Effect of bicarbonate on the water-oxidizing complex of photosystem II in the super-reduced S-states

It is shown that the hydrazine-induced transition of the water-oxidizing complex (WOC) to super-reduced S-states depends on the presence of bicarbonate in the medium so that after a 20 min treatment of isolated spinach thylakoids with 3 mM NH₂NH₂ at 20 °C in the CO₂/HCO₃⁻-depleted buffer the S-state populations are: 42% of S₋₃, 42% of S₋₂, 16% of S₋₁ and even formal S₋₄ state is reached, while in the presence of 2 mM NaHCO₃, the same treatment produces 30% of S₋₃, 38% of S₋₂, and 32% of S₋₁ and there is no indication of the S₋₄ state. Bicarbonate requirement for the oxygen-evolving activity, very low in untreated thylakoids, considerably increases upon the transition of the WOC to the super-reduced S-states, and the requirement becomes low again when the WOC returns back to the normal S-states using pre-illumination. The results are discussed as a possible indication of ligation of bicarbonate to manganese ions within the WOC.     

Bicarbonate stabilizes isolated D1/D2/cytochrome b 559 complex of photosystem 2 against thermoinactivation

It has been shown that thermoinactivation of the isolated D1/D2/cytochrome b ₅₅₉ complex (RC) of photosystem 2 (PS-2) from pea under anaerobic conditions at 35°C in 20 mM Tris-HCl buffer (pH 7.2) depleted of HCO₃⁻, with 35 mM NaCl and 0.05% n-dodecyl-β-maltoside, results in a decrease in photochemical activity measured by photoreduction of the PS-2 primary electron acceptor, pheophytin (by 50% after 3 min of heating), which is accompanied by aggregation of the D1 and D2 proteins. Bicarbonate, formate, and acetate anions added to the sample under these conditions differently influence the maintenance of photochemical activity: a 50% loss of photochemical activity occurs in 11.5 min of heating in the presence of bicarbonate and in 4 and 4.6 min in the presence of formate and acetate, respectively. The addition of bicarbonate completely prevents aggregation of the D1 and D2 proteins as opposed to formate and acetate (their presence has no effect on the aggregation during thermoinactivation). Since the isolated RCs have neither inorganic Mn/Ca-containing core of the water-oxidizing complex nor nonheme Fe²⁺, it is supposed that bicarbonate specifically interacts with the hydrophilic domains of the D1 and D2 proteins, which prevents their structural modification that is a signal for aggregation of these proteins and the loss of photochemical activity.     

Cumulative effect of nitrogen and sulphur on <Emphasis Type="Italic">Brassica juncea</Emphasis> L. genotypes under NaCl stress

In the present study, N and S assimilation, antioxidant enzymes activity, and yield were studied in N and S-treated plants of Brassica juncea (L.) Czern. & Coss. (cvs. Chuutki and Radha) under salt stress. The treatments were given as follows: (1) NaCl₉₀ mM+N₀S₀ mg kg^-1 sand (control), (2) NaCl₉₀ mM+N₆₀S₀ mg kg^-1 sand, (3) NaCl₉₀ mM+N₆₀S₂₀ mg kg^-1 sand, (4) NaCl₉₀ mM+N₆₀S₄₀ mg kg^-1 sand, and (5) NaCl₉₀ mM+N₆₀S₆₀ mg kg^-1 sand. The combined application of N (60 mg kg⁻¹ sand) and S (40 mg kg⁻¹ sand) proved beneficial in alleviating the adverse effect of salt stress on growth attributes (shoot length plant⁻¹, fresh weight plant⁻¹, dry weight plant⁻¹, and area leaf⁻¹), physio-biochemical parameters (carbonic anhydrase activity, total chlorophyll, adenosine triphosphate-sulphurylase activity, leaf N, K and Na content, K/Na ratio, activity of nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, catalase, superoxide dismutase, ascorbate peroxidase and glutathione reductase, and content of glutathione and ascorbate), and yield attributes (pods plant⁻¹, seeds pod⁻¹, and seed yield plant⁻¹). Therefore, it is concluded that combined application of N and S induced the physiological and biochemical mechanisms of Brassica. The stimulation of antioxidant enzymes activity and its synergy with N and S assimilation may be one of the important mechanisms that help the plants to tolerate the salinity stress and resulted in an improved yield.     

Bicarbonate protects the water-oxidizing complex of photosystem II against thermoinactivation in intact <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> cells

Currently available data about bicarbonate (BC) action on the Mn-containing water-oxidizing complex (WOC) of the photosystem II (PSII) were obtained almost solely in vitro, e.g. on subchloroplast membrane fragments enriched with PSII. To investigate the in vivo BC effect on the PSII donor side, we used the method of dark thermoinactivation of intact Chlamydomonas reinhardtii cells. Photosynthetic activity of PSII was measured as photoinduced changes in the PSII chlorophyll fluorescence yield and as the rate of photosynthetic oxygen evolution. To exclude a “direct” effect of the absence of BC on the PSII activity, before measurements of the photosynthetic activity, the concentration of BC in all samples was equalized by addition of NaHCO₃ to each of them (except for those that contained 5 mM of NaHCO₃ during thermoinactivation) to reach the final concentration of 5 mM. This allowed registering only so-called “irreversible” (i.e., not reversible by subsequent addition of BC) effect of the absence of BC during thermoinactivation. It was shown that, if 5 mM NaHCO₃ was added to the medium before thermoinactivation, the rate of inactivation of the PSII donor side was lower than in BC-depleted medium 1.5-to 2-fold. The obtained results are interpreted as an indication that BC protects the donor side of PSII against thermoinactivation in vivo, in intact C. reinhardtii cells. This proves the correctness of the earlier proposition that BC is an integral constituent of the Mn-containing water-oxidizing complex of PSII. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 3, pp. 342–349. The article was translated by the authors.     

Spatial distributions and net deposition rates of mineral elements in the elongating wheat (Triticum aestivum L.) leaf under saline soil conditions

Wheat leaf growth is known to be spatially affected by salinity. The altered spatial distribution of leaf growth under saline conditions may be associated with spatial changes in tissue mineral elements. The objective of this study was to evaluate the spatial distributions of mineral elements and their net deposition rates in the elongating and mature zones of leaf 4 of the main stem of spring wheat (Triticum aestivum L. cv. Lona) during its linear growth phase under saline soil conditions. Plants were grown in an illitic-chloritic silty loam with 0 and 120 mM NaCl. Three days after emergence of leaf 4, sampling was begun at 3 and 13 h into the 16-h light period. Spatial distributions of fresh weight (FW), dry weight (DW), and Na⁺, K⁺, Cl⁻, NO⁻ ₃, Ca²⁺, Mg²⁺, total P, and total N in the elongating and mature tissues were determined on a millimeter scale. The patterns of spatial distribution of Na⁺, Cl⁻, K⁺, NO₃ ⁻, and Ca²⁺ in the growing leaves were affected by salinity, while those of Mg²⁺, total P, and total N were not. Sodium, K⁺, Cl⁻, Ca²⁺, Mg²⁺, and total N concentrations (mmol · kg⁻¹ FW) were consistently higher at 120 mM NaCl than at 0 mM NaCl along the leaf axis from the leaf base, whereas NO₃ ⁻ concentration was lower at 120 mM NaCl. Deposition rates of all nutrients were greatest in the elongation zone. The elongation zone was the strongest sink for mineral elements in the leaf tissues. Local net deposition rates of Na⁺, Cl⁻, Ca²⁺, and Mg²⁺ (mmol · kg⁻¹ FW · h⁻¹) in the most actively elongating zone were enhanced by 120 mM NaCl, whereas for NO₃ ⁻ this was depressed. The lower supply of NO⁻ ₃ to growing leaves may be responsible for the inhibition of growth under saline conditions. Higher tissue concentrations of Na⁺ and Cl⁻ may cause ion imbalance but probably did not result in ion toxicity in the growing leaves. Potassium, Ca²⁺, Mg²⁺, total P, and total N are less plausibly responsible for the reduction in leaf growth in this study. Higher tissue K⁺ and Ca²⁺ concentrations at 120 mM NaCl are probably due to the presence of high Ca²⁺ in the soil of this study. Received: 13 March 1997 / Accepted: 9 June 1997     

Microbial acetate oxidation in horizontal rotating tubular bioreactor

The aim of this work was to investigate the possibility of conducting a continuous aerobic bioprocess in a horizontal rotating tubular bioreactor (HRTB). Aerobic oxidation of acetate by the action of a mixed microbial culture was chosen as a model process. The microbial culture was not only grown in a suspension but also in the form of a biofilm on the interior surface of HRTB. Efficiency of the bioprocess was monitored by determination of the acetate concentration and chemical oxygen demand (COD). While acetate inlet concentration and feeding rate influenced efficiency of acetate oxidation, the bioreactor rotation speed did not influence the bioprocess dynamics significantly. Gradients of acetate concentration and pH along HRTB were more pronounced at lower feeding rates. Volumetric load of acetate was proved to be the most significant parameter. High volumetric loads (above 2 g acetate l⁻¹ h⁻¹) gave poor acetate oxidation efficiency (only 17 to 50%). When the volumetric load was in the range of 0.60–1.75 g acetate l⁻¹ h⁻¹, acetate oxidation efficiency was 50–75%. At lower volumetric loads (0.14–0.58 g acetate l⁻¹ h⁻¹), complete acetate consumption was achieved. On the basis of the obtained results, it can be concluded that HRTB is suitable for conducting aerobic continuous bioprocesses.     

Simultaneous biological removal of sulfur, nitrogen and carbon using EGSB reactor

High-rate biological conversion of sulfide and nitrate in synthetic wastewater to, respectively, elemental sulfur (S⁰) and nitrogen-containing gas (such as N₂) was achieved in an expanded granular sludge bed (EGSB) reactor. A novel strategy was adopted to first cultivate mature granules using anaerobic sludge as seed sludge in sulfate-laden medium. The cultivated granules were then incubated in sulfide-laden medium to acclimate autotrophic denitrifiers. The incubated granules converted sulfide, nitrate, and acetate simultaneously in the same EGSB reactor to S⁰, N-containing gases and CO₂ at loading rates of 3.0 kg S m⁻³ d⁻¹, 1.45 kg N m⁻³ d⁻¹, and 2.77 kg Ac m⁻¹ d⁻¹, respectively, and was not inhibited by sulfide concentrations up to 800 mg l⁻¹. Effects of the C/N ratio on granule performance were identified. The granules cultivated in the sulfide-laden medium have Pseudomonas spp. and Azoarcus sp. presenting the heterotrophs and autotrophs that co-work in the high-rate EGSB-SDD (simultaneous desulfurization and denitrification) reactor.     

Mechanistic and structural aspects of photosynthetic water oxidation

Conclusions on the functional and structural organisation of photosynthetic water oxidation are gathered from a critical survey of the wealth of data reported in the literature and author's own experimental research: (1) the water oxidising complex (WOC) contains a tetranuclear manganese cluster of dimer of dimers' structure and functional heterogeneity of the metal centers, (2) the four step univalent oxidative pathway leading to water oxidation into molecular oxygen and four protons comprises only manganese, tyrosine Y_Z of polypeptide Dl and the substrate as redox active species, (3) the redox transitions S₀→ S₁ and S₁→ S₂ are manganese centered whereas S₂→ S₃ is most likely a ligand-centered reaction, (4) there exist several lines of evidence for a marked structural change that accompanies the redox transition S₂→ S₃, (5) one Ca²⁺ is an indispensible constituent of a functionally competent WOC while the role of Cl is much less clear and a direct participation disputable, (6) substrate water is most likely bound in all redox states S₀,…,S₃ and exchangeable with the bulk phase. The protonation state is determined by the redox state S₁ and the protein microenvironment. A mechanism is proposed for water oxidation in the WOC that is based on three key postulates: (1) water oxidation takes place in the first coordination sphere of one manganese dimer [Mn_aMn_b]; (2) the essential O-O bond is preformed in S₃ as part of a rapid redox isomerism S₃(I)→S₃(II) where in S₃(II) a nuclear geometry and electronic configuration is attained that corresponds to a peroxidic-type species; and (3) S₃(II) is an ‘entatic state’ for the formation of complexed dioxygen triggered by Y_Z^OX induced electron abstraction from the WOC and electronic redistribution to S₀(O₂).     

Effects of NO2? and NO3? on the Fe(III)EDTA reduction in a chemical absorption–biological reduction integrated NOx removal system

The biological reduction of Fe(III) ethylenediaminetetraacetic acid (EDTA) is a key step for NO removal in a chemical absorption–biological reduction integrated process. Since typical flue gas contain oxygen, NO₂ ⁻ and NO₃ ⁻ would be present in the absorption solution after NO absorption. In this paper, the interaction of NO₂ ⁻, NO₃ ⁻, and Fe(III)EDTA reduction was investigated. The experimental results indicate that the Fe(III)EDTA reduction rate decrease with the increase of NO₂ ⁻ or NO₃ ⁻ addition. In the presence of 10 mM NO₂ ⁻ or NO₃ ⁻, the average reduction rate of Fe(III)EDTA during the first 6-h reaction was 0.076 and 0.17 mM h⁻¹, respectively, compared with 1.07 mM h⁻¹ in the absence of NO₂ ⁻ and NO₃ ⁻. Fe(III)EDTA and either NO₂ ⁻ or NO₃ ⁻ reduction occurred simultaneously. Interestingly, the reduction rate of NO₂ ⁻ or NO₃ ⁻ was enhanced in presence of Fe(III)EDTA. The inhibition patterns observed during the effect of NO₂ ⁻ and NO₃ ⁻ on the Fe(III)EDTA reduction experiments suggest that Escherichia coli can utilize NO₂ ⁻, NO₃ ⁻, and Fe(III)EDTA as terminal electron acceptors.     

Production of hexanoic acid from d-galactitol by a newly isolated Clostridium sp. BS-1

In a study screening anaerobic microbes utilizing d-galactitol as a fermentable carbon source, four bacterial strains were isolated from an enrichment culture producing H₂, ethanol, butanol, acetic acid, butyric acid, and hexanoic acid. Among these isolates, strain BS-1 produced hexanoic acid as a major metabolic product of anaerobic fermentation with d-galactitol. Strain BS-1 belonged to the genus Clostridium based on phylogenetic analysis using 16S rRNA gene sequences, and the most closely related strain was Clostridium sporosphaeroides DSM 1294^T, with 94.4% 16S rRNA gene similarity. In batch cultures, Clostridium sp. BS-1 produced 550 ± 31 mL L⁻¹ of H₂, 0.36 ± 0.01 g L⁻¹ of acetic acid, 0.44 ± 0.01 g L⁻¹ of butyric acid, and 0.98 ± 0.03 g L⁻¹ of hexanoic acid in a 4-day cultivation. The production of hexanoic acid increased to 1.22 and 1.73 g L⁻¹ with the addition of 1.5 g L⁻¹ of sodium acetate and 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), respectively. Especially when 1.5 g L⁻¹ of sodium acetate and 100 mM MES were added simultaneously, the production of hexanoic acid increased up to 2.99 g L⁻¹. Without adding sodium acetate, 2.75 g L⁻¹ of hexanoic acid production from d-galactitol was achieved using a coculture of Clostridium sp. BS-1 and one of the isolates, Clostridium sp. BS-7, in the presence of 100 mM MES. In addition, volatile fatty acid (VFA) production by Clostridium sp. BS-1 from d-galactitol and d-glucose was enhanced when a more reduced culture redox potential (CRP) was applied via addition of Na₂S·9H₂O.     

Biokinetic parameters and behavior of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> during anaerobic growth

The biokinetics of glucose metabolism were evaluated in Aeromonas hydrophila during growth in an anaerobic biosystem. After approx 34 h growth, A. hydrophila metabolized 5,000 mg glucose l⁻¹ into the end-products ethanol, acetate, succinate and formate. The maximum growth rate, μ _m, half saturation coefficients, K _s, microbial yield coefficient, Y, cell mass decay rate coefficient, k _d, and substrate inhibition coefficient, K _si were 0.25 ± 0.03 h⁻¹, 118 ± 31 mg glucose l⁻¹, 0.12 μg DNA mg glucose⁻¹, 0.01 h⁻¹, and 3,108 ± 1,152 mg glucose l⁻¹, respectively. These data were used to predict the performance of a continuous growth system with an influent glucose concentration of 5,000 mg l⁻¹. Results of the analysis suggest that A. hydrophila will metabolize glucose at greater than 95% efficiency when hydraulic retention times (HRTs) exceed 7 h, whereas the culture is at risk of washing out at an HRT of 6.7 h.     

Purification,characterization, and potential applications of formate oxidase from <Emphasis Type="Italic">Debaryomyces vanrijiae</Emphasis> MH201

Formate oxidase was found in cell-free extracts of Debaryomyces vanrijiae MH201, a soil isolate. After purification by column chromatography, the preparation showed a protein band corresponding to a molecular mass (MM) of 64 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The MM, estimated by a gel filtration, was 99 kDa. The preparation showed two and three bands on isoelectric focusing under denaturing and native conditions, respectively. These results suggest that the preparation contained three isoforms, each of which might be composed of αα, αβ, and ββ subunits with apparently similar MM. The preparation acted on formate with K _m and V _max values of 11.7 mM and 262 μmol min⁻¹ mg⁻¹, respectively, at pH 4.5 and 25°C, but showed no evidence of activity on the other compounds tested. The optimum pH and temperature were pH 4.0 and 35°C, respectively. The preparation showed activities of 85% of the initial activity after storage at pH 6.0 and 4°C for 8 weeks. When 10 mM formaldehyde was reacted with 2.0 U ml⁻¹ of the enzyme preparation at pH 5.5 and room temperature in the presence of 2.0 U ml⁻¹ of a microbial aldehyde oxidase and 100 U ml⁻¹ of catalase for 180 min, neither of formate nor formaldehyde was detected, suggesting that the reaction involved the quantitative conversion of formaldehyde to carbon dioxide.     

Autotrophic and heterotrophic respiration in needle fir and Quercus-dominated stands in a cool-temperate forest, central Korea

To investigate annual variation in soil respiration (R _S) and its components [autotrophic (R _A) and heterotrophic (R _H)] in relation to seasonal changes in soil temperature (ST) and soil water content (SWC) in an Abies holophylla stand (stand A) and a Quercus-dominated stand (stand Q), we set up trenched plots and measured R _S, ST and SWC for 2 years. The mean annual rate of R _S was 436 mg CO₂ m⁻² h⁻¹, ranging from 76 to 1,170 mg CO₂ m⁻² h⁻¹, in stand A and 376 mg CO₂ m⁻² h⁻¹, ranging from 82 to 1,133 mg CO₂ m⁻² h⁻¹, in stand Q. A significant relationship between R _S and its components and ST was observed over the 2 years in both stands, whereas a significant correlation between R _A and SWC was detected only in stand Q. On average over the 2 years, R _A accounted for approximately 34% (range 17–67%) and 31% (15–82%) of the variation in R _S in stands A and Q, respectively. Our results suggested that vegetation type did not significantly affect the annual mean contributions of R _A or R _H, but did affect the pattern of seasonal change in the contribution of R _A to R _S.     

Identification of random amplified polymorphic DNA (RAPD) markers for self-incompatibility alleles in Corylus avellana L.

 Random amplified polymorphic DNA (RAPD) markers were identified for self-incompatibility (SI) alleles that will allow marker-assisted selection of desired S-alleles in hazelnut (Corylus avellana L.). DNA was extracted from young leaves collected from field-planted parents and 26 progeny of the cross OSU 23.017 (S₁S₁₂)×VR6-28 (S₂S₂₆) (OSU23×VR6). Screening of 10-base oligonucleotide RAPD primers was performed using bulked segregant analysis. DNA samples from 6 trees each were pooled into four ‘bulks’, one for each of the following: S₁ S₂, S₁ S₂₆ ^, S₂ S₁₂, and S₁₂ S₂₆. ‘Super bulks’ of 12 trees each for S₁, S₂, S₁₂, and S₂₆ were then created for each allele by combining the appropriate bulks. The DNA from these four super bulks and from the parents was used as a template in the PCR assays. A total of 250 primers were screened, and one RAPD marker each was identified for alleles S₂ (OPI07₇₅₀) and S₁ (OPJ14₁₇₀₀). OPJ14₁₇₀₀ was identified in 13 of 14 S₁ individuals of the cross OSU23×VR6 used in bulking and yielded a false positive in 1 non-S₁ individual. This same marker was not effective outside the original cross, identifying 4 of 5 S₁ progeny in another cross, ‘Willamette’×VR6-28 (‘Will’×VR6), but yielded false positives in 4 of 9 non-S₁ individuals from the cross ‘Casina’×VR6-28 (‘Cas’×VR6). OPI07₇₅₀ served as an excellent marker for the S₂ allele and was linked closely to this allele, identifying 12 of 13 S₂ individuals in the OSU23×VR6 population with no false positives. OPI07₇₅₀ was found in 4 of 4 S₂ individuals from ‘Will’×VR and 7 of 7 S₂ individuals of ‘Cas’×VR6 with no false positives, as well as 10 of 10 S₂ individuals of the cross OSU 296.082 (S₁S₈)×VR8-32 (S₂S₂₆), with only 1 false positive individual out of 21 progeny. OPI07₇₅₀ was also present in 5 of 5 cultivars carrying the S₂ allele, with no false-positive bands in non-S₂ cultivars, and correctly identified all but 2 S₂ individuals in 57 additional selections in the breeding program. In the OSU23×VR6 population, the recombination rate between the marker OPJ14₁₇₀₀ and the S₁ allele was 7.6% and between the OPI07₇₅₀ marker and the S₂ allele was 3.8%. RAPD marker bands were excised from gels, cloned, and sequenced to enable the production of longer primers (18 or 24 bp) that were used to obtain sequence characterized amplified regions (SCARs). Both the S₁ and S₂ markers were successfully cloned and 18 bp primers yielded the sole OPJ14₁₇₀₀ product, while 24-bp primers yielded OPI07₇₅₀ as well as an additional smaller product (700 bp) that was not polymorphic but was present in all of the S-genotypes examined. Received: 10 January 1998 / Accepted: 26 January 1998     

The reduced S<Subscript>−1</Subscript> and S<Subscript>−2</Subscript> oxidation states of the O<Superscript>2</Superscript>-evolving complex of photosystem II: An EPR microwave power saturation study

Progressive microwave power saturation (P_1/2) measurements have been performed on the tyrosine D radical (Y_D ^•) of photosystem II (PSII) in order to examine its relaxation enhancement by the oxygen-evolving complex (OEC) poised to the reduced S₋₁ and S₋₂ oxidation states by NO treatment. Analysis of the power saturation curves showed that the S₋₁ oxidation state of the OEC does not enhance the relaxation of Y_D ^•: it therefore possesses a diamagnetic ground state. In contrast, the Mn(II)-Mn(III) multiline electron paramagnetic resonance (EPR) signal characteristic of the S₋₂ oxidation state of the OEC was shown to provide a relaxation enhancement pathway for Y_D ^•, however less efficient relative to the one provided by the S₂-state multiline EPR signal. We also examined the Y_D ^• relaxation enhancement characteristics of the EPR-silent oxidation state produced after brief (1–5 min) dark incubation at 0°C of a PSII sample poised to the EPRactive S₋₂ state. This EPR-silent oxidation state denoted as “0°C incubation” state was shown to possess remarkably similar P_1/2 values with the EPR-active S₋₂ state in the overall examined temperature range (6–20 K). In addition, these values remained unchanged after successive cycles of the OEC between the EPR-active S₋₂ state and the “0°C incubation” state. The data presented in this work point to the conclusion that the “0°C incubation” state is indeed an S₋₂ oxidation state with half-integer spin.     

Effect of trace metals on ethanol production from synthesis gas by the ethanologenic acetogen, <Emphasis Type="Italic">Clostridium</Emphasis><Emphasis Type="Italic">ragsdalei</Emphasis>

The effect of trace metal ions (Co²⁺, Cu²⁺, Fe²⁺, Mn²⁺, Mo⁶⁺, Ni²⁺, Zn²⁺, SeO₄ ⁻ and WO₄ ⁻) on growth and ethanol production by an ethanologenic acetogen, Clostridium ragsdalei was investigated in CO:CO₂-grown cells. A standard acetogen medium (ATCC medium no. 1754) was manipulated by varying the concentrations of trace metals in the media. Increasing the individual concentrations of Ni²⁺, Zn²⁺, SeO₄ ⁻ and WO₄ ⁻ from 0.84, 6.96, 1.06, and 0.68 μM in the standard trace metals solution to 8.4, 34.8, 5.3, and 6.8 μM, respectively, increased ethanol production from 35.73 mM under standard metals concentration to 176.5, 187.8, 54.4, and 72.3 mM, respectively. Nickel was necessary for growth of C. ragsdalei. Growth rate (μ) of C. ragsdalei improved from 0.34 to 0.49 (day⁻¹), and carbon monoxide dehydrogenase (CODH) and hydrogenase (H₂ase)-specific activities improved from 38.45 and 0.35 to 48.5 and 1.66 U/mg protein, respectively, at optimum concentration of Ni²⁺. At optimum concentrations of WO₄ ⁻ and SeO₄ ⁻, formate dehydrogenase (FDH) activity improved from 32.3 to 42.6 and 45.4 U/mg protein, respectively. Ethanol production and the activity of FDH reduced from 35 mM and 32.3 U/mg protein to 1.14 mM and 8.79 U/mg protein, respectively, upon elimination of WO₄ ⁻ from the medium. Although increased concentration of Zn²⁺ enhanced growth and ethanol production, the activities of CODH, FDH, H₂ase and alcohol dehydrogenase (ADH) were not affected by varying the Zn²⁺ concentration. Omitting Fe²⁺ from the medium decreased ethanol production from 35.7 to 6.30 mM and decreased activities of CODH, FDH, H₂ase and ADH from 38.5, 32.3, 0.35, and 0.68 U/mg protein to 9.07, 7.01, 0.10, and 0.24 U/mg protein, respectively. Ethanol production improved from 35 to 54 mM when Cu²⁺ was removed from the medium. The optimization of trace metals concentration in the fermentation medium improved enzyme activities (CODH, FDH, and H₂ase), growth and ethanol production by C. ragsdalei.     

Functional Characterization of the Microbial Community in Geothermally Heated Marine Sediments

The microbial population of geothermally heated sediments in a shallow bay of Vulcano Island (Italy) was characterized with respect to metabolic activities and the putatively catalyzing hyperthermophiles. Site-specific anoxic culturing media, most of which were amended with combinations of electron donors (glucose or carboxylic acids) and acceptors (sulfate), were used for selective enrichment of metabolically defined subpopulations. The mostly archaeal chemoautotrophs produced formate at rates of 3.25 and 0.46 fmol cell⁻¹ day⁻¹ with and without sulfate, respectively. The glucose fermenting heterotrophs produced acetate (18 fmol cell⁻¹ day⁻¹) and lactate (2.6 fmol cell⁻¹ day⁻¹) and were identified as predominantly Thermus sp. and coccoid archaea. These archaeal cells also metabolized lactate (5.6 fmol cell⁻¹ day⁻¹), but neither formate nor acetate. The heterotrophic culture enriched on formate/acetate/propionate/sulfate utilized mainly formate (27 fmol cell⁻¹ day⁻¹) and lactate (89–195 fmol cell⁻¹ day⁻¹), and consumed sulfate (38–68 fmol cell⁻¹ day⁻¹). These formate or lactate consuming sulfate reducers were dominated by Archaeoglobales (7% in situ) and unidentified Archaea. The in situ benthic community comprised 15% Crenarchaeota, a significant group only in the autotrophic cultures, and 3% Thermus sp., the putatively predominant group involved in fermentative metabolism. The role of Thermoccales (4% in situ) remained undisclosed in our experiments. This first comprehensive data set established plausible links between several groups of hyperthermophiles in shallow marine hydrothermal systems, their metabolic function within the benthic microbial community, and biogeochemical turnover rates.     

A mass spectrometric investigation of the binding of gold antiarthritic agents and the metabolite [Au(CN)2]− to human serum albumin

Electrospray ionisation (ESI) mass spectrometry was used to examine the reactions of the clinically used antiarthritic agent [Au(S₂O₃)₂]³⁻, and AuPEt₃Cl, a derivative of another clinically used agent auranofin, with human serum albumin (HSA) obtained from a human volunteer. Both compounds reacted readily with HSA to form complexes containing one or more covalently attached gold fragments. In the case of AuPEt₃Cl, binding was accompanied by the loss of the chloride ligand, while for [Au(S₂O₃)₂]³⁻ the mass spectral data indicated binding of Au(S₂O₃) groups. Experiments performed using HSA with Cys34 blocked by reaction with iodoacetamide were consistent with reaction of both gold compounds with this amino acid. Separate blocking experiments using diethylpyrocarbonate and AuPEt₃Cl also provided evidence for histidine residues acting as lower-affinity binding sites for this gold compound. ESI mass spectra of solutions containing [Au(S₂O₃)₂]³⁻ or [Au(CN)₂]⁻, and HSA, provided evidence for the formation of protein complexes in which intact gold molecules were non-covalently bound. In the case of [Au(S₂O₃)₂]³⁻, these non-covalent complexes proved to be transitory in nature. However, for [Au(CN)₂]⁻ a non-covalent complex containing a single gold molecule bound to HSA was found to be stable, and constituted the main adduct formed in solutions containing low-to-medium Au-to-HSA ratios. Evidence was also obtained for the formation of a covalent adduct in which a single Au(CN) moiety was bonded to Cys34 of the protein. AuPEt₃Cl reacted to a much lower extent with HSA that had Cys34 modified by formation of a disulfide bond to added cysteine, than with unmodified HSA. This suggests that the extent of modification of the protein in vivo may have an important influence on the transport and bioavailability of gold antiarthritic drugs.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Glucose Fermentation by <Emphasis Type="Italic">Propionibacterium microaerophilum</Emphasis>: Effect of pH on Metabolism and Bioenergetic

pH affected significantly the growth and the glucose fermentation pattern of Propionibacterium microaerophilum. In neutral conditions (pH 6.5–7.5), growth and glucose fermentation rate (qs) were optimum producing propionate, acetate, CO₂, and formate [which together represented 90% (wt/wt) of the end products], and lactate representing only 10% (wt/wt) of the end products. In acidic conditions, propionate, acetate, and CO₂ represented nearly 100% (wt/wt) of the fermentation end products, whereas in alkaline conditions, a shift of glucose catabolism toward formate and lactate was observed, lactate representing 50% (wt/wt) of the fermentation end products. The energy cellular yields (Y _X/ATP), calculated (i) by taking into account extra ATP synthesized through the reduction of fumarate into succinate, was 6.1–7.2 g mol⁻¹. When this extra ATP was omitted, it was 11.9–13.1 g mol⁻¹. The comparison of these values with those of Y _X/ATP in P. acidipropionici and other anaerobic bacteria suggested that P. microaerophilum could not synthesize ATP through the reduction of fumarate into succinate and therefore differed metabolically from P. acidipropionici. Received: 8 April 2002 / Accepted: 8 May 2002     

Enantioselective hydrolysis of (R)-2, 2-dimethylcyclopropane carboxamide by immobilized cells of an R-amidase-producing bacterium, Delftia tsuruhatensis CCTCC M 205114, on an alginate capsule carrier

Immobilized cells of Delftia tsuruhatensis CCTCC M 205114 harboring R-amidase were applied in asymmetric hydrolysis of (R)-2, 2-dimethylcyclopropane carboxamide (R − 1) from racemic (R, S)-2, 2-dimethylcyclopropane carboxamide to accumulate (S)-2, 2-dimethylcyclopropane carboxamide (S − 1). Maximum R-amidase activity of 13.1 U/g wet cells (0.982 U/g beads) was obtained under conditions of 3% sodium alginate, 2.5% CaCl₂, 15 h crosslinking and 2 mm bead size, which was 53.9% of that of free cells (24.3 U/g wet cells). In addition, characterization of the immobilized cells was examined. The optimum R − 1 hydrolysis conditions were identified as follows: substrate concentration 10 mM, pH 8.5, temperature 35°C and time course 40 min. Under optimum conditions, the maximum yield and enantiomeric excess of (R)-2, 2-dimethylcyclopropanecarboxylic acid were 49.5% and >99%, respectively. This afforded S − 1 with a yield >49% and an e.e. of 97.7%. With good operational stability and excellent enanotioselectivity, the immobilized cells could be potentially utilized in industrial production of S − 1.