid loaded rats as a sign of enhanced amino acid metabolism in the kidney and/or increased amino acid uptake into the tubular cells from the luminal site.     

Incorporation of nitrate nitrogen into amino acids during the anaerobic germination of rice

Summary Incorporation of¹⁵NO₃-into amino acids was studied during the anaerobic germination of rice seeds. In treated coleoptiles, the label was incorporated into glutamine, glutamate, alanine,-aminobutyric acid (Gaba), arginine, aspartate and methionine. These findings are consistent with a primary incorporation of nitrate nitrogen into glutamine, glutamate and aspartate, and their further conversion to alanine, Gaba, arginine and methionine.     

Spray reagent for the detection of amino acids on thin layer chromatography plates

Summary A spray reagent for easy identification of amino acids on thin-layer chromatography plates has been introduced. The reagent is capable of developing various distinguishable colours with many of them. A probable mechanism for such colour formation has also been proposed.