Analysis of methane biodegradation by Methylosinus trichosporium OB3b.

The microbial oxidation of methane in the atmosphere is performed by methanotrophic bacteria that use methane as a unique source of carbon and energy. The objective of this work consisted of the investigation of the best conditions of methane biodegradation by methanotrophic bacteria Methylosinus trichosporium OB3b that oxidize it to carbon dioxide, and the use of these microorganisms in monitoring methods for methane. The results showed that M. trichosporium OB3b was capable to degrade methane in a more effective way with an initial microorganism concentration of 0.0700 g.L(-1), temperature of 30ºC, pH 6.5 and using 1.79 mmol of methane. In these same conditions, there was no bacterial growth when 2.69 mmol of methane was used. The specific rate of microorganism growth, the conversion factor, the efficiency and the volumetric productivity, for the optimized conditions of biodegradation were, respectively, 0.0324 h(-1), 0.6830 gcells/gCH4, 73.73% and 2.7732.10(-3) gcells/L.h. The final product of methane microbiological degradation, carbon dioxide, was quantified through the use of a commercial electrode, and, through this, the grade of methane conversion in carbon dioxide was calculated.

INTRODUCTION

Methane is one of the atmospheric trace gases that participates in photochemical reactions that occur in the atmosphere and it shows a great capacity for absorption of infrared radiation emitted by the surface of the planet (16,18). This gas is produced by methanogenic bacteria Methane is one of the atmospheric trace gases that under anaerobic conditions and it is consumed by methane participates in photochemical reactions that occur in the oxidizing bacteria (methanotrophic bacteria) in aerobic atmosphere and it shows a great capacity for absorption of environments (13,16). Moreover, methane is a greenhouse gas whose atmospheric concentration has been increasing significantly.

ABBREVIATIONS

DO600: optical density at 600 nm;

MMO: methane monooxigenase;

MSf: final amount of substrate (g);

MSo: initial amount of substrate (g);

MXf: final amount of biomass (g);

MXo: initial amount of biomass (g);

P: conversion of CH4 in CO2 (%)

QP: volumetric productivity (g/L.h);

t : time (h);

T: temperature (ºC);

X: cellular concentration (g.L-1);

Xf: final cellular concentration (g.L-1);

Xo initial cellular concentration (g.L-1);

YX/S: biomass yields (gcells/gsubstrate);

ΔX: variation of cellular concentration;

Δtlag: lag phase duration (h);

Δtexp: exponential phase duration (h);

μX: specific growth rate (h-1);

η: efficiency

ΔX: cellular concentration variation (g.L-1);

Δt: time variation (h)

Methane is released into the atmosphere by a number of sources, both natural and anthropogenic (relating to human activity). The anthropogenic sources correspond to approximately 60% of global methane emissions and they include fossil fuel production, animal husbandry (enteric fermentation in livestock and manure management), rice paddies, biomass burning and landfills. The main natural sources of methane include wetlands, oceans, gas hydrates and termites (14,18).

The microbial oxidation of methane in terrestrial environments is the only known net biological methane sink and the process consumes the equivalent of 1-10% of the total global emission of methane. Therefore, microbial oxidation of methane is a key regulator of the atmospheric concentrations of this important trace gas (3,10).

There are many microorganisms that play important role in the natural environment. For instance, they contribute to the geological cycles of elements and transformation of natural chemicals, and they are responsible for decontamination and detoxification of polluted environments (15). Methylotrophic bacteria are among these types of microorganisms. They are able to oxidize one-carbon compounds, such as methane, methanol, methylated amines and halomethanes, as sources of carbon and energy for growth and hence, they are considered to be important regulators of atmospheric methane fluxes in nature (3,5,12).

Methanotrophic bacteria, also named methane-oxidizing bacteria or methanotrophs, are a unique group of methylotrophic bacteria that are capable of using methane as a sole source of cellular carbon and energy. These microorganisms oxidize methane sequentially to methanol, formaldehyde, formate, and finally carbon dioxide (1,5,16): CH

The methanotrophic bacteria are divided into two main groups (types I and II) differing in a variety of aspects such as internal cellular structure, pathway of formaldehyde assimilation, which is the substance assimilated for producing cellular material, and predominant fatty acid chain length. Type I methanotrophs include the genera Methylomonas and Methylobacter and the type II methanotrophs include the genera Methylosinus and Methylocystis. A new group, type X, was added to accommodate methanotrophs similar to Methylococcus capsulatos, which has characteristics of both types I and II (3,5,6).

The oxidation of methane by methanotrophs is initiated by a specific enzyme called methane monooxygenase (MMO). All known methanotrophs are capable to form a particulate or membrane-bound MMO (pMMO). However, the capability to form soluble or cytoplasmic MMO (sMMO) has been observed only in some type II methanotrophs (5). While methanotrophic bacteria are the only group of microorganisms capable to degrade methane, methane monooxigenase is also able to oxidize a variety of environmental pollutants, including trichloroethylene, dicloroethylene and naphthalene. Because of this characteristic, methanotrophic bacteria can be used for remediation of contaminated environments (1,6–9,11).

The purpose of this article is to study the possible application of methanotrophic bacteria Methylosinus trichosporium OB3b in monitoring methods for methane. Thereby, it is important to determine the best conditions for the performance of these cells, and if they remain stable under variations of temperature, pH and amount of substrate, which are found in different environments.

MATERIALS AND METHODS

1. Materials

Methylosinus trichosporium OB3b was obtained from the Biotechnology Research Institute, Canada. The methane 2.5, with purity of 99.5%, was provided by White Martins Industrials Gases SA. All Reagents were purchased from Vetec Química Fina Ltda. (Brazil). Optical densities were determined using a UV-Visible spectrophotometer (CAM SPEC M302). The electrochemical measurements for the reading of the pH values were determined through phmether (Digimed DM20). A commercial CO2 electrode, Model 95-02 (Thermo Orium) was used for CO2 measurements.

2. Medium

Nitrate mineral salts (NMS) medium of Cornish et al. (2) was used throughout the experiments. This medium had the following composition (g.L-1): NaNO3, 0.85; KH2PO4, 0.53; Na2HPO4, 0.86; K2SO4, 0.174; MgSO4.7H2O, 0.037; CaCl2.2H2O, 0.007; ZnSO4.7H2O, 5.74×10-4; MnSO4.4H2O, 4.46×10-4; H3BO3, 1.24×10-4; CuSO4.5H2O, 2.5×10-4; Na2MoO4.2H2O, 9.6×10-5; CoCl2.6H2O, 9.6×10-5; KI, 1.66×10-4; FeSO4.7H2O, 1.12×10-2; H2SO4(2 μM). The pH of this medium was 6.5.

3. Growth Conditions

In all the experiments, the cultivation of the microorganism was performed in 250 mL erlenmeyers flasks containing 50 mL of culture medium (4) and a known volume of methane that was supplied by direct injection on media under specific conditions of temperature and in a rotary shaker (150 rpm). To obtain the initial inoculum, the cultivation was carried out at 30ºC with volume of 40 mL (1.79 mmol) of methane under the same conditions listed above.

The cell growth was followed by measuring the optical density at 600 nm (OD600) in the beginning of the experiment (0 h) and for every six hours. Thus, 1.5 mL aliquots of the medium were collected by a syringe, at time zero and each six hours, until optical density reached a maximum value indicating the beginning of the stationary phase.

The best biodegradation conditions of methane by M. trichosporium OB3b were studied for different initial concentrations of the microorganism, amount of substrate (methane), temperatures and pH values.

The experiments for different initial concentrations of inoculum and the analysis for different conditions of methane amount, temperature and pH were carried out in triplicate. The data were analyzed using Excel 2000 software and the averages values were used to generate the growth curves that were constructed as a function of the incubation time and the cellular concentration of the culture medium.

4. Carbon Dioxide Determination

Carbon dioxide, the end product of methane microbial oxidation, was quantified through a carbon dioxide electrode model 95-02 (Thermo Orion). This electrode uses a gas-permeable membrane to separate the sample solution from the electrode internal solution. Dissolved carbon dioxide in the sample solution diffuses through the membrane until an equilibrium is reached between the partial pressure of CO2 in the sample solution and the CO2 in the internal filling solution.

In all analytical procedures, a buffer solution was added to the standard solutions and to the samples to ensure that the pH was maintained between 4.8 to 5 required for the electrode operation and so that possible interferences were minimized.

For the carbon dioxide determination, the growth of M. trichosporium OB3b was conducted in conditions as ideal for its growth, and samples of 2.5 mL were collected at 0, 18, 24, 36, 48 and 60 hours, during the cell growth.

RESULTS AND DISCUSSION

1. Growth Profiles of Methylosinus trichosporium OB3b

1.1. Growth of M. trichosporium OB3b for Different Initial Concentrations of Inoculum

The aim of this study was to evaluate the effect of the inoculum size (initial concentration of inoculum) on the growth of M. trichosporium OB3b and thereby to determine which inoculum size should be employed to accelerate the process of methane biodegradation by the bacterium. This investigation occurred under specific conditions of methane amount, temperature (30ºC) and pH (6.5).

The proportion methane-air used in this step was 1:5 (v/v), lower than the proportion employed in the works done by Whittenbury et al. (17) and Fox et al. (4). Thus, in each flask of 250 mL, 50 mL was the volume occupied by NMS medium and the cells, and the 200 mL remaining were 40 mL (1.79 mmol) of methane and air.

The profiles of bacterial growth for different initial concentrations of inoculum studied (0.0208 g.L-1; 0.0481 g.L-1; 0.0700 g.L-1 and 0.1493 g.L-1), over a period of 126 hours, were determined by the relationship (Equation 1) between optical density at 600 nm (OD600) and cellular concentration (X) and they are presented in Fig. 1.

Figure 1

Cell concentration (X) obtained for different initial concentrations of inoculum (T = 30ºC, pH = 6.5 and 1.79 mmol methane).

In order to better evaluate and compare the characteristics of growth at differents inoculum size, the variations of cellular concentration (ΔX), the lag phase duration (Δtlag) and the exponential phase duration (Δtexp) were calculated and described on the Table 1. According to ΔX values, it is observed that for the initial inoculum concentration of 0.0700 g.L-1, a larger amount of cells was produced on 126 hours of growth, which may show greater methane degradation at the same period of time.

Table 1

Growth kinetic parameters for different initial concentrations of inoculum (T = 30°C, pH = 6.5, 1,79 mmol of methane; t = 126 h).

Parameters	Initial Concentrations of Inoculum (g.L-1)
	0,0208	0,0481	0,0700	0,1493
DX	0,125	0,138	0,141	0,100
Dtlag	30 h	24 h	18 h	24 h
Dtexp	24 h	36 h	48 h	36 h

Other observations that can be extracted from Table 1 are the duration time of the lag and exponential phases. Again, the best conditions for microorganism adaptation were obtained at initial cellular concentration of 0.0700 g.L-1, temperature of 30ºC, pH = 6.5 and 1.79 mmol methane, once the duration time of the adjustment phase (lag phase) was smaller. As for the variation of the exponential phase, in the same conditions, the Δtexp values showed that the microorganism stayed more time in the exponential phase of growth, demonstrating a greater potential for the degradation of methane. Therefore, the results imply that the inoculum size influences on the kinetics of methane biodegradation.

1.2. Effect of Methane Concentration

The application and use of the bacterium M. trichosporium OB3b for monitoring methane needs an important preliminary study of how this substance can influence the cellular metabolism of the microorganism. Thus, in this assay, the increase of initial methane concentration, from 1.79 to 2.69 mmol, was evaluated for initial cellular concentration of 0.0700 g.L-1, pH 6.5 and temperature of 30ºC. The volume occupied by medium and cells remained the same (50 mL). The results for this both conditions are presented in Fig. 2.

Figure 2

Effect of methane concentrations on the growth of M. trichosporium OB3b (Xo = 0.700 g.L-1; pH = 6.5 and T = 30ºC).

As shown in Fig. 2, the increase of methane was a factor that inhibited the bacterial growth. A relevant explanation to this fact is based on the first stage of methane microbial oxidation by methanotrophic bacteria, which is catalyzed by the enzyme methane monooxigenase (MMO). Only one atom of molecular oxygen is incorporated into methanol; the other atom is released as water (8). This enzyme requires oxygen as a cofactor for performing the initial oxidation of methane (11). Thus, when the proportion methane-air was modified, the amount of available oxygen changed too, and consequently, this modification influenced the stage of methanol formation, which is one of intermediaries belonging to the route of the methane biodegradation by methanotrophs.

Another explanation for the observed behavior can be based on the fact that, with the increase of methane concentration, the methanol production in the growth medium was more accelerated, and according to studies accomplished by Whittenbury et al. (17) and Cornish et al. (2), methanol is toxic to methanotrophic bacteria when presents in high concentrations. Therefore, the methanol generation might have inhibited the cellular metabolism of M. trichosporium OB3b.

1.3. Effect of Temperature

In this assay, the behavior of cellular growth at temperatures of 20ºC, 30ºC and 40ºC was evaluated for an initial cellular concentration of 0.0700 g.L-1, pH 6.5 and with 1.79 mmol of methane.

As shown in Fig. 3, M. trichosporium OB3b was capable to biodegrade methane only at temperatures of 20ºC and 30ºC. At 20ºC, the bacteria took a longer time for adapting to this condition. From Fig. 3, it is observed that the microorganism reached the growth phase (exponential phase) after 30 hours of cultivation, while at 30ºC, such fact happened after 18 hours. Thus, from this observation, it was concluded that the temperature of 30ºC was best suited for the continuity of the experiments with of M. trichosporium OB3b.

Figure 3

Effect of temperature on the growth of M. trichosporium OB3b (Xo = 0.0700 g.L-1; pH = 6.5 and 1.79 mmol of methane).

1.4. Effect of pH

In the biodegradation process of methane by bacteria M. trichosporium OB3b, methane is oxidized to carbon dioxide forming, as intermediaries, methanol, formaldehyde, and formic acid: CH

The employed electrode for CO2 analysis presents as a prerequisite the use of samples in a pH range between 4.8 and 5.2 for ensuring that all bicarbonate or carbonate, possibly present, would be converted to carbon dioxide. Therefore, in these experiments, the cultivation media NMS was acidified before the stage of cellular growth, until those required pH values were reached.

For an initial cell concentration of 0.0700 g.L-1, temperature of 30ºC and 1.79 mmol of methane, the behavior of M. trichosporium OB3b growth was studied on pH values of 4.8 and 5.2, in addition to the pH 6.5, in previous experiments. The results are shown in Fig. 4.

Figure 4

Effect of pH on growth of M. trichosporium OB3b (Xo = 0.0700 g.L-1, T = 30ºC and 1.79 mmol of methane).

From the results, it is noticed that there is no metabolic activity for acid values of pH. According to Whittenbury et al. (17), all methanotroph strains grow over a pH range of 5.8 to 7.4, and pH values of 6.6 to 6.8 appear to be the optimum for growth rate. Studies accomplished by Dianou and Adachi (3) report that M. trichosporium OB3b presents metabolic activity in pH range of 5.5 to 9, and that optimum pH is between 6.5 and 7.0. The results, obtained in this work, are thus in accordance to the related in the literature.

2. Study of Kinetic Parameters for Growth of M. trichosporium OB3b

The characteristic kinetic parameters of growth profile of M. trichosporium OB3b were determined under the conditions of temperature at 30ºC, pH of 6.5 and with 1.79 mmol of methane for each experiment using different initial cellular concentrations of inoculum.

The following parameters were calculated: the specific growth rate (μX), that indicates the increase in biomass per time unit and it is determined from the exponential phase (equation 2); the theoretical and process biomass yields (YX/S), determined as the mass of biomass produced by the mass of methane added to the medium (equation 3); the efficiency (η), obtained through the relationship among the theoretical and process biomass yields (equation 4); and volumetric productivity (QP), determined by dividing the final biomass concentration by the time (h) required to achieve complete methane utilization (equation 5). The calculed results for these growth parameters are reported in Table 2.

Table 2

Calculed kinetic parameters for different initial concentrations of inoculum (pH = 6.5, T = 30°C, and 1.79 mmol of methane).

Parameter	Initial Cellular Concentration of Inoculum (g.L-1)
	0.0208	0.0481	0.0700	0.1493
mx (h-1)	0.0691	0.0407	0.0324	0.0209
YX/S(theoretical) (gcells/gCH4)	1.014	0.9660	0.9279	0.7897
YX/S(process) (gcells/gCH4)	0.8878	0.7762	0.6830	0.3261
h (%)	87.84	80.54	73.73	41.38
Qp (10-3g/L.h)	2.3448	2.8410	2.7732	1.7632

In the analysis of methane biodegradation by M. trichosporium OB3b, the identification of the best conditions shows when this microorganism is indeed capable of the consumption of substrate (methane) in a fast way, reaching the exponential growth phase in smaller time. This observation is of great importance for the development of methane biossensor, since one of desirable characteristics for biosensor application is the obtaining of a fast analysis. Thus, the best conditions of methane biodegradation by M. trichosporium OB3b were observed in the experiments developed with initial cellular concentration of 0.0700 g.L-1, temperature of 30ºC, pH 6.5 and with 1.79 mmol of methane, although, in this situation, the best values do not have been observed for specific growth rate, biomass yield and efficiency.

3. Analysis of Methane Biodegradation Using a CO2 Sensor

In this phase, cultivations of M. trichosporium OB3b were performed under conditions established the best for microorganism growth: initial cellular concentration of 0.0700 g.L-1, temperature of 30ºC, pH 6.5 and 1.79 mmol of methane. The amount of carbon dioxide generated by methane biodegradation, during a growth period of 60 hours, was determined using the carbon dioxide sensor. The CO2 measurements were used for the calculation of methane consumed.

According to Whittenbury et al. (17) for each mol of methane consumed in the biodegradation process, 0.2 to 0.3 mol of carbon dioxide mol is formed. Thus, it was assumed that for each mol of CH4 consumed (16g), 0.25 mol of CO2 was produced (11g). The Equation 6 established this relationship and it was used to determine the amount of methane consumed in 60 hours. In this equation, the amount of CO2 produced was determined using the carbon dioxide sensor.

The conversion percentage (P) of CH4 in CO2 during the biodegradation process was calculated through the relationship between the amount of methane consumed and the initial amount (28.68 mg) of methane used in the experiments (Equation 7). The values obtained for carbon dioxide generated, methane consumed and conversion percentage are described in Table 3.

Table 3

Conversion percentage CH4/CO2 (P) (Xo = 0.0700 g.L'1; T = 30°C; pH = 6.5 and 1.79 mmol of initial methane (28.68 mg)).

Time (h)	Amount of CO2 produced (mg)	Amount of CH4 consumed (mg)	P(%)
0	0.158433	0.229728	0.801
18	0.260014	0.377020	1.31
24	0.463177	0.671606	2.34
36	0.564758	0.818899	2.86
48	0.632479	0.917094	3.20
60	0.683270	0.990741	3.45

The Fig. 5 shows the results of amount of carbon dioxide generated and amount of methane consumed, presented in Table 3, with amount of biomass produced for a period of 60 h, under the conditions studied in this experiment.

Figure 5

Results obtained of CH4 consumed (■),CO2 produced (▲) and biomass produced (□) (Xo = 0.0700 g.L-1, T = 30ºC, pH = 6.5 and 1.79 mmol of methane).

CONCLUSIONS

The best conditions for methane biodegradation by the bacterium M. trichosporium OB3b occurred at an inoculum cellular concentration of 0.0700 g.L-1, a methane-air proportion of 1:5 (for a system with 20% medium), temperature of 30ºC and pH equal to 6.5. In these conditions, a smaller time duration of the lag phase and a wider exponential phase of growth were observed. The maximum and minimum standard deviations did not exceed, respectively, the values of 1.0% and 9.7% in all experiments performed.

The best results for some kinetic parameters were not obtained in the more adequate conditions of methane biodegradation by the bacterium M. trichosporium OB3b. This can be explained by the fact that has an objective to find the conditions of initial cell concentration, amount of methane, temperature and pH which occurred a more efficient adaptation of the bacterium, which was noticed by reducing the duration time of the lag phase. The purpose of employment of microorganism M. trichosporium OB3b in the manufacture of the biosensor is to have a sensitive response in the shortest possible time, which is reached when it shows minor duration time of lag phase. These results should be taken into consideration for the employment of M. trichosporium OB3b in monitoring methods, and its application as biological component in a microbial biossensor of methane.