Aerotolerance of strictly anaerobic microorganisms and factors of defense against oxidative stress: a review

Effects of aerobic conditions on strictly anaerobic microorganisms belonging to diverse taxa (clostridia, acetogenic bacteria, lactic acid bacteria, bacteroids, sulfate-reducing bacteria, and methanogenic archaea) and differing considerably in their oxygen resistance have been reviewed, with emphasis on the role of aerotolerance in the ecology of anaerobes. Consideration is given to components of nutritive media for anaerobe culturing, which decrease the toxic effects of oxygen and there by contribute significantly to maintenance and storage of industrial cultures of strictly anaerobic microorganisms. Physiological and biochemical factors are described, accounting for the relative resistance of many strict anaerobes to oxygen and products of incomplete reduction thereof. Specific attention is given to regulation of enzymes of antioxidative defense, operating in the cells of strict anaerobes under the conditions of oxidative stress caused by oxygen, superoxide anion, or hydrogen peroxide.     

Catalase and Superoxide Dismutase: Distribution, Properties, and Physiological Role in Cells of Strict Anaerobes

This review considers the distribution of the main enzymes of antioxidative defense, superoxide dismutase (SOD) and catalase, in various groups of strictly anaerobic microorganisms: bacteria of the genus Clostridium, Bacteroides, sulfate-reducing and acetogenic bacteria, methanogenic archaea, etc. Molecular and biochemical properties of purified Fe-containing SODs, cambialistic SODs, and heme catalases are presented. The physiological role and origin of the enzymes of antioxidative defense in strict anaerobes are discussed. Physiological responses (induction of SOD and catalase) to factors provoking oxidative stress in the cells of strict anaerobes able to maintain viability under aerobic conditions are also considered.     

Multiple and flexible roles of facultative anaerobic bacteria in microaerophilic oleate degradation

Anaerobic degradation of long-chain fatty acids (LCFA) involves syntrophic bacteria and methanogens, but facultative anaerobic bacteria (FAB) might have a relevant role as well. Here we investigated oleate degradation by a syntrophic synthetic co-culture of Syntrophomonas zehnderi (Sz) and Methanobacterium formicicum (Mf) and FAB (two oleate-degrading Pseudomonas spp. I1 + I2). Sz + Mf were first cultivated in a continuous bioreactor under strict anaerobic conditions. Thereafter, I1 + I2 were inoculated and microaerophilic conditions were provided. Methane and acetate were the main degradation products by Sz + Mf in anaerobiosis and by Sz + Mf + I1 + I2 in microaerophilic conditions. However, acetate production from oleate was higher in microaerophilic conditions (5% O₂) with the four microorganisms together (0.41 ± 0.07 mmol day⁻¹) than in anaerobiosis with Sz + Mf (0.23 ± 0.05 mmol day⁻¹). Oleate degradation in batch assays was faster by Sz + Mf + I1 + I2 (under microaerophilic conditions) than by Sz + Mf alone (under strict anaerobic conditions). I1 + I2 were able to grow with oleate and with intermediates of oleate degradation (hydrogen, acetate and formate). This work highlights the importance of FAB, particularly Pseudomonas sp., in anaerobic reactors treating oleate-based wastewater, because they accelerate oleate conversion to methane, by protecting strict anaerobes from oxygen toxicity and also by acting as alternative hydrogen/formate and acetate scavengers for LCFA-degrading anaerobes.     

THE USE OF MICROBIAL MEMBRANES TO ACHIEVE ANAEROBIOSIS

Several years ago, it was observed that sterile microbial membrane preparations stimulated recovery of certain radiation-injured bacteria. Later it was noted that these same preparations reduce dissolved oxygen to water in a variety of environments, including bacteriological media. This reduction of oxygen is an enzymatic process and is influenced by parameters such as temperature, pH, and the availability of specific oxidizable substrates. Oxygenreducing membrane preparations can be made from several different bacterial species. When added to liquid or solid bacteriological media, membrane preparations rapidly produce and maintain anaerobic conditions favorable for the growth of a wide variety of oxygen-sensitive microorganisms. When used with a specifically designed disposable dish, membrane preparations allow the development of colonies of many anaerobic microorganisms on the surface of agar without the use of anaerobic hoods or other devices. In addition to providing conditions suitable for the growth of anaerobes, membrane preparations stimulate recovery of heat and cold injured bacteria of several different genera including facultative organisms. These results are reminiscent of the early observations regarding the recovery of radiation-injured bacteria. In addition to their usefulness in microbiology, oxygen-reducing membrane preparations have the potential for protecting a wide variety of oxygen-sensitive organic compounds.     

Cultivation of Anaerobic and Facultatively Anaerobic Bacteria from Spacecraft-Associated Clean Rooms

In the course of this biodiversity study, the cultivable microbial community of European spacecraft-associated clean rooms and the Herschel Space Observatory located therein were analyzed during routine assembly operations. Here, we focused on microorganisms capable of growing without oxygen. Anaerobes play a significant role in planetary protection considerations since extraterrestrial environments like Mars probably do not provide enough oxygen for fully aerobic microbial growth. A broad assortment of anaerobic media was used in our cultivation strategies, which focused on microorganisms with special metabolic skills. The majority of the isolated strains grew on anaerobic, complex, nutrient-rich media. Autotrophic microorganisms or microbes capable of fixing nitrogen were also cultivated. A broad range of facultatively anaerobic bacteria was detected during this study and also, for the first time, some strictly anaerobic bacteria (Clostridium and Propionibacterium) were isolated from spacecraft-associated clean rooms. The multiassay cultivation approach was the basis for the detection of several bacteria that had not been cultivated from these special environments before and also led to the discovery of two novel microbial species of Pseudomonas and Paenibacillus.The major issue of planetary protection is to prevent the contamination of extraterrestrial environments by terrestrial biomolecules and life forms. Furthermore, reverse contamination of Earth by extraterrestrial material is also a fundamental concern (1). In order not to affect or even to confound future life detection missions on celestial bodies, which are of interest for their chemical and biological evolution, spacecraft are constructed in so-called clean rooms and are subject to severe cleaning processes and microbiological controls before launch (9). Therefore, these clean rooms are considered extreme environments for microorganisms (47).Detailed planetary protection protocols for missions to Mars were designed for the Viking missions, which were launched in 1975, and about 7,000 samples were taken from the two Viking spacecraft during prelaunch activities in order to determine the cultivable microbial load (37). Besides human-associated bacteria (pathogens and opportunistic pathogens), which were predominant among the microbes detected in these samples, aerobic spore-forming microorganisms (Bacillus) were found frequently on spacecraft and within the facilities.Spores are the resting states of bacteria and are often highly resistant to heat, desiccation, and other abiotic stresses. These multiresistance properties of such spore-forming microorganisms make them perfect candidates for surviving a space flight, and thus, the main focus of attention has been on them. Furthermore, only the detection of aerobic spore-forming bacteria is currently included in space agencies'' planetary protection protocols for the quantitative determination of microbial burden on spacecraft.The presence of extraordinarily (UV-) resistant spores in spacecraft facilities has been reported (31), but it also has been proven that vegetative microbial cells (e.g., Deinococcus radiodurans and Halobacterium sp. strain NRC-1) can resist very harsh conditions, such as extreme doses of (UV and ionizing) radiation and desiccation (8, 11). Recent culture-based and molecular studies have shown that the microbial diversity on spacecraft and within the clean rooms is extraordinarily high and does include extremotolerant bacteria and even archaea (25, 30).The atmospheres of most planets and bodies within the reach of human exploration contain only traces of oxygen (Mars contains 0.13%), probably not enough to support terrestrial aerobic life as we know it (26, 44). Even though Mars'' surface is highly oxidizing and radiation exposed, the Martian subsurface, as well as those of other planets and bodies (like, e.g., Titan), has been discussed as an anaerobic biotope for possible life (4, 40).Therefore, the lack of studies of the existence of anaerobically growing microorganisms in spacecraft-associated clean rooms is quite surprising. One possible reason for this discrepancy might be that the cultivation of anaerobes is challenging. Already in 1969, Hungate published a method for the cultivation of strictly anaerobic methanogenic Archaea (20). Although this technique has undergone a few simplifications during past decades, the cultivation of anaerobes requires specialized and expensive equipment (e.g., anaerobic glove boxes and gas stations), practical experience, and skills in specific methodology. Nevertheless, by the application of anaerobic cultivation strategies, many fascinating microorganisms—such as Nanoarchaeum equitans, the first representative of the new archaeal phylum Nanoarchaeota, or Thermotoga maritima, a hyperthermophilic bacterium growing at up to 90°C (17, 18)—have successfully been isolated from diverse and sometimes extreme biotopes.Generally, there are different types of anaerobic organisms. Facultative anaerobes (like Escherichia coli) are able to adapt their metabolism and can grow under conditions with or without oxygen but prefer aerobic conditions. Aerotolerant anaerobes do not need oxygen for their growth and show no preference, and strict anaerobes (e.g., methanogens) never require oxygen for their reproduction and metabolism. Even more, obligate (strict) anaerobes can be growth inhibited or even killed by oxygen.The presence of anaerobic microorganisms (enriched using the BD GasPak system) in surface samples from U.S. clean rooms has rarely been reported. Members of the facultatively anaerobic genera Paenibacillus and Staphylococcus have been isolated in the course of a study about extremotolerant microorganisms (25). During molecular surveys of U.S. clean rooms, the 16S rRNA genes from strictly anaerobic microorganisms, such as the spore-forming genus Clostridium, have already been detected (29). Nevertheless, the cultivation of these microbes has not yet been successful.With the ExoMars mission impending, the European Space Agency (ESA) is organizing and funding a biodiversity study of the ESA''s clean rooms and the spacecraft therein. The microbiology of these special environments is characterized in detail by a combination of standard procedures, new cultivation approaches, and molecular methods that shall illuminate the presence of planetary protection-relevant microorganisms in these facilities. At the date of sampling, all the clean rooms harbored the Herschel Space Observatory, a spacecraft to be launched together with the Planck satellite in spring 2009, as of this writing. Herschel will be fitted with the largest mirror ever built for a space mission (3.5 m in diameter), and its main goal will be the exploration of the cold universe, i.e., the formation and evolution of proto-galaxies (35). The Herschel Space Observatory does not demand planetary protection requirements, but all clean rooms were in a fully operating state during the construction work. This gave us the opportunity to sample the microbial diversity in these extreme environments without bioburden control but under strict contamination-controlled conditions, with respect to particulates and molecular contamination.This paper presents the results from our attempts to isolate anaerobic and facultatively anaerobic microorganisms from samples of spacecraft and surfaces in European spacecraft-associated clean rooms. For this purpose, we have successfully applied Hungate technology for anaerobic culturing and used an assortment of noncommercial media for the cultivation of a broad variety of microorganisms. Besides the capability of anaerobic growth, many of our isolates revealed special physiological capacities (e.g., nitrogen fixation and autotrophic metabolism) that might be relevant for further planetary protection considerations.     

Variability in survival of Pectinatus cerevisiiphilus, strictly anaerobic bacteria, under different oxygen conditions

Survival of Pectinatus cerevisiiphilus DSM 20466 in pure culture at variable temperatures under different oxygen concentrations was measured. Survival of P. cerevisiiphilus in co-culture with Saccharomyces cerevisiae under both saturated oxygen and brewing conditions was also studied. The survival of strictly anaerobic bacteria to oxygen seems to follow the classical laws of heat resistance. The D(oxy) values of P. cerevisiiphilus , calculated as a function of oxygen level, shows that the oxygen level is important for the survival duration of the bacteria. The temperature greatly influences the oxygen resistance of P. cerevisiiphilus, which increases when the temperature decreases. P. cerevisiiphilus resists better in co-culture than in pure culture under saturated oxygen conditions. Therefore, the oxygenation of the wort does not totally eliminate the risk of beer contamination by this bacterium. Under brewing conditions in co-culture at 8 degrees C, P. cerevisiiphilus grows slowly to reach a final cell concentration up to 10(6) cells/mL in beer, which is undrinkable. Pectinatus is a strictly anaerobic bacterium; however, it is resistant under certain oxygen conditions of incubation. This resistance is considerably higher in the presence of Saccharomyces cerevisiae .     

Isolation and characterization of enteric bacteria from the hindgut of Formosan termite

The Formosan subterranean termite, Coptotermes formosanus Shiraki, is an aggressive, invasive termite species that has caused billions of dollars of damage across the United States for the past 50 years. Termites depend on intestinal microorganisms for cellulose digestion. Symbiotic microorganisms in the termite gut play key physiological functions such as cellulose and hemicellulose digestion, acetogenesis, hydrogenesis, methanogenesis, sulfate reduction, and nitrogen fixation. Additionally, intestinal microbes create suitable conditions for symbiotic protozoans through the production of nutrients and the maintenance of the pH and the anaerobic conditions in the gut. Although extensive research has been done on the symbiotic relationship of these termites and the microbes found in its gut, there is little information available on the role of facultative anaerobes in the gut. We isolated four enteric bacteria from the hindgut of Formosan subterranean termite, C. formosanus. All isolates were facultative anaerobes and G-. The isolates were identified as Serratia marcescens, Enterobacter aerogens, Enterobacter cloacae, and Citrobacter farmeri by using BIOLOG assay and fatty acid methyl ester analysis (FAME). Each isolate was characterized using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and biochemical study. This is the first report on the presence of facultative microbes in termite gut. Results of this first study on facultative microbes in the termite gut indicate that the role of facultative organisms in the Formosan termite gut may be to scavenge oxygen and create anaerobic conditions for the anaerobic microorganisms, which are essential for digestion of cellulose consumed by the termite.     

The occurrence of microorganisms in intraoral abscesses

The aim of this study was to assess the occurrence of microorganisms in 39 intraoral abscesses. The samples were place in transport medium. The materials were inoculated on adequate enrichment and selective media and cultivated under anaerobic and aerobic conditions. The microorganisms were found in all samples (100%), Anaerobic bacteria most frequently were recovered. The predominant anaerobes were from genus Prevotella, Bacteroides, Fusobacterium and Peptostreptococcus. Among aerobic bacteria, the most frequent were gram-positive cocci. The microaerophilic bacteria and fungi most rarely were isolated from pus samples.     

The 'strict' anaerobe Desulfovibrio gigas contains a membrane-bound oxygen-reducing respiratory chain

Sulfate-reducing bacteria are considered as strict anaerobic microorganisms, in spite of the fact that some strains have been shown to tolerate the transient presence of dioxygen. This report shows that membranes from Desulfovibrio gigas grown in fumarate/sulfate contain a respiratory chain fully competent to reduce dioxygen to water. In particular, a membrane-bound terminal oxygen reductase, of the cytochrome bd family, was isolated, characterized, and shown to completely reduce oxygen to water. This oxidase has two subunits with apparent molecular masses of 40 and 29 kDa. Using NADH or succinate as electron donors, the oxygen respiratory rates of D. gigas membranes are comparable to those of aerobic organisms (3.2 and 29 nmol O(2) min(-1) mg protein(-1), respectively). This 'strict anaerobic' bacterium contains all the necessary enzymatic complexes to live aerobically, showing that the relationships between oxygen and anaerobes are much more complex than originally thought.     

Micro-aeration: an attractive strategy to facilitate anaerobic digestion

Micro-aeration can facilitate anaerobic digestion (AD) by regulating microbial communities and promoting the growth of facultative taxa, thereby increasing methane yield and stabilizing the AD process. Additionally, micro-aeration contributes to hydrogen sulfide stripping by oxidization to produce molecular sulfur or sulfuric acid. Although micro-aeration can positively affect AD, it must be strictly regulated to maintain an overall anaerobic environment that permits anaerobic microorganisms to thrive. Even so, obligate anaerobes, especially methanogens, could suffer from oxidative stress during micro-aeration. This review describes the applications of micro-aeration in AD and examines the cutting-edge advances in how methanogens survive under oxygen stress. Moreover, barriers and corresponding solutions are proposed to move micro-aeration technology closer to application at scale.     

严格厌氧菌铁氧还蛋白的研究进展

铁氧还蛋白(ferredoxin,Fd)是一类含有铁硫簇的小分子蛋白质,广泛存在于自然界中,参与生物体内的呼吸、发酵、固氮、二氧化碳固定和制氢等生理过程.Fd对于严格厌氧的细菌尤为重要,是因为这类细菌的能量代谢高度依赖于低氧化还原电势的生物组分,而Fd能够利用铁硫中心灵活调节其氧还电势,适应低电势需求.本文选取厌氧细菌...     

Occurrence of hopanoid lipids in anaerobic Geobacter species

Geobacter metallireducens and G. sulfurreducens have been classified as strictly anaerobic bacteria which grow and thrive in subsurface and sediment environments. Hopanoids are pentacyclic triterpenoid lipids and are important for bacterial membrane stability and functioning. Hopanoids predominantly occur in aerobically growing bacteria of oxic environments. They rarely have been found in facultatively anaerobic bacteria and, to date, not at all in strict anaerobes. Our research shows that anaerobically grown G. metallireducens and G. sulfurreducens bacteria contain a range of hopanoid lipids, such as diploptene (i.e. hop-22(29)-ene) and hop-21-ene, and more complex, elongated hopanoids. In geological formations, diagenetic derivatives of hopanoids are widely used as biomarkers and are recognized as molecular fossils of bacterial origin. To date, these biomarkers have largely been interpreted as those derived from ancient oxic environments. Our findings presented here suggest that this interpretation needs to be re-evaluated. In addition to the origin in oxic environments, 'geohopanoids' may originate from ancient anaerobic environments as well.     

Distribution of the Phosphoenolpyruvate:Glucose Phosphotransferase System in Bacteria

A survey of the occurrence of the phosphoenolpyruvate-dependent glucose phosphotransferase system was carried out in a number of bacteria, representing both gram-positive and gram-negative facultative anaerobic and strictly aerobic types. The system was found to be present in representatives of genera that are characteristically facultative anaerobes, but the system was absent in members of those genera that are strictly aerobic. Thus, although the phosphoenolpyruvate phosphotransferase system is an important system for the transport of sugars in bacteria carrying out anaerobic glycolysis, it plays no role in sugar transport by those organisms having a strictly oxidative physiology. A fundamentally different system, probably not involving phosphorylation during transport, is indicated in this latter group.     

Disulfide bond-dependent mechanism of protection against oxidative stress in pyruvate-ferredoxin oxidoreductase of anaerobic Desulfovibrio bacteria

Oxidative decarboxylation of pyruvate forming acetyl-coenzyme A is a crucial step in many metabolic pathways. In most anaerobes, this reaction is carried out by pyruvate-ferredoxin oxidoreductase (PFOR), an enzyme normally oxygen sensitive except in Desulfovibrio africanus (Da), where it shows an abnormally high oxygen stability. Using site-directed mutagenesis, we have specified a disulfide bond-dependent protective mechanism against oxidative conditions in Da PFOR. Our data demonstrated that the two cysteine residues forming the only disulfide bond in the as-isolated PFOR are crucial for the stability of the enzyme in oxidative conditions. A methionine residue located in the environment of the proximal [4Fe-4S] cluster was also found to be essential for this protective mechanism. In vivo analysis demonstrated unambiguously that PFOR in Da cells as well as two other Desulfovibrio species was efficiently protected against oxidative stress. Importantly, a less active but stable Da PFOR in oxidized cells rapidly reactivated when returned to anaerobic medium. Our work demonstrates the existence of an elegant disulfide bond-dependent reversible mechanism, found in the Desulfovibrio species to protect one of the key enzymes implicated in the central metabolism of these strict anaerobes. This new mechanism could be considered as an adaptation strategy used by sulfate-reducing bacteria to cope with temporary oxidative conditions and to maintain an active dormancy.     

Growth of a facultative anaerobe under oxygen-limiting conditions in pure culture and in co-culture with a sulfate-reducing bacterium

Abstract The occurrence and properties were studied of glucose-metabolizing bacteria present in the anaerobic sediment 5–10 cm below the surface of an estuarine tidal mud-flat. Of all these bacteria (10⁴– 10⁵ per g wet sediment) 80–90% were facultatively anaerobic species. Chemostat enrichments on glucose under aerobic, oxygen-limited and alternately aerobic-anaerobic conditions also yielded cultures dominated by facultative anaerobes. One of the dominant species, tentatively identified as a Vibrio sp., was studied in more detail under oxygen-limiting conditions. Fermentative and respiratory metabolisms were found to operate simultaneously, and the ratio between the two was regulated by the extent of oxygen limitation. A small fraction of the acetate formed under such growth conditions was shown to be subsequently respired. A co-culture was established of the Vibrio sp. and a sulfate-reducing bacterium ( Desulfovibrio HL21 ) in an aerated chemostat. The importance of these observations is discussed in relation to the role of facultative anaerobes in anaerobic habitats.     

New insights in gut microbiota establishment in healthy breast fed neonates

The establishment of a pioneer gut microbiota is increasingly recognized as a crucial stage in neonatal development influencing health throughout life. While current knowledge is mainly based on either culture or molecular analysis of feces, we opted for a comprehensive approach complementing culture with state-of-the-art molecular methods. The bacterial composition in feces from seven healthy vaginally-delivered, breast-fed neonates was analyzed at days 4-6, 9-14 and 25-30 postnatal, using culture, 16S rRNA gene sequencing of isolates, quantitative PCR and pyrosequencing. Anaerobes outnumbered facultative anaerobes in all seven neonates within the first days of life, owing to high levels of Bifidobacterium and unexpectedly also Bacteroides, which were inversely correlated. Four neonates harbored maternal Bacteroides levels, comprising typical adult species, throughout the neonatal period, while in three only subdominant levels were detected. In contrast, the major adult-type butyrate-producing anaerobic populations, Roseburia and Faecalibacterium, remained undetectable during the neonatal period. The presence of Bacteroidetes as pioneer bacteria in the majority of neonates studied demonstrates that adult-type strict anaerobes may reach adult-like population densities within the first week of life. Consequently the switch from facultative to strict anaerobes may occur earlier than previously assumed in breast-fed neonates, and the establishment of the major butyrate-producing populations may be limited by other factors than the absence of anaerobic conditions. The impact of breast milk components on the timing of establishment of anaerobic pioneer bacteria, as well as opportunistic pathogens should be further studied in regard to priming of the gut-associated immune system and consequences on later health.     

Dearomatizing benzene ring reductases

The high resonance energy of the benzene ring is responsible for the relative resistance of aromatic compounds to biodegradation. Nevertheless, bacteria from nearly all physiological groups have been isolated which utilize aromatic growth substrates as the sole source of cell carbon and energy. The enzymatic dearomatization of the benzene nucleus by microorganisms is accomplished in two different manners. In aerobic bacteria the aromatic ring is dearomatized by oxidation, catalyzed by oxygenases. In contrast, anaerobic bacteria attack the aromatic ring by reductive steps. Key intermediates in the anaerobic aromatic metabolism are benzoyl-CoA and compounds with at least two meta-positioned hydroxyl groups (resorcinol, phloroglucinol and hydroxyhydroquinone). In facultative anaerobes, the reductive dearomatization of the key intermediate benzoyl-CoA requires a stoichiometric coupling to ATP hydrolysis, whereas reduction of the other intermediates is readily achieved with suitable electron donors. Obligately anaerobic bacteria appear to use a totally different enzymology for the reductive dearomatization of benzoyl-CoA including selenocysteine- and molybdenum- containing enzymes.     

Factors Related to the Oxygen Tolerance of Anaerobic Bacteria

The effect of atmospheric oxygen on the viability of 13 strains of anaerobic bacteria, two strains of facultative bacteria, and one aerobic organism was examined. There were great variations in oxygen tolerance among the bacteria. All facultative bacteria survived more than 72 h of exposure to atmospheric oxygen. The survival time for anaerobes ranged from less than 45 min for Peptostreptococcus anaerobius to more than 72 h for two Clostridium perfringens strains. An effort was made to relate the degree of oxygen tolerance to the activities of superoxide dismutase, catalase, and peroxidases in cell-free extracts of the bacteria. All facultative bacteria and a number of anaerobic bacteria possessed superoxide dismutase. There was a correlation between superoxide dismutase activity and oxygen tolerance, but there were notable exceptions. Polyacrylamide gel electropherograms stained for superoxide dismutase indicated that many of the anaerobic bacteria contained at least two electrophoretically distinct enzymes with superoxide dismutase activity. All facultative bacteria contained peroxidase, whereas none of the anaerobic bacteria possessed measurable amounts of this enzyme. Catalase activity was variable among the bacteria and showed no relationship to oxygen tolerance. The ability of the bacteria to reduce oxygen was also examined and related to enzyme content and oxygen tolerance. In general, organisms that survived for relatively long periods of time in the presence of oxygen but demonstrated little superoxide dismutase activity reduced little oxygen. The effects of medium composition and conditions of growth were examined for their influence on the level of the three enzymes. Bacteria grown on the surface of an enriched blood agar medium generally had more enzyme activity than bacteria grown in a liquid medium. The data indicate that superoxide dismutase activity and oxygen reduction rates are important determinants related to the tolerance of anaerobic bacteria to oxygen.     

O-Demethylation, dehydroxylation, ring-reduction and cleavage of aromatic substrates by Enterobacteriaceae under anaerobic conditions

Four fermentative facultative anaerobes, members of the genera Enterobacter and Escherichia , were tested for their ability to transform an aromatic lignin derivative, 3-methoxy-4-hydroxy-cinnamic acid (ferulic acid), under anaerobic (fermentative) conditions. The pure cultures studied were shown to O-demethylate, dehydroxylate, reduce the double bond in the side-chain, decarboxylate the aromatic ring to the stage of benzoate and to reduce the ring to an alicyclic acid. Aromatic hydrocarbons (toluene, ethylbenzene and propylbenzene), as well as phenols (phenol, o-cresol, p-cresol, 2-ethylphenol and 3-hydroxy-4-ethylphenol) were also produced. In addition, during 3 months incubation, the cleavage of the aromatic ring occurred, whereby a small fraction of the substrate was converted to straight-chain and branched (methylated, ethylated) five- to eight-carbon aliphatic acids. The results indicate that pure cultures of fermentative facultative anaerobes might be capable of degrading substituted aromatic acids to aliphatic products under strictly anaerobic (fermentative) conditions. These abilities, which have so far been found only in denitrifying pseudomonads among facultative anaerobes, might be common in Enterobacteriaceae. It is conceivable that these bacteria are important as degraders of aromatic compounds in anaerobic ecosystems.