Inhibition of trichloroethylene oxidation by the transformation intermediate carbon monoxide.

Inhibition of trichloroethylene (TCE) oxidation by the transformation intermediate carbon monoxide (CO) was evaluated with the aquifer methanotroph Methylomonas sp. strain MM2. CO was a TCE transformation intermediate. During TCE oxidation, approximately 9 mol% of the TCE was transformed to CO. CO was oxidized by Methylomonas sp. strain MM2, and when formate was provided as an electron donor, the CO oxidation rate doubled. The rate of CO oxidation without formate was 4.6 liter mg (dry weight)-1 day-1, and the rate with formate was 10.2 liter mg (dry weight)-1 day-1. CO inhibited TCE oxidation, both by exerting a demand for reductant and through competitive inhibition. The Ki for CO inhibition of TCE oxidation, 4.2 microM, was much less than the Ki for methane inhibition of TCE oxidation, 116 microM. CO also inhibited methane oxidation, and the degree of inhibition increased with increasing CO concentration. When CO was present, formate amendment was necessary for methane oxidation to occur and both substrates were simultaneously oxidized. CO at a concentration greater than that used in the inhibition studies was not toxic to Methylomonas sp. strain MM2.     

Inhibition of trichloroethylene oxidation by the transformation intermediate carbon monoxide

Inhibition of trichloroethylene (TCE) oxidation by the transformation intermediate carbon monoxide (CO) was evaluated with the aquifer methanotroph Methylomonas sp. strain MM2. CO was a TCE transformation intermediate. During TCE oxidation, approximately 9 mol% of the TCE was transformed to CO. CO was oxidized by Methylomonas sp. strain MM2, and when formate was provided as an electron donor, the CO oxidation rate doubled. The rate of CO oxidation without formate was 4.6 liter mg (dry weight)-1 day-1, and the rate with formate was 10.2 liter mg (dry weight)-1 day-1. CO inhibited TCE oxidation, both by exerting a demand for reductant and through competitive inhibition. The Ki for CO inhibition of TCE oxidation, 4.2 microM, was much less than the Ki for methane inhibition of TCE oxidation, 116 microM. CO also inhibited methane oxidation, and the degree of inhibition increased with increasing CO concentration. When CO was present, formate amendment was necessary for methane oxidation to occur and both substrates were simultaneously oxidized. CO at a concentration greater than that used in the inhibition studies was not toxic to Methylomonas sp. strain MM2.     

Influence of endogenous and exogenous electron donors and trichloroethylene oxidation toxicity on trichloroethylene oxidation by methanotrophic cultures from a groundwater aquifer.

Trichloroethylene (TCE)-transforming aquifer methanotrophs were evaluated for the influence of TCE oxidation toxicity and the effect of reductant availability on TCE transformation rates during methane starvation. TCE oxidation at relatively low (6 mg liter-1) TCE concentrations significantly reduced subsequent methane utilization in mixed and pure cultures tested and reduced the number of viable cells in the pure culture Methylomonas sp. strain MM2 by an order of magnitude. Perchloroethylene, tested at the same concentration, had no effect on the cultures. Neither the TCE itself nor the aqueous intermediates were responsible for the toxic effect, and it is suggested that TCE oxidation toxicity may have resulted from reactive intermediates that attacked cellular macromolecules. During starvation, all methanotrophs tested exhibited a decline in TCE transformation rates, and this decline followed exponential decay. Formate, provided as an exogenous electron donor, increased TCE transformation rates in Methylomonas sp. strain MM2, but not in mixed culture MM1 or unidentified isolate, CSC-1. Mixed culture MM2 did not transform TCE after 15 h of starvation, but mixed cultures MM1 and MM3 did. The methanotrophs in mixed cultures MM1 and MM3, and the unidentified isolate CSC-1 that was isolated from mixed culture MM1 contained lipid inclusions, whereas the methanotrophs of mixed culture MM2 and Methylomonas sp. strain MM2 did not. It is proposed that lipid storage granules serve as an endogenous source of electrons for TCE oxidation during methane starvation.     

Influence of endogenous and exogenous electron donors and trichloroethylene oxidation toxicity on trichloroethylene oxidation by methanotrophic cultures from a groundwater aquifer

Trichloroethylene (TCE)-transforming aquifer methanotrophs were evaluated for the influence of TCE oxidation toxicity and the effect of reductant availability on TCE transformation rates during methane starvation. TCE oxidation at relatively low (6 mg liter-1) TCE concentrations significantly reduced subsequent methane utilization in mixed and pure cultures tested and reduced the number of viable cells in the pure culture Methylomonas sp. strain MM2 by an order of magnitude. Perchloroethylene, tested at the same concentration, had no effect on the cultures. Neither the TCE itself nor the aqueous intermediates were responsible for the toxic effect, and it is suggested that TCE oxidation toxicity may have resulted from reactive intermediates that attacked cellular macromolecules. During starvation, all methanotrophs tested exhibited a decline in TCE transformation rates, and this decline followed exponential decay. Formate, provided as an exogenous electron donor, increased TCE transformation rates in Methylomonas sp. strain MM2, but not in mixed culture MM1 or unidentified isolate, CSC-1. Mixed culture MM2 did not transform TCE after 15 h of starvation, but mixed cultures MM1 and MM3 did. The methanotrophs in mixed cultures MM1 and MM3, and the unidentified isolate CSC-1 that was isolated from mixed culture MM1 contained lipid inclusions, whereas the methanotrophs of mixed culture MM2 and Methylomonas sp. strain MM2 did not. It is proposed that lipid storage granules serve as an endogenous source of electrons for TCE oxidation during methane starvation.     

Optimization of trichloroethylene oxidation by methanotrophs and the use of a colorimetric assay to detect soluble methane monooxygenase activity

Methylosinus trichosporium OB3b biosynthesizes a broad specificity soluble methane monooxygenase that rapidly oxidizes trichloroethylene (TCE). The selective expression of the soluble methane monooxygenase was followed in vivo by a rapid colorimetric assay. Naphthalene was oxidized by purified soluble methane monooxygenase or by cells grown in copper-deficient media to a mixture of 1-naphthol and 2-naphthol. The naphthols were detected by reaction with tetrazotized o-dianisidine to form purple diazo dyes with large molar absorptivities. The rate of color formation with the rapid assay correlated with the velocity of TCE oxidation that was determined by gas chromatography. Both assays were used to optimize conditions for TCE oxidation by M. trichosporium OB3b and to test several methanotrophic bacteria for the ability to oxidize TCE and naphthalene.Abbreviations A₆₀₀ absorbance due to cell density measured at 600 nm - HPLC high pressure liquid chromatography - NADH reduced nicotinamide adenine dinucleotide - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - sMMO soluble methane monooxygenase - TCE trichloroethylene     

濒危植物盘龙参种子的非共生萌发及种苗的快繁研究

以盘龙参种子为材料,筛选离体条件下适宜种子非共生萌发、种苗快繁的培养基。结果表明:盘龙参种子在无植物生长物质的培养基中能够萌发,但不能发育成苗;在含有1.0mg·L-1 KT、0.1mg·L-1 IAA和0.1mg·L-1 GA3的培养基中萌发,并能进一步发育形成种苗;在较高浓度生长素(1.2mg·L-1 NAA)的培养基中不能萌发。种苗在较高浓度细胞分裂素和较低浓度生长素配比的培养基中能够增殖,最高增殖系数可达到2.8,转入壮苗生长培养基中培养80d后可以移栽温室。最适增殖培养基为1/2MS+12.0mg·L-1 6-BA+0.1mg·L-1 NAA+10.0mg·L-1腺嘌呤;最适壮苗生根培养基为1/2MS+1.0mg·L-1 KT+0.1mg·L-1 IAA+10.0mg·L-1腺嘌呤。     

Production of Cytochrome c by an Obligate Methylotroph,Methylomonas sp. YK 1

An obligate methanol-utilizing bacterium, Methylomonas sp. YK 1, was isolated and used as a cytochrome c producer. The strain was mutagenized so as to be resistant to metabolic inhibitors related to the function of cytochrome c. The strain, YK 56, which was derived as a KCN-resistant mutant contained 3 times the cellular level of cytochrome c compared to the parent strain. Optimization of the culture conditions for the mutant to enhance the cytochrome c productivity was performed. Peptone, succinate, l-malate or FeSO₄ · 7H₂O increased the productivity when added to the culture medium. Under the optimal culture conditions, strain YK 56 produced about 60 mg cytochrome c per liter when methanol and peptone were fed to the medium during the cultivation.     

Optimization and maintenance of soluble methane monooxygenase activity inMethylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) maximization studies were carried out as part of a larger effort directed towards the development and optimization of an aqueous phase, multistage, membrane bioreactor system for treatment of polluted groundwater. A modified version of the naphthalene oxidation assay was utilized to determine the effects of methane:oxygen ratio, nutrient supply, and supplementary carbon sources on maximizing and maintaining sMMO activity inMethylosinus trichosporium OB3b.Methylosinus trichosporium OB3b attained peak sMMO activity (275–300 nmol of naphthol formed h^–1 mg of protein^–1 at 25°C) in early stationary growth phase when grown in nitrate mineral salts (NMS) medium. With the onset of methane limitation however, sMMO activity rapidly declined. It was possible to define a simplified nitrate mineral salts (NMS) medium, containing nitrate, phosphate and a source of iron and magnesium, which allowed reasonably high growth rates (_max 0.08 h^–1) and growth yields (0.4–0.5 g cells/g CH₄) and near maximal activities of sMMO. In long term batch culture incubations sMMO activity reached a stable plateau at approximately 45–50% of the initial peak level and this was maintained over several weeks. The addition of d-biotin, pyridoxine, and vitamin B₁₂ (cyanocobalamin) increased the activity level of sMMO in actively growing methanotrophs by 25–75%. The addition of these growth factors to the simplified NMS medium was found to increase the plateau sMMO level in long term batch cultures up to 70% of the original peak activity.Abbreviations sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - NMS nitrate mineral salts - TCE trichloroethene - NADH reduced nicotinamide adenine dinucleotide     

Ethane oxidation by methane-oxidizing bacteria

The relationship between the rates of methane and ethane oxidation by washed suspensions of methane-oxidizing bacteria has been investigated. Considerable differences between bacterial strains were observed. Two closely related Methylomonas strains which differed in their oxidizing capacity were further investigated. The low ethane oxidation rate of one strain could be strongly stimulated by the addition of oxidizable co-substrates, and the presence of ethane stimulated formate oxidation. The other strain had a much higher ethane oxidation rate and stimulation by co-substrates was negligible.Differences between the levels of dissimilative enzymes in cell-free extracts could not be detected. Attempts to produce extracts with methane mono-oxygenase activity failed. When cells were made permeable with chitosan the results suggested that strains with a low ethane oxidizing capacity obtain the required reductant for the mono-oxygenase from endogenous respiration. In strains with a high ethane oxidation rate, the reductant appears to be derived from oxidation of ethanol or acetaldehyde.     

Aerobic cometabolic degradation of trichloroethene by methane and ammonia oxidizing microorganisms naturally associated with <Emphasis Type="Italic">Carex comosa</Emphasis> roots

The degradation potential of trichloroethene by the aerobic methane- and ammonia-oxidizing microorganisms naturally associated with wetland plant (Carex comosa) roots was examined in this study. In bench-scale microcosm experiments with washed (soil free) Carex comosa roots, the activity of root-associated methane- and ammonia-oxidizing microorganisms, which were naturally present on the root surface and/or embedded within the roots, was investigated. Significant methane and ammonia oxidation were observed reproducibly in batch reactors with washed roots incubated in growth media, where methane oxidation developed faster (2 weeks) compared to ammonia oxidation (4 weeks) in live microcosms. After enrichment, the methane oxidizers demonstrated their ability to degrade 150 μg l⁻¹ TCE effectively at 1.9 mg l⁻¹ of aqueous CH₄. In contrast, ammonia oxidizers showed a rapid and complete inhibition of ammonia oxidation with 150 μg l⁻¹ TCE at 20 mg l⁻¹ of NH₄ ⁺-N, which may be attributed to greater sensitivity of ammonia oxidizers to TCE or its degradation product. No such inhibitory effect of TCE degradation was detected on methane oxidation at the above experimental conditions. The results presented here suggest that microorganisms associated with wetland plant roots can assist in the natural attenuation of TCE in contaminated aquatic environments.     

Biodegradation of trichloroethylene and toluene by indigenous microbial populations in vadose sediments

The unsaturated subsurface (vadose zone) receives significant amounts of hazardous chemicals, yet little is known about its microbial communities and their capacity to biodegrade pollutants. Trichloroethylene (TCE) biodegradation occurs readily in surface soils; however, the process usually requires enzyme induction by aromatic compounds, methane, or other cosubstrates. The aerobic biodegradation of toluene and TCE by indigenous microbial populations was measured in samples collected from the vadose zone at unpolluted and gasoline-contaminated sites. Incubation at field moisture levels showed little activity on either TCE or toluene, so samples were tested in soil suspensions. No degradation occurred in samples suspended in water or phosphate buffer solution; however, both toluene and TCE were degraded in samples suspended in mineral salts medium. TCE degradation depended on toluene degradation, and little loss occurred under sterile conditions. Studies with specific nutrients showed that addition of ammonium sulfate was essential for degradation, and addition of other mineral nutrients further enhanced the rate. Additional studies with vadose sediments amended with nutrients showed similar trends to those observed in sediment suspensions. Initial rates of biodegradation in suspensions were faster in uncontaminated samples than in gasolinecontaminated samples, but the same percentages of chemicals were degraded. Biodegradation was slower and less extensive in shallower samples than deeper samples from the uncontaminated site. Two toluene-degrading organisms isolated from a gasoline-contaminated sample were identified as Corynebacterium variabilis SVB74 and Acinetobacter radioresistens SVB65. Inoculation with 10⁶ cells of C. variabilis ml^–1 of soil solution did not enhance the rate of degradation above that of the indigenous population. These results indicate that mineral nutrients limited the rate of TCE and toluene degradation by indigenous populations and that no additional benefit was derived from inoculation with a toluene-degrading bacterial strain. Correspondence to: K.M. Scow     

广西五种绞股蓝属植物离体快繁与种质保存研究

以绞股蓝属植物的带芽茎段为材料,研究不同6-BA浓度与NAA 0.02mg·L-1组合对其诱导、分化和增殖的影响,并建立离体快繁体系。结果表明:MS+6-BA 2.0mg·L-1+NAA 0.02mg·L-1最适宜初代诱导,MS+6-BA 2.0mg·L-1+NAA 0.02mg·L-1最适合扁果绞股蓝的增殖培养,而MS+6-BA 1.5mg·L-1+NAA0.02mg·L-1是其它四种植物增殖的最佳培养基,在1/2MS+NAA 1.0mg·L-1上的生根率均达100%。1/2MS与蔗糖40g·L-1对五种植物的保存效果均最好;添加生长抑制剂能有效减缓生长速度,最佳生长抑制剂为ABA和CCC,浓度均为1.0mg·L-1,其中CCC能适合多个物种,连续保存360d的存活率均在94.5%以上;PP333不适合五种植物的保存。活力检测表明,各种质经保存后增殖、生根能力均未下降。     

Customized media based on miniaturized screening improve growth rate and cell yield of methane-oxidizing bacteria of the genus Methylomonas

The growth of twelve methanotrophic strains within the genus Methylomonas, including the type strains of Methylomonas methanica and Methylomonas koyamae, was evaluated with 40 different variations of standard diluted nitrate mineral salts medium in 96-well microtiter plates. Unique profiles of growth preference were observed for each strain, showing a strong strain dependency for optimal growth conditions, especially with regards to the preferred concentration and nature of the nitrogen source. Based on the miniaturized screening results, a customized medium was designed for each strain, allowing the improvement of the growth of several strains in a batch setup, either by a reduction of the lag phase or by faster biomass accumulation. As such, the maintenance of fastidious strains could be facilitated while the growth of fast-growing Methylomonas strains could be further improved. Methylomonas sp. R-45378 displayed a 50 % increase in cell dry weight when grown in its customized medium and showed the lowest observed nitrogen and oxygen requirement of all tested strains. We demonstrate that the presented miniaturized approach for medium optimization is a simple tool allowing the quick generation of strain-specific growth preference data that can be applied downstream of an isolation campaign. This approach can also be applied as a first step in the search for strains with biotechnological potential, to facilitate cultivation of fastidious strains or to steer future isolation campaigns.     

Improvement of regeneration ability in Phleum pratense L. in vitro culture by dicamba

Calli were induced from mature caryopses of timothy grass (Phleum pratense L.) on MS medium (Murashige and Skoog 1962) supplemented with 500 mg·dm⁻³ casein hydrolysate and 5 mg·dm⁻³ 2,4-D (2,4-dicholorophenoxyacetic acid) or 2 mg·dm⁻³ dicamba (3,6-dichloro-o-anisic acid). Twelve-week-old calli were passaged on media with reduced levels of auxins (2 mg·dm⁻³ 2,4-D or 1 mg·dm⁻³ dicamba). Tissues induced on medium with 2,4-D were transferred on medium with 2,4-D and on medium with dicamba; parallely calli initiated on medium with dicamba were passaged on medium with 2,4-D or dicamba. Calli from various media sequences were used to establish cell suspension cultures in media containing 2 mg·dm⁻³ 2,4-D or 1 mg·dm⁻³ dicamba. An assessment of regeneration ability of calli was made on MS medium containing 0.2 mg·dm⁻³ kinetin. Callus tissue induced and/or subcultured on any of the media with 2,4-D did not regenerate plants while dicamba added to the media was the effective stimulator of regenerability. In the presence of 2,4-D calli and suspensions produced a jelly-like extracellular matrix. In cell suspension this phenomenon was observed 4–5 days after each passage. The measurements of electric potential of calli, growing on MS medium with kinetin were performed. Non-regenerating callus areas had an electric potential close to 0 mV while parts of tissue with meristematic centres were characterized by lower values of electric potential.     

Toxicity of 1,1,1-Trichloroethane and Trichloroethene on a Mixed Culture of Methane-Oxidizing Bacteria

The influence of trichloroethene (TCE; 0 to 65 mg/liter) and 1,1,1-trichloroethane (1,1,1-TCA; 0 to 103 mg/liter) on methane consumption of a mixed culture of methane-oxidizing bacteria was studied in laboratory batch experiments. Increasing concentrations of TCE or 1,1,1-TCA resulted in decreasing methane consumption. Methane consumption was totally inhibited at a concentration of 13 mg of TCE per liter, while methane consumption was still observed at the upper studied concentration of 103 mg of 1,1,1-TCA per liter. The inhibition of methane consumption by TCE depended on the initial concentration of methane. A model accounting for competitive inhibition between methane and TCE or 1,1,1-TCA was used to simulate methane consumption at various concentrations of TCE or 1,1,1-TCA. The simulations indicated that competitive inhibition may be the mechanism causing the inhibitory effect of TCE on methane consumption, while this does not seem to be the case for 1,1,1-TCA.     

The capacity of orotic acid production in pyrimidinedeficient mutants ofBrevibacterium ammoniagenes

Eight uracil-dependent mutants ofBrevibacterium ammoniagenes CCEB 364 and three mutants ofCorynebacterium sp. 9366 were checked for the production of precursors of nucleic acids. Four of the strains liberated into the medium a substantial amount of orotic acid. The production of orotic acid by a mutant ofBrevibacterium ammoniagenes (1043) was examined on mineral media containing varying amounts of glucose in the presence of uracil. The optimum concentration of glucose for the production of orotic acid was found to be 5–8%. On media to which natural substrates were added the orotic acid production increased substantially. The maximum production (6.5 g orotic acid/liter) was reached in a medium containing 0.5% yeast extract and 5% glucose; addition of uracil to this medium had no effect on the production. The maximum rate of production occurred between 24 and 72 h of fermentation. After this period the concentration of orotic acid in the medium decreases.     

Chemically defined media for growth and sporulation of Entomophthora virulenta

Amino acids, salts, and vitamins were combined with dextrose to test their effect on growth and sporulation of Entomophthora virulenta in liquid shake culture. The addition of a vitamin solution to the tested media did not enhance growth or sporulation. MgSO₄·7H₂O was the only salt individually tested that allowed for good growth and sporulation. MgSO₄·7H₂O concentrations exceeding 250 mg/liter in media lacking other salts inhibited sporulation. A simple medium of l-arginine, l-leucine, glycine, and mineral salts allowed high growth and sporulation.     

SIVB 2003 Congress Symposium Proceeding: Plant Growth and Sugar Utilization in an Agitated,Thin-Film Liquid System for Micropropagation

Summary Agitated layers of liquid medium were created on platform shakers in jars with 25–30 ml of medium (similar to conventional agar culture) rotating at 90 rpm. Thin films were scaled up in larger rectangular vessels on tilted shelves that periodically rock. In jars of liquid medium with a density of 180 explants per liter, multiplication rates of Hota tokudama var. ‘Newberry Gold’ were optimal with a media sucrose concentration of 5% [both with and without 1 μM benzyladenine (BA)]. Endogenous levels of soluble sugars were directly related to the concentration of sucrose in the medium. Three Hosta cultivars (‘Striptease’, ‘Minuteman’, and ‘Stiletto’) with plant densities of 40–200 explants per liter of medium were tested in larger, agitated, thin-film vessels in media with 5% sucrose and directly compared to agar medium. Higher rates of multiplication were observed in liquid than agar with the magnitude of the difference dependent on explant density. Pooled results for the three varieties with 200 explants per liter showed multiplication rates of 1.7x and 2.3x for agar and thin-film liquid, respectively. At 40 explants per liter, the multiplication rate was increased to 2.1x for agar and 3.4x for thin-film liquid. Sugar uptake was greater in liquid than agar and was greater in the higher densities, with the magnitude of the effect dependent on plant variety. Increased vessel size in the liquid, thin-film system and greater sugar uptake allowed more, larger plants to be harvested. Alocasia macrorrhizos was cultured in growth medium containing 1μM BA and 5% sucrose with plant densities in the range of 33–330 explants per liter. Dry weight and multiplication rate were greater in the liquid system than agar with the magnitude of the difference dependent on plant density. With approximately 165 explants per liter, and greater at the initiation of culture, plant density limited growth in both agar and liquid thin-film systems. In a multiplication medium (3 μM BA and 3 μM ancymidol) plant size was reduced by 50% and 60% (fresh weight) in liquid and agar, respectively. Initial density in the range of 165–330 explants per liter did not limit growth with the smaller plants in liquid or semisolid multiplication medium. Sugar uptake was greater in liquid than agar. While ample sugar was present in media for growth at any density on agar, sugar depletion was limiting growth at highest densities with the larger plants in liquid growth medium. In semisolid agar medium, sugar uptake by plants was more rapid than diffusion across the agar medium, resulting in non-equilibrium conditions following the culture cycle. In agitated, liquid medium, a greater transfer of sugars to plant tissue was related to accelerated growth.     

Effect of Nitrogen Source on Growth and Trichloroethylene Degradation by Methane-Oxidizing Bacteria

The effect of nitrogen source on methane-oxidizing bacteria with respect to cellular growth and trichloroethylene (TCE) degradation ability were examined. One mixed chemostat culture and two pure type II methane-oxidizing strains, Methylosinus trichosporium OB3b and strain CAC-2, which was isolated from the chemostat culture, were used in this study. All cultures were able to grow with each of three different nitrogen sources: ammonia, nitrate, and molecular nitrogen. Both M. trichosporium OB3b and strain CAC-2 showed slightly lower net cellular growth rates and cell yields but exhibited higher methane uptake rates, levels of poly-β-hydroxybutyrate (PHB) production, and naphthalene oxidation rates when grown under nitrogen-fixing conditions. The TCE-degrading ability of each culture was measured in terms of initial TCE oxidation rates and TCE transformation capacities (mass of TCE degraded/biomass inactivated), measured both with and without external energy sources. Higher initial TCE oxidation rates and TCE transformation capacities were observed in nitrogen-fixing mixed, M. trichosporium OB3b, and CAC-2 cultures than in nitrate- or ammonia-supplied cells. TCE transformation capacities were found to correlate with cellular PHB content in all three cultures. The results of this study suggest that the nitrogen-fixing capabilities of methane-oxidizing bacteria can be used to select for high-activity TCE degraders for the enhancement of bioremediation in fixed-nitrogen-limited environments.     

Optimization of trichloroethylene degradation using soluble methane monooxygenase of Methylosinus trichosporium OB3b expressed in recombinant bacteria

By complementing cell-free extracts of Pseudomonas putida F1/pSMMO20 with purified soluble methane monooxygenase (sMMO) components of Methylosinus trichosporium OB3b, the low cloned-gene sMMO activity in the recombinant strain was found to be due to incomplete activity of the hydroxylase component. To address this incomplete activity, additional sMMO-expressing strains were formed by transferring mmo-containing pSMMO20 and pSMMO50 into various bacterial species including pseudomonads and alpha-2 subdivision strains such as methanotrophs, methylotrophs, Agrobacterium tumefaciens A114, and Rhizobium meliloti 102F34 (11 new strains screened); sMMO activity was detected in the last two strains. To increase plasmid segregational stability, the hok/sok locus originally from Escherichia coli plasmid R1 was inserted downstream of the mmo locus of pSMMO20 (resulting in pSMMO40) and found to enhance plasmid stability in P. putida F1 and R. meliloti 102F34 (first report of hok/sok in Rhizobium). To further increase sMMO activity, a modified Whittenbury minimal medium was selected from various minimal and complex media based on trichloroethylene (TCE) degradation and growth rates and was improved by removing the sMMO-inhibiting metal ions [Cu(II), Ni(II), and Zn(II)] and chloramphenicol from the medium and by supplementing with an iron source (3.6 muM of ferrous ammonium sulfate). Using chemostat-grown P. putida F1/pSMMO40, it was found that sMMO activity was higher for cells grown at higher dilution rates. These optimization efforts resulted in a twofold increase in the extent of TCE degradation and more consistent sMMO activity. (c) 1996 John Wiley & Sons, Inc.