Comparison of recovery of anaerobic bacteria using the Anoxomat, anaerobic chamber, and GasPak jar systems

The Anoxomat system provides an automated evacuation-replacement technique to create an anaerobic or microaerophilic environment in a jar. We evaluated the Anoxomat system for the growth of obligate anaerobes and for the recovery of anaerobic organisms from clinical specimens, and compared its performance to that of an anaerobic chamber and the GasPak System. Of the 54 stock strains tested, the Anoxomat, the chamber, and the GasPak recovered 95%, 95% and 93% at 24 h, respectively. On 29 occasions (51%), the colonies on the Anoxomat plates were slightly larger than those in the chamber and on 17 (30%) occasions larger than the colonies on the GasPak jar plates. At 48 h, the Anoxomat, the chamber, and the GasPak recovered 93.5%, 94.4% and 88.9%, respectively; of 108 anaerobes isolated from 31 clinical specimens. Methylene blue indicators became decolorized (average of 10 tests) within 2 h inside the Anoxomat jars, 2 h 10 min inside the anaerobic chamber, and 2 h 30 min inside the GasPak jars.     

A simplified method for the cultivation of extreme anaerobic Archaea based on the use of sodium sulfite as reducing agent

The extreme sensitivity of many Archaea to oxygen is a major obstacle for their cultivation in the laboratory and the development of archaeal genetic exchange systems. The technique of Balch and Wolfe (1976) is suitable for the cultivation of anaerobic Archaea but involves time-consuming procedures such as the use of air locks and glove boxes. We describe here a procedure for the cultivation of anaerobic Archaea that is more convenient and faster and allows the preparation of liquid media without the use of an anaerobic chamber. When the reducing agent sodium sulfide (Na₂S) was replaced by sodium sulfite (Na₂SO₃), anaerobic media could be prepared without protection from oxygen outside an anaerobic chamber. Exchange of the headspace of serum bottles by appropriate gases was sufficient to maintain anaerobic conditions in the culture media. Organisms that were unable to utilize sulfite as a source for cellular sulfur were supplemented with hydrogen sulfide. H₂S was simply added to the headspace of serum bottles by a syringe. The use of H₂S as a source for sulfur minimized the precipitation of cations by sulfide. Representatives of 12 genera of anaerobic Archaea studied here were able to grow in media prepared by this procedure. For the extremely oxygen-sensitive organism Methanococcus thermolithotrophicus, we show that plates could be prepared outside an anaerobic chamber when sulfite was used as reducing agent. The application of this method may faciliate the cultivation and handling of extreme anaerobic Archaea considerably. Received: January 4, 2000 / Accepted: April 5, 2000     

The anaerobic biosynthesis of vitamin B12

Vitamin B12 (cobalamin) is a cobalt-containing modified tetrapyrrole that is an essential nutrient for higher animals. Its biosynthesis is restricted to certain bacteria and requires approximately 30 enzymatic steps for its complete de novo construction. Remarkably, two distinct biosynthetic pathways exist, which are termed the aerobic and anaerobic routes. The anaerobic pathway has yet to be fully characterized due to the inherent instability of its oxygen-sensitive intermediates. Bacillus megaterium, a bacterium previously used for the commercial production of cobalamin, has a complete anaerobic pathway and this organism is now being used to investigate the anaerobic B12 pathway through the application of recent advances in recombinant protein production. The present paper provides a summary of recent findings in the anaerobic pathway and future perspectives.     

Wireless bioreactor for anaerobic cultivation of bacteria

Anaerobic cultivation methods of bacteria are indispensable in microbiology. One methodology is to cultivate the microbes in anaerobic enclosure with oxygen-adosrbing chemicals. Here, we report an electronic extension of such strategy for facultative anaerobic bacteria. The technique is based a bioreactor with entire operation including turbidity measurement, fluidic mixing, and gas delivery in an anaerobic enclosure. Wireless data transmission is employed and the anaerobic condition is achieved with gas pack. Although the technique is not meant to completely replace the anaerobic chamber for strict anaerobic bacteria, it provides a convenient way to bypass the cumbersome operation in anaerobic chamber for facultative anaerobic bacteria. Such a cultivation strategy is demonstrated with Escherichia coli with different carbon sources and hydrogen as energy source.     

Unusual dehydrations in anaerobic bacteria

Abstract In amino acid fermenting anaerobic bacteria a set of unusual dehydratases is found which use 2-hydroxyacyl-CoA, 4-hydroxybutyryl-CoA or 5-hydroxyvaleryl-CoA as substrates. The extremely oxygen-sensitive 2-hydroxyacyl-CoA dehydratases catalysing the elimination of water from ( R )-lactyl-CoA to acryloyl-CoA or from ( R )-2-hydroxyglutaryl-CoA to glutaconyl-CoA contain iron-sulfur clusters as well as riboflavin and require additional activation by ATP. The dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA is catalysed by a moderately oxygen-sensitive enzyme also containing an iron-sulfur cluster and FAD. In all these reactions a non-activated C-H-bond at C₃ has to be cleaved by mechanisms not yet elucidated. The dehydration of 5-hydroxyvaleryl-CoA to 4-pentenoyl-CoA, however, has been characterised as a redox process mediated by enzyme-bound FAD. Finally, an iron-sulfur cluster-containing but pyridoxal-phosphate-independent l -serine dehydratase is described.     

Carbon and hydrogen isotopic fractionation during anaerobic biodegradation of benzene

Compound-specific isotope analysis has the potential to distinguish physical from biological attenuation processes in the subsurface. In this study, carbon and hydrogen isotopic fractionation effects during biodegradation of benzene under anaerobic conditions with different terminal-electron-accepting processes are reported for the first time. Different enrichment factors (epsilon ) for carbon (range of -1.9 to -3.6 per thousand ) and hydrogen (range of -29 to -79 per thousand ) fractionation were observed during biodegradation of benzene under nitrate-reducing, sulfate-reducing, and methanogenic conditions. These differences are not related to differences in initial biomass or in rates of biodegradation. Carbon isotopic enrichment factors for anaerobic benzene biodegradation in this study are comparable to those previously published for aerobic benzene biodegradation. In contrast, hydrogen enrichment factors determined for anaerobic benzene biodegradation are significantly larger than those previously published for benzene biodegradation under aerobic conditions. A fundamental difference in the previously proposed initial step of aerobic versus proposed anaerobic biodegradation pathways may account for these differences in hydrogen isotopic fractionation. Potentially, C-H bond breakage in the initial step of the anaerobic benzene biodegradation pathway may account for the large fractionation observed compared to that in aerobic benzene biodegradation. Despite some differences in reported enrichment factors between cultures with different terminal-electron-accepting processes, carbon and hydrogen isotope analysis has the potential to provide direct evidence of anaerobic biodegradation of benzene in the field.     

Purification from goat antiserum of immunoglobulins against G6PD from rabbits]

DEAE Affi-Gel Blue (Bio-Rad) provides an efficient and rapid fractionation of human serum proteins by a single chromatographic step. When goat serum is applied to the matrix and chromatography is performed following the procedure utilized for the human serum proteins, the elution pattern changes and the Ig purification is not satisfactory. We achieved a better Ig purification from goat serum by the following improved procedure. We performed first an AS-40 fractionation followed by extensive dialysis in 50 mM Na-citrate pH 5.7. The sample was then loaded onto a P11 column equilibrated in the same buffer. The fraction eluted at Vo contained total IgG and the other serum proteins, except beta-globulins which were eluted with 0.24 M phosphate. Peak 1 concentrated and dialyzed in 20 mM phosphate buffer pH 8 was then applied to a DEAE Affi-Gel Blue column, equilibrated in the same buffer. Two protein peaks were eluted from this column and electrophoretically characterized as: peak 1, containing a pure Ig fraction (70% yield), peak 2 with albumin and other contaminating serum proteins. When goat antiserum is obtained against a specific protein, our technique may be suitably employed to purify polyclonal antibodies for immunoprecipitation studies.     

Purification and characterization of an anabolic fumarate reductase from Methanobacterium thermoautotrophicum

An oxygen-sensitive fumarate reductase has been purified from the cytosol fraction of the cells of the archaebacterium Methanobacterium thermoautotrophicum. A major portion of the purification was performed inside an anaerobic chamber, employing reducing agents to maintain low redox potentials. The apparent molecular weight of the native enzyme is 78,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a minimal subunit molecular weight of about 20,000. Iodoacetamide (1 mM) and copper chloride (5 mM) caused significant loss in the enzyme activity. The optimum temperature for the enzymatic activity was 75 degrees C. The pH optimum was found to be 7.0. The fumarate reductase had an apparent Km of 0.20 mM for fumarate. Purified enzyme was colorless; spectroscopic studies indicated the absence of flavins as a cofactor. The spectral data, however, suggested the presence of an unknown cofactor tightly bound to the enzyme. Fumarate reductase is involved in the anabolic rather than the catabolic metabolism of M. thermoautotrophicum.     

Purification and characterization of an anabolic fumarate reductase from Methanobacterium thermoautotrophicum.

An oxygen-sensitive fumarate reductase has been purified from the cytosol fraction of the cells of the archaebacterium Methanobacterium thermoautotrophicum. A major portion of the purification was performed inside an anaerobic chamber, employing reducing agents to maintain low redox potentials. The apparent molecular weight of the native enzyme is 78,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a minimal subunit molecular weight of about 20,000. Iodoacetamide (1 mM) and copper chloride (5 mM) caused significant loss in the enzyme activity. The optimum temperature for the enzymatic activity was 75 degrees C. The pH optimum was found to be 7.0. The fumarate reductase had an apparent Km of 0.20 mM for fumarate. Purified enzyme was colorless; spectroscopic studies indicated the absence of flavins as a cofactor. The spectral data, however, suggested the presence of an unknown cofactor tightly bound to the enzyme. Fumarate reductase is involved in the anabolic rather than the catabolic metabolism of M. thermoautotrophicum.     

Ether-cleaving enzyme and diol dehydratase involved in anaerobic polyethylene glycol degradation by a new Acetobacterium sp.

A strictly anaerobic, homoacetogenic bacterium was enriched and isolated from anoxic sewage sludge with polyethylene glycol (PEG) 1000 as sole source of carbon and energy, and was assigned to the genus Acetobacterium on the basis of morphological and physiological properties. The new isolate fermented ethylene glycol and PEG's with molecular masses of 106 to 1000 to acetate and small amounts of ethanol. The PEG-degrading activity was not destroyed by proteinase K treatment of whole cells. In cell-free extracts, a diol dehydratase and a PEG-degrading (ether-cleaving) enzyme activity were detected which both formed acetaldehyde as reaction product. The diol dehydratase enzyme was oxygen-sensitive and was stimulated 10–14 fold by added adenosylcobalamine. This enzyme was found mainly in the cytoplasmic fraction (65%) and to some extent (35%) in the membrane fraction. The ether-cleaving enzyme activity reacted with PEG's of molecular masses of 106 to more than 20000. The enzyme was measurable optimally in buffers of high ionic strength (4.0), was extremely oxygen-sensitive, and was inhibited by various corrinoids (adenosylcobalamine, cyanocobalamine, hydroxocobalamine, methylcobalamine). This enzyme was found exclusively in the cytoplasmic fraction. It is concluded that PEG is degraded by this bacterium inside the cytoplasm by a hydroxyl shift reaction, analogous to a diol dehydratase reaction, to form an unstable hemiacetal intermediate. The name polyethylene glycol acetaldehyde lyase is suggested for the responsible enzyme.Abbreviations EG ethylene glycol - DiEG diethylene glycol - TriEG triethylene glycol - TeEG tetraethylene glycol - PEG polyethylene glycol (molecular mass indicated)     

Evaluation of size exclusion chromatography (SEC) for the characterization of extracellular polymeric substances (EPS) in anaerobic granular sludges

The extracellular polymeric substances (EPS) extracted from three granular and one flocculant anaerobic sludges were characterised by size exclusion chromatography (SEC) using two serially linked chromatographic columns in order to obtain more detailed chromatograms. A Superdex peptide 10/300 GL (0.1–7 kDa) and Superdex 20010/300GL (10–600 kDa) from Amersham Biosciences were used in series with a mobile phase at pH 7 with an ionic strength of 0.223 M (phosphate buffer 50 mM and NaCl 150 mM). A part of the EPS molecules displays hydrophobic and/or ionic interactions with the column packing. Interactions could be modified by changing the mobile phase ionic strength or polarity (addition of acetonitrile). The detection wavelength (210 or 280 nm) affects strongly the EPS chromatogram. For a sludge originating from the same type of biofilms (i.e., anaerobic granules), the differences in EPS fingerprints are mainly due to differences in the absorbance of the chromatographic peaks, linked to EPS molecules content and composition. The EPS fingerprint changes significantly when the EPS originate from another type of anaerobic sludges. In addition, EPS fingerprints were affected by the extraction method used (centrifugation only; heat and centrifugation or cationic exchange resin and centrifugation). This phenomenon was observed mainly for the largest and smallest molecules and molecules which display interactions with column packing.     

Separation of asymmetrical hybrid containing hemoglobin F by anaerobic anion-exchange high-performance liquid chromatography

Asymmetrical hybrid hemoglobins formed from mixtures of oxyhemoglobins S and F and A and F were separated by high-performance liquid chromatography on a 4.6 X 250 mm wide-pore polyethyleneimine-silica gel column under anaerobic conditions. The resulting HPLC chromatogram showed three peaks, with the middle peak representing the hybrid hemoglobin. The areas of these three peaks were quantified and the amount of hybrids formed was less than that predicted theoretically. We found that the deviation was due to the equilibrium constant of the FS hybrid hemoglobin differing from that of the parent hemoglobins. In this report, we introduce the anaerobic recycle ion-exchange HPLC method to determine the rate of dissociation of AS and FS hybrid hemoglobins at constant pH buffer conditions. The results obtained by this method demonstrate that FS hybrid hemoglobin is more unstable than AS hybrid hemoglobin. The free energy of association for asymmetrical hybrids containing hemoglobin F is approximately 0.6 Kcal/mol greater than that of the symmetrical parent hemoglobins.     

Large scale preparation and crystallization of neuron-specific enolase

A simple method has been developed for the large scale purification of neuron-specific enolase [EC 4.2.1.11]. The method consists of ammonium sulfate fractionation of brain extract, and two subsequent column chromatography steps on DEAE Sephadex A-50. The chromatography was performed on a short (25 cm height) and thick (8.5 cm inside diameter) column unit that was specially devised for the large scale preparation. The purified enolase was crystallized in 0.05 M imidazole-HCl buffer containing 1.6 M ammonium sulfate (pH 6.39), with a yield of 0.9 g/kg of bovine brain tissue.     

Iron hydrogenases and the evolution of anaerobic eukaryotes

Hydrogenases, oxygen-sensitive enzymes that can make hydrogen gas, are key to the function of hydrogen-producing organelles (hydrogenosomes), which occur in anaerobic protozoa scattered throughout the eukaryotic tree. Hydrogenases also play a central role in the hydrogen and syntrophic hypotheses for eukaryogenesis. Here, we show that sequences related to iron-only hydrogenases ([Fe] hydrogenases) are more widely distributed among eukaryotes than reports of hydrogen production have suggested. Genes encoding small proteins which contain conserved structural features unique to [Fe] hydrogenases were identified on all well-surveyed aerobic eukaryote genomes. Longer sequences encoding [Fe] hydrogenases also occur in the anaerobic eukaryotes Entamoeba histolytica and Spironucleus barkhanus, both of which lack hydrogenosomes. We also identified a new [Fe] hydrogenase sequence from Trichomonas vaginalis, bringing the total of [Fe] hydrogenases reported for this organism to three, all of which may function within its hydrogenosomes. Phylogenetic analysis and hypothesis testing using likelihood ratio tests and parametric bootstrapping suggest that the [Fe] hydrogenases in anaerobic eukaryotes are not monophyletic. Iron-only hydrogenases from Entamoeba, Spironucleus, and Trichomonas are plausibly monophyletic, consistent with the hypothesis that a gene for [Fe] hydrogenase was already present on the genome of the common, perhaps also anaerobic, ancestor of these phylogenetically distinct eukaryotes. Trees where the [Fe] hydrogenase from the hydrogenosomal ciliate Nyctotherus was constrained to be monophyletic with the other eukaryote sequences were rejected using a likelihood ratio test of monophyly. In most analyses, the Nyctotherus sequence formed a sister group with a [Fe] hydrogenase on the genome of the eubacterium Desulfovibrio vulgaris. Thus, it is possible that Nyctotherus obtained its hydrogenosomal [Fe] hydrogenase from a different source from Trichomonas for its hydrogenosomes. We find no support for the hypothesis that components of the Nyctotherus [Fe] hydrogenase fusion protein derive from the mitochondrial respiratory chain.     

A stable organic free radical in anaerobic benzylsuccinate synthase of Azoarcus sp. strain T

The novel enzyme benzylsuccinate synthase initiates anaerobic toluene metabolism by catalyzing the addition of toluene to fumarate, forming benzylsuccinate. Based primarily on its sequence similarity to the glycyl radical enzymes, pyruvate formate-lyase and anaerobic ribonucleotide reductase, benzylsuccinate synthase was speculated to be a glycyl radical enzyme. In this report we use EPR spectroscopy to demonstrate for the first time that active benzylsuccinate synthase from the denitrifying bacterium Azoarcus sp. strain T harbors an oxygen-sensitive stable organic free radical. The EPR signal of the radical was centered at g = 2.0021 and was characterized by a major 2-fold splitting of about 1.5 millitesla. The strong similarities between the EPR signal of the benzylsuccinate synthase radical and that of the glycyl radicals of pyruvate formate-lyase and anaerobic ribonucleotide reductase provide evidence that the benzylsuccinate synthase radical is located on a glycine residue, presumably glycine 828 in Azoarcus sp. strain T benzylsuccinate synthase.     

Aromatizing cyclohexa-1,5-diene-1-carbonyl-coenzyme A oxidase. Characterization and its role in anaerobic aromatic metabolism

Benzoyl-CoA reductases (BCRs) are key enzymes of anaerobic aromatic metabolism in facultatively anaerobic bacteria. The highly oxygen-sensitive enzymes catalyze the ATP-dependent reductive de-aromatization of the substrate, yielding cyclohexa-1,5-diene-1-carbonyl-CoA (1,5-dienoyl-CoA). In extracts from anaerobically grown denitrifying Thauera aromatica, we detected a benzoate-induced, benzoyl-CoA-forming, 1,5-dienoyl-CoA:acceptor oxidoreductase activity. This activity co-purified with BCR but could be partially separated from it by hydroxyapatite chromatography. After activity staining on native gels, a monomeric protein with a subunit molecular weight of M(r) 76,000 was identified. Mass spectrometric analysis of tryptic digests identified peptides from NADH oxidases/2,4-dienoyl-CoA reductases/"old yellow" enzymes. The UV-visible spectrum of the enriched enzyme suggested the presence of flavin and Fe/S-cofactors, and it was bleached upon the addition of 1,5-dienoyl-CoA. The enzyme had a high affinity for dioxygen as electron acceptor (K(m) = 10 microm) and therefore is referred to as 1,5-dienoyl-CoA oxidase (DCO). The likely product formed from dioxygen reduction was H(2)O. DCO was highly specific for 1,5-dienoyl-CoA (K(m) = 27 microm). The initial rate of DCO followed a Nernst curve with half-maximal activity at +10 mV. We propose that DCO provides protection for the extremely oxygen-sensitive BCR enzyme when the bacterium degrades aromatic compounds at the edge of steep oxygen gradients. The redox-dependent switch in DCO guarantees that DCO is only active during oxidative stress and circumvents futile de-aromatization/re-aromatization reactions catalyzed by BCR and DCO.     

Anaerobic multiphasic gel electrophoresis of the molybdoproteins in extracts of Clostridium pasteurianum

Simple procedures using multiphasic buffer systems for anaerobic electrophoresis have been devised to identify oxygen-labile metalloproteins. An anaerobic slab gel apparatus was developed with cooling and design for anaerobic conditions. Included is a procedure to remove sample wells after stacking proteins in a crude extract, to prevent streaking (background) caused by continuous leakage of "nonstacked protein" from the sample wells. Identification of eleven Mo zones in extracts of Clostridium pasteurianum demonstrates the usefulness of the technique in identifying radiolabeled oxygen-labile proteins in cell-free crude lysates.     

Anaerobic column chromatography in the presence of detergents and its application to a bacterial HCN-producing enzyme

An easy method to deoxygenize protein or detergent containing buffer solutions by stirring and sparge flushing with an inert gas is described. The deoxygenation of buffers and the establishment of anaerobic conditions in a column chromatography system was followed continuously with an oxygen electrode at the outlet. All polyethylene tubes were placed inside stainless steel tubes and the interspace flushed with nitrogen. A peristalic pump with silicone rubber tubing, mounted in a small plastic box, was likewise flushed with nitrogen. By this method a very unstable cyanide-producing enzyme preparation, solubilized with Triton X-100 from a Pseudomonas species, could be fractionated on a phenyl Sepharose CL-4B column without loss of activity.     

A new method for liquid nitrogen storage of phototrophic bacteria under anaerobic conditions

A simple, effective and economical method for the long-term preservation of bacteria in liquid nitrogen under anaerobic conditions is described. As a case example anaerobic photosynthetic bacteria were successfully preserved. Gas tight small screw-cap glass ampoules with butyl rubber septa were used for freezing the specimen anaerobically. During experimental manipulations no anaerobic chamber or glove boxes were required. All teste cultures yielded high recoveries after repeated thawing and during storage. After freezing, survival recoveries of Rhodospirillaceae range from 70–100%, whereas with strict anaerobic strains of Chlorobiaceae and Chromatiaceae a maximum loss of 1–2 log₁₀ counts was observed. No further loss in viability occurred after 1–2 years of storage.The small size of the ampoules and the use of single ampoule for 15–20 repeated retrievals proved economical with respect to storage space and costs.The system is compact and suitable for the preservation of anaerobic phototropic bacteria and other fragile anaerobic microorganisms.     

Reductive dechlorination of hexachlorocyclohexane (HCH) isomers in soil under anaerobic conditions

The biological anaerobic reductive dechlorination of beta-hexachlorocyclohexane under methanogenic conditions was tested in a number of contaminated soil samples from two locations in the Netherlands. Soils from a heavily polluted location showed rapid dechlorination of beta-hexachlorocyclohexane to benzene and chlorobenzene with lactate as electron donor. Soils from an adjacent slightly polluted location did not show substantial dechlorination of beta-hexachlorocyclohexane within 4 months. A heavily polluted sample was selected to optimise the dechlorination. All tested hexachlorocyclohexane isomers (alpha-, beta-, gamma-, and delta-), either added separately or simultaneously, were dechlorinated in this soil sample. The most rapid dechlorination was observed at a temperature of 30 degrees C. Dechlorination of beta-hexachlorocyclohexane was observed with acetate, propionate, lactate, methanol, H2, yeast extract and landfill leachate as electron donors. In a soil percolation column, packed with a selected heavily polluted soil sample, the presence of 10 mM sulphate in the influent led to simultaneous dechlorination of beta-hexachlorocyclohexane and sulphate reduction. When the column was fed with 10 mM nitrate instead of sulphate, dechlorination ceased immediately. After omitting nitrate from the influent, dechlorination activity recovered in about 1 month. Also in a separate column, the addition of nitrate from the start of the experiment did not result in dechlorination of beta-HCH. The significance of these experiments for in situ bioremediation of polluted soils is discussed.