Bench-scale fermentation of Trichoderma viride on wastewater sludge: Rheology, lytic enzymes and biocontrol activity

Conidiation and lytic enzyme production by Trichoderma viride at different solids concentration of pre-treated municipal wastewater sludge was examined in a 15-L fermenter. The maximum conidia concentration (5.94 × 10⁷ CFU mL⁻¹ at 96 h) was obtained at 30 g L⁻¹ suspended solids. The maximum lytic enzyme activities were achieved around 12–30 h of fermentation. Bioassay against a fungal phytopathogen, Fusarium sp. showed maximum activity in the sample drawn around 96 h of fermentation at 30 g L⁻¹ suspended solids concentration. Entomotoxicity against spruce budworm larvae showed maximum value ≈17290 SBU μL⁻¹ at 30 g L⁻¹ suspended solids concentration at the end of fermentation (96 h). Plant bioassay showed dual action of T. viride, i.e., disease prevention and growth promotion. The rheological analyses of fermentation sludges showed the pseudoplastic behaviour. In order to maintain required dissolved oxygen concentration ≥30%, the agitation and aeration requirements significantly increased at 35 g L⁻¹ compared to 30 and 25 g L⁻¹. The oxygen uptake rate and volumetric oxygen mass transfer coefficient, k_La at 35 g L⁻¹ did not increase in comparison to 30 g L⁻¹ due to rheological complexity of the broth during fermentation. Thus, the successful fermentation operation of the biocontrol fungus T. viride is a rational indication of its potential for mass-scale production for agriculture and forest sector as a biocontrol agent.     

Effect of oxygen supply on cell growth and saponin production in bioreactor cultures of Panax ginseng

The effects of oxygen supply within the range 20.8–50% (using pure oxygen and air), on cell cultures of Panax ginseng were investigated in a balloon-type bubble bioreactor (5 L capacity, containing 4 L Murashige and Skoog medium, supplemented with 7.0 mg L⁻¹ indolebutyric acid, 0.5 mg L⁻¹ kinetin and 30 g L⁻¹ sucrose). A 40% oxygen supply was found to be optimal for the production of both cell mass and saponin yielding values of 12.8 g (DW) L⁻¹, 4.5 mg (g DW)⁻¹ on day 25, respectively. Low (20.8%, 30%) and high (50%) oxygen concentration supplies were unfavorable to cell growth and saponin accumulation. The results indicate that oxygen supplementation to bioreactor-based ginseng cultures was beneficial for biomass accumulation and saponin production.     

Antimicrobial activity of steady-state cultures of Bacillus sp. CCMI 1051 against wood contaminant fungi

The effect of changing dilution rate (D) on Bacillus sp. CCMI 1051 at dilution rates between 0.1 and 0.55 h⁻¹ in a glucose-limited medium was studied. Biomass values varied between 0.88 and 1.1 g L⁻¹ at D values of 0.15–0.35 h⁻¹. Maximal biomass productivity was found to be 0.39 g L⁻¹ h⁻¹, obtained at D = 0.35 h⁻¹ and corresponding to a 54.4% conversion of the carbon into cell mass. The highest rate of glucose consumption was 4.45 mmol g⁻¹ h⁻¹ occurring at D = 0.4 h⁻¹. The glucose concentration inside the chemostat was below the detection level starting to accumulate around 0.4 h⁻¹. Growth inhibition of fifteen strains of fungi by the broth of the steady-state cell-free supernatants was assessed. Results showed that the relative inhibition differ among the target species but was not influenced by the dilution rate changing.     

Enhancement of growth and gymnodimine production by the marine dinoflagellate, Karenia selliformis

Because of its novel bioactive properties the production of gymnodimine for use as a pharmaceutical precursor has aroused interest. The dinoflagellate, Karenia selliformis produces gymnodimine when grown in bulk culture using GP + selenium medium but the growth rates (μ) and levels of gymnodimine are low (μ, 0.05 days⁻¹; gymnodimine 250 μg L⁻¹ max). We describe the effects of organic acid additions (acetate, glycolate, alanine and glutamate additions and combinations of these) in enhancing growth and gymnodimine production in axenic cultures. The most effective organic acid combinations in decreasing order were: glycolate/alanine > acetate > glycolate. Glycolate/alanine optimised gymnodimine production by prolonging growth (maximum cell yield, 1.76 × 10⁵ cells mL⁻¹; gymnodimine, 1260 μg L⁻¹; growth rate (μ), 0.2 days⁻¹) compared to the control (growth maximum cell yield, 7.8 × 10⁴ cells mL⁻¹; gymnodimine, 780 μg L⁻¹; μ, 0.17 days⁻¹). Acetate enhanced gymnodimine by stimulating growth rate (μ, 0.23 days⁻¹) and the large concentration of gymnodimine per cell (16 pg cell⁻¹ cf. 9.8 pg cell⁻¹ for the control) suggests a role for this compound in gymnodimine biosynthesis. Amending culture media with Mn²⁺ additions resulted in slightly decreased growth in control cultures and increased the gymnodimine while in glycolate/alanine cultures growth was stimulated but gymnodimine production decreased. The results suggest that the organic acid can enhance gymnodimine production by either enhancing growth maximum or the biosynthetic pathway.     

Electrochemical quartz crystal impedance study on the overoxidation of polypyrrole-carbon nanotubes composite film for amperometric detection of dopamine

The electrochemical quartz crystal impedance (EQCI) method was used to study the overoxidation of polypyrrole (PPy)–multiwalled carbon nanotubes (MWCNT) nanocomposite film in neutral and alkaline solutions. The values of molar mass per electron transferred (M/n) obtained during the overoxidation of PPy in 0.10 mol L⁻¹ Na₂SO₄ and 0.20 mol L⁻¹ NaOH aqueous solutions were estimated to be ca. 17 and 22 g mol⁻¹, respectively, suggesting the nucleophilic attack of solution OH⁻ to the pyrrole units during the overoxidation, and the possible partial formation of carboxylic groups after the overoxidation in the NaOH solution. Also, the overoxidized PPy–MWCNT composite film prepared in the NaOH solution showed a notably larger affinity to dopamine (DA) dissolved in a neutral phosphate buffer than that prepared in the Na₂SO₄ solution. The modification of the overoxidized nanocomposite film improved substantially the sensitivity for DA assay in a neutral phosphate buffer, as compared with the modification of overoxidized PPy or MWCNT alone. At a −6 kHz (201-nm thickness) nanocomposite film prepared in a polymerization bath containing 1.0 mg mL⁻¹ MWCNT and overoxidized in 0.20 mol L⁻¹ aqueous NaOH, the peak current response from differential pulse voltammetric assay of DA was linear with DA concentration from 4.0 × 10⁻⁸ to 1.4 × 10⁻⁶ mol L⁻¹, with a lower limit of detection of 1.7 nmol L⁻¹, good anti-interferent ability, as well as good stability and reproducibility.     

Highly efficient polypyridyl-ruthenium(II) photosensitizers with chelating oxygen donor ligands: β-diketonato-bis(dicarboxybipyridine)ruthenium

A series of mono-cationic polypyridyl-ruthenium complexes with strongly electron donating β-diketonate ligands {(dcbp)₂Ru(L)}Cl, where DCBP=4,4′-dicarboxy-2,2′-bipyridine; L=acetylacetonate (1), 3-methyl-2,4-pentanedionate (2), 1,3-diphenyl-1,3-propanedionate (3), have been synthesized as molecular photosensitizers for a nanocrystalline TiO₂ electrode. In alkaline methanol solution, these complexes exhibit intense visible light absorption with low energy MLCT maxima above 517 nm which accompany a significantly enhanced band tail, improving red light absorptivity beyond 600 nm. The photoelectrochemical properties of these three diketonate complexes on a TiO₂ semiconductor have been compared to cis-dithiocyanate complex, (dcbp)₂Ru(NCS)₂, which is one of the most efficient sensitizers reported to date. The diketonate complexes show quite high performances in photoelectrochemical cells containing I⁻/I₃ ⁻ electrolyte. The overall solar light-to-electrical energy conversion efficiencies are in the range of 6.0–3.9% while the dithiocyanate complex yields 5.7% efficiency in our experiments.     

Ethanol production from cheese whey permeate by Kluyveromyces marxianus UFV-3: A flux analysis of oxido-reductive metabolism as a function of lactose concentration and oxygen levels

In order to investigate the effect of lactose concentration and oxygen level on the growth and metabolism of Kluyveromyces marxianus UFV-3 in cheese whey permeate, batch cultures were conducted under aerobic, hypoxic, and anoxic conditions, with lactose at initial concentration ranging from 1 to 240 g L⁻¹. The increase in lactose concentration increased ethanol yield and ethanol volumetric productivity, and has reduced cell yield. When lactose concentration was equal or above 50 g L⁻¹ and the oxygen levels were low, the ethanol yield was close to its theoretical value. Maximum ethanol concentrations attained in this study were 76 and 80 g L⁻¹ in hipoxia and anoxia, respectively. The lactose consumption rate in anoxia was greater than in aerobiosis and hipoxia. However, under anoxia, the lactose consumption rate of K. marxianus followed a saturation kinetics, which was not observed in hypoxia and aerobiosis. All oxygen levels investigated, showed a tendency for saturation of the ethanol production rate above 65 g L⁻¹ lactose. Ethanol production rate was also higher on anoxia.     

Optimisation of β-glucuronidase production from a newly isolated Ganoderma applanatum

Media optimisation was attempted for β-glucuronidase production from a newly and locally isolated (Oxfordshire, UK) fungal strain of Ganoderma applanatum. Both fungal growth and β-glucuronidase activity were found to be greatly affected by varying the carbon or the nitrogen source with gum arabic and yeast extracts being the best carbon and nitrogen sources, respectively. Their concentrations were optimised at 8 g L⁻¹ for the former and 2 g L⁻¹ for the latter.

Work then proceeded to enhance the yield of β-glucuronidase in a controlled environment. Control, batch and fed-batch cultivations were performed in 2-L bioreactors using the optimised medium supplemented with cellobiuronic acid as inducer. Time profiles of biomass dry weight, carbohydrate consumption and β-glucuronidase production were obtained and the results showed that production of β-glucuronidase was noticeably increased by the addition of cellobiuronic acid in both batch and fed-batch fermentations. Although the addition did not produce a variation in the pattern of growth seen between control, and induced fermenters, higher levels of the enzyme were attained when adopting a fed-batch process with 1.09 U mL⁻¹ of culture, corresponding to a 5-fold enhancement in β-glucuronidase production rate compared with batch fermentation.     

Determination of trimethylamine in fish by capillary electrophoresis with electrogenerated tris(2,2'-bipyridyl)ruthenium(II) chemiluminescence detection.

A capillary electrophoresis with electrogenerated chemiluminescence (CE-ECL) method for the determination of trimethylamine (TMA) in fish was studied. In the presence of TMA, ECL from the reaction of analyte and in situ generated tris(2,2'-bipyridyl)ruthenium(III) [Ru(bpy)(3) (3+)] at electrode surface could be produced. The ECL detection was performed using a Pt working electrode biased at 1.23 V (vs. Ag/AgCl) potential in a 10 mmol/L sodium borate buffer solution, pH 9.2, containing 3 mmol/L Ru(bpy)(3) (2+). A linear calibration curve (correlation coefficient = 0.9996) was obtained in the range 8 x 10(-5)-4 x 10(-8) mol/L for TMA concentration. Recoveries obtained were in the range 98.78-101.46%. The method was successfully applied for the assay of TMA in fish, in combination with solid phase extraction (SPE) disks for sample clean-up and enrichment.     

Synthesis, GC selective DNA binding and topoisomerase II inhibition activities of ruthenium(II) polypyridyl complex containing 11-aminopteridino[6,7-f][1,10]phenanthrolin-13(12H)-one

A DNA-intercalating Ru(II) polypyridyl complex [Ru(bpy)₂(appo)]²⁺ (bpy = 2,2′-bipyridine, appo = 11-aminopteridino[6,7-f][1,10]phenanthrolin-13(12H)-one) has been synthesized and characterized by elemental analysis, electrospray mass spectra, ¹H NMR, UV/Vis spectrum, fluorescent spectrum and electrochemistry. The DNA-binding, photocleavage, and topoisomerase inhibition of the complex was studied. Interestingly, the complex binds to DNA via an intercalative mode with preference for GC sequences and cleaves the pBR322 DNA upon irradiation. In addition, the complex shows high inhibition activity against topoisomerase II by interfere the DNA religation.     

Hydroxylation of androstenedione by resting Rhodococcus sp. cells in organic media

The use of organic solvent-based media for microbial 9-hydroxylation of androstenedione (AD) by resting Rhodococcus sp. cells was evaluated. Product yields higher than 90% were observed in a two-phase phthalate-buffer transformation medium (3:1, v/v) containing 1.0 g L⁻¹ AD. The results suggest that in these conditions the reaction of Δ¹-dehydrogenation is considerably inhibited. The successful hydroxylation reaction with a product yield exceeding 60% was found to take place also in a phthalate medium under simultaneous forced aeration and maintenance of a minimum water content of about 3% (v/v).     

Influence of sucrose on the rheology and granule size of cross-linked waxy maize starch dispersions heated at two temperatures

Cross-linked waxy maize (CWM) starch dispersions (STDs) of concentration 50 g kg⁻¹ were heated in sucrose solutions containing 0–600 g kg⁻¹ (g sucrose/kg dispersion) at 85 °C at low shear and in intermittently agitated cans at 110 °C. The STDs heated in 0–300 g kg⁻¹ sucrose exhibited antithixotropic behavior, while those heated in 400–600 g kg⁻¹ sucrose exhibited thixotropic behavior. The mean starch granule diameter of the starch dispersions did not show strong dependence on sucrose concentration. The dispersions, especially those with high sucrose concentrations and heated at 110 °C, exhibited G′ versus frequency (ω) profiles of gels. The STDs exhibited first normal stress differences that increased in magnitude with the concentration of sucrose. Values of the first normal stress coefficient of canned dispersions calculated from dynamic rheological data plotted against ω and experimental values plotted against shear rate of some of the STDs overlapped.     

Effects of feed sugar concentration on continuous ethanol fermentation of cheese whey powder solution (CWP)

Cheese whey powder (CWP) solution with different CWP or sugar concentrations was fermented to ethanol in a continuous fermenter using pure culture of Kluyveromyces marxianus (DSMZ 7239). Sugar concentration of the feed CWP solution varied between 55 and 200 g l⁻¹ while the hydraulic residence time (HRT) was kept constant at 54 h. Ethanol formation, sugar utilization and biomass formation were investigated as functions of the feed sugar concentration. Percent sugar utilization and biomass concentrations decreased and the effluent sugar concentration increased with increasing feed sugar concentrations especially for the feed sugar contents above 100 g l⁻¹. Ethanol concentration and productivity (DP) increased with increasing feed sugar up to 100 g l⁻¹ and then decreased with further increases in the feed sugar content. The highest ethanol concentration (3.7%, v v⁻¹) and productivity (0.54 gE l⁻¹ h⁻¹) were obtained with the feed sugar content of 100 g l⁻¹ or 125 g l⁻¹. The ethanol yield coefficient (Y_P/S) was also maximum (0.49 gE gS⁻¹) when the feed sugar was between 100 and 125 g l⁻¹. The growth yield coefficient (Y_X/S) decreased steadily from 0.123 to 0.063 gX gS⁻¹ when the feed sugar increased from 55 to 200 g l⁻¹ due to adverse effects of high sugar contents on yeast growth. The optimal feed sugar concentration maximizing the ethanol productivity and sugar utilization was between 100 and 125 g l⁻¹ under the specified experimental conditions.     

Kinetics of adsorption of Zn(II) ion on chitosan derivatives

The adsorption of Zn(II) ions from aqueous solution by chitosan derivatives (KCTS and HKCTS) was studied in a batch adsorption system. The adsorption capacities and rates of Zn(II) ions onto chitosan derivatives were evaluated. The adsorption isothermal data could be well interpreted by the Langmuir and Freundlich models. The kinetic experimental data properly correlated with the second-order kinetic model, which indicates that the chemical adsorption is the rate-limiting step. The apparent adsorption activation energy were 25.47 kJ mol⁻ and 5.473 kJ mol⁻, respectively, and the second-order adsorption constant for KCTS and HKCTS were 0.00311 g (mg min)⁻¹ and 0.005 g (mg min)⁻¹, respectively.     

Highly sensitive high-performance liquid chromatographic determination method for a new erythromycin derivative, EM523, and its major metabolites in human plasma and urine using post-column tris(2,2′-bipyridine) ruthenium(III) chemiluminescence detection

A method for the simultaneous determination of de(N-methyl)-N-ethyl-8,9-anhydroerythromycin A 6,9-hemiacetal (EM523, I) and its three metabolites in human plasma and urine has been developed using high-performance liquid chromatography (HPLC) with chemiluminescence (CL) detection. Plasma and urine samples spiked with erythromycin as an internal standard were extracted with a mixture of dichloromethane and diethyl ether under alkaline conditions. The ortanic layer was evaporated under a stream of nitrogen gas. The reconstituted sample was injected into an HPLC apparatus and separated on an ODS column using a gradient elution method. The elute was reacted on-line with a mixture of tris(2,2′-bipyridine) ruthenium(II) and peroxodisulfate, and the generated CL intensity was detected. Optimization of the CL reaction conditions resulted in a sensitive and stable CL intensity for the determination of I and its metabolites. The recovery of each compound from human plasma and urine, and the sensitivity, linearity, accuracy and precision of the method were satisfactory. The lower limits of quantitation for each compound using 0.2 ml of plasma and 0.1 ml of urine were 1 and 00 ng/ml, respectively. This method has been used for the determination of I in samples from clinical trials.     

A comparative study of the spectroscopy and oxidative spectroelectrochemistry of a series of homo- and heterobimetallic Ru(II) and Os(II) polypyridine complexes

A spectroscopic and spectroelectrochemical comparison is made among homo- and heterobimetallic complexes of the form [(bpy)₂Ru(BL)Os(byp)₂]⁴⁺, [(bpy)₂Ru(BL)Ru(bpy)₂]⁴⁺ and [(bpy)₂Os(BL)Os(bpy)₂]⁴⁺ (BL = 2,3,-bis(2′-pyridyl)pyrazzine(dpp),2,3-bis(2′-pyridyl)quinoxaline(dpq) or 2,3-bis(2′-pyridyl)benzoquinoxaline(dpb); bpy = 2,2′-bipyridine). It has been postulated that the spectroscopy of the mixed-metal bimetallic complexes bridged by polyazine bridging ligands can be assigned by comparison to those of the homobimetallic analogs. We have in hand a unique series of complexes where such a postulate can be tested. Utilizing the visible spectra of the homobimetallic Os,Os and Ru,Ru systems, we have been able to generate the spectra of the mixed-metal complexes. Some differences have been seen, particularly in the energy of the Os → dpp ³MLCT. Oxidative spectroelectrochemistry studies on the homobimetallic ruthenium or osmium based systems indicate that upon complete oxidation of both metal centers, transitions in the visible are lost. Hence, partial oxidation of the ruthenium based homobimetallics and Os, Ru mixed-metal bimetallics allows for the direct comparison of the spectroscopic character of the one remaining ruthenium chromophore within these mixed-valence systems. Oxidation to form the Os(III)/Ru(II) species and the Ru(III)/Ru(II) species resulted in similar spectra. This establishes further that the visible spectroscopy of mixed-metal systems of this nature can be accurately interpreted by comparison to the homobimetallic analogs.     

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.     

Biological phosphorus removal in sequencing batch reactor with single-stage oxic process

The performance of biological phosphorus removal (BPR) in a sequencing batch reactor (SBR) with single-stage oxic process was investigated using simulated municipal wastewater. The experimental results showed that BPR could be achieved in a SBR without anaerobic phase, which was conventionally considered as a key phase for BPR. Phosphorus (P) concentration 0.22–1.79 mg L⁻¹ in effluent can be obtained after 4 h aeration when P concentration in influent was about 15–20 mg L⁻¹, the dissolved oxygen (DO) was controlled at 3 ± 0.2 mg L⁻¹ during aerobic phase and pH was maintained 7 ± 0.1, which indicated the efficiencies of P removal were achieved 90% above. Experimental results also showed that P was mainly stored in the form of intracellular storage of polyphosphate (poly-P), and about 207.235 mg phosphates have been removed by the discharge of rich-phosphorus sludge for each SBR cycle. However, the energy storage poly-β-hydroxyalkanoates (PHA) was almost kept constant at a low level (5–6 mg L⁻¹) during the process. Those results showed that phosphate could be transformed to poly-P with single-stage oxic process without PHA accumulation, and BPR could be realized in net phosphate removal.     

Electrochemiluminescence determination of codeine or morphine with an organically modified silicate film immobilizing Ru(bpy)3(2+).

An ECL approach was developed for the determination of codeine or morphine based on tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)(3)(2+)) immobilized in organically modified silicates (ORMOSILs). Tetramethoxysilane (TMOS) and dimethyldimethoxysilane (DiMe-DiMOS) were selected as co-precursors for ORMOSILs, which were then immobilized on a surface of glassy carbon electrode (GCE) by a dip-coating process. Ru(bpy)(3)(2+) was immobilized in the ORMOSIL film via ion-association with poly(p-styrenesulphonate). The ORMOSIL-modified GCE presented good electrochemical and photochemical activities. In a flow system, the eluted codeine or morphine was oxidized on the modified GCE and reacted with immobilized Ru(bpy)(3)(2+) at a potential of +1.20 V (vs. Ag/AgCl). The modified electrode was used for the ECL determination of codeine or morphine and showed high sensitivity. The calibration curves were linear in the range 2 x 10(-8)-5 x 10(-5) mol/L for codeine and 1 x 10(-7)-3 x 10(-4) mol/L for morphine. The detection limit was 5 x 10(-9) mol/L for codeine and 3 x 10(-8) mol/L for morphine, at signal:noise ratio (S:N)=3. Both codeine and morphine showed reproducibility with RSD values <2.5% at 1.0 x 10(-6) mol/L. Furthermore, the modified electrode immobilized Ru(bpy)(3)(2+) was applied to the ECL determination of codeine or morphine in incitant samples.     

Halophila ovalis (R. Br.) Hook. f. from a submarine hot spring in southern Japan

Colonies of the seagrass Halophila ovalis are found growing adjacent to coral Acropora sp. and Seriatopora hystrix in a submarine hot spring (at 15.7 m depth, 28.6°C) at the north coast of Taketomi Island, near the southern tip of Japan. Halophila plants grow in sea water containing sulphide 930 μg S ml⁻¹ and on the substratum with fine precipitates of the submarine hot spring which have sulphide content up to 5400 μg S g⁻¹ DW. The accumulated sulphide concentration reaches as high as 8400 μg S g⁻¹ DW in under ground tissues and 5700 μg S g⁻¹ DW in above-ground tissues, respectively. It is suggested that, not the sulphide concentration but light and possibly water temperature are the limiting factors for the Halophila colonization in the submarine hot spring.