Alkaliphilic sulfidogenesis on cellulose by combined cultures

Soda lakes are characterized by an intense sulfur cycle that begins with sulfidogenesis. Model laboratory experiments that involved combining of pure cultures showed that, during anaerobic decomposition of cellulose by Clostridium alkalicellulosi, the sulfate-reducing bacteria (SRB) of the species Desulfonatronovibrio hydrogenovorans, Desulfonatronum lacustre, and Desulfonatronum cooperativum, different in their nutritional requirements, may directly use the cellulose fermentation products for sulfidogenesis without mediatory microorganisms. In binary cocultures with SRB, the amount of the H₂S formed constituted from one-third to two-thirds of the cellulose [H] equivalents; acetate was among the products formed. When the syntrophic Contubernalis alkalaceticum, capable of acetate oxidation, was incorporated into the trophic chain along with hydrogenotrophic SRB, the amount of the H₂S formed exceeded by 33–42% the amount of the [H] equivalents in the utilized cellulose, water being the source of additional hydrogen. Thus, the trophic pathway from plant residues to sulfide, previously considered to be the longest in the alkaliphilic microbial community, may involve a minimal number of stages and do without intermediate participation of dissipotrophic fermenting organisms.     

Enzymic generation of chiral acetates. A quantitative evaluation of their configurational assay.

1. R-Acetate was generated enzymically from R-acetate in the sequence acetate leads to malate leads to oxaloacetate leads to acetate, and S-acetate likewise from S-acetate. It was concluded that the formation of malate on malate synthase involves the operation of a normal isotopic effect combined with inversion of configuration. The malate synthase kH/k2H was determined as 3.7 +/- 0.5 by a method which yields results independently of the stereochemical purity of the chiral acetates used initially. 2. R-Acetate was also generated from R-acetate in the sequence acetate leads to citrate leads to malate leads to oxaloacetate leads to acetate, and S-acetate likewise from S-acetate. The conclusion is the same as given above, but refers to the formation of citrate on the re-synthase. 3. 2S,3R-[2-2H1,3-2H1,3H1]Malate and 2S,3S-[2-2H1,3-2H1]malate were prepared from 2S-[2,3-2H3]malate by treatment with fumarase in tritiated water and normal water, respectively. It was assumed that these malate specimens were pure with respect to chirality as generated by isotopic labelling. 4. These two malate specimens were partially converted (about 9%) to acetates in conditions where no racemization at the level of transiently formed oxaloacetate occurred. That no racemization took place was demonstrated experimentally. Oxidative enzymic hydrolysis of 2S,3R-[2-2H1,3-2H1,3H1]malate in normal water and of 2S,3S-[2-2H1,3-2H1]malate in tritiated water produced S-[2H1,3H1]acetate and R-[2H1,3H1]acetate, respectively. 5. The isolated R-[2H1,3H1]acetate and S-[2H1,3H1]acetate on configurational analysis yielded malates which in the presence of fumarase retained 79.7 +/- 0.7% and 20.3 +/- 0.9%, respectively, of their total tritium content. The symmetric deviation from the 50% value found with [3H1]acetate strengthens the conclusion that stereochemically pure chiral acetates were analyzed. The malate synthase kH/k2H was determined from the data of this study as 3.9 +/- 0.2. 6. The average of the values given under paragraphs 1 and 5 for the isotopic discrimination on malate synthase corresponds to kH/k2H=3.8 +/- 0.1. It was concluded that the configurational analysis of stereochemically pure R-[2H1,3H1]acetate and S-[2H1,3H1]acetate yields malates which in the presence of fumarase retain 79 +/- 2% and 21 +/- 2%, respectively, of their total tritium content. Hence, a deviation of 29 +/- 2% from the 50% value represents the actual amplitude of the configurational assay. 7. Outlines are given for an enzymic generation of chiral acetates in preparative scale.     

Bioconversion of Cellulose to Acetate with Pure Cultures of Ruminococcus albus and a Hydrogen-Using Acetogen

Bioconversion of cellulose to acetate was accomplished with cocultures of two organisms. One was the cellulolytic species Ruminococcus albus. It ferments crystalline cellulose (Avicel) to acetate, ethanol, CO(inf2), and H(inf2). The other organism (HA) obtains energy for growth by using H(inf2) to reduce CO(inf2) to acetate. HA is a gram-negative coccobacillus that was isolated from horse feces. Coculture of R. albus with HA in batch or continuous culture alters the fermentation products formed from crystalline cellulose by the ruminococcus via interspecies H(inf2) transfer. The major product of the fermentation by R. albus and HA coculture is acetate. High concentrations of acetate (333 mM) were obtained when batch cocultures grown on 5% cellulose were neutralized with Ca(OH)(inf2). Continuous cocultures grown at retention times of 2 and 3.1 days produced 109 and 102 mM acetate, respectively, when fed 1% cellulose with utilization of 84% of the substrate.     

Ca2+-dependent and Ca2+-independent pathways for release of arachidonic acid from phosphatidylinositol in endothelial cells

The pathways for degradation of phosphatidylinositol (PI) were investigated in sonicated suspensions prepared from confluent cultures of bovine pulmonary artery endothelial cells. The time courses of formation of 3H-labeled and 14C-labeled metabolites of phosphatidyl-[3H]inositol ([3H]Ins-PI) and 1-stearoyl-2-[14C] arachidonoyl-PI were determined at 37 degrees C and pH 7.5 in the presence of 2 mM EDTA with or without a 2 mM excess of Ca2+. The rates of formation of lysophosphatidyl-[3H]inositol ([3H]Ins-lyso-PI) and 1-lyso-2-[14C] arachidonoyl-PI were similar in the presence and absence of Ca2+, and the absolute amounts of the two radiolabeled lyso-PI products formed were nearly identical. This indicated that lyso-PI was formed by phospholipase A1, and phospholipase A2 was not measurable. In the presence of EDTA, [14C]arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI paralleled release of glycerophospho-[3H]inositol ([3H]GPI) from [3H]Ins-PI. Formation of [3H]GPI was inhibited by treatment with the specific sulfhydryl reagent, 2,2'-dithiodipyridine, and this was accompanied by an increase in [3H]Ins-lyso-PI. In the presence of Ca2+, [14C] arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI was increased 2-fold and was associated with Ca2+-dependent phospholipase C activity. Under these conditions, [3H]inositol monophosphate production exceeded formation of [14C]arachidonic acid-labeled phospholipase C products, diacylglycerol plus monoacylglycerol, by an amount that was equal to the amount of [14C]arachidonic acid formed in excess of [3H]GPI. Low concentrations of phenylmethanesulfonyl fluoride (15-125 microM) inhibited Ca2+-dependent [14C]arachidonic acid release, and the decrease in [14C] arachidonic acid formed was matched by an equivalent increase in 14C label in diacylglycerol plus monoacyclglycerol. These data supported the existence of two pathways for arachidonic acid release from PI in endothelial cells; a phospholipase A1-lysophospholipase pathway that was Ca2+-independent and a phospholipase C-diacylglycerol lipase pathway that was Ca2+-dependent. The mean percentage of arachidonic acid released from PI via the phospholipase C-diacylglycerol lipase pathway in the presence of Ca2+ was 65 +/- 8%. The mean percentage of nonpolar phospholipase C products of PI metabolized via the diacylglycerol lipase pathway to free arachidonic acid was 28 +/- 3%.     

Phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria inhabiting an oxic-anoxic sewer biofilm determined by combining microautoradiography and fluorescent in situ hybridization.

We simultaneously determined the phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria (SRB) inhabiting a sewer biofilm with oxygen, nitrate, or sulfate as an electron acceptor by combining microautoradiography and fluorescent in situ hybridization (MAR-FISH) with family- and genus-specific 16S rRNA probes. The MAR-FISH analysis revealed that Desulfobulbus hybridized with probe 660 was a dominant SRB subgroup in this sewer biofilm, accounting for 23% of the total SRB. Approximately 9 and 27% of Desulfobulbus cells detected with probe 660 could take up [(14)C]propionate with oxygen and nitrate, respectively, as an electron acceptor, which might explain the high abundance of this species in various oxic environments. Furthermore, more than 40% of Desulfobulbus cells incorporated acetate under anoxic conditions. SRB were also numerically important members of H(2)-utilizing and (14)CO(2)-fixing microbial populations in this sewer biofilm, accounting for roughly 42% of total H(2)-utilizing bacteria hybridized with probe EUB338. A comparative 16S ribosomal DNA analysis revealed that two SRB populations, related to the Desulfomicrobium hypogeium and the Desulfovibrio desulfuricans MB lineages, were found to be important H(2) utilizers in this biofilm. The substrate uptake characteristics of different phylogenetic SRB subgroups were compared with the characteristics described to date. These results provide further insight into the correlation between the 16S rRNA phylogenetic diversity and the physiological diversity of SRB populations inhabiting sewer biofilms.     

Metabolism of acetylcholine in the nervous system of Aplysia californica. III. Studies of an indentified cholinergic neuron

[3H] choline and [3H] acetyl CoA were injected into the cell body of an identified cholinergic neuron, the giant R2 of the Aplysia abdominal ganglion, and the fate and distribution of the radioactivity studied. Direct eveidence was obtained that the availabliity of choline to the enzymatic machinery limits synthesis. [3H] choline injected intrasomatically was converted to acetylcholine far more efficiently than choline taken up into the cell body from the bath. Synthesis from injected [3H] acety CoA was increased more than an order of magnitude when the cosubstrate was injected together with a saturating amount of unlabeled choline. In order to study the kinetics of acetylcholine synthesis in the living neuron, we injected [3H] choline in amounts resulting in a range of intracellular concentrations of about four orders of magnitude. The maximal velocity was 300 pmol of acetylcholine/cell/h and the Michaelis constant was 5.9 mM [3H] choline; these values agreed well with those previously reported for choline acetyltransferase assayed in extracts of Aplysia nervous tissue. [3H] acetylcholine turned over within the injected neuron with a half-life of about 9 h. The ultimate product formed was betaine. Subcellular distribution of [3H] acetylcholine was studied using differential and gradient centrifuagtion, gel filtration, and passage through cellulose acetate filters. A small portion of acetylcholine was contained in particulates the size and density expected of cholinergic vesicles.     

Partial purification and some properties of oxalacetase from Aspergillus niger.

1. Oxalacetase from Asperigillus niger was found to be an inducible enzyme, the induction being dependent not only on neutralisation of the acidic growth medium but also on the presence of carbonate. An explanation is proposed. 2. Three methods were established for the quantitative determination of oxalacetase activity. These are based on the determination of the product acetate, on the absorbance of oxaloacetate and on coupling the hydrolysis of oxaloacetate to the oxidation of malate by NAD in the presence of malate dehydrogenase. 3. Oxalacetase was purified about 50-fold from cell-free extracts of A. niger and used to determine some of its properties such as kinetic constants. 4. 2S-[U-14C, 3-2H2] Malate in the presence of oxalacetase, NAD and malate dehydrogenase was partially converted to acetate and oxalate. The 3H/14C ratio of the isolated acetate was nearly twice as high as that of the malate used initially. The result demonstrates that the keto form of oxaloacetate, not the enol, is the substrate of the enzyme. 5. Equimolecular mixtures of 2S, 3S-[3-2H1] malate + 2S-[2-2H1] malate (mixture 1) and 2S, 3R-[3-2H1, 3H1] malate + 2S, 3R-[2-2H1, 3-3H1] malate (mixture 2) were prepared from 2S-[3-3H2] malate by incubation with fumarase in normal and tritiated water, respectively. The isolated mixture 1, in the presence of oxalacetase, NAD and malate dehydrogenase was incubated in tritiated water for formation of acetate and oxalate; the isolated mixture 2 was treated likewise in normal water. 6. The mixtures of symmetrically labelled [3H1] acetate and chiral acetates thus produced were isolated and the configuration of the [3H1, 3H1] acetate specimens was determined in the sequence acetate leads to malate leads to fumarate, as usual. The [2H1, 3H1] acetate derived from 2S, 3S-[3-2H1] malate (present in mixture 1( yielded a malate which on incubation with fumarase retained 65.0% of its total tritium content. This chiral acetate, therefore, had the R configuration. The [2H1, 3H1] acetate derived from 2S, 3R-[2-2H1, 3-3H1] malate produced a malate which retained 35% of its total tritium content, and therefore had the S configuration. 7. It was concluded that the detachment of the oxaloyl residue from oxaloacetate and its replacement by a proton proceed with inversion of configuration at the methylene group which becomes methyl during the hydrolysis.     

Acetate production from hydrogen and [13C]carbon dioxide by the microflora of human feces.

Fecal suspensions from humans were incubated with 13CO2 and H2. The suspensions were from subjects who harbored 10(8) and 10(10) methanogens per g (dry weight) of feces, respectively, and from a subject who did not harbor methanogens. Quantitative nuclear magnetic resonance spectroscopy showed that acetate labeled in both the methyl and carboxyl groups was formed by suspensions from the subject without methanogens and the subject with the lower concentrations of methanogens. The amounts of labeled acetate formed were in agreement with the amounts expected based on measurements of H2 utilization. No labeled acetate was formed by suspensions from the subject with the higher concentrations of methanogens, and essentially all of the H2 used was accounted for by CH4 production. Suspensions from the subject with lower concentrations of methanogens produced both methane and acetate from H2 and CO2. The results indicate that reduction of CO2 to acetate may be a major pathway for microbial production of acetate in the human colon except when very high concentrations of methanogens (ca. 10(10) per g [dry weight] of feces) are present. Double-labeled acetate was also formed from H2 and 13CO2 by fecal suspensions from nonmethanogenic and moderately methanogenic rats.     

13C and 1H NMR study of cellulose metabolism by Fibrobacter succinogenes S85

Fibrobacter succinogenes S85, a cellulolytic rumen bacterium, is very efficient in degrading lignocellulosic substrates and could be used to develop a biotechnological process for the treatment of wastes. In this work, the metabolism of cellulose by F. succinogenes S85 was investigated using in vivo 13C NMR and 13C-filtered spin-echo difference 1H NMR spectroscopy. The degradation of unlabelled cellulose synthesised by Acetobacter xylinum was studied indirectly, in the presence of [1-13C]glucose, by estimating the isotopic dilution of the final bacterial fermentation products (glycogen, succinate, acetate). During the pre-incubation period of F. succinogenes cells with cellulose fibres, some cells ('non-adherent') did not attach to the solid material. Results for 'adherent' cells showed that about one fourth of the glucose units entering F. succinogenes metabolism originated from cellulose degradation. A huge reversal of succinate metabolism pathway and production of large amounts of unlabelled acetate which was observed during incubation with glucose only, was found to be much decreased in the presence of solid substrate. The synthesis of glucose 6-phophate was slightly increased in the presence of cellulose. Results clearly showed that 'non-adherent' cells were able to metabolise glucose very efficiently; consequently the metabolic state of these cells was not responsible for their 'non-adherence' to cellulose fibre.     

Lipoxygenase products of arachidonic acid modulate biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human neutrophils via phospholipase A2

When human neutrophils, previously labeled in their phospholipids with [14C]arachidonate, were stimulated with the Ca2+-ionophore, A23187, plus Ca2+ in the presence of [3H]acetate, these cells released [14C]arachidonate from membrane phospholipids, produced 5-hydroxy-6,8,11,14-[14C]eicosatetraenoic acid (5-HETE) and 14C-labeled 5S,12R-dihydroxy-6-cis,8,10-trans, 14-cis-eicosatetraenoic acid ([14C]leukotriene B4), and incorporated [3H]acetate into platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Ionophore A23187-induced formation of these radiolabeled products was greatly augmented by submicromolar concentrations of exogenous 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), 5-HETE, and leukotriene B4. In the absence of ionophore A23187, these arachidonic acid metabolites were virtually ineffective. Nordihydroguaiaretic acid (NDGA) and several other lipoxygenase/cyclooxygenase inhibitors (butylated hydroxyanisole, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 1-phenyl-2-pyrazolidinone) caused parallel inhibition of [14C]arachidonate release and [3H]PAF formation in a dose-dependent manner. Specific cyclooxygenase inhibitors, such as indomethacin and naproxen, did not inhibit but rather slightly augmented the formation of these products. Furthermore, addition of 5-HPETE, 5-HETE, or leukotriene B4 (but not 8-HETE or 15-HETE) to neutrophils caused substantial relief of NDGA inhibition of [3H]PAF formation and [14C]arachidonate release. As opposed to [3H]acetate incorporation into PAF, [3H]lyso-PAF incorporation into PAF by activated neutrophils was little affected by NDGA. In addition, NDGA had no effect on lyso-PAF:acetyl-CoA acetyltransferase as measured in neutrophil homogenate preparations. It is concluded that in activated human neutrophils 5-lipoxygenase products can modulate PAF formation by enhancing the expression of phospholipase A2.     

Acetate production from hydrogen and [13C]carbon dioxide by the microflora of human feces

Fecal suspensions from humans were incubated with 13CO2 and H2. The suspensions were from subjects who harbored 10(8) and 10(10) methanogens per g (dry weight) of feces, respectively, and from a subject who did not harbor methanogens. Quantitative nuclear magnetic resonance spectroscopy showed that acetate labeled in both the methyl and carboxyl groups was formed by suspensions from the subject without methanogens and the subject with the lower concentrations of methanogens. The amounts of labeled acetate formed were in agreement with the amounts expected based on measurements of H2 utilization. No labeled acetate was formed by suspensions from the subject with the higher concentrations of methanogens, and essentially all of the H2 used was accounted for by CH4 production. Suspensions from the subject with lower concentrations of methanogens produced both methane and acetate from H2 and CO2. The results indicate that reduction of CO2 to acetate may be a major pathway for microbial production of acetate in the human colon except when very high concentrations of methanogens (ca. 10(10) per g [dry weight] of feces) are present. Double-labeled acetate was also formed from H2 and 13CO2 by fecal suspensions from nonmethanogenic and moderately methanogenic rats.     

Stimulation of platelet-activating factor synthesis by a nonmetabolizable bioactive analog of platelet-activating factor and influence of arachidonic acid metabolites

Platelet-activating factor (PAF) is a potent neutrophil agonist operating through specific receptors located on the cell surface. Binding of PAF to its receptor may also stimulate further PAF synthesis, thus providing a means of amplifying the PAF signal for the cell of origin and/or other responsive cells. In this report we demonstrate that 1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (C-PAF), a nonmetabolizable bioactive analog of PAF, stimulates human neutrophils to synthesize PAF, as detected by [3H]acetate incorporation into PAF. This approach allowed us to conclude that [3H]acetate-labeled PAF was formed from endogenous precursor rather than mere turnover of the stimulatory dose of PAF. PAF's ability to initiate further PAF synthesis was confirmed by measuring the PAF-stimulated conversion of 1-O-[3H]alkyl-2-acylglycerophosphocholine to 1-O-[3H]alkyl-2-acetylglycerophosphocholine by prelabeled human neutrophils and by determining the molecular species of 1-O-alkyl-2-[3H]acetylglycerophosphocholine produced by cells stimulated with a single molecular species of PAF (C15:0). Degradation of exogenously added [3H]PAF was not inhibited by C-PAF/5-hydroxyeicosatetraenoic acid treatment. Thus, inhibition of PAF degradation was ruled out as the mechanism accounting for the appearance of labeled PAF in the stimulated cells. Synthesis of PAF in response to C-PAF was not dependent on cytochalasin B pretreatment but was dramatically potentiated by 5-hydroxyeicosatetraenoic acid, which alone was without effect. Additionally, we have demonstrated that another major arachidonate metabolite of neutrophils, leukotriene B4, stimulates PAF production. Thus, at least three products of activated neutrophils, including PAF itself, can promote PAF synthesis by these cells. This positive feedback effect may amplify autacoid production and the final cellular response.     

Gluconeogenesis in the kidney cortex. Flow of malate between compartments

1. Kidney-cortex slices from starved rats were incubated with l-[U-(14)C]lactate or l-[U-(14)C]malate plus unlabelled acetate and the specific radioactivity of the glucose formed was determined. In parallel experiments the specific radioactivity of the glucose formed from [1-(14)C]acetate plus unlabelled l-lactate and l-malate was determined. 2. By analytical methods the major products formed from the substrates were measured. The glucose formed was purified by paper chromatography for determination of specific radioactivity. 3. The specific radioactivity of the glucose formed from l-[U-(14)C]lactate agrees with predictions of a model based on interaction of the gluconeogenic and the oxidative pathways. 4. The specific radioactivity of the glucose formed from l-[U-(14)C]malate agrees with the predicted value if rapid malate exchange between the cytosol and mitochondria is assumed. 5. The rate of malate exchange between compartments was estimated to be rapid and at least several times the rate of glucose formation. 6. The specific radioactivity of the glucose formed from [1-(14)C]acetate plus unlabelled l-lactate or l-malate agrees with the predictions from the model, again assuming rapid malate exchange between compartments. 7. Malate exchange between compartments together with reversible malate dehydrogenase activity in the mitochondria and cytosol also tends to equilibrate isotopically the NADH pool in these compartments. (3)H from compounds such as l-[2-(3)H]lactate, which form NAD(3)H in the cytosol, appears in part in water; and (3)H from dl-beta-hydroxy[3-(3)H]butyrate, which forms NAD(3)H in the mitochondria, appears in part in glucose, largely on C-4.     

Fermentation of cellulose by Ruminococcus flavefaciens in the presence and absence of Methanobacterium ruminantium

The anaerobic cellulolytic rumen bacterium Ruminococcus flavefaciens normally produces succinic acid as a major fermentation product together with acetic and formic acids, H2, and CO2. When grown on cellulose and in the presence of the methanogenic rumen bacterium Methanobacterium ruminantium, acetate was the major fermentation product; succinate was formed in small amounts; little formate was detected; H2 did not accumulate; and large amounts of CH4 were formed. M. ruminantium depends for growth on the reduction of CO2 to CH4 by H2, which it can obtain directly or by producing H2 and CO2 from formate. In mixed culture, the methanobacterium utilized the H2 and possibly the formate produced by the ruminococcus and in so doing stimulated the flow of electrons generated during glycolysis by the ruminococcus toward H2 formation and away from formation of succinate. This type of interaction may be of significance in determining the flow of cellulose carbon to the normal rumen fermentation products.     

Fermentation of cellulose by Ruminococcus flavefaciens in the presence and absence of Methanobacterium ruminantium.

The anaerobic cellulolytic rumen bacterium Ruminococcus flavefaciens normally produces succinic acid as a major fermentation product together with acetic and formic acids, H2, and CO2. When grown on cellulose and in the presence of the methanogenic rumen bacterium Methanobacterium ruminantium, acetate was the major fermentation product; succinate was formed in small amounts; little formate was detected; H2 did not accumulate; and large amounts of CH4 were formed. M. ruminantium depends for growth on the reduction of CO2 to CH4 by H2, which it can obtain directly or by producing H2 and CO2 from formate. In mixed culture, the methanobacterium utilized the H2 and possibly the formate produced by the ruminococcus and in so doing stimulated the flow of electrons generated during glycolysis by the ruminococcus toward H2 formation and away from formation of succinate. This type of interaction may be of significance in determining the flow of cellulose carbon to the normal rumen fermentation products.     

The metabolism of acetylcarnitine and acetate by bovine and hamster epididymal spermatozoa

Since acetylcarnitine has been identified in the epididymal plasma of many mammalian species, we investigated whether acetylcarnitine could serve as an energy substrate for epididymal bull and hamster spermatozoa. Intact caudal cells from both species oxidized [I-14C]acetyl-l-carnitine to 14CO2, in vitro, and the amount oxidized was dependent on time, substrate concentration, and cell number. Within each species, the rate of oxidation was the same as the rate at which free [1-14C]acetate was oxidized. Spermatozoa incubated with [3H]acetyl-L-carnitine hydrolyzed the compound and [3H]acetate accumulated in the medium. Unlabeled acetate added to the incubation medium competed with cellular uptake of [3H]acetate and resulted in further increase in [3H]acetate accumulation in the medium. Furthermore, the acetyl group of acetylcarnitine was oxidized by spermatozoa without concomitant uptake of the carnitine group. Purified plasma membrane vesicles contained an acetylcarnitine hydrolase activity that was solubilized from whole cells by detergents and that could be distinguished from acetylcholinesterase also present in the cells. The solubilized acetylcarnitine hydrolase activity was inhibited by p-hydroxymercuriphenylsulfonate, but not by the specific acetylcholinesterase inhibitors, eserine or BW63C47. The sulfhydryl blocker also inhibited the production of 14CO2 from [1-14C]acetylcarnitine by intact cells; acetylcholinesterase inhibitors did not. From estimates of sperm energy requirements, our results indicate that extracellular acetylcarnitine serves as a physiologically important energy substrate for maturing sperm cells.     

Competitive reaction kinetics of sulfate-reducing bacteria and methanogenic bacteria in anaerobic filters

A kinetic model for the anaerobic filter (AF) that takes into account the mass fractions of sulfate-reducing bacteria (SRB) (f(SRB)) and methanogenic bacteria (MB) (f(MB)) and an inhibiting effect of H(2)S on bacterial groups is proposed. When the acetate-fed AFs were maintained at the low organic loading rate of 2.5kg COD/m(3)d, variations of the influent COD/SO(4)(2-) ratio (0.5-3.0) does not materially affect the acetate removal efficiency (all varying between 98.1% and 99.7%). With an increase in influent COD/SO(4)(2-) ratio, both the biofilm thickness and the specific substrate utilization rate decreased slightly but f(SRB) decreased markedly. The estimated results of f(SRB) and f(MB) showed that SRB out-competed MB for bacterial growth if the influent COD/SO(4)(2-) ratio was maintained at less than 1.3, whereas MB out-competed SRB for bacterial growth if the influent COD/SO(4)(2-) ratio was maintained at greater than 2.0. The specific substrate utilization rate of SRB (0.19-0.24mg acetate/mg VSSd) was lower than that of MB (0.31-0.59mg acetate/mg VSSd). The estimated kinetic parameters disclosed that the affinity of acetate to MB was higher and unionized H(2)S imposed a greater inhibiting effect on MB. The model simulation results (acetate and sulfate removal) agreed well with the experimental results.     

Containment of biogenic sulfide production in continuous up-flow packed-bed bioreactors with nitrate or nitrite

Produced water from the Coleville oil field in Saskatchewan, Canada was used to inoculate continuous up-flow packed-bed bioreactors. When 7.8 mM sulfate and 25 mM lactate were present in the in-flowing medium, H(2)S production (souring) by sulfate-reducing bacteria (SRB) was prevented by addition of 17.5 mM nitrate or 20 mM nitrite. Changing the sulfate or lactate concentration of the in-flowing medium indicated that the concentrations of nitrate or nitrite required for containment of souring decreased proportionally with a lowered concentration of the electron donor lactate, while the sulfate concentration of the medium had no effect. Microbial communities were dominated by SRB. Nitrate addition did not give rise to changes in community composition, indicating that lactate oxidation and H(2)S removal were caused by the combined action of SRB and nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB). Apparently the nitrite concentrations formed by these NR-SOB did not inhibit the SRB sufficiently to cause community shifts. In contrast, significant community shifts were observed upon direct addition of high concentrations (20 mM) of nitrite. Strains NO3A and NO2B, two newly isolated, nitrate-reducing bacteria (NRB) emerged as major community members. These were found to belong to the epsilon-division of the Proteobacteria, to be most closely related to Campylobacter lari, and to oxidize lactate with nitrate or nitrite as the electron acceptor. Thus the mechanism of microbial H(2)S removal in up-flow packed-bed bioreactors depended on whether nitrate (SRB/NR-SOB) or nitrite (SRB/NR-SOB as well as NRB) was used. However, the amount of nitrate or nitrite needed to completely remove H(2)S was dictated by the electron donor (lactate) concentration, irrespective of mechanism.     

Detection of luciferase gene sequences in nonluminescent bacteria from the Chesapeake Bay

Washed excised roots of rice (Oryza sativa) produced H(2), CH(4) and fatty acids (millimolar concentrations of acetate, propionate, butyrate; micromolar concentrations of isovalerate, valerate) when incubated under anoxic conditions. Surface sterilization of the root material resulted in the inactivation of the production of CH(4), a strong reduction of the production of fatty acids and a transient (75 h) but complete inhibition of the production of H(2). Radioactive bicarbonate was incorporated into CH(4), acetate, propionate and butyrate. About 20-40% of the fatty acid carbon originated from CO(2) reduction. In the presence of phosphate, CH(4) was exclusively produced from H(2)/CO(2), since phosphate selectively inhibited acetoclastic methanogenesis. Acetoclastic methanogenesis was also selectively inhibited by methyl fluoride, while chloroform or 2-bromoethane sulfonate inhibited CH(4) production completely. Production of CH(4), acetate, propionate and butyrate from H(2)/CO(2) was always exergonic with Gibbs free energies <-20 kJ mol(-1) product. Chloroform inhibited the production of acetate and the incorporation of radioactive CO(2) into acetate. Simultaneously, H(2) was no longer consumed and accumulated, indicating that acetate was produced from H(2)/CO(2). Chloroform also resulted in increased production of propionate and butyrate whose formation from CO(2) became more exergonic upon addition of chloroform. Nevertheless, the incorporation of radioactive CO(2) into propionate and butyrate was inhibited by chloroform. The accumulation of propionate and butyrate in the presence of chloroform probably occurred by fermentation of organic matter, rather than by reduction of acetate and CO(2). [U-(14)C]Glucose was indeed converted to acetate, propionate, butyrate, CO(2) and CH(4). Radioactive acetate, CO(2) and CH(4) were also products of the degradation of [U-(14)C]cellulose and [U-(14)C]xylose. Addition of chloroform and methyl fluoride did not affect the product spectrum of [U-(14)C]glucose degradation. The application of combinations of selective inhibitors may be useful to elucidate anaerobic metabolic pathways in mixed microbial cultures and natural microbial communities.     

Influence of hydrogen-consuming bacteria on cellulose degradation by anaerobic fungi.

The presence of methanogens Methanobacterium arboriphilus, Methanobacterium bryantii, or Methanobrevibacter smithii increased the level of cellulose fermentation by 5 to 10% in cultures of several genera of anaerobic fungi. When Neocallimastix sp. strain L2 was grown in coculture with methanogens the rate of cellulose fermentation also increased relative to that for pure cultures of the fungus. Methanogens caused a shift in the fermentation products to more acetate and less lactate, succinate, and ethanol. Formate transfer in cocultures of anaerobic fungi and M. smithii did not result in further stimulation of cellulolysis above the level caused by H2 transfer. When Selenomonas ruminatium was used as a H2-consuming organism in coculture with Neocallimastix sp. strain L2, both the rate and level of cellulolysis increased. The observed influence of the presence of methanogens is interpreted to indicate a shift of electrons from the formation of electron sink carbon products to H2 via reduced pyridine nucleotides, favoring the production of additional acetate and probably ATP. It is not known how S. ruminantium exerts its influence. It might result from a lowered production of electron sink products by the fungus, from consumption of electron sink products or H2 by S. ruminantium, or from competition for free sugars which in pure culture could exert an inhibiting effect on cellulolysis.