Isolation of a dimethylsulfide-utilizingHyphomicrobium species and its application in biofiltration of polluted air

The methylotrophic bacteriumHyphomicrobium VS was enriched and isolated, using activated sewage sludge as inoculum in mineral medium containing dimethylsulfide (DMS) at a low concentration to prevent toxicity. DMS concentrations above 1 mM proved to be growth inhibiting.Hyphomicrobium VS could use DMS, dimethylsulfoxide (DMSO), methanol, formaldehyde, formate, and methylated amines as carbon and energy source. Carbon was assimilated via the serine pathway. DMS-grown cells respired sulfide, thiosulfate, methanethiol, dimethyldisulfide and dimethyltrisulfide.To testHyphomicrobium VS for application in biofiltration of air polluted with volatile sulfur compounds two laboratory scale trickling biofilters with polyurethane and lava stone as carrier material were started up by inoculation with this bacterium. Both methanol- and DMS-grown cells could be used. Only a short adaptation period was needed. Short term experiments showed that high concentrations of DMS (1–2 µmol 1^–1) were removed very efficiently by the biofilters at space velocities up to 100 h^–1.Abbreviations VSC volatile sulfur compounds - DMS dimethylsulfide - DMDS dimethyldisulfide - DMTS dimethyltrisulfide - MT methanethiol - DMSO dimethylsulfoxide     

Oxidation of Dimethyl Sulfide to Dimethyl Sulfoxide by Phototrophic Purple Bacteria

Enrichment cultures of phototrophic purple bacteria rapidly oxidized up to 10 mM dimethyl sulfide (DMS) to dimethyl sulfoxide (DMSO). DMSO was qualitatively identified by proton nuclear magnetic resonance. By using a biological assay, DMSO was always quantitatively recovered from the culture media. DMS oxidation was not detected in cultures incubated in the dark, and it was slow in cultures exposed to full daylight. Under optimal conditions, the second-order rate constant for DMS oxidation was 6 day⁻¹ mg of protein⁻¹ ml⁻¹. The rate constant was reduced in the presence of high concentration of sulfide (>1 mM), but was not affected by the addition of acetate. DMS was also oxidized to DMSO by a pure strain (tentatively identified as a Thiocystis sp.) isolated from the enrichment cultures. DMS supported growth of the enrichment cultures and of the pure strain by serving as an electron source for photosynthesis. A determination of the amount of protein produced in the cultures and an estimation of the electron balance suggested that the two electrons liberated during the oxidation of DMS to DMSO were quantitatively used to reduce carbon dioxide to biomass. The oxidation of DMS by phototrophic purple bacteria may be an important source of DMSO detected in anaerobic ponds and marshes.     

Aerobic turnover of dimethyl sulfide by the anoxygenic phototrophic bacterium Thiocapsa roseopersicina

This is the first report describing the complete oxidation of dimethyl sulfide (DMS) to sulfate by an anoxygenic, phototrophic purple sulfur bacterium. Complete DMS oxidation was observed in cultures of Thiocapsa roseopersicina M11 incubated under oxic/light conditions, resulting in a yield of 30.1 mg protein mmol^–1. No oxidation of DMS occurred under anoxic/light conditions. Chloroform, methyl butyl ether, and 3-amino-1,2,4-triazole, which are specific inhibitors of aerobic DMS oxidation in thiobacilli and hyphomicrobia, did not affect DMS oxidation in strain M11. This could be due to limited transport of the inhibitors through the cell membrane. The growth yield on sulfide as sole electron donor was 22.2 mg protein mmol^–1 under anoxic/light conditions. Since aerobic respiration of sulfide would have resulted in yields lower than 22 mg protein mmol^–1, the higher yield on DMS under oxic/light conditions suggests that the methyl groups of DMS have served as an additional carbon source or as an electron donor in addition to the sulfide moiety. The kinetic parameters V _max and K _m for DMS oxidation under oxic/light conditions were 12.4 ± 1.3 nmol (mg protein)^–1 min^–1 and 2 μM, respectively. T. roseopersicina M11 also produced DMS by cleavage of dimethylsulfoniopropionate (DMSP). Specific DMSP cleavage rates increased with increasing initial substrate concentrations, suggesting that DMSP lyase was only partly induced at lower initial DMSP concentrations. A comparison of T. roseopersicina strains revealed that only strain M11 was able to oxidize DMS and cleave DMSP. Both strain M11 and strain 5811 accumulated DMSP intracellularly during growth, while strain 1711 showed neither of these characteristics. Phylogenetic comparison based on 16S rRNA gene sequence revealed a similarity of 99.0% between strain M11 and strain 5811, and 97.6% between strain M11 and strain 1711. DMS and DMSP utilization thus appear to be strain-specific. Received: 26 March 1999 / Accepted: 18 June 1999     

Effect of PAR irradiance intensity on Phaeocystis antarctica (Prymnesiophyceae) growth and DMSP,DMSO, and acrylate concentrations

Phaeocystis antarctica forms extensive spring blooms in the Southern Ocean that coincide with high concentrations of dimethylsulfoniopropionate (DMSP), dimethylsulfoxide (DMSO), dimethylsulfide (DMS), and acrylate. We determined how concentrations of these compounds changed during the growth of axenic P. antarctica cultures exposed to light-limiting, sub-saturating, and saturating PAR irradiances. Cellular DMSP concentrations per liter cell volume (CV) ranged between 199 and 403 mmol · L_CV⁻¹, with the highest concentrations observed under light-limiting PAR. Cellular acrylate concentrations did not change appreciably with a change in irradiance level or growth, ranging between 18 and 45 mmol · L_CV⁻¹, constituting an estimated 0.2%–2.8% of cellular carbon. Both dissolved acrylate and DMSO increased substantially with irradiance during exponential growth on a per-cell basis, ranging from 0.91 to 3.15 and 0.24 to 1.39 fmol · cell⁻¹, respectively, indicating substantial export of these compounds into the dissolved phase. Average cellular DMSO:DMSP ratios increased 7.6-fold between exponential and stationary phases of batch growth, with a 3- to 13-fold increase in cellular DMSO likely formed from abiotic reactions of DMSP and DMS with reactive oxygen species (ROS). At mM levels, cellular DMSP and acrylate are proposed to serve as de facto antioxidants in P. antarctica not regulated by oxidative stress or changes in ROS. Instead, cellular DMSP concentrations are likely controlled by other physiological processes including an overflow mechanism to remove excess carbon via acrylate, DMS, and DMSO during times of unbalanced growth brought on by physical stress or nutrient limitation. Together, these compounds should aid P. antarctica in adapting to a range of PAR irradiances by maintaining cellular functions and reducing oxidative stress.     

Growth of Rhodopseudomonas capsulata under anaerobic dark conditions with dimethyl sulfoxide

The photosynthetic bacterium, Rhodopseudomonas capsulata, could be cultured anaerobically in the absence of light on a synthetic medium with glucose as the carbon source only when dimethyl sulfoxide (DMSO) was added. The extent of growth was proportional to both DMSO and glucose concentrations. Optimal growth was achieved with 20 mm DMSO and 0.25% glucose. Under the best conditions, cells divided with a doubling time of 12 h. Pyruvate also supported the anaerobic dark growth of R. capsulata when DMSO was present. R. capsulata, R. sphaeroides, and R. palustris strains were all able to grow under anaerobic dark conditions with DMSO. Experiments using [¹⁴C]DMSO showed that more than 95% of the ¹⁴C was converted by cultures of R. capsulata to a volatile compound, identified as dimethyl sulfide (DMS) by gas chromatography, thus demonstrating that DMSO was being reduced to DMS during growth. These results indicate that R. capsulata requires a terminal electron acceptor for anaerobic dark growth and that DMSO can serve that function.     

Microbial Activity in Aquatic Environments Measured by Dimethyl Sulfoxide Reduction and Intercomparison with Commonly Used Methods

A new method to determine microbial (bacterial and fungal) activity in various freshwater habitats is described. Based on microbial reduction of dimethyl sulfoxide (DMSO) to dimethyl sulfide (DMS), our DMSO reduction method allows measurement of the respiratory activity in interstitial water, as well as in the water column. DMSO is added to water samples at a concentration (0.75% [vol/vol] or 106 mM) high enough to compete with other naturally occurring electron acceptors, as determined with oxygen and nitrate, without stimulating or inhibiting microbial activity. Addition of NaN₃, KCN, and formaldehyde, as well as autoclaving, inhibited the production of DMS, which proves that the reduction of DMSO is a biotic process. DMSO reduction is readily detectable via the formation of DMS even at low microbial activities. All water samples showed significant DMSO reduction over several hours. Microbially reduced DMSO is recovered in the form of DMS from water samples by a purge and trap system and is quantified by gas chromatography and detection with a flame photometric detector. The DMSO reduction method was compared with other methods commonly used for assessment of microbial activity. DMSO reduction activity correlated well with bacterial production in predator-free batch cultures. Cell-production-specific DMSO reduction rates did not differ significantly in batch cultures with different nutrient regimes but were different in different growth phases. Overall, a cell-production-specific DMSO reduction rate of 1.26 × 10⁻¹⁷ ± 0.12 × 10⁻¹⁷ mol of DMS per produced cell (mean ± standard error; R² = 0.78) was calculated. We suggest that the relationship of DMSO reduction rates to thymidine and leucine incorporation is linear (the R² values ranged from 0.783 to 0.944), whereas there is an exponential relationship between DMSO reduction rates and glucose uptake, as well as incorporation (the R² values ranged from 0.821 to 0.931). Based on our results, we conclude that the DMSO reduction method is a nonradioactive alternative to other methods commonly used to assess microbial activity.     

Metabolism of DMSP, DMS and DMSO by the cultivable bacterial community associated with the DMSP-producing dinoflagellate Scrippsiella trochoidea

Bacterial species associated with the dimethylsulfoniopropionate (DMSP)-producing phytoplankton Scrippsiella trochoidea were cultured and identified, with the aim of establishing their ability to metabolise DMSP, dimethylsulfide (DMS) and dimethylsulfoxide (DMSO). Results demonstrate that of the cultivable bacteria only α-Proteobacteria were capable of producing DMS from DMSP. The concentration of DMSP was shown to affect the amount of DMS produced. Lower DMSP concentrations (1.5?μmol?dm^?3) were completely assimilated, whereas higher concentrations (10?μmol?dm^?3) resulted in increasing amounts of DMS being produced. By contrast to the restricted set of bacteria that metabolised DMSP,?~?70% of the bacterial isolates were able to ‘consume’ DMS. However, 98-100% of the DMS removed was accounted for as DMSO. Notably, a number of these bacteria would only oxidise DMS in the presence of glucose, including members of the γ-Proteobacteria and Bacteroidetes. The observations from this study, coupled with published field data, identify DMS oxidation to DMSO as a major transformation pathway for DMS, and we speculate that the fate of DMS and DMSP in the field are tightly coupled to the available carbon produced by phytoplankton.     

The role of auxiliary oxidants in maintaining redox balance during phototrophic growth of Rhodobacter capsulatus on propionate or butyrate

Phototrophic growth of Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata) under anaerobic conditions with either butyrate or propionate as carbonsource was dependent on the presence of either CO₂ or an auxiliary oxidant. NO _- ³ , N₂O, trimethylamine-N-oxide (TMAO) or dimethylsulphoxide (DMSO) were effective provided the appropriate anaerobic respiratory pathway was present. NO _- ³ was reduced extensively to NO _- ³ , TMAO to trimethylamine and DMSO to dimethylsulphide under these conditions. Analysis of culture fluids by nuclear magnetic resonance showed that two moles of TMAO or DMSO were reduced per mole of butyrate utilized and one mole of either oxidant was reduced per mole of propionate consumed. The growth rate of Rb. capsulatus on succinate or malate as carbon source was enhanced by TMAO in cultures at low light intensity but not at high light intensities. A new function for anaerobic respiration during photosynthesis is proposed: it permits reducing equivalents from reduced substrates to pass to auxiliary oxidants present in the medium. The use of CO₂ or auxiliary oxidants under phototrophic conditions may be influence by the availability of energy from light. It is suggested that the nuclear magnetic resonance methodology developed could have further applications in studies of bacterial physiology.Abbreviations DMS dimethylsulphide - DMSO dimethylsulphoxide - TMA trimethylamine - TMAO trimethylamine-N-oxide - NMR nuclear magnetic resonance     

Utilization of Dimethyl Sulfide as a Sulfur Source with the Aid of Light by Marinobacterium sp. Strain DMS-S1

Strain DMS-S1 isolated from seawater was able to utilize dimethyl sulfide (DMS) as a sulfur source only in the presence of light in a sulfur-lacking medium. Phylogenetic analysis based on 16S ribosomal DNA genes indicated that the strain was closely related to Marinobacterium georgiense. The strain produced dimethyl sulfoxide (DMSO), which was a main metabolite, and small amounts of formate and formaldehyde when grown on DMS as the sole sulfur source. The cells of the strain grown with succinate as a carbon source were able to use methyl mercaptan or methanesulfonate besides DMS but not DMSO or dimethyl sulfone as a sole sulfur source. DMS was transformed to DMSO primarily at wavelengths between 380 and 480 nm by heat-stable photosensitizers released by the strain. DMS was also degraded to formaldehyde in the presence of light by unidentified heat-stable factors released by the strain, and it appeared that strain DMS-S1 used the degradation products, which should be sulfite, sulfate, or methanesulfonate, as sulfur sources.     

Oxidative stress enhances granulocytic differentiation in HL 60 cells,an acute promyelocytic leukemia cell line

Abstract

This paper studied the effects of physiologically available oxidants on HL 60 differentiation induced by all-trans retinoic acid (ATRA) or dimethyl sulfoxide (DMSO). Hydrogen peroxide (15 μM) and taurine chloramine (200 μM) induced HL 60 differentiation, which was detected by CD11b expression and superoxide production. Cd11b and p67^phox mRNA expression was also augmented by these oxidants. In contrast, reducing chemicals, such as dithiothreitol, 2,3-dimercapto-1-propanol and N-acetylcysteine inhibited CD11b expression. Notably, DMSO inhibited methionine sulfoxide reductase activity, induced heme oxygenase-1 (ho-1) mRNA and enhanced oxidant-induced cell death, which indicated that DMSO intensified oxidative stress. After the addition of oxidants, ho-1 expression preceded the cd11b expression. Vicinal dithiol-reactive phenylarsine oxide (50 nM) also increased CD11b expression induced by DMSO or ATRA. These observations suggested that oxidative stress enhanced granulocytic differentiation of HL 60 cells and that leukaemic cell differentiation was affected by cellular redox status.     

Protection of osteoblastic cells from freeze/thaw cycle-induced oxidative stress by green tea polyphenol

Green tea polyphenol (GTP) together with dimethylsulphoxide (DMSO) were added to a freezing solution of osteoblastic cells (rat calvarial osteoblasts and human osteosarcoma cells) exposed to repeated freeze/thaw cycles (FTC) to induce oxidative stress. When cells were subjected to 3 FTCs, freezing medium containing 10% (v/v) DMSO and 500 μg GTP ml⁻¹ significantly (p < 0.05) suppressed cell detachment and growth inhibition by over 63% and protected cell morphology. Furthermore, the alkaline phosphatase activity of osteoblastic cells was appreciably maintained after 2 and 3 FTCs in this mixture. Polyphenols may thus be of use as a cell cryopreservant and be advantageous in such fields as cell transplantation and tissue engineering.     

A mechanistic and electrochemical study of the interaction between dimethyl sulfide dehydrogenase and its electron transfer partner cytochrome c 2

Dimethyl sulfide dehydrogenase isolated from the photosynthetic bacterium Rhodovulum sulfidophilum is a heterotrimeric enzyme containing a molybdenum cofactor at its catalytic site, as well as five iron–sulfur clusters and a heme b cofactor. It oxidizes dimethyl sulfide (DMS) to dimethyl sulfoxide in its native role and transfers electrons to the photochemical reaction center. There is genetic evidence that cytochrome c ₂ mediates this process, and the steady state kinetics experiments reported here demonstrated that cytochrome c ₂ accepts electrons from DMS dehydrogenase. At saturating concentrations of both substrate (DMS) and cosubstrate (cytochrome c ₂), Michaelis constants, K _M,DMS and K _M,cyt of 53 and 21 μM, respectively, were determined at pH 8. Further kinetic analysis revealed a “ping-pong” enzyme reaction mechanism for DMS dehydrogenase with its two reactants. Direct cyclic voltammetry of cytochrome c ₂ immobilized within a polymer film cast on a glassy carbon electrode revealed a reversible Fe^III/II couple at +328 mV versus the normal hydrogen electrode at pH 8. The Fe^III/II redox potential exhibited only minor pH dependence. In the presence of DMS dehydrogenase and DMS, the peak-shaped voltammogram of cytochrome c ₂ is transformed into a sigmoidal curve consistent with a steady-state (catalytic) reaction. The cytochrome c ₂ effectively mediates electron transfer between the electrode and DMS dehydrogenase during turnover and a significantly lower apparent electrochemical Michaelis constant of 13(±1) μM was obtained. The pH optimum for catalytic DMS oxidation by DMS dehydrogenase with cytochrome c ₂ as the electron acceptor was found to be approximately 8.3.     

Dynamics of Dimethyl Sulfide in a Marine Microbial Mat

Abstract The microbial mat was chosen as a model ecosystem to study dynamics of dimethyl sulfide (DMS) in marine sediments in order to gain insight into key processes and factors which determine emission rates. A practical advantage, compared to open ocean ecosystems, is that microbial mats contain high biomasses of different functional groups of bacteria involved in DMS dynamics, and that DMS concentrations are generally high enough to allow direct measurement of emission rates. Field data showed that, during the seasonal development of microbial mats, concentrations of chlorophyll a corresponded to dimethylsulfoniopropionate (DMSP). DMSP is an important precursor of DMS. It was demonstrated, with laboratory cultures, that various species of benthic diatoms produce substantial amounts of DMSP. The abundances of aerobic and anaerobic DMS- or DMSO-utilizing bacteria were estimated using the most-probable-number technique. Laboratory experiments with relatively undisturbed sediment cores showed that microbial mats act as a sink for DMS under oxic/light (day) conditions, and as a source of DMS under anoxic/dark (night) conditions. Axenic culture studies with Chromatium vinosum M2 and Thiocapsa pfennigii M8 (isolated from a microbial mat) showed that, under anoxic/light conditions, DMS was quantitatively converted to dimethylsulfoxide (DMSO). T. roseopersicina M11 converted DMSP to DMS and acrylate, apparently without use of either substrate. Received: 5 May 1997; Accepted: 21 August 1997     

Nitrate reduction associated with respiration in <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> 2011 is performed by a membrane-bound molybdoenzyme

The purification and biochemical characterization of the respiratory membrane-bound nitrate reductase from Sinorhizobium meliloti 2011 (Sm NR) is reported together with the optimal conditions for cell growth and enzyme production. The best biomass yield was obtained under aerobic conditions in a fed-batch system using Luria–Bertani medium with glucose as carbon source. The highest level of Sm NR production was achieved using microaerobic conditions with the medium supplemented with both nitrate and nitrite. Sm NR is a mononuclear Mo-protein belonging to the DMSO reductase family isolated as a heterodimeric enzyme containing two subunits of 118 and 45 kDa. Protein characterization by mass spectrometry showed homology with respiratory nitrate reductases. UV–Vis spectra of as-isolated and dithionite reduced Sm NR showed characteristic absorption bands of iron-sulfur and heme centers. Kinetic studies indicate that Sm NR follows a Michaelis–Menten mechanism (K _m = 97 ± 11 μM, V = 9.4 ± 0.5 μM min⁻¹, and k _cat = 12.1 ± 0.6 s⁻¹) and is inhibited by azide, chlorate, and cyanide with mixed inhibition patterns. Physiological and kinetic studies indicate that molybdenum is essential for NR activity and that replacement of this metal for tungsten inhibits the enzyme. Although no narGHI gene cluster has been annotated in the genome of rhizobia, the biochemical characterization indicates that Sm NR is a Mo-containing NR enzyme with molecular organization similar to NarGHI.     

Fractionation of stable carbon isotopes by photoautotrophically growing anoxygenic purple and green sulfur bacteria

Fractionation of stable carbon isotopes ¹²C and ¹³C by three pure cultures of photoautotrophic purple sulfur bacteria (Ectothiorhodospira shaposhnikovii, Lamprocystis purpureus, and Thiocapsa sp.) (PSB) and the green sulfur bacterium Prosthecochloris sp. (GSB) was investigated in 13–15-day experiments. The cultivation was carried out in a luminostat (2000 lx) on mineral media with 1–1.5 g/l NaHCO₃ (inoculum) with the subsequent transfer to the medium with up to 10 g/l NaHCO₃. For PSB, the difference in the quantitative characteristics of the isotopic composition of suspended carbon (including bacterial cells) and mineral carbon of the medium (Δ¹³C = δ¹³C_substrate − δ¹³C_biomass) changed from 15.0 to 34.3‰. For GSB, the range of Δ¹³C changes was significantly less (18.3–22.7‰). These data suggested the possibility of a pool of soluble mineral carbon in PSB cells. The pool of intracellular mineral carbon was calculated; depending on the PSB species and growth stage, it varied from 0 to 68% of the total cell carbon. The α coefficients reflecting the carbon isotope fractionation by PSB and GBS and calculated from the changes of the bicarbonate carbon isotopic composition in the medium depending on its consumption were 1.029 ± 0.003 and 1.019 ± 0.001, respectively. These α values did not depend on the growth rate. CO₂ fixation on ribulose-bisphosphate was shown to be the major factor determining the carbon isotope fractionation by PSB; at the stage of CO₂ penetration into the cell, fractionation was insignificant. In GSB, fractionation occurred mostly at CO₂ penetration into the cell, while it was insignificant at the stage of carbon dioxide fixation in the reverse TCA cycle. Analysis of the isotopic data of the photosynthesis by PSB and GSB in meromictic lakes also revealed that in PSB-dominated natural communities suspended organic matter was more enriched with light ¹³C (Δ¹³C = 23.4−24.6‰) than in the communities with more active GSB (Δ¹³C = 10.2−14.0‰)     

Utilization of dimethyl sulfide as a sulfur source with the aid of light by Marinobacterium sp. strain DMS-S1

Strain DMS-S1 isolated from seawater was able to utilize dimethyl sulfide (DMS) as a sulfur source only in the presence of light in a sulfur-lacking medium. Phylogenetic analysis based on 16S ribosomal DNA genes indicated that the strain was closely related to Marinobacterium georgiense. The strain produced dimethyl sulfoxide (DMSO), which was a main metabolite, and small amounts of formate and formaldehyde when grown on DMS as the sole sulfur source. The cells of the strain grown with succinate as a carbon source were able to use methyl mercaptan or methanesulfonate besides DMS but not DMSO or dimethyl sulfone as a sole sulfur source. DMS was transformed to DMSO primarily at wavelengths between 380 and 480 nm by heat-stable photosensitizers released by the strain. DMS was also degraded to formaldehyde in the presence of light by unidentified heat-stable factors released by the strain, and it appeared that strain DMS-S1 used the degradation products, which should be sulfite, sulfate, or methanesulfonate, as sulfur sources.     

Purification and characterization of dimethylsulfide monooxygenase from Hyphomicrobium sulfonivorans

Dimethylsulfide (DMS) is a volatile organosulfur compound which has been implicated in the biogeochemical cycling of sulfur and in climate control. Microbial degradation is a major sink for DMS. DMS metabolism in some bacteria involves its oxidation by a DMS monooxygenase in the first step of the degradation pathway; however, this enzyme has remained uncharacterized until now. We have purified a DMS monooxygenase from Hyphomicrobium sulfonivorans, which was previously isolated from garden soil. The enzyme is a member of the flavin-linked monooxygenases of the luciferase family and is most closely related to nitrilotriacetate monooxygenases. It consists of two subunits: DmoA, a 53-kDa FMNH₂-dependent monooxygenase, and DmoB, a 19-kDa NAD(P)H-dependent flavin oxidoreductase. Enzyme kinetics were investigated with a range of substrates and inhibitors. The enzyme had a K_m of 17.2 (± 0.48) μM for DMS (k_cat = 5.45 s⁻¹) and a V_max of 1.25 (± 0.01) μmol NADH oxidized min⁻¹ (mg protein⁻¹). It was inhibited by umbelliferone, 8-anilinonaphthalenesulfonate, a range of metal-chelating agents, and Hg²⁺, Cd²⁺, and Pb²⁺ ions. The purified enzyme had no activity with the substrates of related enzymes, including alkanesulfonates, aldehydes, nitrilotriacetate, or dibenzothiophenesulfone. The gene encoding the 53-kDa enzyme subunit has been cloned and matched to the enzyme subunit by mass spectrometry. DMS monooxygenase represents a new class of FMNH₂-dependent monooxygenases, based on its specificity for dimethylsulfide and the molecular phylogeny of its predicted amino acid sequence. The gene encoding the large subunit of DMS monooxygenase is colocated with genes encoding putative flavin reductases, homologues of enzymes of inorganic and organic sulfur compound metabolism, and enzymes involved in riboflavin synthesis.Dimethylsulfide (DMS) is a volatile organosulfur compound, important in the biogeochemical cycling of sulfur and global climate regulation (4, 9). Bacterial metabolism of DMS is an important sink of the compound in nature and is thought to account for degradation of over 80% of the DMS produced in the marine environment. Although bacterial pathways of DMS degradation have been studied previously in Hyphomicrobium spp. and in Thiobacillus spp. (12, 36), they remain poorly characterized, and few enzymes of DMS metabolism have been purified (see reference 32). DMS monooxygenase was first reported from an assay of NADH-dependent oxygen uptake in the presence of DMS by cell extracts of Hyphomicrobium S (12), an activity also demonstrated in cell extracts of other Hyphomicrobium, Thiobacillus, and Arthrobacter isolates (6, 7, 34), with specific activities around 30 nmol NADH oxidized min⁻¹ mg protein⁻¹. The enzyme has not previously been purified or characterized.The aims of this study were to purify and characterize the DMS monooxygenase enzyme from a member of the genus Hyphomicrobium. Since Hyphomicrobium S is no longer available, studies were undertaken using the type strain of H. sulfonivorans. The strain was originally isolated from garden soil and grows on DMS, as well as the related compounds dimethyl sulfoxide (DMSO) and dimethylsulfone (DMSO₂). During growth on DMSO₂, H. sulfonivorans first reduces DMSO₂ to DMSO by a dimethylsulfone reductase, and subsequently a DMSO reductase converts DMSO to DMS, which is further oxidized to methanethiol and formaldehyde by a DMS monooxygenase. Oxidation of methanethiol to formaldehyde by methanethiol oxidase yields another mole of formaldehyde, which is either assimilated into biomass or oxidized to carbon dioxide to provide reducing equivalents (Fig. ​(Fig.1).1). DMS monooxygenase activity is present in the soluble protein fraction during growth on these compounds (6, 7). A 53-kDa polypeptide was previously observed in organisms grown on DMS, DMSO, and DMSO₂ (6, 7), but its significance in the metabolism of these compounds was unknown.Open in a separate windowFIG. 1.Pathway and enzymes of dimethylsulfone degradation in Hyphomicrobium sulfonivorans S1. Reduction of dimethylsulfone [DMSO₂; (CH₃)₂SO₂] to dimethyl sulfoxide [DMSO; (CH₃)₂SO] and further reduction of DMSO to dimethylsulfide provides the substrate for DMS monooxygenase. Formaldehyde is either assimilated (via the serine cycle) or oxidized to CO₂ providing reducing equivalents. Sulfide is oxidized to sulfate; see reference 7 for further details.     

Carbon dioxide and biearbonate in cell division

Summary The effects of carbon dioxide and of bicarbonate on cell division were studied on synchronized cells of the high-temperature green alga, Chlorella 7-11-05. After 7 hours of growth in nutrient medium in light, cells were centrifuged and resuspended in distilled water or in bicarbonate and placed in darkness. Atmospheric air, or a mixture of carbon dioxide and air, was bubbled through algal suspensions during the dark period. In distilled water cells readily divided in atmospheric air but not in 1% (2.6·10^-4 M) or in higher concentrations of carbon dioxide. The suspension of cells in bicarbonate counteracted the inhibitory action of carbon dioxide. A minimum molar concentration of bicarbonate necessary to counteract the inhibitory effect of carbon dioxide was found to be equal to the molar concentration of carbon dioxide in the suspending fluid. The highest concentration of carbon dioxide, the adverse effect of which could not be balanced by any concentration of bicarbonate, was found to be in the vicinity of 1.3·10^-2 M (50% CO₂ in air). Possible effects on cell division of the change in Ph and the implicated role of carbon dioxide in normal and neoplastic growth were discussed.     

Growth and production characteristics of permeabilized Coleus blumei cells in immobilized fed-batch culture

Summary Permeabilized Coleus blumei cells were cultivated in an immobilized state to study the effect of dimethyl sulfoxide (DMSO) concentrations and growth regulators on cell growth and rosmarinic acid (RA) production characteristics. Luffa (the fibrous skeleton of mature fruit of Luffa cylindrica) was a good support matrix for cell immobilization because of its high void volume. Maximum cell loading capacity was 1.33 g dry cell weight (DCW)/g dry Luffa. The experiments were done in shake flasks with no free medium. The medium was supplied in a fed-batch mode to avoid the flotation of Luffa pieces. The sucrose in the medium was completely hydrolyzed to glucose and fructose without any sugar accumulation in the medium. The cell viability was slightly higher in the cells on top of the Luffa than those in the middle. Cell growth rate and rosmarinic acid (RA) production were approximately half that obtained in cell suspension cultures. Cell yield (g DCW/g glucose) was similar to that of cell suspension cultures. The absence of growth regulators did not promote an increase of RA production but did decrease the cell mass. The second step preconditioning with 0.5% DMSO did not improve the cell's adaptability to higher DMSO concentrations and the cell mass did not increase with 2.5% DMSO.     

Improved growth and methane production conditions for Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum was grown on a defined mineral salts medium under strictly anaerobic conditions with H₂ and CO₂ as the sole energy and carbon sources, respectively. The cultivation medium was optimized with respect to non-organic components including Se(IV), W(VI), N, Ni(II), Fe(II), Co(II) and Mo(VI). Sulphide concentration in the medium was maintained constant using an on-line regulatory system by the addition of 0.5 M Na₂S. A maximum supply rate of 0.6 vvm of a mixture of 80% H₂ and 20% CO₂ was achieved for the gaseous substrates. Under these conditions a specific growth rate of 0.30 h^–1 and a cell concentration of 4.8 g cell dry weight (DW) l^–1, representing a 140% increase over previously published results, were obtained. The growth yield of 2.3 g DW mol^–1 CH₄ was similar to published values. However, the overall specific productivity was enhanced from 11 mmol CH₄ g^–1 DW h^–1 to 24 mmol CH₄ g^–1 DW h^–1, corresponding to an improvement of 120%. Correspondence to: U. von Stockar