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Biotechnology and Bioprocess Engineering 18: 827-833 (2013). DOI 10.1007/s12257-013-0083-0. Ethanethiol Degradation by Ralstonia eutropha.

Biotechnology and Bioprocess Engineering 18: 827-833 (2013) DOI 10.1007/s12257-013-0083-0

RESEARCH PAPER

Ethanethiol Degradation by Ralstonia eutropha Mahsa Sedighi, Farzaneh Vahabzadeh, Seyed Morteza Zamir, and Abbas Naderifar

Received: 1 February 2013 / Accepted: 11 February 2013 © The Korean Society for Biotechnology and Bioengineering and Springer 2013

Abstract In the present study, a pure culture of Ralstonia eutropha was used to degrade gaseous ethanethiol. Ethane thiol at various initial concentrations ranging from 115 to 320 mg/m3 was degraded almost completely within 120 ~ 168 h, while at higher concentrations up to 452 mg/m3, removal efficiency declined. It was likely that ethanethiol was used as the source of energy by R. eutropha, since no clear increase in the biomass concentration was observed. Kinetic data of ethanethiol bidegradation could be fitted using the Monod model. The kinetic parameters were qm = 0.23 (mg ethanethiol/g biomass/h), and Ks = 1.379 (mg/L). The mineralization pathway of ethanethiol through sulphate, as the detected product, and the energy production were discussed in some detail. Keywords: ethanethiol, biodegradation, kinetics, Ralstonia eutropha, sulphate

1. Introduction Odorous compounds, including volatile organic compounds (VOCs), volatile fatty acids, H2S, and amines, as well as volatile organic sulfide compounds (VOSCs, e.g. ethanethiol (ET), dimethyl disulfide (DMDS) and methanethiol), are emitted from various industries, including kraft pulp mills, petroleum refineries, tanneries, waste water treatment plants, landfills, composting, and some food industries [1,2]. It is noted that H2S and other sulfur containing compounds are Mahsa Sedighi, Farzaneh Vahabzadeh*, Abbas Naderifar Chemical Engineering Department, Amirkabir University of Technology, Tehran, Iran Tel: +98-216-454-3161; Fax: +98-216-640-5847 E-mail: [email protected] Seyed Morteza Zamir Biotechnology Group, Chemical Engineering Department, Tarbiat Modares University, Tehran, Iran

the major odor causing substances, and are also recognized as important pollutants in air. These compounds have low odor threshold values, and are detectable by humans at extremely low concentrations [2,3]. VOSCs not only cause nuisance, because of their special odor, but also have negative impacts on human health [2-4]. Therefore, control of sulfur emission to the atmosphere is becoming a growing concern nowadays, and various physical, chemical and biological abatement technologies have been developed for this purpose [2]. In most cases, physical-chemical techniques are often unsatisfactory, due to high investment and operation costs, and possible generation of secondary waste streams. However, biological processes have been found to be very promising technologies for the removal of toxic volatile organic compounds, because of many intrinsic advantages, such as low cost, high efficiency, operational simplicity, as well as no by-products produced for further treatment [2,5]. Among the VOSCs, ethanethiol (CH3-CH2-SH) is significantly toxic to human health [6]. It is a colorless liquid, with a low odor threshold of 0.7 µg/L. The maximum allowable concentration in an environment for ethanethiol should not exceed 10.0 mg/L [7]. Thus, it is desirable to develop an effective biodegradation route for the removal of odorous organic pollutants, such as ethanethiol. Mixed cultures, often originating from wastewater treatment plants or from similar origin, have been used in many cases of waste gas treatment, though pure strains have sometimes also been used [8]. The target in the use of a monoculture system is to have access to a known (pure) culture, where obtaining repeatable experimental data would be most likely. Use of a test system under sterilized condition is the preferred choice, although performing biodegradation experiments under unsterilized condition could be a reasonable approach, mainly on the basis of the operational cost. Growth/inhibition characteristics of the microbial cells in the case of using a known culture could

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be modeled appropriately, and relevant kinetic description would then be highly informative. Compared with the microorganisms for the treatment of VOCs, the species isolated for the biodegradation of VOSCs are very rare [7]. Although some aerobic microorganisms, such as Hyphomicrobium MS3, Hyphomicrobium S, Thiobacillus thioparus E6 and DW44, were isolated to degrade VOSCs, the use of these microorganisms for the biological treatment of wastewater containing ethanethiol or ethanethiol off gas has seldom been reported [6]. Degradation of ethanethiol during microbial biodegradation, as a single organic compound or as a waste gas mixture, has been claimed in previous reports [6,7,9-11]; however, biological oxidation of alkylsulfides is still an under developed field of research. It should be mentioned that no individual bacterial strain capable of decomposing ethanethiol has been reported, except a new bacterial strain isolated from activated sludge obtained from a domestic wastewater treatment plant [6]. As the single strain is isolated from domestic wastewater, the degradation experiments are hardly universal. Therefore, it is interesting to introduce a known single strain, capable of degrading ethanethiol. In the present study, biodegradation of ethanethiol by using the pure culture of Ralstonia eutropha (formerly called Alcaligenes eutrophus) is reported. In our previous works, biodegradation of phenol and formaldehyde by R. eutropha has been reported [12,13]. Another strain of this bacterial genus, Alcaligenes xylosoxidans, was also used by Kim et al. for the biodegradation of thiodiglycol (TDG), the hydrolysis product of sulfur mustard [14]. As far as we are aware, this is the first use of R. eutropha as a purenonpathogenic culture for the biological oxidation of an alkylsulfide. The reaction kinetics for the biodegradation of ethanethiol by the bacterial strain, and the oxidative pathway according to the sulfate production, were also investigated.

2. Materials and Methods 2.1. Microorganism and cultivation medium Ralstonia eutropha (PTCC 1615) was purchased from the Iranian Research Organization for Science and Technology, and was maintained at low temperature 4oC, on slants containing nutrient medium. Subcultures were routinely made every 2 months. The nutrient medium contained the following ingredients (in g/L); glucose, 3; yeast extract, 2; peptone, 2; KH2PO4, 1; K2HPO4, 1; (NH4)2SO4, 1; MgSO4.7H2O, 0.05; 15 g/L agar was used for solid growth media. The pH of the medium was adjusted to 7, and it was sterilized, by autoclaving at 121oC for 20 min. The mineral salts medium (MSM) used for investigating the bacterial biodegradation of ethanethiol contained (g/L) 1 KH2PO4,

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1 K2HPO4, 1 (NH4)2SO4, and 0.05 MgSO4.7H2O. 2.2. Chemicals Ethanethiol (97%) was provided by Sigma-Aldrich. All other chemicals used for the preparation of aqueous medium and biochemical experiments were of analytical grade, with the highest purities. 2.3. Experiments Batch experiments were conducted using 20 mL of MSM in 120 mL sterile serum bottles, sealed with Teflon-coated silicone septa and aluminum crimp caps (to avoid the volatilization of ethanethiol during the experiments), in a rotary shaker at 150 rpm. The temperature and pH of the medium were fixed at 30oC and 7, respectively, and all experiments were carried out in triplicate. Before the experiment, inoculum was prepared by suspending bacterial cells in distilled water. The optical density of the cell suspension was adjusted to 1.0 at 600 nm. 5 mL of the cell suspension was inoculated into 20 mL MSM. Different volumes of ethanethiol were injected into the cultures through Teflon-coated silicone septum, by using a 10 µL syringe. Ethanethiol balances in vapor-liquid twophases within 60 min. At equilibrium, the partition between gas and liquid phase is generally described by Henry’s law, which is given by the equation: Cgi = HiCli

(1)

where Cgi and Cli are the concentration of the test substrate in the gas and liquid phase, and Hi is Henry’s coefficient [15]. By analyzing the headspace gaseous samples concentrations, and using the partition coefficient (Henry’s coefficient) of ethanethiol, the concentration of ethanethiol in the liquid phase was estimated. The concentration of ethanethiol in the gas phase was selected as the initial concentration after equilibrium. Samplings from the gas and liquid phases were carried out by the insertion of syringes into the septum. To determine the possibility of chemical degradation and volatilization of ethanethiol, parallel control tests were conducted in serum bottles, without the introduction of biomass. Results showed negligible change in ethanethiol concentration under the studied conditions, for all initial concentrations. 2.4. Analytical techniques The ethanethiol concentrations in the gas phase of serum bottles were analyzed by using a gas chromatograph (GC) (Young Lin, ACME-61000 equipped with Helium Ionization Detector (HID), and a capillary column TRACSIL TRB-5 (30 m × 0.53 mm × 3.0 µm). The oven temperature was maintained at 80oC for 2 min, and raised to 150oC at

Ethanethiol Degradation by Ralstonia eutropha

10oC/min, and finally maintained at 150oC for 1 min. The temperature of injector and detector were fixed at 150 and 240oC, respectively. After various test time intervals, 0.5 mL of the gas sample was extracted from each of the serum bottles, by means of a 2.5 mL gas-tight syringe. A calibration curve was used to determine the ethanethiol concentration. The concentration of ethanethiol in the liquid phase was estimated using the partition coefficient (Henry’s coefficient) of ethanethiol over ethanethiol/ MSM at 30oC. To evaluate the partition coefficient, defined volumes of liquid ethanethiol were injected into 120 mL serum bottles containing 25 mL MSM. The bottles were sealed by Teflon-coated silicone septa and aluminum crimp caps. After reaching vaporliquid equilibrium, the ethanethiol concentration in the gas phase was determined by gas chromatography. A mass balance was performed to estimate the partition coefficient, considering that the difference between the initial and final concentrations of ethanethiol in the headspace (Vheadspace • ∆Cgas), corresponds to the absorbed ethanethiol in the liquid phase (Vliq • Cliq). Biomass concentration in the serum bottles was determined by the measurement of the OD (Optical Density) of the liquid inside the serum bottles at 600 nm, using a spectrophotometer (Jasco V-550). A calibration curve was used to relate the absorbance of the culture to the biomass concentration. The concentrations of sulfate were determined by means of an Ion Chromatograph (Waters 2695, alliance), equipped with Conductivity Detector (Waters 432), and an IC-Pak Anion HR column. All measurements were carried out in triplicate, and the error in these experiments was less than 5%.

3. Results and Discussions 3.1. R. eutropha capacity in using ethanethiol as the sole carbon source The extent of an organic pollutant removal in any waste treatment process (biological or nonbiological), depends on the initial concentration of the test pollutant. To study the effects of initial concentration on the bacterial degradative capacity, ethanethiol at ten different concentrations, ranged from 115 to 452 mg/m3, was introduced to the bacterial culture. The results of the equilibrium test showed that ethanethiol vapor-liquid balance was established within 60 min. Biodegradation study at different initial concentrations was determined with the same initial biomass concentration. Ethanethiol concentration in the gaseous phase was monitored during incubation by the headspace analyses. Fig. 1 shows the degradation trends of ethanethiol in the gas phase during the batch experiments. The removal

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Fig. 1. Effects of initial concentration of ethanethiol on the removal efficiencies in batch culture at 30oC and at pH value 7 for 168 h.

efficiency (RE) of ethanethiol increased with increasing the time, at fixed initial concentration. Complete ethanethiol removal was achieved at concentrations below 320 mg/m3. In contrast, the bacterial culture could not degrade higher concentrations of ethanethiol (355, 395, 427, and 452 mg/m3) completely during 168 h. With increasing the initial concentration, the RE decreased. It should be pointed out that the RE of ethanethiol was also higher at lower concentrations within initial time intervals, than at concentrations above 320 mg/m3. For example, almost 70% of ethanethiol at 180 mg/m3 was degraded within the first 48 h, while 37% of ethanethiol at 452 mg/m3 was removed. Afterwards, all the degradation curves smoothly dropped with increasing the reaction time. The reduction of REs at high initial ethanethiol concentrations (above 320 mg/m3) could be due to the substrate inhibition and overloading for the bacteria. At lower initial concentrations, the bacteria were not saturated by the substrate, and slight increase of the concentration had a greater effect on the duration of degradation time, than on the maximum REs. Thus, ethanethiol can almost be completely degraded by R. eutropha, as the initial concentrations range from 115 to 320 mg/m3. At higher initial concentrations, mass transfer of ethanethiol from gas phase to the liquid phase was improved due to higher concentration gradients, and a kinetic limitation occurred. Therefore, overloading of substrate for the bacteria may result in the decrease of RE

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with the increase in initial ethanethiol concentration, and more time was needed to achieve the same RE, which was obtained at concentrations below 320 mg/m3. 3.2. Kinetic modeling for ethanethiol biodegradation Catalytic (enzymatic) degradation of a particular substrate considering the microbial cells as the catalyst, follow the saturation kinetics that have been well described by the Michaelis-Menten and Monod models. Saturation kinetics suggest that at low substrate concentrations (relative to the half-saturation constant), the degradation rate is proportional to the substrate concentration (first-order equation), while at high substrate concentrations, the degradation rate is independent of the substrate concentration (zero-order equation). In various features of biodegradation kinetics (i.e. growth-linked systems or no growth situations), different models, including first-order, zero-order, logistic, Monod (with and without growth), and logarithmic, can be used to describe biodegradation [16-18]. In order to obtain the specific degradation rate (SDR) by the bacterial culture and incorporate it into a model, the variation of liquid-phase concentration was monitored during the incubation. By dividing the degradation rate to the initial biomass, the SDR can be verified. The SDRs for various initial ethanethiol concentrations are shown in Fig. 2. The SDR curve can be divided into two parts. At low ethanethiol concentrations, SDR increased with the increase in the ethanethiol concentration (first order equation); however, when ethanethiol concentration exceeded a threshold value (i.e. 2.561 mg ethanethiol/L), SDR remained almost constant (zero order equation). Regarding the SDR trend, the Monod model was fitted to the experimental data, which is represented by Eq. (2): qm S SDR = -----------Ks + S

(2)

Fig. 2. Specific degradation rates as a function of initial ethanethiol concentration.

where, S is the concentration of substrate (mg/L), qm is the maximum specific degradation rate (mg/g/h), and Ks is the half velocity constant (mg/L). Application of Monod equation to the experimental data gave a good fit, as R2 was observed to be 0.94. The model parameters were evaluated by using GraphPad Prism 5, which obtained as follows: qm = 0.23 Ks = 1.379 Finally, the Monod equation for the biodegradation of ethanethiol by R.eutropha can be represented as follows (Eq. (3)): qmSX 0.23SX dS = ------------------------- = –SDR.X = –-----------Ks + S 1.379 + S dt

(3)

In Eq. (3), X represents the biomass concentration (g/L). R. eutropha is the most widely studied bacterium, due to its ability to accumulate large amounts of Poly-βhydroxybutyric acid (PHB), as an energy reserve material, when nutrients such as nitrogen or phosphorous sources are available in limiting concentrations, in the presence of excess carbon sources [19,20]. By considering the conditions mentioned above, the formation of PHB was very unlikely (i.e. ethanethiol, at the concentrations used in this study, was not a suitable carbon source for synthesis of this biodegradable polymer). Various models have been fitted to the biodegradation kinetics data for sulfuric compounds in batch mode. Most of them were considered as pseudo-kinetic models, i.e. pseudo-first or second order models, which have a strong ability to predict the kinetic behavior of biodegradation. However, they are not general, and can only be applied in distinct cases, which limits their capability of comparing their results with other kinetic data. Application of the general kinetic models, such as Monod or Haldane, provides an opportunity to compare different biodegradation kinetic behaviors. Unfortunately, the kinetic results for ethanethiol biodegradation have commonly been presented by pseudotype models. Wan et al. used Lysinibacillus sphaericus strain RG-1, isolated from activated sludge obtained from a domestic wastewater treatment plant, for the biodegradation of ethanethiol in aqueous medium [6]. They described the degradation reaction by pseudo first-order equation, in which the maximum degradation rate constant and the minimum half-life were calculated to be 0.0308/h and 22.5 h under the optimized conditions, respectively, i.e. 4 mg ethanethiol/L, 30oC, and pH = 7. Schreiber and Pavlostathis used the Monod-based model for the thiosulfate concentration profiles ranging between 100 and 600 mg/L (as S) [21]. The values of biokinetic coefficients k (the maximum

Ethanethiol Degradation by Ralstonia eutropha

specific substrate utilization rate) and Ks (half-velocity coefficient) for the oxidation of thiosulfate in mixed (i.e.

Fig. 3. Time-course variation in (A) ethanethiol concentration, and (B) OD at 320 (○) and 452 (■ ) mg/m3 initial ethanethiol concentration.

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heterotrophic and autotrophic) microbial systems were estimated as equal to 19.8 mg thiosulfate-S/mg biomass-d and 14 mg thiosulfate-S/L, respectively. It was interesting that at all ethanethiol concentrations, no increase in biomass was observed, although the ethanethiol removal occurred during the plateau period. Data of the ethanethiol concentration and the bacteria concentration at 320 mg/m3 (the highest concentration with RE > 95%) and 452 mg/m3 (the highest ethanethiol concentration) are shown in Fig. 3. It is possible that cell division was stopped owing to the repairable substrate-induced damage to DNA, as was reported in previous studies [22,23]. Energy metabolism and cell synthesis are the major biochemical events in microbial performance. No increase in biomass can be used as an index of the uncoupling of dissimilation from assimilation. This may be to the bacterial cells advantage, to survive and sacrifice biomass yield to the removal of substrate. Similar kinetic results were also observed by Schmidtet al. [24]. Their results indicated that in the presence of an adequate number of cells, the rate of metabolism of concentrations of substrate that do not support growth is first order (simple form of the Monod model, at low concentrations of substrate). 3.3. Sulphate production and degradation pathway of ethanethiol by R. eutropha Ethanethiol can be converted to diethyl disulphide through chemical oxidation. It can also be transformed to ethyl methyl sulphide, or consumed by the bacterial culture to produce sulphate. These pathways were proposed for ethanethiol degradation with Pseudomonas sp. by Barreiros et al. [25]. Their study on mineralization of the thiocarbamate

Fig. 4. Proposed oxidative pathways in aerobic degradation of ethanethiol.

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Fig. 5. Trend of sulfate formation during biodegradation of ethanethiol at 320 mg/m3.

herbicide molinate showed that ethyl methyl sulphide could be formed through ethanethiol transformation. But the researchers did not observe any accumulation of this product, although the rate of transformation was high. Therefore, they concluded that ethanethiol was enzymatically oxidized, with the likely production of sulphate, along with CO2 (complete mineralization). The findings of the present study can be interpreted in terms of the above suggested mechanism, since there are performance similarities between Pseudomonas sp. and Ralstonia sp. in degrading environmental pollutants (Fig. 4). As mentioned before, results showed negligible change in ethanethiol concentration in control tests. Since there was no abiotic conversion, ethanethiol could be transformed to ethyl methyl sulphide, or consumed by the bacterial culture to produce sulphate. Considering an obvious increase in the concentration of sulphate during the biodegradation process (Fig. 5), transformation of ethanethiol was also not probable, since the transformation rate is high. Appearance of sulphate in the present study directs the focusing point towards enzymatic reactions involved in the energy production, either through sulphite oxidation (sulphite oxidase pathway), or a reversal of the activity of adenosine phophosulfate reductase (Fig. 4). Obtaining energy from the oxidation of reduced inorganic/ organic sulphur compounds to sulphate has been well described in the literature [26-28]. In these enzymatic reactions, production of sulphite is the first step through which the electrons transfer from sulphide, as the electron donor, to the electron transport system, located in the cell membrane, and subsequently to the final electron acceptor, which is oxygen in aerobic microorganisms. Continuation of these reactions occurs through assimilatory sulphate reduction pathway, which requires the expenditure of ATP and NADPH (as the reducing power). The catalytic role of

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sulphite reductase in these reductive types of reactions is conversion of sulphite to sulphide, which is usable by the bacterial cells, for biosynthesis of amino acids and proteins (Fig. 4). In the present study, R. eutropha performed as an ethanethiol oxidizer. The results were in agreement with the literature, where appearance of sulphate (Fig. 5) would be with the concomitant formation of ATP (dissimilatory pathway). According to Fig. 5, the most sulphate production was detected during the first day, whereas the most decrease in ethanethiol concentration was also observed. The fluctuations in sulphate concentration clearly indicate the ability of R. eutrophato perform the assimilatory reduction of sulphate. Similarly, An et al. reported sulfate production for ethanethiol oxidation under aerobic conditions, which indicated that ethanethiol oxidation takes place following an oxidative pathway via H2S, once the S-C bonds have been broken [7,10,29]. Visscher and Taylor also reported ethanethiol degradation to sulfate and CO2 with Thiobacillus sp. [30].

4. Conclusion Ethanethiol can be efficiently degraded by the pure culture of R. eutropha, in various initial concentrations. The Monod model successfully predicted kinetic data obtained from batch experiments. Ethanethiol was consumed as an energy source by R. eutropha, as there was no significant growth of biomass during the process. Detection of sulphate in the reaction mixture was indicative of biological oxidative pathway, through which the energy requirement is supplied.

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