Reductive, Coenzyme A-Mediated Pathway for 3-Chlorobenzoate Degradation in the Phototrophic Bacterium Rhodopseudomonas palustris

We isolated a strain of Rhodopseudomonas palustris (RCB100) by selective enrichment in light on 3-chlorobenzoate to investigate the steps that it uses to accomplish anaerobic dechlorination. Analyses of metabolite pools as well as enzyme assays suggest that R. palustris grows on 3-chlorobenzoate by (i) converting it to 3-chlorobenzoyl coenzyme A (3-chlorobenzoyl–CoA), (ii) reductively dehalogenating 3-chlorobenzoyl–CoA to benzoyl-CoA, and (iii) degrading benzoyl-CoA to acetyl-CoA and carbon dioxide. R. palustris uses 3-chlorobenzoate only as a carbon source and thus incorporates the acetyl-CoA that is produced into cell material. The reductive dechlorination route used by R. palustris for 3-chlorobenzoate degradation differs from those previously described in that a CoA thioester, rather than an unmodified aromatic acid, is the substrate for complete dehalogenation.     

Degradation of Polycyclic Aromatic Hydrocarbons at Low Temperature under Aerobic and Nitrate-Reducing Conditions in Enrichment Cultures from Northern Soils

The potential for biodegradation of polycyclic aromatic hydrocarbons (PAHs) at low temperature and under anaerobic conditions is not well understood, but such biodegradation would be very useful for remediation of polluted sites. Biodegradation of a mixture of 11 different PAHs with two to five aromatic rings, each at a concentration of 10 μg/ml, was studied in enrichment cultures inoculated with samples of four northern soils. Under aerobic conditions, low temperature severely limited PAH biodegradation. After 90 days, aerobic cultures at 20°C removed 52 to 88% of the PAHs. The most extensive PAH degradation under aerobic conditions at 7°C, 53% removal, occurred in a culture from creosote-contaminated soil. Low temperature did not substantially limit PAH biodegradation under nitrate-reducing conditions. Under nitrate-reducing conditions, naphthalene, 2-methylnaphthalene, fluorene, and phenanthrene were degraded. The most extensive PAH degradation under nitrate-reducing conditions at 7°C, 39% removal, occurred in a culture from fuel-contaminated Arctic soil. In separate transfer cultures from the above Arctic soil, incubated anaerobically at 7°C, removal of 2-methylnaphthalene and fluorene was stoichiometrically coupled to nitrate removal. Ribosomal intergenic spacer analysis suggested that enrichment resulted in a few predominant bacterial populations, including members of the genera Acidovorax, Bordetella, Pseudomonas, Sphingomonas, and Variovorax. Predominant populations from different soils often included phylotypes with nearly identical partial 16S rRNA gene sequences (i.e., same genus) but never included phylotypes with identical ribosomal intergenic spacers (i.e., different species or subspecies). The composition of the enriched communities appeared to be more affected by presence of oxygen, than by temperature or source of the inoculum.     

Microbial Degradation of Crude Oil: Factors Affecting the Dispersion in Sea Water by Mixed and Pure Cultures

By means of the enrichment culture technique, a mixed population of microorganisms was obtained which catalyzed the dispersion of crude oil in supplemented sea water. From this enrichment culture, eight pure cultures were isolated and studied. Only one of the isolates (RAG-1) brought about a significant dispersion of crude oil. RAG-1 has been tentatively characterized as a member of the genus Arthrobacter. The other seven isolates gave rise to colonies on supplemented oil agar, but were neither able to disperse oil nor to stimulate the dispersion catalyzed by RAG-1. The dispersion of crude oil by either RAG-1 or the enrichment culture was absolutely dependent on exogenous sources of nitrogen and phosphorous and completely inhibited by 10⁻²m azide. The increase in cell number of RAG-1 was directly proportional to the concentration of crude oil added to the medium over the range 0.05 to 1.0 mg/ml. Within this linear region, 1.0 mg of crude oil yielded 9 × 10⁷ cells and approximately 65% of the oil was converted into a nonbenzene extractable form. Accompanying the emulsification was a decrease in the pH from 7.6 to 5.0. Acidic conditions, however, were neither necessary nor sufficient for oil dispersion. When sea water was supplemented with 0.029 mm K₂HPO₄ and 3.8 mm (NH₄)₂SO₄ and inoculated with RAG-1, oil dispersion occurred within 1 day. This dispersion could also be brought about by the supernatant following separation of the cells from the medium. Similarly, the supernatant obtained following growth of RAG-1 on hexadecane was capable of emulsifying crude oil in 60 min.     

Utilization of Organic Nitrogen Sources by Two Phytoplankton Species and a Bacterial Isolate in Pure and Mixed Cultures

Algal production of dissolved organic carbon and the regeneration of nutrients from dissolved organic carbon by bacteria are important aspects of nutrient cycling in the sea, especially when inorganic nitrogen is limiting. Dissolved free amino acids are a major carbon source for bacteria and can be used by phytoplankton as a nitrogen source. We examined the interactions between the phytoplankton species Emiliania huxleyi and Thalassiosira pseudonana and a bacterial isolate from the North Sea. The organisms were cultured with eight different amino acids and a protein as the only nitrogen sources, in pure and mixed cultures. Of the two algae, only E. huxleyi was able to grow on amino acids. The bacterium MD1 used all substrates supplied, except serine. During growth of MD1 in pure culture, ammonium accumulated in the medium. Contrary to the expectation, the percentage of ammonium regenerated from the amino acids taken up showed no correlation with the substrate C/N ratio. In mixed culture, the algae grew well in those cultures in which the bacteria grew well. The bacterial yields (cell number) were also higher in mixed culture than in pure culture. In the cultures of MD1 and T. pseudonana, the increase in bacterial yield (number of cells) over that of the pure culture was comparable to the bacterial yield in mixed culture on a mineral medium. This result suggests that T. pseudonana excreted a more-or-less-constant amount of carbon. The bacterial yields in mixed cultures with E. huxleyi showed a smaller and less consistent difference than those of the pure cultures of MD1. It is possible that the ability of E. huxleyi to use amino acids influenced the bacterial yield. The results suggest that interactions between algae and bacteria influence the regeneration of nitrogen from organic carbon and that this influence differs from one species to another.     

Dissimilatory Nitrate and Nitrite Reduction under Aerobic Conditions by an Aerobically and Anaerobically Grown Alcaligenes sp. and by Activated Sludge

The oxygen uptake of an Alcaligenes sp., isolated from activated sludge, was inhibited by small amounts of nitric oxide. The occurrence of this inhibition was dependent on the growth conditions and the pretreatment of the cells. Anaerobically grown cells, which had subsequently been aerated in a nitrogen-free medium, accumulated nitric oxide, after the addition of nitrate or nitrite. When the oxygen uptake was inhibited by nitric oxide, dissimilatory reduction of nitrate and nitrite proceeded under aerobic conditions at the same rate as in the absence of oxygen. Activated sludge removed nitric oxide actively under aerobic conditions and as a consequence the oxygen uptake of the sludge was not inhibited in the presence of nitrite. The rate of nitrate reduction under aerobic conditions was about 20% of that in the absence of oxygen.     

Fate of TNT and Its Transformation Products in Mixed Aerobic Cultures

The fate of 2,4,6-trinitrotoluene (TNT) and TNT transformation products in two aerobic enrichment cultures was investigated. Contaminant fate was assessed through analysis of TNT and its oxygen-stable aminated derivatives using capillary electrophoresis and by tracking the distribution of ¹⁴C-labeled products in either the dissolved, mineralized, or biomass fractions. TNT transformation products were generated by reduction with Fe(0), reduction by S^2-, and transformation by Clostridium acetobutylicum and by Eichornia crassipies (water hyacinth). Enrichment cultures varied in the growth substrate and nitrogen source supplied. The dextrose-fed mixed culture (DMC) was enriched on dextrose with yeast extract providing nitrogen for growth, whereas the anthranilic acid-fed mixed culture (AMC) received anthranilic acid as its source of both energy and nitrogen. Each culture transformed TNT, but their product distributions varied. The DMC exhibited higher levels of biomass association, whereas the AMC produced higher levels of aminated nitrotoluenes and unidentified water-soluble products. Neither mineralized TNT to a significant degree. TNT disappearance was observed in all transformation systems, along with the formation of water-soluble products; however, formation of aminated nitrotoluenes was observed only in the sulfide systems. Neither aerobic culture was capable of mineralizing the TNT transformation products introduced, regardless of the transformation method used to prepare them. The distribution of products between the aqueous phase and the biomass did vary between cultures and was affected by the transformation system used.     

Some Experiments and Conclusions about the Influence of Pure H2 Gason the Anaerobic Degradation of Propionate by Mixed Cultures

Reaction enthalpy for propionate degradationΔG₀ is only negative when the partial pressure ofhydrogen pH₂ is less than 10^—4 bar. This means that for pH₂ more than 10^—4 bar, a total anaerobic degradation of propionate is impossible for thermodynamic reasons. Therefore, with increasing pH₂, the anaerobic degradation rate of propionate via acetate is inhibited. There are two ways to investigate the inhibitory effect of pH₂: to keep the concentration of hydrogen consuming bacteria low or to increase the mass transfer by feeding the hydrogen at higher flow rates. The author used an extended fixed bed reactor filled with polyurethane particles as a carrier for the bacteria, aerated with pure H₂ gas. The results, compared with the literature by using model equations in order to standardize the data, correspond well: The addition of pure H₂ gas has no observable effects on propionate degradation.In the fixed bed reactor with immobilized bacteria, it was not possible to reach an inhibitory concentration of H₂ and high process stability could be maintained.     

Perspectives in the Modeling and Optimization of PHB Production by Pure and Mixed Cultures

ABSTRACT

Poly(β-hydroxybutyrate) or PHB is an important member of the family of polyhydroxyalkanoates with properties that make it potentially competitive with synthetic polymers. In addition, PHB is biodegradable. While the biochemistry of PHB synthesis by microorganisms is well known, improvement of large-scale productivity requires good fermentation modeling and optimization. The latter aspect is reviewed here.

Current models are of two types: (i) mechanistic and (ii) cybernetic. The models may be unstructured or structured, and they have been applied to single cultures and co-cultures. However, neither class of models expresses adequately all the important features of large-scale non-ideal fermentations. Model-independent neural networks provide faithful representations of observations, but they can be difficult to design. So hybrid models, combining mechanistic, cybernetic and neural models, offer a useful compromise. All three kinds of basic models are discussed with applications and directions toward hybrid model development.     

Degradation of Alpha-, Beta-, and Gamma-Hexachlorocyclohexane by a Soil Bacterium under Aerobic Conditions

A Pseudomonas sp., isolated from sugarcane rhizosphere soil, readily metabolized not only alpha and gamma isomers of hexachlorocyclohexane, but also the thermodynamically more stable beta isomer, under aerobic conditions. Bacterial degradation of the three isomers led to the accumulation of a transitory metabolite and eventual release of covalently linked chlorine as chloride in stoichiometric amounts.     

Effect of Fermentation Conditions on Growth of Streptococcus cremoris AM2 and Leuconostoc lactis CNRZ 1091 in Pure and Mixed Cultures

Two strains of mesophilic lactic acid bacteria, Streptococcus cremoris AM2 and Leuconostoc lactis CNRZ 1091, were grown in pure and mixed cultures in the presence or absence of citrate (15 mM) and at controlled (pH 6.5) or uncontrolled pH. Microbial cell densities at the end of growth, maximum growth rates, the pH decrease of the medium resulting from growth, and the corresponding acidification rates were determined to establish comparisons. The control of pH in pure cultures had no effect on L. lactis CNRZ 1091 populations. The final populations of S. cremoris AM2, however, were at least five times higher than when the pH was not controlled (4 × 10⁸ vs. 2 × 10⁹ CFU · ml⁻¹). The pH had no effect on the growth rate of either strain. That of S. cremoris AM2 (0.8 h⁻¹) was about twice that of L. lactis CNRZ 1091. When the pH fell below 5, the growth of both strains decreased or stopped altogether. Citrate had no effect on S. cremoris AM2, while final populations of L. lactis CNRZ 1091 were two to three times higher (3 × 10⁸ CFU · ml⁻¹); it had no effect on the maximum growth rates of the two strains. Citrate attenuated the pH decrease of the medium and reduced the maximum acidification rate of the culture by 50%, due to the growth of S. cremoris AM2. Acidification due to L. lactis CNRZ 1091, however, was very slight. Regardless of the conditions of pH and citrate, the total bacterial population in mixed culture was lower (by 39%) than that of the sum of each pure culture. Mixed culture improved the maximum growth rate of L. lactis CNRZ 1091 (0.6 h⁻¹) by 50%, while that of S. cremoris AM2 was unaffected. The acidification rate of the growth medium in mixed culture, affected by the presence of citrate, resulted from the development and activity of S. cremoris AM2.     

Leaching of Pyrite by Acidophilic Heterotrophic Iron-Oxidizing Bacteria in Pure and Mixed Cultures

Seven strains of heterotrophic iron-oxidizing acidophilic bacteria were examined to determine their abilities to promote oxidative dissolution of pyrite (FeS₂) when they were grown in pure cultures and in mixed cultures with sulfur-oxidizing Thiobacillus spp. Only one of the isolates (strain T-24) oxidized pyrite when it was grown in pyrite-basal salts medium. However, when pyrite-containing cultures were supplemented with 0.02% (wt/vol) yeast extract, most of the isolates oxidized pyrite, and one (strain T-24) promoted rates of mineral dissolution similar to the rates observed with the iron-oxidizing autotroph Thiobacillus ferrooxidans. Pyrite oxidation by another isolate (strain T-21) occurred in cultures containing between 0.005 and 0.05% (wt/vol) yeast extract but was completely inhibited in cultures containing 0.5% yeast extract. Ferrous iron was also needed for mineral dissolution by the iron-oxidizing heterotrophs, indicating that these organisms oxidize pyrite via the “indirect” mechanism. Mixed cultures of three isolates (strains T-21, T-23, and T-24) and the sulfur-oxidizing autotroph Thiobacillus thiooxidans promoted pyrite dissolution; since neither strains T-21 and T-23 nor T. thiooxidans could oxidize this mineral in yeast extract-free media, this was a novel example of bacterial synergism. Mixed cultures of strains T-21 and T-23 and the sulfur-oxidizing mixotroph Thiobacillus acidophilus also oxidized pyrite but to a lesser extent than did mixed cultures containing T. thiooxidans. Pyrite leaching by strain T-23 grown in an organic compound-rich medium and incubated either shaken or unshaken was also assessed. The potential environmental significance of iron-oxidizing heterotrophs in accelerating pyrite oxidation is discussed.     

Growth of Pure and Mixed Cultures of Micro-organisms Concerned in the Treatment of Carbonization Waste Liquors

S ummary : Three strains of bacteria responsible for the destruction of the major constituents of carbonization waste liquor were isolated from a laboratory scale, activated sludge plant successfully treating such a liquor. Of the 3 strains one was able to grow on thiocyanate; the other 2 strains grew well on phenol. Behaviour of these organisms in pure and mixed culture showed marked differences: in pure culture, growth of the thiocyanate-degrading strain was unaffected by the presence of 100 mg of phenol/l, but in mixed culture, active growth of another organism on the phenol completely inhibited growth on the thiocyanate. Batch and continuous culture experiments were made with 2 organisms competing for phenol. Both stimulation and inhibition of growth were found, dependent on the ratio between the concentrations of organisms present.     

O-Methyl sugars in lipopolysaccharides of Rhodospirillacea. Identification of 3-O-methyl-d-mannose in Rhodopseudomonas viridis and of 4-O-methyl-d-xylose and 3-O-methyl-6-deoxy-d-talose in Rhodopseudomonas palustris respectively

1. This paper deals with the identification of three O-methyl sugars in lipopolysaccharides isolated from strains of the Gram-negative photosynthetic family Rhodospirillaceae. In addition to the previously described 3-O-methyl-l-xylose, a second O-methyl sugar was encountered in the lipopolysaccharide of Rhodopseudomonas viridis F, namely 3-O-methyl-d-mannose. The lipopolysaccharides of two strains of Rhodopseudomonas palustris (strain 1e5 and 8/1) contain two O-methylsugars, 4-O-methyl-d-xylose and 3-O-methyl-6-deoxy-d-talose (d-acovenose). 4-O-Methyl-d-xylose, but not 3-O-methyl-6-deoxy-d-talose, could be identified in the lipopolysaccharides of the strains K/1 and 2/2 of the same species. 2. The O-methyl sugars described in this communication were isolated by paper chromatography and identified by g.l.c., paper chromatography, high-voltage electrophoresis and mass spectrometry. Besides the genuine sugars, their alditol acetates and their demethylated (parental) forms were investigated. Optical rotation measurements and, in one case, enzymic reactions were used to establish the optical configuration of the sugars under investigation.     

Regulation of carbon flows in the tricarboxylic acid cycle-glyoxylate bypass system in Rhodopseudomonas palustris under different growth conditions

The functional roles of the malate dehydrogenase (MDH) tetrameric and dimeric isoforms in the metabolism of the purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris, strain f-8pt, was studied with the use of specific inhibitors. It was shown that the enzyme tetrameric form allows the functioning of the glyoxylate cycle and the dimeric form provides for the operation of the tricarboxylic acid cycle.     

Nitric Oxide Is Required for Homeostasis of Oxygen and Reactive Oxygen Species in Barley Roots under Aerobic Conditions

Oxygen, the terminal electron acceptor for mitochondrial electron transport, is vital for plants because of its role in the production of ATP by oxidative phosphorylation. While photosynthetic oxygen production contributes to the oxygen supply in leaves, reducing the risk of oxygen limitation of mitochondrial metabolism under most conditions, root tissues often suffer oxygen deprivation during normal development due to the lack of an endogenous supply and isolation from atmospheric oxygen.     

Determination and comparison of the baseline proteomes of the versatile microbe Rhodopseudomonas palustris under its major metabolic states

Rhodopseudomonas palustris is a purple nonsulfur anoxygenic phototrophic bacterium that is ubiquitous in soil and water. R. palustris is metabolically versatile with respect to energy generation and carbon and nitrogen metabolism. We have characterized and compared the baseline proteome of a R. palustris wild-type strain grown under six metabolic conditions. The methodology for proteome analysis involved protein fractionation by centrifugation, subsequent digestion with trypsin, and analysis of peptides by liquid chromatography coupled with tandem mass spectrometry. Using these methods, we identified 1664 proteins out of 4836 predicted proteins with conservative filtering constraints. A total of 107 novel hypothetical proteins and 218 conserved hypothetical proteins were detected. Qualitative analyses revealed over 311 proteins exhibiting marked differences between conditions, many of these being hypothetical or conserved hypothetical proteins showing strong correlations with different metabolic modes. For example, five proteins encoded by genes from a novel operon appeared only after anaerobic growth with no evidence of these proteins in extracts of aerobically grown cells. Proteins known to be associated with specialized growth states such as nitrogen fixation, photoautotrophic, or growth on benzoate, were observed to be up-regulated under those states.     

Response of Aerobic Anoxygenic Phototrophic Bacterial Diversity to Environment Conditions in Saline Lakes and Daotang River on the Tibetan Plateau,NW China

Variations of the pufM gene [encoding the M subunit of the photosynthetic reaction center in aerobic anoxygenic phototrophic (AAnP) bacteria] diversity in response to environmental changes were investigated in waters of six aqueous regimes (including Daotang River and five saline/hypersaline lakes on the Tibetan Plateau) representing a full salinity gradient from freshwater to NaCl-saturation. AAnP bacterial community structures responded to salinity change: Gamma-like AAnP community was predominant in freshwater Daotang River (0.01% salinity). AAnP community structure shifted from Loktanella-like sequences of the Alphaproteobacteria in saline Qinghai Lake to Roseobacter-like sequences in hypersaline lakes (Gahai, Xiaochaidan and Charhan). In addition to salinity, other environmental variables (e.g. N and P availability, TOC and/or DOC, and HCO^? ₃/CO^2? ₃ ions) were also important in affecting the pufM gene diversity in hypersaline lakes. These data have important implications for our understanding of the response of AAnP bacterial community to environmental variables in high-altitude aquatic ecosystems.