Inorganic C-sources for Lemanea,Cladophora and Ranunculus in a fast-flowing stream: Measurements of gas exchange and of carbon isotope ratio and their ecological implications

Summary CO₂-and O₂-exchange characteristics and ¹³C values have been measured in a rhodophycean haptophyte (Lemanea mamillosa), a chlorophycean haptophyte (Cladophora glomerata) and a magnoliophyte rhizophyte (Ranunculus sp.) from a 5 m stretch of the Dichty Burn near Dundee. Light-and CO₂-saturated rates of photosynthesis are greatest on a dry weight basis for Cladophora and lowest for Lemanea; the order is reversed on a surface area basis. The CO₂ concentration at pH 6.5 at which photosynthesis is half-saturated is 25–40 M, with Lemanea rather lower than Cladophora or Ranunculus; these half-saturation values are similar to the free CO₂ concentration in the Burn water. Lemanea cannot use HCO ₃ ^- in photosynthesis, while Cladophora and Ranunculus can. Despite being within a factor or two of saturation with free CO₂ in terms of the bulk water concentration, the growth habit of Cladophora and, particularly, Ranunculus means that the high water velocity in the Burn does not necessarily prevent C depletion effects around the plants, thus providing a possible role for HCO ₃ ^- use by these plants. Lemanea lives in the fastest-growing parts of the Burn, and its growth habit insures that it is exposed to this high water velocity, thus minimising CO₂ depletion during photosynthesis despite the low surface/volume ratio for this plant. ¹³C measurements on the inorganic C in the Burn water are consistent with at least part of its excess (above air-equilibrium) inorganic C levels coming from heterotrophic activity. Lemanea has the most negative ¹³C value of the three plants, consistent with CO₂ use and small diffusion resistances. Ranunculus has the least negative ¹³C value, consistent with some CO₂ depletion and/or HCO ₃ ^- use in situ related to a high diffusion resistance in a rhizophyte which does not have to obtain all of its N and P from the bulk water but can obtain some from the sediments. Cladophora is intermediate, suggesting some CO₂ depletion and/or HCO ₃ ^- use in this densely growing haptophyte.Abbreviations RuBPc-o Ribulose bisphosphate carboxylase-oxygenese (E. C. 4.1.1.39) - PEPc Phosphenolpyruvate carboxylase (E.C 4.1.1.31) - PEPck Phosphoenolpyruvate carboxykinase (ATP) (E.C. 4.1.1.48)     

C, N and P nutrition of Lemanea mamillosa Kütz. (Batrachospermales, Rhodophyta) in the Dighty Burn, Angus, U.K.

Abstract. Field measurements of the growth rate of the red freshwater macroalga Lemanca mamillosa Kutz, in the Dighty Burn, together with measurements of water velocity, [CO₂], [NO₃], [NH₃+ NH₄⁺] and [phosphate], have been made between February and July. This period covers the growth of the erect gametophyte and later of the carposporophyte inside the gametophyte. Hydrodynamic studies in the laboratory on benzoic acid models of the gametophyte suggest an average in situ unstirred layer some 12 μm thick. For growth of the gametophyte, this estimated boundary layer thickness, together with the measured inorganic C transport pathway within the plant, suggest that growth is not significantly restricted by CO₂ transport from the bulk phase to the plastids. δ¹³C measurements on source CO₂ and on plant organic C bear this out. Habitat choice (low temperatures: CO₂ enrichment from ground-water input: rapid water flow), plant morphology and anatomy (turbulence-generating ‘knobbles’ on the nodes; plastids close to the outside of the plant), and plant biochemistry (high CO₂ affinity of the RUBISCO carboxylase; quite high carbonic anhydrase activity) are responsible for this lack of limitation by inorganic C transport in the growing gametophyte which lacks HCO₃ transport and a CO₂ concentrating mechanism. Transport through the boundary layer does not significantly restrict acquisition by the plant of N (probably as NH₄⁺, despite the preponderance of NO₃ in the environment) or of P (as orthophosphate) in the field. The membrane transporters, which have high substrate affinities (K½'s about 2 mmol NH₄⁺ m^-3 and < 2 mmol inorganic phosphate m^?3), probably impose the major limitation. The development of the carposporophyte later in the season, and an increase in the thickness of the cortex of the gametophyte, result in an increased (less negative) δ¹³C, suggesting a significant diffusion limitation to CO₂ transport. This conclusion is reinforced by consideration of the opposing effect on Δδ¹³ C of the decreased demand for products of phosphoenolpyruvate carboxylase activity as the N/C ratio decreases late in the growing season.     

Present and potential uses of the natural abundance of stable isotopes in plant science, with illustrations from the marine environment

The use of stable isotope natural abundance measurements in plant ecophysiological research is discussed in the context of studies of ¹³C/¹²C ratios in marine plants, with emphasis on the uniqueness of the information given by natural abundance measurements and of the importance of complementary data obtained by other techniques in making full use of the natural abundance data. (1) Inorganic C acquisition and assimilation in marine plants can involve diffusive entry of CO₂, or the occurrence of a CO₂-concentrating mechanism frequently involving active HCO₃^? influx. For diffusive CO₂ entry, the δ¹³C measurements can give unique information on the fractional limitation of photosynthesis by CO₂ transport which, with photosynthetic rate measurements, can be used to compute transport conductances. For active HCO₃^?, influx, the δ¹³C values uniquely permit computation of the ratio of the bidirection fluxes (influx/efflux) which, with photon yield data, can be used to given information on the mechanism of the efflux. The analyses are absolutely dependent on external (non-δ¹³C) data distinguishing between diffusive CO₂ entry and the occurrence of a CO₂ concentrating mechanism. (2) δ¹³C measurements on marine photolithotrophs and on members of other trophic levels collected from the sea can give unique data on food webs, with measurements of δ values for other isotopes and compositional data adding precision to the interpretations. (3) Measurements of in situδ¹³C values for extant marine photolithotrophs, compared with δ¹³C values for ancient atmospheric CO₂, can give unique information on the mechanism of atmospheric CO₂ draw-down at the start of glacials; other information permits more concrete conclusions to be drawn.     

Minor stable carbon isotope fractionation between respired carbon dioxide and bulk soil organic matter during laboratory incubation of topsoil

A common assumption in paleoenvironmental reconstructions using soils is that the carbon isotope composition of soil-respired CO₂ is equivalent to the carbon isotope composition of bulk soil organic matter (SOM). However, the occurrence of a non-zero per mil carbon isotope enrichment factor between CO₂ and SOM (\(\varepsilon_{{{\text{CO}}_{ 2} - {\text{SOM}}}}\)) during soil respiration is the most widely accepted explanation for the down-profile increase in SOM δ¹³C values commonly observed in well-drained soils. In order to shed light on this apparent discrepancy, we incubated soil samples collected from the top 2 cm of soils with pure C₃ vegetation and compared the δ¹³C values of soil-respired CO₂ to the δ¹³C values of bulk SOM. Our results show near-zero \(\varepsilon_{{{\text{CO}}_{ 2} - {\text{SOM}}}}\) values (?0.3 to 0.4 ‰), supporting the use of paleosol organic matter as a proxy for paleo soil-respired CO₂. Significantly more negative \(\varepsilon_{{{\text{CO}}_{ 2} - {\text{SOM}}}}\) values are required to explain the typical δ¹³C profiles of SOM in well-drained soils. Therefore our results also suggest that typical SOM δ¹³C profiles result from either (1) a process other than carbon isotope fractionation between CO₂ and SOM during soil respiration or (2) \(\varepsilon_{{{\text{CO}}_{ 2} - {\text{SOM}}}}\) values that become increasingly negative as SOM matures.     

Measurements of substrate oxidation using 13CO2-breath testing reveals shifts in fuel mix during starvation

Most fasting animals are believed to sequentially switch from predominantly utilizing one metabolic substrate to another from carbohydrates, to lipids, then to proteins. The timing of these physiological transitions has been estimated using measures of substrate oxidation including changes in respiratory exchange ratios, blood metabolites, nitrogen excretion, or enzyme activities in tissues. Here, we demonstrate how ¹³CO₂-breath testing can be used to partition among the oxidation of distinct nutrient pools in the body (i.e., carbohydrates, lipids, and proteins) that have become artificially enriched in ¹³C. Seventy-two Swiss Webster mice were raised to adulthood on diets supplemented with ¹³C-1-l-leucine, ¹³C-1-palmitic acid, ¹³C-1-d-glucose, or no tracer. Mice were then fasted for 72 h during which $ \dot{V}{\text{O}}_{2} $ , $ \dot{V}{\text{CO}}_{2} $ , δ¹³C of exhaled CO₂, body temperature, body mass, and blood metabolites (i.e., glucose, ketone bodies, and triacylglycerols) were measured. The fasting mice exhibited reductions in body mass (29 %), body temperature (3.3 °C), minimum observed metabolic rates (24 %), and respiratory exchange ratio (0.18), as well as significant changes in blood metabolites; but these responses were not particularly indicative of changes in oxidative fuel mixture. Measurements of endogenous nutrient oxidation by way of ¹³CO₂-breath testing revealed a decrease in the rate of oxidation of carbohydrates from 61 to 10 % of the total energy expenditure during the first 6 h without food. This response was mirrored by a coincidental increase in rate of endogenous lipid oxidation from 18 to 64 %. A transient peak in carbohydrate oxidation occurred between 8 and 14 h, presumably during increased glycogen mobilization. A well-defined period of protein sparing between 8 and 12 h was observed where endogenous protein oxidation accounted for as little as 8 % of the total energy expenditure. Thereafter, protein oxidation continually increased accounting for as much as 24 % of the total energy expenditure by 72 h. This study demonstrates that ¹³CO₂-breath testing may provide a complementary approach to characterizing the timing and magnitude of sequential changes in substrate oxidation that occur during prolonged fasting and starvation.     

Carbon: freshwater plants

δ¹³C values for freshwater aquatic plant matter varies from ?11 to ?50‰ and is not a clear indicator of photosynthetic pathway as in terrestrial plants. Several factors affect δ¹³C of aquatic plant matter. These include: (1) The δ¹³C signature of the source carbon has been observed to range from +1‰ for HCO₃^? derived from limestone to ?30‰ for CO₂ derived from respiration. (2) Some plants assimilate HCO₃^?, which is –7 to –11‰ less negative than CO₂. (3) C₃, C₄, and CAM photosynthetic pathways are present in aquatic plants. (4) Diffusional resistances are orders of magnitude greater in the aquatic environment than in the aerial environment. The greater viscosity of water acts to reduce mixing of the carbon pool in the boundary layer with that of the bulk solution. In effect, many aquatic plants draw from a finite carbon pool, and as in terrestrial plants growing in a closed system, biochemical discrimination is reduced. In standing water, this factor results in most aquatic plants having a δ¹³C value similar to the source carbon. Using Farquhar's equation and other physiological data, it is possible to use δ¹³C values to evaluate various parameters affecting photosynthesis, such as limitations imposed by CO₂ diffusion and carbon source.     

Isotopic biosignatures in carbonate‐rich,cyanobacteria‐dominated microbial mats of the Cariboo Plateau,B.C.

Photosynthetic activity in carbonate‐rich benthic microbial mats located in saline, alkaline lakes on the Cariboo Plateau, B.C. resulted in pCO₂ below equilibrium and δ¹³C_DIC values up to +6.0‰ above predicted carbon dioxide (CO₂) equilibrium values, representing a biosignature of photosynthesis. Mat‐associated δ¹³C_carb values ranged from ~4 to 8‰ within any individual lake, with observations of both enrichments (up to 3.8‰) and depletions (up to 11.6‰) relative to the concurrent dissolved inorganic carbon (DIC). Seasonal and annual variations in δ¹³C values reflected the balance between photosynthetic ¹³C‐enrichment and heterotrophic inputs of ¹³C‐depleted DIC. Mat microelectrode profiles identified oxic zones where δ¹³C_carb was within 0.2‰ of surface DIC overlying anoxic zones associated with sulphate reduction where δ¹³C_carb was depleted by up to 5‰ relative to surface DIC reflecting inputs of ¹³C‐depleted DIC. δ¹³C values of sulphate reducing bacteria biomarker phospholipid fatty acids (PLFA) were depleted relative to the bulk organic matter by ~4‰, consistent with heterotrophic synthesis, while the majority of PLFA had larger offsets consistent with autotrophy. Mean δ¹³C_org values ranged from ?18.7 ± 0.1 to ?25.3 ± 1.0‰ with mean Δ¹³C_inorg‐org values ranging from 21.1 to 24.2‰, consistent with non‐CO₂‐limited photosynthesis, suggesting that Precambrian δ¹³C_org values of ~?26‰ do not necessitate higher atmospheric CO₂ concentrations. Rather, it is likely that the high DIC and carbonate content of these systems provide a non‐limiting carbon source allowing for expression of large photosynthetic offsets, in contrast to the smaller offsets observed in saline, organic‐rich and hot spring microbial mats.     

Purification and Properties of α-d-Galactosidase Produced by Corticium rolfsii

An α-d-galactosidase was purified from the culture filtrate of Corticium rolfsii IFO 6146 by a combination of QAE-Sephadex A-50 and SE-Sephadex C-50 chromatography. The purified enzyme was demonstrated to be free of other possibly interfering glycosidases and glycanases. The maximum activity of the enzyme towards p-nitrophenyl α-d-galactopyrano-side was found to be at pH 2.5 to 4.5, and the enzyme was fairly active at pH 1.1 to 2.0. The enzyme was stable over a pH range 4.0 to 7.0 at 5°C for 72 hr and relatively unstable at pH 1.1 to 2.0 as compared with endo-polygalacturonase, α-l-arabinofuranosidase and β-d-galactosidase produced by C. rolfsii. The enzymic activity was completely inhibited by Hg²⁺ and Ag⁺ ions, respectively. Km values were determined to be 0.16 × 10^?3 m for p-nitrophenyl α-d-galactopyranoside and 0.26 × 10^?3m for o-nitrophenyl α-d-galactopyranoside. The values of V_max were also determined to be 26.6 μmoles and 28.6 μmoles per min per mg for p- and o-nitrophenyl α-d-galactopyranoside, respectively.     

Syntheses of 14C-Labeled Pyrethrin I,Allethrin, Phthalthrin,and Dimethrin on a Submillimole Scale

Studies on the metabolic fate and degradation chemistry of pyrethroid insecticide chemicals are greatly facilitated by the use of compounds radiolabeled, in separate preparations, in the acid and alcohol moieties. Acid-labeled preparations were made by converting d-trans-chrysanthemic acid-1-¹⁴C (88 mg, 1.3 mCi/mm) into d-trans-d-pyrethrin-1-¹⁴C (68 mg, 1.3 mCi/mm), d-trans-d-allethrin-¹⁴C (43 mg, 1.3 mCi/mm), d-trans-dimethrin-¹⁴C (54 mg, 0.294 mCi/mm), and d-trans-phthalthrin-¹⁴C (47 mg, 0.294 mCi/mm), incorporating approximately 81% of the starting radiocarbon into the four pyrethroid preparations. Alcohol-labeled preparations were made by converting acetone-1,3-¹⁴C into d-trans-dl-ailethrin-¹⁴C (146 mg, 0.162 mCi/mm) and formaldehyde-¹⁴C into d-trans-phthalthrin-¹⁴C (299 mg, 0.276 mCi/mm). Each labeled compound had a high stereochemical purity and a radiochemical purity of greater than 99%. Detailed procedures were worked out for all conversions which took place in high yields except in one case: the synthesis of allethrin labeled in the alcohol moiety.     

Enhanced growth of the red algaPorphyra yezoensis Ueda in high CO2 concentrations

Leafy thalli of the red algaPorphyra yezoensis Ueda, initiated from conchospores released from free-living conchocelis, were cultured using aeration with high CO₂. It was found that the higher the CO₂ concentration, the faster the growth of the thalli. Aeration with elevated CO₂ lowered pH in dark, but raised pH remarkably in light with the thalli, because the photosynthetic conversion of HCO ₃ ^? to OH^? and CO₂ proceeded much faster than the dissociation of hydrated CO₂ releasing H⁺. Photosynthesis of the alga was found to be enhanced in the seawater of elevated dissolved inorganic carbon (DIC, CO₂ + HCO ₃ ^? + CO ₃ ^? ). It is concluded that the increased pH in the light resulted in the increase of DIC in the culture media, thus enhancing photosynthesis and growth. The relevance of the results to removal of atmospheric CO₂ by marine algae is discussed.     

Enhanced inorganic carbon uptake by Chlorella sp. IMMTCC-2 under autotrophic conditions for lipid production and CO2 sequestration

To achieve sustainable production of biofuel from microalgae, a well-optimized and sustained biomass production is prerequisite. The major factor determining the higher productivity of algae is the availability and uptake of CO₂ for biomass growth. In this study, an improved CO₂ sequestration method leading to improved biomass yields has been investigated. The ability of OH^? ions in fixing dissolved CO₂ in form of HCO ₃ ^? in algal growth medium was studied using a Chlorella sp. and scaled-up in a photobioreactor. It was observed that a critical concentration of 0.005?M OH^? is required for HCO ₃ ^? formation and utilization by algae. HCO ₃ ^? uptake was enhanced by 70.8% (in presence of 0.01?M NaOH) with a sixfold increase in growth rate compared with only CO₂ system. In mineral carbon systems such as NaHCO₃ and Na₂CO₃, increase in HCO ₃ ^? uptake was enhanced by 65.4% and 63.4%, respectively. The maximum rate of CO₂ fixation of 6.6?mg?L^?1?h^?1 was obtained with 0.01?M NaOH which was 1.5 times compared with mineral carbon sources. The biomass from scale-up experiment contained 16.3% lipid (by weight) of which 75% is unsaturated fatty acids (in total lipids). This supports the idea that fixing the dissolved CO₂ in the form of bicarbonate using alkali helps in increased biomass productivity rather than CO₂ itself, forms a precursor for biodiesel, and increases CO₂ sequestration in a cyclic process.     

Preparation of [1-13C]D-Glucose 6-Phosphate from [13C]Methanol and D-Ribose 5-Phosphate with Methylotrophic Enzymes

[¹³C]Formaldehyde was selectively incorporated into the C-1 position of D-fructose 6-phosphate by condensation with D-ribulose 5-phosphate catalyzed by a partially purified enzyme system for formaldehyde fixation in Methylomonas aminofaciens 77a. Much of the [1-¹³C]D-fructose 6-phosphate produced in this reaction was converted to [1-¹³C]D-glucose 6-phosphate by the addition of glucose-6-phosphate isomerase. A fed-batch reaction with periodic additions of the substrates afforded 56.2 g/liter D-glucose 6-phosphate and 26.8g/liter D-fructose 6-phosphate. When [¹³C]methanol was used as the C₁-donor, the yield of [1-¹³C]D-glucose 6-phosphate was high when alcohol oxidase was added. The optimum conditions for sugar phosphate production in the fed-batch reaction gave 45.6g/liter [1-¹³C]D-glucose 6-phosphate and 16.4g/liter [1-¹³C]D-fructose 6-phosphate in 165min. The molar yield of the total sugar phosphates to methanol added was 95%. The addition of H₂O₂ and catalase to the reaction system supplied molecular oxygen for methanol oxidation to formaldehyde by alcohol oxidase.     

Biological Activities of Fundamental,Carbohydrate Skeleton of Lipid A Containing Amide-linked 3-Hydroxytetradecanoic Acid

In the initial stage of hydrolysis, exo-ß-(l-→3)-d-glucanase from Basidiomycetes sp. QM 806 cleaved laminaran from Eisenia bicyclis with a pattern resembling an endo-hydrolase. Five kinds of intermediate gluco-oligosaccharides were separated by a combination of gel filtration and HPLC. They were shown to be 3²-O-ß-d-glucosyl-gentiobiose, 3²-O-ß-d-gentiobiosyl-gentiobiose, 3³O-ß-d-glucosyl-gentiotriose, 3⁴-ß-d-glucosyl-3²-O-gentiobiosyl-gentiobiose, and 3³-O-ß-d-gentio-biosylgentiotriose by enzymic hydrolysis and methylation analysis as well as by ¹³C NMR spectroscopy. As a result, such kinds of ß-(l → 3)-ß-(l→6)-linked oligosaccharides could be accounted for in the initial cleavage, and they were hydrolyzed ultimately to glucose, gentiobiose, and gentio-triose. It suggests that a single (1 → 3)-linkage on a block of (1 → 6)-links show some resistance to attack by this enzyme.     

Effect of SO32- on the activity of ribulose bisphosphate carboxylase from seedlings of Pinus silvestris

Abstract The effect of sulphite on ribulose bisphosphate carboxylase, extracted from needles of Pinus silvestris L., was studied in vitro at pH 8.15 and 25°C. 1 mM and higher concentrations of SO₃^2- inhibited the enzyme. The enzyme was activated either in the assay medium (2.5 – 20 mM HCO_3, 20 mM MgCl₂) or in 10 or 20 mM HCO₃^- and 20–25 mM MgCl₂. Linear reciprocal plots of the activity versus the substrate concentration were obtained, when the HCO₃^- concentration during activation was 4 mM or higher. When the enzyme was activated at high HCO₃^- and Mg²⁺ concentrations, the K_m(CO₂) was c. 27 μM. With respect to HCO₃^-. SO₃^2- inhibited the enzyme in a non-competitive fashion. The inhibition was similar, whether SO₃^2- was present during activation or not. Apparently. SO₃^2- did not interfere with the binding of CO₂ and Mg²⁺ at the activating site. The K₁ was 11–13 mM SO₃^2-. With respect to ribulose bisphosphate the inhibition was also noncompetitive. Similar results with respect to HCO₃^- were obtained for spinach, Spinacia oleracea L., which is contrary to earlier reports.     

Deontostoma species from subantarctic coasts (Nematoda,Leptosomatidae)

The major cyanobacteria in Crawford Lake are benthic mat forming Lyngbya and Oscillatoria and not phytoplankton. The eutrophication of the lake has resulted in a decline in the mat forming cyanobacteria as inferred from palaeopigment analyses of the core from this lake. In previous palaeolimnological studies the concentrations of oxillaxanthin and myxoxanthophyll have been used as correlates with lake trophic levels. High concentration of oscillaxanthin and myxoxanthophyll were interpreted as indicating eutrophic conditions prevailed. Our results indicated that when Crawford Lake was most eutrophic little oscillaxanthin and myxoxanthophyll was produced. High values of ¹³C at the depth of 34–48 cm (1500–1760 A. D.) were related to a dense population of benthic Oscillatoria and Lyngbya living on the bottom of the lake during that period. The Oscillatoria and Lyngbya utilize bicarbonate as a source of inorganic carbon. Carbonate has a high ¹³C value. Very low ¹³C values were found at 0–7 cm (1955–1992 A. D.) in the Crawford sediment core at the time when phyto- plankton dominated the core. Phytoplankton are enriched in ¹²C by photosynthetic assimilation of CO₂.     

Carbonate ions appear to neither inhibit nor stimulate use of bicarbonate ions in photosynthesis by Ulva lactuca

Rates of photosynthesis by the marine macroalga Ulva lactuca were measured in a factorial experiment at five concentrations of HCO₃^? and CO₃^2- between 0·20 and 1·26 mol m^?3, but very low concentrations of CO₂. The results demonstrated that HCO₃^? was available for use, but an analysis of variance showed that CO₃^2- had neither an inhibiting nor a stimulating effect on rates of photosynthesis over this concentration range. Over the experiment, pH varied from 8·46 to 10·06 and this also had no significant effect on rates of photosynthesis. The lack of a stimulatory effect of high concentrations of CO₃^2- on the rate of photosynthesis at low concentrations of HCO₃^? was taken as circumstantial evidence for direct uptake of HCO₃^? rather than proton extrusion and external production of CO₂. In the rockpools in which U. lactuca grows, pH values up to 10·35 have been recorded, and for much of the time, CO₃^2- was the major form of inorganic carbon available. The apparent lack of an ability to use CO₃^2- under these conditions suggests that direct use of CO₃^2- as a source of inorganic carbon for photosynthesis is unlikely to be widespread.     

Factors controlling the stable isotope composition and C:N ratio of seston and periphyton in shallow lake mesocosms with contrasting nutrient loadings and temperatures

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Biosynthesis of Fukinolic Acid Isolated from Petasites japonicus

The biosynthesis of fukinolic acid, which had been isolated from the Japanese fuki vegetable, Petasites japonicus, was investigated by feeding selected ¹³C-labeled compounds to axenic cultures of P. japonicus. [1,2-¹³C₂] sodium acetate and [1-¹³C] L-tyrosine were incorporated into the fukiic acid sub group, while [3-¹³C] L-phenylalanine was incorporated into the caffeic acid moiety.     

Isotope discrimination by form IC RubisCO from Ralstonia eutropha and Rhodobacter sphaeroides,metabolically versatile members of ‘Proteobacteria’ from aquatic and soil habitats

RubisCO, the CO₂ fixing enzyme of the Calvin–Benson–Bassham (CBB) cycle, is responsible for the majority of carbon fixation on Earth. RubisCO fixes ¹²CO₂ faster than ¹³CO₂ resulting in ¹³C-depleted biomass, enabling the use of δ¹³C values to trace CBB activity in contemporary and ancient environments. Enzymatic fractionation is expressed as an ε value, and is routinely used in modelling, for example, the global carbon cycle and climate change, and for interpreting trophic interactions. Although values for spinach RubisCO (ε = ~29‰) have routinely been used in such efforts, there are five different forms of RubisCO utilized by diverse photolithoautotrophs and chemolithoautotrophs and ε values, now known for four forms (IA, B, D and II), vary substantially with ε = 11‰ to 27‰. Given the importance of ε values in δ¹³C evaluation, we measured enzymatic fractionation of the fifth form, form IC RubisCO, which is found widely in aquatic and terrestrial environments. Values were determined for two model organisms, the ‘Proteobacteria’ Ralstonia eutropha (ε = 19.0‰) and Rhodobacter sphaeroides (ε = 22.4‰). It is apparent from these measurements that all RubisCO forms measured to date discriminate less than commonly assumed based on spinach, and that enzyme ε values must be considered when interpreting and modelling variability of δ¹³C values in nature.     

Reverse Reaction of Malic Enzyme for HCO3 − Fixation into Pyruvic Acid to Synthesize L-Malic Acid with Enzymatic Coenzyme Regeneration

Malic enzyme [L-malate: NAD(P)⁺ oxidoreductase (EC 1.1.1.39)] catalyzes the oxidative decarboxylation of L-malic acid to produce pyruvic acid using the oxidized form of NAD(P) (NAD(P)⁺). We used a reverse reaction of the malic enzyme of Pseudomonas diminuta IFO 13182 for HCO₃ ^? fixation into pyruvic acid to produce L-malic acid with coenzyme (NADH) generation. Glucose-6-phosphate dehydrogenase (EC1.1.1.49) of Leuconostoc mesenteroides was suitable for coenzyme regeneration. Optimum conditions for the carboxylation of pyruvic acid were examined, including pyruvic acid, NAD⁺, and both malic enzyme and glucose-6-phosphate dehydrogenase concentrations. Under optimal conditions, the ratio of HCO₃ ^? and pyruvic acid to malic acid was about 38% after 24 h of incubation at 30 °C, and the concentration of the accumulated L-malic acid in the reaction mixture was 38 mM. The malic enzyme reverse reaction was also carried out by the conjugated redox enzyme reaction with water-soluble polymer-bound NAD⁺.