Resonant Raman quantification of zeaxanthin production from Flavobacterium multivorum

Resonant Raman scattering was used as a novel, rapid, non-destructive optical technique to measure zeaxanthin levels in Flavobacterium multivorum ATCC 55238. Culture broth, after bacterial growth for 40 h, exhibited characteristic resonance Raman vibrational modes at 1159 cm^–1 (C-C stretch) and 1525 cm^–1 (C=C stretch) upon excitation at 488 nm. A striking correlation was observed between the carotenoid level as estimated by HPLC and by resonance Raman spectroscopy.     

Production of Deuterated Lutein by Chlorella protothecoides and its Detection by Mass Spectrometric Methods

Chlorella protothecoides, a lutein-producing microalga, was grown aerobically in a mineral medium prepared with 70% (v/v) deuterated water. HPLC/atmospheric pressure chemical ionization-mass spectrometry (HPLC/APCI-MS) analysis revealed 58% replacement of hydrogen by deuterium atoms as indicated by the molecular mass cluster at around m/z 599. The rapidly growing microalga had much higher levels (58%) of deuterium substitution relative to previously reported (9–15%) natural sources of lutein.     

The biosynthetic pathway of carotenoids in the astaxanthin-producing green alga <Emphasis Type="Italic">Chlorella zofingiensis</Emphasis>

The carotenoid composition of the astaxanthin-producing green alga Chlorella zofingiensis was investigated using high-performance liquid chromatography. Astaxanthin, adonixanthin, and zeaxanthin are the major carotenoids in this alga. The pigment pattern was characterized during the accumulation period, and in response to diphenylamine (DPA), an inhibitor of carotenoid biosynthesis. An increase in zeaxanthin followed by a decrease in xanthophyll was seen after the induction of astaxanthin biosynthesis by glucose. This biphasic kinetics of zeaxanthin was parallel to the marked increase in adonixanthin (from 0 mg g⁻¹ to 0.21 mg g⁻¹) and astaxanthin (from 0.05 mg g⁻¹ to 0.35 mg g⁻¹) and decrease of β-carotene (from 0.30 mg g⁻¹ to 0.03 mg g⁻¹). More importantly, unlike the Haematococcus alga, in which there was a high β-carotene accumulation after DPA treatment, C. zofingiensis showed an accumulation of extra zeaxanthin instead of β-carotene after treatment of the cells with DPA. All these results observed in vivo studies corroborate the observations in vitro studies at the enzyme level that zeaxanthin is a substrate for the carotenoid oxygenase in C. zofingiensis. It is suggested that zeaxanthin might be an important intermediate and not an end product of the biosynthetic pathway of astaxanthin. Therefore, a new pathway for astaxanthin formation by C. zofingiensis, which is different from that of the other astaxanthin-producing microorganisms, is proposed. An understanding of the astaxanthin biosynthetic pathway may yield important information toward the optimization of astaxanthin production, especially for the improvement of astaxanthin through genetic manipulations.     

Dynamic Changes of Photosynthetic Pigments in Soybean Callus under High Irradiance

Dynamic changes of neoxanthin (NEO), violaxanthin (VIO), anteraxanthin (ANT), zeaxanthin (ZEA), chlorophyll (Chl) a, Chl b, α-carotene, β-carotene, and their behaviour under increasing duration of high irradiance (HI) were investigated in the soybean hypocotyl callus culture. The calli were induced on solid (1.1 % agar) MS medium (pH 5.8) supplemented with 4.52 μM 2,4-D, 2.32 μM kinetin, and 3 % sucrose. After 30 d of culture, the green calli were irradiated with “white light” (133W m⁻²) for 0, 3.5, and 24 h. HPLC profiles were separated on a C₁₈ column. With increasing duration of HI, the content of total carotenoids (Cars) increased, but the ratio of Chl a+b/Cars decreased. With lengthening the duration of HI, there was induction of ZEA. Contents of ANT, α-carotene, and β-carotene remained nearly constant, but ratio of ZEA/Chl a+b increased with lengthening the HI duration. This revised version was published online in August 2006 with corrections to the Cover Date.     

A resonance Raman band assignable to the O–O stretching mode in the resting oxidized state of bovine heart cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase

In the resting oxidized state (the fully oxidized “as-isolated” state) of cytochrome c oxidase (CcO) preparation, a resonance Raman band is observed at 755 cm^-1 upon 647.1 nm excitation in resonance with an absorption band at 655 nm. Addition of cyanide eliminates the Raman band concomitant with loss of the absorption band at 655 nm. These results strongly suggest that the Raman band at 755 cm^-1 originates from the O−O stretching mode of the bridging peroxide (Fe−O^-−O^-−Cu) in the O₂ reduction site of the fully oxidized “as-isolated” CcO. Although the peroxide bridged structure has been proposed on the basis of X-ray crystallography and reductive titration experiments, the present vibrational spectroscopic analyses reveal conclusively the chemical nature of the bridging ligand at the O₂ reduction site of the fully oxidized “as-isolated” bovine heart CcO.     

High‐performance liquid chromatography study of gatifloxacin and sparfloxacin using erythrosine as post‐column resonance Rayleigh scattering reagent and mechanism study

Herein, a highly selective high‐performance liquid chromatography (HPLC) coupled with resonance Rayleigh scattering (RRS) method was developed to detect gatifloxacin (GFLX) and sparfloxacin (SPLX). GFLX and SPLX were first separated by HPLC, then, in pH 4.4 Britton–Robinson (BR) buffer medium, protonic quaternary ammonia cation of GFLX and SPLX reacted with erythrosine (ERY) to form 1:1 ion‐association complexes, which resulted in a significant enhancement of RRS signal. The experimental conditions of HPLC and post‐column RRS have been investigated, including detection wavelength, flow rate, pH, reacting tube length and reaction temperature. Reaction mechanism were studied in detail by calculating the distribution fraction. The maximum RRS signals for GFLX and SPLX were recorded at λ_ex = λ_em = 330 nm. The detection limits were 3.8 ng ml^?1 for GFLX and 17.5 ng ml^?1 for SPLX at a signal‐to‐noise ratio of 3. The developed method was successfully applied to the determination of GFLX and SPLX in water samples. Recoveries from spiked water samples were 97.56–98.85%.     

Adaptation of the photosynthetic apparatus of Nitellopsis obtusa to changing light intensity at the molecular level: different pathways of a singlet excitation quenching

The effect of prolonged illumination (60 min) with photosynthetically active monochromatic radiation of low intensity (3 μmol m⁻² s⁻¹) and high intensity (60 μmol m⁻² s⁻¹), corresponding to the physiological conditions and light stress conditions, respectively, was studied in the algae Nitellopsis obtusa. Illumination of Nitellopsis obtusa cells with strong light was associated with activation of the xanthophyll cycle, manifested by the deepoxidation of violaxanthin and accumulation of antheraxanthin and zeaxanthin. At the same time, the efficient singlet excitation quenching in the photosynthetic apparatus was activated, as demonstrated by the decrease in the intensity of the chlorophyll a fluorescence emission by ca 50 %. The difference of the fluorescence excitation spectra recorded before and after the light treatment match the difference absorption spectrum of the xanthophyll cycle pigments. The illumination with low light intensity resulted also in the chlorophyll a fluorescence quenching but the effect was very small (less than 10 %). The fluorescence quenching is interpreted in terms of the energy transfer between the Q_y energy level of chlorophyll a and the 2¹ A_g ⁻ energy level of zeaxanthin. The singlet energy levels of carotenoids, corresponding to the green spectral region, are also taken into consideration in the interpretation of the excitation energy exchange between the carotenoids and chlorophylls. Possible molecular mechanisms involved in the activation of the strong and the weak excitation quenching, including violaxanthin isomerization, and possible physiological functions of such pathways of energy transfer are discussed.     

Heterologous expression of polyhydroxyalkanoate depolymerase from <Emphasis Type="Italic">Thermobifida</Emphasis> sp. in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and catalytic analysis by surface plasmon resonance

A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His₆-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase containing a proline–serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V _max and K _m of 3.63 ± 0.16 μmol min⁻¹ mg⁻¹ and 0.79 ± 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50–55°C and pH 7–8. Surface plasmon resonance (SPR) analysis demonstrated that PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm⁻² h⁻¹ for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique will be of utility for further study of bioplastic degradation.     

Cell growth and nutritive value of the tropical benthic diatom, <Emphasis Type="Italic">Amphora</Emphasis> sp., at varying levels of nutrients and light intensity,and different culture locations

Two series of experiments were conducted to determine suitable growth factors for the mass propagation of the local algal isolate Amphora sp. A higher growth rate of 0.2 doubling (μ) day⁻¹ was attained at a lower photosynthetic photon flux density (PPFD; 11.4 μmol photon m⁻²s⁻¹) compared to cultures exposed to higher levels of PPFD (16.1 μmol photon m⁻²s⁻¹, −0.1 μ day ⁻¹; 31.3 μmol photon m⁻²s⁻¹, 0.0 μ day⁻¹). Cultures located inside the laboratory had a significantly higher cell density (133 × 10⁴ cells cm⁻²) and growth rate (0.3 μ day⁻¹) compared to those located outdoors (100 × 10⁴ cells cm⁻², 0.2 μ day⁻¹). A comparison of nutrient medium across two locations showed that lipid content was significantly higher in cultures enriched with F/2MTM (macronutrients + trace metals) and F/2MV (macronutrients + vitamins). Saturated fatty acids were also present in high concentrations in cultures enriched with F/2M (macronutrients only). Significantly higher amounts of saturated fatty acids were observed in cultures located outdoors (33.1%) compared to those located indoors (26.6%). The protein, carbohydrates and n-6 fatty acid content of Amphora sp. were influenced by the location and enrichment of the cultures. This study has identified growth conditions for mass culture of Amphora sp. and determined biochemical composition under those culture conditions. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Acaricidal effects of <Emphasis Type="Italic">Corymbia citriodora</Emphasis> oil containing <Emphasis Type="Italic">para</Emphasis>-menthane-3,8-diol against nymphs of <Emphasis Type="Italic">Ixodes ricinus</Emphasis> (Acari: Ixodidae)

The toxicity of para-menthane-3,8-diol (PMD), the main arthropod-repellent compound in the oil of the lemon eucalyptus, Corymbia citriodora, was evaluated against nymphs of Ixodes ricinus using five methods (A–E) of a contact toxicity bioassay. Mortality rates were estimated by recording numbers of dead nymphs at 30 min intervals during the first 5 h after the start of exposure and at longer intervals thereafter. The mortality rate increased with increasing concentration of PMD and duration of exposure with a distinct effect after 3.5 h. From the results obtained by methods A, C and E, the LC₅₀ range was 0.035–0.037 mg PMD/cm² and the LC₉₅ range was 0.095–0.097 mg PMD/cm² at 4 h of exposure; the LT₅₀ range was 2.1–2.8 h and the LT₉₅ range was 3.9–4.2 h at 0.1 mg PMD/cm². To determine the duration of toxic activity of PMD, different concentrations (0.002, 0.01, 0.1 mg PMD/cm²) were tested and mortality was recorded at each concentration after 1 h; thereafter new ticks were tested. This test revealed that the lethal activity of PMD remained for 24 h but appeared absent after 48 h. The overall results show that PMD is toxic to nymphs of I. ricinus and may be useful for tick control.     

Tissue-specific accumulation of carotenoids in carrot roots

Raman spectroscopy can be used for sensitive detection of carotenoids in living tissue and Raman mapping provides further information about their spatial distribution in the measured plant sample. In this work, the relative content and distribution of the main carrot (Daucus carota L.) root carotenoids, α-, β-carotene, lutein and lycopene were assessed using near-infrared Fourier transform Raman spectroscopy. The pigments were measured simultaneously in situ in root sections without any preliminary sample preparation. The Raman spectra obtained from carrots of different origin and root colour had intensive bands of carotenoids that could be assigned to β-carotene (1,520 cm⁻¹), lycopene (1,510 cm⁻¹) and α-carotene/lutein (1,527 cm⁻¹). The Raman mapping technique revealed detailed information regarding the relative content and distribution of these carotenoids. The level of β-carotene was heterogeneous across root sections of orange, yellow, red and purple roots, and in the secondary phloem increased gradually from periderm towards the core, but declined fast in cells close to the vascular cambium. α-carotene/lutein were deposited in younger cells with a higher rate than β-carotene while lycopene in red carrots accumulated throughout the whole secondary phloem at the same level. The results indicate developmental regulation of carotenoid genes in carrot root and that Raman spectroscopy can supply essential information on carotenogenesis useful for molecular investigations on gene expression and regulation.     

The Z-Conformation of Poly(dA-dT) · Poly(dA-dT) in Solution as Studied by Ultraviolet Resonance Raman Spectroscopy

Abstract

Poly(dA-dT) poly(dA-dT) structures in aqueous solutions with high NaCl concentrations and in the presence of Ni²⁺ ions have been studied with resonance Raman spectroscopy (RRS). In low water activity the effects of added 95 mM NiCl₂ in solution stabilize the syn geometry of the purines and reorganize the water distribution via local interactions of Ni-water charged complexes with the adenine N7 position. It is shown that RRS provides good marker bands for a left-handed helix: i) a purine ring breathing mode around 630 cm″^?1coupled to the deoxyribose vibration in the syn geometry, ii) a 1300-1340 cm^?1 region characterizing local chemical interactions of the Ni²⁺ ions with the adenien N7 position, iii) lines at about 1483-and 1582 cm^?1 correlated to the anti/syn reorientation of the adenine residues on B-Z structure transition, iv) marker bands of the thymidine carbonyl group couplings at 1680-and 1733 cm^?1 due to the disposition of the thymidine residues in the Z helix specific geometry. Hence poly(dA-dT) poly(dA-dT) can adopt a Z form in solution. The Z form observed in alternate purine-pyrimidine sequences does not require G-C base pairs.     

A steady-state and pre-steady-state kinetics study of the tungstoenzyme formaldehyde ferredoxin oxidoreductase from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis>

Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a homotetrameric protein with one tungstodipterin and one [4Fe–4S] cubane per 69-kDa subunit. The enzyme kinetics have been studied under steady-state conditions at 80 °C and pre-steady state conditions at 50 °C, in the latter case via monitoring of the relatively weak (ε ≈ 2 mM⁻¹ cm⁻¹) optical spectrum of the tungsten cofactor. The steady-state data are consistent with a substrate substituted-enzyme mechanism for three substrates (formaldehyde plus two ferredoxin molecules). The K _M value for free formaldehyde (21 μM) with ferredoxin as an electron acceptor is approximately 3 times lower than the value measured when benzyl viologen is used as an acceptor. The K _M of ferredoxin (14 μM) is an order of magnitude less than previously reported values. An explanation for this discrepancy may be the fact that high concentrations of substrate are inhibitory and denaturing to the enzyme. Pre-steady-state difference spectra reveal peak shifts and a lack of isosbestic points, an indication that several processes happen in the first seconds of the reaction. Two fast processes (k _obs1 = 4.7 s⁻¹, k _obs2 = 1.9 s⁻¹) are interpreted as oxidation of the substrate followed by rearrangement of the active site. Alternatively, these processes could be the entry/binding of the substrate followed by its oxidation. The release of the product and the electron shuffling over the tungsten and iron–sulfur center in the absence of an external electron acceptor are slower (k _obs3 = 6.10 × 10⁻² s⁻¹, k _obs4 = 2.18 × 10⁻² s⁻¹). On the basis of these results in combination with results from previous electron paramagnetic resonance studies an activation route plus catalytic redox cycle is proposed.     

1H NMR and GC/MS metabolomics of earthworm responses to sub-lethal DDT and endosulfan exposure

The metabolic response of the earthworm Eisenia fetida to two pesticides, dichlorodiphenyltrichloroethane (DDT) and endosulfan, was characterized in contact tests using proton nuclear magnetic resonance (¹H NMR) and principal component analysis (PCA). PCA loading plots suggested that maltose, leucine and alanine were important metabolites contributing to the differences in dosed and control earthworms for both compounds at doses of 0.5, 1.0 and 2.0 μg/cm². Gas chromatography/mass spectrometry (GC/MS) was used to quantify the metabolites identified in E. fetida and determine if the changes in maltose, leucine and alanine following exposure to DDT and endosulfan (at 0.5 and 1.0 μg/cm²) were reproducible and greater than the natural variability. Quantification by GC/MS suggested that maltose was not a reliable biomarker since it both increased and decreased in earthworms exposed to DDT and increased by just 3% with exposure to endosulfan. Leucine was not stable with the GC/MS derivitization method used in this study and could not be confirmed as a reliable biomarker. However, alanine consistently increased for both DDT and endosulfan exposed E. fetida. Alanine showed considerable variability in control earthworms (±41.6%), yet the variability in alanine to glycine ratios was just ±10.5%. Increases in the alanine to glycine ratio were statistically significant at the P = 0.05 level for the 1.0 μg/cm² DDT dose and both the 0.5 and 1.0 μg/cm² endosulfan doses, suggesting that deviations from the normal homeostatic ratio of 1.5 for alanine to glycine is a potential biomarker of DDT and endosulfan exposure warranting further study. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Environmental Metabolomics Special Issue of Metabolomics.     

Self-hydroxylation of taurine/α-ketoglutarate dioxygenase: evidence for more than one oxygen activation mechanism

2-Aminoethanesulfonic acid (taurine)/α-ketoglutarate (αKG) dioxygenase (TauD) is a mononuclear non-heme iron enzyme that catalyzes the hydroxylation of taurine to generate sulfite and aminoacetaldehyde in the presence of O₂, αKG, and Fe(II). Fe(II)TauD complexed with αKG or succinate, the decarboxylated product of αKG, reacts with O₂ in the absence of prime substrate to generate 550- and 720-nm chromophores, respectively, that are interconvertible by the addition or removal of bound bicarbonate and have resonance Raman features characteristic of an Fe(III)–catecholate complex. Mutagenesis studies suggest that both reactions result in the self-hydroxylation of the active-site residue Tyr73, and liquid chromatography nano-spray mass spectrometry/mass spectrometry evidence corroborates this result for the succinate reaction. Furthermore, isotope-labeling resonance Raman studies demonstrate that the oxygen atom incorporated into the tyrosyl residue derives from H₂ ¹⁸O and ¹⁸O₂ for the αKG and succinate reactions, respectively, suggesting distinct mechanistic pathways. Whereas the αKG-dependent hydroxylation likely proceeds via an Fe(IV)=O intermediate that is known to be generated during substrate hydroxylation, we propose Fe(III)–OOH (or Fe(V)=O) as the oxygenating species in the succinate-dependent reaction. These results demonstrate the two oxygenating mechanisms available to enzymes with a 2-His-1-carboxylate triad, depending on whether the electron source donates one or two electrons.     

Resonance Raman spectroscopy of cytochrome<Emphasis Type="Italic"> c</Emphasis> peroxidase variants that mimic manganese peroxidase

Cytochrome c peroxidase (CcP) variants with an engineered Mn(II) binding site, including MnCcP [CcP(MI, G41E, V45E, H181D)], MnCcP(W191F), and MnCcP(W191F, W51F), that mimic manganese peroxidase (MnP), have been characterized by resonance Raman (RR) spectroscopy. Analysis of the Raman bands in the 200–700 cm^–1 and 1300–1650 cm^–1 regions indicates that both the coordination and spin state of the heme iron in the variants differ from that of CcP(MI), the recombinant yeast CcP containing additional Met-Ile residues at the N-terminus. At neutral pH the frequencies of the ₃ mode indicate that a pure five-coordinate heme iron exists in CcP(MI) whereas a six-coordinate low-spin iron is the dominant species in the CcP variants with the engineered Mn(II) binding site. The H181D mutation, which weakens the proximal linkage to the heme iron, may be responsible for these spectral and structural changes. Raman spectra of the variants CcP(MI, W191F) and CcP(MI, W191F, W51F) were also obtained to clarify the structural and functional roles of mutations at two tryptophan sites. The W51F mutation was found to disrupt H-bonding to the distal water molecules and the resulting variants tended to form transitional or mixed coordination states that possess spectral and structural features similar to that of MnP. Such structural features, with a loosened distal water, may facilitate the binding of H₂O₂ and increase the rate constant for compound I formation. This effect, in addition to the elimination of an H-bond to ferryl oxygen by the same mutation, accounts for the increased MnP specific activity of MnCcP(W191F, W51F).Electronic Supplementary Material Supplementary material is available in the online version of this article at .Abbreviations CcP cytochrome c peroxidase - CcP(MI) recombinant yeast CcP containing Met-Ile at the N-terminus in addition to the normal wild-type CcP sequence - HRP horseradish peroxidase - MnCcP CcP(MI, G41E, V45E, H181D) - MnCcP(W191F) CcP(MI, G41E, V45E, H181D, W191F) - MnCcP(W191F, W51F) CcP(MI, G41E, V45E, H181D, W191F, W51F) - MnP manganese peroxidase - RR resonance Raman - WtCcP wild-type cytochrome c peroxidase     

Influence of ultraviolet-C on structure and function of <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7942 photolyase

In this work, an over-expressed cyclobutane pyrimidine dimer (CPD) photolyase of Synechococcus sp. PCC 7942 was used to investigate UV-C (ultraviolet irradiation of C-region) influence on photoreactivation. In vivo photoreactivation experiments indicated that the survival rate decreased from 100 to 2.6% when the UV-C flux was increased from 1.1 to 68.5 μW/cm². It seemed that the photolyase was easily inactivated at UV-C intensities ≥25.5 μW/cm². Spectrometric analysis indicated that tertiary structure of the photolyase changed evidently when the UV-C fluxes were ≥25.5 μW/cm², while the secondary structure was almost unchanged even at 170 μW/cm². Band shift assay indicated that catalytic activity of the photolyase was impaired at fluxes ≥25.5 μW/cm², but no significant influence on DNA-binding activity was observed. These results suggest that photoreactivation is efficient at UV-C fluxes ≤25.5 μW/cm², but would be impaired by intense UV-C irradiation due to structure changes of the photolyase. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 5, pp. 668–673.     

Structure identification and fermentation characteristics of pinoresinol diglucoside produced by <Emphasis Type="Italic">Phomopsis</Emphasis> sp. isolated from <Emphasis Type="Italic">Eucommia ulmoides</Emphasis> Oliv

Pinoresinol diglucoside (PDG) is the important antihypertensive compound in Eucommia ulmoides Oliv., a traditional Chinese herb medicine. The research objective was to certify the possibility of producing PDG through fermentation. PDG-producing endophytic fungi were isolated from E. ulmoides Oliv., and the highest PDG-yielding (11.65 mg/L) isolate, XP-8, was identified as Phomopsis sp. according to the morphological characteristics and the phylogenetic tree constructed on the basis of the gene sequence in the internal transcribed spacers district. The microbial PDG was isolated by using S-8 resin and purified to a purity of 98.7% using preparative high-performance liquid chromatography (HPLC). Information obtained from the UV spectrum (277 and 227 nm, in water solution), infra-red spectrum (3,428; 2,930; 2,877; 1,637; 1,600; and 1,513; 1,460; 1,421; 1,269; 1,223; 1,075; 658 cm⁻¹, in powder), molecular weight (682 Da, measured using HPLC-electrospray ionization mass spectrometry (ESI/MS) and tandem mass spectrometry), and nuclear magnetic resonance analysis show the microbial PDG is (+)-1-pinoresinol 4,4′-di-O-β-d-glucopyranoside, same as the plant-derived PDG. The microbial PDG is stable in pH range from 3 to 11 but less stable at temperature higher than 90 °C and in light exposure. During the fermentation, PDG production outside cells starts at the later stage of cell growth when the residual sugar in the medium was low. The study reveals the possibility for production of PDG by fermentation.     

Quantification of water and biomass in small colony variant PAO1 biofilms by confocal Raman microspectroscopy

The Raman spectra, water content, and biomass density of wild-type (WT) Pseudomonas aeruginosa PAO1, small colony variant (SCV) PAO1, and Pseudoalteromonas sp. NCIMB 2021 biofilms were compared in order to determine their variation with strain and species. Living, fully submerged biofilms were analyzed in situ by confocal Raman microspectroscopy for up to 2 weeks. Water to biomass ratios (W/BRs), which are the ratios of the O–H stretching vibration of water at 3,450 cm⁻¹ to the C–H stretching band characteristic of biomass at 2,950 cm⁻¹, were used to estimate the biomass density and cell density by comparison with W/BRs of protein solutions and bacterial suspensions, respectively, on calibration curves. The hydration within SCV biofilm colonies was extremely heterogeneous whereas W/BRs were generally constant in young WT biofilm colonies. The mean biomass in biofilm colonies of WT or colony cores of SCV was typically equivalent to 16% to 27% protein (w/v), but was 10% or less for NCIMB 2021. The corresponding cell densities were 7.5 to >10 × 10¹⁰ cfu mL⁻¹ for SCV, while the maximum cell density for NCIMB biofilms was 2.8 × 10¹⁰ cfu mL⁻¹.     

Production and structural analysis of the polysaccharide secreted by <Emphasis Type="Italic">Trametes (Coriolus) versicolor</Emphasis> ATCC 200801

Trametes versicolor ATCC 200801 secretes 4.1 g L⁻¹ of exopolysaccharide (EPS) when synthetic minimal medium and low-shear bioreactor cultivation technique are used. Structural and compositional analyses by thin layer chromatography, gas chromatography–mass spectrometry, electrospray ionization tandem mass spectrometry, and nuclear magnetic resonance spectroscopy yielded predominantly glucose and small amounts of galactose, mannose, arabinose, and xylose. The main EPS is composed of β-1,3/β-1,6-linked d-glucose molecules which is identical with Schizophyllan but does not possess a triple helical arrangement as secondary structure. Two molar mass fractions were detected by size exclusion chromatography yielding weight-average molecular weights of 4,100 and 2.6 kDa. Protein content varies between 2–3.6% (w/w). The exopolysaccharide is different in the nature of the glycosidic linkage, composition of monosaccharides, protein content, and weight-average molecular weight compared to the well-known polysaccharopeptide (PSP) and polysaccharopeptide Krestin (PSK).