Thermo-alkali-stable catalases from newly isolated Bacillus sp. for the treatment and recycling of textile bleaching effluents

Three thermoalkaliphilic bacteria, which were grown at pH 9.3–10 and 60–65 °C were isolated out of a textile wastewater drain. The unknown micro-organisms were identified as thermoalkaliphilic Bacillus sp. Growth conditions were studied and catalase activities and stabilities compared. Catalases from Bacillus SF showed high stabilities at 60 °C and pH 9 (t_1/2=38 h) and thus this strain was chosen for further investigations, such as electron microscopy, immobilization of catalase and hydrogen peroxide degradation studies. Degradation of hydrogen peroxide with an immobilized catalase from Bacillus SF enabled the reuse of the water for the dyeing process. In contrast, application of the free enzyme for treatment of bleaching effluents, caused interaction between the denaturated protein and the dye, resulting in reduced dye uptake, and a higher color difference of 1.3 ΔE* of dyed fabrics compared to 0.9 ΔE* when using the immobilized enzyme.     

Improved high thermal stability of pullulanase from a newly isolated thermophilic Bacillus sp. AN-7

A thermophilic Bacillus sp. strain AN-7, isolated from a soil in India, produced an extracellular pullulanase upon growth on starch–peptone medium. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The optimum temperature and pH for activity was 90 °C and 6.0. With half-life time longer than one day at 80 °C the enzyme proves to be thermostable in the pH range 4.5–7.0. The pullulanase from Bacillus strain lost activity rapidly when incubated at temperature higher than 105 °C or at pH lower than 4.5. Pullulanase was completely inhibited by the Hg²⁺ ions. Ca²⁺, dithiothreitol, and Mn²⁺ stimulated the pullulanase activity. Kinetic experiments at 80 °C and pH 6.0 gave V_max and K_m values of 154 U mg⁻¹ and 1.3 mg ml⁻¹. The products of pullulan were maltotriose and maltose. This proved that the purified pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) from Bacillus sp. AN-7 is classified under pullulanase type I. To our knowledge, this Bacillus pullulanase is the most highly thermostable type I pullulanase known to date.     

Hydrogen peroxide degradation by immobilized cells of alkaliphilic Bacillus halodurans

Whole cells of Bacillus halodurans LBK 261 were used as a source of catalase for degradation of hydrogen peroxide. The organism, B. halodurans grown at 55°C and pH 10, yielded a maximum catalase activity of 275 U g^-1 (wet wt.) cells. The catalase in the whole cells was active over a broad range of pH with a maximum at pH 8-9. The enzyme was optimally active at 55°C, but had low stability above 40°C. The whole cell biocatalyst exhibited a K_m of 6.6 mM for H₂O₂ and V_max of 707 mM H₂O₂ min^-1 g^-1 wet wt. cells, and showed saturation kinetics at 50 mM H₂O₂. The cells were entrapped in calcium alginate and used for H₂O₂ degradation at pH 9 in batch and continuous mode. In the batch process, the immobilized preparation containing 1.5 g (wet wt.) cells could be recycled at least four times for complete degradation of the peroxide in 50 mL solution at 25°C. An excess of immobilized biocatalyst could be used in a continuous stirred tank reactor for an average of 9 days at temperatures upto 55°C, and in a packed bed reactor (PBR) for 5 days before the beads started to deform.     

嗜麦芽寡养单胞菌PX1的降解芘特性及定殖效能

为丰富多环芳烃降解菌菌种库、降低农作物的污染风险,本研究对一株可高效降解多环芳烃(PAHs)的植物内生菌进行筛选鉴定,并初步探究其降解途径以及定殖效能。结果表明: 菌株PX1为嗜麦芽寡养单胞菌。该菌株对多环芳烃的降解具有广谱性,7 d几乎可彻底降解PAH无机盐培养基中的萘,在分别含有50.0 mg·L^-1菲、20.0 mg·L^-1芘、20.0 mg·L^-1荧蒽和10.0 mg·L^-1苯并[a]芘的培养体系中,对菲、芘、荧蒽、苯并[a]芘的降解率分别为72.6%、50.7%、31.9%和12.9%。选取芘作为PAHs模型研究菌株PX1的降解特性。酶活性试验表明,芘可诱导菌株PX1体内邻苯二甲酸双加氧酶、邻苯二酚-1,2-双加氧酶和邻苯二酚-2,3-双加氧酶的活性。在芘降解过程中检测到4,5-环氧化芘、4,5-二羟基芘、龙胆酸/原茶儿酸、水杨酸、顺-己二烯二酸/2-羟粘糠酸半醛、顺-2′-羧基苯丙酮酸、1-羟基-2-萘甲酸、水杨醛等中间产物。浸种定殖试验表明,菌株PX1可高效定殖到空心菜和小麦体内,显著促进空心菜和小麦生长,并能够将空心菜、小麦体内及其生长基质中的芘浓度分别降低29.8%～50.7%、52.4%～67.1%和8.0%～15.3%。表明菌株PX1主要通过“水杨酸途径”和“邻苯二甲酸途径”降解芘,且可以定殖到植物体内,促进植物生长。     

Hydrogen and polyhydroxybutyrate producing abilities of microbes from diverse habitats by dark fermentative process

Thirty five bacterial isolates from diverse environmental sources such as contaminated food, nitrogen rich soil, activated sludges from pesticide and oil refineries effluent treatment plants were found to belong to Bacillus, Bordetella, Enterobacter, Proteus, and Pseudomonas sp. on the basis of 16S rRNA gene sequence analysis. Under dark fermentative conditions, maximum hydrogen (H₂) yields (mol/mol of glucose added) were recorded to be 0.68 with Enterobacter aerogenes EGU16 followed by 0.63 with Bacillus cereus EGU43 and Bacillus thuringiensis EGU45. H₂ constituted 63–69% of the total biogas evolved. Out of these 35 microbes, 18 isolates had the ability to produce polyhydroxybutyrate (PHB), which varied up to 500 mg/l of medium, equivalent to a yield of 66.6%. The highest PHB yield was recorded with B. cereus strain EGU3. Nine strains had high hydrolytic activities (zone of hydrolysis): lipase (34–38 mm) – Bacillus sphaericus strains EGU385, EGU399 and EGU542; protease (56–62 mm) – Bacillus sp. strains EGU444, EGU447 and EGU445; amylase (23 mm) – B. thuringiensis EGU378, marine bacterium strain EGU409 and Pseudomonas sp. strain EGU448. These strains with high hydrolytic activities had relatively low H₂ producing abilities in the range of 0.26–0.42 mol/mol of glucose added and only B. thuringiensis strain EGU378 had the ability to produce PHB. This is the first report among the non-photosynthetic microbes, where the same organism(s) – B. cereus strain EGU43 and B. thuringiensis strain EGU45, have been shown to produce H₂ – 0.63 mol/mol of glucose added and PHB – 420–435 mg/l medium.     

Characterization of growth hormone-releasing hormone (GHRH) binding to cloned porcine GHRH receptor

To study structure-activity relationships of growth hormone-releasing hormone (GHRH), a competitive binding assay was developed using cloned porcine adenopituitary GHRH receptors expressed in human kidney 293 cells. Specific binding of [His¹,¹²⁵I-Tyr¹⁰,Nle²⁷]hGHRH(1–32)-NH₂ increased linearly with protein concentration (10–45 μg protein/tube). Binding reached equilibrium after 90 min at 30°C and remained constant for at least 240 min. Binding was reversible to one class of high-affinity sites (K_d = 1.04 ± 0.19 nM, B_max = 3.9 ± 0.53 pmol/mg protein). Binding was selective with a rank order of affinity (IC₅₀) for porcine GHRH (2.8 ± 0.51 nM), rat GHRH (3.1 ± 0.69 nM), [N-Ac-Tyr¹, -Arg²]hGHRH(3–29)-NH₂ (3.9 ± 0.58 nM), and [ -Thr⁷]GHRH(1–29)-NH₂ (189.7 ± 14.3 nM), consistent with their binding to a GHRH receptor. Nonhydrolyzable guanine nucleotides inhibited binding. These data describe a selective and reliable method for a competitive GHRH binding assay that for the first time utilizes rapid filtration to terminate the binding assay.     

Purification and characterization of a Na/K dependent alginate lyase from turban shell gut Vibrio sp. YKW-34

An alginate lyase with high specific enzyme activity was purified from Vibrio sp. YKW-34, which was newly isolated from turban shell gut. The alginate lyase was purified by in order of ion exchange, hydrophobic and gel filtration chromatographies to homogeneity with a recovery of 7% and a fold of 25. This alginate lyase was composed of a single polypeptide chain with molecular mass of 60 kDa and isoelectric point of 5.5–5.7. The optimal pH and temperature for alginate lyase activity were pH 7.0 and 40 °C, respectively. The alginate lyase was stable over pH 7.0–10.0 and at temperature below 50 °C. The alginate lyase had substrate specificity for both poly-guluronate and poly-mannuronate units. The k_cat/K_m value for alginate (heterotype) was 1.7 × 10⁶ s⁻¹ M⁻¹. The enzyme activity was completely lost by dialysis and restored by addition of Na⁺ or K⁺. The optimal activity exhibited in 0.1 M of Na⁺ or K⁺. This enzyme was resistant to denaturing reagents (SDS and urea), reducing reagents (β-mercaptoethanol and DTT) and chelating reagents (EGTA and EDTA).     

来源于嗜碱芽孢杆菌N16-5甘露聚糖利用基因簇的乙酰酯酶AesA的克隆及性质分析*

天然来源的多糖底物上常存在乙酰基取代,特异性的乙酰酯酶能够切割这些底物上的乙酰基,从而有利于聚糖底物的进一步降解.对Bacillus sp. N16-5甘露聚糖利用基因簇上编码的乙酰酯酶AesA进行了基因克隆和异源表达,并对其酶学性质进行了研究.aesA基因长957bp,编码318个氨基酸,属于碳水化合物酯酶第7家族.AesA对4-甲基伞形酮乙酸酯(4-methylumbelliferyl-acetate)表现出较好的催化活性,金属离子Fe³⁺,Fe²⁺,Mn²⁺及Cu²⁺对AesA活性均有不同程度的促进作用.AesA与甘露聚糖酶ManA对乙酰化的甘露聚糖底物具有显著的协同作用.此项研究有助于理解嗜碱芽孢杆菌Bacillus sp.N16-5对甘露聚糖的水解机制,并且在甘露聚糖降解中具有潜在的应用前景.     

Determination of the maximum and minimum lethal temperatures (LT50) for Loxosceles intermedia Mello-Leitão, 1934 and L. laeta (Nicolet, 1849) (Araneae, Sicariidae)

In this study, the maximum and minimum lethal temperatures (LT₅₀) of L. intermedia and L. laeta were determined in two treatments: gradual heating (25–50°C) and cooling (25°C to −5°C), and 1 h at a constant temperature. In gradual temperatures change, L. intermedia mortality started at 40°C and the LT₅₀ was 42°C; for L. laeta, mortality began at 35°C and the LT₅₀ was 40°C. At low temperatures, mortality was registered only at −5°C for both species. In the constant temperature L. intermedia showed a maximum LT₅₀ at 35°C and L. laeta at 32°C; the minimum LT for both species was −7°C.     

Studies on composition and stability of a large membered bacterial consortium degrading phenol

A ten member microbial consortium (AS) consisting of eight phenol-degrading and two non-phenol-degrading strains of bacteria was developed and maintained in a fed-batch reactor by feeding 500 mg l⁻¹ phenol for four years at 28 ± 3 °C. The consortium could degrade 99% of 500 mg l⁻¹ phenol after 24 hours incubation with a biomass increase of 2.6 × 10⁷ to 4 × 10¹² CFU ml⁻¹. Characterization of the members revealed that it consisted of 4 principal genera, Bacillus, Pseudomonas, Rhodococcus, Streptomyces and an unidentified bacterium. Phenol degradation by the mixed culture and Bacillus subtilis, an isolate from the consortium was compared using a range of phenol concentrations (400 to 700 mg l⁻¹) and by mixing with either 160 mg l⁻¹ glucose or 50 mg l⁻¹ of 2,4-dichlorophenol in the medium. Simultaneous utilization of unrelated mixed substrates (glucose/2,4-dichlorophenol) by the consortium and Bacillus subtilis, indicated the diauxic growth pattern of the organisms. A unique characteristic of the members of the consortia was their ability to oxidize chloro aromatic compounds via meta pathway and methyl aromatic compounds via ortho cleavage pathway. The ability of a large membered microbial consortia to maintain its stability with respect to its composition and effectiveness in phenol degradation indicated its suitability for bioremediation applications.     

Plant growth-promoting Pseudomonas bearing catabolic plasmids: Naphthalene degradation and effect on plants

Two IncP-9 naphthalene degradative plasmids pOV17 and pBS216 were transferred into plant growth-promoting Pseudomonas which were represented by species P. aureofaciens, P. chlororaphis, P. fluorescens, and P. putida. The strains with the same plasmid differed significantly by their growth parameters, stability of the plasmid and plant protective effect from naphthalene action. Strains P. putida 53a(pOV17) and P. chlororaphis PCL1391(pOV17) demonstrated higher population number in the rhizosphere. Moreover, they protected the mustard plants from naphthalene toxic influence more effectively than the wild type strain P. aureofaciens OV17(pOV17). The activity of catechol-2,3-dioxygenase in the strains with the plasmid pOV17 was higher than that in strains with the plasmid pBS216. The strain P. putida 53a(pOV17) with high catechol-2,3-dioxygenase activity has been demonstrated to have the best protective effect. The strain P. putida 53a(pBS216) without catechol dioxygenases activities did not have protective effect but suppressed the plant germination.     

Biodegradation of trichloroethylene and involvement of an aromatic biodegradative pathway

Biodegradation of trichloroethylene (TCE) by bacterial strain G4 resulted in complete dechlorination of the compound, as indicated by the production of inorganic chloride. A component of the water from which strain G4 was isolated that was required for TCE degradation was identified as phenol. Strain G4 degraded TCE in the presence of chloramphenicol only when preinduced with phenol. Toluene, o-cresol. and m-cresol could replace the phenol requirement. Two of the inducers of TCE metabolism, phenol and toluene, apparently induced the same aromatic degradative pathway that cleaved the aromatic ring by meta fission. Cells induced with either phenol or toluene had similar oxidation rates for several aromatic compounds and had similar levels of catechol-2,3-dioxygenase. The results indicate that one or more enzymes of an inducible pathway for aromatic degradation in strain G4 are responsible for the degradation of TCE.     

Biodegradation of trichloroethylene and involvement of an aromatic biodegradative pathway.

Biodegradation of trichloroethylene (TCE) by bacterial strain G4 resulted in complete dechlorination of the compound, as indicated by the production of inorganic chloride. A component of the water from which strain G4 was isolated that was required for TCE degradation was identified as phenol. Strain G4 degraded TCE in the presence of chloramphenicol only when preinduced with phenol. Toluene, o-cresol. and m-cresol could replace the phenol requirement. Two of the inducers of TCE metabolism, phenol and toluene, apparently induced the same aromatic degradative pathway that cleaved the aromatic ring by meta fission. Cells induced with either phenol or toluene had similar oxidation rates for several aromatic compounds and had similar levels of catechol-2,3-dioxygenase. The results indicate that one or more enzymes of an inducible pathway for aromatic degradation in strain G4 are responsible for the degradation of TCE.     

Estrone sulfate (E1S), a prognosis marker for tumor aggressiveness in prostate cancer (PCa)

Seeking insight into the possible role of estrogens in prostate cancer (PCa) evolution, we assayed serum E₂, estrone (E₁), and estrone sulfate (E₁S) in 349 PCa and 100 benign prostatic hyperplasia (BPH) patients, and in 208 control subjects in the same age range (50–74 years).

E₁ (pmol/L ± S.D.) and E₁S (nmol/L ± S.D.) in the PCa and BPH patients (respectively 126.1 ± 66.1 and 2.82 ± 1.78, and 127.8 ± 56.4 and 2.78 ± 2.12) were significantly higher than in the controls (113.8 ± 47.6 and 2.11 ± 0.96). E₂ was not significantly different among the PCa, BPH, and control groups. These assays were also carried out in PCa patients after partition by prognosis (PSA, Gleason score (GS), histological stage, and surgical margins (SM)). Significantly higher E₁S levels were found in PCa with: PSA > 10 ng/L (3.05 ± 1.92) versus PSA ≤ 10 ng/mL (2.60 ± 1.55), stage pT3-T4 (2.99 ± 1.80) versus pT2 (2.58 ± 1.58), and positive (3.26 ± 1.95) versus negative margins (2.52 ± 1.48). E₁ was higher in poor- than in better-prognosis PCa. E₂ was significantly higher in PCa with GS ≥ 4 + 3 (109.5 ± 43.8) versus GS ≤ 3 + 4 (100.6 ± 36.5) and increased significantly when GS increased from 3 + 3 to 4 + 4. Estrogens, especially E₁S appeared to be possible markers of PCa progression.

Attempting to identify potential sources of E₂ in PCa according to prognosis, as well as in BPH, we found a significant correlation coefficient between E₁S and E₂ (0.266–0.347) in poor-prognosis PCa and no correlation in BPH (0.026) and better-prognosis PCa (0.013–0.104).

It is as though during progression of PCa from good to poor prognosis there were a shift in the E₁ to E₂ metabolic pathway from predominantly oxidative to predominantly reductive.     

Characterization of esterases in malathion-resistant and susceptible strains of the pteromalid parasitoid Anisopteromalus calandrae

General esterase, malathion-specific carboxylesterase, phosphotriesterase, glutathione S-transferase, cytochrome P-450-dependent monooxygenase activity, and target site sensitivity were compared in malathion-resistant (R) and malathion-susceptible (S) strains of the parasitoid Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae). Activity against -naphthyl acetate was not significantly different in male and female wasps for either strain. General esterase activity ranged from 1.2-fold to 2.5-fold higher in the R strain compared with the S strain, but these differences between strains were not consistent. Based on V_max/K_m ratios estimated for a number of analogs of four substrates (-naphthyl acetate, β-naphthyl acetate, 4-methylumbelliferyl acetate, and p-nitrophenyl acetate) there was no evidence that general esterase activity was elevated or reduced in the R strain. Malathion-specific carboxylesterase (MCE) activity, determined by using 2,3-¹⁴C-malathion as substrate, was 10- to 30-fold higher in the R strain compared with that in the S strain. The MCE has a pH optima at about pH 7, is cytosolic, and is labile upon storage at −80°C. MCE activity could be recovered from native 10% PAGE gels and IEF–PAGE gels (pI=5.2), but the peak of MCE activity also contained the major peak of activity against -naphthyl acetate. There was no evidence for major involvement of phosphotriesterase, glutathione S-transferase, monooxygenase, or altered acetylcholinesterase in the resistance. These data suggest that an increased activity of a MCE in the R strain is the probable major mechanism conferring resistance to malathion in A. calandrae. This study provides the first characterization of a biochemical resistance mechanism in a parasitoid with a high level of resistance to an organophosphate insecticide.     

Production d'NH4 par la crevette Crangon crangon L. dans deux écosystémes côtiers. approche expérimentale et étude de l'influence du sédiment sur le taux d'excrétion

Estimation of the ammonia production of the shrimp C. crangon in two littoral ecosystems (oligotrophic sand and eutrophic mud) was determined in winter and summer conditions from laboratory observations in experimental microcosms. The ammonia excretion rate of C. crangon was not influenced by either the sediment type or the ammonia concentration of the overlying water; on the other hand, the mean excretion rate and the response to initial handling stress increased markedly as shrimp were deprived of soft substratum.

The daily ammonia production of C. crangon was 16 μmol NH₃ · g ⁻¹ wet wt · day ⁻¹ in winter and 40 μmol in summer. A gross production of 12 μmol NH₃ · m⁻² · day ⁻¹ and 300–700 μmol μ m⁻² · day⁻¹, respectively, could be expected in the two ecosystems studied. This would account for 5% (winter) and 2–4% (summer) of the total NH⁺₄ flux at the sediment-water interface. The contribution of the excretion of all macrofauna to the NH⁺₄ flux from the sediment is discussed.     

An isolated Candida albicans TL3 capable of degrading phenol at large concentration

An isolated yeast strain was grown aerobically on phenol as a sole carbon source up to 24 mM; the rate of degradation of phenol at 30 degrees C was greater than other microorganisms at the comparable phenol concentrations. This microorganism was further identified and is designated Candida albicans TL3. The catabolic activity of C. albicans TL3 for degradation of phenol was evaluated with the K(s) and V(max) values of 1.7 +/- 0.1 mM and 0.66 +/- 0.02 micromol/min/mg of protein, respectively. With application of enzymatic, chromatographic and mass-spectrometric analyses, we confirmed that catechol and cis,cis-muconic acid were produced during the biodegradation of phenol performed by C. albicans TL3, indicating the occurrence of an ortho-fission pathway. The maximum activity of phenol hydroxylase and catechol-1,2-dioxygenase were induced when this strain grew in phenol culture media at 22 mM and 10 mM, respectively. In addition to phenol, C. albicans TL3 was effective in degrading formaldehyde, which is another major pollutant in waste water from a factory producing phenolic resin. The promising result from the bio-treatment of such factory effluent makes Candida albicans TL3 be a potentially useful strain for industrial application.     

Structural determinations, magnetic and EPR studies of complexes involving the Cr(OH)2Cr unit

Five heterometallic compounds with formulae [Ba(H₂O)₄Cr₂(μ-OH)₂(nta)₂] · 3H₂O (I), [M(bpy)₂(H₂O)₂] [Cr₂(OH)₂(nta)₂] · 7H₂O, where M²⁺ = Zn, (II); Ni, (III); Co, (IV) and [Mn(H₂O)₃(bpy)Cr₂(OH)₂(nta)₂] · (bpy) · 5H₂O (V); bpy = 2,2′-bipyridine, (nta = nitrilotriacetate ion) have been prepared by reaction of I with the corresponding M^II-sulfates in the presence of 2,2′-bipyridine. Substances I–V have been characterized by magnetic susceptibility measurements, EPR and X-ray determinations. I represents a 2D coordination polymer formed by coordination of centrosymmetrical dimeric chromium(III) units and Barium cations. The 10-coordinate Ba polyhedron is completed by four water molecules. Compounds II–IV are isostructural and consist of non-centrosymmetric dimeric anions [Cr₂(μ-OH)₂(nta)₂]²⁻, complex cations [M^II(bpy)₂(H₂O)₂]²⁺ and solvate water molecules. The octahedral coordination of chromium atoms implies four donor atoms of the nta³⁻ ligands and two bridging OH groups. Multiple hydrogen bonds of coordinated and solvate water molecules link anions and cations in a 3D network. A similar [Cr₂(μ-OH)₂(nta)₂]²⁻ unit is found in V. The bridging function is performed by a carboxylate oxygen atom of the nta ligand that leads to the formation of a trinuclear complex [Mn(bpy)(H₂O)₂Cr₂(μ-OH)₂(nta)₂]. Experimental and calculated frequency and temperature dependences of EPR spectra of these compounds are presented. The fine structure appearing on the EPR spectra of compound V is analyzed in detail at different temperatures. It is established that the main part of the EPR signals is due to the transitions in the spin states of a spin multiplet with S = 2. Analyses of experimental and calculated spectra confirm the absence of interaction between metal ions (M^II) and Cr-dimers in complexes III and IV and the presence of weak Mn–Cr interactions in V. The temperature dependence of magnetic susceptibilities for I–V was fitted on the basis of the expression derived from isotropic Hamiltonian including a bi-quadratic exchange term.     

Heterobimetallic aggregates of copper(I) with thiotungstates and thiomolybdates. Synthesis and characterization of polymeric (NEt4)3[Cu4(NCS)5WS4], (NEt4)2[(CuNCS)3WS4] and (NEt4)2[(CuNCS)4WS4], M = Mo, W

Reaction of (NEt₄)₂MS₄ (M = Mo, W) with CuCl and KSCN (or NH₄SCN) in acetone or acetonitrile affords a new set of mixed metal–sulfur compounds: infinite anionic chains Cu₄(NCS)₅MS_4³⁻ (1,2), (CuNCS)₃WS_4²⁻ (3) and two dimensional polymeric dianions (CuNCS)₄MS_4²⁻ (4,5). Crystal of 1 (M = W) and 3 are triclinic, space group P1(1:a = 10.356(2),b = 15.039(1),c = 17.356(2)Å, = 78.27(1)°, β = 88.89(2)° and γ = 88.60(1)°,Z = 2,R = 0.04 for 3915 independent data;3:a = 8.449(2),b = 14.622(4),c = 15.809(8)Å, = 61.84(3)°, β = 73.67(3)° and γ = 78.23(2)°,Z = 2,R = 0.029 for 6585 independent data). Crystals of 4 (M = W) and 5 (M = Mo) are monoclinic, space group P2₁/m,Z = 2 (4:a = 12.296(4),b = 14.794(4),c = 10.260(3)Åand β = 101.88(3)°,R = 0.034 for 4450 independent data;5:a = 12.306(2),b = 14.809(3),c = 10.257(2)Åand β = 101.99(3)°,R = 0.043 for 3078 independent data). The crystal structure determinations of 4 and 5 show that four edges of the tetrahedral MS_4²⁻ core are coordinated by copper atoms forming WS₄Cu₄ aggregates linked by eight-membered Cu(NCS)₂Cu rings. A two-dimensional network is thus formed in the diagonal (101) plane. The space between the anionic two-dimensional networks is filled with the NEt_4⁺ cations. Additional NCS groups lead to the [Cu₄(NCS)₅WS₄]³⁻ (1) trianion connected by NCS bridges forming pseudo-dimers. These latter are held together by weak CuS(NCS) interactions giving rise to infinite chains along a direction parallel to [100]. In contrast complex3 develops infinite chains from WS₄Cu₃ aggregates with the same Cu(NCS)₂Cu bridges as in 4 and 5. These chains are running along a direction parallel to [010]. The structural data of the different types of polymeric compounds containing MS_4²⁻ and CuNCS have been used to interpret vibrational spectroscopic data of the thiocyanate groups.     

Probing fluorescence induction in chloroplasts on a nanosecond time scale utilizing picosecond laser pulse pairs

The fluorescence induction and other fluorescence properties of spinach chloroplasts at room temperature were probed utilizing two 30-ps wide laser pulses (530 nm) spaced Δt (s) apart in time (Δt = 5–110 ns). The energy of the first pulse (P₁) was varied (10¹²–10¹⁶ photons · cm⁻²), while the energy of the second (probe) pulse (P₂) was held constant (5 · 10¹³ photons · cm⁻²). A gated (10 ns) optical multichannel analyzer-spectrograph system allowed for the detection of the fluorescence generated either by P₁ alone, or by P₂ alone (preceded by P₁). The dominant effect observed for the fluorescence yield generated by P₁ alone is the usual singlet-singlet exciton annihilation which gives rise to a decrease in the yield at high energies. However, when the fluorescence yield of dark-adapted chloroplasts is measured utilizing P₂ (preceded by pulse P₁) an increase in this yield is observed. The magnitude of this increase depends on Δt, and is characterized by a time constant of 28 ± 4 ns. This rise in the fluorescence yield is attributed to a reduction of the oxidized (by P₁) reaction center P-680⁺ by a primary donor. At high pulse energies (P₁ = 4 · 10¹⁴ photons · cm⁻²) the magnitude of this fluorescence induction is diminished by another quenching effect which is attributed to triplet excited states generated by intense P₁ pulses. Assuming that the P₁ pulse energy dependence of the fluorescence yield rise reflects the closing of the reaction centers, it is estimated that about 3–4 photon hits per reaction center are required to close completely the reaction centers, and that there are 185–210 chlorophyll molecules per Photosystem II reaction center.