13C and 1H Nuclear Magnetic Resonance Study of Glycogen Futile Cycling in Strains of the Genus Fibrobacter

We investigated the carbon metabolism of three strains of Fibrobacter succinogenes and one strain of Fibrobacter intestinalis. The four strains produced the same amounts of the metabolites succinate, acetate, and formate in approximately the same ratio (3.7/1/0.3). The four strains similarly stored glycogen during all growth phases, and the glycogen-to-protein ratio was close to 0.6 during the exponential growth phase. ¹³C nuclear magnetic resonance (NMR) analysis of [1-¹³C]glucose utilization by resting cells of the four strains revealed a reversal of glycolysis at the triose phosphate level and the same metabolic pathways. Glycogen futile cycling was demonstrated by ¹³C NMR by following the simultaneous metabolism of labeled [¹³C]glycogen and exogenous unlabeled glucose. The isotopic dilutions of the CH₂ of succinate and the CH₃ of acetate when the resting cells were metabolizing [1-¹³C]glucose and unlabeled glycogen were precisely quantified by using ¹³C-filtered spin-echo difference ¹H NMR spectroscopy. The measured isotopic dilutions were not the same for succinate and acetate; in the case of succinate, the dilutions reflected only the contribution of glycogen futile cycling, while in the case of acetate, another mechanism was also involved. Results obtained in complementary experiments are consistent with reversal of the succinate synthesis pathway. Our results indicated that for all of the strains, from 12 to 16% of the glucose entering the metabolic pathway originated from prestored glycogen. Although genetically diverse, the four Fibrobacter strains studied had very similar carbon metabolism characteristics.     

Measurement of Monosaccharides and Conversion of Glucose to Acetate in Anoxic Rice Field Soil

Degradation of glucose has been implicated in acetate production in rice field soil, but the abundance of glucose, the temporal change of glucose turnover, and the relationship between glucose and acetate catabolism are not well understood. We therefore measured the pool sizes of glucose and acetate in rice field soil and investigated the turnover of [U-¹⁴C]glucose and [2-¹⁴C]acetate. Acetate accumulated up to about 2 mM during days 5 to 10 after flooding of the soil. Subsequently, methanogenesis started and the acetate concentration decreased to about 100 to 200 μM. Glucose always made up >50% of the total monosaccharides detected. Glucose concentrations decreased during the first 10 days from 90 μM initially to about 3 μM after 40 days of incubation. With the exception at day 0 when glucose consumption was slow, the glucose turnover time was in the range of minutes, while the acetate turnover time was in the range of hours. Anaerobic degradation of [U-¹⁴C]glucose released [¹⁴C]acetate and ¹⁴CO₂ as the main products, with [¹⁴C]acetate being released faster than ¹⁴CO₂. The products of [2-¹⁴C]acetate metabolism, on the other hand, were ¹⁴CO₂ during the reduction phase of soil incubation (days 0 to 15) and ¹⁴CH₄ during the methanogenic phase (after day 15). Except during the accumulation period of acetate (days 5 to 10), approximately 50 to 80% of the acetate consumed was produced from glucose catabolism. However, during the accumulation period of acetate, the rate of acetate production from glucose greatly exceeded that of acetate consumption. Under steady-state conditions, up to 67% of the CH₄ was produced from acetate, of which up to 56% was produced from glucose degradation.     

Interactions between carbon and nitrogen metabolism in Fibrobacter succinogenes S85: a 1H and 13C nuclear magnetic resonance and enzymatic study

The effect of the presence of ammonia on [1-13C]glucose metabolism in the rumen fibrolytic bacterium Fibrobacter succinogenes S85 was studied by 13C and 1H nuclear magnetic resonance (NMR). Ammonia halved the level of glycogen storage and increased the rate of glucose conversion into acetate and succinate 2.2-fold and 1.4-fold, respectively, reducing the succinate-to-acetate ratio. The 13C enrichment of succinate and acetate was precisely quantified by 13C-filtered spin-echo difference 1H-NMR spectroscopy. The presence of ammonia did not modify the 13C enrichment of succinate C-2 (without ammonia, 20.8%, and with ammonia, 21.6%), indicating that the isotopic dilution of metabolites due to utilization of endogenous glycogen was not affected. In contrast, the presence of ammonia markedly decreased the 13C enrichment of acetate C-2 (from 40 to 31%), reflecting enhanced reversal of the succinate synthesis pathway. The reversal of glycolysis was unaffected by the presence of ammonia as shown by 13C-NMR analysis. Study of cell extracts showed that the main pathways of ammonia assimilation in F. succinogenes were glutamate dehydrogenase and alanine dehydrogenase. Glutamine synthetase activity was not detected. Glutamate dehydrogenase was active with both NAD and NADP as cofactors and was not repressed under ammonia limitation in the culture. Glutamate-pyruvate and glutamate-oxaloacetate transaminase activities were evidenced by spectrophotometry and 1H NMR. When cells were incubated in vivo with [1-13C]glucose, only 13C-labeled aspartate, glutamate, alanine, and valine were detected. Their labelings were consistent with the proposed amino acid synthesis pathway and with the reversal of the succinate synthesis pathway.     

Pathway of Propionate Oxidation by a Syntrophic Culture of Smithella propionica and Methanospirillum hungatei

The pathway of propionate conversion in a syntrophic coculture of Smithella propionica and Methanospirillum hungatei JF1 was investigated by ¹³C-NMR spectroscopy. Cocultures produced acetate and butyrate from propionate. [3-¹³C]propionate was converted to [2-¹³C]acetate, with no [1-¹³C]acetate formed. Butyrate from [3-¹³C]propionate was labeled at the C2 and C4 positions in a ratio of about 1:1.5. Double-labeled propionate (2,3-¹³C) yielded not only double-labeled acetate but also single-labeled acetate at the C1 or C2 position. Most butyrate formed from [2,3-¹³C]propionate was also double labeled in either the C1 and C2 atoms or the C3 and C4 atoms in a ratio of about 1:1.5. Smaller amounts of single-labeled butyrate and other combinations were also produced. 1-¹³C-labeled propionate yielded both [1-¹³C]acetate and [2-¹³C]acetate. When ¹³C-labeled bicarbonate was present, label was not incorporated into acetate, propionate, or butyrate. In each of the incubations described above, ¹³C was never recovered in bicarbonate or methane. These results indicate that S. propionica does not degrade propionate via the methyl-malonyl-coenzyme A (CoA) pathway or any other of the known pathways, such as the acryloyl-CoA pathway or the reductive carboxylation pathway. Our results strongly suggest that propionate is dismutated to acetate and butyrate via a six-carbon intermediate.     

Changes of Fermentation Pathways of Fecal Microbial Communities Associated with a Drug Treatment That Increases Dietary Starch in the Human Colon

Acarbose inhibits starch digestion in the human small intestine. This increases the amount of starch available for microbial fermentation to acetate, propionate, and butyrate in the colon. Relatively large amounts of butyrate are produced from starch by colonic microbes. Colonic epithelial cells use butyrate as an energy source, and butyrate causes the differentiation of colon cancer cells. In this study we investigated whether colonic fermentation pathways changed during treatment with acarbose. We examined fermentations by fecal suspensions obtained from subjects who participated in an acarbose-placebo crossover trial. After incubation with [1-¹³C]glucose and ¹²CO₂ or with unlabeled glucose and ¹³CO₂, the distribution of ¹³C in product C atoms was determined by nuclear magnetic resonance spectrometry and gas chromatography-mass spectrometry. Regardless of the treatment, acetate, propionate, and butyrate were produced from pyruvate formed by the Embden-Meyerhof-Parnas pathway. Considerable amounts of acetate were also formed by the reduction of CO₂. Butyrate formation from glucose increased and propionate formation decreased with acarbose treatment. Concomitantly, the amounts of CO₂ reduced to acetate were 30% of the total acetate in untreated subjects and 17% of the total acetate in the treated subjects. The acetate, propionate, and butyrate concentrations were 57, 20, and 23% of the total final concentrations, respectively, for the untreated subjects and 57, 13, and 30% of the total final concentrations, respectively, for the treated subjects.     

Glucose Metabolism and Kinetics of Phosphorus Removal by the Fermentative Bacterium Microlunatus phosphovorus

Phosphorus and carbon metabolism in Microlunatus phosphovorus was investigated by using a batch reactor to study the kinetics of uptake and release of extracellular compounds, in combination with ³¹P and ¹³C nuclear magnetic resonance (NMR) to characterize intracellular pools and to trace the fate of carbon substrates through the anaerobic and aerobic cycles. The organism was subjected to repetitive anaerobic and aerobic cycles to induce phosphorus release and uptake in a sequencial batch reactor; an ultrafiltration membrane module was required since cell suspensions did not sediment. M. phosphovorus fermented glucose to acetate via an Embden-Meyerhof pathway but was unable to grow under anaerobic conditions. A remarkable time shift was observed between the uptake of glucose and excretion of acetate, resulting in an intracellular accumulation of acetate. The acetate produced was oxidized in the subsequent aerobic stage. Very high phosphorus release and uptake rates were measured, 3.34 mmol g of cell⁻¹ h⁻¹ and 1.56 mmol g of cell⁻¹ h⁻¹, respectively, values only comparable with those determined in activated sludge. In the aerobic period, growth was strictly dependent on the availability of external phosphate. Natural abundance ¹³C NMR showed the presence of reserves of glutamate and trehalose in cell suspensions. Unexpectedly, [1-¹³C]glucose was not significantly channeled to the synthesis of internal reserves in the anaerobic phase, and acetate was not during the aerobic stage, although the glutamate pool became labeled via the exchange with intermediates of the tricarboxylic acid cycle at the level of glutamate dehydrogenase. The intracellular pool of glutamate increased under anaerobic conditions and decreased during the aerobic period. The contribution of M. phosphovorus for phosphorus removal in wastewater treatment plants is discussed on the basis of the metabolic features disclosed by this study.     

Stress Induction of Mitochondrial Formate Dehydrogenase in Potato Leaves

The metabolism of [1-¹³C]glucose in Pisolithus tinctorius cv Coker & Couch, in uninoculated seedlings of Eucalyptus globulus bicostata ex Maiden cv Kirkp., and in the E. globulus-P. tinctorius ectomycorrhiza was studied using nuclear magnetic resonance spectroscopy. In roots of uninoculated seedlings, the ¹³C label was mainly incorporated into sucrose and glutamine. The ratio (¹³C3 + ¹³C2)/¹³C4 of glutamine was approximately 1.0 during the time-course experiment, indicating equivalent contributions of phosphoenolpyruvate carboxylase and pyruvate dehydrogenase to the production of α-ketoglutarate used for synthesis of this amino acid. In free-living P. tinctorius, most of the ¹³C label was incorporated into mannitol, trehalose, glutamine, and alanine, whereas arabitol, erythritol, and glutamate were weakly labeled. Amino acid biosynthesis was an important sink of assimilated ¹³C (43%), and anaplerotic CO₂ fixation contributed 42% of the C flux entering the Krebs cycle. In ectomycorrhizae, sucrose accumulation was decreased in the colonized roots compared with uninoculated control plants, whereas ¹³C incorporation into arabitol and erythritol was nearly 4-fold higher in the symbiotic mycelium than in the free-living fungus. It appears that fungal utilization of glucose in the symbiotic state is altered and oriented toward the synthesis of short-chain polyols.     

Vertical Distribution of Methanogens in the Anoxic Sediment of Rotsee (Switzerland)

Anoxic sediments from Rotsee (Switzerland) were analyzed for the presence and diversity of methanogens by using molecular tools and for methanogenic activity by using radiotracer techniques, in addition to the measurement of chemical profiles. After PCR-assisted sequence retrieval of the 16S rRNA genes (16S rDNA) from the anoxic sediment of Rotsee, cloning, and sequencing, a phylogenetic analysis identified two clusters of sequences and four separated clones. The sequences in cluster 1 grouped with those of Methanosaeta spp., whereas the sequences in cluster 2 comprised the methanogenic endosymbiont of Plagiopyla nasuta. Discriminative oligonucleotide probes were constructed against both clusters and two of the separated clones. These probes were used subsequently for the analysis of indigenous methanogens in a core of the sediment, in addition to domain-specific probes against members of the domains Bacteria and Archaea and the fluorescent stain 4′,6-diamidino-2-phenylindole (DAPI), by fluorescent in situ hybridization. After DAPI staining, the highest microbial density was obtained in the upper sediment layer; this density decreased with depth from (1.01 ± 0.25) × 10¹⁰ to (2.62 ± 0.58) × 10¹⁰ cells per g of sediment (dry weight). This zone corresponded to that of highest metabolic activity, as indicated by the ammonia, alkalinity, and pH profiles, whereas the methane profile was constant. Probes Eub338 and Arch915 detected on average 16 and 6% of the DAPI-stained cells as members of the domains Bacteria and Archaea, respectively. Probe Rotcl1 identified on average 4% of the DAPI-stained cells as Methanosaeta spp., which were present throughout the whole core. In contrast, probe Rotcl2 identified only 0.7% of the DAPI-stained cells as relatives of the methanogenic endosymbiont of P. nasuta, which was present exclusively in the upper 2 cm of the sediment. Probes Rotp13 and Rotp17 did not detect any cells. The spatial distribution of the two methanogenic populations corresponded well to the methane production rates determined by incubation with either [¹⁴C]acetate or [¹⁴C]bicarbonate. Methanogenesis from acetate accounted for almost all of the total methane production, which concurs with the predominance of acetoclastic Methanosaeta spp. that represented on average 91% of the archaeal population. Significant hydrogenotrophic methanogenesis was found only in the organically enriched upper 2 cm of the sediment, where the probably hydrogenotrophic relatives of the methanogenic endosymbiont of P. nasuta, accounting on average for 7% of the archaeal population, were also detected.     

Biosynthetic Pathway of Citrinin in the Filamentous Fungus Monascus ruber as Revealed by 13C Nuclear Magnetic Resonance

Carbon isotope distribution of [¹³C]citrinin from Monascus ruber incubated with [¹³C]acetate revealed that the biosynthesis of the toxin originated from a tetraketide, instead of a pentaketide as has been shown for Penicillium and Aspergillus species. The production of polyketide red pigments and citrinin by M. ruber may therefore be regulated at the level of the tetraketide branch point.     

Anaerobic Mineralization of Stable-Isotope-Labeled 2-Methylnaphthalene

An active sulfate-reducing consortium that degrades 2-methylnaphthalene (2-MNAP) at rates of up to 25 μM day⁻¹ was established. Degradation was inhibited in the presence of molybdate and ceased in the absence of sulfate. As much as 87% of 2-[¹⁴C]MNAP was mineralized to ¹⁴CO₂. 2-Naphthoic acid (2-NA) was detected as a metabolite, and incubation with either deuterated 2-MNAP or [¹³C]bicarbonate indicates that 2-NA is the result of oxidation of the methyl group. Also detected were carboxylated 2-MNAPs, suggesting the presence of an alternative pathway for 2-MNAP degradation.     

Medium-Chain Fatty Acids Affect Citrinin Production in the Filamentous Fungus Monascus ruber

During submerged culture in the presence of glucose and glutamate, the filamentous fungus Monascus ruber produces water-soluble red pigments together with citrinin, a mycotoxin with nephrotoxic and hepatoxic effects on animals. Analysis of the ¹³C-pigment molecules from mycelia cultivated with [1-¹³C]-, [2-¹³C]-, or [1,2-¹³C]acetate by ¹³C nuclear magnetic resonance indicated that the biosynthesis of the red pigments used both the polyketide pathway, to generate the chromophore structure, and the fatty acid synthesis pathway, to produce a medium-chain fatty acid (octanoic acid) which was then bound to the chromophore by a trans-esterification reaction. Hence, to enhance pigment production, we tried to short-circuit the de novo synthesis of medium-chain fatty acids by adding them to the culture broth. Of fatty acids with carbon chains ranging from 6 to 18 carbon atoms, only octanoic acid showed a 30 to 50% stimulation of red pigment production, by a mechanism which, in contrast to expectation, did not involve its direct trans-esterification on the chromophore backbone. However, the medium- and long-chain fatty acids tested were readily assimilated by the fungus, and in the case of fatty acids ranging from 8 to 12 carbon atoms, 30 to 40% of their initial amount transiently accumulated in the growth medium in the form of the corresponding methylketone 1 carbon unit shorter. Very interestingly, these fatty acids or their corresponding methylketones caused a strong reduction in, or even a complete inhibition of, citrinin production by M. ruber when they were added to the medium. Several data indicated that this effect could be due to the degradation of the newly synthesized citrinin (or an intermediate in the citrinin pathway) by hydrogen peroxide resulting from peroxisome proliferation induced by medium-chain fatty acids or methylketones.     

Metabolic Analysis of Escherichia coli in the Presence and Absence of the Carboxylating Enzymes Phosphoenolpyruvate Carboxylase and Pyruvate Carboxylase

Fermentation patterns of Escherichia coli with and without the phosphoenolpyruvate carboxylase (PPC) and pyruvate carboxylase (PYC) enzymes were compared under anaerobic conditions with glucose as a carbon source. Time profiles of glucose and fermentation product concentrations were determined and used to calculate metabolic fluxes through central carbon pathways during exponential cell growth. The presence of the Rhizobium etli pyc gene in E. coli (JCL1242/pTrc99A-pyc) restored the succinate producing ability of E. coli ppc null mutants (JCL1242), with PYC competing favorably with both pyruvate formate lyase and lactate dehydrogenase. Succinate formation was slightly greater by JCL1242/pTrc99A-pyc than by cells which overproduced PPC (JCL1242/pPC201, ppc⁺), even though PPC activity in cell extracts of JCL1242/pPC201 (ppc⁺) was 40-fold greater than PYC activity in extracts of JCL1242/pTrc99a-pyc. Flux calculations indicate that during anaerobic metabolism the pyc⁺ strain had a 34% greater specific glucose consumption rate, a 37% greater specific rate of ATP formation, and a 6% greater specific growth rate compared to the ppc⁺ strain. In light of the important position of pyruvate at the juncture of NADH-generating pathways and NADH-dissimilating branches, the results show that when PPC or PYC is expressed, the metabolic network adapts by altering the flux to lactate and the molar ratio of ethanol to acetate formation.     

Enzymatic Combustion of Aromatic and Aliphatic Compounds by Manganese Peroxidase from Nematoloma frowardii

The direct involvement of manganese peroxidase (MnP) in the mineralization of natural and xenobiotic compounds was evaluated. A broad spectrum of aromatic substances were partially mineralized by the MnP system of the white rot fungus Nematoloma frowardii. The cell-free MnP system partially converted several aromatic compounds, including [U-¹⁴C]pentachlorophenol ([U-¹⁴C]PCP), [U-¹⁴C]catechol, [U-¹⁴C]tyrosine, [U-¹⁴C]tryptophan, [4,5,9,10-¹⁴C]pyrene, and [ring U-¹⁴C]2-amino-4,6-dinitrotoluene ([¹⁴C]2-AmDNT), to ¹⁴CO₂. Mineralization was dependent on the ratio of MnP activity to concentration of reduced glutathione (thiol-mediated oxidation), a finding which was demonstrated by using [¹⁴C]2-AmDNT as an example. At [¹⁴C]2-AmDNT concentrations ranging from 2 to 120 μM, the amount of released ¹⁴CO₂ was directly proportional to the concentration of [¹⁴C]2-AmDNT. The formation of highly polar products was also observed with [¹⁴C]2-AmDNT and [U-¹⁴C]PCP; these products were probably low-molecular-weight carboxylic acids. Among the aliphatic compounds tested, glyoxalate was mineralized to the greatest extent. Eighty-six percent of the ¹⁴COOH-glyoxalate and 9% of the ¹⁴CHO-glyoxalate were converted to ¹⁴CO₂, indicating that decarboxylation reactions may be the final step in MnP-catalyzed mineralization. The extracellular enzymatic combustion catalyzed by MnP could represent an important pathway for the formation of carbon dioxide from recalcitrant xenobiotic compounds and may also have general significance in the overall biodegradation of resistant natural macromolecules, such as lignins and humic substances.     

Incorporation of [15N]Ammonia by the Cellulolytic Ruminal Bacteria Fibrobacter succinogenes BL2, Ruminococcus albus SY3, and Ruminococcus flavefaciens 17

The origin of cell nitrogen and amino acid nitrogen during growth of ruminal cellulolytic bacteria in different growth media was investigated by using ¹⁵NH₃. At high concentrations of peptides (Trypticase, 10 g/liter) and amino acids (15.5 g/liter), significant amounts of cell nitrogen of Fibrobacter succinogenes BL2 (51%), Ruminococcus flavefaciens 17 (43%), and Ruminococcus albus SY3 (46%) were derived from non-NH₃-N. With peptides at 1 g/liter, a mean of 80% of cell nitrogen was from NH₃. More cell nitrogen was formed from NH₃ during growth on cellobiose compared with growth on cellulose in all media. Phenylalanine was essential for F. succinogenes, and its ¹⁵N enrichment declined more than that of other amino acids in all species when amino acids were added to the medium.     

Engineering of Acetate Recycling and Citrate Synthase to Improve Aerobic Succinate Production in Corynebacterium glutamicum

Corynebacterium glutamicum lacking the succinate dehydrogenase complex can produce succinate aerobically with acetate representing the major byproduct. Efforts to increase succinate production involved deletion of acetate formation pathways and overexpression of anaplerotic pathways, but acetate formation could not be completely eliminated. To address this issue, we constructed a pathway for recycling wasted carbon in succinate-producing C. glutamicum. The acetyl-CoA synthetase from Bacillus subtilis was heterologously introduced into C. glutamicum for the first time. The engineered strain ZX1 (pEacsA) did not secrete acetate and produced succinate with a yield of 0.50 mol (mol glucose)⁻¹. Moreover, in order to drive more carbon towards succinate biosynthesis, the native citrate synthase encoded by gltA was overexpressed, leading to strain ZX1 (pEacsAgltA), which showed a 22% increase in succinate yield and a 62% decrease in pyruvate yield compared to strain ZX1 (pEacsA). In fed-batch cultivations, strain ZX1 (pEacsAgltA) produced 241 mM succinate with an average volumetric productivity of 3.55 mM h⁻¹ and an average yield of 0.63 mol (mol glucose) ⁻¹, making it a promising platform for the aerobic production of succinate at large scale.     

Study of the 3-hydroxy eicosanoyl-coenzyme A dehydratase and (E)-2,3 enoyl-coenzyme A reductase involved in acyl-coenzyme A elongation in etiolated leek seedlings

(R,S)-[1-¹⁴C]3-Hydroxy eicosanoyl-coenzyme A (CoA) has been chemically synthesized to study the 3-hydroxy acyl-CoA dehydratase involved in the acyl-CoA elongase of etiolated leek (Allium porrum L.) seedling microsomes. 3-Hydroxy eicosanoyl-CoA (3-OH C20:0-CoA) dehydration led to the formation of (E)-2,3 eicosanoyl-CoA, which has been characterized. Our kinetic studies have determined the optimal conditions of the dehydration and also resolved the stereospecificity requirement of the dehydratase for (R)-3-OH C20:0-CoA. Isotopic dilution experiments showed that 3-hydroxy acyl-CoA dehydratase had a marked preference for (R)-3-OH C20:0-CoA. Moreover, the very-long-chain synthesis using (R)-3-OH C20:0-CoA isomer and [2-¹⁴C]malonyl-CoA was higher than that using the (S) isomer, whatever the malonyl-CoA and the 3-OH C20:0-CoA concentrations. We have also used [1-¹⁴C]3-OH C20:0-CoA to investigate the reductant requirement of the enoyl-CoA reductase of the acyl-CoA elongase complex. In the presence of NADPH, [1-¹⁴C]3-OH C20:0-CoA conversion was stimulated. Aside from the product of dehydration, i.e. (E)-2,3 eicosanoyl-CoA, we detected eicosanoyl-CoA resulting from the reduction of (E)-2,3 eicosanoyl-CoA. When we replaced NADPH with NADH, the eicosanoyl-CoA was 8- to 10-fold less abundant. Finally, in the presence of malonyl-CoA and NADPH or NADH, [1-¹⁴C]3-OH C20:0-CoA led to the synthesis of very-long-chain fatty acids. This synthesis was measured using [1-¹⁴C]3-OH C20:0-CoA and malonyl-CoA or (E)-2,3 eicosanoyl-CoA and [2-¹⁴C]malonyl-CoA. In both conditions and in the presence of NADPH, the acyl-CoA elongation activity was about 60 nmol mg⁻¹ h⁻¹, which is the highest ever reported for a plant system.     

Anaerobic Mineralization of Toluene by Enriched Sediments with Quinones and Humus as Terminal Electron Acceptors

The anaerobic microbial oxidation of toluene to CO₂ coupled to humus respiration was demonstrated by use of enriched anaerobic sediments from the Amsterdam petroleum harbor (APH) and the Rhine River. Both highly purified soil humic acids (HPSHA) and the humic quinone moiety model compound anthraquinone-2,6-disulfonate (AQDS) were utilized as terminal electron acceptors. After 2 weeks of incubation, 50 and 85% of added uniformly labeled [¹³C]toluene were recovered as ¹³CO₂ in HPSHA- and AQDS-supplemented APH sediment enrichment cultures, respectively; negligible recovery occurred in unsupplemented cultures. The conversion of [¹³C]toluene agreed with the high level of recovery of electrons as reduced humus or as anthrahydroquinone-2,6-disulfonate. APH sediment was also able to use nitrate and amorphous manganese dioxide as terminal electron acceptors to support the anaerobic biodegradation of toluene. The addition of substoichiometric amounts of humic acids to bioassay reaction mixtures containing amorphous ferric oxyhydroxide as a terminal electron acceptor led to more than 65% conversion of toluene (1 mM) after 11 weeks of incubation, a result which paralleled the partial recovery of electron equivalents as acid-extractable Fe(II). Negligible conversion of toluene and reduction of Fe(III) occurred in these bioassay reaction mixtures when humic acids were omitted. The present study provides clear quantitative evidence for the mineralization of an aromatic hydrocarbon by humus-respiring microorganisms. The results indicate that humic substances may significantly contribute to the intrinsic bioremediation of anaerobic sites contaminated with priority pollutants by serving as terminal electron acceptors.     

Effects of Eliminating Pyruvate Node Pathways and of Coexpression of Heterogeneous Carboxylation Enzymes on Succinate Production by Enterobacter aerogenes

Lowering the pH in bacterium-based succinate fermentation is considered a feasible approach to reduce total production costs. Newly isolated Enterobacter aerogenes strain AJ110637, a rapid carbon source assimilator under weakly acidic (pH 5.0) conditions, was selected as a platform for succinate production. Our previous work showed that the ΔadhE/PCK strain, developed from AJ110637 with inactivated ethanol dehydrogenase and introduced Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PCK), generated succinate as a major product of anaerobic mixed-acid fermentation from glucose under weakly acidic conditions (pH <6.2). To further improve the production of succinate by the ΔadhE/PCK strain, metabolically engineered strains were designed based on the elimination of pathways that produced undesirable products and the introduction of two carboxylation pathways from phosphoenolpyruvate and pyruvate to oxaloacetate. The highest production of succinate was observed with strain ES04/PCK+PYC, which had inactivated ethanol, lactate, acetate, and 2,3-butanediol pathways and coexpressed PCK and Corynebacterium glutamicum pyruvate carboxylase (PYC). This strain produced succinate from glucose with over 70% yield (gram per gram) without any measurable formation of ethanol, lactate, or 2,3-butanediol under weakly acidic conditions. The impact of lowering the pH from 7.0 to 5.5 on succinate production in this strain was evaluated under pH-controlled batch culture conditions and showed that the lower pH decreased the succinate titer but increased its yield. These findings can be applied to identify additional engineering targets to increase succinate production.     

Degradation of Phthalate and Di-(2-Ethylhexyl)phthalate by Indigenous and Inoculated Microorganisms in Sludge-Amended Soil

The metabolism of phthalic acid (PA) and di-(2-ethylhexyl)phthalate (DEHP) in sludge-amended agricultural soil was studied with radiotracer techniques. The initial rates of metabolism of PA and DEHP (4.1 nmol/g [dry weight]) were estimated to be 731.8 and 25.6 pmol/g (dry weight) per day, respectively. Indigenous microorganisms assimilated 28 and 17% of the carbon in [¹⁴C]PA and [¹⁴C]DEHP, respectively, into microbial biomass. The rates of DEHP metabolism were much greater in sludge assays without soil than in assays with sludge-amended soil. Mineralization of [¹⁴C]DEHP to ¹⁴CO₂ increased fourfold after inoculation of sludge and soil samples with DEHP-degrading strain SDE 2. The elevated mineralization potential was maintained for more than 27 days. Experiments performed with strain SDE 2 suggested that the bioavailability and mineralization of DEHP decreased substantially in the presence of soil and sludge components. The microorganisms metabolizing PA and DEHP in sludge and sludge-amended soil were characterized by substrate-specific radiolabelling, followed by analysis of ¹⁴C-labelled phospholipid ester-linked fatty acids (¹⁴C-PLFAs). This assay provided a radioactive fingerprint of the organisms actively metabolizing [¹⁴C]PA and [¹⁴C]DEHP. The ¹⁴C-PLFA fingerprints showed that organisms with different PLFA compositions metabolized PA and DEHP in sludge-amended soil. In contrast, microorganisms with comparable ¹⁴C-PLFA fingerprints were found to dominate DEHP metabolism in sludge and sludge-amended soil. Our results suggested that indigenous sludge microorganisms dominated DEHP degradation in sludge-amended soil. Mineralization of DEHP and PA followed complex kinetics that could not be described by simple first-order equations. The initial mineralization activity was described by an exponential function; this was followed by a second phase that was described best by a fractional power function. In the initial phase, the half times for PA and DEHP in sludge-amended soil were 2 and 58 days, respectively. In the late phase of incubation, the apparent half times for PA and DEHP increased to 15 and 147 days, respectively. In the second phase (after more than 28 days), the half time for DEHP was 2.9 times longer in sludge-amended soil assays than in sludge assays without soil. Experiments with radiolabelled DEHP degraders suggested that a significant fraction of the ¹⁴CO₂ produced in long-term degradation assays may have originated from turnover of labelled microbial biomass rather than mineralization of [¹⁴C]PA or [¹⁴C]DEHP. It was estimated that a significant amount of DEHP with poor biodegradability and extractability remains in sludge-amended soil for extended periods of time despite the presence of microorganisms capable of degrading the compound (e.g., more than 40% of the DEHP added is not mineralized after 1 year).     

Successive Mineralization and Detoxification of Benzo[a]pyrene by the White Rot Fungus Bjerkandera sp. Strain BOS55 and Indigenous Microflora

White rot fungi can oxidize high-molecular-weight polycyclic aromatic hydrocarbons (PAH) rapidly to polar metabolites, but only limited mineralization takes place. The objectives of this study were to determine if the polar metabolites can be readily mineralized by indigenous microflora from several inoculum sources, such as activated sludge, forest soils, and PAH-adapted sediment sludge, and to determine if such metabolites have decreased mutagenicity compared to the mutagenicity of the parent PAH. ¹⁴C-radiolabeled benzo[a]pyrene was subjected to oxidation by the white rot fungus Bjerkandera sp. strain BOS55. After 15 days, up to 8.5% of the [¹⁴C]benzo[a]pyrene was recovered as ¹⁴CO₂ in fungal cultures, up to 73% was recovered as water-soluble metabolites, and only 4% remained soluble in dibutyl ether. Thin-layer chromatography analysis revealed that many polar fluorescent metabolites accumulated. Addition of indigenous microflora to fungal cultures with oxidized benzo[a]pyrene on day 15 resulted in an initially rapid increase in the level of ¹⁴CO₂ recovery to a maximal value of 34% by the end of the experiments (>150 days), and the level of water-soluble label decreased to 16% of the initial level. In fungal cultures not inoculated with microflora, the level of ¹⁴CO₂ recovery increased to 13.5%, while the level of recovery of water-soluble metabolites remained as high as 61%. No large differences in ¹⁴CO₂ production were observed with several inocula, showing that some polar metabolites of fungal benzo[a]pyrene oxidation were readily degraded by indigenous microorganisms, while other metabolites were not. Of the inocula tested, only PAH-adapted sediment sludge was capable of directly mineralizing intact benzo[a]pyrene, albeit at a lower rate and to a lesser extent than the mineralization observed after combined treatment with white rot fungi and indigenous microflora. Fungal oxidation of benzo[a]pyrene resulted in rapid and almost complete elimination of its high mutagenic potential, as observed in the Salmonella typhimurium revertant test performed with strains TA100 and TA98. Moreover, no direct mutagenic metabolite could be detected during fungal oxidation. The remaining weak mutagenic activity of fungal cultures containing benzo[a]pyrene metabolites towards strain TA98 was further decreased by subsequent incubations with indigenous microflora.