STUDIES ON THE METABOLISM OF THE AUTOTROPHIC BACTERIA : III. THE NATURE OF THE ENERGY STORAGE MATERIAL ACTIVE IN THE CHEMOSYNTHETIC PROCESS

In the autotrophic bacterium, Thiobacillus thiooxidans, the oxidation of sulfur is coupled to transfers of phosphate from the medium to the cells. CO₂ fixation is coupled to transfers of inorganic phosphate from the cells to the medium and is dependent, in the absence of concomitant sulfur oxidation, upon the amount of phosphate previously taken up during sulfur oxidation. The energy reservoir, which is formed by sulfur oxidation in the absence of CO₂ and which can be released for the fixation of CO₂ under conditions which do not permit sulfur oxidation, is a phosphorylated compound and the data suggest that the energy is stored in the cell as phosphate bond energy. It is possible to oxidize sulfur at a constant rate for hours in the absence of CO₂. The phosphate energy formed during this process is probably released by cell phosphotases. It is possible to inhibit these phosphotases by means of inorganic phosphate and thus to inhibit sulfur oxidation in the absence of CO₂. In the presence of CO₂, where alternative uses for the phosphate energy are available, the inhibition is relieved. Sulfur oxidation (energy input) is coupled, not to CO₂ fixation, but to phosphate esterification. CO₂ fixation (energy utilization) is coupled with phosphate release.     

STUDIES ON THE METABOLISM OF AUTOTROPHIC BACTERIA : II. THE NATURE OF THE CHEMOSYNTHETIC REACTION

In a study of chemosynthesis (the fixation of CO₂ by autotrophic bacteria in the dark) in Thiobacillus thiooxidans, the data obtained support the following conclusions: 1. CO₂ can be fixed by "resting cells" of Thiobacillus thiooxidans; the fixation is not "growth bound." 2. The physiological condition of the cell is of considerable importance in determining CO₂ fixation. 3. CO₂ fixation can occur in the absence of oxidizable sulfur in "young" cells. The extent of this fixation appears to be dependent upon the pCO₂. 4. CO₂ fixation can also occur under anaerobic conditions and the presence of sulfur does not influence such fixation. 5. However, in the CO₂ fixation by cells in the absence of sulfur, only a limited amount of CO₂ can be fixed. This amount is approximately 40 µl. CO₂ per 100 micrograms bacterial nitrogen. After a culture has utilized this amount of CO₂ it no longer has the ability to fix CO₂ but releases it during its respiration. 6. Relatively short periods of sulfur oxidation can restore the ability of cells to fix CO₂ under conditions where sulfur oxidation is prevented. 7. It is possible to oxidize sulfur in the absence of CO₂ and to store the energy thus formed within the cell. It is then possible to use this energy at a later time for the fixation of CO₂ in the entire absence of sulfur oxidation. 8. Cultures of Thiobacillus thiooxidans respiring on sulfur utilize CO₂ in a reaction which proceeds to a zero concentration of CO₂ in the atmosphere. 9. CO₂ may act as an oxidizing agent for sulfur. 10. Hydrogen is not utilized by the organism. 11. It is possible to selectively inhibit sulfur oxidation and CO₂ fixation.     

Dynamics of Metabolic Activities and Gene Expression in the Roseobacter Clade Bacterium Phaeobacter sp. Strain MED193 during Growth with Thiosulfate

Metagenomic analyses of surface seawater reveal that genes for sulfur oxidation are widespread in bacterioplankton communities. However, little is known about the metabolic processes used to exploit the energy potentially gained from inorganic sulfur oxidation in oxic seawater. We therefore studied the sox gene system containing Roseobacter clade isolate Phaeobacter sp. strain MED193 in acetate minimal medium with and without thiosulfate. The addition of thiosulfate enhanced the bacterial growth yields up to 40% in this strain. Concomitantly, soxB and soxY gene expression increased about 8-fold with thiosulfate and remained 11-fold higher than that in controls through stationary phase. At stationary phase, thiosulfate stimulated protein synthesis and anaplerotic CO₂ fixation rates up to 5- and 35-fold, respectively. Several genes involved in anaplerotic CO₂ fixation (i.e., pyruvate carboxylase, propionyl coenzyme A [CoA], and crotonyl-CoA carboxylase) were highly expressed during active growth, coinciding with high CO₂ fixation rates. The high expression of key genes in the ethylmalonyl-CoA pathway suggests that this is an important pathway for the utilization of two-carbon compounds in Phaeobacter sp. MED193. Overall, our findings imply that Roseobacter clade bacteria carrying sox genes can use their lithotrophic potential to gain additional energy from sulfur oxidation for both increasing their growth capacity and improving their long-term survival.     

Microbes mediating the sulfur cycle in the Atlantic Ocean and their link to chemolithoautotrophy

Only about 10%–30% of the organic matter produced in the epipelagic layers reaches the dark ocean. Under these limiting conditions, reduced inorganic substrates might be used as an energy source to fuel prokaryotic chemoautotrophic and/or mixotrophic activity. The aprA gene encodes the alpha subunit of the adenosine-5′-phosphosulfate (APS) reductase, present in sulfate-reducing (SRP) and sulfur-oxidizing prokaryotes (SOP). The sulfur-oxidizing pathway can be coupled to inorganic carbon fixation via the Calvin–Benson–Bassham cycle. The abundances of aprA and cbbM, encoding RuBisCO form II (the key CO₂ fixing enzyme), were determined over the entire water column along a latitudinal transect in the Atlantic from 64°N to 50°S covering six oceanic provinces. The abundance of aprA and cbbM genes significantly increased with depth reaching the highest abundances in meso- and upper bathypelagic layers. The contribution of cells containing these genes also increased from mesotrophic towards oligotrophic provinces, suggesting that under nutrient limiting conditions alternative energy sources are advantageous. However, the aprA/cbbM ratios indicated that only a fraction of the SOP is associated with inorganic carbon fixation. The aprA harbouring prokaryotic community was dominated by Pelagibacterales in surface and mesopelagic waters, while Candidatus Thioglobus, Chromatiales and the Deltaproteobacterium_SCGC dominated the bathypelagic realm. Noticeably, the contribution of the SRP to the prokaryotic community harbouring aprA gene was low, suggesting a major utilization of inorganic sulfur compounds either as an energy source (occasionally coupled with inorganic carbon fixation) or in biosynthesis pathways.     

Active Transport of Inorganic Carbon Increases the Rate of O(2) Photoreduction by the Cyanobacterium Synechococcus UTEX 625

Chlorophyll a fluorescence of Synechococcus UTEX 625 was quenched during the transport of inorganic carbon, even when CO₂ fixation was inhibited by iodoacetamide. Measurements with a pulse modulation fluorometer showed that at least 75% of the quenching was due to oxidation of Q_a, the primary acceptor of photosystem II. Mass spectrometry revealed that transport of inorganic carbon increased the rate of O₂ photoreduction. Hence, O₂ could serve as an electron acceptor to allow oxidation of Q_a even in the absence of CO₂ fixation.     

Significance of Phosphoenolpyruvate Carboxylase during Ammonium Assimilation: Carbon Isotope Discrimination in Photosynthesis and Respiration by the N-Limited Green Alga Selenastrum minutum

The effect of N-assimilation on the partitioning of carbon fixation between phosphoenolpyruvate carboxylase (PEPcase) and ribulose bisphosphate carboxylase/oxygenase (Rubisco) was determined by measuring stable carbon isotope discrimination during photosynthesis by an N-limited green alga, Selenastrum minutum (Naeg.) Collins. This was facilitated by a two process model accounting for simultaneous CO₂ fixation and respiratory CO₂ release. Discrimination by control cells was consistent with the majority of carbon being fixed by Rubisco. During nitrogen assimilation however, discrimination was greatly reduced indicating an enhanced flux through PEPcase which accounted for upward of 70% of total carbon fixation. This shift toward anaplerotic metabolism supports a large increase in tricarboxylic acid cycle activity primarily between oxaloacetate and α-ketoglutarate thereby facilitating the provision of carbon skeletons for amino acid synthesis. This provides an example of a unique set of conditions under which anaplerotic carbon fixation by PEPcase exceeds photosynthetic carbon fixation by Rubisco in a C₃ organism.     

In Vivo Studies in Rhodospirillum rubrum Indicate That Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Catalyzes Two Obligatorily Required and Physiologically Significant Reactions for Distinct Carbon and Sulfur Metabolic Pathways

All organisms possess fundamental metabolic pathways to ensure that needed carbon and sulfur compounds are provided to the cell in the proper chemical form and oxidation state. For most organisms capable of using CO₂ as sole source of carbon, ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) catalyzes primary carbon dioxide assimilation. In addition, sulfur salvage pathways are necessary to ensure that key sulfur-containing compounds are both available and, where necessary, detoxified in the cell. Using knock-out mutations and metabolomics in the bacterium Rhodospirillum rubrum, we show here that Rubisco concurrently catalyzes key and essential reactions for seemingly unrelated but physiologically essential central carbon and sulfur salvage metabolic pathways of the cell. In this study, complementation and mutagenesis studies indicated that representatives of all known extant functional Rubisco forms found in nature are capable of simultaneously catalyzing reactions required for both CO₂-dependent growth as well as growth using 5-methylthioadenosine as sole sulfur source under anaerobic photosynthetic conditions. Moreover, specific inactivation of the CO₂ fixation reaction did not affect the ability of Rubisco to support anaerobic 5-methylthioadenosine metabolism, suggesting that the active site of Rubisco has evolved to ensure that this enzyme maintains both key functions. Thus, despite the coevolution of both functions, the active site of this protein may be differentially modified to affect only one of its key functions.     

Connecting biodiversity and potential functional role in modern euxinic environments by microbial metagenomics

Stratified sulfurous lakes are appropriate environments for studying the links between composition and functionality in microbial communities and are potentially modern analogs of anoxic conditions prevailing in the ancient ocean. We explored these aspects in the Lake Banyoles karstic area (NE Spain) through metagenomics and in silico reconstruction of carbon, nitrogen and sulfur metabolic pathways that were tightly coupled through a few bacterial groups. The potential for nitrogen fixation and denitrification was detected in both autotrophs and heterotrophs, with a major role for nitrogen and carbon fixations in Chlorobiaceae. Campylobacterales accounted for a large percentage of denitrification genes, while Gallionellales were putatively involved in denitrification, iron oxidation and carbon fixation and may have a major role in the biogeochemistry of the iron cycle. Bacteroidales were also abundant and showed potential for dissimilatory nitrate reduction to ammonium. The very low abundance of genes for nitrification, the minor presence of anammox genes, the high potential for nitrogen fixation and mineralization and the potential for chemotrophic CO₂ fixation and CO oxidation all provide potential clues on the anoxic zones functioning. We observed higher gene abundance of ammonia-oxidizing bacteria than ammonia-oxidizing archaea that may have a geochemical and evolutionary link related to the dominance of Fe in these environments. Overall, these results offer a more detailed perspective on the microbial ecology of anoxic environments and may help to develop new geochemical proxies to infer biology and chemistry interactions in ancient ecosystems.     

Comparison analysis on the energy efficiencies and biomass yields in microbial CO2 fixation

CO₂ fixation by a hydrogen-oxidizing bacterium, Cupriavidus necator, was evaluated in a packed bed bioreactor under a constant flow rate of gas mixtures (H₂, O₂, CO₂). The overall energy efficiency depends on the efficiencies of CO₂ fixation into carbohydrate and the reduced carbon into biomass and bioproducts, respectively. The efficiencies varied with the limiting gas substrate. Under O₂ limitation, the efficiency (20–30%) of CO₂ fixation increased with time and was higher than the overall efficiency (12–18%). Under H₂ limitation, the efficiency of CO₂ fixation declined with time while the biomass yield was quite similar to that under O₂ limitation. A cellular metabolic model was suggested for the lithoautotrophic growth of C. necator, including CO₂ fixation into carbohydrate followed by the main metabolic pathway of reduced carbon. Under CO₂ limitation, most H₂ energy was wasted, resulting in a very low biomass yield. Under a dual limitation of O₂ and nitrogen, biosynthesis of poly(3-hydroxybutyrate) was triggered, and the energy efficiency or yield of biopolyester was lower than those of microbial cell mass. Compared with a green microalga Neochloris oleoabundans that produces lipid under nutrient limitation, C. necator exhibited a much higher (3–6 times) energy efficiency in producing biomass and bioproducts from CO₂.     

Photochemical disproportionation of sulfur into sulfide and sulfate byChlorobium limicola formathiosulfatophilum

The anaerobic bacteriumChlorobium assimilates carbon dioxide in the light with various sulfur compounds as electron donors. The well-known metabolic pathway proceeds from the oxidation of sulfide via sulfur to sulfate. In the dark the reaction is partially reversed when sulfur is reduced to hydrogen sulfide. The fermenting cells thereby release an excess of reductant. We have now found a hydrogen sulfide production from sulfur, which is light-dependent. It is more than ten times faster than the dark reaction. This appears in experiments where the cell suspension is illuminated in absence of CO₂ and flushed continuously with H₂ or Ar. The H₂S is trapped with ZnCl₂ and the S^2- titrated with iodine. The total amount of H₂S evolved in the light increases proportionally with the amount of sulfur added, and about one-half of the added sulfur is converted to H₂S. Another part of the metabolized sulfur appears at the same time as sulfate, but all the sulfur oxidized to sulfate does not account for the larger amount of sulfur reduced to hydrogen sulfide. Very likely other unanalyzed oxidized sulfur compounds must also have been produced. Use of H₂ instead of Ar as the anaerobic gas phase does not increase the amount of H₂S produced, nor does the addition of thiosulfate; sulfur itself is the preferred electron donor for the sulfur reduction. Up to a light intensity of 10000 ergs cm^-2sec^-1 CO₂ does not affect H₂S production. Without CO₂, saturation of the light-dependent evolution of H₂S is reached at about 40000 ergs cm^-2sec^-1. In contrast, presence of CO₂ at this light intensity makes the sulfide production disappear completely. On application of mass spectrometry to the gas exchange upon illumination, at high light intensity a H₂S gush is found during the first 3 min. This is followed by CO₂ fixation, while simultaneously the reductant H₂S is now taken up. WithRhodospirillum rubrum, the addition of sulfur leads to a moderate evolution of H₂S. In contrast toChlorobium this reaction inR. rubrum is not light-sensitive, nor does it produce detectable amounts of sulfate. After addition of malate the rate of H₂S evolution does increase in the light, since the cells use malate as an electron donor during their photochemical metabolism.     

Regional Variability and Drivers of Below Ice CO<Subscript>2</Subscript> in Boreal and Subarctic Lakes

Northern lakes are ice-covered for considerable portions of the year, where carbon dioxide (CO₂) can accumulate below ice, subsequently leading to high CO₂ emissions at ice-melt. Current knowledge on the regional control and variability of below ice partial pressure of carbon dioxide (pCO₂) is lacking, creating a gap in our understanding of how ice cover dynamics affect the CO₂ accumulation below ice and therefore CO₂ emissions from inland waters during the ice-melt period. To narrow this gap, we identified the drivers of below ice pCO₂ variation across 506 Swedish and Finnish lakes using water chemistry, lake morphometry, catchment characteristics, lake position, and climate variables. We found that lake depth and trophic status were the most important variables explaining variations in below ice pCO₂ across the 506 lakes_. Together, lake morphometry and water chemistry explained 53% of the site-to-site variation in below ice pCO₂. Regional climate (including ice cover duration) and latitude only explained 7% of the variation in below ice pCO₂. Thus, our results suggest that on a regional scale a shortening of the ice cover period on lakes may not directly affect the accumulation of CO₂ below ice but rather indirectly through increased mobility of nutrients and carbon loading to lakes. Thus, given that climate-induced changes are most evident in northern ecosystems, adequately predicting the consequences of a changing climate on future CO₂ emission estimates from northern lakes involves monitoring changes not only to ice cover but also to changes in the trophic status of lakes.     

Corrosion of Pipe Steel Samples and Conjugated Conversion of Sulfur Compounds by Thionic Bacteria <Emphasis Type="Italic">Halothiobacillus neapolitanus</Emphasis> DSM 15147

The kinetics of conversion of sulfur compounds by Halothiobacillus neapolitanus DSM 15147 bacteria was studied in the presence of steel samples. It was shown that the presence of steel altered the known pathway of sulfur compound oxidation by thionic bacteria. Production of atomic hydrogen via the interaction between biogenic sulfuric acid and steel enhanced secondary production of intermediates and decreased the content of sulfate produced previously. The process was accompanied by pH elevation and continuation of intense growth of the thionic bacterium culture. Thionic bacteria formed a corrosive medium, which caused metal destruction. The protective properties of anticorrosive coatings 225 LS and 640 mk were tested. It was shown that these coatings protected steel from the destructive effect of biogenic sulfuric acid.     

ON THE GROWTH AND RESPIRATION OF SULFUR-OXIDIZING BACTERIA

1. It is shown that Sulfomonas thiooxidans oxidizes elementary sulfur completely to sulfuric acid. Sodium thiosulfate is oxidized by this organism completely to sulfate. Sulfomonas thiooxidans differs, in this respect, from various other sulfur-oxidizing bacilli which either produce elementary sulfur, from the thiosulfate, or convert it into sulfates and persulfates. 2. The organism derives its carbon from the CO₂ of the atmosphere, but is incapable of deriving the carbon from carbonates or organic matter. 3. The S:C, or ratio between the amount of sulfur oxidized to sulfate and amount of carbon assimilated chemosynthetically from the CO₂ of the atmosphere, is, with elementary sulfur as a source of energy, 31.8, and with thiosulfate 64.2. The higher ratio in the case of the thiosulfate is due to the smaller amount of energy liberated in the oxidation of sulfur compound than in the elementary form. 4. Of the total energy made available in the oxidation of the sulfur to sulfuric acid, only 6.65 per cent is used by the organism for the reduction of atmospheric CO₂ and assimilation of carbon. 5. Sulfates do not exert any injurious effect upon sulfur oxidation by Sulfomonas thiooxidans. Any effect obtained is due to the cation rather than the sulfate radical. Nitrates exert a distinctly injurious action both on the growth and respiration of the organism. 6. There is a definite correlation between the amount of sulfur present and velocity of oxidation, very similar to that found in the growth of yeasts and nitrifying bacteria. Oxidation reaches a maximum with about 25 gm. of sulfur added to 100 cc. of medium. However, larger amounts of sulfur have no injurious effect. 7. Dextrose does not exert any appreciable injurious effect in concentrations less than 5 per cent. The injurious effect of peptone sets in at 0.1 per cent concentration and brings sulfur oxidation almost to a standstill in 1 per cent concentration. Dextrose does not exert any appreciable influence upon sulfur oxidation and carbon assimilation from the carbon dioxide of the atmosphere. 8. Sulfomonas thiooxidans can withstand large concentrations of sulfuric acid. The oxidation of sulfur is affected only to a small extent even by 0.25 molar initial concentration of the acid. In 0.5 molar solutions, the injurious effect becomes marked. The organism may produce as much as 1.5 molar acid, without being destroyed. 9. Growth is at an optimum at a hydrogen ion concentration equivalent to pH 2.0 to 5.5, dropping down rapidly on the alkaline side, but not to such an extent on the acid, particularly when a pure culture is employed. 10. Respiration of the sulfur-oxidizing bacteria can be studied by using the filtrate of a vigorously growing culture, to which a definite amount of sulfur is added, and incubating for 12 to 24 hours.