Production of soluble methane monooxygenase during growth of Methylosinus trichosporium on methanol

Soluble methane monooxygenase (sMMO) can degrade many chlorinated and aromatic pollutants. It is produced by certain methanotrophs such as Methylosinus trichosporium when grown on methane under copper limitation but, due to its low aqueous solubility, methane cannot support dense biomass growth. Since it is water soluble, methanol may be a more attractive growth substrate, but it is widely believed that sMMO is not produced on methanol. In this study, when the growth-limiting substrate was switched from methane to methanol, in the presence of the particulate MMO inhibitor, allylthiourea, growth of M. trichosporium OB3b continued unabated and sMMO activity was completely retained. When allylthiourea was then removed, sMMO activity was maintained for an additional 24 generations, albeit at a slightly lower level due to the presence of 0.70 microM of Cu(2+) in the feed medium. While a biomass density of only 2 g l(-1) could be obtained on methane, 7.4 g l(-1) was achieved by feeding methanol exponentially, and 29 g l(-1) was obtained using a modified feeding strategy employing on-line carbon dioxide production measurement. It was concluded that methanol can be employed to produce large amounts of M. trichosporium biomass containing sMMO.     

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     

Cerium Regulates Expression of Alternative Methanol Dehydrogenases in Methylosinus trichosporium OB3b

Methanotrophs have multiple methane monooxygenases that are well known to be regulated by copper, i.e., a “copper switch.” At low copper/biomass ratios the soluble methane monooxygenase (sMMO) is expressed while expression and activity of the particulate methane monooxygenase (pMMO) increases with increasing availability of copper. In many methanotrophs there are also multiple methanol dehydrogenases (MeDHs), one based on Mxa and another based on Xox. Mxa-MeDH is known to have calcium in its active site, while Xox-MeDHs have been shown to have rare earth elements in their active site. We show here that the expression levels of Mxa-MeDH and Xox-MeDH in Methylosinus trichosporium OB3b significantly decreased and increased, respectively, when grown in the presence of cerium but the absence of copper compared to the absence of both metals. Expression of sMMO and pMMO was not affected. In the presence of copper, the effect of cerium on gene expression was less significant, i.e., expression of Mxa-MeDH in the presence of copper and cerium was slightly lower than in the presence of copper alone, but Xox-MeDH was again found to increase significantly. As expected, the addition of copper caused sMMO and pMMO expression levels to significantly decrease and increase, respectively, but the simultaneous addition of cerium had no discernible effect on MMO expression. As a result, it appears Mxa-MeDH can be uncoupled from methane oxidation by sMMO in M. trichosporium OB3b but not from pMMO.     

Effect of Copper Speciation on Whole-Cell Soluble Methane Monooxygenase Activity in Methylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) activity in Methylosinus trichosporium OB3b was found to be more strongly affected as copper-to-biomass ratios changed in a newly developed medium, M2M, which uses pyrophosphate for metal chelation, than in nitrate mineral salts (NMS), which uses EDTA. When M2M medium was amended with EDTA, sMMO activity was similar to that in NMS medium, indicating that EDTA-bound copper had lower bioavailability than pyrophosphate-bound copper. EDTA did not limit the association of copper with the cells; rather, copper was sequestered in a form which did not affect sMMO activity.     

Methylosinus trichosporium OB3b Mutants Having Constitutive Expression of Soluble Methane Monooxygenase in the Presence of High Levels of Copper

The methanotrophic bacterium Methylosinus trichosporium OB3b is unusually active in degrading recalcitrant haloalkanes such as trichloroethylene (TCE). The first and rate-limiting step in the degradation of TCE is catalyzed by a soluble methane monooxygenase (sMMO). This enzyme is not expressed when the cells are grown in the presence of copper at concentrations typically found in polluted groundwater. Under these conditions, M. trichosporium OB3b expresses a particulate form of the enzyme (pMMO), which has a narrow substrate specificity and does not degrade TCE at any significant rate. We have isolated M. trichosporium OB3b mutants that are deficient in pMMO and express sMMO constitutively in the presence of elevated concentrations of copper. One mutant (PP358) exhibited a TCE degradation rate which was almost twice as high as that of the wild-type strain grown under optimal conditions (without copper). All of the mutants lost the ability to express pMMO activity and to form stacked intracellular membranes characteristic of wild-type cells expressing pMMO.     

Evaluation of transformation capacity for degradation of ethylene chlorides by<Emphasis Type="Italic">Methylosinus trichosporium</Emphasis> OB3b

The transformation capacity (T_c) ofMethylosinus trichosporium OB3b in the degradation of ethylene chlorides was determined by measuring the decrease of soluble methane monooxygenase (sMMO) activity of resting cells in batch experiments. All measurements of sMMO activity were taken in the presence of 20 mM formate to avoid the deficiency of reducing power, and within 2 hrs to avoid the effect of natural inactivation from instability of the resting cells. The constant T_c values of 0.58±0.132 and 0.80±0.17 μmol/mg cell were obtained for trichloroethylene (TCE) and 1,2-dichloroethylene (cis andtrans-1,2-DCE), respectively, regardless of their concentrations. The transformation capacity measured by this method can be used to predict the amount of cells that should be stimulated inin-situ bioremediation.     

Isolation of Copper Biochelates from Methylosinus trichosporium OB3b and Soluble Methane Monooxygenase Mutants

Methylosinus trichosporium OB3b produces an extracellular copper-binding ligand (CBL) with high affinity for copper. Wild-type cells and mutants that express soluble methane monooxygenase (sMMO) in the presence and absence of copper (sMMO^c) were used to obtain cell exudates that were separated and analyzed by size exclusion high-performance liquid chromatography. A single chromatographic peak, when present, contained most of the aqueous-phase Cu(II) present in the culture medium. In mutant cultures that were unable to acquire copper, extracellular CBL accumulated to high levels both in the presence and in the absence of copper. Conversely, in wild-type cultures containing 5 μM Cu(II), extracellular CBL was maintained at a low, steady level during exponential growth, after which the external ligand was rapidly consumed. When Cu(II) was omitted from the growth medium, the wild-type organism produced the CBL at a rate that was proportional to cell density. After copper was added to this previously Cu-deprived culture, the CBL and copper concentrations in the medium decreased at approximately the same rate. Apparently, the extracellular CBL was produced throughout the period of cell growth, in the presence and absence of Cu(II), by both the mutant and wild-type cultures and was reinternalized or otherwise utilized by the wild-type cultures when it was bound to copper. CBL produced by the mutant strain facilitated copper uptake by wild-type cells, indicating that the extracellular CBLs produced by the mutant and wild-type organisms are functionally indistinguishable. CBL from the wild-type strain did not promote copper uptake by the mutant. The molecular weight of the CBL was estimated to be 500, and its association constant with copper was 1.4 × 10¹⁶ M⁻¹. CBL exhibited a preference for copper, even in the presence of 20-fold higher concentrations of nickel. External complexation may play a role in normal copper acquisition by M. trichosporium OB3b. The sMMO^c phenotype is probably related to the mutant’s inability to take up CBL-complexed copper, not to a defective CBL structure.     

Inoculum size and auxin concentration influence the growth of adventitious roots and accumulation of ginsenosides in suspension cultures of ginseng ( Panax ginseng C.A. Meyer)

The effect of the root-inoculum size and axuin concentration on growth of adventitious roots and accumulation of ginsenosides were studied during suspension cultures of ginseng (Panax ginseng C.A. Meyer). Of the various concentrations of indole-3-butyric acid (IBA) and γ-naphthaleneacetic acid (NAA) used as supplementary growth regulators along with Murashige and Skoog medium, 25 μM IBA was found suitable for lateral root induction and growth, as well as accumulation of ginsenosides. Inoculum size of 5 g L⁻¹ was found suitable for optimal biomass (10.5 g L⁻¹ dry biomass) and ginsenosides (5.4 mg g⁻¹ DW) accumulation. Of the various length of root inocula tested (chopped to 1–3, 4–6, 7–10 mm and un-chopped), root inocula of 7–10 mm was found suitable for biomass and ginsenoside accumulation.     

Paclobutrazol and plant-growth promoting bacterial endophyte Pantoea sp. enhance copper tolerance of guinea grass (Panicum maximum) in hydroponic culture

As most gramineous plants, guinea grass (Panicum maximum) comprise cellulosic biomass, which may be used as a feedstock for bioenergy. In order to develop such potential energy plants on copper-polluted lands, the hydroponic experiments with Cu, Paclobutrazol (PP333, a kind of antigibberellin) and plant growth-promoting bacterial endophyte (PGPB) treatments were carried out in a greenhouse. The seedlings of two cultivars of guinea grass, GG1 (P. maximum var. Natsukomaki) and GG2 (P. maximum var. Natsukaze) in 3 weeks old were treated, respectively, with different Cu treatments [0(CK), 100, 200, 300, 400, 500 μM l⁻¹ Cu] for estimating Cu toxicity. The results showed that elevated Cu restrained plant growth and reduced biomass. According to the EC50 value [the Cu concentration when the relative gain in fresh weight ratio was 50% of control] of two tested cultivars, the concentration of Cu for further experiments was decided as 300 μM l⁻¹. Both pretreatments of PP333 (200, 400, 600 mg l⁻¹) and PGPB (Pantoea sp.) significantly alleviated the negative affect caused by stress of 300 μM l⁻¹ Cu. The pretreatment of 400 mg l⁻¹ PP333 promoted both two cultivars in biomass, compared to 300 μM l⁻¹ Cu treat. The inoculation of Pantoea sp. Jp3-3 increased shoot dry weight, compared to Cu treat. The results suggested that the main reason for both PP333 and Pantoea sp. Jp3-3 enhanced Cu tolerance in guinea grass was that their pretreatments significantly decreased Cu absorption and accumulation under excessive Cu stress. The present study has provided a new insight into the exploitation of energy plant in heavy metal polluted condition by the way of plant growth regulation for increasing heavy metal tolerance.     

Factors affecting competition between type I and type II methanotrophs in two-organism,continuous-flow reactors

Competition experiments were performed in a continuous-flow reactor using Methylosinus trichosporium OB3b, a type II methanotroph, and Methylomonas albus BG8, a type I methanotroph. The experiments were designed to establish conditions under which type II methanotrophs, which have significant cometabolic potential, prevail over type I strains. The primary determinants of species selection were dissolved methane, copper, and nitrate concentrations. Dissolved oxygen and methanol concentrations played secondary roles. M. trichosporium OB3b proved dominant under copper and nitratelimited conditions. The ratio of M. trichosporium to M. albus in the reactor increased ten-fold in less than 100 hours following the removal of copper from the reactor feed. Numbers of M. albus declined to levels that were below detection limits (<106/ml) under nitrogen-limited conditions. In the latter experiment, the competitive success of M. trichosporiumdepended on the maintenance of an ambient dissolved oxygen level below about 7.5 × 10^–5 M, or 30% of saturation with air. The ability of M. trichosporium to express soluble methane monooxygenase under copper limitation and nitrogenase under nitrate limitation was very significant. M. albus predominated under methane-limited conditions, especially when low levels of methanol were simultaneously added with methane to the reactor. The results imply that nitrogen limitation can be used to select for type II strains such as M. trichosporium OB3b. Offprint requests to: Pierre Servais     

Paraffin oil as a “methane vector” for rapid and high cell density cultivation of <Emphasis Type="Italic">Methylosinus trichosporium</Emphasis> OB3b

Slow growth and relatively low cell densities of methanotrophs have limited their uses in industrial applications. In this study, a novel method for rapid cultivation of Methylosinus trichosporium OB3b was studied by adding a water-immiscible organic solvent in the medium. Paraffin oil was the most effective at enhancing cell growth and final cell density. This is at least partially due to the increase of methane gas transfer between gas and medium phases since methane solubility is higher in paraffin than in water/nitrate minimal salt medium. During cultivation with paraffin oil at 5% (v/v) in the medium, M. trichosporium OB3b cells also showed higher concentrations of the intermediary metabolites, such as formic acid and pyruvic acid, and consumed more methane compared with the control. Paraffin as methane vector to improve methanotroph growth was further studied in a 5-L fermentor at three concentrations (i.e., 2.5%, 5%, and 10%). Cell density reached about 14 g dry weight per liter with 5% paraffin, around seven times higher than that of the control (without paraffin). Cells cultivated with paraffin tended to accumulate around the interface between oil droplets and the water phase and could exist in oil phase in the case of 10% (v/v) paraffin. These results indicated that paraffin could enhance methanotroph growth, which is potentially useful in cultivation of methanotrophs in large scale in industry. Bing Han and Tao Su contributed equally to this work.     

Methanobactin from Methylocystis sp. Strain SB2 Affects Gene Expression and Methane Monooxygenase Activity in Methylosinus trichosporium OB3b

Methanotrophs can express a cytoplasmic (soluble) methane monooxygenase (sMMO) or membrane-bound (particulate) methane monooxygenase (pMMO). Expression of these MMOs is strongly regulated by the availability of copper. Many methanotrophs have been found to synthesize a novel compound, methanobactin (Mb), that is responsible for the uptake of copper, and methanobactin produced by Methylosinus trichosporium OB3b plays a key role in controlling expression of MMO genes in this strain. As all known forms of methanobactin are structurally similar, it was hypothesized that methanobactin from one methanotroph may alter gene expression in another. When Methylosinus trichosporium OB3b was grown in the presence of 1 μM CuCl₂, expression of mmoX, encoding a subunit of the hydroxylase component of sMMO, was very low. mmoX expression increased, however, when methanobactin from Methylocystis sp. strain SB2 (SB2-Mb) was added, as did whole-cell sMMO activity, but there was no significant change in the amount of copper associated with M. trichosporium OB3b. If M. trichosporium OB3b was grown in the absence of CuCl₂, the mmoX expression level was high but decreased by several orders of magnitude if copper prebound to SB2-Mb (Cu-SB2-Mb) was added, and biomass-associated copper was increased. Exposure of Methylosinus trichosporium OB3b to SB2-Mb had no effect on expression of mbnA, encoding the polypeptide precursor of methanobactin in either the presence or absence of CuCl₂. mbnA expression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl₂. These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin “piracy” may be commonplace.     

Establishment of in vitro plants, cell and tissue cultures from Oldenlandia affinis for the production of cyclic peptides

In vitro cultured plants from Oldenlandia affinis were established from seeds and grown on a hormone-free medium. In vitro plants produced the cyclic peptide kalata B1 in concentrations of 0.67 mg g⁻¹ dry weight after growth of 30 days. This was approximately 50% of the concentration analysed in green house plants (shoot tips), where different concentrations have been determined in leaves (1.82 mg g⁻¹), shoot tips (1.36 mg g⁻¹), stems (0.36 mg g⁻¹), and in flowers (0.16 mg g⁻¹). Callus and cell suspension cultures could be initiated from aseptic root, stem and leaf explants of O. affinis seedlings and plants. Different light intensities were shown to affect culture growth as well as chlorophyll synthesis. The friable callus was then used for the establishment of a cell suspension culture. Fresh and dry weight measurements showed that growth was optimal on MS medium supplemented with 0.4 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-d). Leaf suspensions cultured on this medium showed a 4-fold increase of biomass by the first week of incubation. No quantifiable amounts of kalata B1 were produced under these conditions. Morphological differentiation seems to be essential for cyclic peptide production. Therefore, several undifferentiated as well as organised cell lines of O. affinis have been developed. These cell lines will constitute a worthwhile starting point for the optimisation of kalata B1 synthesis in liquid media to the objective of producing cyclic peptides under controlled and defined conditions in bioreactors.     

Large-scale cultivation of adventitious roots of Echinacea purpurea in airlift bioreactors for the production of chichoric acid, chlorogenic acid and caftaric acid

Adventitious roots of Echinacea purpurea were cultured in airlift bioreactors (20 l, 500 l balloon-type, bubble bioreactors and 1,000 l drum-type bubble bioreactor) using Murashige and Skoog (MS) medium with 2 mg indole butyric acid l⁻¹ and 50 g sucrose l⁻¹ for the production of chichoric acid, chlorogenic acid and caftaric acid. In the 20 l bioreactor (containing 14 l MS medium) a maximum yield of 11 g dry biomass l⁻¹ was achieved after 60 days. However, the amount of total phenolics (57 mg g⁻¹ DW), flavonoids (34 mg g⁻¹ DW) and caffeic acid derivatives (38 mg g⁻¹ DW) were highest after 50 days. Based on these studies, pilot-scale cultures were established and 3.6 kg and 5.1 kg dry biomass were achieved in the 500 l and 1,000 l bioreactors, respectively. The accumulation of 5 mg chlorogenic acid g⁻¹ DW, 22 mg chichoric acid g⁻¹ DW and 4 mg caftaric acids g⁻¹ DW were achieved with adventitious roots grown in 1,000 l bioreactors.     

Quasi steady state growth of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> in glucose-limited acceleration stat (A-stat) cultures

Quasi steady state growth of Lactococcus lactis IL 1403 was studied in glucose-limited A-stat cultivation experiments with acceleration rates (a) from 0.003 to 0.06 h⁻² after initial stabilization of the cultures in chemostat at D = 0.2–0.3 h⁻¹. It was shown that the high limit of quasi steady state growth rate depended on the acceleration rate used—at an acceleration rate 0.003 h⁻² the quasi steady state growth was observed until μ _crit = 0.59 h⁻¹, which is also the μ _max value for the culture. Lower values of μ _crit were observed at higher acceleration rates. The steady state growth of bacteria stabilized at dilution rate 0.2 h⁻¹ was immediately disrupted after initiating acceleration at the highest acceleration rate studied—0.06 h⁻². Observation was made that differences [Δ(μ − D)] of the specific growth rates from pre-programmed dilution rates were the lowest using an acceleration rate of 0.003 h⁻² (< 4% of preset changing growth rate). The adaptability of cells to follow preprogrammed growth rate was found to decrease with increasing dilution rate—it was shown that lower acceleration rates should be applied at higher growth rates to maintain the culture in the quasi steady state. The critical specific growth rate and the biomass yields based on glucose consumption were higher if the medium contained S ₀ = 5 g L⁻¹ glucose instead of S ₀ = 10 g L⁻¹. It was assumed that this was due to the inhibitory effect of lactate accumulating at higher concentrations in the latter cultures. Parallel A-stat experiments at the same acceleration and dilution rates showed good reproducibility—Δ(μ − D) was less than 5%, standard deviations of biomass yields per ATP produced (Y _ATP), and biomass yields per glucose consumed (Y _XS) were less than 15%.     

Co-metabolic biodegradation of trichloroethylene by Methylosinus trichosporium is stimulated by low concentrations methane or methanol

Co-metabolic biodegradation of trichloroethylene by Methylosinus trichosporium OB3b was stimulated by low concentrations of methane (up to 70 M) or methanol (up to 0.4 mM) but inhibited at higher concentrations of them. A kinetic equation describing the dual effects of methane or methanol is proposed and the relevant kinetic constants have been determined.     

Nonvascular contribution to ecosystem NPP in a subarctic heath during early and late growing season

Bryophytes and lichens abound in many arctic ecosystems and can contribute substantially to the ecosystem net primary production (NPP). Because of their growth seasonality and their potential for growth out of the growing season peak, bryophyte and lichen contribution to NPP may be particularly significant when vascular plants are less active and ecosystems act as a source of carbon (C). To clarify these dynamics, nonvascular and vascular aboveground NPP was compared for a subarctic heath during two contrasting periods of the growing season, viz. early-mid summer and late summer-early autumn. Nonvascular NPP was determined by assessing shoot biomass increment of three moss species (Hylocomium splendens, Pleurozium schreberi and Dicranum elongatum) and by scaling to ecosystem level using average standing crop. For D. elongatum, these estimates were compared with production estimates obtained from measurements of shoot length increase. Vascular NPP was determined by harvesting shrub and herb apical growth and considering production due to stem secondary growth of shrubs. Hylocomium splendens and Pleurozium schreberi showed highest biomass growth in late summer, whereas for D. elongatum this occurred in early summer. Maximum relative growth rates were ca. 0.003–0.007 g g⁻¹ d⁻¹. For D. elongatum, production estimates from length growth differed from estimations from biomass growth, likely because of an uncoupling between length growth and biomass shoot growth. Nonvascular NPP was 0.37 and 0.46 g dry weight m⁻² d⁻¹, in early and late summer, respectively, whereas in the same periods vascular NPP was 3.6 and 1.1 g dry weight m⁻² d⁻¹. The contribution of nonvascular NPP to total aboveground NPP was therefore minor in early summer but substantial in late summer, when 25% of the C accumulated by the vegetation was incorporated into nonvascular plant tissue. The expected global change-induced reduction of nonvascular plant biomass in subarctic heath is likely therefore to enhance C release during the late part of the growing season.     

Development and mathematical modeling of a two-stage reactor system for trichloroethylene degradation using <Emphasis Type="Italic">Methylosinus trichosporium</Emphasis> OB3b

A two-stage reactor system was developed for the continuous degradation of gas-phase trichloroethylene (TCE). Methylosinus trichosporium OB3b was immobilized on activated carbon in a TCE degradation reactor, trickling biofilter (TBF). The TBF was coupled with a continuous stirred tank reactor (CSTR) to allow recirculation of microbial cells from/to the TBF for the reactivation of inactivated cells during TCE degradation. The mass transfer aspect of the TBF was analyzed, and mass transfer coefficient of 3.9 h⁻¹ was estimated. The loss of soluble methane monooxygenase (sMMO) activity was modeled based on a material balance on the CSTR and TBF, and transformation capacity (T _c) was determined to be 20.2 mol mg⁻¹. Maximum TCE degradation rate of 525 mg 1⁻¹ d⁻¹ was obtained and reactor has been stably operated for more than 270 days.