EP1073714A1

EP1073714A1 - Ethylene oxidizing microorganism and use thereof

Info

Publication number: EP1073714A1
Application number: EP99902512A
Authority: EP
Inventors: Lars Elsgaard
Original assignee: Danmarks JordbrugsForskning
Current assignee: Danmarks JordbrugsForskning
Priority date: 1998-02-06
Filing date: 1999-02-08
Publication date: 2001-02-07
Also published as: AU2265899A; WO1999040177A1

Abstract

Ethylene oxidizing microorganisms which are capable of reducing the ethylene content in an ethylene-containing gas to levels below 0.1 ppm and which have an ethylene removal efficiency which exceeds 90 % even at temperatures around 10 °C. The microorganisms show a high degree of operational stability when used in a bioreactor for extended periods of time in the presence of ethylene and they can, when mixed with a carrier material, be stored for several weeks in the absence of ethylene without significant loss of their ethylene removal capacity. A bioreactor comprising the microorganism is used for controlling the ethylene content in storage facilities for agricultural or horticultural products.

Description

ETHYLENE OXIDIZING MICROORGANISM AND USE THEREOF

FIELD OF INVENTION

The present invention relates to the field of biological control of ethylene in a gas, in particular in storage facilities for agricultural/horticultural products.

TECHNICAL BACKGROUND AND PRIOR ART

Ethylene (C₂H₄) is a gaseous plant hormone which is generated and used by the plant to control a variety of naturally occurring phenomena such as growth, flowering, organ initiation, ripening and aging. It is physiologically active in trace amounts, i.e. less than 0.1 parts per million (ppm). Being produced by plants, ethylene can accumulate in storage facilities where fruits, flowers and vegetables are stored. As an aging hormone, ethylene accelerates fruit ripening, floral senescence and leaf abscissions. However, such aging phenomena may be undesirable and may give rise to economically significant losses e.g. resulting from overripe fruit, misshapen or senescent flowers and defoliated vegetation.

In order to keep agricultural (including horticultural) products fresh during storage, it is therefore required to control the level of ethylene in the atmosphere of the storage facilities. The threshold ethylene concentration for post-harvest agricultural crops varies among plant species, but in general there is no detectable effect below 0.01 ppm. Thus, keeping the concentration of ethylene at such a low level in storage facilities is an important requirement.

Several ethylene removal techniques for use in storage facilities for agricultural products are commercially available. The presently most commonly used technique is to reduce accumulation of ethylene by ventilation. However, ventilation is impractical in storage facilities where the temperature and/or the composition of the atmosphere has to be controlled or when the inlet open air contains too much ethylene.

Chemical ethylene scrubbers are widely used to control the ethylene content in storage facilities for agricultural products. Such scrubbers contain e.g. potassium permanganate (KMnO₄) which is absorbed on a suitable carrier. A serious drawback of such scrubbers is 2

their high cost and the fact, that they are only useful in small storage rooms since KMnO₄ is rapidly consumed and must be renewed several times during a conservation period.

There is therefore an industrial need for a method for continuous removal of ethylene in storage facilities which fulfils the following criteria: (i) it does not affect the general ventilation conditions, (ii) it can be operated in storage facilities independently of the size hereof, (iii) it is capable of reducing the concentration of ethylene in the atmosphere of the facilities to levels significantly below 0.1 ppm, preferably to levels approaching, at or below 0.01 ppm, (iv) it has a high operational stability over a prolonged period of time, and (v) it has a high ethylene removal efficiency, preferably a removal efficiency in excess of 90%. Furthermore, it is substantial to have a system which is capable of reducing ethylene to the above low levels at low temperature such as around 10°C or lower, since agricultural products are usually stored at such low temperatures.

The use of ethylene oxidizing microorganisms as biological catalysts for ethylene removal has been suggested as an interesting alternative to the above conventional mechanical or chemical methods. So far, however, no biological system for ethylene removal has been disclosed which fulfils the above criteria.

Thus, in a study by van Ginkel et. al. (2), it was reported that ethylene was consumed by the Mycobactehum strain E3 immobilized on lava or perlite under bioreactor conditions. However, under these conditions, the operational stability of that organism was unsatisfactory and the ethylene concentration was only reduced to about 1 ppm. Furthermore, it was observed that the activity of the immobilized bacteria declined rapidly below 10°C and was almost absent at 4°C. The highest removal efficiency reported in this study was only 68%.

In a subsequent study, van Ginkel et al. (3) reported the removal of ethylene using a gas/solid bioreactor with the above Mycobacte um strain immobilized on compost. Under these conditions, this bacterium was only capable of reducing the ethylene concentration to 0.25 ppm during the 8 weeks of the experiment. The highest removal efficiency was 90%.

De Heyder et al. (1 ) studied ethylene removal by a packed granular activated carbon biobed (at 19 to 23°C), which was inoculated with the above Mycobacteήum strain. Although the ethylene mass removal rate of the biobed was found to be relatively high, the 3

lowest outlet concentration was as high as 19 ppm and the highest removal efficiency found was 84,5%.

In Japanese patent application, Kokai 8-196208, is disclosed a method of reducing the concentration of ethylene by culturing Pseυdomonas cepacia, Bacillus cereus and/or Rhodococcus erythropolis on a soil material and enclosing the culture material in bags of bonded fibre fabric and placing such bags in containers with vegetables and fruits. However, the minimum ethylene concentration in the containers obtained by this method was as high as 1.1 ppm. Furthermore, it was shown that at temperatures below 20°C the capability of the bacteria to reduce ethylene declined and was almost absent at 10°C. At room temperature, the highest removal efficiency was 82%.

Thus, the prior art is not aware of any microorganism which is capable of reducing the concentration of ethylene to levels approaching the threshold level of 0.01 ppm. Further- more, none of the known ethylene removing microorganisms have the capability to reduce the concentration of ethylene to low levels at temperatures at or below 10°C and the ethylene removal efficiency of these microorganisms is at the most 90% under optimum growth conditions. It is therefore evident, that the known ethylene consuming microorganism are of limited industrial interest for use as biological catalysts for ethylene removal in agricultural/horticultural storage facilities.

The present invention, however, provides novel ethylene consuming microorganisms which are capable of reducing ethylene concentrations to levels significantly below 0.1 ppm and which additionally, has an ethylene removal efficiency which exceeds 90% even at temperatures around 10°C.

Additionally, the microorganisms according to the invention show a high degree of operational stability when used in a bioreactor for extended periods of time such as more than 80 days in the presence of ethylene. Furthermore, the microorganisms can, when mixed with a suitable carrier material, be stored for several weeks in the absence of ethylene without significant loss of their ethylene removal capacity. 4

SUMMARY OF THE INVENTION

Accordingly, the present invention relates in a first aspect to an isolated ethylene oxidizing microorganism which, when immobilized on a solid earner material, is capable of con- tinuously removing in excess of 90% of the content of ethylene in a gas stream being contacted with said material.

In further aspects, there is provided a solid material carrying an ethylene oxidizing microorganism as defined above and an ethylene removing bioreactor comprising the ethylene oxidizing microorganism or a solid organic or inorganic carrier mateπal carrying the microorganism, said bioreactor comprising gas stream inlet means and outlet means and optionally, means for collecting gas samples.

In a still further aspect there is provided a method of controlling the ethylene content in a gas by contacting the gas with a solid carrier material as defined above or by passing the gas through the bioreactor of the invention.

DETAILED DISCLOSURE OF THE INVENTION

Thus, it is an important objective of the present invention to provide an ethylene oxidizing microorganism which, when it is immobilized on a earner material, is capable of continuously removing in excess of 90% of the content of ethylene in a gas stream being contacted with such a material. As used herein the term "immobilized" refers to any mode of association of the microorganism with inner and outer surfaces of the carrier material.

Although presently preferred carrier materials are solid materials, the use of fluid carriers for the ethylene oxidizing microorganism is also encompassed within the scope of the invention.

Solid carrier materials which are useful in the present invention may be any organic or inorganic material which permits the microorganism according to the invention to be associated with it and which permits the microorganism to be metabolically active. Such mateπals include organic materials selected from peat soil, resins, activated carbon, plant mateπal particles such as saw dust, compost, naturally occurring polymers such as 5 polysaccharides e.g. starch, cellulose, pectin, or proteins and synthetic polymers such as e.g. polyolefins. Useful inorganic materials include as examples soil, sand, clay, periite, lava and particulate inorganic materials.

In the context of an ethylene oxidizing microorganism, the term "oxidizing" is used interchangeably with the term "removing", "reducing" and "consuming", and relates to any effect resulting in a decrease of the ethylene concentration in an atmosphere or in a gas stream.

In useful embodiments, the microorganism of the present invention is one which is capable of continuously removing at least 95% of the content of ethylene present in a gas which is contacted with the microorganism. It is, however, preferred that ethylene is removed at an even higher efficiency such as at least 96% removal e.g. at least 97% including at least 98% or at least 99% removal. In particularly preferred embodiments at least 99,9% of the ethylene content of the gas is removed. This effect of continuously removing ethylene occurs at appropriate temperature conditions for the particular microbial strain, typically at temperatures in the range of 0 to 60°C such as the range of 5 to 50°C, including 10 to 30°C, e.g. at 20°C or lower such as at 15°C or lower including at 10°C or lower.

It will be understood that the ethylene removal efficiency as referred to herein is preferably independent of the initial content of ethylene in the gas which is contacted with the ethylene oxidizing microorganism according to the invention. Accordingly, the microorganism is preferably capable of having the above removal efficiencies at any relevant level of ethylene. Thus, in useful embodiments of the invention the microorganism is capable of effectively removing ethylene even if this carbon source is supplied at low concentrations such as below 10 ppm such as 2 ppm. This characteristic is of considerable industrial interest, since, as it is explained above, even low levels of ethylene such as e.g. 2 ppm can result in severe damages on post-harvest products in storage facilities.

It is, as it is mentioned above, of considerable commercial interest to provide an ethylene oxidizing microorganism having the capability of reducing the concentrations of ethylene to levels approaching 0.01 ppm which is generally considered to be the threshold level for post-harvest agricultural or horticultural products. Accordingly, the present invention provides an ethylene oxidizing microorganism which is capable of continuously reducing the content of ethylene in a gas to at the most 1 ppm such as at the most 0.5 ppm including 6 at the most 0.2 ppm e.g. at the most 0.1 ppm. In particularly preferred embodiments, the microorganism is one which is capable of reducing the ethylene content to at the most 0.05 ppm such as at the most 0.025 ppm including at the most 0.01 ppm.

As used herein, the expression "post-harvest agricultural/horticultural products" refers to harvested agricultural or horticultural crops such as vegetables like e.g. carrots, cauliflower, cabbage, broccoli or potatoes. However, the term also encompasses harvested fruits such as e.g. apples, pears, bananas, oranges or grapes, or the term may relate to flowers like e.g. cut flowers or pot plants.

In one useful specific embodiment of the invention the ethylene oxidizing microorganism is one which, when it is used in a bioreactor according to the invention having a volume of about 700 cm³ and loaded with 0.44 g cm^"3 of a mixture of peat soil, distilled water and a suspension of the microorganism containing about 2x10⁸ cells mL^"1, in a weight ratio of about 3:2:1 , at a gas flow rate which is in the range of 71.4 to 75.9 mL min^"1, is capable of removing in excess of 90% such as in excess of 95% including in excess of 99% of an ethylene content of up to 117 ppm ethylene present in the inlet gas.

In other useful embodiments, the ethylene oxidizing microorganism is one which under the above specific bioreactor conditions is capable of retaining its ethylene removal efficiency during long-term continuous operation. Thus, the ethylene removal efficiency of the microorganism under these operation conditions is preferably retained for at ieast 10 days such as at Ieast 20 days including at Ieast 50 days e.g. at Ieast 80 days. The expression "continuous operation of the bioreactor" as used herein, refers to a substantially unin- terrupted flow of gas having an ethylene content of up to 117 ppm through the bioreactor.

An advantageous feature of the microorganism according to the invention is that it can be stored for several weeks in the absence of ethylene without significant loss of its ethylene removal capacity. In one particularly useful embodiment, the microorganism is one which, when it has been stored for 2 weeks or longer at a temperature in the range of 2 to 22°C i in a bioreactor having a volume of about 700 cm³ and loaded with 0.44 g cm^"3 of a mixture of peat soil, distilled water and a suspension of the microorganism containing about 2x10⁸ cells mL^'1, in a weight ratio of about 3:2:1, has retained at Ieast about 15% of its ethylene oxidizing capacity such as at Ieast 20% and preferably at Ieast 50% e.g. at Ieast 75% of its pre-storage ethylene oxidizing capacity. In addition to being capable of retaining a substantial part of its ethylene removal efficiency during storage under ethylene starvation conditions, the microorganism according to the invention is in a further useful embodiment a microorganism which is capable of substantially regenerating its full ethylene oxidizing capacity after it has been stored for 2 weeks or longer under ethylene starvation at the above conditions, the regeneration occurring within a short period of time following adding ethylene to the inlet gas stream, such as within 1 day, e.g. within 2 days including within 3 days or within 4 days. In the present context, the term "full ethylene oxidizing capacity" indicates that the microorganism after storage under the specific conditions regains it ability to continuously remove in excess of 90% of the content of ethylene in a gas.

Presently preferred ethylene oxidizing microorganisms according to the invention are the Rhodococcus erythropolis strain RD-4 and strain RD-4a deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Mascheroder Weg, 1b, D- 38124 Braunschweig on 2 February 1998 under the accession No. DSM 11980, and on 4 February 1999 under the accession number No. 12668, respectively.

In a further aspect of the invention there is provided a solid material carrying the ethylene oxidizing microorganism of the invention. Such an inoculated material is useful in a bioreactor as described in the following, or it may be used as such, optionally provided in a container or packaging e.g. for household use or for use in retail stores where the above agricultural or horticultural product are stored or displayed for sale. The number of viable cells of the microorganism being associated with the solid material can vary according to the particular use and the amount of ethylene to be removed. However, a suitable cell concentration is typically in the range of 10⁵ to 10¹⁰ viable cells per g of the solid material, such as e.g. in the range of 10⁶ to 10⁸ per g including the range of 5 x 10° to 5 x 10⁷ per g solid material.

As the solid carrier material may be used any of the materials which are mentioned above. A suitable pH of the material used according to the invention will depend on the type of microorganism, but is typically in the range of 4 to 9, such as between 5 and 8. The material must have a moisture content and an a_w which permits the microorganism to be metabolically active when it is associated with the material. 8

In a still further aspect, the invention pertains to an ethylene removing bioreactor compπsing the ethyiene oxidizing microorganism of the invention or a solid organic or inorganic carrier material according to the invention onto which the ethylene oxidizing microorganism is immobilized. As used herein the term "bioreactor" refers to any device which comprises a housing enclosing the ethyiene oxidizing microorganism in a form where it is metabolicaily active when a gas containing ethylene is fed to the reactor. The reactor housing can be of any suitable material such as metal, glass, ceramics, wood or a polymeric material. The volume of the reactor can be selected so as to have a ethylene removing capacity which is appropriate under the particular conditions where it is to be used.

Although the bioreactor according to the invention may be used without the gas flow being passed actively under pressure, i e. passive, it is preferred that the ethylene containing gas is fed actively to the bioreactor Accordingly, the bioreactor is in useful embodiments provided with or connected to means for regulating the flow rate of the gas stream. Such means include as examples air pumps and fans operably connected with the inlet means. Optionally, the flow rate regulating means are provided with a flowmeter to monitor the gas flow.

In useful embodiments, the bioreactor of the present invention, when operating under appropπate conditions, is capable of reducing the ethylene content of the inlet gas to at the most 1 ppm such as at the most 0.5 ppm including at the most 0.2 ppm e.g. at the most 0.1 ppm. In particularly preferred embodiments, the ethylene content is reduced to at the most 0.05 ppm such as at the most 0.025 ppm including at the most 0.01 ppm.

It will be understood that "appropriate conditions" include the temperature conditions under which the bioreactor is operated and which should support the highest possible rate of ethylene oxidation by the microorganism. Since, as it is mentioned above, the usual storage temperature in storage facilities for agricultural product is around 10°C, it is preferred that the bioreactor operates effectively at such lower temperatures Accordingly, the bioreactor is preferably capable of reducing the ethylene content of a gas with the removal efficiency as defined above when operated at or below 20°C such as at or below 15°C including about 10°C or lower.

In addition to measuring the ethylene removal efficiency as an indication of the function of 9 the bioreactor, one alternative way of characterizing the ethylene removal capability of the bioreactor is by determining the "ethyiene removal rate" as descnbed in the below examples. Another parameter which can be used for characterization of the bioreactor efficiency is the ethylene mass removal as also described in the examples below.

In a still further aspect of the invention there is provided a method of controlling the ethylene content in a gas by contacting the gas with a solid earner material as described above or by passing the gas through the bioreactor of the invention.

In one useful embodiment of this method a solid material carrying the ethylene oxidizing microorganism is present in a package of a plant product or is placed at a distance to such a product which permits the material to at Ieast partially remove the ethylene content in the gas surrounding the product For such a use, the solid material is conveniently enclosed in a container made of a solid material or a bag made of a flexible material such as a woven or non-woven fabric or sheet material, the enclosure for the carrier material being permeable to gas to permit the ethylene containing atmosphere in the package to be brought into contact with the ethylene oxidizing microorganism.

In another useful embodiment, a bioreactor of the invention is operated in plant product storage facility or transportation vehicle or is operatively connected to such facility or vehicle

The invention is further illustrated by the following examples and the drawings where

Fig. 1 shows the growth (^■) and the C₂H₄ consumption (•) by the microbial strain RD-4 at 30°C. The studies were done by use of stoppered 5-L bottles with 2 Lof medium (SMAG) An inoculum of 1 % was transferred to the medium and C₂H₄ was added to a headspace concentration of 1%. The cultures were incubated at 30°C on a water bath with orbital shaking (120rpm). Ethylene was assayed by GC analysis of headspace gas samples and was replenished to 1% when the concentration dropped below 0.5%. Growth was quantified by measurements of the optical density at 550 nm (OD₅₅o). Total growth was equivalent to an OD₅₅₀ of 0.673. Total C₂H consumption was 357 ml.

Fig. 2 shows a laboratory scale bioreactor with butyl stoppers (BS) for gas sampling at the inlet (In) for each 5 cm depth increment, and at the outlet (Out). A flow of humidified air with 10

C₂H₄ was applied to the inlet from two mass flow controllers. GP, glass wool plugger; RS, rubber stopper; TC, T-connector,

Fig. 3 illustrates the concentration of ethylene in the gas flow at the inlet (o) and at the outlet (•) of the bioreactor during operation for 16 days with 2.05 ppm C₂H₄ (73.3 mL min^'1). Data represent the mean ± SE of 3 to 4 samples. Note the scale break on the y-axis,

Fig. 4 illustrates the concentration of ethyiene at different soil depths of the bioreactor after operation with 2.05 ppm C₂H₄ for 1 h (•), 5 days (■), and 16 days ( ^ ). Soil depths of 0 and 35 cm represent the bioreactor inlet and outlet, respectively,

Fig. 5 illustrates the ethylene removal rates by individual 5-cm segments of the bioreactor during operation for 16 days with 2.05 ppm C₂H₄. Data are shown for the segments from 0 to 5 cm (•), 5 to 10 cm (^■), and 30 to 35 cm ( ^ ),

Fig. 6 illustrates the removal of ethylene by the bioreactor after transition of the inlet C₂H₄ concentration from 0 to 2 ppm (^■) and from 2 to 117 ppm (•). The dotted line indicates the latter transition. Prior to time 0, the bioreactor was operated with atmospheric air for 25 days. Ethylene removal was expressed as the ratio between the outlet concentration (C_out) and the inlet concentration (C_ιn). Data are the mean ± SE of 3 pairs of analyses,

Fig. 7 illustrates the concentration of ethylene in the gas flow of the bioreactor during operation for 80 days with 117 ppm C₂H₄. Data are shown for the 5-cm depth (-*^• ), the 10- cm depth (^■), and the outlet (•). Prior to time 0, the inlet C₂H₄ concentration was 2 ppm. Data for the outlet concentrations represent the mean ± SE (n = 3),

Fig. 8 illustrates the concentration of ethyiene in the gas flow at the inlet (o) and the outlet (•) of the bioreactor after transition from 21 to 10°C. Temperature equilibrium at 10°C was reached after operation for 5 h. Data are the mean ± SE (n = 3). Note the scale break at the y-axis,

Fig. 9 shows a time course of C₂H₄ removal in batch experiments with inoculated or uninoculated peat soil. Ethylene was added at about 20 ppm. Results are shown for freshly inoculated peat soil ( ^ ), inoculated peat soil stored at 20°C for 28 days ( -^*■ ), uninoculated peat soil prior to storage (•), and uninoculated peat soil after storage at 20°C for 28 days 1 1

(■). Data represent the mean ± SE (n = 3), and

Fig. 10 illustrates the initial C₂H₄ removal rates in batch experiments with inoculated peat soil. Soil from three batches were assayed prior to storage and after storage at 2, 8 or 20°C, respectively for 2, 3 and 4 weeks. Data represent the mean ± SE of rates calculated for triplicate assays.

EXAMPLES

Materials and Methods

Gas analysis

Gas samples for analysis of C₂H , CO₂ and O₂ were collected by use of 1 mL gas-tight syringes (Pressure-Lok). The C₂H₄ concentration (0.6 mL samples) was determined using a Hewlett-Packard GC model 5840A equipped with a flame ionization detector. Gases were separated on a glass column packed with Poropak N (80 to 100 mesh). The carrier gas was N₂ (20 mL min^'1) and the column temperature was 95°C. Injection and detection temperatures were 105 and 150°C, respectively. Calibration of the GC was done on a daily basis by injection of 10 ppm C₂H standards. For injection of a 0.6 mL gas sample, the C₂H₄ detection limit was 0.04 ppm for a signal-to-noise ratio of 3.

Occasionally, samples from the bioreactor were collected by passing the outlet flow through a stoppered 120 mL serum vial for 1 to 2 h by use of two hypodermic needles. The serum vial was then removed from the gas stream and headspace samples of 1.0 mL were analyzed for C₂H₄ by use of a Photovac 10S plus portable digital GC equipped with a photoionization detector. Gases were separated on a packed HayeSep Q column operated at 45°C with synthetic air as carrier gas (14.5 mL min^"1). The detection limit with this equipment was 0.002 ppm for injection of a 1.0 mL C₂H₄ standard.

Oxygen (0.5 mL samples) was measured on a Varian 3700 GC with a thermal conductivity detector and a Molecular Sieve 5A column. The oven temperature was 30°C and the carrier gas was He (76 mL min^"1). Carbon dioxide (0.2 mL samples) was measured on Mikrolab GC 82 (Mikrolab, Højbjerg, Denmark) with a thermal conductivity detector and a 12

Poropak N column operated at 60°C with He as carrier gas (43 mL mm^"1)

Statistics

Data from replicate samples are presented as mean ± SE (standard error) with indication of the number of samples (n). Ratios of outlet-to-inlet concentrations are presented as mean ± SE of replicate pairs of consecutive measurements of outlet and inlet concentrations. The significance of differences between data from the bioreactor inlet and outlet was tested by use of a two-tailed Student's t-test.

Calculation of the removal rate (RR)

For the calculation of the removal rate (RR) the following equation 1 was used RR(s) =ΔC₂H₄ (s) x F x p(C₂H₄), where RR(s) is the C₂H₄ removal rate (μg C₂H₄ h^"1) by a given 5 cm segment (s), ΔC₂H₄ (s) is the difference between the inlet and outlet C₂H₄ concentration of the segment (μL L^"1), F is the flow rate (4.4 L h^' ), and p(C₂H ) is the density of C₂H-, at 21°CJ1.16 μg μL-¹).

Calculation of the mass removal (MR)

For the calculation of the maximum mass removal (MR) the following equation 4 was used MR = ΔC₂H₄ x F x V^"1 x p(C₂H ), where MR is the mass removal of C₂H₄, ΔC₂H₄ is the difference between inlet and outlet C₂H₄ concentrations (μL L^" ), F is the flow rate (L d^"1, where d=day), V is the bioreactor volume (m³) and ρ(C₂H₄) is the density of C₂H₄ at 21 °C (1.16 10^'9 kg μL^'1).

EXAMPLE 1

Isolation and preliminary characterization of an ethylene oxidizing microbial strain RD-4 and preparation of cell suspensions hereof

Sandy sediment samples were collected in March 1995 from an oligotrophic lake (Tjele Langsø) near Fouium, Denmark. After sampling, 10 g subsamples of sediment were incubated in serum bottles (120 ml) that were closed by butyl rubber stoppers (10 mm 13 thick). The serum bottles were amended with 2 ml of 1000 ppm C H (AGA SpecialGas, Lidingo, Sweden) and incubated at 25°C. The time course of C₂H₄ and O₂ turn-over was followed by GC analysis of headspace gas samples during an incubation period of up to 8 weeks.

After the time course experiments, the C₂H₄ degrading sediment samples were biended with sterile water and used as inoculum for purification of ethylene-oxidizing microorganism. Medium for cultivation of ethylene-oxidizing microorganism was prepared from a basal salts solution which contained the following (in g L^"1 of distilled water): NH₄CI, 0.6; KCI, 0.6; MgSO₄-7 H₂O, 0.4; KH₂PO₄, 0.3; K₂HPO₄, 0.3; CaCI₂ 2 H₂O, 0.1. The pH of the medium was adjusted to 6.8 For preparation of agar plates, the medium was supplemented with 17 g of agar (Difco) that were rinsed 3 times in distilled water The medium was autoclaved (121°C, 20 m ), cooied, and supplemented with nonchelated trace elements (1 ml L^"1) and vitamin mixture (1 ml L^"1) including thiamine and vitamin B12 A second vitamin solution was added (1 ml L^"1), which contained (in mg L^"1 of 10 mM phosphate buffer, pH 7.1). Iipoic acid, 10; folic acid, 20; menadione, 100. Both vitamin solutions were filter-steriiized (0.2 μm) and kept at 5°C. The final pH of the medium (referred to as SMAG) was 6.7 to 7.0. Two complex media, SMAG-Y and SMAG-YPG, respectively, were prepared by addition to the SMAG medium of yeast extract (0 1 g L^'1), or a mixture of yeast extract (2 g L^"1), peptone (2 g L^"1) and glucose (1 g L^"1)

Inoculum from the ethylene degrading sediment samples were streaked on SMAG agar plates The inoculated agar plates were placed in air-tight 1-L jars with a butyl rubber stopper inserted in the lid Through the stopper, 10 ml of pure C H₄ (99.5%) was added to the jars, that were incubated at 25°C. The appearance of colonies was checked regularly and individual colonies were picked and streaked on fresh agar plates. After 3 to 4 rounds of purification with C₂H₄ as the sole source of energy and carbon, the resulting strains were transferred to agar plates with acetate and incubated under atmospheric air Purity of the cultures were checked by phase-contrast microscopy (Leitz Laborlux-S) and by the appearance of one or more types of colonies during incubation for up to 6 weeks on agar plates with SMAG-Y or SMAG-YPG, respectively. The former were incubated with C₂H₄ as previously described, while the latter was incubated in ambient air without C₂H₄

One of the isolated strains was RD-4, which appeared to be the most efficient of the ethylene degrading microorganism The strain was an aerobic, immotile, rodshaped 14

microorganism with a yellow pigmentation seen in the cell pellets after centrifugation. The time course of C₂H₄ consumption and growth of strain RD-4 in liquid SMAG medium is shown in Fig. 1. From these data it was calculated that the strain had a doubling time of about 20 h.

For the present purpose, the strain RD-4 was grown in four 5 L vessels with 2 L of defined mineral medium without added C-sources. The medium in each bottle was inoculated with 20 mL of a pre-grown culture and C₂H₄ was added to a headspace concentration of 1%. The cultures were incubated at 30°C on a water bath with orbital shaking (120 rpm). During a 5-days incubation period the C₂H₄ concentration was replenished to 1 % when it dropped below 0.5% (assayed by GC analysis). After 5 days of growth, the ethyiene oxidizing microorganisms were harvested by centrifugation (16.000 x g, 15 min) and resuspended in 1.5 L of autoclaved tap water. The density of the cell suspension was estimated to 2 x 10⁸ cells mL^"1 by direct microscopic counts in a Bϋrker-Tϋrk counting chamber with a depth of 50μm. The cell suspension was stored at 0 to 2°C until needed for the experiments described in examples 3-8 (one week later) and for the experiments described in example 9 (two weeks later).

EXAMPLE 2

Construction of a laboratory scale bioreactor for reducing ethylene in atmospheric air

A laboratory scale bioreactor was constructed from an acrylic core with an inner diameter of 5 cm and a length of 40 cm (Fig. 2). Six butyl rubber stoppers were inserted for gas sampling in holes, that were made at a vertical distance of 5 cm, starting 7.5 cm from the bottom of the core. The core was stoppered at both ends with punctured rubber stoppers, each equipped with a T-connector. At one orifice the T-connectors were stoppered by a butyl rubber stopper for gas sampling, while the other orifice served as a channel for gas flow (Fig. 2). In the mode of operation, the bioreactor could be sampled at depths from 0 to 35 cm (inlet and outlet, respectively) with increments of 5 cm (Fig. 2).

In the mode of operation, the bioreactor had a volume of 687 cm³ and a peat soil content of 300 g. The volume of the solid peat-soil phase, estimated by soaking in water, was 307 cm³ and thus the gas phase (V) in the bioreactor was equal to 380 cm³. 15

A mixture of atmospheric air and C₂H in N₂ (AGA SpecialGas, Lidingo, Sweden) was supplied to the bioreactor by use of two mass flow controllers (Side-Trak 840, Sierra Instruments, Inc , Monterey, Calif ) with a resulting output rate of 73 mL mm ¹ This rate was kept constant throughout the expeπments Before the inlet, the gas mixture was humidified by bubbling through a flask with distilled water To verify that a constant gas flow occurred during the expeπments, a Jour Digital Fiowmeter (Jour Research, Onsaia, Sweden) was connected to the outlet of the bioreactor The readings of the fiowmeter were checked on a daily to weekly basis

During the expeπments, the flow rate measured at the outlet of the bioreactor ranged from 71 4 to 75.9 mL mm^"1 with a mean ± SE of 73.3 ± 0 3 mL mm^"1 (n = 23). In a control experiment it was demonstrated that the flow rates at the inlet and the outlet were identical (76 5 to 76 8 mL mm ¹) and equalled the sum of the flow rates for the two gaseous components that were mixed (67 9 and 8 7 mL m ¹) Furthermore, it was demonstrated that with an empty bioreactor, the outlet C₂H₄ concentrations (2 19 ± 0.03 ppm, n = 3) were equal to the inlet concentrations (2.18 ± 0.03 ppm, n = 3) This showed that no significant leakage or adsorption occurred

As the present experiments were done at flow rates (F) of 73.3 mL mm^"1 it was calculated that the dilution rate (D = F/V) of the gaseous atmosphere in the bioreactor was 0 19 mm^'1 Applying the equation (equation 3) dC/dt = -DC to the proportion (0 to 100%) of atmospheric air in the bioreactor, it results from integration that (equation 4). C* = C₀ exp (- Dt), where C_t and C₀ is the proportion of atmospheric air at time t and 0, respectively, D is the dilution rate (0 19 mm^"1), and t is the operation time For the upstart situation (i e., C₀ = 100%) it was calculated from equation 3 that 99.9% and 99 999% of the atmosphere in the bioreactor should be replaced by the C₂H₄ gas mixture after 36 and 60 mm of operation, respectively Thus, a lower outlet than inlet C₂H₄ concentration after 1 h of operation could positively be attributed to C H removal by the inoculated soil This could be concluded because no leakage or adsorption occurred in control experiments. Similarly, subsequent changes made to the inlet C₂H₄ concentration should be prevailing at the outiet within 1 h of operation unless C₂H removal by the soil occurred 16

EXAMPLE 3

Analysis of the ethylene removal efficiency of the bioreactor when operating with 2 ppm C7H4

Immobilized ethylene-oxidizing microorganism were prepared by mixing 300 g of peat soil (Sphagnum no. 2, Pindstrup Mosebrug, Pindstrup, Denmark) with 200 mL of distilled water and 100 mL of microbial cell suspension (2 x 10⁸ cells mL^"1). The soil sample was thoroughly mixed and allowed to equilibrate at room temperature (20 to 22°C) for 1 h. The inoculated peat soil was loosely packed in the bioreactor to a density of 0 44 g cm^'3

A mixture of 10 ppm C₂H₄ and atmospheric air (1 -4) was applied to the bioreactor that was operated at room temperature. During an operation period of 16 days, gas samples (0.2 to 0.6 mL) were collected from the bioreactor and analyzed for C₂H and occasionally for O₂ and CO₂

During the expeπment, a stable inlet concentration of 2.05 ± 0.01 ppm (n = 57) was applied to the bioreactor (Fig. 3). Measurements of the outlet C₂H₄ concentration after 1 h of operation (0.23 ± 0.01 ppm, n = 4) demonstrated that 89% of the incoming C₂H₄ was actively removed by the bioreactor already at this early stage (Fig. 3). During the expeπment, the capacity of C₂H removal gradually improved and increased to 99% of the incoming concentration Thus, at the end of the experiment the bioreactor had a stable performance with an outlet concentration of only 0.017 to 0.020 ppm C₂H₄ (Fig.3)

EXAMPLE 4

Analysis of the gas composition in different soil depths of the bioreactor. and measurement of the ethylene removal stability of the bioreactor when operated with 2 ppm ethylene

Supplemental to the experiment described in Example 3, the gas composition at different soil depths of the bioreactor was analyzed The gas analysis showed that after 1 h of operation all soil layers were exposed to C₂H at concentrations ranging from 2.05 to 0.23 ppm (Fig. 4). However, as the capacity of C H₄ removal increased in the soil layers close to the inlet, removal of the incoming C₂H₄ ultimately occurred within the first 15 cm of the 17

bioreactor (Fig. 4). Further characterization of the C₂H removal by the 5 cm soil segments was done by calculation of the individual C₂H₄ removal rates according to equation 1 (Materials and Methods). These calculations showed that the initial C₂H removal rate in each segment (after 1 h of operation) depended linearly on the C₂H₄ concentration (data not shown).

Data from the 0 to 5 cm segment demonstrates that with the same inlet concentration of 2.05 ppm, there was an increase in the C₂H₄ removal rate from 3.1 to 7.9 μg C₂H₄ h^"1 during operation for 16 days (Fig. 5). This could be due to (i) microbial adaptation to the occurring C₂H concentration or (ii) an increase in cell number in the segment, or (iii) a combination of these effects. If the rate increase was due to an increase in the number of microorganisms (with a constant C₂H₄ removal rate per cell), it was calculated that the population in the 0 to 5 cm segment should increase from the initial number of 1.5 x 10⁷ cells cm^"3 to 3.8 x 10⁷ cells cm^"3. This could be obtained just by slow growth of the RD-4 strain, which had a doubling time of ~-22 h when cultivated in mineral medium with a headspace of 1% C₂H₄ (data not shown). No increases in the C₂H₄ removal rate occurred in the soil layers above the 0 to 5 cm segment, indicating that no growth of the added microorganism occurred in these layers (Fig. 5).

The linearity in the correlation between the initial C₂H₄ removal rate and the incoming C₂H₄ concentration for each 5-cm segment suggested that a concentration dependent C₂H removal occurred when microbial numbers were equal. In terms of reaction kinetics this indicated that the inlet C₂H concentration (2.05 ppm) was below the concentration that would give the maximal C₂H₄ removal rate in the 0 to 5 cm segment. No detailed kinetic experiments were done in the present study, and therefore calculation of kinetic parameters for RD-4 was not attempted. However, a high affinity for C₂H₄ (low K_m) could be involved in the depletion of C₂H to the low level of 0.017 ppm.

EXAMPLE 5

Analysis of the oxygen and carbon dioxide concentration in the bioreactor when operated with 2 ppm C7H

Supplementary to the experiments described in Example 3 and 4 the oxygen was measured after 1 day of operation. The oxygen measurements showed that the O₂ content 18 of the inlet gas and the outlet gas was 15.6 ± 0.1% (n = 3) and 15.2 ± 0.1 % (n = 3), respectively. This demonstrated that oxic conditions prevailed throughout the whole soil column. At the same time, the CO₂ concentrations at the inlet and the outlet were 0.018 ± 0.001% (n = 4) and 0.024 ± 0.001% (n = 3), respectively. Thus, the outlet CO₂ concentration was significantly higher than the inlet CO₂ concentration (P < 10^"4), demonstrating that a net mineralization of organic carbon occurred in the bioreactor.

EXAMPLE 6

Analysis of the removal efficiency of the bioreactor when adjusting the inlet ethylene concentration from 2 ppm to 117 ppm after 25 days of C7H starvation.

Following the operation with 2 ppm C₂H , as described in Example 3, the bioreactor was operated with atmospheric air for 25 days. Subsequently the inlet level was adjusted to 2 ppm C₂H₄ for 8 days, and then to 117 ppm for 80 days. The latter concentration was obtained by mixing 1000 ppm C₂H₄ and atmospheric air (1:8). Gas samples for C₂H₄ analysis were withdrawn regularly.

The results in Fig. 6 show that operation for 25 days without C₂H₄ in the inlet gas caused a decrease in the bioreactor capacity for C₂H removal. When 2 ppm C₂H₄ was reapplied, only 11 to 15% of the incoming C₂H was removed after 0.5 to 3 h of operation (Fig. 6). However, during operation for 8 days the capacity for C₂H₄ removal was recovered and >98% of the incoming C₂H was actively removed by the soil-bed reactor (Fig. 6).

Transition of the inlet C₂H₄ level from 2 to 117 ppm similarly caused a transient decrease in the efficiency of the bioreactor, which initially (after 0.5 h) removed 10% of the incoming C₂H (after transition the inlet concentration was 117.2 ± 0.4 ppm, n = 76, during the subsequent experiments). After operation with 117 ppm C₂H₄ for 4 days, the C₂H₄ concentration at the outlet of the bioreactor was below the detection limit of 0.04 ppm (Fig. 6). Thus, more than 99.9% of the incoming C₂H₄ was removed by the bioreactor.

This demonstrates that the microorganism is able to survive in the peat soil carrier material without an external source of C₂H₄. Furthermore, this shows that the microorganism is capable of substantially regenerating its full ethylene oxidizing capacity after ethylene starvation for a period of time. 19

EXAMPLE 7

Analysis of the long-term operational stability of the bioreactor comprising the immobilized microorganism

In a subsequent experiment to the experiment described in Example 6, the long-term operational stability of the soil-bed reactor was tested with the inlet level of 117 ppm C₂H₄ (Fig. 7). It was demonstrated that for more than 75 days of constant operation (Day 5 to 80), the soil-bed reactor was able to reduce the incoming C₂H₄ concentration to less than 0.04 ppm. The main part of the C₂H removal was accomplished during passage through the first 0 to 5 cm of the bioreactor (Fig. 7). However, after 70 days of operation, the C₂H₄ removal by the 0 to 5 cm segment started to decrease. The decrease in the efficiency of the 0 to 5 cm segment had no influence on the overall performance of the bioreactor, because the next soil layers (5 to 10 cm segment) were able to remove the incoming ethylene to less than 0.04 ppm (Fig. 7).

EXAMPLE 8

Analysis of the removal efficiency of the bioreactor when transferred from 21 to 10°C

During continuous operation with 117 ppm C₂H₄, as described in the experiment in Example 7, the influence of temperature was tested by placing the bioreactor in a thermostated incubator (10°C) adapted with holes for the inlet and outlet tubing. Control experiments showed that temperature equilibrium at 10°C was reached in the center of the bioreactor within 5 h. Ethylene concentrations were measured at the bioreactor inlet and outlet during operation for 18 days.

The results in Fig. 8 show that when the bioreactor was transferred from 21 to 10°C (Fig. 8), the outlet C₂H concentration gradually increased from <0.04 ppm to 46.6 ± 0.3 ppm (n = 3). This corresponded to a decrease in the C₂H₄ removal rates from 597 to 376 μg C₂H₄ h^"1, as calculated from equation 1 in "Method and Materials" (using p(C₂H₄) = 1.20 μg μL^"1 at 10°C). Applying the integrated Arrhenius equation it was calculated from these data that the apparent activation energy, E_a, for the C₂H₄ removal process was 29 kJ mol^"1. 20

However, after 2 days of operation at 10°C, the outlet concentration started to decrease with a linear time course (Fig. 8). Thus, when the experiment was stopped after 18 days, the outlet concentration was 1.6 ± 0.1 ppm C₂H₄ (n = 3), which was equivalent to a removal efficiency of 98.6%. These results indicates that a proliferation of the C₂H₄ reducing microorganism still occurs at 10°C.

EXAMPLE 9

Batch experiments to analyze the effect of storage on the C?H₄ reducing capacity of the inoculated solid material

A sample of immobilized microorganism was prepared as described in Example 3. while a control sample was prepared by mixing 100 g of peat soil with 100 mL of distilled water. Each of the soil samples were split in to three portions that were placed at 2, 8, and 20°C, respectively. To test the C₂H₄ removal capacity prior to storage, triplicate soil samples of 10 g were transferred to 120 mL serum vials that were purged with atmospheric air and closed by butyl rubber stoppers. Ethylene was added to a headspace concentration of ~20 ppm and the time course of C₂H₄ removal was followed at room temperature (20 to 22°C) by GC analysis of withdrawn gas samples (0.4 mL). Similar assays were performed with the stored soil samples after 2, 3 and 4 weeks of storage. Prior to these assays the triplicate 10 g subsamples were allowed to equilibrate at room temperature for 1 h.

In assays with freshly inoculated peat soil, a rapid depletion of the added -20 ppm C₂H was observed (Fig. 9). The C H₄ removal occurred constitutively at a rate of 17.1 ppm h^"1, as calculated by using linear regression of the data of the first 1 h of the experiment (Fig. 9). After storage of the inoculated peat soil for 28 days at 20°C, a constitutive C₂H₄ removal still occurred, but at a lower rate than for the freshly inoculated soil (Fig. 9). Thus, the data for the first 1.5 h of the experiment indicated a removal rate of 3.3 ppm C₂H₄ h^'1. Within the assay time of 5 h, no C₂H₄ removal occurred in samples of peat soil without added microorganism, neither initially or after 28 days of storage at 20°C (Fig. 9).

All time courses of C₂H₄ removal by inoculated peat-soil stored at 2, 8, or 20°C for 0, 2, 3 and 4 weeks fell within the range depicted by Day 0 and Day 28 in Fig. 9. Thus, for comparison, the initial C₂H₄ removal rate for each assay was calculated from the first 2 to 3 21 data points obtained during 0 to 1.5 h of incubation. These rates demonstrated that the C₂H₄ removal capacity decreased as a result of longer storage time (Fig. 10). However, the extent of the decrease caused by the storage time was almost unaffected by the storage temperature (Fig. 10). Thus, after 4 weeks of storage at all three temperatures, the C₂H₄ removal rate represented 15 to 21 % of the removal rate observed prior to storage.

Concerning the application of inoculated peat soil for purposes of C₂H₄ removal in bioreactors, the results demonstrated that cold storage did not improve the keeping quality of the inoculated peat soil. Rather, the results suggested that, even at 20°C, the inoculated peat-soil could be starved for C₂H₄ for up to 2 weeks and still retain half of the original activity. This would allow a certain time lap between the preparation of a bioreactor and its subsequent use for C₂H₄ removal.

22

REFERENCES

1. De Heyder, B., A. Overmeire, H. Van Langenhove, and W. Verstraete. 1994. Ethene removal from a synthetic waste gas using a dry biobed. Biotechnol. Bioeng. 44:642-648.

2. van Ginkel, C. G., H. G. J. Welten, J. A. M. de Bont, and H. A. M. Boerrigter. 1986. Removal of ethene to very low concentrations by immobilized Mycobacterium E3. J. Chem. Tech. Biotechnol. 36:593-598.

3. van Ginkel, C. G., H. G. J. Welten, and J. A. M. de Bont. 1987. Growth and stability of ethene-utilizing bacteria on compost at very low substrate concentrations. FEMS Microbiol. Ecol. 45:65-69.

4. Japanese patent application, Kokai No. 8-196208.

23

Applicant's or agent* file Inlen-alιαnaiappiical-onr* θ. reference number 19844 PC 1 P C T/D K 9 9 / 000 5 9

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule I3bis)

A. The indications made below relate to the microorganism referred to in the description on page 7 , line 14

B. IDENTIFICATION OF DEPOS-TT Further deposits are identified on an additional sheet | X|

Name of depositary institution

DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH

Address of depositary institution (including postal code and country)

Mascheroder Weg IB D-38124 Braunschweig Germany

Date of deposit Accession Number

2 February 1998 DSM 11980

C ADDITIONA-C INDICATIONS (leareblankifnetappBcaUc) This information is cxintinued on an additional sheet Q]

As regards the respective Patent Offices of the respective designated states, the applicants request that a sample of the deposited microorganisms only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn.

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (iftheindicaύoiu re otforβttieάgna dStaieή

E. SEPARATE FURNISHING OF lNDICATIONS (ieavcblank if not applicable)

The indications luted below will be submitted to the international Bureau later (spcάfy the general nature ofϋxeinΔcatwns eg, 'λceanon Number ofDepotW)

For receiving Office use onl For international Bureau use only

This sheet was received with tbe international application I I This sheet was received by tbe International Bureau on:

Authorized officer Authorized officer

Vnπ- prrrorxntΛ 24

| A Apiplicant's or agent* Gle InternalionalapplicationN*

19844 PC 1 reference number 1Η/0K 99 / 00059

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule 13bis)

A. The indications made below relate to the microorganism referred to in the description on page - line 14

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet I -I

Name of depositary institution

DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH

Address of depositary institution (including postal code and country)

Mascheroder eg IB D-38124 Braunschweig Germany

Date of deposit Accession Number

4 February 1999 DSM 12668

C ADDITIONAL. INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet ~~~[

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (?/ the indications are not for all designated States)

E. SEPARATE FURNISHING OF^INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the international Bureau l ter (specif ihegeneralnatureofuininύcations e.y, 'Accession Number of Deposit'

For receiving Office use only For international Bureau use only

This sheet was received with the international application I I This sheet was received by tbe International Bureau on:

Authorized officer Authorized officer

Fnrm Pf ntn/l*U /Inlw 1θα-»\

Claims

25CLAIMS

1. An isolated ethyiene oxidizing microorganism which, when immobilized on a solid carrier material, is capable of continuously removing in excess of 90%, preferably at Ieast 95%, of

5 the content of ethylene in a gas stream being contacted with said material, including the microorganisms which are the Rhodococcus erythropolis strain RD-4 and strain RD-4a deposited under the accession numbers DSM 11980 and DSM 12668, respectively.

2. A microorganism according to claim 1 which is capable of continuously removing at Ieast 10 90%, preferably at ieast 95%, of the content of ethylene at a temperature of 20┬░C or lower such as at 15┬░C or lower including at 10┬░C or lower.

3. A microorganism according to claim 1 which, when it is used in a bioreactor having a volume of about 700 cm³ and loaded with 0.44 g cm^"3 of a mixture of peat soil, distilled

15 water and a suspension of the microorganism containing about 2x10⁸ ceils mL^"1, in a weight ratio of about 3:2: 1 , at a gas flow rate which is in the range of 71.4 to 75.9 mL min^'1 is capable of removing at Ieast 90%, preferably at Ieast 95% and most preferably at Ieast 99%, of an ethylene content of up to 117 ppm ethyiene present in the inlet gas.

20 4. A microorganism according to claim 1 which retains its ethyiene removal efficiency during continuous operation of the bioreactor for at Ieast 10 days such as at Ieast 20 days including at 50 days e.g. at Ieast 80 days.

5. A microorganism according to claim 1 which, when it has been stored for 2 weeks or 25 longer at a temperature in the range of 2 to 22┬░C in a bioreactor having a volume of about

700 cm³ and loaded with 0.44 g cm^"3 of a mixture of peat soil, distilled water and a suspension of the microorganism containing about 2x10⁸ cells mL^'1, in a weight ratio of about 3:2:1, has retained at Ieast about 15% of its ethylene oxidizing capacity such as at Ieast 20% and preferably at Ieast 50% of its ethyiene oxidizing capacity. 30

6. A microorganism according to claim 1 which is capable of substantially regenerating its full ethylene oxidizing capacity after it has been stored for 2 weeks or longer under ethylene starvation conditions at a temperature in the range of 2 to 22┬░C in a bioreactor having a volume of about 700 cm³ and loaded with 0.44 g cm^"3 of a mixture of peat soil,

35 distilled water and a suspension of the microorganism containing about 2x10⁸ cells mL^"1, i in 26 a weight ratio of about 3:2:1 , the regeneration occurring within 4 days of adding ethylene to the inlet gas stream.

7. A solid material, including an organic material selected from the group consisting of peat soil, a resin, activated carbon, compost, a naturally occurring polymer and a synthetic polymer and an inorganic material including a material selected from the group consisting of soil, sand, clay, perlite, lava and a particulate inorganic material, carrying a microorganism according to any of claims 1-6.

8. An ethylene removing bioreactor comprising the microorganism according to any of claims 1-6 or a solid material according to claim 7, the bioreactor comprising gas stream inlet means and outlet means and optionally, means for collecting gas samples and optionally being provided with or connected to means for regulating the flow rate of the gas stream, the bioreactor preferably being a bioreactor which under appropriate operation conditions is capable of reducing the ethyiene content of the inlet gas to at the most 1 ppm such as at the most 0.5 ppm including at the most 0.2 ppm e.g. at the most 0.1 ppm or more preferably capable of reducing the ethylene content to at the most 0.05 ppm such as at the most 0.025 ppm including at the most 0.01 ppm.

9. A bioreactor according to claim 8 which is capable of reducing the ethylene content when operated at a temperature at or below 20┬░C such as at or below 15┬░C including about 10┬░C or lower.

10. A method of controlling the ethyiene content in a gas by contacting the gas with a solid material according to claim 7 or by passing the gas through the bioreactor according to claim 8 or 9, including a method wherein the solid material is present in a package of a plant product or is placed at a distance to such a product which permits the material to at Ieast partially remove the ethylene content in the gas surrounding the product or a method wherein the bioreactor is operated in a plant product storage facility or transportation vehicle or is operatively connected to such facility or vehicle.