Physiology of Geobacter metallireducens under excess and limitation of electron donors. Part II. Mimicking environmental conditions during cultivation in retentostats

The strict anaerobe Geobacter metallireducens was cultivated in retentostats under acetate and acetate plus benzoate limitation in the presence of Fe(III) citrate in order to investigate its physiology under close to natural conditions. Growth rates below 0.003 h⁻¹ were achieved in the course of cultivation. A nano-liquid chromatography–tandem mass spectrometry-based proteomic approach (nano-LC–MS/MS) with subsequent label-free quantification was performed on proteins extracted from cells sampled at different time points during retentostat cultivation. Proteins detected at low (0.002 h⁻¹) and high (0.06 h⁻¹) growth rates were compared between corresponding growth conditions (acetate or acetate plus benzoate). Carbon limitation significantly increased the abundances of several catabolic proteins involved in the degradation of substrates not present in the medium (ethanol, butyrate, fatty acids, and aromatic compounds). Growth rate-specific physiology was reflected in the changed abundances of energy-, chemotaxis-, oxidative stress-, and transport-related proteins. Mimicking natural conditions by extremely slow bacterial growth allowed to show how G. metallireducens optimized its physiology in order to survive in its natural habitats, since it was prepared to consume several carbon sources simultaneously and to withstand various environmental stresses.     

Methane Fermentation of Ferulate and Benzoate: Anaerobic Degradation Pathways

The anaerobic biodegradation of ferulate and benzoate in stabilized methanogenic consortia was examined in detail. Up to 99% of the ferulate and 98% of the benzoate were converted to carbon dioxide and methane. Methanogenesis was inhibited with 2-bromoethanesulfonic acid, which reduced the gas production and enhanced the buildup of intermediates. Use of high-performance liquid chromatography and two gas chromatographic procedures yielded identification of the following compounds: caffeate, p-hydroxycinnamate, cinnamate, phenylpropionate, phenylacetate, benzoate, and toluene during ferulate degradation; and benzene, cyclohexane, methylcyclohexane, cyclohexanecarboxylate, cyclohexanone, 1-methylcyclohexanone, pimelate, adipate, succinate, lactate, heptanoate, caproate, isocaproate, valerate, butyrate, isobutyrate, propionate, and acetate during the degradation of either benzoate or ferulate. Based on the identification of the above compounds, more complete reductive pathways for ferulate and benzoate are proposed.     

Isolation and physiological characterization of Syntrophus buswellii strain GA from a syntrophic benzoate-degrading,strictly anaerobic coculture

A stable, syntrophic benzoate-degrading bacterial consortium was enriched from sewage sludge. It oxidized benzoate or 3-phenylpropionate to acetate, H₂ and CO₂. As hydrogen scavengers Methanospirillum hungatei and Desulfovibrio sp. were present. The benzoate-degrading bacteria of this syntrophic culture and of Syntrophus buswelli were able to grow with benzoate/crotonate or crotonate alone in the absence of a hydrogen-utilizing partner organism. If crotonate was the only substrate, acetate and butyrate were produced, while during growth on benzoate or 3-phenylpropionate crotonate served as a reducible co-substrate and was exclusively converted to butyrate. In the presence of crotonate interspecies hydrogen transfer was not necessary as a hydrogen sink. The benzoate degrader was isolated as a pure culture with crotonate as the only carbon source. The pure culture could also grow with benzoate/crotonate or 3-phenylpropionate/crotonate. The effect of high concentrations of crotonate and of acetate or butyrate on growth of the benzoate degrader was investigated. The benzoate degrader was compared with S. buswellii for its morphology, physiology and DNA base composition. Except for the fact that S. buswellii was also able to grow on cinnamate, no differences between the two organisms were detected. The isolate is named S. buswelli, strain GA.     

Identification of volatiles from differently aged red clover (Trifolium pratense) root extracts and behavioural responses of clover root borer (Hylastinus obscurus) (Marsham) (Coleoptera: Scolytidae) to them

Root extracts from 1.5 and 2.5-year-old red clover (Trifolium pratense) were obtained using supercritical fluid extraction (SFE). GC–MS analysis and Kovats indices allowed identification of the volatile compounds as butyl acetate, E-2-hexenal, α-pinene, benzaldehyde, 6-methyl-5-hepten-2-one, limonene, acetophenone, methyl benzoate, nonanal, octanoic acid and decanal.     

Anaerobic Benzene Oxidation via Phenol in Geobacter metallireducens

Anaerobic activation of benzene is expected to represent a novel biochemistry of environmental significance. Therefore, benzene metabolism was investigated in Geobacter metallireducens, the only genetically tractable organism known to anaerobically degrade benzene. Trace amounts (<0.5 μM) of phenol accumulated in cultures of Geobacter metallireducens anaerobically oxidizing benzene to carbon dioxide with the reduction of Fe(III). Phenol was not detected in cell-free controls or in Fe(II)- and benzene-containing cultures of Geobacter sulfurreducens, a Geobacter species that cannot metabolize benzene. The phenol produced in G. metallireducens cultures was labeled with ¹⁸O during growth in H₂¹⁸O, as expected for anaerobic conversion of benzene to phenol. Analysis of whole-genome gene expression patterns indicated that genes for phenol metabolism were upregulated during growth on benzene but that genes for benzoate or toluene metabolism were not, further suggesting that phenol was an intermediate in benzene metabolism. Deletion of the genes for PpsA or PpcB, subunits of two enzymes specifically required for the metabolism of phenol, removed the capacity for benzene metabolism. These results demonstrate that benzene hydroxylation to phenol is an alternative to carboxylation for anaerobic benzene activation and suggest that this may be an important metabolic route for benzene removal in petroleum-contaminated groundwaters, in which Geobacter species are considered to play an important role in anaerobic benzene degradation.     

Effects of inhibition and repression on the utilization of substrates by heterogeneous bacterial communities

This investigation attempts to evaluate to what extent enzyme inhibition and repression by metabolites, indigenous to the cell, are significant phenomena in natural microbial communities. Three case histories of the kinetics of substrate utilization and growth in multisubstrate media by heterogeneous bacterial populations are presented: (i) concurrent substrate utilization and growth on both substrates simultaneously (glucose plus benzoate); (ii) sequential substrate elimination accompanied by diauxic growth as a result of inhibition of enzyme activity (glucose plus galactose); (iii) sequential substrate utilization accompanied by diauxic growth caused by repression of enzyme formation (glucose plus l-phenylalanine, benzoate plus l-phenylalanine). It is shown that enzyme inhibition was observed in two-substrate media as well as in multisubstrate media and was maintained at low substrate concentrations (few milligrams per liter). A special attempt has been made to maintain the diversity of the experimental microbial population during the adaptation and enrichment period. All substrates were determined with sensitive analytical methods specific for the individual substrates. The results obtained confirm that catabolite repression and the resulting sequential substrate utilization are observed in heterogeneous bacterial populations.     

Effect of ethanol, acetate, and phenol on toluene degradation activity and tod-lux expression in Pseudomonas putida TOD102: evaluation of the metabolic flux dilution model

The reporter strain Pseudomonas putida TOD102 (with a tod-lux fusion) was used in chemostat experiments with binary substrate mixtures to investigate the effect of potentially occurring cosubstrates on toluene degradation activity. Although toluene was simultaneously utilized with other cosubstrates, its metabolic flux (defined as the toluene utilization rate per cell) decreased with increasing influent concentrations of ethanol, acetate, or phenol. Three inhibitory mechanisms were considered to explain these trends: (1) repression of the tod gene (coding for toluene dioxygenase) by acetate and ethanol, which was quantified by a decrease in specific bioluminescence; (2) competitive inhibition of toluene dioxygenase by phenol; and (3) metabolic flux dilution (MFD) by all three cosubstrates. Based on experimental observations, MFD was modeled without any fitting parameters by assuming that the metabolic flux of a substrate in a mixture is proportional to its relative availability (expressed as a fraction of the influent total organic carbon). Thus, increasing concentrations of alternative carbon sources "dilute" the metabolic flux of toluene without necessarily repressing tod, as observed with phenol (a known tod inducer). For all cosubstrates, the MFD model slightly overpredicted the measured toluene metabolic flux. Incorporating catabolite repression (for experiments with acetate or ethanol) or competitive inhibition (for experiments with phenol) with independently obtained parameters resulted in more accurate fits of the observed decrease in toluene metabolic flux with increasing cosubstrate concentration. These results imply that alternative carbon sources (including inducers) are likely to hinder toluene utilization per unit cell, and that these effects can be accurately predicted with simple mathematical models.     

Link between microbial composition and carbon substrate-uptake preferences in a PHA-storing community

The microbial community of a fermented molasses-fed sequencing batch reactor (SBR) operated under feast and famine conditions for production of polyhydroxyalkanoates (PHAs) was identified and quantified through a 16 S rRNA gene clone library and fluorescence in situ hybridization (FISH). The microbial enrichment was found to be composed of PHA-storing populations (84% of the microbial community), comprising members of the genera Azoarcus, Thauera and Paracoccus. The dominant PHA-storing populations ensured the high functional stability of the system (characterized by high PHA-storage efficiency, up to 60% PHA content). The fermented molasses contained primarily acetate, propionate, butyrate and valerate. The substrate preferences were determined by microautoradiography-FISH and differences in the substrate-uptake capabilities for the various probe-defined populations were found. The results showed that in the presence of multiple substrates, microbial populations specialized in different substrates were selected, thereby co-existing in the SBR by adapting to different niches. Azoarcus and Thauera, primarily consumed acetate and butyrate, respectively. Paracoccus consumed a broader range of substrates and had a higher cell-specific substrate uptake. The relative species composition and their substrate specialization were reflected in the substrate removal rates of different volatile fatty acids in the SBR reactor.     

Substrate interactions during the biodegradation of BTEX and THF mixtures by Pseudomonas oleovorans DT4

The efficient tetrahydrofuran (THF)-degrading bacterium, Pseudomonas oleovorans DT4 was used to investigate the substrate interactions during the aerobic biotransformation of THF and BTEX mixtures. Benzene and toluene could be utilized as growth substrates by DT4, whereas cometabolism of m-xylene, p-xylene and ethylbenzene occurred with THF. In binary mixtures, THF degradation was delayed by xylene, ethylbenzene, toluene and benzene in descending order of inhibitory effects. Conversely, benzene (or toluene) degradation was greatly enhanced by THF leading to a higher degradation rate of 39.68 mg/(h g dry weight) and a shorter complete degradation time about 21 h, possibly because THF acted as an “energy generator”. Additionally, the induction experiments suggested that BTEX and THF degradation was initiated by independent and inducible enzymes. The transient intermediate hydroquinone was detected in benzene biodegradation with THF while catechol in the process without THF, suggesting that P. oleovorans DT4 possessed two distinguished benzene pathways.     

Regulation of acetate and acetyl-CoA converting enzymes during growth on acetate and/or glucose in the halophilic archaeon Haloarcula marismortui

Haloarcula marismortui formed acetate during aerobic growth on glucose and utilized acetate as growth substrate. On glucose/acetate mixtures diauxic growth was observed with glucose as the preferred substrate. Regulation of enzyme activities, related to glucose and acetate metabolism was analyzed. It was found that both glucose dehydrogenase (GDH) and ADP-forming acetyl-CoA synthetase (ACD) were upregulated during periods of glucose consumption and acetate formation, whereas both AMP-forming acetyl-CoA synthetase (ACS) and malate synthase (MS) were downregulated. Conversely, upregulation of ACS and MS and downregulation of ACD and GDH were observed during periods of acetate consumption. MS was also upregulated during growth on peptides in the absence of acetate. From the data we conclude that a glucose-inducible ACD catalyzes acetate formation whereas acetate activation is catalyzed by an acetate-inducible ACS; both ACS and MS are apparently induced by acetate and repressed by glucose.     

Studies on the substrate range of Clostridium kluyveri; the use of propanol and succinate

The metabolism of Clostridium kluyveri has been extensively studied, but the range of substrates C. kluyveri can use for growth has not been fully explored. The use of propanol and succinate as growth substrates were established. C. kluyveri grows on acetate with propanol replacing ethanol. The principle carbon containing products were propionate, valerate, butyrate and hexanoate with traces of heptanoate. When grown with ethanol and succinate the principle carbon-containing products were acetate, butyrate and hexanoate. Hexanol was found as a product when incubated with ethanol and succinate 4-hydroxybutyrate or 3-butenoate. 5-Hexenoate was also a product of 3-butenoate and ethanol metabolism. The splitting of succinate into 2 acetates was indicated with ethanol providing the necessary reducing equivalents. Hydrogen was also found as a source of reducing equivalents but could not replace ethanol. A mechanism of succinate metabolism to acetate was proposed which accounts for growth yield, energetics considerations, carbon balances, production of side products and intermediates.Contribution No. 3619     

Fermentation of cinnamate by a mesophilic strict anaerobe,Acetivibrio multivorans sp. nov.

A cinnamate-fermenting bacterium (strain PeC1) was isolated in pure culture from anoxic sludge of an oil refinery wastewater treatment facility. It was a mesophilic gram-negative non-sporing actively motile rod. It did not reduce nitrate, sulfte, or other sulfur compounds as electron acceptors. It fermented cinnamate to 3-phenylpropionate, benzoate, and acetate; crotonate to butyrate and acetate; pyruvate to lactate and acetate; acetoin to ethanol and acetate; and carbohydrates to ethanol, formate, and acetate. The DNA base ratio of the strain was 44 mol% guanine plus cytosine. It is described as a new species of the genus Acetivibrio, A. multivorans sp. nov.     

Fluxes of carbon, phosphorylation, and redox intermediates during growth of saccharomyces cerevisiae on different carbon sources

In the present work we develop a method for estimating anabolic fluxes when yeast are growing on various carbon substrates (glucose, glycerol, lactate, pyruvate, acetate, or ethanol) in minimal medium. Fluxes through the central amphibolic pathways were calculated from the product of the total required amount of a specified carbon intermediate times the growth rate. The required amount of each carbon intermediate was estimated from the experimentally determined macromolecular composition of cells grown in each carbon source and the monomer composition of macromolecules.Substrates sharing most metabolic pathways such as ethanol and acetate, despite changes in the macromolecular composition, namely carbohydrate content (34% +/- 1 and 21% +/- 3, respectively), did not show large variations in the overall fluxes through the main amphibolic pathways. For instance, in order to supply anabolic precursors to sustain growth rates in the range of 0.16/h to 0.205/h, similar large fluxes through Acetyl CoA synthase were required by acetate (4.2 mmol/hr g dw) or ethanol (5.2 mmol/h g dw).The V(max) activities of key enzymes of the main amphibolic pathways measured in permeabilized yeast cells allowed to confirm, qualitatively, the operation of those pathways for all substrates and were consistent on most substrates with the estimated fluxes required to sustain growth.When ATP produced from oxidation of the NADH synthesized along with the key intermediary metabolites was taken into account, higher Y(ATP) (max) values (36 with respect to 24 g dw/mol ATP) were obtained for glucose. The same result was obtained for glycerol, ethanol, and acetate. A yield index (YI) was defined as the ratio of the theoretically estimated substrate flux required to sustain a given growth rate over the experimentally measured flux of substrate consumption. Comparison of Yl between growth on various carbon sources led us to conclude that ethanol (Yl = 0.84), acetate (Yl = 0.77), and lactate (Yl = 0.77) displayed the most efficient use of substrate for biomass production. For the other substrates, the Yl decayed in the following order: pyruvate > glycerol > glucose.An improvement of the quantitative understanding of yeast metabolism, energetics, and physiology is provided by the present analysis. The methodology proposed can be applied to other eukaryotic organisms of known chemical composition. (c) 1995 John Wiley & Sons, Inc.     

Regulation of pathways degrading aromatic substrates in Pseudomonas putida. Enzymic response to binary mixtures of substrates

1. Induction constants (K(ind)) and repression constants (K(rep)), which are a measure of the affinity of the inducers or repressors for the induction systems, were measured for mandelate, benzoate and p-hydroxybenzoate in Pseudomonas putida. 2. From these results, the enzymic response of the organism to media containing pairs of these substrates was predicted. Nitrogen-limited chemostats, operated at high growth rates, were used to investigate these predictions in cells grown first on one aromatic substrate with the second added later. 3. In general, the values of K(ind) and K(rep) predicted quite accurately the response to substrate mixtures. Thus, in the presence of mandelate and either benzoate or p-hydroxybenzoate, the enzymes of mandelate metabolism were repressed almost completely, and the bacteria were fully induced for the alternative substrate (benzoate or p-hydroxybenzoate), which was preferentially utilized for growth. When benzoate and p-hydroxybenzoate were the two substrates in the mixture, the enzymes for metabolism of the latter were strongly repressed and growth took place mainly on benzoate. 4. The enzymic response to mixed substrates did not result in the metabolism of the better growth substrate, but in the substrate requiring the synthesis of fewer enzymes. Thus benzoate is used in preference to mandelate although the latter supports a faster growth rate. It is nevertheless considered that, with our present knowledge of the natural habitat of the organism, it is impossible to decide whether protein economy or growth rate was the factor determining the evolution of this control system.     

己酸菌LⅡ己酸发酵过程中生理及代谢特点

在了解了产己酸细菌ＬＩＩ的发酵最适条件的基础上,进一步对己酸发酵过程中的生理及代谢特性作了较详细的分析。结果表明,在己酸发酵过程中除产生正常代谢产物己酸外,还产生丁酸、乙酸、戊酸等其它酸类和甲烷,氢气和ＣＯ_２等气体。用１—^１３Ｃ－乙酸钠和未标记的乙醇作为底物发酵,用色质联用仪（ＧＣ／ＭＳ）分析,结果表明：在己酸合成中碳的来源为乙醇和乙酸,中间代谢产物为丁酸。     

Growth performance and pathway flux determine substrate preference of Alcaligenes eutrophus during growth on acetate plus aromatic compound mixtures

During batch growth of Alcaligenes eutrophus on various aromatic compounds in the presence of acetate, several distinct behaviour patterns were observed. The utilization of substrates of the meta pathway (phenol or p-cresol) was inhibited by acetate. When the aromatic was a substrate of the p-hydroxybenzoate branch of the ortho pathway, growth was mixotrophic, i.e. both substrates were consumed simultaneously. For the substrates of the gentisate pathway or the benzoate branch of the ortho pathway, substrate preference was governed by growth performance. Aromatic compounds enabling growth rate and yields higher than those obtained on acetate alone (i.e. benzoate, benzaldehyde, m-hydroxybenzoate and gentisate) inhibited acetate utilization, while acetate was the substrate consumed preferentially in mixtures containing aromatic compounds supporting only slow growth (i.e. benzoyl formate and 4-fluorobenzoate). Received: 18 April 1996 / Received revision: 9 July 1996 / Accepted: 15 July 1996     

Regulation of methylotrophic and heterotrophic metabolism in Arthrobacter P1. Growth on mixtures of methylamine and acetate in batch and continuous cultures

Incubations of Arthrobacter P1 in batch culture in media with mixtures of acetate and methylamine resulted in sequential utilization of the two carbon substrates, but not in diauxic growth. Irrespective of the way cells were pregrown, acetate was the preferred substrate and subsequent studies showed that this is due to the fact that acetate is a strong inhibitor of the methylamine transport system and amine oxidase in Arthrobacter P1. An analysis of enzyme activities in cell-free extracts showed that synthesis of amine oxidase occurred already in the first growth phase with acetate, whereas rapid synthesis of hexulose phosphate synthase was only observed once methylamine utilization started. It is therefore concluded that in Arthrobacter P1 the synthesis of the enzymes specific for methylamine oxidation is not regulated co-ordinately with those involved in formaldehyde fixation, but induced sequentially by methylamine and formaldehyde, respectively.During growth of Arthrobacter P1 on the same mixture in carbon- and energy source-limited continuous cultures both substrates were used simultaneously and completely at dilution rates below the _max on either of these substrates. Addition of methylamine, in concentrations as low as 0.5 mM, to the medium reservoir of an acetate-limited continuous culture (D=0.10 h^-1) already resulted in synthesis of both amine oxidase and hexulose phosphate synthase. In the reverse experiment, addition of acetate to the medium reservoir of a methylamine-limited continuous culture (D=0.10 h^-1), acetate was initially only used as an energy source. Synthesis of the glyoxylate cycle enzymes, however, did occur at acetate concentration in the feed above 7.5–10 mM. This indicates that at acetate concentrations below 10 mM the metabolism of the C₁ substrate methylamine is able to cause a complete repression of the synthesis of the enzymes involved in carbon assimilation from the C₂ substrate acetate.Abbreviations HPS Hexulose phosphate synthase - MS mineral salts - RuMP ribulose monophosphate