Degradation of cyanobacterial biomass in anoxic tidal-flat sediments: a microcosm study of metabolic processes and community changes

To follow the anaerobic degradation of organic matter in tidal-flat sediments, a stimulation experiment with ¹³C-labeled Spirulina biomass (130 mg per 21 g sediment slurry) was conducted over a period of 24 days. A combination of microcalorimetry to record process kinetics, chemical analyses of fermentation products and RNA-based stable-isotope probing (SIP) to follow community changes was applied. Different degradation phases could be identified by microcalorimetry: Within 2 days, heat output reached its maximum (55 μW), while primary fermentation products were formed (in μmol) as follows: acetate 440, ethanol 195, butyrate 128, propionate 112, H₂ 127 and smaller amounts of valerate, propanol and butanol. Sulfate was depleted within 7 days. Thereafter, methanogenesis was observed and secondary fermentation proceeded. H₂ and alcohols disappeared completely, whereas fatty acids decreased in concentration. Three main degraders were identified by RNA-based SIP and denaturant gradient gel electrophoresis. After 12 h, two phylotypes clearly enriched in ¹³C: (i) Psychrilyobacter atlanticus, a fermenter known to produce hydrogen and acetate and (ii) bacteria distantly related to Propionigenium. A Cytophaga-related bacterium was highly abundant after day 3. Sulfate reduction appeared to be performed by incompletely oxidizing species, as only sulfate-reducing bacteria related to Desulfovibrio were labeled as long as sulfate was available.     

Multiple growth inhibition of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in 1,3-propanediol fermentation

The inhibition of substrate and product on the growth of Klebsiella pneumoniae in anaerobic and aerobic batch fermentation for the production of 1,3-propanediol was studied. The cells under anaerobic conditions had a higher maximum specific growth rate of 0.19 h^–1 and lower tolerance to 110 g glycerol l^–1, compared to the maximum specific growth rate of 0.17 h^–1 and tolerance to 133 g glycerol l^–1 under aerobic conditions. Acetate was the main inhibitory metabolite during the fermentation under anaerobic conditions, with lactate and ethanol the next most inhibitory. The critical concentrations of acetate, lactate and ethanol were assessed to be 15, 19, 26 g l^–1, respectively. However, cells grown under aerobic conditions were more resistant to acetate and lactate but less resistant to ethanol. The critical concentrations of acetate, lactate and ethanol were assessed to be 24, 26, and 17 g l^–1, respectivelyRevisions requested 8 september; Revisions received 2 November 2004     

Understanding aerobic/anaerobic metabolism in Caldibacillus debilis through a comparison with model organisms

Caldibacillus debilis GB1 is a facultative anaerobe isolated from a thermophilic aero-tolerant cellulolytic enrichment culture. There is a lack of representative proteomes of facultative anaerobic thermophilic Bacillaceae, exploring aerobic/anaerobic expression. The C. debilis GB1 genome was sequenced and annotated, and the proteome characterized under aerobic and anaerobic conditions while grown on cellobiose. The draft sequence of C. debilis GB1 contains a 3,340,752 bp chromosome and a 5,386 bp plasmid distributed over 49 contigs. Two-dimensional liquid chromatography mass spectrometry/mass spectrometry was used with Isobaric Tags for Relative and Absolute Quantification (iTRAQ) to compare protein expression profiles, focusing on energy production and conversion pathways. Under aerobic conditions, proteins in glycolysis and pyruvate fermentation pathways were down-regulated. Simultaneously, proteins within the tricarboxylic acid cycle, pyruvate dehydrogenase, the electron transport chain, and oxygen scavenging pathways showed increased amounts. Under anaerobic conditions, protein levels in fermentation pathways were consistent with the generated end-products: formate, acetate, ethanol, lactate, and CO₂. Under aerobic conditions CO₂ and acetate production was consistent with incomplete respiration. Through a direct comparison with gene expression profiles from Escherichia coli, we show that global regulation of core metabolism pathways is similar in thermophilic and mesophilic facultative anaerobes of the Phylum Proteobacteria and Firmicutes.     

Aerobic denitrification: a controversy revived

During studies on the denitrifying mixotroph, Thiosphaera pantotropha, it has been found that this organism is capable of simultaneously utilizing nitrate and oxygen as terminal electron acceptors in respiration. This phenomenon, termed aerobic denitrification, has been found in cultures maintained at dissolved oxygen concentrations up to 90% of air saturation.The evidence for aerobic denitrification was obtained from a number of independant experiments. Denitrifying enzymes were present even in organisms growing aerobically without nitrate. Aerobic yields on acetate were higher (8.1 g protein/mol) without than with (6.0 g protein/mol) nitrate, while the anaerobic yield with nitrate was even lower (4 g protein/mol). The maximum specific growth rate of Tsa. pantotropha was higher (0.34 h^-1) in the presence of both oxygen (>80% air saturation) and nitrate than in similar cultures not supplied with nitrate (0.27 h^-1), indicating that the rate of electron transport to oxygen was limiting. This was confirmed by oxygen uptake experiments which showed that although the rate of respiration on acetate was not affected by nitrate, the total oxygen uptake was reduced in its presence. The original oxygen uptake could be restored by the addition of denitrification inhibitors.Dedicated to Professor Dr. H.-G. Schlegel on the occasion of his 60th birthday     

Oxygen and temperature-dependent structural and redox changes in a novel cytochrome c(4) from the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina

A novel cytochrome c₄, the first of this type in purple phototrophic bacteria has been discovered in Thiocapsa roseopersicina. The fact that cytochrome c₄ has been found in an anaerobic organism puts in question the up hereto suggested role of cytochromes c₄ in the aerobic respiratory metabolism. The structure of cytochrome c₄ was studied under both aerobic and anaerobic conditions, using differential scanning calorimetry and a combination of redox potentiostatic measurements with CD and UV-Vis absorption techniques. Cytochrome c₄ maintained its functional capability at high temperature (60 °C) if it was kept under anaerobic conditions. With increasing temperature under aerobic conditions, however, there are dramatic conformational changes in the protein and coordination changes on the iron side. Presumably oxygen binds to the iron at the position left vacant by the methionine and facilitates conformational changes with low reversibility.     

Identification of a novel acetate-utilizing bacterium belonging to Synergistes group 4 in anaerobic digester sludge

Major acetate-utilizing bacterial and archaeal populations in methanogenic anaerobic digester sludge were identified and quantified by radioisotope- and stable-isotope-based functional analyses, microautoradiography-fluorescence in situ hybridization (MAR-FISH) and stable-isotope probing of 16S rRNA (RNA-SIP) that can directly link 16S rRNA phylogeny with in situ metabolic function. First, MAR-FISH with ¹⁴C-acetate indicated the significant utilization of acetate by only two major groups, unidentified bacterial cells and Methanosaeta-like filamentous archaeal cells, in the digester sludge. To identify the acetate-utilizing unidentified bacteria, RNA-SIP was conducted with ¹³C₆-glucose and ¹³C₃-propionate as sole carbon source, which were followed by phylogenetic analysis of 16S rRNA. We found that bacteria belonging to Synergistes group 4 were commonly detected in both 16S rRNA clone libraries derived from the sludge incubated with ¹³C-glucose and ¹³C-propionate. To confirm that this bacterial group can utilize acetate, specific FISH probe targeting for Synergistes group 4 was newly designed and applied to the sludge incubated with ¹⁴C-acetate for MAR-FISH. The MAR-FISH result showed that bacteria belonging to Synergistes group 4 significantly took up acetate and their active population size was comparable to that of Methanosaeta in this sludge. In addition, as bacteria belonging to Synergistes group 4 had high K_m for acetate and maximum utilization rate, they are more competitive for acetate over Methanosaeta at high acetate concentrations (2.5–10 m). To our knowledge, it is the first time to report the acetate-utilizing activity of uncultured bacteria belonging to Synergistes group 4 and its competitive significance to acetoclastic methanogen, Methanosaeta.     

The effects of the oxygen transfer coefficient and substrate concentration on the xylose fermentation by Debaryomyces hansenii

Relevant production of xylitol by Debaryomyces hansenii requires semiaerobic conditions since in aerobic conditions the accumulated reduced adenine dinucleotide coenzyme is fully reoxidized leading to the conversion of xylitol into xylulose. For oxygen transfer coefficient values from 0.24 to 1.88 min^-1, in shake flasks experiments, biomass formation increased proportionally to the aeration rate as shown in the oxygen transfer coefficient and xylose concentration isoresponse contours. The metabolic products under study, xylitol and ethanol were mainly growth associated. However, for oxygen transfer coefficient above 0.5 min^-1 higher initial xylose concentration stimulated the rate of production of xylitol. This fact was less evident for ethanol production. The direct relationship between increased biomass and products formation rate, indicated that the experimental domain in respect to the aeration rate was below the threshold level before the decreasing in metabolic production rates reported in literature for xylose-fermenting yeasts. The fact that ethanol was produced, albeit in low levels, throughout the experimental design indicated that the semiaerobic conditions were always attained. Debaryomyces hansenii showed to be an important xylitol producer exhibiting a xylitol/ethanol ratio above four and a carbon conversion of 54% for xylitol.Abbreviations K_La oxygen transfer coefficient - DO(T) dissolved oxygen (tension) - OUR oxygen uptake rate - NAD(H) oxidised (reduced) nicotinamide adenine dinucleotide - NADP(H) oxidised (reduced) nicotinamide adenine dinucleotide phosphate - CRC catabolic reduction charge - C oxygen concentration in the culture medium - C^* oxygen concentration at saturation conditions - Y_i response from experiment i - parameters of the polynomial model - x experimental factor level (coded units) - R² coefficient of multiple determination - t time     

High-light induced singlet oxygen formation in cytochrome b6f complex from Bryopsis corticulans as detected by EPR spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy was used to detect the light-induced formation of singlet oxygen (¹O₂*) in the intact and the Rieske-depleted cytochrome b₆f complexes (Cyt b₆f) from Bryopsis corticulans, as well as in the isolated Rieske Fe–S protein. It is shown that, under white-light illumination and aerobic conditions, chlorophyll a (Chl a) bound in the intact Cyt b₆f can be bleached by light-induced ¹O₂*, and that the ¹O₂* production can be promoted by D₂O or scavenged by extraneous antioxidants such as l-histidine, ascorbate, β-carotene and glutathione. Under similar experimental conditions, ¹O₂* was also detected in the Rieske-depleted Cyt b₆f complex, but not in the isolated Rieske Fe–S protein. The results prove that Chl a cofactor, rather than Rieske Fe–S protein, is the specific site of ¹O₂* formation, a conclusion which draws further support from the generation of ¹O₂* with selective excitation of Chl a using monocolor red light.     

Differential mechanism of light-induced and oxygen-dependent restoration of the high-potential form of cytochrome b559 in Tris-treated Photosystem II membranes

The effect of illumination and molecular oxygen on the redox and the redox potential changes of cytochrome b₅₅₉ (cyt b₅₅₉) has been studied in Tris-treated spinach photosystem II (PSII) membranes. It has been demonstrated that the illumination of Tris-treated PSII membranes induced the conversion of the intermediate-potential (IP) to the reduced high-potential (HP^Fe2+) form of cyt b₅₅₉, whereas the removal of molecular oxygen resulted in the conversion of the IP form to the oxidized high-potential (HP^Fe3+) form of cyt b₅₅₉. Light-induced conversion of cyt b₅₅₉ from the IP to the HP form was completely inhibited above pH 8 or by the modification of histidine ligand that prevents its protonation. Interestingly, no effect of high pH or histidine modification was observed during the conversion of the IP to the HP form of cyt b₅₅₉ after the removal of molecular oxygen. These results indicate that conversion from the IP to the HP form of cyt b₅₅₉ proceeds via different mechanisms. Under illumination, conversion of the IP to the HP form of cyt b₅₅₉ depends primarily on the protonation of the histidine residue, whereas under anaerobic conditions, the conversion of the IP to the HP form of cyt b₅₅₉ is driven by higher hydrophobicity of the environment around the heme iron resulting from the absence of molecular oxygen.     

Deciphering the Genome of Polyphosphate Accumulating Actinobacterium Microlunatus phosphovorus

Polyphosphate accumulating organisms (PAOs) belong mostly to Proteobacteria and Actinobacteria and are quite divergent. Under aerobic conditions, they accumulate intracellular polyphosphate (polyP), while they typically synthesize polyhydroxyalkanoates (PHAs) under anaerobic conditions. Many ecological, physiological, and genomic analyses have been performed with proteobacterial PAOs, but few with actinobacterial PAOs. In this study, the whole genome sequence of an actinobacterial PAO, Microlunatus phosphovorus NM-1^T (NBRC 101784^T), was determined. The number of genes for polyP metabolism was greater in M. phosphovorus than in other actinobacteria; it possesses genes for four polyP kinases (ppks), two polyP-dependent glucokinases (ppgks), and three phosphate transporters (pits). In contrast, it harbours only a single ppx gene for exopolyphosphatase, although two copies of ppx are generally present in other actinobacteria. Furthermore, M. phosphovorus lacks the phaABC genes for PHA synthesis and the actP gene encoding an acetate/H⁺ symporter, both of which play crucial roles in anaerobic PHA accumulation in proteobacterial PAOs. Thus, while the general features of M. phosphovorus regarding aerobic polyP accumulation are similar to those of proteobacterial PAOs, its anaerobic polyP use and PHA synthesis appear to be different.     

Inorganic carbon fixation by chemosynthetic ectosymbionts and nutritional transfers to the hydrothermal vent host-shrimp Rimicaris exoculata

The shrimp Rimicaris exoculata dominates several hydrothermal vent ecosystems of the Mid-Atlantic Ridge and is thought to be a primary consumer harbouring a chemoautotrophic bacterial community in its gill chamber. The aim of the present study was to test current hypotheses concerning the epibiont''s chemoautotrophy, and the mutualistic character of this association. In-vivo experiments were carried out in a pressurised aquarium with isotope-labelled inorganic carbon (NaH¹³CO₃ and NaH¹⁴CO₃) in the presence of two different electron donors (Na₂S₂O₃ and Fe²⁺) and with radiolabelled organic compounds (¹⁴C-acetate and ³H-lysine) chosen as potential bacterial substrates and/or metabolic by-products in experiments mimicking transfer of small biomolecules from epibionts to host. The bacterial epibionts were found to assimilate inorganic carbon by chemoautotrophy, but many of them (thick filaments of epsilonproteobacteria) appeared versatile and able to switch between electron donors, including organic compounds (heterotrophic acetate and lysine uptake). At least some of them (thin filamentous gammaproteobacteria) also seem capable of internal energy storage that could supply chemosynthetic metabolism for hours under conditions of electron donor deprivation. As direct nutritional transfer from bacteria to host was detected, the association appears as true mutualism. Import of soluble bacterial products occurs by permeation across the gill chamber integument, rather than via the digestive tract. This first demonstration of such capabilities in a decapod crustacean supports the previously discarded hypothesis of transtegumental absorption of dissolved organic matter or carbon as a common nutritional pathway.     

Glucose Metabolism in Lactococcus lactis MG1363 under Different Aeration Conditions: Requirement of Acetate To Sustain Growth under Microaerobic Conditions

Lactococcus lactis subsp. lactis MG1363 was grown in batch cultures on a defined medium with glucose as the energy source under different aeration conditions, namely, anaerobic conditions, aerobic conditions, and microaerobic conditions with a dissolved oxygen tension of 5% (when saturation with air was used as the reference). The maximum specific growth rate was high (0.78 to 0.91 h⁻¹) under all aeration conditions but decreased with increasing aeration, and more than 90% of the glucose was converted to lactate. However, a shift in by-product formation was observed. Increasing aeration resulted in acetate, CO₂, and acetoin replacing formate and ethanol as end products. Under microaerobic conditions, growth came to a gradual halt, although more than 60% of the glucose was still left. A decline in growth was not observed during microaerobic cultivation when acetate was added to the medium. We hypothesize that the decline in growth was due to a lack of acetyl coenzyme A (acetyl-CoA) needed for fatty acid synthesis since acetyl-CoA can be synthesized from acetate by means of acetate kinase and phosphotransacetylase activities.     

Reductive dioxygen scavenging by flavo-diiron proteins of Clostridium acetobutylicum

Two flavo-diiron proteins (FDPs), FprA1 and FprA2, are up-regulated when the strictly anaerobic solvent producer, Clostridium acetobutylicum, is exposed to dioxygen. These two FDPs were purified following heterologous overexpression in Escherichia coli as N-terminal Strep-tag fusion proteins. The recombinant FprA1 and FprA2 were found to be homodimeric and homotetrameric, respectively, and both FDPs functioned as terminal components of NADH oxidases (NADH:O₂ oxidoreductases) when using C. acetobutylicum NADH:rubredoxin oxidoreductase (NROR) and rubredoxin (Rd) as electron transport intermediaries. Both FDPs catalyzed the four-electron reduction of molecular oxygen to water with similar specific activities. The results are consistent with these FDPs functioning as efficient scavengers of intracellular dioxygen under aerobic or microoxic growth conditions.     

Inhibition of nitric oxide production in LPS-stimulated RAW 264.7 cells by stem bark of Ulmus pumila L.

This study was designed to isolate and identify a potent inhibitory compound against nitric oxide (NO) production from the stem bark of Ulmus pumila L. Ethyl acetate fraction of hot water extract registered a higher level of total phenolics (756.93 mg GAE/g) and also showed strong DPPH (IC₅₀ at 5.6 μg/mL) and ABTS (TEAC value 0.9703) radical scavenging activities than other fractions. Crude extract and its fractions significantly decreased nitrite accumulation in LPS-stimulated RAW 264.7 cells indicating that they potentially inhibited the NO production in a concentration dependent manner. Based on higher inhibitory activity, the ethyl acetate fraction was subjected to Sephadex LH-20 column chromatography and yielded seven fractions and all these fractions registered appreciable levels of inhibitory activity on NO production. The most effective fraction F1 was further purified and subjected to ¹H, ¹³C-NMR and mass spectrometry analysis and the compound was identified as icariside E₄. The results suggest that the U. pumila extract and the isolated compound icariside E₄ effectively inhibited the NO production and may be useful in preventing inflammatory diseases mediated by excessive production of NO.     

Effect of hydrogen peroxide production and the Fenton reaction on membrane composition of Streptococcus pneumoniae

As part of its aerobic metabolism, Streptococcus pneumoniae generates high levels of H₂O₂ by pyruvate oxidase (SpxB), which can be further reduced to yield the damaging hydroxyl radicals via the Fenton reaction. A universal conserved adaptation response observed among bacteria is the adjustment of the membrane fatty acids to various growth conditions. The aim of the present study was to reveal the effect of endogenous reactive oxygen species (ROS) formation on membrane composition of S. pneumoniae. Blocking carbon aerobic metabolism, by growing the bacteria at anaerobic conditions or by the truncation of the spxB gene, resulted in a significant enhancement in fatty acid unsaturation, mainly cis-vaccenic acid. Moreover, reducing the level of OH^· by growing the bacteria at acidic pH, or in the presence of an OH^· scavenger (salicylate), resulted in increased fatty acid unsaturation, similar to that obtained under anaerobic conditions. RT-PCR results demonstrated that this change does not originate from a change in mRNA expression level of the fatty acid synthase II genes. We suggest that endogenous ROS play an important regulatory role in membrane adaptation, allowing the survival of this anaerobic organism at aerobic environments of the host.     

Development of an industrial ethanol-producing yeast strain for efficient utilization of cellobiose

The BGL1 gene, encoding β-glucosidase in Saccharomycopsis fibuligera, was intracellular, secreted or cell-wall associated expressed in an industrial strain of Saccharomyces cerevisiae. The obtained recombinant strains were studied under aerobic and anaerobic conditions. The results indicated that both the wild type and recombinant strain expressing intracellular β-glucosidase cannot grow in medium using cellobiose as sole carbon source. As for the recombinant EB1 expressing secreted enzyme and WB1 expressing cell-wall associated enzyme, the maximum specific growth rates (μ_max) could reach 0.03 and 0.05 h⁻¹ under anaerobic conditions, respectively. Meanwhile, the surface-engineered S. cerevisiae utilized 5.2 g cellobiose L⁻¹ and produced 2.3 g ethanol L⁻¹ in 48 h, while S. cerevisiae secreting β-glucosidase into culture broth used 3.6 g cellobiose L⁻¹ and produced 1.5 g ethanol L⁻¹ over the same period, but no-full depletion of cellobiose were observed for both the used recombinant strains. The results suggest that S. cerevisiae used in industrial ethanol production is deficient in cellobiose transporter. However, when β-glucoside permease and β-glucosidase were co-expressed in this strain, it could uptake cellobiose and showed higher growth rate (0.11 h⁻¹) on cellobiose.     

Heterotrophic nitrification and aerobic denitrification inAlcaligenes faecalis strain TUD

Heterotrophic nitrification and aerobic and anaerobic denitrification byAlcaligenes faecalis strain TUD were studied in continuous cultures under various environmental conditions. Both nitrification and denitrification activities increased with the dilution rate. At dissolved oxygen concentrations above 46% air saturation, hydroxylamine, nitrite and nitrate accumulated, indicating that both the nitrification and denitrification were less efficient. The overall nitrification activity was, however, essentially unaffected by the oxygen concentration. The nitrification rate increased with increasing ammonia concentration, but was lower in the presence of nitrate or nitrite. When present, hydroxylamine, was nitrified preferentially. Relatively low concentrations of acetate caused substrate inhibition (K_I=109 M acetate). Denitrifying or assimilatory nitrate reductases were not detected, and the copper nitrite reductase, rather than cytochrome cd, was present. Thiosulphate (a potential inhibitor of heterotrophic nitrification) was oxidized byA. faecalis strain TUD, with a maximum oxygen uptake rate of 140–170nmol O₂·min^-1·mg prot^-1. Comparison of the behaviour ofA. faecalis TUD with that of other bacteria capable of heterotrophic nitrification and aerobic denitrification established that the response of these organisms to environmental parameters is not uniform. Similarities were found in their responses to dissolved oxygen concentrations, growth rate and ammonia concentration. However, they differed in their responses to externally supplied nitrite and nitrate.     

Identification of succinate exporter in Corynebacterium glutamicum and its physiological roles under anaerobic conditions

Corynebacterium glutamicum produces succinate from glucose via the reductive tricarboxylic acid cycle under microaerobic and anaerobic conditions. We identified a NCgl2130 gene of C. glutamicum as a novel succinate exporter that functions in succinate production, and designated sucE1. sucE1 expression levels were higher under microaerobic conditions than aerobic conditions, and overexpression or disruption of sucE1 respectively increased or decreased succinate productivity during fermentation. Under microaerobic conditions, the sucE1 disruptant sucE1Δ showed 30% less succinate productivity and a lower sugar-consumption rate than the parental strain. Under anaerobic conditions, succinate production by sucE1Δ ceased. The intracellular succinate and fructose-1,6-bisphosphate levels of sucE1Δ under microaerobic conditions were respectively 1.7-fold and 1.6-fold higher than those of the parental strain, suggesting that loss of SucE1 function caused a failure of succinate removal from the cells, leading to intracellular accumulation that inhibited upstream sugar metabolism. Homology and transmembrane helix searches identified SucE1 as a membrane protein belonging to the aspartate:alanine exchanger (AAE) family. Partially purified 6x-histidine-tagged SucE1 (SucE1-[His]₆) reconstituted in succinate-loaded liposomes clearly demonstrated counterflow and self-exchange activities for succinate. Together, these findings suggest that sucE1 encodes a novel succinate exporter that is induced under microaerobic conditions, and is important for succinate production under both microaerobic and anaerobic conditions.