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.     

Reactions of dimethylsulfoxide reductase from Rhodobacter capsulatus with dimethyl sulfide and with dimethyl sulfoxide: complexities revealed by conventional and stopped-flow spectrophotometry.

Improved assays for the molybdenum enzyme dimethylsulfoxide reductase (DMSOR) with dimethyl sulfoxide (DMSO) and with dimethyl sulfide (DMS) as substrates are described. Maximum activity was observed at pH 6.5 and below and at 8.3, respectively. Rapid-scan stopped-flow spectrophotometry has been used to investigate the reduction of the enzyme by DMS to a species previously characterized by its UV-visible spectrum [McAlpine, A. S., McEwan, A. G., and Bailey, S. (1998) J. Mol. Biol. 275, 613-623], and its subsequent reoxidation by DMSO. Both these two-electron reactions were faster than enzyme turnover under steady-state conditions, indicating that one-electron reactions with artificial dyes were rate-limiting. Second-order rate constants for the two-electron reduction and reoxidation reactions at pH 5.5 were (1.9 +/- 0.1) x 10(5) and (4.3 +/- 0.3) x 10(2) M-1 s-1, respectively, while at pH 8.0, the catalytic step was rate-limiting (62 s-1). Kinetically, for the two-electron reactions, the enzyme is more effective in DMS oxidation than in DMSO reduction. Reduction of DMSOR by DMS was incomplete below approximately 1 mM DMS but complete at higher concentrations, implying that the enzyme's redox potential is slightly higher than that of the DMS-DMSO couple. In contrast, reoxidation of the DMS-reduced state by DMSO was always incomplete, regardless of the DMSO concentration. Evidence for the existence of a spectroscopically indistinguishable reduced state, which could not be reoxidized by DMSO, was obtained. Brief reaction (less than approximately 15 min) of DMS with DMSOR was fully reversible on removal of the DMS. However, in the presence of excess DMS, a further slow reaction occurred aerobically, but not anaerobically, to yield a stable enzyme form having a lambdamax at 660 mn. This state (DMSORmod) retained full activity in steady-state assays with DMSO, but was inactive toward DMS. It could however be reconverted to the original resting state by reduction with methyl viologen radical and reoxidation with DMSO. We suggest that in this enzyme form two of the dithiolene ligands of the molybdenum have dissociated and formed a disulfide. The implications of this new species are discussed in relation both to conflicting published information for DMSOR from X-ray crystallography and to previous spectroscopic data for its reduced forms.     

Inactivation of MXR1 Abolishes Formation of Dimethyl Sulfide from Dimethyl Sulfoxide in Saccharomyces cerevisiae

Dimethyl sulfide (DMS) is a sulfur compound of importance for the organoleptic properties of beer, especially some lager beers. Synthesis of DMS during beer production occurs partly during wort production and partly during fermentation. Methionine sulfoxide reductases are the enzymes responsible for reduction of oxidized cellular methionines. These enzymes have been suggested to be able to reduce dimethyl sulfoxide (DMSO) as well, with DMS as the product. A gene for an enzymatic activity leading to methionine sulfoxide reduction in Saccharomyces yeast was recently identified. We confirmed that the Saccharomyces cerevisiae open reading frame YER042w appears to encode a methionine sulfoxide reductase, and propose the name MXR1 for the gene. We found that Mxr1p catalyzes reduction of DMSO to DMS and that an mxr1 disruption mutant cannot reduce DMSO to DMS. Mutant strains appear to have unchanged fitness under several laboratory conditions, and in this paper I hypothesize that disruption of MXR1 in brewing yeasts would neutralize the contribution of the yeast to the DMS content in beer.     

Dimethyl sulphide and methanethiol formation in microbial mats: potential pathways for biogenic signatures

Mechanisms of dimethyl sulphide (DMS) and methanethiol (MT) production and consumption were determined in moderately hypersaline mats, Guerrero Negro, Mexico. Biological pathways regulated the net flux of DMS and MT as revealed by increases in flux resulting from decreased salinity, increased temperature and the removal of oxygen. Dimethylsulphoniopropionate (DMSP) was not present in these microbial mats and DMS and MT are probably formed by the reaction of photosynthetically produced low-molecular weight organic carbon and biogenic hydrogen sulphide derived from sulphate reduction. These observations provide an alternative to the notion that DMSP or S-containing amino acids are the dominant precursors of DMS in intertidal sediment systems. The major sink for DMS in the microbial mats was biological consumption, whereas photochemical oxidation to dimethylsulphoxide was the major sink for DMS in the overlying water column. Diel flux measurements demonstrated that significantly more DMS is released from the system during the night than during the day. The major consumers of DMS in the presence of oxygen were monooxygenase-utilizing bacteria, whereas under anoxic conditions, DMS was predominantly consumed by sulphate-reducing bacteria and methanethiol was consumed by methanogenic bacteria. Aerobic and anaerobic consumption rates of DMS were nearly identical. Mass balance estimates suggest that the consumption in the water column is likely to be smaller than net the flux from the mats. Volatile organic sulphur compounds are thus indicators of high rates of carbon fixation and sulphate reduction in these laminated sediment ecosystems, and atmospheric sulphur can be generated as a biogenic signature of the microbial mat community.     

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     

Reactions of dimethylsulfoxide reductase in the presence of dimethyl sulfide and the structure of the dimethyl sulfide-modified enzyme

The bis-molybdopterin enzyme dimethylsulfoxide reductase (DMSOR) from Rhodobacter capsulatus catalyzes the conversion of dimethyl sulfoxide (DMSO) to dimethyl sulfide (DMS), reversibly, in the presence of suitable e(-)-donors or e(-)-acceptors. The catalytically significant intermediate formed by reaction of DMSOR with DMS ('the DMS species') and a damaged enzyme form derived by reaction of the latter with O(2) (DMS-modified enzyme, DMSOR(mod)D) have been investigated. Evidence is presented that Mo in the DMS species is not, as widely assumed, Mo(IV). Formation of the DMS species is reversed on removing DMS or by addition of an excess of DMSO. Equilibrium constants for the competing reactions of DMS and DMSO with the oxidized enzyme (K(d) = 0.07 +/- 0.01 and 21 +/- 5 mM, respectively) that control these processes indicate formation of the DMS species occurs at a redox potential that is 80 mV higher than that required, according to the literature, for reduction of Mo(VI) to Mo(IV) in the free enzyme. Specificity studies show that with dimethyl selenide, DMSOR yields a species analogous to the DMS species but with the 550 nm peak blue-shifted by 27 nm. It is concluded from published redox potential data that this band is due to metal-to-ligand charge transfer from Mo(V) to the chalcogenide. Since the DMS species gives no EPR signal in the normal or parallel mode, a free radical is presumed to be in close proximity to the metal, most likely on the S. The species is thus formulated as Mo(V)-O-S(*)Me(2). Existing X-ray crystallographic and Raman data are consistent with this structure. Furthermore, 1e(-) oxidation of the DMS species with phenazine ethosulfate yields a Mo(V) form without an -OH ligand, since its EPR signal shows no proton splittings. This form presumably arises via dissociation of DMSO. The structure of DMSOR(mod)D has been determined by X-ray crystallography. All four thiolate ligands and Ogamma of serine-147 remain coordinated to Mo, but there are no terminal oxygen ligands and Mo is Mo(VI). Thus, it is a dead-end species, neither oxo group acceptance nor e(-)-donation being possible. O(2)-dependent formation of DMSOR(mod)D represents noncatalytic breakdown of the DMS species by a pathway alternative to that in turnover, with oxidation to Mo(VI) presumably preceding product release. Steps in the forward and backward catalytic cycles are discussed in relation to earlier stopped-flow data. The finding that in the back-assay the Mo(IV) state may at least in part be by-passed via two successive 1e(-) reactions of the DMS species with the e(-)-acceptor, may have implications in relation to the existence of separate molybdopterin enzymes catalyzing DMSO reduction and DMS oxidation, respectively.     

A new fungus which degrades hydrogen sulfide,methanethiol, dimethyl sulfide and dimethyl disulfide

Summary A new Basidiomycete showed significantly higher degradation rates, 10,000 times for H₂S,40 times for dimethyl sulfide(DMS),15 times for methanethiol(MT) and 4 times for dimethyl disulfide(DMDS) than any reported previously. The optimal pH for degradation activity was around 7. Degradation rate for each gas when mixed gases of H₂S,MT and DMS were supplied was almost the same as that for single gas supply. H₂S was oxidized to SO₄ via SO₃ and DMS was stoichiometrically converted to dimethyl sulfoxide(DMSO).     

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.     

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.     

Dimethyl sulfoxide reduction by a hyperhermophilic archaeon Thermococcus onnurineus NA1 via a cysteine-cystine redox shuttle

A variety of microbes grow by respiration with dimethyl sulfoxide (DMSO) as an electron acceptor, and several distinct DMSO respiratory systems, consisting of electron carriers and a terminal DMSO reductase, have been characterized. The heterotrophic growth of a hyperthermophilic archaeon Thermococcus onnurineus NA1 was enhanced by the addition of DMSO, but the archaeon was not capable of reducing DMSO to DMS directly using a DMSO reductase. Instead, the archaeon reduced DMSO via a cysteine-cystine redox shuttle through a mechanism whereby cystine is microbially reduced to cysteine, which is then reoxidized by DMSO reduction. A thioredoxin reductase-protein disulfide oxidoreductase redox couple was identified to have intracellular cystine-reducing activity, permitting recycle of cysteine. This study presents the first example of DMSO reduction via an electron shuttle. Several Thermococcales species also exhibited enhanced growth coupled with DMSO reduction, probably by disposing of excess reducing power rather than conserving energy.     

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.     

Marinobacterium sp. strain DMS-S1 uses dimethyl sulphide as a sulphur source after light-dependent transformation by excreted flavins

Marinobacterium sp. strain DMS-S1 is a unique marine bacterium that can use dimethyl sulphide (DMS) as a sulphur source only in the presence of light. High-performance liquid chromatography (HPLC) analyses of the culture supernatant revealed that excreted factors, which could transform DMS to dimethyl sulphoxide (DMSO) under light, are FAD and riboflavin. In addition, FAD appeared to catalyse the photolysis of DMS to not only DMSO but also methanesulphonate (MSA), formate, formaldehyde and sulphate. As strain DMS-S1 can use sulphate and MSA as a sole sulphur source independently of light, the excretion of flavins appeared to support the growth on DMS under light. Furthermore, three out of 12 marine bacteria from IAM culture collection were found to be able to grow on DMS with the aid of photolysis by the flavins excreted. This is the first report that bacteria can use light to assimilate oceanic organic sulphur compounds outside the cells by excreting flavins as photosensitizers.     

Dimethylsulphoxide and trimethylamine-N-oxide as bacterial electron transport acceptors: use of nuclear magnetic resonance to assay and characterise the reductase system in Rhodobacter capsulatus

Nuclear magnetic resonance is established as a sensitive and specific method for following the reduction of dimethylsulphoxide and trimethylamine-N-oxide by bacteria. Using this method it has been shown that cells of Rhodobacter capsulatus reduce both dimethylsulphoxide and trimethylamine-N-oxide at linear rates at all concentrations of these acceptors that can be conveniently detected during a continuous assay. The rate of reduction of trimethylamine-N-oxide was eightfold higher than the rate of dimethylsulphoxide reduction. An upper limit of approximately 0.1 mM may be placed upon the apparent K _m value for each acceptor, but the value for dimethylsulphoxide is deduced to be lower than that for trimethylamine-N-oxide on the basis of the strong inhibitory effect of the former on the reduction of the latter. Reduction of trimethylamine-N-oxide by Rb. capsulatus was inhibited by illumination and by oxygen, but only the former effect was relieved following dissipation of the proton electrochemical gradient across the cytoplasmic membrane. Rotenone inhibited the reduction of trimethylamine-N-oxide whereas myxothiazol did not, consistent with a pathway of electrons to the reductase from NADH dehydrogenase that does not involve the cytochrome bc ₁complex.Abbreviations DMS dimethyl sulphide - DMSO dimethyl sulphoxide - DSS 3-(trimethylsilyl)-1-propane-sulphonic acid - FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - TMA trimethylamine - TMAO trimethylamine-N-oxide     

Temperature-dependent perturbation of phospholipid bilayers by dimethylsulfoxide.

Dimethylsulfoxide (DMSO) is known to protect isolated enzymes during freezing while destabilizing proteins at high temperatures. This apparent paradox is the subject of a review by Arakawa et al. ((1990) Cryobiology 27, 401-415), who present evidence for a temperature-dependent, hydrophobic interaction between DMSO and non-polar moieties of proteins. The present study investigates the interaction of DMSO with phospholipid bilayers. Phospholipid vesicles containing carboxyfluorescein were exposed to several concentrations of DMSO at various temperatures. Leakage rates increased with DMSO concentration and temperature. This effect was not reduced in the presence of solutes that have been shown to neutralize DMSO toxicity in tissues. The increased leakage rates correlate well with the increased partitioning of DMSO from water to octanol at higher temperatures. Additionally, reductions in the CH2 vibrations of the bilayer are also shown to depend on DMSO concentration and temperature. A similar reduction in CH2 vibrations was observed in solutions of octanol and DMSO, suggesting that this effect is not mediated through an interaction with water. Furthermore, investigation of sulfoxide vibrations indicate that DMSO is not hydrogen bonded to the alcohol moiety of octanol, and therefore the interaction between DMSO and octanol is most likely due to a hydrophobic association. These results are consistent with a destabilization of phospholipid membranes at higher temperatures due to a hydrophobic association between DMSO and the bilayer.     

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.     

Methylated sulfur compounds in microbial mats: in situ concentrations and metabolism by a colorless sulfur bacterium.

The concentrations of the volatile organic sulfur compounds methanethiol, dimethyl disulfide, and dimethyl sulfide (DMS) and the viable population capable of DMS utilization in laminated microbial ecosystems were evaluated. Significant levels of DMS and dimethyl disulfide (maximum concentrations of 220 and 24 nmol cm3 of sediment-1, respectively) could be detected only at the top 20 mm of the microbial mat, whereas methanethiol was found only at depth horizons from 20 to 50 mm (maximum concentration of 42 nmol cm3 of sediment-1). DMS concentrations in the surface layer doubled after cold hydrolysis of its precursor, dimethylsulfoniopropionate. Most-probable-number counts revealed 2.2 x 10(5) cells cm3 of sediment-1, in the 0- to 5-mm depth horizon, capable of growth on DMS as the sole source of energy. An obligately chemolithoautotrophic bacillus designated strain T5 was isolated from the top layer of the marine sediment. Continuous culture studies in which DMS was the growth-limiting substrate revealed a maximum specific growth rate of 0.10 h-1 and a saturation constant of 90 mumol liter-1 for aerobic growth on this substrate.     

Electron donors and the quinone involved in dimethyl sulfoxide reduction inEscherichia coli

Escherichia coli can use dimethyl sulfoxide (DMSO) as an electron acceptor during anaerobic growth on the oxidizable substrate, glycerol. During growth, the DMSO is reduced to dimethyl sulfide (DMS). For the reduction of DMSO, NADH, formate, lactate, reduced benzyl viologen, reduced methyl viologen, and dithionite can serve as electron donors. The terminal reductase and the dehydrogenases linking the various electron donors to the electron transport chain were found to be membrane bound. Chlorate-resistant mutants (chl) were unable to grow and reduce DMSO. However, in the case of thechlD mutant, growth and DMSO reduction can be restored by growth in the presence of high concentrations of molybdate. Mutants ofE. coli blocked in menaquinone (vitamin K₂) biosynthesis—menB, menC, andmenD—were unable to grow with DMSO as an electron acceptor, even though the terminal reductase is present in these mutants. Both growth and DMSO reduction could be restored in these mutants by growth in the presence of the menaquinone intermediates,o-succinylbenzoate and 1,4-dihydroxy-2-naphthoate, depending on the metabolic block of the mutant. Thus menaquinone is involved in electron transport during DMSO reduction.     

Methylated sulfur compounds in microbial mats: in situ concentrations and metabolism by a colorless sulfur bacterium

The concentrations of the volatile organic sulfur compounds methanethiol, dimethyl disulfide, and dimethyl sulfide (DMS) and the viable population capable of DMS utilization in laminated microbial ecosystems were evaluated. Significant levels of DMS and dimethyl disulfide (maximum concentrations of 220 and 24 nmol cm3 of sediment-1, respectively) could be detected only at the top 20 mm of the microbial mat, whereas methanethiol was found only at depth horizons from 20 to 50 mm (maximum concentration of 42 nmol cm3 of sediment-1). DMS concentrations in the surface layer doubled after cold hydrolysis of its precursor, dimethylsulfoniopropionate. Most-probable-number counts revealed 2.2 x 10(5) cells cm3 of sediment-1, in the 0- to 5-mm depth horizon, capable of growth on DMS as the sole source of energy. An obligately chemolithoautotrophic bacillus designated strain T5 was isolated from the top layer of the marine sediment. Continuous culture studies in which DMS was the growth-limiting substrate revealed a maximum specific growth rate of 0.10 h-1 and a saturation constant of 90 mumol liter-1 for aerobic growth on this substrate.     

Enhanced production of monomeric interferon-beta by CHO cells through the control of culture conditions

The enhancement of recombinant protein expression of a transfected cell line is essential for the development of an efficient large-scale bioprocess. The effect of various media additives and temperature conditions were studied in an attempt to optimize protein production, stability, and protein glycosylation from a Chinese hamster ovary (CHO) cell line producing human beta-interferon (Hu-beta-IFN). We observed a decrease in the ELISA response of the glycoprotein in the later stages of batch cultures, which was attributed to molecular aggregation. Cells were subjected to various concentrations of glycerol, dimethyl sulfoxide (DMSO), and sodium butyrate (NaBu) in a variety of culture systems and conditions. The addition of both NaBu and DMSO resulted in higher specific productivities but reduced growth rates that resulted in a net reduction of interferon produced. Glycerol appeared to stabilize the secreted beta-IFN, resulting in reduced aggregation, despite a decrease in cell growth rate. Glycosylation analysis of isolated beta-IFN showed a time-dependent decrease in sialylation in batch culture that was ameliorated by the presence of glycerol. Low-temperature conditions (30 degrees C) had the greatest effect on productivity with a significant increase in beta-IFN titer as well as a reduction in the degree of molecular aggregation.