Ethylene Removal at Low Temperatures under Biofilter and Batch Conditions

Abstract

Removal of the plant hormone ethylene (C₂H₄) is often required by horticultural storage facilities, which are operated at temperatures below 10°C. The aim of this study was to demonstrate an efficient, biological C₂H₄ removal under such low-temperature conditions. Peat-soil, acclimated to degradation of C₂H₄, was packed in a biofilter (687 cm³) and subjected to an airflow (∼73 ml min⁻¹) with 2 ppm (μl liter⁻¹) C₂H₄. The C₂H₄ removal efficiencies achieved at 20, 10, and 5°C, respectively, were 99.0, 98.8, and 98.4%. This corresponded to C₂H₄ levels of 0.022 to 0.032 ppm in the biofilter outlet air. At 2°C, the average C₂H₄ removal efficiency dropped to 83%. The detailed temperature response of C₂H₄ removal was tested under batch conditions by incubation of 1-g soil samples in a temperature gradient ranging from 0 to 29°C with increments of 1°C. The C₂H₄ removal rate was highest at 26°C (0.85 μg of C₂H₄ g [dry weight]⁻¹ h⁻¹), but remained at levels of 0.14 to 0.28 μg of C₂H₄ g (dry weight)⁻¹ h⁻¹ at 0 to 10°C. At 35 to 40°C, the C₂H₄ removal rate was negligible (0.02 to 0.06 μg of C₂H₄ g [dry weight]⁻¹ h⁻¹). The Q₁₀ (i.e., the ratio of rates 10°C apart) for C₂H₄ removal was 1.9 for the interval 0 to 10°C. In conclusion, the present results demonstrated microbial C₂H₄ removal, which proceeded at 0 to 2°C and produced a moderately psychrophilic temperature response.

The alkene C₂H₄ (common name, ethylene; International Union of Pure and Applied Chemistry name, ethene) is unique among atmosphere-polluting hydrocarbons because it is a plant hormone. Biological effects of C₂H₄ occur at concentrations below 0.1 ppm, which is generally more than 100-fold lower than those for other short-chain hydrocarbons (13).

Air purification by biological filters (20) has been suggested as a method of C₂H₄ removal from industrial waste gas and from horticultural storage facilities, where plant-produced C₂H₄ may accumulate to levels that cause a premature ripening or senescence of the plant material (2, 6, 7, 8, 10, 29, 30). During storage and transport of horticultural produce, temperatures below 10°C are often obligatory; for instance, 4 to 6°C is optimal for 35% (and acceptable for 85%) of the transport volume of potted plants produced in Denmark (17). A prerequisite for successful biofiltration under such conditions is the existence of C₂H₄-degrading microorganisms with sufficient activity at low temperatures. So far, however, biocatalysts for efficient C₂H₄ removal at temperatures below 10°C have not been described.

In the present report, microbial C₂H₄ removal at temperatures as low as 0 to 2°C was shown to occur indigenously in horticultural peat-soil under biofilter and batch conditions.

MATERIALS AND METHODS

Acclimated peat-soil.

Horticultural peat-soil (Pindstrup Blend 2; Pindstrup Mosebrug, Pindstrup, Denmark) was acclimated to C₂H₄ degradation by incubation of 800 g of soil (185 g [dry weight]) in a gastight glass bottle (5.5 liters) with a headspace concentration of ∼500 ppm C₂H₄. Through a butyl rubber stopper, gas samples (0.5 ml) for C₂H₄ analysis were withdrawn regularly during incubation at room temperature (∼20°C) for 28 days. After depletion of the C₂H₄, the bottle was purged with atmospheric air and new C₂H₄ was added. This was done repeatedly (six times) upon subsequent depletions.

Biofilter experiment.

A biofilter was made from an acrylic core (inner diameter, 5 cm; effective length, 35 cm) as previously described (10). Acclimated peat-soil was packed in the biofilter (∼0.15 g [dry weight] cm⁻³), and a humidified mixture of atmospheric air and 10 ppm C₂H₄ (4:1) was supplied to the inlet of the biofilter by two mass flow controllers (10). The flow rate (∼73 ml min⁻¹) was verified daily with a digital flowmeter at the biofilter outlet. The biofilter was first operated at room temperature (∼20°C) and then at 10, 5, and 2°C in a thermostatted incubator (Refritherm 6E; Struers, Rødovre, Denmark). During operation at 2°C (and 5 and 10°C in control experiments), temperatures at biofilter soil depths of 5 and 25 cm (i.e., distances from the inlet) were measured with permanently installed thermometers with steel penetration probes. During operation for 20 days, gas samples (20 ml) for C₂H₄ analysis were regularly collected. Thus, at each of 36 sampling occasions, the C₂H₄ removal efficiency was determined from three pairs of inlet (C_in) and outlet (C_out) C₂H₄ concentrations (removal efficiency = [1 − C_out/C_in] × 100%). Additional gas samples (duplicates) were collected through butyl rubber stoppers at biofilter soil depths of 5, 10, 15, 20, 25, and 30 cm.

Temperature gradient batch experiments.

Peat-soil from the biofilter was sieved (2 mm; final dry matter content, 18.7%) and weighed (1 g) portions were placed into 90 test tubes (28 ml), which were closed with butyl rubber stoppers and crimp seals. Triplicate test tubes were equilibrated (1 to 2 h) at each of 30 different temperatures, ranging from 0 to 29°C, in a temperature gradient incubator. Basically, this device was an insulated aluminum bar with 30 rows of six sample wells that fit the 28-ml test tubes. Cooling and heating were applied (and automatically regulated) at opposite ends of the aluminum bar, thereby producing a thermal gradient with increments of 1°C. The temperatures of 15 sample rows in the aluminum bar were measured by permanently installed Pt-100 sensors, and the recordings were logged on a personal computer at 5-min intervals. Temperatures of intermediate sample rows were calculated by linear regression.

After temperature equilibration, each test tube was injected with C₂H₄ to an initial headspace concentration of ∼500 ppm (511 ± 14 ppm; mean ± standard deviation; n = 30). Over a time course of 3 to 7 days, the C₂H₄ concentration in each test tube was measured four to six times, and the rates of C₂H₄ removal were calculated by linear regression.

By using the same approach described above, the C₂H₄ removal rates at 25, 35, and 40°C were tested with biofilter soil samples that had been stored at 2°C and reacclimated (15°C) under a headspace of ∼500 ppm C₂H₄.

Fate of C₂H₄.

The transformation of C₂H₄ to CO₂ was tested with acclimated peat-soil (4 g) incubated in stoppered 120-ml serum bottles with or without ∼750 ppm C₂H₄. For each treatment (i.e., with or without C₂H₄), three soil samples were incubated at 5 and 15°C. Gas samples (0.2 ml) for analysis of C₂H₄ and CO₂ were withdrawn during incubation for 2 weeks.

Analyses and statistics.

C₂H₄ was quantified with a Shimadzu GC-14B gas chromatograph with a flame ionization detector. Gases were separated on a Poropak Q (100 to 120 mesh) steel column (inner diameter, 2 mm; length, 1.9 m) at 95°C. The injection and detection temperatures were 150 and 200°C, respectively. During the biofilter experiments, gas samples were injected through a 2.5-ml sample loop that was purged with a sample volume of 20 ml. With this configuration, the C₂H₄ detection limit was 0.013 ppm for a signal/noise ratio of 3. During the remaining experiments, gas samples (0.5 ml) were injected directly by using a 1-ml gastight syringe.

CO₂ was quantified with a GC 82 gas chromatograph (Mikrolab, Højbjerg, Denmark) with a temperature conductivity detector. The column (Poropak N) was operated at 60°C with He as the carrier gas (flow rate, 43 ml min⁻¹). The injected sample volumes were 0.2 to 0.5 ml.

Dry matter content was determined gravimetrically after drying of three to six soil samples overnight at 105°C. Soil pH was measured by a glass electrode in soil-water suspensions (1:5).

Unless otherwise stated, the results of replicate samples are presented as means ± standard deviations for the number of samples (n) indicated.

RESULTS

Acclimated peat-soil.

Degradation of C₂H₄ was initially preceded by an acclimation period of ∼11 days, and thereafter, the C₂H₄ pool was depleted within 6 days (data not shown). New C₂H₄ was depleted within 1 to 2 days with no further acclimation period. After the last C₂H₄ addition, the rate of headspace C₂H₄ removal (HR) was 36 ppm h⁻¹ (data not shown). For the headspace volume (V) of ∼5 liters and the soil content (M) of 185 g (dry weight), this corresponded to a specific removal rate (SR) of 1.13 μg of C₂H₄ g (dry weight)⁻¹ h⁻¹ as calculated by SR = HR × V × ρ(C₂H₄) × M⁻¹, where ρ(C₂H₄) is the density of C₂H₄ at 20°C (1.16 μg μl⁻¹).

Biofilter experiment.

During the biofilter experiment (20 days), the flow rate ranged from 72.0 to 73.4 ml min⁻¹ (73.0 ± 0.4 ml min⁻¹; n = 23). The inlet C₂H₄ concentration ranged from 1.92 to 2.13 ppm (2.01 ± 0.05 ppm; n = 36). The soil pHs before and after the biofilter experiment were 5.7 ± 0.1 and 5.9 ± 0.1, respectively (n = 6).

Measurements of the outlet C₂H₄ concentration after 0.5 h of operation showed that 82.0% of the incoming C₂H₄ was initially removed (Fig. 1). After 2 days of operation, the efficiency of C₂H₄ removal increased to 99.0% ± 0.2% (n = 4). Hence, during operation for 2 to 6 days at 20°C, the biofilter had an outlet concentration of only 0.022 ± 0.005 ppm C₂H₄ (n = 4). When the biofilter was transferred from 20 to 10°C, no significant change in the C₂H₄ removal efficiency (98.8% ± 0.6%; n = 6) occurred during operation for 2 days (Fig. 1). Lowering the incubation temperature to 5°C caused a transient decrease in the C₂H₄ removal efficiency to 95.0%. Then, during operation for 3 to 6 days at 5°C, the removal efficiency increased to 98.4% ± 0.5% (n = 5), with an average outlet C₂H₄ concentration of 0.032 ± 0.010 ppm (n = 5). Further lowering of the incubation temperature to 2°C caused a permanent decrease in the average removal efficiency to 83.0% during 6 days of operation (Fig. 1). This corresponded to an outlet C₂H₄ level of 0.338 ± 0.030 ppm (n = 13).

FIG. 1.

C₂H₄ removal efficiency of the biofilter during operation at 20, 10, 5, and 2°C with 2 ppm C₂H₄. The dotted lines indicate the temperature transitions. The data represent the mean of three determinations. Coefficients of variation ranged from 0 to 3%. Note the y axis starts at 80%.

C₂H₄ measurements at different biofilter soil depths showed that all soil layers were initially exposed to C₂H₄ levels declining from 2.03 ppm at the inlet (i.e., 0-cm soil depth) to 0.37 ppm at the outlet (i.e., 35-cm soil depth). However, after 5 days of operation at 20°C, the C₂H₄ removal occurred almost completely within the first 0 to 10 cm of the biofilter (Fig. 2). Similarly, after 5 days of operation at 5 and 2°C, the C₂H₄ removal occurred within the first 0 to 15 cm of the biofilter (Fig. 2). At 2°C, however, a relatively high C₂H₄ concentration (∼0.34 ppm) passed through the 15- to 35-cm soil layer without further removal (Fig. 2).

FIG. 2.

C₂H₄ concentrations at different biofilter soil depths after operation for 5 days at 20°C (●), 5°C (■), and 2°C (▴). Soil depths of 0 and 35 cm represent the biofilter inlet and outlet, respectively.

The temperatures measured in the center of the biofilter during operation at 2°C (6 days) were 2.7 ± 0.3°C at a 5-cm soil depth and 1.8 ± 0.3°C at a 25-cm soil depth (n = 18). Control experiments showed that incubation of the biofilter at 10°C resulted in constant temperatures of 10.4 and 9.9°C at soil depths of 5 and 25 cm, respectively. During control incubation at 5°C, the temperature of the inlet gas was 6.1°C, and constant temperatures of 5.3 and 4.9°C were measured at soil depths of 5 and 25 cm, respectively.

Temperature gradient batch experiments.

Table 1 shows the highly constant incubation temperatures in the thermal gradient during the batch incubation period of 7 days. Linear regression between the temperature and the sample row position (y = −1.00x + 30.13) had a regression coefficient (r²) of 1.000 (n = 15).

TABLE 1.

Mean, minimum, and maximum temperatures for individual sample rows during a 7-day temperature gradient incubation

Sample row position	Temp (°C)a
	Meanb	Minimum	Maximum
1	29.0	29.0	29.0
3	27.2	27.2	27.3
5	25.0	25.0	25.1
7	23.1	23.0	23.2
9	21.1	21.1	21.2
11	19.2	19.2	19.4
15	15.2	15.1	15.3
16	14.2	14.2	14.4
18	12.3	12.2	12.4
20	10.2	10.2	10.4
22	8.2	8.1	8.4
24	6.2	6.2	6.4
26	4.1	4.0	4.3
28	1.9	1.9	2.1
30	0.0	0.0	0.1

^an = 1,975 readings. 

^bStandard deviations around the mean were <0.04°C. 

A linear time course of C₂H₄ removal was observed at all temperatures during the temperature gradient incubation. Linear regression coefficients (r²) ranged from 0.84 to 1.00, with a mean of 0.98 (n = 90). The highest C₂H₄ removal rate (0.85 μg of C₂H₄ g [dry weight]⁻¹ h⁻¹) occurred at 26°C (Fig. 3). At lower temperatures, the C₂H₄ removal rate decreased, but remained at levels of 0.14 to 0.28 μg of C₂H₄ g (dry weight)⁻¹ h⁻¹, even at 0 to 10°C (Fig. 3). For soil samples incubated at 25, 35, and 40°C (after storage and reacclimation), the C₂H₄ removal rates were 0.72 ± 0.02, 0.06 ± 0.03, and 0.02 ± 0.01 μg of C₂H₄ g (dry weight)⁻¹ h⁻¹, respectively (n = 3).

FIG. 3.

Temperature dependence of C₂H₄ removal in batch incubations of biofilter soil. The data represent the mean ± standard error (n = 3). The insert shows an Arrhenius plot with linear regression of data below the optimum temperature (≤26°C).

Below the optimum temperature of 26°C, the temperature dependence of C₂H₄ removal fitted the Arrhenius equation: ln (rate) = ln A + E_a/RT, where A is a constant, E_a is the apparent activation energy, R is the gas constant, and T is the absolute temperature (3). From the slope of the Arrhenius plot (Fig. 3, insert), E_a was found to 45 kJ mol⁻¹. This E_a corresponded to a Q₁₀ of 1.9, as calculated for the temperature interval from 0 to 10°C {Q₁₀ = exp[E_a × 10/RT × (T + 10)]}.

Fate of C₂H₄.

In batch experiments at 15°C, added C₂H₄ (equivalent to 3.7 ± 0.1 μmol; n = 3) was depleted in 4 days with a concurrent CO₂ production of 34.1 ± 2.8 μmol of CO₂ (n = 3). In samples without added C₂H₄, the CO₂ production was 27.5 ± 3.2 μmol of CO₂ (n = 3). Thus, on average, the additional CO₂ production in samples with C₂H₄ was equivalent to 6.6 μmol of CO₂. At 5°C, added C₂H₄ (equivalent to 3.7 ± 0.1 μmol of C₂H₄) was depleted after 12 days with a concurrent CO₂ production of 25.0 ± 2.0 μmol of CO₂ (n = 3). In samples without added C₂H₄, the CO₂ production corresponded to 22.8 ± 0.8 μmol of CO₂. Thus, the additional CO₂ production in samples with C₂H₄ was equivalent to 2.2 μmol of CO₂.

DISCUSSION

C₂H₄ removal by soil samples was first reported by Abeles et al. (1) and has been documented by several authors (5, 26, 27, 35). The process is mainly mediated by ethylene-degrading bacteria, which have been isolated from various soil types (5, 16, 24, 31). Thus, the capacity for C₂H₄ degradation has been found in such genera as Xanthobacter, Nocardia, Mycobacterium, Rhodococcus, Bacillus, and Pseudomonas (25, 31). In some soil types, including the present peat-soil, an acclimation period of several days to weeks may precede the C₂H₄ removal (5, 11). This is most likely explained by enzyme activation or growth of the ethylene-degrading bacteria, which initially may occur in low numbers (5, 34). Indeed, the microbiological nature of the C₂H₄ removal in the present experiments was demonstrated by the initial acclimation period of ∼11 days, which disappeared for subsequent additions of C₂H₄. Also, the microbial mediation of the C₂H₄ removal was suggested from the close association of optimum (26°C) and maximum (35 to 40°C) temperatures, which are characteristic of enzymatic processes. Finally, in batch experiments with the present peat-soil type, it was shown that no C₂H₄ removal occurred when the peat-soil was sterilized by autoclaving on 2 consecutive days prior to incubation (data not shown).

C₂H₄ removal by biofilters.

C₂H₄ removal by inoculated biofilters (for examples, see references 7 and 29) has been surveyed previously (10, 11). However, a comparison of different filters is difficult or even impossible unless they have been operated under exactly the same conditions (e.g., inlet C₂H₄ concentration and volume/flow ratio). The present biofilter was operated under similar conditions to a peat-soil biofilter, which was inoculated with ethylene-oxidizing bacteria (10). This allowed a comparison of acclimated peat-soil and inoculated peat-soil as biofilter materials. The present biofilter showed a lowest outlet level of 0.022 ppm of C₂H₄ within few days of operation at 20°C. This was similar to the lowest outlet concentration (0.017 to 0.020 ppm C₂H₄) observed for the inoculated biofilter (10). Also, it was shown that C₂H₄ removal occurred primarily within the first 10-cm soil segment of each of the biofilters. Thus, a similar removal efficiency apparently could be obtained with acclimated and inoculated peat-soil under biofilter conditions with 2 ppm C₂H₄. For experiments with much higher loads of C₂H₄ (inlet concentration of ∼117 ppm C₂H₄), both biofilter types also showed an efficient C₂H₄ removal, but differed in operational stability, which was highest for the inoculated biofilter (10, 12).

Using a biofilter based on indigenous microorganisms in compost, van Ginkel et al. (30) showed that C₂H₄ removal was induced after ca. 4 weeks of operation with 2 ppm C₂H₄ (flow rate, 1.3 ml min⁻¹; reactor volume, 15 cm³; temperature, 30°C). At the end of an 8-week operation period, the C₂H₄ removal efficiency corresponded to ∼45%, but a stable (and maximal) efficiency was not attained during the experiment (30). Therefore, a comparison between the acclimated compost biofilter and the present peat-soil biofilter was not possible.

Temperature dependence of C₂H₄ removal.

The effect of low temperature on C₂H₄ removal has only been tested in few studies. Thus, van Ginkel et al. (29) concluded that C₂H₄ removal by liquid cultures of a known biocatalyst, Mycobacterium sp. strain E3, declined rapidly below 10°C and was almost absent at 4°C. Also, de Bont (5) found that no appreciable C₂H₄ removal occurred when a clay soil was incubated at 4°C, and, likewise, forest soil samples removed almost no C₂H₄ when incubated at 0 and 10°C (25). A 1998 study (10) showed that an inoculated biofilter could be adapted to efficient C₂H₄ removal at 10°C, but the performance at lower temperatures was not tested. The present study for the first time has demonstrated microbial C₂H₄ removal at temperatures as low as 0 to 2°C. Furthermore, the increase in C₂H₄ removal efficiency that occurred during biofilter operation at 5°C indicated adaptation (or possibly growth) of ethylene-degrading microorganisms at this temperature (Fig. 1).

C₂H₄ removal by indigenous microorganisms in peat-soil in principle could be mediated by different groups of microorganisms with different temperature responses. However, the present data did not reveal the occurrence of such different temperature groups, because only one temperature optimum was identified from the fine-scale temperature response of C₂H₄ removal. Thus, the activated peat-soil seemed to be dominated by only one group of ethylene-degrading bacteria or, alternatively, by different groups with similar temperature responses. The present temperature response was characterized by a relatively low Q₁₀ of 1.9, which was in accordance with low temperature sensitivity and consequently a relatively high rate at low temperature (23).

Microbial temperature groups.

Although no categorical definitions exist (for examples, see references 4, 14, 18, and 33), microorganisms may be divided into thermal groups, such as psychrophiles and mesophiles, based on minimum, optimum, and maximum temperatures for growth (T_min, T_opt, and T_max, respectively). Organisms which are not truly psychrophilic (21), but still able to grow at ≤5°C, may be considered as psychrotrophs or (more correctly) moderate psychrophiles (9; H. W. Jannasch, Letter, ASM News 64:185, 1998). A current definition of this group was adopted by Wiegel (33), who used the criteria T_min <5°C, T_opt >15°C, and T_max >20°C.

While the definitions of thermal groups apply to microbial growth temperatures, the same terminology is often used to characterize the temperature dependence of metabolic processes, such as respiration or substrate degradation. Growth and metabolic rates may not scale directly (22, 32), however, and therefore the growth response of the present microorganisms was uncertain, although the temperature response of microbial C₂H₄ degradation could be characterized as moderately psychrophilic. However, a moderately psychrophilic growth response could not be excluded, because the T_opt and T_max for growth may actually be lower than those for metabolic processes in cold-adapted bacteria (15, 18, 19, 28).

Fate of C₂H₄.

Based on the relationship between C₂H₄ removal and CO₂ accumulation, an attempt was made to determine the fate of C₂H₄ in the acclimated peat-soil. At 15°C, removal of 3.7 μmol of C₂H₄ was accompanied by increased production of CO₂ (equivalent to 6.6 μmol). These data were in reasonable agreement with the following stoichiometry of complete C₂H₄ oxidation:

At 5°C, removal of 3.7 μmol of C₂H₄ was accompanied by increased CO₂ production (equivalent to 2.2 μmol). Rather than a complete C₂H₄ oxidation to CO₂, these data indicated the accumulation of intermediate products during the C₂H₄ depletion. However, no gaseous intermediates were detected during the experiments, and therefore the existence of intermediates was not confirmed. Indeed, the peat-soil system may not be ideal for mass balance studies due to the abundance of organic carbon substrates. Thus, it cannot be excluded that ethylene-oxidizing bacteria may shift from utilization of C₂H₄ to other carbon substrates when C₂H₄ is not available. Therefore, an apparent lack of CO₂ production in response to C₂H₄ removal may be caused by a higher CO₂ production from other carbon substrates in the absence of C₂H₄ rather than in its presence. In conclusion, the present C₂H₄ removal was mediated by ethylene-oxidizing microorganisms, which at least at 15°C caused a complete oxidation of C₂H₄ to CO₂.